University of Wollongong Research Online University of Wollongong Thesis Collection 1954-2016 University of Wollongong Thesis Collections 1987 Flowering phenologies and reproductive success of co-occurring Banksia species Brian James Copland University of Wollongong Follow this and additional works at: https://ro.uow.edu.au/theses University of Wollongong Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorise you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part of this work may be reproduced by any process, nor may any other exclusive right be exercised, without the permission of the author. Copyright owners are entitled to take legal action against persons who infringe their copyright. 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For further information contact the UOW Library: research-pubs@uow.edu.au FLOW ERING PHENOLOGIES AND REPRODUCTIVE SUCCESS OF CO-OCCURRING BANKSIA SPECIES.A thesis submitted in partial fulfilment of the requirements of the degreeofMASTER OF SCIENCE ( HONOURS )fromTHE UNIVERSITY OF WOLLONGONGbyBRIAN JAMES COPLAND B.A. Dip. Teach.UNIVERSITY OF 3 WOLLONGONG Ilibrary I Biology DepartmentJune, 1987.7**1 ¿5DECLARATION.The work presented in this thesis, in fulfilment of conditions governing the award of the degree of Master of Science ( Honours ), was carried out by me between April 19S2 and December 1986 through the Biology Department, University of Wollongong, Wollongong, New South Wales and has not been submitted to any other university or institution for a higher degreeiSUMMARYThere has been an expansion of interest, in recent years, in the nature of plant/ pollinator interactions, especially with respect to competition. Many studies lack generality, however, for two r e a s o n s ( i) they were completed without knowledge of precise flowering phenologies and (ii) they were short-term studies, usually completed within one flowering season. When dealing with perennials, subject to temporal variation in factors that influence their reproductive success, studies should be carried out over a number of flowering seasons.Four co-occurring species of the genus Banksia ( Proteaceae ) were studied over a period of four years in order to determine (i) their precise flowering phenologies and (ii) factors that influence their reproductive success Three of the species, Banksia paludosa, B ericifo lia var. ericifo lla , and B. spinulosa var spinulosa flowered simultaneously over winter, while the summer-flowering B\ serrala was phenologically isolated from the others. Because the three simultaneously-flowering species shared the same guild of pollinators, it was considered likely that they would compete for pollinator services if pollination were a limited resourceTo assess whether pollination was limiting reproductive success in my study, natural fruit-set was determined and compared to fruit-set after experimental pollen supplementation. Natural fruit-set, measured by the proportion of inflorescences that set some fruit, was variable both within a year and between years The mean number of follicles that developed per non-barren cone, however, was relatively constant throughout the study. Pollen supplementation experiments suggested that pollination limited fruit-set in B ericifo lia only on one occasion, namely during a period of superabundant inflorescence production. Both B, paludosa and B, spinulosa appeared to be quite severely limited by pollination service in a site where they co- occurrediíwith the much more prolific flowering B, e r id fo lia , If these Banksia species are competing for pollinators, as the data presented here implies, then B. paludosa and B spin ulosa are the poorer competitors.Pollen supplementation had no effect on the mean number of follicles per non-barren cone, but had a significant effect on the spatial distribution of these follicles. More of the earlier flowers to open ( located at the top of the spike in B e rid fo lia ) set fruit in the pollen supplemented sample compared to the control sample, in which very few early opening flowers set fruit This suggests that there is a threshold number of flowers that must be open on an inflorescence before potential pollinators are attracted to it.In 1986, fewer pollen grains germinated on the stigmas of late season B e rid fo lia flowers compared to the number germinated in early and peak season flowers. However, there was no difference in the proportion of pollen-bearing stigmas at late flowering and an increase in the number of pollen grains per pollen-bearing stigma at this time. Reproductive success in B. e rid fo lia , as measured by fruit-set, was not diminished at late-flowering in 19S6, compared with earlier in the season, ( as it had been in 1984 and 1985 ) suggesting that factors more fundamental than pollen availability were responsible for the ultimate level of fruit-set during that year.An experiment on resource allocation suggested that B erid fo lia may be able to shunt resources between inflorescences. Results, although equivocal, lend support to an hypothesis invoking resource limitation as the primary cause of fruit- set limitation in B, e rid fo liaillACKNOWLEDGEMENTS.I would like to express my sincere gratitude to my supervisor, Dr Rob Whelan for his guidance, encouragement and many hours of helpful discussion throughout this study.The assistance of the following people is also gratefully acknowledged ; Ross Goldingay, Biology Department, University of Wollongong, for discussion and for commenting on an earlier draft of this thesis; Sue Carthew, Biology Department, University of Wollongong, for help with the work on pollen tube germination; Dr Ken Russell, Mathematics Department, University of Wollongong, for help with some of the statistical analyses; Dr, Brian Collins, Department of Biology, Curtin University of Technology, for access to unpublished material presented in Collins and Rebelo ( in press ); Richard and Pat Jordan, Wardens at Barren Grounds Nature Reserve, for the use of meteorological data, help in identifying understory species, discussions and for their hospitalityFinally, I would like to sincerely thank my wife, Gale, without whose patience, encouragement and understanding this work would never have been completedIVTABLE OF CONTENTS.SUMMARY ,ACKNOWLEDGEMENTS ¿iiTABLE OF CONTENTS ivLIST OF TABLES viLIST OF FIGURES Vlil1. INTRODUCTION1.1 Flowering Phenology 31.2 Reproductive Success 21 3 Pollination and the Proteaceae 31.4 Characteristics of the Genus Banksia 41.5 Co-occurring Banksias in Eastern Australia 101 6 The Aims of this Study 212 STUDY AREAS AND SPECIES2.1 Budderoo Plateau 222 2 Study Sites 223 FLOWERING PHENOLOGY31 Introduction 293.2 Materials and Methods 3033 Results 323 4 Discussion 374. FRUIT-SET AND FACTORS INFLUENCING IT : AN INTRODUCTION 455. VARIATION IN NATURAL LEVELS OF FRUIT-SET5.1 Introduction 485.2 Materials and Methods 4953 Results 505.4 DiscussionPage60V6. POLLEN ADDITION EXPERIMENTS6.1 Introduction 656.2 Materials and Methods 666.3 Results 676.4 Discussion 717. NUMBERS OF POLLEN GRAINS AND POLLEN TUBES PER STIGMA7.1 Introduction 757.2 Materials and Methods 757.3 Results 767.4 Discussion 768. RESOURCE ALLOCATION8.1 Introduction 798.2 Materials and Methods 808.3 Results 818.4 Discussion 819. INTEGRATIVE DISCUSSION 85BIBLIOGRAPHY 93APPENDIX 103TABLE OF CONTENTS ( Continued )PageviLIST OF TABLES.Table 1.1: Dimensions of inflorescences and flowers of the four Banksia 20species on the Budderoo PlateauTable 31 Months of peak-flowering of B serrata, B e ricifo lia B paludosa 36 and B. spiautosa over the study periodTable 3 2 : Differences in the distributions of flowering times of two groups 36 of 15 plants of the same species in two study sitesTable 3 3 : Comparison of flowering phenologies reported by George ( 198-4 ), 39McFarland ( 1985 ) and this study.Table 5 1 : Natural fruit-set. 53Table 5 2 : Inter- and intra-year differences in natural fruit-set 54Table 5 3 : ANOVA showing the relationship between B ericifo lia mean 56follicles per non-barren cone, time of flowering and yearTable 5 4 : ANOVA showing the relationship between B paludosa mean 56follicles per non-barren cone, time of flowering and yearTable 5 5 : Estimated seed output for 30 B, ericifo lia plants during 1984, 581985 and 1986.Table 5 6 : Estimates of percentage of B\ ericifo lia flowers producing follicles 59during the early, peak and late parts of the 1986 flowering seasonTable 5 7 : Proportions of non-barren cones reported for B spinulosa in 611983/84 by Whelan and Goldingay ( 1986 ) and in 1985/86 ( this study ).Table 6.1 : Comparison of control and pollen-added fruit-sets as measured 68by the percentage of non-barren cones.Table 6.2 : Comparison of control and pollen-added fruit-sets as measured 70by mean number of follicles per non-barren conePagevuTable 7.1 ; (A) Numbers of pollen-grains and pollen-tubes per stigma in B e ricifo lia during 1986. (B) Significance of differences in numbers of pollen-grains and pollen-tubes Table 7.2 : Proportions of B. e ricifo lia flowers that contained pollen- tubes and produced follicles during the 1986 flowering season Table 8.1 : Fruit production by treatment plants ( early and peak inflorescences removed ) and control plantsLIST OF TABLES ( Continued ) Page777$82V i l iLIST OF FIGURES. PageFigure 1.1 : G revillea sp. showing prominent styles and stigmas with terminal 5 pollen presenters, characteristic of most Proteaceae Figure 1.2 : a. Straight style, b. Hooked style. 5Figure 1 3 : Scanning electron micrograph of a B. spinulosa pollen 6presenter showing the stigmatic groove.Figure 1.4 a : B. ericifo lia seeds and separator. 8Figure 1.4 b : Banksia ericifo lia infructescence showing separators 9emerging from partially open follicles.Figure 1.5 a . B. serrata habit. 11Figure 1.5 b : B. serrata inflorescence. 12Figure 1.6 a : 3 . paludosa habit. 13Figure 1.6 b : B. paludosa inflorescence. 14Figure 1.7 a : B. ericifo lia var. ericifo lia habit. 16Figure 1.7 b : B. e ricifo lia var. ericifo lia inflorescence. 17Figure 1.8 a : B .spinulosa var. spinulosa habit. 18Figure 1.8 b : B .spinulosa var. spinulosa inflorescence. 19Figure 2.1 : Location of the study sites. 23Figure 2.2 : Vegetation typical of the study sites on the Budderoo Plateau. 24Figure 2.3 : Monthly rainfall at Barren Grounds Nature Reserve. 25Figure 2.4 : Annual rainfall at Barren Grounds Nature Reserve. 25Figure 2.5 : Temperature variation at Barren Grounds Nature Reserve. 26Figure 2.6 : Distribution of Banksia species in study site 1. 27Figure 2.7 : Distribution of Banksia species in study site 2. 28Figure 31 : Flowering phenology - study site 1. 33Figure 3.2 : Flowering phenology - study site 2. 33IXFigure 3 3 a : Cumulative percentages of spent B. serrate and B paJudosa 34inflorescences during the years 1983-1986Figure 3 3 b : Cumulative percentages of spent B e ricifo iia and B spinuJosa 33 inflorescences during the years 1983-1986Figure 3 4 : Mean numbers of inflorescences per plant per year 3bSignificance of between year differences,Figure 51 Frequency distribution of follicles produced by B paJudosa 51inflorescences in 1986.Figure 5 2 : Frequency distribution of follicles produced by B ericifo iia 51inflorescences in 1986.Figure 5 3 : Frequency distribution of follicles produced by B. spinuJosa 52inflorescences in 1986Figure 5 4 : Distribution of follicles on fruiting cones in 1986 60Figure 6.1: Distribution of follicles on fruiting cones in 1986 ( control and 71 pollen-added )Figure 9.1 : Factors affecting fruit-set in B, e riclfo lia , B. spinulosa and 91B. paJudosaLIST OF FIGURES ( Continued ) Pagei1. INTRODUCTION1.1 FLOWERING PHENOLOGY.Detailed analyses of spatial and temporal aspects of flowering have been surpnsingly scarce ( Wyatt 1980 ) despite the widespread recognition of their importance in plant population and community ecology ( Harper 1977, Janzen 1977 ) This paucity of detailed information is particularly acute in the Australian context where flowering phenologies of the majority of species are known only in the most general of terms, if at ail. Although there has been an increasing interest in the reproductive biology of the genus BanJcsJa ( Family Proteaceae ) in recent years ( see Collins and Rebelo 1987 for review ), there has yet to be any detailed, or long-term assessment of the flowering phenologies of any of the species that occur in eastern AustraliaThe variation in flowering patterns displayed by plants has significance for many aspects of community ecology ( Bawa 1983 ), such as the organisation and structure of communities ( Stiles 1978 ), optimal foraging theory ( Pyke e ta l 1977 ), and the evolution of reproductive strategies ( Schaffer and Gadgil 1975 ) Flowering phenologies need to be known precisely if inferences are to be made from the patterns observed ( Rathcke 1984 ), For example, competition for pollinators has often been inferred from the sequential flowering phenologies observed in many plant communities ( Robertson 1895, Mosquin 1971, Carpenter 1976, Stiles 19/7.1978, Feinsinger 1978, Waser 1978a, Pleasants 1980 ). These displaced flowering times have been interpreted as the result of past selection to reduce interspecific competition for pollinators. A major difficulty, however, is that it is impossible to test for the underlying competitive process when the flowering patterns are the historical result of long periods of selection, or as Connell ( 1980 ) put it the ghosts of competition past." Another problem lies in the fact that although species in single communities can always be arranged from the earliest flowering to the latest flowering, mereC09026984422inspection cannot tell us the reasons for these patterns or even whether they are different from random expectations ( Vaser 1983 )In spite of all of these caveats, a knowledge of precise flowering phenologies is essential, not as an end in itself, but as a prerequisite for all other studies concerned with plant reproductive success1.2 REPRODUCTIVE SUCCESS.A great deal of ecological research in recent times ( see Real 1983, Jones and Little 1983 and Collins and Rebelo 1987 for reviews ) has centered around argumen ts about the causes of low fruit-set in many plant species Most authors have regarded low conversion ratios of flowers into fruits a result of either pollination limitation or resource limitation. Some ( eg. Stephenson 1981, Stephenson and Berlin 1983, and Bawa and Webb 1984 ) have argued, in addition, for the importance of sexual selection In this case, large numbers of flowers would be advantageous in an evolutionary sense because of the two-fold benefits of increased pollen dispersal and the ability of the maternal plant to select, from a larger number of zygotes, those that will share the available resources ( ie. by selective abortion of those that are less desirable ) Fruit-set is a convenient and reliable indicator of reproductive success ( Zimmerman 1980a, Augspurger 1981, Rathcke 1983 ) My investigation of reproductive success in co-occurring Banks/» species documented the relationship between natural fruit-set and flowering time and compared it to fruit-set resulting from the supplementation of natural pollen levels. Because of the likelihood of temporal variation in the factors that influence fruit-set ( Primack 1980. Gross and Werner 1983, Rathcke 1983 ), it was essential to ensure that natural and pollen-added fruit-sets were compared at various times during a flowering season and over a number of years. Pollination limitation of reproductive success in a given year can be claimed if pollen-added fruit-set is consistently greater than natural fruit-set1.3 POLLINATION AND THE PROTEACEAE.The Australian angiosperm flora consists of over 12,000 species ( Ride 1978, Beadle 1981 ). An important part of this flora consists of the Proteaceae - a family of flowering trees and shrubs with major generic diversity (seventy-five genera are now recognised) and endemism in Australia ( Johnson and Briggs 1975, Coiiins and Rebelo 1987 ). Although morphologically diverse, the Australian Proteaceae exhibit a number of common characteristics of importance in the study of their pollination biologyAlmost all species of the Proteaceae produce nectar. Many species produce copious amounts, especially in cases where either inflorescence-structure or other observations indicate adaptations for pollination by vertebrates ( Johnson and Briggs 1975 ) Nectar is the primary reward for floral visitorsMost genera in the Proteaceae share an unusual floral characteristic , a specialised style tip surrounding the initially non-receptive stigmatic surface is used as a ' pollen-presenter" ( Johnson and Briggs 1975 )( Figure 1.1 ) The staminaJ filaments are fused to the perianth, the anthers dehisce as the flower is opening, depositing their pollen on the stigma, and then rolling back with the perianth segments. The prominent style and stigma then serve to transfer pollen to potential pollinators as they visit the flower to harvest nectar. Protan dry ( flowers having male function then female function ) is said to be the rule rather than the exception in the Proteaceae ( Carolin 1%1, Johnson and Briggs 1975 ), although there is little experimental evidence of this.The concentration of large numbers of flowers in simple or compound heads, usually in conjunction with yellow or red colouration of the flowers, is widespread in the two largest subfamilies ( the Proteoideae, eg. P etrophlle, Isopogon and Conosperm um and the Grevilleoideae, eg. G revillea , Hakea, Banksia, Telopea and4Lam bertla). These conditions are thought to be secondary, convergent adaptations to bird and small mammal pollinators ( Johnson and Briggs 1975, Carpenter 1978, Ford et a J 1979, Whelan and Burbidge 1980. Recher 1981, Paton and Turner 1985 )1.4 CHARACTERISTICS OF THE GENUS BAMKSIA.Banksias are woody evergreen plants, ranging in habit from prostrate shrubs to tall trees, in many of their characteristics they are typical of the Proteaceae. Of the seventy-three species currently recognised ( George 1981 ), thirty-six have a thick, fire-tolerant trunk or a woody lignotuber from which they are able to resprout after fire. Established plants of the other species are killed by fire and populations must regenerate from seed (George 1984 ).Perhaps the most readily identifiable characteristics of Banksia species are their inflorescences and the fruiting "cones" ( infructescences ) that subsequently develop from them. The flowers are produced in dense spikes, with hundreds or thousands of flowers per spike. The flowers are arranged in a complex pattern around a central woody axis. They occur in pairs and are located in a double-spiral pattern and often in vertical rows as well ( George 1984 ). Each individual flower has a floral-tube or perianth made up of four segments called tepals. Each tepai has an anther near its apex which sheds its pollen just before the flower opens. The style is wiry; straight in some species and curved or hooked in others ( Figure 1.2 ). At the proximal end of the style is an ovary containing two ovules and at the distal end there is the microscopic stigmatic groove ( Figure 1.3 ). Below the stigmatic groove is the section of the style modified to form a pollen-presenter. Just before the flower opens, the style straightens or elongates and the pollen-presenter receives the pollen from the anthers. After opening, the pollen-presenter protrudes well beyond the perianth.Many species ofBanksia have flowers that produce a superabundance ofF ig u re i . l . Greviliea sp. showing prominent styles and stigmas with club-shaped terminal pollen presenters ( circled ), characteristic of most ProteaceaeFigure 1.2 : a. Straigh t style ( eg. B. serrate, B paludosa ), b. Hooked style ( eg. Be rici folia, B spinulosa )6F igu re 1.3 : Scanning electron micrograph of a B spinulosa pollen presentershowing the stigmatic groove ( X 100 ).nectar. This has been interpreted by many workers ( eg Paton and Ford 1977, Carpenter 1978, Ford e ta l 1979, Keighery 1982, Turner 1982, Paton and Turner 1985 ) as an adaptation for pollination by birds and possibly small mammals As in most Proteaceae, the flowers are protandrous and the stigma becomes receptive to pollen from other flowers after the self-pollen on the pollen- presenter has had the opportunity to be removed ( George 1984, Collins and Spice 1986 ) in most species, the flowers closest to the base of the spike open first and this continues acropetaiiy giving rise to a zone that can be clearly distinguished from the rest of the spike ( Collins and Spice 1986 ). The reverse applies in all except one of the species that have hooked styles : ie. the flowers mature basipetally ( George 1984 )The banksia "cone", which develops after fertilisation, comprises the central woody axis of the flower spike along with the woody follicles that form from the ovaries surrounding successfully fertilised ovules and remnants of the spent flowers The number of follicles that develop on each cone is quite variable but is always very small compared to the number of flowers produced ( see Lamont e ta l 1985 and Collins and Rebelo 1987 for review ).Serotiny, or more correctly, bradyspory' ( Pate and Beard 1984 ) ( the retention of unopened follicles on a plant between fires ) is common in banksias , with approximately 90% of species showing some degree of it ( George 1981 ), The extent to which a particular species is serotinous, however, shows considerable intra-specific variation and appears to be correlated with plant height and rainfall ( Cowling and Lamont 1985 a ) and with intensity of predation ( A. Hunt pers. comm ). When a follicle opens, either spontaneously when mature or after fire, there are generally two winged seeds with a "separator" between them ( Figures 1,4 a tx b ). In some species, however, ( eg, Banksia g ra n d is) most follicles contain only one seed even though there is room for two in a follicle ( Abbott 1985 ); in others ( e g . B. e r id fo lia ) only one of the two seeds produced is viable and has an embryo ( R.8Figure 1.4 a : Bunksia ericifolia seeds ( left and r i g h t ) and separator ( middle )9Figure 1.4 b : Banks/* eriafotis infructescence showing separators em ergingfrom partially open follicles.JOWhelan pers. comm.). The separator has two wings which are exposed when the follicle ruptures. Cowling and Lamont ( 1985 b) suggested that the wings are hydroscopic, moving together when wet and spreading apart when dry. This action would allow the separator to act like a lever, gradually extracting the seeds from the loilicie in response to a number of wet-dry cycles. This seed-release mechanism was also found to be more effective at lower temperatures ( ie. 15-20°C ), ensuring that seeds are released in the greatest numbers when the optimal conditions for germination are present.1.5 CO-OCCURRING BANES IAS IN EASTERN AUSTRALIA.All of the seventy-three species of Banksia now recognised occur in Australia. Of these, fifty-eight or 79% are found only in Western Australia, mainly in the south-west. In south-eastern and eastern Australia the diversity is not nearly as great, with only fourteen species occurring between Eyre Peninsula in the south central coast and Cape York Peninsula, north Queensland ( George 1984 ). Many of the east coast species, however, co-occur in the same sites ( personal observations ).The four species of Banksia investigated in this study are common in the open woodland of the coastal plain and adjacent ranges in eastern N S W. from the central coast to Jervis Bay ( George 1984 ). Two study sites ( see Chapter 2 ) were chosen in an area where ail four species co-occur.Banksia serrata and B. paiudosa ( Figures 1.5 and 1.6 ) both have flowers with straight styles ( Section : Banksia; George 1984 ), whereas B. ericifo lia var. ericifo lia and B. spinuiosa var. spinuJosa ( Figures 1.7 and 1.8 ) have flowers with hooked styles and are classified within Section :Oncostylis, ( George 1984 ).Banksia serrata, the "Old-Man Banksia" of May Gibbs’ children s stories, occurs as a large shrub or small tree to 10m (Wrigley and Fagg 1979 ). Grey flowers with cream styles are borne on terminal, cylindrical inflorescences ranging in size from11Figure 1.5 a : Banksia serrate habit. H eigh t; 6 metres, canopy diameter 4 metres12Figure 1.5 b : Banksiaserrate inflorescence. Length : 15 cm, width 10 cm13Figure i .6 a : Baaksmpaludosa habit. Last burnt in 1968,18 years prior to photograph. H eight: 0.5 metres, diameter . 2 metres14Figure 1.6 b : BanksiapaJudosa inflorescence Length 10 cm, width cO157- 15 cm. long and 9-12 cm. wide. Anthesis occurs acropetaliy. Fruiting cones have persistent old flowers and large elliptical follicles, 25-35 mm. long, 20-25 mm. high, and 15-22 mm. wide ( George 1984 ). B. serra/a has thick bark capable of resisting fire, after which it resprouts by epicormic shoots.Banksiapaludosa is an open, branching shrub with a variable number of stems originating from a lignotuber. Although individuals can reach 2 m , generally they were below 0.5 m. high in my study sites and up to 2.5 m. in diameter but usually much less. Grey to brown flowers are held on cylindrical inflorescences 7-13 cm. long and 3-4 cm. wide ( George 1984 ). Anthesis occurs acropetaliy. Fruiting cones contain narrow, elliptical follicles ( 9-18 mm. long, 1-5 mm. high, and 3-7 mm wide ( George 1984 )) and the old persistent flower parts. B. paludosa is fire tolerant, resprouting from the lignotuber.Banksia spinulosa var. spin ulosa , or "Hairpin Banksia", is a small shrub, rarely exceeding 2 m. x 2 m. in my plots. Several stems originate from a lignotuber as in B. paludosa . Flowers are golden yellow with reddish-purple to black, hooked styles, 30-40 mm. long. Inflorescences are cylindrical and terminal, ranging in size from 6-12 cm. long and 6-7 cm. wide. Anthesis occurs basipetaiiy. Fruiting cones contain a widely variable number of follicles (range : 0 - 104, mean = 45, personal observations) which are elliptical to rhombic in shape, 10-24 mm. long, 5-7 mm. high, and 3-14 mm. wide ( George 1984). Old perianths are usually persistent on the cones for some years but styles soon fall after flowering. B. splnulosa var. splnulosa is fire tolerant and resprouts from the lignotuber ( Carpenter andRecher 1979 ).Banksia ericlfo lia var. e ric ifo lia , the "Heath Banksia", is a single-stemmed, dense shrub or small tree to 6 m.. Flowers are usually brownish-yellow with orange or orange-red styles. Styles are 30-35 mm. long and hooked at the apex ( George 1984 ). Inflorescences are cylindrical, terminal, 4-5 cm. wide and vary in length from about8- 26 cm. ( Paton and Turner 1985 ). Anthesis occurs basipetaiiy, as in B, spin u losa .16Figure 1.7 * : Banksia ericifolia var. ericifolia habit. Last burnt in 1% 8,18 years prior to photograph. H eight: 2.5 metres, diameter : 3 metres.17Figure 1.7 b : Ban ksia eri c i folia va r. ericifolia inflorescence Length 12 cm,width : 4 cm18Figure 1.8 a : Banksia spinulosa var. spinulosa habit. Last burnt in i% 8 ,18 yearsprior to photograph. H eigh t: 1 metre, diameter : 2 metres19Figure 1.8 b : Banksmspinuiosa var spinulosa inflorescence Length 9 cmwidth 6 cm20Follicle number per fruiting cone is quite variable, ranging from 0 to about 100 with a mean of about 40 ( personal observation ). Follicles are oblong to rhombic in shape, 13-22 mm. long, 6-10 mm. high and 4-13 mm. wide ( George 1984 ). Old perianths are persistent, but styles fall after flowering. B. e n cifo lia is killed by fire, after which the follicles open and the seed-bank, previously retained on the plant, is released, resulting in a new, even-aged cohort of individuals (Bradstock and Myerscough 1981 ). It has been argued that because B. e ricifo lia is an obligate reseeder, it tends to produce bigger inflorescences, more inflorescences per plant, and, due to its subsequent attractiveness to potential pollinators, sets more seeds per cone than resprouting congenerics with which it co-occurs ( Carpenter and Recher 1979 ). My preliminary observations confirmed prolific flowering in B ericifo lia compared to the other Banksia species at my study sites and for this reason much of the research described in this thesis has been concentrated on it.The floral characteristics of the four Banks/# species that are sympatric in my study sites are summarised in Table 1.1.Table 1 . 1: Dimensions of inflorescences and flowers of the four Banks/a species at study sites on the Budderoo Plateau. Data include standard errors of means; sample size in parentheses. ( Data of R. Whelan, pers comm.)Species Ht. of inflorescence (cm) No, flowers Length of style (rom)B. paludosa 8.5 ± 0.3 (50 ) 972.3 ± 28.3 (50) 16.9 ± 0.2 (50)B sp in u tosa 11.8 + 0.5(15) 934.91 42.0(15) 31.9 + 0.3(152)B. e ricifo lia 13.8 ±0.3 (50) 826.01 18.9(50) 33.8 ± 0.1 (138)B .serrata 14.2 ±0.5 (8) 1721.6 ± 61.9 (5 ) 42.9 1 0.6 (40)Each species produces inflorescences typical of the kind thought to be pollinated by birds, especially honeyeaters ( Meliphagidae ), and small mammals such as R atios fu scip es, A n tech in u sstu a rtii and Petaurus b revicep s ( Carpenter 1978. Hopper and Burbidge 1978, Veins and Rourke 1978, Ford e ta l 1979, Veins e ta l 1979, Hopper 1980, Vhelan and Burbidge 1980, Clifford and Drake 1981. Turner 1982, Wooller e l a l 1983,21Lamont e ta l 1985, Paton and Turner 1985, Whelan and Goldingay 1986, Collins and Rebelo 1987, Goldingay e ta l. 1987 ).1.6 THE AIMS OF THIS STUDY.The purposes of this study were to assess whether B serrata, B paludosa, B. spinulosa and B, e rlc lfo lia flower simultaneously in a location where all four species co-occur, and to investigate reproductive success, as measured by fruit-set. Few studies on perennial plants have attempted to monitor factors affecting reproductive success for much more than one year. When the plants under investigation are long-lived, there is an obvious need for more long-term data before conclusions about such things as the adaptive nature of flowering phenologies can be drawn with any confidence.As this project was carried out on a part-time basis, there was an opportunity to investigate reproductive success over an extended period of time In order to do this a number of questions needed to be answered :-(1) What are the precise flowering phenologies of the four Banksia species?(2) What are their natural levels of fruit-set?(3) Is fruit-set limited by the amount of pollen received, ie. is pollination in short supply?(4) Is fruit-set limited by the resources available to an inflorescence?(5) Do any of the above vary from year to year?222. STUDY AREAS AND SPECIES.2.1 BUDDEROO PLATEAU.The study was conducted between January 1983 and December 1986, in two study sites in an area of dry sclerophyll woodland on the Budderoo Plateau, approximately 20 km south-east of Robertson, New South Wales ( Figure 2.1 ). The study sites were selected to contain reasonable abundances of the four species under investigation and because one of the closely adjacent sites contained Banksia ericifo lla while the other did not ( as discussed in Chapter 3, Flowering Phenology )The study sites ( Figure 2.2 ) are part of a gently undulating plateau forming the top of the Illawarra Escarpment, at an elevation of 650 metres, overlooking Jamberoo and the coast between Lake Illawarra and Kiama. The plateau, although formed from very uniform Hawkesbury Sandstone, has a variety of vegetation types ranging from closed heath and sedgeland to woodland. The character of the vegetation is largely determined by local variations in rainfall and drainage ( Burrough e ta l 1977 ).Climatic conditions in the area are typified by warm summers, cold winters and plentiful rainfall ( 16 yr. mean = 2250 mm. per annum ) throughout the year ( Figures 2.3.2.4 and 2.5 )2.2 STUDY SITES.Study site 1,( 34° 39' 10" S, 150° 42’ 15" E ) was an area of open woodland dominated by Eucalyptus gum m ifera, £ sieb eri and Banksia serra ta . Banksia serrata occurred throughout the study site as isolated individual trees and also as an almost pure stand in one area ( Figure 2.6 ). Banksiapaludosa made up a large part of the understory in the moister parts of the study area with B. spinulosa var. spinulosa more prominent in the drier areas. A llocasuarinapaludosa , E pacris Io n g iflo ra , D illw ynia floribu n da , A cacia suaveolens, Leptosperm um attenuatum , M elaleuca squarrosa, HakeaL3Figure 2.1 Location of the Study Sites24F ig u re 2.2 : Photograph shows vegetation typical of the study sites on the BudderuoPlateau251983 1984 1985 1Q86YEARF igu re 2.3 : Monthly rainfall at Barren Grounds Nature Reserve, 1 km from study sitesFigure 2.4 : Annual rainfall at Barren Grounds Nature Reserve, 1 km. from study sites.261983 1984 1985 1986Figu re 2.5 : Temperature variation at Barren Grounds Nature Reserve, 1 km. from study sites.dactyloides and Boron la thujona were also common in the understory throughout the study site.Study site 2, ( 34° 38' 50" S, 150° 42' 15" E ), some 500 metres to the north of site 1, and separated from it by an area of low heath.was virtually identical except for the presence of an extensive stand of Banksia ericifolia var. ericifolia ( Figure 2.7 ). Banksia ericifolia occurs in a poorly drained part of the site,which was often water-logged for a number of days following rain. It is not clear whether the presence of B, ericifolia in site 2 and its absence in site 1 is a result of different drainage patterns, fire histories, or some combination of these and other factors.EscarpmentKEY:B paJudosa B spin ulossB. serrateN20 m.F ig u re 2.6: Distribution of Banksia species in study site 1EscarpmentN +KEY:~j B, paiudosa B spm ulostt ^ B serrate m B ericifolia20 mFigure 2 .7 ; Distribution oiBanksia species in study site 2293- FLOWERING PHENOLOGY31 INTRODUCTION.Members of plant communities do not all flower at the same time. Robertson ( 1895 ) was the first to propose that differences in flowering time might be caused or maintained by competition for pollination. If fruit-set is limited by pollination, it seems logical that natural selection should favour a timing of flowering which maximises pollination ( Agren and FagerstrOm 1980 ). Evidence from a number of field studies has led to the hypothesis that interspecific competition for pollinators should promote temporal segregation of flowering times among co-occurring species ( Mosquin 1971, Stiles 1975, Waser 1978 a ). This hypothesis has been further analysed in a number of theoretical studies ( Levin and Anderson 1970, Waser 1978 b, Waser 1983 ). Arguments that competition for pollination should lead to offset flowering times rest on the assumption that this trait can be changed readily by selection. This appears to be a reasonable assumption because flowering time has been shown to change rapidly under various regimes of artificial and natural selection ( see Waser 1983 for review ). Some studies indicate that flowering time is an extremely flexible trait which suggests that close examination of the flowering phenology of a species may reveal many differences in flowering time among spatially separated populations reflecting local variation in the intensity of competition ( Waser 1983 )In south-eastern Australia, Banksia species ( Proteaceae ) are a conspicuous part of the flora because of their large numbers and also because of the showy inflorescences that they produce. In many sites, a number of Banksia species co-occur and utilise the same guild of, predominately, vertebrate pollinators. For these reasons, banksias are appropriate subjects for an investigation of flowering phenology and the possible adaptive significance of competition for pollinators.Before the late 1970’s there was little reported work on the breeding systems of30the Proteaceae ( Scott 1980 ). In recent years, however, there has been an expansion of interest in the pollination of Australian plants in general ( Hopper 1980 ) and of the genus Banksia in particular, as described in the previous chapters In spite of this, no long-term studies have been done and there is still little detail known about the breeding systems of BanksiaFor most Banksia species, flowering phenologies are known only in the most general terms ( George 1984 ) without any details of variations between different areas and from year to year.The principal purpose of this part of the study was to determine the precise flowering phenologies of the four Banksia species that are sympatric on the Budderoo plateau. Once precise phenologies are known, a number of questions then arise (1) are there significant differences between the two study sites3 *7(2) are inflorescence numbers significantly different in successive years and, i f so, are these differences related to abiotic factors such as temperature and rainfall?(3) how do the data compare with the limited amount available from other studies?(4) do the phenologies suggest that competition between any of the species, for the services of pollinators, is likely?3 2 MATERIALS AND METHODS.LA) Flowering Phenoloaies.In January 1983,15 B. serrata, B. paiudosa and B. spinulosa plants, in each plot, werechosen at random by tagging the first 40 plants ( with evidence of previousflowering ) of each species encountered and then using random numbers to select 15 of the 40. Banksia e ricifo lia was present only in plot 2 so 30 plants were selected in this plot by first tagging 80 plants and then generating 30 random numbers between311 and 80. These 30 plants of each species were used throughout the four years of the study. At intervals of 4-5 weeks between January 1983 and December 1986 each plant was scored for the number of inflorescences in flower. "In flower" was defined as at least 10 pollen-presenters on an inflorescence exserted from their perianths Initially, individual inflorescences were tagged to ensure that they would not be counted twice i f their period of flowering exceeded four to five weeks It soon became obvious that this procedure was unnecessary because (1) although B. paludosa and B. splnulosa can take up to 6-8 weeks per inflorescence to complete flowering ( McFarland 1985, Whelan and Goldingay 1986, and personal observations ), if a cumulative total of inflorescences "in flower" was kept for each plant, it was easy to check whether an inflorescence had been counted previously ( due to the small size of B. paludosa and B. sp in u Joss plants and the relatively small numbers of inflorescences per plant ), and (2) B. serrate and B, erlcifo lla inflorescences take less than four weeks to complete flowering ( Paton and Turner 1985, and personal observations ). The possibility of failing to account for inflorescences of B serrate and B. e ricifo lia that flowered between monthly visits was guarded against by checking cumulative totals wherever plant size and inflorescence number made it feasible. Few, if any, inflorescences were missed or double-counted in this wayAlthough the dispersion of flowering times of species can sometimes he analysed statistically by using random models that can generate null hypotheses ( eg Parrish and Bazzaz 1979, Poole and Rathcke 1979, Rabinowitz e ta l 1981 ), this technique is not statistically applicable to small subsets of species ( Rath cke 1984 )The dispersion of flowering times of the four BanksJa species in this study was determined by simple inspection of the phenological distributions generated from monthly totals of numbers of inflorescences "in flower"32(B) Differences Between Plots.Differences in the phenologies of B. serra te, BpaJudosa and B spinulosa in each of the two plots, were analysed by computing the cumulative percentages of inflorescences of each species that had already flowered by each sample date in a particular year and then using a two-sample Kolmogorov-Smirnov test to assess the difference in the distributions of the two samples.LC) Annual Variation in Inflorescence Production.Differences in numbers of inflorescences in each of the four flowering seasons were analysed using paired t-tests and the results compared with temporal variations in climate ( see Figures 2.3,2.4 and 2.5 )3 3 RESULTS.(A) Flowering Phenologies.Graphs of flowering phenologies over a 4-year period ( Figures 31 and 3 2 ) show that B! serrate flowered during summer, from January to March or April and was therefore phenologically isolated from the other three co-occurring Banksia species Banksiapaludosa f B. spinulosa var sp in u losa , and B, e r ic l fo lia var ericlfo lia all flowered during winter and displayed considerable overlap in both peak flowering and spread of their flowering times. Both onset of flowering and peak-flowering dates showed little variation from year to year ( Figures 3 3 a&b, and Table 3 1 ) Banksia ericifo lia was in flower for the longest period, averaging slightly more than 7 months per year, followed by B, paludosa and B spinulosa both with average flowering periods of about 5 5 months per year All three species were in flower simultaneously for about 5 months each year, although the peak-flowering date of B. spinulosa was always later than the other two species ( Table 3 1)33iMean no. inflors. per plant1086420H B, paludosa B B. serrala Za B.spinuiosaJ M M J S N J M M J S N 1985 1986F igu re 3-1 : Flowering phenology - study site 1Mean No. inflors. per plant14J M M J S N J M M J S N J M M J S N J M M J O D 1983 1984 1985 1986Figure 3.2 : Flowering phenology - study site 2.34dank sw serrataBanksia paludosaFigure 3-3 » . : Cumulative percentage of spent Banksia serrata and B paludosa inflorescences during the years 1983-198635datisi# enct /M aBotisi# spinuhs#Figure 3 .3 b. : Cumulative percentage of spent Banksia en ei folia and D spinulosainflorescences during the years 1983-198636Table 3-1: Months of peak-flowering of B. serrate, B. ericifo lia , B. paJudosa and B spin ulosa over the study period1983 1984 1985 1986Banksia serrate February February January FebruaryBanksia e ric ifo lia June July June June-JulyBanksia paludosa June July June JulyBanksia spinulosa August July-August July-August July-AugustLB) Differences Between Study Sites.Analysis of the distributions of inflorescence numbers ( expressed as cumulative percentages of the years total ) in each of the study sites using the two-sample Kolmogorov-Smirnov test ( Table 3 2) showed that flowering times of B. paJudosa , B spinulosa and B. serrate were not statistically different between sites ( B. ericifo lia occurred in only one of the plots ).Table 3-2 : Differences in the distributions of flowering times of two groups of 15 plants of the same species in two study sites on the Budderoo Plateau.Species/Year Probability that both samples cc population ( H0 ) . ( KolmogorovB serm ta/ 1983 P =0.419B serrate/ 1984 P =0.419B. serrate/ 1985 P = 0.419B serrate/ 1986 P =0.419B spin ulosa/ 1983 P =0.419B\ spin ulosa/ 1984 P = 0.342B spin ulosa/ 1985 P = 0.419B spin ulosa/ 1986 P = 0.342B paludosa / 1983 P = 0.342B paludosa / 1984 P = 0.342B, paludosa / 1985 P =0.419B paludosa / 1986 P = 0.34237ICLAnnual Variation in Inflorescence ProductionPaired t-tests indicate that there were highly significant differences in inflorescence numbers for all species when alternate years were compared ( Figure 3.4 ) There was no significant difference between numbers of inflorescences produced in the two poor-flowering years ( 1984 and 1986 ) or between numbers produced in the two prolific years (1983 and 1985 ), except in the case of B. e ric ifo lia , which produced such a large crop of inflorescences in 1985 that it was significantly different to all other years in which this study was carried out3.4 DISCUSSION.Of the four species of Banksia that co-occur on the Budderoo Plateau, one flowers during summer and the other three show considerable overlap in their flowering during winter. This situation is very similar to that reported by Whelan and Burbidge ( 1980 ) and Lewis and Bell ( 1981 ) in Western Australia, where the summer-flowering species, Banksia attenuate, was phenologically isolated from the three species ( B m enziesii, B. liU o ra lis and B. telm atiaea ) with which it co-occurred in their study areas. Flowering time for each species was remarkably consistent with the dates for onset of flowering and peak-flowering varying by no more than 4 weeks throughout the four years of the study ( Figures 3 3 a, 3 3 b and Table 3-1 ) ( the variation may have been less than 4 weeks, but phenology data, collected monthly, do not allow more precision than this ). Length of flowering season appeared to bear no relationship to the number of inflorescences produced by individual plants in a particular year This contrasts with the data of McFarland ( 1985 ) for B. oblongifolia and B spinulosa var. co llin a which suggested that duration of flowering was positively correlated with the number of inflorescences produced by plants. Banksia serrate the summer-flower in g species, was phenologically isolated from B. ericifo lia , B paiudosa38(A) Banks/* serrât*(B) Banks/apaludosa(C) Banks/* sp/nu/osa(B) Banks** arid folia1984 **#1985 N.S * * * ' - - -1986 * * * N.S. * * *1983 1984 19851984 ###1985 N.S. * * *1986 *#* N.S. * * *1983 1984 19851984 #1985 N.S. * * *1986 N.S. N.S. 1* *!1983 1984 19851984 * * *1985 ###1986 *## N.S. * * *1983 1984 1985Figure 3.4 : Left-hand side - mean number of inflorescences per plant per year. Bars represent standard errors.Right-hand side - significance of betT/een-year differences using paired t-tests ( * * P < 0.05, ** * P < 0.01, * * * * P < 0.001 ).39and B sp in u los». all o f which flowered between late autumn and early spring The period when all three winter-flowering species flowered together was usually as long as five months, although peak flowering times did not usually coincide ( Table 3 1 ) In most years Banksiapaludosa reached peak flowering first in May-June, followed by B e ricifo lia in June-July and finally B. spinulosa in July-AugustInformation on flowering phenologies with which to compare my results is limited to a general treatment given in George ( 1984 ) for all Banksia species and to a study by McFarland ( 1985 ) of B, spinulosa var. collin a ( Table 3 3 ) Limited though it may be, the available data suggest that although the flowering phenologies for these Banksia species may be consistent from year to year at the same site, there is considerable variation in their flowering times across their geographical rangeTable 3-3 : Comparison of flowering phenologies reported by George ( 1984 ), McFarland ( 1985 ) and this study.Month.Species. Ja n . Feb. Mar. Apr. May Ju n . Ju i. Aug. Sep. Oct. Nov. Dec.B serrate _____________________________________________( George 1984)B. serrate _____________________( this study)B. paJudosa ----------------------------( George 1984 )B. paJudosa ---------------------------------------------------------( this study )B e ric ifo lia var. e r i c i f o l i a -----------------------------------( George 1984 )B e r ic i fo lia var. e r ic i fo lia -------------------------------------------( this study)B. spinulosa var. spinulosa -----------------------------------( George 1984 )B spin ulosa var. spinulosa ------------------------------------( this study)B spin ulosa var collin a --------------------------------------------------------- -( McFarland 1985 )40McFarland ( 1985 ) found that a B! in teg rifo lia population in the New England National Park ( elevation 1400 m .) had a shorter flowering season than coastal populations of the same species. The summer-flowering Banksia serrate population in my study sites ( at an elevation of 650 metres ) showed a similarly shortened flowering season compared to that displayed by populations of B. serrate at sea-level at the same latitude ( R. Whelan pers. comm. ). Populations of win ter-flowering B. paludosa and B ericifo lia in my study sites, in contrast, had flowering periods that were longer than those reported by George ( 1984 ) for coastal populations of the same species McFarland ( 1985 ) reported a similar finding for the winter-flowering B spinulosa va,r. co llin a at his study sites and suggested that the extended flowering season was caused by low temperatures at the higher altitudes, which slowed down the rate of anthesis of flowers. My study sites experienced temperatures that were usually 5-10 °C lower than those of the adjacent coastal plain and winter frosts were not uncommon If McFarland ( 1985 ) was correct, longer flowering times would be expected in banksias subjected to these lower temperatures. It may also be expected that B spinulosa var. spinulosa, flowering at an elevation of 650 m. in my study sites, would have a flowering season intermediate in length between that reported by McFarland ( 1985 ) for B. spinulosa var. cotlina growing at 1400 m. and George ( 1984 ) for B spinulosa var. spinulosa growing atsea-level ( Table 3 3 ) ( assuming that low temperatures have the same effect on both sub-species ). Although I found no marked increase in the length of the flowering season compared to that reported by George ( 1984 ) it is clear that B. spinulosa continues to flower until later in the year in my study sites compared to those at sea-level, but not as late as those at 1400 m. in the New England National Park.Phenology and Competition forJPollin&tion.Banksia e ric ifo lia B ,spinulosa, and B paludosa have phenologies that suggest that competition for pollinators is possible between them. This possibility is further41strengthened by a consideration of the floral morphology of the three species and the pollinators that they attract. Each of these banksias produces inflorescences typical of the kind thought to be pollinated by birds, especially honeyeaters ( Meliphagidae )( Hopper and Burbidge 1978, Ford e ta l. 1979, Hopper 1980, Whelan and Burbidge 1980, Clifford and Drake 1981, Wooller e ta l. 1983, Patón and Turner 1985, Collins and Rebelo 1987 ); non-flying mammals such as RalUts fu scip e s, A n tech in u sstu a rtii, and Cercartetusnanus ( Carpenter 1978, Weins and Rourke 1978, Weins e ta l 1979, Turner 1982, Whelan and Goldingay 1986 ); gliding marsupials such as Petaurus breviceps ( Goldingay e ta l. 1987 ); and possibly introduced honeybees, A p is m elíifera ( Patón and Turner 1985 ) Banksiapaludosa has relatively narrow (7-13 cm. long, 3-4 cm wide ( George 1984 )), straight-styled inflorescences which are often cryptic and closeto the ground. Banksia ericifo lia and B spinulosa have very similar inflorescences ( see Figures 1.7 b and 1.8 b ). Flowers of both these species have hooked styles and are of similar length ( B .ericifotia :- 30-35 mm., B. spinulosa :- 30-40 mm. ( George 1984 )) and shape. In view of these similarities, it would appear that if there is competition for pollinators amongst these co-occurring Banksia species, it is most likely to be between B! e ricifo lia and B. sp in u losa . Competition for pollinators may eventually result in the evolution of offset flowering times due to the decline in reproductive success experienced by the less-successful competitors when the species sharing a particular suite of pollinators are in sympatry ( Levin and Anderson 1970, Mosquin 1971, Stiles 1977).Flowering phenologies for both study sites were very similar ( Table 32). The literature on flowering phenology as an evolutionary response to competition for pollination leads to the suggestion that flowering times of an inferior competitor would be different in the presence and in absence of a superior competitor ( Waser 1983 )• Thus it was hypothesised that the presence or absence of the prolific flowering B. e ricifo lia may have led to local differences in the flowering times of B spinulosa. This was not the case: B. spinulosa phenologies were not detectably different between42the study sites in any of the four years. This is not a surprising finding, however, for various reasons (1) The close proximity of the plots ( 500 m .), allowing gene flow between them via pollen and/or seeds. (2) Consideration of the relative abilities of the species involved to change their flowering phenologies over time. There is evidence that flowering time may be heritable ( Gross and Werner 1983 ), but for changes to occur, plants that have an evolutionary advantage due to their particular phenology must have the opportunity to produce large numbers of offspring. These better adapted individuals can then compete with an increasingly diminishing population of plants that flower at some other, less fortuitous, time. Of the three Banksia species mooted as possible competitors in this study, B. ericifo lia is suggested as the most likely to be able to modify its phenology over a given period of time. Banksia e ricifo lia is an obligate re-seeder, producing large numbers of new seedlings after fires that kill the parent plants. This rapid turnover of the population can occur in as short a time as 5-10 years. Banksiapaludosa and B spinulosa are very long-lived plants, re-sprouting from lignotubers time and again after fire. Exact life-expectancies of these resprouters are not known, but it may well be that some individuals live for centuries. With low levels of seed-set and continued dominance of parent plants after fire, it seems unlikely that there can be any rapid adjustment in the flowering times of populations of B.paludosa and B. spin ulosa . (3) The impact of a co-occurring population of B. e rie ifo lla may often be transient, relative to the lifetimes of B. spinulosa and B. paludosa individuals, especially if fires are common and consecutive fires occur within a few years of each other, before the new, even-aged population has an opportunity to establish a seed-bank. So B. paludosa and B spin ulosa appear doomed to suffer from competition from B ericifo lia whenever it co-occurs. It is also possible that the study-sites used in this project were on a narrow zone of overlap between the largely disjunct ranges of B. ericifo lia and B spinulosa Competition for pollination may, therefore, explain past evolution of different site requirements for the two species.43Annual Variation in Flowering Intensities.Inflorescence production ( Figures 3.1,32 and 3.4) appears to follow a two-year cycle of abundance followed by scarcity. Years of apparent superabundance, such as in 1985, may also follow a cycle but many more years of observation will be needed to determine whether this is so. Examples of regular patterns of flowering are not uncommon. However, little work has been done on this aspect of plant biology in Australia. Ashton ( 1973 ), in a long-term study on Eucalyptus regnaas > found distinct flowering periodicity with a four-year cycle of prolific flowering in the 1950 s and a 2-year cycle in the same plants in the early 1960‘s. Abbott ( 1985 ) also found year-to year fluctuation in flower numbers in a four-year study on Baokslagrandis McFarland ( 1985 ) found yearly differences in inflorescence numbers produced by the same group of Banksia in teg rifo lia and B spinulosa plants These differences were ascribed to climatic factors such as rainfall and temperature Consideration of temperature and rainfall data at my study sites ( Figures 2.3,2.4 and 2 5 ) indicates that there is no clear link between them and inflorescence production Temperature ranges were very similar in all years of the study Rainfall was highest in 1985, the year of superabundant flowering ( especially for B. ericifo lia ), but was also higher than average in 1984, a poor flowering year. The other prolific flowering year, 1983, was a year of only average rainfall. Similarly, monthly distribution of rainfall in each of the four years did not suggest any pattern that could be related to inflorescence productionThe reasons for a regular flowering rhythm are complex and both carbohydrate storage and carbon/nitrogen ratios have been proposed as determining factors ( Klebs 1910, Ashton 1973 )• Mooney eta lX 1975 ) have shown that carbohydrate levels in most species determine the number of floral primordia laid down and that carbon gain is correlated with precipitation in perennial shrubs Keeley ( 1977 ) showed a correlation between flower and fruit production in one year and rainfall in the previous year in four species of evergreen chaparral shrubs This44does not explain why, in my study, there was such a large fluctuation in inflorescence numbers over four years when rainfall was above average for three years in a row ( Figure 2.4 ). It may well be that when rainfall is exceptionally low, it becomes the limiting factor determining inflorescence numbers. However, when rainfall is consistently at, or above some threshold, then other factors such as the levels of phosphorus and/or nitrogen may become limiting such that the plant needs a greater period of time than one year to accumulate sufficient nutrients to allow an episode of prolific flowering, especially after a season of high fruit-set. This is, of course, purely speculative and in need of much more research because little is known about the relative contribution of stored versus current assimilates for flower production and fruit maturation ( Stephenson 1981 ). However, evidence that makes ita reasonable hypothesis comes from the fact that these banksias are growing in soil that is low in both nitrogen and phosphorus ( Beadle 1966 ), and thatZammit( 1986 ) demonstrated, by adding fertilisers, that available phosphorus can limit the number of inflorescences produced per plant in Baaksia eririfo lia and B obioagifoliaConclusionsIn this chapter I have shown that three of the four co-occurring Baaksia species in my study sites had substantially overlapping flowering times. If these species share the same suite of pollinators, as suggested by all available evidence, then it is likely that they will compete for the services of these pollinators, i f pollination service is in short supply. One way to determine whether pollination is limiting reproductive success is to test the effect on natural levels of fruit-set ( an indicator of reproductive success ) of supplementing the amount of pollen available to inflorescences. Natural fruit-set and the effect of pollen supplementation are reported in the following chapters.454, FRUIT-SET AMR FACTORS INFLUENCING IT : AN INTRODUCTIONFruit-set can be used as an indicator of plant reproductive success or fitness ( Zimmerman 1980 a, Augspurger 1981,Rathcke 1983 ) It is not uncommon for plants to produce many more Hovers than fruits ( Stephenson and Bertin 1983 ) and lov fruit-sets have been reported in the literature for a number of flowering plant species ( eg. Schemske e ta l. 1978, Willson e ta l 1979, Bierzychudek 1981, Stephenson 1981 ). The Australian Proteaceae also have very low fruit-sets ( Johnson and Briggs 1975, Rourke and Weins 1977, Whelan and Burbidge 1980, Pyke 1981, Pyke 1982, Lamont e ta l. 1985, Collins and Rebelo 1987 ). Lamont e ta l. ( 1985 ) reported a mean value for fruit-set, relative to Hovers produced, of 3 4%, based on data from 9 genera of Australian Proteaceae. Baaksia species have the lowest fruit-sets of all the genera of Proteaceae for which there are data, with a mean of 2 3% ( Hopper 1980, Whelan and Burbidge 1980, Levis and Bell 1981, Wooller e ta l. 1983, Lamont e ta l 1985, Collins and Spice 1986, Collins and Rebelo 1987 )Various hypotheses have been advanced to explain low levels of fruit-set, suggesting that (i) pollination is insufficient ( Waser 1979, Hopper 1980, Zimmerman 1980 a, Bierzychudek 1981, Gross and Werner 1983, Rathcke 1983, Wooller e ta l 1983, Whelan and Goldingay 1986 ), (ii) nutrients are in short supply ( Willson and Rathcke 1974, Schemske e ta l. 1978, Motten e ta l. 1981, Stephenson 1981, Lamont e ta l 1985, Paton and Turner 1985, Collins and Spice 1986 ), (iii) space is insufficient for all fertilised ovules to develop ( Collins and Spice 1986 ), (iv) there are numerous physiological and genetic barriers such as low pollen viability, ovule abortion, high levels of self in g leading to the expression of recessive lethality and andromonoecious Hovers ( Johnson and Briggs 1975, Stephenson 1981, Collins and Rebelo 1987, Carthew e ta l. 1987, S. James ( pers. comm.)), (v) predators are removing, or arresting the development of, large numbers of seeds ( Scott 1982, Zammitand Hood 1986 ), (vi) pollinator attraction requires the production of many Hovers to provide a reasonable46floral display, and (vii) large numbers of flowers are produced to give tiie plant a choice of which zygotes to develop It is important to note that more than one factor can limit fruit-set ( Gross and Werner 1983 ) ( eg. Zammit ( 1986 ) displayed both nutrient and ’ predation" limits to fruit-set), and that the interaction of a suite of factors may change over space and time, or even during the flowering period of an individual flower ( Primack 1980, Rathcke 1983 ). This obviously cautions against using single-sample surveys for making generalisations ( Wiens 1981 ). as has often been the case in studies of fruit-set limitationGross and Werner ( 1983 ) suggested that physiological or environmental causes for differences in fruit-set can be ruled out by performing hand-pollinations on plants which flower at different times throughout the flowering season Natural fruit-set per flower must be compared with that of a simultaneously hand-pollinated sample because of the potential for temporal variation in other factors such as abortion rates, self-fertility, or pollen viability that affect fruit-set ( Rathcke 1983 ) Pollination would be seen as limiting if addition of extra pollen ( over and above natural levels of pollen received by flowers ) resulted in a consistent increase in fruit-set. Pollination limitation of fruit-set can result from a shortage of suitable pollinators, failure of pollinators to deliver sufficient suitable pollen, or clogging of stigmas by inappropriate pollen.The purpose of this part of my study was to assess the degree to which fruit-set in three co-occurring, simultaneously flowering Banksia species was limited by pollination. In order to do this, it was first necessary to establish the level of natural fruit-set in each species and this is the subject of Chapter 5. In Chapter 6 ,1 report on the effect on fruit-set of experimentally supplementing natural pollination Because of its prolific flowering and long flowering season, Banksia ericifo lla was selected for additional experiments on (i) the determination of levels of pollination, by counting numbers of pollen grains and tubes per stigma ( Chapter 7 ); and (ii) a manipulation to assess whether resources are differentially allocated to ovules that47are pollinated earlier in the flowering season ( Chapter $ )485. VARIATION IN NATURAL LEVELS OF FRUIT-SET.5.1 INTRODUCTION.In the Australian Proteaceae, percentages of flowers per inflorescence that set fruit are very low. Values for individual species range from 01% in Banksia cham aephyton and 3. eJegans to 16.3% in Hakea erinacea ( Collins and Rebelo 1987 ) The figures given for percentage fruit-sets are generally based on studies completed in one particular year and usually, for convenience, at the time of peak flowering of the species in question. However, differences in fruit-set at different times during the flowering season of a plant species have been reported by a number of researchers ( eg. Carpenter 1976, Schemske 1977, Schemske eta l. 1978, Waser 1978a, Meiampy and Hayworth 1980, Zimmerman 1980a, b ). In view of this, it is important to know whether figures for fruit-set are consistent throughout the flowering season, or whether they vary depending on the part of the season in which the flowers were functional.The three co-occurring, simultaneously flowering Banksia species, B, ericifo Jia , B. spinulosa and B. paludosa all have quite long flowering seasons ( Chapter 3 ) and, consequently, there is opportunity for the myriad factors that can influence fruit-set to vary during this time, with the result that there may be variation in the numbers of fruits produced per flower within the season. In addition, i f one is interested in the factors that impinge on fruit-set such as pollination and nutrient availability, it is essential to realise that the relative importance of each can change within a flowering season and also between seasons ( Gross and Werner 1983). In this chapter I report on the results of a three-year study on natural fruit-set in Banksia e ricifo lia ,B . spinulosa and 3 . paludosa.495 2 MATERIALS AND METHODS.After initially assessing flowering phenology in 1983 ( see Chapter 3 ), a study of fruit-set in B e n d fo lia commenced in 1984. The flowering season of B e n d fo lia was divided into three parts regarded as early ( April ). peak ( July ) and late ( September ). During each part of the season, functional inflorescences ( inflorescences which had just begun to open flowers ) on 15-20 randomly selected trees were marked with aluminium tags and left until the following January when they were harvested and scored for number of mature follicles. Numbers tagged at each time depended on the availability of inflorescences within the study site and varied between 28 and 117 during the three years of the investigationDuring 1985 the study was extended to include both B. paludosa and B. spin ulosa Although 1985 was a relatively prolific flowering year for all Banksia species in my study sites, there were nevertheless insufficient B spinulosa inflorescences to allow three separate investigations of fruit-set, so only one was completed at the peak of flowering. The same was true of B. spinulosa in 1986. Banksia paludosa flowered in large numbers in 1985, allowing early, peak and late tagging of inflorescences, but flowering was so poor in 1986 ( see Chapter 3 ) that no further work on this species was feasible during this year.A study on the effects of supplementing natural levels of pollination was also started in 1985 ( Chapter 6 ) and a separate group of control inflorescences was tagged for comparison with pollen-added inflorescences. This sample of control inflorescences did not differ statistically from the inflorescences used in the natural fruit-set determination so it was considered appropriate to add data from both groups together to increase the sample sizes in this study of natural fruit-setIn 1986, as well as scoring number of mature follicles per B. ericifo lia and B. spin ulosa infructescence ( fruiting “cone" ), data were also collected on the spatial distribution of follicles on non-barren cones ( "nbc" ) of B e n d fo lia , B. spinulosa50and B, paludosa. Following the method of McFarland ( 1985 ), follicle distribution was described as either “whole" ( follicles present over the entire cone ), "top" ( follicles on the upper two-thirds of the cone ) or "bottom" ( follicles on the lower two-thirds of the cone ). The lengths of all B. e ricifo lia cones were also measured From these lengths, the number of flowers that were present on each cone can be calculated using the regression equation, y = 53 5x + 92.6 , where y is number of flowers and x the length of the inflorescence (cm.), suggested by Patón and Turner ( 1985 ) for B e ric ifo lia . This allows fruit-set to be expressed in three ways :- ( 1 ) as a percentage of the estimate of number of flowers produced, (2) as a proportion of non-barren cones to total cones, and (3) as the mean number of follicles per non-barren cone Using known values for the percentage of non-barren cones, mean number of follicles per non-barren cone and the number of inflorescences produced on individual plants during a particular year, seed output per plant per year can be calculated Baaksia ericifo lia produces only one viable seed per follicle ( R Whelan pers comm ) so yearly seed output in this species can be estimated as : S = no inflors x proportion non-barren x mean no. follicles per non-barren cone5.3 RESULTS.The considerable annual variation in numbers of inflorescences ( see Chapter 3 ). was also evident in fruit-set. Fruit-set was always low throughout the three years of the study ( eg. in 1986, on average, only 2.5% of the estimated number of flowers of B. ericifo lia set fruit) ( see Table 5 6).The production of seed-bearing follicles per inflorescence in B ericifo lia , B spinuJosa and B. paludosa in 1986 showed a distinct bimodal pattern ( Figures 5 1,5 2, and 5 3 ) . This pattern has already been reported for B. ericifo lia ( Patón and Turner 1985 ; Zammit and Hood 1986 ) and for B. oblon gifolia ( Zammit and Hood 1986 ) It results from the large numbers of Banksia inflorescences that set no seed51No. of Follicles.Figure 5.1 : Frequency distribution of follicles produced by B.paludosa inflorescences in 1986,No.of FolliclesFigure 5.2 : Frequency distribution of follicles produced by B. ericifo lia inflorescences in 1986.52100 110 120No, of FolliclesF igu re 5-3 : Frequency distribution of follicles produced by B. spinulosa inflorescences in 1986.Furthermore, it is this bimodality which led Whelan and Burbidge ( 1980 ) and Whelan and Goldingay ( 1986 ) to measure fruit-set by a combination of two measures the proportion of non-barren cones and the mean number of follicles per non-barren cone. The percentage of non-barren cones developing from inflorescences ( %nbc), showed significant within-year and between-year differences during the three years of this study ( Tables 51 and 5.2 ). In 1984, late-flowering B. erlcifolla inflorescences produced a significantly lower proportion of non-barren cones ( 27,7% ) than bothearly ( 63 89% ), ( X2j = 10.03, P < 0.01 ), and peak-flowering ( 68 38% ) inflorescences53Table 5.1 : Natural fruit-set : "n" « number of inflorescences; "nbc" * non-barren cone; means are ± s.e.m; ( size of sample used to calculate mean)(À) B. e r ic ifo li* .Early Peak Laten Xnbc mean follicles per nbc n Xnbc mean follicles per nbc n Xnbc mean follicles per nbc1984 36 6389 44.17 ± 3 30(23) 117 68.38 45.86+ 1.86(80) 47 27.70 41.38 + 4.27(13)1985 53 50.94 39.78 + 2.94(27) 58 32.76 40.37 ± 2.11(19) 58 12.07 43.71 +6.63(7)1986 28 32.10 24.22 ± 3 44(9) 28 57.10 47.00 ± 2.78(16) 29 41.40 49.00 ±8.49(12)(B) B. p ila d o s* .Earlymean follicles n X nbc per nbcPeakmean follicles n X nbc per nbcLatemean follicles n X nbc per nbc1985 61 22.95 32.86 ± 3-86(14) 56 42.85 40.46 + 4.55(24) 30 20.00 44.00 ± 5 32(6)(C) B. sp m u losa Peakn Xnbc mean follicles per nbc1985 56 10.71 46.00 ± 4.63(6)1986 110 42.73 52.92 + 3.64(47)Table 5.2 : Inter- and intra-year differences in natural fruit-set as measured by the proportion of non-barren cones produced from inflorescences ( ’̂ nbc" ), using chi-square tests for dependence between barrenness and season ( E * early, P = peak, L = late; * = P<0.05, ** = P<0.01, *** -P <0.001, N.S = not significant. )(À) B. e r ic ifo li*.Differences in %nbc.E vsP P v s L E v sL1984 N.S. * * * * *1985 N.S. ♦ * ***1986 N.S. N.S. N.S.Differences in %nbc84vs'85 85vs 86 ‘84vs'86Early N.S. N.S *Peak * * * * N.S.Late * * * N.S.(B) B. pm ludos*.Differences in %nbc. E ys P P ys L E vs L 1985 * N.S.(C) B. spinuJos*.Differences in %nbc. '85vs'86Peak * * *55( X2| = 18.29, P <0.001 ). The same was true in 1985, late-flowering inflorescences being significantly less successful ( 12.07% ) at producing non-barren cones than either early-flowering ( 50.94% ), ( X2j = 19.82, P < 0.001 ), or peak-flowering( 32.76% ), ( X2 j * 7.14, P < 0.01 ) inflorescences. In 1986, a particularly poor flowering year, there were no statistically significant differences in %nbc between early and peak (X 2 J =3 54 ), peak and late ( X2 j =1.49 ), or early and late (X 2j =0.51 ) inflorescences. Percentages of non-barren cones, resulting from inflorescences at peak flowering, were significantly different in successive years ( 1984/1985, X2 j *19.93, P < 0.001 ; 1985/1986, X2 j = 4.63, P < 0.05 ), as were those developing from lateinflorescences ( 1984/1985, X2, = 4.01, P <0.05 ; 1985/1986, X2, =9 87, P <0 01 ) Thelimited data collected for B. paludosa in 1985 show a similar variation in %nbc during its flowering season ( Tables 51 and 5 2). Percentage of non-barren cones resulting from peak-flowering B. paludosa inflorescences ( 42.85% ) was significantly higherthan that from early-flowering ( 22.95% ), ( X2 j = 5 26, P < 0.05 ) and late-flowering( 20.0% ), ( X2, = 4.56, P < 0.05 ) inflorescences. As previously mentioned, low numbersof B. spinulosa inflorescences in my study sites precluded the division of its flowering season into three meaningful parts so data are available only for ‘peak'-flowering in 1985 and 1986 The percentages of non-barren cones produced inthese successive years ( 1985 :10.71%, 1986 :42.73% ) differed significantly ( X2 j =17 5, P< 0.001 ).Mean number of mature follicles on non-barren cones was much less variable than %nbc. Analysis of the data for B erlclfo lia using 2-way ANOVA ( Table 5 3 ) showed no consistent difference in mean follicles per non-barren cone at different parts of the flowering season ( P = 0.063 ) or from year to year ( P = 0 .486 ) There was,56Table 5-3 : ANOVA table showing the relationship between B ericifo lia meanfollicles per non-barren cone, time of the flowering season ( early, peak and late ) and year ( 1984 - 1986 ).Source Sum of Squares Deg. of Freedom Mean Squares F-Ratio Prob>FBetweenTime 1476.514 2 738.257 2.806 0.063BetweenYear 381.194 2 190 597 0 724 0 486Interaction 3116.566 4 779.141 2.961 0.021Error 51837.941 197 263.137Total 56812.214 205Table 5.4 : ANOVA table showing the relationship between B\ paludosa mean follicles per non-barren cone and time of the flowering season ( early, peak and late ) in 1985Source Sum of Squares Deg. of Freedom Mean Squares F-Ratio Prob>FBetweenTime 719.305 2 359.652 0 985 0.382Error 14969 673 41 365 114Total 15688.977 4357however, a significant interaction effect ( P = 0.021 ). Inspection of the data ( Table 51 ) indicates that the source of this interaction effect is the low value ( 24 22 ) for mean follicles per non-barren cone at early- flowering in 1986 This mean was significantly lower than that obtained at peak-flowering ( 47.0 follicles per nbc). ( t = 5 046, P < 0.001 ) and late-flowering ( 49.0 follicles per nbc ), ( t = 2 406, P < 0 05 ) in 1986. It was also significantly below the early-flowering data in 1984 ( 44 17 follicles per nbc ), ( t = 3 482, P < 0.01 ) and 1985 ( 39.78 follicles per nbc ), ( t - 2.831, P < 0.01 ) Banksiapaludosa did not differ significantly in mean number of follicles per nbc during 1985 ( 1-way ANOVA, P = 0.382 ) at the study sites ( Tables 51 and 5 4 ) Banksia spinuJosa also showed no significant differences in mean follicles per non-barren cone between 1985 and 1986, ( t = 0.665, P = 0.509 )Estimation of the yearly seed output of 30 B ericifo lia plants ( Table 5 5 ) shows that, although the proportion of non-barren cones was significantly lower during 1985 than in 1984 or 1986, and mean numbers of follicles per non-barren cone were virtually constant from year to year, total seed output in 1985 was still very much greater than in 1984 and 1986. Note, however, that in the calculation of these figures, the proportion of non-barren cones is an average value for the year, and that it proved to be quite variable within most years. The huge contribution to the seed-bank in 1985 reflects the prolific inflorescence production of B. ericifo lia in that year which, in almost every plant, more than compensated for the lower percentage of fruiting cones produced from inflorescencesA highly significant relationship between the length of a Banksia ericifo lia inflorescence and the number of flowers produced on it was expected ( Patón and Turner 1985 ) The percentage of B. ericifo lia flowers forming follicles during the 1986 flowering season ( Table 5 6 ) was estimated using the regression equation derived by Patón and Turner ( 1985 )58Table 5-5 : Estimated seed outputfor 30 taggedB .ericifo lia plants during 1984,1985 and 1986.( seed output = no. inflorescences x %nbc x mean follicles per nbc ). N.B. These data take no account of seed consumption by insect larvae within cones ( see Zammitand Hood 1986 )1984 1985 1986Plant No. No. inflors. Est. seed output No. inflors. Est. seed output No. inflors. Est. seed output1 1 255 47 616.4 3 5232 7 178.7 39 5115 0 03 7 178.7 62 8132 1 17.44 1 255 53 695.1 7 122.15 3 76.6 118 1547.7 1 17.46 0 0 21 275 2 14 244.27 10 2553 55 721.4 16 279.18 6 153.2 54 708.3 1 17.49 0 0 40 524.6 33 575 610 18 459,5 106 1390.3 8 139.511 1 255 33 432.8 0 012 0 0 11 144.3 1 17413 14 357.4 48 629.5 0 014 0 0 37 485 3 5 87215 30 765 8 76 996.8 20 348.916 4 102.1 61 800.1 14 244.217 10 2553 27 354.1 2 34918 7 178.7 22 288.5 0 019 2 51 44 577.1 2 34.920 17 4339 61 800.1 11 191 921 1 255 38 498.4 3 52322 8 204.2 52 682 0 023 12 306.3 16 209.8 1 17424 10 2553 8 1049 0 025 1 25 5 27 354.1 0 026 0 0 21 275 4 5 87 227 4 102.1 58 760.7 0 028 0 0 46 603 3 2 34,929 2 51 33 432 8 12 209330 11 280.8 72 9443 2 349Mean+ s.e m 6 2 ± 13 159 1 ± 32 5 46.2 ± 4 6 605 9 ± 59.9 5-5 ±1.4 95 3 + 24.459Tabic 5.6 : Estimates of percentage of B. ericifo lia flowers producing follicles during the early, peak and late parts of the 1986 flowering season(% ) EARLY PEAK LATE0.98 3 64 2 96Mean lengths of inflorescences ± s.ern. at early ( 13 14 ± 0.46 cm (N = 28)), peak ( 12.04 ± 0.54 cm.(N «28)) and late ( 11.52 ± 0.69 cm.(N =29)) flowering times were not significantly different ( 1-way ANOVA; P = 0.129 ) ( see Appendix ), so the difference in percentages of flowers setting fruit at early, peak and late flowering times is wholly attributable to the significant difference in mean follicles per non-barren cone during early-flowering, and not to any difference in mean number of flowers per inflorescence.Large B. ericifo lia inflorescences ( > 14 cm. ) were more likely to producenon-barren cones than small inflorescences ( í 14 cm ), ( X2j = 8.59, P <0 01 ) Fornon-barren cones, analysis by simple linear regression showed that there was also a significant positive correlation between size of inflorescence and number of follicles ( Regression coeff. R = 0.497, df. * 36, P < 0.01 ) in 1986Follicles on fruiting cones of B ericifo lia , B. spinulosa and B paludosa ( Figure 5 4 ) were positioned, with few exceptions, either on the bottom two-thirds of the spike or all over it. Approximately 75% of B. spinulosa and B. paludosa non-barren cones have follicles spread over the entire cone whereas, in B. ericifo lia , the figure is closer to 50%.60PercentageofConesB. eric i folia ( N * 1 0 9 ) B. spinulosa ( N * 47 ) B. paludosa ( A *■ /1 0 JWhole Cone None on Top 1/3 None on Bottom 1/3Figure 5-4 : Distribution of follicles on fruiting cones in 1986.5.4 DISCUSSION.Fruit-set in the genus Banksia is generally very low ( Hopper 1980, Whelan and Burbidge 1980, Levis and Bell 1981, Wooller etal. 1983. Lamont etal. 1985, Collins and Spice 1986, Collins and Rebelo 1987 ).The three co-occurring, simultaneously flovering Banksia species in this study proved to be no exception to this general rule. Banksia ericifoiia, for example, during the 1986 flovering season, converted, on average, only 2,5% of its flovers into mature follicles,A surprise finding ( also recently reported by Whelan and Goldin gay ( 1986 ) ) is that the percentage of inflorescences producing non-barren cones varied61considerably both within and between years ( Tables 51 and 5.2 ), whereas the mean number of follicles produced per non-barren cone was much more constantComparison with data from other studies suggests similar temporal variation in fruit-set, measured by the proportion of non-barren cones produced. Although the value of 10.7 for %nbc obtained in this study for B. sp in alosa in 1985 is not significantly different from the 19% reported by Whelan and Goldingay ( 1986 ) during 1983 for B, sp ln a losg in study sites less than one kilometre away from mine ( Table 5 7 ), it is significantly different to their 1984 figure of 25%. The 1986 figure of 42.7 for %nbc ( this study ) is significantly different to both figures obtained by Whelan and Goldingay ( 1986 ).McFarland ( 1985 ) provided the only other published data for B. spinulosa ( although his study was on var. co llln a , not var. spin alosa), finding that 65% of cones were non-barren in New England National Park during the years 1981 to 1983Table 5.7 : Proportions of non-barren cones reported for Banksm spm ulosa in 1983/84 by Whelan and Goldingay ( 1986 ) and in 1985/86 ( this study )BARREN NON-BARREN X NON-BARREN SIGNIFICANCE1CONES CONES1983 ( Whelan and Goldingay ) 97 23 19.2 a, b1984 ( Whelan and Goldingay ) 54 18 250 a1985 ( This Study ) 50 6 10.7 b1986 ( This Study ) 63 47 42 7 c1 Samples with the same letter are not significantly different at the 0 05 level62The number of follicles per non-barren cone in each of the three species was almost constant, both within and between years, throughout this study. Means for B e ricifo lia ( Table 5-1 ) varied little over the three years and were very similar to the mean of 47.8 follicles per non-barren cone found by Patón and Turner ( 1985 ) for B e ric ifo lia . Whelan and Goldingay ( 1986 ) also reported consistency in mean follicles per nbc within the same year for B, spin u/osa and B. paludosa, and between years for B sp in u losa . In the case of B. paludosa, however, they found a significant difference in mean follicles per nbc between 1983 ( mean = 30.2 ) and 1984 ( mean = 19.3 ) The mean of 46.0 follicles per nbc for B. spinulosa in my study sites during 1985 was not significantly different to the 1986 mean of 52.92 follicles per nbc ( t = 0.665, df - 51 ) Both means are, however, quite low compared to the mean of 80.7 follicles per nbc produced consistently in 1983 and 1984 by B, spinulosa ( Whelan and Goldingay 1986 ) in study sites less than a kilometre away from those used in my study. All of this suggests that although mean number of follicles per non-barren cone is constant for a species within a particular year and is often constant between years at a particular site, it can vary quite markedly from one location to anotherProlific flowering of B. ericifo lia in certain years leads to a greater contribution to the seed-bank as a consequence, in spite of the fact that the proportion of non-barren cones is much smaller than in "normal" years. This is because mean follicles per non-barren cone is constant from year to year and although the percentage of inflorescences that produce fruiting cones is less in a prolific flowering year, the total number of fruiting cones is much greater. For example, in 1984,30 B. ericifo lia plants produced a total of 187 inflorescences On average, 58% of them or 108 inflorescences produced fruiting cones In 1985 the same 30 plants produced 1386 inflorescences of which 31% ( or 430 ) produced fruiting cones. In other words, an increase in number of inflorescences by a factor of 7.4 produced a four-fold increase in number of fruiting cones and an estimated seed63output 3.8 times as great.Large B. ericifo lia inflorescences ( >14 cm. ) produced significantly more non-barren cones than small ones ( í 14 cm. ). For non-barren cones there vas also a positive relationship between size of inflorescence and the number of follicles Increase in fruit-set per inflorescence as a result of increase in the number of flowers per inflorescence has also been reported by Willson and Rath eke ( 1974 ), Willson and Price ( 1977 ) and Pyke ( 1982 ). Such trends can result either from the differential effects of plant traits on pollinator attraction ( larger inflorescences being more attractive to potential pollinators ) or from patterns of resource allocation within the plants ( Pyke 1982 ). I f pollination is not limiting fruit-set, the increase in fruit-set in large inflorescences will be due to the larger amount of resources channelled into them by the plant. Patón and Turner ( 1985) also found a significant relationship between size of inflorescence and %nbc for B. e ric ifo lia , but failed to find one between size and number of follicles per nbc. This is, perhaps, not surprising in view of the large amount of variability that appears to be a feature of the reproduction of these plants.McFarland ( 1985 ) found that about 70% of B. spinulosa non-barren cones had follicles restricted to the bottom two-thirds of the cone, and argued that this reflects inflorescence morphology and the consequent activities of avian pollinators His hypothesis is not supported by the data presented here ( Figure 5 4). Two species with quite dissimilar inflorescence morphology ( B. spinulosa and B paludosa ) had almost identical follicle distributions, whereas the two species with very similar inflorescence morphology, and presumably similar pollinators, ( B e ric i fo lia and B. spinulosa ), had follicle distributions that were quite disparateAre low, variable levels of fruit-set caused by fluctuations in pollen availability, resource availability or some combination of both? In the next chapter I64report on the results of experiments designed to show whether fruit-set in Banksia e ric ifo lia B spinulosa and B paludosa is limited by pollination656. POLLEN ADDITION EXPERIMENTS6.1 INTRODUCTION.Studies of resource allocation in plants are generally based on the unstated assumption that the reproductive output of a plant is limited by the availability of resources( Bierzychudek 1981 ). However, it is possible that reproduction may be limited by pollinator effectiveness. The yield of many crop plants has long been known to be pollinator- limited, although this may be entirely due to the planting of vast numbers of a single species in the same location. How common is pollination- limitation in natural populations? Bierzychudek ( 1981 ) cited six cases in which natural populations of plants were pollination limited; Erythronium albidum ,( Schemske e ta l. 1978 ), P h lox divarcata, ( Willson eta l. 1979 ), Combretum fruticosum , ( Schemske 1980b ), Brass&voia nodosa, ( Schemske 1980a ), Lithosperm um caroiin len se, ( Weller 1980 ) and E n cyclia cordigera, ( Janzen eta l. 1980 ); as well as her own study on Arlsaem a trlphyllum , in which she managed to increase seed production by a factor of 40 by hand pollination. Other plants in which reproductive output was increased by hand pollination experiments include Andira in erm is ( Frankie e ta l. 1976 ), C h ilo p slslin ea ris, ( Peterson e ta l 1982 ), 26 of 34 tree species in Costa Rica ( Bawa 1974 ), E ichornla crassipes, ( Barrett 1980 ), Isopyrum biternatum ,( Melampy and Hayworth 1980 ) and 2 of 3 Solldago species ( Gross and Werner 1983 ) Consideration of these and other studies has led Bierzychudek ( 1981 ), Rath eke ( 1983 ) and others to conclude that pollination limitation of reproductive output does not appear to be a rare phenomenon in natural populations. Many studies do indicate, however, that this limitation varies over space and timePollination-limitation could result from competition among plants for limited pollinators, from pollen loss to non-conspecifics, from stigma-clogging with foreign pollen, or from a paucity of pollinators that exists for other reasons ( Rath eke 1983 ) To determine whether plants are pollination-limited, natural fruit-set per flower or66per inflorescence must be compared with that of a simultaneously hand-pollinated group of inflorescences because of potential temporal variation in abortion rates ( Stephenson 1981 ), self-fertility ( Pinthus 1959 ), and pollen viability ( Woodell et a l. 1977). Hopper and Burbidge ( 1978 ) reported that red wattlebirds in south-western Australia foraged almost exclusively on whichever Anigozanthos species was at peak flowering and ignored the species at other times. It seemed reasonable that similar pollinator behaviour may occur within Banksia dominated woodland in eastern Australia, so it was considered that pollen availability was more likely to be limiting fruit-set during the early and late parts of the flowering season. Here I report on the effects on fruit-set of supplementing the amount of pollen received by inflorescences of Banksia ericifo lia , B. spinuJosa and B. paludosa.6.2 MATERIALS AND METHODS.At the same time as the investigation of natural levels of fruit-set, a comparative study was commenced on the effects on fruit-set of supplementing pollen by hand. In June 1984, during the period of peak flowering, 100 B. ericifo lia inflorescences were tagged on a range of plants throughout the study site. Pollen, from sources within and outside the study site, was added by hand to the odd-numbered inflorescences on five occasions over a ten-day period. The inflorescences were left uncovered throughout the study, so pollen added by hand supplemented natural levels of pollination. Even-numbered inflorescences were left untreated as a control sample Inflorescences were harvested in January of the following year and scored for number of mature follicles. At the end of 1984 it became apparent that fruit-set, as measured by the percentage of non-barren cones, had not been consistent throughout the flowering season . For this reason it was considered appropriate to add pollen to populations of early, peak and late flowering inflorescences of B. ericifo liain 198567Numbers of inflorescences in treatments were reduced from 100 to 60 ( 30 pollen-added, 30 control ) due to difficulties encountered in finding sufficient inflorescences during the early and late flowering times. As before, either odd or even-numbered inflorescences were randomly selected for pollen-addition The number of hand pollinations was increased to between seven and ten during the life of an inflorescence. During 1985, an identical study was conducted on early and peak B. paludosa, inflorescences ( there were insufficient late flowering inflorescences to include in the study ), and on one group of B. spinulosa inflorescences during the "peak" of its flowering season. As mentioned above ( Chapter 3 ), 1986 was such a poor flowering season that B. e ricifo lia was the only species that produced sufficient inflorescences to allow continuation of the pollen-added study in that year. In 1986, both control and pollen-added inflorescences were measured so that flower numbers could be estimated ( see 5 2 ) and patterns of distribution of follicles on non-barren cones were noted.The proportion of non-barren cones produced from inflorescences and mean numbers of follicles per non-barren cone found for those inflorescences used to determine natural levels of fruit-set in 1984 and 1985 ( Chapter 5 ) were not significantly different to that from the control group used in this part of the study so these data were combined to increase the sample size. In 1986, the same inflorescences were used as a control in the pollen-added study and for determination of natural level of fruit-set. Control and pollen-added cones were compared on the basis of percentage of non-barren cones ( using X2 test of independence ), number of follicles per non-barren cone ( using unpaired t-tests ) and percentage of flowers that set seeds.6.3 RESULTS.Pollen additions to B. ericifo lia inflorescences resulted in a significant increase ( X2j = 3,97, P < 0.05 ) in the percentage of non-barren cones on only one occasion - duringTable 6.1 Comparison of control and pollen-added fruit-sets as measured by the percentage of non-barren cones ( %nhc ) ! N - total inflorescences in \ sample; a =X2 test of independence; N S = not significant * = P * 0 iñ -* <0.01, * * * - P <0.001 )(A) B. e r ic ifo lia . Percentage Non-Barren Cones ( N )Flowering Time Control Pollen Added Significance3Peak 1984 68.38(117) 79 17(48) NSEarly 1985 50.94(53) 52 00(25) NSPeak 19S5 32.76(58) 55 17 (29)Late 1985 12 07(58) 370(27) N.SEarly 1986 32 10(28) 18.52 (27) N S.Peak 1986 57 10 (28) 58 62 (29! > T C !'l *,Late 1986 4140(29) 20 69(29) HI <•(B) B. paludosa. Percentage Non-Barren Cones ( N )Flowering Time Control Pollen Added Significance3Early 1985 22.95 (61) 43.33(30) *Peak 1985 42.86 (56) 48.28 (29) N.S.(C) B. sp in u losa. Percentage Non-Barren Cones ( N )Flowering Time Control Pollen Added Significance3Peak 1985 10.71 (56) 43 33(30) *69peak-flowering in 1985 ( Table 6.1 ). At all other times during the study, pollination of B. e ric ifo lia vas not a limiting factor.Supplementation of pollen to inflorescences of B. paludosa during the early part of its flowering season in 1985 resulted in a significant increase ( X2j = 4.01, P <0.05 ) in %nbc from 22.95% in control inflorescences to 43.33% in the pollen-added group. These figures are very similar to the 20.2% nbc for controls and 45 2% nbc for pollen-added inflorescences obtained by Whelan and Goldingay ( 1986 ) for experiments on B paludosa in 1984.In B sp in u losa , the very low natural fruit-set in 1985 ( 10.71% nbc ) wasincreased dramatically by addition of pollen to 43 33% n b c(X 2j = 12.19, P< 0.001 )indicating that the reproductive success of B, spinulosa was severely limited by shortage of appropriate pollen during this yearFruit-set, measured by the mean number of follicles per non-barren cone, was independent of the treatment! Table 6.2 ). Addition of pollen made no significant difference to this indicator of reproductive success in any of the three species at any time during the study. This rather unexpected result was also found by Whelan and Goldingay ( 1986 ) for both B paludosa and B spinulosaPercentages of pollen-added B e ricifo lia flowers forming follicles in 1986 were similar to the controls. Early-flowering pollen-added flowers were the least successful with 0.69% setting seed, compared to 2.87% of peak flowers and 1 34% of late flowers.The distribution of follicles on pollen-added B. ericifo lia fruiting cones ( Figure 6.1 ) showed a significantly different pattern to that observed in untreated cones, with 10.7% of treated cones having follicles present on the top two-thirds ofthe cone compared to 0% in 109 untreated cones ( X22 = 12.37, P < 0 01 ). Although thenumber of pollen-added cones observed was very small, it appears that addition of pollen by hand throughout the life of an inflorescence results in fruit-set by more of70Table 6.2 : Comparison of control and pollen-added fruit-sets as measured by mean number of follicles per non-barren cone. ( a unpaired t-tests, N.S. « notsignificant, * = P <0.05, ** = P <0.01, *** = P < 0 .0 0 1 ).(A) B. e r ic ifolim . Mean Number of Follicles per Non-Barren Cone ± s e m (N)Flowering Time Control Pollen Added Significan ceaPeak 1984 45 86 1 1.86 (80) 42.37 + 2.78(38) N.S.Early 1985 39.78 ± 2.94 (27) 46.23 + 3.79(13) N.S.Peak 1985 40.37 + 2.11 (19) 44.81 + 325(16) N.S.Late 1985 43 71 + 6.63 (7) 32 (1) N.S.Early 1986 24.22 + 3.44 (9) 28.60 + 550 (5) N.S.Peak 1986 47.00 ± 2.77 (16) 37.47+ 3.67(17) N.S.Late 1986 49.00 + 8.49(12) 44.67+ 11.66(6) N.S.(B) B. peludosm . Mean Number of Follicles per Non-Barren Cone ± s.e.m. (N)Flowering Time Control Pollen Added SignificanceaEarly 1985 32.86+ 3.86(14) 38.15 ±5.03 (13) N.S.Peak 1985 40.46 + 4.55 (24) 32.43+ 5 38(14) N.S.(C)B. spinulosm . Mean Number of Follicles per Non-Barren Cone t s.e m (N)Flowering Time Control Pollen Added Significan ceaPeak 1985 46.00 + 4.63 (6) 55.69 ±7.01(13) N.S.71the first flowers to open ( the ones at the top of a B ericifolia inflorescence )80Ü 3. eric i folia ( N - 109 ) E Pollen AddedB. ericifoliaHone on Top 1/3 None on Bottom 1/3Figure 6.1 : Distribution of follicles on fruiting cones in 1986.6.4 DISCUSSION.Addition of pollen to B. ericifolia inflorescences produced an increase in fruit-set ( measured by the percentage of non-barren cones ) on only one occasion, during peak flowering in 1985. Results of pollen-addition experiments show that, at all other times during the study, pollen availability was not a limiting factor for fruit-set in B. ericifolia The proportion of non-barren cones in B. paludosa was increased significantly from 22.9% to 43 .3% by addition of extra pollen in the early part of its 1985 flowering season. Banksia spinuJosa, during the same year, displayed a highly significant, four-fold increase in this proportion from 10.7% in the controls to 43.3%72when pollen was added by hand.Banksia e ricifo lia , B. sp in u lo ss and B paludosa are pollinated by the same suite of pollinators - native birds, especially honeyeaters ( Meliphagidae ); non-flying mammals, such as Rattus fu scip es, Ante chin us stu a rtii an d Cercartetus n a n u s, gliding marsupials such as Petaurus breviceps', and possibly introduced honeybees, A p is m ellifera ( Carpenter 1978, Paton and Turner 1985, Whelan and Goldingay 1986, Goldingay e ta l. 1987 and personal observations ). The pollination service that a suite of pollinators can provide is a finite resource for which there may be interspecific and/or intraspecific competition. Although not demonstrated conclusively in this study, competition for pollination between B. ericifo lia and B spinulosa is inferred here from the results of pollen supplementation to B spinulosa inflorescences. When B. spinulosa inflorescences in study site 2 ( which contains a large concentration of B ericifo lia ( Figure 2.7 )) were supplemented with pollen in 1985, there was a four-fold increase in the proportion of inflorescences setting fruit In comparison Whelan and Goldingay ( 1986 ), working in nearby study sites ( without B. e ric ifo lia ), were unable to increase fruit-set in B. spinulosa by pollen- supplementation in 1984, suggesting that without the competitive influence of B e ric ifo lia , B, spinulosa received sufficient natural pollination to maximise its fruit-set at the level predetermined by other factors such as resource-availability Other results from several years of pollen-addition experiments in this study suggest that this inferred competition may vary in intensity both within a particular flowering season and between seasons. When B. ericifo lia produced a huge number of inflorescences in 1985, pollinator service was limiting, resulting in increased fruit-set in all three simultaneously-flowering Banksia species when pollen was added to inflorescences by hand. Pollen availability was limiting at this time probably because either there were insufficient pollinators to fully service the huge number of inflorescences on offer, or pollinators made a large proportion of intra-plant visits which resulted in an increase in the clogging of stigmas by inappropriate pollen and73pollen wastage, as B. e ririfo lia is self-incompatible. In other years, when the flowering of B. ericifo lm was less prolific, pollination did not limit fruit-set, as evidenced by the failure to increase the fruit-set of B e ririfo lia by pollen additions during these years and also by the increase in %nbc of control B. spinulosa inflorescences from 10.7% in 1985 ( when B. eririfo lm inflorescences were prolific ) to 42.7% in 1986 ( a poor flowering year for B eririfo lia ) It is interesting to note that when pollen was added to B. spinulosa inflorescences in 1985 the %nbc increased to 43-3%, almost identical to the levels achieved naturally in 1986 when B. eririfo lia had a very poor flowering year.Pollen supplementation to inflorescences failed to produce any increase in numbers of follicles, measured for non-barren cones, in any of the three species investigated over the three years of the study. Whelan and Goldin gay ( 1986 ) suggested that this may indicate a mechanism, operating at the level of the inflorescence, that sets an upper limit to the number of follicles that can be supported. They argued that this upper limit could be flexible, depending on the availability of nutrients and would result in little difference between control and pollen-added inflorescences in a particular year but there may be marked between-year differences.Support for this hypothesis comes from follicle distributions on B eririfo lia non-barren cones ( Figure 6.1 ). None of 109 control cones had follicles present only on the top two-thirds of the cone, but 10.7% of the pollen-added cones had folliclesdistributed in this way, a highly significant difference ( X22 * 12.37, P < 0.01 ). Thepollen-added group had extra cross pollen added to all of the flowers on the inflorescence, starting with the first ones to open, which, in B. e ririfo lia , are at the top. Coupled with the lack of intra- and inter-year variation in the mean number of follicles per non-barren cone that is apparent in this species, this would explain the restriction in follicle position to the upper two-thirds of the cone - i.e. if there is a maximum number of follicles that can be supported on a fruiting cone, by the timethe flowers at the bottom of a pollen-added inflorescence are mature, the maximum number of fertilised ovules has already been reached and fruit-set is complete The absence of control cones with follicles restricted to the upper two-thirds of the cone can be explained by assuming that there is a threshold number of flowers that must be open on an inflorescence before pollinators are attracted to it By the time pollinators are attracted to an inflorescence, the first group of flowers to open are already senescent and their ovules remain unfertilised, resulting in no follicle development at the top end of these inflorescences in which an thesis occurs basipetally. This pattern of follicle development might also be explained by assuming that inflorescences are capable of differential abortion, in which the plant is able to favour the development of outcrossed ovules at the expense of those that are selfed ( Bawaand Webb 1984 ). This mechanism would cause early self-fertilised ovules to be aborted if sufficient numbers of outcrosses occur later in the life of the inflorescence.74757. HUMBEKS OF POLLEN GRAINS AND POLLEN TUBES PER STIGMA.7.1 INTRODUCTION.During 1984 and 1985, natural fruit-set in Banksia e rlc ifo lia , measured by the proportion of inflorescences that produced non-barren cones, was significantJy lower in inflorescences that flowered during the late part of the flowering season compared to those that flowered at early and peak times. Pollen-addition experiments in 1985 failed to produce an increase in the proportion of non-barren cones during this late flowering period, indicating that the reason for reduced reproductive success at this time was probably not shortage of suitable pollen. In order to provide confirmatory evidence for rejection of a pollination-limitation hypothesis, it was considered appropriate to investigate the numbers of pollen grains that were present on the stigmas of B. e ricifo lla flowers and the pollen tubes that developed from them at various times during the 1986 flowering season.7.2 M ATERIALS AND METHODS.During 1986, B. e ricifo lia styles were collected during the early, peak and late parts of the flowering season. On each occasion, 5 styles from 2 inflorescences on each of 10 plants were collected, giving a total of 100 styles. Styles were removed with forceps from the area on the inflorescences just above the advancing front of dehiscing flowers ( ie. flowers that had been open for a day or two ) and fixed in a solution of 3 parts ethanol and 1 part acetic acid for 1-2 hours. After fixing, styles were washed thoroughly in running water and transferred to 70% ethanol for storage. Preparation for microscopic examination involved cutting off the terminal 2-3 mm of styles containing the stigmatic region, placing in a solution of 50 gL~* sodium sulfite and autoclaving for 10 minutes at 121°C. Stigmas were then placed in a solution of decolourised aniline blue containing 10% glycerol for 2-3 hours ( Bernhardt and Calder 1981 ). Finally, stigmas were placed on a microscope slide, squashed with asecond slide, covered with a cover slip and viewed under a fluorescence microscope Under these conditions, pollen grains are clearly seen as light-grey/cream coloured structures and pollen tubes possess a light-green fluorescence that makes them obvious against the dark-blue fluorescence of the style. Each stigma was observed under high-power ( 400X ) magnification and numbers of pollen grains per stigma and pollen tubes per style were counted. Numbers of pollen grains and tubes at early. peak and late flowering times were compared.7.3 RESULTS.The proportion of B. e ricifo lla stigmas containing pollen grains did not vary significantly throughout 1986 ( Table 7.1 ), remaining consistently at about 50% of early, peak and late-flowering stigmas. However, only 28% of stigmas from late-flowering inflorescences contained pollen-tubes, compared to 46% of those frompeak-flowering inflorescences; a highly significant difference ( X2 j = 6.96, P < 0.01 ).Mean number of pollen grains per pollen-bearing stigma increased slightly during the flowering season from 2.07 at early-flowering to 2.67 at peak-flowering and 2.73 at late-flowering. The increase in number of pollen grains per pollen-bearing stigma between early and late-flowering was significant ( t = 2.081, df = 90, P < 0 05 ) Despite this increase, there were significantly fewer pollen-tubes per pollen-bearing stigma at late-flowering compared to peak-flowering ( t = 3 589, df = 100, P < 0.01 )7.4 DISCUSSION.Analysis of numbers of pollen grains per stigma and pollen tubes per style ( Table7.1 ) provides additional information that may be of importance in understanding the breeding system of B. e ricifo lia . Although there were no differences in the percentages of stigmas containing pollen throughout the 1986 flowering season and, in fact, a significant increase in the number of pollen grains per pollen-bearing stigma at late flowering time, there were significantly fewer pollen tubes growing in7677Table 7.1 : (A) Numbers of pollen-grains and pollen-tubes per stigma in B. ericifo lia during 1986. (B) Significance of differences in numbers of pollen-grains and pollen-tubes in B. ericifo lia during 1986. ( a using chi-square test for independence,® using unpaired t-tests, E = early, P « peak, L = late, * * P < 0.05, ** = P < 0.01, *** = P < 0.001, N.S. = not significant. )(A) Pollen Grains/Tubes per Stigm a.Early Peak Late% stigmas bearing pollen ( n * 100 ). 44 53 49% stigmas bearing pollen tubes ( n * 100 ) 37 46 28mean grains per pollen-bearing stigma 2 .07 ± 0 .21 2.67 ± 0.23 2.73 ±0.23mean pollen-tubes per pollen-bearing stigma 1.41 ± 0.16 1.96 ± 0.22 0.98 i 0.16(B) Sig n ifica n ce o f Y ith in -y e a r D ifferences.E v sP P v sL E vs L% stigmas bearing pollen a N.S. N.S. N.S% stigmas bearing pollen tubes a N.S. * * N.S.mean grains per pollen-bearing stigma® N.S. N.S. *mean pollen-tubes per pollen-bearing stigma® N.S. * * N.S.?8styles of Hovers from late inflorescences. Furthermore the proportion of styles containing pollen tubes was lower in late-season than in other samples This result appears to confirm what might have been expected based on the natural ami pollen-added fruit-set data for 1984 and 1985, ie, a significantly lower number of pollen-tubes produced at late-Howering resulting in more barren cones at this time During 1986 however, when the data on numbers of pollen tubes were collected t here was no reduced reproductive success evident in late-flowering B en a foh a inflorescences This result suggests that, at least in this year of poor flowerin g even the lowest levels of pollination and pollen tube numbers were adequate to reach the limit imposed by other factors. Further evidence to support this proposal comes from a comparison of the proportion of Howers that contained pollen tubes and the proportion that produced follicles in 1986 ( Table 7.2 )Table 7.2 : Proportions of B erjcJfo tia flowers that contained pollen tubes and produced follicles during the early, peak and late parts of the 1986 flowering seasonEARLY PEAK LATE% flowers with pollen tubes 37 0 46 0 28 0% flowers producing follicles 0 98 3 64 2 %The table clearly indicates that very few successful pollinations become successful fruit-sets; once again showing that reproductive success in Banksia ericifo h a , as measured by fruit-set, was determined, at least in 1986, by factors other than pollination serviceStudies such as this have potential value for advancing our understanding oi the breeding systems of B&nksia species. To be of maximum use, however tbev would need to be carried out at early, peak and late flowering time over a number of prolific and poor-flowering years8. RESOURCE ALLOCATION.8.1 INTRODUCTION.Stephenson ( 1981 ) suggested that for plants that consistently produce mature i runs from only a small fraction of their flowers, resources, rather than the number of female flowers, determine the upper limit to fruit production In some cases, it is possible that extrinsic factors such as lack of pollination, predation or climatic factors depress fruit production below the upper limit, but generally it is the intrinsic resource availability that limits reproductive output When this occurs a number of pollinated flowers and young fruits either abscise or fail to develop further until the fruit and/or seed number is matched to the available resourcesEvidence from Stephenson ( 1981 ) and Bawaand Webb t 1984 l clearly indicates that there is competition for limited resources within a plant resulting in U an increase in the probability that remaining flowers will set fruit when the number of earlier-pollinated flowers is decreased and (ii) a temporal decline in fruit set as those flowers that are pollinated first use up the available resources For example, Bawaand Webb found that in Caesalpiniaerîostachys the late- maturing flowers in the upper one third of the inflorescence generally only function as pollen donors but if no fruits have been set in the lower part of the inflorescence then these late flowers are more likely to set fruits Stephenson ( 1983 ) also reported m his review of flower and fruit abortion, that the temporal decline in fruit-set jn plants that are resource limited can be averted if early blooming flowers are thinned immediately before or after pollination Under these conditions fruit-set in the remaining Flowers reaches levels similar to those expected from the earliest flowers I have also reported ( Chapter 6 ) that follicle distribution in pollen added B cn a fo lia inflorescences suggests a similar kind of competition for resources within individual inflorescences. Paton and Turner ( 1985 ) also inferred that B cn cifo Jia may becapable of shunting resources between inflorescences within the same plantIn Baaksia e ric ifo lia , reproductive output is variable between and within years, but was rarely limited by pollination ( Chapter 6 ) Rather t han simply claiming that fruit-set in B ericifo lia must be, by default resource limited an experiment was set up during the early part of the flowering season in 19$n to determine whether resources within the plant are differentially allocated to those ovules that are fertilised first, at the expense of ovules that are fertilised later in the season.8.2 MATERIALS AND METHODS.Thirty B. ericifo lia plants were tagged. All plants chosen had astern circumference at ground level between 30 and 40 cm. Tagged plants were monitored throughout the early and peak parts of the flowering season and all inflorescences that developed during these times were removed with secateurs, to prevent any investment of resources by the plants in the development of fertilised ovules It was considered that the amount of resources committed to these early and peak inflorescences would have been minimal as floral structures are relatively inexpensive to produce ( Chaplin and Walker 1982 ). Inflorescences that matured in the late part of the flowering season were left untouched so that they could be compared to a population of late flowering inflorescences present on control plants of the same general size The controls were plants that had already produced early and/or peak-flowering inflorescences and had therefore allocated resources to their development If resources were limiting and could be shunted to later flowering inflorescences, then a higher proportion of the late-flowering treatment inflorescences would be expected to produce fo llic les than the late-flowering inflorescences in the control population818.3 RESULTS.Of the original 30 treatment plants tagged for inflorescence removal, only 7 continued to produce inflorescences during late flowering time This greatly reduced the sample size of treatment inflorescences Table 8 1 shows the differences between the two samples. The treatment ( early and peak inflorescences removed ) population produced 40.9% non-barren cones which was greater than the control population ( 20% nbc ). However, fruit-set on cones and treatment were, nevertheless,independent ( j = 2.69, P = 0.1 ), most likely because of small sample size. Meannumber of follicles per non-barren cone for the treatment population was 36 8 ±4.2 s.e. which is not significantly different ( t = 0.852, df = 13 ) from the 42.2 ± 4.4 follicles per non-barren cone obtained for the controls.8.4 DISCUSSION.If amount of pollination is rarely limiting in B. e ricifo lia , then is low fruit-set caused by resource restriction for an inflorescence? Patón and Turner ( 1985 ) suggested that the bimodal distribution of follicles on B. ericifo lia infructescences was a result of conservation of scarce resources within a plant - ie. there is less cost in providing few cones with many follicles rather than many cones with few follicles. Whelan and Goldingay ( 1986 ) argued that resource limitation within a cone may have been responsible for their inability to increase the number of follicles on non-barren cones by pollen supplementation.Unfortunately the results obtained in this experiment ( Table 8.1 ) are equivocal. The treatment plants, whose available resources were conserved until the late part of the flowering season by cutting off all early and peak inflorescences, produced a percentage of non-barren cones ( 40.9% ) that was twice that of the control plants ( 20% ). This may suggest that resources were indeed the factors limiting reproductive success, at least in these plants in this year. However, the82Table 8.1 . Fruit production by treatment plants ( early and peak inflorescences removed ) and control plants ( early and peak inflorescences alloyed to develop into fruiting cones )A . Treatm ent Population.Plant No. Circum.(cm) No.inflorsremoved No.late inflors remaining No.barren No.iollicles/nbc1 40 24 3 1 51,282 36 5 2 ou3 38 18 2 1 2b4 40 3 6 1L. 35 33 45. 185 35 7 3 0 586 39 3 2 2/ 39 12 4 *7J 3 7B. Control PopulationPlant No. Circum.(cm) No.inflorsremoved No.late inflors. No.barren No.iollicles/nbc1 37 - 0 9 -2 37 - 6 5 473 33 - 2 ! 3 C)4 40 - 5 4 455 39 - 1 16 34 - 3 2 2h7 39 - 4 -7 40 3>.S3number of plants available for study in this experiment was small and tbe proportion of inflorescences setting fruit was statistically independent of the treatmentImplicit in this experiment is the assumption that the plant is able to shunt resources between flowers on the same plant; eg the tendency for plants to mature fruit on early pollinated flowers at the expense of flowers pollinated later in the flowering season ( Stephenson 1981 ). Whelan and Goldingay ( 1986 ) were aware that dividing the total number of inflorescences on a Banksia paludosa plant among three treatments ( control, autogamy and pollen-added ) may result in a plant developing inflorescences with the best quality pollen at the expense of the remaining ones ( Pyke 1981 ), if these plants could shunt resources to different inflorescences However, when they assessed inflorescences on a further sample of untreated plants asimilar pattern of fruit-set to that of control inflorescences on treatment plants was revealed. Similarly, Zammitand Hood ( 1986 ) found that untreated inflorescences on plants that had inflorescences treated with insecticide to exclude flower and seed predators, produced the same number of seeds as inflorescences on neighbouring non- experimental plantsThe data presented here provide some suggestion that B en cifoh a may be able to direct resources to particular inflorescences, although the evidence is lar irom compelling. A clearer picture would be gained by matching more rigorously lor the numbers of early and peak flowering inflorescences on plants used as treatments and controls, because this may be important in determining how much of the available resources have been pre-empted by early inflorescencesThe experiment was carried out in 1986, a very poor flowering year for B erlclfo lia ^ h tn , in fact, there was no difference in the proportion of inflorescences forming non-barren cones at early, peak and late flowering ( Table 5 2 ) This suggests that 1986 was a good year as far as the ratio of resources to inflorescen ees is concerned and appears atypical when compared to the two years that preceeded it84The treatment sample in this experiment had a proportion of non-barren rones (40.9% ) which was virtually identical to that produced by the random sample at inflorescences used to assess degree of barrenness at late flowering in 1986 (414% )( Table 5 1 ) This suggests that the poor fruit-set (20.0% ) of the control sample used in this experiment may have resulted from factors other than, or in addition to shortage of resourcesTo determine the importance of resource limitation satisfactorily in these plants, experiments such as this will need to be carried out over a number ol years to ensure that any patterns that are detected remain consistent and are not the result nj one unusual flowering period.9. INTEGRATIVE DISCUSSION.Four species of the genus Banksia ( B serrala B. paludosa, B ericifo lia and B spinulosa ) are sympatric in study sites on the Budderoo Plateau near Robertson N S W Of these species, three ( B paJudosa, B ericifo lia and B spinulosa ) flower simultaneously overwinter, while the fourth ( B. serrala ) flowers during summer and is therefore phenologically isolated from the othersFruit-set in the three species for which data were collected is typical of the low levels experienced by most members of the Proteaceae in Australia. For example Banksia e r ic i fo lia converted an average of 2.5% of its flowers into fruits in my study sites in 1986. This lack of reproductive success in Banksias has been largely attributed to either limited pollination or limited resources ( Whelan and Burhidge 1980, Lamont e la l, 1985, Paton and Turner 1985, Abbott 1985, Collins and Spice 1986 Whelan and Goldingay 1986, Collins and Rebelo 1987 )The four co-occurring Banksia species in my study sites are likely to share the same pollinator guild ( Carpenter 1978, Paton and Turner 1985, Whelan and Goldingay 1986, Goldingay e la l 1987 and personal observations ) so it was reasonable to hypothesise at the commencement of this study that pollination may limit the reproductive success of some or all of the simultaneously flowering speciesPollen supplementation experiments revealed that fruit-set in Banksia e ricifo lia was rarely limited by pollination service At most times in most years B e ric ifo lia received at least sufficient natural pollination and fruit-set was unaf fected by experimental pollen supplementation. This finding is the same as that reported by Paton and Turner ( 1985 ). However, during a period of prolific flowering, namely peak flowering time in 1985, fruit-set in B ericifo lia was pollination-limited evidenced by the increase in the percentage of non-barren cones produced from inflorescences (%nbc achieved when natural pollination levels were supplemented by hand. Pollinators were apparently unable to deliver sufficient pollen to all of the86inflorescences on offer at this time, or perhaps there were so many inf lorescences that pollinator movement between plants was greatly reduced, resulting in a large increase in self-pollination,The episode of prolific flowering by Baoksia ericifo lia in 1985 in my study sites also had implications for fruit-set in the other two simultaneously flowering congenerics. Data for B. spm ulosa and B. paludosa are much less complete, making any conclusions about control of fruit-set in these species rather tenuous However it does seem that, for these two species, pollination limitation of fruit-set was important in some years. Baoksia spin u losa , for example, which always produced lew inflorescences per plant compared to the other species ( Figure 3-d ). had a natural level of fruit-set ( as measured by %nbc ) of 107% in 1985 ( a prolific flowering year for B. e ric ifo lia ) which was increased to 43 3% by supplementation of pollen In 1986 a poor flowering year for B. e ric ifo lia , the natural level of %nbc for B spinulosa was 42.7%, almost identical to that achieved in the previous year by adding extra pollen by hand. So it seems at my study sites that the temporal variation in importance of pollination limitation of fruit-set in B, spinulosa may depend upon the intensity of flowering of the much more prolific B. ericifo lia , Data for B paludosa are limited but, at least during the early part of the 1985 flowering season at my study sites, fruit-set was limited by pollination; increasing from the natural level of 22 95% nbc lo 43-33% nbc when pollen was supplemented by handFurther evidence to counter the notion that fruit-set in Baoksia ericifo lia was primarily pollination-limited comes from an investigation of the numbers ol pollen grains and pollen tubes present on stigmas during the early, peak and late flowerin g periods in 1986. Variation in pollination was not matched by variation in reproductive success throughout the season. In spite of the fact that the proportion of stigmas bearing pollen did not vary to any extent throughout the year and the mean number of grains per pollen-bearing stigma actually increased from early through to late flowering, there was a significantly lower proportion of stigmas containing pollen87tubes and also a lover mean number of pollen tubes per pollen bearing stigma in the late flowering group. This suggested that fruit-set did not depend simply on the number of appropriate pollen grains that reached the stigma and that there was some form of pollen/style incompatibility mechanism at work here These results initially appeared consistent with the lover proportion of non-barren cones expected at late flowering in 1986, based on previous experience of late-flowering B ericifoU a inflorescences in 1984 and 1985 However, in 1986 there was no decrease in the fruit-set of late flowering inflorescences. This suggested that the ultimate level of reproductive success in B\ ericifoU a in that year was controlled by factors more fundamental than the number of pollen tubes that reach ovariesFruit-set in Banksia ericifoU a is not primarily pollination limited Does this mean that fruit-set in B. ericifoU a is, by default, resource limited? A manipulation to address this question more fully produced equivocal results Late flowering inflorescences on two groups of trees were compared The treatment group had all of their early and peak blooming inflorescences removed to prevent the allocation of resources to the development of any ovules that may have been fertilised before late flowering time. The control group consisted of trees that possessed late flowering inflorescences and had also produced a number of early and/or peak flowering inflorescences into which, presumably, resources were being channeled to allow the development of any fertilised ovules. The treatment group produced double the percentage of non-barren cones of the control group ( treatment =■■ 40 9% ; control ^20% ), however, the result was not statistically significant ( X2 j = 2 69, P * 0 1 ) Thiswas due, most likely, to the small sample size used here because very few treatment plants continued to produce inflorescences during the late flowering periodThe proportion of barren inflorescences produced by the control sample in this experiment was much greater than that found in a random sample of late inflorescences ( most of which occurred on plants with spent inflorescences from earlier in the season ) used to assess natural fruit-set during the period of lateflovering in 1986 ( Table 5.1 ). The reasons for the difference are not clear, but are probably, once again, related to the small sample sizeThere is, in addition, some slight support for a conclusion invoking availability of resources as the primary cause of fruit-set limitation in Banksia e ric ifo iia , Although no statistical significance vas obtained, the effect of removal of early and peak-flovering inHorescences on fruit-set in late-flovering inflorescences vould appear to be worth pursuing over a number of years, with larger sample sizes, for B ericifo iia and other Banksia speciesThe conclusion that fruit-set in B, ericifo iia is primarily resource limited vould not be a surprising one, because Banksia seeds contain between S and 500 times more phosphorus, nitrogen, potassium, copper and zinc than other plant tissues ( Kuo e tal, 1982, Patón and Turner 1985 ). In addition, the plants in this study were growing in shallow, nutrient-poor sandy-loam derived from Hawkesbury Sandstone ( Burrough et a i\ 1977 )As veil as this background level of resource limitation, B. ericifo iia can also be pollination limited whenever resource availability is above a certain threshold, allowing the development of large numbers of inflorescences The net result ol the interplay of these limiting factors varies from year to year, depending largely upon the availability of resources, Temporal variation in the relative importance n! limiting factors cautions, as Weins ( 1981 ) outlined, against the use of single-sample surveys to imply causation. If this study had been carried out only during peak flowering in 1985, very different conclusions would have been reachedIf total fruit-set in these Banksia species is not generally limited by the amount of pollination, are pollinators still able to determine which inflorescences sei seed? Patón and Turner ( 1985 ) found that large B. ericifo iia inflorescences produced more non-barren cones than small inflorescences and argued that this was because large inflorescences are more attractive to pollinators. My study also found that significantly more non-barren cones resulted from large ( > 14 cm ) inflorescences89in 1986. In addition, large inflorescences were found to produce significantly more follicles per non-barren cone, hardly a surprising resultas large inflorescences have proportionally more of everything ( eg. flowers ) than small ones Patón and Turner ( 1985 ), however, found that the number of follicles per non-barren cone was unrelated to inflorescence size in B. e ric ifo lia . When small ( s 14 cm ) control and pollen-added inflorescences were compared there was no significant difference inthe proportions of non-barren cones ( X2j = 0.85 ) produced, suggesting that thediminished reproductive success of small inflorescences is related to availability of resources rather than reduced attractiveness to pollinators.If reproductive success, measured by levels of fruit-set, is primarily limited by the availability of resources, then it should be possible to increase fruit-set by the addition of resources in the form of fertilisers. Zammit ( 1986 ) was able to increase the amount of fruit-set per plant in Banksia ericifo lia and B oblongifolia by addition of phosphorus. He found that the increases were apparent only after a year had elapsed following the application of the fertiliser, indicating that stored phosphorus, rather than phosphorus available during flowering, was responsibleEven if resource availability was hypothetically limitless and pollination levels were more than adequate, there would remain an upper limit, well below 100%, to the proportion of flowers that are converted to fruits. The arguments for this are at least three-fold. Firstly, excess flowers may be produced in response to selection for male function, to increase pollen dispersal ( Stephenson 1981, Bawa and Webb 1984 ). Obviously, if andromonoecious flowers are produced ( as is suspected in Banksias ( Johnson and Briggs 1975, Collins and Spice 1986 )) flower to fruit ratios will always be lower than otherwise possible. Secondly, it has been argued ( Stephenson 1981, Bawa and Webb 1984 ) that extra fruits are produced so that those that are less fit in terms of their paternity can be selectively aborted. Thirdly, the space available on a Bank?** infructescence will always limit the number of follicles that can develop.Throughout my study, mean number of follicles per non-barren cone did not90vary significantly in any of the species at any time. Variation in fruit-set occurred only in terms of the proportion of inflorescences that produced non-barren cones This finding suggests that there was an upper limit to the number of follicles that can be supported on an inflorescence and that this limit is not controlled by the amount of pollination received, except perhaps in exceptional circumstances when pollination levels are very low. Whelan and Goldingay ( 1986 ) have already reported this phenomenon in Banksiapaludosa and B. spinulosa and have further suggested that this indicates that resource limitation in these banksias is operating at the level of the inflorescence and that there may be competition among fertilised ovules for the available resources. My results suggest that this mechanism is operating in B ericifo lia as well as ini?. paludosa and B. spinulosa The bimodal distributions of follicle numbers on cones ( Figures 5.1» 5 2 and 5 3 ) show that, in almost every case, inflorescences produce either no follicles or more than ten. This suggests that there is also a lower limit to the number of follicles that will be supported on a fruiting cone. These plants appear to need a certain minimum number of ovules fertilised successfully before an inflorescence will supply resources for their development, Patón and Turner ( 1985 ) interpreted this as an adaptation that would conserve resources by not providing supporting structures for small numbers of follicles. They also argued that larger numbers of follicles clustered together on a fruiting cone would be more likely to survive high intensity fires than small numbers of follicles widely spaced and subsequently this pattern of follicle distribution would be selected for in areas subject to wildfire.Conclusion.I propose that the ultimate level of reproductive success achieved by these plants is determined by a heirarchy of factors, each of which has the potential to impinge on fruit-set if the other, more fundamental, factors have been satisfied ( Figure 9.1 ).The most fundamental of factors is resource availability - maternal investment is9ÎNumber of Inflorescences per Plant ------ ►* Level of resources.| • 1 Fruit-set limited by pollination.F ig u re 9.1 : Factors affecting fruit-set in Banksia en cifo h a , B, spinulosa and B paJudosamatched to this by variable fruit abortion rates ( Stephenson 1981, Bawa and Webb 1984 ). If all of the ovules can be supported by the available resources, then fruit-set may be limited by pollination in a number of ways. Waser and Price ( 1983 ) suggested that optimal outcrossing distances, often as short as a few metres, exist for many plants. Pollen from sources closer than this optimal distance will cause increased inbreeding depression, the extreme of which is selling. Pollen from sources beyond the optimal distance will result in outbreeding depression. The distances that pollinators will travel will depend on the availability of floral rewards, so inflorescence density will be a major factor in determining the proportion of92pollinator visits that result in successful pollinations, ie. the proportion that bring pollen from the optimal outcrossing distance. In other words, one would expect depression in fruit-set per plant at both extremes of an inflorescence-density continuum ( Figure 9.1 ).In addition to its important role in determining the degree of intraspecific competition for pollination, the inflorescence density of a prolific-flowering species such as B. ericifo Jia may influence fruit-set in simultaneously flowering, co-occurring Banksia species by affecting the intensity of interspecific competition for pollination.On rare occasions, when inflorescence numbers are very high, pollination limits fruit-set in B. ericifoiiaK Figure 9.1 ). Pollination may be limiting only when all of the other more fundamental conditions that impinge on fruit-set are fulfilled. Further, even if resources, pollination and other factors were not limiting, 100% conversion of flowers into fruit is not possible because of the spatial limitations imposed by the size of the developing follicles on a Banksia infructescence, the presence of some flowers that may perform only male function and the proposal that plants can selectively abort a proportion of fertilised ovules that are in some way less fit than others.Although carried out over four flowering seasons, it is apparent that continued work over many more years will be necessary before the cyclical nature of flowering intensity in these Banksia species and its relationship, if any, to climatic factors, is fully understood. More work is also needed on experiments that test specifically for the ability of these plants to partition their available resources into particular inflorescences and into particular follicles within these inflorescences. 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( 1980 b ) Reproduction in Polem oaium pre-dispersal seed predationEcology 61.502-506mAPPEÄDIX.Lengths of B. erlcifoh& inflorescences (cm) during early, peak and late flowering in1986EARLY EEAK L A U95 10.5 ‘.1.5125 95 75145 14.0 90170 12.0 10.513 0 180 95105 18.0 11 012.0 9.0 13-512 0 105 7.510.0 95 14 011.0 135 15513 0 15 0 11.0170 10.5 9012.0 55 11.5160 10.0 10.015 0 16.0 5.090 140 8.512.0 125 95125 150 6.0130 135 18.0110 115 12512.0 130 90130 12.5 18 0120 8 5 9 0140 105 15513.5 9.5 13.5185 13.0 10 5155 11.0 17.017.0 11 0 20 011.0Length of Inflorescences ANOVASource Sum of Squares Deg of Freedom Mean Squares F-Ratio Proh>fBetweenTime 39.119 2 19559 2.103 0.129Error 762 534 82 9 300Total 801 753 84Allbook91 Ryedale Road West Ryde 211 ̂Phone: 807 6026

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