Restoration Genetics of Murray Mallee

and Neotropical Forests

Martin F. Breed

A thesis submitted in fulfilment of the

requirements for the degree of

Doctor of Philosophy

School of Earth & Environmental Sciences

University of Adelaide, Australia

February 2013

Tree Restoration Genetics M. F. Breed

Table of Contents

SUMMARY ...... 3 DECLARATION ...... 6 PUBLISHED AND SUBMITTED WORKS INCLUDED IN THESIS ...... 7 ACKNOWLEDGEMENTS ...... 8 CHAPTER 1: INTRODUCTION ...... 12 Thesis structure ...... 12 Thesis aims and objectives ...... 19 CHAPTER 2: FOREST FRAGMENTATION GENETICS ...... 20 Breed M.F., Ottewell K.M., Gardner M.G., Dormontt, E.E., Lowe A.J. (submitted). Mating patterns and pollinator mobility are critical traits in forest fragmentation genetics. Heredity...... 20 CHAPTER 3: REPRODUCTIVE ASSURANCE SHIFTS IN MAHOGANY .... 63 Breed M.F., Gardner M.G., Ottewell K.M., Navarro C., Lowe A.J. (2012). Shifts in reproductive assurance strategies and inbreeding costs associated with habitat fragmentation in Central American mahogany. Ecol. Lett. 15(5), 444-452...... 63 Media uptake ...... 81 CHAPTER 4: POLLEN DIVERSITY MATTERS ...... 82 Breed M.F., Marklund M.H.K., Ottewell K.M., Gardner M.G., Harris J.C.B., Lowe A.J. (2012). Pollen diversity matters: revealing the neglected effect of pollen diversity on fitness in fragmented landscapes. Mol. Ecol. 21(24), 5955-5968...... 82 CHAPTER 5: EUCALYPT RESISTANCE TO FRAGMENTATION ...... 109 Breed M.F., Ottewell K.M., Gardner M.G., Marklund M.H.K., Stead M.G., Harris J.C.B., Lowe A.J. (in press). Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt. Heredity. DOI: 10.1038/hdy.2012.72 ...... 109 CHAPTER 6: ADAPTATION AND SCATTERED TREES ...... 126 Breed M.F., Ottewell K.M., Gardner M.G., Lowe A.J. (2011). Clarifying climate change adaptation responses for scattered trees in modified landscapes. J. Appl. Ecol. 48(3), 637-641...... 126 Faculty of 1000 Recommended Paper ...... 139 CHAPTER 7: WHICH PROVENANCE AND WHERE? ...... 141 Breed M.F., Stead M.G., Ottewell K.M., Gardner M.G., Lowe A.J. (2013). Which provenance and where? Seed sourcing strategies for revegetation in a changing environment. Cons. Genet. 14(1), 1-10...... 141 CONCLUSIONS ...... 153 COMPLETE LIST OF PUBLICATIONS ...... 157 BIBLIOGRAPHY ...... 159

Pg 2 Tree Restoration Genetics M. F. Breed

Summary

Fragmented tree populations are not expected to be as susceptible to small population paradigm effects (e.g. genetic drift) that generally dominate conservation genetics and restoration as many other taxa.

The reasons for this are that trees tend to (1) undergo regular far- reaching gene flow, even in fragmented landscapes, (2) have many overlapping generations and (3) be long lived relative to when most habitat fragmentation occurred. These traits result in tree populations having great genetic inertia and thus they tend to maintain genetic diversity (as measured by numbers of alleles) despite significant habitat fragmentation.

However, trees are not resistant to changes in the genetic diversity of their progeny (as measured by observed heterozygosity) as a result of habitat fragmentation. Habitat fragmentation can alter the mating patterns of individual trees by changing pollination dynamics

(e.g. levels of selfing, pollen diversity) and these mating patterns directly influence the genetic makeup of progeny. Tree progeny are predicted to be particularly sensitive to mating pattern changes of their maternal plant since most tree species predominantly outcross, leading them to accumulate more genetic load than plants that regularly self- pollinate. Consequently, reduced pollen diversity is likely to reduce pollen competition or reduce heterosis effects within the observed generation; more selfing is expected to increase inbreeding depression.

Pg 3 Tree Restoration Genetics M. F. Breed

Furthermore, for trees, these patterns remain to be examined in an experimental or quantitative way. Furthermore, discussions of these trends have often relied on theoretical arguments, rather than empirical data, paving the way for experimental investigations. Consequently, it was the primary goal of this thesis to examine some of these gaps in knowledge in an experimental and quantitative way.

Specifically the aims of this thesis were to:

1. Examine and quantify the impact of fragmentation and tree

density on mating patterns, and how this may vary with

pollinators of differing mobility

2. Determine the theoretical expectations and perform empirical

tests of mating pattern-fitness relationships in trees

3. Explore the plant genetic resource management implications

that arise from the observations in aims 1 and 2

In general the results showed that stands of trees that have experienced habitat fragmentation or are present in lower densities express a quantifiable negative shift in their mating patterns (i.e. they tend to self more and receive less pollen diversity). More mobile pollinators appear to buffer trees from these negative shifts in their mating patterns.

I present a theoretical guide to the mating pattern-fitness relationships in terms of habitat fragmentation. I found that an increase in selfing and a decrease in pollen diversity are both important factors that could be quantified as impacting on fitness of established seedlings.

Pg 4 Tree Restoration Genetics M. F. Breed

Taken together, these findings suggest that seeds collected from larger, less fragmented and higher density stands have higher fitness.

Consequently, collecting seeds from these stands should lead to better outcomes of ex situ and in situ conservation, restoration and revegetation plantings.

Pg 5 Tree Restoration Genetics M. F. Breed

Declaration

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution to Martin Breed and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.

I give consent to this copy of my thesis when deposited in the

University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis (as listed below) resides with the copyright holder(s) of those works.

I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue, the Australian Digital Thesis Program

(ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

Signed …………………………….. Date: 12 March 2013

Pg 6 Tree Restoration Genetics M. F. Breed

Published and submitted works included in thesis

1. Breed M.F., Ottewell K.M., Gardner M.G., Dormontt E.E., Lowe A.J. (submitted). Mating patterns and pollinator mobility are the critical traits in forest fragmentation genetics: no longer a paradox, just looking in the wrong place. Heredity.

2. Breed M.F., Gardner M.G., Ottewell K.M., Navarro C., Lowe A.J. (2012). Shifts in reproductive assurance strategies and inbreeding costs associated with habitat fragmentation in Central American mahogany. Ecol. Lett. 15(5), 444-452.

http://onlinelibrary.wiley.com/doi/10.1111/j.1461- 0248.2012.01752.x/abstract

3. Breed M.F., Marklund M.H.K., Ottewell K.M., Gardner M.G., Harris J.C.B., Lowe A.J. (2012). Pollen diversity matters: revealing the neglected effect of pollen diversity on fitness in fragmented landscapes. Mol. Ecol. 21(24), 5955-5968.

http://onlinelibrary.wiley.com.proxy.library.adelaide.edu.au/doi/10.11 11/mec.12056/abstract

4. Breed M.F., Ottewell K.M., Gardner M.G., Marklund M.H.K., Stead M.G., Harris J.C.B., Lowe A.J. (in press). Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt. Heredity. DOI: 10.1038/hdy.2012.72

http://www.nature.com/hdy/journal/vaop/ncurrent/full/hdy201272a.ht ml

5. Breed M.F., Ottewell K.M., Gardner M.G., Lowe A.J. (2011). Clarifying climate change adaptation responses for scattered trees in modified landscapes. J. Appl. Ecol. 48(3), 637-641.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 2664.2011.01969.x/abstract

6. Breed M.F., Stead M.G., Ottewell K.M., Gardner M.G., Lowe A.J. (2013). Which provenance and where? Seed sourcing strategies for revegetation in a changing environment. Cons. Genet. 14(1), 1-10.

http://link.springer.com/article/10.1007%2Fs10592-012-0425-z

Pg 7 Tree Restoration Genetics M. F. Breed

Acknowledgements

Firstly I’d like to thank my supervisor Andy Lowe. He has been an excellent mentor over the years and I thank him for accepting me as a

PhD student. He never stopped his dynamic style of encouragement and guidance, while also letting me plan my work quite freely.

Thanks also to Mike Gardner and Kym Ottewell, my co- supervisors. You have both provided excellent support and experienced guidance throughout my candidature.

There are a number of people with whom I spent significant amounts of time discussing the development of the Australian components of my

PhD. These include:

• Rob Murphy from Rural Solutions, Martin O’Leary from the State

Herbarium of South Australia, Bruce Smith from Trees for Life

and Michael Stead from the University of Adelaide. Rob, Martin,

Bruce and Michael were instrumental in teaching me the skills of

identifying mallee eucalypts in the field.

• Matt Coulter and Tina Miljanovic from Mt Lofty Botanic Gardens

and Bruce Smith and Dennis Hayles from Trees for Life taught

me how to overcome the challenges of rearing mallee eucalypt

seedlings.

• Dennis Hayles from Trees for Life, Rob Murphy from Rural

Solutions, Joe Stephens from Australian Wildlife Conservancy

and Matt Kilby from Global Land Repairs taught me the skills of Pg 8 Tree Restoration Genetics M. F. Breed

how to design and implement a revegetation trial in semi-arid

areas of Australia.

• Rob Murphy from Rural Solutions and Joe Stephens, Phil Scully,

Kerryn Herman and Matt Hayward from Australian Wildlife

Conservancy were very accommodating and flexible in helping

me find locations to plant my trees.

• Next I must thank all those volunteers who helped me rear, plant

and monitor >2500 eucalypt seedlings. These people include:

Tom Hunt, Susie Pendle, Michael Stead, Duncan Jardine, Carlos

Navarro, Kym Abrams, Bert Harris, Sarah Smith, Tessa Bradford,

Bianca Dunker, Annabel Smith, Julie and Jack Paine, Andrea

Ramirez, Catalina Sanchez, Jolene Scoble, Ian Matthews, Maria

Marklund and my parents, Bill and Esther Breed.

I feel that I have many to thank for the academic development offered at

The University of Adelaide over the last 3 years. In particular I would like to thank ACEBB and Environment Institute management committees for offering such stimulating seminars, workshops and symposia. Additionally, I would like to thank those in the Darling building, especially members of the Lowe Lab Group, for the many discussions about lab, field and general science topics. Thanks also to the TERN team on level 12 of the Schulz building for the entertaining and thought provoking weekly meetings.

Pg 9 Tree Restoration Genetics M. F. Breed

I have also been lucky enough to spend a proportion of my PhD with researchers at Uppsala University, Sweden and Plant Genetics

Institute, Florence, Italy. In Uppsala, I’m very grateful for the generous offer to be a visiting student during the Swedish summer of 2011.

Specifically I thank Professor Jon Ågren and Professor Martin Lascoux for helping to organise my stay. Thanks again to Jon, as well as

Yoshiaki Tsuda, Lára Hallson, Andreas Rudh and Shaun Boye for the many insightful discussions that took place during my visit to Sweden. I also thank Beppe Vendramin for his warm invitation to Florence and the many discussions about my PhD and beyond.

The Native Vegetation Council of South Australia (grant

09/10/27), Estate of the late Winifred Violet Scott Charitable Trust, Sir

Mark Mitchell Foundation, Nature Foundation SA Inc., Field Naturalist

Society of South Australia, National Climate Change Adaptation

Research Facility, Australian Geographic Society, Biological Society of

South Australia, Wildlife Preservation Society of Australia, Environment

Institute and the Noel and Vivian Lothian Scholarships of 2010 and

2012 are gratefully acknowledged for their financial support. The

University of Adelaide research branch staff was instrumental in helping me coordinate my grant applications.

Away from work, there have been four things that have kept my sanity over the last 3 years: rockclimbing, mountain biking, lunchtime volleyball and the support from my family. Thanks Bert, Nik, Tom and

Matt for the many sessions out in the Adelaide Hills and those great

Pg 10 Tree Restoration Genetics M. F. Breed

trips to the Gramps and Araps; long live the mulled wine, long weekends and Kachoong whippers. Thanks Shawn, Håkan, Lára and

Pablo for the granite cranking around Uppsala and Stockholm. Thanks

Duncan and Matt for those MTB sessions in the hills, long may they continue. Thanks so much to the SA Museum lunchtime volleyballers -

Laurie, Ralph, Remko, Mark, Michael, David, David, Cam, Flavia, Ben,

Em, Thierry - you are the highlight of my Mondays and Thursdays.

To my family:

Mum, Dad, Andrew and Matt: thank you all so much for providing such a stimulating life. Nature and science (not the journals!) are what come to mind when I think of my earliest and fondest childhood memories.

Andrew and Matt, you have always inspired and motivated me to be the best I can be. Thank you both so much for the continued challenges of my views, sporting ability and knowledge.

Min älskling Maria: Du betyder allt för mig. Jag är ledsen för all tid min doktorsavhandling tog från dig. Ditt stöd och din kärlek gör mitt liv fantastiskt. Du är min kärlek, mitt hjärta, mitt allt!

Pg 11 Chapter 1: Introduction M. F. Breed

Chapter 1: Introduction

Thesis structure

The main body of this thesis comprises six papers that have either been published or have been submitted for publication. They are presented in the format of the relevant journal or as the published version of the paper, preceded by a title page and statements of authorship.

Supplementary information is provided at the end of each chapter if present in the corresponding paper.

Here in Chapter 1 I go through the composition of my thesis. I briefly summarise each of the subsequent chapters. In this chapter I justify the flow of ideas from the review (Chapter 2), through the empirical case studies (also Chapter 2 as well as Chapters 3 to 5) to the logical conclusion of developing a body of work that has implications beyond generating knowledge for knowledge’s sake - harnessing this information to better manage native plant genetic resources (Chapters 6 and 7). A final chapter presents a broader synthesis of my work, highlighting key advances in our knowledge, some limitations of the approaches taken and identifying areas for future research.

Chapter 2 is a manuscript submitted to Heredity that develops the background and expected trends that are investigated in detail in

Chapters 3 to 5. It is a review with meta-analysis that summarises the state of the scientific field at this point in time. In brief, the review

Pg 12 Chapter 1: Introduction M. F. Breed

explores how the mating patterns of animal-pollinated woody plants shift with habitat fragmentation.

Woody plants generally live for a long time (tens to hundreds of years), have many overlapping generations within populations, perform far-reaching pollen flow (even in fragmented landscapes), are monoecious or hermaphroditic and most are pollinated by animals

(Barrett 1998; Lowe et al. 2005; Petit & Hampe 2006; Kramer et al.

2008; Ollerton et al. 2011).

The mating patterns of plants describe how plants transfer genes from one generation to the next through sexual reproduction (Barrett

1998). Since the statistical developments of the 1990s (Ritland 1989;

Ritland 1990, 2002), three summary statistics have come to dominate the description of plant mating patterns. They typically describe the degree of maternal (ovule) selfing (s, often described as the outcrossing rate tm, and 1-tm = s), the average maternal relatedness to received outcrossed pollen (biparental inbreeding, tm-ts) and the pollen diversity received by a female (often measured by correlated paternity, rp, where

-1 rp = the effective number of pollen donors of a given mother tree). I use these statistics to compare the mean effect of fragmentation on mating patterns across published studies in the meta-analysis presented in Chapter 2 (Arnqvist & Wooster 1995; Borenstein et al.

2009).

Questions posed in Chapter 2 are:

Pg 13 Chapter 1: Introduction M. F. Breed

1. What are the general effects of habitat fragmentation on the

mating patterns of animal-pollinated woody plants?

2. Are certain mating patterns more susceptible to change than

others?

3. Are there life history traits that expose or protect species from

mating pattern changes in fragmented systems?

In Chapter 2 I also present a case study of mating pattern shifts with habitat fragmentation from three species of Murray-Darling Basin mallee eucalypts. In this case study, the degree of habitat fragmentation was correlated with conspecific density, where greater fragmentation leads to lower density (a pattern that is generally accepted; Ghazoul

2005; Lowe et al. 2005; Ward et al. 2005; Eckert et al. 2010). Two of these mallee eucalypts will be investigated further in Chapters 4 and 5.

In this data section of Chapter 2 I ask:

1. How do the mating patterns of mallee eucalypts relate to

density?

2. Are certain mating patterns more sensitive to changes in

density than others?

3. Does the mobility of pollinators change these relationships?

Knowledge gaps identified in Chapter 2 are primary foci for the remainder of my thesis. They are:

Pg 14 Chapter 1: Introduction M. F. Breed

1. Do changes to mating patterns caused by anthropogenic

habitat fragmentation lead to fitness impacts? This question is

investigated in Chapters 3 to 5.

2. How can this information on the mechanisms underlying

changes to plant fitness be harnessed to improve the future

health of natural plant population and develop restoration

outcomes? This question is investigated in Chapters 6 and 7.

Chapter 3 is a paper published in Ecology Letters that develops a framework of expected mating pattern changes with habitat fragmentation and how these changes should theoretically impact offspring fitness (Fig. 1 of Chapter 3). This is a direct extension of ideas proposed in Chapter 2.

In Chapter 3 we explore a case study that demonstrates a shift in mating patterns due to habitat fragmentation in 16 natural populations of mahogany from Central America. Furthermore, we observe that these changes to mating patterns impact on the fitness of mahogany saplings in quite different ways, which relates to the ecology of the maternal plants. We discuss what these different responses may mean for future studies of this kind.

Chapter 4 is currently accepted pending revisions in Molecular Ecology and is a case study of shifting mating patterns caused by habitat fragmentation in a population of a eucalypt species from the Murray-

Pg 15 Chapter 1: Introduction M. F. Breed

Darling Basin in South Australia. This chapter develops ideas discussed in Chapter 2 and 3; how habitat fragmentation may impact mating patterns of plants, what this means for plant genetic resource management as well as developing ideas and data on how different mating patterns may impact plant fitness. We observed that shifting mating patterns impact on tree progeny fitness in ways that are unexpected and, consequently, have not been included in native plant genetic resource management plans. Specifically, we found that pollen diversity, not inbreeding, was the most important predictor of tree progeny fitness; this was an important finding because inbreeding- related effects are most often attributed to fitness declines of tree progeny (Lowe et al. 2005; Eckert et al. 2010), and this bias is represented in native plant genetic resource management plans

(Broadhurst et al. 2008; Sgrò et al. 2011; Weeks et al. 2011).

Chapter 5 is accepted in the journal Heredity and is a second case study of shifting mating patterns caused by habitat fragmentation in a eucalypt species from the Murray-Darling Basin in South Australia.

However in this case, we observe that mating patterns do not shift despite severe habitat fragmentation unlike the trends reported in

Chapters 3 and 4. This chapter develops the idea raised in Chapter 2 that pollinator mobility may be an important factor in determining the susceptibility of plants to mating pattern shifts with habitat fragmentation.

Pg 16 Chapter 1: Introduction M. F. Breed

In Chapters 6 and 7 I discuss how to harness these mating pattern data to improve the future health of natural plant populations and develop restoration outcomes. In each of these chapters I integrate information on mating pattern shifts in fragmented systems (as reviewed in Chapter

2 and discussed in Chapters 2 through 5) together with information on topics that are complementary or necessary to consider in these fragmented systems (e.g. adaptation, gene flow, genetic drift, climate change, selection pressures, outbreeding depression).

In Chapter 6, a paper published in the Journal of Applied Ecology,

I discuss the possible involvement of trees in their most highly fragmented sense (i.e. scattered or paddock trees) in adaptation of these trees to climate change. I then discuss specifically how scattered trees could be foci for restoration: either as starting points for restoration projects or as possible seed sources. The use of scattered trees (and trees in other landscape contexts) as seed sources is developed in much more detail in Chapter 7.

In Chapter 7, a paper submitted to Conservation Genetics, I bring together numerous ecological and evolutionary issues relating to seed sourcing for revegetation, including the expected mating pattern and seed quality shifts with habitat fragmentation as discussed in Chapters

2 to 5. I tie together these biological issues with a review of recent seed- sourcing developments (e.g. Broadhurst et al. 2008; Byrne et al. 2011;

Sgrò et al. 2011; Weeks et al. 2011). I develop a decision-making

Pg 17 Chapter 1: Introduction M. F. Breed

framework for a suite of different seed-sourcing approaches that is based on the biological issues raised in recent literature as well as my thesis.

In my conclusion, Chapter 8, I reflect on the contribution Chapters 2 to 7 have had to the field. I propose some future experiments and analyses that would be useful in light of the findings from my thesis.

Pg 18 Chapter 1: Introduction M. F. Breed

Thesis aims and objectives

The primary aim of my thesis is to determine the effect of anthropogenic habitat fragmentation on the mating patterns and fitness of trees. The secondary aim is to evaluate how to better harness this information to improve the future health of natural plant populations and improve restoration outcomes.

From these aims, a series of more specific objectives were derived:

1. Evaluate the general trends between anthropogenic habitat

fragmentation and density changes with the mating patterns of

trees

2. Identify groups of trees that are more susceptible to shifts in

mating patterns with anthropogenic habitat fragmentation and

density changes

3. Discover how these shifts in mating patterns can impact fitness

and seed quality.

Then, in light of evidence developed in response to objectives 1 to 3

4. Evaluate how best to mitigate these seed quality issues for seed

sourcing for restoration

5. Gauge the utility of harnessing this information to improve the

future health of natural plant population.

Pg 19 Chapter 2: Forest fragmentation genetics M. F. Breed

Chapter 2: Forest fragmentation genetics

Statement of Authorship

Breed M.F., Ottewell K.M., Gardner M.G., Dormontt, E.E., Lowe A.J.

(submitted). Mating patterns and pollinator mobility are critical traits in forest fragmentation genetics. Heredity.

Martin F. Breed (Candidate) Designed the study, collected and generated data and wrote manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Gardner (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 20 Chapter 2: Forest fragmentation genetics M. F. Breed

Maria H. K. Marklund (Co-author) Involved with developing ideas, generated field and molecular data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Eleanor D. Dormontt (Co-author) Involved with developing ideas, collected and generated data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis. !

Signed …………………………….. Date: 12 March 2013

Andrew J. Lowe (Supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 21 Chapter 2: Forest fragmentation genetics M. F. Breed

Mating patterns and pollinator mobility are critical traits in forest

fragmentation genetics

Running title: forest fragmentation genetics

Martin F. Breed1,2, Kym M. Ottewell1,3, Michael G. Gardner1,4,

Maria H. K. Marklund1,5, Eleanor E. Dormontt1, Andrew J. Lowe1,6*

1Australian Centre for Evolutionary Biology and Biodiversity (ACEBB) and School of Earth and Environmental Sciences, University of

Adelaide, North Terrace, SA 5005, Australia; 2Plant Ecology and

Evolution, Department of Ecology and Genetics, Evolutionary Biology

Centre, Uppsala University, Norbyvägen 18D, SE-75236 Uppsala,

Sweden; 3Science Division, Department of Environment and

Conservation, WA 6983, Australia; 4School of Biological Sciences,

Flinders University of South Australia, GPO Box 2100, Adelaide, SA

5001, Australia; 5Department of Limnology, Evolutionary Biology Centre,

Uppsala University, Norbyvägen 18D, SE-75236 Uppsala,

Sweden;6State Herbarium of South Australia, Science Resource Centre,

Department of Environment, Water and Natural Resources, North

Terrace, SA 5005, Australia.

Pg 22 Chapter 2: Forest fragmentation genetics M. F. Breed

*Author for correspondence: Andrew J. Lowe, tel + 61 8 8313 1140, fax: + 61 8 8313 4364, Email: [email protected]

Type of article: Original Article

Keywords: mating system; plant genetic resources; plant-pollinator mutualisms; pollen competition; pollen discounting

Word count: 4200

Pg 23 Chapter 2: Forest fragmentation genetics M. F. Breed

ABSTRACT

Most woody plants are animal-pollinated, but the global problem of habitat fragmentation is changing pollination dynamics. Consequently, the genetic diversity and fitness of progeny of animal-pollinated woody plants sired in fragmented landscapes tend to decline due to shifts in plant mating patterns (e.g. reduced outcrossing rate, pollen diversity).

However, the magnitude of this mating pattern shift should theoretically be a function of pollinator mobility. We first test this hypothesis by exploring the mating patterns of three ecologically divergent eucalypts sampled across a habitat fragmentation gradient in southern Australia.

We demonstrate increased selfing and decreased pollen diversity with increased fragmentation for two small-insect pollinated eucalypts, but no such relationship for the mobile-bird pollinated eucalypt. In a meta- analysis, we then show that fragmentation generally does increase selfing rates and decrease pollen diversity, and that more mobile pollinators tended to dampen these mating pattern shifts. Together, our findings support the premise that variation in pollinator form contributes to the diversity of mating pattern responses to habitat fragmentation.

Pg 24 Chapter 2: Forest fragmentation genetics M. F. Breed

INTRODUCTION

Habitat fragmentation is a globally pervasive problem that continues to drive changes to woody plant ecosystems (FAO, 2012). Since most species of woody plants are animal-pollinated (Ollerton et al, 2011), studying the impacts of fragmentation on woody plant-pollinator interactions seems particularly important, especially because significant amounts of biodiversity rely on these plant-pollinator interactions.

Fragmentation causes the spatial arrangement of plants to change (Young et al, 1996; Aguilar et al, 2008) and may change the abundance and foraging behaviour of pollinators (Schleuning et al,

2011; Hadley and Betts, 2012). However, populations of animal- pollinated woody plants do not appear to be as susceptible as many other taxa to the small population paradigm effects resulting from habitat fragmentation (e.g. genetic drift), which dominate conservation genetics - the ‘paradox of forest fragmentation genetics’ (Kramer et al,

2008; Vranckx et al, 2011).

The reasons for this population-level robustness are that woody plants tend to have many overlapping generations within populations; be long-lived in relation to when most habitat fragmentation occurred; and undergo regular far-reaching gene flow, mediated by their pollinators, even in fragmented landscapes (Petit and Hampe, 2006;

Kremer et al, 2012). These traits result in substantial genetic inertia within populations of animal-pollinated woody plants, which generally maintains genetic diversity within fragmented populations (Vranckx et al,

Pg 25 Chapter 2: Forest fragmentation genetics M. F. Breed

2011). This paradox of forest fragmentation genetics also identifies that animal-pollinated woody plants are not sensitive to fitness effects resulting from post-fragmentation genetic drift.

However, recent work has shown that the progeny of animal- pollinated woody plants in fragmented forests is the life stage where fitness effects are expected to manifest (Yates et al, 2007; Breed et al,

2012a; Breed et al, 2012b; Vranckx et al, 2011). Reduced conspecific density following habitat fragmentation has been demonstrated to change the mating patterns of standing adult animal-pollinated woody plants (e.g. increasing inbreeding, reducing pollen diversity; Eckert et al,

2010; Breed et al, 2012a). These changes in mating patterns drive an immediate gain or loss of genetic diversity in the progeny generation

(Lowe et al, 2005). Furthermore, fitness of the progeny generation is expected to be directly impacted by mating pattern changes via increased inbreeding depression (Keller and Waller, 2002) and reduced pollen competition and/or reduced female choice (Skogsmyr and

Lankinen, 2002; Breed et al, 2012b). Both these effects should increase the frequency of expression of low-fitness phenotypes resulting from high genetic load (Breed et al, 2012a).

Additionally, species with less mobile pollinators should theoretically be more sensitive to the drivers of these fitness effects

(Heinrich and Raven, 1972; Charnov, 1976; Hadley and Betts, 2012).

As plant densities decline, animal pollinators are less likely to shift from one plant to another due to the increased costs of doing so - the theory

Pg 26 Chapter 2: Forest fragmentation genetics M. F. Breed

of optimal foraging (Heinrich and Raven, 1972; Charnov, 1976; Ottewell et al, 2009). A pollinator foraging on a plant for longer periods of time increases the probability of selfing (either via autogamy or geitonogamy; i.e. pollination from the same or a different flower on the same plant) for plants that are hermaphrodites and that are not strictly self-incompatible

(Karron et al, 2009). As a consequence of increased self-pollen being received, an increase in pollen discounting is expected (Barrett, 1998).

A recent review of outcrossing rates in undisturbed vs. disturbed plant populations across 27 species confirmed the expectation of decreased outcrossing rate in disturbed plant populations (Eckert et al, 2010).

Mobility of pollinators has been hypothesised to play an important role in this apparent resilience to decreased outcrossing in fragmented systems (Lowe et al, 2005; Ottewell et al, 2009).

Consequently, the generic expectation of decreased outcrossing with increased habitat fragmentation may need refining to be a function of pollinator mobility. In cases where pollinators have limited mobility (e.g. small insects), these pollinators will tend to shift from plant to plant less freely than mobile pollinators, increasing the degree of pollen discounting and reducing the diversity of available pollen (Heinrich and

Raven, 1972; Charnov, 1976).

Pollen diversity (often measured by correlated paternity, rp) is a parameter that also describes the mating patterns of plants, but is reported less often than outcrossing rate. This is surprising since it can be a strong predictor of offspring fitness (Breed et al, 2012a). Pollen

Pg 27 Chapter 2: Forest fragmentation genetics M. F. Breed

diversity should decline with lower conspecific density, because as density declines the number of different pollen sources received into a given canopy is also expected to decline (Bianchi and Cunningham,

2012; Breed et al, 2012b). This relationship is due to reduced numbers of pollen donors in the landscape and the fact that animal pollinators are less likely to shift from one plant to another due to the imposed costs of movement (Bianchi and Cunningham, 2012). As a consequence of reduced numbers of pollen sources, the correlated paternity within a given progeny array should increase. The effect of density on correlated paternity should again be a function of pollinator mobility, as more mobile pollinators should overcome greater distances between canopies more easily than less mobile pollinators, resulting in larger pollination neighbourhoods (Bianchi and Cunningham, 2012;

Breed et al, in press).

In this paper, we examine the relationships between habitat fragmentation and mating patterns in animal-pollinated woody plants.

We present an in-depth study of mating patterns of three closely related eucalypt species that vary in the mobility of their pollinators. The effect of pollinator mobility on mating pattern shifts in fragmented systems has been highlighted as a research gap (Ghazoul, 2005; Lowe et al, 2005;

Eckert et al, 2010), but to the best of our knowledge no previous study has comprehensively explored this topic. We sampled 199 progeny arrays from 13 groups of maternal plants within a single landscape in southern Australia across a habitat fragmentation gradient (Figure 1).

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We then investigate the generality of habitat fragmentation- mating pattern relationships of animal-pollinated woody plants with a meta-analysis. No previous review has performed a quantitative assessment of habitat fragmentation or density effects on mating patterns of plants. Furthermore, previously published qualitative reviews have focussed on outcrossing rates of Neotropical trees, despite other mating pattern data, taxa and regions being well represented (e.g. pollen diversity, shrubs, southern Australia, east Asia).

We predict that for animal-pollinated woody plants: outcrossing rate will negatively associate with habitat fragmentation and lower plant density; biparental inbreeding will positively associate with habitat fragmentation and lower plant density, though this relationship should be weaker due to variation in spatial genetic structure across populations; correlated paternity will negatively associate with habitat fragmentation and lower plant density; and these predicted relationships will be dampened by increased pollinator mobility, which should buffer the impacts of reduced plant density or losses of conspecifics due to habitat fragmentation.

MATERIAL AND METHODS

Mating patterns and density in mallee eucalypts

Study species

Eucalyptus gracilis F.Muell., E. incrassata Labill. and E. socialis F.Muell. ex. Miq. are multi-stemmed, sclerophyllous trees common throughout

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the sand and sand-over-limestone soils of the Murray-Darling Basin, southern Australia (Nicolle, 1997; Figure 1). They generally grow from 2 to 8 metres high. E. socialis and E. gracilis have small white hermaphroditic flowers (diameter of mature flowers with reflexed stamens: <15 mm) and are pollinated primarily by small insects and, to a lesser degree, by birds and small marsupials (Slee et al, 2006). E. incrassata has larger reddish-white hermaphroditic flowers (diameter of mature flowers with reflexed stamens: <35 mm) and is pollinated primarily by birds (Family Meliphagidae) and to a lesser degree insects

(Bond and Brown, 1979).

Eucalypt flowers are protandrous (i.e. male reproductive phase precedes female phase within flowers) and flower development within and between inflorescences is sequential and gradual. Therefore, flowers in male or female phase may be in close proximity, allowing geitonogamous selfing to occur (House, 1997). Data from closely related eucalypts suggest that the species investigated here probably have late-acting self-incompatibility mechanisms, resulting in mixed mating to preferential outcrossing (tm generally >0.60; Horsley and

Johnson, 2007). Serotinous fruit (i.e. seeds released in response to an environmental trigger) are held over numerous years, with drying triggering seed-release. Seeds are small (<2 mm diameter) and gravity dispersed. Based on data from E. incrassata and our field observations, ants generally exhaust soil seed banks, except during particularly heavy seed release such as post fire (Wellington and Noble, 1985).

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Seed collection

Open-pollinated seeds were collected from maternal plants (n = 199) across six sites in the Murray-Darling Basin (Figure 1). At Scotia

Sanctuary, Bakara Conservation Park (CP), Brookfield CP, Yookamurra

Sanctuary and Lowan CP sites maternal plants were from generally higher density woodlands with no history of known habitat fragmentation (Figure 1 inset maps c, d, e, f, g respectively; Appendix

S1 in supplementary information for sampling information). Monarto maternal plants (Figure 1 inset maps h, i, j) were located from either higher density small remnant woodlands or were isolated pasture trees

(see Appendix S1 for sampling information). Small remnant woodlands were natural habitats surrounded by agricultural land. Isolated pasture trees were in very small clusters of vegetation (often only a single tree) either in agricultural land or between public roads and agricultural land.

In all cases, care was taken to avoid sampling near neighbours. We attempted to include each species from all sites and to sample populations of each species that had similar fragmentation histories and/or stand densities (for more details see Appendix S1). A subset of the genotype data included in this study has been included in previous studies. This includes E. socialis data from Yookamurra Sanctuary and

Monarto (n = 47 maternal plants; Breed et al, 2012b) and E. incrassata data from Monarto (n = 37 maternal plants; Breed et al, in press).

We used two methods to measure conspecific density. For isolated pasture trees we counted the number of conspecifics within a

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30 m radius of each tree (i.e. nearest-neighbours), then extrapolated this to trees per hectare. The 30 m radius was a manageable distance given the reasonably evenly spaced distribution of the isolated pasture trees. To account for potential clumped distribution of conspecifics in large intact and small remnant woodlands, we estimated conspecific density by counting all conspecifics in 8 replicates of 30 m x 10 m transects and extrapolated this to trees per hectare.

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¯ a) b) Scotia Sanctuary c) Bakara Conservation Park d)

c

b 0 1000 2000 km ((( e E f d DDD

! h g i j

DD 0 25 50 100 km

Brookfield Conservation Park e) Yookarmurra Sanctuary f) Lowan Conservation Park g)

DD (

(( (( ( ( E

ED E 0 2 4 8 km E D (!(! DED (!(!(! EE h) i) E j) Monarto ( Monarto Monarto ( ED E E (! D D E ED ( ( (( (((

ED DD DEDDEE D DD D (( ((( ED E E D EDED E E Eucalyptus incrassata ( Eucalyptus gracilis D Eucalyptus socialis Eucalypt woodland

Figure 1. Map showing regional overview of the six sites where seeds were collected from maternal plants from three eucalypt species across a density gradient caused by habitat fragmentation (b). Inset maps c to g show intact woodland sites: Scotia Sanctuary (c), Bakara

Conservation Park (d), Brookfield Conservation Park (e), Yookamurra

Sanctuary (f), Lowan Conservation Park (g). Sampling from the more fragmented Monarto region is shown in inset maps h to j.

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Genotyping

Leaf tissue and seeds were collected from all maternal plants. Seeds were germinated under semi-controlled glasshouse conditions and we sampled their leaf tissue. DNA was extracted from leaf tissue using

Machery-Nagel Nucleospin Plant II Kit at the Australian Genome

Research Facility (Adelaide, Australia). Nine direct-labelled microsatellite markers were selected from the set of EST-derived markers by Faria et al. (2010; EMBRA914; EMBRA1284; EMBRA1363;

EMBRA1382; EMBRA1445; EMBRA1468; EMBRA1928; EMBRA1990;

EMBRA2002). PCR was performed in a single 10 μL multiplex PCR following standard Qiagen Multiplex PCR conditions (Qiagen, Hilden,

Germany; see Appendix S2 for PCR conditions).

Analysis

Maternal genotypes were used to estimate genetic diversity of maternal plants, which were presumed to reflect pre-clearance dynamics since all trees sampled were estimated to be >80 years old and most land clearance occurred <80 years ago. Maternal genotypes were also used to screen for null alleles in MICRO-CHECKER (Oosterhout et al, 2004).

GENEPOP (http://genepop.curtin.edu.au) was used to test for heterozygote deficit/excess and linkage disequilibrium, applying sequential Bonferroni correction for multiple testing where appropriate.

Additionally, per-locus probability of paternity exclusion (Q) and

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combined probability of paternity exclusion (QC) were estimated in

GENALEX (Peakall and Smouse, 2006).

We estimated the following genetic diversity parameters for maternal plants and progeny groups using GENALEX: number of alleles

(A), Nei’s unbiased expected and observed heterozygosity (HE and HO, respectively; Nei, 1973) and fixation index (F). To account for differences in sample size, we rarefied the mean number of alleles per locus (AR) using HP-RARE (Kalinowski, 2005).

We estimated the following mating system parameters for each group of maternal plants from progeny array genotypes in MLTR

(Ritland, 2002): multilocus outcrossing rate (tm; where the selfing rate S

= 1-tm), biparental inbreeding (tm-ts) and multilocus correlated paternity

(rp). Families were bootstrapped 1000 times to calculate variance estimates for each parameter.

Meta-analysis

Data

We searched for relevant articles on the ISI Web of Knowledge database (http://apps.webofknowledge.com) on 12 December 2012 using the following search terms: (density OR habitat disturbance OR habitat frag* OR logging) AND (inbreed* OR mating system OR mating pattern OR outcross* OR selfing OR pollen diversity OR correlated paternity OR correlation of paternity). From this list, we reviewed articles and retained those that reported at least one mating system parameter

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for groups of trees or shrubs that differed in their history of habitat fragmentation or density. We recorded the mating system parameters observed in the intact forest or ‘best on offer’ locations (e.g. unlogged habitat) and the fragmented or disturbed habitat. If these data were presented in a figure, we extracted values using DATATHIEF

(http://www.datatheif.org). We recorded the species’ form (tree or shrub), pollinator mobility (mobile vs. less mobile) and all mating system parameters reported. Pollinator mobility was classified as ‘mobile’ if the authors specified that pollination was conducted by mobile vertebrates

(e.g. birds, bats or large insects). Pollinator mobility was classified as

‘less mobile’ if the authors specified that small insects did most of the pollination (e.g. small moths, bees). We also included mating pattern data presented in this study (see Appendix S3 for datasets included in meta-analysis).

Analysis

We conducted our meta-analysis following methods described by

Borenstein et al. (2009). For each study, we calculated Hedges’ g; the difference between forest or ‘best on offer’ and disturbed habitat mean mating system parameters standardised using the pooled standard deviation of the two groups (Borenstein et al, 2009). Because Hedges’ g is a biased estimator of effect size, we used a conversion factor to compute a bias-corrected metric, Hedges’ g*. We then calculated the average effect size using the random-effects model, where effect sizes

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of individual comparisons are weighted by the inverse of within-study variance plus between-study variance. We calculated the effect size of the entire dataset and for subgroups of data divided into pollinator mobility (mobile vs. less mobile). We tested for publication bias by visually examining funnel plots of effect sizes plotted against standard errors to assess the symmetry of study precision around effect size.

Relatively symmetrical funnel plots suggest there were no relationships between effect size and study size, and that studies with small effect sizes do not have a lower probability of being published (see Appendix

S4 for funnel plots).

RESULTS

Mating patterns and density in mallee eucalypts

Marker quality

We genotyped 199 open-pollinated progeny arrays (= 2867 progeny) from 13 groups of maternal plants from six sites across the Murray-

Darling Basin (see Appendix S1 for sampling information). The combined probability of paternity exclusion if neither parent is known indicates good resolution for genetic markers used across all maternal plant groups (QC = 1.00 in all cases; see Appendix S5 for detailed microsatellite data). No significant excesses or deficits of heterozygotes were observed in the groups of maternal plants (Appendix S1). We found no significant levels of null alleles at any loci within any maternal

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plant group. No significant linkage disequilibrium was observed between pairs of loci.

Genetic diversity

No significant differences in allelic richness and expected heterozygosity between progeny and maternal plants were observed across species (all t-test p-values > 0.05; Appendix S1). However, progeny were significantly more homozygous than maternal plants, particularly progeny from isolated pasture trees (isolated pasture trees:

E. socialis t = 3.24, d.f. = 16, p-value < 0.01; E. gracilis t = 2.42, d.f. =

16, p-value < 0.05; E. incrassata t = 5.25, d.f. = 14, p-value < 0.01;

Appendix S1).

Mating patterns and density

Each species experienced low levels of selfing (all groups s < 0.30; see

Appendix S6 for mating pattern results), but we observed a significant shifts towards increased selfing, biparental inbreeding and correlated paternity in the insect-pollinated E. socialis and E. gracilis maternal plants sampled from lower density stands (linear regression of density

2 (log) effect: tm ß = 0.21 to 0.25, r = 0.79 to 0.91, p-value < 0.001; tm-ts ß

2 = -0.05 to -0.09, r = 0.36 to 0.70, p-value < 0.05; rp ß = -0.24 to -0.28, r2 = 0.84 to 0.94, p-value < 0.001; Figure 2). The bird-pollinated E. incrassata showed no significant change in mating patterns across a

30-fold decrease in density (from 1.7 trees ha-1 to 51 trees ha-1, linear

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2 2 regression of density(log) effect: tm ß = 0.01, r = 0.10; tm-ts ß = -0.05, r

2 = 0.52; rp ß = -0.05, r = 0.47; all p-values > 0.05).

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(a) Eucalyptus gracilis 1.00!

ß = 0.25 2 0.80! r = 0.79

0.60!

0.40!

ß = -0.05 2

Mating system parameter value ! r = 0.36 0.20!

ß = -0.28 2 r = 0.84 0.00! 0.00 0.50 1.00 1.50 2.00 log density (trees ha-1)! 1.001.00! (b) Eucalyptus socialis

0.80! ß = 0.20 2 r = 0.91

0.60!

0.40! ß = -0.09 2 r = 0.70

Mating system parameter value ! 0.20!

ß = -0.24 2 r = 0.94 0.00! 0.00!0.50!1.00!1.50! (c) loglogEucalyptus density (trees incrassata hhaa-1-1)! 1.00!

ß = 0.01 2 0.80! r = 0.10

0.60!

0.40!

ß = -0.05 2 Mating system parameter value ! 0.20! r = 0.52 ß = -0.05 2 r = 0.47 0.00! 0.00!0.50!1.00!1.50!2.00! log density (trees ha-1)!

Figure 2. Relationships between mating patterns and density of three mallee eucalypts. Insect pollinated Eucalyptus gracilis (a) and E. socialis (b) outcrossing rate (tm; where the selfing rate s = 1-tm) shown by filled squares, biparental inbreeding (tm-ts) by filled triangles and correlated paternity (rp) by filled circles. The bird-pollinated E. incrassata mating patterns are shown by corresponding open shapes (c). The Pg 40 Chapter 2: Forest fragmentation genetics M. F. Breed

trendlines show slopes (ß) and goodness of fit (r2) of linear regressions between log density and mating patterns. 95% CI are not shown as they fall within the outer edge of each symbol.

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Meta-analysis

Our analysis involved 36 studies that presented 44 comparisons of selfing rate, 30 biparental inbreeding comparisons and 29 correlated paternity comparisons (Appendix S3).

Habitat fragmentation had a significant effect increasing selfing rate (Hedges’ g* = 1.04; 95% CI, 1.39 to 0.69; z = 5.33, p-value <

0.0001) and correlated paternity (Hedges’ g* = 0.74; 95% CI, 0.35 to

1.13; z = 3.73, p-value < 0.001; Figure 3 panel a). Habitat fragmentation did not significantly change biparental inbreeding, but a general increase was observed (Hedges’ g* = 0.32; 95% CI, 0.00 to 0.65; z =

1.96, p-value = 0.05).

When data were analysed separately for species with mobile vs. less mobile pollinators, habitat fragmentation still had a significant positive effect on selfing rates and correlated paternity in both groups

(mobile and less mobile pollinated species: selfing rate p-value < 0.001 and < 0.0001; correlated paternity p-value < 0.05 and < 0.01). In both cases, the effect was generally stronger for plants with less mobile pollinators, though the mean effects were not statistically different between groups (Figure 3 panel b). No significant effect of habitat fragmentation on biparental inbreeding was apparent when the data were analysed separately by pollinator groups (both p-values > 0.05).

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(a) Overall effect

Selfing rate (n = 44)

Biparental inbreeding (n = 30)

Correlated paternity (n = 29)

-0.3!0.0!0.3!0.6!0.9!1.2!1.5!1.8! Hedge's d*!

(b) Pollinator mobility Selfing rate Mobile (n = 21) Less mobile (n = 23)

Biparental inbreeding Mobile (n = 16) Less mobile (n = 14)

Correlated paternity Mobile (n = 14) Less mobile (n = 15)

-0.3!0.0!0.3!0.6!0.9!1.2!1.5!1.8! Hedge'sHedges’ d*d * !

Figure 3. Mean effect sizes (Hedges’ d*) of habitat fragmentation on animal-pollinated woody plant mating patterns. Overall mean effect shown in panel (a) and effects separated by pollinator mobility in panel

(b). Error bars show bias-corrected 95% bootstrap CIs. A mean effect size is significantly different from zero when its 95% CI does not overlap zero. Positive mean effect sizes indicate that the fragmented group of maternal plants had on average larger values for the given mating pattern. The number of studies is shown in parentheses.

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DISCUSSION

Our study aimed to test whether human mediated stand density changes were correlated with changes in selfing rates, biparental inbreeding and correlated paternity. Additionally, we wanted to test whether greater pollinator mobility acted as a buffer against mating pattern shifts as a consequence of reduced stand density. To the best of our knowledge, our eucalypt case study is the most in-depth study performed into these predictions conducted thus far (Appendix S3) and our meta-analysis is the first quantitative review of this topic.

Generality of habitat fragmentation-mating pattern interactions

Our meta-analysis of the available literature (44 datasets) demonstrates that the negative effect of reduced stand density on mating patterns, which we observed in our eucalypt study, is a general one for animal- pollinated woody plants. We observed a general negative trend between habitat fragmentation (a surrogate for lower stand density;

Ghazoul, 2005; Lowe et al, 2005) and less pollen diversity received into animal-pollinated woody plant canopies. This general trend has not been previously reported. In addition, we report that habitat fragmentation has a quantifiable effect on selfing rates, which is consistent with previous qualitative reviews (Ghazoul, 2005; Lowe et al,

2005; Eckert et al, 2010).

Our meta-analysis is the first review to investigate the general impact of habitat fragmentation on pollen diversity of plants. There are

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numerous studies that demonstrate how reduced pollen diversity received into plant canopies can associate with reduced offspring fitness in fragmented systems (Hoebee and Young, 2001; Cascante et al, 2002; Fuchs et al, 2003; González-Varo et al, 2010; Breed et al,

2012a), however more work is required to investigate the mechanism of this fitness impact. Increased correlated paternity has been hypothesised to associate with less pollen competition to fertilize receptive ovules (Breed et al, 2012a). However, pollen grain size and tube growth rates were not measured in these studies, leaving the possibility that alternative hypotheses may explain the relationship between progeny vigour and pollen diversity (e.g. heterosis effect from more genetically diverse pollen; Skogsmyr and Lankinen, 2002).

Additionally, there is uncertainty about how pollen diversity received onto a stigma relates to correlated paternity after fertilisation, but we assume that these parameters are inversely proportional (i.e. more pollen diversity received on stigma = lower correlated paternity). This relationship may breakdown in the presence of female choice for specific pollen grains or if certain males tend to provide more competitive pollen - ‘supermales’ (Skogsmyr and Lankinen, 2002).

There are numerous cases in the literature that demonstrate a link between the offspring fitness of animal-pollinated woody plants and increasing selfing caused by habitat fragmentation (Cascante et al,

2002; Fuchs et al, 2003; Quesada et al, 2004; Hirayama et al, 2007;

González-Varo et al, 2010; Breed et al, 2012a). The mechanism

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whereby fitness is impacted due to increased selfing has been clearly articulated in the literature on inbreeding and inbreeding depression (e.g.

Keller and Waller, 2002). Animal-pollinated woody plants should be particularly susceptible to inbreeding depression since most predominantly outcross, leading them to accumulate genetic load

(Klekowski, 1988). Much work has been conducted on the evolutionary patterns of plant outcrossing (Barrett, 1998). However, more studies are needed to investigate what types of habitat fragmentation make plants most susceptible to induced increases in inbreeding (e.g. fragmentation, logging, disruption to pollinator community). The future viability of populations that produce high levels of low fitness offspring (i.e. inbred or poor pollen-ovule combinations) also requires further investigation.

Natural selection generally purges low fitness offspring as a cohort ages; this has been documented in both non-fragmented (e.g. Ueno et al, 2002) and fragmented systems (Vranckx et al, 2011). However, the viability of fragmented populations that produce these high levels of low fitness offspring remains unknown, but a tendency of demographic decline would be expected.

Pollinator mobility interactions with habitat disturbance

As predicted, we observed that the mating patterns of two insect- pollinated mallee eucalypts were negatively correlated with stand density. This is an important finding because a change in mating patterns is expected to result in progeny of lower fitness (Breed et al,

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2012a). Furthermore, as predicted, there was no relationship between stand density and mating patterns for Eucalyptus incrassata, which is primarily pollinated by highly mobile birds.

Our study highlights how pollinator mobility may affect a species’ susceptibility to shifts in mating patterns in response to fragmentation.

Pollination neighbourhoods of small-insect pollinated woody plants appear to decline as density declines. These reduced pollination neighbourhoods are expected to lead to greater pollen discounting (i.e. selfing increases due to a reduction in outcrossed siring success;

Barrett, 1998), provision of less diverse pollen (increasing correlated paternity; Bianchi and Cunningham, 2012) and proportionally more pollen from close relatives (increasing biparental inbreeding; Dubreuil et al, 2010). The bird-pollinated E. incrassata appeared not to be suffering from these pollen limitation and pollination neighbourhood effects, likely because its bird pollinators have the ability to sample larger and more diverse pollination neighbourhoods, even in fragmented landscapes

(Paton, 2004).

The results from our meta-analysis generally support the findings of our eucalypt study where mating patterns shifted in fragmented habitats as a function of pollinator mobility. More mobile pollinators (e.g. birds, bats) should function within larger pollination neighbourhoods than less mobile pollinators (e.g. small insects; Bianchi and

Cunningham, 2012). Thus, the pollen provision of more mobile pollinators should be less affected by changes in stand density than

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less mobile pollinators. However, the pollinator mobility effect was not significant in our meta-analysis. Additionally, it is possible that the strong effect of pollinator mobility we observed in our eucalypt study might have been unusual because the bird-pollinators of E. incrassata are particularly mobile (Family Meliphagidae; Paton, 2004). A factor that may be influencing the size of the pollinator mobility effect in our meta- analysis is the binary grouping of pollinators into mobility categories

(mobile vs. less mobile). This grouping seems to over-simplify reality, since birds may indeed be susceptible to fragmentation and, as a consequence, may experience contractions of their foraging areas

(Ibarra-Macias et al, 2011) or declines in their populations (Turner,

1996). Furthermore, other studies have demonstrated far-reaching pollination of small-insects in fragmented landscapes (>1 km; Dick et al,

2003; Byrne et al, 2008). However, there are many cases where birds have been shown to maintain or even increase their foraging in fragmented landscapes (Paton, 2004; Byrne et al, 2007). Other studies have demonstrated that insect-pollination decreases in fragmented systems (Aizen and Feinsinger, 1994). Despite the expectation that selfing and pollen diversity should be a function of pollinator mobility, in line with landscape ecology models (Charnov, 1976; Karron et al,

2009), more experimental studies into woody plant density, pollen movement and mating patterns are required to further understand these associations.

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A central argument for the paradox of forest fragmentation genetics is that average pollen flow distances may locally increase in fragmented animal-pollinated woody plant populations, genetically buffering these populations (Kramer et al, 2008). This may indeed be the case in some populations, however there are now two well- supported general patterns that are inconsistent with this genetic buffering effect. Firstly, genetic diversity of progeny from animal- pollinated woody plant populations declines with fragmentation

(Vranckx et al, 2011). Secondly, as we show here, animal-pollinated woody plant mating patterns are impacted by habitat fragmentation. We suggest that when pollen neighbourhoods decline due to fragmentation, mean gene flow will locally increase because of increased spacing between plants. However, as a consequence of this increased spacing, pollinators will spend more time in individual canopies (increasing selfing rates) and decrease the provision of different pollen sources

(increasing correlated paternity). Together, these effects reduce progeny genetic diversity and impact on adult mating patterns, which both have fitness consequences for the progeny generation.

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ACKNOWLEDGEMENTS

This work was supported by ARC Linkage project (LP110200805) and

SA Premier's Science and Research Fund awarded to A.J.L.; Native

Vegetation Council SA (grant 09/10/27), Nature Foundation SA Inc.,

Australian Geographic Society, Biological Society of SA, Field Naturalist

Society of SA, Wildlife Preservation Society of Australia, NCCARF

Travel Grants awarded to M.F.B. The authors thank Rob Murphy from

Rural Solutions SA, Matt Hayward and Phil Scully from Australian

Wildlife Conservancy and volunteers for fieldwork assistance.

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Supplementary information

Supplementary information is available at Heredity’s website.

Conflict of interest

The authors declare no conflict of interest.

Data archiving

Data for this study are available at: to be completed after manuscript is accepted for publication.

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Table S1. Sampling and genetic parameter estimates for each group of maternal plants

Genetic variability of groups of maternal plants from three eucalypt species collected in the southern Australia. Progeny array size, density and growth data are also reported (n, number of samples; AR, rarefied allelic richness; HE and HO, unbiased expected and observed heterozygosity, respectively; F, Fixation index; progeny array, mean number of offspring per progeny array; standard deviations in parentheses).

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- 18 15 262 205 .65) 7 (0.19) 7 0.7 (0.2) 0.7 20.4 (3.2) 20.4 (0.2) 4.92 (3.3) 15.4 Sanctuary 4.95 (0 4.95 (0.11) 0.83 (0.08) 0.83 (0.11) 0.04 (0.11) 0.82 (0.17) 0.16 (1.43) 4.84 (0.20) 0.79 (0.22) 0.79 (0.21) 0.05 (2.83) 12.2 (1.41) 4.69 (0.21) 0.76 (0.24) 0.64 0.1

- - 14.75 (0.59) 14.75 Scotia

- 20 18 293 197 2 (0.25) 2 (1.39) 9 49.3 (4.6) 49.3 (0.14) 0.8 (3.2) 23.3 (1.4) 4.69 Sanctuary 4.96 (0.88) 4.96 (0.14) 0.81 (0.08) 0.86 (0.14) 0.11 (0.23) 4.89 0.7 (0.25) 0.14 5.1 (0.19) 0.80 (0.16) 0.81 (0.22) 0.06 (0.21) 0.75 (0.22) 0.66 (0.16) 0.12

- - 13.44 (0.89) 13.44 (3.24) 10.94 Yookamurra

2) 10 148

31.3 (3.0) 31.3 servation servation 4.03 (1.06) 4.03 (0.1 0.71 (0.13) 0.73 (0.14) 0.09 (1.03) 4.31 (0.12) 0.73 (0.14) 0.66 (0.16) 0.08 - 14.80 (0.63) 14.80 Park Con Bakara Bakara

8 110

51.1 (3.4) 51.1 4.07 (1.05) 4.07 (0.20) 0.74 (0.23) 0.79 (0.14) 0.14 (1.14) 4.43 (0.19) 0.73 (0.19) 0.68 (0.17) 0.06 - 13.75 (2.82) 13.75 Park Conservation Lowan

12 171

23.00 (2.3) 23.00 (1.21) 4.72 (0.15) 0.79 (0.15) 0.83 (0.06) 0.11 (1.15) 4.85 (0.15) 0.80 (0.24) 0.67 (0.26) 0.19 - 14.25 (2.60) 14.25 Conservation Park Brookfield Brookfield

2) 20 16 21 288 197 357 23.6 (2.3) 23.6 (1.9) 11.4 (4.5) 12.6 5.31 (0.82) 5.31 (0.11) 0.85 (0.11) 0.85 (0.14) 0.03 (0.23) 5.17 (0.14) 0.83 (0.23) 0.71 (0.23) 0.17 (1.45) 4.95 (0.22) 0.78 (0.17) 0.76 (0.20) 0.04 (1.33) 4.75 (0.20) 0.76 (0.23) 0.60 (0.16) 0.22 (1.22) 4.65 (0.12) 0.79 (0.14) 0.88 (0.12) 0.16 (1.2 4.55 (0.16) 0.76 (0.15) 0.61 (0.20) 0.18 - - - 14.70 (0.58) 14.70 (5.45) 12.31 (4.66) 17.00 high density high - Monarto

12 13 16 172 175 292 .59) 5.3 (4.1) 5.3 (2.9) 2.2 (3.2) 1.7 5.10 (1.40) 5.10 (0.15) 0.82 (0.15) 0.82 (0.09) 0.05 (1.19) 4.86 (0.13) 0.81 (0.18) 0.62 (0.17) 0.24 (1.54) 5.01 (0.16) 0.80 (0.19) 0.80 (0.19) 0.06 (1.46) 4.72 (0.23) 0.75 (0.23) 0.55 (0.19) 0.26 (1.26) 4.62 (0.11) 0.80 (0.06) 0.92 (0.17) 0.21 (1.19) 4.56 (0.11) 0.79 (0.14) 0.64 (0.16) 0.19 - - - low density 14.33 (2.31) 14.33 (4 13.46 (4.27) 18.25 - Monarto

) ) ) 1 1 1

- - -

y array size array y

E O E O E O E O E O E O

n Density (trees ha AR H H F n Progen AR H H F n Density (trees ha AR H H F n arraysize Progeny AR H H F n Density (trees ha AR H H F n arraysize Progeny AR H H F Adults Progeny Adults Progeny Adults Progeny Species, cohort and parameter gracilis Eucalyptus socialis Eucalyptus incrassata Eucalyptus

Pg 53 Chapter 2: Forest fragmentation genetics M. F. Breed

Appendix S2. Detailed PCR conditions

Each 10 μL multiplex PCR contained 1 μL template DNA (ca. 20 ng μL-

1), 5 μL 2X Qiagen Multiplex PCR Master Mix (Qiagen, Hilden,

Germany), 3 μL of nuclease-free water, 1 μL of primer mix with each primer at 2 μM concentration. Standard Qiagen Multiplex PCR conditions were used with an initial activation step at 95°C for 15 minutes, 40 cycles of denaturation at 94°C for 30 seconds, annealing at

60°C for 90 seconds and extension at 60°C for 60 seconds, with final extension at 60°C for 30 minutes. LIZ500 size standard was added to samples and fragments were separated on an AB3730 genetic analyser with a 36 cm capillary array (Applied Biosystems, Foster City, MA, USA) at AGRF. Alleles were sized using GeneMapper software (Applied

Biosystems) and double-checked manually. EMBRA1363 produced two unlinked and scoreable PCR products (see Appendix S5 for details on microsatellite loci scored per species).

Pg 54 Chapter 2: Forest fragmentation genetics M. F. Breed

Table S3. Details of datasets included in meta-analysis

Plant species, pollinator mobility, life form and mating system information included in the meta-analysis (nfam, total number of families per group; tm, multilocus outcrossing rate; tm-ts, biparental inbreeding estimate; rp, multilocus correlated paternity; ± one standard deviation).

Forest or 'best on offer' Fragmented group group Pollinator Life Species n t t -t r n t t -t r Reference mobility form fam m m s p fam m m s p Acacia Non- 0.89 ± 0.01 ± 0.15 ± 0.85 ± 0.05 ± 0.06 ± (Coates et Shrub 10 10 anfractuosa mobile 0.20 0.20 0.20 0.20 0.20 0.20 al. 2007) Banksia 1.00 ± 0.01 ± 0.09 ± 0.98 ± 0.03 ± 0.03 ± (Coates et Mobile Shrub 10 10 oligantha 0.20 0.20 0.20 0.20 0.20 0.20 al. 2007) Banksia 0.99 ± 0.04 ± 0.33 ± 0.91 ± 0.04 ± 0.34 ± (Llorens et sphaerocarpa Mobile Shrub 11 10 0.34 0.13 0.10 0.04 0.19 0.10 al. 2011) var. caesia Calothamnus 0.70 ± 0.02 ± 0.27 ± 0.54 ± 0.01 ± 0.08 ± (Yates et al. Mobile Shrub 20 20 quadrifidus 0.22 0.09 0.27 0.45 0.13 0.09 2007a) Carapa Non- 0.94 ± 0.02 ± 0.05 ± 0.93 ± 0.03 ± 0.05 ± (Cloutier et Tree 21 21 guianensis mobile 0.12 0.07 0.06 0.15 0.08 0.06 al. 2007) Carapa Non- 0.97 ± 0.99 ± (Hall et al. Tree 20 20 guianensis mobile 0.10 0.13 1994) Carapa Non- 0.85 ± 0.11 ± 0.63 ± 0.06 ± (Doligez & Tree 33 14 procera mobile 0.05 0.02 0.08 0.02 Joly 1997) Caryocar 1.00 ± 0.21 ± 0.19 ± 1.00 ± 0.23 ± 0.17 ± (Collevatti Mobile Tree 14 10 brasiliense 0.00 0.03 0.19 0.00 0.03 0.05 et al. 2001) (Murawski Cavanillesia 0.57 ± 0.35 ± Mobile Tree 20 11 & Hamrick platanifolia 0.11 0.08 1992) Ceiba 0.96 ± 0.97 ± (Quesada Mobile Tree 36 31 ausculifolia 0.27 0.03 et al. 2004) Ceiba 0.90 ± 0.91 ± (Quesada Mobile Tree 12 18 grandiflora 0.59 0.04 et al. 2004) Dinizia 0.90 ± 0.86 ± (Dick et al. Mobile Tree 14 6 excelsa 0.08 0.01 2003) Dipteryx 0.93 ± 0.03 ± 0.87 ± 0.06 ± (Hanson et Mobile Tree 31 31 panamensis 0.15 0.13 0.18 0.15 al. 2008) Dryobalanop Non- 0.86 ± 0.79 ± (Hall et al. Tree 10 29 s aromatica mobile 0.34 0.04 1994) Dryobalanop Non- 0.92 ± 0.08 ± 0.11 ± 0.77 ± 0.04 ± 0.39 ± Tree 10 10 (Lee 2000) s aromatica mobile 0.04 0.03 0.06 0.06 0.01 0.08 Embotrium 0.84 ± 0.08 ± 0.18 ± 0.73 ± 0.00 ± 0.07 ± (Mathiasen Mobile Tree 18 2 coccineum 0.34 0.88 0.38 0.27 0.16 0.51 et al. 2007) (Rocha & Enterolobium 1.00 ± 0.02 ± 0.10 ± 1.00 ± 0.02 ± 0.19 ± Mobile Tree 20 20 Aguilar cyclocarpum 0.16 0.10 0.16 0.25 0.10 0.30 2001) Eucalyptus Non- 0.68 ± 0.17 ± 0.43 ± 0.50 ± 0.31 ± 0.29 ± (Butcher et Tree 16 11 benthamii mobile 0.05 0.11 0.35 0.12 0.30 0.11 al. 2005) Eucalyptus Non- 0.89 ± 0.04 ± 0.12 ± 0.65 ± 0.06 ± 0.06 ± (Mimura et globulus Tree 20 20 mobile 0.09 0.06 0.09 0.21 0.07 0.07 al. 2009) (Tasmania) Eucalyptus Non- 0.86 ± 0.06 ± 0.20 ± 0.79 ± 0.11 ± 0.03 ± (Mimura et globulus Tree 20 20 mobile 0.14 0.07 0.14 0.27 0.09 0.05 al. 2009) (Victoria) Eucalyptus Non- 0.98 ± 0.11 ± 0.06 ± 0.74 ± 0.16 ± 0.32 ± Tree 20 12 this study gracilis mobile 0.01 0.02 0.01 0.08 0.03 0.06 Eucalyptus 0.96 ± 0.08 ± 0.10 ± 0.94 ± 0.19 ± 0.16 ± Mobile Tree 8 16 this study incrassata 0.07 0.04 0.03 0.03 0.03 0.03

Pg 55 Chapter 2: Forest fragmentation genetics M. F. Breed

Eucalyptus Non- 0.80 ± 0.08 ± 0.92 ± 0.71 ± 0.14 ± 0.78 ± (Millar et al. Tree 10 10 marginata mobile 0.28 0.06 0.32 0.35 0.13 0.28 2000) Eucalyptus 0.93 ± 0.05 ± 0.11 ± 0.51 ± 0.04 ± 0.13 ± (Coates et Mobile Tree 10 10 rameliana 0.20 0.20 0.20 0.20 0.20 0.20 al. 2007) Eucalyptus Non- 0.84 ± 0.20 ± 0.32 ± 0.73 ± 0.23 ± 0.13 ± Tree 16 13 this study socialis mobile 0.04 0.03 0.10 0.09 0.05 0.04 (Hoebee & Grevillea 1.00 ± 0.04 ± 0.44 ± 0.96 ± 0.03 ± 0.31 ± Mobile Shrub 10 10 Young iaspicula 0.05 0.05 0.05 0.05 0.05 0.05 2001) (Starr & Hakea Non- 0.12 ± 0.09 ± Shrub 6 8 Carthew carinata mobile 0.50 0.50 1998) (Franceschi Helicteres 0.65 ± 0.02 ± 0.49 ± 0.02 ± Mobile Shrub 19 22 nelli & brevispira 0.05 0.02 0.04 0.02 Bawa 2000) Lambertia 0.65 ± 0.05 ± 0.11 ± 0.38 ± 0.03 ± 0.12 ± (Coates et Mobile Shrub 10 10 orbifolia 0.20 0.20 0.20 0.20 0.20 0.20 al. 2007) Magnolia Non- 0.74 ± 0.04 ± 0.62 ± 0.08 ± (Hirayama Tree 20 10 stellata mobile 0.55 0.66 0.10 0.12 et al. 2007) Magnolia Non- 0.68 ± 0.09 ± 0.33 ± 0.65 ± 0.11 ± 0.05 ± (Tamaki et Tree 35 39 stellata mobile 0.69 0.27 0.67 0.09 0.60 0.10 al. 2009) (González- Myrtus Non- 0.34 ± 0.02 ± 0.19 ± 0.13 ± 0.02 ± 0.11 ± Shrub 22 10 Varo et al. communis mobile 0.09 0.02 0.42 0.10 0.05 0.13 2010) Pachira 0.92 ± 0.74 ± 0.78 ± 0.47 ± (Fuchs et Mobile Tree 15 15 quinata 0.17 0.46 0.07 0.33 al. 2003) (Murawski Platypodium Non- 0.90 ± 0.92 ± Tree 5 5 & Hamrick elegans mobile 0.10 0.10 1991) Non- 0.14 ± 0.09 ± (Jolivet et Prunus avium Tree 10 10 mobile 0.05 0.05 al. 2011) Psychotria Non- 0.50 ± 0.07 ± 0.15 ± 0.37 ± 0.05 ± 0.07 ± (Ramos et Shrub 18 24 tenuinervis mobile 0.44 0.13 0.24 0.05 0.10 0.21 al. 2008) Samanea 0.99 ± 0.12 ± 0.91 ± 0.03 ± (Cascante Mobile Tree 20 17 saman 0.01 0.41 0.01 0.19 et al. 2002) (Ribeiro & Senna 0.54 ± 0.05 ± 0.31 ± 0.84 ± 0.11 ± 0.25 ± Mobile Tree 22 14 Lovato miltijuga 0.34 0.08 0.58 0.04 0.13 0.72 2004) Shorea Non- 0.96 ± 0.52 ± (Obayashi Tree 11 5 curtisii mobile 0.03 0.02 et al. 2002) Shorea 0.87 ± 0.71 ± (Murawski Mobile Tree 8 22 megistophylla 0.16 0.10 et al. 1994) (Murawski Sorocea Non- 0.97 ± 1.00 ± Tree 7 8 & Hamrick affinis mobile 0.05 0.13 1991) Swietenia macrophylla Non- 0.99 ± 0.15 ± 0.28 ± 0.99 ± 0.21 ± 0.15 ± (Breed et al. Tree 23 12 (dry mobile 0.05 0.06 0.08 0.10 0.09 0.03 2012a) provenances) Swietenia macrophylla Non- 0.99 ± 0.15 ± 0.45 ± 0.85 ± 0.31 ± 0.25 ± (Breed et al. Tree 24 12 (mesic mobile 0.04 0.05 0.09 0.04 0.03 0.05 2012a) provenances) (Aldrich & Symphonia 0.85 ± 0.74 ± Mobile Tree 30 30 Hamrick globulifera 0.20 0.20 1998) Verticordia Non- 0.73 ± 0.11 ± 0.14 ± 0.62 ± 0.06 ± 0.29 ± (Coates et Shrub 10 10 fimbrilepis mobile 0.20 0.20 0.20 0.20 0.20 0.20 al. 2007)

Pg 56 Chapter 2: Forest fragmentation genetics M. F. Breed

Figure S4. Meta-analysis funnel plots

0.0 (A) 0.5 1.0 1.5 Standard error 2.0 2.5 3.0

-15 -10 -5 0 5 (B) 0.0 0.2 0.4 Standard error 0.6

-2 -1 0 1 2 3 (C) 0.0 0.2 0.4 0.6 Standard error 0.8 1.0

-4 -2 0 2 4 6 Standardised mean difference

Funnel plots of standardised mean differences and standard error for

(A) multilocus outcrossing rate, (B) biparental inbreeding (C) and multilocus correlated paternity. The relative symmetry and left-right evenness of the funnel plots signifies no problem with publication bias. Pg 57 Chapter 2: Forest fragmentation genetics M. F. Breed

Table S5. Details on microsatellite loci scored for each species

separated by maternal plant group

Genetic variability of groups of maternal plants for three eucalypt

species scored at 8 or 9 microsatellite markers (A, number of alleles; HE

and HO, unbiased expected and observed heterozygosity, respectively;

F, fixation index; Q, probability of paternity exclusion; QC, combined

probability of paternity exclusion if no parents are known; all F

estimates were not significantly different from zero after sequential

Bonferroni adjustment of p-values with a nominal level of 0.05).

Species and maternal plant group Locus A HE HO F Q

Eucalyptus incrassata Monarto-low density EMBRA1382 9 0.88 0.94 -0.11 0.87 Monarto-low density EMBRA2002 7 0.72 0.94 -0.35 0.65 Monarto-low density EMBRA1445 6 0.69 0.94 -0.40 0.63 Monarto-low density EMBRA914 4 0.64 0.88 -0.42 0.50 Monarto-low density EMBRA1990 7 0.79 0.81 -0.06 0.75 Monarto-low density EMBRA1284 7 0.82 1.00 -0.26 0.78 Monarto-low density EMBRA1928 11 0.90 0.88 -0.01 0.89 Monarto-low density EMBRA1468 18 0.95 1.00 -0.08 0.96 8.63 0.80 0.92 -0.21 QC = Monarto-low density Mean (4.31) (0.11) (0.06) (0.17) 1.00 Lowan CP EMBRA1382 7.00 0.83 1.00 -0.28 0.78 Lowan CP EMBRA2002 5.00 0.65 0.75 -0.23 0.55 Lowan CP EMBRA1445 5.00 0.76 0.88 -0.23 0.67 Lowan CP EMBRA1990 7.00 0.89 1.00 -0.20 0.84 Lowan CP EMBRA1284 6.00 0.79 0.75 -0.01 0.74 Lowan CP EMBRA1928 6.00 0.82 0.75 0.02 0.74 Lowan CP EMBRA1468 6.00 0.84 0.75 0.05 0.77 Lowan CP EMBRA1363a 3.00 0.24 0.25 -0.10 0.21 Lowan CP EMBRA1363b 6.00 0.81 1.00 -0.32 0.74 5.67 0.74 0.79 -0.14 QC = Lowan CP Mean (1.22) (0.20) (0.23) (0.14) 1.00 Bakara CP EMBRA1382 6.00 0.74 0.70 0.00 0.71 Bakara CP EMBRA2002 5.00 0.78 0.80 -0.08 0.70 Bakara CP EMBRA1445 4.00 0.70 0.80 -0.20 0.56 Bakara CP EMBRA1990 9.00 0.89 0.80 0.06 0.87 Bakara CP EMBRA1284 3.00 0.58 0.60 -0.08 0.44

Pg 58 Chapter 2: Forest fragmentation genetics M. F. Breed

Bakara CP EMBRA1928 5.00 0.69 0.60 0.09 0.58 Bakara CP EMBRA1468 6.00 0.85 1.00 -0.24 0.80 Bakara CP EMBRA1363a 4.00 0.55 0.70 -0.33 0.46 Bakara CP EMBRA1363b 4.00 0.61 0.60 -0.03 0.52 5.11 0.71 0.73 -0.09 QC = Bakara CP Mean (1.76) (0.12) (0.13) (0.14) 1.00 Monarto-high density EMBRA1382 12 0.91 0.95 -0.08 0.92 Monarto-high density EMBRA2002 7 0.75 0.95 -0.29 0.71 Monarto-high density EMBRA1445 6 0.68 0.90 -0.37 0.60 Monarto-high density EMBRA914 6 0.58 0.57 -0.01 0.53 Monarto-high density EMBRA1990 8 0.83 0.95 -0.17 0.81 Monarto-high density EMBRA1284 7 0.74 0.81 -0.12 0.71 Monarto-high density EMBRA1928 11 0.90 1.00 -0.14 0.91 Monarto-high density EMBRA1468 15 0.94 1.00 -0.09 0.95 9.00 0.79 0.89 -0.16 QC = Monarto-high density Mean (3.30) (0.12) (0.14) (0.12) 1.00

Eucalyptus gracilis Monarto-low density EMBRA1990 11.00 0.89 0.83 0.02 0.88 Monarto-low density EMBRA2002 7.00 0.85 0.92 -0.13 0.81 Monarto-low density EMBRA1445 10.00 0.85 0.75 0.08 0.83 Monarto-low density EMBRA1284 9.00 0.85 0.92 -0.12 0.82 Monarto-low density EMBRA1928 15.00 0.94 0.83 0.08 0.94 Monarto-low density EMBRA1468 17.00 0.95 1.00 -0.10 0.95 Monarto-low density EMBRA1363a 4.00 0.47 0.50 -0.11 0.38 Monarto-low density EMBRA1363b 5.00 0.78 0.83 -0.12 0.70 9.57 0.81 0.82 -0.06 QC = Monarto-low density Mean (4.89) (0.16) (0.16) (0.09) 1.00 Monarto-high density EMBRA1382 12 0.89 1.00 -0.15 0.90 Monarto-high density EMBRA2002 8 0.88 0.82 0.04 0.87 Monarto-high density EMBRA1445 13 0.92 0.94 -0.05 0.93 Monarto-high density EMBRA1284 12 0.91 0.82 0.07 0.92 Monarto-high density EMBRA1928 15 0.93 1.00 -0.11 0.94 Monarto-high density EMBRA1468 14 0.94 0.94 -0.04 0.94 Monarto-high density EMBRA1363a 5 0.63 0.76 -0.24 0.51 Monarto-high density EMBRA1363b 5 0.70 0.65 0.05 0.62 10.50 0.85 0.87 -0.06 QC = Monarto-high density Mean (6.71) (0.18) (0.18) (0.23) 1.00 Brookfield CP EMBRA1990 10.00 0.90 1.00 -0.16 0.89 Brookfield CP EMBRA2002 6.00 0.79 0.75 0.00 0.75 Brookfield CP EMBRA1445 6.00 0.70 0.75 -0.13 0.62 Brookfield CP EMBRA1284 8.00 0.88 0.92 -0.09 0.86 Brookfield CP EMBRA1928 12.00 0.94 1.00 -0.11 0.94 Brookfield CP EMBRA1468 10.00 0.87 0.92 -0.10 0.86 Brookfield CP EMBRA1363a 6.00 0.50 0.58 -0.22 0.49 Brookfield CP EMBRA1363b 6.00 0.70 0.75 -0.11 0.60 7.71 0.77 0.81 -0.11 QC = Brookfield CP Mean (2.43) (0.15) (0.14) (0.07) 1.00 Yookamurra Sanctuary EMBRA1382 12 0.88 0.85 0.01 0.88

Pg 59 Chapter 2: Forest fragmentation genetics M. F. Breed

Yookamurra Sanctuary EMBRA2002 10 0.85 0.95 -0.14 0.86 Yookamurra Sanctuary EMBRA1445 10 0.75 0.85 -0.16 0.74 Yookamurra Sanctuary EMBRA1284 14 0.91 0.90 -0.01 0.92 Yookamurra Sanctuary EMBRA1928 15 0.93 0.95 -0.04 0.95 Yookamurra Sanctuary EMBRA1468 16 0.92 0.90 0.00 0.94 Yookamurra Sanctuary EMBRA1363a 4 0.54 0.75 -0.41 0.40 Yookamurra Sanctuary EMBRA1363b 5 0.70 0.75 -0.09 0.63 10.75 0.81 0.86 -0.11 QC = Yookamurra Sanctuary Mean (3.58) (0.22) (0.13) (0.22) 1.00 Scotia Sanctuary EMBRA1382 10 0.87 0.83 0.01 0.86 Scotia Sanctuary EMBRA2002 9 0.87 0.94 -0.12 0.86 Scotia Sanctuary EMBRA1445 10 0.80 0.72 0.07 0.80 Scotia Sanctuary EMBRA1284 11 0.90 0.83 0.05 0.90 Scotia Sanctuary EMBRA1928 13 0.90 0.94 -0.08 0.91 Scotia Sanctuary EMBRA1468 11 0.88 0.83 0.03 0.89 Scotia Sanctuary EMBRA1363a 5 0.58 0.72 -0.29 0.46 Scotia Sanctuary EMBRA1363b 8 0.80 0.78 0.00 0.77 9.63 0.83 0.83 -0.04 QC = Scotia Sanctuary Mean (7.01) (0.16) (0.13) (0.18) 1.00

Eucalyptus socialis Monarto-low density EMBRA1382 6 0.60 0.69 -0.21 0.55 Monarto-low density EMBRA2002 5 0.77 0.92 -0.25 0.68 Monarto-low density EMBRA914 6 0.78 0.69 0.08 0.72 Monarto-low density EMBRA1990 9 0.86 1.00 -0.20 0.85 Monarto-low density EMBRA1284 13 0.94 1.00 -0.11 0.93 Monarto-low density EMBRA1928 14 0.94 0.85 0.07 0.94 Monarto-low density EMBRA1468 11 0.88 0.85 0.00 0.87 Monarto-low density EMBRA1363a 7 0.83 0.62 0.23 0.79 Monarto-low density EMBRA1363b 4 0.57 0.62 -0.13 0.41 8.33 0.80 0.80 -0.06 QC = Monarto-low density Mean (4.33) (0.16) (0.19) (0.19) 1.00 Monarto-high density EMBRA1382 5 0.53 0.69 -0.33 0.45 Monarto-high density EMBRA2002 8 0.77 0.75 -0.01 0.71 Monarto-high density EMBRA914 8 0.84 0.94 -0.15 0.82 Monarto-high density EMBRA1990 10 0.86 0.75 0.10 0.85 Monarto-high density EMBRA1284 12 0.91 0.88 0.01 0.92 Monarto-high density EMBRA1928 12 0.91 0.81 0.08 0.91 Monarto-high density EMBRA1468 14 0.91 0.75 0.15 0.92 Monarto-high density EMBRA1363a 6 0.77 0.81 -0.09 0.71 Monarto-high density EMBRA1363b 5 0.47 0.50 -0.10 0.42 8.89 0.78 0.76 -0.04 QC = Monarto-high density Mean (4.39) (0.22) (0.17) (0.20) 1.00 Yookamurra Sanctuary EMBRA1382 6 0.66 0.89 -0.39 0.55 Yookamurra Sanctuary EMBRA2002 9 0.81 0.83 -0.06 0.81 Yookamurra Sanctuary EMBRA914 7 0.69 0.61 0.09 0.67 Yookamurra Sanctuary EMBRA1990 10 0.90 0.89 -0.02 0.90 Yookamurra Sanctuary EMBRA1284 14 0.93 0.94 -0.05 0.93

Pg 60 Chapter 2: Forest fragmentation genetics M. F. Breed

Yookamurra Sanctuary EMBRA1928 10 0.91 0.89 0.00 0.92 Yookamurra Sanctuary EMBRA1468 14 0.93 0.83 0.08 0.94 Yookamurra Sanctuary EMBRA1363a 9 0.83 0.78 0.04 0.81 Yookamurra Sanctuary EMBRA1363b 6 0.56 0.67 -0.22 0.51 9.44 0.80 0.81 -0.06 QC = Yookamurra Sanctuary Mean (4.25) (0.19) (0.16) (0.22) 1.00 Scotia Sanctuary EMBRA1382 5.00 0.56 0.67 -0.24 0.47 Scotia Sanctuary EMBRA2002 5.00 0.76 0.93 -0.27 0.67 Scotia Sanctuary EMBRA914 7.00 0.78 0.67 0.12 0.74 Scotia Sanctuary EMBRA1990 10.00 0.88 1.00 -0.18 0.87 Scotia Sanctuary EMBRA1284 13.00 0.93 1.00 -0.11 0.93 Scotia Sanctuary EMBRA1928 13.00 0.94 0.80 0.12 0.94 Scotia Sanctuary EMBRA1468 12.00 0.88 0.87 -0.02 0.88 Scotia Sanctuary EMBRA1363a 7.00 0.82 0.67 0.15 0.76 Scotia Sanctuary EMBRA1363b 4.00 0.52 0.53 -0.05 0.38 8.44 0.79 0.79 -0.05 QC = Scotia Sanctuary Mean (3.61) (0.15) (0.17) (0.16) 1.00

Pg 61 Chapter 2: Forest fragmentation genetics M. F. Breed

Table S6. Detailed mating pattern trends

Detailed mating pattern trends of three eucalypt species sampled from across southern Australia from varying stand densities caused by increasing habitat fragmentation (tm, multilocus outcrossing rate; tm-ts, biparental inbreeding estimate; rp, multilocus correlated paternity; standard deviations in parentheses; 95% confidence interval homogeneous subgroups indicated by ‘A’, ‘B’ and ‘C’).

-1 Species and group Adult density (trees ha ) tm tm-ts rp E. gracilis [insect pollinated] 0.74 0.16 0.32 Monarto-low density 5.3 (4.1) (0.08) C (0.03) B (0.06) A 0.95 0.16 0.11 Scotia Sanctuary 20.4 (3.2) (0.02) A (0.03) B (0.04) BC 0.87 0.12 0.06 Brookfield Conservation Park 23.00 (2.3) (0.04) B (0.10) A (0.02) C 0.97 0.15 0.12 Monarto-high density 23.7 (2.3) (0.02) A (0.01) B (0.02) BC 0.98 0.11 0.06 Yookamurra Sanctuary 49.3 (4.6) (0.01) A (0.02) A (0.01) C

E. socialis [insect pollinated] 0.73 0.23 0.32 Monarto-low density 2.2 (2.9) (0.09) C (0.05) C (0.10) C 0.84 0.2 0.13 Monarto-high density 11.4 (1.9) (0.04) B (0.03) C (0.04) A 0.93 0.18 0.16 Scotia Sanctuary 15.4 (3.3) (0.03) A (0.03) BC (0.05) BC 0.92 0.11 0.06 Yookamurra Sanctuary 23.3 (3.2) (0.04) A (0.05) A (0.04) B

E. incrassata [bird pollinated] 1.7 (3.2) 0.94 0.16 0.16 Monarto-low density (0.02) A (0.01) BC (0.03) B 12.6 (4.5) 0.94 0.18 0.18 Monarto-high density (0.03) A (0.03) BC (0.05) B 0.93 0.11 0.08 Bakara Conservation Park 31.3 (3.0) (0.06) A (0.03) A (0.01) C 0.96 0.08 0.10 Lowan Conservation Park 51.1 (3.4) (0.07) A (0.04) A (0.03) C

Pg 62 Chapter 3: Reproductive assurance shifts in mahogany M. F. Breed

Chapter 3: Reproductive assurance shifts in mahogany

Statement of Authorship

Breed M.F., Gardner M.G., Ottewell K.M., Navarro C., Lowe A.J. (2012).

Shifts in reproductive assurance strategies and inbreeding costs associated with habitat fragmentation in Central American mahogany.

Ecol. Lett. 15(5), 444-452.

http://onlinelibrary.wiley.com/doi/10.1111/j.1461-0248.2012.01752.x/abstract

Martin F. Breed (Candidate) Performed field, molecular and simulation analyses, wrote manuscript.

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Gardner (Co-supervisor) Generated molecular data, involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 63

Chapter 3: Reproductive assurance shifts in mahogany M. F. Breed

Carlos Navarro (Co-author) Designed the study, collected and generated field and molecular data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Andrew J. Lowe (Supervisor) Designed the study and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 64

Chapter 3: Reproductive assurance shifts in mahogany M. F. Breed

A Breed, M.F., Gardner, M.G., Ottewell, K.M., Navarro, C. & Lowe, A.J. (2012). Shifts in reproductive assurance strategies and inbreeding costs associated with habitat fragmentation in Central American mahogany. Ecology Letters, v. 15 (5), pp. 444-452.

NOTE: This publication is included on pages 65-80 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/j.1461-0248.2012.01752.x

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Chapter 3: Reproductive assurance shifts in mahogany M. F. Breed

Media uptake

Bio-medicine: http://www.bio-medicine.org/biology-news-1/Pollen-can-protect- mahogany-from-extinction-24374-1/

ECOS Magazine: http://www.ecosmagazine.com/?paper=EC12261

Environment Institute: http://environmentinstitute.wordpress.com/2012/04/04/pollen-can- protect-mahogany-from-extinction/

Eurekalert: http://www.eurekalert.org/pub_releases/2012-04/uoa-pcp040312.php

Newsecology: http://www.newsecology.com/agriculture-and-food/seeds/17577-pollen- can-protect-mahogany-from-extinction.html

Physorg: http://www.physorg.com/news/2012-04-pollen-mahogany- extinction.html

Redorbit: http://www.redorbit.com/news/science/1112507327/pollen-can-protect- mahogany-from-extinction/

University of Adelaide: http://www.adelaide.edu.au/news/print51881.html

Pg 81

Chapter 4: Pollen diversity matters M. F. Breed

Chapter 4: Pollen diversity matters

Statement of Authorship

Breed M.F., Marklund M.H.K., Ottewell K.M., Gardner M.G., Harris

J.C.B., Lowe A.J. (2012). Pollen diversity matters: revealing the neglected effect of pollen diversity on fitness in fragmented landscapes.

Mol. Ecol. 21(24), 5955-5968.

http://onlinelibrary.wiley.com/doi/10.1111/mec.12056/abstract

Martin F. Breed (Candidate) Designed the study, generated field and molecular data, analysed data and wrote manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Maria H. K. Marklund (Co-author) Designed the study, generated field and molecular data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 82

Chapter 4: Pollen diversity matters M. F. Breed

Michael G. Gardner (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

J. Berton H. Harris (Co-author) Generated field data, analysed data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Andrew J. Lowe (Supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 83

Chapter 4: Pollen diversity matters M. F. Breed

A Breed, M.F., Marklund, M.H.K., Ottewell, K.M., Gardner, M.G., Harris, J.C.B. & Lowe, A.J. (2012). Pollen diversity matters: revealing the neglected effect of pollen diversity on fitness in fragmented landscapes. Molecular Ecology, v. 21 (24), pp. 5955-5968.

NOTE: This publication is included on pages 84-108 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/mec.12056

Pg 84

Chapter 5: Eucalypt resistance to fragmentation M. F. Breed

Chapter 5: Eucalypt resistance to fragmentation

Statement of Authorship

Breed M.F., Ottewell K.M., Gardner M.G., Marklund M.H.K., Stead M.G.,

Harris J.C.B., Lowe A.J. (in press). Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt.

Heredity. DOI: 10.1038/hdy.2012.72

http://www.nature.com/hdy/journal/vaop/ncurrent/full/hdy201272a.html

Martin F. Breed (Candidate) Designed the study, generated field and molecular data, analysed data and wrote manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Contributed to study design and to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Gardner (Co-supervisor) Contributed to study design and to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 109

Chapter 5: Eucalypt resistance to fragmentation M. F. Breed

Maria H. K. Marklund (Co-author) Designed the study, generated field and molecular data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Stead (Co-author) Generated field data, analysed data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

J. Berton H. Harris (Co-author) Generated field data, analysed data and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Andrew J. Lowe (Supervisor) Designed study and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

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Chapter 5: Eucalypt resistance to fragmentation M. F. Breed

A Breed, M.F., Ottewell, K.M., Gardner, M.G., Marklund, M.H.K., Stead, M.G., Harris, J.C.B. & Lowe, A.J. (in press). Mating system and early viability resistance to habitat fragmentation in a bird- pollinated eucalypt. Heredity, online article, pp. 1-8

NOTE: This publication is included on pages 111-125 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1038/hdy.2012.72

Pg 111

Chapter 6: Adaptation and scattered trees M. F. Breed

Chapter 6: Adaptation and scattered trees

Statement of Authorship

Breed M.F., Ottewell K.M., Gardner M.G., Lowe A.J. (2011). Clarifying climate change adaptation responses for scattered trees in modified landscapes. J. Appl. Ecol. 48(3), 637-641.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2664.2011.01969.x/abstract

Breed, Martin F. (Candidate) Initially developed ideas and wrote manuscript.

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Gardner (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 126

Chapter 6: Adaptation and scattered trees M. F. Breed

Andrew J. Lowe (Supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 127

Chapter 6: Adaptation and scattered trees M. F. Breed

A Breed, M.F., Ottewell, K.M., Gardner, M.G. & Lowe, A.J. (2011). Clarifying climate change adaptation responses for scattered trees in modified landscapes. Journal of Applied Ecology, v. 48 (3), pp. 637-641.

NOTE: This publication is included on pages 128-140 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/j.1365-2664.2011.01969.x

Pg 128

Chapter 7: Which provenance and where? M. F. Breed

Chapter 7: Which provenance and where?

Statement of Authorship

Breed M.F., Stead M.G., Ottewell K.M., Gardner M.G., Lowe A.J. (2013).

Which provenance and where? Seed sourcing strategies for revegetation in a changing environment. Cons. Genet. 14(1), 1-10.

http://link.springer.com/article/10.1007/s10592-012-0425-z/fulltext.html

Martin F. Breed (Candidate) Initially developed ideas and wrote manuscript.

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Michael G. Stead (Co-author) Initially developed ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Kym M. Ottewell (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

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Chapter 7: Which provenance and where? M. F. Breed

Michael G. Gardner (Co-supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Andrew J. Lowe (Supervisor) Involved with developing ideas and contributed to revisions of the manuscript

I hereby certify that the statement of contribution is inclusion of the paper in the thesis.

Signed …………………………….. Date: 12 March 2013

Pg 142

Chapter 7: Which provenance and where? M. F. Breed

A Breed, M.F., Stead, M.G., Ottewell, K.M., Gardner, M.G. & Lowe A.J. (2013). Which provenance and where? Seed sourcing strategies for revegetation in a changing environment. Conservation Genetics, v. 14 (1), pp. 1-10.

NOTE: This publication is included on pages 143-152 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1007/s10592-012-0425-z

Pg 143

Conclusion M. F. Breed

Conclusions

This work has advanced our understanding of the impacts of habitat fragmentation on the mating patterns and fitness of animal-pollinated trees. It is now apparent that forest fragmentation generally does have negative impacts on the mating patterns of animal-pollinated trees. This appears to be not just in terms of increasing selfing rates, but also other aspects of plant mating systems such as reproduction among close relatives (biparental inbreeding) and pollen diversity. The latter two parameters have been neglected from previous studies, despite the fact that they should be good predictors of offspring fitness. Additionally, through a series of common garden experiments and mating pattern assessments across several species, data presented in this thesis demonstrate that these shifts in mating patterns impact on seedling fitness - linking predictions from forest fragmentation studies and mating system theory (Skogsmyr & Lankinen 2002; Lowe et al. 2005; Eckert et al. 2010).

Future work needs to identify what ecological traits expose species to or buffer animal-pollinated trees from these negative mating pattern and fitness effects. For example, in our meta-analysis of mating system studies in Chapter 2 we found a positive, though non-significant, association between pollinator mobility and mating system changes.

Mobile pollinators (e.g. birds, bats) potentially buffered trees from significant shifts in mating patterns with habitat fragmentation.

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Conclusion M. F. Breed

Unfortunately, when the data in the meta-analysis were separated into mobile vs. non-mobile groups, sample sizes were insufficient to detect significant trends, but the effect seems intuitive (as shown in the eucalypt case study in Chapter 2) and deserves further investigation.

Furthermore, only a crude pollinator mobility classification was possible because very little description of pollinator ecology and, in particular, dispersal distances were presented across the studies included in the meta-analysis. Future work is also required to determine the link between mating pattern-fragmentation trends with changes in the spatial arrangement of the trees themselves (e.g. density) compared to changes in the pollinator community that services the trees (e.g. pollinator declines; pollinator community shifts). This is important because research conducted on mating patterns of trees is nearly always conducted without assessments of the pollinators themselves and the opposite is also true.

It is often assumed that selfing rate changes are most important to observe in forest fragmentation studies because trees generally outcross (Ghazoul 2005; Lowe et al. 2005; Ward et al. 2005; Eckert et al. 2010), thus augmenting the accumulation of genetic load that should be exposed to strong negative selection in the presence of inbreeding

(Crnokrak & Barrett 2002; Szulkin et al. 2010). However, as shown in

Chapters 3 and 4, this is not necessarily the case. In these chapters, pollen diversity was often a better predictor of offspring fitness than outcrossing or biparental inbreeding. Future work is required to explore Pg 154

Conclusion M. F. Breed

the genetic or physiological mechanisms of how lower pollen diversity reduces progeny fitness (as discussed in detail in chapter 2). In

Chapters 3 and 4, the mechanisms causing the fitness impact were predicted to be due to less pollen competition or a weaker heterosis effect (Yasui 1998; Skogsmyr & Lankinen 2002). Future work could involve observations of pollen tube growth to directly investigate pollen competition. Additionally, future work could perform controlled crosses, candidate gene resequencing or QTL mapping to identify the gene(s) that could be influenced by a heterosis effect.

We identified numerous areas of native plant genetic resource management where this mating pattern-fragmentation information could be usefully integrated. This is particularly relevant because native plant genetic resource management policies are generally biased towards concerns relating to issues of the Small Population Paradigm (sensu

Caughley 1994; i.e. avoiding populations that have undergone strong genetic drift; Kanowski & Boshier 1997; Falk et al. 2001; Guerrant Jr. et al. 2004; Kramer & Havens 2009; Maschinski & Haskins 2012). For example, in Chapter 6, we outline why habitat corridor planning in agricultural landscapes needs to consider the benefits of connecting the remnant pasture trees with genetically diverse surrounding vegetation to improve the quality of recruits back into these cleared areas.

Furthermore, in Chapter 7 a decision-making framework is proposed on how to avoid collecting poor quality seeds resulting from habitat fragmentation. Additionally, in Chapter 7, seed sourcing strategies are Pg 155

Conclusion M. F. Breed

reviewed and their suitability to a variety of different environmental conditions, data availability and genetic risk management situations is discussed. Thus, policy refinement is clearly required to incorporate the potential changes in mating patterns, and not genetic drift, as the main genetic concerns in native tree genetic resource management.

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Tree Restoration Genetics M. F. Breed

Complete list of publications

This list of publications comprises those that were either published as a result of work conducted in this thesis or were published as a result of work conducted alongside the research presented in this thesis.

• McCallum K.P., Guerin G.R., Breed M.F., Lowe A.J. (accepted

January 2013). Combining population genetics, species

distribution modelling and field assessments to understand a

species’ vulnerability to climate change. Aust. Ecol.

• Breed M.F., Ottewell K.M., Gardner M.G., Marklund M.H.K.,

Stead M.G. Harris J.B.C. & Lowe A.J. (in press) Mating system

and early viability resistance to habitat fragmentation of a bird-

pollinated eucalypt. Heredity. DOI: 10.1038/hdy.2012.72.

• Breed M.F., Stead M.G., Ottewell K.M., Gardner M.G., Lowe A.J.

(2013). Which provenance and where? Seed sourcing strategies

for revegetation in a changing environment. Conservation

Genetics. 14(1), 1-10.

• Rudh A., Breed M.F., Qvarnström A. (2013). Does aggression

and explorative behaviour decrease with lost warning

colouration? Biol. J. Linnean Soc. 108(1), 116-126.

• Breed M.F., Marklund M.H.K., Ottewell K.M., Gardner M.G.,

Harris J.C.B., Lowe A.J. (2012). Pollen diversity matters:

revealing the neglected effect of pollen diversity on fitness in

fragmented landscapes. Mol. Ecol. 21(24), 5955-5968.

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Tree Restoration Genetics M. F. Breed

• Murphy N.P., Breed M.F., Guzik M., Cooper S., Austin A. (2012)

Trapped in desert springs: phylogeography of Australian desert

spring snails. J. Biogeog. 39(9), 1573-1582.

• Breed M.F., Gardner M.G., Ottewell K.M., Navarro C. & Lowe

A.J. (2012). Shifts in reproductive assurance strategies and

inbreeding costs associated with habitat fragmentation in Central

American mahogany. Ecol. Lett. 15(5), 444-452.

• Breed A.C., Breed M.F., Meers J., Field H.E. (2011). Evidence of

endemic Hendra virus infection in flying-foxes (Pteropus

conspicillatus) - implications for disease risk management. PLoS

One. 6, e28816.

• Weeks A.R., Sgro C.M., Young A.G., Frankham R., Mitchell N.J.,

Miller K.A., Byrne M., Coates D.J., Eldridge M.D.B., Sunnucks P.,

Breed M.F., James E.A. & Hoffmann A.A. (2011). Assessing the

benefits and risks of translocations in changing environments: a

genetic perspective. Evol. Appl. 4(6), 709-725.

• Breed M.F., Ottewell K.M., Gardner M.G., Lowe A.J. (2011).

Clarifying climate change adaptation responses for scattered

trees in modified landscapes. J. Appl. Ecol. 48(3), 637-641.

Pg 158

Tree Restoration Genetics M. F. Breed

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