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2002 Effects of European honeybees (Apis mellifera) on the pollination ecology of bird-and insect-adapted Australian plants Thomas Martin Celebrezze University of Wollongong
Recommended Citation Celebrezze, Thomas Martin, Effects of European honeybees (Apis mellifera) on the pollination ecology of bird-and insect-adapted Australian plants, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1046
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Effects of European Honeybees (Apis mellifera) on the
Pollination Ecology of Bird- and Insect-adapted
Australian Plants
*A thesis submitted in fulfilment of the requirements for the award of the degree DOCTOR OF PHILOSOPHY
from
UNIVERSITY OF WOLLONGONG
by THOMAS MARTIN CELEBREZZE, BAS, MSc DEPARTMENT OF BIOLOGICAL SCIENCES
2002 CERTIFICATION
I, Thomas M. Celebrezze, declare that this thesis, submitted in fulfilment of the requirements for the award of Doctor of Philosophy, in the Department of Biological
Sciences, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution.
Thomas M. Celebrezze
18 October 2002
1 TABLE OF CONTENTS
CHAPTER 1. GENERAL INTRODUCTION 1
1.1 GLOBAL TRENDS IN POLLINATION DISRUPTION 1
1.2 ENDEMIC AUSTRALIAN POLLINATION 3
1.3 THE POTENTIAL ROLE OF HONEYBEES IN AUSTRALIA 5
1.4 INTERACTIONS OF HONEYBEES AND HABITAT FRAGMENTATION 7
1.5 EVIDENCE FOR HONEYBEE EFFECTS IN AUSTRALIA 7
1.6 NEED FOR AN INTEGRATED, COMPARATIVE STUDY 8
1.7 STUDY DESIGN AND AIMS 10
CHAPTER 2. COMPARISON IN MYRTACEAE 14
2.1 INTRODUCTION 14
2.1.1 Myrtaceae pollination systems 14
2.1.2 Study aims and predicted results 15
2.1.3 Study species 16
2.1.4 Study sites 18
2.2 METHODS 21
2.2.1 Potential pollinator foraging frequency and behaviour 21
2.2.2 Selective pollinator exposure experiments 24
2.2.3 Germination experiments 25
2.3 RESULTS 26
2.3.1 Potential pollinators and their visitation frequency. 26
2.3.2 Honeybee behaviour 29 ii 2.3.3 Pollinator exposure experiments 30
2.3.4 Germination experiments 33
2.4 DISCUSSION 36
2.4.1 Predicted and observed outcomes 36
2.4.2 Pollination systems 37
2.4.3 Pollinator frequency and behaviour 39
2.4.4 Plant sexual systems 40
2.4.5 Potential inbreeding depression 41
2.4.6 Conservation significance and evolutionary implications 42
CHAPTER 3. COMPARISON IN EPACRIDACEAE 45
3.1 INTRODUCTION 45
3.1.1 Epacridaceae pollination systems 45
3.1.2 Study aims and predicted results 45
3.1.3 Study species. 48
3.1.4 Study sites 51
3.2 METHODS 52
3.2.1 Floral longevity 52
3.2.2 Potential pollinators 52
3.2.3 Insect floral visitors and visitation frequency 53
3.2.4 Honeybee and other insect foraging behaviour 54
3.2.5 Bird floral visitor frequency 55
3.2.6 Bird foraging behaviour 56
3.2.7 Other evidence of pollinator visits 56
3.2.8 Breeding systems 57 iii 3.2.9 Selective pollinator exposure experiments 58
3.2.10 Potential cage effects 60
3.3 RESULTS 61
3.3.1 Epacris microphylla 61
3.3.2 Styphelia tubiflora 72
3.4 DISCUSSION 81
3.4.1 Predicted and observed outcomes 81
3.4.2 Breeding systems 82
3.4.3 Foraging behaviour of birds and honeybees 82
3.4.4 Confounding effect of floral robbers 83
3.4.5 Bird visitation 84
3.4.6 Generalism in Epacris microphylla 85
3.4.7 Cage effect in Epacris microphylla populations 85
CHAPTER 4. COMPARISON IN PROTEACEAE 87
4.1 INTRODUCTION 87
4.1.1 Proteaceae pollination systems 87
4.1.2 Study aims and predicted results 88
4.2 METHODS 89
4.2.1 Study species 91
4.2.2 Study sites 95
4.2.3 Potential pollinator foraging frequency and behaviour 97
4.2.4 Breeding System 99
4.2.5 Selective pollinator exposure experiments 100
4.2.6 Genetic assessment 102 iv 4.3 RESULTS 107
4.3.1 Potential pollinators 107
4.3.2 Foraging behaviour of floral visitors 113
4.3.3 Breeding systems 116
4.3.4 Selective pollinator exposure experiments 122
4.3.5 Genetic assessment 125
4.4 DISCUSSION 134
4.4.1 Breeding and mating systems 134
4.4.2 Effect of honeybees on reproductive success 136
4.4.3 Potential pollinators and their apparent relative effectiveness 138
4.4.4 Evidence for honeybee-mediated geitonogamy in G. sphacelata 139
4.4.5 Conformity of genetic data with assumptions 142
4.4.6 Comparative effectiveness ofb ird and honeybee pollination 142
4.4.7 Genetic neighbourhood size 144
4.4.8 Evolutionary consequences and conservation implications 146
CHAPTER 5. GENERAL DISCUSSION 148
5.1 SUMMARY OF OUTCOMES 148
5.2 OVERVIEW OF RESULTS 149
5.3 THE STATUS OF KNOWLEDGE ON HONEYBEE EFFECTS IN AUSTRALIA 150
5.4 THE ROLE OF POLLINATION ADAPTATIONS 150
5.5 EVOLUTIONARY CONSEQUENCES AND CONSERVATION IMPLICATIONS 153
5.6 ASSESSMENT OF POLICY IMPLICATIONS 154
REFERENCES 156 v LIST OF TABLES
Table 1.1 A comparison of the social pollinating insects by biogeographic region and a selection of examples of bird-pollination adaptations 2
Table 1.2 Comparative investigations undertaken in this study on the effects of honeybees (Apis mellifera) on the pollination ecology of bird-adapted versus insect-adapted Australian plants in three plant families 13
Table 2.1 Honeybee and bird foraging in populations of Callistemon citrinus, C. linearis andC. linearifolius 20
Table 2.2 Insect morphospecies observed visiting flowers of Baeckea imbricata and Callistemon citrinus 28
Table 2.3 Frequency of insects visits to Baeckea imbricata 29
Table 2.4 Honeybee foraging behaviour at flowers and plants oi Baeckea imbricata and Callistemon citrinus 30
Table 2.5 The effect of selective pollinator exposure on components of plant reproductive output in two populations of Callistemon citrinus near
Sydney, Australia 32
Table 2.6 The proportion of capsules produced per flower by selective pollinator exclusion treatment in two populations of Baeckea imbricata 33
Table 3.1 The experimental power of selective pollinator exclusion experiments in
Epacris microphylla and Styphelia tubiflora 60
Table 3.2 Insect morphospecies observed visiting flowers of Epacris microphylla in two populations in Royal National Park, Australia 62
Table 3.3 Pollinator behaviour while foraging among flowers and plants of two species of Epacridaceae 64
vi 3.4 Estimated average daily visits per flower and per plant by honeybees and native insects 67
Table 3.5 Results of selective pollinator exclusions and self- and cross-pollination experiments on reproduction of Epacris microphylla in two populations in two years 70
Table 3.6 Results of x tests of the presence of honeybees and native insects in caged inflorescences during hourly censuses 71
Table 3.7 Average number of among-plant movements per day by eastern spinebills on Styphelia tubiflora 75
Table 3.8 Percent of flowers which initiated fruitset and then aborted for open- pollinated, bird-excluded, bagging (autogamy), self-pollinated and cross- pollinated treatments for Styphelia tubiflora 81
Table 4.1 Investigations into potential effects of honeybees on the pollination systems of two species of Grevillea 90
Table 4.2 Insect morphospecies observed visiting Grevillea acanthifolia and G sphacelata flowers 108
Table 4.3 Floral visitor movement rates among inflorescences and plants in two
Grevillea species 114
Table 4.4 Estimated daily visits per inflorescence and per plant 116
Table 4.5 The breeding systems of Grevillea acanthifolia and G. sphacelata from cross-and self-pollination experiments 117
Table 4.6 The proportion of fertile inflorescences and mean number of fruits per fertile inflorescences in Grevillea acanthifolia and G. sphacelata 124
Table 4.7 The allelic richness of all adults sampled and the total allelic richness of microsatellite loci in Grevillea acanthifolia in two populations 128 vii Table 4.8 Proportion of Grevillea acanthifolia seed that was detectably outcrossed and outcrossing rate estimates (t) for seed produced through open pollination and vertebrate exclusion in two populations 131
Table 4.9 Comparison of observed heterozygosity (H0) in adults in two Grevillea acanthifolia populations with expected values if populations are at
Hardy-Weinberg equilibrium (HE) 133
viii LIST OF FIGURES
Figure 2.1 Results of Callistemon citrinus germination experiments of seed produced selective pollinator exposures 35
Figure 3.1 The proportion of Epacris microphylla plants with visiting honeybees and native insects 65
Figure 3.2 The proportion of Styphelia tubiflora plants observed being visited by honeybees 74
Figure 3.3 The proportion of Styphelia tubiflora plants visited per hour by nectar- foraging eastern spinebills and/or New Holland honeyeaters during half- hour or one-hour observation periods 76
Figure 3.4 Proportion of flowers that produced fruit in two Styphelia tubiflora populations 79
Figure 4.1 Average proportion of Grevillea sphacelata plants observed being visited by honeybees and native insects 109
Figure 4.2 Average proportion of Grevillea acanthifolia plants observed being visited by honeybees 110
Figure 4.3 Proportion of Grevillea acanthifolia plants observed being visited by
New Holland honeyeaters Ill
Figure 4.4 Between plant movement distances of birds and honeybees foraging on
Grevillea acanthifolia and honeybees foraging on G. sphacelata 115
Figure 4.5 The distribution of fruit set per inflorescence by experimental treatment in population 1 of Grevillea acanthifolia 118
Figure 4.6 The distribution of fruit set per inflorescence by experimental treatment in population 2 of Grevillea acanthifolia 119
ix Figure 4.7 The distribution of fruit set per inflorescence by experimental treatment in population 1 of Grevillea sphacelata 120
Figure 4.8 The distribution of fruit set per inflorescence by experimental treatment in population 2 of Grevillea sphacelata 121
Figure 4.9 Proportion of Grevillea acanthifolia seed that was apparently outcrossed when one, two or three loci were considered 129
x LIST OF ILLUSTRATIONS
Illustration 2.1 The "bottlebrush" inflorescence of Callistemon citrinus 16
Illustration 2.2 Baeckea imbricata in coastal heath 17
Illustration 3.1 The foraging behaviour of honeybees and native pollinators at flowers of Epacris microphylla and Styphelia tubiflora 47
Illustration 3.2 Epacris microphylla at Royal National Park, Australia with cage for the exclusion of birds 49
Illustration 3.3 Styphelia tubiflora 50
Illustration 4.1 Grevillea acanthifolia inflorescence in partial bloom, with several flowers exposing pollen presenters 92
Illustration 4.2 The foraging behaviour of honeybees at flowers of Grevillea sphacelata and honeybees and birds at Grevillea acanthifolia 93
Illustration 4.3 Grevillea sphacelata inflorescence 94
xi ABSTRACT
European honeybees were introduced to Australia in the 1820s and are now widespread as feral and domestic colonies in the temperate region, foraging for nectar and pollen from plants in at least two hundred genera. Many of the plant species visited by honeybees are among the nearly 1,000 species that havefloral structure s that facilitate pollination by birds or mammals. Numerous ecologists have expressed concerns that the pollination systems of such plants, which evolved in the absence of winged social insects, are at risk of disruption by honeybees, particularly because anthers, nectaries and/or stigmas are often separated by several centimetres. At the same time, honeybee industry advocates continue to argue for greater access to natural areas, citing a lack of evidence of negative impacts. To date, experimental studies of potential honeybee disruption of
Australian pollination systems have produced a range of outcomes apparently demonstrating that honeybees can enhance, decrease or have little effect on the quantity of fruit or seed produced in different vertebrate-adapted species.
Less apparently, honeybees may be altering plant fitness by reducing seed quality.
Such an effect would be expected if short-distance honeybee movement facilitate geitonogamy and inbreeding in contrast to longer-distance movements by native pollinators. As a result, honeybees could be expected to be a significant evolutionary pressure for some plant species.
A comparative approach is needed to determine what types of pollination systems are most at risk of negative honeybee effects such as severe reproductive decline or inbreeding depression. In this study, I compared the roles of honeybees and native animal visitors in the pollination ecology of bird- and insect-adapted plant species using confamilial pairs of plant species in three plant families: Callistemon citrinus and
Baeckea imbricata (Myrtaceae); Styphelia tubiflora and Epacris microphylla
xii (Epacridaceae); and Grevillea acanthifolia and G. sphacelata (Proteaceae). In all six species, I identified the suites of probable pollinators and observed their foraging behaviour at flowers and among plants. I compared the proportion of flowers that produced fruit among selective pollination exposure treatments - exclusion of birds, exclusion of all pollinators, and open pollination - to deduce the likely role of honeybees.
In the Epacridaceae and Proteaceae, I determined if plants were self-compatible and compared fruit set of manually pollinated flowers with that of open flowers to determine if plants were pollinator limited. Finally, in order to determine whether honeybees may be reducing seed quality, I compared seed produced from selective pollinator exposures for viability and genriination rate (in Callistemon citrinus) and outcrossing rate (in
Grevillea acanthifolia).
Overall, the results of this study did not support the hypothesis that pollination of bird-adapted plants by honeybees alone would produce less fruit than open pollination.
Rather, fruit set in such species following exposure to honeybees alone was equivalent
(Callistemon citrinus), marginally lower (Grevillea acanthifolia) or higher (Styphelia tubiflora) than fruit set following exposure to both honeybees and birds. The last outcome appeared to be an artefact of the exclusion cages, which excluded native bird nectar robbers; such birds appeared to be responsible for damaging more than 60% of flowers on 60% of exposed plants.
This study showed that honeybees might interfere with the pollination of insect- adapted plant species, even if they brush pollen onto stigmas. In two populations of
Grevillea sphacelata, each flower was likely to receive more than 20 honeybee visits per day, whereas native insect visitors were so infrequent that a comparable measure could not be reliably calculated. Individual honeybees moved infrequently among plants and pollen was nearly always removed from open flowers by previous visitors; of 870 flower
xiii visits, only 5 resulted in visible amounts of pollen deposited on the honeybee.
Honeybees appeared to promote geitonogamous pollination in the self-incompatible G. sphacelata, pre-empting potentially more effective pollination by native insects and thus limiting fruit set. Hand-pollination studies revealed that cross-pollination produced 4 to
13 times more fruit than open pollination. In contrast, honeybees appeared to contribute to fruit set along with numerous native insect species in two self-compatible species
(Epacris microphylla and Baeckea imbricata).
Honeybees may not provide pollinations of equivalent quality to native pollinators in some bird-adapted species. In the self-compatible Callistemon citrinus seed germination experiments suggested that seed produced following pollination by honeyeaters and honeybees germinated more rapidly (80 % cumulative germination at 4.6 days) than seed produced by honeybee pollination alone (6.5 days). Seed produced by autogamy germinated slowest (9.4 days). In two Grevillea acanthifolia populations, the outcrossing rates of seed produced by open pollination were not significantly different from those of seeds produced by exposure to insects alone, suggesting that honeybees do not cause inbreeding. Nevertheless, behavioural differences between honeybees and birds suggest that the social insect may be decreasing genetic neighbourhood size. Taken together, the results of studies in these two species suggest that honeybees may be facilitating changes in plant populations which have not yet been detected, including inbreeding, character shifts (e.g. shift toward self-compatibility) and decreasing genetic neighbourhood size.
Pollination "syndromes" are not a good predictor of honeybee effects. It is also evident that honeybees may have little effect on the pollination systems of some species.
However, this study demonstrates that honeybees may be disrupting reproductive success in self-incompatible species, even if they are adapted to insect pollination, and may be xiv causing genetic shifts in species with mixed mating systems, even if they do not limit seed production. Based upon the evidence in this study, the conservation consequences for many plant species might include severe pollinator limitation of fruit set, a shift toward self-compatibility in plants with mixed mating systems and a decline in population viability because of loss of genetic variability.
xv ACKNOWLEDGEMENTS
I received financial support for this research through an Australian
Commonwealth Overseas Postgraduate Research Scholarship and a University of
Wollongong University Postgraduate Award. This research was also supported by an
Australian Research Council grant to David J. Ayre and Robert J. Whelan, an
Australian Flora Foundation grant, and the University of Wollongong's Institute for
Conservation Biology. The NSW National Parks and Wildlife Service provided a permit for field studies as well as study leave to complete this thesis.
I thank Simon Watson, Carlo Pisanu, Andrew Stanton, Warren Speicher, Samatha
Bandi, Eunice Subba, Steve Hollis and Glenn Johnstone for their assistance in the field.
I am indebted to Kyoko Oda, who counted and sorted 60,880 tiny particles. I am grateful to my good friend Robert Parkinson, who provided the illustrations. Phillip
England provided advice on some of the perils of microsatellites, and Dave Roberts helped overcome crucial data gaps. I also thank Mark Dowton, Tanya Llorens, Annette
Usher, Raellie Patterson, Dave Roberts and Chris Howard for assistance with genetic laboratory work. I am grateful for stimulating and encouraging conversations with
David Paton, Maile Neel, Susan Hoebee, Glenda Wardell, and many others. Samantha
Lloyd, Patricia Hogbin, Meredith Henderson, Mark Ooi, and Andrew Denham provided feedback on drafts. I thank my examiners Dr. Steven Hopper and Professor James
Thomson for their positive comments and constructive criticisms.
I am privileged to have had Professors Rob Whelan and David Ayre as my supervisors; they have provided even-handed guidance. For the many sacrifices of my parents, who have always encouraged my intellectual pursuits, I am forever grateful.
Finally, I dedicate this work to Simon Watson, who has been marvellous since the day we met. "Only one sweeter end can readily be recalled - the delicious death of an Ohio honey- hunter, who seeking honey in the crotch of a hollow tree, found such exceeding store of it, that leaning too far over, it sucked him in, so that he died embalmed. How many, think ye, have likewise fallen into Plato's honeyhead and sweetly perished there?"
Herman Melville, Moby Dick
xvii CHAPTER 1. GENERAL INTRODUCTION
1.1 Global trends in pollination disruption
Anthropogenic disruption of native pollination systems has recently been identified in several continents as a phenomenon with potentially cascading conservation implications (Buchmann and Nabhan 1996, Butz Huryn 1997, Allan-
Wardell et al. 1998, Kremen and Ricketts 2000). With the recent precipitous decline in honeybee populations in North America, for instance (Allen-Wardell et al. 1998), ecologists there have focused on threats to native insect pollinators such as population declines due to habitat fragmentation and pesticide drift (e.g. Kearns et al. 1998). On the other hand, the ongoing presence of exotic social insect pollinators, such as honeybees and bumblebees, is likely to be an equally important force in disrupting many co-evolved pollination systems (Aizen and Feinsinger 1994, Buchmann and
Nabhan 1996, Butz Huryn 1997). Potential impacts include changes in native pollinator behaviour or abundance, reductions in seed production, and decline in seed quality (Paton 1996, Butz Huryn 1997, Kearns et al. 1998, England et al. 2001).
Alteration of pollination systems may be widespread in other biogeograpbic regions where social insects have been introduced (Table 1.1). These may have eluded detection or study because they are not as apparent as other factors such as habitat destruction and because baseline measurements prior to the introduction of honeybees are virtually non-existent.
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Australia has been isolated for more than 40 million years in the virtual absence of social pollinating insects (Michener 1965, Paton 1986a, b) (Table 1.1). Michener
(1965) noted the very high diversity of the Australian bee fauna in relation to other continents, and he attributed this, in part, to this absence. This isolation has resulted in an endemic flora with a great diversity of biotic pollination systems (Armstrong 1979) including pollination by mammals (e.g. Carthew and Goldingay 1997), birds (e.g. Ford et al. 1979, Paton 1986b) and thousands of species of pollinating solitary flies, bees and other insects (e.g. Michener 1965, Armstrong 1979). Despite the wide variety of native pollination systems, few highly specific pollination relationships have been identified.
The most notable of these is the pollination of orchids (Prasophyllum ssp.) by thynnid wasps through pseudocopulation (Stoutamire 1974). Ford et al. (1979) estimated that more than 100 bird species forage for nectar at flowers for part of their diet and about
1,000 Australian plant species arepollinated by birds. These plant species possess characteristics, such as tubular flowers (e.g. Epacridaceae), bundles of large cup-like flowers (Myrtaceae), and brush-like inflorescences (Myrtaceae and Proteaceae) that facilitate pollination by various bird species.
As suggested above, many plant species have apparent characteristics that promote pollination by one guild of animals or another and such characters have contributed to adaptive radiation in plants (Stebbins 1970). I avoid the term
"pollination syndromes" (Faegri and van der Pijl 1979) to describe these characteristics because this concept has contributed to the tacit assumption that specialisation in pollination is an evolutionary norm (Waser et al. 1996) and the neglect of direct pollinator observations (Fester and Dudash 2001). These features now contribute to fitness regardless of their origins, but may represent a composite of characters shaped
3 by natural selection for their current use (adaptations) and other characters (exaptations)
(Gould and Vrba 1982). Exaptations are features coopted from previous functions that had some other adaptive use, or from characters which did not arise from natural selection and had no previous utility. Strictly speaking, adaptations and exaptations are collectively termed "aptations" (Gould and Vrba 1982), and clarifying which characters were strictly adaptations would require functional, heritability, fitness, and phylogenetic testing (Mishler 2000). However, it is generally accepted that floral characters are likely to be subject to adaptive pressure (Stebbins 1970), so I will use the terms "adaptations", "bird-adapted" and "insect-adapted" to describe existing floral characters.
Arguably, southern Australian bird pollination adaptations are different from their counterparts in other biogeographic regions because they have not evolved mechanisms to prevent nectar or pollen theft by nectar- and pollen-gathering social insects such as honeybees or bumblebees (Table 1.1). Because of their high population densities and methodical foraging, honeybees were likely to be a significant force in floral evolution in their natural range, and plants that benefit from bird pollination in those regions may have evolved mechanisms to prevent insects from accessing flowers (Skead 1967) or to avoid detection (Raven 1972). For example, bird-adapted plants in North America typically secrete nectar in narrow tubular flowers readily accessed by hummingbirds but not bumblebees (e.g. 8 of 9 species illustrated in Kodric-Brown and Brown 1979).
Likewise, in Southern Africa, many bird-pollinated plants have physical structures restricting access by insect pollinators, such as the hinge mechanism of species of
Strelitzia flowers that excludes the endemic honeybees but ensures that the sunbird visitors have access to nectar while being brushed with pollen (Frost and Frost 1981) or the bracts of Protea which may exclude insects as noted by Stead (1967). For many Australian plants that are visited by birds, however, both pollen and nectar are readily accessible to flying insects (pers. obs.). This potentially controversial distinction has not been examined in the pollination literature.
1.3 The potential role of honeybees in Australia
There is no doubt that honeybees are a highly successful introduced species in
Australia and have the potential to significantly alter pollination systems and their evolutionary trajectory. Since their introduction in the 1820s, honeybees have populated much of the temperate, subtropical and tropical (Hopper 1980) zones as both domestic and feral hives. Hopper (1987) identified the potential impacts of honeybees on plant and animal populations and several key areas for study, including the potential effect of honeybees as competitors of native insects. Honeybees collect pollen and/or nectar from more than 1,000 plant species across at least 200 plant genera (Paton 1996), and can remove in excess of 80% of the nectar or pollen produced by some species of plants (Paton 1990, 1993). Honeybees may be ineffective pollinators of many
Australian plant species that have floral adaptations to pollination by vertebrates. In many of these species, the nectary, pollen and stigmas are too widely separated for bees to contact with pollen and/or stigma when foraging for nectar (Ford and Paton 1986,
Taylor and Whelan 1988, Paton 1993, Faulks 1999). Because of this morphological mismatch, many ecologists believe that honeybees will be disrupting the pollination ecology of these species (Taylor and Whelan 1988, Pyke 1990, Vaughton 1992, Paton
1993, Paton 1996, Pyke 1999). Honeybees may indirectly decrease pollination by competing with or displacing native flower visitors (Hopper 1987, Paton 1996), by pre emptively removing pollen from flowers (Vaughton 1996, Celebrezze and Paton
5 submitted), and by collecting pollen that has already been deposited on stigmas (Gross and Mackay 1998).
In addition to decreasing fruit and/or seed set, honeybees may also alter pollen quality (and therefore potentially seed quality) by moving pollen among flowers on the same plant (geitonogamy) or among neighbouring plants which are likely to be related
(Ayre et al. 1994, Richardson et al. 2000). Native pollinators such as birds (Ford and
Paton 1986) and some insects appear to move more frequently and/or over greater distances among plants than honeybees do (pers. obs.; Paton, pers. comm; Wardell, pers. comm.). Only England et al. (2001) have experimentally tested the genetic effects of honeybees in natural populations of an Australian plant species to determine if such inbreeding was taking place. They found that honeybees were the only important insect visitor, and that outcrossing rates in the self-compatible G macleayana were significantly lower in inflorescences from which vertebrates were selectively excluded than in inflorescences accessible to both insects and vertebrates.
Honeybee-mediated inbreeding is a significant conservation concern because inbreeding is expected to have profound negative effect on fitness in some plant species
(Charlesworth and Charlesworth 1987, Waser 1993, Keller and Waller 2002). Potential negative effects of inbreeding, collectively termed inbreeding depression, include decreased fitness of offspring through the expression of lethal recessive mutations; loss of heterosis (with its associated fitness advantage); and genetic drift and fixation resulting in a loss of potentially adaptive variation (Charlesworth and Charlesworth
1987). These effects are all likely to increase the risk of local extinction.
6 1.4 Interactions of honeybees and habitat fragmentation
While honeybees are likely to be a factor in intact conservation areas, they are also present in fragmented habitats which are now the focus of conservation management agendas because these habitat types are often underrepresented in the formal reserve system. Honeybee effects are likely to have complex interactions with habitat fragmentation. If native birds are absent from small native vegetation remnants
(Paton 1996, Paton 2000), honeybees may provide an important service as a
"substitute" pollinator. Alternatively, honeybee invasion of habitat fragments may contribute to declines in native insect pollinator diversity (Aizen and Feinsinger 1994) although this verdict is premature (Thomson 2001). In Australia, Cunningham (2000) found that plants in linear fragments of habitat sometimes had lower reproductive success than those in non-linear reserves, but the relative role of honeybees was not examined, particularly in small populations. Honeybees may contribute to inbreeding in fragmented populations but are unlikely to offset this effect through gene flow among populations.
1.5 Evidence for honeybee effects in Australia
The ideal experiment to measure the role of honeybees would be to remove them from some areas and monitor the consequences. Feral honeybees, however, cannot be locally extirpated easily (Oldroyd 1998), and such experiments have not been conducted. Instead, the role of honeybees as pollinators of Australian plants has usually been extrapolated from measurements of fruit set of plants adapted to bird or marsupial pollination when large pollinators (but not honeybees) were excluded with mesh cages. Paton (1996) reviewed the evidence of honeybee impacts in Australia and found that these impacts were likely to be broad-ranging and highly variable for both
7 native plants and animals. Butz Huryn (1997), on the other hand, emphasised that there was little evidence that introduced honeybees were likely to be a significant conservation concern. These studies found that honeybees can pollinate a range of vertebrate-pollinated plants including species of Banksia, Grevillea, Callistemon,
Correa, Cyanthodes and Brachyloma. In many cases, though, the quantity of fruit and/or seed produced was significantly lower than when vertebrates also had access to flowers (Paton and Turner 1985, Taylor and Whelan 1988, Vaughton 1992, Paton 1993,
Paton 1996, Vaughton 1996, Faulks 1999, Higham and McQuillan 2000, Celebrezze and Paton submitted). Paton (1996,2000) increased the population density of domestic honeybee hives in an area where native pollinators have declined and found that honeybees contributed to increased fruit set, indicating that honeybees can be important as pollinators in fragmented plant populations which do not receive sufficient pollination from native animals. Likewise, Gross (2001) calculated, from visitation rates, that honeybees may contribute to pollination in some years when native insects do not provide adequate service to Dillwyniajuniperina populations.
1.6 Need for an integrated, comparative study
Previous studies have generally focused on reproductive outputs without assessing pollinator suites, pollinator behaviour and seed quality and taking into account natural variation in these parameters among populations and across years.
Despite the variety of studies by numerous ecologists illustrating that honeybees alter pollination systems in Australia, none has demonstrated that bird-adapted plants are more susceptible to perturbation than insect-adapted ones by comparing the effects of selective pollinator exposure experiments in bird- and insect-adapted species in several plant families. Such an approach is needed to guide appropriate conservation
8 management of honeybees because it will help determine the likely variation, breadth and importance of honeybee effects to the conservation of biodiversity.
Hopper (1987) identified the need for research on the effect of honeybee in species-specific pollination systems. However, while such research is clearly needed for the management of particular target threatened species, such negative impacts are not likely to result in the establishment of policies which manage honeybees on the broad scale. For example, the New South Wales National Parks and Wildlife Service
Beekeeping policy (September 1999) states that "The European honey bee is an exotic species that has adverse impacts on some native biota" but does not recommend the control of honeybees. The Minister for the Environment at that time (Allan, pers. comm.) identified the lack of any clear pattern of impacts on any class of plant or animal species as the primary factor limiting any recommendation to control honeybees in conservation areas. Because of its policy importance, the effects of honeybees on common plant species adapted to generalist vertebrate in contrast with insect pollination is of greater urgency than the study of individual species most at risk.
Previous research has demonstrated that honeybees may alter pollination systems in numerous ways and has suggested that seed quality may also be affected, but these studies have not demonstrated a link between pollinator behavioural differences and seed quality. Restricted honeybee movement within populations may facilitate geitonogamy and therefore inbreeding. A comparative approach is needed to determine which plant species are most at risk of negative effects such as severe reproductive decline or inbreeding depression.
9 1.7 Study design and aims
To address these problems, I compared the relative impact of honeybees on bird- adapted shrubs and related insect-adapted shrubs in three large plant families of
Gondwanan origin (Raven and Axelrod 1972, Johnson and Briggs 1981). These families,
Epacridaceae, Myrtaceae, and Proteaceae, have the highest incidence of bird adaptation in Australia (Ford et al. 1979).
I compared the roles of honeybees and native animal visitors in the pollination ecology of bird- and insect-adapted plant species using confamilial pairs of plant species in three plant families: Callistemon citrinus and Baeckea imbricata (Myrtaceae),
Styphelia tubiflora and Epacris microphylla (Epacridaceae), and Grevillea acanthifolia and G sphacelata (Proteaceae). All of these are hermaphroditic woody perennial shrubs in populations east of the Great Dividing Range in south eastern Australia. I chose these study species for pragmatic reasons: they had characteristics of bird- or insect-adaptations and flowered at times that were logistically compatible for experimental study. I was able to locate at least one population of each where honeybees were foraging and established study sites were readily accessible. Ideally, I would have paired plants with similar breeding systems, but this was not possible because breeding systems are known for so few Australian plant species (Armstrong 1979) and, in any case, may vary among populations (Hermanutz et al. 1998). With the exception of Grevillea sphacelata
(Proteaceae) (Hermanutz et al. 1998), there were no published accounts of the breeding systems of the species selected at the time that the study was initiated in 1997.
I used selective pollinator exposure experiments to determine the likely role of honeybees and native visitors as pollinators. Differences in pollinator effectiveness are likely to be a driving force in the evolution of floral forms adapted to particular guilds of visitors (Stebbins 1970), but precise measurement of effectiveness requires detailed study of pollen deposition of individual pollinators visiting individual flowers (e.g.
Herrera 1987). Because of time constraints, I compared the frequency of animal visits to flowers and assumed that visitors were potentially effective pollinators if they appeared to contact the pollen-bearing surface and stigma during some visits. I assumed that visitors which were frequent and potentially effective pollinators were
"important" pollinators. Where possible I quantified floral visitor frequency and behaviour and repeated the exposure experiments in at least two flowering seasons in two populations (Table 1.2). I predicted that:
(i) of the species selected for study, birds would be the most frequent native pollinators of species with apparent bird-pollination adaptations and insects would be the most frequent visitors of those with insect pollination adaptations;
(ii) open-pollinated flowers and flowers caged in 1.52 cm mesh would produce equivalent fruit set in the insect-adapted species;
(iii) bird-exclusion would result in lower reproductive success than open pollination in the bird-adapted species;
(iv) honeybees would move less frequently and shorter distances than birds in populations of the bird-adapted species; and
(v) seeds of bird-adapted plants produced by exposure to honeybees alone would have lower fitness and lower outcrossing rates than those produced through open pollination.
To test these predictions, I conducted a series of studies in each pair of species
linking behavioural observations of pollinators, selective pollinator exposure 11 experiments, and ideally, measures of seed quality (Table 1.2). I performed selective pollinator exposure experiments in all cases. I observed floral visitor abundance and behaviour in both species of Proteaceae and Epacridaceae and one of the Myrtaceae species, Baeckea imbricata (I obtained preliminary observations for Callistemon citrinus). I tested breeding systems in Epacridaceae and Proteaceae, but not in Myrtaceae because of time constraints during the flowering period.
I subsequently compared seed quality of open-pollinated and bird-excluded seed in bird-adapted species to test whether honeybee-mediated pollination alone produced lower outcrossing rates than open pollination. I examined germination rates in C. citrinus seed produced by exposure to birds and insects compared to those produced by insects alone
(honeybees were by far the most frequent insect floral visitor). I measured the genetic effect of exclusion of birds from the bird-adapted Grevillea acanthifolia in two populations using microsatellite markers (Queller et al. 1993). I was unable to germinate or determine genotypes of seeds of the Epacridaceae species Styphelia tubiflora.
Except where otherwise noted, I report means and standard errors for all results.
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K CO CO _c CO CO X X XXX 3 T3 X X X X 0 < C .El | d TJ 0 0 CO c *-* CO 3 Q. 0 CO o uonejndod CM CN CM CM CM CM CO T3 o c CO 3 -4—< to g O 0 O CJ 0 o CU "E re > uoijeidepe "E CU COD _5 CO cp co o la CO CO 3 c c *-* c 3 -a co -2 fD co CO co •2 0 0 8a. ^ •a*. •»_. > _i; O CO eg > T3 2_ -c •C: 0 0 E Q. -5 o o cu i > 0 3 •Q o c 8 Haw CO 0 •4-. c "0 CD oS .CD co CO t_ >co> •^ CO CO CO co ro E o CD a> 71 CU 2.1 Introduction 2.1.1 Myrtaceae pollination systems The plant family Myrtaceae includes over 1,400 species in 70 genera in Australia (Harden 1990) with a wide range of floral types including generalist, bird-adapted, and insect-adapted systems (Hopper and Moran 1981, Paton and Turner 1985, Beardsell et al. 1993). This diversity provides ample opportunity to compare the effect of honeybees on bird- versus insect-adapted species (Paton 1996). Nevertheless, the role of honeybees as pollinators of Myrtaceae has only been examined in detail in a few studies, with a range of results. Behavioural observations suggest that honeybees were likely to brush pollen onto stigmas in E. costata (Horskins and Turner 1999) but not Calothamnus quadrifidus (Collins et al. 1984), but these observations were not substantiated with evidence of reproductive success or failure following honeybee exposure. Moncur and Kleinschmidt (1992), in their study of plantation Eucalyptus species, stated that honeybees supplemented pollination because native pollinators such as birds, insects and possibly bats were absent. More recently Paton (1993, 1996) presented experimental evidence that honeybees had a negative effect on the pollination systems of the bird-adapted Callistemon rugulosus in natural populations, and Hingston (2002) found that visits to flowers by swift parrots produced significantly greater seed set than honeybee visits in a population of E. globulus. 14 2.1.2 Study aims and predicted results I compared the role of honeybee floral visitors in the pollination system of a bird- adapted and an insect-adapted shrub species. The overall design aimed to link pollinator observations, selective pollinator exposure experiments, and germination experiments in Callistemon citrinus and Baeckea imbricata (Table 1.2). I made the following predictions: (i) birds would be the most frequent native pollinators of C. citrinus and insects the most frequent visitors of B. imbricata; (ii) open pollinated and bird-excluded flowers would produce equivalent fruit set in B. imbricata; (iii) bird-exclusion would result in lower reproductive success than open pollination in C. citrinus; and (iv) C. citrinus seeds produced by exposure to honeybees alone would have low viability or delayed germination relative to those produced through open pollination (suggesting inbreeding depression). To test these predictions, I identified floral visitors to both species, observed their behaviour at flowers, the frequency of their visits and their movements among plants. I compared reproductive success following selective exposure of flowers to (i) no pollinators, (ii) insects alone and (iii) all pollinators. Finally, for the bird-adapted species, I also designed seed germination experiments to compare the relative viability and time to germination of C. citrinus seed. 15 2.1.3 Study species Callistemon citrinus is a widespread erect shrub to 4 m forming dense stands. It occurs at sites with poor drainage in sclerophyll woodlands near the coast and along watercourses from Victoria to Queensland (Harden 1990). Flowering is greatest in November and December (Carolin 1987, Harden 1990) although it can occur sporadically year round. Individual flowers are sessile to the stem with circular petals approximately 5 mm long and numerous, free, red filaments up to 25 mm long with a versatile anther at the same level as the stigma, together forming a "bottlebrush" inflorescence and remain open for several days. Inflorescences with numerous red flowers form terminal or near-terminal spikes six to 12 cm long (Illustration 2.1). Flowers secrete abundant nectar that attracts several bird species, particularly honeyeaters (Meliphagidae) and lorikeets (Loriidae). Illustration 2.1 The "bottlebrush" inflorescence of Callistemon citrinus. Photograph by M. Fagg, © Australian National Herbarium. 16 Baeckea imbricata (Illustration 2.2) occurs on coastal cliffs and heaths on shallow soils in New South Wales and southern Queensland, often forming dense stands from 0.5 to 3 m in height. Plants bloom from spring to late summer when, typically, 5 to 10 cm lengths of stem will display a few hundred flowers (analogous to an inflorescence). The axial flowers are approximately 4 mm in diameter, with four circular white petals less than 2 mm long, 5 to 7 mm long stamens and a shorter pistil. Individual flowers produce little nectar but the surface of the ovary appears sticky after anthesis. Flowers remain open for several days (pers. obs.). Illustration 2.2 Baeckea imbricata in coastal heath near Kurnell, New South Wales. I initially selected two other Myrtaceae species, Darwinia fascicularis and Leptospermum laevigatum, for study but terminated observations of these species when it became apparent that they were not appropriate. Darwinia fascicularis received abundant bird and honeybee visits. However, fertile and barren seed were indistinguishable under a dissecting microscope (pers. obs. and R. Whelan, pers. comm.) or with tetrazolium (Kearns and Inouye 1993)) so I could not determine its breeding system or the effects of pollinators on reproductive success. Leptospermum laevegatum received visits from numerous species of insects and, from its floral characteristics, appeared to be insect-adapted. However, following the set up of experimental pollinator exposures, I observed brush wattlebirds (Anthochaera chrysoptera) occasionally visiting flowers. Moreover, high winds in the coastal habitat of the species often resulted in dislodgment of bird-exclusion cages, damaging seed capsules. 2.1.4 Study sites I conducted field experiments during one flowering season in two populations of both species, each with honeybees foraging at flowers. Both Baeckea imbricata populations were large stands of several thousand individuals in Botany Bay National Park near Kurnell, New South Wales (population 1,151° 14' E, 34° 20' S) and La Perouse, New South Wales (population 2, 151° 14' E, 34° 1' S), but were separated from each other by the mouth of Botany Bay. Population 1 was approximately 200 m from the coast on shallow soils (two to ten cm) over sandstone bedrock. In this population, I selected plants haphazardly and randomly assigned these to experimental treatments. Population 2 was a band about 50 m wide and plants were more variable in size than population 1. In this population, I selected the larger individuals (about 3 m height versus about 0.3 m in other parts of the population) for study because these plants were likely to be safer from vandalism than the small individuals (because cages on them were not visible from the coastal path). The larger plants selected were randomly assigned to experimental treatments. I later discuss how this conscious bias was likely to effect the results. 18 To identify natural populations where honeybees were foraging, I visited eight natural populations of C. citrinus, two of C. linearifolius and one of C. linearis on warm days when honeybees were active (Table 2.1). I recorded the number of flowering inflorescences on all flowering individuals, and observed individual plants for 3 minutes, noting when honeybees were seen foraging. I also noted birds foraging for nectar. In total, I observed a total of 93 haphazardly-selected plants for a total of 4.65 hours over 14 days. Table 2.1 Honeybee and bird foraging in populations of Callistemon citrinus, C. linearis and C. linearifolius. Honeybees were observed foraging only on C. citrinus and only at two of the populations. inflor- total time Site date plants escences (minutes) honeybees birds* C. citrinus Berowa Gully 23/12/1999 6 23 18 4 Bundeen/Malabar Road 26/10/1999 11 98 33 0 Carrington Falls 14/12/1999 1 5 3 0 Darkes Forest 2/12/1999 5 12 15 0 Garie-Tonoom Falls 22/11/1999 4 100 12 7 Jennifer St. 3/11/1999 4 75 12 0 23/11/1999 8 71 24 0 Kurnell 19/11/1999 1 9 3 0 North Head 1/12/1999 16 76 48 0 Tonoum Falls 22/11/1999 1 1 3 0 C. linearis BundeenaRoad 26/10/1999 3 7 9 0 0 La Perouse 3/11/1999 4 17 12 0 0 Royal N.P. Visitors Centre 5/11/1999 9 45 27 0 0 C. linearifolius Kurnell 19/10/1999 10 66 30 0 1 19/11/1999 10 54 30 0 1 *ln total, 3 brush wattlebirds (Anthochaera chrysoptera) and 4 New Holland honeyeaters (Phylidonyris novaehollandiae) were observed. I frequently observed honeybees foraging on Callistemon hybrids planted in suburban and urban street trees in the Sydney and Illawarra regions and some species of Callistemon are planted as nectar sources for commercial apiaries in India (Gupta and Kumar 1993). However, honeybees were highly variable in their utilisation of flowers in natural 20 Callistemon populations (Table 2.1). Of the 14 populations, I observed honeybees foraging at only 2 populations, both C. citrinus. For this study, I selected only one of two populations where I detected honeybees foraging because, at the other, individual plants were too large for me to access inflorescences. I subsequently included another population where honeybees were reportedly active (R. Whelan, pers. comm.). Both were small C. citrinus populations (approximately ten to 30 reproductive individuals to three m in height) in wet heathy woodlands at the Royal National Park near Toonoum Falls (151° 2' E, 34° 10' S at approximately 100 m elevation) and at Barren Grounds Nature Reserve between the Warden's Residence and the Illawarra Lookout (150° 43' E, 34° 40' S, at approximately 1,200 m elevation). 2.2 Methods 2.2.1 Potential pollinator foraging frequency and behaviour 2.2.1.1 Foraging behaviour of honeybees To obtain useful estimates of potential pollinator suites and behaviour during the experimental period, I needed rapid techniques that would be repeated for each plant species in order to sample variation throughout the day, among days, among seasons and not least among plant species but not limit my capacity to conduct other experiments and observations. Therefore, to estimate how frequently flowers were visited by honeybees, I observed "bouts" by eight individual honeybees in B. imbricata population 1, six in B. imbricata population 2 and eight in C. citrinus population 1. For each of these bouts, I noted the resource collected (pollen and/or nectar), the number of plants and inflorescences or flowers (for C. citrinus and B. imbricata respectively), and the number of seconds I observed the bout. I calculated the proportion of honeybee movements between flowers that were also between plants as an indication of potential for pollen transfer among plants. 2.2.1.2 Foraging frequency of honeybees Pollinator effectiveness is most accurately determined by examining deposition of pollen on individual stigmas and measuring the frequency and duration of visits by individual foraging animals to flowers (e.g. Hererra 1987). Such observations were not logistically feasible in this study so to obtain an indicative measure of the frequency of visits to flowers, I examined 30 B. imbricata plants at each population for 30 seconds at hourly intervals on two days. Because I initially observed insects to be very infrequent visitors on cold, windy or rainy days, I made these observations on days that were sunny, still and warm. I noted the number of honeybees and/or native insects at each of these plants and, where possible, identified native insects to family and morphospecies. I also estimated body length (tip of head to end of thorax) as an indication of potential to brush both anthers and stigma and assumed that insects less than 2 mm in length were unlikely to brush both anthers and stigmas of B. imbricata flowers. To estimate the likelihood that flowers received visits in population 1,1 counted the number of flowers on each plant observed. The number of flowers was too high in population 2 to make such counts and flowering stems were at different stages of development within plants so that simple estimates based upon plant size were not appropriate. I also noted native insects on flowers opportunistically and where possible captured them in a sweep net for identification to family. 22 I combined foraging rates from bouts of individual honeybees and the proportion of plants and flowers visited in censuses to estimate the frequency that plants and flowers were visited. I assumed that the average number of honeybees visiting plants during my instantaneous censuses represented the average number of honeybees foraging continuously on plants when they were not being observed. I also assumed that the average foraging rate I obtained from honeybee bout observations were indicative of average foraging rates of honeybees foraging at plants when not being observed. I calculated the average daily number of insect visits per plant at both populations and per flower in population 1 as the average number of honeybees foraging per plant or per flower observed in censuses multiplied by the foraging rate obtained from observing individual honeybee bouts (averaged among individual bouts).. I determined hourly foraging rates of honeybees from bouts observed between 10:45 and 16:40 for B. imbricata. For C. citrinus, all bouts observed were after 15:00 and might be expected to overestimate the relative frequency of honeybee movements among plants because nectar may have been depleted by that time of day. 2.2.1.3 Bird visitors I noted the species of birds foraging for nectar on C. citrinus during set up of exclusion experiments, but was subsequently unable to record behavioural data because of concerns by the management authority (P. Hay, pers. comm.). 23 2.2.2 Selective pollinator exposure experiments I determined the relative role of all pollinators, insects alone, and autogamy in producing fruit in all of the study populations through selective pollinator exposures. The three treatments I used were: (i) autogamy (flowers bagged with polyester "liner" sleeves ); (ii) open pollination (flowers exposed to all pollinators); or (iii) bird-exclusion, (flowers caged with 1.5 cm2 plastic mesh over a cylindrical chicken-wire frame (20 cm diameter, 30 to 40 cm length)). The polyester used in the bagging treatment appeared to dry quickly following rain. In order to minimise differences in honeybee behaviour resulting from cages used to exclude birds, the dimensions of these cages were not varied between populations or species. I intended experimental units to be individual plants but both C. citrinus populations were too small (fewer than 20 flowering individuals) to provide sufficient independent replication among the three treatments. Instead, I assigned individual inflorescences within plants to treatments alternately, if plants held more than one inflorescence. For B. imbricata, I randomly assigned individual plants to treatments. This resulted in inclusion of all flowers in the experiment in population 1, but to only a small fraction of the thousands of flowers presented in population 2. For C. citrinus, I used all available inflorescences in each population. For the plants with all treatments (five in population 1, ten in population 2), I tested the proportions of flowers which set fruit using Friedman's Two-Way ANOVA. However, because this analysis neglected many of the data, I also tested all data as if they were independent using one-way ANOVA and post-hoc Student Newman Keuls tests (using the arcsine of the square root of the raw data to correct for non-normal distribution). In the one case where the same treatment was repeated more than once on the same plant (e.g. the plant had four inflorescences), I included the average proportion of capsules per flower, number of seeds per capsule, and time to germination in the analysis to avoid pseudoreplication. I did not pair treatments in B. imbricata because my sample was a small proportion of the total number of adults in the population. I randomly assigned each of 29 plants in population 1 and 23 in population 2 to one of the three treatments. I haphazardly selected individual flowering branches approximately 20 to 30 cm long for one of the three treatments. For each replicate, I recorded the number of buds, and four to six weeks after blooming, bagged the plants in order to capture mature capsules that fell from the stem upon maturation. The seed was too miniscule to be captured with these bags. I tested for differences in the proportion of buds which produced capsules among treatments (after transforming the data to correct for non-normal distribution (arcsine of the squareroot)) using one-way ANOVA and non-parametric multiple comparisons for unequal sample sizes (Zar 1984). 2.2.3 Germination experiments To determine whether experimental pollinator exposure treatments affected seed viability and time to germination, I supervised a student, Kyoko Oda, in counting and germinating seed from population 1. Oda determined the number of seeds per capsule in five capsules for each infractescence 17 months after flowering. She dried capsules in a heating oven at 200°C for one minute to maximise germination (Whelan and Brown 1998). She placed 100 seeds on moistened blotting paper and observed germination for 20 days. Subsequently, she distinguished "filled" and "empty" seed under a dissecting microscope; all empty seed were inviable. I used the remaining, filled seed (3 to 53 filled seeds per inflorescence) to test for differences in viability and germination rate. I calculated the proportion of filled seed that eventually germinated and the time to 50 and 80% germination of viable seed and tested the significance of differences among treatments using Kruskal-Wallis and Mann-Whitney tests (because the data were not normally distributed and variances were not equal among groups). We attempted to conduct the same experiment using seed from population 2, but six months following flowering, fruits had not matured and capsules collected did not release seed following air drying or heat treatment. 2.3 Results 2.3.1 Potential pollinators and their visitation frequency I observed at least 25 species of native insects that visited flowers of B. imbricata (Table 2.2). Thirteen species of flies (Diptera) made up the largest family group, followed by six bees or wasps (Hymenoptera), five beetles (Coleoptera), two bugs (Hemiptera) and a butterfly (Lepidoptera). Two Hymenoptera morphospecies occurred in both populations. Judging from body lengths, all of these visitors were likely to brush both the anthers and the stigma while foraging for nectar and/or pollen. 26 I rarely detected honeybees during censuses of either B. imbricata population (Table 2.3). None were present on tagged plants during censuses in population 1 on the 17th of November 1999 or in population 2 on the 4th or 23rd of November 1999 (Table 2.3). In 30- second observations of 30 plants over eight hours on the 19th of October 1999, eight honeybees were foraging (Table 2.3). Nevertheless, I found that honeybees were by far the most numerous single species of floral visitor (Table 2.3), making up 35% of the 23 insects observed in censuses. Native insects were collectively more frequent in censuses but no single species was predominant (pers. obs.). Because other insects were so infrequent, I was unable to obtain data for foraging bouts of these. Some individual native insects remained on flowers for several minutes and then flew to another plant several metres away (pers. obs.). I saw three native wasps and one ant gathering nectar on one occasion on C. citrinus flowers but none of these brushed the anthers or stigma (Table 2.2) but birds were the predominant native visitors to C. citrinus inflorescences. I noted three species of honeyeaters (Meliphagidaea) - brush wattlebirds (Anthochaera chrysoptera), New Holland honeyeaters (Phylidonyris novaehollandiae) and eastern spinebills (Acanthorhynchus tenuirostris) - foraging for nectar on inflorescences in population 1. During initial censuses of honeybee visitation in populations of three Callistemon species, I observed New Holland honeyeaters and brush wattlebirds foraged on four of 93 plants observed during three-minute censuses (Table 2.1). 27 Table 2.2 Insect morphospecies observed visiting flowers of Baeckea imbricata and Callistemon citrinus in the Sydney region, Australia, during flowering seasons in 1999 and 2000. Body length was from the tip of head to end of thorax. Order Family and morphospecies body length (mm) Baeckea imbricata population 1 Coleoptera Elateridae Coleoptera Bi1 5 Coleoptera Bi2 Diptera Bi1 1.5 Diptera Bi2 2 Diptera Bi3 4 Diptera Stratiomyidae 5 Diptera Syrphidae 5 Diptera Bi4 8 Diptera Bi5 10 Diptera Bi6 Diptera Syrphidae Hemiptera Miridae 3 Hemiptera Pentatomidae 7 Hymenoptera Apoidea, Apis mellifera 10 Hymenoptera Formicidae Hymenoptera Vespidae 8 Hymenoptera Vespidae 10 population 2 Coleoptera Lycidae 11 Coleoptera SF Cantharidae? Diptera Syrphidae Diptera Bibionidae 8 Diptera Tipulidae 20 Diptera Muridae 10 Hymenoptera Apoidea, Apis mellifera 10 Hymenoptera Vespidae 8 Hymenoptera Vespidae 12 Hymenoptera Vespidae Lepidoptera Bi1 30 Callistemon citrinus population 1 Hymenoptera Apoidea, Apis mellifera 10 Hymenoptera Formicidae Hymenoptera Vespidae 3 population 2 Hymenoptera Apoidea, Apis mellifera 10 Hymenoptera Vespidae 6 Hymenoptera Vespidae 10 28 Table 2.3 Frequency of honeybees and native insects visiting Baeckea imbricata. Total number of insects observed foraging on plants in 30 second censuses at intervals between 10:00 and 18:00. Census intervals were every hour or two hours. The average number of visits per flower and plant were estimated as the product of the number of honeybees foraging and the honeybee foraging rate (735 flowers/hr and 269 plants/hr for population 1, Table 2.4), divided by the total number of plants or flowers observed. Flower counts were not feasible in population 2. honeybe e visits number observed Ferda y native date censuses plants flowers honeybees insects per pisin t per flower population 1 19/10/1999 8 30 1319 8 2 72 4.8 17/11/1999 7 28 1588 0 7 0 0 population 2 4/11/1999 5 30 no data 0 5 0 0 23/11/1999 4 30 no data 0 1 0 0 2.3.2 Honeybee behaviour Despite the low frequency of detection in censuses, honeybees were foraging in both populations on eachfield day . Honeybees generally foraged for nectar (63% of bouts observed in population 1, n = 8; 83% in population 2, n = 6) although the remainder foraged for pollen (Table 2.4). On 19 October 1999,1 estimated that each plant received an average of more than 70 visits and each flower more than four visits from honeybees (Table 2.3). In population 2, however, there were more flowers on individual plants, which may account for the low proportion of movements among plants. Table 2.4 Honeybee foraging behaviour at flowers and plants of Baeckea imbricata and Callistemon citrinus. foraging rate proportion proportion of number foraging for flowers per no. plants no. flowers movements of bouts nectar plant per hour per hour among plants Baeckea imbricata population 1 8 0.63 4.2+1.6 269 + 44 735 + 59 0.32 + 0.05 population 2 6 0.83 34.1 + 11.9 81+47 1080+166 0.05 + 0.02 Callistemon citrinus population 1 8 1 7.2+1.3 70 + 15 396 + 37 0.37 + 0.18 Honeybees were frequent visitors to C. citrinus inflorescences in population 1, where they typically foraged for nectar by crawling around the base of the flowers. Contrary to my expectations, honeybees often brushed anthers and stigmas while arriving at or departing from inflorescences. In population 1, honeybees moved frequently among plants, visiting on average only seven flowers on a given plant (Table 2.3) even though inflorescences presented 14 to 92 flowers each. 2.3.3 Pollinator exposure experiments Callistemon citrinus appeared to be fully self-compatible and capable of setting seed by autogamy at both populations, with no apparent contribution from pollinators to the quantity of capsules produced. Between 59 and 80% of C. citrinus flowers set capsules; there were no significant differences among treatments in either population (Table 2.5; one- 30 way ANOVA population 1 df = 28, F = 2.136, P = 0.138; population 2 df = 52, F = 2.413, P = 0.10). Baeckea imbricata produced some seed via autogamy at both populations but pollinators contributed to fruit set in population 1. Bird-exclusion did not appear to limit B. imbricata fruit set at either population but exclusion of all pollinators restricted fruit set in population 1. The proportion of flowers which produced capsules did not differ significantly between open-pollination and bird-exclusion treatments in either population of B. imbricata (range, 28 to 39%, Table 2.6). Open-pollinated flowers were more likely to produce capsules than flowers in the autogamy treatment in B. imbricata population 1 (Q = 3.096, F = 3, P < 0.05) but not in population 2 (Kruskal-Wallis %2 = 2.462, df = 2, P = 0.29). 31 CD CD c H— CD o XI § CO CD ^5 CO O) co .c CD CD CD • — > ^- •*-< ^-< c CD o in CO •^ o CD ro CD Q. c o T^ d T^ TJ T3 T3 c *-< co o xs + + + o CD ^ '.£5 CD co O O O *J v—" CD CD •CD CD E O 3 XI _ CD TO rt "CD T3 o >> CO ro 05 O C c O > co CD CD CD CD CD ro CNJ o CO r^ in CD CD 3 CD o O T3 T3 CO 0) -•— cr ci + o O O c -•-• _CD: c E m + + o _J -#— o •*-* co c C '•4— CO CD •^r ro CD CD co 3 O CD co n ro O •* Q. CO T3 o "a i- O CN o la. CD CD ro CD a. CD | CO ^ oa. Q. •-"• c 9i CN CD ro ro 3 «~" ro o + + + D D T3 CO 00 O 05 c: CO _£ _. 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E C O O co CO CD CD CD CD CDO o1 "at ^ m m > 3 O .Q 1— u— co in i^- z O «: O CD CD ro CO 3 CD ro CO CO C *-t w o oX O CO 3 c CD C C < CD CO cn O t> CD g c O g ro =5 C3) o O ro CN ro CD CD CO to CD 3 CN CD d _3 CD E o o c E o _: >> CD CD c ro X (- CZ> > CO g a. g CD Q. CO CD CO c ro o c XJ ro o 0 CD 3 +-< .b 3 3 Q- c\i Q. Q. 3 -D ct.ro O Q) O ro O o JQ Q. CD Q. h- 32 Table 2.6 The proportion of capsules produced per flower by selective pollinator exclusion treatment in two populations of Baeckea imbricata. Pairs with the same superscript letter are not significantly different at a = 0.05 (nonparametric multiple comparison, Zar 1984). proportion of capsules treatment # plants # flowers per flower population 1 autogamy 8 1,528 0.13 + 0.043 open pollination 10 1,166 0.39 + 0.06b bird exclusion 11 1,233 0.32 + 0.07" population 2 autogamy 20 1,891 0.2 + 0.083 open pollination 7 1,341 0.28 + 0.083 bird exclusion 8 1,241 0.28 + 0.09a 2.3.4 Germination experiments Callistemon citrinus seed produced by autogamy and exposure to insects alone was equally viable but slower to germinate than seed produced by open pollination. Although the average percentage of viable autogamous C. citrinus seeds (71%) was lower than either seed produced by bird exclusion (59%) or open pollination (63%, Table 2.5), this difference was not statistically significant (Kruskal-Wallis Test, %2 = 3.934, P = 0.140). Although the patterns of germination were not radically different among treatments, viable autogamous seed took significantly longer to reach 50% germination than open pollinated seed (Mann- Whitney U = 8.00, P = 0.043) and significantly longer than either bird-excluded (U = 8.0, P = 0.043) or open-pollinated seed (U = 7.5, P = 0.004) to reach 80% germination. This lag was also apparent from plots of cumulative viable seed germinated (Figure 2.1 a, b, c). In calculating both viability and time to germination, I excluded a replicate in which only 4% of filled seed germinated, because this very low germination seems likely to have resulted from factors other than the genetic composition of seeds. The data for this plant are graphed in Figure 2.1. 1 1 1 3 1 5 1 7 1 9 number of days Results of Callistemon citrinus germination experiments of seed produced from (a) exposure of inflorescences to all available pollinators including birds and honeybees, (b) exposure to honeybees (and potentially other insects), and (c) bagging treatment. Lines represent the cumulative proportion of seed germinating (ovals indicate mean). 2.4 Discussion 2.4.1 Predicted and observed outcomes The results of this study did not support the predicted contrast in fruit set between bird- and insect-adapted species. Both C. citrinus and B. imbricata had equivalent fruit set following open- and bird-exclusion pollination treatments. However, because the presence of honeybees might have interfered with bird pollination in this study, I cannot eliminate the possibility that seed set in C. citrinus was limited by the presence of honeybees. My other findings suggest that honeybee pollination may result in inferior seed than bird pollination in bird-adapted species. Inbred (autogamous) C. citrinus seed were slightly but significantly slower to germinate than open-pollinated seed, but not than insect- pollinated seed. Similar results were found in Protea by Wright (1994). Numerous studies (reviewed in Pollack and Roos 1972) have demonstrated a correlation between time to germination and subsequent adult fitness. It is not clear that the self-compatible C. citrinus would naturally produce comparable levels of seed in the absence of pollinators (autogamy) because the potential confounding effect of enhanced geitonogamy from pollen rubbing on the sleeves, but in both populations bagged inflorescences produced comparable proportions of seeds per capsule as flowers exposed to all pollinators or to insects alone. These results for C. citrinus may have been confounded by within-plant treatments being treated as independent but this is likely to have exaggerated rather than minimised the power of these tests to detect real differences in fruit set. Although inflorescences on the same plant may not be independent in their production of fruit or seed because, for example, plants may direct resources to inflorescences which have received higher quality pollen, this type of effect would be expected to increase the difference among treatments if there were differences in pollen quality compared to non-paired treatments. There is apparently no difference infruit set or seed set among inflorescences pollinated by honeybees and those exposed to all pollinators. 2.4.2 Pollination systems Direct observations of pollinator suites indicate that the two species provided a genuine contrast between bird and insect adaptation for this study. As predicted, honeybees and a wide variety of native insect species were apparently pollinating B. imbricata flowers while birds were apparently the most effective native pollinator of C. citrinus, visiting flowers frequently, brushing the stigmas and anthers, and moving frequently among plants. Callistemon citrinus is a classic bird (and potentially nocturnal mammal) pollinated plant species, producing copious nectar rewards in densely packed arrays (inflorescences). Recent studies have confirmed that birds are the predominant native pollinators of several red, brush-flowered species in the genera Callistemon (Paton 1997), Darwinia (Celebrezze, unpublished data) and Metrosideros in New Zealand (see references in Schmidt-Adams et al. 1999). Keighery (1982) identified 12 genera of Myrtaceae in Western Australia which each contain at least one species of bird-adapted plant. Numerous studies have shown that Eucalyptus nectar flows are important sources of energy for Australian birds (Ford and Paton 1977, Ford et al. 1979, Ford 1981, Paton 1982, Collins et al. 1985, Collins and Newland 1986, Mac Nally and McGoldrick 1997). Probably the majority of the hundreds of Eucalyptus species, as well as many Myrtaceous shrub species with small white flowers (e.g. Leptospermum spp.) are generalists, pollinated by birds, insects and marsupials (Ford et al. 1979). 37 In a few cases, Eucalyptus flowers have developed specialist structures. For example, E. stoatei (Hopper and Moran 1981) and E. conferruminata (Keighery 1982) have elongated stamens and red floral parts that promote effective pollination by birds. Sampson (1998) reported genetic evidence of high outcrossing rates in E. rameliana and attributed this to specialist structures promoting bird pollination. Honeybees may be ineffective at moving pollen among self-incompatible trees: Hingston (2002) recently demonstrated that pollination by swift parrots (Lathamus discolor) causes seed production in the self- incompatible E. globulus ssp. globulus, while honeybees and bumblebees (which have recently been introduced to Tasmania) did not. Baeckea imbricata appeared to be generalist insect pollinated, attracting numerous species of insects with large but diffuse floral displays. Flowers are small, ensuring that visitors are brushed with pollen when they are collecting nectar. The pollination ecology of insect-adapted Myrtaceae is poorly known, but many species are apparently generalists. The diversity of insect pollinators visiting B. imbricata is typical of many Myrtaceae (Beardsell et al. 1993). Little is known about the relative effectiveness of insect visitors, including the quantities of pollen they are likely to deposit on stigmas with each visit, but Armstrong (1979) listed insect floral visitors at Callistemon, Melaleuca, Leptospermum and other Myrtaceae genera. Horskins and Turner (1999) listed families of invertebrate visitors to Eucalyptus costata flowers but did not quantify the role of vertebrates as pollinators. More intensive observations would be needed to establish a comprehensive list of insect species that contribute to B. imbricata pollination, and the variation among their effectiveness. 38 2.4.3 Pollinator frequency and behaviour I reported the frequency of honeybee and native insect visitors to B. imbricata plants on two days in each population (Table 2.3). and the rate of foraging for a few individual honeybees (Table 2.4) in both B. imbricata and C. citrinus. These small samples are not likely to have captured the natural variation in insect pollination foraging behaviour, and are likely to overestimate the overall rates of visits because they were taken on still, warm days, when insect activity is greatest I did not account for variation within years and populations or at different times of day. Despite these limitations, these observations show that both honeybees and native insects are likely to visit most B. imbricata flowers at least a few times over the several days they remain open. The role of honeybees as pollinators differed between the study populations, presumably because of differences in plant size. For example, only 5% of honeybee movements among flowers were among plants in population 2. where plants had thousands of flowers, compared to 32% in population 1, where plants had much small floral displays (Table 2.4). In C. citrinus, an unexpectedly high proportion of honeybee movements were between plants (37 + 18%), so honeybees might have contributed to cross-pollination, but these movements were nearly always between neighbouring plants, while birds often flew several metres among plants. Such behavioural differences may have long term consequences to the genetic composition and structure of populations. I did not measure the floral constancy of birds compared to honeybees; this factor might be expected to influence the relative pollinator effectiveness of these visitors and warrant further study. 39 2.4.4 Plant sexual systems Both species in this study were self-compatible and produced fruit autogamously. In fact, reproductive success did not differ significantly between open pollination and autogamy in C. citrinus at either population or in B. imbricata population 1, suggesting that animals contributed little to the quantity of fruit set in these cases. Animal behaviour, floral adaptations and breeding systems will all contribute to the plants' sexual system; considering the large floral displays and apparent copious nectar produced by C. citrinus flowers, autogamous fruit set indicates the species has a "facultatively xenogamous" sexual system (Cruden and Lyon 1989). This contrasts with other self-incompatible Myrtaceae such as Eucalyptus spathulata, which produced the greatest proportion of capsule set through cross-pollination (Sedgley and Granger 1996) and the many instances of near- complete self-incompatibility in Eucalyptus (reviewed in Potts 1987). Pollinators may also contribute to fruit set of self-compatible species if the species has a preferentially outcrossing mating system. For example, Burrows (2000) reported lower seed production in isolated Eucalyptus melliodora trees in comparison to woodland individuals, presumably because woodland trees receive more outcrossed pollen than isolated trees. This study does not clarify what factors limited fruit set or how self- pollination occurred. Self-pollination and cross-pollination experiments are necessary to test whether either species is preferentially outcrossing and whether the observed levels of open fruit set are pollinator limited. Apparent "autogamy" may have been mediated by small insect "in-fauna" which could have transferred pollen from stigma to pistil while moving within the flowers (although I did not observe such insects). More likely, the 40 bagged treatment itself may have facilitated autogamy; I noted pollen adhering to the inside of the polyester exclusion bags and wind may have brushed this onto receptive stigmas. 2.4.5 Potential inbreeding depression For C. citrinus, the observed decreases in seed quality for honeybee-mediated and autogamous seeds relative to open pollinated inflorescences are consistent with inbreeding depression, the expression of mutational load (Charlesworth 1989). This result is consistent with the expectation of early-acting inbreeding depression in predominantly outcrossed species in general (Husband and Schemske 1996) and with numerous studies in Myrtaceae (particularly Eucalyptus) which have revealed evidence for inbreeding depression (presumably because they are well studied as a group rather than because inbreeding depression is a familial trait). For example, Sedgley and Granger (1996) examined embryos in two Eucalyptus species and noted that most self-pollinated ovules did not develop. Kennington and James (1997) found that the self-compatible E. argutifolia was nevertheless highly outcrossed, which they attributed to seed abortion caused in part by early expression of lethal recessive genes. Outcrossed seed in E. obliqua (Brown et al. 1975), E. pauciflora (Phillips and Brown 1977) and E. stoatei (Hopper and Moran 1981) were more likely to germinate and survive than inbred seed. Inbreeding decreased adult fitness in both E. gunnii (Potts et al. 1987) and E. globulus ssp. globulus (Hardner and Potts 1995). On the other hand, Schmidt-Adam et al. (1999) interpreted high germination rates among selfed Metrosideros seed to indicate a lack of early-acting inbreeding depression. The behaviour of birds and honeybees might account for differences in seed quality between open- and insect-pollinated C. citrinus inflorescences. Birds are likely to move 41 pollen greater distances among plants resulting in high levels of outcrossing, as was found by Sampson (1998) in populations of the bird-adapted E. ramiliana. The intermediate level of inbreeding implied by the seed germination experiments in the current study as would be expected if honeybees generally transferred pollen either geitonogamously or to neighbouring plants. Birds were likely to be important pollinators of E. cosmophylla, C. macropuncus, and C. rugulosus (Paton 1982, 1993). Alternatively, factors other than inbreeding may have contributed to the significant delay in germination of C. citrinus in two of the three treatments. For example, polyester bags may have created an unfavourable microclimate that decreased the viability of seeds in the autogamy treatment. I attempted to minimise this potential effect by removing bags when flowering was completed. 2.4.6 Conservation significance and evolutionary implications The conflict between the evolutionary benefits and costs of inbreeding and outcrossing in plants is one of the oldest evolutionary debates and remains unresolved (Darwin 1876, Wright 1931, Jain 1976, Waller 1984, Charlesworth 1989), with no single theoretical model explaining the range of known mating and breeding systems observed in actual plant populations. Theoretically there must be evolutionary advantages to inbreeding, which is very common among plants (Jain 1976, Holsinger 1988). For instance, some degree of inbreeding may be optimal for plant fitness because it conserves local adaptations and prevents the breakdown of co-adapted gene complexes (Shields 1982). 42 Inbreeding may have evolutionary consequences because it may favour certain traits (such as self-compatibility) while at the same time facilitating genetic drift in general. However, inbreeding depression may be irrelevant to population viability if early life stages contribute little to population growth (Haldane 1957, Mitchell-Olds and Waller 1985). In the long run, inbreeding will facilitate genetic drift, decreasing genetic diversity and, particularly for small plant populations, increasing the risk of loss of genetic diversity and local extinction (Wright 1931, Ellstrand and Elam 1993). Inbreeding may also have negative consequences for individual plant fitness if other individuals produce higher quality outcrossed seed. The persistence of honeybees might favour individuals with some degree of self-incompatibility. Callistemon citrinus may be buffered from the negative consequences of honeybee- mediated inbreeding by several factors. Open pollinated flowers, despite the visitation of honeybees, showed no evidence of inbreeding, suggesting that birds are providing outcross pollen to inflorescences. Adult plants typically hold several years accumulated aerial seed bank, which are released en masse following fire, so some of these seed are likely to avoid predation and germinate. Adult plants also resprout after moderate fires (pers. obs.). The conservation consequences of honeybee-mediated inbreeding on C. citrinus is uncertain, but the results of this study, based upon a haphazardly selected bird-pollinated species, suggest that the effect may be more widespread than previously believed. The demography of shrub species generally is poorly known (Harper 1977), even though such plants make up an important structural and ecological component of many Australian vegetation communities. The results of this first attempt in examining the germination rates of honeybee- and open-pollinated seed in an Australian bird-adapted shrub suggest that this is an expedient way to examine the breadth of hidden changes in seed quality as a result of honeybee visitation. Other Australian bird-adapted shrub species with similar characteristics - small populations, no soil-stored seed bank, and an inability to resprout - would be expected to suffer from negative consequences of this inbreeding and should be among the next to be studied using this method. Ideally, the fitness of offspring should be tested in actual field conditions, and followed through to measure fecundity of offspring and the F2 generation, to provide a genuine test of the importance of pollinators to seed quality (Charlesworth and Charlesworth 1987, Herrera 1987). CHAPTER 3. COMPARISON IN EPACRIDACEAE 3.1 Introduction 3.1.1 Epacridaceae pollination systems The plant family Epacridaceae, though relatively large and diverse in the Australian flora and believed to be a clade of the family Ericaceae (Crayn et al. 1998), has received relatively little detailed attention by pollination ecologists. Keighery (1996) summarises the range of animals visiting various species of Western Australian species, noting a number of species which received only bird visits and others visited by moths, flies, bees and butterflies (see also Brown et al. 1997). He describes and illustrates the floral morphology of "specialised" tubular flowers visited by birds, compared to "unspecialised" flowers visited by many species of insect. A few studies suggest that honeybees interfere with bird-pollination systems in Epacridaceae by removing pollen before birds visit flowers, moving pollen to flowers on the same plant (geitonogamy) or moving pollen short distances among plants (Higham and McQuillan 2000, Celebrezze and Paton submitted). 3.1.2 Study aims and predicted results In this study I compared the role of honeybees in the current pollination systems of an insect-adapted and bird-adapted Epacridaceae species. I evaluated the floral visitors of Epacris microphylla and Styphelia tubiflora, perennial shrubs that often co-occur. I linked these data to results of selective pollinator exposure experiments. I made the following predictions: 45 (i) birds would be the most frequent native pollinators of S. tubiflora and insects would be the most frequent visitors of E. microphylla; (ii) open-pollinated and bird-excluded flowers would produce equivalent fruit set in E. microphylla; (iii) bird-exclusion would result in lower reproductive success than open- pollination in S. tubiflora; and (iv) honeybees would move less frequently and shorter distances than birds in populations of S. tubiflora. In order to determine whether the observed patterns or fruit set were consistent among years and populations, I replicated the experiment at two sites in two years. I assessed the outcome of selective pollinator exposure experiments in the light of data on floral longevity, pollen removal, and nectar robbery. Ideally, I would have included an assessment of the quality of seeds produced by various selective pollinator exposure treatments (Table 1.2), but I found that seeds of both species appeared to have dormancy mechanisms preventing germination and both were too small to be used for isozyme analysis of genetic variability. (Research on germination cues is the next step to facilitate this line of research.) 46 (a) (b) Illustration 3.1 The foraging behaviour of honeybees and native pollinators at flowers of Epacris microphylla (a) and Styphelia tubiflora (b). Honeybees, native flies and numerous other insects visit and potentially pollinate the small flowers of E. microphylla. Honeybees collect pollen from S. tubiflora flowers, simultaneously brushing the stigmas, while Eastern spinebills (Acanthorhynchus tenuirostris, shown) and New Holland Honeyeaters (Phylidonyris novaehollandiae) collect nectar through the corolla tube, which results in pollen being brushed onto facial feathers and potentially transferred to stigmas. Illustration by Robert Parkinson. In thefirst flowering season I visited numerous sites with habitat attributes of S. tubiflora but was able to locate only one population where honeybees were foraging, so I compared the effect of exclusion experiments in this population with another population where honeybees were not seen visiting flowers. This approach offered the advantage of contrasting the residual effect of exclusion of birds as in Chapter 2 as well as comparing the results of pollination by honeybees alone at one population (cage selective exposure experiments) to birds alone (open pollination) in another population. 3.1.3 Study species Epacris microphylla is widespread in sandy soils from heath to woodlands throughout south eastern Australia (Fairley and Moore 2000). Plants are single- or multi- stemmed erect wiry shrubs (Illustrations 3.1 and 3.2), typically 30 to 100 cm in height and have shallow fibrous roots. Individual plants produce numerous small white flowers (5 to 7 mm diameter) with no corolla tube, axial to the scale-like leaves (3 to 4 mm length). Flowering is predominantly during the Southern Hemisphere winter and spring (Harden 1990) and individual plants may bloom more than once per year (pers. obs.). Flowers open sequentially along the stem, so that stems appear to be analogous to spike inflorescences (Figure 3.1) (Harden 1990). Upon anthesis, anthers dehisce, releasing pink pollen. The surface of the ovary is green and sticky with a scant amount of nectar; ovaries dry and turn red four to eight days later (pers. obs.). The fruit is a papery five-locular capsule which is retained on the stem and opens upon ripening, releasing numerous tiny seeds (0.1 mm in diameter). Musgrave (1951) observed several species of flies on E. microphylla flowers during August and September at Royal National Park, and T. D. Auld (pers. comm.) 48 suggested the seeds are released gradually from capsules when stems are rattled by the wind. Plants did not resprout from stems after being burned by an intense fire (pers. obs., March 2002). Illustration 3.2 Epacris microphylla at Royal National Park, Australia. Styphelia tubiflora occurs in heathlands on sandy soils and near streams in open woodlands from Jervis Bay to the Central Coast, New South Wales (Harden 1990-1992). Plants are wiry or lax spreading shrubs with rigid, sharply pointed leaves typically 15 mm in length (Illustration 3.1). The axial flowers and nearly mature flower buds are red tubes 20 to 25 mm long (Illustration 3.3) dangling from a flexible pedicel (characteristic of many bird-adapted plant species (Hurlbert et al. 1996)). 49 Mature flower buds can be triggered to open by a touch or pinch to the tip, although they will eventually open without visitation (pers. obs.). The exerted anthers dehisce prior to flower opening and pollen is deposited on the hairy, curled petals. The distance between anther and stigma ranges from approximately one to 15 mm (pers. obs.); further investigation is needed to determine if this results from the growth of pistil or styles during the life of the flower or heterostyly among individual plants. Droplets of nectar are visible through the translucent base of the corolla tube in most mature flower buds and recently opened flowers. The fruit is a hard capsule that is dropped upon maturing and dehisces when heated (pers. obs.) releasing up to five oblong seeds (approximately 2 mm long). Plants are killed by fires (pers. obs. March 2002). Ford et al. (1979) predicted that Styphelia species were exclusively bird pollinated. Styphelia tubiflora flowers only once per year in winter (Harden 1992, Fairley and Moore 2000). Illustration 3.3 Styphelia tubiflora © the Australian National Herbarium. 50 I initially selected a plant species that subsequently did not conform to my experimental design. I observed honeybees in one population of the apparently insect- adapted Woolsia pungens on one occasion but subsequently recorded only a few honeybees at either of two populations (Royal National Park and Botany Bay National Park), so that they did not appear to be common visitors. Flowers opened in the evening and produced a strong nutty odour, suggesting pollination by crepuscular or nocturnal moths (Armstrong 1979). Such pollinators were likely to be excluded by 1.5 cm2 mesh used in experimental exclusions, biasing the results of exclusion in comparison to open controls. For these reasons, I terminated study of this species. 3.1.4 Study sites I observed potential pollinators and performed field experiments in two populations for each species over two years. I studied populations of E. microphylla at Toonoum Falls (population 1, 151° 2' E, 34° 10' S) and Curra Moors (population 2, 151° 4' E, 34° 8' S), Royal National Park in 1999 and 2000. Likewise, I studied S. tubiflora at Toonoum Falls, Royal National Park (population 1, 151° 2' E, 34° 10' S) in 1998 and 2000 and at Bundanoon Creek, Meryla State Forest (population 2, 150° 25' E, 34° 37' S) in 1999 and 2000. The vegetation at Curra Moors was heath (Siddiqi et al. 1976). The Toonoum Falls location was a mosaic of heathland and woodland. Bundanoon Creek was woodland with a heathy understorey. Both Royal National Parks sites burned in 1994, while the Bundanoon Creek site had not burned since at least 1983 (N. Cowley pers. comm.) 51 I conducted experiments and bird observations on S. tubiflorafrom May to August in Royal National Park and July to November at the more elevated Bundanoon Creek site. I studied E. microphylla at Royal National Park from July to September. 3.2 Methods 3.2.1 Floral longevity I estimated floral longevity of both species in order to calculate, based upon pollinator behaviour data, how many visits a flower was likely to receive before fading. I collected stems from ten plants from population 1 of each species, placed immediately in weak sugar solution in a room with indirect sunlight, and followed the fate of the cohort of flowers which opened. Although this method is not ideal, I noted that flower buds continued to open for several days after stems were cut. Styphelia tubiflora flowers, on the other hand, wilted within an hour of cutting. Epacris microphylla stems collected on 8 September 2000 produced and held flowers through to 25 September 2000 (one to 27 flowers per plant). I used the average floral longevity to calculate the number of visits each flower was likely to receive. 3.2.2 Potential pollinators To evaluate whether pollinator suites corresponded to the apparent bird- and insect- adapted floral morphologies of the two study species, and to estimate the likely effectiveness of floral visitors as pollinators, I used the methods of recording insect behaviour described in Section 2.2.1. I observed floral visitors at each population and recorded the following data, where possible: 52 (i) the species (or morphospecies for native insects) of visitor; (ii) what floral resource each collected (pollen and/or nectar); (iii) the rate of movement while foraging (number of flowers and plant visited per minute); (iv) the proportion of total movements among flowers that were movements among plants; and (v) the distance of movements between consecutive plant visits. 3.2.3 Insect floral visitors and visitation frequency To measure the diversity of potential pollinators and the frequency of their visits to flowers and plants, I censused insect floral visitors at intervals throughout the day on several days during the flowering period for both plant species. I included in each census flowering individuals within a set of plants I had selected haphazardly on a previous day (when they were budding). On census days, some previously tagged plants were not flowering, so the number of plants observed varied from day to day ranging from 14 to 48 plants in each E. microphylla population and 10 to 48 plants in S. tubiflora. I counted the number of open flowers on each of the plants observed and, at each one- or two-hourly interval, I recorded the number of individual insects foraging at flowers on each plant. Initially, I used instantaneous censuses but I soon realised that, although honeybees were foraging in the populations, I was very unlikely to detect them with this technique, so I increased my observations to 30 seconds per plant (timed on a stopwatch). 53 In order to describe the range of species potentially pollinating each species of plant, I identified native insects to family and morphospecies. Where possible, I captured specimens of each morphospecies and measured their body length as an indication of their potential to brush both stigmas and anthers during flower visits. I made these observations on warm days when insects were likely to be most active. As a result, my estimates of visitation frequency were likely to exaggerate the actual average number of visits a flower received, since some inclement days would be likely during the lifetime of any given flower. 3.2.4 Honeybee and other insect foraging behaviour To estimate the daily number of insect visits to each flower and plant, I timed honeybee foraging bouts opportunistically throughout the flowering period as with Myrtaceae (Section 2.2.1), recording 31 individual bouts for S. tubiflora and 44 for E. microphylla. I could not track individual insects but I assumed that each bout was a separate, independent individual for purposes of analysis because bouts were collected throughout the study season in each year. I calculated the daily number of all insect visits per flower and per plant using the method described in Chapter 2, by combining the frequency with species visited groups of plants during censuses throughout the day (number of visits per flower or plant per day) and the rate this species foraged (from observations of individual bouts) (as per Paton, pers. comm.). 54 3.2.5 Bird floral visitor frequency To determine how frequently S. tubiflora plants were visited by nectar-feeding birds, I selected vantage points from which I could clearly observe bird visits to a set of flowering S. tubiflora plants (ranging from six to 31 individuals) using binoculars and observed these groups of plants for periods of 30 to 60 minutes (timed on a stop watch). During these periods, I recorded the number of flowers on each plant, the duration of each bird visit within the observation area and the proportion of plants visited. I made these observations on two days in each population during the first year (1998 and 1999) and on four and three days respectively in population 1 and 2 in 2000, with the assistance of volunteers. I grouped results into three time periods: morning (6:00 to 10:00), midday (10:00 to 12:00), and late afternoon (14:00 to 18:00). I calculated the average proportion of plants visited per hour for a total of 51.25 hours of direct observation. Increased effort in 2000 did not appear to result in an increase in the rate of detection of bird floral visitors, suggesting that birds were genuinely infrequent visitors (Figure 3.3). The proportions of visits were not normally distributed (Kolmogorov-Smirnov P < 0.005, Shapiro-Wilk test P = 0.01) and variances were not homogeneous (based on the mean proportion of plants visited, Levene Statistic = 6.09, P = 0.004), so parametric ANOVA and t-tests are not applicable (Zar 1984). I used a non-parametric Kruskal-Wallis test and non-parametric multiple comparisons (Zar 1984) to determine if differences in visitation rates among time of day were significant. 55 3.2.6 Bird foraging behaviour I examined the foraging behaviour of eastern spinebills (Acanthorhynchus tenuirostris) foraged in S. tubiflora by observing the movements of individual birds. I made these observations on five dates at both populations. I recorded the number of flowers and plants individual birds visited, the duration of visits and the distance moved among plants. Because of the tangled condition of the vegetation, I had to observe birds at close range to obtain these data; in total, I recorded 10 bouts on 5 days in 1998 and 2000. I combined the foraging rate data with census data and divided by the average number of plants I was observing in total to estimate the average number of visits to plants each day. (I could not calculate visits per flower per day because the census data occurred across the range of the species' floral phenology but was pooled.) As with honeybees, I noted, where possible, whether bird movements among plants were between neighbouring plants, one to five m, five to ten m, or > ten m. 3.2.7 Other evidence of pollinator visits Direct observations may underestimate the total visitation to flowers during floral lifetimes because of poor visibility and because birds might visit at times when observations are not being made. Therefore, I examined S. tubiflora flowers for evidence that nectar and pollen were being removed from open flowers. I measured volume of S. tubiflora nectar and the presence or absence of pollen for 108 flowers on 23 open plants and 147 flowers on 12 bagged plants to indirectly quantify the removal of nectar by birds and pollen by birds and honeybees. I measured nectar concentration in both groups to ensure that the differences in volume observed could not be accounted for by differences in 56 evaporation. Bagged plants had significantly more open flowers than exposed plants, possibly because I had selected larger than average plants for bagged treatment to ensure that some flowers would be open or because bagged plants tended to retain flowers longer. After recording some initial nectar data, I noted that some flowers on unbagged plants appeared to have damage resulting from nectar robbery by birds (sensu Inouye 1980). I recorded the number of flowers with this damage on 11 haphazardly selected plants that were spatially intermingled with the selective pollinator exposure experiment (I did not examine the experimental plants in order to avoid confounding the experiment through incidental pollination or other effects). I used t-tests to determine if mean nectar volumes, nectar concentration and pollen removal differed between open and bagged plants. 3.2.8 Breeding systems I used cross- and self-pollination treatments to test whether the two plant species were self-compatible or self-incompatible, and, by comparing the results of these treatments with open pollination, to determine if they pollinators may be a limiting factor in fruit production in the study populations. I randomly assigned five to 18 plants to treatments in each population of both species in 2000 and in S. tubiflora in 1998. I bagged budding plants with 1 mm2 nylon mesh in the first year but in the second year I used polyester fabric bags to avoid inadvertent cross pollination. For both species, I transferred pollen to stigmas of all open flowers within the treatment from anthers of flowers picked from a bagged source on the same plant (selfed) or from haphazardly selected donor plants 5 to 10 metres away (crossed). I labelled treated flowers with a permanent marker and when possible I repeated treatments on more than one day to try to minimise the effect of variation in 57 stigmatic receptivity or pollen viability onfruit set . I report the self-compatibility index (SI) as the proportion of fruits per flower (taken at maturation) to facilitate comparison with other studies (Kendrick and Knox 1989, Becerra and Lloyd 1992, Vaughton 1996, Hermanutz et al. 1998). I analysed the results of these tests along with the results of selective pollinator exposure experiments below. 3.2.9 Selective pollinator exposure experiments In order to measure the relative roles of pollinator suites in determining fruit set for both species, I randomly assigned individual plants to one of three pollinator exposure treatments as described in Chapter 2. For all treatments (including the breeding experiment), I returned four to six weeks after flowering and recorded the proportion of flowers that produced capsules. For a sample often capsules per plant, I counted the number of seeds per capsule. I harvested these capsules haphazardly along the whole length of the flowering stems to avoid confounding effects of floral position on seed set. The ratio of capsules to flowers and the average number of seeds per capsule were approximately normally distributed (homogeneity of variance tests P > 0.05) and variances did not differ significantly across treatments (Levene test 2-tailed P > 0.05) for both selective pollinator exposure experiments and breeding system tests. I compared these values among treatments using one-way ANOVA and Tukey's HSD test. I calculated the percentage of initiated fruit that later aborted for 5. tubiflora to determine if plants appeared to be selectively aborting the fruit resulting from any of the treatments (tested using one-way ANOVA and Tukey's HSD test). Unlike S. tubiflora, 58 there was no clear visual cue for E. microphyllafruit initiation , so I could not determine whether fruit was aborted subsequent to initiation for this species. I used the method described by Zar (1984, p. 173) to calculate the power of the experiments to detect differences among treatments in fruit set. I report "phi", which is related to the noncentrality parameter and is plotted against for various alpha levels (Zar 1984, Appendix Fig. B. 1); I could detect a significant difference of 5% with the power shown in Table 3.1.1 report power for a = 0.05. In some populations and years experimental power was very low because of the small sample sizes and high variances in fruit set (Table 3.1). Sample sizes were generally restricted by the available time, but in population 2 of S. tubiflora I used all adults plants I could locate in both years (in 2000,1 found additional plants). Standard deviations were greater in the second year, outweighing my attempts to increase power in population 1 of E. microphylla in the second year (power decreased from 0.80 to 0.50 despite an increase in sample size from 8 to 10). A slight decrease in sample size, combined with an increase in standard deviation, shrank the power of experiments in E. microphylla population 2 from 0.90 to 0.70. 59 Table 3.1 The experimental power of selective pollinator exclusion experiments in two populations of Epacris microphylla and Styphelia tubiflora. Because samples sizes were not equal in all treatments in most years and populations, power at phi for a = 0.05 was calculated based upon an average sample size. The power reported is therefore approximate. average approximate population year n s Phi power Epacris microphylla 1 1999 8 0.243 2.00 0.80 2000 10 0.354 2.52 0.50 2 1999 8 0.188 2.16 0.90 2000 6 0.285 0.75 0.70 Styphelia tubiflora 1 1998 6 0.193 0.52 0.30 2000 20 0.423 2.36 0.97 2 1999 5 0.228 1.22 0.35 2000 15 0.229 2.50 0.90 3.2.10 Potential cage effects I observed honeybees and native insects moving through bird-exclusion mesh at both populations of E. microphylla and population 1 of S. tubiflora to determine if honeybees and native insects visitation rates were reduced by the presence of the cages. I compared visitation rates to caged versus open plants during hourly censuses using one- tailed x2 tests- 1 noticed that S. tubiflora flowers sometimes poked out of cages where they may have been contacted by birds in 1998, so in 1999 and 2000 I modified the design of the bird-exclusion cages to prevent this. I report the results of all three years and examine the potential confounding effect of the earlier treatment in the discussion. 60 3.3 Results 3.3.1 Epacris microphylla 3.3.1.1 Floral longevity In both years, E. microphylla bloomed for four to six weeks beginning in early September in both populations. Flowers were relatively long-lived compared to examples reviewed by Primack (1985), remaining open for six to 15 days (n = 10 plants, 101 flowers). 3.3.1.2 Pollinators and their behaviour I observed at least 27 species of native insects visiting flowers of E. microphylla (Table 3.2). Nineteen of these were flying species of insects greater than 2 mm in length and were therefore likely to brush the anthers and/or stigma of a typical flower (Figure 3.1). Flies were predominant and appeared to seek to obtain nectar from the sticky surface of the ovary, as did five species of bees. I observed two species of native bees move among flowers and plants at a slower rate than honeybees (Table 3.3). Beetles appeared to be eating pollen or anthers or to be resting on flowers. 61 Table 3.2 Insect morphospecies observed visitingflowers of Epacris microphylla in two populations in Royal National Park, New South Wales, during winter flowering periods in 1998, 1999 and 2000. Order Family and morphospecies body lenc: population 1 Coleoptera Buprestidae 4.0 Diptera Muscidae sp. Em1 4.0 Diptera Muscidae sp. Em2 5.0 Diptera Muscidae sp. Em3 10.0 Diptera Muscidae sp. Em4 12.0 Diptera Series Schizophora 1.0 Diptera Syrphidae 9.0 Diptera Tipulindae 25.0 Hemiptera Pentatomidae sp. Em1 8.0 Hemiptera Miridae 8.0 Hymenoptera Formicidae sp. Em1 2.0 Hymenoptera Formicidae sp. Em2 20.0 Hymenoptera Apoidea sp. Em1 5.0 Hymenoptera Apoidea sp. Em2 10.0 Hymenoptera Apoidea Apis mellifera 10.0 Thysanoptera Aeolothripidae? 0.1 population 2 Coleoptera sp. Em1 2.0 Coleoptera sp. Em2 0.1 Diptera sp. Em1 3.0 Diptera sp. Em2 2.0 Diptera Muscidae sp. Em5 9.0 Diptera Muscidae sp. Em3 10.0 Diptera Muscidae sp. Em6 15.0 Hemiptera Pentatomidae sp. Em1 8.0 Hemiptera Pentatomidae sp. Em2 10.0 Hymenoptera Apoidea sp. Em3 4.0 Hymenoptera Apoidea sp. Em4 6.0 Hymenoptera Apoidea sp. Em5 10.0 Hymenoptera Apoidea Apis mellifera 10.0 Hymenoptera Formicidae sp. 1 2.0 Hymenoptera Formicidae sp. 3 4.0 Lycaenidae Nymphalidae 40.0 62 I observed honeybees visiting E. microphyllaflowers mor efrequently than any other species of insect, although native insects in general were still frequent visitors (Figure 3.3). I did not detect honeybees in censuses on 1 September 2000, which was abbreviated due to a sudden storm (Figure 3.3), but honeybees were foraging in both populations on this day (pers. obs.). Fifty percent of honeybees that I observed (22 of 44) foraged only for nectar, while 47.7% foraged for pollen alone and 2.3% (one individual) foraged for both. (Two individuals also gathered nectar from Leucopogon microphallus (Epacridaceae) during foraging bouts.) 63 Table 3.3 Pollinator behaviour while foraging among flowers and plants of two species of Epacridaceae in New South Wales during winter flowering periods, including the average number of flowers visited on each plant per minute of observation, and the proportion of total movements which were among plants for birds, honeybees and native insects, with standard errors. Honeybee bout observations ranged from 30 seconds to more than 5 minutes each. movements per minute proportion of total among among flowers movements bouts flowers plants per plant among plants observed Epacris microphylla honeybees foraging for nectar 14.8 + 1.2 4.8 + 0.5 3.3 + 0.4 0.36 + 0.04 22 foraging for pollen 17.4 + 1.1 3.9 + 0.5 4.3 + 0.5 0.22 + 0.03 21 overall 16.0 + 0.8 4.4 + 0.3 3.8 + 0.4 0.29 + 0.03 43 Apoidea sp. Em1 11.9 + 0.9 3.1+1.1 3.3 + 0.7 0.26 + 0.08 2 Apoidea sp. Em4 5.7 1.4 2.5 0.25 1 Diptera Muscidae sp Em4 8 5 1 Diptera sp Em1 3.9 7 1 Formicidae sp. Em2 1.1 0.19 3.5 0.17 1 Styphelia tubiflora honeybees 4.1+0.9 1.8 + 0.3 2.9 + 0.5 0.48 + 0.07 28 eastern spinebilis 13.3 + 2.0 3.5+1.1 5.6 + 1.8 0.26 + 0.04 10 64 c — o c 3 .*: o CoD _(:0 .Q CU CO CD O •D 0) CU CO _ CU CD CO _3 '•a^>a O CC CO c c •o CD c CL TO >% O CU CO l_ la- cn CD *•—' _3 CO CU E CU 3 "O _3 c cu CD cu C h- CD o Cfl CD CO _z __ c CD C/) CO CO O 3 c > < cn _: J- Q. 5 u c CO QC_D 5^ CcD CD CD Q. c T3 CD M— CD o -^ 7 cu Q. 7D E o i- a: .W •c~ CO cCJ c CD o Q. -«—• UJ CD 3 % O Q_ CO c O X °o.'<% a. CD 'cPx XLo o X O CU 03 Q. £ O r~ a i— o 55 Q- cn 3 C- o 0 CJ O CD a CD CN °o. CU o CN •«_• c ill % CD CD CN T- CO CO o CN T— CO CO a °0.: co CD cu o o 1 O 0) CD +- o, L_ T3 3 CD O spasui 6u;)!SiA q,iM sjue|d \o uoijjodojd CO CD u. .>£ a. 65 When visiting plants, honeybees typically visited only 4 flowers per plant (Table 3.3), even though plants on average displayed 20 and 68 flowers on the days on which bouts were recorded. Overall, 29 ± 3% (s.e.) of all honeybee movements were between plants (Table 3.3), and these plants were almost always neighbours less than a metre apart (97.7%, n = 482 movements). Honeybees foraging for pollen moved significantly less frequently between plants (t = 2.71, df = 42, one-tailed P = 0.004), and visited significantly more flowers on each plant (t = 1.65, df = 42, one-tailed P > 0.05) than honeybees foraging for nectar. More than 90% of the 118 honeybee movements among plants were to the nearest neighbouring plant, while 3.4% were within two to five m and 5.9% were 5 to 10 m. A greater proportion of plants received visits in the morning than later in the day (Figure 3.3). 3.3.1.3 Frequency of honeybee and native insect visits The extrapolated daily visits per flower and plant (Table 3.4) for honeybees versus native insects suggest that honeybees and native insects were visiting plants and flowers with comparable frequency. I used the average foraging rates from 43 honeybee bouts and the highest foraging rate of a native insect (Table 3.3) because I was able to obtain only a few foraging rates for native insects. This may exaggerate native insect foraging frequencies and in any case can only be interpreted as a rough estimate. 66 CN O CO CD T3 •— LO cu CD c "~ oo SZ X> ro > CO CD. X) o 1— o CD •a-< COD a. o CO O3 ro CD CO '" CN co T~ O X) > D. § i— c co CO 5 CU CD > o x CN _> c >•- CO CO ro ClaD. CD CO •a Q. X> CD xs CU 3u. CD CD CO CO *. CD CN co o CD O T- CN O 00 CN 3 C CD •f L^ E CD oo "fr CO IO CM CO IoD CO co co CD- CD CD CD L o CO CD CD co" 3 X> CD- +-* CO c >, o CD CD CD 00 •* CD o CO O CO LO O CO CO CD C CO o c o 1 >CD CO sz CD c 8. c CaD. T3 T— CN CO c o co o o o o o o o •o CD i2 CD Q. CD CO 3 •J>= CoD CD O _ CD CO CD I— CD J_ CO CO c c >-. CD -3 CD O CO CN co CN o co o oo TJ- o co co c ro CO CD L_ xo CO XC)D c -»-o» >-. XI CO CO CO CD CD > C _! ro CO 3 o CD CO sz CD- aCO c L- CD o T— CD U cn CD CJ) o •*»• o •«* CM T- co D. ID a—• O) CO c co r- oo m io o CN o io r«- CD co co X3 c x> o CN »- CM CO t CO ^ TT c •c CN CD ro _ o 1 CD CD o CO CD OO CO •<* 5 XI c CO oo CD S O CO O) 67 3.3.1.4 Breeding system Experimental self-pollination produced fruit in both E. microphylla populations in 2000 (population 1 SI = 0.68, population 2 SI = 0.88). Both treatments produced a significantly lower proportion of fruits to flowers than open pollinated or bird-excluded plants (Table 3.5). Self-pollinated flowers produced fewer seeds (2.5 + 0.8) than cross- pollinated ones (10.5 + 4.5) in population 2 (Table 3.5). This lower seed set could not have resulted from differences in the quantity of pollen deposited on stigmas, since I used the same technique for both self- and cross-pollination treatments. Early-acting inbreeding depression (e.g. embryo abortion) or partial self-incompatibility mechanisms may have contributed to this result. Autogamy treatments resulted in significantly fewer capsules per flower than bird- exclusion as well as open-pollination (with the exception of population 2 in 2000). These plants also had the lowest average number of seeds per capsule in population 2 in 2000 (Table 3.5). Data suggests that honeybees were not significantly deterred by cages (Table 3.5) but the power for these tests to detect deterrence was weak because of the infrequency of visits overall and the small sample sizes. 3.3.1.5 Selective pollinator exposure experiments Caged E. microphylla flowers produced fewer capsules per flower (0.60 + 0.09) than open pollinated plants (0.85 + 0.5) in population 1 in 2000, but not in the other three experiments (Table 3.5). There was no apparent partem in seeds per capsule between these treatments. 68 Honeybees were significantly less likely to be present on caged plants than open plants in population 1 in 2000 (Table 3.6) but not in any of the other case. 69 Table 3.5 Results of selective pollinator exclusions and self- and cross-pollination experiments on reproduction of Epacris microphylla in two populations in two years. Within each population and year, proportions marked with the same symbol or letter are not significantly different (P > 0.05, Tukey's HSD test). Within populations and years, there were no significant differences in seeds per capsule (P > 0.05, one way ANOVA). The number of seeds per flower is derived from the average number of seeds per capsule in a sample of capsules. Proportion of Seeds per Treatment capsules per Number of capsule (sample seeds per flower (+ s. e.) plants size) flower population 1 1999 open pollinated 0.53 + 0.07a g 9.0+1.0(9) 4.8 bird excluded 0.36 + 0.06b 10 5.4+1.8(6) 1.9 autogamy 0.11 + 0.27° 5 6.7 + 4.7(2) 0.7 2000 open pollinated 0.85 + 0.05a 11 - - bird excluded 0.60 + 0.09ab 9 - - autogamy 0.18 + 0.06° 10 - - self-pollination 0.24 + 0.06° 8 - - cross-pollination 0.32 + 0.09bc 10 - - population 2 1999 open pollinated 0.32 + 0.05a 10 4.6+1.0(8) 1.5 bird excluded 0.22 + 0.03b 9 5.1 + 1.5(4) 1.1 autogamy 0.05 + 0.01° 8 - - 2000 open pollinated 0.50 + 0.10a 7 7.0+1.5(5) 3.5 bird excluded 0.56 + 0.083 7 6.2 + 0.8 (6) 3.5 autogamy 0.09 + 0.04" 5 2.0 + 0.6 (3) 0.2 self-pollination 0.29 + 0.10° 9 2.5 + 0.8(7) 0.7 cross-pollination 0.33 + 0.08a 5 10.5 + 4.5(5) 3.5 Table 3.6 Results of % tests of the presence of honeybees in caged inflorescences during hourly censuses. Total observations (number of times individual plants were censused) are reported. The values were significantly different at a = 0.05 (df = 1) only in Epacris microphylla population 1 in 2000 . open exclusion pollinated treatment chi-square Epacris microphylla population 1 1999 honeybees 12 0 total 280 56 2.39 2000 honeybees 4 0 total 68 63 3.89 population 2 1999 honeybees 11 2 total 224 74 0.57 2000 honeybees 0 0 total 28 28 0 Styphelia tubiflora population 1 2000 honeybees 12 28 total 327 731 0.02 3.3.2 Styphelia tubiflora 3.3.2.1 Bird visitors Of the 25 individual birds observed visiting S. tubiflora, 92% of birds visiting were eastern spinebills (Acanthorhynchus tenuirostris) and the remaining 8% were New Holland honeyeaters (Phylidonyris novaehollandiae, only seen visiting S. tubiflora in population 1). Honeyeaters landed on stems and probed corollas of S. tubiflora resulting in pollen being brushed on the face and head (Illustration 3.1). Eastern spinebills moved frequently among plants while foraging, and typically probed about six flowers on each plant (Table 3.2) before moving to another plant. Many bird movements were obscured by vegetation, but of those noted (n = 18 movements), 66.6% were among neighbouring plants, 5.5% were to a perch within 5 m, and 27.8% were greater than 10 m. Unlike honeybees, which forage locally until they return to the hive, birds appeared to move through the population sporadically, stopping to forage briefly before moving on, typically to nearby flowering Banksia plants (pers. obs.). Bird visits were highly variable among populations (Figure 3.3). Only about 10% of plants were apparently visited by birds each day in population 1, compared to two visits per plant per day in population 2 (Table 3.4). Bird visits were more frequently earlier in the day and no birds were detected visiting observed groups of S. tubiflora plants after 10:00 in population 1 in 1998 or in any other instance after 14:00 (Figure 3.4). When results were pooled across population and years, the proportion of plants visited was significantly less during the period from 14:00 to 18:00 than either 6:00 to 10:00 (Mann-Whitney U = 215.5, P = 0.021) or 10:00 to 14:00 (U = 191.5, P = 0.016). 72 3.3.2.2 Honeybees foraging frequency and behaviour At population 1, honeybees collected pollen from S. tubiflora by clasping the anthers and stigma of a flower in a bundle (Illustration 3.1). In 1998,1 observed only one honeybee out of a total of 297 instantaneous censuses of plants on 12 June and 31 July. In 2000,1 increased the amount of time I observed each plant for floral visitors to thirty seconds; these thirty-second censuses in 2000 revealed that honeybees foraged between 9:00 and 15:00 with a peak around 11:00 (Figure 3.2). Honeybees typically collected pollen from 2.6 + 0.2 flowers at each plant they visited. Of the among-plant movements I recorded (n = 34 movements), honeybees nearly always moved among neighbouring plants (91.1%) although I did observe individual honeybee movements from 2 to 5 m and from 5 to 10 m. By measuring the number of movements among flowers and plants per minute, I determined that honeybees foraged much more slowly among flowers and half as slowly among plants than birds. However, honeybees moved more frequently than birds among plants (Table 3.3). Extrapolated visitation rates (Table 3.4) suggest that each plant was likely to receive many honeybee visits daily. Most flowers are likely to receive several honeybee visits daily. Although I occasionally saw honeybees foraging on other plant species in population 2,1 never observed honeybees visiting S. tubiflora at this location during hourly censuses (3 days between 8:00 and 16:00, 10 plants observed), nor did any of four other field workers on eight other days (14 person days). Therefore, the two populations provided a useful contrast between the reproductive success of flowers when exposed to birds and honeybees versus honeybees alone. 73 9:00(111,3) 11:00(207,5) 13:00(207,5) 15:00(207,5) time of day Figure 3.2 The proportion of Styphelia tubiflora plants in population 1 observed being visited by honeybees at intervals throughout the day, with standard error among days (total number of plants and days shown in parentheses). Table 3.7 Average number of among plant movements per day by eastern spinebills on Styphelia tubiflora based on an average foraging rate of 3.5 plants per minute and total foraging time (adjusted for average observation time per 4 hour period). Visits per plant per day were calculated from the total foraging time multiplied by the foraging rate, divided by the average number of plants observed. average average number average number number of total visits per observation of plants visits foraging plant per time period time (hrs) ot•serve d obsserve d time (sec) day population 1 1998 06:00 to 10:00 1 16.6 8 32 10:00 to 14:00 1 20 0 0 14:00 to 18:00 0.75 20 0 0 average daily total 20 32 0.1 2000 06:00 to 10:00 0.78 19.8 9 28 10:00 to 14:00 0.69 21.6 10 28 14:00 to 18:00 0.66 24 0 0 average daily total 21.8 56 0.1 population 2 1999 06:00 to 10:00 0.67 9 28 75 10:00 to 14:00 0.75 9 36 108 14:00 to 18:00 0.88 9 17 60 average daily total 9 243 1.6 2000 06:00 to 10:00 0.58 8.5 74 171 10:00 to 14:00 0.58 8 50 116 14:00 to 18:00 0.75 8.8 0 0 average daily total 8.4 287 2 o o 5 cu CO P. oP^ S >, •*t «- oo CL. CD o U TJ O ° O ° CD X o ~ •* ^ CD CD CO JD := X 3F o o CU C c o o P. co Q. CO o CO "~ O ^ CO c o O T- t~ o Cl) CD o CO 3 ro Q. 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Nectar concentration did not differ among treatments, suggesting that the result was not an artefact of differential evaporation (26.5 ± 5.5% sugar for bagged, versus 22.6 ± 7.4% for open pollinated). Pollen was often absent from flowers. Of eleven unmanipulated plants examined in detail, nine (81.2%) had some flowers with pollen absent (average 80 ± 9% flowers without pollen). However, a sample of three bagged plants revealed that 66.9 ± 20.5% of flowers were missing pollen, suggesting that the powdery pollen may be easily brushed off by bags or drop off after several days. Seven of the eleven exposed plants (63.6%) had damaged flowers. On these plants, an average of 61.7 ± 34.3% of flowers were damaged. Damage included broken anthers, pierced corolla (typically near the nectaries at the base), and crushed corolla, and appeared to be the result of a bird's beak, rather than an insect's mandibles. (In bagged plants, only 1.9 ± 1.9% flowers on one of three plants appeared damaged.) The sample of exposed plants may have been unrepresentative of the population as a whole because these plants had fewer flowers than bagged plants. Since pollinators are likely to be attracted to larger plants, the indirect visitation reported here is likely to underestimate the proportion of the plant population that were visited by birds. How this would affect the proportion of flowers damaged overall is not clear. 77 3.3.2.4 Breeding system Styphelia tubiflora was self-compatible at both populations in 2000 and in population 1 in 1998 (population 1 SI = 0.74, population 2 SI - 0.65). Fruit set did not differ significantly with self-, cross- or open- pollination treatments (Figure 3.4). In 2000, fruit set in both populations was limited by the effectiveness of pollinators; cross pollination resulted in a significantly higher proportion of fruit set (Tukey's HSD P < 0.05, Figure 3.4) than either autogamous or open pollinated treatments (Figure 3.4). I found that S. tubiflora has an unusual mechanism which appears to promote outcrossing but ensure fruit set through delayed autogamy. Dense hairs on the petals and interior of the corolla are dusted with pollen when flowers open. These hairs also brush anthers and then the stigma during abscission of the corolla upon completion of flowering. Autogamous fruit set is nevertheless lower than fruit set through self-pollination (significant in population 2 in 2000, Figure 3.4) and sometimes fails (population 2 in 1999). This may have resulted from poor pollen viability and/or stigmatic receptivity by the time flowers drop and brush pollen on the stigma. Individual cross- and self-pollinated flowers were retained on stems for up to eight days (pers. obs.). 78 °h o o X! % -o<9,.. <*. o *V o i-H c CN CD W-6 CD o W o X! o CM CD O AX "a£ re I CD 3 s % ^ a. c" 1 1 i 1 I oo h- CD IO •0- co CN I o5 o o o o o o o "Sfc. *o •%, Q- in.y paonpojd ipiuM sjaMoy jo uojjjodojd X 3.3.2.5 Selective pollinator exposure experiments Honeybees were present in equal frequencies on caged and uncaged S. tubiflora plants (for population 1 in 2000, %2 = 0 02, not significant at a = 0.05), suggesting that they were not deterred by cages (Table 3.5). Honeybees can apparently pollinate S. tubiflora (Figure 3.4). Exclusion of birds but not insects resulted in significantly greater fruit set than open pollination in the presence of honeybees (population 1, 2000, Tukey's HSD P < 0.05), but not in the site where honeybees were absent (population 2 both years, Figure 3.4). There were no significant differences in the abortion rates of fruits among treatments in population 1 (Table 3.8). 80 Table 3.8 Percent of flowers which initiated fruit set and then aborted (with standard error) for open-pollinated, bird- excluded, bagging (autogamy), self-pollinated and cross-pollinated treatments for Styphelia tubiflora population 1. There were no significant differences at a = 0.05 using one-way ANOVA and Tukey's HSD tests. percent of flowers which initiated number of plants treatment fruitset, then in sample aborted (+ s.e.) open pollinated 13.5 + 2.9 19 bird excluded 12.1+3.0 20 autogamy 16.8 + 5.2 9 self-pollinated 7.3 + 2.4 6 cross-pollinated 3.5+1.7 6 3.4 Discussion 3.4.1 Predicted and observed outcomes Direct observations of nativefloral visitors to E. microphylla and S. tubiflora support the expectation (prediction i) that these species provide a genuine comparison of the effects of honeybees between these two pollination adaptations. Epacris microphylla was visited by 20 species of potentially pollinating native insects, predominantly flies and bees as noted by Musgrave (1951), while S. tubiflora flowers were probed by long-billed honeyeaters (Illustration 3.1). As expected (prediction ii), exclusion of birds had no apparent effect on fruit set in E. microphylla. Surprisingly, exclusion of birds resulted in significantly greater 81 fruit set than open pollination in the S. tubiflora population visited by honeybees (contra prediction iii). 3.4.2 Breeding systems Both species were self-compatible and somewhat autogamous, and breeding system tests provided no evidence that either species was preferentially outcrossing. Overall, E. microphylla plants from which all potential pollinators were excluded (autogamy treatment, Table 3.5), produced significantly fewer capsules per flower plants exposed to pollinators, indicating that pollinators contributed to fruit set. Close examination of the flowers of S. tubiflora revealed that corolla abscission could facilitate delayed self-pollination, as has been found in Mimulus guttatus (Dole 1990, 1992). Hairs on the internal surface of the corolla tube brush past anthers and then the stigma as flowers drop. This suggests that reproductive assurance may have been an important evolutionary factor for S. tubiflora because bird pollinators may not provide an effective service in every flowering season, although these hypotheses requires experimental testing (Lloyd 1992, Eckert 2002). 3.4.3 Foraging behaviour of birds and honeybees In the population where honeybees gathered pollen from S. tubiflora, these insects were much more frequent visitors than birds. Each day, eastern spinebills and New Holland honeyeaters visited an estimated 10% of flowering S. tubiflora plants in population 1 (Table 3.4) compared to dozens of visits per day by honeybees (Table 3.3). Honeybees moved relatively frequently among plants compared to eastern spinebills (48.4% versus 82 26.1%, Table 3.2). However, honeybees nearly always moved to a near neighbouring plant (91.1% < 1 m), while more than 27% of bird movements were greater than two metres. As a result, honeybee pollination may be causing inbreeding if neighbouring plants are related. Honeybee foraging behaviour may be interrupting the evolutionary advantages of the bird- adapted S. tubiflora "facultative xenogamous" sexual system (Cruden and Lyon 1989), which may have assured reproductive success in years when bird visitation was low, but promoted long-distance outcrossing in other years. 3.4.4 Confounding effect of floral robbers The current study is unique in demonstrating that exclusion of birds but not insects may result in greater fruit set than open pollination in an Australian bird-adapted plant species (Figure 3.4). This counterintuitive effect may have resulted from the incidental exclusion of bird nectar robbers as well as legitimate bird pollinators from caged (and bagged) flowers. This view is supported by evidence of damage to flowers. In population 1 in 2000, nectar robbers apparently damaged 62% of flowers on 7 of 11 plants assessed. Short-billed nectar eating birds may pierce, rip, or crush tubular flowers or flower buds to access nectar, as has been found in cultivated Nicotiana glauca and Antholiza sp. in Australia (white-eyes, Zosterops sp. and white-plumed honeyeaters, Lichenostomus penicillatus, Paton pers. comm.) and in wild populations of Fuschia megellanica in South America (Traveset et al. 1998). This behaviour is likely to damage ovaries or pistils, reducing fruit set. Alternatively, birds may be depositing pollen from other species which they are visiting (e.g. Banksia) on S. tubiflora pistils, resulting in pollen clogging (Vaughton 1992, 1996). 83 3.4.5 Bird visitation Honeyeaters appeared to be very infrequent visitors to S. tubiflora in both years and in both populations relative to other Australian bird-adapted species, such as Banksia sp. (Armstrong 1991) and G. acanthifolia (Chapter 4). Birds may have neglected S. tubiflora at the time of this study in favour of more prolific nectar sources that were concurrently available, such as several other winter flowering shrubs that co-occur in heath communities including Banksia spp. A typical S. tubiflora plant in population 1, for example, might present a standing crop of 321.5 kJ of nectar per day (22.6% by weight sugar = 250 mg/mL, multiplied by 0.0077 mL per flower, 10 flowers/plant and 16.7 kJ/mg sugar) compared to an individual Banksia ericifolia or B. spinulosa inflorescence, which yields typically more than 1000 kJ each per day (McFarland 1985, Lloyd 1998). Armstrong (1991) found that B. ericifolia accounted for 95% of the nectar energy available from May to July in 1987 and 1988, and territorial male New Holland and white-cheeked honeyeaters relied almost entirely on B. ericifolia for nectar during those months in both years. Armstrong's tallies (Table 1 of Armstrong 1991) suggest that honeyeaters were visiting Banksia more than 400 times more frequently than alternative sources, including S. tubiflora. Nectar robbery may have exacerbated the neglect by legitimate bird pollinators, as was found in Central American hummingbird-pollinated vines (McDade and Kinsman 1980). Interactions among flowering plant species are complex (Feinsinger 1987) and neither competition among plants for pollinators nor "magnet species" theory has been tested in the current study. 84 Styphelia tubiflora may compensate for its relatively negligible energy rewards through floral longevity. This may explain why, despite the apparent infrequency of bird visits most flowers appeared to have received bird visits. 3.4.6 Generalism in Epacris microphylla My data probably underestimates the diversity of pollinators foraging at E. microphylla flowers because of the relatively low sampling effort and because I did not monitor nocturnal visitors such as moths. Collectively, pollinators appeared to be providing "full service" in both populations. Open- and cross-pollination of E. microphylla resulted in equivalent fruit set, suggesting that pollination by numerous insect species did not limit fruit set. Although there may be differences in pollinator effectiveness among insect species (Primack and Silander 1975), it is plausible that these differences are negligible in E. microphylla, which is self-compatible and produces nectar, pollen and stigma in very close proximity (2 mm). Some studies have found negligible differences in pollinator effectiveness among species within a guild (e.g. moths) (Pettersson 1991) or among groups (e.g. bees versus butterflies, Eckert 2002), despite apparent differences in the insects' morphology and behaviour. 3.4.7 Cage effect in Epacris microphylla populations Insects may be deterred or may alter their behaviour at caged flowers relative to open pollinated flowers, which may explain why caged E. microphylla plants produced significantly fewer fruit in population 2 in 2000 than open pollinated plants (32% versus 22% fruit set, Tukey's HSD P < 0.05). Such effects have been detected in some studies 85 (e.g. Paton and Turner 1985, Armstrong 1991) and not in others (Robertson, pers. comm.) and may depend upon such factors as cage aperture size (Armstrong 1991), increased standing crops on caged plants (Paton and Turner 1985) or the average distance between flowering plants in the population (pers. obs.) I sometimes observed honeybees to be apparently deterred by cages as they approached flowering E. microphylla stems, but only in one case (population 1, 2000, Table 3.6) did I detect significantly fewer honeybees at caged than uncaged plants, even though sample sizes were increased in other cases. The power of hourly censuses to detect such deterrence was low because honeybees were infrequent and only a few caged plants were censused. Cages may have been a significant barrier for some insect species relative to the proximity of other, uncaged plants in the dense E. microphylla populations, where flowers occur in a nearly continuous array but each offers relatively little nectar. In most other species where cages similar to those in this study were used, large quantities of floral rewards were concentrated in inflorescences and honeybees did not appear to be deterred (e.g. Grevillea, Banksia and Callistemon Paton 1996, Celebrezze, unpublished data). 86 CHAPTER 4. COMPARISON IN PROTEACEAE 4.1 Introduction 4.1.1 Proteaceae pollination systems The plant family Proteaceae presents a powerful system in which to examine the effects of honeybees on the reproductive success (measured by fruit set) and outcrossing rate of plants adapted to visitation by vertebrates versus insects. The family includes 1100 species across 46 genera in Australia (CSERO 1995), and most species have secondary pollen presentation, with pollen deposited on and/or adjacent to the stigmatic surface prior to anthesis (Collins and Rebelo 1987, Ayre and Whelan 1989). The length and shape of the "pollen presenter", as well as the arrangement of flowers in inflorescences with as many as 1000 flowers, have evolved into several distinct configurations (Collins and Rebelo 1987, Taylor and Whelan 1988). Congeneric species with marsupial, bird and insect-adapted inflorescences occur in many genera within the family. Members of the family have particularly low fruit set, potentially resulting from mate choice, pollen limitation and resource scarcity (Copland and Whelan 1989, Whelan and Goldingay 1989, Harriss and Whelan 1993, Goldingay and Carthew 1998, Hermanutz et al. 1998). Taylor and Whelan (1988) predicted that honeybees were interfering in the pollination ecology of bird pollinated Grevillea species, "stealing" pollen and/or nectar without causing pollination. Several studies in Proteaceae have linked the interaction between pollinator behaviour and breeding systems with reproductive and genetic outcomes (Ayre et al. 1994, Goldingay and Carthew 1998, Richardson et al. 2000, England et al. 2001). 4.1.2 Study aims and predicted results My primary objective was to compare the effects of European honeybees on the reproductive success (fertility and fruit set of inflorescences) of two closely related species of Proteaceae with bird- and insect-adapted pollination systems to test the hypothesis that honeybees are detrimental to pollination systems of bird-adapted but not insect-adapted species. The ideal experiment to test honeybee effects would be to exclude honeybees from some plants but not others but, because feral honeybees cannot be easily manipulated, I examined the residual effect of excluding bird (and other vertebrate pollinators, hereafter "bird exclusion") to determine if honeybees alone caused comparable fruit set and outcrossing rates as exposure to birds and honeybees in the bird-adapted species. In the insect-adapted G. sphacelata, insects are likely to brush pollen presenters while collecting nectar at flowers, and flowers are arranged in whorl-like "spiderflower" inflorescences (Illustration 4.2). In the bird-adapted G. acanthifolia, 30 to 100 flowers are arranged in a toothbrush-like bundle and birds are likely to be brushed with pollen from recently opened pollen presenters while collecting nectar. In this study I conducted nearly the full spectrum of investigations on honeybee effects with replication in populations and years (Table 1.2). I made the following predictions: (i) exposure to honeybees would result in fertility and fruit set comparable to maximum fruit set achieved through cross-pollination in the insect-adapted species; (ii) exposure to honeybees alone would result in fertility and fruit set comparable to autogamy rates and significantly less than open-pollination in the bird-adapted species; (iii) honeybees would move less frequently and over shorter distances between plants than birds in populations of the bird-adapted species; (iv) for the bird-adapted species, seeds produced by exposure to honeybees alone would be significantly less outcrossed than those produced through open- pollination. 4.2 Methods Table 4.1 outlines the scope of the experiments and observations in this study including; behavioural assessment of potential pollinators, breeding system tests, selective pollination exposure experiments, and, for the bird-adapted G. acanthifolia, measurements of outcrossing rates using microsatellite genetic markers. 89 cn c CO CO CO c & o o CNJ •=^= sCD c 3 Q.CD x k I CD CO o W """ X CO O 0 '( 1 CD "5 ro 3 c c £ The bird-pollinated G. acanthifolia occurs in peaty sedge meadows, hanging swamps and along watercourses above 800 m elevation in the Blue Mountains west of Sydney, New South Wales (McGillivray 1993, Olde and Marriott 1994). It is an erect, spreading, perennial shrub, 0.5 to three m tall, with red racemose inflorescences 5 to 10 cm long containing 30 to 90 flowers (mean = 53), each with a style 2 to 2.5 cm long (Illustrations 4.1 and 4.2). Stems may grow horizontally and eventually be covered by a thick mat of peat so that separate stems on the same plant may be difficult to identify. Inflorescences open from base to apex. In mid-bloom, flowers of various stages are present on the single toothbrush like inflorescence (Illustrations 4.1 and 4.2). Fruits contain one or two seeds. Flower visitors include birds such as New Holland honeyeaters (Phylidonyris noveahollandiae), as well as European honeybees and a variety of native ants (pers. obs.). Because of the physical arrangement of pollen presenters and nectar rewards, birds generally must lean over the inflorescence to obtain nectar, brushing pollen onto the neck and head (Illustration 4.2). 91 Illustration 4.1 Grevillea acanthifolia inflorescence with a visiting honeybee foraging for nectar. Several flowers are open, exposing pollen presenters. 92 Illustration 4.2 The foraging behaviour of honeybees at flowers of Grevillea sphacelata (a) and honeybees and birds at Grevillea acanthifolia (b). Honeybees, as well as several species of native insects such as flies, collect nectar from the flowers of G. sphacelata and are likely to brush the pollen presenter in the process. The pollen presenter of G. acanthifolia, on the other hand, is unlikely to brush honeybees crawling among flowers gathering nectar, whereas the presenter is likely to contact the heads of birds as they lean over inflorescences probing for nectar. Illustration by Robert Parkinson. 93 Illustration 4.3 Grevillea sphacelata showing a budding inflorescence in which pollen presenters have not yet opened (left) and a ripening fruit (right), (courtesy NPWS). I cut 12 inflorescences at various stages of opening from separate plants and left them in water for 24 hours away from pollinators but in full sunlight. These inflorescences produced 10 + 3 pi of nectar of 20 + 1% concentration (standard errors shown, concentration on BRIX scale, mg of sugar per mg solution). Buds that were nearly open were red and produced 17% of the standing crop of nectar but the remaining 83% was in flowers that had opened in the previous 24 hours. Most flowers (55 to 93%) appear, based upon a reaction with hydrogen peroxide, to be receptive immediately upon opening and for the subsequent 48 hours (unpublished data). Grevillea sphacelata (syn. G. buxifolia ssp. sphacelata) occurs from Sydney Harbour south to Mittagong and Dapto in the Sydney Basin and Illawarra regions (Fairley and Moore 2000). It is a perennial shrub, 0.5 to 2 m tall, which grows in dry sclerophyll woodlands on well-drained soils (Olde and Marriott 1994). Each 1 to 3 cm diameter inflorescence produces 10 to 20 pink to purple 0.5 to 1.5 cm long flowers radiating from a central pedicle, typical of the 'spider-flowered' members of the genus (Illustrations 4.2 and 4.3). Inflorescences sampled in mid-day in November 1997 had small volumes of (0.8 + 0.2 pi, n = 23 plants) of very sweet nectar (mean = 64%, or 840 g/L, n = 12 plants). Like G. acanthifolia, inflorescences typically have flowers of various stages when they are in mid-bloom. Hermanutz et al. (1998) and Richardson et al. (2000) reported that G. sphacelata was self-incompatible at three other populations. 4.2.2 Study sites I observed pollinators and conducted field experiments in two populations for each species, in two years for G. sphacelata and three years for G. acanthifolia. The additional year for G. acanthifolia was necessary to increase sample sizes for genetic assessment and to increase experimental power through experiments paired on plants. The G. acanthifolia sites were in the Blue Mountains National Park above 800 m elevation, 90 kilometres west of Sydney, New South Wales. Population 1 is located at Blackheath (251 089.55 E, 6 273 606.65 N) and population 2 at Wentworth Falls (255 410.68 E, 6 265 109.49 N). The G. sphacelata sites were located in the Royal National Park, 30 km south of Sydney on the coastal plateau (50 to 100 m elevation) near Waterfall (population 1, 320 596.91 E, 6 224 460.80 N) and population 2 at the China Helipad (315 583.67 E, 6 220 589.2 N). (These locations are Australian Map Grid 56 latitudes and longitudes.) The G. sphacelata study sites appeared to be part of much more widespread populations which occur along ridge systems in Royal National Park, probably comprising of many thousands of adults, while the G. acanthifolia populations are geographically restricted to swamps and creeklines. 95 Population 1 was relatively large, with an estimated 1,000 to 5,000 adults, while population 2 contained approximately 80 to 160 adults. 96 4.2.3 Potential pollinator foraging frequency and behaviour 4.2.3.1 Potential insect pollinators To determine what species of insects were visiting flowers, and to measure the abundance of diurnal insects foraging at inflorescences, I tagged a set of 15 to 30 plants and scored the number of insects foraging on each of those that were flowering. Each day, I observed each plant at four to six hourly or two-hourly intervals between 7:00 to 19:00, scanning each inflorescence and recorded the total number of insects foraging on any open inflorescences ("instantaneous censuses"). Where possible, I identified insects to family and morphospecies and noted approximate body length as an indication of the likely match between the size of pollinators and floral anatomy. I also recorded additional species of native insect that I observed visiting flowers of these Grevillea species while setting up selective pollinator exposure experiments or conducting other field work. I repeated these "snapshot" censuses in G. acanthifolia population 1 on 3 days in 1998 (13 to 25 plants flowering) and 6 days in 2000 for G. acanthifolia (15 plants) and, for G. sphacelata, over 3 days in population 1 in 1997 (25 plants) and 2 days in each population (29 to 30 plants) in spring 1999 (Table 4.1). 4.2.3.2 Honeybee behaviour To determine whether honeybees were likely to pollinate flowers and move pollen among plants, I measured honeybee behaviour as described in Chapter 3. I quantified the behaviour of 24 honeybees foraging in G. acanthifolia population 1 in 2000. I recorded distances of movement among plants for 17 individual honeybees in 1998 in population 1. The site of population 2 was too steep and densely vegetated to effectively or safely collect these data. For G. sphacelata, sample sizes were as follows (number of bouts where movement distances were noted in parentheses): population 1, 1998, 12 (0) and 2000, 27 (15); population 2, spring 1998,25 (5). In order to gauge whether honeybees brushing pollen presenters were likely to encounter pollen, and to determine subsequent pollen carryover, I collected pollen carryover data for 10 honeybee bouts on 26 September 1999 at G. sphacelata population 2. Honeybees nearly always brushed pollen presenters while foraging, resulting in accumulation of bright orange pollen on the thorax. I noted when visible pollen was brushed on the dorsal thorax of the honeybee during foraging and when this pollen was no longer visible (e.g. how many flowers, inflorescences, and plants may have been brushed with this pollen.) I observed these individuals from approximately 0.5 m. This method did not appear to alter the honeybees' foraging behaviour compared to honeybees observed at a greater distance (pers. obs., D. Paton, pers. comm.). 4.2.3.3 Bird visitors To determine whether birds were likely to pollinate G. acanthifolia, which species visited, and how frequently they visited, I observed bird behaviour over three days in 1999 and six days in 2000. I observed groups of G. acanthifolia plants in population 1 (25 and 86 plants, respectively) for 30 or 60 minute periods to determine what proportion of plants were receiving visits (per hour). Because nectar availability and bird behaviour generally varies through the day (e.g. (Paton 1985), I divided my results into four time periods; morning (06:00 to 10:00), midday (10:00 to 14:00), afternoon (14:00 to 16:00) and early evening (16:00 to sunset). I summarised the 140 among-plant movement distances recorded during hourly censuses including movements out of the observation area if honeyeaters were observed to proceed to foraging on G. acanthifolia plants (but excluding movements to a perch or groom). I made additional opportunistic observations of foraging birds to determine the rate at which birds probed inflorescences for nectar (inflorescences per second, n = 129 birds). To determine whether birds (and potentially nocturnal mammals) were likely to remove pollen from pollen presenters during the first day of flower opening, I examined the removal of pollen from recently opened flowers on inflorescences which were exposed when honeybees were not actively foraging (evening, night and morning) and when both birds and honeybees were actively foraging (9:00 to 17:00) over 3 days in population 1 in 2000 (24, 25 and 26 January 2000). 4.2.3.4 Other potential pollinators I looked for evidence of nocturnal mammal visits by painting stems of 10 plants with relatively large floral displays with white acrylic paint mixed in equal measure with paint drying retardant and revisiting these plants to examine stems for footprints based upon the technique used by Cunningham (1991). 4.2.4 Breeding System I tested breeding systems through self- and cross-pollination experiments for G. acanthifolia in population 1 in 1998 and 2000 and in G. sphacelata in population 1 in 1997 and spring 1999 and population 2 in spring 1999 (Table 4.1). For G. acanthifolia, I bagged one inflorescence on each of 15 plants using 1 mm2 plastic mesh. For G. sphacelata I bagged three to 10 budding inflorescences on each plant in population 1 (n = 12) and population 2 (n = 10). In both species, I randomly assigned plants to self- or cross- pollination treatments and bagged budding inflorescences. About a week later I removed all visible pollen from all open pollen presenters with a cotton tip and brushed self- or cross-pollen over the stigmatic surface and returned three to five times over two weeks to repeat this treatment. I collected cross pollen from haphazardly selected plants five to 20 m from the treated plant. Neither species produced fruit in the first trial of this treatment potentially because of poor receptivity or pollen viability at the time of the treatment, so, in spring 1999,1 treated inflorescences two to seven times over six weeks for G. sphacelata (both populations) and in 20001 treated flowers six times over two weeks for G. acanthifolia (population 1) to ensure that the period of stigma receptivity was intercepted. I recorded fruit initiation (determined by a swelling and reddening of ovaries two to three weeks after flowering) and final fruit set about eight weeks after flowering and compared these results with selective pollination exclusion treatments using non-parametric ANOVAs discussed below. 4.2.5 Selective pollinator exposure experiments I selectively exposed a total of 307 G. acanthifolia and 651 G. sphacelata inflorescences (71 plants) in the summers between 1997 and 2000 using the treatments described in Chapter 2 (Table 4.1). I attempted to develop a fourth treatment, exclusion of honeybees, in the bird-adapted species, by bagging inflorescences and exposing them when 100 only birds were active (evening and early morning). This resulted in no fruit set and may have been confounded by the repeated opening and closing of the bags. In order to increase the power of experiments to detect differences in fruit set between bird- and insect-exposed inflorescences in G. acanthifolia in 2000,1 paired treatments among inflorescences on the same plant where more than one inflorescence was available. I compared the fruit set and proportion of inflorescences setting fruit among these treatments using paired non-parametric ANOVA (Freidman's Two Way test). I excluded birds from G. acanthifolia inflorescences with 1.5 cm2 plastic mesh wrapped around a cylinder of 10 cm2 wire fencing 30 to 40 cm long and 15 cm in diameter. I used a single layer of 1 mm2 mesh (tulle) to exclude all pollinators in 1998, but some pollen presenters were potentially exposed to animal-mediated pollination because they sometimes poked through the mesh. In 19991 used a double layer of mesh but some pollen presenters still poked through, so in 2000,1 switched to polyester sleeves. I report the results of all three years and examine the potential confounding effect of the earlier treatment in the discussion. To control for potential cage effects among plant species, I used cages of the same dimensions to exclude birds from G. sphacelata in 1997 and Autumn 1999. In contrast to a prior study at another population (Richardson et al. 2000), fruit set at these study populations was so low that I needed to maximise the number of inflorescences treated on each plant to improve the observed power of the experiments, so I constructed frames of PVC piping around each plant randomly assigned to bird exclusion, and spread 1.5 cm2 nylon mesh of these frames. 101 I examinedfruit se t one to two weeks followingflowering, and again upon completion of fruit set at six to eight weeks following flowering. Because data were non- normal (and could not be readily transformed because of the large number of zero values), and variances were sometimes significantly different among treatments, I used non- parametric ANOVA to compare, by treatment, the proportion of inflorescences which produced fruit (hereafter "fertile inflorescences"), the number of fruit per fertile inflorescence, the proportion of fruit which initiated and then aborted, and the number of fruits per inflorescence. For G. acanthifolia in 2000,1 used paired non-parametric tests (Friedman's Two Way Test) to compare fruit set among treatments. 4.2.6 Genetic assessment To examine whether pollinator suites affected the genetic composition of seeds, I extracted G. acanthifolia DNA from seeds and leaves and determined a genetic fingerprint for microsatellite markers. I used three microsatellite marker primers developed for closely related (McGillivray 1993) Grevillea species (GmlO and Gm25 (England et al. 1999) and Gi9 (Hoebee unpubl. data), following the procedure of England (England et al. 1999, England et al. 2002). The resulting data were the genotypes of each seed, maternal parent, and several other adult individuals within each of the two populations. 4.2.6.1 Sample sizes Because sample sizes during the first and second years of the study were small, I pooled genetic data from all years of the selective pollination exposure experiments to estimate outcrossing rates and make direct estimates of levels of outcrossing. For population 1,1 determined the genotypes of 36 progeny arrays, including 31 maternal plants and a total of 171 seeds (106 from open pollination, 65 from exclusion of vertebrates). I genotyped an additional 30 adults that were not used in the pollinator exposure experiments because these contributed to the accuracy of the outcrossing rate estimates (Section 4.3.6.2). For population 2,1 genotyped 29 progeny arrays (77 open- pollinated seed and 29 exclusion treatment seeds), as well as 25 additional adults. I determined the genotypes for the two most powerful loci (see Section 4.4.5.2) for 242 seed in 13 to 24 progeny arrays. However, some gaps in my genetic dataset arose in part from a conscious decision to exclude marker GmlO from outcrossing estimates (see Section 4.4.5.2) and from problems in DNA extraction and storage. Some adult leaf tissue samples did not yield DNA using the CTAB (as per England et al. 1999, England et al. 2002), DNeze™ or Quiagen™ extraction methods, resulting in the discrepancy between the number of maternal families and the number of maternal adults genotyped. In some other cases, adult plants could not be relocated in the field after the first extraction method failed. In addition, some seed genotypes were not obtained for all loci because DNA apparently degraded in storage (confirmed using agarose gel elecrophoresis). These problems illustrate the importance of optimising DNA extraction purity for each tissue type for the long-term storage and consistent amplification. Sample sizes used in each analysis by genotype are shown in Tables 4.7, 4.8 and 4.9. I verified the reliability of scoring by repeating genotyping for one or more locus in 25 adults and 59 seeds in population 1 and 15 adults and 57 seeds in population 2. 103 4.2.6.2 Outcrossing rates I measured "detectable outcrossing" by comparing the two-locus microsatellite genotypes (Gm25 and Gi9) of adults and their seed. This comparison will reveal a minimum estimate of the outcrossing rate. For example, if a maternal parent is homozygous, almost all heterozygous seeds it produces are assumed to result from outcrossing (mutation rates being exceedingly low). However, a proportion of apparently identical homozygous seed should also be expected to result from outcrossing if that allele is present in the surrounding population of adult plants or if there are any null alleles within the population. Better estimates are obtained using single and multilocus outcrossing estimates, because, assuming random dispersal of pollen, population allele frequencies provide an estimate of the probability that a homozygous pair actually resulted from an outcrossing event. I calculated and report both direct measures and estimates of outcrossing rates. Inclusion of adults which had not produced seed (30 in population 1, 25 in population 2) increased the power of such estimates because it provided a more accurate estimate of adult population allele frequencies and pollen allele frequencies which are used in Ritland's revised multilocus estimation program (MTLR Version 1.1, 1996; hftp://genetics.forestry.ubc.ca/ritland/programs.html accessed October 2001). Multilocus outcrossing estimates rely upon several assumptions. This analysis assumes that (i) each adult contributes equally to the pollen pool, (ii) maternal adults are a random sample of the population; and (iii) all alleles are segregating independently. To test the first assumption I used x2-tests to compare allele frequencies of the adult plants with pollen allele frequency estimates produced by MLTR. (MLTR produces these allele frequencies by comparing maternal adult and seed genotypes and factoring in overall seed allele frequencies with a bootstrapping method.) I tested whether the maternal adult population was a random sample of the larger population by comparing allele frequencies of the maternal adults with the allele frequencies of all of the adults sampled. I used Genepop (Raymond and Rousset 1995) to test the assumption that, for the pair of loci used in the assessment, genotypes were sorting independently from genotypes at the other locus in each population using the "linkage disequilibrium" function. Outcrossing rates (i) were estimated with standard deviations using both single and multi-locus data with MLTR. In brief, this program nominates the value of t that corresponds to the observed deviation from random mating by a given treatment, and produces standard deviations based on 1,000 bootstraps across progeny arrays (Ritland and Jain 1981, Ritland 1990), reviewed in Luikart and England (1999). MLTR does not directly test for significant differences among treatment group or from unity (Ritland and Jain 1981), although this may be inferred as a difference of at least two standard deviations. 4.2.6.3 Evidence for limited pollen dispersal Subpopulational structure might be expected if pollen flow and/or seed dispersal was restricted in the past (Levin and Kerster 1971). Regardless of whether honeybees cause inbreeding, they may decrease genetic neighbourhood size through restricted foraging movements among neighbouring plants. To determine whether there may be non-random movement of pollen in the populations, I used x2-tests to compare the allele frequencies of the pollen pool (estimated by MTLR) and both seed produced by open-pollination and seed produced by exclusion of vertebrates. I calculated the indirect fixation index (FiS), a measure of subpopulational genetic structure in the adult populations (which might be expected based upon past episodes of restricted pollen flow or seed dispersal) as the difference in observed and expected heterozygosities divided by the expected heterozygosity. I used Genepop to determine if the adult populations genotype frequencies departed from expected values at Hardy-Weinberg equilibrium. 106 4.3 Results 4.3.1 Potential pollinators Honeybees were visiting inflorescences of both Grevillea species in all years and populations, with New Holland honeyeaters (Phylidonyris novaehollandiae) in G. acanthifolia (Table 4.2). Instantaneous hourly censuses demonstrated that honeybees were the most frequent forager, present on 4 to 15% of G. sphacelata (Figure 4.1) and 2 to 18% of G. acanthifolia plants (Figure 4.2). Native insects were present on 2 to 4% of G. sphacelata flowers during censuses (Figure 4.1), while New Holland honeyeaters were never observed visiting at these times but foraged on 0 to 25% of plants during half hour observations of G. acanthifolia (Figure 4.3). Of native visitors, at least six species of insects were large enough to be likely to contact pollen presenters while foraging on G. sphacelata flowers. 107 Table 4.2 Insect morphospecies observed visiting Grevillea acanthifolia and 6. sphacelata flowers in two populations. Insects in at least five families were likely to contact G. sphacelata pollen presenters. Order family (morphospecies) body length (mm) likely pollinator? Grevillea acanthifolia population 1 Hymenoptera Formicidae sp. Ga1 3 no Formicidae sp. Ga2 4 no Formicidae sp. Ga3 5 no Formicidae sp. Ga4 8 no Formicidae sp. Ga5 2 no Formicidae sp. Ga6 no Apoidea sp. Ga1 10 no Apoidea Apis mellifera 10 ? Vespidae? sp. Ga1 5 no Vespidae* 15 yes population 2 Hymenoptera Apoidea Apis mellifera 10 ? Lepidoptera Hesperiidae sp. Ga1 20 yes Grevillea sphacelata population 1 Coleoptera Curculionidae sp Gs1 1.5 no Diptera sp. Gs1 10 yes sp. Gs2 2 no Hemiptera Miridae sp. Gs1 9 yes Hymenoptera Apoidea sp. Gs1 8 yes Apoidea Apis mellifera 10 yes Formicidae sp. Gs1 no Formicidae sp. Gs2 15 no Vepidae (European wasp) 15 yes Vespidae? sp. Gs1 8 yes Vespidae? sp. Gs2 5 no Vespidae? sp. Gs3 4 no Vespidae? sp. Gs4 3 no Vespidae? sp. Gs5 no population 2 Coleoptera Tenebrionidae sp. Gs1 11 no Diptera sp. Gs1 10 yes Hemiptera Miridae sp. Gs1 9 yes Hymenoptera Apoidea sp. Gs1 8 yes Apoidea Apis mellifera 10 yes Evaniidae sp. Gs1 8 yes Formicidae sp. Gs3 3 no Vespidae sp. Gs6 no Vespidae? sp. Gs2 5 no c XI g CD -. ro CD Q. CO -3 O O Q. •— c ro 0 a. 3 o CD o JD fa Or, CD E CO 3 c c CD ro > O n ro x> CN c c -o ro ro c CO g _: o c ro o CD g CD cfl '•4—' CO CO ro JO CU CD CD O cu £ co w -CD CD CO >* cCoO CD c CD _>a. CO o 10 o. c ro c xs ID o CD XI CD CJ o XI JD CO CD 3 ^-i E -d CO o _i CD CD > c ._ CO CD c CD C ro CD ^-» CO •a O CD c c_ CD ro CA •o CD >. CD *-» CO ro CroO CD XI JD cro TCJD O c ro ro E .a a> Q c oCO E ro CD CO 3 a. ro Oi CD C CJ) SZ Q> i- 110 Honeybees were frequent visitors to both species and foraged for nectar throughout the day, with peaks for G. acanthifolia around midday (11:00) and late afternoon (15:00 - 16:00) and midday for G. sphacelata (Figures 4.2 and 4.3). Honeybees typically landed on G. acanthifolia inflorescences from above, collected nectar from flowers or buds (Illustration 4.2), continued collecting nectar on the same inflorescence, and then flew off. Honeybees were never observed collecting pollen, but six of 26 individual foraging honeybees (23%) brushed one or more pollen presenters on an inflorescence during landing or take-off. Three of these individuals went on to brush another pollen presenter on another plant. 0.4 1998 2000 "35 > 42 ~~ c £ro• 0.2 o c o ti o *± r*i a J$> # J$> Jt- o .<£ .<$> .<$> # K N*- N* N* J* e& e& *& ^ r& J? ^ ^ -£• •£• N*- NP time of day Figure 4.3 Proportion of Grevillea acanthifolia plants visited by New Holland honeyeaters during half-hour observations in population 1 over three days in 1998 and six days in 2000. The mean number of plants observed in 1998 was 25 and in 2000, 86. Ill New Holland honeyeaters appeared to be the most frequent native pollinators of G. acanthifolia. The birds foraged for nectar on inflorescences throughout the day, with peak frequency in early morning in both 1999 and 2000 in population 1. During visits, honeyeaters typically brushed against rows of open flowers on an inflorescence while probing closed red flower buds (which held 83% of the nectar available), so that pollen presenters were brushed on the chin, head or cheeks (Illustration 4.2). On one occasion, a butterfly was observed to brush pollen presenters while collecting nectar at G. acanthifolia. All other native insect visitors (Table 4.2) appeared to be nectar robbers, thieves or flower predators. Two flying Hymenoptera species collected nectar on a few occasions, but also failed to contact pollen presenters. At least six species of ants were found repeatedly on the same individual inflorescences, foraging on nectar and in some cases defending these nectar resources when disturbed, but were never observed brushing pollen presenters. Honeybees visiting G. sphacelata nearly always made full contact with pollen presenters while foraging on nectar (Illustration 4.2; see also Richardson et al. 2000). In G. sphacelata, honeybees usually visited all open flowers on each inflorescence, and then proceeded to the nearest inflorescence, generally on the same plant. However, honeybees rarely encountered pollen presenters with visible amounts of pollen in G. sphacelata; ten honeybees were observed foraging at a total of 870 flowers on 26 September 1999 in population 2, but only five were brushed with visible amounts of pollen, at a total of six flowers. I followed these individuals to determine the potential pollen carryover and noted when pollen was no longer visible. On average, pollen remained visible for visits to 26 + 13 flowers, at 9.2 ±3.4 inflorescences, but never more than to the neighbouring plant (1.7 + 0.2 plants). 112 No single native insect species accounted for the majority of potential pollination events for G. sphacelata (Table 4.2), but rather native visitors were infrequent, moved erratically when in flight, and seldom foraged at all available flowers on an inflorescence. Of 11 species of flying Hymenoptera foraging at G. sphacelata nectaries, four were large enough to brush pollen presenters. A large fly (Family Diptera) and true bug (Hemiptera) species brushed pollen presenters while probing nectaries at both populations, while a small fly and two ant species apparently did not. On two occasions during instantaneous censuses honeybees were preyed upon while foraging, once by a native wasp, and once by a spider. 4.3.2 Foraging behaviour of floral visitors Twenty nine percent of honeybee movements between inflorescences of G. sphacelata were between plants (Table 4.3). In G. acanthifolia, 70% of between- inflorescence movements by honeybees were among plants compared to 89% for honeyeaters (Table 4.3). Birds and honeybees foraged at about the same rate among inflorescences and plants for G. acanthifolia (Table 4.3), but, because honeybees were much more frequent visitors than birds throughout the day (Figure 4.2 versus Figure 4.3), I estimated that individual inflorescences were receiving more than 40 honeybee visits each day on average compared to less than one bird visit (Table 4.4). 113 Table 4.3 Floral visitor movement rates among inflorescences and plants in two Grevillea species, the average number of inflorescences visited on each plant, and the percentage of total movements that were among plants for birds, honeybees and native insects. Honeybees moved significantly more frequently among inflorescences at G. sphacelata population 2 than population 1 (one way ANOVA, df = 64, F = 17.3, P < 0.000). Honeybees visited more inflorescences on each plant in population 2 than population 1 (one way ANOVA, df = 64, F = 4.13, P < 0.046). movements proportion movements among which were among plants inflorescences # inflorescences between population (per minute) (per minute) visited per plant plants Grevillea sphacelata Apis mellffera 1 1.7 + 0.2(30) 5.4 + 0.4 (40) 4.1 + 0.5 0.32 2 2.1+0.6(19) 8.4 + 0.7(25) 7.5 + 2.0 0.25 pooled 1.9 + 0.2(49) 6.5 + 0.4(65) 5.4 + 0.8 0.29 Grevillea acanthifolia Apis mellifera 1 1.4 + 0.2(8) 2.0 + 0.5(19) 1.7 + 0.1(32) 0.7 New Holland honeyeaters 1 2.7 + 0.4(19) 2.4 + 0.8(18) 1.7 + 0.3(20) 0.89 Honeyeaters sometimes (15%) moved further than 10 m while foraging among G. acanthifolia plants. In contrast, only 6% of observed honeybee interplant movements were greater than 10 m, significantly fewer than long-distance movements by birds (Figure 4.4; %2 = 20.6, df = 2, P < 0.001). Honeybees nearly always moved to near neighbours among G. sphacelata plants (Figure 4.4), and I never observed them moving more than 10 m despite the relatively open condition of the vegetation in these populations. 114 TJ 0 i_ *-C» TJ 3 0 L. CO roc SZo ro 0 0 k_ co 0 JD D. en JD CO CD 0 CO sz 0 sz 3 c CO C .co" C c o o o 3 TJ I S3 c Ero -g 0 0 JG g ICO XI 0 c JD «» 0 c Oi 0 >0 > ro OS o CO T- TJ 0 3 O CJ C 8 -o « Q. CO *E— O c o o CD szo _ S, c Q. CD "W_a CO >, ^ 0 C GL C0O O 0 CO k— ro 8 Li- D. 0 co 5 .CD c 0 co 0 c c o CN 3 O C E X 2 a E g >O CD co ro o0 X ro 3 CD J: > 0 *-* 3 0 CD CL JD Eo sz 0c CO O CO E Q. 0 o TJ CD c 0 C 0 0CO oCO 8 ro >> 0 CO > CD 0 JD 0 o c CO >. CO C 0 0 E oCO o o JC 0 TJ c c 0 c sz szc ro> ro ro a> hro- 5_ szro a. "ro £tn cn CO c Eo CO • daily visits per n visitor per plant inflorescence (days) Grevillea sphacelata population 1 honeybees 103 + 30 29 + 2 4 population 2 honeybees 88 + 24 23 + 9 2 Grevillea acanthifolia population 1 honeybees 91+23 43 + 9 8 New Holland Honeyeater 2.0 + 0.5 0.7 + 0.2 8 I found no evidence of climbing vertebrate visitors to any of the ten G. acanthifolia plants painted with white acrylic paint. Pollen was removed from 34.9 + 6.1% of G. acanthifolia inflorescences (n = 17) that were exposed to pollinators from about 16:00 to 09:00 over three nights, which was not significantly different from the proportion removed during the day (37.4 + 5.8%, n = 22). A few inflorescences (n = 6) were exposed only for three hours on the evening of 26 January 2000 (17:00 to 20:00); 12.8 ± 1.0% of these had pollen removed. Such pollen removal is consistent with the behaviour of foraging birds because birds appeared to be especially active in early mornings and at dusk. 4.3.3 Breeding systems Grevillea acanthifolia was at least partly self-compatible at both populations. Although cross- and self-pollination of 10 inflorescences resulted in no fruit set in 1998 116 (Figure 4.5), two of seven self-pollinated G acanthifolia inflorescence produced fruit in population 1 in 2000 (SI = 0.68, Figure 4.6). About 20% of individual plants at both populations (Figures 4.5 and 4.6, population 1 in 2000, n = 15, population 2 in 1999 n = 5 and in 2000 n = 23) produced somefruit vi a autogamy in 2000. Three of eight cross pollinated inflorescences producedfruit (Figures 4.5 and 4.6, Table 4.6). Table 4.5 The breeding systems of Grevillea acanthifolia and G. sphacelata from cross- and self- pollination experiments. Proportion fertile inflorescences Fruits per fertile inflorescence population self n cross n self n cross n G. acanthifolia 1 2000 0.28 7 0.25 6 1.5 + 0.5 2 1.7 + 0.3 3 G. sphacelata 1 Spring 1999 0 6 0.52 + 0.08 4 no data 0 1.6 + 0.5 4 2 Spring 1999 0 5 0.16 + 0.07 5 no data 0 1 3 For G. acanthifolia,fruit set in both cross- and self-pollinated inflorescences was not significantly different from autogamous fruit set. These results suggest that my method of pollen transfer was not effective and neither manual pollination treatment reached the levels of fruit set obtained through open-pollination (Figures 4.5 and 4.6). In 1999, self- and cross-pollinations in population 1 resulted in significantly lowerfruit set than open- pollinatedfruit set (Figure 4.5; Kruskal-Wallis P < 0.005). I may not have pollinated receptive stigmas with viable pollen. Alternatively, bagging or repeated removal and replacement of bags may have removed pollen from the pollen presenters or created microclimatic conditions that reduced fruit set. 117 oo o 0 CO 0 •4—' ro CD 1— "o O O UJ TJ M— CD0 . CO C CO E CU CD CO CO 0 0 10 0 CO < >% rox CO 0 o 0 i CO 0 X ro 3 l_ CD TJ 0 sz sz O CU 5 0 c •4—» ro JD >.- 5 J_ CO < cu .ro CO o0 3 G5 in JD CU I£ _co C II c 0 O" ' ro JD T" CO «« 4 4 <**< JD O o "co X E d ro 3 V 40— _a> ro I D a. E TJ re V 0 0 cu 2 c: CO CD CO Q..E TJ «< <4 4 0 0 S CL 0 o = TJ 0 C«4D— —C—D d 3 3 o O 3 0 T- TJ O O CJ l a. X 0 C '> c 0 >. I— o 4 « o TJ ro CO 0 o E ro c cj CD 3 'c CO ro c CO 5 TJ o CL ro ^. i cn o c CO c 0 CM o Q. 0 0 J: ro CO _ c E 0 CO ro c +-. Ck—O C0D 0 _ 0 c ro ••—' sz CD 0 E E 0 ro E 0 E 4 21 0 CO ro ro 0 4 •4-*CD co I • JD U 0 E X *^ TJ 0 ro 0 CU CD 3 c _: _c < ^C_O T3 0 CO ro CU JE sz ro ro Oj E 0 4 4 4 CQ I, _-.E _. 0 TJ 'I 0 0 o = 0X o IO CM CD o 0 0c O tz 0 Q. 0 > a. 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CO TJ CD ro c 0 3 TJ TJ 2CD 0 2 > v^ TJ JD TJ 9 C. co" 3 CD -•— O o C CO X CD 0 0 C c O :\-co 4 4 44 I o — '+-> c CD ro 0 <> TJ 3 CL E CD 0 ro -_: 0 JS o CL •*-0 < 3CO a. .£ _C_D 1— o = c c 444 1 0 CO cn o E *-< c a. 'co cn ro 5 2 0 L3O cn >. •*-» CJ O ro c0 d E c 0 11 Q. CD CO CO O) 0 a 02 -•-« O E H— CD 3 0 C W CD Xa . __ C 0 0 0 >* Q. JD a>-< E 0 '3 ro O *1-— c M— *-2< 0 O c CJ 1— 0 TJ CO 0 0 0 0 JD TJ 1— E 0 3 o JD O 3 CO 1— 0 X 0 cC O 0 4 0 CD. ro (cJ 0 __. 4 4 CO 2 0> *40- *-> CD *— '3 0 TJ TJ _. SZ 0 M— *—. +-» c c m O CO ro 0 ™ +-0< c ro 121 Cross-pollination in spring 1999 consistently resulted in far greater proportions of inflorescences setting fruit (16 to 52%) than self-pollination (4%) indicating that the species was preferentially outcrossing. Grevillea sphacelata was generally self- incompatible but I detected some variation in the breeding system; a few individual plants produced selfed seed in each populations (SI = 0.10 population 1, SI = 0 population 2). In population 1, one inflorescence produced a fruit via autogamous self-pollination in spring 1999 (Figure 4.7) and 2 of 20 autogamy treatments also resulted in fruit in population 1 in 1997 and in population 2 in Autumn 1999 (Figure 4.8). 4.3.4 Selective pollinator exposure experiments Open pollinated fruit/flower ratios were extremely low (1.7 to 1.9% for G. acanthifolia and 0.5 to 0.6% for G. sphacelata). The average number of fruits each fertile inflorescence produced was low and relatively constant (G. acanthifolia 1.5 to 2 fruits, G sphacelata nearly always one, Table 4.6), while the proportion of fertile inflorescences accounted for most of the variance in fruit set observed (G. acanthifolia, 33 to 76%; G. sphacelata 4 to 20% Table 4.6). Grevillea sphacelata fertility and fruit set were not significantly lower when birds were excluded than open-pollinated; both treatments resulted in a significantly greater proportion of flowers producing fruit than autogamy, suggesting that pollen vectors were contributing to fruit set (Table 4.6). However, pollinators were evidently not fully servicing flowers; in population 1 in spring 1999, open-pollinated plants produced lower 122 fruit set than cross-pollinated ones (0.52 + 0.08, n = 4 cross-pollinated; 0.04 + 0.02, n = 9 open-pollinated; Kruskal-Wallis Test, P < 0.001, Figures 4.7 and 4.8). In G. acanthifolia, the contrast between autogamous fruit set and the other exposure treatments indicate that pollinators contributed to fruit set. Bagged inflorescences were significantly less likely to be fertile and fertile inflorescences produced significantly fewer fruit than open-pollinated ones in population 1 in 1999 and in both populations in 2000 (Friedman's Two Way Test, P < 0.05), when fruit set also was significantly lower in both populations (Figures 4.5 and 4.6, Table 4.3; population 1, Friedman's Two Way Test 0.05 < P < 0.10; population 2, P < 0.05). Genetic analysis (see Section 4.3.5) demonstrated that some seeds from the 1998 autogamy treatment were, in fact, cross-pollinated; the 1998 autogamy treatment was excluded from the analysis of selective pollinator exposure experiments. Breeding system tests resulted in lower fruit set than open pollination so do not provide an indication of whether pollinators are limiting or maximising fruit set in open-pollinated plants. 123 ro 0 o c CD & TJ CN CO CM CO in 0 d d ca d d CD "E TJ + 1 d TJ 3 +1 +1 * + 1 -- +1 JD O co •M" + 1 C 0 X CO in CD CJ 0 c\i CM .CD 0c CJ CO •3- 10 CM I 0 _: L. C Mo— CD TJ CO CO 00 CO 0 CO CO CJ c. 0 0 0 C CD 0 d d 0 ro +1 +1 + 1 + 1 •E 0a + 1 +1 ro cn LO •>«; 0 0 O CM cq cn ro M— Q. 1— 5 0 cn a 0 CO tz 2 •4-« TJ '3 0 CD _ u. CO cu CD c m CM CD CO E CL d O d d CD 0 ro + 1 + 1 + 1 CM T- TJ CJ + 1 CD O) CO c in co m O 0 T- T- 0 d ,_ c +-> to | o 3 cCO ro S H 00 TJ CO •E 00 co CM CM £ s _! -- CO CO CD J9 0 TJ 0 o CD. , © 0 co CM CO CM m d d 0 "- w •a "2 CO CO +1 + co co + 1 MCO I? '5 JD O co 0 c CO X d cn CO Q) d o 3 0 CO 4.6). When these treatments were paired on plants in 2000 though, the number of fruits per inflorescence was significantly lower in one population (population 2, Friedman's Two Way Test, P < 0.05) (Figure 4.6); this trend was nearly significant at a = 0.05 in population 1 (Figure 4.5; Friedman's Two Way test, 0.05 < P < 0.10). The weight and size of seeds did not reveal differences in seed quality among treatments. In G. acanthifolia, 25 to 83% of initiated fruit was later aborted, but this did not vary significantly among treatments. There was no difference among treatments in mean seed weight or length. 4.3.5 Genetic assessment Microsatellite markers allowed the examination of the current mating system and genotypic structure of adults in two G. acanthifolia populations. Genotypes for the two most powerful markers were determined (see Section 4.4.5.2) for seed within 13 to 24 progeny arrays in each of the treatment groups in the two populations (Table 4.7), with an average of 2.2 + 1.2 to 4.4 ± 0.7 seed per array in population 1 and 2 respectively. 4.3.5.1 Conformity with assumptions Most of the assumptions of the multilocus estimation procedures appeared to be satisfied by my data. There was no evidence of linkage disequilibrium for the Gm25 and Gi9 loci among genotypes in population 2 (genotypic disequilibrium test P = 0.44), while in population 1 this test was marginally significant (P = 0.052). Maternal adult allele frequencies did not differ significantly from the allele frequencies of the non-maternal plants genotyped, suggesting that the maternal plants were a random sample of the adult population. In population 2, allele frequencies did not differ between adults and pollen estimated allele frequencies for open-pollinated seed, which implies that the pollen pool was a nearly random sample of available alleles. However, in population 1, allele frequencies of adults and open-pollinated seed for Gm25 differed significantly (x2 = 20.5, df = 8, 0.01 > P > 0.005). This difference might have arisen from disproportionate contribution to the seed genotype estimates by maternal plants. For example, the genotypes of the two adults with the largest progeny arrays of open pollinated seed (29 and 17) were the same (232/238); these two alleles were also the ones which differed the most in frequency between the adult and seed arrays (allele 238, 13% in adults, 23% in seed; allele 232,24 versus 32%). These progeny arrays were by far the largest (the next largest had 9 seed) but may not have had the same impact on Gi9 allele frequency comparisons because one was homozygous and the other heterozygous for the most common allele. Despite this potential bias resulting from two maternal adults, these progeny arrays were produced by numerous independent pollination events (6 and 5 inflorescences respectively, in two years each). Such a bias would not be expected to alter estimated outcrossing rates which take into consideration maternal genotypes before producing pollen allele frequency estimates; the adult population genotypes and pollen pool estimates produced by MTLR for Gm25 did not differ significantly (x2 = 9.8, df = 9). Alternatively, differences in allele frequencies of adults and open-pollinated seed for Gm25 may have resulted from the adult allele frequencies being unrepresentative of the population as a whole. For this locus, the pollen pool included two alleles not detected in the adult population. This is not surprising since, particularly in population 1, the majority of adult plants were not genotyped and in any case the frequencies of alleles were only slightly different between these two groups. 4.3.5.2 Relative power of microsatellite marker loci Microsatellite loci differed in their allelic richness (Table 4.7) suggesting that their contributions to the power of outcrossing estimates were unequal. I tested the relative power of loci in detecting outcrossing rates by plotting the proportion of detectably outcrossed seeds for one, two, or all three loci for the subset of seeds for which I obtained all three genotypes (Figure 4.9). This plot demonstrates that GmlO added very little power to the test, presumably because this locus had fewer alleles than the other two loci (Table 4.7). As a result, I did not genotype all samples for this locus and the results of outcrossing measures using microsatellite markers Gm25 and Gi9 are shown in Table 4.8. 127 Table 4.7 The allelic richness of all adults sampled and the total allelic richness of microsatellite loci in Grevillea acanthifolia in two populations. Adults include maternal plants from progeny arrays and additional flowering individuals (sample sizes shown in Table 4.8). loci Gi9 Gm25 Gm10 alleles n alleles n alleles n population 1 adults 10 60 9 50 3 28 seed & adults 12 227 10 210 4 111 population 2 adults 5 44 6 40 3 9 seed & adults 10 141 7 135 4 57 0.9 2 0.8 o 3 O 0.7 0 J~D 0.6 CD -w 0.5CJ 0 ! 0.40 TJ >+— 0.3O c 0.2o '•& 0.1o CL 2 0CL 1 2 number of loci Figure 4.9 Proportion of Grevillea acanthifolia seed that was apparently outcrossed when one, two or three loci were considered. The data shown are for the 102 seed for which all three genotypes were determined. Locus Gm10 contributed relatively little power. Detectable outcrosses using Gi9 and Gm25 combined (marked with the arrow) was 0.65, compared to 0.70 when all three loci are used. Triangles indicate values using Gm10 either alone or in combination with other loci. 4.3.5.3 Outcrossing rates Outcrossing, regardless of how it was measured, was uniformly high for both open- pollinated and bird-excluded treatments in both populations (Table 4.8). The proportion of observable outcrosses did not differ significantly between treatments in either population (Mann-Whitney test P > 0.20). Multilocus outcrossing rates (t^) were less than unity in all cases regardless of treatment but this departure was less than two standard deviations in every case (Table 4.8). Single locus estimates for Gm25 (ts) in open pollination and bird- exclusion treatment were more than two standard deviations lower than unity in population 1 whereas only open-pollinated seeds were significantly less outcrossed than random mating for Gm25 in population 2. In that population, estimates based on Gi9 differed significantly from unity in the bird-exclusion treatment group. Estimates using Gi9 alone were not significantly different from unity in both treatments in population 1. The lack of a consistent pattern in the single locus results may have been a consequence of small sample sizes or may indicate that in general, seed were slightly less outcrossed than would be expected under random mating. In any event, at least 50% of all seed were detectably outcrossed regardless of treatment. 130 0 CO CO LO CO LO CO CO CM JD OJ LO CN CM CN c CD TJ g TJ 0 0 +-* 0 CO CO O) _- CO 0 o •M" (0 0 CL 0 o o O CO 9 3 TJ CM ro 0 >. d d d d © 0 O d -}-^ c TJ + 1 + 1 + 1 + 1 0 >N A O 0 co CO CJ CO ft! 5 0. c OJ §8 ICO i TJ 0c o OJ TJ co 0 + 1 •^0= JD 0 3 CO Q. 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The inbreeding coefficient Fis did not depart consistently from zero, and was small (population 1, 0.064; population 2, -0.062). Tests based upon Markov chain resampling (using Genepop) indicate that genotype frequencies conformed to expectations for Hardy-Weinberg equilibrium (P > 0.10 in all cases for Hardy-Weinberg exact test, Hardy-Weinberg test for heterozygosity deficit, and Hardy-Weinberg test for heterozygosity excess). These results provide no indication of genetic population substructure that might result from non-random movement of pollen within the populations (as would be expected if honeybees were decreasing genetic neighbourhoods relative to birds) but these tests are not direct measures of such potential effects. 132 Table 4.9 Comparison of observed heterozygosity (H0) in adults in two Grevillea acanthifolia populations with expected values if populations are at Hardy-Weinberg equilibrium (HE). F|S is the inbreeding coefficient (H0-HE /HE). Gm25 Gi9 Gm10 population 1 H0 0.719 0.804 0.407 HE 0.813 0.714 0.344 F,s -0.116 0.126 0.183 n (adults) of 61 total 60 50 28 population 2 H0 0.756 0.628 0.3 HE 0.684 0.709 0.364 F,s 0.105 -0.114 -0.176 n (adults) of 49 total 44 40 9 133 4.4 Discussion This study provided little support for the hypothesis that exotic honeybees are inferior pollinators of the bird-adapted G. acanthifolia or that honeybees are likely to pollinate the insect-adapted G. sphacelata. In some cases, honeybees may be limiting fruit set of the insect-adapted G. sphacelata by transferring pollen primarily among flowers within plants of this self-incompatible species. Exposure of G. acanthifolia to insects alone (predominantly honeybees) resulted in only a slight decline in reproductive success, relative to exposure to both birds and insects, and seed produced by this treatment were equally outcrossed to open pollinated seed. The most parsimonious explanation for these results is that honeybees moved pollen as effectively among plants or over the same range of distances among plants as native pollinators. 4.4.1 Breeding and mating systems Grevillea sphacelata appears to be generally self-incompatible, with self-pollination resulting in very little fruit set in either of the study populations reported here, as well as in the populations examined by Hermanutz et al. (1998) and Richardson et al. (2000). In this study, some individual plants were self-compatible or that self-incompatibility mechanisms broke down in some cases; only 3 of 21 plants that were not exposed to potential cross- pollination produced fruit (Figures 4.7 and 4.8). Thomson (pers. comm.) noted that this pattern would be expected if inconspicuous pollinators breached the polyester bags. However, in one case I observed fruit set of individual flowers which I pollinated with self pollen, suggesting that there was some variation in the breeding system. Although open-pollination resulted in greater than 90% estimated outcrossing rates (Table 4.7), G. acanthifolia produced fruit via autogamy treatments and self-pollinations in a few of the many plants tested (Figures 4.6 and 4.7, Table 4.6). Gleeson (1994) found that the self-incompatibility of G acanthifolia varied, with two populations which were slightly self-compatible (SI = 0.24 and 0.15) and a third, located near population 2 in this study, which was highly self-compatible (SI =1). Gleeson used small sample sizes (3 plants at each population) so this result could be a function of variation in self-compatibility among plants within populations. Nevertheless, Gleeson also found that fruit set from cross- pollination did not reach or exceed open-pollination fruit set despite several repeated cross- pollinations of some flowers at each treated inflorescence in order to ensure that the timing of pollen deposition intersected the receptivity of the stigmatic surface (Whelan and Goldingay 1986). These results indicate that G. acanthifolia has a range of mating systems; honeybee behaviour among inflorescences might favour reproduction in self- compatible individuals over time. The mechanisms underlying the mating system of this species are also not known and require further examination (Goldingay and Carthew 1998). Cross pollinations may be favoured without precluding self-fertilisation; for example, Harriss and Whelan (1993) found that pollen tube growth following self-pollination of G. macleayana was slower than that following cross-pollination. The relatively high frequency of barren inflorescences in G. acanthifolia, suggests other mechanisms, such as thresholds for the number or quality of pollinations at the inflorescence level, might have limited reproductive success in these treatments, although more detailed work would be necessary to examine this and other potential self-incompatibility mechanisms in Grevillea. Bagging may have produced a detrimental microclimate that inhibited seed production although one particular G. sphacelata individual produced numerous selfed fruit within the bags. Another possibility is that differences in self- and cross- pollen tube growth, as has been found in Grevillea (Harriss and Whelan 1993). Whole-plant treatments were not used to test pollinator limitation so the significantly higher fruit set in cross-pollination treatment than after open-pollination might have compensated for a higher rate of abortions elsewhere within plants as a result of resource limitation (Whelan and Goldingay 1986). 4.4.2 Effect of honeybees on reproductive success Contrary to expectations, fruit set was only slightly higher in open-pollination than bird-exclusion. If birds had been important to the production of fruit, a greater disparity between open-pollination and bird-exclusion would be expected. There are several possible explanations for this result: (i) Visiting birds may not have deposited pollen on stigmas. This is not consistent with my observations of New Holland honeyeaters in population 1, which nearly always brushed pollen presenters while foraging and so should have deposited pollen onto stigmas. In general, foraging honeyeaters are likely to have relatively large pollen loads (Paton and Ford 1977). (ii) Honeybees may have interfered with bird pollination, making birds ineffective (Paton 1996, Gross and Mackay 1998). The kind of interference observed by Gross (1998) seems unlikely because honeybees appeared to only incidentally brush pollen 136 presenters while foraging for nectar. Although birds were frequent visitors to G. acanthifolia inflorescences, comparison of bird foraging territories and honeybee foraging intensity among plant populations might reveal evidence of competition for nectar among these species (Paton 1996). (iii) Birds and honeybees might have differed in the quality of pollen they deliver to flowers but this possibility was not borne out in fruit set or in genetic evidence (paternity tests - with more powerful data - would be needed to detect changes in genetic neighbourhood). (iv) Fruit set may have been limited by internal factors and bees provided sufficient pollination. There is some evidence for resource limitation in other Grevillea which, collectively, have among the lowest fruit flower ratios reported in any genera (Ayre and Whelan 1989; Hermanutz et al. 1998). In field trials I could not identify a repellent that excluded honeybees but not birds (unpubl. data), and I am not aware of G. acanthifolia populations where honeybees do not forage for nectar. Removal of feral honeybees in populations previously studied would allow a clear determination on whether honeybees interfere with effective bird pollination, and whether bird pollination alone provides enough pollen for other factors, such as resources, to limit fruit set. The impact of honeybees may depend as much on whether they are actively collecting pollen as on whether inflorescences are adapted to bird- or insect-pollination. Honeybee behaviour differs markedly among plant species and populations. The minimal effect of honeybees at G. acanthifolia contrasts with the results of comparable experiments on the nearly identical inflorescences of G. macleayana, in which honeybees collected pollen but produced half the fruit set obtained through exposure to all potential pollinators (Vaughton 1996). 4.4.3 Potential pollinators and their apparent relative effectiveness Honeybees were the most frequent visitors to flowers of both species in this study. On some days, they were likely to visit G. sphacelata inflorescences more than 20 times and G. acanthifolia inflorescences more than 60 times (Table 4.4). Some honeybees contacted pollen presenters of G. acanthifolia flowers, providing an opportunity for cross-pollination by honeybees but birds appeared to be the more effective pollinator. New Holland honeyeaters were more likely than honeybees to brush several pollen presenters when visiting inflorescences and other studies have found that the birds accumulate large pollen loads (Paton and Ford 1977). I estimated that birds visited each G. acanthifolia inflorescence less than once per day and always brushed pollen presenters during visits and pollen was removed from 35% of inflorescences exposed when honeybees were not active. I also found that birds occasionally moved much greater distances than honeybees when moving among inflorescences. (Although I found no evidence of terrestrial mammals, sample sizes were small. I also made no nocturnal insect observations.) Honeybees nearly always contacted pollen presenters when collecting nectar from flowers and were much more frequent visitors than native insects (Table 4.4). Solitary native insects greater than 1 cm in length appeared to be the most effective native pollinators of G. sphacelata. Despite their infrequency, native insects might be more effective pollinators than honeybees because the native species appeared to move more erratically among inflorescences and plants than honeybees (D. Paton, pers. comm.; G. Wardell, pers. comm.). These behaviours may have evolved as a result of the high risk of predation at flowers that is likely to be a strong selective pressure on solitary insects (Charnov 1976, Frankie et al. 1976, Hassell and Southwood 1978). Arguably, this risk is likely to be a weaker selective pressure in social insect species because the hive may benefit from methodical behaviour even if it puts individual foraging workers at a greater risk (although this model requires testing). The actions of honeybees may restrict fruit-set below the resource limit of fruit set (Haig and Westoby 1988, Stock et al. 1989, Trueman and Wallace 1999, Goldingay 2000, Celebrezze and Paton submitted). Breeding systems and open pollination experiments in this and the other studies (Hermanutz et al. 1998, Richardson et al. 2000) in a total of six populations found that the species is generally self-incompatible and apparently pollen-limited. Cross-pollination resulted in greater fruit set than open-pollination, suggesting that pollinators were limiting fruit set overall in those years and populations (Haig and Westoby 1988, Ayre and Whelan 1989, Burd 1994) as has been observed in other Proteaceae (Copland and Whelan 1989). The end result for the self-incompatible G. sphacelata would be greater among plant pollen movement resulting in greater fruit set, although this requires testing in the absence of honeybees. 4.4.4 Evidence for honeybee-mediated geitonogamy in G. sphacelata Honeybees appeared to limit fruit set in G. sphacelata through geitonogamy sensu (de Jong et al. 1993) and near-neighbour matings in some seasons. In nearly all honeybee bouts observed at close range, visible pollen was already removed from the flower being visited, presumably by a previous visitor. When pollen was deposited on a honeybee, it was generally no longer visible before the honeybee moved to another plant to forage, and was never still visible at a visit to a third plant. My observations probably underestimated maximum pollen carryover because pollen carryover was not directly measured (by examining deposition on virgin stigmas). Schaal (1980) found that gene flow was greater than expected pollen carryover in Lupinus texensis. In a study of Erythronium, direct observations of pollen deposition by foraging pollinators suggest that carryover will vary among plant species and pollinator, but even with this variation pollen was not generally carried over to more than 20 flower visits (Thomson 1986). Thomson (1986) found that, for insects which groom frequently (such as honeybees), carryover drops steeply after a few flower visits. However, visually clean pollinators can deposit significant pollen loads (Thomson, pers. comm.), and some G. sphacelata pollen carryover by honeybees was probably occurring despite the infrequency of movements among plants. Nevertheless, genetic evidence from another G. sphacelata study corroborates my view that honeybee behaviour facilitates near-neighbour matings; Richardson et al. (2000) found that G sphacelata seed arrays in homozygous plants were uniformly heterozygous for genotypes found in adjacent plants, and honeybees were the only floral visitor observed in their study populations. Collectively, these data suggest that the breeding system of G. sphacelata is compromised by the methodical foraging behaviour and high frequency of honeybee visits. 140 While several studies in Australian Proteaceae have suggested that a low frequency of pollinator visits may cause pollen limitation (Goldingay and Carthew 1998), the results of this study of G. sphacelata strongly suggest that too frequent visits by an ineffective pollinator may interfere with less frequent but more effective pollinator visits (Gross and Mackay 1998, Faulks 1999, Celebrezze and Paton submitted). In contrast, native solitary insects are thought to forage more erratically among plants (Paton, pers. comm.; Wardell, pers. comm.; Celebrezze, pers. obs.). Such non-linear interactions among different types of pollinators are not uncommon (Thomson 2001). I predict that fruit set would increase as a result of greater outcrossing if honeybees but not native insects were removed from any of these study populations. An alternative explanation for pollen limited fruit set is that native insect populations have declined as a result of honeybee competition, but this hypothesis is difficult to test (Thomson 2001). Geitonogamy may be a potent selective force in the evolution of self- incompatibility mechanisms in plants generally (Arroyo 1976), including in G. sphacelata populations. Combined with demographic factors such as fire, populations of G. sphacelata may become smaller and relatively inbred over time. Seeds lacking self- incompatibility genes might be disproportionately represented in the seed bank because self-compatible maternal plants may produce more fruits than other plants. At the same time, the total seed bank may decline in size and these relatively small seed banks may be depleted if fires kill the adult population. Such processes may be occurring in some populations; for example, population 1 burned in 1994 and again in December 2001. Although some plants may resprout following fires, the December 2001 fire killed all of 141 the adults in population 1 (pers. obs.). These speculations require further study, particularly in populations with high fire frequencies. 4.4.5 Conformity of genetic data with assumptions I examined the genetic consequences of honeybee pollination more directly in G. acanthifolia. The genetic data conformed to most of the assumptions upon which the multilocus outcrossing estimates were based. The marginal value of the linkage disequilibrium test in population 1 suggests that the two microsatellite loci used may be in linkage disequilibria, which would be expected to weaken the power of MLTR to detect outcross events (Ritland and Jain 1981), not bias estimates in favour of one or another treatment. Even if these loci were linked, MLTR multilocus outcrossing estimates were very high for both treatments in population 1. Likewise, the difference in seed and adult allele frequencies appears to be the result of the contribution of a total of 40 seed from two adults, but MLTR produces pollen allele frequencies taking maternal genotypes into account, and these did not differ from adult genotypes. 4.4.6 Comparative effectiveness of bird and honeybee pollination Genetic markers were used to directly assess whether honeybees were likely to cause inbreeding or change subpopulational genetic structure in populations. Breeding system experiments in this and another study (Gleeson 1994) indicated that G. acanthifolia produced seed from self- and cross-pollination and was an appropriate species for studying the effects of pollinator suites on the mating system. In population 1,67% of seed was detectably outcrossed, with a multilocus t of 0.92 ± 0.08 (s.d.). At least 53% of seed was produced by outcross pollination in population 2. These results are surprising because pollen movement by honeybees seemed to be limited compared to birds, which sometimes moved greater than 50 m among plants (Figure 4.4) and because honeybees appear to be causing inbreeding in the current seed generations of G. macleayana (Ayre et al. 1994, England et al. 2001). These data may indicate that birds and honeybees are equally effective in pollinating G. acanthifolia. Alternatively, honeybees might have interfered with more effective bird-pollinations and/or honeybees might have been less effective in moving pollen among plants but the tests for such effects were not powerful enough to detect them. Interference would be expected if honeybee foraging limited the effectiveness of birds in long-distance pollen dispersal. Such a result is predicted by models of interactions of pollinators which differ in their effectiveness in picking up or deposing pollen at flowers (Thomson et al. 2000). Thomson et al. (2000) expected birds to deposit relatively large amounts of pollen on stigmas, while grooming insects would deposit less. Because honeybees groom regularly they would be less likely to deposit pollen at flowers and may therefore create a context in which birds deposit fewer pollen grains on stigmas than they would be in the absence of honeybees. This model is consistent with the observed behaviour of birds and honeybees at inflorescences. Incidental contact of honeybees with pollen presenters on arrival or departure from inflorescences may be frequent enough to remove pollen before bird visit inflorescences, potentially decreasing the effectiveness of bird pollinators. Birds, however, are likely to brush many open flowers at each inflorescence they visit. 143 Although this study provides no genetic evidence that honeybee pollination produces inferior seed to open pollination in G. acanthifolia during the three years of this study, honeybees may still be having an effect on population genetic structure in the long term. Birds sometimes moved over greater distances when foraging than the maximum honeybee movements observed (Figure 4.4), but in general birds and honeybees moved comparable distances between plants when foraging. Significantly more bird than honeybee movements, however, were greater than 10 m. A more intensive genetic sampling method would be necessary to determine if ongoing honeybee pollination may be reducing genetic neighbourhood size in G. acanthifolia populations. Although the two loci I used had relatively high allelic richness (Table 4.9), they did not provide unique genotypes for all adults sampled. Other microsatellite loci have subsequently been developed for Grevillea species that might permit such identification and subsequent paternity analysis of seed (P. England pers. comm., A. Usher, pers. comm.). This method would permit the assignment of both maternal and paternal seed parents, coupled with mapping of the populations and spatial autocorrelation analysis. Such analyses could test the hypothesis that honeybees alone move pollen shorter distances than all pollinators combined. 4.4.7 Genetic neighbourhood size Evidence of population genetic structure varies in the few reported cases in Proteaceae. I found no evidence of subpopulation structure in either population of G. acanthifolia, suggesting that the adult population was produced through random mating and was at Hardy-Weinberg equilibrium. The bird-adapted G. macleayana had high levels of selfing in populations where honeybees collected pollen (Ayre et al. 1994, England et al. 1999); the potential effect of honeybees on genetic neighbourhoods in this species warrants examination (Whelan et al. 2000). In contrast, self-incompatibility may be buffering G. acanthifolia from high levels of honeybee-mediated self-pollination although further breeding system tests are needed. Carthew (1993) found a slight departure from panmixia in a population of Banksia spinulosa, another vertebrate-adapted species which, in the study population, was somewhat self-compatible but predominantly outcrossed. In contrast, the results of Hoebee and Young (2001) detected relatively small neighbourhood sizes and highly correlated paternity in the endangered bird-pollinated G. iaspicula. Although I did not have sufficient sample sizes to gauge these measures, such patterns would be expected to result in a departure from Hardy-Weinberg equilibrium. Habitat fragmentation may be restricting pollinator movements within small patches for G. iaspicula. As in the G. iaspicula study, bird movements were most frequent among neighbouring plants, but unlike the rare Grevillia, G. acanthifolia occurs in relatively large populations in intact native vegetation. Whelan et al. (2001) examine the consequences of population fragmentation on the genetic structure of Grevillea populations more generally. Although there is no evidence of population structure in the current adult G. acanthifolia population, honeybees may still cause such structure in the long term by restricted movements among plants. This effect may not have surfaced as yet in the adult population because honeybees were introduced relatively recently in relation to the lifespan of adult plants (assuming this is 30 years, about 4 to 5 generations). Recruitment may require fire, which may be relatively infrequent in the swamps where G. acanthifolia occurs, and outcrossed seed stored in the seed bank may also buffer the adult population from the effects of inbreeding. Eventually, however, persistent honeybee foraging may be 145 expressed in a reduction of genetic neighbourhoods because seed has no apparent long distance dispersal mechanism. Detecting such changes would require more intensive sampling methods than used in this study (Feinsinger et al. 1986, Golenberg 1987). 4.4.8 Evolutionary consequences and conservation implications Honeybees might be causing the direct decline in reproductive success in some populations of G. sphacelata even if they typically contact the sexual structures of flowers during foraging. It is alarming that honeybees appear to sharply limit the reproductive success of this insect-adapted species. Further research could determine the role of the seed bank in the demography of this species. Honeybees do not appear to be a first order threat to G. acanthifolia population viability in the short term. I found no evidence for pollinator limitation (although this result requires confirmation, preferably using mixed pollen loads). The probable life history of the species, including resprouting following fires and a soil-stored seed bank, should buffer it from the risks posed to population viability through inbreeding in the short term. Nevertheless, honeybee pollination may place directional selective pressure on some traits of this bird-adapted species, resulting in higher fecundity in individuals with these traits which might be expressed in recruitment events in the near future. Floral preaptations that promote honeybee pollination might be favoured, such as short pollen presenters and large pollen presenter surfaces (Cruden 2000). The mating system could plausibly be expected to shift toward self-compatibility since at least some individuals produced seed from self-pollen. In the long-term, honeybee-mediated inbreeding might also pose a 146 significant risk of population extinction before such evolutionary shifts can take hold, particularly for small populations (Ellstrand and Elam 1993). 147 CHAPTER 5. GENERAL DISCUSSION 5.1 Summary of outcomes This is the first comparative study of the effects of honeybees on bird- and insect- adapted Australian pollination systems, and is also among the few studies to link pollinator behaviour, plant reproductive response and in some cases the assessment of seed quality. The experimental and behavioural data reported here from paired species in the three plant families Myrtaceae, Epacridaceae and Proteaceae demonstrate an unexpected lack of correspondence between pollination adaptations and the quantity of seed produced when birds but not honeybees were excluded from flowers. Seed germination data from one species hinted that honeybees may facilitate inbreeding depression while genetic data revealed no clear evidence of inbreeding in another species. Overall the study results reiterate the remarkable complexity of honeybee interactions with Australian pollination systems found by previous researchers but clarifies that apparent pollination adaptations alone cannot be used to as a predictor of honeybee impacts. Despite this complexity, the study should alert conservation managers to the possibility that honeybees may pose a widespread threat to the conservation of plant genetic biodiversity, especially (but not only) for bird-adapted Australian plants. Honeybee-mediated inbreeding is likely to contribute to a decline in population viability and genetic diversity. I suspect that "facultative xenogamous" plants - such as self- compatible bird-adapted species - which occur in small populations are particularly at risk of such effects. 148 Similarly, the results of this study suggest that honeybees may limitfruit set in insect-adapted plants even if they appear to pollinate flowers during foraging, by interfering with the effectiveness of native pollinators (as was found in other systems such as Thomson and Thomson 1992) and/or by geitonogamy in self-incompatible species. Experiments in which honeybee were removed would clarify the relative effectiveness of native pollinators without honeybee interference, and further study of self-incompatible plant species should also be emphasised. 5.2 Overview of results The most severe decline in fruit production relative to cross-pollinated breeding system tests was found in the self-incompatible G. sphacelata, a species adapted to native insect visits. I argue that honeybee foraging behaviour results in geitonogamous pollen transfer and pre-emption of pollen from more effective native pollinator visits. For G. acanthifolia (Proteaceae), the proportion of flowers producing fruits dropped slightly when birds were excluded. Honeybees had no effect on fruit set or seed set of Callistemon citrinus (Myrtaceae). In the family Epacridaceae, visitation by honeybees but not birds augmented fruit set in Styphelia tubiflora, suggesting that honeybees contribute to pollination and some native bird species may be reducing fruit set through nectar robbery and flower damage. In all of these cases, honeybees may still have had an overall negative effect on fruit set and the possibility that honeybees effects may have been masked by indirect interactions with native pollinators (Thomson et al. 2000) or a variety of potential cage effects discussed below cannot be excluded (Paton and Turner 1985). 149 5.3 The status of knowledge on honeybee effects in Australia In addition to being the first broad comparative study of the effects of honeybees on bird- versus insect-adapted plants, this study fills several gaps in our understanding of the effects of introduced honeybees. I assessed pollen removal by honeybees and linked behavioural observations with reproductive outputs, an approach absent in some of the previous studies (e.g. Taylor and Whelan 1988, Gross 1993 but see Vaughton 1992, Paton 1993, Hingston 2002). In two of the three pairs of species I determined the breeding systems of plants, measured the rate and variation in seed production and determined if seed production was limited by pollination. My results suggest that predominantly outcrossing but self-compatible species, such as C. citrinus, may be particularly at risk of the long-term consequences of inbreeding depression through honeybee-mediated geitonogamy. 5.4 The role of pollination adaptations Apparent pollination adaptations can be deceiving (Waser et al. 1996). In the current study, the selection and subsequent rejection of two plant species from the experimental design may itself be seen as a test of the predictive value of floral characteristics in inferring the pollinators of species before direct field observations. Leptospermum laevegatum has open white flowers with no apparent specialised structures, and produce relatively little nectar, so they might be assumed to be fly, bee or beetle pollinated, but bird pollinators visit it as well, as has been found in many Eucalyptus species (Ford et al. 1979). Even moth pollination "syndromes", considered one of the most distinctive (e.g. white tubular corollas and a strong nocturnal scent, (Faegri and Pijl 1979, Ollerton 1998), do not exclude potential pollination by diurnal insects, as I found for Woolsia pungens (unpublished data). Although apparent adaptations can be deceiving, generalist insect pollination systems may be the norm among Australian plants (Armstrong 1979) and, in combination with self-compatibility, buffer these plants from negative impacts due to honeybees. I found no evidence that honeybees limited fruit set in the self-compatible, generalist insect pollinated species in this study: cross-pollination fruit-set was equivalent to open pollination in E. microphylla and B. imbricata. These species were visited by many morphospecies in several insect families. Although many comparable species might also be generalist insect pollinated, the breeding systems of these Australian plants are poorly known so the assumption that all generalist insect pollinated species are buffered from negative honeybee effects is premature. Although honeybees might not be threatening true generalists that are self- compatible, the exotic insects might be disrupting specialist insect pollinationadaptations. Grevillea sphacelata received visits from a relatively few potentially pollinating insect morphospecies and might specialise in large, solitary native bees or flies which occur in population densities. Likewise, honeybees may be restricting the effectiveness of birds as pollinators in G. acanthifolia. Finally, although honeybees can pollinate many apparently bird-adapted species, they may be altering pollination systems in less apparent but equally important ways; the resulting impact may be very widespread. The presence of social pollinating insects is thought to have led to the predominance of generalisation in Europe and northern North America (Johnson and Steiner 2000). Vertebrate specialist pollination adaptations are relatively common in the Australian flora compared to many Northern Hemisphere regions, and arguably the absence of social honeybees may have 151 contributed to this relative abundance of vertebrate specialists. Mammal and marsupial pollination appears to be more common than previously understood (Carthew and Goldingay 1997), and bird adaptations are found in approximately 1,000 Australian plant species (Ford et al. 1979). These adaptations corresponded to pollinator suites in this study: birds were the predominant native visitors to all three bird-adapted plant species (although I cannot rule out a contribution by nocturnal mammals). Shifts in pollinators can result in diversifying selection on floral characters (e.g. Pauw 1998). Thomson (2000) found strong evidence of bird- and bee-pollination adaptations in several pairs of closely related Penstemon spp., including differences in pollen presentation. The interruption of mutualistic bird-pollination systems by honeybee in Australia might therefore be expected to result in selection for floral traits promoting generalist pollination, although no studies have as yet examined this potential effect. In this study I have grouped three very different floral designs into the category of "bird-adapted", including cylindrical and one-sided brush inflorescences (C. citrinus and G. acanthifolia respectively) and solitary tubular flowers (S. tubiflora). In the context of this study "insect-adapted " species were primarily visited by honeybees and a range of native solitary insects. These native insects are profoundly different from honeybees in behaviour and life history. Floral displays also varied, from very numerous flowers providing tiny rewards (B. imbricata and E. microphylla), small inflorescences with small amounts of very sweet nectar (G. sphacelata), or large bright arrays of flowers (S. tubiflora, G acanthifolia). These various ways of packaging rewards may influence pollinator movements among flowers. 5.5 Evolutionary consequences and conservation implications Honeybees are clearly capable of major disruption to Australian plant pollination systems at several levels, but the species and populations most at risk are obscured by the complex interactions of pollinator behaviour, pollination system and breeding system. Morphological surrogates of "pollination syndromes" are not good predictors of honeybee effects. Overall, the results of this study echoed Butz Huryn (1997), who did not find "simple patterns of consistently positive or negative pollinator effects". This makes interpretation of the conservation implications of honeybee persistence in Australian vegetation complex. Although there was no clear relationship to pollination adaptations generally, the impact of honeybees on reproductive success in one of six common shrub species indicates that honeybees are likely to be having negative effects in many other species that have not been studied. This very small sample of the Australian flora revealed evidence of honeybee changes to reproductive success and seed quality. The long term consequences of these changes, and their probable frequency in the vast majority of Australian plant species which have not been studied, is unknown but may be severe. For example, the long-term consequences of persistent inbreeding in small populations is genetic fixation and in some cases, local extinction, both serious threats to genetic and species biodiversity (Ellstrand and Elam 1993). This may also be the case in other biogeographic regions, such as New Zealand and Hawaii, where honeybees have been introduced. Because honeybees are so abundant and stereotyped in their behaviour, they could represent a genuinely strong selective pressure in many bird-adapted Australian plant species, although such effects are likely to be difficult to detect in the short term. 153 The current study supports recent observations of others who have found that pollinator behaviour may be more important than morphological pollination adaptations in explaining real pollination systems (Murcia and Feinsinger 1996). Vaughton (1996) suggested that, in the long term, the introduction of honeybees would cause evolutionary changes within the Australian bird-adapted flora. This is expected as native pollinators are rare and honeybees can cause some pollination (Thomson et al. 1994). This process, though gradual, could lead to a loss of mechanisms that promote bird-mediated cross-pollination, especially if these mechanisms are costly in terms of reproductive success (e.g. Armbruster and Baldwin 1998). Other traits which might be altered include nectar volume and concentration (Pyke 1981), floral longevity (Ashman and Schoen 1994, Charnov 1996), floral morphology (Paton 1986a, Galen 1996), or floral display (Vaughton and Ramsey 1998), which are all variable and partially heritable characters. Honeybee pollination might advantage self-compatible individuals in predominantly self-incompatible populations, resulting, in the long-term, in a shift in mating system (Schemske and Lande 1985) and population genetic structure (Levin and Kerster 1971). However, honeybee-mediated inbreeding might also may pose a significant risk of population extinction before such evolutionary shifts can take hold, particularly for small populations (Ellstrand and Elam 1993). 5.6 Assessment of policy implications This study did not address the potential competitive effects of honeybees on other pollinators, but the breadth and variety of potential impacts has led to the listing of "competition from feral honeybees" as a "key threatening process" within the New South Wales Threatened Species Conservation Act 1995. This listing triggers the requirement for the production of a "threat abatement plan". This may include 154 management actions such as the selective suppression of honeybees in conservation areas, particularly where listed threatened plant species are present. This status presents an opportunity to learn more about the role of honeybees in Australian pollination through experimental removal in some regions. Several techniques for feral honeybee removal from Australian native vegetation have been developed, but all are costly and temporary (Oldroyd 1998). Honeybee industry advocates have argued that because researchers have not found definitive evidence of negative honeybee effects, such effects do not exist (Sommerville 1999) despite the potential breadth of effects which would appear to trigger the precautionary principle (Paton 1996). Divergence in values between beekeepers and conservationists has led to policy gridlock and further scientific study in isolation from this conflict will not resolve the issue (Pyke 1999). This conflict is counterproductive; for example, honeybees may be important pollinators of native species in fragmented habitats where native pollinators are no longer providing full service and further scientific study of this role is needed (Cunningham 2000, Paton 2000, Gross 2001). 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