Phylogeography and population genetics of Taiwanese macaque (Macaca cyclopis)

摘 要 本論文規劃為五個章節，分別探討台灣獼猴(Macaca cyclopis)與近緣種間、種內及族群間相關之遺傳研究。第一章簡介：(1)台灣獼猴與其近緣種 (馬來猴系，the fascicularis group) 間的親緣關係；(2)更新世冰河進退可能造成的親緣地理模式；(3)台灣地質形成史；(4)台灣物種呈現的親緣地理模式；及(5)低侵略性及非侵略性採樣法在靈長類遺傳研究上的應用。這些主題說明與本研究相關的背景知識。 在第二章中，利用粒線體DNA控制區域的序列分析，研究恆河猴系（the mulatta group）內三種近緣種，包括恆河猴（M. mulatta）、台灣獼猴（M. cyclopis）、與日本獼猴（M. fuscata）間的演化關係。本文描述並檢驗Fooden and Albrecht （1999）所提出的恆河猴演化及播遷假設。研究結果顯示，遺傳證據支持此假設中所提亞洲東部恆河猴族群曾經發生南撤及北拓事件，但是南撤北拓事件發生的時間可回溯至比最後一次冰期更早的年代。此外，本文推估這三種近緣種分化的時間約在十七萬年前。 第三章主要研究台灣獼猴的親緣地理模式。對採樣自31個地方族群的個體進行粒線體控制區域的分析，共發現5個不同的親緣分支（包括北部、東北部、中部、南部、及東部分支）。這些分支內的成員主要依其分布的地理區域而分群，並於中央山脈的東側形成不同親緣分支的交會帶。這表示對台灣獼猴來說，中央山脈並非有效的地理障蔽。雖然台灣獼猴的遺傳結構可能受距離隔離所影響，但由親緣分支間顯著的分化程度顯示，不同的粒線體DNA分支系可能曾被隔離於不同的冰期避難所。本研究同時根據mismatch distribution analysis及nested clade analysis的結果去推導台灣獼猴的族群歷史。 第四章應用以個體判別為基礎的分析步驟，偵測壽山獼猴族群內是否有次結構的存在，並藉以判別雄性播遷者的來源社群。本研究共分析126隻成年獼猴，9組微隨體基因型資料。研究結果顯示，社群間具低至中度(FST=0.023-0.098)但顯著的遺傳分化，而分化可能與獼猴雌性居留原地及社群內非隨機交配有關。根據Cornuet et al. （1999）所建議獲得不同程度個體判別正確率所要求的條件，本研究所得的個體判別分析正確率可達85％。判別分析的正確率可藉由增加個體樣本數，或微隨體基因座數目來提升。相較於傳統田野觀察，判別分析可成為瞭解雄性播遷模式的一項捷徑工具。 第五章為一項前導實驗，用以瞭解利用非侵略性採樣法研究台灣獼猴族群遺傳學的適用性。本研究採用排遺樣本為DNA來源，實驗結果顯示微隨體PCR成功率低（42.4%，n=181），而粒線體DNA PCR成功率則可接受 （66.5%，n=334）。由於微隨體PCR成功率低，加上對偶基因的再現性差（高對偶基因遺失率及高鬼帶出現率），使獲得可信度高的微隨體基因資料困難重重。但近來報導的各項新技術，或許有助於克服此困難。本文並針對非侵略性採樣法的益處及缺點進行討論。Abstract The dissertation is organized into 5 chapters, which include aspects of inter-specific, intra-specific, and intra-populational genetic studies of Formosan macaques (Macaca cyclopis). Chapter One gives general introductions of 1) phylogenetic relationship of Formosan macaque and its sister species (the fascicularis group); 2) phylogeographic patterns associated with Pleistocene effect; 3) geodynamic history of Taiwan; 4) phylogeographic patterns and possible locations of Pleistocene refugia for species in Taiwan; and 5) the application of low- and non-invasive sampling method in primate genetic studies. These topics illustrate general background associated with the study. In Chapter Two, evolutionary relationship of the closely related species in the mulatta group (including M. mulatta, M. cyclopis and M. fuscata) was investigated by sequences of mtDNA control region (CR). The hypothetical scenario for evolution and dispersal of M. mulatta suggested by Fooden and Albrecht (1999) was described and tested. The results indicate that the southward retreat and the northward recolonization of M. mulatta populations in the eastern Asia proposed in the scenario were supported by the genetic evidence. However, the age of retreat and recolonization may be dated back to a more ancient past during the middle Pleistocene instead of during the last glacial period. In addition, a more recent age of mtDNA lineage isolation, 0.17 mya, among the 3 species is suggested. In Chapter Three, phylogeographic pattern of M. cyclopis was studies. Samples from 31 local populations were incorporated in the mtDNA CR analyses. Five distinct phylogenetic clades (North, Northeast, Central, South, and East clades) were found to be clustered mainly according to their geographic regions with a major suture zone located in the eastern side of the central mountain range (CMR). It indicates that the CMR may not be a serious physical barrier for M. cyclopis. Although genetic pattern of isolation by distance is accepted in the species, the deep divergence between phylogenetic clades suggests that different mtDNA lineages might have been isolated in different refugia during glacial times. Inferences of population histories were also derived from results analyzed by mismatch distribution analysis (MDA) and nested clad analysis (NCA). Chapter Four describes an application of assignment-based approaches to detect population substructure and to identify origins of male migrants in the Shoushan macaque population. Microsatellite genotypes (9 loci) from 126 adult individuals were analyzed. Significant but low to moderate genetic differentiation (FST =0.023-0.098) was found between social troops, which may be associated with female philopatry and non-random mating. An acceptable accuracy of the results (~85%) can be obtained according to the criteria suggested by Cornuet et al. (1999). The accuracy can be improved by increasing number of samples or microsatellite loci. The assignment approaches thus provide a quick alternative to field observation in understanding patterns of male dispersal. Chapter Five illustrates a pilot study that was conducted to test the applicability of non-invasive sampling approach in the population genetic studies of Taiwanese macaques. Feces were used as DNA sources in the study. Success rate of microsatellite PCR amplification is low (42.4%, n=181) while that of mtDNA PCR amplification is acceptable (66.5%, n=334). The low PCR success rate and poor PCR repeatability of microsatellite alleles (high allelic dropout rate and appearance of false alleles) makes it difficult to obtain a reliable microsatellite data set. However, the difficulties may be overcome by new techniques. The putative benefits and drawbacks of the approach are also discussed in this chapter.Contents 摘要…………………………………….………………………. I Abstract..……………………………………………………III Chapter One General Introduction……………………………………………….1 1.1 Phylogenetic studies in Macaca cyclopis…………………………………………1 1.2 Pleistocene effect and phylogeography…………………….………..……………4 1.3 Geodynamic evolution of Taiwan…………………………………….…………..6 1.4 Phylogeography of species in Taiwan………………………………….…………7 1.5 Application of low-invasive and non-invasive sampling methods in wild primate populations…………………………………………………………….….……….8 1.6 Organization of the dissertation…………………………………………………...9 1.7 References………………………………………………………………………..10 Tables…………………………………………………………………………...……16 Chapter Two Evolution and dispersal of the mulatta group …………………...18 2.1 Introduction…………………………………………………………………..…..18 2.2 Material and methods…………………………………………………………….22 2.3 Results……………………………………………………………………………27 2.4 Discussion………………………………………………………………………..31 2.5 References………………………………………………………………………..38 Figure legends………………………………………………………………………..42 Figures……………………………………………………………………….……….44 Tables………………………………………………………………………..……….52 Chapter Three Intraspecife phylogeography of Macaca cyclopis…………...…58 3.1 Introduction………………………………………………………………………58 3.2 Material and methods…………………………………………………….………60 3.3 Results……………………………………………………………………..….….67 3.4 Discussion…………………………………………………………………….….70 3.5 References……………………………………………………………………..…76 Figure legends………………………………………………………………………..80 Figures………………………………………………………………………………..82 Tables…………………………………………………………………...……………91 Chapter Four Detection of population structuring and male transfer in Shoushan monkeys by assignment-based approaches……….……………….96 4.1 Introduction………………………………………………………………………96 4.2 Material and methods…………………………………………………..………...99 4.3 Results………………………………………………………………………..…104 4.4 Discussion………………………………………………………………...…….106 4.5 References………………………………………………………………..……..110 Figure legend..………………………………………………………………...……114 Figure.……………………………………………………………………………....115 Tables……………………………………………………………...………………..116 Chapter Five Examination for applicability of non-invasive sampling in population genetic study of Formosan macaques (Macaca cyclopis)……....122 5.1 Introduction………………………………………………………………..……122 5.2 Material and methods…………………………………………….……………..124 5.3 Results…………………………………………………………….…………….129 5.4 Discussion………………………………………………………………………131 5.5 References…………………………………..…………………………………..136 Tables……………………………………………………………………………….14