Recovery of the endangered Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys) Hanna Lee Mounce A thesis submitted for the degree of Doctor of Philosophy in Biodiversity Management Durrell Institute of Conservation and Ecology School of Anthropology and Conservation University of Kent August 2015 Recovery of the Endangered Maui Parrotbill 2 Acknowledgements To begin with, I would like to thank Carl Jones for introducing me to Jim Groombridge at a Durrell WCT workshop and suggesting the partnership of his supervision of my thesis. While Carl barely knew me at the time, his introduction and recommendation got this entire project underway. Secondly, I would like to thank Jim Groombridge for not only his willingness to take me on as a student but also for his enthusiasm for my project. Jim’s encouragement and guidance throughout this entire process was above and beyond my expectations for an advisor. I also appreciated his flexibility in advising a student based 11,000 km away and who decided not only to start other new research projects during this time period but also start a family. I would also like to thank David Leonard who instigated the funding for this project to cover a PhD student and also gave heaps of advice throughout the writing of every chapter of this thesis. I thank Kirsty Swinnerton, who served not only as an advisor and editor throughout this entire process but also as an amazing support and friend. I also must thank Kirsty for initially hiring me as a temporary field technician on Maui many years ago and introducing me to the amazing Hawaiian ecosystem and birds for which I am forever grateful. I also thank Jon Plissner. Jon has been a great mentor through my professional career, and when I was first offered a job in Maui I thought that it sounded too challenging and was considering turning it down. Jon told me to stop doubting myself and to take the job, thus kickstarting my conservation career in Hawaii. I thank David Duffy for supporting my thesis while I was employed with him through the University of Hawaii and for helping to coordinate and supervise a remote defense from UH. I thank Samit Kundu for getting me started in the genetics lab and Siobhan Simpson and Rachel Bristol for their continued help through my lab work as well as their support during my time in the UK. I also thank Claire Raisin for her help not only in the genetics lab but in getting the structure of my thesis started, helping me sort a place to live in the UK, Recovery of the Endangered Maui Parrotbill 3 teaching me how to salt the roads to be able to get to the lab, and helping me bridge the adjustment gap in coming to the UK. I thank all of the field crews of Maui Forest Bird Recovery Project for their dedicated hard work in strenuous field conditions which has provided much of the data used within this thesis. I thank my sisters, Nora Mounce and Emily Scholz, and my aunt, Caroline Tepley, for proofing my final thesis. I thank my son, Nolan Te Kahurangi Davidson, who showed up towards the end of my admission period, for being such a wonderful baby and letting me get this submission completed. I also thank my sister, Nora Mounce, and my mother, Charlene Mounce, for coming out to Maui to help watch Nolan while I was finishing writing up this thesis. I thank my entire family for their support and encouragement, especially my brother Phil (RIP) who left us during the end of my admission period and whom with I will miss celebrating this submission. Lastly I thank my husband, Jamie Davidson, who might not have known the substantial weight of this project as it was already in the works when we were engaged and married. I appreciate all of his support as well as taking care of our house and all my critters during my long trips to the UK. Acknowledgements specific to each self-standing chapter are located at the end of each chapter. Recovery of the Endangered Maui Parrotbill 4 Author’s Declaration All chapters of this thesis were written by HL Mounce with editorial suggestions from my supervisor, JJ Groombridge. All work was conducted while HL Mounce was employed by Maui Forest Bird Recovery Project, a part of Pacific Cooperative Studies Unit of Research Corporation of the University of Hawaii and supported through State of Hawaii Division of Forestry and Wildlife and United States Fish and Wildlife Service grants. There were numerous people that provided input and comments during the research and publication phases of this thesis. Individual contributions are listed in the chapters below. Co-author affiliations are listed as the institutions at the time of collaboration. Chapter 2 Determining productivity of the Maui Parrotbill, an endangered Hawaiian honeycreeper: The data collected for this chapter was a collaborative effort by the seasonal staff at Maui Forest Bird Recovery Project. Analysis was completed by HL Mounce with assistance of SR Wheeler, CL Rutt and J Milikowsky for the nest survival data. Co-authors all commented on drafts of the manuscript written by HL Mounce before it was submitted for publication. This chapter has been published, Mounce HL, Leonard DL, Swinnerton KJ, Becker CD, Berthold LK, Iknayan KJ, Groombridge JJ (2013) Determining productivity of Maui Parrotbills, an endangered Hawaiian honeycreeper. Journal of Field Ornithology 84: 32-39. Chapter 3 Management implications derived from long term re-sight data: annual survival of the Maui Parrotbill (Pseudonestor xanthophrys): This chapter used an 18 year encounter dataset from mark-resight data maintained by Maui Forest Bird Recovery Project. HL Mounce contributed field re-sights from 2006-2011. All other re-sights were provided by seasonal staff at Maui Forest Bird Recovery Project: TK Pratt, JC Simon, PE Baker, and SL Pimm. Analyses Recovery of the Endangered Maui Parrotbill 5 were undertaken by HL Mounce with input from EH Paxton. All co-authors reviewed and provided guidance and comments on the manuscript written by HL Mounce before submission for publication. This chapter has been published, Mounce HL, Iknayan KJ, Leonard DL, Swinnerton KJ, Groombridge JJ (2014). Management implications derived from long term re-sight data: annual survival of the Maui Parrotbill Pseudonestor xanthophrys. Bird Conservation International 24:316-326. Chapter 4 Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies: Blood samples were collected by HL Mounce 2006-2012 and the contemporary samples prior to 2006 were provided by Maui Forest Bird Recovery Project. Museum toe-pad samples were provided by the museums listed in the acknowledgements. The microsatellite library was developed by Genetic Identification Services, Inc. (California, USA). Guidance and training in laboratory techniques were provided by C Raisin and S Kundu. Microsatellite optimization was assisted by H Wickenden at University of Kent. STRUCTURE and GENELAND analyses were assisted by S Simpson at University of Kent. KJ Iknayan, CC Warren, and RL Hammond assisted with GIS mapping. All other lab work, analyses, and writing were undertaken by HL Mounce. AC Frantz provided R code for the simulation to model proportion of alleles that could theoretically be transferred with differing numbers of founder individuals. All co-authors provided comments and edits on a draft manuscript prior to submission for publication. This chapter has been published, Mounce HL, Raisin C, Leonard DL, Wickenden H, Swinnerton KJ, Groombridge JJ (2015) Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies. Conservation Genetics 16:71-84. Recovery of the Endangered Maui Parrotbill 6 Chapter 5 Extinction risk and reintroduction options for the endangered Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys): The data used in these models is a culmination of the chapters 2-4. Introduction to PVA methods and the use of their applications was initiated by KJ Swinnerton. CP McGowan and EH Paxton provided guidance on analyses and, in combination with the other co-authors, provided comments and edits. The R code used to evaluate translocation options was developed by CP McGowan and optimized by HL Mounce and CC Warren. This chapter has been submitted for publication and is currently under review. Recovery of the Endangered Maui Parrotbill 7 This research was approved and supported by American Bird Conservancy, Haleakala National Park, Hawaii Audubon Society, Maui Forest Bird Recovery Project, Pacific Cooperative Studies Unit (as a part of the Research Corporation of the University of Hawaii), The Nature Conservancy Hawaii, The Sidney Perry Foundation, Tri-Isle RC&D, Inc., San Diego Zoo Global, State of Hawaii Division of Forestry and Wildlife, and United States Fish and Wildlife Service. Recovery of the Endangered Maui Parrotbill 8 © The copyright of this thesis rests with the author. No quotation or information derived from it may be published without the prior written consent of the author and any such use should be explicitly acknowledged. Recovery of the Endangered Maui Parrotbill 9 Abstract Species recovery programs are tasked with reversing the declines of threatened and endangered speices and mitigating the threats to their populations. These goals must be accomplished in the face of a human dominated global landscape where habitat destruction and alteration is still increasing at an alarming rate. Hawaii, as common on many islands, has one of the highest historical extinction rates in the world. Here I use the Maui Parrotbill (Kiwikiu; Pseudonestor xanthophrys) to explore population demographics, genetics, population viability, and recovery options for one of Hawaii’s most critically endangered passerines (Maui Island endemic, pop. ~500). The accurate estimation of key demographic parameters is invaluable for making decisions about the management of endangered wildlife. Due to the challenges of data collection on a rare and cryptic species that inhabits remote terrain, such estimates are often difficult to obtain and reliable basic demographic data was not before available for parrotbills. First I look at parrotbill productivity estimates through both nest success and annual reproductive success measures. Secondly, I look at annual survival based on an 18 year encounter history. These studies both suggest population limitations may be coming from fecundity, and juvenile and female survival. Maui Parrotbill once inhabited a variety of forest types throughout Maui Nui but are now restricted to a single strip of wet forest 40-50 km2 in size. I quantified the levels of contemporary genetic diversity and structure in wild and captive Kiwikiu populations, and compared these genetic patterns to those observed within historical nuclear diversity derived from 100-year old museum samples enabling the design of a conservation translocation strategy that is tailored to the patterns of genetic structure across the species’ range. Lastly, I combine these data into a comprehensive population viability model to assess the risks to this population and evaluate the impacts of recovery options to the overall viability trajectory of a species. In planning for a reintroduction of parrotbills to areas of their former range, this model provides managers with demographic benchmarks that the new population will need to meet in order for the reintroduction to be successful. Recovery of the endangered Maui Parrotbill 10 TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................... 2 AUTHOR’S DECLARATION ............................................................... 4 ABSTRACT ................................................................................. 9 LIST OF TABLES .......................................................................... 13 LIST OF FIGURES ........................................................................ 15 CHAPTER 1. INTRODUCTION .......................................................................... 18 1.1 REINTRODUCTION AS A TOOL FOR SPECIES CONSERVATION .............................. 18 1.2 ECOLOGICAL AND GENETIC CONSIDERATIONS FOR REINTRODUCTIONS .................... 18 1.3 ADAPTIVE RADIATIONS OF BIRDS AND CONSERVATION OF ISLAND ENDEMICS ............... 22 1.4 HAWAII AS EXTINCTION CAPITAL OF THE WORLD ........................................ 23 1.5 CONSERVATION OF AVIAN SPECIES ..................................................... 24 1.6 ONGOING EXTINCTIONS ............................................................... 26 1.7 MAUI AND ITS ENDEMIC AVIFAUNA ...................................................... 28 1.8 THE MAUI PARROTBILL- A STUDY SYSTEM AND STRATEGY FOR SPECIES RECOVERY ....... 30 1.9 OVERALL AIM OF THE THESIS .......................................................... 34 1.10 THESIS OUTLINE ..................................................................... 35 CHAPTER 2. DETERMINING PRODUCTIVITY OF THE MAUI PARROTBILL, AN ENDANGERED HAWAIIAN HONEYCREEPER ............................................................ 37 ABSTRACT ................................................................................. 38 2.1 INTRODUCTION ....................................................................... 39 2.2 METHODS ............................................................................ 41 2.3 RESULTS ............................................................................. 45 2.4 DISCUSSION ........................................................................... 46 Recovery of the endangered Maui Parrotbill 11 2.5 FIGURES AND TABLES ................................................................. 49 CHAPTER 3. MANAGEMENT IMPLICATIONS DERIVED FROM LONG TERM RE-SIGHT DATA: ANNUAL SURVIVAL OF THE MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS) ......................................................................... 52 ABSTRACT ................................................................................. 53 3.1 INTRODUCTION ....................................................................... 54 3.2 METHODS ............................................................................ 56 3.3 RESULTS ............................................................................. 58 3.4 DISCUSSION ........................................................................... 59 3.5 FIGURES AND TABLES ................................................................. 64 CHAPTER 4. SPATIAL GENETIC ARCHITECTURE OF THE CRITICALLY-ENDANGERED MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS): MANAGEMENT CONSIDERATIONS FOR REINTRODUCTION STRATEGIES ........................... 71 ABSTRACT ................................................................................. 72 4.1 INTRODUCTION ....................................................................... 73 4.2 METHODS ............................................................................ 76 4.3 RESULTS ............................................................................. 83 4.4 DISCUSSION ........................................................................... 86 4.5 FIGURES AND TABLES ................................................................. 93 CHAPTER 5. EXTINCTION RISK AND REINTRODUCTION OPTIONS FOR THE ENDANGERED MAUI PARROTBILL (KIWIKIU, PSEUDONESTOR XANTHOPHRYS) .............. 101 ABSTRACT ................................................................................ 102 5.1 INTRODUCTION ...................................................................... 103 Recovery of the endangered Maui Parrotbill 12 5.2 METHODS ........................................................................... 106 5.3 RESULTS ............................................................................ 114 5.4 DISCUSSION .......................................................................... 115 5.5 FIGURES AND TABLES ................................................................ 121 CHAPTER 6. SYNOPSIS ............................................................................... 130 6.1 SUMMARY OF KEY FINDINGS .......................................................... 131 6.2 RECOMMENDATIONS FOR CONSERVATION MANAGEMENT ................................ 133 6.3 PROGRESS IN THE NEXT STEPS TO RECOVERY .......................................... 135 6.4 FUTURE RESEARCH .................................................................. 136 REFERENCES ........................................................................... 137 APPENDICES ............................................................................. 1-A APPENDIX A. CHAPTER 3 SUPPLEMENTARY MATERIALS .................................... 2-A APPENDIX B. CHAPTER 4 SUPPLEMENTARY MATERIALS .................................. 14-A APPENDIX C. CHAPTER 5 SUPPLEMENTARY MATERIALS .................................. 17-A APPENDIX D. EUROPEAN MOLECULAR BIOLOGY LABORATORY DEPOSITS .................. 26-A APPENDIX E. MAPA SAMPLE HISTORIES FOR GENETIC ANALYSES ......................... 47-A APPENDIX F. MAUI ALAUAHIO HAPLOTYPE DIVERSITY.................................... 53-A APPENDIX G. LEEWARD HALEAKALA EXPERIMENTAL RESTORATION PLAN ................. 60-A APPENDIX H. SUPPLEMENTAL FEEDING TRIALS FOR MAUI PARROTBILLS .................. 73-A APPENDIX I. PROGRESS IN NAKULA EXPERIMENTAL RESTORATION EFFORTS .............. 80-A APPENDIX J. HOME RANGE PATTERNS OF MAUI PARROTBILL AND MAUI ‘ALAUAHIO: IMPLICATIONS FOR PROPOSED TRANSLOCATIONS EFFORTS ................................ 87-A Recovery of the endangered Maui Parrotbill 13 LIST OF TABLES TABLE 1.6.1 RECENT (1968-1983) AND CURRENT (2014) STATUS OF ENDANGERED AND RECENTLY EXTINCT HAWAIIAN FOREST BIRDS .................................................. 27 TABLE 1.8.1 CURRENT AND HISTORICAL INVENTORY OF KIWIKIU IN CAPTIVITY ................. 35 TABLE 2.1 MAUI PARROTBILL ANNUAL REPRODUCTIVE SUCCESS BASED ON NUMBER OF PAIRS OBSERVED WITH FLEDGLINGS AT TWO STUDY SITES (FSB AND HR3) IN HANAWI NAR, 2008-2011 ............................................................................................. 52 TABLE 3.1 APPARENT SURVIVAL (Φ) AND ENCOUNTER PROBABILITY (Ρ) MODELS FOR MAUI PARROTBILLS GROUPED BY AGE (JUVENILE AND ADULT). SUBSCRIPTS INDICATE WHETHER PARAMETERS DIFFERED AMONG GROUPS (E.G. ΦAGE) OR TIME (ΦT) OR WERE CONSTANT (Φ.). OVERDISPERSION IS CORRECTED TO 1.605 (Ĉ) BASED ON GOODNESS OF FIT TEST ON GLOBAL MODEL (ΦAGE*TΡAGE*T). ,QAICC IS THE DIFFERENCE FROM THE BEST (LOWEST AICC) MODEL. AICC WEIGHT IS THE RELATIVE LIKELIHOOD OF EACH MODEL .................................................... 67 TABLE 3.2 APPARENT SURVIVAL (Φ) AND ENCOUNTER PROBABILITY (Ρ) MODELS FOR MAUI PARROTBILLS GROUPED BY SEX (JUVENILE BIRDS OMITTED). SUBSCRIPTS INDICATE WHETHER PARAMETERS DIFFERED AMONG GROUPS (E.G. ΦSEX) OR TIME (ΦT) OR WERE CONSTANT (Φ.). DATA UNDERDISPERSED (Ĉ = 0.900) BASED ON GOODNESS OF FIT TEST ON GLOBAL MODEL (ΦSEX*TΡSEX* T), Ĉ LEFT AT 1.00. ,QAICC IS THE DIFFERENCE FROM THE BEST (LOWEST AICC) MODEL. AICC WEIGHT IS THE RELATIVE LIKELIHOOD OF EACH MODEL .................................................... 68 TABLE 4.1 SAMPLE SIZE (N), NUMBER OF HAPLOTYPES (HP), HAPLOTYPE (GENE) DIVERSITY (HD), NUCLEOTIDE DIVERSITY (Π) AND THE FREQUENCIES (F) OF HAPLOTYPES A, B AND C FOR THE MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS) MTDNA ANALYSES .............................. 97 Recovery of the endangered Maui Parrotbill 14 TABLE 4.2 CHARACTERISTIZATION OF MAUI PARRTOBILL (PSEUDONESTOR XANTHOPHRYS) MICROSATELLITE LOCI. ANNEALING TEMPERATURE (TA), ALLELIC DIVERSITY (A), OBSERVED HETEROZYGOSITY (HO), EXPECTED HETEROZYGOSITY (HE), HARDY-WEINBERG EQUILIBRIUM TEST P VALUES AS IDENTIFIED BY GENEPOP V4.0.10 (PHWE) FOR EAST POPULATION. CHROMOSOME LOCATION ASSIGNMENTS ARE MAPPED AGAINST THE ZEBRA FINCH (TAENIOPYGIA GUTTATA) ..... 98 TABLE 4.3 ALLELIC AND HETEROZYGOSITY PATTERNS BASED ON MICROSATELLITE DATA ACROSS THE EAST, WEST, AND CAPTIVE MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS) POPULATIONS ... 99 TABLE 5.1 PARAMETER INPUT VALUES FOR THE BASE AND MODIFIED BASE VORTEX PVA MODEL USED FOR MAUI PARROTBILLS. INPUT PARAMETERS DERIVED FROM MOUNCE ET AL. 2013, MOUNCE ET AL. 2014, AND MOUNCE ET AL. 2015. VALUES IN BOLD HIGHLIGHT CHANGES BETWEEN THE BASE AND MODIFIED BASE MODELS ..................................................................... 132 TABLE 5.2 VORTEX MODEL RESULTS FOR THE BASE AND MODIFIED BASE MODEL FOR THE MAUI PARROTBILL POPULATION(S) WITH THE VIABILITY MEASURES OF STOCH-R (MEAN POPULATION GROWTH RATE), Λ (MEAN INTRINSIC RATE OF GROWTH), R0 (NET REPLACEMENT RATE), PS (PROBABILITY OF SURVIVAL), N-ALL (MEAN POPULATION SIZE FROM ALL ITERATIONS), N-EXTANT (MEAN POPULATION SIZE FROM EXTANT POPULATIONS), AND GD (GENE DIVERSITY) ............ 133 TABLE 5.3 RESULTS OF THE SENSITIVITY ANALYSIS IN VORTEX FOR THE MAUI PARROTBIILL METAPOPULATION BASED ON PARAMETER CHANGES IN THE “MODIFIED BASE MODEL.” BASE MODEL VALUES ARE IN GRAYSCALE IN EACH PARAMETER CATEGORY ................................... 134 TABLE 5.4 THE NUMBER OF MAUI PARROTBILL NEEDED FROM THE EAST, WEST, AND CAPTIVE POPULATIONS FOR REINTRODUCTION EFFORTS ACROSS THREE-YEAR REINTRODUCTION SCENARIOS ............................................................................................ 135 Recovery of the endangered Maui Parrotbill 15 LIST OF FIGURES FIGURE 1.3.1 ADAPTIVE RADIATION DIAGRAM OF HAWAIIAN HONEYCREEPERS FROM PRATT 2005 AND GALAPAGOS FINCHES FROM DARWIN 1959 ............................................... 23 FIGURE 1.7.1 THE HAWAIIAN ARCHIPELAGO WITH MAUI ISLAND OUTLINED .................... 29 FIGURE 1.7.2 MEAN ANNUAL RAINFALL FOR MAUI NUI HIGHLIGHTING THE DIFFERENT ECOSYSTEMS FORMED ON THE ISLAND OF MAUI ............................................................. 30 FIGURE 1.8.1 ADULT MALE MAUI PARROTBILL AND AN ADULT FEMALE ........................... 32 FIGURE 1.8.2 US FISH AND WILDLIFE SERVICE CRITICAL HABITAT FOR PSEUDONESTOR XANTHOPHRYS ON THE ISLAND OF MAUI WITHIN MONTANE MESIC FOREST ....................... 34 FIGURE 2.1 STUDY SITES WHERE THE PRODUCTIVITY OF MAUI PARROTBILLS WAS EXAMINED IN OUR STUDY. BOTH FRISBEE MEADOWS (FSB, 77 HA) AND POOULI CAMP (HR3, 56 HA) ARE LOCATED IN THE HANAWI NATURAL AREA RESERVE, ISLAND OF MAUI, HAWAII ............................. 53 FIGURE 3.1 STUDY AREA WITHIN THE HANAWI NATURAL AREA RESERVE, EAST MAUI, HAWAII . 69 FIGURE 3.2 DETECTION PROBABILITY VARIATION OVER TIME IN MAUI PARROTBILL SURVIVAL ANALYSES FOR AGE-SPECIFIC ANALYSIS (A) AND SEX-SPECIFIC ANALYSIS (B). ERROR BARS INDICATE STANDARD ERROR FOR EACH YEAR 1996-2011 ................................................ 70 FIGURE 3.3 APPARENT SURVIVAL PROBABILITY IN MAUI PARROTBILL BY SEX (A) AND AGE (B). ERROR BARS INDICATE STANDARD ERROR FOR EACH INDIVIDUAL GROUP ......................... 71 Recovery of the endangered Maui Parrotbill 16 FIGURE 3.4 APPARENT SURVIVAL FOR JUVENILE MAUI PARROTBILL VARIED MUCH MORE THROUGH TIME THAN DID ADULT APPARENT SURVIVAL ILLUSTRATING THE LIMITATIONS OF USING A SMALLER DATA SET MAY DISTORT THE RESULTS TO SUGGEST HIGHER JUVENILE SURVIVAL .................. 72 FIGURE 4.1 MAP OF HALEAKALA VOLCANO, EASTERN MAUI, HAWAII, USA, WITH THE CURRENT MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS) POPULATION RANGE, THE HANAWI NATURAL AREA RESERVE [MANAGED FOR FOREST BIRD PROTECTION BY THE STATE OF HAWAII, DIVISION OF FORESTRY AND WILDLIFE (DOFAW)] AND THE WAIKAMOI PRESERVE (MANAGED FOR FOREST BIRD PROTECTION BY THE NATURE CONSERVANCY HAWAII). THE NAKULA NATURAL AREA RESERVE ON THE LEEWARD SLOPE HAS BEEN IDENTIFIED AS A RESTORATION PRIORITY BY DOFAW AS WELL AS THE MOST SUITABLE LOCATION TO ESTABLISH A SECOND MAUI PARROTBILL POPULATION. THE CURRENT MAUI PARROTBILL RANGE GIVEN SUITABLE HABITAT IS 40 KM2, SHOWING THE SAMPLING LOCATION OF ALL WILD INDIVIDUALS INCLUDED IN THE GENETIC ANALYSES. THE PREDICTED RANGE CONSTRICTS TO 9 KM2 BY 2090 BASED ON CLIMATE CHANGE MODELS. THE POTENTIAL SUITABLE HABITAT IN 2090 GIVEN THE SAME CLIMATE CHANGE MODELS BUT LANDSCAPE LEVEL HABITAT RESTORATION WOULD MAINTAIN 40 KM2 ........................................................................... 100 FIGURE 4.2 MEAN LIKELIHOODS, LIKELIHOODS RATE OF CHANGE, AND ,K VALUES CALCULATED FROM NUMBER OF GENETIC CLUSTERS (K = 1-5) IN STRUCTURE BEFORE AND AFTER APPLYING THE EVANNO ET AL. (2005) CORRECTION TO THE OUTPUT IN STRUCTURE HARVESTER ........ 102 FIGURE 4.3 MAUI PARROTBILL (PSEUDONESTOR XANTHOPHRYS) ASSIGNMENT PROBABILITIES TO EACH POPULATION CLUSTER FROM GENELAND .............................................. 103 FIGURE 4.4 A POSTERIOR DISTRIBUTION OF THE TEMPORAL CHANGE IN HISTORICAL AND CONTEMPORARY EFFECTIVE POPULATION SIZE (NE) FOR THE PARROTBILL POPULATION AS DERIVED FROM TMVP ANALYSES FOLLOWING THE METHODS OF BEAUMONT (2003) ...................... 104 FIGURE 4.5 PROBABILITY VALUES FOR CAPTURING DIFFERENT PERCENTAGES OF THE TOTAL GENETIC DIVERSITY AVAILABLE IN THE EAST, WEST AND TOTAL WILD MAUI PARROTBILL (PSEUDONESTOR Recovery of the endangered Maui Parrotbill 17 XANTHOPHRYS) POPULATION MODELLED WHILE USING DIFFERENT NUMBERS OF INDIVIDUALS IN TRANSLOCATION EFFORTS ................................................................... 105 FIGURE 5.1 EAST MAUI, HAWAII (HALEAKALA VOLCANO) LAND PROTECTIONS THAT MAY BENEFIT NATIVE FOREST BIRDS. THE CURRENT MAUI PARROTBILL POPULATION RANGE OVERLAYS THE WINDWARD (NORTHEAST) RESERVES, HANAWI NAR AND TNC WAIKAMOI PRESERVE, AND THE NEWEST RESERVE, NAKULA NAR, IS SHOWN ON THE LEEWARD (SOUTHERN) SLOPE ............. 128 FIGURE 5.2 PROJECTED MEAN FINAL POPULATION SIZES (N-ALL) FOR MAUI PARROTBILL UNDER BASE AND MODIFIED BASE MODELS IN VORTEX. SOLID BLACK LINE REPRESENTS THE “BASE MODEL POPULATION METAPOPULATION”, SOLID BLACK LINE WITH DOT REPRESENTS THE “MODIFIED BASE MODEL METAPOPULATION”, AND BROKEN GRAY LINE WITH BOX REPRESENTS THE “MODIFIED BASE MODEL EAST POPULATION”, AND DOTTED GRAY LINE WITH DIAMOND REPRESENTS THE “MODIFIED BASE MODEL WEST POPULATION”. WHERE N-ALL BECOMES ZERO INDICATES POPULATION COLLAPSE IN 100% OF SIMULATIONS (1000 RUNS) ..................................................... 129 FIGURE 5.3 FEMALE MAUI PARROTBILL POPULATION TRAJECTORIES FOR THE THREE EXISTING POPULATIONS (A- EAST, B- WEST, AND D- CAPTIVE) AND THE PROPOSED REINTRODUCED LEEWARD POPULATION (C- LEEWARD). POPULATION ESTIMATES IN FOR A, B, C, AND D ARE BASED ON A PROPOSED THREE-YEAR REINTRODUCTION SCENARIO WHEREIN THE CAPTIVE FLOCK IS AUGMENTED TO SOURCE 1 FEMALE/YEAR IN COMBINATION WITH TRANSLOCATIONS FROM EXISTING WILD POPULATIONS (SCENARIO IV). DEMOGRAPHIC PARAMETERS FOR WILD POPULATIONS ARE SET TO VALUES FROM MOUNCE ET AL. 2013 AND MOUNCE ET AL. 2014 ........................................... 130 FIGURE 5.4 MAUI PARROTBILL POPULATION TRAJECTORIES FOR THE REINTRODUCED LEEWARD POPULATION BASED ON REINTRODUCTION SCENARIO IV. SHOWN FIRST ARE POPULATION TRAJECTORIES PREDICTING TRENDS WITH ANNUAL FECUNDITY, FEMALE SURVIVORSHIP, AND YOUNG OF THE YEAR SET AS IN MOUNCE ET AL. 2013 AND MOUNCE ET AL. 2014 (A). NEXT (B, C, AND D) ARE SHOWN WITH PARAMETERS INCREASED BY 5% (B), 10% (C), AND 20% (D) BASED ON POTENTIAL BENEFITS OF THE LEEWARD MESIC HABITAT. VALUES SHOWN ARE THE NUMBER OF ADULT FEMALES IN THE POPULATION ............................................................................................ 131 Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 18 Chapter 1 Introduction 1.1 REINTRODUCTION AS A TOOL FOR SPECIES CONSERVATION In order to recover endangered species, their populations need to be returned to areas of their former range. More often than not, these species have been extirpated from former areas of their range. The release of such organisms back into historical habitats is known as a reintroduction (IUCN 1987). As this conservation tool is a varied and complex method of recovery, there are a variety of terms that must be defined to cover these actions. When moving threatened and endangered species where conservation is the main objective, these reintroductions are considered also conservation translocations (Hodder and Bullock 1997). For simplicity from here on, we will use the terms reintroduction to refer to both reintroductions and conservation translocations. These actions are not new to species recovery efforts. Reintroductions have been widely used throughout conservation programs worldwide and the number of such efforts is growing exponentially each year (Seddon et al. 2012). There are many specific reasons for using reintroduction as a conservation tool in a given species, but put in its simplest form, restoring a species to an area where it has been extirpated will increase the total number of individuals for that given species over time and reduce extinction risk. Still, reintroductions should be approached with caution as despite notable successes, there is also a high rate of failure (Griffith et al. 1989; Wolf et al. 1996). This thesis introduces a system and an endangered species that could benefit from a well-planned and implemented reintroduction. Each section within is aimed at synthesizing data to be used to design such a reintroduction and aims to provide managers with the best information to facilitate responsible decision-making and an increased chance of success. Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 19 1.2 ECOLOGICAL AND GENETIC CONSIDERATIONS FOR REINTRODUCTIONS Reintroductions are now widely applied as a conservation tool yet despite their extensive use, the ecological and genetic implications of using reintroductions as a tool for species recovery are still poorly understood (Robichaux et al. 1997; Groombridge et al. 2012). Furthermore, reintroductions are still often unsuccessful. The increasing use of reintroductions is not unexpected due to the accelerating rate of global ecological change (habitat loss and fragmentation, biological invasions and climate change) and the corresponding pressure on biodiversity. The International Union for Conservation of Nature (IUCN) Reintroduction Specialist Group recently updated their guidelines for reintroductions as a response to these increases in usage (IUCN/SSC 2013). The original guidelines were developed out of necessity in the face of rising numbers of global reintroductions taking place and many of these reintroductions observed to be failing (IUCN 1998). Reasons for these failures have been attributed to poor quality habitat at release sites (Moorhouse et al. 2009; White et al. 2012), too few individuals being released (Wolf et al. 1998; Fischer and Lindenmayer 2000), captive sourced individuals being less suitable than wild individuals (Jule et al. 2008; Aaltonen et al. 2009), depredation risks (Moorhouse et al. 2009; White et al. 2012), and failure to remedy the original causes for the species’ decline (Fischer and Lindenmayer 2000). Unfortunately, the lack of post-release monitoring after many reintroductions leaves the causes and timing of many failures still unknown (Seddon et al. 2007). While some of these ecological consequences may be difficult to predict, we can attempt to account for the genetic considerations pre-release. Genetic factors play an important role in assessing a species extinction risk, and thus genetic management too should be an important consideration for designing a successful reintroduction program (Groombridge et al. 2012). We can assume that reintroduced populations that are founded Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 20 from small numbers of individuals may suffer the same associated genetic problems as other small or bottlenecked populations. These processes such as loss of genetic variation, inbreeding and inbreeding depression have all been widely accepted as increasing the risk of extinction in small populations and would therefore be expected to operate in the same manner on a reintroduced population (Keller and Waller 2002; Frankham 2005). Although we have knowledge on the expected genetic mechanisms at play, we cannot always predict how these genetic processes will interact with the ecological processes at a release site. In other words, how a certain level of inbreeding affects the trajectory and/or fitness of a population may be predictable in the species’ current range; but, once they are faced with lower quality habitat and/or increased depredation risk and/or naïve birds sourced from captivity, these effects become much more difficult to predict. Every reintroduction is unique and the incorporation of high quality data on genetic and ecological factors must be considered together in order to formulate a well-designed reintroduction. Even with the best available data, the ideal reintroduction scenarios for a given species are not often possible. There are frequently various threats that managers are unable to mitigate in selected release sites. For a species to be successful in the long-term, a reintroduced population needs to be able to survive these threats while also retaining adequate levels of genetic diversity to adapt to future environmental change (Reed and Frankham 2003; Keller et al. 2012). When attempting to restore small and declining populations, models often predict idealized numbers of individuals adequate to maintain this genetic diversity that are as large as the total global population itself. In these cases, we may have to manage the genetics of the reintroduced populations in order to maximize the retention of genetic diversity but know that we are unlikely to achieve the ideal population sizes. Most species conservation programs do not have the luxury of time and are often faced with rapidly declining populations. These programs will most Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 21 often be faced with the need to make a decision to reintroduce a population with a lower genetic potential. However, this option may be preferable to taking no action at all. Many of these actions have been taken on islands, which is not unexpected given that islands hold a large percentage of our global avifauna diversity (Stattersfield et al. 1998). These actions also are trying to counteract some of the most rapidly declining avian populations. Some estimating that more than half of island bird species may be functionally extinct by the turn of the century (Şekercioğlu et al. 2004). Many of these islands have already suffered massive extinctions (ex. New Zealand, Craig et al. 2000; Mauritius, Cheke and Hume 2008), and reintroductions have become a common management practice for recovering the remaining island species (Jones and Merton 2012). Due to the inherent nature of island populations, these species do not have the luxury of large numbers of individuals that continental conservation programs might have and often rely on small numbers of founding individuals. Although historically the success of reintroductions has been positively correlated with the numbers of animals released (Griffith et al. 1989; Wolf et al. 1996. Wolf et al. 1998), there are many examples of successful and well-known island reintroductions around the world that started from low numbers of individuals. Some examples of these include but are not limited to: • The Echo Parakeet (Psittacula echo) in Mauritius which recovered from ~10 individuals to more than 550 (Jones and Merton 2012). • The South Island Saddleback (Philesturnus carunculatus) in New Zealand which recovered from ~36 individuals to more than 2000 (Masuda and Jamieson 2013). • The Black Robin (Petroica travers) in New Zealand which recovered from ~5 individuals to ~200 (Butler and Merton 1992). • The Laysan Duck (Anas laysanensis) in Hawaii which recovered from 42 founders on a new island to a breeding population of more than 500 (Reynolds et al. 2013). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 22 • The Mauritius Kestrel (Falco punctatus) in Mauritius which recovered from ~4 individuals to more than 500 (Jones and Merton 2012). • The Nihoa Millerbird (Acrocephalus familiaris kingi) in Hawaii which has recovered from 50 founders on a new island to a breeding population of more than 160 (Dalton et al. 2014) 1.3 ADAPTIVE RADIATIONS OF BIRDS AND CONSERVATION OF ISLAND ENDEMICS Adaptive radiation is defined as “the evolution of ecological diversity within a rapidly multiplying lineage” (Schulter 2000). While naturalists have documented adaptive radiations of birds on islands across the globe, nowhere else in the world has there ever been such a complex diversity of avian species that have evolved in such a small place, in such a short amount of time as in the Hawaiian Islands. The Hawaiian Islands are large and extremely isolated. Thus, the evolutionary processes that occurred on them created a diverse flora and fauna and a multitude of new species from relatively very few founders. The Hawaiian Honeycreepers comprise more than 50 species and the present count is continuing to grow as new information is contributed from the recent fossil record (James and Olson 2005; Pratt 2005; James and Olson 2006). The classic example of adaptive radiation popularly used in literature is that of the Galapagos finches, a group of only 14 species (Figure 1.3.1). Hawaiian Honeycreepers are the only diverse radiation of birds in the Hawaiian Islands, a fact that may account for the high number of species. While there are a few lineages of crows, monarchs, and thrushes across the Hawaiian Islands, they evolved to produce comparatively few species (Pratt 2009). Therefore, the honeycreepers were exhibiting such an array of different morphological and physical traits as to exploit the majority of different environments in the islands available for avian species. Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 23 At the heart of the evolutionary mechanism of adaptive radiation is the process of competition (Schulter 2000). The pressure of intense intraspecific competition leads individuals to exploit novel food resources, precipitating specialization in foraging strategies and life history changes (Pratt 2009). While this specialization may have allowed high densities of forest birds to coexist in relatively small areas/islands, these narrow feeding niches also may set up Hawaiian Honeycreepers to be highly vulnerable to environmental change. 1.4 HAWAII AS THE ‘EXINCTION CAPITAL’ OF THE WORLD The main Hawaiian Islands sit about 4000 km from the nearest continent and about 3200 km from any other high-island groups of any size (Howarth et al. 1988). The Hawaiian Islands, the most isolated archipelago in the world, often are referred to as the extinction capital of the world, having lost more bird species than anywhere else on earth. Similar as in other oceanic islands, the arrival of humans in Hawaii, and the non-native animals, diseases, and plants brought with them, had devastating effects on native flora and fauna which continue today. Figure 1.3.1 Adaptive radiation diagram of Hawaiian Honeycreepers from Pratt 2005 (right) and Galapagos finches from Darwin 1959 (left).Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 24 The Hawaiian ecosystems were profoundly transformed first by the Polynesians approximately a millennium ago and then again by Westerners in the past two centuries. As little as 10% of the original avifauna of Hawaii persists today (Pimm et al. 1995), and almost all of the species that remain have undergone drastic population declines and range contractions (Banko and Banko 2009). Small ranges and minimal numbers of individuals, as exist in these island birds, predispose such species to extinction, and thus they are highly vulnerable to anthropogenic changes in their ecosystems (Hughes 2004; Sodhi et al. 2004). Each of these human arrivals brought a range of threats to the environment. These threats included island-wide habitat destruction and degradation. Forest size, structure, and compositions were all altered after Polynesian contact by the introduction of rats (Rattus exulans), fire and agriculture (Pratt and Jacobi 2009). Nearly all bird species are impacted by massive deforestation (Davies et al. 2000; Zanette et al. 2000). These forests were further degraded by the European introduction of ungulates (hoofed-mammals). In places where these animals did not destroy the forest entirely, they removed the understory vegetation and disrupted the overall plant communities (Pratt and Jacobi 2009). Compounding these habitat alterations was the introduction of a variety of non-native predators and competitors (Lindsey et al. 2009). Following the introduction of mammals to the Hawaiian Islands, almost all flightless species disappeared (Olson and James 1991) and those avian species that survived nested as high in the trees as possible (Woodworth and Pratt 2009). Possibly the most devastating introduction was that of foreign disease. Avian malaria (Plasmodium relictum) is thought to have arrived to the islands around 1920 and the widespread susceptibility of native forest birds was devastating with only a few lowland populations developing resistance (Woodworth et al. 2005; Woodworth and Pratt 2009). Avian malaria and its vector, mosquitoes (Culex quinquefasciatus), are limited by cool Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 25 temperatures and thus have restricted native birds to high elevation refuges. Elsewhere these introductions of disease, predation, habitat degradation and competition have reduced bird populations (Savidge 1987; Mack et al. 2000), but the culminating effects these had on small bodies of land and high levels of endemism in Hawaii were devastating. 1.5 CONSERVATION OF AVIAN SPECIES Birds offer a unique opportunity to track global changes in biodiversity. This is because birds occur in nearly every habitat on Earth, and they are often one of the most visible forms of wildlife within a given area. As a result of this visibility, humans are often very familiar with the avian species around them. Despite our species awareness and often affinity for such species, human activities are undoubtedly to blame for the global biodiversity crisis that we are currently facing (Issac et al. 2007; Jones and Merton 2012). Dubbed the “sixth extinction” (Barnosky et al. 2011), this is the only global mass extinction that has been triggered by human activities (Leakey and Lewin 1996). One in eight of the world’s bird species is globally threatened (BirdLife International 2014a), and with predicted increases in extinction rates, estimates forecast that we could lose up to 20% of all remaining vertebrate species within the next century (Baillie et al. 2010; Sinervo et al. 2010). Islands, being particularly vulnerable, may suffer even higher losses than these predictions. While island species deserve immediate conservation attention, there are not only numerous biological challenges facing their recovery but social and political barriers as well. The reductions in numbers of island species have in many cases isolated the birds from people. This lack of awareness and identity of such species has become a key social factor that affects support for avian conservation (Leonard 2009). Furthermore, the isolation of islands does not facilitate the appreciation and understanding of rare and endangered island fauna to a larger global audience (Pratt et al. 2009b). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 26 Conserving avian biodiversity is an overwhelming task and it can be easy to become overly pessimistic in thinking about the future of endangered and imperiled species throughout the world. We are in a state of increasing human populations and the associated increasing human resource demands make this even more challenging. Avian conservation programs should focus on removing and lessening the adverse effects on these species in the immediate future. By minimizing these measurable threats, recovery programs can reverse the trajectory of decline in these species one at a time. As discussed earlier, this approach has successfully brought numerous species back from the brink of extinction (see examples in previous section 1.2). Furthermore, there are many reasons for guarded optimism when considering the conservation of Hawaiian species in particular. Despite the many that have been lost, the surviving Hawaiian Honeycreeper populations are still large enough to recover and there are significant areas of protected native habitat remaining for these species (Pratt et al. 2009b). There has also been a marked increase in public awareness since the first Hawaiian Forest Bird Surveys (1976-1983) brought international attention to the plight of Hawaiian birds (Scott et al. 1986) and with this, more support for conservation efforts (Dayer et al. 2006). However, these facts can only provide guarded optimism because there are still major obstacles to overcome as these species battle to survive in the face of ongoing alien introductions, climate change, and growing costs of implementing recovery actions (an estimated $4.6 million per species per year; Leonard 2009). 1.6 ONGOING EXTICTIONS Although there are multitudes of global efforts in avian conservation and the preservation of individual species, many still think of extinctions as things that happened in the past. School children are often taught to associate extinction with the Jurassic period but this ecological phenomenon Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 27 is still a very current and ongoing process, especially on islands. While extinction rates have varied through time, the past 400 years have experienced a vertebrate extinction rate of 20-200 times that of “natural” or “background” extinction rates throughout history (Groombridge and Jenkins 2002) and as much as 100 times greater over the past 100 years (Mace et al. 2009). As discussed above and illustrated with the incidents of mass extinctions in the Hawaiian Islands, the process of extinction has turned from a natural process to a human-induced one. An exact estimate of lost species is difficult to calculate as many Hawaiian species have not been seen in many years are still listed as endangered with the United States Fish and Wildlife Service. Three such forest bird species exist on Maui. The Maui Akepa (Loxops ochraceus), endangered, was last heard (but not seen) in 1995 (Reynolds and Snetsinger 2001), the Nukupuu (Hemignathus lucidus), endangered, has not been seen since 1996 (Reynolds and Snetsinger 2001), and the last known Poouli (Melamprosops phaeosoma) died in captivity at the end of 2004 (BirdLife International 2014b). The recent population declines and extinctions that have occurred in Hawaii in less than a century are numerous (Table 1.6.1). Of the avian species that are left in the Hawaiian Islands, only 11 are common enough to suggest their future may be secure. Although habitat still exists for other Maui species which have not been seen in many years, extensive work being done on the conservation of other Maui avifauna has failed to yield any more recent sightings of these species (MFBRP unpublished data). Table 1.6.1 Recent (1968-1983) and Current (2014) Status of Endangered and Recently Extinct Hawaiian forest birds (Banko and Banko 2009, Gorreson et al. 2009, VanderWerf 2013). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 28 Species Hawaiian Island Recent Population 1968-1983 Current Population Last Seen USFWS Listing Alala (Corvus hawaiiensis) Hawaii Rare Extinct in the wild 2002 Endangered Ooaa (Moho braccatus) Kauai Rare Extinct 1987? Endangered Bishop's Oo (Moho bishopi) Maui Rare? Extinct 1981? Millerbird (Acrocephalus familiaris) Nihoa Abundant ~380 . Endangered Kamao (Myadestes woahensis) Kauai Rare Extinct 1985 Endangered Olomao (Myadestes lanaiensis) Molokai Rare Extinct 1980 Endangered Puaiohi (Myadestes palmeri) Kauai Rare ~500 . Endangered Laysan Finch (Telespiza cantans) Laysan Abundant > 10,000 . Endangered Nihoa Finch (Telespiza ultima) Nihoa Abundant ~3,000 . Endangered Ou (Psittirostrata psittacea) Kauai Rare Extinct 1989 Endangered Ou (Psittirostrata psittacea) Hawaii Rare Extinct 1987 Endangered Palila (Loxioides bailleui) Hawaii Rare ~1,260 . Endangered Maui Parrotbill (Pseudonestor xanthophrys) Maui Rare ~500 . Endangered Greater Akialoa (Hemignathus obscurus) Kauai Extinct Extinct 1969 Endangered Nukupuu (Hemignatus lucidus) Maui Rare Extinct 1996 Endangered Nukupuu (Hemignatus lucidus) Kauai Rare Extinct 1990s? Endangered Akiapolaau (Hemignathus munroi) Hawaii Rare ~ 1,900 . Endangered Akikiki (Oreomystis bairdi) Kauai Rare ~1,300 . Endangered Hawaii creeper (Oreomystis mana) Hawaii Rare ~14,000 . Endangered Oahu Alauahio (Paroreomyza maculata) Oahu Rare Extinct 1978 Endangered Kakawahie (Paroreomyza flammea) Molokai Extinct Extinct 1963 Endangered Akekee (Loxops caeruleirostris) Kauai Rare ~3,100 . Endangered Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 29 Maui Akepa (Loxops ochraceus) Maui Rare Extinct 1980 Endangered Hawaii Akepa (Loxops coccineus) Hawaii Rare ~12,000 . Endangered Akohekohe (Palmeria dolei) Maui Rare ~6,700 . Endangered Poouli (Melamprosops phaeosoma) Maui Rare Extinct 2004 Endangered 1.7 MAUI AND ITS ENDEMIC AVIFAUNA Maui is the second largest island in the Hawaiian archipelago (1,883 km2) and is estimated to be around 800,000 years old (Howarth et al. 1988) (Figure 1.7.1). This volcanic island was formed from two volcanoes that overlapped one another. The last, erupting around 1790, is considered a dormant volcano but not extinct. The mountain of Haleakala forms the eastern half of the island. This summit rises 10,023 feet above sea level and has a peak exposed to both wet windward trade winds and drier leeward air (Giambelluca et al. 2013; Figure 1.7.2). This allows one mountain to host a wide variety of different ecosystems from dry shrub lands to wet rainforests in a relatively small area. Figure 1.7.1. The Hawaiian archipelago with Maui Island outlined in yellow. Today Maui is primarily a tourist destination. Visitors come seeking a lush tropical paradise and assume that the lowland vegetation that they are seeing is native to Hawaii. This belief is far from the truth, and much of the native vegetation on Maui has been destroyed or highly degraded. Likely due to its inaccessibility, the windward forests of Haleakala house some of the most pristine wet forests left in Hawaii. Contrastingly, the leeward forests Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 30 have all but disappeared since the initial arrival of Polynesians. It is estimated that less than 10% of the original forests of leeward Haleakala remain today. In the Hawaiian archipelago, no island has had more extinctions than Maui. Of those that remain, Maui has three forest bird species that are found across multiple islands, the Apapane (Himatione sanguinea), the Hawaii Amakihi (Chlorodrepanis virens), and the Iiwi (Drepanis coccinea). It also has three extant endemic forest bird species. The Maui Alauahio (Paroreomyza montana) is the most abundant with a population estimate of at least 55,000 (Brinck et al. 2012). The critically endangered Akohekohe, or Crested Honeycreeper (Palmeria dolei), is the next most abundant with a population estimate of 3,753 (± 373) (IUCN 2011; Scott et al. 1986). Lastly, the critically endangered Maui Parrotbill, or Kiwikiu, (Pseudonestor xanthophrys) is the rarest of endemic Maui forest birds. Maui Parrotbills have a population estimate of 502 (272-732 95% CI) individuals (Scott et al. 1986) across their current range of approximately 50 km2 (or 421 (209-674 95% CI) individuals within a single 40 km2 area (Brinck et al. 2012)) on windward Haleakala. Figure 1.7.2 Mean annual rainfall for Maui Nui highlighting the different ecosystems formed on the Island of Maui (Giambelluca et al. 2013). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 31 1.8 THE MAUI PARROTBILL- A STUDY SYSTEM AND STRATEGY FOR SPECIES RECOVERY It is incredible to think that on an island as small as Maui, a species could go undetected for long periods of time. Indeed the Maui Parrotbill disappeared after its initial descriptions by Rothschild in 1893-1900 and was thought to be extinct until it was rediscovered in 1950 (Richards and Baldwin 1953). Because of this extreme rarity, Maui Parrotbill had no surviving Hawaiian name as Hawaiian was not a written language, and it had been lost over time. In 2010, the Hawaiian Lexicon Committee gave Maui Parrotbills a new Hawaiian name, Kiwikiu. Kiwikiu is named for its curved sickle-shaped bill, its whistle, and the weather on Haleakala, the mountain on which it now survives. The natural history of this species was still largely unknown until the first active nest was discovered 1993 (Van Gelder 1993; Lockwood et al. 1994). Further ecological studies did not begin until the 2000s after the formation of Maui Forest Bird Recovery Project, an organization focused on the development and implementation of techniques that recover Maui's endangered birds and restoration of their habitats through research, development, and application of conservation techniques. Maui Parrotbills are part of the subfamily Drepanidinae, of Fringillidae (finch family), with the other Hawaiian Honeycreepers. They were previously considered part of the “Hawaiian finches” like the Palila (Loxioides bailleui) and Laysan Finch (Telespiza cantans) (Berger 1981), but more recent phylogenetic studies have revealed the Akiapolaau (Hemignathus munroi) on the Island of Hawaii as the parrotbill’s closest extant relative (Lerner et al. 2011). Maui Parrotbills are is one of the largest extant honeycreepers (length 14 cm, males 25 g, females 20 g) with a relatively large, parrot-like bill. It uses Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 32 this bill to search for concealed invertebrates as well as rip into branches, bark, and stems, and to bite open fruit. Parrotbills are also one of the most sexually dimorphic of the honeycreepers with the males being approximately 20% larger than the females (Simon et al. 1997) (Figure 1.8.1). Figure 1.8.1 Adult male Maui Parrotbill (right) and an adult female (left). Parrotbills have very unique breeding behaviors. Males and females remain in established pairs year-round and only tend to re-pair after the death of a mate (MFBRP unpublished data). Pairs maintain home ranges of 9-11 ha throughout their range (Warren and Mounce 2014). Most nesting occurs January-July, but nests have been found in all months of the year except September, and pairs defend their territories year-round (MFBRP unpublished data). Pairs often will nest up to three times until they are successful, laying a single egg, although there have been several observations made of pairs caring for two offspring (Simon et al. 1997, MFBRP unpublished data). Once the chick fledges, it has a juvenile dependence period of up to 18 months (Simon et al. 1997, MFBRP unpublished data). To balance this low reproductive potential, parrotbills have a long life. The oldest known parrotbill is a minimum of 16 years old (Mounce et al. 2012). Parrotbills occur in mesic and wet native montane forests. These remnant Maui forests are dominated by ohia (Metrosideros polymorpha), olapa (Cheirodendron trigynum), kolea (Myrsine lessertiana), and kawau (Ilex anomala). More important for their feeding ecology than this overstory, is the diverse understory of native plants including akala (Rubus hawaiensis), ohelo Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 33 (Vaccinium calycinum), alani (Melicope spp.), pilo (Coprosma spp.), and kanawao (Broussaisia arguta) (Simon et al. 1997, Stein 2007). Parrotbills forage mainly on the woody portions of native shrubs and trees using their powerful bills to excavate bark and wood for insects and other arthropods. Feeding primarily on the larvae and pupae of beetles and moths (as much as 90% coming from Lepidoptera larvae (Peck et al. 2015), they also forage on soft fruits to extract invertebrates (Perkins 1903, Mountainspring 1987, Simon et al. 1997, Stein 2007). Parrotbills are range restricted to high elevation forests above the “malaria line” on east Maui. This area gives the species only 50 km2 of available habitat (USFWS 2006). This is a massive constriction of their historical habitat as they were once found on the island of Molokai as well as in low elevation forests (Gorreson et al. 2009). The most comprehensive population survey for parrotbills was done in 1980 as part of Hawaii Forest Bird Surveys, run by the State of Hawaii. The population estimate at that time was 502 ± 116 individuals (Scott et al. 1986). While some subsequent surveys have shown densities in certain portions of their range as similar to 1980 (Simon et al. 2002), none have been able to conclusively show that the population is stable across their range (Gorreson et al. 2009). An intense population survey within 36.9 km2 of parrotbill habitat in 2011 estimated 421 individuals (209-674 95% CI), but excluded some additional areas of known parrotbill habitat (Brinck et al. 2012). Regardless of exact numbers, recent distribution surveys have suggested that this habitat area is “full” (MFBRP unpublished data). Creating a second population of Maui Parrotbills has been identified as key to their long-term survival (USFWS 2006). Furthermore, USFWS has identified areas across Maui Nui that they consider “critical habitat” for the recovery of the species. In addition to the wet forest where the species is currently found, this includes montane mesic forest around Haleakala (USFWS 2012) (Figure 1.8.2). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 34 In addition to the wild population, a captive population of Maui Parrotbills was initiated in 1997. A total of seven founding individuals were collected from the wild in 1997, 1999, 2001, and 2005. Two of the seven founders have never produced offspring, and thus the captive population is composed of five genetic founders. As of August 2015, the captive flock consisted seven males and five females (Table 1.8.1). Current breeding potential in captivity appears to be limited by the relatively small number of breeding females. Four of the five females currently in captivity appear to be non-reproductive due to obvious physical ailments (e.g. blindness) or based on their poor reproductive history. Although the remaining female (MP011) has been productive by producing six offspring throughout her 11 year life, she has laid a relatively large number eggs over this period and may be starting to show signs of senecence. During the 2015 breeding season, none of MP015’s eggs successfully hatched for the first time since the 2011 breeding season. Figure 1.8.2. US Fish and Wildlife Service critical habitat for Pseudonestor xanthophrys on the Island of Maui within montane mesic forest (USFWS 2012). Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 35 Table 1.8.1. Current and historical inventory of Kiwikiu in captivity. Studbook Sex Founder/ Descendant Hatch Date Status Reproductive status MP003 Female Founder 4/30/1999 Alive Blind MP009 Female Founder 6/12/2001 Alive Never laid an egg MP011 Female Descendant 5/17/2004 Alive Productive female MP012 Female Descendant 6/21/2004 Alive Never laid fertile egg MP015 Female Descendant 3/5/2005 Alive No descendants MP017 Male Founder 1/1/2005* Alive - MP018 Male Founder 1/1/2005* Alive - MP022 Male Descendant 3/2/2012 Alive - MP023 Male Descendant 3/2/2012 Alive - MP024 Male Descendant 4/2/2012 Alive - MP026 Male Descendant 4/15/2013 Alive - MP027 Male Descendant 3/23/2014 Alive - MP001 Male Founder 1/27/1997 Dead - MP002 Female Founder 3/21/1999 Dead - MP004 Female Descendant 7/21/2000 Dead - MP005 Male Descendant 9/18/2000 Dead - MP006 Female Descendant 5/2/2001 Dead - MP007 Female Descendant 6/17/2001 Dead - MP008 Female Descendant 7/19/2001 Dead - MP010 Male Founder 5/1/2001* Dead - MP013 Unknown Descendant 8/16/2004 Dead - MP014 Unknown Descendant 9/13/2004 Dead - MP016 Female Descendant 9/12/2005 Dead - MP019 Unknown Descendant 7/4/2007 Dead - MP020 Male Descendant 7/9/2008 Dead - MP021 Female Descendant 3/21/2009 Dead - MP025 Female Descendant 12/18/2012 Dead - *Estimated hatch date. Adult bird collected from the wild. Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 36 1.9 OVERALL AIM OF THE THESIS Many species around the world are disappearing at a much faster rate than recovery efforts can be designed, including Hawaiian honeycreepers. There are more species becoming endangered every year and fewer resources to address the threats against them. The resources needed to tackle the biodiversity crisis in Hawaii are large, and Hawaii’s isolation from the mainland United States limits support for species conservation as compared to continental species (Leonard 2008). With 17 species of Hawaiian birds of fewer than 1000 individuals remaining, conservation efforts need to be well planned and have a high rate of success. As conservation managers, we need to be able to identify the genetic and ecological constraints in each species and be able to design studies to evaluate these constraints before reintroduction or other recovery efforts are implemented. However, many species do not have the luxury of time before conservation efforts must be undertaken. I undertook this research focused on Maui Parrotbill in order to inform conservation managers of the next best steps in planning a reintroduction for this species. This research made use of all past ecological data that had been gathered on this species in order to synthesize everything that is known about Maui Parrotbills within this thesis. This includes: o An evaluation of demographics for Maui Parrotbills, a rare and cryptic species for which large sample sizes are impracticable and conclusions must be carefully drawn from all quantitative and anecdotal data available o An assessment of the current genetic profile of Maui Parrotbills o The use of available knowledge on Maui Parrotbills to inform conservation managers as to the benefits and risks to conservation actions and no action for this species Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 37 THESIS OUTLINE My thesis starts with examining nest success and annual reproductive success for Maui Parrotbill, Chapter 2. While productivity is at the heart of the demographics driving the trajectory of any population, this information was mainly unknown for this species due to the difficult field conditions for collecting it and the small sample sizes that we were able to achieve per given person efforts. Although nest success is a commonly used metric of productivity, I compared the more labour intensive annual reproductive success methods in order to legitimize the information we ascertained from the nest success alone and present both of these analyses. In Chapter 3 I examine the second largest factor driving population trajectories, survival. Parameters such as survival rates are an important component to understanding population ecology and informing management decisions but can be quite difficult to determine for rare species. This chapter makes use of a long-term dataset of mark-re-sight information for Maui Parrotbill. This 18-year encounter history allowed a comprehensive analysis that accounted for annual variations in survival and detection probabilities through time. In Chapter 4 I examine the genetic profile of the current Maui Parrotbill population. I used nuclear and mitochondrial DNA to quantify the levels of contemporary genetic diversity and structure in wild and captive parrotbill populations. I further compared these genetic patterns to those observed within historical nuclear diversity derived from 100-year old museum samples. While the contemporary population structure lends valuable information on how to manage this wild population, how to manage the captive populations, and how to plan reintroduction efforts for the species, the historical data yeilds a better perspective on the overall effects of the different threats that have harmed this species through time. Recovery of the endangered Maui Parrotbill – Chapter 1 – Introduction 38 In Chapter 5 I use all the information derived from the previous chapters to build a comprehensive population viability analysis for Maui Parrotbill. This analysis examined population growth rates and probabilities of extinction within the current population. Using a sensitivity analysis, I identified key parameters limiting the probability of persistence in the next 25 years. With few management options available to implement in their current range, I then explored various options for establishment of an additional new population and the cost/benefit of such for the population as a whole. At the conclusion of my thesis in Chapter 6, I provide a synopsis of the key findings throughout these studies and use these to provide guidance on the best recovery strategiy for this endangered species. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Chapter 2 Determining productivity of the Maui Parrotbill, an endangered Hawaiian honeycreeper HANNA L. MOUNCE1, 2, DAVID L. LEONARD3, KIRSTY J. SWINNERTON4, C. DUSTIN BECKER5, LAURA K. BERTHOLD1, KELLY J. IKNAYAN1, and JIM J. GROOMBRIDGE2 1 Maui Forest Bird Recovery Project, Makawao, Hawaii 96768, USA 2 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent CT2 7NR, UK 3 Pacific Cooperative Studies Unit, Division of Forestry and Wildlife, Honolulu, Hawaii 96813, USA 4 Island Conservation, Santa Cruz, California 95060, USA 5 Life Net Nature, Wilcox, Arizona 85643, USA Key words: annual reproductive success, demography, nest success, population monitoring Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity ABSTRACT Maui Parrotbills (Pseudonestor xanthophrys), critically endangered Hawaiian honeycreepers endemic to the island of Maui, are restricted to a single population of ~500 individuals located in remote, mountainous terrain. January to June 2006-2011, we located nests and fledglings in the Hanawi Natural Area Reserve (NAR) in east Maui, Hawaii, to document nest success and annual reproductive success. Nest success is a commonly used measure of productivity and is a central component of many demographic studies. Annual reproductive success is less frequently documented because greater effort is required to monitor the reproductive success of breeding pairs through time. However, for species whose nests are difficult to locate or access, such as Maui Parrotbills, the presence or absence of fledged young may provide a more accurate measure of breeding success than monitoring nests. During our study, we located and determined the outcome of 30 nests to document nest success, and monitored 106 territories for the presence or absence of fledglings to calculate annual reproductive success. Nest success probability was 19% (N = 30) and seasonal nest success was 46%. During our monitoring efforts, 49 of 106 breeding pairs produced a single fledged young. Because parrotbills typically have single egg clutches and only re-nest after nests fail, the presence or absence of a fledgling is an indication of a pair’s overall reproductive success for a breeding season. The number of fledglings per pair produced an annual reproductive success estimate of 46%, confirming our initial productivity estimate from nests. Thus, our results indicate that the two methods, determining annual reproductive success by monitoring fledglings and calculating nest success, provide similar estimates of annual productivity for Maui Parrotbills. Based on our estimates, the parrotbill population appears to be demographically stable. However, our productivity estimate was based only on the population at Hanawi, an area representing just 3% of the total range of parrotbills. Thus, our results may not accurately reflect the status of parrotbills over their entire range. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity 2.1 INTRODUCTION Maui Parrotbills (Pseudonestor xanthophrys) are a federally endangered and red-listed critically endangered species of Hawaiian honeycreeper (U.S. Fish and Wildlife Service 1967, IUCN 2011). The species is restricted to a single population occupying an area of ~50 km2 on the northeastern slopes of Haleakala, Maui, Hawaii (Scott et al. 1986; U.S. Fish and Wildlife Service 2006). Historically, Maui Parrotbills (hereafter referred to as parrotbills) were distributed across the islands of Maui and Molokai (James and Olson 1991), where they may have preferred native koa (Acacia koa) forests (Perkins 1903). Clearing of lowland forests and introduction of alien diseases (i.e., avian malaria and pox) drastically reduced the range of parrotbills, and they are now restricted to high-elevation (1200 - 2350 m) wet montane forests, where cool temperatures limit disease vectors (i.e., mosquitoes) and consequently the spread of avian malaria (Scott et al. 1986; Mountainspring 1987; Simon et al. 1997). Population estimates of parrotbills based on data collected in the 1980s suggested a stable population of 502 ± 230 (95% CI) individuals (Scott et al. 1986). More recent surveys, however, have been inadequate to produce an accurate population estimate. Whereas range-wide surveys through 2001 yielded densities similar to those in the 1980s, the trend assessment was inconclusive regarding the stability of the population (Gorreson et al. 2009; Camp et al. 2009). Parrotbills are insectivorous honeycreepers that defend year-round territories (Pratt et al. 2001) and frequently occur in family groups, in which young remain with parents for five to eighteen months after fledging (Simon et al. 1997; MFBRP unpublished data). Parrotbills breed from November to June, with most breeding between February and June. Males and females form long-term monogamous pair bonds, typically foraging together year round. Females typically lay single-egg clutches and only re-nest after nest failure, which often Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity occurs during periods of heavy rain (Lockwood et al. 1994; Simon et al. 1997). Due to their rarity and tendency to nest high in the forest canopy, information about parrotbill reproductive success is limited and no recruitment data are available. In the absence of a conclusive population estimate, population modeling may be crucial in guiding management efforts for this species. For example, population viability analyses (PVAs) provide managers with information about extinction risk that is useful in developing management strategies for endangered species (Boyce 1992; Akçakaya and Atwood 1997; Brook et al. 2000). However, all population models rely on accurate demographic data. Unfortunately, the quality of such data is often poorest for endangered species – species that are most commonly in greatest need of accurate PVAs to inform their conservation management (Beissinger and Westphal 1998). One key demographic component of all population models is productivity, and nest success is a commonly used metric for estimating this variable (Woodworth et al. 2001; Renner and McCaffery 2008; Hartman and Oring 2009; Nappi and Drapeau 2009). The Mayfield estimator or more recently developed methods implemented in Program MARK (White and Burnham 1999) and SAS/STAT® software have been used to standardize data from nests found (Mayfield 1961, 1975; Rotella et al. 2004), but information about the success of individual nests does not always reflect reproductive output at the population level (Murray 2000; Jones et al. 2005), especially when it is not possible to monitor all nesting attempts (Thompson et al. 2001). This problem can be particularly acute for cryptic species that are difficult to locate and monitor. Therefore, alternative methods for estimating productivity are necessary for some species. One potential alternative method is the intensive monitoring of breeding pairs and calculation of annual reproductive success (ARS) based on the number of fledged young per pair. Given the difficulty of monitoring individual Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity birds through an entire breeding season, few investigators have quantified productivity using this method (Porneluzi and Faaborg 1999; Jones et al. 2005, Vanderwerf 2009; Rogers 2011). The choice of reproductive measure and the resulting fecundity estimates that different estimators produce can have far-reaching effects when determining population viability. Furthermore, models of population dynamics have been shown to be sensitive to small changes in such estimates (Powell et al. 1999; Woodworth 1999). Consequently, we estimated the productivity of breeding parrotbills using both nest success and annual reproductive success, and compared estimates to evaluate their relative performance in the Hanawi Natural Area Reserve. 2.2 METHODS Study Area The Hanawi Natural Area Reserve (NAR) covers 3036 ha on the windward slopes of Haleakala Volcano. Within the reserve, 800 ha above 1600 m in elevation are fenced and ungulate free; this is the core area used by the current parrotbill population (U.S. Fish and Wildlife Service 2006). We used two study areas in the Reserve, Frisbee Meadows (FSB) and Poouli Camp (HR3). The FSB study area (77 ha) is between 1600 and 2200 m asl, and the HR3 study area (56 ha) between 1550 and 1950 m asl (Figure 2.1). Non-native rodents are controlled on 35 ha of the HR3 site (Malcolm et al. 2008; Figure1). The area is characterized by steep, rugged terrain and supports a thick montane, wet forest dominated by ohia (Metrosideros polymorpha) and olapa (Cheirodendron trigynum; Jacobi 1989). The forest has an intact native understory and sub-canopy that provides high-quality foraging habitat for parrotbills. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Parrotbills were captured prior to and throughout the duration of our study using passive mist-netting and targeted mist-netting using playback. Of 212 adult (ASY) birds monitored over the 4-yr period, 130 were marked with a unique color band combination. Unmarked birds could be accounted for when paired with a banded individual during a single breeding season, but could not be identified between years. Territories were defined by the presence of singing males, males counter-singing with neighboring males, and regular presence of foraging adults; little overlap was observed between adjacent territories. Birds were assumed to be paired if they were observed foraging and travelling together, occupied the same territory, and demonstrated typical breeding behaviors such as mutual preening, mutual feeding, and nest building. To prevent possible double-counting, pairs where both adults were unbanded were only classified as discrete pairs when their territories bordered those of marked individuals. We searched for nests and fledglings along trails at each study site. Trails were 50 to 100 m apart in a network web that covered the entire study area and were systematically searched at least once per week from 07:00 to 17:30, each observer covering ~2 km per day. We conducted searches along 32.5 km of trails in the two study areas. Once an adult was detected, observers stayed for several hours to identify the individual and note behavioral activity. Three to six observers searched each site daily, except during severe weather. In addition to regular trail coverage, all territories in each study area were visited weekly to locate adults. We located fledglings either using their incessant begging calls (Simon et al. 1997) or by following parents to offspring. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Nest success From January to June 2006-2011, nests were located by observing adults carrying nesting material and the location of courtship displays, copulations, and pair feedings, all of which usually occurred near nest sites. Because of individual variation in the timing of breeding and the length of the breeding season, we could not determine if nests we monitored were first, second, or third nesting attempts for the year. Nests were usually monitored daily for 3 to 6 hrs using spotting scopes or binoculars from a distance of ~30 m until chicks fledged or nest failure was confirmed. Because nest contents were usually not visible, parental behavior at nests was used to determine nesting stage (e.g., constructing, incubating, brooding, or fledged; see Becker et al. 2010). Only nests where an egg was presumed to have been laid, based on observation of apparent incubation, brooding, or food delivery, were included in our analyses. Nests were classified as successful if fledglings were observed, with young considered to have fledged when they left nest trees. Previously active nests where no activity was documented for ≥3 h were classified as failures. Over a 3-h time period, adults typically visit nests two to three times (Becker et al. 2010). All failed nests were checked at least once more 1-3 days after failure was documented. Causes and timing (nest stage) of failures could not be determined for most nests because nests were located high (~ 11 m) in the canopy. When possible, we used mirrors or climbed nest trees to view nest contents. Parrotbill nest success was calculated using PROC GENMOD (SAS Institute 2008) to fit a logistic-exposure model (Shaffer 2004). This generalized linear model with a modified link function uses the appropriate likelihood estimator for interval data, avoiding assumptions about when failure occurs and allowing variable intervals between observations. We pooled nest data across all years to Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity increase our sample size because there was no apparent annual variation (Kershner et al. 2001). For nests found under construction, the first day of incubation was determined by female behavior (i.e., when first observed incubating). Because only single-egg clutches have been documented (U.S. Fish and Wildlife Service 2006), we assumed incubation began immediately after an egg was laid. Because we were unable to determine the contents of most nests, we did not differentiate between egg and nestling survival. Annual reproductive success From January to June 2008 - 2011, we systematically monitored the territories of 106 pairs of parrotbills for the presence of fledglings to calculate annual reproductive success. Because parrotbills typically have single-egg clutches and only re-nest after nest failure, the presence or absence of a fledgling is an indication of a pair’s reproductive success for a breeding season (Simon et al. 1997). Therefore, annual reproductive success was estimated by dividing the number of pairs with offspring by the total number of pairs observed during a breeding season. Population growth model To determine the overall effect of each estimate of productivity (nest success and annual reproductive success), we calculated the finite rate of population growth (λ) using the formula: λ = PA + PJβ(0.5), with PA = adult survival, PJ = juvenile survival, and β = average productivity per pair. Values of λ > 1 indicate a population increase and values of λ < 1 indicate decline. Adult and juvenile survival estimates derived from the same study population were drawn from Vetter et al. (2012). Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Due to re-nesting, our nest success estimate did not reflect seasonal productivity (Streby and Anderson 2011). Parrotbills have been observed to make up to three nesting attempts per season after nest failures (MFBRP unpublished data). We adjusted our β value for nest success with the following equation to have comparable seasonal productivity estimates based on each method: Seasonal nest success = Observed nest success + (Observed nest success *(1- Observed nest success)) + (Observed nest success *((1- Observed nest success)*(1- Observed nest success))) 2.3 RESULTS Nest success During six breeding seasons (2006-2011), we located 30 Maui Parrotbill nests (24 at HR3 and six at FSB). Eight nests either did not progress past the nest-building stage or nest outcome could not be determined; these nests were not included in our analyses. All nests were located in ohia trees, most in outer canopy branches 5.2 to 18.2 m above ground (mean = 10.9 m). Fifteen of 22 nests failed (68.2%). The logistic-exposure method resulted in a nest success probability of 0.185 ± 0.056 and a daily nest survival probability of 0.953 ± 0.007. One egg that did not hatch after 31 days of incubation was presumed to be infertile. Seven of the 15 failures occurred during the first 10 days of the nestling period, and one chick was predated by a Pueo (Asio flammeus sandwichensis; Mounce 2008). The cause of failure of the other 14 nests could not be determined. Seasonal nest success, adjusted for re-nesting, was 46% (N = 22 nests). Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Annual reproductive success During four breeding seasons (2008-2011), we monitored 43 pairs at HR3 and 63 pairs at FSB. Annual reproductive success estimates were 51% and 43% for HR3 and FSB, respectively, resulting in an overall estimate of 46% (Table 2.1). For all four years combined, we found no difference in productivity between the two study sites (χ² = 6.5, k = 3, P = 0.10). Population growth model According to Vetter et al. (2012), adult survival in our population of parrotbills was estimated at 0.84 ± 0.04 and juvenile survival at 0.76 ± 0.09. Based on both our seasonal nest success estimate and our annual reproductive success estimate, our model predicts a stable population (λ = 1.02 ± 0.07). 2.4 DISCUSSION Demographic modeling relies on accurate estimates of reproductive success. Therefore, using the reproductive monitoring method that provides the most accurate productivity data is critical. We suspected that our estimates of annual reproductive success based on observations of family groups would be the superior method because we calculated annual reproductive success using a larger subset of the population than for nest success, and because this method more accurately reflected season-long productivity. However, our results suggest that using either method (seasonal nest success or annual reproductive success) to estimate annual productivity is adequate for studies of this species. Both productivity calculations are indeed confirmations of one another, both resulting in a 46% annual productivity estimate. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Our productivity estimate suggests a stable or potentially increasing population. This is supported by results from population monitoring using point transect distance-sampling throughout the species’ range that were unable to detect any recent changes in population size (Gorreson et al. 2009; Camp et al. 2009). However, these transect surveys are only repeated every 5 yrs and, because parrotbills are long-lived birds (up to 16 yrs), any changes in population could take several years to detect. Thus, our productivity values may be more valuable to managers than range-wide survey data. For species like parrotbills that occur at low densities (as few as 10 birds/km²) and have difficult-to-locate nests, determining productivity by documenting the number of young fledged per pair confirmed the validity of our seasonal nest success estimates despite low sample sizes. However, these two methods may not yield similar results for all species. Although monitoring nests is critical for identifying factors that might limit productivity (i.e., weather or predation; Jones et al. 2005), nest success has been shown to provide inaccurate estimates of productivity in other passerines (Murray 2000; Underwood and Roth 2002; Grzybowski and Pease 2005). In a review of methods for estimating productivity, Anders and Marshall (2005) noted that quantifying the season-long productivity of individuals in a population provides the most accurate estimate of population productivity. When obtaining such data is not logistically practical, productivity can still be estimated more accurately by incorporating other variables into population models (Anders and Marshall 2005). For example, for species where nests are difficult to locate or access, like those of Maui Parrotbills in our study, surveying territories for the presence of fledglings can be less time-consuming than locating and monitoring nests because fledglings often beg loudly and adults give alarm calls or chips (contact calls) when potential predators approach (Anders and Marshall 2005). Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Although our data suggest some variation in annual reproductive success of parrotbills between our two study areas, we found no significant spatial and temporal differences, even though predator control was conducted at over 62% of the HR3 site during our study, but not conducted at FSB. Populations of non-native mammalian predators, including rats (Rattus spp.) and mongooses (Herpestes javanicus), were controlled using bait stations loaded with rodenticide, snap traps, and mongoose body traps (Malcolm et al. 2008). These non-native mammals are considered major threats to Hawaiian birds and, although rats have not been documented as predators of parrotbill nests, they have been documented predating Akohekohe nests at heights similar to those of parrotbill nests (Scott et al. 1986; Simon et al. 2001). Predator control efforts may need to be expanded to cover at least 100% of each pairs’ home range, with perhaps an additional buffer of control around each home range, if any resulting difference in nest success is to be realized from these management efforts. Based on our annual productivity estimate, the parrotbill population appears to be demographically stable at Hanawi, close to the core of their range. However, our productivity estimate was based only on the population at Hanawi, an area that represents just 3% of the total range of parrotbills. Thus, our results may not accurately reflect the status of parrotbills over their entire range. Survey efforts using point transect distance-sampling throughout their range indicate that parrotbill densities may be lower outside of Hanawi (Maui Forest Bird Recovery Project unpublished data), but there has been no detailed demographic monitoring in other areas of the species’ range. Expanding our productivity estimate techniques to the outer edges of the species’ range will enhance the utility of population modeling studies and will help managers to develop a more sophisticated assessment of population-wide levels of productivity. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity ACKNOWLEDGMENTS Maui Forest Bird Recovery Project operates under the sponsorship of Pacific Cooperative Studies Unit and Research Corporation of the University of Hawaii. State of Hawaii Division of Forestry and Wildlife, and Pacific Island Office of the U.S. Fish and Wildlife Service provided funding, oversight, scientific input, access into the Hanawi Natural Area Reserve and the required state and federal permits. We would like to thank the following partners for logistical support and/or access to their properties: Haleakala National Park, The Nature Conservancy Hawaii, Tri-Isle Research, Conservation & Development, Pacific Helicopters, and Windward Aviation. S. Wheeler, C. Rutt, and J. Milikowsky assisted with data analyses. Finally, we thank all of the seasonal biologists who contributed to the data set used in this study. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity 2.5 TABLES AND FIGURES Table 2.1. Maui Parrotbill annual reproductive success based on number of pairs observed with fledglings at two study sites (FSB and HR3) in Hanawi NAR 2008-2011. Site Year Number of pairs observed Number of pairs with juveniles Percent success FSB 2008 11 3 27.3% 2009 15 8 53.3% 2010 18 6 33.3% 2011 19 10 52.6% HR3 2008 10 4 40.0% 2009 8 6 75.0% 2010 9 5 55.6% 2011 16 7 43.8% FSB totals 63 27 42.9% HR3 totals 43 22 51.2% Totals 106 49 46.2% Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Figure. 2.1. Study sites where the productivity of Maui Parrotbills was examined in our study. Both Frisbee Meadows (FSB, 77 ha) and Poouli Camp (HR3, 56 ha) are located in the Hanawi Natural Area Reserve, Island of Maui, Hawaii. Recovery of the endangered Maui Parrotbill – Chapter 2 - Productivity Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 55 Chapter 3 Management implications derived from long term re-sight data: annual survival of the Maui Parrotbill (Pseudonestor xanthophrys) HANNA L. MOUNCE1,2, KELLY J. IKNAYAN3, DAVID L. LEONARD3, KIRSTY J. SWINNERTON4 and JIM J. GROOMBRIDGE2 1 Maui Forest Bird Recovery Project, Makawao, Hawaii 96768, USA 2 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent CT2 7NR, UK 3 UC Berkeley Department of Environmental Science, Policy, & Management, Berkeley, California, 94720, USA 4 U.S. Fish and Wildlife Service, Portland, Oregon 97232, USA 5 Island Conservation, Santa Cruz, California, USA Keywords: apparent survival, endangered species, demography, Maui Parrotbill, population monitoring Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 56 ABSTRACT The accurate estimation of key demographic parameters is invaluable for making decisions about the management of endangered wildlife but such estimates are often difficult to obtain. Parameters such as species-specific apparent survival rates are an important component to understanding population ecology and informing management decisions. Maui Parrotbills (Pseudonestor xanthophrys) are ‘Critically Endangered’ Hawaiian honeycreepers endemic to the Island of Maui. We used an 18 year encounter history dataset comprising 146 marked individuals to estimate apparent survival between sexes and age classes (juvenile, adult). A difference in survival rates between sexes was strongly supported; 0.72 ± 0.04 for adult females and 0.82 ± 0.03 for adult males. This difference may be a reflection of either reproductive costs or additional risks of incubation and brooding, such as depredation. We also found support for age- biased survival, but limited information for juveniles did not provide a well-supported model fit for our data (juvenile survival = 0.17 ± 0.15; adults = 0.78 ± 0.02). However, apparent adult survival was similar to that of other Hawaiian passerines (mean 0.78 ± 0.03, n = 16). These results suggest that efforts to prevent the extinction of this species may benefit from future management strategies focused on increasing female survival such as predator reduction. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 57 3.1 INTRODUCTION Extinction risk is high for many Hawaiian bird species and this situation creates an urgent need for reliable assessment of their density and distribution. Maui Parrotbill (hereafter parrotbill) (Pseudonestor xanthophrys) is a critically endangered insectivorous forest bird endemic to the island of Maui (U.S. Fish and Wildlife Service 2006; IUCN 2012). Limited range, combined with small population size and low densities (U.S. Fish and Wildlife Service 2006) puts parrotbills at particular risk of extinction. Like most Hawaiian forest birds, parrotbills are currently limited to high elevation forests that are relatively free of mosquitoes and avian malaria (Plasmodium sp.) (Scott et al. 1986; Mountainspring 1987; Atkinson and LaPointe 2009), but these forests are otherwise likely suboptimal habitat (Simon et al. 1997; U.S. Fish and Wildlife Service 2006; Becker et al. 2010). In addition, widespread habitat loss, especially of koa (Acacia koa) forests, parrotbills’ preferred foraging habitat (Perkins 1903), has contributed to their current limited distribution on the windward slopes of east Maui (U.S. Fish and Wildlife Service 2006). The depredation of nests, juveniles, and adults also may limit the parrotbill population and, while the cause and importance of depredation is unclear, rodents (Rattus spp.), feral cats (Felis catus), and the invasive small Indian mongoose (Herpestes auropunctatus) are present throughout the area where the current population persists (Sugihara 1997; Malcolm et al. 2008). Based on the 1980 Hawaii Forest Bird Survey that used island wide point counts, the parrotbill population was estimated at 502 ± 116 (Scott et al. 1986). As a rare species with low detection rates, more recent surveys have been unable to confirm the stability of the parrotbill population (Gorreson et al. 2009). Indeed, Brinck et al. (2012) found that the repeated sampling frequencies and number of visits that would be necessary to increase the power of these surveys to detect trends in the parrotbill population would be particularly high and therefore logistically unfeasible. Due to the limitations of these survey Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 58 efforts for estimating the population size of a rare species, demographic analysis is perhaps the only alternative means for providing a better insight into the population dynamics of the parrotbill. An understanding of population demography, coupled with an understanding of the factors limiting population growth, is essential for recovering populations of endangered species, designing effective conservation strategies and making informed management decisions (Anders and Marshall 2005). While studies of population dynamics depend heavily on mortality and recruitment rates (Lebreton et al. 1993), such information is often lacking for endangered species, but it is often these same species of conservation focus that would benefit the most from such studies (Beissinger and Westphal 1998). Fundamental to population demography is an understanding of the variability in survival among individuals (Lack 1954; Eberhardt 1985). Accurate measurements of population-specific survival are essential for estimating reliable rates of population change, as many models of population dynamics are sensitive to small deviations in estimates of demographic measures (Noon and Sauer 1992; Porneluzi and Faaborg 1999; Woodworth 1999). Constant effort mist-netting and banding has historically been a common method used to estimate survival rates of passerines (DeSante and Burton 1994). This technique is limited by the fact that previously banded individuals are not always recaptured even though they may still be alive (Chase et al. 1997). Indeed, whether or not a banded bird is subsequently detected is a function of probabilities: survival, emigration, and detection. Re-sighting and re-capturing marked individuals has since improved this method for generating the most accurate estimations of survival in forest bird species (Sandercock et al. 2000; Gardali and Nur 2006; Johnson et al. 2006). Furthermore, understanding variation in age-specific and sex-specific survival can provide valuable insights to inform the ecology and conservation of a species (Sandercock et al. 2000; Martin 2002). Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 59 Considering the limitations of accurately estimating the population of rare species, accurate demographic data would provide managers with a yardstick to monitor the population trajectory of a species. However, the low densities and few individuals indicative of a rare and endangered species results in mark-recapture studies requiring large amounts of time and effort. In addition, parrotbills inhabit very rugged and remote terrain. Both these characteristics make collecting demographic data a challenge. Indeed, long-term demographic data for rare species inhabiting remote areas are uncommonly available for managers. Here we improve upon previous demographic estimates for this species (Vetter et. al 2012) by summarizing survival probability of parrotbills in the core of their population range using 18 years of encounter data, and we examine differences in age- and sex-specific survival probabilities. 3.2 METHODS Study Area We conducted this study within the Hanawi Natural Area Reserve (NAR) on the windward slope of Haleakala volcano, Maui, Hawaii (Figure 3.1). Our 180- hectare (ha) study site extended from 1600 to 2100 metres in elevation. This study area is located within an 800 ha portion of the reserve, managed by the State of Hawaii and has been fenced and free from invasive ungulates since 1997; it protects some of the most pristine native forest remaining in Hawaii. The area is mainly a montane wet forest characterized by rugged and steep terrain. Ohia (Metrosideros polymorpha) and olapa (Cheirodendron trigynum) are the dominant canopy species, although subalpine scrub and subalpine grassland occur at the highest elevations (Jacobi 1989). The study site supports the highest known density of parrotbills (Scott et al. 1986; Pratt et al. 2009a). Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 60 Mark-Recapture Mark-recapture was a combination of recapture and re-sight efforts which varied across years, beginning at the higher elevations in 1994-1997 and resuming in 2006-2011 ( Simon 1998; Simon et al. 2000; Berlin et al. 2001a; Berlin et al. 2001b; Pratt et al. 2001; Simon et al. 2001; Simon et al. 2002) and beginning at the lower elevations in 1998 and continuing through 2011. Most recapture and re-sight effort has occurred from January - June during the peak of the breeding season. Banding occurred across two field sites connected by an extensive trail system (Figure 3.1). Individuals were initially captured in mist-nets and banded with a unique combination of a US Fish and Wildlife Service numbered band and three darvic plastic colored leg bands. To increase the capture rates above that of passive mist net efforts, playbacks were used in areas where unbanded individuals had been located. Once captured, parrotbills were aged and sexed using plumage and morphometric criteria (Berlin et al. 2001a). Both passive and targeted banding continued annually in different locations covering each study site. Re-sights were obtained by searching for banded individuals systematically across all trails, during each breeding season, as well as opportunistically in the same areas throughout the rest of the year. Subsequent re-sights were documented along with GPS locations. Data Analyses Based on capture, recapture, and re-sight histories from 1994-2011 for individually-marked parrotbills, we used Cormack-Jolly-Seber (CJS) models of live recaptures in program MARK, version 6.0, (White and Burnham 1999) to estimate apparent annual survival (φ) and encounter probability (ρ). While not explicitly designed for the combination of re-sight and recapture data, CJS is the most appropriate mark-recapture model for this type of data and has been Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 61 widely used with similar data sets (Nur and Sydeman 1999; Sandercock et al. 2000; Vanderwerf 2009). We used an encounter period of one year given that (i) many individuals were only detected once a year and (ii) that subsequent encounters were often 10-12 months apart. Because of the rugged terrain, the fate of subsequently undetected individuals was unknown, thus the sampled population was defined as open, and survival estimates represent apparent survival. Using dates for the initial capture, and all subsequent recaptures and re-sights, we compiled an encounter history for each individual across the 18- year period. In separate analyses, parrotbills were grouped by sex (male or female) and age class (juvenile or adult). Hatch-year (HY, juvenile) parrotbills cannot be conclusively sexed and were excluded from the sex-specific analysis. For each analysis, we started with the simplest model in which φ and ρ were both constant. Using standard model notation, this model is represented as φ(.)ρ(.) for each model set (Lebreton et al. 1992). Each model was compared with Akaike’s Information Criterion corrected for small sample size using the quasi-likelihood adjustment (QAICc), as calculated by Program MARK. The model with the lowest QAICc value was considered to have the best fit (Burnham and Anderson 2002). To test that the arrangement of our data met expectations based on the assumptions underlying the model, we evaluated goodness-of-fit of our global (highest parameterized) model using the Program RELEASE GOF provided in Program MARK. We adjusted both analyses to the goodness-of-fit calculated value of ĉ (variance inflation factor or lack of fit) from 1000 simulations before model selection. For both age and sex, we present the most parsimonious model and all models with QAICc weight in addition to the null (φ(.)ρ(.)), global (φ(g*t)ρ(g*t)), and fully time dependent (φ(t)ρ(t)) models. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 62 3.3 RESULTS Between 1994 and 2011, 146 individual parrotbills were banded in the study area (see Appendix A) and included in the age-specific analysis. Of these, 136 (64 females, 72 males) were included in our sex-specific analysis. Ten were HY birds and were excluded from the sex analysis. The number of individuals recaptured and re-sighted varied each year (see Appendix A for recapture histories), an average of 18.11 unique individuals were detected annually. The best-fit age-specific and sex-specific models for parrotbills showed apparent survival that varied with age and sex but was constant across years and showed an encounter probability that varied with time (Table 3.2, Model 1; Table 3.3, Model 1; Figure 3.2). No other models were of a reasonable fit for either group. We found strong support for sex-specific differences in survival with males showing higher survival rates than females (males 0.82 ± 0.03; females 0.72 ± 0.04). We also found juveniles to show lower survival rates than adults (juveniles 0.17 ± 0.15; adults 0.78 ± 0.02) (Figure 3.3). While our sex-specific model had good fit to the CJS model selected (GOF Test 2 + Test 3 χ2=51.320, df=57, p-value=0.687, ĉ=0.900), our age-specific model did not (GOF Test 2 + Test 3 χ2=56.164, df=35, p-value=0.013, ĉ=1.605) due to insufficient data in the HY group to calculate independent χ2 results. 3.4 DISCUSSION As with most survival studies of an open population, mortality and emigration cannot be separated and thus survival is likely to be underestimated (Cilimburg et al. 2002). This effect is especially true in rugged terrain which limits detectability. Even so, adult apparent survival was similar to that of other Hawaiian avifauna; Akohekoke (Palmeria dolei) show the highest annual survival of any Hawaiian passerine at 0.95 (Simon et al. 2001) but the average annual adult survival of 16 Hawaiian passerines averaged 0.78 ± 0.03 (Pratt et al. 2009a; Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 63 Woodworth and Pratt 2009). Conversely, juvenile apparent survival was lower than expected. Although the greater dispersal of young can contribute to differences in adult and juvenile survival (Greenwood and Harvey 1982), juvenile parrotbill survival was lower than that demonstrated for other Hawaiian birds. Woodworth and Pratt (2009) reported that the average annual juvenile survival of 13 other Hawaiian passerines was 0.32 ± 0.03. However, the juvenile parrotbill survival estimate was based on only 10 individuals, which contributes to the large standard error and only a moderate fit to our CJS model. While acknowledging that our estimate lacks precision, juvenile parrotbill survival is certainly lower than that of adults and it would not be surprising if juvenile survival of this species is particularly low. Unpublished data on territory occupancy in the Hanawi study site indicates that most available habitat is occupied. Young birds may be forced into poorer quality habitat, limiting their chances of survival. Although, we currently have little information on juvenile dispersal, and a larger sample size is necessary for a more precise survival estimate, it should be noted that the juvenile individuals included in Vetter et al. (2012) were never re-sighted in the subsequent years of this study. The lack of detection for any of these juvenile individuals in the years that followed, combined with high estimates of juvenile survival during years of low detection probability early on in this study, accounts for the large difference in juvenile survival estimates between the two datasets (Figure 3.4). Several re-sights of juvenile individuals during years of low overall detection probabilities artificially inflated the early juvenile survival estimates used in Vetter et al. (2012). We appreciate that both data sets are still sparse in their data on juvenile individuals but given that none of the juveniles marked during the years of high detection probabilities were seen again, we believe that this brought the average juvenile survival down to a more representative value. Vetter et al. (2012) also did not detect strong differences in apparent survival between male and female parrotbil. As both analyses were conducted using similar methods, the increased detection probability in the survey years Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 64 2008 to 2011 in this study provided a more robust sample with which to demonstrate the sex-specific apparent survival. We found that parrotbills maintain strong pair bonds throughout the year and are often seen together. We found little bias for sex-specific encounter probabilities and re-sights were nearly evenly distributed between the sexes (females 410, males 465). Additionally, if we consider the Φsex.ρsex model (QAICc weight = 0.000), and assume that sex-specific encounter rates had a larger effect than the models suggested, males had a detection probability of 0.457 (± 0.043) and females 0.666 (± 0.060) (see Appendix A for more details). As with many species, the difference in apparent survival between males and females is more likely a result of higher reproductive costs for females, a higher rate of depredation, and/or higher emigration rates. While we can only speculate on the latter due to a lack of data, females do incur high energetic demands associated with egg production and incubation and have a higher risk of being depredated on the nest (Nur 1998; Ghalambor and Martin 2001; Fontaine and Martin 2006). Although female parrotbills alone incubate eggs and brood nestlings, the cost of reproduction for females may be similar to the cost to males of establishing and defending territories as well as provisioning females and offspring. Owens and Bennett (1994) found that provisioning chicks can have a higher direct mortality cost to adults than nest building and incubation. In parrotbills, higher female mortality is more likely the result of higher rates of depredation. Rodents (Rattus spp.) are predators of native island birds and have been documented depredating incubating and brooding females (Atkinson 1977; Moors et al. 1992; Robertson et al. 1994). In Hawaii, rats are responsible for the high female mortality in the Oahu Elepaio (Chasiempis ibidis) (Vanderwerf and Smith 2002) and may account for the sex-specific survival difference noted in the parrotbills. Our highest selected models all incorporated a detection probability that varied considerably through time. Annual survey effort was influenced by the remoteness of our field sites and the rugged terrain. Access to the study area (by helicopter and on foot) was typically influenced by weather. Poor weather Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 65 further influenced the probability of detecting individuals as re-sighting individuals in rain or mist was difficult. Despite uneven detection probabilities, the differences in survival estimates between this study and Vetter et al. (2012) illustrates the importance of long-term data sets for rare and cryptic species as well as those which may provide a scarcity of data within any period of years. We analyzed just the 2003-2011 datasets using the same methodologies to better understand the differences between these two studies. While the first portion of this data set used in Vetter et al. (2012) may have overestimated juvenile survival and was not able to resolve sex-based differences in apparent survival, the same is true of the latter half of the dataset when considered independently. Although these years had very high detection probabilities associated with them, and found similarly low juvenile survival, this subset of data had unresolved model rankings. Our results suggest that conservation management focused on increasing female (and possibly juvenile) survival would likely benefit the recovery of the parrotbill population. Male survival would appear to be high for a small passerine, but although not necessarily so for a tropical species (see VanderWerf 2009). Landscape-scale rodent control would likely benefit the parrotbill and other native forest birds on Maui. Female survival of the Oahu Elepaio has been shown to increase following rodent control (Vanderwerf and Smith 2002). Other strategies could include intensive management such as supplemental feeding. Food supplementation has been a successful strategy in recovering endangered birds including the San Clemente Loggerhead Shrike (Lanius ludovicianus mearnsi) (Heath et al. 2008), Florida Scrub Jay (Aphelocoma coerulescens) (Schoech et al. 2008), Hihi (Notiomystis cincta) (Castro et al. 2003), and Kakapo (Strigops habroptila) (Clout et al. 2002) and has served as a short-term measure to support populations while longer-term habitat restoration occurs. Finally, restoring high elevation forests that are buffered from extreme weather and that have a high abundance of koa trees may provide the greatest Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 66 opportunity to increase the parrotbill population size (Simon et al. 1997; U.S. Fish and Wildlife Service 2006; Becker et al. 2010). More data may be able to resolve our juvenile survival estimates for this model. If juvenile survival is indeed low as a result of all suitable habitats being already occupied, the addition of new high quality habitat may be the only management strategy capable of increasing juvenile survival. Currently an 1100-ha area of mesic koa forest is being restored on leeward east Maui, possibly the single most significant conservation action taken for the parrotbill since the exclusion of feral ungulates from Hanawi in 1997. Experimental releases of parrotbills into this habitat are scheduled to occur in the next five years. ACKNOWLEDGEMENTS This study was conducted by Maui Forest Bird Recovery Project, a project administered and funded by Pacific Cooperative Studies Unit, University of Hawaii, State of Hawaii Department of Land and Natural Resources, Division of Forestry and Wildlife, and the U.S. Fish and Wildlife Service. The Division of Forestry and Wildlife, and Pacific Island Office of U.S. Fish and Wildlife Service provided scientific input, access into Hanawi Natural Area Reserve and the required state and federal permits. We would like to thank the following partners for logistical support and access to their properties: Hawaii Natural Area Reserves System Commission, Haleakala National Park, Haleakala Ranch, The Nature Conservancy, Tri-Isle RC & D, Pacific Helicopters, and Windward Aviation. We would like to thank Thane Pratt, John Simon, Paul Baker, and Stuart Pimm for use of their banding and re-sight data. We are grateful to Rick Camp and Hawaii Forest Bird Interagency Database Project for providing population estimates and densities from historical and recent bird surveys. This manuscript was greatly improved by the help of Eben Paxton and one anonymous reviewer. Finally, we thank all of the seasonal biologists who assisted in the strenuous data collection for this study. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 67 3.5 TABLES AND FIGURES Table 3.1. Apparent survival (φ) and encounter probability (ρ) models for Maui Parrotbills grouped by age (juvenile and adult). Subscripts indicate whether parameters differed among groups (e.g. φage) or time (φt) or were constant (φ.). Overdispersion is corrected to 1.605 (ĉ) based on goodness of fit test on global model (φage*tρage*t). RQAICc is the difference from the best (lowest AICc) model. AICc weight is the relative likelihood of each model. # Model ∆QAICc QAICc weight No. Parameters Deviance 1 Φageρt 0.8756 19 153.1149 2 Φ.ρt 4.6759 0.0845 18 160.1123 3 Φageρ. 7.5464 0.0201 3 195.6714 4 Φ.ρage 8.8355 0.0156 3 196.9604 5 Φageρage 9.4988 0.0076 4 195.5618 6 Φ.ρ. 12.5860 0.0016 2 202.7570 7 Φtρt 39.1413 0.0000 33 157.6439 8 Φage*tρage*t 47.3016 0.0000 48 149.6518 Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 68 Table 3.2. Apparent survival (φ) and encounter probability (ρ) models for Maui Parrotbills grouped by sex (juvenile birds omitted). Subscripts indicate whether parameters differed among groups (e.g. φsex) or time (φt) or were constant (φ.). Data underdispersed (ĉ = 0.900) based on goodness of fit test on global model (φsex*tρsex* t), ĉ left at 1.00. RQAICc is the difference from the best (lowest AICc) model. AICc weight is the relative likelihood of each model. # Model ∆QAICc QAICc weight No. Parameters Deviance 1 Φsexρt 0.7923 19 349.9260 2 Φ.ρt 2.6782 0.2077 18 354.9384 3 Φ.ρ. 55.8461 0.0000 2 442.9450 4 Φtρt 34.1433 0.0000 32 351.8188 5 Φsex*tρsex*t 92.2258 0.0000 64 311.8119 Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 69 Figure 3.1. Study area within the Hanawi Natural Area Reserve, east Maui, Hawaii. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 70 Figure 3.2. Detection probability variation over time in Maui Parrotbill survival analyses for age-specific analysis (a) and sex-specific analysis (b). Error bars indicate standard error for each year 1996-2011. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 71 Figure 3.3. Apparent survival probability in Maui Parrotbill by sex (a) and age (b). Error bars indicate standard error for each individual group. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 72 Figure 3.4. Apparent survival for juvenile Maui Parrotbill varied much more through time than did adult apparent survival illustrating the limitations of using a smaller data set may distort the results to suggest higher juvenile survival. Recovery of the endangered Maui Parrotbill – Chapter 3 – Survival 73 Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 74 Chapter 4 Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies HANNA L. MOUNCE1, 2, CLAIRE RAISIN1,3, DAVID L. LEONARD4,5, HANNAH WICKENDEN6, KIRSTY J. SWINNERTON7 and JIM J. GROMMBRIDGE1 1 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury CT2 7NR UK 2 Maui Forest Bird Recovery Project, Makawao, Hawaii 96768 USA. 3 Size of Wales, Llandaff, Cardiff CF5 2DT UK 4 Pacific Cooperative Studies Unit, Division of Forestry and Wildlife, Honolulu, Hawaii 96813, USA 5 Current: U.S. Fish and Wildlife Service, Portland, Oregon 97232, USA 6 Oxford Brookes University, Department of Biological and Medical Sciences, Headington Oxford OX3 0BP UK 7 Island Conservation, Santa Cruz, California 95060, USA Keywords: ancestral DNA, control region, genetic structure, Maui Parrotbill, microsatellites, reintroduction Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 75 ABSTRACT Conservation translocations are an important tool to circumvent extinctions on oceanic islands. A thorough understanding of all components of a species’ biology, including genetic diversity and structure, can maximize their likelihood of success. The Maui Parrotbill (Pseudonestor xanthophrys) is an endangered Hawaiian honeycreeper endemic to the island of Maui. With a population of approximately 500 individuals restricted to 50 km2 of habitat, this species is at high risk of extinction. Using nuclear and mitochdondrial DNA, this study quantified the levels of contemporary genetic diversity and structure in wild and captive parrotbill populations, and compared these genetic patterns to those observed within historical nuclear diversity derived from 100-year old museum samples. Substantial differences in the effective population sizes estimated between contemporary and historical parrotbill populations highlight the impact that introduced disease had on this species just before the turn of the century. Contemporary parrotbill diversity was low (global Fst = 0.056), and there has been a 96% reduction in genetic effective population size between contemporary and historical samples. This should not eliminate a conservation translocation (or reintroduction) as a viable recovery option. Measures of population differentiation (pairwise Fst and Rst) between different sections of the current population on either side of the Koolau Gap suggest that current genetic structure may be the result of this topographic barrier to gene flow. These data can enable the design of a conservation translocation strategy that is tailored to the patterns of genetic structure across the species’ range. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 76 4.1 INTRODUCTION Reintroductions are a form of conservation translocation used to re-establish self-sustaining populations in areas from which species have been extirpated (Griffith et al. 1989; Armstrong et al. 2002; IUCN/SSC 2013). Such reintroductions have been particularly important on oceanic islands where exposure to extinction risk is higher than on continents (Steadman 2006; Jones and Merton 2012). Island species continue to be impacted by numerous anthropogenic threats; and, as threats are removed, reintroductions are often essential to re-establish species into areas of their former range (Castro et al. 1995; Armstrong et al. 2002; Cristinacce et al. 2009). To maximize success, reintroductions need to be carefully planned and have a solid understanding of the species, their threats, and habitat requirements. Historically low reintroduction success within species conservation (Griffith et al. 1989; Wolf et al. 1996; Fischer and Lindenmayer 2000;) has stimulated interest in integrating genetic information into the design of reintroductions to improve long-term viability of the resulting new populations (Seddon et al. 2007; Ewen et al. 2012). Maximizing retention of genetic diversity to enhance long-term evolutionary potential is one way of achieving a successful reintroduction, an aim widely accepted as a fundamental component of population management for conservation (Frankel and Soule 1981; Frankham et al. 2010). There is a need to incorporate this more explicitly into the design of reintroduction programs (Groombridge et al. 2012; Keller et al. 2012). Loss of genetic diversity, increased levels of inbreeding, and accumulation of deleterious mutations in small populations are factors likely to have important consequences for the long-term persistence of reintroduced populations (Groombridge et al. 2012), and therefore it is important to ensure that a reintroduced population genetically represents the source population (Beck et al. 1994; Fischer and Lindenmayer 2000). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 77 Before undertaking reintroductions, conservation managers should ideally know not only the genetic diversity of the source population, but also how that diversity is distributed spatially and temporally within a historical framework. Genetic structure arising from natural processes such as dispersal barriers or habitat gradients can then be distinguished from those that have arisen as a consequence of anthropogenic factors such as habitat loss and fragmentation. In addition to the benefits of applying this information at a practical level, an understanding of the ecological and historical context is also valuable for interpreting observed patterns of genetic diversity as knowledge of historical levels can often provide valuable perspective on contemporary genetic diversity (Groombridge et al. 2012). The Maui Parrotbill (Kiwikiu; Pseudonestor xanthophrys) is an endangered insectivorous forest bird endemic to Maui, Hawaii, at high risk of extinction (U.S. Fish and Wildlife Service 2006; IUCN 2011; IUCN/SSC 2013). The population is estimated at 502 ± 116 (Scott et al. 1986). Due to the relatively low detection rates of parrotbills, more recent surveys have been unable to generate a more precise range-wide population estimate (Brinck et al. 2012) or to confirm the stability of the population (Gorreson et al. 2009). The current range of parrotbills is small, 40-50 km2 on the windward slopes of Haleakala volcano (Simon et al. 1997; Brinck et al. 2012) and is likely an artifact of past habitat loss, especially of the native koa (Acacia koa) forests, the possibly preferred foraging habitat of parrotbills (Perkins 1903), and current disease distribution (U.S. Fish and Wildlife Service 2006). Sub-fossils indicate a species’ range that included the entire islands of Maui and Molokai (James et al. 1987; James and Olson 1991; Olson and James 1991; Simon et al. 1997; U.S. Fish and Wildlife Service 2006; Becker et al. 2010). Parrotbills are currently restricted to high elevation wet forests that are relatively free of avian malaria (Plasmodium relictum) and its primary vector, the non-native southern house mosquito (Culex quinquefasciatus) Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 78 (Scott et al. 1986; Mountainspring 1987; Atkinson and LaPointe 2009). Based on productivity and nest survival data (Mountainspring 1987; Mounce et al. 2013), these forests may be suboptimal habitats for this species. The Koolau Gap, a 2 km-wide valley, cuts through a portion of the current parrotbill population range (Figure 4.1). The floor of Koolau Gap averages 600 m lower in elevation than the adjacent uplands. This feature is one of two such valleys that were formed as deep dissections in the landscape, which channeled lava into the ocean when the Haleakala volcano was formed prior to avian inhabitants (Morgan 1996). Since avian malaria and its mosquito vector are cold intolerant, and normally do not occur at elevations over 1500 m (Atkinson and LaPointe 2009), the Koolau Gap is thought to provide suitable habitat for these disease threats within an otherwise malaria-free forest. Although some Hawaiian forest birds have exhibited resistance to avian malaria (Foster et al. 2007), parrotbills have no known tolerance (Atkinson and LaPointe 2009); this is considered a primary factor limiting the habitat available for the parrotbill population (Scott et al. 1986; LaPointe et al. 2009). The Koolau Gap therefore presents a potential dispersal barrier within the current parrotbill population. Furthermore, climate change predictions suggest that high elevation rain forest habitats will become degraded (Loope and Giambelluca 1998; Giambelluca et al. 2008;) and unsuitable for parrotbills as warmer temperatures facilitate the persistence of avian malaria at higher elevations, such that up to 75% of current parrotbill habitat may become unsuitable in the future (Benning et al. 2002) (Figure 4.1). As a consequence of these predictions, there is a need to establish a second population of parrotbills in other high-elevation disease-free habitats on Maui. Doing so would re-establish the species in its former range, and fulfill a critical recovery action as outlined in the species’ recovery plan (U.S. Fish and Wildlife Service 2006). One potential location for reintroduction is Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 79 the leeward side of Haleakala. Historically, these areas supported mesic and dry forests dominated by koa and state-owned lands have been prioritized for large-scale habitat restoration (Nakula NAR, Figure 4.1) specifically to provide habitat for parrotbills. Establishment of a reintroduced population will require translocation of individuals from the windward population and/or from the small captive breeding population in Hawaii (Mounce and Leonard 2012). This paper describes the first population-level genetic study of parrotbills, quantifying levels of genetic diversity across both wild and captive populations using a suite of species-specific microsatellite markers and avian mitochondrial DNA (mtDNA) control region primers. In addition, we use microsatellite markers to quantify historical levels of genetic diversity in the parrotbill population by genotyping 100-year-old museum samples. We use these genetic data to: (i) characterize loss of historical genetic diversity and interpret it alongside the known and suspected ecological history of the species, (ii) utilize observed spatial patterns of genetic structure to suggest a reintroduction strategy optimized to maximize retention of genetic diversity, and (iii) estimate, based on contemporary levels of genetic diversity, how many individuals would be required for reintroduction in order to capture the majority of diversity. 4.2 METHODS Samples Contemporary genetic samples (n=129, comprising 118 wild individuals and 11 captive birds) were collected across the species’ range between 1996 and 2011 and within two captive breeding facilities managed by San Diego Zoo Global (Figure 4.1). Wild birds were caught opportunistically throughout the year using mist nets and recorded playbacks. Blood samples were Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 80 collected from the brachial vein using a 27 G needle and a 1.2 mm x 75 mm capillary tube. Blood was stored in a ‘Queen’s Lysis buffer’ (0.01 M Tris, 0.01 M NaCl, 0.01 M EDTA, and 1% n-lauroylsarcosine, pH 7.5) (Seutin et al. 1991) solution at 4 oC. Feather samples consisting of 4-6 breast feathers were collected per individual and were also stored dry at 4 oC. Birds were banded with unique combinations of metal and plastic color-bands to facilitate ongoing demographic studies (Vetter et al. 2012; Mounce et al. 2013; 2014) as well as to prevent re-sampling. Geospatial location data was collected for each capture. Individuals were referred to as captive or wild. The latter were further grouped geographically as those captured east or west of the Koolau Gap (hereafter east and west; Figure 4.1). Historical samples (n = 34) were obtained by sampling toe-pad tissue of all known museum specimens (see Appendix B). Laboratory Methods DNA extraction Genomic DNA was extracted from blood samples using an ammonium acetate precipitation method (Nicholls et al. 2000) and from feathers using DNeasy® Blood and Tissue Kit (Qiagen, West Sussex, UK) protocols for animal tissues. The DNA concentration was estimated using HyperLadder™ 100 base-pair (bp; Bioline, London, UK) on 1.0% agarose gels stained with either ethidium bromide (Fisher Scientific, Leicestershire, UK) or SYBR® Safe (Fisher Scientific, Leicestershire, UK). The laboratory work on museum samples was carried out in a dedicated museum DNA laboratory where no contemporary avian DNA had been present. Furthermore, all work with museum samples/DNA was carried out in a UV-irradiated fume hood to further eliminate any potentially contaminating DNA before and after each laboratory session. Genomic DNA was extracted from museum samples in batches of three-five samples, with Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 81 negative controls, using QIAamp DNA Micro kits (Qiagen, UK), following the manufacturer’s protocol for isolation of genomic DNA from forensic case work samples, with an extended overnight step of incubation with Proteinase K. mtDNA amplification For contemporary samples, we used polymerase chain reaction (PCR) to amplify a 677 bp fragment of the mitochondrial (mtDNA) control-region using primers LCRL1 (5’-CGCTATGACCCTCCACGAA-3’) and HCR1045 (5’-GAGACGACCTTATCCGCAAA-3’) (Tarr 1995; Tarr and Fleischer 1995). In each sample, the fragment was amplified in 50 µl reactions containing 1x Taq reaction buffer [160mM (NH4)SO4, 670 mM Tris-HCI], 1.5 mM MgCl2, 200 µM each dNTP, 0.2 µM each primer, 2 units Taq DNA polymerase (Bioline, London, UK), and 40 ng template DNA. Amplification was performed under mineral oil using the following cycling conditions: 94 oC for 7 min; then 30 cycles of 94 oC for 30 sec, 52 oC for 30 sec and 72 oC for 1 min; followed by a final step of 10 min at 72 oC. All reaction products were checked using electrophoresis on agarose gels and those that had contamination in the negative controls or did not show strong amplifications were discarded and re-amplified. PCR products were sequenced using an ABI 377 DNA sequencer (Macrogen Genomics, South Korea, and Source BioScience, Nottingham, United Kingdom). Characterization and amplification of microsatellite markers Microsatellites were isolated from an enriched genomic library developed for parrotbill by Genetic Identification Services (Chatsworth, CA USA) following procedures in and Jones et al. (2002) and Cristinacce et al. (2009). Recombinant plasmids were produced by ligating restriction fragments (350-700 bp) from pooled genomic DNA from five individual wild parrotbills [two males (#1371-04952 and #1371-04937) and three females Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 82 (#1371-04951, #1371-04954 and #1371-04948)] into the Hind III (AAGCTT) cut site of the pUC19 plasmid. DNA sequencing was accomplished using Amersham’s DYEnamic™ ET Terminator Cycle Sequencing Kit (Amersham Biosciences P/N US81050) with an M-13 forward primer (5'- AGGAAACAGCTATGACCATG -3'), followed by electrophoresis on an Applied BioSystems Model 377 DNA Sequencer. PCR primers were designed to the flanking regions of 24 microsatellites using DesignerPCR, version 1.03 (Research Genetics, Inc.). These primers were used to amplify five assumed unrelated parrotbill individuals to assess polymorphism. Sixteen loci (13 polymorphic and 3 monomorphic) were selected for further optimization. Using the previously extracted and diluted genomic DNA for contemporary samples, a range of annealing temperatures (50-68 oC) were tested, and the temperature producing the cleanest PCR product, as observed on 1.0% agarose gels stained with ethidium bromide, was selected for subsequent PCRs. Each locus was amplified separately in 12.5 µl reactions containing 1x Taq reaction buffer [160mM (NH4)SO4, 670 mM Tris-HCI], 1.5 mM MgCl2, 200 µM each dNTP, 0.2 µM each primer, 0.5 u Taq, and 10 ng template DNA. PCR amplification was performed under mineral oil using the following cycling conditions: 94 oC for 5 min; then 30 cycles of 94 oC for 30 s, 57 or 62 oC for 30 s and 72 oC for 1 min; followed by a final step of 10 min at 72 oC. All amplifications were checked on agarose gel and weak amplifications were discarded and run again. Twenty percent of the samples were also amplified a second time. Multiplexing was performed post PCR amplification. Fluorescently labelled DNA fragments were detected using an Applied Biosystems 3730 DNA Analyzer with GeneScan ROX-500 size standard (DBS Genomics, Durham, United Kingdom). Historical samples were amplified for each locus separately in 4 µl PCR reactions containing 1 x Qiagen multiplex PCR master mix (Qiagen, UK), 1 µM of each primer and 2 µl of template DNA at unknown concentrations following Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 83 Kenta et al. (2008). PCR amplification was performed for each locus under mineral oil using the following cycling conditions: 95 oC for 15 min; then 46 cycles of 94 oC for 30 s, 60 oC for 90 s and 72 oC for 60 s; followed by a final period of 60 oC for 30 min. PCR products from museum samples were genotyped individually for each locus using the same ABI 3730 DNA Analyser as used for the contemporary samples. While there is a greater potential for allelic dropout when genotyping ancient and degraded DNA (Taberlet et al. 1996; Nielsen et al. 1999), re-amplifications were not possible for extracted museum samples due to the small quantities of DNA extracted from each tissue sample. Data analysis mtDNA PCR products obtained from the first 10 DNA extractions (8.3% of the total samples) were sequenced in both the forward and the reverse directions. After end clipping and careful visual examination of the sequence reads, each was aligned. Alignments for each individual showed no discrepancies in base calls between the forward and reverse strands and subsequent samples were sequenced using the forward primer only. Chromatograms were edited using FINCHTV (Geospiza Inc.). Sequences were aligned in CLUSTAL X v2 (Larkin et al. 2007). Each polymorphism was visually scrutinized on the chromatograms. Samples with new or rare polymorphisms were sequenced twice, in both forward and reverse directions, by Macrogen Genomics Inc. and Source BioScience to confirm polymorphisms and haplotypes. Individuals were referred to as captive or wild. The latter were further grouped geographically as those captured east or west of the Koolau Gap (Figure 4.1). Standard DNA polymorphism and genetic differentiation measures were calculated using DNASP v4.00 (Rozas et al. 2003). All sequences have been deposited in European Nucleotide Archive (see Appendix D). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 84 Microsatellite genotyping Genotypes were scored using GENEMAPPER v3.7 (Applied Biosystems, Inc.). Twenty percent of the samples were scored a second time from subsequent PCR amplifications to check for allelic dropout. We calculated allelic and heterozygosity patterns using GENALEX 6 (Peakall and Smouse 2006) and tested for significant deviations from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium at each locus for each sampling locality using GENEPOP v4.0.10 (Raymond and Rousset 1995). We corrected the linkage disequilibrium tests for multiple comparisons using a sequential Bonferroni correction (Rice 1989). We calculated allelic richness per locus for each sampling site using FSTAT 2.9 (Goudet 1995). Null allele frequencies were estimated using CERVUS (Marshall et al. 1998). Patterns of genetic structure For the entire contemporary population, we applied an analysis of molecular variance (AMOVA) using GENALEX 6 (Excoffier et al. 1992). AMOVA provides estimates of traditional F-statistics (Weir and Cockerham 1984), as well as their analogues (Rst and ɸpt) to examine subpopulation structuring and calculate pairwise Fst and Rst between the east, west, and captive populations. Given that a limitation of the AMOVA framework is that it requires a priori clustering of samples, microsatellite genotypes were grouped according to their geographic location (east, west and captive). As a source for comparison, we also generated similar population differentiation results using FSTAT 2.9 (Goudet 1995) incorporating an exact G-test (Goudet et al. 1996), and to account for sample size bias we calculated allelic richness and private allelic richness for each population using the rarefaction technique in HP-RARE 1.1 (Kalinowski 2005). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 85 To further describe genetic structure of the wild population, a Bayesian clustering method that did not take spatial location designations into account using STRUCTURE v2.3.3 (Pritchard et al. 2000) was used. STRUCTURE implements a Bayesian approach to estimate the most likely number of population clusters (K) based on the genotypes of the individuals included in the analysis. We specified an admixture model, whereby a proportion of the genome of each individual is probabilistically assigned to each cluster according to allele frequency by minimising deviations from Hardy-Weinberg equilibrium. The data were grouped geographically (see Figure 4.1) but genotypes from all wild individuals were pooled into a single dataset and analyzed for signals of genetic structure. STRUCTURE allows the input of predefined populations to enable easier comparison with the allele frequency based structure, but it does not use this information prior to analyses. This approach allowed a comparison between signals of structure in the ecologically-inferred east and west populations and any signal of structure evident from allele frequencies alone. Geographic grouping was delineated using the natural topography of east Maui. Since the overall range for parrotbills is small (40-50 km2), and other than the Koolau Gap, there are no geographic barriers to the existing population, we did not believe that high K values would be biologically feasible in this population. Therefore, we considered a maximum value for K of 1-5. We completed 20 runs of 1000000 iterations with thinning of 100 after an initial burn-in of 100000 as per revised recommendations in Gilbert et al. 2012. The assignment values, log likelihood scores, likelihood rate of change, and V K (Evanno et al.2005), were examined using STRUCTURE HARVESTER (Earl and vonHoldt 2012) to infer the optimal number of clusters. GENELAND v3.3.0, a spatial Bayesian clustering program, also was used to assess population structure in the wild population (Guillot et al. 2005). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 86 Unlike STRUCTURE, GENELAND assumes that genotypes are spatially correlated. This assumption is based on a hidden partition model that estimates the number of populations (based on genetic criteria) within the study area, assigns individuals to their populations of origin, and potentially detects immigrants. Inferences from spatial Bayesian clustering programs can be reasonably robust when the number of available polymorphic loci in a study is limited (Chen et al. 2007). A mixed analysis was first implemented to identify the number of clusters in the data considering a maximum of K = 1-5, with five repetitions. The results of these mixture analyses were then used to conduct the admixture analysis (Corander and Marttinen 2006). Tests for patterns of isolation by distance for the wild population were performed using ISOLDE program within GENEPOP v4.0.10 (Rousset 1997; Rousset 2000). This program regresses estimates of Fst/(1-Fst) to the natural log of the geographic distance between populations and compares the distance with a simple Mantel test (Rousset 1997). Geographic distances between each were calculated as linear distance between UTM (Universal Transverse Mercator geographic coordinate system) locations for each captured individual. Estimation of temporal change in effective population size To estimate changes in effective populations size (Ne) between contemporary and historical populations, we used TMVP (Beaumont 2003) and NEESTIMATOR (Ovenden et al. 2007). Using a Markov chain Monte Carlo (MCMC) approach in a Bayesian framework, TMVP samples independent genealogical histories using importance sampling. The program makes explicit use of collection dates for all individuals and combines historical and contemporary data to obtain a posterior distribution of Ne at the time of the oldest sample and at the most recent sample (NA and N0 respectively; Beaumont 2003). We specified a mean generation time of three years based on known reproductive Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 87 biology, and field observations of parrotbills, and after confirming the robustness of the TMVP program by running several sensitivity analyses at generation times of 1-4. We ran 20000 MCMC updates (an initial 10% were discarded as burn-in) with 10 updates between estimate outputs and used a rectangular prior of 0-1000 for both NA and N0. NEESTIMATOR uses a linkage disequilibrium approach and provides an estimate of Ne between two generations (NA and N0; Ovenden et al. 2007). Estimating sufficient individuals for reintroduction To assess the proportion of genetic diversity that could be captured within a given number of individuals, we performed a simulation in R (Team 2010). This calculated the range in proportion of alleles that could theoretically be transferred to a new population with differing numbers of founder individuals. The model first randomly selected numbers of founders in increments of five for up to 30 birds from the east and west populations and ran 1000 replicates. The number of founders was relatively small due to the low availability of potential founding individuals in either population. Second to look at what might be genetically desirable albeit biologically improbable, the model was repeated for numbers of founder individuals between 5-120 birds for 1000 replicates for each number of individuals. These estimated individuals to be translocated for a probability of capturing 100% of the genetic diversity in the founders of the reintroduced population. 4.3 RESULTS mtDNA Sequences from 85 individuals (56 east, 18 west, and 11 captive) identified three haplotypes (A, B, and C). It was decided that it was unlikely that the remaining 44 parrotbill samples would identify additional Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 88 haplotypes. Each haplotype was defined by a single bp change. Haplotype B had a G/A replacement and haplotype C had a C/T replacement. While haplotypes A and B were found across the species range and captivity, haplotype C was restricted to one adult female captured in the east. Haplotype diversity (Hd) was 0.382 overall, nucleotide diversity (pi) was 0.001 and haplotype frequencies varied (Table 4.1). Restricting the analysis to the wild individuals resulted in a slightly lower Hd (0.365), while nucleotide diversity remained the same. There was no significant difference in the mtDNA genetic differentiation between the wild and captive samples (χ2 = 1.792, df = 2, p = 0.408) or between the wild east and west samples (χ2 = 0.810, df = 2, p = 0.667; Table 4.1). Characterization and amplification of microsatellite markers We found 15 polymorphic loci. Based on sequence homology, all loci could be assigned a chromosome location on the Zebra Finch (Taeniopygia guttata) genome and mapped using MapChart 2.2 (Voorrips 2002) to identify Z-linked or other closely aligned loci based on chromosome locations (see Appendix B). Two loci were found to be Z-linked (sex-linked), therefore only males were characterized at these loci as females were always homozygous. One locus could not be scored cleanly due to spurious bands, one locus deviated from Hardy-Weinberg in all populations, and one locus was found in linkage disequilibrium with two other loci. These three loci were excluded, leaving 12 loci for final analyses (Table 4.2). Sequences and primer information for these 12 loci have been deposited in European Nucleotide Archive (See Appendix D). A total of 86% of loci across contemporary samples were amplified and genotyped successfully. No evidence suggestive of allelic dropout was apparent in re-amplification runs. A total of 56% of loci across historical samples were amplified and genotyped successfully. Five historical samples yielded no accessible DNA which may be a result of past storage Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 89 conditions and preservation treatment of specimens (Lindahl 1993; Wandeler et al. 2003). Genotyping analyses Mean allelic richness for the contemporary population was 4.05 (1.67-6.04). The differences between observed, expected, and unbiased expected heterozygosity patterns were similar across each of the three populations (Table 4.3). The east population had the highest level of heterozygosity and a significantly higher number of private alleles per loci (Table 4.3). Rarefaction analyses found similar patterns of allelic richness and private allelic richness with analyses across only 10, 20, and 30 genes (see Appendix B). Global Fst for the entire dataset was low (Fst = 0.06). There were significant differentiations in pairwise Fst and Rst between east and west (Fst = 0.05; Rst = 0.06) and between west and captive populations (Fst = 0.10; Rst = 0.16) but not between east and captive birds. The mean numbers of alleles per locus, effective alleles per locus, and private alleles per locus were all higher in the historical samples compared to the contemporary population (Table 4.3). Population patterns of genetic diversity STRUCTURE and GENELAND analyses resolved a clear pattern of population structure in the wild. Applying the Evanno et al. (2005) correction to the STRUCTURE output yielded a signal of V K= 4 (Figure 4.2). Individuals were mapped according to the probability of assignment to each cluster from GENELAND (K = 2 and 4; Figure 4.3). No significant evidence for isolation by distance was detected within the genotype dataset. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 90 Estimates of historical and contemporary Ne The temporal change in effective population size (Ne) from TMVP analyses is given in Figure 4.4. The density of points is proportional to the probability density of population size at the time of the oldest sample and the most recent sample. An off diagonal distribution therefore indicates a change in Ne. The resulting output for the contemporary population provides strong evidence for a severe decline in Ne across the past 110 years. The modal mean for the contemporary population in 2011 is 37 (95% HPD limits 22-71) compared to a modal mean of 991 in 1892 (95% HPD limits 575->1000). NEESTIMATOR calculated a contemporary Ne estimate of 52.5 (45.7-61 95% CI). Estimating sufficient individuals for reintroduction Random capture of 25 individuals from the east would ensure the inclusion of 80% of the genetic diversity. Ten individuals would capture the equivalent genetic diversity from the west. A random selection of 30 individuals from across the species’ entire range would capture 80% of the total contemporary genetic diversity, 60 individuals would capture 90% and 105 individuals would have to be selected to capture 100% of the genetic diversity (Figure 4.5). 4.4 DISCUSSION Our results reveal a 96% reduction in genetic Ne over the past 110 years, as well as a signature of spatial structuring of the current parrotbill population that is underpinned by topographical features and reflected in a skewed distribution of private alleles. Together, these data provide a valuable new perspective on the conservation management of this species and an important framework for the design of a reintroduction strategy to maximise future retention of genetic diversity. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 91 Contemporary levels of genetic diversity Contemporary mitochondrial DNA haplotype (Hd) and nucleotide (pi) diversity was low compared to other more common Hawaiian honeycreeper species (Hawaii Akepa Hd = 0.980, pi = 0.006 Reding et al. 2010, Hawaii Amakihi Hd = 0.882, pi = 0.008 Foster et al. 2007). Nuclear DNA revealed similar levels of observed and expected heterozygosity (0.574 and 0.534, respectively), which were within the expected range for a population with a history of small population size (Frankham et al. 2002). Island species often exhibit low levels of genetic diversity (Frankham 1997; 1998), commonly interpreted as being a consequence of their having a history of long-term isolation and restricted population size. Historical loss of genetic diversity While it is impossible to assume that any historical museum samples are representative of a panmictic population, knowledge of historical patterns of genetic diversity can provide insight into a population’s demographic trajectory and can reveal indications of genetic impoverishment. Nuclear DNA markers showed temporal losses in genetic diversity across all diversity measures. Mean number of alleles per locus and proportion of polymorphic loci were both reduced by 12%, mean number of effective alleles per locus was reduced by 30%, mean number of private alleles was reduced by 10% amongst the east and by 100% amongst the west population, whilst expected heterozygosity was reduced by 19%. These reductions are not unsubstantial and suggest that the current parrotbill population has experienced severe reduction in size over the last century. The species has also experienced a reduction in Ne of approximately 96%, from an ancestral Ne of 991 to a contemporary Ne of 37. The estimate of current census population size (N) for parrotbills in the area of their range from which our samples were taken is 421 (209-674 95% CI) individuals (Brinck Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 92 et al. 2012). This estimate of N aligns closely with our genetic estimates of contemporary Ne, assuming a Ne /N ratio of 10% for wildlife populations (Frankham et al. 2002). The finding of an historical reduction in Ne of at least an order of magnitude across the past 110 years enables some interpretation of what ecological or environmental mechanisms may have been responsible for this decline. While Hawaiian forest birds have suffered a plethora of threats that have reduced their populations over time (Banko and Banko 2009), it is perhaps most likely that the bottleneck required to precipitate the observed reduction in Ne since 1894 occurred relatively quickly, imposing a reduction in population size within a short period of time and to a level that subsequently induced the substantial loss of genetic diversity (Frankham et al. 2010). Such an interpretation and the timing of this event could be explained by the arrival of avian malaria on the Hawaiian Islands (Atkinson and LaPointe 2009). Mosquitoes were introduced to Maui in 1826 (Dine 1904; Hardy 1960), providing a mechanism for spreading disease (avian malaria, Plasmodium sp.), which is believed to have arrived to the islands sometime in the early twentieth century and effected non-native passerines soon thereafter (Atkinson and LaPointe 2009). While Hawaii extinctions have been attributed to post Polynesian (James and Olson 1991; Olson and James 1982; James et al. 1987) and post European (Banko and Banko 2009) contact, the arrival of mosquitoes and avian malaria was potentially the most serious threat to the parrotbill population and the resulting population contraction. Patterns of contemporary genetic differentiation and structure Fst and Rst values showed significant deviations on either side of the Koolau Gap, a division supported by both STRUCTURE and GENELAND analyses. This suggests that the Koolau Gap is acting as a topographic barrier that is limiting gene flow between these two sections of the current population. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 93 Further subdivision within the east and measures of genetic diversity calculated per population lend support to this result. These higher levels of overall diversity and allele privatization in the east cannot be explained by sample size alone. While sampled disproportionally, overall population estimates for these areas (the global population comprises ~¾ on the east and ~¼ on the west) show that our sample sizes represented ~20% of the west and ~22% of the east population. Optimizing reintroduction strategy for genetic diversity The low detection rate during field surveys has made it difficult to confirm the apparent stability of this species’ population over the past 30 years (Gorreson et al. 2009). This fact, coupled with the low Ne and the presence of a rare mtDNA haplotype, emphasizes the need to move forward the recovery program for this species. There are a number of different recovery options; below, we discuss how integration of genetic data into each of them can maximize the retention of genetic diversity within the restored population and potentially enhance the success of reintroduction efforts. (i) The first recovery option would be to raise the quality of the current habitat to increase population density and in turn increase the global population and reduce the continued loss of genetic diversity. Rat (Rattus spp.) densities are very high across much of the current population’s habitat (Sugihara 1997; Malcolm et al. 2008) and intense rodent reduction efforts might increase survival and/or productivity by reducing depredation risk. However, recent demographic work suggests that although low fecundity and productivity may complicate this species’ recovery, it is not the main factor limiting the population (Mounce et al. 2013). (ii) The second recovery option would be to establish a second population through the release of captive bred individuals. The significant Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 94 deviation in Fst and Rst values for the west and captive populations raises concerns as to whether the captive population should play a significant role in future reintroduction efforts. If managers choose to rely heavily on the captive population for reintroduction, the overall genetic diversity of the captive flock would need to be increased to reflect the overall diversity in the wild. We would suggest that this include a combination of new genes from the east to incorporate the private alleles not represented in captivity, as well as genes from the west. Due to the low reproductive potential of this species and the minimal breeding success that they have had in captivity thus far, we do not recommend this strategy as the time line for developing a genetically diverse captive population would greatly delay the recovery efforts. (iii) Lastly, we feel the recovery option that has the greatest chance of success and maximizes the potential for retention of genetic diversity, would be to establish a second population through translocating wild individuals from both the east and the west to leeward east Maui. There is the potential of capturing varying levels of genetic diversity in the reintroduction design and the presence of low levels of genetic diversity observed highlights the importance of capturing as much diversity as possible amongst the founding individuals (Groombridge et al. 2012) in order to retain maximum evolutionary potential to adapt to environmental change. The patterns of population structure are designed to allow managers to weight the genetic considerations in moving different numbers of individuals from different areas of the population. Furthermore, the estimation of sufficient individuals for reintroduction informs managers of the numbers necessary in structuring a reintroduction through translocations. With a small and endangered population like Maui Parrotbill, the numbers of individuals needed to capture 100% of the available genetic diversity are probably unrealistic as they represent up to one quarter of the global population. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 95 Instead, managers will need to determine what an acceptable amount of genetic diversity is and how to best design efforts to attain that goal as no one single strategy to cope with the genetic trade-offs in reintroduction designs has been universally accepted (Groombridge et al. 2012). We feel that the reintroduction design should attempt to capture all available genetic diversity in the wild while considering that the global population size itself may limit the number of individuals that can be taken from any one area. This population is unlikely to suffer a reduced level of fitness from outbreeding depression and the loss of local adaptations (Frankham et al. 2010) given that parrotbills have been extirpated from the koa-dominated mesic forest similar to that of the reintroduction sites available (U.S. Fish and Wildlife Service 2006), and it is unlikely that adaptations to the drier forest still exist in the current genetic diversity of the parrotbill population. Although these data reveal the dramatic affect avian malaria has had on the parrotbill population, there are currently no management techniques available to control its spread in Hawaiian honeycreepers. Fortunately, the mosquitoes and Plasmodium do not thrive at higher elevation habitats, and thus the current wild population is relatively secure from their effects at these high elevation refugia. This not only explains the current distribution of parrotbills across Haleakala above ~1500 m in elevation, but also enforces future management efforts to be restricted to these high-elevations areas. Reintroductions and recovery actions will be limited to currently “malaria free” zones on Haleakala. Currently 1100 ha of mesic koa forest is being restored on leeward east Maui and is the best suitable location for the reintroduction. Knowledge of population genetics can inform the choices managers will have to make, but genetics is just one of a variety of ecological and other factors needed in order to best design these reintroductions whether through conservation translocations of wild individuals, the release of captive-bred birds, or a combination of both. While the best reintroduction design will most likely incorporate components from several of the options Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 96 outlined, the reintroduction of wild-sourced individuals selected from across the different genetically structured parrotbill populations provides the greatest potential for success going forward. ACKNOWLEDGEMENTS Funding from the United States Fish and Wildlife Service and the State of Hawaii Division of Forestry and Wildlife helped to make this work possible. We thank San Diego Zoo Global for their assistance and permissions in sampling the captive Maui Parrotbill flock as well as National Museum of Natural History (Smithsonian, D.C., USA), Museum of Comparative Zoology (Cambridge, UK), Cambridge University Museum of Zoology (Cambridge, UK), British Museum of Natural History (London, UK), Bernice Pauahi Bishop Museum (Honolulu, USA), and American Museum of Natural History (New York, USA) for access to the museum specimens for sampling. We thank Samit Kundu for laboratory assistance and guidance, Kelly Iknayan, Christopher Warren and Ruby Hammond for assistance with GIS analyses, and Siobhan Simpson and Rachel Bristol for assistance in microsatellite analyses. We also thank Carl Jones for connecting this team of researchers with one another, as well as Eben Paxton, David Duffy, and John Vetter for initial reviews of this manuscript. Lastly, we thank Maui Forest Recovery Project field teams for their arduous work in collecting blood and feather samples and The Nature Conservancy Hawaii and State of Hawaii Division of Forestry and Wildlife for access to public and private lands to collect such samples. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 97 4.5 TABLES AND FIGURES Table 4.1. Sample size (n), number of haplotypes (Hp), haplotype (gene) diversity (Hd), nucleotide diversity (pi) and the frequencies (f) of haplotypes A, B and C for Maui Parrotbill (Pseudonestor xanthophrys) mtDNA analyses. n Hp Hd ± SD pi f (A) f (B) f (C) East 56 3 0.350 ± 0.067 0.001 0.786 0.196 0.018 West 18 2 0.425 ± 0.099 0.001 0.722 0.278 0.000 Captive 11 2 0.509 ± 0.010 0.001 0.636 0.364 0.000 Total 85 3 0.382 ± 0.050 0.001 0.753 0.235 0.012 Table 4.2. Characteristization of Maui Parrtobill (Pseudonestor xanthophrys) microsatellite loci. Annealing temperature (Ta), allelic diversity (A), observed heterozygosity (Ho), expected heterozygosity (He), Hardy-Weinberg equilibrium test P values as identified by GenePop v4.0.10 (PHWE) for east population. Chromosome location assignments are mapped against the zebra finch (Taeniopygia guttata). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 98 LocusChromosome locationChromosome location (bp)BLAST P(N)Nucleo-tidetypeRepeat motifFluro-label (F)Primer sequences (5'-3')Optimized Ta (oC)AExp. Allele sizes (bp)Obs. allele sizesrange (bp)HoHePHWENull allele frequencyPxa01Tgu757290010.0tri(GTT)8 5'-HEXF: CAACTGATGCTGTTAGCAGATG575271260-2780.100.140.030.13R: AGGTTCCCAAAGTCCTTTCTCPxa02Tgu370905375E-142tetra(TAGA) 1(GTA) 1(TAGA) 2(GACA)1(TAGA) 3(GACA) 1(TAGA) 5 5'-FAMF: TTTCCACTTGTCCACTTCACC579286273-3330.800.830.380.02R: GCATCCATCCATCTATTCACCPxa03Tgu676890050.0tri(GTT) 145'-FAMF: CTGAGGGGAGAGGTTGTCAT579265246-2680.610.630.230.01R: TGGGGAAATAGTTGGAGGAGPxa04Tgu237940853E-143tri(CAA) 105'-HEXF: GGGAATAATGAAGCCTAAGAGG576257243-2780.670.640.030.03R: AACTGTGGGGCAACTAATGAPxa05Tgu2374306180.0tri(CAT) 2(CAC) 1(CAT)65'-HEXF: ATTGCTGCCATTTTCCTCTC572129121-1260.130.140.570.02R: GAGTGCTCTCCACACCACACPxa06Tgu1A10561894E-107tetra(CTAT) 125'-FAMF: GGCACTGGATGATCTTGAAG5711158128-1710.880.820.770.04R: ATTTCCTCCTCTTGGAGCACPxa07Tgu3158942090.0tetra(CTAT) 1(CAT)1(CTAT) 115'-FAMF: ACTGCACTTGTGGTGAAAGAC576240201-2430.630.720.040.06R: GTTTTCCTTCAGACTGGAACAGPxa08TguZ31129640.0tetra(CTAT) 20(GCAT)1(CTAT)115'-HEXF: CAGTGCAGGACTGTGTATTATG6211247168-2400.610.730.070.08R: GCAAAGGAAAAAGGAGAAATAGPxa09Tgu174006841E-97tetra(TAGA) 4(TA)2(TAGA) 55'-FAMF: CGTGACTCAGCACCTACAAC627142139-1690.680.710.080.01R: TGCACAAACAAACATTCTGTCPxa10Tgu2165260071E-127tri(GTT) 105'-HEXF: GCTTCAGCAAACAGCATTC576262238-2650.450.420.250.04R: CTGCGTGAGTTCATTTCATCPxa11Tgu114812431E-177tetra(CTAT)1(CAT) 1(CTAT) 14(CTAAT) 1(CTAT) 45'-HEXF: GTGGCAGTGTAGCAGAGTCT628201202-2340.830.780.380.03R: CCATTCTTAGGCTGAGGTAACPxa12Tgu4114044433E-75tetra(CTAT) 18(CAT) 1(CTAT) 15'-HEXF: GCAGCACAAGTGGAACTG577259244-2700.830.750.610.06R: TTGAGCCAGAAGGGATAACRecovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 99 Table 4.3. Allelic and heterozygosity patterns based on microsatellite data across the east, west, and captive Maui Parrotbill (Pseudonestor xanthophrys) populations. Standard errors are shown in parentheses. !" #$ #$ %" #$ & ' Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 100 Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 101 Figure 4.1. Map of Haleakala Volcano, eastern Maui, Hawaii, USA, with the current Maui Parrotbill (Pseudonestor xanthophrys) population range, the Hanawi Natural Area Reserve [managed for forest bird protection by the State of Hawaii, Division of Forestry and Wildlife (DOFAW)] and the Waikamoi Preserve (managed for forest bird protection by The Nature Conservancy Hawaii). The Nakula Natural Area Reserve on the leeward slope has been identified as a restoration priority by DOFAW as well as the most suitable location to establish a second Maui Parrotbill population. The current Maui Parrotbill range given suitable habitat is 40 km2, showing the sampling location of all wild individuals included in the genetic analyses. The predicted range constricts to 9 km2 by 2090 based on climate change models. The potential suitable habitat in 2090 given the same climate change models but landscape level habitat restoration would maintain 40 km2. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 102 Figure 4.2. Mean likelihoods, likelihoods rate of change, and VK values calculated from number of genetic clusters (K = 1-5) in STRUCTURE before and after applying the Evanno et al.(2005) correction to the output in STRUCTURE HARVESTER. Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 103 Figure 4.3. Maui Parrotbill (Pseudonestor xanthophrys) assignment probabilities to each population cluster Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 104 from GENELAND results of K = 2 (right) and K = 3 (left). Figure 4.4. A posterior distribution of the temporal change in historical and contemporary effective population size (Ne) for the parrotbill population as derived from TMVP analyses following the methods of Beaumont (2003). Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 105 Figure 4.5. Probability values for capturing different percentages of the total genetic diversity available in the east, west and total wild Maui Parrotbill (Pseudonestor xanthophrys) population modelled while using different numbers of individuals in translocation efforts.Recovery of the endangered Maui Parrotbill – Chapter 4 – Genetics 106 Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 107 Chapter 5 Extinction risk and reintroduction options for the endangered Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys) HANNA L. MOUNCE1, 2, CHRISTOPHER C. WARREN1, CONOR P. McGOWAN3, EBEN H. PAXTON4, and JIM J. GROOMBRIDGE2 1 Maui Forest Bird Recovery Project, Makawao, Hawaii 96768, USA 2 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent CT2 7NR, UK 3 U.S. Geological Survey, Alabama Cooperative Fish and Wildlife Research Unit, School of Forestry and Wildlife Sciences, Auburn University, Auburn, Alabama, USA 4 U.S. Geological Survey Pacific Island Ecosystems Research Center, Hawaii National Park, Hawaii, USA Keywords: extinction risk, Hawaiian Honeycreepers, population viability modelling, reintroduction, VORTEX Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 108 ABSTRACT Extinction rates for island birds around the world have been historically high and are increasing. For forest passerines, the Hawaiian archipelago has suffered some of the highest of these extinction rates. Population viability analyses (PVA) can be used to assess risks to vulnerable populations and evaluate the relative benefits of various conservation strategies. Here we present a PVA to assess the long term viability for Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys), an endangered passerine on the Hawaiian island of Maui. Modeling the current demographic trajectory of the wild population, Maui Parrotbill are predicted to not persist beyond 25 years. Female mortality and fecundity are two factors driving this decline. To evaluate and compare management options involving captive rearing and translocation strategies we built a female-only age-structured, meta-population simulation model. Due to the low reproductive potential of parrotbills in captivity, the number of individuals (as many as 20% of the global population) needed to source a reintroduction from captive reared birds is unrealistic. A reintroduction model that incorporates a minimal contribution from captivity and instead translocates mostly wild individuals was found to be the most favorable option for managers. New habitat is being restored on leeward east Maui which may provide more favorable climate and habitat conditions for the species and lead increased reproductive output. Our model provides managers with benchmarks for fecundity and survival to maximize the likelihood of reintroduction success, and highlights the importance of establishing a new population in potentially favorable habitat to ensure long-term persistence of the species. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 109 5.1 INTRODUCTION One in eight of the world’s bird species is globally threatened (BirdLife International 2014a) and extinction rates are highest on islands (Gilpin and Soulé 1986; Steadman 2006). There is a broad suite of ecological characteristics that can explain extinction risk of small populations of threatened species. Amongst these are a host of stochastic threats can that interact with deterministic factors to lead to population extinction (Shaffer 1987), including demographic, environmental, genetic, and catastrophic threats (Shaffer 1981). Together, these factors form the ‘extinction VORTEX’ and their interacting effects increase extinction probability (Gilpin and Soulé 1986; Soulé and Mills 1998; Mills 2007). Population viability analysis (PVA) is an analytical tool used to measure and ultimately combat the processes that can lead to extinction, whereby data can be applied to a suite of models that combine the effects of deterministic and stochastic factors to estimate a population’s probability of future persistence across specified time frames (Gilpin and Soulé 1986; Caughley 1994; Beissinger 2002). Ideally, a PVA should incorporate every aspect of a population’s biology, and when correctly parameterized it can provide insights into what factors constitute the greatest threats to the population’s survival (Mills 2007). While PVA is often used to quantify absolute risk of extinction, their real value is in an applied context, to examine the relative benefits of alternative management actions and estimate relative probability of extinction under different strategies (Akçakaya and Sjogren-Gulve 2000; Ellner and Fieberg 2003). The species-specific information needed to calculate a population’s absolute risk of extinction with precision and to compare relative extinction risk under different management scenarios is rarely achievable for endangered species, due to their low density and often cryptic habits. However, in those instances where endangered species have been sufficiently well-studied, PVA becomes a more useful tool to conservation managers (Ralls et al. 2002). Indeed, predicting time to extinction under a variety of scenarios can inform conservation decisions, help guide management efforts, and prioritize Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 110 management options (Clark et al. 1991; Cook et al. 2012). For example, if an endangered species is predicted to go extinct within a decade, recovery actions will need to differ substantially from those for a population predicted to decline slowly over a century. Furthermore, by comparing the model output for different scenarios, managers can evaluate which strategies are most effective to reverse the trajectory of a species’ decline versus other approaches that might be capable of achieving the same goal but for which the necessary techniques are unavailable or less developed. One species of immediate conservation concern is the Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys), a critically endangered Hawaiian honeycreeper endemic to Maui Nui (the islands of Maui and Molokai) in the Hawaiian Islands. The Maui Parrotbill is a feeding specialist with a parrot-like beak for extracting insect prey from bark and decaying wood (U.S. Fish and Wildlife Service 1967; Simon et al. 1997; IUCN 2012). Extensive demographic studies have been carried out on productivity and survival in two separate areas of the species’ range (Becker et al. 2010; Mounce et al. 2013; Mounce et al. 2014), indicating parrotbills are long-lived, strongly monogamous passerines which can reproduce for a minimum of 15 years. Breeding pairs typically produce only one offspring per year, exhibit prolonged parental care (5-18 months), and defend multi-purpose territories averaging 6-8 ha (Simon et al. 2000; Mounce et al. 2013; Warren and Mounce 2014). Parrotbills were once abundant on the islands of Maui and Molokai (James and Olson 1991), but have undergone substantial declines since the arrival of humans approximately 800-1000 years ago (Mounce et al. 2015). Today the wild population comprises approximately 500-600 individuals which occupy less than 50 km2 on windward east Maui (Scott et al. 1986; U.S. Fish and Wildlife Service 2006; Camp et al. 2009). While population-wide surveys have been unable to assess the stability of the current population (Gorresen et al. 2009), a spatiotemporal analysis of genetic diversity indicates a severe historical decline in population size over the past century (Mounce et al. 2015). Due to a lack of resistance by the native forest birds to mosquito borne diseases, such as avian malaria (Plasmodium relictum), forests Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 111 above 1500 m elevation provides the only existing refuge for most native Hawaiian honeycreepers (Scott et al. 1986; Mountainspring 1987; Simon et al. 1997) including Maui Parrotbills. Unfortunately, avian malaria is now moving into higher elevations, coincident with increasing average temperatures in Hawaii and gradually eroding available habitat for these species (Giambelluca et al. 2008; Benning et al. 2002; Harvell et al. 2002). Moreover, these high elevation windward habitats are likely suboptimal for parrotbills as 1) these habitats lack a perhaps key foraging substrate, koa (Acacia koa) (Perkins 1903), and 2) the prevalence of nest failures, frequently attributable to severe weather, in these areas are high (U.S. Fish and Wildlife Service 2006; Becker et al. 2010; Mounce et al. 2013). The historically forested island of Maui once provided almost island-wide habitat for parrotbills including lowland and leeward (southeast) forests. Sadly, little suitable habitat exists beyond the species’ current range with the exception of a few remnant forest tracts on leeward east Maui, such as those found in Nakula Natural Area Reserve (NAR). In addition to the wild population, there is a small captive flock of parrotbills (currently 15 individuals) that was established in 1997 and is managed by San Diego Zoo Global on the islands of Maui and Hawaii. Together, the captive flock and habitat restoration efforts have paved the way for a number of potential conservation strategies for this species, prompting the need for the evaluation. We applied PVA models using detailed data from demographic, genetic, and environmental/ecological studies recently completed for this species (Mounce et al. 2013, 2014 and 2015), to assess long-term viability of Maui Parrotbills and evaluate conservation strategies that may be applied to increase the long-term viability of the species. We used an individual based simulation model (VORTEX) to understand key limiting factors for the current population by determining which demographic variable(s) were most influential for population growth and long-term viability. In addition, we created a custom demographic model to examine the effects of (1) different management strategies to improve productivity and survival in the species’ current range, (2) removing individuals Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 112 from the wild for reintroduction elsewhere, and (3) establishing an additional geographically-distinct population in the leeward forests, currently unoccupied by parrotbills, which may provide a long-term refugia for the species. 5.2 METHODS Wild Population Viability To explore different viability scenarios on the current wild population, we used VORTEX V10.0.7.3 (Lacy and Pollak 2014). VORTEX is a widely used and flexible simulation program that runs individual-based Monte Carlo simulations while combining the effects of deterministic values and stochastic (demographic, environmental, and genetic) events. To model the extinction processes that threaten small populations, VORTEX relies upon user-specified demographic values (including variance) and then models population dynamics as discrete, sequential events that incorporate stochasticity through random draws from probability distributions. VORTEX runs multiple simulations (set at 1000) to generate a distribution of possible fates that a population might experience under a given set of parameters. Each run steps through a series of events that describes an annual cycle (reproduction, mortality, dispersal among populations, removals, supplementation, and if necessary, truncation to the carrying capacity) (Lacy 2000; Miller and Lacy 2005). VORTEX is appropriate for modelling the parrotbill population as it was initially designed for vertebrate populations with low fecundity and long life spans. Although VORTEX has the further ability to include catastrophes in its models, we did not use this feature for simulations on Maui Parrotbills. Though there are limits to the biological and stochastic complexity that VORTEX can simulate compared to other PVA software packages, this potential shortcoming was not an issue for the analysis of the current wild population because estimates for all of the fundamental parameters were available. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 113 Measures of viability PVAs are limited by the quality of the input parameters available for a given species under each given scenario and do not identify absolute probabilities of extinction in a given time frame (Akçakaya and Sjogren-Gulve 2000; Reed et al. 2002). Therefore, it is important to evaluate a variety of quantitative measures that are available to evaluate population viability across all models rather than relying solely on the viability measures most commonly presented in PVA studies such as extinction probability, population size, and time to extinction (Pe’er et al. 2013). Consequently, for each model, we present mean population growth rate (stoch-r ), mean intrinsic rate of growth (λ), net replacement rate (i.e. per generation rate of change; R0), probability of survival (PS), mean population size from all iterations (N-all), mean population size from extant populations (N-extant), and gene diversity (GD), the expected heterozygosity in extant populations. Base Model Our base model was parameterized using estimates of parrotbill vital rates from Mounce et al. (2013, 2014, and 2015) (Table 5.1). The correlation of environmental variation (EV; defined as the annual variation in the probabilities of reproduction and survival that arise from random variation in environmental conditions) was set at zero as reproduction and survival parameters already averaged temporal variation as they were derived from long-term data sets. This base model was designed to represent the parrotbill population in its current state without incorporation of any change in threats (besides normal demographic and stochastic effects of small population size) and thus produces simulations of a probable population trajectory without any management actions. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 114 We made four changes to this base model for subsequent scenarios. First, two of the key input parameters for these models appeared problematic for the generation of an accurate PVA. The estimate of juvenile survival presented in Mounce et al. (2014) was markedly lower than previously reported annual juvenile survival rates for parrotbills (0.76 ± 0.09; Vetter et al. 2012) and for other Hawaiian passerines (average 0.32 ± 0.03; Woodworth and Pratt 2009). Furthermore, this estimate was generated from only 10 individuals (the estimate produced by Vetter et al. 2012 was generated from a subset of seven of these same individuals) and had a large standard error (0.15; Mounce et al. 2014). Second, the estimate of annual reproductive success (ARS) presented in Mounce et al. (2013) was derived from the core breeding season for parrotbills (January-June). Breeding attempts for this species have been observed during 11 of 12 months. Therefore, it is probable that while this ARS estimate may capture the majority of the success in a given year, it is likely to be an underestimate of the true ARS over the entire calendar year. Third, an annual decline in carrying capacity (K) for this species in their current habitat is inevitable due to the current and future upslope movement of avian malaria and its vector due to climate change (Benning et al. 2002; Harvell et al. 2002; Giambelluca et al. 2008). Fourth, genetic analysis has shown that the parrotbill population is not contiguous across their range likely due to limited dispersal between two subpopulations (Mounce et al. 2015). To incorporate these four discrepancies in to our models, we (1) decreased juvenile mortality from 83% to 68% to reflect values found in the other Hawaiian passerines (equal to 39-44% of adult survival), (2) increased by 10% the percentage of breeding females to account for a reasonable estimate of less well-documented ARS in the months outside of January-June, (3) added a decrease in K of -1% per year to account for the influx of avian malaria resulting from climate changes (Giambelluca et al. 2008), and (4) divided the population into two sub-populations with an associated K for each calculated from Warren and Mounce (2014) (Modified base model in Table 5.1). Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 115 General model settings For all individual simulations we used 1000 iterations spanning 25 years for a long-term monogamous species. Although longer time frames are more appropriate for assessing the predicted longevity of a species, for this exercise our focus was on the immediate viability risk and the effects of conservation actions that can be implemented to prevent imminent extinction. Due to persistent problems associated with introduced predators, continued loss of habitat, invasive species, and the inherent risks of a critically endangered organism, modeling population dynamics for this species on a longer timeframe would not provide any additional insight for critical management needs. Testing demographic sensitivity To test the demographic sensitivity of the current wild populations, all parameters were kept constant at the Modified Base Model parameter settings (Table 5.1) while one parameter in turn was manipulated (Jørgensen and Fath 2011). This sensitivity analysis was designed to identify which parameters were most sensitive to the survival of the metapopulation and to be able to compare sensitivities across parameters. To accomplish this we used a sensitivity index calculation (Sx) from Pertoldi et al. (2013): Sx = (O X/X) / (O Y/Y), where O X is the change in the observed measure of viability, and O Y is the change in the parameter of interest. Population Viability with Management To evaluate and compare management options in more detail involving captive rearing and translocation strategies tailored to the recovery requirements for this species, we built a female-only age-structured, meta-Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 116 population simulation model in R 3.0.1 (The R Foundation for Statistical Computing 2013). The viability models produced using R and VORTEX were nearly identical. However, while VORTEX allowed for easily repeatable modeling of population viability, R provided a more flexible framework for simulating management scenarios. We included an immature age class and a breeding adult age class in the model to reflect known age at maturation and differential survival rates of immature and adult birds (Mounce et al. 2014). Future immature bird abundance was modeled as a product of the number of breeding adults (), the fecundity rate () and the survival rate of young of the year (), as follows: , The number of adults in the future was a product of the number of adults () and their annual survival rate (), plus the product of the number of immature birds ( ) and their annual survival rate ( ), as follows: . Adult survival rates in the simulations were stochastic and drawn from a beta distribution where the alpha and beta shape parameters were derived from the survival estimates reported in Mounce et al. (2014) using the ‘method of moments’ calculations (see Morris and Doak 2002). As with the VORTEX model parameters described above, the juvenile survival rates from Mounce et al. (2014) were extremely low and based on a small sample size. Using those survival rates in our model predicted rapid and near certain extinction for the population, which seems unreasonable given the apparent population stability documented over the last 20 years based on repeated population and territory counts (Gorresen et al. 2009). We investigated juvenile survival estimates from other endemic Hawaiian forest bird species, but most suffer from the same low sample size issues and exhibit low survival estimates. Consistent with our VORTEX models, therefore, we set immature survival rate at 0.72 (SD 0.02) because at Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 117 that rate population projections in the wild populations roughly reflected the observed population growth rates from population surveys and territory counts. We encountered the same issues with hatch-year survival rates and set the mean at 0.32 (SD 0.03). Again, these mean and standard deviations were converted to alpha and beta shape parameters for a beta distribution using the ‘method of moments’ calculations (see Morris and Doak 2002). Fecundity rate was incorporated as the number of female offspring fledged per breeding female and was modeled as a log-normally distributed random variable. Mean annual fecundity was set at 0.588 with a 0.15 coefficient of variation. In our model we also included a ceiling type density dependent function whereby if a specified abundance threshold (885 females) was exceeded, the fecundity rate for that year was set to 0. We created a meta-population with implicit spatial structure to the population, by creating four separate sub-populations in the simulations. Two populations represent the existing east (Hanawi NAR) and west (TNC Waikamoi Preserve) populations (Mounce et al. 2015) on the windward slopes of Haleakala (Figure 5.1). Another population represents the proposed third population that will be established on the leeward slopes of Haleakala (Nakula NAR; Figure 5.1), and a fourth population represents the experimental captive breeding population that may serve as a source of individuals for release into the wild populations. The two wild populations use the projection equations described above. For the third (not yet established) reintroduced population we tested the effects of increased survival and fecundity rates on the probability of successfully establishing a wild self-sustaining population and on overall species extinction probability. We input demographic rates 5-20% higher for the third population to reflect the hypothesis that these drier forested habitats will provide higher quality habitat than the extremely wet windward rainforests, some demographic values that would more closely reflect those found in other Hawaiian honeycreepers (Woodworth and Pratt 2009). Abundance in the east wild population was initially set at 238 females, the west wild population was Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 118 initially set at 53 females (we used density estimates from Camp et al. 2009 and extrapolated to generate population estimates across all current occupied habitat from MFBRP unpublished data), and the third wild, yet to be established population was initially set at 0. The captive population was modelled differently from the wild populations since in captivity the birds are not subject to the same ecological processes. Instead, once established, the future abundance in captivity ( ) is the current number of individuals (), plus the number successfully reared (), minus the number that died (), which were modelled as Poisson distributed random variables with a mean of 2.0, and incorporated into the projection as follows: . We set initial abundance in captive population at 7 females to reflect current conditions of the captive flock. Movements between the sub-populations were restricted to translocations, meaning there was no natural rate of immigration between sub-populations. The projected abundance in a subpopulation was a function of natural population dynamics (as described above), and the number of individuals added to and subtracted from the population as follows: where T indicates the number of birds moved, i represents the current sub-population being projected and j indicates the other sub-populations to or from which individuals can be translocated. The model was written in a generalized Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 119 form so that birds could be moved from any sub-population to another (see Appendix C), but in our simulations management actions were limited to establishing a new sub-population and/or contributing to the small captive population. Translocations of a specific number of birds between sub-populations could be specified for a limited number of years such that if abundance in the west and east sub-populations fell below 25 or 100, respectively (excluding the captive population), removing individuals from that sub-population was prohibited. Lastly, individuals introduced to the wild from captivity are typically less successful (Fischer and Lindenmayer 2000). The fact that Maui Parrotbills will be re-established in a different habitat type increases the uncertainly regarding their survival. To reflect that uncertainty in our model, we made first year survival of captive released birds an annually varying uniformly distributed random number bounded between 0.4 and 0.9. The reintroduction scenarios varied mainly on account of where birds are sourced, namely the east and west wild populations, and the captive population. The goal of the captive breeding program from its onset has been to develop a sustainable breeding program for the species in the event of a collapse of the wild populations and/or to act as a source for reintroduction as new habitat became available. However, the captive program has only been moderately successful. The population remains small and few birds are recruited each year. As of 2015, the captive population consisted of seven females and eight males, which together produce an average of one bird each year. Given a sex ratio of 50:50 this represents a rate of 0.07 females produced per female per year. Realistic options for sourcing birds for reintroduction from the captive population include: a) Releasing a large proportion of the existing captive birds (e.g., 5 females and 5 males) in a single year. b) Releasing a minimal subset of the captive population (e.g., 1 female and 1 male) over the course of a few years. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 120 c) Augmenting the captive flock with wild birds, allowing for the release of a larger number of captive birds over the course of a few years. A working group of researchers and managers (Maui Parrotbill Reintroduction Working Group) have developed a plan to reintroduce parrotbills to Nakula NAR over a three year period, a strategy designed to balance probability of success and the best use of resources. Data on parrotbills home range sizes has suggested that the Nakula NAR may be able to support approximately 15 parrotbill pairs in the first few years of a reintroduction program (Warren and Mounce 2014). Considering these restrictions in the total numbers of individuals the area can immediately support, we therefore tested a variety of reintroduction scenarios whereby six pairs are released each year. While there are many possible scenarios that could be tested; we selected six that we felt were realistic given current management opportunities as follows: i. Release only captive birds currently available to establish a second population; ii. Augment the captive flock with wild birds such that the captive flock alone would source a second population; iii. Augment the captive flock with wild birds such that the captive flock would provide half the individuals needed for reintroduction with the other half from translocated wild individuals; iv. Augment the captive flock with wild birds such that the captive flock would provide 1 female per year in combination with translocated wild individuals; v. Release only captive birds currently available in combination with wild translocations to establish a second population; and, vi. Release wild translocated individuals to establish a second population with no input from the captive population. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 121 5.3 RESULTS Wild Population Viability All models predicted a negative population growth rate (Table 5.2). None of the population trajectories for the east, west, and metapopulations predicted persistence beyond 25 years (Figure 5.2, Figure 5.3A; B). The sensitivity tests identified multiple parameters as being particularly important in driving the overall population trajectory, specifically female mortality at all ages and fecundity (both the % of females producing more than one offspring and the % of females successfully producing per year; Table 5.3). Several parameters were not heavily weighted in determining the overall model results due to high levels of uncertainty surrounding them. These included initial population size, predicted annual habitat loss predicted, maximum lifespan as well as reproductive lifespan, and the age of first reproduction. Population Viability with Management Assuming no changes to the fecundity amongst the captive flock, sourcing the reintroduction using only captive birds would require either using those birds currently available (i), using six females (leaving one female in captivity), or moving 79 females from the wild into captivity to increase the captive populations’ productivity (ii). The third option (iii) would still require 36 additional females to be brought into captivity to supplement the captive flock in order to source 50% of the translocations (Table 5.4). The fourth option (iv) requires that seven additional birds are brought into captivity so that the captive flock could consistently supply one female per year for reintroduction efforts. Population trajectories among the last three scenarios (iv, v, and vi) are the same as they use the same input parameters, scenarios differed in where the birds came from (Figure 5.3). Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 122 Increasing important demographic parameters under the assumption of a more beneficial environment in the mesic leeward forests results in different population trajectories (under reintroduction scenario iv) after the initial three-year reintroduction timeframe (Figure 5.4). All reintroduction scenario models show that a 5% increase in demographic rates may not be sufficient to maintain the reintroduced population. However, a 10% increase results in a stable or increasing leeward population and a 20% increase results in an exponential increase in this population. Using reintroduction scenario iv, Figure 5.4 demonstrates the effects of increased demographic rates on the fate of the reintroduced population. 5.4 DISCUSSION The PVA models presented here further underline the perilous status of Maui Parrotbill. The precise timeline of extinction is beyond the scope of these analyses. However, the rapid decline projected by these models highlights the fact that certain aspects of the species’ biology and ecology (e.g. single egg clutch, prolonged parental investment) make this species in its current state (e.g. small, contracting range, occupying potentially suboptimal habitat) highly vulnerable to rapid extinction. This modeling exercise allowed us to identify the demographic traits most limiting the species to explore potential management solutions. Herein we further identified the most promising scenarios for reintroducing the species to previously occupied leeward mesic forests. We found that a reintroduction scenario that incorporates a minimal contribution from captivity and instead translocated mostly wild individuals to be most favorable for managers. Our population models highlight the strength and weakness of several conservation strategies that managers could implement given existing resources and capabilities in attempts to recover the wild population. Given that the population model does not reach carrying capacity, simply increasing available habitat in their current range may not have any impact on increasing the total Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 123 population. Alternatively, if managers are able to augment the current habitat (e.g., threat mitigation, such as control of predators) to increase quality, and thus increase some of the more sensitive parameters (i.e. female survival) within the current populations, they may be able to increase the viability of the population. Unfortunately, for parrotbills these options appear to be quite limited for a number of reasons. First, the forest currently occupied by parrotbills is native Hawaiian rainforest. This habitat is likely already as good as it is going to get as it is already closely protected (i.e. fenced and free of ungulates) and actively managed by the State of Hawaii and private conservation organizations. Second, there are limited options for other management interventions. Weather has been identified as a key limiting factor to reproductive success of parrotbills, with high incidence of nest failure in heavy rain events (Becker et al. 2010). Although weather cannot be manipulated, there have been numerous other unsuccessful attempts to manage parrotbills within their current forest habitat. These efforts have included trying to increase productivity and/or survival by providing supplemental food to wild individuals (see Appendix H), decreasing predation risk through control of invasive mammalian predators, and decreasing nest predation by protecting nest trees from mammalian predation (MFBRP unpublished data). Our simulations and resulting extinction probabilities are limited by the precision of the demographic parameters. While this study has used the most comprehensive data available on Maui Parrotbill, there is still uncertainty in several critical parameters. The variables with the most uncertainty included initial population size, annual habitat loss as predicted through climate change models, maximum lifespan and reproductive lifespan of individual parrotbill, and age of first reproduction. The effort necessary to collect additional data for these variables would require a substantial investment (see Brinck et al. 2012). An alternative approach could be to model parametric uncertainty directly into our simulation models and evaluate the benefits of increased parametric Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 124 precision through simulations (McGowan et al. 2011). However, our sensitivity analysis identified female mortality, annual reproductive success, and percentages of nests with two offspring as those parameters most responsible for driving the population changes observed - all parameters that we have high confidence in. Without clear strategies for increasing population viability of parrotbills in existing reserves, an alternative strategy would be to establish new populations in potentially superior habitat. Historically, parrotbills may have preferred habitats containing a higher proportion of koa, a preferred food source (Perkins 1903), which tend to occur in drier, leeward areas on Maui. Furthermore, sub-fossil records show a distribution of this species across the island, not restricted to the high elevation wet windward forests where they are found currently (James and Olson 1991). Today, there are no parrotbills in koa dominated forests on Maui. We do not know whether parrotbills were historically distributed at higher densities in the wet windward forests, but it may be that these areas were always marginal habitat. Regardless, if managers do not have the tools to successfully manage this species in currently occupied habitats, then increasing the range of habitats occupied may provide a viable long-term conservation strategy. Furthermore, our models did not include possibility of catastrophic events, but having an entire global population of any species within one 40-50 km2 area puts it at greater risk of extinction in the event of a severe hurricane or other weather event. Given the apparent limitation of management options in currently occupied habitats, we simulated reintroduction scenarios to explore moving birds from existing populations to the leeward side of Haleakala, Nakula NAR, a drier and more mesic koa dominated habitat. Furthermore, reintroducing birds to an area where they might be able to benefit from increased survival and productivity may be key to the species long-term success. Our models evaluated moving birds from the wild, using captive bred birds, and a combination of these Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 125 alternatives, building in uncertainty on immediate survival of the reintroduced birds since parrotbills have never been translocated to a new habitat before. Using captive-bred individuals has ecological consequences such as behavioral deficiencies, high susceptibility to starvation and disease, high post-release predation rates, and overall low reintroduction success rates that have been widely documented (Curio 1996, Fischer and Lindenmayer 2000, Jule et al. 2008, Rantanen et al. 2010). Captive parrotbills have the additional disadvantage of reduced genetic variation and significant genetic differentiation compared to some wild individuals (pairwise Fst and Rst between west and captive populations [Fst = 0.10; Rst = 0.16] Mounce et al. 2015). Furthermore, given the low reproduction of captive parrotbills, using only captive bred birds would 1) hinder the current captive population, 2) establish a new population with genetic variation from few females (i), or 3) require that a very large number of wild individuals be brought into captivity (ii and iii requiring 29% or 13%, respectively, of all wild females). Without considering potential effects to the wild populations, the resources necessary to capture and care for these high numbers of a critically endangered species in captivity is unrealistic with current conservation support available in Hawaii (Leonard 2008). Conversely, if the availability of resources for this sort of hands-on management greatly improved, there may be some advantages, namely that captive birds may anchor any wild birds to the release area which would facilitate monitoring. A major obstacle in translocations of wild individuals is they often reject the habitat close to release sites and travel long distances before settling (Stamps and Swaisgood 2007), exhibiting preferences that captive individuals may not have. Scenario iv models a reintroduction that incorporates a minimal contribution from captive individuals and has the advantage of potentially being among the least expensive scenarios. The ultimate monetary costs of many of the key steps involved in these scenarios remain unknown and in some cases are impossible to predict (e.g. the amount of field time required to capture 79 females (scenario ii)). Without these figures a cost comparison among all Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 126 scenarios is not an option at this time. However, scenario iv calls for the least amount of effort devoted to capturing wild individuals to be added to the captive population, a benefit over ii and iii, while also making use of the investment already made toward maintaining the captive population, an advantage over vi. This scenario also does not deplete the already small captive population, unlike i and v, and minimizes the addition of new birds to captivity and thus the costs in maintaining the larger captive population. Scenario iv would also likely provide the new populations with the most solid genetic foundation considering the genetic differentiation observed between the east/captive and west wild parrotbill populations (Mounce et al. 2015). In order for the leeward population to be considered genetically viable (Foose 1993) birds from both the east and the west should ideally need to be incorporated in the releases; captive birds were sourced from the east population only. We further explored scenario iv by looking at the reintroduced population’s viability using parameter values from the current wild population (Figure 5.4, Mounce et al. 2013, 2014) as well as predicted trends in annual fecundity, female survivorship, and hatch-year survivorship increased by 5%, 10%, and 20%. These increased demographics were examined based on potential benefits the leeward mesic habitat may have for the species. The exact limitations of the wetter windward habitats are unknown but parrotbills in the mesic forest may have increased nest success and/or increased foraging success in the drier habitat. Parrotbills may also have reduced predation pressure in a habitat with lower invasive mammal densities (MFBRP unpublished data). Our results demonstrate that the persistence of the reintroduced population is largely predicated on there being an increase in key demographic parameters in the new and potentially favorable environment. Fortunately an increase in these demographic traits is predicted and demographic rates could be assessed through post release monitoring of individuals. Given the importance of high demographic rates for a new leeward population, a reintroduction strategy that includes an adaptive management Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 127 plan to increase demographic rates by 10-20% through management and monitoring is likely to be the most successful approach (Williams et al. 2007). Without having any Maui Parrotbills currently inhabiting a koa dominated landscape, it is impossible to know how individuals of this species will respond once released. Yet, the increases in demographic values for the leeward population seem justifiable given it may be more suitable habitat (Perkins 1903) and the ability to conduct active management like reforestation and predator control. However, we do not know whether this species is plastic enough in their ecology to rebound from the release of those constraints or not. Therefore, managers could use these demographic parameter values as benchmarks for the monitoring of the reintroduced population in order to have a successful and viable population in the future. ACKNOWLEDGEMENTS Funding from the United States Fish and Wildlife Service, State of Hawaii Division of Forestry and Wildlife, and American Bird Conservancy helped to make this work possible. We thank San Diego Zoo Global for their assistance with data regarding the captive breeding facilities. We thank David Duffy, Chris Farmer, Bryce Masuda, and John Vetter for initial reviews of this manuscript. Lastly, we thank Maui Forest Bird Recovery Project field teams for their arduous work in collecting all of the field data used from previous publications in these analyses as well as The Nature Conservancy Hawaii and State of Hawaii Division of Forestry and Wildlife for access to public and private lands to collect such data. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 128 FIGURES AND TABLES Figure 5.1. East Maui, Hawaii (Haleakala Volcano) land protections that may benefit native forest birds. The current Maui Parrotbill population range overlays the windward (northeast) reserves, Hanawi NAR and TNC Waikamoi Preserve, and the newest reserve, Nakula NAR, is shown on the leeward (southern) slope. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 129 Figure 5.2. Projected mean final population sizes (N-all) for Maui Parrotbill under base and modified base models in VORTEX. Solid black line represents the “Base Model Population Metapopulation”, solid black line with dot represents the “Modified Base Model Metapopulation”, and broken gray line with box represents the “Modified Base Model East Population”, and dotted gray line with diamond represents the “Modified Base Model West Population”. Where N-all becomes zero indicates population collapse in 100% of simulations (1000 runs). Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 130 Figure 5.3. Female Maui Parrotbill population trajectories for the three existing populations (A- East, B- West, and D- Captive) and the proposed reintroduced leeward population (C- Leeward). Population estimates in for A, B, C, and D are based on a proposed three-year reintroduction scenario wherein the captive flock is augmented to source 1 female/year in combination with translocations from existing wild populations (scenario iv). Demographic parameters for wild populations are set to values from Mounce et al. 2013 and Mounce et al. 2014. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 131 Figure 5.4. Maui Parrotbill population trajectories for the reintroduced leeward population based on reintroduction scenario iv. Shown first are population trajectories predicting trends with annual fecundity, female survivorship, and young of the year set as in Mounce et al. 2013 and Mounce et al. 2014 (A). Next (B, C, and D) are shown with parameters increased by 5% (B), 10% (C), and 20% (D) based on potential benefits of the leeward mesic habitat. Values shown are the number of adult females in the population. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 132 Table 5.1. Parameter input values for the base and modified base VORTEX PVA model used for Maui Parrotbills. Input parameters derived from Mounce et al. 2013, Mounce et al. 2014, and Mounce et al. 2015. Values in bold highlight changes between the Base and Modified Base models. Base Model Modified Base Model East Pop (1) West Pop (2) Dispersal No Yes Age range . 1-5 Dispersing sexes . M and F % survival of dispersers . 100 % dispersing between pops . 2 Age of 1st offspring 2 2 Max age of reproduction 15 15 Max lifespan 20 20 Max broods per year 1 1 Max progeny per brood 2 2 Sex ratio at birth 50 50 % adult females breeding (EV) 46 ( 0.25) 56 (± 0.25) % 1 offspring (% 2 offspring) 95 (5) 95 (5) ♀/♂ Mortality rates 0-1 83 (± 0.15) 68 (± 0.10) ♀ Mortality rates after age 1 28 (± 0.03) 28 (± 0.03) ♂ Mortality rates after age 1 18 (± 0.04) 18 (± 0.04) Catastrophes None None % ♂ in breeding pool 100 100 Initial population size 583 477 106 Age distribution Stable Stable Carrying capacity (K) (SD due to EV) 885 (44) 724 (36) 161 (8) Future change in K? No Yes Over how many years? . 75 % annual increase of decrease . -1 Harvest None None Supplementation None None Genetics Known Known Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 133 Table 5.2. VORTEX model results for the base and modified base model for the Maui Parrotbill population(s) with the viability measures of stoch-r (mean population growth rate), λ (mean intrinsic rate of growth), R0 (net replacement rate), PS (probability of survival), N-all (mean population size from all iterations), N-extant (mean population size from extant populations), and GD (gene diversity). Base model Modified base model stoch-r -0.326 -0.234 SD r 0.125 0.123 λ 0.725 0.791 R0 0.105 0.241 PS 0.003 0.116 N-all 0.080 1.200 SD N-all 0.300 1.360 N-extant 2.000 3.380 SD N-extant 0.000 1.710 GD 0.413 0.516 SD GD 0.013 0.091 Table 5.3. Results of the sensitivity analysis in VORTEX for the Maui Parrotbiill metapopulation based on parameter changes in the “Modified base model.” Base model values are in grayscale in each parameter category. Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 134 Sensitivity testing index Model values stoch-r Sx of r N- extant East West Base Age of 1st offspring 2 -0.247 . 2.94 Age of 1st offspring + 1 yr. 3 -0.259 0.14 2.85 Age of 1st offspring + 2 yrs. 4 -0.267 0.15 2.56 Max age of reproduction - 3 yrs. 12 -0.260 0.20 2.66 Base max age of reproduction 15 -0.247 . 2.94 Max age of reproduction + 3 yrs. 18 -0.239 0.20 3.38 Max lifespan - 5 yrs. 15 -0.252 0.06 3.12 Base max lifespan 20 -0.247 . 2.94 Max lifespan + 5 yrs. 25 -0.239 0.17 3.29 % adult females breeding - 10 % (EV) 46 (± 0.25) -0.268 0.36 2.93 % adult females breeding - 5 % (EV) 51 (± 0.25) -0.256 0.36 2.71 Base % adult females breeding 56 (± 0.25) -0.247 . 2.94 % adult females breeding + 5 % (EV) 61 (± 0.25) -0.238 0.46 3.56 % adult females breeding + 10 % (EV) 66 (± 0.25) -0.228 0.55 3.72 % 1 offspring + 5 % 100 -0.254 0.55 3.12 Base % 1 offspring 95 -0.247 . 2.94 % 1 offspring - 5 % 90 -0.241 0.45 3.18 % 1 offspring - 10 % 85 -0.237 0.36 3.33 ♀/♂ Mortality rates (0-1) - 10 % 58 (± 0.10) -0.217 0.80 4.39 ♀/♂ Mortality rates (0-1) - 5 % 63 (± 0.10) -0.231 0.87 3.84 Base ♀/♂ Mortality rates (0-1) 68 (± 0.10) -0.247 . 2.94 ♀/♂ Mortality rates (0-1) + 5 % 73 (± 0.10) -0.265 0.99 2.57 ♀/♂ Mortality rates (0-1) + 10 % 78 (± 0.10) -0.286 1.06 2.67 ♀ Mortality rates after age 1 - 10 % 18 (± 0.03) -0.151 1.14 15.19 ♀ Mortality rates after age 1 - 5 % 23 (± 0.03) -0.201 1.05 5.37 Base ♀ Mortality rates after age 1 28 (± 0.03) -0.247 . 2.94 ♀ Mortality rates after age 1 + 5 % 33 (± 0.03) -0.298 1.13 2.00 ♀ Mortality rates after age 1 + 10 % 38 (± 0.03) -0.363 1.21 0.00 ♂ Mortality rates after age 1 - 10 % 8 (± 0.04) -0.235 0.04 3.89 ♂ Mortality rates after age 1 - 5 % 13 (± 0.04) -0.240 0.08 3.18 Base ♂ Mortality rates after age 1 18 (± 0.04) -0.247 . 2.94 ♂ Mortality rates after age 1 + 5 % 23 (± 0.04) -0.256 0.16 2.73 ♂ Mortality rates after age 1 + 10 % 28 (± 0.04) -0.266 -0.20 2.51 Initial population size - 25 % 358 80 -0.246 0.01 3.43 Base initial population size 477 106 -0.247 . 2.94 Initial population size + 25 % 596 133 -0.248 0.02 3.26 Annual increase in habitat loss 0 % 0 % -0.247 0.00 3.19 Base annual increase in habitat loss 1 % -0.247 . 2.94 Annual increase in habitat loss + 1 % 2 % -0.247 0.00 2.90 Annual increase in habitat loss + 3 % 4 % -0.246 0.01 2.88 Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 135 Table 5.4. The number of Maui Parrotbill needed from the east, west, and captive populations for reintroduction efforts across three-year reintroduction scenarios. Reintroduction Scenarios: Wild Females to Captivity (Year 1) Captive Females to Nakula (per year x 3) East Females to Nakula (per year x 3) West Females to Nakula (per year x 3) i. Release only captive birds currently available to establish a second population 0 2 0 0 ii. Augment the captive flock with wild birds such that the captive flock would source a second population 79 6 0 0 iii. Augment the captive flock with wild birds such that the captive flock would provide ½ the individuals needed for reintroduction with the other half from translocating wild individuals 36 3 2 1 iv. Augment the captive flock with wild birds such that the captive flock would provide 1 female per year in combination with translocating wild individuals 7 1 3 2 v. Release only captive birds currently available in combination with wild translocations to establish a second population 0 2 3 1 vi. Release wild translocated individuals to establish a second population 0 0 4 2 Recovery of the endangered Maui Parrotbill – Chapter 5 – Population Viability Modeling 136 Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis Chapter 6 Synopsis HANNA L. MOUNCE1, 2 1 Maui Forest Bird Recovery Project, Makawao, Hawaii 96768, USA 2 Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent CT2 7NR, UK Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis 6.1 SUMMARY OF KEY FINDINGS The biodiversity of the Hawaiian Islands have suffered widespread extinctions. Consequently, they have lost over half of their native forest birds. The Maui Parrotbill (Kiwikiu; Pseudonestor xanthophrys) is a critically endangered Hawaiian honeycreeper endemic to the island of Maui. These birds are restricted to a single population of ~500 individuals located in remote wet rainforest on windward east Maui. This thesis is aimed at providing information needed to recover this species and to interrupt its path towards extinction. Prior to this research, parrotbills were a largely understudied species with little known about their ecology. To this end, this thesis synthesizes the best data available in order to increase our knowledge about the biology of parrotbills. Through an intensive field effort studying fecundity, I provide estimates of annual productivity for Maui Parrotbills (46 %). I found that determining annual reproductive success by monitoring fledglings and calculating nest success through nest monitoring, both yielded similar results. More enlightening than the actual estimate, were the productivity limitations that were revealed. The limitations imposed by weather patterns on the windward slope were particularly worrisome. Parrotbill nests most often failed in heavy rain and storm events. While birds were observed to re-nest up to two additional times after failures, these events are common in the forest area to which species has been restricted to and not indicative of Hawaiian forests as a whole. The same field efforts tracking parrotbills to estimate productivity provided data on adult and juvenile survival. These data were combined with previously collected field data to construct an 18-year encounter history for the species. A difference in survival rates between sexes was strongly supported; 0.72 ± 0.04 for adult females and 0.82 ± 0.03 for adult males. This difference may be a reflection of either reproductive costs or additional risks of incubation Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis and brooding, such as depredation. Additionally, I found support for age- biased survival (juvenile survival = 0.17 ± 0.15; adults = 0.78 ± 0.02), but had very limited information for juveniles (Njuvenile = 10, Nadult = 136). With a population of approximately 500 individuals restricted to 50 km2 of habitat, Maui Parrotbill are at high risk of extinction. The recovery of this species will likely require intensive hands-on management. Based on these demographic data, this can be expected to involve reintroductions to additional and/or more suitable habitat areas. A thorough understanding of all components of a species’ biology, including genetic diversity and structure, are required to maximize the likelihood of success in such reintroductions. Examining differences in genetic diversity (calculated through effective population sizes) amongst contemporary and historical parrotbill populations, this thesis highlights the impact that introduced disease had on this species just before the turn of the century. Contemporary genetic diversity in parrotbill was low (global Fst = 0.056), and there was a 96% reduction in genetic effective population size between contemporary and historical samples. Furthermore, measures of population differentiation (pairwise Fst and Rst) suggest a fragmented genetic structure with compromised gene flow within the global population. The distribution of the two major segments of the current population suggests that current genetic structure may be the result of a topographic barrier, an erosional depression, the Koolau Gap. Combining this new information on Maui Parotbills, I built a comprehensive population viability model. The current population demographics predicted extinction of the species within 25 years. Among the top components driving this decline in our evaluation were female mortality and productivity. Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis Unfortunately for managers, techniques available to mitigate the threats to female survival and productivity in the wild are limited if not impossible. Alternatively, there is historically occupied habitat currently being restored on leeward east Maui for the benefit of Maui Parrotbill and other imperiled Hawaiian biota on Maui. The evaluation of management options suggests that parrotbills’ success and viability in this new habitat will hinge on the birds’ demographic plasticity and their ability to increase productivity and survival values there. This habitat is a mesic forest that appreciates lower annual rainfall forest than the habitat in the birds’ current range. While considering translocating both captive breeding and wild individuals as management options, this model provides managers with benchmark values for fecundity and survival that will need to be met amongst the new population in order for a reintroduction to be successful and sustainable. 6.2 RECOMMENDATIONS FOR CONSERVATION MANAGEMENT OF THE KIWIKIU AND OTHER ENDEMIC HAWAIIAN HONEYCREEPERS ON MAUI The purpose of this thesis is to provide managers with the information necessary to prevent the extinction of Maui Parrotbill and to facilitate the development of practical recovery strategies and techniques. As with most Hawaiian Honeycreepers, parrotbills are surviving in a fragment of the habitat that makes up a fragment of their former historical range. The combined impacts of habitat destruction and introduced threats (i.e. mammalian predators, ungulates, invasive plants, and disease) have all drastically reduced the forest habitats available to these birds. While habitat reclamation and restoration is possible under normal circumstances, the introduction of non-native disease, avian malaria (Plasmodium relictum) and its vector, mosquitoes (Culex quinquefasciatus), Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis make lowland forests uninhabitable by native forest birds. There is hope for the future with some species of Hawaiian Honeycreepers showing signs of disease resistance (Woodworth et al. 2005; Woodworth and Pratt 2009). Unfortunately, however, the most critically endangered species in Hawaii may not have the time or the genetic diversity necessary to persist in the face of these threats. Without intervention, extinction for these critically endangered species may be imminent. While the disease-free high elevation refugia on Maui and Hawaii Island are perhaps the best places for preserving Hawaii’s avian biodiversity, the windward forests on Maui may not be the most suitable habitat. It is not clear from historical accounts of Hawaiian passerines (Perkins 1903) whether these species actually preferred drier more mesic forest areas over the wet forests they are currently inhabit; or whether the few accessible areas to which these early naturalists could observe the birds happened to be mesic forest areas. Despite the limited historical data, the demographic studies on parrotbills presented here do not predict a sustainable population in the future. The small size of the parrotbill population puts the species at a high risk of extinction and coupled with the unsustainable demographic parameters observed in the wild, almost certainly forecasts their extinction. Management options to mitigate the threats that were found to be most limiting to the parrotbill population do not exist. Reducing predation risks to these birds would perhaps increase survival. However, the native habitat is too mountainous, fragile, and remote to make wide-scale rodent reduction a viable option for managers. Secondly, increasing nest success may also increase productivity. However, with nests failing in heavy weather, there are no management options to counteract the storm patterns in this habitat. Furthermore, the birds have not responded to management techniques that are available in this area like supplemental feeding to try to increase survival and/or productivity. Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis The combination of all these factors and limitations make moving these birds to a new habitat the most immediately viable option to secure their survival. Reintroductions are widely applied as a conservation tool and should be carefully implemented for Maui Parrotbill. Using the most up-to-date species biology data provided here is the first step towards a successful reintroduction. One of the biggest challenges for this species and other Hawaiian forest birds is the lack of information available about how the birds used or will use habitat areas other than those in which they are currently found. However, subfossil records confirm parrotbills existence in a wide variety of habitats (James and Olson 1991). While I might predict that these birds may demonstrate higher productivity or survival rates in areas with lower predation risk and drier weather patterns, there are no historical demographic data to evaluate this. Additionally, as discussed with the diminished genetic diversity within the species, these birds may not have the capacity to adapt to new environments in the same ways a more diverse species would. Nonetheless, the low observed levels of genetic diversity should not prevent managers from undertaking a reintroduction of Maui Parrotbill to leeward east Maui. A well planned reintroduction can establish a population with the best genetic structure available. The models presented here provide a benchmark for how to assess the viability of the new population over time once they are on the landscape. The success of the reintroduction will largely hinge upon how plastic these birds still are in their ability to survive in novel habitats after being restricted to the wet forests for some time. 6.3 PROGRESS IN THE NEXT STEPS TO RECOVERY Using the data presented here, I have identified that a reintroduction to historical habitat on leeward Haleakala is crucial to the long-term recovery of Maui Parrotbill. A Maui Parrotbill Reintroduction Working Group was formed in Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis 2014 to discuss the options for reintroduction. The biggest challenge in selecting a site for a parrotbill reintroduction is that there are no pristine high elevation native forests remaining on Maui outside of the 40-50 km2 area where the species currently exists. However, a remnant strip of degraded mesic forest still exists on leeward Maui. The Nakula Natural Area Reserve (NAR) on leeward east Maui has been selected as the site for a trial reintroduction in 2017. Many steps are currently underway to prepare the Nakula NAR for parrotbills by 2017. The first was landscape level fencing and ungulate removal. The native habitat within the reserve is degraded and is expected to require significant restoration before it will be suitable to support a viable parrotbill population. Maui Forest Bird Recovery Project initiated an experimental restoration experiment in 2012 to identify the most efficient and effective method(s) of restoring forest in an approximate 170-ha section of Nakula (see Appendix G). In addition to these experimental plots, landscape-level planting has begun. This is focused especially on planting corridors to connect existing vegetation across the landscape. Many challenges lay ahead for both restoration efforts and reintroduction planning. These include, but are not limited to, controlling weeds, sourcing seeds, herbicide restrictions in a forested landscape, and applying the experimental restoration results to the larger Nakula-Kahikinui area. Despite these challenges, the Maui Parrotbill Reintroduction Working Group has begun to draft a plan for the reintroduction in 2015. 6.4 FUTURE RESEARCH There will be future research needed on demographics, behavior, and particularly habitat use by the new reintroduced Maui Parrotbill population before we can assure their viability and sustainability into the future. In addition, other areas of Maui, particularly on the leeward and western slopes of Haleakala, should be identified as possible restoration areas to provide more high elevation forest habitats. Hopefully, the lessons learned in the Nakula NAR Recovery of the endangered Maui Parrotbill – Chapter 6 – Synopsis restoration project will be able to be applied to larger landscape areas of Maui in the future and increase the habitat available for parrotbills and other native honeycreepers on Maui. While this reintroduction is the next step needed to diminish an immediate extinction risk, it is focused on a single species. There is very limited ecosystem restoration implemented in Hawaii. With so many forest birds already lost, their ecosystem functions have also been lost. Unless these functions are replaced by an introduced non-native species, these ecosystems will never be restored to their original health. 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Tarr CL, Fleischer RC (1995) Evolutionary relationships of the Hawaiian honeycreepers (Aves, Drepanidinae). In: Wagner WL, Funk VA (eds) Hawaiian biogeography. Evolution on a hot spot archipelago. Smithson. Inst. Press, Washington, USA pp 147-158. Team RDC (2010) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Retrieved from http://www.R-project.org. Thompson BC, Knadle GE, Brubaker DL, Brubaker KS (2001) Nest success is not an adequate comparative estimate of avian reproduction. Journal of Field Ornithology 72:527-536.Underwood TJ, Roth RR (2002) Demographic variables are poor indicators of Wood Thrush productivity. Condor 104: 92-102. United States Fish and Wildlife Service (2012) Endangered and threatened Wildlife and plants; listing 38 species on Molokai, Lanai, and Maui as endangered and designating critical habitat on Molokai, Lanai, Maui, and Kahoolawe for 135 species. Federal Register 77:112, pp 34464-344775. United States Fish and Wildlife Service (2006) Revised recovery plan for the Hawaiian forest birds. Region 1, Portland, USA, pp 1-622. United States Fish and Wildlife Service (1967) Office of the Secretary; Native Fish and Wildlife; Endangered Species. Federal Register 32: 4001. VanderWerf EA (2013) Hawaiian Bird Conservation Action Plan. Pacific Rim Conservation, Honolulu, USA. Vanderwerf EA (2009) Importance of nest predation by alien rodents and avian poxvirus in conservation of Oahu Elepaio. Journal of Wildlife Management 73: 737-746. Vanderwerf EA, Smith DG (2002) Effects of alien rodent control on demography of the O'ahu 'Elepaio, an endangered Hawaiian forest bird. Pacific Conservation Biology 8: 73-81. Recovery of the endangered Maui Parrotbill – References 168 Van Dine DL (1904) Mosquitoes in Hawaii. Hawaii Agricultural Experiment Station Bulletin 6:1-30. Van Gelder E (1993) First Maui Parrotbill nests found. Hawaii’s Forests and Wildlife 8(1): 9. Vetter JP, Swinnerton KJ, VanderWerf EA, Garvin JC, Mounce HL, Breniser HE, Leonard DL, Fretz JS (2012) Survival estimates for two Hawaiian honeycreepers. Pacific Science 66(3): 299-309. Voorrips RE (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. Journal of Heredity 93: 77-78. Wandeler P, Smith S, Morin PA, Pettifor RA, Funk SM (2003) Patterns of nuclear DNA degeneration over time–a case study in historic teeth samples. Molecular Ecology 12: 1087-1093. Warren C, Mounce H (2014) Home-range patterns of two Hawaiian honeycreepers, Kiwikiu (Psuedonestor xanthophrys) and Maui Alauahio (Paroeomyza montana). Presentation, Island Biology Conference, Honolulu, USA. Williams BK, Szaro RC, Shapiro CD (2007) Adaptive Management: the US Department of the Interior Technical Guide. Adaptive Management Working Group. US Department of the Interior, Washington DC. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370. White GC, Burnham KP (1999) Program MARK: survival estimation from populations of marked animals. Bird Study 46 (Suppl.): 120-138. White TH Jr., Collar NJ, Moorhouse RJ, Sanz V, Stolen ED, Brightsmith DJ (2012) Psittacine reintroductions: common denominators of success. Biological Conservation, 148: 106-115. Recovery of the endangered Maui Parrotbill – References 169 Wolf CM, Garland T, Griffith B (1998) Predictors of avian and mammalian translocation success: reanalysis with phylogenetically independent contrasts. Biological Conservation, 86: 243-255. Wolf CM, Griffith B, Reed C, Temple SA (1996) Avian and mammalian translocations: update and reanalysis of 1987 survey data. Conservation Biology 10: 1142-1154. Woodworth BL, Pratt TK (2009) Life History and Demography. In: Pratt TK, Atkinson CT, Banko PC, Jacobi JD, Woodworth B (eds) Conservation biology of Hawaiian forest birds: implications for island avifauna. Yale University Press, New Haven, USA pp 194-233. Woodworth, BL, Atkinson, CT, LaPointe, DA, Hart, PJ, Spiegel, CS, Tweed, EJ, Henneman, C, LeBraun, J, Denette, T, DeMots, R, Kozar, KL, Triglia, D, Lease, D, Gregor, A, Smith, T, Duffy, D (2005) Host populations persistence in the face of introduced vector-borne diseases: Hawaii Amakihi and avian malaria. Proceedings of the National Academy of Sciences 102: 1531-1536. Woodworth BL, Nelson JT, Tweed EJ, Fancy SG, Moore MP, Cohen EB, Collins MS (2001) Breeding productivity and survival of the endangered Hawaii Creeper in a wet forest refuge on Mauna Kea, Hawaii. Studies in Avian Biology 22: 164-172. Woodworth BL (1999) Modeling population dynamics of a songbird exposed to parasitism and predation and evaluating management options. Conservation Biology 13: 67-76. Recovery of the endangered Maui Parrotbill – Appendices 1-AAppendices A. CHAPTER 3 SUPPLEMENTARY MATERIALS ............................. 2-A B. CHAPTER 4 SUPPLEMENTARY MATERIALS ............................ 14-A C. CHAPTER 5 SUPPLEMENTARY MATERIALS ............................ 17-A D. EUROPEAN MOLECULAR BIOLOGY LABORATORY DEPOSITS ........... 26-A E. MAPA SAMPLE HISTORIES FOR GENETIC ANALYSES .................. 47-A F. MAUI ALAUAHIO HAPLOTYPE DIVERSITY ............................. 53-A G. LEEWARD HALEAKALA EXPERIMENTAL RESTORATION PLAN .......... 60-A H. SUPPLEMENTAL FEEDING TRIALS FOR WILD MAUI PARROTBILLS ...... 73-A I. PROGRESS IN NAKULA EXPERIMENTAL RESTORATION EFFORTS ....... 80-A J. HOME RANGE PATTERNS OF MAUI PARROTBILL AND MAUI `ALAUAHIO: IMPLICATIONS FOR PROPOSED TRANSLOCATIONS EFFORTS ........... 87-A Recovery of the endangered Maui Parrotbill – Appendices 2-AAppendix A. Supplementary materials for Chapter 3: Management implications derived from long term re-sight data: annual survival of the Maui Parrotbill (Pseudonestor xanthophrys) Table A.1. Capture histories for male and female Maui Parrotbills 1994-2011 Ages: ASY = After Second Year bird, AHY = After Hatch Year bird, HY = Hatch Year bird Sexes: F = Female, M = Male, U = Unknown Site: HR3 = Home Range 3 or more eastern portion of study site, FSB = Frisbee Meadows or more western portion of study site /* MAPA Data, Recaptures and Resights, 17 occasions, 2 groups Group 1=Males Group 2=Females */ /*1181-80089 1/8/2011 ASY F HR3 */000000000000000001 0 1 /*1181-80096 1/9/2011 ASY F HR3 */000000000000000001 0 1 /*1371-04807 7/14/1994 AHY F FSB */110000000000000000 0 1 /*1371-04808 7/16/1994 AHY M FSB */111000000000000000 1 0 /*1371-04809 7/20/1994 ASY M FSB */100000000000000000 1 0 /*1371-04810 7/30/1994 AHY F FSB */110000000000000000 0 1 /*1371-04811 8/2/1994 AHY M FSB */111100000000010000 1 0 /*1371-04812 8/18/1994 AHY M FSB */110111110000000000 1 0 /*1371-04813 9/5/1994 AHY F FSB */110100000000000000 0 1 /*1371-04814 9/7/1994 AHY M FSB */100000000000000000 1 0 /*1371-04815 2/15/1995 ASY M FSB */011010000000000000 1 0 /*1371-04816 2/15/1995 AHY F FSB */011000000000000000 0 1 /*1371-04817 10/5/1994 ASY F FSB */111100000000000000 0 1 /*1371-04818 2/15/1995 ASY M FSB */011100000100000000 1 0 /*1371-04819 2/16/1995 ASY M FSB */011100000000000000 1 0 /*1371-04820 2/22/1995 ASY F FSB */010000000000000000 0 1 /*1371-04821 7/1/1995 AHY F FSB */011000000000000000 0 1 /*1371-04822 8/13/1995 AHY F FSB */011110000000000000 0 1 /*1371-04823 9/6/1995 AHY F FSB */011100000000000000 0 1 /*1371-04824 9/21/1995 AHY M FSB */011100000000000000 1 0 /*1371-04825 9/21/1995 AHY F FSB */010000000000000000 0 1 /*1371-04826 11/1/1995 AHY M FSB */011100000000000000 1 0 /*1371-04828 1/29/1996 ASY F FSB */001110000000000000 0 1 Recovery of the endangered Maui Parrotbill – Appendices 3-A/*1371-04829 3/11/1996 AHY F FSB */001000000000000000 0 1 /*1371-04830 7/1/1996 AHY F FSB */001000000000000000 0 1 /*1371-04831 2/19/1997 AHY M FSB */000100000000000000 1 0 /*1371-04903 3/31/1996 ASY M HR3 */001000000000000000 1 0 /*1371-04906 4/3/1996 ASY M HR3 */001100000000000000 1 0 /*1371-04907 4/3/1996 SY M HR3 */001000000000000000 1 0 /*1371-04915 2/17/1997 ASY M HR3 */000110000000000000 1 0 /*1371-04916 2/17/1997 ASY F HR3 */000100000000000000 0 1 /*1371-04918 2/20/1997 ASY F HR3 */000100000000000000 0 1 /*1371-04919 2/20/1997 ASY M HR3 */000100000000000000 1 0 /*1371-04920 2/20/1997 SY M HR3 */000100000000000000 1 0 /*1371-04921 2/20/1997 ASY M HR3 */000100000000000000 1 0 /*1371-04922 2/20/1997 AHY F HR3 */000100000000000111 0 1 /*1371-04929 12/11/1997 AHY M HR1 */000100000000000001 1 0 /*1371-04932 12/14/1997 AHY M HR1 */000100000100000010 1 0 /*1371-04933 1/13/1998 SY F HR3 */000010000000000000 0 1 /*1371-04934 1/13/1998 AHY F HR3 */000011000000000000 0 1 /*1371-04937 2/6/1998 AHY M HR3 */000011010100000000 1 0 /*1371-04938 2/6/1998 AHY F HR3 */000010000000000000 0 1 /*1371-04939 2/8/1998 AHY M HR3 */000010000000000101 1 0 /*1371-04940 2/11/1998 SY F HR3 */000011000000000000 0 1 /*1371-04941 2/21/1998 AHY F HR3 */000011000000000000 0 1 /*1371-04942 2/21/1998 AHY M HR3 */000011010011000000 1 0 /*1371-04944 4/4/1998 AHY F HR3 */000010000000000000 0 1 /*1371-04947 6/12/2001 AHY M FSB */000000000000000100 1 0 /*1371-04948 1/8/2002 SY F HR3 */000000010000000000 0 1 /*1371-04949 1/11/2002 ASY M HR3 */000000010000000000 1 0 /*1371-04950 1/11/2002 ASY F HR3 */000000010111000000 0 1 /*1371-04951 1/14/2002 ASY F HR3 */000000010000000000 0 1 /*1371-04952 1/14/2002 ASY M HR3 */000000010111110000 1 0 /*1371-04953 1/14/2002 SY F HR3 */000000010000000000 0 1 /*1371-04954 1/25/2002 ASY F HR3 */000000010110000000 0 1 /*1371-04955 2/5/2002 AHY F HR3 */000000010110000000 0 1 /*1371-04956 2/4/2003 SY M HR1 */000000000100000000 1 0 /*1371-04959 5/27/2003 AHY M HR3 */000000000100000000 1 0 /*1371-04960 10/22/2003 AHY M HR3 */000000000111111111 1 0 Recovery of the endangered Maui Parrotbill – Appendices 4-A/*1371-04961 10/24/2003 AHY M HR3 */000000000100000000 1 0 /*1371-04962 10/25/2003 AHY F HR3 */000000000111111100 0 1 /*1371-04963 10/26/2003 AHY F HR3 */000000000110100000 0 1 /*1371-04964 10/31/2003 SY F HR3 */000000000100000000 0 1 /*1371-04967 3/12/2004 SY F HR3 */000000000010000000 0 1 /*1371-04968 3/12/2004 SY M HR3 */000000000011110000 1 0 /*1371-04969 3/13/2004 SY F HR3 */000000000010000000 0 1 /*1371-04970 3/13/2004 ASY M HR3 */000000000010000000 1 0 /*1371-04971 3/13/2004 ASY F HR3 */000000000011111111 0 1 /*1371-04976 11/18/2006 AHY F FSB */000000000000110000 0 1 /*1371-04977 11/18/2006 AHY M FSB */000000000000100000 1 0 /*1371-04978 12/4/2006 AHY M FSB */000000000000100000 1 0 /*1371-04979 1/17/2007 ASY M HR3 */000000000000010000 1 0 /*1371-04980 1/17/2007 ASY F HR3 */000000000000011010 0 1 /*1371-04981 1/20/2007 ASY F HR3 */000000000000010000 0 1 /*1371-04982 1/20/2007 ASY M HR3 */000000000000011111 1 0 /*1371-04983 2/3/2007 AHY F HR3 */000000000000011110 0 1 /*1371-04984 2/4/2007 SY M HR3 */000000000000010001 1 0 /*1371-04985 2/4/2007 SY F HR3 */000000000000010000 0 1 /*1371-04986 5/4/2007 SY F HR3 */000000000000010110 0 1 /*1371-04987 5/4/2007 ASY M HR3 */000000000000010110 1 0 /*1371-04988 11/17/2006 AHY M FSB */000000000000100000 1 0 /*1371-04989 5/21/2003 AHY M HR3 */000000000100110110 1 0 /*1401-47511 11/16/2007 AHY F FSB */000000000000010000 0 1 /*1401-47512 11/16/2007 AHY M FSB */000000000000011000 1 0 /*1401-47513 11/18/2007 AHY M FSB */000000000000010110 1 0 /*1401-47514 11/19/2007 AHY M FSB */000000000000011000 1 0 /*1401-47515 11/30/2007 AHY F FSB */000000000000011100 0 1 /*1401-47516 12/1/2007 AHY M FSB */000000000000011100 1 0 /*1401-47517 12/2/2007 AHY M FSB */000000000000011111 1 0 /*1401-47518 2/27/2008 SY F FSB */000000000000001000 0 1 /*1401-47519 2/27/2008 AHY F FSB */000000000000001000 0 1 /*1401-47520 4/24/2008 ASY M HR3 */000000000000001000 1 0 /*1401-47521 4/25/2008 AHY M HR3 */000000000000001110 1 0 /*1401-47523 2/24/2008 ASY F HR3 */000000000000001100 0 1 /*1401-47525 4/30/2008 AHY F FSB */000000000000001101 0 1 Recovery of the endangered Maui Parrotbill – Appendices 5-A/*1401-47528 12/7/2008 AHY F FSB */000000000000001100 0 1 /*1401-47530 12/7/2008 AHY M FSB */000000000000001000 1 0 /*1401-47531 12/9/2008 AHY M FSB */000000000000001111 1 0 /*1401-47532 12/9/2008 AHY F FSB */000000000000001100 0 1 /*1401-47533 1/30/2009 AHY M HR3 */000000000000000110 1 0 /*1401-47534 1/30/2009 AHY F HR3 */000000000000000111 0 1 /*1401-47535 1/27/2009 AHY M HR3 */000000000000000111 1 0 /*1401-47536 2/16/2009 AHY F FSB */000000000000000100 0 1 /*1401-47537 3/19/2009 AHY M HR3 */000000000000000110 1 0 /*1401-47539 8/17/2009 AHY F HR3 */000000000000000100 0 1 /*1401-47540 4/16/2009 AHY F FSB */000000000000000111 0 1 /*1401-47541 4/16/2009 ASY M FSB */000000000000000111 1 0 /*1401-47548 11/24/2009 AHY F HR3 */000000000000000110 0 1 /*1401-47549 12/4/2009 AHY F FSB */000000000000000111 0 1 /*1401-47550 12/4/2009 AHY F FSB */000000000000000111 0 1 /*1401-47551 12/8/2009 SY M FSB */000000000000000100 1 0 /*1401-47552 1/14/2010 AHY F HR3 */000000000000000011 0 1 /*1401-47553 1/16/2010 ASY M HR3 */000000000000000010 1 0 /*1401-47555 1/26/2010 ASY F FSB */000000000000000011 0 1 /*1401-47556 1/26/2010 ASY M FSB */000000000000000010 1 0 /*1401-47557 1/27/2010 ASY M FSB */000000000000000011 1 0 /*1401-47558 1/31/2010 ASY M FSB */000000000000000011 1 0 /*1401-47559 2/21/2010 ASY M FSB */000000000000000011 1 0 /*1401-47560 2/23/2010 ASY M FSB */000000000000000011 1 0 /*1401-47561 2/23/2010 ASY M FSB */000000000000000011 1 0 /*1401-47566 7/26/2010 AHY M FSB */000000000000000011 1 0 /*1401-47587 3/2/2011 AHY M HR3 */000000000000000001 1 0 /*1541-80268 6/17/2011 ASY M FSB */000000000000000001 1 0 /*1541-80269 6/19/2011 ASY M FSB */000000000000000001 1 0 /*1541-80270 6/19/2011 ASY F FSB */000000000000000001 0 1 /*1541-80271 6/22/2011 ASY M FSB */000000000000000001 1 0 /*1791-16901 5/3/2011 ASY F HR3 */000000000000000001 0 1 /*1791-16902 6/14/2011 AHY F HR3 */000000000000000001 0 1 /*1791-16904 6/15/2011 ASY M HR3 */000000000000000001 1 0 /*1821-17705 3/3/2011 SY M FSB */000000000000000001 1 0 /*1871-17701 2/27/2011 ASY M FSB */000000000000000001 1 0 Recovery of the endangered Maui Parrotbill – Appendices 6-A/*1871-17702 3/1/2011 ASY F FSB */000000000000000001 0 1 /*1871-17703 3/2/2011 ASY M FSB */000000000000000001 1 0 /*1871-17704 3/2/2011 ASY M FSB */000000000000000001 1 0 /*1871-17708 3/15/2011 AHY F FSB */000000000000000001 0 1 /*1871-17709 4/28/2011 ASY M FSB */000000000000000001 1 0 Table A.2. Capture histories for adult and juvenile Maui Parrotbills 1994-2011 /* MAPA Data, Recaptures and Resights, 17 occasions, 2 groups Group 1=HY Group 2=Adult */ /*1181-80089 1/8/2011 ASY F HR3 */000000000000000001 0 1 /*1181-80096 1/9/2011 ASY F HR3 */000000000000000001 0 1 /*1371-04807 7/14/1994 AHY F FSB */110000000000000000 0 1 /*1371-04808 7/16/1994 AHY M FSB */111000000000000000 0 1 /*1371-04809 7/20/1994 ASY M FSB */100000000000000000 0 1 /*1371-04810 7/30/1994 AHY F FSB */110000000000000000 0 1 /*1371-04811 8/2/1994 AHY M FSB */111100000000010000 0 1 /*1371-04812 8/18/1994 AHY M FSB */110111110000000000 0 1 /*1371-04813 9/5/1994 AHY F FSB */110100000000000000 0 1 /*1371-04814 9/7/1994 AHY M FSB */100000000000000000 0 1 /*1371-04815 2/15/1995 ASY M FSB */011010000000000000 0 1 /*1371-04816 2/15/1995 AHY F FSB */011000000000000000 0 1 /*1371-04817 10/5/1994 ASY F FSB */111100000000000000 0 1 /*1371-04818 2/15/1995 ASY M FSB */011100000100000000 0 1 /*1371-04819 2/16/1995 ASY M FSB */011100000000000000 0 1 /*1371-04820 2/22/1995 ASY F FSB */010000000000000000 0 1 /*1371-04821 7/1/1995 AHY F FSB */011000000000000000 0 1 /*1371-04822 8/13/1995 AHY F FSB */011110000000000000 0 1 /*1371-04823 9/6/1995 AHY F FSB */011100000000000000 0 1 /*1371-04824 9/21/1995 AHY M FSB */011100000000000000 0 1 /*1371-04825 9/21/1995 AHY F FSB */010000000000000000 0 1 /*1371-04826 11/1/1995 AHY M FSB */011100000000000000 0 1 /*1371-04827 1/26/1996 HY U FSB */001000000000000000 1 0 /*1371-04828 1/29/1996 ASY F FSB */001110000000000000 0 1 /*1371-04829 3/11/1996 AHY F FSB */001000000000000000 0 1 Recovery of the endangered Maui Parrotbill – Appendices 7-A/*1371-04830 7/1/1996 AHY F FSB */001000000000000000 0 1 /*1371-04831 2/19/1997 AHY M FSB */000100000000000000 0 1 /*1371-04903 3/31/1996 ASY M HR3 */001000000000000000 0 1 /*1371-04906 4/3/1996 ASY M HR3 */001100000000000000 0 1 /*1371-04907 4/3/1996 SY M HR3 */001000000000000000 0 1 /*1371-04908 4/29/1996 HY F HR1 */001100000000000000 1 0 /*1371-04915 2/17/1997 ASY M HR3 */000110000000000000 0 1 /*1371-04916 2/17/1997 ASY F HR3 */000100000000000000 0 1 /*1371-04918 2/20/1997 ASY F HR3 */000100000000000000 0 1 /*1371-04919 2/20/1997 ASY M HR3 */000100000000000000 0 1 /*1371-04920 2/20/1997 SY M HR3 */000100000000000000 0 1 /*1371-04921 2/20/1997 ASY M HR3 */000100000000000000 0 1 /*1371-04922 2/20/1997 AHY F HR3 */000100000000000111 0 1 /*1371-04923 5/21/1997 HY M HR3 */000100000000000000 1 0 /*1371-04929 12/11/1997 AHY M HR1 */000100000000000001 0 1 /*1371-04932 12/14/1997 AHY M HR1 */000100000100000010 0 1 /*1371-04933 1/13/1998 SY F HR3 */000010000000000000 0 1 /*1371-04934 1/13/1998 AHY F HR3 */000011000000000000 0 1 /*1371-04937 2/6/1998 AHY M HR3 */000011001100000000 0 1 /*1371-04938 2/6/1998 AHY F HR3 */000010000000000000 0 1 /*1371-04939 2/8/1998 AHY M HR3 */000010000000000101 0 1 /*1371-04940 2/11/1998 SY F HR3 */000011000000000000 0 1 /*1371-04941 2/21/1998 AHY F HR3 */000011000000000000 0 1 /*1371-04942 2/21/1998 AHY M HR3 */000011001011000000 0 1 /*1371-04944 4/4/1998 AHY F HR3 */000010000000000000 0 1 /*1371-04947 6/12/2001 AHY M FSB */000000000000000100 0 1 /*1371-04948 1/8/2002 SY F HR3 */000000001000000000 0 1 /*1371-04949 1/11/2002 ASY M HR3 */000000001000000000 0 1 /*1371-04950 1/11/2002 ASY F HR3 */000000001111000000 0 1 /*1371-04951 1/14/2002 ASY F HR3 */000000001000000000 0 1 /*1371-04952 1/14/2002 ASY M HR3 */000000001111110000 0 1 /*1371-04953 1/14/2002 SY F HR3 */000000001000000000 0 1 /*1371-04954 1/25/2002 ASY F HR3 */000000001110000000 0 1 /*1371-04955 2/5/2002 AHY F HR3 */000000001110000000 0 1 /*1371-04956 2/4/2003 SY M HR1 */000000000100000000 0 1 /*1371-04959 5/27/2003 AHY M HR3 */000000000100000000 0 1 Recovery of the endangered Maui Parrotbill – Appendices 8-A/*1371-04960 10/22/2003 AHY M HR3 */000000000111111111 0 1 /*1371-04961 10/24/2003 AHY M HR3 */000000000100000000 0 1 /*1371-04962 10/25/2003 AHY F HR3 */000000000111111100 0 1 /*1371-04963 10/26/2003 AHY F HR3 */000000000110100000 0 1 /*1371-04964 10/31/2003 SY F HR3 */000000000100000000 0 1 /*1371-04966 3/10/2004 HY M HR3 */ 00000000010000000 1 0 /*1371-04967 3/12/2004 SY F HR3 */000000000010000000 0 1 /*1371-04968 3/12/2004 SY M HR3 */000000000011110000 0 1 /*1371-04969 3/13/2004 SY F HR3 */000000000010000000 0 1 /*1371-04970 3/13/2004 ASY M HR3 */000000000010000000 0 1 /*1371-04971 3/13/2004 ASY F HR3 */000000000011111111 0 1 /*1371-04975 6/10/2006 HY U HR3 */000000000000100000 1 0 /*1371-04976 11/18/2006 AHY F FSB */000000000000110000 0 1 /*1371-04977 11/18/2006 AHY M FSB */000000000000100000 0 1 /*1371-04978 12/4/2006 AHY M FSB */000000000000100000 0 1 /*1371-04979 1/17/2007 ASY M HR3 */000000000000010000 0 1 /*1371-04980 1/17/2007 ASY F HR3 */000000000000011010 0 1 /*1371-04981 1/20/2007 ASY F HR3 */000000000000010000 0 1 /*1371-04982 1/20/2007 ASY M HR3 */000000000000011111 0 1 /*1371-04983 2/3/2007 AHY F HR3 */000000000000011110 0 1 /*1371-04984 2/4/2007 SY M HR3 */000000000000010001 0 1 /*1371-04985 2/4/2007 SY F HR3 */000000000000010000 0 1 /*1371-04986 5/4/2007 SY F HR3 */000000000000010110 0 1 /*1371-04987 5/4/2007 ASY M HR3 */000000000000010110 0 1 /*1371-04988 11/17/2006 AHY M FSB */000000000000100000 0 1 /*1371-04989 5/21/2003 AHY M HR3 */000000000100110110 0 1 /*1401-47511 11/16/2007 AHY F FSB */000000000000010000 0 1 /*1401-47512 11/16/2007 AHY M FSB */000000000000011000 0 1 /*1401-47513 11/18/2007 AHY M FSB */000000000000010110 0 1 /*1401-47514 11/19/2007 AHY M FSB */000000000000011000 0 1 /*1401-47515 11/30/2007 AHY F FSB */000000000000011100 0 1 /*1401-47516 12/1/2007 AHY M FSB */000000000000011100 0 1 /*1401-47517 12/2/2007 AHY M FSB */000000000000011111 0 1 /*1401-47518 2/27/2008 SY F FSB */000000000000001000 0 1 /*1401-47519 2/27/2008 AHY F FSB */000000000000001000 0 1 /*1401-47520 4/24/2008 ASY M HR3 */000000000000001000 0 1 Recovery of the endangered Maui Parrotbill – Appendices 9-A/*1401-47521 4/25/2008 AHY M HR3 */000000000000001110 0 1 /*1401-47523 2/24/2008 ASY F HR3 */000000000000001100 0 1 /*1401-47525 4/30/2008 AHY F FSB */000000000000001101 0 1 /*1401-47526 9/12/2008 HY M FSB */000000000000001000 1 0 /*1401-47527 12/5/2008 HY U FSB */000000000000001000 1 0 /*1401-47528 12/7/2008 AHY F FSB */000000000000001100 0 1 /*1401-47530 12/7/2008 AHY M FSB */000000000000001000 0 1 /*1401-47531 12/9/2008 AHY M FSB */000000000000001111 0 1 /*1401-47532 12/9/2008 AHY F FSB */000000000000001100 0 1 /*1401-47533 1/30/2009 AHY M HR3 */000000000000000110 0 1 /*1401-47534 1/30/2009 AHY F HR3 */000000000000000111 0 1 /*1401-47535 1/27/2009 AHY M HR3 */000000000000000111 0 1 /*1401-47536 2/16/2009 AHY F FSB */000000000000000100 0 1 /*1401-47537 3/19/2009 AHY M HR3 */000000000000000110 0 1 /*1401-47539 8/17/2009 AHY F HR3 */000000000000000100 0 1 /*1401-47540 4/16/2009 AHY F FSB */000000000000000111 0 1 /*1401-47541 4/16/2009 ASY M FSB */000000000000000111 0 1 /*1401-47547 11/24/2009 HY U HR3 */000000000000000100 1 0 /*1401-47548 11/24/2009 AHY F HR3 */000000000000000110 0 1 /*1401-47549 12/4/2009 AHY F FSB */000000000000000111 0 1 /*1401-47550 12/4/2009 AHY F FSB */000000000000000111 0 1 /*1401-47551 12/8/2009 SY M FSB */000000000000000100 0 1 /*1401-47552 1/14/2010 AHY F HR3 */000000000000000011 0 1 /*1401-47553 1/16/2010 ASY M HR3 */000000000000000010 0 1 /*1401-47555 1/26/2010 ASY F FSB */000000000000000011 0 1 /*1401-47556 1/26/2010 ASY M FSB */000000000000000010 0 1 /*1401-47557 1/27/2010 ASY M FSB */000000000000000011 0 1 /*1401-47558 1/31/2010 ASY M FSB */000000000000000011 0 1 /*1401-47559 2/21/2010 ASY M FSB */000000000000000011 0 1 /*1401-47560 2/23/2010 ASY M FSB */000000000000000011 0 1 /*1401-47561 2/23/2010 ASY M FSB */000000000000000011 0 1 /*1401-47566 7/26/2010 AHY M FSB */000000000000000011 0 1 /*1401-47587 3/2/2011 AHY M HR3 */000000000000000001 0 1 /*1541-80268 6/17/2011 ASY M FSB */000000000000000001 0 1 /*1541-80269 6/19/2011 ASY M FSB */000000000000000001 0 1 /*1541-80270 6/19/2011 ASY F FSB */000000000000000001 0 1 Recovery of the endangered Maui Parrotbill – Appendices 10-A/*1541-80271 6/22/2011 ASY M FSB */000000000000000001 0 1 /*1791-16901 5/3/2011 ASY F HR3 */000000000000000001 0 1 /*1791-16902 6/14/2011 AHY F HR3 */000000000000000001 0 1 /*1791-16904 6/15/2011 ASY M HR3 */000000000000000001 0 1 /*1791-16905 6/16/2011 HY U HR3 */000000000000000001 1 0 /*1791-16906 6/15/2011 HY U HR3 */000000000000000001 1 0 /*1821-17705 3/3/2011 SY M FSB */000000000000000001 0 1 /*1871-17701 2/27/2011 ASY M FSB */000000000000000001 0 1 /*1871-17702 3/1/2011 ASY F FSB */000000000000000001 0 1 /*1871-17703 3/2/2011 ASY M FSB */000000000000000001 0 1 /*1871-17704 3/2/2011 ASY M FSB */000000000000000001 0 1 /*1871-17708 3/15/2011 AHY F FSB */000000000000000001 0 1 /*1871-17709 4/28/2011 ASY M FSB */000000000000000001 0 1 Recovery of the endangered Maui Parrotbill – Appendices 11-ATable A.3. Maui Parrotbill individuals included in the MARK analyses 1994-2011. Banded represents birds marked during that year, re-sighted includes both observed and recaptured individuals. Year Banded birds Re-sighted birds Total Encounters 1994 9 0 9 1995 11 7 18 1996 8 12 26 1997 11 15 36 1998 9 10 22 1999 0 13 13 2000 0 4 6 2001 1 0 1 2002 8 4 12 2003 8 10 21 2004 6 12 19 2005 0 7 7 2006 5 7 12 2007 16 8 24 2008 12 11 23 2009 13 21 35 2010 10 23 33 2011 19 16 33 Total 146 180 326 Recovery of the endangered Maui Parrotbill – Appendices 12-ATable A.4. Goodness of fit results for all tests runs in Program RELEASE (within Program MARK). χ2 df p-value Sex-Specific Model TEST 1 15.046 29.000 0.985 TEST 2 34.895 22.000 0.040 TEST 3 16.425 35.000 0.997 TEST 2 + TEST 3 51.320 57.000 0.687 Age-Specific Model TEST 1 5.238 7.000 0.631 TEST 2 40.592 13.000 0.000 TEST 3 15.572 22.000 0.837 TEST 2 + TEST 3 56.164 35.000 0.013 Recovery of the endangered Maui Parrotbill – Appendices 13-A Figure A.1. Detection probability for male (a) and female (b) adult Maui Parrotbills over time. Error bars indicate standard error for each year 1996-2011. In model Φsex.ρsex model (QAICc weight = 0.000), males had a detection probability of 0.457 (± 0.043) and females 0.666 (± 0.060). Recovery of the endangered Maui Parrotbill – Appendices 14-AAppendix B. Supplementary materials for Chapter 4: Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies Table B.1. Historical Maui Parrotbill museum sample information from American Museum of Natural History, New York, USA (AMNH), Museum of Comparative Zoology, Cambridge, MA, USA (MCZ), Cambridge University Museum of Zoology, Cambridge, UK (CMZ), British Museum of Natural History, London, UK (BMNH UK), Bernice Pauahi Bishop Museum, Honolulu, USA (BMNH US), and Smithsonian National Museum of Natural History, Washington DC, US (USNM). Museum specimens were collected across the period from 1892-1901. All specimens were presumed to be collected from the western-most region of the parrotbill’s range on Haleakala as per collectors’ notes on locations. Museum Sample ID Date of Collection Collector Location Age Sex AMNH 193408 05/1896 Perkins Haleakala AHY Male AMNH 193409 6/17/1901 Henshaw Olinda AHY Female AMNH 453556 10/1896 Palmer Haleakala AHY Male AMNH 453557 08/4/1892 Palmer AHY Male AMNH 453558 08/3/1892 Palmer AHY Male AMNH 453559 08/16/1892 Palmer AHY Male AMNH 453560 08/15/1892 Palmer AHY Female AMNH 453561 08/3/1892 Palmer AHY Female AMNH 453562 08/03/1892 Palmer AHY Unknown AMNH 453563 Palmer AHY Female MCZ 134719 1894 Perkins AHY Male MCZ 47905 1896 Perkins AHY Male Recovery of the endangered Maui Parrotbill – Appendices 15-ACMZ 27/DRE/9/a/1 1894 Perkins AHY Female CMZ 27/DRE/9/a/2 1896 Perkins AHY Male CMZ 27/DRE/9/a/3 1896 Perkins AHY Female CMZ 27/DRE/9/a/4 1896 Perkins HY Male CMZ 27/DRE/9/a/5 1896 Perkins SY Male BMNH UK 1939.12.9.53 1892 Rothschild AHY Male BMNH UK 1939.12.9.57 08/05/1892 Rothschild HY Unknown BMNH US 95.7.20.170 1894 Perkins AHY Male BMNH US 95.7.20.171 1894 Perkins AHY Female BMNH US 97.10.28.22 1896 Perkins AHY Female BMNH US 97.10.28.23 1896 Perkins AHY Male BMNH US 97.10.28.24 1896 Perkins SY Female BMNH US 97.10.28.25 1896 Perkins AHY Male USNM 177971 1901 Henshaw Ukulele AHY Male USNM 177972 1901 Henshaw Ukulele AHY Female Table B.2. Average allelic and private allelic richness using rarefaction technique over all loci. Includes results for 10, 20, 50, and 110 genes based on microsatellite data across the east (n=110), west (n=19), and captive (n=11) Maui Parrotbill (Pseudonestor xanthophrys) populations. Allelic Richness 10 genes 20 genes 50 genes 110 genes Captive 3.27 3.82 3.83 3.83 East 3.74 4.71 5.83 6.66 West 3.04 3.72 4.08 4.08 Private Allelic Richness Captive 0.47 0.28 0.09 0.00 East 0.71 0.89 1.47 2.10 West 0.45 0.43 0.35 0.26 Recovery of the endangered Maui Parrotbill – Appendices 16-A Figure B.1. Chromosome locations in the zebra finch (Taeniopygia guttata) genone of the 12 microsatellite loci characterized in the Maui Parrotbill (Pseudonestor xanthophrys) compiled by the National Library of Medicine, http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_PROG_DEF=megaBlast&BLAST_SPEC=OGP__59729__12898). 17-AAppendix C. Supplementary materials for Chapter 5: Using population viability analysis to model extinction risk and recovery options for a critically endangered Hawaiian honeycreeper: Maui Parrotbill (Kiwikiu, Pseudonestor xanthophrys) MAPA PVA Model Code in Program R It = 1000 yrs=25 #Model parameters for Pops 1 & 2 (East & West) F=0.588 #Annual fecundity ((0.95*1)+(0.05*2) SDF=F*0.15 #SD Annual fecundity AS=0.72 #Adult female survival from Mounce et al. 2014 SDAS=0.02 #SD Adult female survival IS=0.72 #Immature (1 year old) survival from Mounce et al. 2014 SDIS=0.02 #SDImmature survival YS=0.32 #Woodworth et al. 2009 SDYS=0.03 #Woodworth et al. 2009 #Max and Min survival of newly released (captive released or translocated) RSmax=0.9 RSmin=0.4 RS=matrix(runif(yrs*It,RSmin,RSmax),It,yrs) #Model parameters for pop 3 (shown are 5% increase in demographics) 18-AF3=0.631 #Annual fecundity ((0.95*1)+(0.05*2), boosted 5% SDF3=F3*0.15 #SD Annual fecundity AS3=0.78 #Adult female survival from Mounce et al. 2014 boosted 5% SDAS3=0.02 #SD Adult female survival IS3=0.78 #Immature (1 year old) survival from Mounce et al. 2014 boosted 5% SDIS3=0.02 #SDImmature survival YS3=0.37 #Woodworth et al. 2009 boosted 5% SDYS3=0.03 #Woodworth et al. 2009 # Alpha shape parameter for each iteration to be used in annual variable selection process aAS = AS*((AS*(1-AS)/SDAS)-1) bAS = (1-AS)*((AS*(1-AS)/SDAS)-1) aIS = IS*((IS*(1-IS)/SDIS)-1) bIS = (1-IS)*((IS*(1-IS)/SDIS)-1) aYS = YS*((YS*(1-YS)/SDYS)-1) bYS = (1-YS)*((YS*(1-YS)/SDYS)-1) aF = log(F)-1/2*SDF bF = log((SDF^2)/(F^2) + 1) aAS3 = AS3*((AS3*(1-AS3)/SDAS3)-1) bAS3 = (1-AS3)*((AS3*(1-AS3)/SDAS3)-1) aIS3 = IS3*((IS3*(1-IS3)/SDIS3)-1) bIS3 = (1-IS3)*((IS3*(1-IS3)/SDIS3)-1) aYS3 = YS3*((YS3*(1-YS3)/SDYS3)-1) bYS3 = (1-YS3)*((YS3*(1-YS3)/SDYS3)-1) aF3 = log(F3)-1/2*SDF3 19-AbF3 = log((SDF3^2)/(F3^2) + 1) CD = 0.25 #mean number of deaths in the captive population (CD = captive deaths) CN = 0.5 #mean number of successful rearings in the captive population (CN = captive nubes) YM = 3 #Max years of management (e.g. translocation or captive releases) M12 = matrix(0,It,yrs) #Move individuals from pop 1 to pop 2 M13 = matrix(3,It,yrs) #Move individuals from pop 1 to pop 3 M21 = matrix(0,It,yrs) #Move individuals from pop 2 to pop 1 M23 = matrix(2,It,yrs) #Move individuals from pop 2 to pop 3 M31 = matrix(0,It,yrs) #Move individuals from pop 3 to pop 1 M32 = matrix(0,It,yrs) #Move individuals from pop 3 to pop 2 M1c = matrix(4,It,yrs) #Move individuals from pop 1 to captivity M2c = matrix(3,It,yrs) #Move individuals from pop 2 to captivity M3c = matrix(0,It,yrs) #Move individuals from pop 3 to captivity Mc1 = matrix(0,It,yrs) #Move individuals from captivity to pop 1 Mc2 = matrix(0,It,yrs) #Move individuals from captivity to pop 2 Mc3 = matrix(1,It,yrs) #Move individuals from captivity to pop 3 #Limits management to years less than the Max M12[,(YM+1):yrs]=0 #Move individuals each year up until the max allowed M13[,(YM+1):yrs]=0 M21[,(YM+1):yrs]=0 M23[,(YM+1):yrs]=0 M31[,(YM+1):yrs]=0 M32[,(YM+1):yrs]=0 20-AM1c[,2:yrs]=0 #Move individuals in year 1 only M2c[,2:yrs]=0 M3c[,(YM+1):yrs]=0 Mc1[,(YM+1):yrs]=0 Mc2[,(YM+1):yrs]=0 Mc3[,(YM+1):yrs]=0 #Data arrays for stochastic iterations A=matrix(0,It,yrs) I=matrix(0,It,yrs) Y=matrix(0,It,yrs) f=matrix(0,It,yrs) A3=matrix(0,It,yrs) I3=matrix(0,It,yrs) Y3=matrix(0,It,yrs) f3=matrix(0,It,yrs) MLam1=matrix(0,It,yrs) MLam2=matrix(0,It,yrs) MLam3=matrix(0,It,yrs) MLamc=matrix(0,It,yrs) et =matrix(0,It,yrs) e1 =matrix(0,It,yrs) e2 =matrix(0,It,yrs) e3 =matrix(0,It,yrs) ec =matrix(0,It,yrs) 21-A#data arrays fors abundance projections Na1=matrix(0,It,yrs) #number of adults in region 1 Nj1=matrix(0,It,yrs) #number of adults in region 1 Na2=matrix(0,It,yrs)#number of adults in region 2 Nj2=matrix(0,It,yrs)#number of adults in region 2 Nj3=matrix(0,It,yrs)#number of adults in region 3 Na3=matrix(0,It,yrs)#number of adults in region 3 Nc=matrix(0,It,yrs)#number of adults in captivity Na1i = 238.5 #initial number of birds in pop 1 Na2i = 53 #initial number of birds in pop 2 Na3i = 0 #initial number of birds in pop 3 Nci = 7 #initial number of birds in captivity #Begin Stochastic simulation for(i in 1:It){ for(j in 1:yrs){ #Parameter values for pop 1&2 A[i,j]=rbeta(1,aAS,bAS) I[i,j]=rbeta(1,aIS,bIS) Y[i,j]=rbeta(1,aYS,bYS) f[i,j]=rlnorm(1,aF,bF) #Parameter values for pop 3 A3[i,j]=rbeta(1,aAS3,bAS3) I3[i,j]=rbeta(1,aIS3,bIS3) Y3[i,j]=rbeta(1,aYS3,bYS3) f3[i,j]=rlnorm(1,aF3,bF3) 22-A #Abundance projection split by separate populations #projects current years crop of juvenile age class if(j==1) Nj1[i,j]=round(Na1i*.2) else Nj1[i,j]=round((Na1[i,j-1]*f[i,j-1])*Y[i,j-1]) #projects current years crop of adult age class, include translocation and captive releases if(j==1) Na1[i,j]=round(Na1i*.8) else Na1[i,j]= round(((Na1[i,j-1]*A[i,j-1])+(Mc1[i,j-1]+M21[i,j-1]+M31[i,j-1])*RS[i,j])+((Nj1[i,j-1]-(M1c[i,j-1]+M12[i,j-1]+M13[i,j-1]))*I[i,j-1])) #sets any abundance less than 1 to extinct if (Na1[i,j]<1) Na1[i,j]=0 #resets fecundity to 0 if abundance exceeds 885 (carrying capacity) if (Na1[i,j]>885) f[i,j]=0 #restrict management actions to 0 movements if abundance is below 100 if (Na1[i,j]<100) M12[i,j] = 0 else M12[i,j]=M12[i,j] if (Na1[i,j]<100) M13[i,j] = 0 else M13[i,j]=M13[i,j] if (Na1[i,j]<100) M1c[i,j] = 0 else M1c[i,j]=M1c[i,j] if(j==1) Nj2[i,j]=Na2i*.2 else Nj2[i,j]=round((Na2[i,j-1]*f[i,j-1])*Y[i,j-1]) if(j==1) Na2[i,j]=Na2i*.8 else Na2[i,j]= round(((Na2[i,j-1]*A[i,j-1])+(Mc2[i,j-1]+M12[i,j-1]+M32[i,j-1])*RS[i,j])+((Nj2[i,j-1]-(M2c[i,j-1]+M21[i,j-1]+M23[i,j-1]))*I[i,j-1])) if (Na2[i,j]<1) Na2[i,j]=0 if (Na2[i,j]>885) f[i,j]=0 if (Na2[i,j]<25) M21[i,j] = 0 else M21[i,j]=M21[i,j] if (Na2[i,j]<25) M23[i,j] = 0 else M23[i,j]=M23[i,j] if (Na2[i,j]<25) M2c[i,j] = 0 else M2c[i,j]=M2c[i,j] if(j==1) Nj3[i,j]=Na3i*.2 else Nj3[i,j]=round((Na3[i,j-1]*f3[i,j-1])*Y3[i,j-1]) 23-Aif(j==1) Na3[i,j]=Na3i*.8 else Na3[i,j]= round(((Na3[i,j-1]*A3[i,j-1])+(Mc3[i,j-1]+M13[i,j-1]+M23[i,j-1])*RS[i,j])+((Nj3[i,j-1]-(M3c[i,j-1]+M31[i,j-1]+M32[i,j-1]))*I3[i,j-1])) if (Na3[i,j]<1) Na3[i,j]=0 if (Na3[i,j]>885) f[i,j]=0 if (Na3[i,j]<100) M31[i,j] = 0 else M31[i,j]=M31[i,j] if (Na3[i,j]<100) M32[i,j] = 0 else M32[i,j]=M32[i,j] if (Na3[i,j]<100) M3c[i,j] = 0 else M3c[i,j]=M3c[i,j] #To add births and deaths to the captive population we used a random poisson distributed function and there is not carrying capacity if(j==1) Nc[i,j]=Nci else if(Nc[i,j-1]==0) Nc[i,j]=round(0+(M1c[i,j-1]+M2c[i,j-1]+M3c[i,j-1])-(Mc1[i,j-1]+Mc2[i,j-1]+Mc3[i,j-1])) else Nc[i,j]=round(Nc[i,j-1]+rpois(1,CN)-rpois(1,CD)+(M1c[i,j-1]+M2c[i,j-1]+M3c[i,j-1])-(Mc1[i,j-1]+Mc2[i,j-1]+Mc3[i,j-1])) if (Nc[i,j]<1) Nc[i,j]=0 if (Nc[i,j]<20) Mc1[i,j] = 0 else Mc1[i,j]=Mc1[i,j] if (Nc[i,j]<20) Mc2[i,j] = 0 else Mc2[i,j]=Mc2[i,j] if (Nc[i,j]<5) Mc3[i,j] = 0 else Mc3[i,j]=Mc3[i,j] if(j>1)MLam1[i,j]=Na1[i,j]/Na1[i,j-1] if(j>1)MLam2[i,j]=Na2[i,j]/Na2[i,j-1] if(j>1)MLam3[i,j]=Na3[i,j]/Na3[i,j-1] if(j>1)MLamc[i,j]=Nc[i,j]/Nc[i,j-1] #Calculate quasi extinction probability if ((Na1[i,j]+Na2[i,j]+Na3[i,j]) < 10) et[i,j]=1 if (Na1[i,j] < 10) e1[i,j]=1 if (Na2[i,j] < 10) e2[i,j]=1 24-Aif (Na3[i,j] < 10) e3[i,j]=1 if (Nc[i,j] < 2) ec[i,j]=1 }} #Calculate median population size and 95% quantiles for each population MNa1=apply(Na1,2,median) LBNa1=apply(Na1,2,quantile, probs=(.025)) UBNa1=apply(Na1,2,quantile, probs=(.95)) MNa2=apply(Na2,2,median) LBNa2=apply(Na2,2,quantile, probs=(.025)) UBNa2=apply(Na2,2,quantile, probs=(.95)) MNa3=apply(Na3,2,median) LBNa3=apply(Na3,2,quantile, probs=(.025)) UBNa3=apply(Na3,2,quantile, probs=(.95)) MNc=apply(Nc,2,median) LBNc=apply(Nc,2,quantile, probs=(.025)) UBNc=apply(Nc,2,quantile, probs=(.95)) #calculate median population growth median(MLam1[,2:yrs]) median(MLam2[,2:yrs]) median(MLam3[,2:yrs]) median(MLamc[,2:yrs]) #Calculate and print quasi extinction for each region and captivity set = apply(et,2,sum) 25-Apet = set/It pet se1 = apply(e1,2,sum) pe1 = se1/It pe1 se2 = apply(e2,2,sum) pe2 = se2/It pe2 se3 = apply(e3,2,sum) pe3 = se3/It pe3 sec = apply(ec,2,sum) pec = sec/It pec 26-AAppendix D. European Molecular Biology Laboratory deposits Accession#: LM993639 Status: not confidential Description: Pseudonestor xanthophrys mitochondrial partial D-loop LM993639; SV 1; linear; genomic DNA; STD; VRT; 252 BP. 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 1) Pseudonestor xanthophrys mitochondrial partial D-loop Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. Mitochondrion 1-252 Mounce H.; Submitted (25-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. Mounce H.L., Raisin C., Swinnerton K.J., Leonard D.L., Wickenden H., Groombridge J.J.; "Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies"; Unpublished. 27-A MD5; db95fcd47c0cdd21852b4eaf478b0310. Key Location/Qualifiers source 1..252 /organism="Pseudonestor xanthophrys" /organelle="mitochondrion" /mol_type="genomic DNA" /country="USA:Hawaii" /isolation_source="Blood" /collection_date="08-Oct-2009" /sex="Male" /db_xref="taxon:64813" D-loop <1..>252 Sequence 252 BP; 80 A; 91 C; 36 G; 45 T; 0 other; acgaacagcc caaacattat ctccaaaacg gacctcatac ggccaataca cccaccagag acattcttgt ttcaggtacc atatagccca aatgctccta cctacagcca agccgcaagc gtcacccaaa gacccaggaa cttacctact ataccccaaa cccaaccaag gaaacgaggg atgtcccagt acacctttgc attcccctag accactgaat tcgcccacct cctaggcaag attctcctcc aa Accession#: LM993640 Status: not confidential Description: Pseudonestor xanthophrys mitochondrial partial D-loop LM993640; SV 1; linear; genomic DNA; STD; VRT; 250 BP. 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 1) Pseudonestor xanthophrys mitochondrial partial D-loop Pseudonestor xanthophrys 28-A Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. Mitochondrion 1-250 Mounce H.; Submitted (25-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. Mounce H.L., Raisin C., Swinnerton K.J., Leonard D.L., Wickenden H., Groombridge J.J.; "Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies"; Unpublished. MD5; 6471aaa6f02172b6b9b011171799a096. Key Location/Qualifiers source 1..250 /organism="Pseudonestor xanthophrys" /organelle="mitochondrion" /mol_type="genomic DNA" /country="USA:Hawaii" /isolation_source="Blood" /collection_date="16-Apr-2009" /sex="Female" /db_xref="taxon:64813" D-loop <1..>250 29-A Sequence 250 BP; 79 A; 90 C; 36 G; 45 T; 0 other; gaacagccca aacattatct ccaaaacgga cctcatacgg ccaatacacc caccagagac attcttgttt caggtaccat atagcccaaa tgctcctacc tacagccaag ccgcaagcgt cacccaaaga cccaggaact tacctactat accccaaacc caaccaagga aacgagggat gtcccagtac acctttgcat tcccctagac cactgaattc gcccacctcc taggcaagat tctcctccaa Accession#: LM993641 Status: not confidential Description: Pseudonestor xanthophrys mitochondrial partial D-loop LM993641; SV 1; linear; genomic DNA; STD; VRT; 131 BP. 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 1) Pseudonestor xanthophrys mitochondrial partial D-loop Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. Mitochondrion 1-131 Mounce H.; Submitted (25-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, HI 96768, USA. 30-A Mounce H.L., Raisin C., Swinnerton K.J., Leonard D.L., Wickenden H., Groombridge J.J.; "Spatial genetic architecture of the critically-endangered Maui Parrotbill (Pseudonestor xanthophrys): management considerations for reintroduction strategies"; Unpublished. MD5; 75289cd5f0aab6ba6b4601faf0fce329. Key Location/Qualifiers source 1..131 /organism="Pseudonestor xanthophrys" /organelle="mitochondrion" /mol_type="genomic DNA" /country="USA:Hawaii" /isolation_source="Blood" /collection_date="13-May-2009" /sex="Male" /db_xref="taxon:64813" D-loop <1..>131 Sequence 131 BP; 40 A; 49 C; 18 G; 24 T; 0 other; tcacccaaag acccaggaac ttacctacta taccccaaac ccaaccaagg aaacgaggga tgtcccagta cacctttgca ttcccctaga ccactgaatt cgcccacctc ctaggcaaga ttctcctcca a Accession#: LM993642 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa01 ID LM993642; SV 1; linear; genomic DNA; STD; VRT; 819 BP. public 31-A08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa01 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-819 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; 3f200caea7d107e9c1d3a6cbc7a4d120. Key Location/Qualifiers source 1..819 /organism="Pseudonestor xanthophrys"/chromosome="7"/mol_type="genomic DNA"/country="USA:Hawaii, Hanawi NAR"/collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 223..244/rpt_type=TANDEM/rpt_unit_seq="GTT" /satellite="microsatellite:Pxa01" Sequence 819 BP; 229 A; 162 C; 195 G; 219 T; 14 other; ctgaggtaaa aaagccattc agtgacaggc cacataaagc attttatgac agaaaacaaa 60 ggagcaggtc tttcatccaa agagtggagg aggcagaatg ccttgaaggt atggtccatg 120 tatgtgcaac tgatgctgtt agcagatgca aaaaaaaaac ttagagtatc agccctagaa 180 ttctatggta tttcgtgttg ctattttact tagtattatt attgttgttg ttgttgttgt 240 tgttggtttt gctgctattg ttgtggttag tagatatgat gtgctagata ttgtccatta 300 gtagaaaaaa tggatgagga aaaaagtgca gtgcaggctt ttatgtgcaa gacaattcaa 360 ggtaagtagc ataatagaga aaggactttg ggaacctcat gggcgaggcc agggaggact 420 tgtaggctgc tacaggagaa cctgaacagt tgaaattggg atagcagatg taaaaggggt 480 gggtaaggca aaagagaatc cttttcttaa tgcctccata aaacagtcca ttgccagagt 540 32-A gtttttctgt tgcactgcag acagttcgtc tctggaccct gaagtctcca aggcaaatgc 600 ctatggttca ctggctgcct gtcctctgtt cagtacaacc acatctcctc cccctgaagg 660 ctggccttac gccatcccca agcngttngc tccccgttga actgtnccaa nggttnccct 720 ggaacaanaa attccaaaag nctttgnnat tgnccctggc nagggtccga acttcctaaa 780 aaaggaattc cccccggggt taacccnaan ncctcccaa 819 Accession#: LM993643 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa02 ID LM993643; SV 1; linear; genomic DNA; STD; VRT; 671 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa02 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-671 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA.. MD5; db5b2d91822845b713072b0f8e324bf3. Key Location/Qualifiers source 1..671 /organism="Pseudonestor xanthophrys"/chromosome="3"/mol_type="genomic DNA"/country="USA:Hawaii, Hanawi 33-ANAR"/collection_date="2001"/tissue_type="blood"/db_xref="taxon:64813"/repeat_region 249..280/rpt_type=TANDEM/rpt_unit_seq="TAGA"/satellite="microsatellite:Pxa02" Sequence 671 BP; 190 A; 136 C; 134 G; 211 T; 0 other; aatacacaaa atccccctct tggaggccca cctctaagtg ttctatgcac cccaattctc 60 catttgccaa gccatgggat gcactgggtt tagtggatga gccacttatg gcagcctaaa 120 tatttgaaaa cttctgtttt tttctttttt tcccagattt tttcagtacc cctttccact 180 tgtccacttc accagtcaga tgttcttgtt ccctctattt tttttctgga tgaggcaaga 240 catgagagat agatgataga tagacagata gatagataga cagatagata gatagataga 300 taatatatct gtatcatcta caatggtatt tatctggaga gatatccatg catttatatc 360 tatatataaa gagagatgga tggatggatg atggatggat gggtgggtgg atggatggat 420 aaatgtatta ataaatgggt gaatagatgg atggatgcaa taccacagca gagactttgc 480 acatggtctt acaacatcta tatcttaata tgtccttgga ctgagtgcca gaccttttcc 540 tgcttctcca tattcctgcc tcagcagtca tcccgagaca agaaacctgc attttgaccc 600 ctctatacct tcagaaacag atttagagat aatttcagcc tcccaaacag ttgcagaggt 660 gcttctcttg c 671 Accession#: LM993644 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa03 ID LM993644; SV 1; linear; genomic DNA; STD; VRT; 643 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa03 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 34-A1-643 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; f51646bc11d5c2767bcc40c217f93015. Key Location/Qualifiers source 1..643 /organism="Pseudonestor xanthophrys"/chromosome="6"/mol_type="genomic DNA"/country="USA:Hawaii, Hanawi NAR"/collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 521..563/rpt_type=TANDEM/rpt_unit_seq="GTT"/satellite="microsatellite:Pxa03" Sequence 643 BP; 150 A; 132 C; 140 G; 221 T; 0 other; tctggagagt ttctgctgtt tttcttcacc cctctttctt taacaagaag aaaaaaagta 60 gggctttgga agattttttt tgagcaaaac aacctttcaa gctgaatctg tgtttatagt 120 tggaactgat ctatgctgag ccccagcacc ctatgctaaa ccacgtgtgc aaaaggacct 180 cattatcatg tactgtgggt ggatgaaatg gctcccaaca cacctccctg ccccagggct 240 ctgctcagat gtgagagtat catccatcac gttctgttgc ctctgtgttt gcagtcttgg 300 tgaatctgca cagacattag gactgttact tcacatataa ttgtggctaa agtacccaga 360 aaccctaatt ggtttctgag gggagaggtt gtcatggatt ttgtagagcc aagagggacc 420 ttcataatca cacattgttc tgttctgtat aattcggatt ggagagtttg atcagtacct 480 tctgctttat ttaacttttt gtgactaggg caggacctga cgttgttgtt gttgttgttg 540 ttgttgttgt tgttgttgtt gttttgcatt aataattttc ctgagatgat ggaaaatgca 600 ctgcttgtcc ttctgaccat ctcctccaac tatttcccca cag 643 Accession#: LM993645 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa04 ID LM993645; SV 1; linear; genomic DNA; STD; VRT; 557 BP. public 35-A08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa04 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-557 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. DR MD5; 38dcb47b3132bd424a0344eeeba6c9bb. Key Location/Qualifiers source 1..557 /organism="Pseudonestor xanthophrys"/chromosome="2" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR" /collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 217..247 /rpt_type=TANDEM /rpt_unit_seq="CAA" /satellite="microsatellite:Pxa04" Sequence 557 BP; 180 A; 129 C; 106 G; 142 T; 0 other; gcactgaata ttaacctaca tattcaacag gtgaactttc ccatttcaga agggaataat 60 gaagcctaag aggccaaatg ttctggaaaa aagcacaaca aacccaattg tttacccaaa 120 catgcccaga gagatgacat ttaaattgtt tattttcagc ctgtttaaaa tgtctaaatt 180 agcattcaaa ttgtattgac cactgtgatg gggaaaacaa caacaacaac aacaacaaca 240 acaacaaatc ctgaagatct ggatgttaac aaatagcaga agccaatttc attagttgcc 300 ccacagttca gcatctgatc acactggggc ttccaggcat ccaaagggct gtcgctcaga 360 gcctgacctg tcactgggtt ctggctacca ggccaccatc tggtttatga taccagtggc 420 tctaacacct ccagattttg gtgccctcct ctggagcacg cttacagtct aatgcagccc 480 agacatgcag atgtgggtat gtcagatatg caatctgatc aatgcttttc caaaattcca 540 36-A taaaatgatg tagtgag 557 Accession#: LM993646 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa05 ID LM993646; SV 1; linear; genomic DNA; STD; VRT; 687 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa05 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-687 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; ccc3273b14a53f30ef1021f99850d794. Key Location/Qualifiers source 1..687 /organism="Pseudonestor xanthophrys"/chromosome="2"/mol_type="genomic DNA"/country="USA:Hawaii, Hanawi NAR"/collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 130..157 /rpt_type=TANDEM/rpt_unit_seq="CAT" /satellite="microsatellite:Pxa05" Sequence 687 BP; 161 A; 162 C; 141 G; 223 T; 0 other; 37-A gagagcatat gttcttgagc agttaatcct tgcacaggct taggcaggaa aagaaaaatg 60 aattgtatat gctatgacta ttggagttga attatgtatt tagtgtgatt gctgccattt 120 tcctctcaat catcatcacc atcatcatca tcatcatcac ctccctccct gctcctgcca 180 tgcctcatcc tcctggatgt acatctccag gcccatgtgt ggtgtggaga gcactctgac 240 tcctttgccc gtggactcac taacgcttct ctcccaggca gtgagaatgc cttgtcacac 300 ttgaaccaca agtggattct ggattctccc gcttggagtt catggatcta acctggcagc 360 caccaatctg tatgtcttct gtaaatgatc ctccaagggt ttacctgtag cagtgctgtc 420 atgctcccag gaataatcac aaggtttgtg ttcatttttg tagctcttta ttctgtgatg 480 tgcccttgaa acccgtgagg ctccaacttt gtctataaat cttgttaaaa gggtgtgggt 540 tgcctgttcc acacagcaaa aaaagcaatt tttaagttat ttagatacta ttcatcttct 600 ccctgaagcc tggtcaaaac ttctgtttca acattgtcct ttgttataga gtgaggaatg 660 gtggcaattt tcctgcatgt gctgtgg 687 Accession#: LM993647 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa06 ID LM993647; SV 1; linear; genomic DNA; STD; VRT; 480 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa06 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-480 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 38-A96768, USA. DR MD5; d8d296d3eda052317bf1f56b17e9216c. Key Location/Qualifiers source 1..480 /organism="Pseudonestor xanthophrys"/chromosome="1A"/mol_type="genomic DNA"/country="USA:Hawaii, Hanawi NAR"/collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 367..415/rpt_type=TANDEM /rpt_unit_seq="CTAT" /satellite="microsatellite:Pxa06" Sequence 480 BP; 114 A; 86 C; 119 G; 161 T; 0 other; gggtctcacc tcacttgtca cttctgtaaa ctgggagtga acttgactga aagttttaca 60 tgggcaggta ttgacagaac aagggtgttt tgaagtttta aagggtgttt taagcgtttt 120 aaactaggaa aggagacatt cagattggtt attaaccaga aatacttctg agggtggtga 180 ggtcctggca cagggtgctt agacaagttg tggctgttcc tggaagtgtc caaggccagg 240 ttggacattt gggcttggag caacctggga tggtggaagg tgtccctgcc catggcaggg 300 ggtggcactg gatgatcttg aagatcaaac tattgtctga ttttgagaac tttatctatc 360 tgtctgtcta tctatctatc tatctatcta tctatctatc tatctatcta tctatcttat 420 ctctctcttt ctctctctct agtgctccaa gaggaggaaa taactctgat tatgttgttt 480 Accession#: LM993648 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa07 ID LM993648; SV 1; linear; genomic DNA; STD; VRT; 643 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa07 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; 39-AFringillidae; Drepanidinae; Pseudonestor. 1-643 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; 2c124f2fdced76f7c53b4ad1010914fb. Key Location/Qualifiers source 1..643 /organism="Pseudonestor xanthophrys"/chromosome="3"/mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR"/collection_date="2001"/tissue_type="blood" /db_xref="taxon:64813"/repeat_region 60..111/rpt_type=TANDEM/rpt_unit_seq="CTAT" /satellite="microsatellite:Pxa07" Sequence 643 BP; 192 A; 127 C; 124 G; 200 T; 0 other; aacaccagga ctgcacttgt ggtgaaagac ttctcattag agaaaagtat tatatctatt 60 ctatcatcta tctatctatc tatctatcta tctatctatc tatctatcta tatgaataat 120 gtactatggg gatttcagaa gaagattttg ctttgaagca ctccaaaatc attgatataa 180 agagacattt ttgaaaaaag gctctgtctg tacaatattt aacttttctg ttccagtctg 240 aaggaaaaca ttaatgaata aaaatattat ttccaagcat caaactgaag tacatagcag 300 aattggctgg agcacttgac acagtccatg ttcttcaggt tttgtagaat cagttctagg 360 aaacaaaccc attcacaatg gctaggacag gttggaagag acagatcacc aacacattaa 420 cttgttcttt tcccttctac ttatcagtaa gactactgtg atgaccctac cagtgtcctc 480 ctggactgcc ctgtccccat agcaaggagg ttgagtggag aatccctgtg ctgacttgcg 540 tcctctgtgg gcatcttttc ttctgcagga tctggatgct tacagattaa atttggtggg 600 agtaactctc ttagggtagg acatagttac gagccggaca gag 643 Accession#: LM993649 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa08 ID LM993649; SV 1; linear; genomic DNA; STD; VRT; 733 BP. public 40-A 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa08 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-733 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; a68aa617e1b484d65847c7f04f84495a. Key Location/Qualifiers source 1..733 /organism="Pseudonestor xanthophrys" /chromosome="Z" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR" /collection_date="2001" /tissue_type="blood" /db_xref="taxon:64813"/repeat_region 393..521 /rpt_type=TANDEM /rpt_unit_seq="CTAT" /satellite="microsatellite:Pxa08" Sequence 733 BP; 214 A; 171 C; 101 G; 247 T; 0 other; cttctactaa ggaaccatct ttaattggag acagagggga ggttttttgt gctgaactct 60 ttcccacccc ataagcaacc attgtacaac tgcaaaactc agaaaattta cccagaaact 120 ttgtgtagaa actttgtcca ggcacacctg gacagacaga cacacaccct cacctgctct 180 cttgatgtca acaaatttcc agcatcttcc agagagaagt ttgagccagt ccaaaactcc 240 ttacagagga tgtactgggg aatttactag aagaaccagg aaagccaatt ttctatacat 300 ccatgggccc ctacccctga cagtgcagga ctgtgtatta tgactacagg ccatgatcac 360 acatgtgtca ctggaggcat gggtagtgat tatctatcta tctatctatc tatctatcta 420 tctatctatc tatctatcta tctatctatc tatctatcta tctatctatc tatgcatcta 480 41-A tctatctatc tatctatcta tctatctatc tatctatcta tgcatctatc taatctcttc 540 tctctctatt tctccttttt cctttgcaat tttcctctca tgggatgatc ataaacacca 600 gaatggctat agatcccttt tacaatcctt acagtttttt ccagatagat ttcaaagcaa 660 tttctcaaat aaaattaatt ttaatataat ttagatagag taatttgttt taagacatcc 720 caaagaatct gtt 733 Accession#: LM993650 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa09 ID LM993650; SV 1; linear; genomic DNA; STD; VRT; 462 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa09 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-462 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; 2ac28a4f0ac216741a0b06a3b0da6872. Key Location/Qualifiers source 1..462 /organism="Pseudonestor xanthophrys" /chromosome="1" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR" /collection_date="2001" /tissue_type="blood" 42-A/db_xref="taxon:64813"/repeat_region 110..150 /rpt_type=TANDEM /rpt_unit_seq="TAGA" /satellite="microsatellite:Pxa09" Sequence 462 BP; 133 A; 123 C; 113 G; 93 T; 0 other; gctgcgagag ggctgctgcc tgagcagcct gcttccctgc cttccctgaa ctgcctcagc 60 acccacgtga ctcagcacct acaacatggt tagaaacata aatacacaga tagatagata 120 gatagatata tagatagata gatagataga tatggtaagc aaatgcactg cgcctatgca 180 ctgatggaca gaatgtttgt ttgtgcacct ggcattctga tgcacgaaca gccacgaggc 240 gtggcattgg cacgctgagg caatgagtca gggcaccact ggtgaagcaa agcccacagc 300 ctggcaacac agcgagttgg acaactagca gcatcggcac catgacagca tcacggcgtc 360 attcaggttg gagaagactt aggaggtcac tgagtccaac tgttatccca ggactgtcaa 420 gtccaacact aaaccacgtc cctcactgcc acttctacac ag 462 Accession#: LM993651 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa10 ID LM993651; SV 1; linear; genomic DNA; STD; VRT; 799 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa10 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-799 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 43-A96768, USA. DR MD5; 396f44e41b5a371f6ff0f511ed91f1d7. Key Location/Qualifiers source 1..799 /organism="Pseudonestor xanthophrys" /chromosome="2" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR" /collection_date="2001" /tissue_type="blood"/db_xref="taxon:64813"/repeat_region 384..423 /rpt_type=TANDEM /rpt_unit_seq="GTT"/satellite="microsatellite:Pxa10" Sequence 799 BP; 229 A; 177 C; 160 G; 233 T; 0 other; gtctaattac acataaccta gttctgactt atttggcatg tttatgctgc ctcctgatcc 60 agcaccacac agccagcatg tgcaggactg agtcccacaa gcagccctga ttcccagaca 120 cctcaaggtc ttagtgaggc tgacaaaacg ctgcatcccc ctaagcatct ctcaggctca 180 gatcacctct gtgcagaagt ccaggggact gatggggaag ggtttctgtg aggcaaagta 240 ggaggaagaa accagcccca ctggttaggt gatcaccaac atgaactgac tcaatccttt 300 aaaacactgt gcttcagcaa acagcattcc ttgaccctcc tcttaaccct cagtgttaga 360 gagtgatttt tttttttttt ttttgttgtt gctgttgttg ttgttgttgt tgttgttgtt 420 gttgaaaaag ctcttgacca aacctgagaa gtcttcaggg aaaaggtgat ttggcttttc 480 aaataacttt ttttcataac tcctctctga ctgtcagttt agtttattta tatcataaat 540 caaaaggtta aagatgaaat gaactcacgc agagaattac ttaaagactc atagcaacat 600 catcagtact ttatacaaaa gccaaaaaat cagcaaaaca ttaattttaa gtgtgtagac 660 aaataagtta ttttagccag taatcagaag agaattccac ctccataaaa gcttgcatgc 720 ctgcaggtcg actctagagg atccccgggt accgagctcg aattcactgg ccgtcgtttt 780 acaacgtcgt gactgggaa 799 Accession#: LM993652 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa11 ID LM993652; SV 1; linear; genomic DNA; STD; VRT; 542 BP. public 44-A08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa11 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-542 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; 8db518c3885dd5d3f9f925b62ecc2d2b. Key Location/Qualifiers source 1..542 /organism="Pseudonestor xanthophrys" /chromosome="1" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR" /collection_date="2001" /tissue_type="blood" /db_xref="taxon:64813"/repeat_region 94..178 /rpt_type=TANDEM /rpt_unit_seq="CTAT" /satellite="microsatellite:Pxa11" Sequence 542 BP; 159 A; 115 C; 111 G; 157 T; 0 other; tctttcctca acacatatga gcaaacacat atgagcagag aagtaacatt atacctaatt 60 aaacctggct gacagtgtgg cagtgtagca gagtctatca tctatctatc tatctatcta 120 tctatctatc tatctatcta tctatctatc tatctatcta atctatctat ctatctatct 180 aatctacctg tctatctatc tatcgtccat ctatttccat gtaagttgta gttctatgaa 240 tactccagta tttcaagtta cctcagccta agaatggcta acctggacac acaggtcact 300 gcatgaaagt gaagtggaag tgagattcct caaatgtggt gcagttggag gactgggcaa 360 atcagtggct gctagaagaa gccatcaaat tgtgggcagg tatttcaatc cagagaatct 420 ctggcatgca ggagtggttg atccctggta tgaagtgcaa agcccaggag aatggctggc 480 ttcagaagag actccataaa actctacata tgtgtggggc acagcaggga ccaaggttcc 540 45-A ag 542 Accession#: LM993653 Status: not confidential Description: Pseudonestor xanthophrys microsatellite DNA locus Pxa12 ID LM993653; SV 1; linear; genomic DNA; STD; VRT; 759 BP. public 08-AUG-2014 (Rel. 121, Created) 08-AUG-2014 (Rel. 121, Last updated, Version 0) Pseudonestor xanthophrys microsatellite DNA locus Pxa12 Pseudonestor xanthophrys Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Testudines + Archosauria group; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Passeriformes; Passeroidea; Fringillidae; Drepanidinae; Pseudonestor. 1-759 Mounce H.; Submitted (26-JUL-2014) to the INSDC. Dr. Jim Groombridge, Maui Forest Bird Recovery Project, 2465 Olinda Road, 96768, USA. MD5; 883ddef7ce7050834383448daba4f011. Key Location/Qualifiers source 1..759 /organism="Pseudonestor xanthophrys" /chromosome="4" /mol_type="genomic DNA" /country="USA:Hawaii, Hanawi NAR"/collection_date="2001" /tissue_type="blood" /db_xref="taxon:64813"/repeat_region 417..509 /rpt_type=TANDEM /rpt_unit_seq="CTAT" /satellite="microsatellite:Pxa12" Sequence 759 BP; 212 A; 179 C; 146 G; 222 T; 0 other; 46-A ggaggatggt atattcatat tcatattcat attcatattc atgttcatat tcatattcat 60 attcatattc atattcacca gtgtttttga ttatgtaacc ttaatacact atttccaaac 120 cccctggcta catctgtgtc agtatgagag aagagagggg aatttccttt gcaccttgct 180 ggaggatgat aaaggtgtaa cctgcatgtg accaaaatgc tgctgtgact gagagaagcc 240 tttcatcact ctcagagaaa aaagcaaggc atgctgagtg taagtgggac acttccctgt 300 gcttctccta cagcagcaca agtggaactg gtgcaatggg aggcccttcc agccttcctg 360 cctgtgttgc catggtgtga acaggcagag atatctgccc acatgcacct tgtatatcta 420 tctatctaat ctatctatct atctatctat ctatctatct atctatctat ctatctatct 480 atctatctat ctatctatct atcatctatc tcagcatact atcagcattt tgcaagggca 540 gaattgcagc atgttatccc ttctggctca aggaactgag cctctctccg tgaccgaccc 600 tctctcttta ggcatcagtg tgcatccgaa acaacagtac taaaaacagg ctgaagaaaa 660 gagcaaaagt atttccagca acaagaacaa aaggttttta ttcccaagca tttcgagtga 720 gaggcccctg ccagcaaggc accaaggcag ccgcgggag 47-AAppendix E. MAPA sample histories for genetic analyses Table D.1 Individual histories for all Maui Parrotbill (Pseudonestor xanthophrys) samples used in genetic analyses MAPA Samples Collected Label Sample Type Band Number Color Bands Date Location Age Sex MAPA001 Blood/Feather 1401-47543 AL/BL, BL/YE 10/8/2009 WAIK AHY M MAPA002 Blood/Feather 1401-47542 AL/BK, BK/YE 10/2/2009 WAIK AHY M MAPA003 Blood/Feather 1401-47539 AL/BL, BL/RD 8/17/2009 HR3 AHY F MAPA004 Blood/Feather 1401-47538 AL/GR, BL/YE 5/13/2009 HR2 AHY M MAPA004 Blood/Feather 1401-47538 AL/GR BL/YE 5/13/2009 HR2 AHY M MAPA005 Blood/Feather 1401-47541 GR/AL,BK/YE 4/16/2009 FSB ASY M MAPA005 Blood/Feather 1401-47541 GR/AL, BK/YE 4/16/2009 FSB ASY M MAPA006 Blood/Feather 1401-47540 GR/BK,AL/YE 4/16/2009 FSB AHY F MAPA007 Blood/Feather 1401-47537 BK/AL, BL/RD 3/19/2009 HR3 AHY M MAPA008 Blood/Feather 1371-04971 GR/YE, RD/AL 3/18/2009 HR3 ASY F MAPA008 Blood/Feather 1371-04971 GR/YE, RD/AL 3/13/2004 HR3 ASY F MAPA009 Blood/Feather 1401-47536 RD/AL, BL/BL 2/16/2009 FSB AHY F MAPA010 Blood/Feather 1401-47533 RD/AL,RD/BL 1/30/2009 HR3 AHY M MAPA011 Blood/Feather 1401-47534 AL/RD,RD/GR 1/30/2009 HR3 AHY F MAPA012 Blood/Feather 1401-47535 AL/BL,YE/RD 1/27/2009 HR3 AHY M MAPA013 Blood/Feather 1401-47532 AL/GR,GR/BK 12/9/2008 FSB AHY F MAPA014 Blood/Feather 1401-47531 BK/YE,AL/YE 12/9/2008 FSB AHY M MAPA015 Blood/Feather 1401-47528 BL/YE,BL/AL 12/7/2008 FSB AHY F MAPA016 Blood/Feather 1401-47530 YE/BL,AL/YE 12/7/2008 FSB AHY M MAPA017 Blood/Feather 1401-47526 AL/RD, YE/BK 9/12/2008 FSB HY M MAPA018 Feather 1401-47512 AL/BL, YE/BK 11/16/2007 FSB AHY M 48-AMAPA019 Blood/Feather 1401-47516 AL/BL, BK/RD 12/1/2007 FSB AHY M MAPA020 Blood/Feather 1401-47525 AL/BL, GR/YE 4/30/2008 FSB AHY F MAPA021 Feather 1401-47521 YE/YE, RD/AL 4/25/2008 HR3 AHY M MAPA022 Blood/Feather 1401-47520 YE/RD, BL/AL 4/24/2008 HR3 ASY M MAPA023 Blood/Feather 1401-47544 AL/GR , RD/GR 10/22/2009 WAIK AHY F MAPA024 Blood/Feather 1401-47545 AL/BL, YE/GR 10/28/2009 WAIK AHY F MAPA025 Blood/Feather 1401-47546 AL/GR, GR/YE 10/28/2009 WAIK AHY M MAPA026 Blood/Feather 1371-04982 BL/BL, BL/AL 1/20/2007 HR3 ASY M MAPA026 Blood/Feather 1371-04982 BL/BL, BL/AL 11/23/2009 HR3 ASY M MAPA027 Blood/Feather 1371-04983 BL/YE, AL/RD 2/3/2007 HR3 AHY F MAPA027 Blood/Feather 1371-04983 BL/YE, AL/RD 11/24/2009 HR3 AHY F MAPA028 Blood/Feather 1401-47548 AL/GR, GR/RD 11/24/2009 HR3 AHY F MAPA029 Blood/Feather 1371-04989 GR/GR, RD/AL 11/24/2009 HR3 ASY M MAPA030 Blood/Feather 1401-47550 AL/BL, BL/GR 12/4/2009 FSB AHY F MAPA031 Blood/Feather 1401-47549 AL/BK, BK/BL 12/4/2009 FSB AHY F MAPA032 Blood/Feather 1401-47551 AL/E, GR/RD 12/8/2009 FSB SY M MAPA033 Blood/Feather 1401-47552 RD/GR, GR/AL 1/14/2010 HR3 AHY F MAPA034 Blood/Feather 1401-47553 GR/AL, GR/BK 1/16/2010 HR3 ASY M MAPA035 Blood/Feather 1401-47554 GR/YE, AL/BL 1/21/2010 WAIK ASY M MAPA036 Blood/Feather 1401-47555 RD/YE, AL/BL 1/26/2010 FSB ASY F MAPA037 Blood/Feather 1401-47556 RD/BL, AL/BL 1/26/2010 FSB ASY M MAPA038 Blood/Feather 1401-47557 GR/AL, WH/GR 1/27/2010 FSB ASY M MAPA039 Blood/Feather 1401-47558 AL/RD, GR/GR 1/31/2010 FSB ASY M MAPA040 Blood/Feather 1401-47559 GR/BK, GR/AL 2/21/2010 FSB ASY M MAPA041 Blood/Feather 1401-47560 YE/BK, BL/AL 2/23/2010 FSB ASY M MAPA042 Blood/Feather 1401-47561 YE/BK, AL/BK 2/23/2010 FSB ASY M MAPA043 Blood/Feather 1371-04922 GR/AL, WH/BK 2/23/2010 FSB ASY M MAPA044 Blood/Feather 1401-47562 AL/GR, BL/RD 4/22/2010 WAIK SY F MAPA045 Blood/Feather 1401-47563 GR/AL, BL/WH 4/27/2010 WAIK ASY M MAPA046 Blood/Feather 1401-47564 GR/BL, BL/AL 5/17/2010 KIPA SY F 49-AMAPA047 Blood/Feather 1401-47565 BL/RD, AL/GR 5/18/2010 KIPA ASY M MAPA048 Feather 1401-47566 GR/GR, AL/RD 7/26/2010 FSB AHY M MAPA049 Feather 1401-47511 YE/AL, YE/YE 11/16/2007 FSB AHY F MAPA050 Feather 1401-47513 GR/BK, BL/AL 11/18/2007 FSB AHY M MAPA051 Feather 1401-47514 AL/BK, GR/GR 11/19/2007 FSB AHY M MAPA052 Feather 1401-47515 AL/BK, RD/BK 11/30/2007 FSB AHY F MAPA053 Feather 1401-47517 AL/RD, RD/YE 12/2/2007 FSB AHY M MAPA054 Feather 1401-47518 RD/GR, AL/GR 2/27/2008 FSB SY F MAPA055 Feather 1401-47523 BK/AL, RD/RD 2/24/2008 HR3 ASY F MAPA056 Feather 1401-47527 RD/AL,BL/GR 12/5/2008 FSB HY U MAPA057 Feather 1401-47547 AL/BL, BK/YE 11/24/2009 HR3 HY U MAPA058 Blood/Feather Captive SB#1 . 7/6/2010 KBCC ASY M MAPA059 Blood/Feather Captive SB#20 . 4/24/2010 KBCC ASY M MAPA060 Feather 1371-04981 YE/AL, YE/BL 1/20/2007 HR3 ASY F MAPA061 Feather 1371-04987 GR/AL, BK/RD 5/4/2007 HR3 ASY M MAPA062 Feather 1371-04979 GR/BK, AL/BL 1/17/2007 HR3 ASY M MAPA063 Feather 1371-04978 AL/RD, RD/BK 12/4/2006 FSB AHY M MAPA064 Feather 1371-04977 . 11/18/2006 FSB AHY M MAPA065 Feather 1371-04985 AL/BK, BL/RD 2/4/2007 HR3 SY F MAPA066 Feather 1371-04986 AL/RD, GR/RD 5/4/2007 HR3 SY F MAPA067 Feather 1371-04976 YE/RD, YE/AL 11/18/2006 FSB AHY F MAPA068 Feather 1371-04980 BK/AL, BK/GR 1/17/2007 HR3 ASY F MAPA069 Feather 1371-04984 GR/YE, BK/AL 2/4/2007 HR3 SY M MAPA070 Feather 1371-04830 AL/RD, WH/BL 6/1/1996 FSB AHY F MAPA071 Feather 1371-04962 GR/GR,AL./GR 5/11/2006 HR3 AHY F MAPA072 Feather 1401-47519 BK/BK,BL/AL 2/27/2008 FSB AHY F MAPA073 Blood 1371-04951 BK/RD, AL/BK 1/14/2002 HR3 ASY F MAPA074 Blood 1371-04954 AL/RD, OR/WH 1/25/2002 HR3 ASY F MAPA075 Blood 1371-04952 AL/WH, GR/RD 1/14/2002 HR3 ASY M MAPA076 Blood 1371-04948 BK/AL, BL/GR 1/8/2002 HR3 SY F 50-AMAPA077 Blood 1371-04937 BL/RD, AL/WH 12/12/2003 ECF ASY M MAPA078 Blood Captive SB#3 . 6/15/2011 MBCC . F MAPA079 Blood Captive SB#10 . 6/15/2011 MBCC . M MAPA080 Blood Captive SB#18 . 6/15/2011 MBCC . M MAPA081 Blood Captive SB#11 . 6/15/2011 MBCC . F MAPA082 Feather (Pins) 1181-80096 AL/WH, WH/YE 1/9/2011 HR3 ASY F MAPA083 Blood 1371-04960 AL/GR, BL/BL 1/9/2011 HR3 ASY M MAPA084 Blood 1181-80089 BL/RD, YE/AL 1/8/2011 HR3 ASY F MAPA085 Blood 1401-47586 RD/AL, RD/GR 2/15/2011 WAIK ASY F MAPA086 Blood 1401-47587 AL/BK, RD/BL 3/2/2011 HR3 ASY M MAPA087 Blood 1871-17701 YE/GR, AL/RD 2/27/2011 FSB ASY M MAPA088 Blood 1871-17702 YE/AL, GR/RD 3/1/2011 FSB ASY F MAPA089 Blood 1871-17703 YE/GR, AL/WH 3/2/2011 FSB ASY M MAPA090 Blood 1871-17704 WH/AL, BL/BK 3/2/2011 FSB ASY M MAPA091 Blood 1871-17705 GR/BK, AL/GR 3/3/2011 FSB SY M MAPA092 Blood 1871-17708 GR/BL, AL/BL 3/15/2011 FSB AHY F MAPA093 Blood 1791-16901 RD/RD,RD/AL 5/3/2011 HR3 ASY F MAPA094 Blood 1871-17709 YE/BL, YE/AL 4/28/2011 FSB ASY M MAPA095 Blood 1371-04939 WH/AL, WH/RD 5/19/2011 FSB ASY M MAPA096 Blood 1541-80237 GR/WH, YE/AL 5/30/2011 WAIK ASY M MAPA097 Blood 1541-80243 GR/AL, RD/YE 5/30/2011 WAIK HY . MAPA098 Blood Captive SB#9 . 6/15/2011 MBCC . F MAPA099 Blood 1791-16902 RD/BL, AL/RD 6/14/2011 HR3 AHY F MAPA100 Feather (Pins) 1791-16905 BL/BK, AL/GR 6/16/2011 HR3 HY . MAPA101 Blood 1791-16906 YE/GR, AL/YE 6/15/2011 HR3 SY . MAPA102 Blood 1541-80271 YE/AL, YE/GR 6/22/2011 FSB ASY M MAPA103 Blood 1541-80268 AL/RD, YE/RD 6/17/2011 FSB ASY M MAPA104 Blood 1791-16904 WH/GR, RD/AL 6/15/2011 HR3 ASY M MAPA105 Blood 1541-80270 RD/AL, BL/BK 6/19/2011 FSB ASY F MAPA106 Blood/Feather 1541-80269 GR/AL, RD/BK 6/19/2011 FSB ASY M 51-AMAPA107 Blood Captive SB#15 . 7/26/2011 KBCC . F MAPA108 Blood Captive SB#17 . 7/26/2011 KBCC . M MAPA109 Blood 1791-16907 BL/YE, YE/AL 8/24/2011 WAIK ASY F MAPA110 Blood Captive SB#12 . . KBCC ASY F MAPA111 Blood Captive SB#4 . . KBCC ASY F MAPA112 Tissue . . 5/12/2011 FSB nestling . MAPA113 Blood slide . . 2001 HR3 . . MAPA119 Blood 1871-17927 BL/AL, YE/BK 9/1/2011 WAIK AHY M MAPA120 Blood 1871-17928 RD/YE, AL/YE 9/2/2011 WAIK AHY F MAPA121 Blood 1871-17935 GR/AL, RD/GR . WAIK AHY M MAPA122 Blood 1871-17958 AL/GR, RD/RD 9/19/2011 WAIK AHY M MAPA123 Blood 1871-17957 GR/AL, RD/BL 9/18/2011 WAIK AHY F MAPA124 Blood 1791-16909 YE/BL, AL/BK 10/7/2011 WAIK AHY M MAPA125 Blood 1401-47568 AL/YE, RD/RD 10/23/2011 WAIK AHY M MAPA201 Toe pad AMNH #193408 . 05/1896 Perkins . M MAPA202 Toe pad AMNH #193409 . 6/17/1901 Henshaw . F MAPA203 Toe pad AMNH #453556 . 10/1896 Palmer . M MAPA204 Toe pad AMNH #453557 . 8/4/1892 Palmer . M MAPA205 Toe pad AMNH #453558 . 8/3/1892 Palmer . M MAPA206 Toe pad AMNH #453559 . 8/16/1892 Palmer . M MAPA207 Toe pad AMNH #453560 . 8/15/1892 Palmer . F MAPA208 Toe pad AMNH #453561 . 8/3/1892 Palmer . F MAPA209 Toe pad AMNH #453562 . 8/03/1892 Palmer . . MAPA210 Toe pad AMNH #453563 . . Palmer . F MAPA211 Toe pad BMNH 95.7.20.170 . 1894 Perkins . M MAPA212 Toe pad BMNH 1939.12.9.57 . 8/5/1892 Rothschild HY M? MAPA213 Toe pad USNM 177972 . 1901 Henshaw F MAPA214 Toe pad MCZ 47905 . 1896 Perkins . M MAPA215 Toe pad MCZ 134719 . 1894 Perkins . M MAPA216 Toe pad USNM 177971 . 1901 Henshaw . M 52-AMAPA217 Toe pad BMNH 1939.12.9.53 . 1892 Rothschild . M MAPA218 Toe pad BMNH 95.7.20.171 . 1894 Perkins . F MAPA219 Toe pad BMNH 97.10.28.22 . 1896 Perkins . F MAPA220 Toe pad BMNH 97.10.28.23 . 1896 Perkins . M MAPA221 Toe pad BMNH 97.10.28.25 . 1896 Perkins . M MAPA222 Toe pad BMNH 97.10.28.24 . 1896 Perkins . M MAPA223 Toe pad CMZ 27/DRE/9/a/1 . 1894 Perkins . M MAPA224 Toe pad CMZ 27/DRE/9/a/2 . 1896 Perkins . M MAPA225 Toe pad CMZ 27/DRE/9/a/3 . 1896 Perkins . F MAPA226 Toe pad CMZ 27/DRE/9/a/4 . 1896 Perkins HY M MAPA227 Toe pad CMZ 27/DRE/9/a/5 . 1896 Perkins . M MAPA228 Toe pad BBM 241 . . . . MAPA229 Toe pad BBM 4094 . . . . 53-AAppendix F. Maui Alauahio haplotype diversity INTRODUCTION The endangered Maui Parrotbill (Kiwikiu; Psuedonestor xanthophyrs) and Maui Alauahio (Paroreomyza montana) are two extant insectivorous honeycreepers endemic to the island of Maui. Both have suffered severe range contractions from the destruction of habitat and the compounding impacts of exotic species and diseases (Scott et al. 1986). Avian malaria has decimated low and mid-elevation bird populations throughout the islands and is a primary threat because of its potential rise in elevation (Benning et al. 2002). Currently, 421 (209-674 with 95% CI) Maui Parrotbills and 55,262 (52,729-57,921 with 95% CI) Alauahios persist within 40-50 km2 of native forest (Brinck et al. 2011). Differences in life history traits may be responsible for the difference in abundance of these species, which have suffered similar range contractions and are susceptible to the same threats within their habitat. Maui Parrotbill (left) and Maui Alauahio (right). The low population number for Maui Parrotbills suggests that genetic factors may be increasing their risk of extinction as populations with low diversity are susceptible to the impacts of inbreeding depression, which increases their susceptibility to novel diseases (Frankham et al. 2002). The evolution of a resistance to malaria is essential for the long-term survival of Hawaii’s honeycreepers, and a high genetic diversity will likely facilitate this 54-Aevolution (Kilpatrick 2006; Foster et al. 2007). We investigated the mitochondrial DNA diversity in Maui Parrotbills and Maui Alauahios using control region sequence data. METHODS Study Sites and Sample Collection Maui Parrotbills and Maui Alauahios were sampled from Hanawi Natural Area Reserve, Kipahulu Valley (Haleakala National Park) and Waikamoi Preserve (The Nature Conservancy) (Figure E.1). Birds were caught with mist-nets and blood samples were collected from the brachial vein. mtDNA Extraction and Amplification Genomic DNA was extracted from blood samples using an ammonium acetate precipitation method (Nicholls et al. 2000). We used the control-region primers LCRL1 (5’-CGCTATGACCCTCCACGAA-3’) and HCR1045 (5’- GAGACGACCTTATCCGCAAA-3’) (Tarr 1995) for Maui Parrotbills and L16743 (5’-TTCTCCGAGATCTACGGCCT-3’) (Tarr 1995) and CH1 (5’-CCAATAGCGCAAAAGAGCAA-3’) (Marthinsen et al. 2008) for Maui Alauahios. PCR products were sequenced by Macrogen Genomics and Source BioScience. The first 10 DNA extractions were sequenced off both the forward and the reverse primers and showed no differences in base calls between the two. Subsequent samples were sequenced on the forward primer only. Chromatographs were edited using FinchTV (Geospiza Inc.). Sequences were aligned in ClustalX Version 2 (Larkin et al. 2005). Samples with rare polymorphisms were sequenced twice by Macrogen Inc. and Source BioScience. Standard DNA polymorphism and genetic differentiation measures were calculated in DnaSP Version 4.00 (Rozas et al. 2003). 55-AFigure E.1. Current ranges of the Maui Parrotbill and the Maui Alauahio overlaying East Maui’s protected areas. RESULTS Figure E.2: Locations of sampled Maui Parrotbills and Maui Alauahios and the distribution of unique haplotypes, each represented by a different color. The pie chart displays the proportion of the sampled birds expressing each of these haplotypes. 56-AI sequenced 74 Maui Parrotbills across the population. These data defined 3 haplotypes (A, B, and C) of 667 bp in length. While haplotypes A and B were found in all 3 parts of the populations, haplotype C was only found in one individual. Haplotype diversity (Hd) was 0.365, nucleotide diversity (pi) 0.001. I sequenced 32 Maui Alauahios within the range of Maui Parrotbills. These data defined 14 haplotypes of 519 bp in length. There was wide variation in base pair changes for Alauahios. (Hd) was 0.901 and (pi) was 0.006. DISCUSSION Maui Parrotbills have a much lower genetic diversity than Maui Alauahios. Although this is expected due to their smaller population size, the high level of diversity in Alauahio and the comparison between these two species was surprising. An earlier evolutionary divergence, a lesser degree of specialization and a higher fecundity are likely responsible for the larger population and the higher level of genetic diversity in Alauahio. The small population size and low genetic diversity of Maui Parrotbills makes the reestablishment of a second wild population a high priority for their recovery (USFWS 2006). In the radiation of the Hawaiian honeycreepers, Maui Alauahio diverged 1.18 million years earlier than Maui Parrotbill (Lerner et al. 2011). Mitochodrial DNA is theorized to mutate at a mean rate of about 2% sequence divergence per million years, so this earlier divergence may have benefitted Alauahio some, but other factors must have contributed (Lovette 2004). Maui Parrotbills, with their powerful hooked bill, are more specialized, and do not persist in the exotic forests occupied by Alauahios. This suggests a higher level of adaptability and behavioral plasticity in Alauahios. A higher fecundity also may help Alauahios maintain higher densities and a larger population than Maui Parrotbills (Simon et al. 1997, Baker and Baker 2000). Evidence suggests that Maui Parrotbills have saturated their available habitat and that young disperse to lower elevations where they are more susceptible to avian malaria (MFBRP unpubl.) An expansion of habitat would allow an increase in population and genetic diversity 57-Aand this may facilitate the evolution of a resistance to malaria. This resistance has been demonstrated in Hawaii Amakihis (Hemignathus virens) on the island of Hawaii (Foster et al. 2007). Restoration efforts have begun in Nakula Natural Area Reserve (NAR). Nakula NAR lies in Kahikinui Forest Reserve on the leeward side of Haleakala. Fossil evidence suggests that Maui Parrotbills and Alauahios once persisted there (Scott et al. 1986, Baker and Baker 2000). Managers plan to translocate Maui Parrotbills to this new habitat within five years. Although the impacts of founder effects may be severe for Maui Parrotbills, their translocation to the expanded range may be their only hope for increasing populations and genetic diversity. Maui Alauahios have a greater potential to develop a resistance to malaria and will become even greater if introduced to the new habitat because of their higher diversity. REFERENCES Baker H, Baker PE (200) Maui Alauahio (Paroremyza montana) In: Poole A, Gill F (eds) The birds of North America, no. 504, Birds of North America Inc., Philiadelphia, USA. Benning TL, LaPointe D, Atkinson CT, Vitousek PM (2002) Interactions of climate change with biological invasions and land use in the Hawaiian Islands: modeling the fate of endemic birds using a geogrpahical information system. Proceedings of the National Academy of Sciences, USA 99f=:14246-14249. Brinck KW, Camp RJ, Gorresen PM, Leonard DL, Mounce HL, Iknayan KJ, Paxton EH (2012) 2011 Kiwikiu (Maui Parrotbill) and Maui Alauahio abundance estimates and the effect of sampling effort on power to detect a trend. Technical Report HCSU-035. Foster JT, Woodworth BL, Eggert LE, Hart PJ, Palmer D, Duffy DC, Fleischer RC (2007) Genetic structure and evolved malaria resistance in Hawaiian honeycreepers. Molecular Ecology 16:4738-4746. Frankham R, Ballou JD, Briscoe DA (2002) Introduction to conservation genetics. Cambridge University Press, Cambridge. 58-AKilpatrick AM (2006) Facilitating the evolution of resistance to avian malaria in Hawaiian birds. Biological Conservation 128:475-485. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948. Lerner HRL, Meyer M, Janes HF, Hofreiter M, Fleischer RC (2011) Multilocus resolution of phylogeny and timescale in the extant adaptive radiation of Hawaiian honeycreepers. Current Biology 21:1838-1844. Lovette IJ (2004) Mitochondrial dating and mixed support for the “2% rule” in birds. Auk 121:106. Marthinsen G, Wennerber L, Lifjeld T (2008) Low support for separate species within the redpoll complex (Carduelis flammea-hornemanni-cabaret) from analyses of mtDNA and microsatellite markers. Molecular Phylogenetics and Evolution 47:1005-1017. Nicholls JA, Double MC, Rowell DM, Magrath RD (2000) The evolution of cooperative and pair breeding in thornbills Acanthiza (Pardalotidae). Journal of Avian Biology 31:165. Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496-2497. Scott JM, Mountainspring S, Ramsey FL, Kepler CB (1986) Forest bird communities of the Hawaiian Islands: their dynamics, ecology, and conservation. Studies in Avian Biology, Lawrence, USA. 9:i-xii. Simon JC, Baker PE, Baker H (1997) Maui Parrotbill (Psuedonestor xanthophyrs). In: Poole A, Gill F (eds) The birds of North America, no. 311. Academy of Natural Sciences, Philiadelphia, USA and the American Ornithologists’ Union, Washington DC, USA, pp 1-16. Tarr CL (1995) Primers for amplification and determination of mitochondrial control-region sequences in oscine passerines. Molecular Ecology 4:527-530. U.S. Fish and Wildlife Service (2006) Revised recovery plan for the Hawaiian forest birds. Region 1, Portland, OR, pp 1-622. 59-AAppendix G. Leeward Haleakala Experimental Restoration Plan Protocols for Restoration Trials in the Nakula Natural Area Reserve This report was generated for internal use and for distribution to partners of Maui Forest Bird Recovery Project, State of Hawaii Division of Forestry and Wildlife and American Bird Conservancy by Chris Farmer, David Leonard, and Hanna Mounce NAKULA NATURAL AREA RESERVE The Nakula Natural Area Reserve (NAR; 614 ha) on the leeward slope of Haleakalā is continuous with the 925 ha Kahikinui Forest Reserve (FR) (Figure 1). The dramatic elevation change across the NAR (> 1,700 vertical m in 4 km) and the corresponding moisture gradient has compressed several native habitats into a relatively small area. Nakula NAR has numerous small gullies, which provide moist, shady habitat and support several rare plant species in what is now a harsh, dry environment. These gulches also protect native plants from ungulates. Leeward koa forests on the slopes of the larger Hawaiian volcanoes are unique in that they depend largely on precipitation and fog drip from clouds created by convection and diurnal heating. They differ markedly from koa forests on the windward slopes of the islands. Figure 1. Location of Nakula NAR, Kahikinui FR, and existing and proposed fence lines. 60-ABetween the top of the NAR at 2,830 m and approximately 2,461 m a relatively intact Pūkiawe / ‘Ōhelo Dry Subalpine Shrubland (plant community classifications follow Gagne and Cuddihy 1999) predominates and is characterized by a dense pūkiawe shrubland matrix, interspersed with native grass and fern patches. Although feral goats and pigs are present, their impact on this community has been minimal. Between 2,461 m and 2,000 m, goats have mostly denuded the native vegetation. However, remnants of ‘Ōhi‘a Subalpine Dry Forest and Māmane Subalpine Dry Forest persists in some of the larger gulches, or in areas where the underlying substrate has resisted erosion. Below the temperature inversion layer at about 2,000 m ungulates have mostly converted the Koa / ‘Ōhi‘a Montane Mesic Forest to an open grassland dominated by non-native pasture grasses. Prior to the invasion of ungulates, many rare plants occurred in this community. At the upper reaches of this community, a dry subtype of this forest exists, with a koa canopy and an understory of tall ‘a‘ali‘i shrubs. As moisture increases with decreasing elevation, species diversity and tree size increase, and this community is best represented between 1,077 m and 1,385 m elevation. This portion of the NAR has many gulches and cliff faces, which provide protected microhabitats, as well as springs and seeps that feed intermittent streams. In areas inaccessible to ungulates, a diverse assemblage of native ferns and understory plants persist. Below 1,077 m elevation, moisture decreases, and the vegetation grades into a severely degraded remnant of what was once a diverse assemblage of dryland trees; classified loosely as Olopua Montane Mesic Forest. A 150 ha parcel between 1,108 and 1,785 m has been fenced, and ungulates will be removed in the fall of 2012 (Figure 2). This area historically supported a koa-‘ōhi‘a montane mesic forest, which declined markedly between 1890 and 1930 due to feral ungulates (Hosmer 1912). The area is now mostly pasture dominated by kikuyu grass (Pennisetum clandestinum) with widely scattered native trees, although a diverse assemblage of native ferns and other understory plants are present in areas inaccessible to ungulates. This area receives over 1,000 mm of rainfall annually, with 70% falling between November and March. Temperatures rarely fall below 8° C (Minyard et al. 1994). Site access is by helicopter or a rugged 4.8 km hike. 61-A Figure 2. Location of the trial restoration site within the Nakula NAR. This plan outlines experimental trials that will be conducted within the restoration area (Figure 2, delineated by the red and blue completed fencelines) to determine cost-effective protocols for the eventual restoration of native forest within the entire Nakula NAR. The preliminary data from these trials will be incorporated into an overall Nakula Restoration Plan that will expand and implement these results to the entire NAR. The ultimate goal is to restore the ecosystem so that the landscape is capable of supporting a self-sustaining Maui Parrotbill population. MAUI PARROTBILL The endangered Maui Parrotbill (Pseudonestor xanthophrys) numbers ~500 individuals and is restricted to ~40 km2 of high elevation ‘ōhi‘a (see Table 1 for scientific names) forest on windward east Maui. Their current distribution is an artifact of habitat loss and the past and current distribution of alien diseases (e.g., avian malaria [Plasmodium relictum]) and alien disease vectors (i.e., mosquitoes; USFWS 2006). However, this habitat is likely suboptimal partly because winter storms are a significant cause of nest failure (Becker et al. 2010). Historically parrotbills occurred in mesic koa forest and this may be the species’ preferred habitat (Perkins 1903). Restoring remnant koa forest on leeward east Maui in the 62-ANakula NAR and the Kahikinui FR and establishing a second population is a high priority recovery action (USFWS 2006). Leeward east Maui is drier than windward habitats and storm frequency is lower and storms are less severe (i.e., more suitable; Mawdsley et al. 2009). In addition, leeward areas support few mosquitoes and once restored will provide additional high elevation, disease-free forest, which is critical as climate change will facilitate the upward movement of malaria (Benning et al. 2002). Restoring degraded habitat for rare species is a common sense approach to addressing climate change (Hunter et al. 2010, Lindenmayer et al. 2010). Creating an additional population and increasing the number of individuals, could facilitate the development of disease resistance (Kilpatrick 2006) and robust populations, well distributed across suitable habitat, are most likely to persist despite climate change (Schwartz et al. 2006). Table 1. Maui Parrotbill food plants, important canopy, or midstory species, the difficulty of successfully growing large numbers of individuals (1 easiest, 5 most difficult), and the number of months required for seedlings to mature in SC10 dibble tubes. 63-A Common name Scientific name MAPA food plant Difficulty SC10 ‘A‘ali‘i Dodonaea viscosa No 1 10 Ākala Rubus hawaiensis Yes 1 5 Alani Melicope clusiifolia Yes 3 10 Kanawao Broussaisia arguta Yes 5 na Kāwa‘ū Ilex anomala Yes 4 8 Koa Acacia koa Yes 2 5 Kōlea Myrsine lessertiana Yes 1 6 Mamaki Pipterus albidus No 2 5 Māmane Sophora chrysophylla No 1 10 ‘Ōhelo Vaccinium calycinum Yes 5 na ‘Ōhi‘a Metrosideros polymorpha Yes 3 10 ‘Ōlapa Cheirodendron trigynum Yes 3 9 Pilo Coprosma spp. Yes 1 6 Pūkiawe Styphelia tameiameiae Yes 5 na NAKULA RESTORATION TRIALS The entire Nakula NAR and 376 ha of the Kahikinui FR will be fenced by February 2014. What is learned from these trials may have some applicability to the Kahikinui FR, but additional methods will likely have to be developed to restore the most degraded areas. Trials will focus on dominant canopy (e.g., koa, ‘ōhi‘a) and subcanopy (māmane, ‘a‘ali‘i) species as well as important Maui Parrotbill food plants. Seed availability, germination and growing success will limit the species available for outplanting. Initially, the nine species that are easiest to germinate and grow in the nursery (Table 1, species with a difficulty ranking of 1–3) will be attempted. Seeds of the remaining species will be collected opportunistically and provided to Native Nursery, LCC (1267 Na‘alae Road, Kula, HI 96790; the nursery with which the state has a standing contract) so that propagation protocols can continue to be refined. Additional seeds from all species will be collected, processed, and stored with the Maui 64-AForest Bird Recovery Project (MFBRP) until ready to be used for seed scatter trials. A weather station that collects daily temperature highs and lows and rainfall will be installed on-site. Plot placement All the plots will be placed in open areas (i.e., no canopy), and with a relatively even slope (i.e., not super steep or dead-flat) at approximately 1,500–1,600 m elevation (Figure 2). The plots will be in the large, relatively open grassy area in the central western section of the parcel. Plot locations for the three experimental trials (outplanting, seed scatter, and natural regeneration) will be determined during the July and October seed collection trips. Plots to quantify natural regeneration will be monitored as soon as the area is ungulate-free. Seed scattering within plots will be initiated as soon as sufficient quantities of seeds have been collected and the area is ungulate-free. Treatments will be applied to outplanting plots based on the availability of seedlings. All plots will be permanently marked and GPS coordinates collected. Outplanting To develop outplanting protocols that maximize survival and minimize costs, twenty-seven 10 x 15 m plots will be established (Table 2). Three replicates of three treatments will be deployed for nine species (see below): 1) No treatment, living grass (i.e., Control; Treatment P1), 2) grass killed with herbicide (i.e., Treatment P2), and 3) grass killed with herbicide cleared with a weed-eater or mattock (i.e., Treatment P3). The species mix outplanted may change based on seedling availability. Details of treatments Treatment 2 (herbicide) will use a mixture of two non-restricted use pesticides, 0.8% Honcho Plus (glyphosphate; EPA Reg. No. 524-454) and 0.15% Polaris AC (imazapyr; EPA Reg. No. 228-570). These will be combined with and 0.5% Can-Hance surfactant, and applied 80–110 days prior to outplanting at a rate of about 757.1 liters/ha. These herbicides have been used effectively at Ulupalakua Ranch, Maui (J. Leary pers. comm.) and Pu‘u Mali, Hawai‘i Island (R. Stephens, pers. comm.) to prepare sites dominated by exotic grasses for restoration. For Treatment 3, an approximately 0.5 m2 area will be cleared with a weed-eater for each seedling. 65-APlanting of seedlings Seedlings will be planted using a planting stick (‘ō‘ō) or pick. Staff from Leeward Haleakala Watershed Restoration Partnership will provide instructions on proper planting techniques prior to the first planting trip. One hundred and fifty seedlings will be planted, at 1 m spacing, in each plot. Kōlea, māmane, and mamaki (species group 1) will be planted at nine plots; ‘a‘ali‘i, ākala, and ‘ōhi‘a (species group 2) will be planted at nine plots; and koa, ‘ōhi‘a, and pilo (species group 3) will be planted at nine plots. The remaining species (alani, kanawao, kāwa‘ū, ‘ōhelo, and pūkiawe) were not selected due to germination difficulty and low seed availability (Table 1). ‘Ōlapa seeds were collected and planned for planting group 3. However, poor germination meant that the species had to be replaced by ‘ōhi‘a in this planting group. Poor germination also meant that fewer ākala will be ready for planting; 38 individuals / plot instead of 50 / plot. As a result additional ‘a‘ali‘i and ‘ōhi‘a will be planted per plot in group 2, 56 / species/ plot. In planting group 3, 28 ‘ōhi‘a / plot will be planted, taking the place of the planned ‘olapa, and 61 koa and pilo / plot will be planted to make up the difference. All the seedlings cannot be planted at once, so each group will be planted in alternate, staggered weeks. In each treatment 1,350 seedlings will be planted (Table 2). Seedlings will be planted systematically, alternating species for subsequent identification and monitoring. Blue-X tree shelters will be installed on 20 randomly selected seedlings / species / plot for species with ≥ 50 individuals per plot and ½ of plants will receive shelters for species with < 50 individuals per plot; 60 tree shelters per plot in G1, 59 shelters per plot in G2, and 54 shelters per plot in G3. Survival of all seedlings per species per plot will be tracked throughout the experiment. Survival will be assessed at 6, 12, 18, and 24 mo. The following information will be collected and examined for an effect on survival: slope, aspect, presence of shelter, rainfall, and temperature. This information will allow subsequent planting protocols and locations to be fine-tuned to increase the survival of seedlings. 66-A Table 2. Number of seedlings per replicated plot, per treatment, and overall in outplanting trials. Twenty individuals of each species will receive Blue-X tree shelters for all species with ≥ 50 individuals per plot and ½ of plants will receive shelters for species with < 50 individuals per plot; 60 tree shelters per plot in G1, 59 shelters per plot in G2, and 54 shelters per plot in G3. Each plot will be 10 x 15 m. 1 Kl = Kōlea, Sc = Māmane, Pa = Mamaki; Dv = ‘A‘ali‘i, Rh = Ākala, Mp = ‘Ōhi‘a; Ak = Koa, Cs = Pilo. 67-A Outplanting Corridors Uneven germination between species has meant that fewer than 50 individuals / species / plot will be available for planting, as discussed above. However, for other species far more individuals germinated than expected resulting in more individuals ready for planting than are necessary for the experimental plots. Due to the value of these seedlings that were sourced from within the study site, one or more “planting corridors” will be established running along contours, effectively connecting drainages. These corridors will serve as repositories for any “extra” seedlings not required for the restoration trial experiment. Priority of “extra” seedlings will be given to tree canopy plots and then the planting corridors. Plants will be planted at 3 m intervals throughout the corridor in a systematic manner among species. Planting locations will be prepared with herbicide in the same fashion as the herbicide treatment outplanting plots. Survival and height will be recorded for all individuals planted in corridors. Seed Scatter To evaluate the efficacy of seed scatter, sixteen 5 x 10 m plots will be established. Four replicates of four treatments will be deployed: 1) No treatment; living grass (i.e., Control; Treatment S1), 2) grass killed with herbicide (i.e., Treatment S2), 3) grass killed with herbicide and removed with a rake (i.e., Treatment S3), 4) plot scarified with a mattocks, and no herbicide applied (i.e., Treatment S4). The more intensive treatments (S3, S4) are not feasible to conduct over the entire 614 ha NAR, but the results will indicate the maximum restoration potential at the site. The results will also indicate the practicality and utility of creating small, restored “habitat islands” across the landscape. These restored “islands” could serve as seed sources, and also potentially make the microclimate more hospitable for seedling establishment and growth. These changes could serve to jump start the restoration across the NAR, and increase the effectiveness of the less labor-intensive techniques (e.g., S2). A mix of all available native species’ seeds, but most likely the 14 species in Table 1, will be hand broadcast over the area and gently raked as appropriate. The amount of seed per species broadcasted will be carefully documented. The exact mix and amount of seeds will depend on the amount of seed collected, but the minimum threshold will be 320 seeds per species (20 seeds per plot, 16 scatter plots). The seed plots will be searched every 3 mo. for seedlings, and an appropriate number of seedlings will be marked and tracked to determine germination and survival. We will request that Native Nursery provides photographs of 68-Aseedlings of all species or staff will visit the nursery to become familiar with the species. See below for protocols for treating seeds prior to scatter. Seed Collection and Growing Seedlings Seeds will be collected on site, or as near to the site as practical. As many seeds as possible will be collected following the protocols outlined below. All seeds will be germinated and grown by Native Nursery. Seeds for scatter will be processed and stored with MFBRP. Natural Regeneration To evaluate the presence of a seed bank and the rate of natural recruitment, twenty-four 10 x 10 m plots will be established. These plots also will provide data on the number of seedlings expected to germinate if no seeds were scattered). Six replicates (Table 3) of four treatments will be monitored: 1) No treatment; living grass (i.e., Control; Treatment R1), 2) grass killed with herbicide (i.e., Treatment R2), 3) grass killed with herbicide and removed with a rake (i.e., Treatment R3), 4) plot scarified with a mattocks and no herbicide applied (i.e., Treatment R4). Similar to the seed scatter trials, the more labor intensive treatments (R3, R4) will indicate the maximum restoration potential of these techniques, and their potential utility in creating smaller, restored “islands” across the greater landscape. To examine distance effects from mature koa trees, the edge of plots will be placed 5 and 25 m from the edge of the crown of living koa trees. Three plots per distance per treatment will be deployed. Plots will be searched semi-annually for recruitment and an appropriate number of seedlings will be flagged to track survival and to determine factors associated with germination (i.e., distance from mother tree, seed bed [mineral or organic]). Table 3. Number of natural regeneration plots for each treatment (R1–4). 5 m1 25 m Treatment R1. control 3 3 Treatment R2. herbicide 3 3 Treatment R3. herbicide + cleared 3 3 Treatment R4. scarified 3 3 1 Distance from the edge of the crown of the largest living koa tree in the area. Tree canopy 69-A To evaluate the effect of growing under the canopy of mature trees, eighteen mature, living koa trees will be selected as “tree canopy plots”. Plot borders will be defined by the drip-line of selected trees. Trees will be non-randomly selected to reduce variation in plot area but an effort will be made to select trees throughout the restoration trial area at the same elevation as the other treatment plots. These plots will provide data on the potential benefits of the comparatively more mesic microhabitat that exists below the drip-line of mature trees. Four replicates of four treatments will be applied: 1) No treatments; living grass (i.e. Control; Treatment T1), 2) grass killed with herbicide (i.e. Treatment T2), 3) grass killed with herbicide and removed with a weed-eater and/or rakes (i.e. Treatment T3), 4) aboveground grass removed with a weed-eater (i.e. Treatment T4). Two additional plots will be sprayed with fertilizer (i.e. Fertilizer, Treatment T5) to potentially promote growth and stimulate the natural seed bank. Plots in Treatments T1-T4 will be divided in half creating two equal sections with respect to canopy cover and slope. Half of each plot will receive outplantings (in a similar manner to the outplanting plots) and the other half will not (similar to natural regeneration plots). Dividing plots in half will help account for variation in plot size and slope. All outplantings will be flagged. The fertilizer plots will not receive outplantings. The following information will be collected and examined for an effect on survival: slope, aspect, diameter at breast height of plot tree, height of plot tree, plot area, rainfall, and temperature. Timeline Seed collecting trips Based on documented phenology (Lamoureux 1973, Medeiros 1998, Berlin et al. 2000) seed collecting trips to Nakula are scheduled for July 2012, October 2012, and January 2013. However, additional trips will be scheduled based on information from the field regarding seed availability. Outplanting The timing of outplanting will be dependent on having a sufficient number of seedlings, but are tentatively scheduled to start in September 2013. Planting should proceed as soon as possible, although the driest months (June–August) should be avoided. Seed Scatter 70-ASeed scatter should be conducted several times during the winter rainy season, but will be dependent on having sufficient seeds. Natural Regeneration Monitoring the natural regeneration within the plots will begin as soon as the plots are established and the majority of the ungulates are removed, e.g., fall 2012. Seed collection and treatment protocols Alvin Yoshinga (2001, 2007, 2010) has written extensively about seed collection, preparation and storage for native Hawaiian plants. We will be following his recommended protocols for most species. REFERENCES Baker, P. J., P. G. Scowcroft, and J. J Ewel. 2009. Koa (Acacia koa) ecology and silviculture. Gen. Tech. Rep. PSW-GTR-211. Albany, CA: U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station. 129 p. Baskin, J. M., B. H. Davis, C. C. Baskin, S. M. Gleason and S. Cordell. 2004. 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L. 1990. Introduction to xerophytic native Hawaiian plants. The Bulletin of the National Tropical Botanical Garden 20:49-54. Elevitch, C .R., K. M. Wilkinson, and J. B. Friday. 2006. Acacia koa (koa) and Acacia koaia (koai‘a), ver. 3. In: C.R. Elevitch (ed.). Species Profiles for Pacific Island Agroforestry. Permanent Agriculture Resources (PAR), Hōlualoa, Hawai‘i (http://www.traditionaltree.org). 71-AFriday, J. B., and D. A. Herbert. 2006. Metrosideros polymorpha (‘ōhi‘a), ver. 3.2. In: Elevitch, C.R. (ed.). Species Profiles for Pacific Island Agroforestry. Permanent Agriculture Resources (PAR), Hōlualoa, Hawai‘i (http://www.traditionaltree. org). Gagne, W. C., and L. W. Cuddihy. 1999. Vegetation. In: The Manual of the Flowering Plants of Hawai‘i, W.L. Wagner, D.R. Herbst, and S.H. Sohmer (eds.), University of Hawai‘i Press and Bishop Museum Press, Honolulu. Hosmer, R. S. 1912. Creation of the Kula Forest Reserve. The Hawaiian Forester and Agriculturalist 9:274-281. 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Flowering plant and gymnosperms of Haleakala National Park. Cooperative National Park Resources Studies Unit, University of Hawaii at Manoa, Department of Botany. PCSU Technical Report, 120. Mawdsley, J. R, R. O’Malley, and D. S. Ojima. 2009. A review of climate-change adaptation strategies for wildlife management and biodiversity conservation. Conservation Biology 23:1080-1089. Minyard W. P., T. W. Giambelluca, and D. Nullet. 1994. Elevational patterns of climate on the leeward slope of East Maui, Hawaii. Honolulu (HI): Cooperative National Park Resources Studies Unit, University of Hawaii at Manoa, Department of Botany. PCSU Technical Report 92. National Tropical Botanical Garden (NTBG). 1996. Ten native Hawaiian trees for urban landscapes. Lawai, Hawaii: Education and Plant Science Departments. National Tropical Botanical Garden. Obata, J. K. 1967. Seed germination in native Hawaiian plants. Newsletter of the Hawaiian Botanical Society 6:13-20. Schwartz, M. W., L. R. Iverson, A. M. Prasad, S. N. Matthews, and R. J. O’Connor. 2006. Predicting extinctions as a result of climate change. Ecology 87:1611-1615. Scowcroft, P. G. 1992. Role of decaying logs and other organic seedbeds in natural regeneration of Hawaiian forest species on abandoned montane pasture. In: C.E. Conrad, and L. Newell. (Tech. Coord.), Proceedings of the Session on Tropical Forestry for People of the Pacific, XVII Pacific 72-AScience Congress. General Technical Report PSW-129 Pacific Southwest Research Station, USDA Forest Service, Albany, CA, pp. 67-73. U.S. Fish and Wildlife Service. 2006. Revised recovery plan for Hawaiian forest birds. U.S. Fish and Wildlife Service, Portland. Yoshinaga, A. 1998a. Growing native. Newsletter of the Hawaiian Botanical Society 37:6-7. Yoshinaga, A. 1998b. Storing seeds of some native rain forest plants: some simple methods. Newsletter of the Hawaiian Botanical Society 37:28-32. Yoshinaga, A. 2001. Seed storage practices for native Hawaiian plants. Accessed 10 July 2012. http://hawaiiconservation.org/resources/publications/general_conservation_resources Yoshinaga, A. 2007. Seed conservation notes: research on seed germination and seed storage 2002 to 2007. Accessed 10 July 2012. http://flemingarboretum.org/2011/preservation-efforts/seed-conservation-notes-2/ Yoshinaga, A. 2010. Guidelines for successful seed storage. Accessed 10 July 2012. http://www.ctahr.hawaii.edu/ougc/downloads/Guidelines_for_Storage.pdf 73-AAppendix H. Supplemental Feeding Trials for Wild Maui Parrotbills 2012 and 2013 Experiments with Developing and Using Supplemental Feeders for Kiwikiu (Maui Parrotbill; Psuedonestor xanthophrys): Potentials for translocation efforts and increasing productivity This report was generated for internal use and to share with partners at Maui Forest Bird Recovery Project by Hanna L. Mounce and Laura K. Berthold Introduction The Kiwikiu is a critically endangered insectivorous Hawaiian honeycreeper, with a population of ~500 individuals found only on the windward side of east Maui (Simon et al. 1997). Lack of habitat due to invasive species destruction and the presence of avian malaria and non-native predators are some of the reasons why Kiwikiu are endangered. Additionally, recent research has indicated that productivity may be inadequate to increase the population, and available habitat may decrease with climate change. Two management strategies that could be critical towards recovery are: 1) Determining a method of population management that will increase reproductive output 2) Expanding available habitat through restoration and invasive species management in addition to creating a second population through reintroductions on the leeward side of east Maui Providing supplemental food to birds has been found to increase reproductive output by boosting clutch size, number of breeding attempts, nestling weight, and the number of independent young produced, e.g. Song Sparrows (Melospiza melodi) (Arcese and Smith 1988) and Florida Scrub Jay (Aphelocoma coerulescens) (Schoech et al. 2007). Supplemental food has also been used when reintroducing a population, such as the endangered Hihi (Notiomystis cincta) in New Zealand (Castro et al. 2003). 74-ADuring the 2012 and 2013 breeding seasons, we experimented with providing supplemental food to closely monitored Kiwikiu breeding pairs. If Kiwikiu use supplemental feeding stations this could increase productivity of the existing population and assist with the re-establishment of a second population. Materials and Methods Kiwikiu pairs were located and monitored February through June in 158 ha of The Nature Conservancy’s Waikamoi Preserve, between 1600 and 1900 m in elevation (Fig. H.1). Feeding stations were set up in April of 2012 and February of 2013 based on pair activities. We provided commercially raised mealworms on a feeder tray situated 1 m above ground in 2012 and 2.5 m above the ground in 2013 (Fig. H.2). The feeding apparatus was designed to be rat-proof and to slowly dispense mealworms over time. Stations were monitored with remote trail cameras (Reconyx PC800 HyperFire Professional Semi-Covert IR) and visited every few days for maintenance. Since Maui Alauahio (Paroreomyza montana, MAAL) and Kiwikiu (MAPA) forage together, we attempted to lure both species to the stations with playback and bird decoys. We also camouflaged half of the feeders with native vegetation. Results 2012 Six feeder stations were installed in 2012 (Figure H.1, Table H.1). Five were located in an area where pairs with a hatch-year (HY) regularly foraged and one where a pair was nest building. Cameras were set up at four of six feeders. The only species detected using the feeders were Red-billed Leiothrix (Leiothrix lutea, RBLE). Rats were also captured on camera but were never successful at getting onto the feeder trays. When playback was used, Alauahio would chip (contact call) above the feeder but would not visit it. 75-A2013 Six feeder stations were installed in 2013 (Figure H.2). Each were set out as a pair of feeders in areas of high MAPA activity. Cameras were set up at all feeders. The only species detected using the feeders were Red-billed Leiothrix. Figure H.1. Locations of feeders and pairs targeted in 2012. 76-A Figure H.2. Locations of feeders and pairs targeted in 2013. 77-AFigure H.3. Maui Parrotbill supplemental feeder design. Figure H.4. Maui Parrotbill supplemental feeders installed in the field. 78-A Discussion Wild Kiwikiu have never visited the supplemental feeding stations; however, it can take time for target birds to find and use supplemented arthropod food (Podolsky et al. 2004). Even though we attempted to place the feeders where Kiwikiu were foraging, pairs typically forage throughout their homes ranges, which are fairly large (average ~5 ha/pair in the core of their population) (MFBRP unpublished data). It is possible that because of this, they did not find the feeders in the time that they were available. In order to increase the chances of Kiwikiu finding these feeders, in 2013 we positioned the feeders in a more clustered arrangement, but it did not change the results we observed. After the 2012 season, we also thought that more experimentation could be done with making the feeders look more natural, such as making the feeder appearance mimic a preferred plant. This was heavily focused on in 2013 again with no difference in the observed results. Red-billed Leiothrix forage low in the understory, have higher densities, and have smaller home ranges (3 ha) (Male et al. 1998), which may have pre-disposed them to discovering the feeders before native species. Leiothrix pose several problems in that they quickly remove all the mealworms, may chase off other birds from the feeder, and may transmit avian disease and/or parasites via the feeder. To dissuade leiothrix from using the feeders after 2012 we increased the height of the station above the common feeding height of leiothrix but still in the foraging range of Kiwikiu but this did not discourage the leothrix use of the feeders. Another alternative might be to try the feeders outside the core of the leiothrix breeding season (April-August) (Male et al. 1998), but this would also be attempting to supplement Kiwikiu outside of their breeding season as well. We would also like to work with captive Kiwikiu and perhaps released captive birds could “teach” wild birds to use the stations. When this feeder design was installed in an aviary with captive Kiwikiu, they used the feeder immediately. This would be advantageous in designing the reintroduction protocols for Kiwikiu to leeward Haleakala. 79-A References Arcese, P. and J.N.M. Smith. 1988. Effects of population density and supplemental food on reproduction in Song Sparrows. Journal of Animal Ecology 57:119-136. Castro, I., D.H. Brunton, K. M. Mason, B. Ebert, R. Griffiths. 2003. Life history traits and food supplementation affect productivity in a translocated population of the endangered Hihi (Stitchbirds, Notiomystis cincta). Biological Conservation 114:271-280. Male, T.D., S.G. Fancy, C.J. Ralph. 1998. Red-billed Leiothrix (Leiothrix lutea). The Birds of North America Online (A. Poole, Ed.). Ithaca: Cornell Lab of Ornithology. Podolsky, A.L., T.R. Simons, J.A. Collazo. 2004. A method of food supplementation for ground foraging insectivorous songbirds. Journal of Field Ornithology 75:296-302. Schoech, S.J., E.S. Bridge, R.K. Boughton, S.J. Reynolds, J.W. Atwell, R. Bowman. 2007. Food supplementation: a tool to increase reproductive output? A case study in the threatened Florida Scrub-Jay. Biological Conservation 141: 162-173. Simon, J.C., P.E. Baker, and H.Baker. 1997. Maui Parrotbill (Psedonestor xanthophrys). The Birds of North America Online (A. Poole, Ed.). Ithaca: Cornell Lab of Ornithology. 80-AAppendix I. Progress in Nakula Experimental Restoration Efforts 2013-2014 Results of Experimental Restoration Efforts in Nakula NAR This report was created by Hanna Mounce and Chris Warren for distribution to Maui Forest Bird Recvoery Project Partners and financial sponsors (American Bird Conservancy and State of Hawaii Division of Forestry and Wildlife) Outplanting Monitoring – May 2014 Monitoring protocols were designed to assess the density, diversity and survival of woody plant species ≥ 15 cm in height. The presence of key non-native species is also being recorded to assess the threat of invasive species. Initial six-month survival rates were very high with 97% of planted individuals surviving 6 months post-planting. All species had survival rates above 97% with the exception of mamaki (92%) and akala (87%). See Figure 1 for survival rates per species. Blue-X tree shelters were retained on 87% of plants where they were installed; the remainder had blown off. However, a fair number of retained shelters malfunctioned wherein they blew down but stayed on the plant. We recorded the number of malfunctioning shelters that likely had killed or were likely to kill the plant and these shelters were removed. These accounted for 11% of all shelters installed. Approximately 25-50% of remaining shelters needed adjusting (e.g. adding additional stake) per plot. Additional statistics per plot type have not yet been analyzed. Figure 1. Six month out-planting plot survival statistics 81-ANatural Regeneration Monitoring - July 2014 As of July 2014 the MFBRP restoration experiment entered the 24-month mark since plots were assigned and the 12-month mark for monitoring the first plots in which treatments were applied (natural regeneration treatment plots). In July, we monitored 40 restoration plots including all plots of the natural regeneration and seed scatter plots. This represented the 6-month monitoring (first round) for seed scatter plots and 12-month monitoring (second round) for natural regeneration plots. Within these two plot types, four treatments were applied; control, herbicide, herbicide and weed-whack and ground scarification. At the 12-month mark since treatment application in the natural regeneration plots we are able to make some preliminary conclusions about the outcomes of the four treatments applied to these plots; control, herbicide-only, herbicide + weed-whack, and ground scarification. It is important to note that long-term success of the different treatments may change as the plots mature, particularly in regards to grass re-growth. Figure 2. Ground cover of natural regeneration plots. This figure shows ground cover of natural regeneration plots at the 6- and 12-month marks. We recorded cover as the percentage (out of 100%) each of five categories covered each entire plot (10 m × 10 m). (Notes: 1) Rock/stick and tree/shrub make up a very small proportion of the ground cover at even the 12-month mark. Tree/shrub will likely remain somewhat low because each tree/shrub generally does not cover a large area and therefore there would need to be a very large number of mature trees/shrubs to add up to 82-Amuch cover. 2) Grass cover recovered similarly in the herbicide and herbicide + weed-whack treatments, rebounding to 50% cover by 12-months. All plots likely had ~90% grass cover prior to treatment application. 3) Forbs (mainly non-native annuals) generally do not amount to a large proportion of the ground cover but appear to be increasing in the herbicide-only treatment. This includes anecdotal evidence that some weedy species, e.g. Cirsium vulgare, are increasing in the herbicide-only treatment and single plants can account for a fair percentage of ground cover. 4) The ground scarification method used (described below) was insufficient in suppressing the overall dominance of grass within experimental plots. By 6-months, the first monitoring time period, these plots showed little to no difference compared to the control plots in overall ground cover patterns. By 12-months the exposed topsoil sections were still visible but were quickly being overtopped by grass, shading most of these sections if not overgrowing them entirely. Overall, the herbicide in combination with weed-whacking treatment within the natural regeneration plots has produced the largest number of seedlings by far. Figure 3. Mean number of individual plants per treatment. This figure compares the number of individual wood plants (>=15 cm in height) present within the natural regeneration treatment experimental plots at the 6- and 12-month marks. Note that the y-axis scales differently at 40. [Notes: 1) Herbicide-only and ground scarification (without herbicide) did not result in a higher number of individual plants than the control treatment. 2) Herbicide + Weed-whack treatment plots had significantly more individual plants present at the 6-month mark and vastly more at the 12-month mark. 3) The vast majority (89%) of recorded individual plants measured were a`ali`i (Dodonea viscosa). Most of the remaining plants recorded besides a`ali`i were koa with a very small minority being pukiawe and thimbleberry. 4) The large standard error indicated in the 12-month monitoring of the ground scarification treatment is largely due to a single plot. This plot, 83-AR06, was the first to receive the scarification treatment. Originally, the plan had been to use a rototiller to remove the grass mat but this plan was scrapped after it became clear that using a rototiller out there was not going to be possible. The alternative strategy was to physically remove the grass from the plots by hand with pulaskis and rakes. This was done on R06 but this method took about 8 hrs to complete this one plot. Because of this we moved to the strategy used on all other scarification plots in the natural regeneration and seed scatter plots wherein we manually removed grass mats in 1-m2 sections throughout the plot with a pulaski, exposing the topsoil within a total of ~25% of the plot. The number of seedlings in R06 is much higher than all other scarification plots. This may suggest that the physical removal of grass, exposing the topsoil provides the biggest benefit to germinating native plants. The results from the herbicide + weed-whack treatment may indicate that the addition of herbicide may help suppress grass re-growth after the physical removal of the grass biomass. This may explain the large difference between the herbicide-only and herbicide + weed-whack treatments.] By 12-months the average number of seedlings in this treatment was ~ 20 × the number of seedlings in the control and herbicide-only treatments. Average height of these seedlings in the herbicide + weed-whack plots is shorter than those in the other treatments. Figure 4. The mean height of plants per species per treatment. This figure shows the height of recorded woody plants (>=15cm) within natural regeneration treatment experimental plots at the 12-month mark. Error bars represent ± SE. No error bars are present on pukiawe averages as these represent single individuals and no variance could be calculated. [Notes: 1) The comparatively shorter average in the herbicide + weed-whack treatment is likely the result of three main factors: i. Plants in the control, herbicide-only, and ground scarification plots may be older than 12-months as these plants were not disturbed by treatment application. We observed very little herbicide-related death of 84-Awoody plants in the herbicide-only plots. ii. All individuals recorded in the herbicide + weed-whack treatment present at the time of the treatment application were cut down during weed-whacking. Thus, all recorded individuals represented in the above figure are not older than 12-months. iii. We recorded a much higher percentage of small seedlings (15-18 cm) in the herbicide + weed-whack treatment which likely drew the average down. In total 16% of all a`ali`i recorded were 15-18 cm in size and all but one individual were recorded in the herbicide + weed-whack treatment (20% of all a`ali`i in the H + WW treatment). This is reflected in part by the average number of individuals in the previous figure. 2) Many (most?) koa seedlings appear to be root-shoots, thereby benefiting from the resources of a mother tree. The heights attained from root-shoot individuals undoubtedly are greater than those germinating from seed. Thus, the heights in all treatments represented here are likely taller than seeded individuals. 3) We did not record average grass height during monitoring. In retrospect this may have been smart as we could then speak more directly to what heights need to be achieved in a native seedling to not be suppressed by future grass growth. Comparative ground cover (shown in following figure) shows that grass suppression in terms of soil coverage is approximately equal for the herbicide-only and the herbicide + weed-whack treatments. However, anecdotally the grass height appears to be shorter in the herbicide + weed-whack treatment. In most places grass height in this treatment is < 15 cm. Thus, an average height of >20 cm shown in all species has allowed the seedlings at 12-months to achieve a height greater than the grass. This may change as exponential grass growth continues.] This is likely the result of the fact that all plants were cut down during the weed-whack application while plants present in the other plots were largely unaffected by treatment application. Additionally, 20% of seedlings recorded in the herbicide + weed-whack treatment were between 15-18 cm in height. This size class was largely absent in the other treatments. Therefore the average height of seedlings in the herbicide + weed-whack plots was drawn down by the presence of many, small seedlings. Across all plots we have only recorded four woody species to date; a`ali`i (Dodonea viscosa, 89% of all seedlings recorded), koa (Acacia koa, 20%), pukiawe (Styphelia tameiameiae, <1%), and thimbleberry (Rubus rosifoilus,1 individual). The 85-Alack of diversity by 12-months in the natural regeneration plots is a bit concerning and may indicate a depauperate seed bank within much of Nakula. However, in many areas outside the plots we have noted the presence of seedling growth of pilo (Comprosma foliosa), ohelo (Vaccinium reticulatum) and others largely in gulches (pilo) and eroded areas (ohelo) free from grass. This may indicate that the poor seed bank may be restricted to the most heavily grazed areas now dominated by grasses, also where our experimental plots are located. The relative abundance of naturally regenerating a`ali`i seedlings even in the control plots may indicate that significant outplanting of this species is not the most pressing restoration need in Nakula. It seems likely that this of all species has the ability to recover on its own to a large degree. We should focus our outplanting efforts and resources toward adding more diversity to the area. Despite the observed regeneration of koas in Nakula, outplanting this species may still be needed as many (most?) seedlings recorded in our plots appear to be root-shoots (Figure 5). Outplanting genetically distinct individuals will greatly increase the genetic diversity in the area and make the species more robust and disease/pest resistant. We do not know the number of koa genets in Nakula at present and the number of root-shoots and low seed sets that we have observed in the last two years may indicate that this number may be low. This highlights the need for outplantings of individuals sourced from the largest number of genets possible. We have yet to observe any natural regeneration in the form of seedlings, in and outside experimental plots, of many important native tree and shrub species that are common within Nakula. This includes ohia (Metrosideros polymorpha), kolea (Myrsine lessertiana), and iliahi (Santalum haleakalae, much rarer than others). Many mature ohias and some koas are showing signs of lateral growth from the trunks. This may increase the “bushiness” of these species and add shade to the understory. Extremely limited regeneration of kawau (Ilex anomala) and olapa (Cheirodendron trigynum) has been observed and is highly localized in gulches. Therefore it is very 86-Aimportant to the overall diversity and health of the forest that these species continue to be outplanted. Additionally, we monitored 1,291 seedlings planted between 10/08/14 and 01/25/14 for survival, representing 7-9 month survival data (Figure 5). These plants form a corridor connecting existing vegetation and are additional to the restoration plots. Overall survival was high at 87% for all 10 species combined. All spp. showed 7-9-mo survival ~ 90% except mamaki (43.9%) and kawau (81.8%). Figure 5. Seven-nine month survival of corridor plantings.87-AAppendix J: Home range Patterns of Maui Parrotbill and Maui `Alauahio: Implications for Proposed Translocations Efforts Christopher C. Warren, Peter J. Motyka, and Hanna L. Mounce Manuscript under review with Journal of Wildife Management, March 2015. ABSTRACT Once occupying a variety of habitats on the islands of Maui and Moloka`i, the critically endangered Maui Parrotbill (Kiwikiu; Pseudonestor xanthophrys) is now restricted to native, wet forest on the windward slopes of east Maui above 1200 m in elevation. Parrotbills are restricted to this fraction of their former range mainly through habitat loss and disease. Continued range contraction is expected. In order to prevent extinction, reintroducing parrotbills to historically occupied native, mesic forest on the leeward slopes of Haleakalā is considered a critical recovery action. Managers have selected the newly established Nakula Natural Area Reserve (NAR) as the site for reintroduction, and restoration efforts are currently underway to support this goal. It is also expected that other extirpated species, including the endemic Maui `Alauahio (Paroreomyza montana), may recolonize these forests naturally as the habitat improves.We estimated home range size (area) of parrotbills and `alauahios at three sites within the birds’ current ranges to provide a measure of area required by individuals of these species. We then used these estimates to calculate the potential abundance of both species in five planned restoration areas where future populations may occur on leeward Haleakalā. We calculated home ranges using minimum convex polygons and kernel density estimators from resighting data of color-banded birds from 2007-2014. Parrotbill home ranges were estimated to be between 8.48 ± 1.18 ha and 8.76 ± 1.05 ha (± SE) depending on estimation technique and `alauahio home ranges were between 0.8 ± 0.08 ha and 0.95 ± 0.08 ha. These estimates are the first to be derived from such a large dataset and date range for these species. The relative homogeneity of home range sizes among study sites may support the use of this metric, estimated in the species’ current ranges, to predict potential abundance on leeward Haleakalā. Though we do not know how these species 88-Awill behave in the new habitat, the estimates of home range size presented here provide guidance in planning the reintroduction of parrotbills to Nakula NAR. KEY WORDS Home range, Hawaii, Kernel density estimators, Maui `alauahio, Maui Parrotbill, Minimum convex polygon, Paroreomyza montana, Potential Abundance, Pseudonestor xanthophrys, Translocation INTRODUCTION As in the entire Hawaiian archipelago, the native avifauna of the island of Maui has suffered widespread extinctions and range contractions (Warner 1968, Scott et al. 1986, Pratt et al. 2009). Of the more than 20 species of forest passerine known to have existed prior to the arrival of humans (James and Olson 1991) only six species remain. Of these six, three are endemic to Maui and two are federally listed as endangered. The global wild populations of the endemic species, Maui Parrotbill (Kiwikiu; henceforth parrotbill; Pseudonestor xanthophrys), Maui `Alauahio (henceforth `alauahio; Paroreomyza montana), and `Ākohekohe (Palmeria dolei), are restricted to a single strip of native forest (the largest tract remaining) on Haleakalā Volcano in east Maui above 1200 m in elevation (85 km2) with the exception of a small, relictual population of `alauahio in Kula Forest Reserve (FR; henceforth Kula) (Figure J.1). Continued range contraction is expected for these species driven by habitat destruction and disease (Benning et al. 2002, U.S. Fish and Wildlife Service 2006). Establishing a second population of parrotbills, the most critically endangered of the three, to once occupied habitat on leeward Haleakalā is considered the highest priority for the long-term persistence and viability of the species (U.S. Fish and Wildlife Service 2006). A leeward population of parrotbills will provide protection from loss due to stochastic events in the single extant population, increased genetic diversity for the species as a whole, and the drier leeward habitat may provide some refuge from disease-carrying mosquitos. 89-APlanning a reintroduction of this kind requires a good understanding of the expected ecology of the organism within the release site (Griffith et al. 1989, Seddon et al. 2007, International Union for Conservation of Nature 2013). An estimate of home range size (Burt 1943), the area that an individual requires for survival and reproduction, allows conservation managers to estimate the number of individuals that may be supported within a reserve (i.e. carrying capacity) and may provide a benchmark for success of translocation efforts. To estimate home range in birds requires observations of individuals across an extended time period. Spot mapping (or territory mapping) was designed to investigate local densities of a species by marking known locations of individuals from repeated visits to a site (Verner 1985, Bibby et al. 1992). Spot mapping is a robust method for estimating home range that produces fine-scale, spatially explicit estimates of space use and density within localized areas (Verner 1985). Even if an individual temporarily emigrates from its home range, the repeated surveys capture the core area(s) and thus presumably the most important area(s) to that individual. Using marked individuals for these methods, such as through color-banding, allows an observer to confidently track individuals across extended time periods providing estimates of both among and within individual variation in home range size. In 2011, the State of Hawaii Division of Land and Natural Resources established Nakula Natural Area Reserve (NAR; henceforth Nakula) on leeward Haleakalā in part for the protection of the parrotbill and has been designated as the reintroduction site for the species (Division of Forestry and Wildlife 2010; Figure 1). The habitat is classified as mesic forest with a canopy dominated by koa (Acacia koa), `a`ali`i (Dodonea viscosa) and `ōhi`a (Metrosideros polymorpha). Although koa is not present in much of the current parrotbill range, some of the earliest observations of this species noted a strong affinity for koa (Henshaw 1902, Perkins 1903) suggesting that mesic, koa-dominated forests may have been a preferred habitat for the species. Subfossils also show the species to have been historically present within the Kahikinui region (leeward Haleakalā west of the Kaupō Gap) (James and Olson 1991). Unfortunately, the remaining native mesic forest has been greatly denuded as a result of heavy 90-Agrazing by ungulates. Restoration efforts are currently in place within a 170-ha fenced, ungulate-free area of Nakula in preparation for the reintroduction. Complete restoration of the area is expected to take decades but portions of the area are considered currently suitable for a small population of parrotbills until more of the forest regenerates and/or are restored. However, the number of individual parrotbills that can be supported within this area is unknown. Although the habitat composition and structure within Nakula will likely remain different in many aspects from the habitat that the species currently occupies, estimates of space use from the current range provide a baseline estimate of the amount of area required per individual and therefore the number of individuals this area may support. This method of predicting space use for conservation efforts has been previously conducted for multiple species including (red squirrels; Rodriguez and Andren 1999) (Eurasian lynx; Schadt et al. 2002). The total population of parrotbills has been estimated at 500-600 individuals (502 ± 116 [Scott et al. 1986], 590 ± 208 [Camp et al. 2009]) and occurs at low density compared to sympatrics throughout its range (Scott et al. 1986, Camp et al. 2009, Brinck et al. 2011). Little is known about how much area is required for an individual parrotbill to survive and reproduce (Simon et al. 1997). Using the best data available at the time, Pratt et al. (2001) estimated home range size (area) of the parrotbill to be 2.26 ha based on a limited sample size of individuals (n = 7) in one study site at the core of the species’ range (Hanawi NAR; also included in the present study). Herein we utilized similar methods to include seven years of study and a second study site at the western edge of the species’ range. Although the entire range of parrotbills is approximately 50 km2 on the windward slopes of east Maui (Simon et al. 1997), Mounce et al. (2015) found evidence suggesting that the Ko`olau Gap, a large erosional depression in the center of the species’ range, acts as an east-west dispersal barrier shaping the genetic population structure of the species. This genetic variation, combined with variation in habitat and climate across the species’ range, suggests that the biology of the species may also vary spatially. 91-AAdditionally, we estimated home range size for the `alauahio; another endemic insectivore that shares many habitats and foraging substrates with parrotbills. The global population of `alauahi is significantly larger than that of parrotbills (>55,000 [Brinck et al. 2012]) and the population on windward Haleakalā may be increasing (Camp et al. 2009). Home range size of the species is reported to be 1–2 ha on windward Haleakalā (Baker and Baker 2000). `Alauahio were likely extirpated from the Kahikinui region sometime before 1980 and no definitive contemporary records exist for the species in this region. However, subfossil evidence shows the species to have been historically present (James and Olson 1991). Few surveys have been conducted in the leeward region and the status and distribution of the species beyond its current known range remains unknown. However, `alauahios are known to exist in some marginal habitats (e.g. scrubland, non-native forest) and may have the potential to recolonize Kahikinui as the habitat improves (Scott et al 1986). The abundance of `alauahios in the small, disjunct population in Kula is unknown but this represents the closest known population of the species to the restoration areas of Kahikinui (approximately 4 km). STUDY AREA We investigated home range size of parrotbills and `alauahios at two and three study sites, respectively, within Hanawi NAR (henceforth Hanawi; 20°44’N, 156°7’W), The Nature Conservancy’s Waikamoi Preserve (henceforth Waikamoi; 20°46’N, 156°13’W), and Kula (26°42’N, 156°18’W) in east Maui, Hawaii, USA (Figure J.1). Both Hanawi and Waikamoi contained some of the most pristine remaining native forest on Maui; primarily dense, montane rainforests dominated by `ōhi`a and `ōlapa (Cheirodendron trigynum; Jacobi 1989). Kula in contrast was dominated by non-native tree species including various conifers (Families: Pinaceae and Cupressaceae), eucalyptus (Eucalyptus spp.), and tropical ash (Fraxinus uhdei). Hanawi is situated in the eastern portion of both parrotbill and `alauahio ranges while Waikamoi is situated at the western edge of their ranges (Figure J.1). Rainfall was greatest in Hanawi receiving ≥ 92-A10,000 mm on average each year, moderate in Waikamoi at ≥ 2,000 mm per year, and comparatively low in Kula with around 900 mm each year (Giambelluca et al. 2013). The protected lands on leeward Haleakalā that have been identified as areas for forest restoration fall within three land management units, Nakula (20°41’N, 156°13’W), Kahikinui FR (20°41’N, 156°12’W), and State of Hawaii Department of Hawaiian Home Lands (henceforth Hawaiian Home Lands)–Kahikinui unit (20°40’N, 156°15’W) (Figure 1). It is unclear what proportion of these areas is currently suitable to support these species or how long it may take to be restored to high quality native forest bird habitat. The time-scale associated with protecting these areas varies as by size of management unit and the associated costs of fencing and restoring each section. Consequently each section of habitat will become available to native forest bird species at different times. On account of the different timelines associated with each unit, we estimated the number of parrotbills and `alauahios in areas equivalent to the sizes of five management units (Figure J.1, Table J.2). METHODS Observation Data Maui Forest Bird Recovery Project (MFBRP) conducted intensive spot mapping surveys (Verner 1985, Bibby et al. 1992) for parrotbills and `alauahios annually from 2007-2011 in Hanawi, 2012-2014 in Waikamoi and 2013-2014 in Kula. At each site individuals were captured and fitted with a unique combination of colored leg bands. Each year from 1 February to 1 July three to seven observers systematically searched study sites and recorded locations of all color-banded individuals encountered, using handheld Global Positioning System (GPS) units in Universal Transverse Mercator (UTM) coordinates. The difficulty in traversing the terrain, as well as the sensitivity of the forest to disturbance, forced observers to stay largely on established trails in Hanawi and Waikamoi. Extensive trail systems allowed for comprehensive coverage of each study site with minimal damage to the forest. Observers were not as limited to trails within 93-Athe non-native forests of Kula. However, banding efforts and spot mapping were concentrated in areas with the highest densities of `alauahios. Areas covered were 184 ha in Waikamoi, 133 ha in Hanawi, and 220 ha in Kula. Survey effort was similar across all three study sites and averaged 2,504.7 ± 480.5 survey hours per year (survey effort recorded 2010-2014). Home Range Metrics In general, despite the high survey effort, sample size of resight points per individual per year was low for the use of estimating home ranges (parrotbill = 7.23 ± 0.93 resights/bird/yr, `alauahio = 5.38 ± 0.47 resights/bird/yr). The low number of observations per individual is to be expected for rare and low density species. As sample size of observations per individual may influence the size and shape of a home range we chose to use two methods to delineate home ranges; minimum convex polygons (MCP) (Mohr 1947, Hayne 1949) and kernel density estimators (KDE) (Worton 1989) (Figure J.2). Both MCP and KDE use a set of repeated observations to estimate a home range area. These methods predict areas where an animal was likely to occur during the survey period based on proximity to known locations (Bibby et al. 1992). In this way each observation is used as an index of the movement patterns of an individual animal. Outlying points (i.e. resighting points separated from the main cluster) in this case may represent two possibilities: 1) an individual travelling outside its core range or 2) an artifact of uneven survey effort within the localized area (e.g. wide-spread trails). In this example using KDE limits the impact of outlying points on the metrics of a given home range by weighting contours by frequency of occurrence thereby targeting the core area(s) of a home range. An MCP for the same individual would incorporate all points and the space between as part of the home range, thereby accounting for potential missed observations in the interstitial space between the apparent main cluster of observations and an outlier due to uneven survey effort. For 94-Athese reasons we chose to estimate home ranges using both MCP and KDE to obtain two estimates based on these different techniques. We restricted delineation of home ranges to individuals with sufficient data for accurate home range construction; ≥ 10 resights per year (28.7% of resighted parrotbill and 20.8% of resighted `alauahio) (Pratt et al. 2001). Some individuals were resighted more than once within a given day and these were only included if the bird was resighted ≥ 15 minutes after and/or was seen ≥ 50 m between consecutive points. To reduce the influence of single days on the size of home ranges we also restricted our analyses to individuals resighted on a minimum of three days. Only one `alauahio was removed from our analyses based on this parameter. In rare cases (n = 10) a single outlying resight point was excluded from construction of an `alauahio home range. This was done only for single points that were clearly the result of a GPS error or a band misidentification resulting in a point distantly disjunct (e.g. > 1 km) from the main cluster of resight points for an individual (e.g. outside the study site). One additional `alauahio was excluded from all analyses because the loss of an outlier point meant that they no longer met the 10 resight minimum. No parrotbill individuals were excluded from analyses due to the date minimum or outlying points. We estimated MCP and KDE home ranges of both species in Geospatial Modeling Environment version 0.7.2.0 (Beyer 2012) using the ‘genmcp’ and ‘kde’ tools (Appendix J.1). We used smoothed cross validation to estimate bandwidth and a raster cell size of 10 per recommendations of Beyer (2012). We estimated 100% MCP home ranges rather than eliminating a certain proportion of outer points because we were interested in an estimate of the entire area an individual utilized during the study period. We chose to delineate 50%, 75% and 90% KDE isopleths (contour intervals) for each individual. The isopleths contain a percentage of the volume of the contour raster created by the KDE. In effect these represent different levels of confidence in the size of a home range, 50% being the most conservative and 90% being the most liberal. The 90% isopleths ultimately contain the largest areas within an approximated home range that do not contain observation points. 95-APair Home Ranges As parrotbills are known to be socially monogamous (Simon et al. 1997), an estimate of the area used by a mated pair of individuals may be more appropriate for conservation planning. This follows the assumption that a mated pair would occupy a smaller amount of habitat than the sum of two unrelated individuals. Pairing status and identity was recorded for all banded parrotbill within each study site each year. We compared home ranges between paired individuals and estimated a combined home range for each known pair within each year. We compared home range size and overlap between known paired individuals where both individuals were resighted ≥ 10 times within a year. This analysis was not conducted for `alauahios as pairing status was not recorded for this species. To estimate pair home ranges we clipped (‘Clip’ tool in ArcMap 10.0 [Environmental Systems Research Institute, Inc., Redlands, CA]) the MCP and 70% KDE home range polygon of each individual of a pair by their respective mate’s home range polygons. We then added the shared (overlapped), male- and female-only areas of each pair to estimate a collective pair home range. From this result we calculated the area and proportion of overlap among pairs’ home ranges. We then compared the sizes of the pair home ranges to the home ranges of the same paired individuals independent of their mates. Estimating home range size for pairs provided a way to adjust mean home range size for all individuals as if we had been able to measure home range for all mated pairs. Although pairing status is not always established for all individuals within a study site each year, unpaired adult parrotbills were exceptionally rare during these spot mapping surveys (MFBRP unpublished data). All individuals for which a home range was estimated in this study were known to be paired with either a banded or unbanded bird, justification for adjusting individual home range size to pair home ranges for use in predicting the number of parrotbills that may inhabit a given area. Estimating Potential Abundance 96-AWe calculated an adjusted mean home range size for all parrotbill home ranges as though they were all paired by multiplying the mean home range size of all individuals (Hi) by the quotient of mean pair home range size (Hp) and mean individual home range size of the known paired individuals (Hm; i.e the individuals used to estimate Hp). Home range size could not be adjusted to pair home range size for `alauahios because pair identity was not recorded for this species. However, `alauahios are highly gregarious and live in small family groups usually consisting of a male, female and two to four sub-adults (often helpers at the nest) (Baker and Baker 2000). This may mean that every `alauahio home range may actually represents approximately three individuals, so we calculated the number of individuals that could potentially occupy an area as though each home range was equivalent to three individuals, a “family home range”. We estimated the potential abundance of both species across a range of habitat sizes equivalent to the sizes of five planned restoration units within the elevational range of parrotbills (1200 – 2150 m, Camp et al. 2009), Nakula 1 (162 ha), Nakula 2 (98 ha), Nakula 3 (120 ha), Kahikinui (264 ha), and Hawaiian Home Lands (1052 ha). These areas are all contiguous and differ only in administration and/or restoration timeline. To estimate the potential number of individual parrotbills and `alauahios ( that may inhabit restoration areas in the future we divided area (A) relevant to size of available habitat on leeward Haleakalā by the adjusted pair home range of parrotbills (Equation J.1) and the individual home range size of `alauahios (Equation J.2). Equation J.1 Equation J.2. 97-A Statistical Analysis To assess the effect of the number of resight points and resight dates on home range size we performed separate linear regressions for these factors and species. To test for variation in home range size between study sites we performed separate repeated measures analyses of variance (ANOVA) for each species. We did this using linear mixed effects modeling blocking for individual bird ID followed by Type III ANOVA in R 3.0.1 (The R Foundation for Statistical Computing, Vienna, Austria). For the analysis of parrotbill home range we included site and sex as fixed factors. Determining sex of `alauahios was not possible in the field unless birds were in breeding condition. As a result only a small subset of individual `alauahios were of known sex and therefore sex was not included as a factor of home range for this species in these analyses. Only site was included as a fixed factor in the `alauahio models. We used a two-tailed t-test to compare the percentage home range overlap between parrotbill mates. RESULTS Of the 223 parrotbills and 1287 `alauahios banded by MFBRP from 1992–2014, 51.5% and 56.7% were resighted between 2007 and 2014, respectively. After excluding individuals with < 10 observations and < 3 observation dates, 33 parrotbill and 152 `alauahio individuals were available for analyses. We were able to calculate home ranges for an average of 5 (± 3.2 SD) (range 0 [2010] to 10 [2011]) parrotbills and 26.4 (± 22.1) (range 10 [2007 & 2012] to 75 [2014]) `alauahios per year. We estimated home range size of 17 parrotbills from Hanawi and 16 from Waikamoi. We estimated home range size for 59, 54, and 39 `alauahios from Hanawi, Waikamoi and Kula study sites, respectively. Minimum known age of individuals included in analyses ranged from 2-10 years old in parrotbills and 1-10 years old in `alauahios. We were able to estimate the home ranges of a total of 19 male and 14 female parrotbills. A total of eight parrotbill pairs were available for analysis of pair home range. 98-AOf the 33 parrotbill individuals analyzed, we repeatedly measured home range size of five individuals (15%) (i.e. more than one year) and two of these individuals were measured in three separate years. Repeatedly measured `alauahios accounted for 27.6% of individuals (n = 42) and 7% of individuals (n = 12) were measured more than two years. We were able to estimate a home range for a single individual `alauahio in Hanawi in five separate years from 2007–2011. `Alauahio MCP home range size was not correlated with the number of resight points (R = 0.08) or the number of resighting dates (R = 0.17) per year. Home range size of `alauahio calculated by KDE was also not correlated to the number of resights (R = 0.09) or number of resighting dates (R = 0.006). In contrast, MCP-described parrotbill home range size was positively correlated with both the number of resight points (R = 0.36, P < 0.001) and resighting dates (R = 0.30, P < 0.001). However, parrotbill home ranges measured with KDE were not correlated with the number of resights (R = 0.05) or resight dates (R = 0.01). As a result all means are presented unweighted except MCP home ranges of parrotbills presented as an average weighted by the number of resights per individual. Maui Parrotbill Home range Size and Overlap Mean MCP home range of parrotbill was 8.76 ± 1.05 (SE) ha and ranged from 0.45–31.23 ha across all years and study sites (Figures J.3A, J.4A). Mean KDE home range of parrotbill was 4.69 ± 0.69 ha among 50% isopleths and 16.39 ± 2.21 ha among 90% isopleths. Of the 70% isopleths mean home range size was 8.48 ± 1.18 ha. This contour level captured the most resight points while also minimizing the amount of “extrapolated” area beyond the cluster of observation points. Outlier home ranges (> 2 × SD) using all methods were rare (5% of home ranges) (Figure J.3A). Parrotbill home range size did not vary among study sites or sex among MCPs or at any KDE contour levels (Table J.1, Figure J.4A). Parrotbill pairs share home ranges to a large degree; sharing an average of 74.84 ± 9.35% (MCP) or 64.6 ± 6.91% (KDE) of their home range with their mate’s home range. 99-AMales and females overlapped their mate’s home ranges to the same degree (MCP: t = 0.63, P = 0.548; KDE: t = 0.70, P = 0.508). The mean pair home range size was 15.48 ± 3.74 ha (MCP) and 17.62 ± 4.03 ha (KDE). The mean area of the additive pair home range was between 20.75 % (MCP) and 44.69% (KDE) larger than the mean home range size of the individuals included in the pair analysis. The adjusted pair home range size of parrotbill pairs using data from all individuals was 10.21 ha (MCP) and 11.54 ha (KDE). Maui `Alauahio Home Range Size Mean home range size of `alauahio was 0.8 ± 0.08 (SE) ha and ranged from 0.02–9.08 ha using the MCP method across all years and study sites (Figures J.3B, J.4B). Mean KDE home range of `alauahios was 0.52 ± 0.04 ha among 50% isopleths and 1.86 ± 0.15 ha among 90% isopleths. Among 70% isopleths, the contour producing most realistic home ranges, mean home range was 0.95 ± 0.08 ha and ranged from 0.3 to 8.87 ha (Figure J.3B, J.4B). As in parrotbills, outlier home ranges were rare (4%) (Figure J.3B). Home range size of `alauahios varied among study sites based on the MCP method and the 50% and 90% isopleths of KDE home ranges but not the 70% isopleths (Table J.1). Using the MCP method `alauahio home ranges were significantly larger in Hanawi than the other two sites (Waikamoi: t = 3.51, p < 0.001; Kula: t = 3.42, P < 0.001). Home ranges in Waikamoi and Kula did not differ (t = -0.24, P = 0.811). Estimating Abundance on Leeward Haleakalā To estimate the number of parrotbill and `alauahio individuals on leeward Haleakalā we used the mean home range size based on MCP and 70% KDE polygons. Table J.2 presents the predicted number of non-overlapping home ranges within areas equivalent to the size of five highlighted restoration areas. By size of area we predict that between 33.3–37.7 pairs or 66.7–75.3 individual parrotbills and 405.3–481.6 `alauahio family groups or 1,215.9–1,441.8 individual `alauahio could exist in Nakula 100-A(sections 1–3), identified as the first reintroduction site, depending on home range estimation technique. We also predict that the fenced section of Hawaiian Home Lands will support an additional 182.3–206 parrotbills and 3,325.6–3,951.4 `alauahios. Although the forest in the Kahikinui section (outside of the small section fenced in as part of Nakula 2) has the longest restoration timeline, this area may support an additional 41.3–46.7 parrotbills and 753.1–894.8 `alauahios. DISCUSSION For decades, the Kahikinui region of Maui has been identified as an area in need of conservation and restoration for both watershed health and the preservation of threatened and endangered organisms (Scott et al. 1986, Division of Forestry and Wildlife 2010). Preparation for the planned reintroduction of parrotbills to the area has provided the impetus for significant restoration efforts in Nakula NAR. Concurrently, large sections of contiguous areas in Hawaiian Home Lands and Kahikinui FR have been fenced (or soon will be) and restoration actions are underway. Together these areas contain the majority of the remaining forest on leeward Haleakalā, providing a large area for reintroduced parrotbills and dispersing individuals to occupy. Before the reintroduction can proceed, however, conservation managers require an estimate of the number of individual parrotbills the area can support to plan the number and density of released individuals. To this end we estimated the home range size of parrotbills and the `alauahios in their current ranges. By considering these estimates measure of the amount of area individuals or pairs require we extrapolated this space requirement to areas equivalent to restoration zones to estimate the number of individuals that can potentially be supported by the Kahikinui region. While much remains unknown as to how these species will behave in this new habitat, the estimates presented here provide a method to broadly predict the potential abundance of these species in areas that may be occupied in the future, critical for conservation planning and recovery of these species. 101-AOne of the challenging aspects of designing a reintroduction plan for parrotbills, similar to other Hawaiian birds, is the relative paucity of published information about the biology and behavioral ecology of this species. The first estimates of parrotbill abundance were presented in 1986 (Scott et al.) and the first active nest was not described until 1993 (Van Gelder). While robust demographic and behavioral information is crucial to designing a successful reintroduction, collecting these data is challenging for a species that even historically was described as “local and rare” (Rothschild 1900) and exists at low densities throughout its native habitat (Scott et al. 1986, Brinck et al. 2011). An estimate of home range size and potential abundance throughout the recovery region (U.S. Fish and Wildlife Service 2006) is a key piece of this information. The previous estimate of home range size by Pratt et al. (2001) was based on a limited number of individuals at a single study site. Our 8.76 ha estimate of individual parrotbill home range size was much larger than the estimate provided by Pratt et al. (6.67 ha based on 100% MCP, 2.26 ha based on 80% MCP), likely a result of the increased sample size and/or the inclusion of a second study site. We found no difference in parrotbill home range size between the two study sites. While both sites are dominated by the same native tree species, Hanawi receives significantly more annual rainfall compared to Waikamoi (Giambelluca et al. 2013) and the structure and composition of the plant community differs slightly between the two sites. This result may indicate little variation or plasticity in home range size of the species in response to variation in habitat characteristics. However with only two sites containing some of the best remaining habitat for the species, our data have limited capacity to speak to the overall variation that may exist throughout the entire species’ range (including marginal habitat) or may have existed historically (including other habitat types no longer available). The relative abundance of `alauahios masks the threats that the species faces. As in parrotbills, limited behavioral and demographic information is available for `alauahios and the overall distribution of the species beyond the range described here remains in question. The current study presents the first estimates of home range size based on such large sample sizes across both of these species’ ranges. We estimated home 102-Arange size of `alauahios at 0.8–0.95 ha, similar to the 1–2 ha reported by Baker and Baker (2000). However, these authors reported home range size to be smaller in wet native forest where we found the opposite pattern; home range size was largest at our wettest native forest site. The structure of heterogeneity in `alauahio home range size throughout its range does not fall along an apparent rainfall or habitat gradient. Rainfall amounts generally decrease from an east to west direction within the area encompassing these three study sites (Giambelluca et al. 2013). Home range size was largest in Hanawi and smallest in Waikamoi, both dominated by native forest, with the non-native-dominated Kula site having intermediate home range size. The data suggest that several factors likely influence home range size of `alauahios and that the species can exist in a wide range of habitats and climates. Waikamoi may present more favorable conditions for a variety of reasons allowing individuals to maintain smaller home ranges. The habitat on leeward Haleakalā differs markedly from the habitat that either species currently inhabits (e.g. koa- rather than ohia-dominant canopy) and no information exists on habitat utilization for these species outside their current ranges. For the purpose of reintroduction planning we need to predict the number of individuals that may inhabit an area. In order to do this using these data we make two important assumptions. 1) We assume no overlap among individual home ranges. Although home ranges do overlap to an unknown degree, assuming no overlap means that predictions of abundance for a given size of habitat are conservative; greater overlap equals higher potential abundance. 2) We assume no variation in home range size as a function of habitat. We tested this hypothesis indirectly by sampling both species at multiple study sites throughout their ranges. The actual future populations will be influenced by home range overlap and habitat consistency within conservation areas. These estimates represent a maximum number of individuals given 100% saturation of suitable, equivalent habitat within Nakula and surrounding areas and a minimum number of individuals from the perspective of home range overlap. Home range size in the leeward habitat will undoubtedly be driven in part by food resource availability (Schoener 1971). Both parrotbills and `alauahios are primarily 103-Ainsectivorous, gleaning or extracting insects from tree and shrub branches (Simon et al. 1997, Baker and Baker 2000). While insect density and diversity per stem is similar in Nakula to that in Hanawi, stem density is lower in Nakula (Peck et al. 2015) thereby reducing food resource density. Additionally, the historic observations of the parrotbills’ preference for koa as a foraging substrate (Henshaw 1902, Perkins 1903) may indicate a qualitative benefit to a habitat dominated by koa. Our assumption that home range size will be similar to that within the current range may hold particularly if qualitative differences in food resources (“preferred” habitat) balance out the reduction in quantity of resources (stem density). MANAGEMENT IMPLICATIONS While the habitat across the Kahikinui region varies in quality and is in various stages of restoration, this region has perhaps the greatest potential to increase the range and population size of many of the rarest birds on Maui (U.S. Fish and Wildlife Service 2006). Based on home range size and the sizes of restoration areas our analyses show the potential to increase the size of parrotbills range by 33% and the overall population by 58–66%. Of most concern to the preservation of all native passerines on Maui, global climate change is predicted to allow disease-carrying mosquitos to breed at higher elevations, thereby reducing the current ranges of these species (Benning et al. 2002). If we are to mitigate loss of habitat due to a rising “mosquito line” more habitat must be restored at higher elevations outside of the current ranges of these species. If enough habitat is restored at high elevations around Haleakalā Volcano, theoretically parrotbills may maintain a similar range and population size as exists today well into the future regardless of the climate-change-influenced habitat contractions. Establishing new populations of these species in Kahikinui is the first step toward protecting them. Given the time scale of habitat loss due to disease prevalence combined with the length of time it takes to fully restore forested habitats, attention should also be given to restoring additional available lands at high elevation beyond Kahikinui, particularly the western slopes of Haleakalā. 104-A ACKNOWLEDGMENTS We gratefully acknowledge all the countless field assistants and volunteers that helped collect these data who are too numerous to list. We also thank J Vetter, S Fretz, D Duffy, C Farmer, and F Duvall for editorial input and advice. LITERATURE CITED Baker, H., and P. E. Baker. 2000. 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The Condor 70:101-120 Worton, B. J. 1989. Kernel methods for estimating the utilization distribution in home range studies. Ecology 70:164-168.109-A Figure J.1. Study sites where home ranges of Maui Parrotbill and Maui `Alauahio were examined as well as protected lands for future populations of both species. Highlighted are the five conservation areas within the elevational range of parrotbills where potential abundance was predicted.110-A Figure J.2. Examples of home ranges of individual (A) Maui Parrotbill and (B) Maui `Alauahio for a single year constructed using minimum convex polygons (MCP) and kernel density estimators (KDE). Each KDE home range is shown with 50%, 70% and 90% isopleths. Scale differs between panels. 111-A Figure J.3. Histogram showing frequencies of minimum convex polygon (MCP) and 70% isopleth kernel density estimated (KDE) home range sizes for (A) Maui Parrotbill and (B) Maui `Alauahio. Axes differ between panels. 112-A Figure J.4. Mean home range size per study site. (A) Mean Maui Parrotbill home range in WAI, KUL, all sites and pairs (all sites). (B) Mean Maui `Alauahio home ranges at the three study sites. Axes differ between panels. 113-ATable J.1. Analysis of Variance Type III results for Maui `Alauahio and Maui Parrotbill. Fixed factors included were site for `alauahios and site and sex for parrotbills. Results are given for Kernel Density Estimator (KDE) and Minimum Convex Polygon (MCP) home ranges. F P df F P df F P df F P df50% KDE 0.98 0.323 1,1 0.10 0.754 1,1 0.19 0.667 1,1 7.29 0.026 1,270% KDE 0.90 0.342 1,1 0.06 0.808 1,1 0.11 0.744 1,1 4.47 0.107 1,290% KDE 0.78 0.376 1,1 0.03 0.853 1,1 0.03 0.867 1,1 43.88 ≤ 0.001 1,2MCP 0.23 0.633 1,1 0.04 0.850 1,1 0.22 0.638 1,1 17.04 ≤ 0.001 1,2Maui `alauahioMaui parrotbillSiteSite Sex Site × Sex 114-ATable J.2. Predicted abundance of Maui Parrotbill and Maui `Alauahio within habitat equivalent to the sizes (ha) of five restoration segments on leeward Haleakalā, Maui, USA (Figure 1). Managing agencies are Department of Hawaiian Home Lands (DHHL) and Division of Forestry and Wildlife (DOFAW). Restoration segments are listed based on approximate restoration timeline, Nakula 1 having the shortest timeline. Predictions are based on estimated pair home range size of 10.21 ha (MCP) and 11.54 ha (KDE) for parrotbills and individual home range size of `alauahios of 0.8 ha (MCP) and 0.95 ha (KDE). The percentage increase in total population size is based on a global population size of 500 parrotbills and 55000 `alauahios. Name Managing AgencyProtected AreaYear fenced MCP KDE MCP KDENakula 1 NARS 162.32 2012 31.8 28.1 609.6 513.0Nakula 2 NARS 98.01 2015a 19.2 17.0 368.1 309.8Hawaiian HomelandsDHHL 1052.21 2015a 206.0 182.3 3951.4 3325.6Nakula 3 NARS 120.72 TBD 23.6 20.9 453.4 381.6Kahikinui FR 238.26 TBD 46.7 41.3 894.8 753.1Total 1671.53 327.3 289.7 6277.2 5283.0% increase of Total Population 65.5% 57.9% 11.4% 9.6%MAPA individuals (N̂) MAAL individuals (N̂)115-A APPENDIX J.1. GEOSPATIAL MODELLING CODE. The full code used to create of minimum convex polygons and kernel density estimated polygons in Geospatial Modelling Environment; adapted from Beyer (2012). Prior to these analyses we created separate shapefiles containing only individuals with ≥ 10 resight points. These analyses were run for each species separately by year. n = individuals for a given year. setwd(in="file", out="outfile"); splitdataset(in="resight_shapefile.shp", uidfield="ANIMID", outws="outfile", prefix="ANIM"); for(i in 1:n){ genmcp(in=paste("ANIM",i,".shp"), out=paste("mcp_","ANIM","#year#",i,".shp")) }; for(i in 1:n){ kde(in=paste("ANIM",i,".shp"), out=paste("kde","scv","#year#","ANIM",i,".img"), bandwidth="SCV", cellsize=10) }; for(i in 1:n){ isopleth(in=paste("kdescv#year#ANIM",i,".img"), out=paste("iso#year#_ANIM",i,".shp"), quantiles=c(0.5,0.7,0.9), poly=paste("isopoly#year#_ANIM",i,".shp")) }; for(i in 1:n){ addarea(in=paste("mcp_","ANIM","#year#",i,".shp"), area="AREA_ha", areaunits="hect") }; for(i in 1:n){ addarea(in=paste("isopoly#year#_ANIM",i,".shp"), area="AREA_ha", areaunits="hect") }; for(i in 1:n){ 116-A addcodedfield(in=paste("mcp_","ANIM","#year#",i,".shp"), field="ANIMID", fieldtype="SHORT", constant=i) }; for(i in 1:n){ addcodedfield(in=paste("isopoly#year#_ANIM",i,".shp"), field="ANIMID", fieldtype="SHORT", constant=i) }

Taking off

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Taking off

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