Trollius Europaeus in a Fragmented Landscape Reproductive Success, Genetic Diversity and Trait Differentiation in a Nursery Pollinated Plant

Research Collection

Doctoral Thesis

Trollius Europaeus in a fragmented landscape reproductive success, genetic diversity and trait differentiation in a nursery pollinated plant

Author(s): Klank, Charlotte

Publication Date: 2011

Permanent Link: https://doi.org/10.3929/ethz-a-007195830

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ETH Library o Diss. ETH N 20049

TROLLIUS EUROPAEUS IN A FRAGMENTED LANDSCAPE: REPRODUCTIVE SUCCESS, GENETIC

DIVERSITY AND TRAIT DIFFERENTIATION IN A NURSERY POLLINATED PLANT

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by

Charlotte Klank

Dipl. Biol., University of Goettingen

born 03.09.1979

citizen of Germany

accepted on the recommendation of Prof. Jaboury Ghazoul Prof. William E. Kunin Prof. Alex Widmer Dr. Andrea R. Pluess

2011

CONTENTS

SUMMARY

English 1 German 2

CHAPTER 1: Introduction

Introduction 3 Trollius europaeus and Chiastocheta 7 References 9

CHAPTER 2: Effects of population size on plant reproduction and pollinator abundance in a specialized pollination system.

Summary 14 Introduction 15 Material & Methods 17 Results 23 Discussion 28 References 31

CHAPTER 3: Genetic diversity and plant performance in fragmented populations of globeflowers (Trollius europaeus) within agricultural landscapes

Summary 36 Introduction 37 Material & Methods 40 Results 46 Discussion 50 References 53 Appendix 58 CONTENTS

CHAPTER 4: Conservation implications based on neutral and quantitative genetic differentiation in Swiss Trollius europaeus populations

Summary 62 Introduction 63 Material & Methods 65 Results 68 Discussion 71 References 74 Appendix 77

CHAPTER 5: Conclusion

Conclusions 84 References 87

ACKNOWLEDGEMENTS 89

CURRICULUM VITAE 91

SUMMARY

Summary Many plant species are currently experiencing negative consequences of habitat fragmentation as a result of reductions in population size and disruptions in pollination services. Plants in specialized pollination systems might be especially vulnerable to changes in plant population size and density resulting from land-use changes. Representing such a system, we chose the globeflower Trollius europaeus L. with its pollinating fly Chiastocheta, which also acts as a seed predator at its larval stage as our study system. We performed an assessment of reproductive success and pollinator abundance, neutral genetic diversity survey and a greenhouse experiment to determine the competitive ability and quantitative genetic variation using 19 globeflower populations located in the Swiss Plateau to investigate the effects of habitat fragmentation on reproductive success, genetic diversity and trait differentiation. Our results show that population size negatively influenced reproductive success, while it plant population densities had an increasing effect. Chiastocheta abundance within T. europaeus flowers was independent of plant population size, though Chiastocheta numbers within flowers were inversely correlated to local T. europaeus flower density.

Regarding neutral genetic diversity, the overall genetic differentiation was low (FST = 0.033) with a marginal significant isolation by distance (P = 0.060), and no significant effect for population size, population density or pollinator abundance on neutral genetic diversity was found. Regarding the greenhouse experiment to assess the competitive ability and quantitative genetic variation, no negative effects of habitat fragmentation were found. Overall, Chiastocheta abundance was the main driver of reproductive output of T. europaeus through its dual role as an obligate pollinator and seed predator: at least some Chiastocheta flies were needed to secure pollination, but a continued increase in Chiastocheta flies within a flower incurred seed predation costs that greatly reduced reproductive success. Thus high local flower density contributed positively to per capita reproductive output by diluting Chiastocheta abundance within flowers independently of overall population size. Populations of T. europaeus existing within the highly modified landscape of the Swiss Plateau are currently able to persist and reproduce and show no signs of negative habitat fragmentation effects. Our study highlights the importance to carefully assess what determines the quality of population persistence – in our case rather plant density than pure plant number – and shows that a good understanding of the pollination system and population dynamics is needed to make sound management decision.

ZUSAMMENFASSUNG

Zusammenfassung Viele Pflanzenarten sind von den Auswirkungen der Habitatfragmentation betroffen, da diese häufig entweder die Populationsgrösse reduzieren oder die Bestäubungsmechanismen gestört werden. Pflanzen mit einem besonders spezialisierten Bestäubungssystem werden im Allgemeinen als besonders anfällig für solche Störungen erachtet. Die Trollblume Trollius europaeus ist durch solch ein System charakterisiert: Die Fliegen der Gattung Chiastocheta sind die alleinigen Bestäuber, gleichzeitig ernähren sich ihre Larven auch von den Samen der Trollblume. In dieser Studie untersuchten wir die Auswirkungen der Landschaftszersiedelung anhand einer Feldstudie zur Bestimmungen der pflanzlichen Reproduktion und der Anzahl der Bestäuber, einer Studie zur Bestimmung der neutralen genetischen Diversität und Gewächshausexperimenten zur Konkurrenzfähigkeit und quantitativen genetischen Differenzierung in 19 T. europaeus Populationen im Schweizer Mitteland. Unsere Ergebnisse zeigen dass die Samenproduktion negativ durch die Populationsgrösse beeinflusst wurde, während die Pflanzendichte einen positiven Effekt hatte. Die Anzahl der Chiastocheta war unabhängig von der Populationsgrösse, allerdings kleinräumig positiv mit der Pflanzendichte korreliert. Die neutrale genetische Differenzierung war niedrig (FST = 0.033), und zeigte auch keine signifikante isolation-by-distance (P = 0.060). Weiter waren Populationsgrösse, Pflanzendichte und Bestäuberanzahl nicht mit der genetischen Diversität korreliert. Die Gewächshausexperimente zur Konkurrenzfähigkeit und quantitativen genetischen Differenzierung zeigten ebenfalls keine signifikanten Effekte, die auf negative Auswirkungen der Habitatfragmentation schliessen lassen. Insgesamt zeigen unsere Ergebnisse dass Chiastocheta der Hauptfaktor für den Fortpflanzungserfolg der Trollblume aufgrund ihrer doppelten Rolle als Bestäuber und Räuber ist. Wenigstens einige Fliegen sind nötig um eine ausreichende Bestäubung zu sichern, aber ein kontinuierlicher Anstieg innerhalb der Blüten hat einen erhöhten Verlust an Samen durch die Larven zur Folge. Daher haben höhere Pflanzendichten einen positiven Effekt auf die per capita Samenproduktion, da sie die Chiastocheta Dichte unabhängig von der gesamten Populationsgrösse verringern.Populationen der Trollblume können derzeit gut in der bereits stark veränderten Landschaft existieren und zeigen bisher keine negativen Auswirkungen der Landschaftszerteilung. Unsere Studie zeigt die Bedeutung einer genauen Betrachtung der Faktoren, die den Fortpflanzungserfolg einer Art bestimmen. Diese sind nicht immer nur die allgemeine Populationsgrösse, sondern können auch andere sein, wie zum Beispiel in diesem Fall Pflanzendichte

CHAPTER 1 - INTRODUCTION

INTRODUCTION

CHAPTER 1 - INTRODUCTION

Biodiversity in a changing world

For many centuries, human activities have been changing ecosystems and landscapes, and today about one half of the entire global land surface can be considered to be altered by anthropogenic causes (Vitousek et al., 1997, Saunders et al., 1991, Ehrlich and Wilson, 1991). Besides climate change and invasive species, habitat fragmentation caused by human modification of natural landscapes is the main threat to biodiversity and terrestrial ecosystems worldwide (Sala et al., 2000, Fahrig, 2003, Reed, 2004). While biodiversity can be seen as a value in itself, it also has considerable economic value through various services. Indeed, current biodiversity declines have been estimated to be causing economic losses of up to €50 billion each year (Braat et al., 2008). While most of these losses occur in tropical rainforest regions, developed countries are also strongly affected by habitat fragmentation and landscape alteration, often caused by expanding settlement areas, urban sprawl and intensive agriculture (Antrop, 1998, Tilman et al., 2001). In consequence, species extinction rates are now considerably higher than before such widespread landscape and ecosystem alteration (Ehrlich and Wilson, 1991, Thompson and Jones, 1999, Saunders and Briggs, 2002, Hanski and Ovaskainen, 2002, Loreau et al., 2001). Specifically, many regional and local plant extinctions have been caused by direct habitat loss or altered ecosystem processes within and across small and recently fragmented populations (Drayton and Primack, 1996, Fischer and Stöcklin, 1997, Thompson and Jones, 1999, Stehlik et al., 2007). In Switzerland, where urbanization is expanding rapidly (Schulz and Dosch, 2005), wet habitats have undergone especially drastic changes during the last century. Up to 90% of the former wet meadow areas have been lost due to drainage, deterioration and fertilization (Broggi and Schlegel, 1989, Bowman et al., 2008), with many localised plant extinctions (Stehlik et al., 2007, Lienert et al., 2002a, Lienert et al., 2002b, Lienert and Fischer, 2003, Jules, 1998, Hooftman et al., 2003). Habitat fragmentation has three main consequences for plant populations: (1) the direct loss of suitable habitat, changes in population structure and/or size, (2) increasing spatial isolation between remnant populations, causing negative Allee effects (Stephens et al., 1999) which (3) potentially limits a species’ ability to adapt to new environments (Sultan, 2000, Schlichting, 1986).

CHAPTER 1 - INTRODUCTION

Effects of habitat fragmentation on plant fitness and pollination

Plants restricted to small populations are susceptible to changes in fecundity, mortality and recruitment rates, all of which limit reproductive success, plant performance and population viability (Bowman et al., 2008, Fischer and Matthies, 1998, Ghazoul, 2005, Leimu et al., 2006, Schleuning et al., 2009). A cause for such negative effects is the disruption of plant – pollinator interactions, which have been widely documented (Aizen and Feinsinger, 2003, Harris and Johnson, 2004, Ghazoul, 2005, Aguilar et al., 2006) and which arise from changes in the pollinator assemblage or pollinator behaviour (Steffan-Dewenter and Tscharntke, 1999, Steffan-Dewenter and Westphal, 2008, Harris and Johnson, 2004, Gonzalez-Varo et al., 2009, Jakobsson et al., 2009). Both might alter the quantity and quality of pollination events, thereby decreasing plant fitness (Goverde et al., 2002, Peterson et al., 2008). These effects can have considerable importance for plants involved in highly specialised and obligate pollination interactions (Bond, 1994, Ghazoul, 2005, Johnson and Steiner, 2000, Potts et al., 2010), as any impact to the pollinator would directly affect the plant and vice versa.

Population genetics in fragmented landscapes

A further factor limiting population viability and increasing extinction risks in fragmented populations is the loss of genetic variation (see e.g. Ellstrand and Elam, 1993, Young et al., 1996, Lynch et al., 1995, Leimu et al., 2006). Reductions in migration rates and a subsequently limited gene pool are common consequences, increasing inbreeding rates and homozygosity within a population (Conner and Hartl, 2004). Genetic drift is further enhanced in small populations, reducing genetic variation within populations by a loss of heterozygosity and the fixation of alleles (Ellstrand and Elam, 1993). As a result, differentiation between population increases, and variation within these populations decreases. Genetic bottlenecks and founder events further reduce the available pool of alleles in a population, and plants in recently fragmented populations might have lost their ability to respond to new environments (Sultan, 2000), if e.g. fixed alleles affect plasticity through deleterious mutations (Lynch et al., 1995). Such detrimental effects on plant fitness have been documented frequently (e.g. Leimu et al., 2006, Ellstrand and Elam, 1993, Keller and Waller, 2002), and are often caused by the loss of heterozygote advantages and the expression and accumulation of deleterious mutations. Genetic erosion resulting from 5

CHAPTER 1 - INTRODUCTION population fragmentation can therefore limit a species’ ability to adapt to new environments, as a species potential to cope with changed conditions depends also on the amount of genetic variation underlying adaptive traits (Podolsky, 2001, Booy et al., 2000, Young et al., 1996).

Motivation

Ecological and genetic processes often interact, and so it is important to explore how these processes in combination affect fragmented natural populations. Our expectation is that plant species that obligately rely on specialist pollinators would be particularly susceptible to such changes as a decline in the plant is expected to be paralleled by a decline in the pollinator and vice-versa. We therefore focussed on the globeflower (Trollius europaeus) and its pollinating Chiastocheta flies (Diptera: Anthomyiidae). Trollius europaeus occurs mainly in moist habitats, which have undergone substantial reductions throughout Europe, and has a nursery pollination system in which the development of the Chiastocheta larval occurs within the globeflower carpels. Although such specialised systems are relatively rare (Waser et al., 1996), they may nevertheless provide valuable insight and early warnings of wider changes at the community level. To evaluate how habitat fragmentation affects we investigated how Trollius europaeus population size affects Chiastocheta abundance, reproductive success, plant performance and genetic diversity in 19 populations located on nature reserves in northeastern Switzerland and conducted the following sets of experiments: • Field studies to quantify reproductive success, seed predation and pollinator abundance were carried out. • Using a neutral molecular approach (AFLP: Amplified fragment length

polymorphisms), we calculated genetic diversity and population differentiation (FST) from leaf material collected in all study populations. • A greenhouse study was carried out to determine plant fitness measured as the

absolute severity of competition and to calculate QST to determine whether genetic drift or natural selection were shaping population differentiation.

CHAPTER 1 - INTRODUCTION

Trollius europaeus and Chiastocheta: A nursery pollination system

One of the few known obligate pollination mutualisms is the interaction between the globeflower Trollius europaeus and its pollinators which comprise several species of Chiastocheta flies (Diptera: Anthomyiidae). The hermaphroditic, self-incompatible perennial T. europaeus occurs mainly on moist meadows across northern and mid-Europe (Lauber and Wagner, 2001). Flowering usually commences simultaneously within a population, and flowers have a mean lifespan of seven days and are 1.5 to 5.0 cm in diameter, with ~12 ovules per carpel and 6-15 petals and 10-15 nectariferous staminoida (Jaeger and Després, 1998, Jaeger et al., 2001, Pellmyr, 1989). Trollius europaeus is solely pollinated by Chiastocheta flies as the tightly closed, globose flower excludes access to almost all other pollinators, while Chiastocheta flies are able to enter by moving between the sepals. The flies use the globe shaped flowers to forage, mate, shelter and oviposit, and in the course of doing so they pollinate the flowers. The developing larvae feed on the ripening seeds during fruit maturation (three to four weeks), before falling onto the ground to pupate and overwinter to emerge the following spring (Pellmyr, 1992, Jaeger and Després, 1998, Pellmyr, 1989). References to this association date back to 1895 (Mik, 1895) and further descriptions of the system were given in the 1950s (Hennig, 1953, Collin, 1954), but the full extent of the mutualistic association was not described in detail until the late 1980s (Pellmyr, 1989). Since then several studies have described the mutualism and the involved mechanisms in detail. In northern Europe six Chiastocheta species (C. rotundiventris, C. trollii, C. inermella, C. macropyga, C. setifera and C. dentifera) are associated with T. europaeus (Jaeger and Després, 1998, Pellmyr, 1989), which seem to have diversified on the host plant through sympatric speciation during the Pleistocene (Després and Jaeger, 1999, Després et al., 2002, Ferdy et al., 2002, Després and Cherif, 2004). Species vary from mutualistic to antagonistic in their interaction, and differences stem from either varying temporal (different oviposition strategies) or spatial patterns (larval mining patterns of seeds) (Pompanon et al., 2006, Pellmyr, 1989). The most antagonistic species, C. dentifera, has the latest oviposition and hardly contributes to pollination, as most flowers are already saturated with pollen when visited. In addition to its low pollination service, C. dentifera lays clumps of eggs at a late flowering stage, exerting a higher predation pressure on the T. europaeus fruits than other Chiastocheta species. In contrast, the mutualistic species C. rotundiventris shows an early oviposition during flowering and a small number of eggs is laid (usually one); thus females 7

CHAPTER 1 - INTRODUCTION have to visit many unpollinated or only moderately pollinated flowers before laying all eggs. The remaining four species oviposit sequentially throughout the flowering phase (Pompanon et al., 2006, Després and Jaeger, 1999, Pellmyr, 1989, Johannesen and Loeschcke, 1996). Due to the underlying conflict-of-interest in such a nursery pollination system, the amount of seeds consumed by Chiastocheta larvae in relation to the pollination service provided is of crucial importance for the survival of the mutualism (Després and Jaeger, 1999). It is thought that larvae do not consume all seeds in a fruit, even though they can move freely between carpels (Pellmyr, 1989). The percentage of seeds lost to predation was found to be limited, ranging between 10% to 93% (Jaeger et al., 2001, Després and Cherif, 2004, Hemborg and Després, 1999, Jaeger et al., 2000), which might be due to larval competition. Jaeger et al. (2001) showed that seed consumption decreased with increasing larval presence, presumably due to larval interference. This was further supported by Despres et al. (2007), showing that density-dependent larval competition was responsible for the stability of the mutualism across its range and diverse ecological and geographical conditions. Pollination services by male flies are another factor balancing the detrimental effects of seed predation in T. europaeus. Even though males have a smaller body size and thus lower pollen loads than females, their higher visitation rates compensates this difference (Després, 2003). Pollination by males is especially important given that there is no direct cost imposed by larval seed consumption. As larvae can consume up to 76 developing seeds, while females only pollinate around 37 seeds (Jaeger et al., 2001), male pollination appears to contribute substantially towards balancing antagonistic effects (Després and Cherif, 2004). Sole pollination by females could lead to substantial seed consumption by the developing larvae, possibly leading to the starvation of larvae and collapsing pollinator populations (Després, 2003, Després and Cherif, 2004, Pompanon et al., 2006).

CHAPTER 1 - INTRODUCTION

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CHAPTER 1 - INTRODUCTION

ecosystem boundaries? An Australian example. Landscape and Urban Planning, 61, 71-82. Saunders, D.A., Hobbs, R.J. & Margules, C.R. (1991) Biological Consequences of Ecosystem Fragmentation - a Review. Conservation Biology, 5, 18-32. Schleuning, M., Niggemann, M., Becker, U. & Matthies, D. (2009) Negative effects of habitat degradation and fragmentation on the declining grassland plant Trifolium montanum. Basic and Applied Ecology, 10, 61-69. Schlichting, C.D. (1986) The evolution of phenotypic plasticity in plants. Annual Review of Ecology and Systematics, 17, 667-693. Schulz, B. & Dosch, F. (2005) Trends der Siedlungsflächenentwicklung und ihre Steuerung in der Schweiz und Deutschland. DISP, 160, 5-15. Steffan-Dewenter, I. & Tscharntke, T. (1999) Effects of habitat isolation on pollinator communities and seed set. Oecologia, 121, 432-440. Steffan-Dewenter, I. & Westphal, C. (2008) The interplay of pollinator diversity, pollination services and landscape change. Journal of Applied Ecology, 45, 737-741. Stehlik, I., Caspersen, J.P., Wirth, L. & Holderegger, R. (2007) Floral free fall in the Swiss lowlands: environmental determinants of local plant extinction in a peri-urban landscape. Journal of Ecology, 95, 734-744. Stephens, P.A., Sutherland, W.J. & Freckleton, R.P. (1999) What is the Allee effect? Oikos, 87, 185-190. Sultan, S.E. (2000) Phenotypic plasticity for plant development, function and life history. Trends in Plant Science, 5, 537-542. Thompson, K. & Jones, A. (1999) Human population density and prediction of local plant extinction in Britain. Conservation Biology, 13, 185-189. Tilman, D., Fargione, J., Wolff, B., D'Antonio, C., Dobson, A., Howarth, R., Schindler, D., Schlesinger, W.H., Simberloff, D. & Swackhamer, D. (2001) Forecasting agriculturally driven global environmental change. Science, 292, 281-284. Vitousek, P.M., Mooney, H.A., Lubchenco, J. & Melillo, J.M. (1997) Human domination of Earth's ecosystems. Science, 277, 494-499. Waser, N.M., Chittka, L., Price, M.V., Williams, N.M. & Ollerton, J. (1996) Generalization in pollination systems, and why it matters. Ecology, 77, 1043-1060. Young, A., Boyle, T. & Brown, T. (1996) The population genetic consequences of habitat fragmentation for plants. Trends in Ecology & Evolution, 11, 413-418.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

EFFECTS OF POPULATION SIZE ON PLANT REPRODUCTION

AND POLLINATOR ABUNDANCE IN A SPECIALIZED

POLLINATION SYSTEM

Charlotte Klank, Andrea R. Pluess & Jaboury Ghazoul

published in the Journal of Ecology (2010), 98, 1389–1397

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Summary

Many plant species are currently experiencing negative consequences of habitat fragmentation as a result of reductions in population size and disruptions in pollination services. Plants in specialized pollination systems might be especially vulnerable to changes in plant population size and density resulting from land-use changes. Representing such a system, we chose the globeflower Trollius europaeus L. with its pollinating fly Chiastocheta, which also acts as a seed predator at its larval stage, to investigate the effects of small plant population size on reproductive success and pollinator abundance. Reproductive output of T. europaeus declined with increasing plant population size, while Chiastocheta abundance within T. europaeus flowers was independent of plant population size. However, at the local level, Chiastocheta numbers within flowers were inversely correlated to local T. europaeus flower density. We further found that increasing floral densities increased plant reproductive success at the population level. Chiastocheta abundance was the main driver of reproductive output of T. europaeus through its dual role as an obligate pollinator and seed predator: at least some Chiastocheta flies were needed to secure pollination, but a continued increase in Chiastocheta flies within a flower incurred seed predation costs that greatly reduced reproductive success. Thus high local flower density contributed positively to per capita reproductive output by diluting Chiastocheta abundance within flowers independently of overall population size. Our findings highlight that plant population size is not always the main determinant of reproductive success for populations, but that other factors such as plant density and the specific ecology of a pollinator and its interplay with other population parameters can be more important in determining the fate of a population. Furthermore, the effects of plant population size and floral density on pollinator visitation in T. europaeus vary across scales, with implications for plant fitness. It is therefore important not to focus solely on pure plant population size in determining population viability. Thus, from a conservation perspective, even small and isolated T. europaeus populations may be viable and resistant to pollination- associated vulnerabilities depending on plant density at local (subpopulation) scales.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Introduction

During the last century human activities have greatly changed Earth’s ecosystems and landscapes. In consequence, regional and local plant extinctions have been caused by direct habitat loss and by altered ecosystem processes within and across recently fragmented, small populations (Stehlik et al., 2007, Drayton and Primack, 1996, Fischer and Stöcklin, 1997, Thompson and Jones, 1999). Mechanisms driving these changes are diverse. Habitat fragmentation directly reduces plant population size and increases population isolation, which makes plant populations more susceptible to environmental stochasticity (Kery et al., 2000, Hobbs and Yates, 2003) and disrupts ecological processes that underlie fecundity and recruitment (Fischer and Matthies, 1998, Ghazoul, 2005, Leimu et al., 2006, Bowman et al., 2008, Schleuning et al., 2009). Specifically, negative effects of habitat fragmentation on plant–pollinator interactions have been widely documented (Aizen and Feinsinger, 2003, Harris and Johnson, 2004, Ghazoul, 2005, Aguilar et al., 2006). Such negative effects may arise from disruptions affecting either the pollinator assemblage or pollinator behaviour (Steffan-Dewenter and Tscharntke, 1999, Steffan-Dewenter and Westphal, 2008, Harris and Johnson, 2004, Gonzalez-Varo et al., 2009, Jakobsson et al., 2009), both of which might alter the quantity and quality of pollination events, thereby decreasing plant fitness (Goverde et al., 2002, Peterson et al., 2008). Further factors influencing pollination rates are population and sub-population plant densities. High densities might enhance pollination by attracting larger numbers of pollinators (Bernhardt et al., 2008, Hegland et al., 2009). On the other hand, high flower densities might increase competition for a limited number of pollinators, hence reduce per capita pollination rates and reproductive success (Campbell and Husband, 2007, Gunton and Kunin, 2009, Spigler and Chang, 2009). The outcome of plant–pollinator interactions with respect to plant density, population size and population isolation may vary across spatial scales depending on the scale at which particular pollinators respond to floral resources (Dauber et al., 2010, Gunton and Kunin, 2009). Plants that have highly specialized and obligate interactions with their pollinators are thought be most susceptible to habitat fragmentation effects (Johnson and Steiner, 2000, Bond, 1994, Ghazoul, 2005), as any impact on the pollinator would directly affect the plant and vice versa. Although specialized plant–pollinator systems are relatively rare (Waser et al., 1996), they may nevertheless provide insight and early warnings of wider changes at the community level. For these reasons, this study focussed on globeflower (Trollius europaeus) 15

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE and its pollinating flies Chiastocheta spp. (Diptera: Anthomyiidae), an example of a specialized pollination mutualism that could be reproductively affected by habitat fragmentation, which has negatively affected population levels of T. europaeus across much of its European range. While still common in many alpine areas of Switzerland, T. europaeus has experienced substantial reductions in the Swiss lowlands due to drainage and agricultural intensification. The tightly closed, globose flower of T. europaeus effectively excludes access to the inner flower for insects other than Chiastocheta, rendering the plant almost entirely dependent on up to six Chiastocheta species for pollination (Pellmyr, 1989, Jaeger and Després, 1998). The flies forage, mate, shelter and oviposit within the globe-shaped flower, effecting pollination in the process. Their larvae feed and develop on the ripening seeds during fruit maturation (three to four weeks) and then, after completion of larval development, fall to the ground where they pupate and overwinter to emerge the following spring (Pellmyr, 1989, Pellmyr, 1992, Jaeger and Després, 1998). This double role of Chiastocheta as the main pollinator during its adult stage and seed predator during larval development adds further complexity to the reproductive success of T. europaeus in terms of possible trade-offs between pollination services and predation costs.

We thus hypothesized that a) Chiastocheta abundance increases with T. europaeus population sizes and that b) reproductive success is positively related to T. europaeus population size. Specifically, we seek to (i) determine the effects of plant population size and local plant density on Chiastocheta fly abundance in T. europaeus flowers, (ii) determine the reproductive output of T. europaeus across a range of plant population sizes, and (iii) to investigate the reproductive success of T. europaeus in the absence of Chiastocheta flies.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Material and Methods

Study Species Trollius europaeus L. (Ranunculaceae) is a perennial, arctic-alpine, hermaphroditic plant, distributed throughout Europe, Asia and North America, occurring in moist habitats throughout northern and central Europe (Lauber and Wagner, 2001). It usually has one yellow flower per stem (Doroszewska, 1974), with sometimes two or more flowers branching off a stem. Flowering usually commences simultaneously within a population (Pellmyr, 1989, Jaeger and Després, 1998, Lauber and Wagner, 2001). The self-incompatible flowers have c. 12 ovules per carpel and a mean lifespan of 7 days (Pellmyr, 1989, Jaeger and Després, 1998, Jaeger et al., 2001). All seventeen species of Chiastocheta (Anthomyiidae) flies, distributed throughout Europe and Asia (Pellmyr, 1992), are associated with flowers of the genus Trollius (Collin, 1954, Pellmyr, 1992). Pollination systems vary from facultative to obligate, depending on the Trollius species (Després et al., 2002). In Europe, six species (C. inermella, C. dentifera, C. macropyga, C. setifera, C. rotundiventris and C. trollii.) have been described, and all are associated obligately with T. europaeus (Jaeger and Després, 1998).

Data collection From 2006 to 2008, 19 T. europaeus populations were studied in eastern Switzerland. Eight populations were sampled over three consecutive years (2006, 2007, 2008), and an additional 11 populations in 2007 and 2008. Elevations ranged from 537 to 1250 m a.s.l.. Seventeen populations were situated in the greater area around Lake Zurich, while two populations were situated further away (Fig. 1). All but one of the populations were located in small nature protection areas, in regions which have been subject to considerable changes in land use over the last century. It is therefore very likely that these populations are a subset of populations that once occurred in the region. To determine plant population size, exhaustive counts of all T. europaeus flowers were done in the ten smallest populations at the end of the flowering season (July – August). For the nine larger populations the population size was extrapolated based on the number of flowers within 70 to 74 randomly selected 5-m radius plots recorded in 2007 and 2008 (Table 1). Numbers of flowers were converted to density as flowers m-2 for analysis. Population-level plant density as flowers m-2 was calculated by dividing population size (total number of flowers) by the population area.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Pollinator abundance To estimate the abundance of the pollinating flies in a population we recorded the number of Chiastocheta in all flowers for the ten small populations, and in at least 200 flowers for large populations. Counts were done once during the flowering season in 2006 and 2007, and twice in 2008. For later inclusion in the analysis of reproductive ability we calculated fly abundance per flower in each population for each year, using the mean across all sampled flowers. In 2007 and 2008, we also determined Chiastocheta abundance in relation to local T. europaeus density by scoring Chiastocheta numbers in a focal plant together with the number of T. europaeus flowers within a 5-m radius of that plant. In the smallest population, which covered an area of 80 m2, we were only able to sample six focal flowers in this way, while avoiding overlapping 5-m radius circles. At other populations we used a larger focal flower sample size of up to a maximum of 51 flowers per population.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Fig. 1: Location of the 19 Trollius europaeus populations in Switzerland. The grey areas are lakes; the largest lake in the centre is Lake Zurich. Reproduced with the permission of swisstopo (JA100120).

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

) -2 10 10 1.2 1.2 1.4 6.9 0.15 0.15 0.32 0.18 1.24 0.11 1.72 5.24 2.34 2.73 2.11 3.98 2.77 1.79 4.93 14.44 density (flowers m 140 230 230 310 800 840 1120 1140 1920 3930 4260 12110 24350 27120 43360 57780 86460 238040 820700 total no. flowers total no.

100 100 100 100 100 100 100 100 100 100 100 49.5 49.5 6.81 6.74 3.87 19.08 32.28 14.04 12.72 % of population% of sampled ] 2 52 52 935 718 250 651 950 750 1291 7567 1374 1822 2198 2198 2198 2198 2198 2229 2324 2198 populations sampled in this study Area sampled [m sampled Area

1 . 2 70 70 70 70 70 70 71 74 70 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. Trollius europaeus no. of no. sampling areas 80 80 940 720 250 650 950 750 1290 7570 1370 1820 4440 6810 11520 15650 32260 17520 34500 56840 Each sampling area is 31.42 m 31.42 is area Each sampling 1 population area Characteristics of the 19 Characteristics 740 678 745 712 612 656 612 764 732 639 771 852 703 800 597 537 752 1050 1250 elevation Table 1: F02 F15 F11 F13 F24 F01 F30 F07 F23 F29 F34 F41 B11 B15 B03 Kafer Kafer Kriens Kriens F25_27 Arvenbuel Arvenbuel population

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Reproductive success and seed predation in natural populations Reproductive success and seed predation were quantified by counting the number of carpels, intact seeds and seeds with signs of predation in developed seed heads. For each population 35 seed heads were bagged after flowering with fine mesh during each season and collected after seed maturation. As a measure of reproductive success and seed predation the proportion of seeds (intact or predated) was calculated by dividing the number of seeds by the maximum number of seeds that could have been produced by one flower. To obtain the maximum number of seeds, we multiplied the number of carpels per seed head by the mean number of ovules per carpel. We justify this on account of the highly variable number of carpels across globeflowers (range 4-78 from 1575 flower heads) which contrasts with the marked consistency of ovules per carpel across flowers and populations (mean of 11.5 ± 1.5, range 5-18, based on a sample of five carpels per seed head from 10 individuals each drawn from five populations; no significant differences between populations). These values are almost identical to those obtained by Jaeger & Després (1998) and Després et al. (2007): range of 10-69 carpels sampled across 1710 flower heads, with mean 11.6 ± 1.6 ovules per carpel.

Reproductive success and seed predation in the absence of Chiastocheta To account for possible pollination of T. europaeus by other insects in the absence of Chiastocheta, we placed groups of around 20 T. europaeus plants bought from nurseries in locations with no naturally occurring populations in the vicinity that might act as Chiastocheta sources. In 2007 plants were placed at six locations, in 2008 at two locations. To ensure that no Chiastocheta flies were present, plants were checked regularly during the flowering season. Seed heads were bagged and numbers of carpels and seeds were counted as in the natural populations. To verify self incompatibility of T. europaeus, plants reared from seeds collected from natural populations were kept in a greenhouse. A total of 89 plants were used to test for selfing. Developing flower buds were bagged with fine mesh to prevent insect access to flowers. Of the 89 plants, 28 were tested for apomixis by removing the developing anthers, 30 flowers received no treatment, and 31 flowers were self-pollinated by hand.

Statistical Data Analysis All statistical analyses were carried out in R, version 2.10.1 (RDevelopmentCoreTeam, 2009). All data sets were analysed with generalized linear mixed-effects models (GLMM) to

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE allow for random effects caused by the repetition of years and population, fitting them as populations nested in years in the random-effect term, following the advice in Bolker et al. (2008) and O’Hara (2009).We applied the lmer function from the lme4 package (Bates, 2005); fixed effects showing strong outliers were log-transformed before fitting the model and those showing a pairwise correlation coefficient above 0.8 were centred (Zuur et al., 2009). Data on pollinator abundance was analysed with a Poisson error family. On the population level fixed explanatory variables were plant population size (log-transformed) and population-level plant density. Data on fly abundance in relation to plant densities in a 5- m radius were analysed separately, with plant density at the 5-m scale being log- transformed. Data sets on reproductive success and seed predation were analysed using a binomial error family to compare proportions. The fixed explanatory variables were plant population size (log-transformed), population-level plant density, and fly abundance. We also included two interaction terms in the model, average fly abundance : plant population density and average fly abundance : plant population size. Using the Akaike Information Criterion (AIC), model reduction and selection was tested based on significant differences (χ2 test) between models, selecting the model with the lowest AIC. To analyse whether Chiastocheta-free flowers produced significantly different proportions of intact or predated seed than plants in natural populations, a binomial test to compare proportions was carried out (Crawley, 2007).

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Results

The 19 populations measured in this study varied greatly in plant population size (Table 1). The smallest population produced a total of 140 flowers during the recorded flowering season, the largest population had an estimated 820 690 flowers. Average plant density for each population ranged from 0.11 to 14.44 plants m-2 (Table 1).

Pollinator abundance Estimates of the abundance of Chiastocheta spp. showed that flies were present in all years and populations except 2006, when no Chiastocheta flies were recorded in one population (F30). Fly numbers differed between years (Kruskal–Wallis test: χ2 = 570, d.f. = 2, P <0.001) and populations (Kruskal–Wallis test: χ2 = 489, d.f. = 18, P <0.001). In 2006, no Chiastocheta were found in 92.5% of the 2042 flowers sampled, with a maximum of four flies found in the rest; in 2007, from a total of 3684 flowers 75.6% lacked Chiastocheta, with a maximum of six flies per flower; and in 2008, 89.6% of 11 141 flowers lacked Chiastocheta, with a maximum of 8 flies per flower. Neither plant population size nor population-level plant density had a significant effect on the number of Chiastocheta found per flower (Table 2a). Chiastocheta abundance in relation to the 5-m scale T. europaeus density (Fig. 2) decreased with increasing flower density around the focal plant (p = 0.0003, Table 2b), which varied between 0 and 17.88 flowers m-2.

8 7 6 5

per focal flower 4 3 2 1 Chiastocheta 0

0 0.5 1 1.5 2 mean local flower density [flowers m-2 ]

Fig. 2: Mean local flower density versus number of Chiastocheta found in a focal flower, averaged for each level of Chiastocheta for each year (2007: filled circle, 2008: crossed circle). Error bars represent the standard error of the local density. Sample size exceed 50 except for fly presence of 3 (n=17), 4 (n=4), 5 (n=3), 6 (n=0), 7 (n=2) and 8 (n=1).

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Table 2: Chiastocheta abundance per flower in relation to a) population size and density of Trollius europaeus on the population level and b) the number of T. europaeus in a 5-m radius around the focal flower. Both data sets were analysed using a generalized linear mixed-effects model with Poisson error distribution and year and population fitted as random effects. a) large scale effects (n = 16867) random effects SD year 0.6202 population:year 0.6966 fixed effects estimate SE Z - value P - value intercept -2.1949 0.5627 -3.901 <0.001 log(plant population size) 0.0395 0.0553 0.714 0.48 plant population density -0.0600 0.0400 -1.498 0.13 b) small scale effect (n = 998) random effects SD year 0.4442 population:year 0.4701 fixed effects estimate SE Z- value P - value intercept -1.0144 0.3294 -3.080 0.0021 log(local density + 0.1) -0.1991 0.0543 -3.666 0.0003

Reproductive success The median proportion of intact seeds over all years was 0.369 (inter quantile range (IQR) 0.288) and varied significantly between years (Kruskal–Wallis test: χ2 = 99.5, d.f. = 2, P <0.001) and populations (Kruskal–Wallis test: χ2 = 209.7, d.f. = 18, P <0.001). Median values were 0.424 in 2006 (IQR 0.281), 0.304 in 2007 (IQR 0.284) and 0.412 in 2008 (IQR 0.276). Out of the 1575 seed heads collected, only 18 samples did not produce intact seeds. Including an interaction of fly abundance with either population-level plant density (model selection: χ2 = 2.817, d.f. = 1, P = 0.09) or plant population size (model selection: χ2 = 2.156, d.f. = 1, P = 0.14) did not significantly improve the model based on the AIC value. We thus used the simple GLMM without interactions. We found a significantly negative relationship between reproductive success and plant population size averaged over all years (P = 0.0032, Table 3), although there were differences between years (Fig. 3a) with a strong effect only in 2007. Reproductive success was also negatively associated with Chiastocheta abundance over all years (P <0.001; Table 3), with each year showing negative slopes (Fig. 3b). Population-level plant density was the only variable showing a positive correlation with reproductive success (P = 0.0025, Table3, Fig. 3c).

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Seed Predation Seed predation had a median of 0.102 (IQR 0.137) and was significantly different between years (Kruskal–Wallis test: χ2 = 72.1, d.f. = 2, P <0.001) and populations (Kruskal–Wallis test: χ2 = 416.8, d.f. = 18, P <0.001). We used a simple GLMM without interaction terms, as including interactions between fly abundance and population-level plant density (model selection: χ2 = 2.8665, d.f. = 1, P = 0.09) or plant population size (model selection: χ2 = 0.7783, d.f. = 1, P = 0.38) did not significantly improve the model. Chiastocheta abundance was positively correlated with seed predation (p <0.001, Table 4) in all years (Fig. 4a). Population-level plant density showed a marginally significant decreasing effect (P = 0.052; Table 4, Fig. 4b) on seed predation.

Table 3: Effects of plant population size, fly abundance and population-level plant density on reproductive success of Trollius europaeus derived from a binomial generalized linear mixed-effects model with year and population fitted as random effects. random effects SD year 0.0000 population:year 0.3312 fixed effects estimate SE Z - value P - value intercept -0.3634 0.0946 -3.840 <0.001 log(plant population size) 1 -0.0744 0.0253 -2.944 0.0032 fly abundance -1.5144 0.2334 -6.490 <0.001 plant population density 0.0554 0.0183 3.022 0.0025 1log(population size) was centred.

Table 4: Effects of plant population size, fly abundance and population-level plant density on seed predation of Trollius europaeus derived from a binomial generalized linear mixed-effects model with year and population fitted as random effects. random effects SD year 0.0000 population:year 0.5060 fixed effects estimate SE Z - value P - value intercept -2.2457 0.1446 -15.956 <0.001 log(plant population size) 1 0.0673 0.0386 1.739 0.082 fly abundance 1.4724 0.3561 4.133 <0.001 plant population density -0.0546 0.0280 -1.946 0.052 1log(population size) was centred.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Reproductive success and seed predation in the absence of Chiastocheta Plants placed in artificial groups at a total of eight Chiastocheta-free locations in 2007 and 2008 produced a total of 278 flowers, of which 46 (16.5%) produced no seeds. The proportion of intact seeds (2.6%) was significantly lower than in the natural populations in this study (χ2 = 19488.45, d.f. = 1, P < 0.001). Predated seeds were only found in 8 of the 278 samples (2.9%), which was also much lower than in natural populations (χ2 = 10293.96, d.f. = 1, P < 0.001). No seeds were produced by apomixis. Seeds were produced in 10.9% of artificially selfed flowers and in 4.2% of flowers that received no treatment (i.e. self- pollination in the absence of a pollen vector). a) b) 0.6

0.5

0.4

0.3

reproductive succuess 0.2

0.1

100 1000 10000 100000 800000 0 0.2 0.4 0.6 0.8 1.0 1.2 population size [total no. of flowers] average no. of Chiastocheta per flower

c) 0.6

0.5

0.4

0.3

reproductive success 0.2

0.1

0246810 12 14 16 -2 population density [flower m ]

Fig. 3: Reproductive success (proportion of intact seeds, mean per population year-1) as a function of a) Trollius europaeus population size, b) Chiastocheta abundance and c) plant population density (2006: open circle and dotted line, 2007: filled circle and solid line, 2008: crossed circle and dashed line). Error bars represent the standard error of the mean.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

a) b) seed predation 0 0.1 0.2 0.3

0 0.2 0.4 0.6 0.8 1.0 1.2 082 4 6 10 12 14 16

average no. of Chiastocheta per flower population density [flower m -2 ]

Fig. 4: Seed predation (proportion of intact seeds, mean per population year-1) as a function of a) Chiastocheta abundance and b) Trollius europaeus population density (2006: open circle and dotted line, 2007: filled circle and solid line, 2008: crossed circle and dashed line). Error bars represent the standard error of the mean.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

Discussion

Trollius europaeus within small populations was expected to have reduced reproductive ability compared to large populations on account of a reduced ability to support sufficiently large numbers of the obligate Chiastocheta pollinator. Contrary to our expectation, we found no direct effects of plant population size or population-level plant density on the abundance of Chiastocheta at the population level (Table 2a). Furthermore, there was a decline in seed production with increasing plant population size, contrary to our expectation that seed production would decline in smaller plant populations. Chiastocheta-free plants in both natural and greenhouse settings did not produce substantial numbers of intact seeds, confirming the dependency of T. europaeus on Chiastocheta and the limited availability of alternative pollinators (see also Ibanez et al., 2009). We further found that reproductive success (seed set per flower) decreased when fly abundance per flower was high, on account of higher seed predation (Fig. 4a), a result that is similar to that found for figs and fig wasps, another nursery pollination system (Herre and West, 1997). As fly abundance per flower was negatively associated with 5-m scale plant density (Table 2b), a local dilution effect of flies among flowers in high-plant-density patches appears to reduce the cost to flowers in terms of predated ovules. Thus, while Chiastocheta abundance was not impacted by overall population size, plants growing in high-plant-density patches appear to benefit from the availability of sufficient flies for pollination while avoiding high costs associated with seed predation. A study of the senita cactus–senita moth reproductive mutualism (another example of a nursery pollination system similar to that in T. europaeus) revealed no consistent relationship of either fruit or seed consumption by senita moth larvae or fruit set with variation in cactus population density (Holland and Fleming, 1999). This study was, however, conducted at only five populations distributed across two widely separated regions. Further, density was scored only at the population scale and possible local density effects were not investigated. Despite the many reported instances of declining reproductive output in smaller populations (e.g. Fischer and Matthies, 1998, Kery et al., 2000, Wolf and Harrison, 2001, Johnson et al., 2004, Winter et al., 2008, Schleuning et al., 2009, and reviews by Hobbs and Yates, 2003, Ghazoul, 2005, Aguilar et al., 2006, Leimu et al., 2006), other recent studies have found no clear association between population size and reproductive output, or even declines in per capita seed set in large populations (Brys et al., 2008, Spigler and Chang, 2008, Spigler and Chang, 2009, Rabasa et al., 2009, Tsaliki and Diekmann, 2009). Intraspecific resource 28

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE competition and, alternatively, pollinator limitation, have been proposed as explanations for limited or declining seed set within large populations. In our study, pollen limitation is unlikely because we did not find a relationship between fly abundance per flower and plant population size. Trollius distributions within a population are, however, often variable and patchy and flies might respond to smaller scales which could turn individual patches into relatively distinct sub-populations. The negative effect of plant population size on reproductive success can be explained by the double role of Chiastocheta as the main pollinator and pre-dispersal seed predator. As fly abundance per flower increased, reproductive success declined due to seed predation. A similar effect has been suggested by Morris et al. (2003) as a regulatory mechanism for coexistence between mutualistic partners, whereby high pollinator densities depress plant reproduction, thus imposing a negative feedback. Trollius europaeus therefore benefited from modest Chiastocheta visitation, but as visitation continued to increase, the cost to seed predation increased more rapidly than the gains from pollination. This outcome further provides empirical support for the unimodal functional response model developed for the senita cactus–senita moth nursery pollination system where modelled cactus populations decreased when moth populations were either too low (insufficient pollination) or too high (high fruit consumption by larvae) (Holland et al., 2002). In contrast to our study, Despres et al. (2007) did not find a relationship between fly density and seed production across 38 globeflower populations. These populations were, however, sampled from different years and across wide altitudinal (400 – 2500 m a.s.l.) and latitudinal ranges (Swedish Lapland and French Alps). Substantial inter-year differences (see Figs 3a, 3b and 4b) and much variation among populations are found in our own results, despite the populations being from similar geographical and altitudinal localities. Thus differences in year, altitude and locality across populations might, possibly, confound the detection of such responses in the Despres et al. (2007) study. On the other hand, their data for higher predation at high fly densities (i.e. four or more eggs per flower head) confirmed our own results. Fly abundance per flower was itself an inverse function of the density of flowers within local patches (i.e. at the 5-m radius scale). Local flower density therefore had an important mitigating effect on seed predation as the probability of harbouring large numbers of Chiastocheta adults (and therefore presumably larvae) per flower decreased at higher flower densities. Our data do not allow us to test this directly, but a similar dilution effect (a form of predator satiation) has been proposed for dipterocarp trees in Asian rain forests, where high densities of seed increase per capita seed survival through the dilution of seed predation

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

(Ghazoul and Satake, 2009). In the Trollius–Chiastocheta system the process unfolds with respect to local small-scale flower (rather than seed) density, but the mechanism remains the same. Overall, even though T. europaeus relies on a highly specialized pollination system, no evidence was found to suggest that the relatively small populations in the Swiss lowlands are experiencing negative consequences of habitat fragmentation and reductions in plant population size. Other nursery pollination mutualisms have been suggested to be vulnerable to Allee effects at small population sizes. Anstett et al. (1997, 1995) note that at low densities asynchronously flowering figs may not be able to sustain viable pollinator populations and suggest that fig–wasp systems are especially sensitive on account of the highly specialized pollination systems. In our system even the smallest Trollius populations showed no sign of Allee effects — if anything the opposite was true in that reproductive ability in small populations might be enhanced due to a more optimal trade-off between pollination services and seed predation, mediated through per capita Chiastocheta abundance, which itself is a function of T. europaeus flower density at local scales (5-m scale). While some flies are needed to ensure pollination of T. europaeus, seed predation by Chiastocheta larvae can greatly decrease seed set as abundance per flower increases (Fig. 3b). While T. europaeus populations are still large and continuous at higher altitudes, other populations in the Swiss lowlands only occur on relatively small nature protection areas that are often surrounded by farmland or urban areas. Our results indicate that these small and isolated populations remain viable, but are more likely to be successful if the plants are distributed in locally dense patches, rather than being uniformly distributed across the habitat. Conservation attention should therefore prioritize populations with high local plant densities of T. europaeus, as it is these that are likely to be most reproductively secure. Further, the protection of even small pockets of suitable habitat with existing T. europaeus populations, as is often currently the case, appears both feasible and even desirable from a plant reproductive perspective.

CHAPTER 2 – PLANT REPRODUCTION AND POLLINATOR ABUNDANCE

References

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Johnson, S.D., Collin, C.L., Wissman, H.J., Halvarsson, E. & Agren, J. (2004) Factors contributing to variation in seed production among remnant populations of the endangered daisy Gerbera aurantiaca. Biotropica, 36, 148-155. Johnson, S.D. & Steiner, K.E. (2000) Generalization versus specialization in plant pollination systems. Trends in Ecology & Evolution, 15, 140-143. Kery, M., Matthies, D. & Spillmann, H.H. (2000) Reduced fecundity and offspring performance in small populations of the declining grassland plants Primula veris and Gentiana lutea. Journal of Ecology, 88, 17-30. Lauber, K. & Wagner, G. (2001) Flora Helvetica: Farbfotos von 3000 Blüten- und Farnpflanzen der Schweiz, Artenbeschreibungen und Bestimmungsschlüssel. Paul Haupt Bern. Leimu, R., Mutikainen, P., Koricheva, J. & Fischer, M. (2006) How general are positive relationships between plant population size, fitness and genetic variation? Journal of Ecology, 94, 942-952. Morris, W.F., Bronstein, J.L. & Wilson, W.G. (2003) Three-way coexistence in obligate mutualist-exploiter interactions: The potential role of competition. American Naturalist, 161, 860-875. O'Hara, R.B. (2009) How to make models add up - a primer on GLMMs. Annales Zoologici Fennici, 46, 124-137. Pellmyr, O. (1989) The cost of mutualism - interactions between Trollius europaeus and its pollinating parasites. Oecologia, 78, 53-59. Pellmyr, O. (1992) The phylogeny of a mutualism - evolution and coadaptation between Trollius and its seed-parasitic pollinators. Biological Journal of the Linnean Society, 47, 337-365. Peterson, A., Bartish, I.V. & Peterson, J. (2008) Effects of population size on genetic diversity, fitness and pollinator community composition in fragmented populations of Anthericum liliago L. Plant Ecology, 198, 101-110. Rabasa, S.G., Gutierrez, D. & Escudero, A. (2009) Temporal variation in the effects of habitat fragmentation on reproduction of the Mediterranean shrub Colutea hispanica. Plant Ecology, 200, 241-254. RDevelopmentCoreTeam (2009) R: A language and environment for statistical computing. Vienna, Austria. Schleuning, M., Niggemann, M., Becker, U. & Matthies, D. (2009) Negative effects of habitat degradation and fragmentation on the declining grassland plant Trifolium montanum. Basic and Applied Ecology, 10, 61-69. Spigler, R.B. & Chang, S.M. (2008) Effects of plant abundance on reproductive success in the biennial Sabatia angularis (Gentianaceae): spatial scale matters. Journal of Ecology, 96, 323-333. Spigler, R.B. & Chang, S.M. (2009) Pollen limitation and reproduction varies with population size in experimental populations of Sabatia angularis (Gentianaceae). Botany, 87, 330-338. Steffan-Dewenter, I. & Tscharntke, T. (1999) Effects of habitat isolation on pollinator communities and seed set. Oecologia, 121, 432-440. Steffan-Dewenter, I. & Westphal, C. (2008) The interplay of pollinator diversity, pollination services and landscape change. Journal of Applied Ecology, 45, 737-741. Stehlik, I., Caspersen, J.P., Wirth, L. & Holderegger, R. (2007) Floral free fall in the Swiss lowlands: environmental determinants of local plant extinction in a peri-urban landscape. Journal of Ecology, 95, 734-744. Thompson, K. & Jones, A. (1999) Human population density and prediction of local plant extinction in Britain. Conservation Biology, 13, 185-189.

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Tsaliki, M. & Diekmann, M. (2009) Fitness and survival in fragmented populations of Narthecium ossifragum at the species' range margin. Acta Oecologica-International Journal of Ecology, 35, 415-421. Waser, N.M., Chittka, L., Price, M.V., Williams, N.M. & Ollerton, J. (1996) Generalization in pollination systems, and why it matters. Ecology, 77, 1043-1060. Winter, C., Lehmann, S. & Diekmann, M. (2008) Determinants of reproductive success: A comparative study of five endangered river corridor plants in fragmented habitats. Biological Conservation, 141, 1095-1104. Wolf, A.T. & Harrison, S.P. (2001) Effects of habitat size and patch isolation on reproductive success of the serpentine morning glory. Conservation Biology, 15, 111- 121. Zuur, A.F., Ieno, E.N., Walker, N.J., Saveliev, A.A. & Smith, G.M. (2009) Mixed Effects Models and Extensions in Ecology with R. Springer, New York.

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

GENETIC VARIATION AND PLANT PERFORMANCE IN

FRAGMENTED POPULATIONS OF GLOBEFLOWERS

(TROLLIUS EUROPAEUS) WITHIN AGRICULTURAL

LANDSCAPES.

Charlotte Klank, Jaboury Ghazoul and Andrea R. Pluess

In review

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Summary

The management of remnant populations in highly fragmented landscapes requires a thorough understanding of the processes shaping population persistence. We investigated relationships between population characteristics (i.e. size, density and pollinator abundance), offspring performance, genetic diversity and differentiation in Trollius europaeus, a plant with a nursery pollination system. In 19 populations of different sizes and located in north- east Switzerland, an area which underwent vast developmental changes over the last decades, we assessed neutral genetic diversity (Ntotal = 383) using AFLPs and plant performance in a greenhouse experiment (Ntotal = 584) using competition and control treatments.

Overall genetic differentiation was low (FST = 0.033) with a marginal significant isolation by distance effect (P = 0.06) indicating (historical) genetic connectivity among the populations.

Mean expected heterozygosity was HE of 0.309 (0.0257 – 0.393) while inbreeding coefficients (FIS) were significant in only three populations. Genetic diversity was not related to population size, plant density or pollinator abundance. Plant performance was reduced under competition (P < 0.001) but the severity of competition was independent of genetic diversity and population size. In summary, remnant populations of T. europaeus appear to retain genetic diversity and seem capable of persisting under the present conditions within an agricultural matrix. While T. europaeus has a highly specialized pollination system and limited seed dispersal, and thus an expected genetic and reproductive vulnerability to fragmentation, the persistence of T. europaeus bodes well for other perennial herbal species in such communities and landscapes.

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Introduction

Habitat fragmentation caused by human modification of landscapes is a principal threat to biodiversity and terrestrial ecosystems worldwide (Sala et al., 2000, Fahrig, 2003, Reed, 2004, Thomas et al., 2004). Today approximately half of the total land surface has been altered by human activities (Ehrlich and Wilson, 1991, Saunders et al., 1991, Vitousek et al., 1997), especially in Western Europe where natural habitats have become increasingly isolated within a matrix of intensive agriculture and urbanization (Thompson and Jones, 1999). Habitat fragmentation has three main consequences for plant populations: the direct loss of suitable habitat, reductions in population size and increasing spatial isolation between remnant populations. These changes in the population distribution, structure and size often cause Allee effects (Stephens et al., 1999), limiting reproductive success (Leimu et al., 2006), plant performance and reducing population viability (Fischer and Matthies, 1998, Bowman et al., 2008, Schleuning et al., 2009). The resulting small populations are also increasingly vulnerable to environmental and demographic stochasticity (Shaffer, 1981, Kery et al., 2000, Hobbs and Yates, 2003), rendering them prone to local extinction. The vulnerability of fragmented populations might be exacerbated by reductions in genetic variation associated with a reduced population size and increased spatial isolation (Ellstrand and Elam, 1993, Lynch et al., 1995, Young et al., 1996, Leimu et al., 2006). In addition, genetic drift increases in small populations as well as inbreeding, reducing genetic variation within populations through a loss of heterozygosity and the fixation of alleles (Ellstrand and Elam, 1993). Both processes are enhanced through reduced immigration rates in isolated populations, promoting increased genetic differentiation between populations as well as reduced genetic variation within the single population and limiting the available pool of alleles in a population. Such reduced genetic variation, commonly associated with reduced population size, can lower plant growth and reproduction (Keller and Waller, 2002, Leimu et al., 2006). Genetic erosion may also limit a species’ ability to adapt to new environments, as its ability to cope with changed conditions depends also on the amount of genetic variation underlying adaptive traits (Young et al., 1996, Booy et al., 2000, Podolsky, 2001). Aside from habitat fragmentation, land use changes through an intensification of agriculture cause nutrient enrichment, which leads to increased productivity and hence competition for space and light among component species (Gough et al., 2000, Mittelbach et al., 2001, Schippers and Joenje, 2002, Suding et al., 2005). If plants then have a reduced ability to respond to 37

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE changed environmental conditions due to genetic erosion, their extinction risk might be particularly high (Lynch et al., 1995, Sultan, 2000). The genetic effects of habitat fragmentation and its consequences might be mediated by a plants’ pollination system. Highly specialized pollination systems might render plants especially prone to fragmentation effects as pollination can easily collapse in the event that fragmentation affects the specialised pollinators or if plant populations drop below a threshold and become unable to support sufficient numbers of pollinators (Bond, 1994, Waser et al., 1996, Johnson and Steiner, 2000, Ghazoul, 2005). Globeflower Trollius europaeus (Ranunculaceae), a plant with a specialised pollination system that has been subject to widespread habitat fragmentation, represents an interesting case study for the effects of habitat fragmentation on population genetics and performance. While still common in many European alpine regions, the species is becoming increasingly threatened in lowland regions due to its requirement for moist meadows (Muncaciu et al., 2010, Lemke, 2011). In Switzerland moist habitats have undergone drastic changes during the last century, with up to 90% of the former areas being lost due to drainage, deterioration and fertilization (Broggi and Schlegel, 1989, Bowman et al., 2008). In our main study region, T. europaeus populations have undergone substantial reduction in the last decades, dropping from frequent to rare occurrences (Artendatenbank Canton Zurich, http://www.aln.zh.ch), as many habitats are undergoing considerable changes due to urban sprawl and agricultural intensification (pers. communication with local farmers). The remaining populations in the Canton Zurich are now mainly found on nature protection sites. In addition, T. europaeus has a highly specialized nursery pollination system in which Chiastocheta flies act almost as exclusive pollinators. Chiastocheta depends on T. europaeus as their larvae develop within the flower and consume a portion of the developing seeds (Pellmyr, 1989). Since plant population size, density and pollinator abundance are all potentially influenced by habitat fragmentation, influencing plant population persistence, we tested how these variables affect neutral genetic variation in plant populations, plant performance in a greenhouse experiment and their interaction. We chose to explore such interactions on T. europaeus not only because it is a species of conservation concern in many regions of Europe (including Switzerland), but also because its pollination system might render this species particularly vulnerable to land use changes. As Chiastocheta is expected to have short flight ranges (Després, 2003, Johannesen and Loeschcke, 1996), this pollination system might have resulted in highly differentiated plant populations. Such differentiation might

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE either add negatively due to increased population isolation or alleviate ongoing fragmentation processes due to earlier purging effects (Byers and Waller, 1999, Ouborg and Vantreuren, 1994). Besides genetic differentiation we tested whether small populations of Trollius europaeus exhibit reduced genetic variation and plant performance and whether plant performance is positively correlated with genetic diversity. As it has been suggested that small populations might show a reduced competition ability (Fenster and Dudash, 1994, Young et al., 1996), we assessed plant performance in a competition treatment as well as a control treatment to determine the severity of competition.

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Material & Methods

Study Species Trollius europaeus L. (Ranunculaceae) is a long-lived perennial, self-incompatible plant, occurring in moist habitats throughout northern and mid-Europe (Lauber and Wagner, 2001). It usually has one (sometimes two or more) yellow globose and tightly closed flowers per stalk (Doroszewska, 1974), and reaches first bloom after approx. 2-3 years. The flower shape excludes insects other than a few species of Chiastocheta flies, rendering pollination dependent on up to six Chiastocheta species (Pellmyr, 1989, Jaeger and Després, 1998). While adult Chiastocheta flies use the flowers to forage, mate, shelter and oviposit; the larval stage feeds and develops on ovules within the ripening seed heads, forming a nursery pollination system (Pellmyr, 1989, Pellmyr, 1992, Jaeger and Després, 1998, Pompanon et al., 2006, Dufay and Anstett, 2003).

Figure 1: Location of the 19 Trollius europaeus populations in Switzerland. The grey areas are lakes; the largest lake in the centre is Lake Zurich. Reproduced with the permission of swisstopo (JA100120).

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE ) E H 0.017 0.017 0.014 0.018 0.019 0.013 0.017 0.015 0.018 0.018 0.017 0.019 0.017 0.019 0.018 0.017 0.017 0.018 0.017 0.018 SE (

E H 0.3 0.3 0.3 0.3 0.33 0.33 0.36 0.31 0.26 0.39 0.32 0.37 0.28 0.28 0.28 0.28 0.29 0.32 0.31 0.32 0.29

IS F 0.419 0.419 0.749 0.238 0.080 0.752 0.296 0.492 0.305 0.234 0.169 0.118 0.210 0.307 0.184 0.168 0.258 0.536 0.322 0.194 upper CI

IS F 0.006 0.006 0.035 0.016 -0.050 -0.050 -0.265 -0.040 -0.184 -0.031 -0.063 -0.109 -0.125 -0.187 -0.069 -0.184 -0.158 -0.058 -0.087 -0.081 -0.040 ower CI

IS are significant. significant. are F 0.184 0.184 0.242 0.099 0.361 0.151 0.263 0.121 0.125 0.030 0.012 0.119 0.000 0.005 0.100 0.224 0.120 0.077 IS -0.052 -0.052 -0.003 F 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 98.7 98.7 98.7 97.4 98.7 PLP 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 76 # loci flies per flower), location of the studied studied the of location flower), per flies 21 21 20 20 21 20 22 19 15 22 20 20 22 22 21 15 21 21 19 22 (AFLP) intervals (Bonferroni corrected), corrected), (Bonferroni intervals N 0.2 0.2 Chiastocheta 0.13 0.13 0.119 0.119 0.201 0.393 0.267 0.219 0.101 0.128 0.253 0.071 0.129 0.078 0.292 0.069 0.236 0.091 0.239 0.113 = 0.117 (95% CI: 0.064 – 0.170). Bold Bold – 0.170). 0.064 CI: (95% = 0.117 pollinator IS abundance F ) -2 10 10 1.2 1.2 1.4 6.9 1.24 1.24 0.18 2.11 1.72 3.98 2.34 2.77 4.93 2.73 0.15 0.32 0.32 0.11 1.79 5.24 ) at overall overall ) at 14.44 14.44 flower E H density (m for each population, 95% confidence IS 230 230 840 800 800 310 230 140 size 3930 3930 1140 1920 1920 1120 4260 57'780 57'780 24'350 24'350 27'120 43'360 86'460 12'110 F 820'700 820'700 238'040 238'040 population

1 ), pollinator), abundance(mean number of -2 coordinates 732'336/223'768 732'336/223'768 683'267/235'908 712'323/236'612 702'884/244'312 680'308/236'502 705'296/241'768 705'729/245'752 705'852/257'047 682'861/235'022 707'460/244'360 708'000/250'470 707'931/254'335 708'330/242'391 709'023/253'436 709'868/257'254 711'868/241'711 694'443/225'287 708'454/252'875 662'502/206'591 678 678 656 537 639 612 764 712 745 703 732 612 800 771 597 752 852 740 1250 1250 1050 (m s.s.l.) Elevation Elevation populations, inbreeding coefficient populations, inbreeding Population size, flower density (m density flower size, Population europaeus

F1 F2 F7 B3 F11 F13 F15 F23 F24 F29 F30 F34 F41 B11 B11 B15 ARV Kafer Kriens F25_27 F25_27 population ercentage of polymorphic loci (PLP) and expected heterozygosity ( heterozygosity expected and (PLP) loci polymorphic of ercentage Trollius Table 1: p Switzerland) Wabern, Landestopographie, für (Bundesamt maps topographical Swiss the to according meters in 1 Coordinates

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Study sites Plant material was collected from 19 Trollius europaeus populations in northeast Switzerland, on elevations ranging from 537 to 1’250 m a.s.l (Table 1). Populations were situated north-east and south-west of Lake Zurich (Fig. 1). Pollinator abundance, plant population size and density (Table 1) were determined in a parallel study during 2006 to 2008, where we investigated amongst others the effect of pollinator abundance on seed set (Klank et al., 2010). Flower number as a surrogate for population size was found to range from 140 to 820 700 flowers (median 3930), with densities from 0.11 to 14.44 flowers m-2 (median 2.11). Pollinator abundance was determined by counting Chiastocheta flies in either all flowers in the small populations or at least 200 flowers in large populations over three years at one of two time points each (2006-2008, see Klank et al. (2010) for a detailed description). On average 0.069 to 0.393 Chiastocheta flies per flower (median 0.13) were found.

Genetic survey Genetic diversity and differentiation were assessed with AFLPs (amplified fragment length polymorphisms). Leaves from 22 randomly sampled individuals were collected from each population in 2006 and 2007 with a minimum inter–individual distance of ~4 meters to avoid sampling from the same individual. Leaf material was dried in silica gel immediately after collection. DNA extractions were done with the DNeasy Plant Maxi Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s protocol using 10 mg of dried plant material. DNA concentrations were measured with a Nanodrop 1000 (Thermo Scientific) and averaged at ~24 ng/µl. Selective AFLP primer pairs were Eco-ATC – Mse-CAG, Eco- ATC – Mse-CAT and Eco-AGA – Mse-CTC as used by Després et al. (Després, Loriot et al. 2002). For the digestion-ligation step, first 10 µl extracted DNA (~240 ng) was digested for 1 h at 37°C in a 27 µl restriction mix consisting of 2 U EcoR1, 2 U Mse1, 0.4 µl bovine serum albumin (10 mg/ml), and 4 µl NEB2 buffer (10x, BioLabs). Second, PCR adaptors were ligated to the DNA fragments for 3 h at room temperature by adding 14 µl ligation mix consisting of 15 pmol EcoR1 adaptor, 136.35 pmol Mse1 adaptor, 0,1 µl bovine serum albumin, 5 µl ATP (10 mM), 1 µl NEB2 (10x, BioLabs), and 20 U T4 Ligase (BioLabs). This step was finished by inactivating the ligase for 10 min at 65° C. For preselective amplification 1.5 µl restriction-ligation mix were added to 19 µl PCR reaction mix containing 157.5 ng of each preselective primer Eco-A and Mse-C, 0.3 µl dNTP mix (25 mM), 1.5 µl MgCl2 (25mM), 4 µl Promega GoTaq© reaction buffer (5x, no MgCl2) and 42

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

0.375 U GoTaq polymerase (Promega). PCR reactions cycling conditions were as follows: 1 cycle (94°C for 3 min), 20 cycles (94°C for 30 s, 60° for 30 s, 72°C for 60 s) and 1 cycle (72°C for 1 min). This preselective PCR product was then 1:20 times diluted with 1x TE buffer. Selective amplifications were performed by adding 3 µl of the diluted preselective

PCR product to 11 µl selective PCR mix containing 1.5 µl MgCl2 (25 mM), 0.5 µl dNTPs

(25 mM), 3 µl GoTaq reaction buffer (5x, no MgCl2) and 0,375 U GoTaq polymerase. PCR cycling conditions were 1 cycle (94°C for 3 min), 10 cycles (94°C for 30 s, 65°C for 30 s with a reduction of the annealing temperature of -1°C from 65° to 56°), then 25 cycles (94°C for 30 s, 54°C for 30 s, 72°C for 1 min) and 1 cycle (72°C for 60 s). All PCRs were carried out using a Bio-Rad dyad cycler. Fragments were separated on an ABI3720xl (GeneScanTM 500 LIZ® sizing standard) and Genemapper 3.5 software (Applied Biosystems) used for fragment reading. We used the following procedure to generate the fragment absence/presence matrix: Preliminary peak positions were selected manually for each primer pair in Genemapper 3.5 (ABI) and peak height data were recorded. Using AFLPScore 1.4 (Whitlock et al., 2008) we retained those loci that had an error rate of < 5% using 28 duplicated samples, resulting in a total of 101 loci. As recommended by Lynch and Milligan (1994), we retained only loci with a frequency smaller than 1-(3/n) with n being the number of total samples, leading to a set of 76 polymorphic loci for analysis.

Greenhouse experiment To test plant performance in relation to population origin we determined the absolute severity of competition in a greenhouse setting. We selected the fast growing yellow oat- grass Trisetum flavescens (Poaceae) as the competitor species as it occurs naturally with T. europaeus (Ellenberg, 1996). In 2007, one mature seed head per individual from 16 plants (i.e. seed families) per population were collected. The seeds were placed on standard germination soil in petri dishes and cold stratified for four months in the dark at 4°C and then placed in growth cabinets (MLR-351H, SANYO Electric Co. Ltd.) for germination. The cabinets were run with an alternating 12 h light cycle at 15°C, 50% relative humidity and 14.4 k lx. The petri dishes were left in the growth cabinets for 10 weeks and seedlings were then transplanted into trays in the greenhouse. The greenhouse compartments were run with 20 k lx from 08:00 to 18:00 and temperatures of 20°C during daytime and 16°C at night. Plants were watered automatically every day using watering mats. After two weeks two similar sized seedlings per seed familiy were planted in 1 L pots (13.5 cm height, Ø 11 cm) filled with standard garden soil. Trisetum flavescens seeds were sown into half of the pots

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE two days after transplanting the seedlings. Thus from each seed family one seedling was grown with T. flavescens and another was grown alone (i.e. control treatment). Per treatment, plants were randomly assigned to two blocks which were kept in two different greenhouse compartments. The blocks of each treatment were formed to avoid interference of above- ground competition by T. flavescens between the two treatments. Blocks were exchanged biweekly between compartments and plants within blocks repositioned randomly. We recorded leaf width and number of leaves per seedling at the start of the experiment. After 10 weeks, above and below ground biomass were harvested. We measured the total leaf area per plant using a LI3000C leaf scanner with a LI-3050C Transparent Belt Conveyer (Li-Cor Biosciences). We further measured stalk lengths and recorded the dry weight of above and below ground biomass. We also assessed the influence of the competition treatment: According to Snaydon (1991), the proportional reduction in plant performance due to the competition treatment for each parameter (leaf area, average stalk length within an individual, above and below ground biomass) is expressed as the absolute severity of competition (ASC) for each seed family, calculated as log10 (trait measure without competition) minus log10 (trait measure with competition). Positive ASC values indicate a better performance in the control treatment, while negative values indicate a higher performance for plants grown in competition.

Statistical data analysis Genetic data Given that the Hardy-Weinberg equilibrium conditions cannot be readily assumed in remnant populations that have been subjected to fragmentation and increasing isolation, we applied the approach of Damashapatra et al. (2008) by estimating the inbreeding coefficient

(FIS) of each individual and calculating the overall mean and mean per population for later analysis. Mean FIS were tested for significance by calculating 95% Bonferroni corrected confidence intervals (CI) with the null hypothesis of no difference. The derived overall FIS value was implemented in AFLPSURV (Vekemans et al., 2002) for further analysis. We calculated population differentiation FST (mean overall FST and population pairwise FST) as well as expected heterozygosity (HE). Calculations were done using the Bayesian method with non-uniform prior distribution of allele frequencies. Permutations (N = 4999) of individuals among populations were done to test the hypothesis of no genetic differentiation among populations. Significances for the observed pairwise FST were evaluated using 95%

Bonferroni-corrected CI of the boostraped data. In case of mainly significant pairwise FST, a

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Mantel Test to test for isolation by distance based on pairwise population genetic distances

(FST / (1-FST)); calculated in AFLPSURV) and log transformed geographic distances (Rousset, 1997) with 4999 permutations available in IBD (Bohonak, 2002) was carried out.

We further evaluated the relationship between HE and population size (log-transformed), population density and pollinator abundance by applying a linear regression in R (RDevelopmentCoreTeam, 2009).

Greenhouse data To test whether plant performance was reduced in the competition treatment and if populations differed in their response as well as whether greenhouse compartments had an effect we first analysed the harvest data of each parameter. Eleven seedlings from eight populations independent of population size died during the experiment, which were removed from the analysis with their half-sib counterparts. We applied a type III ANCOVA with populations included as a random factor and treatment, compartment, as well as their interaction with population as fixed factor. Initial leaf width was also fitted as a fixed factor to account for variations in seedling size at the beginning of the experiment. Treatment was tested on the interaction term of treatment : population, and compartment was tested on the interaction term of compartment : population, whereas all other terms were tested on the residual. After calculating the absolute severity of competition (ASC) we used a linear mixed-effects model for analysis. As individual data points are not independent and nested within populations, we fitted the factorized population origin as a random factor. We used the ASC of the initial leaf width as a covariable, and population size (log-transformed), population density and expected heterozygosity as fixed effects. Both analyses were carried out using R (RDevelopmentCoreTeam, 2009) and fixed effects showing a pairwise correlation coefficient above 0.8 were centred (Zuur et al., 2009). Varying degrees of freedom result from the exclusion of missing data points.

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Results

Genetic survey Calculating the overall inbreeding coefficient over all 383 individuals used in this study gave a significant mean FIS of 0.117 (SE = 0.018, 95% CI [0.064 – 0.170]). Mean FIS for each population were non-significant expect for three populations (Table 1).

Using the overall mean FIS value (0.117), a mean expected heterozygosity (HE) of 0.309 (SE

0.008) was calculated, with HE ranging from 0.260 to 0.393 (Table 1). The percentage of polymorphic loci (PLP) varied between 97.4% - 100% (Table 1).

We further found our study populations to have a relatively low overall FST of 0.033 and the 95% CI of the permuted data set of randomly assigned individuals being [-0.006 – 0.002] showing that our populations were significantly differentiated. As all population pairwise

FST were significant (Table A1) we carried out a Mantel test for isolation by distance and found a marginally significant correlation coefficient of 0.209 (P = 0.0598, Fig. 2). Expected heterozygosity showed no significant relationship with either population size, population density or pollinator abundance (Table 2).

Table 2: Multiple linear regression for expected heterozygosity HE in 19 Trollius europaeus populations.

estimate std. error t value P intercept 0.3320 0.0333 9.968 <0.0001 population size1 -0.0062 0.0039 -1.601 0.1302 population density 0.0052 0.0028 1.887 0.0786 pollinator abundance 0.0721 0.0902 0.799 0.4369 1 log-transformed

Greenhouse experiment All measured traits were influenced by the treatment and the population origin (P > 0.001, Table 3). The compartment assignment was not significant (P > 0.18) except for average stalk length (P = 0.004, Table 3). Interactions between treatment : population and compartment : population were non-significant, except for below-ground biomass which showed a compartment: population interaction (P = 0.005; Table 3). The total leaf area of Trollius europaeus generally decreased when grown with Trisetum flavescens (ranging between -12.47% and - 44.42%). The same was found for above and below ground biomass (btw. -9.87% and -41.37%, btw. -19.65% and -46.00%, respectively). Average stalk length showed an increase of 15.50% to 69.12% when grown in combination with T. flavescens. An 46

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE overview of all population means and differences between treatments can be found in Table A2 in the Appendix. The absolute severity of competition (ASC) showed no significant correlations with population size, population density or expected heterozygosity (Table 4). The only significant effect came from the initial leaf width at the start of the experiment (P > 0.0001), and population density was marginally significant for below ground ASC (Table 4).

Figure 2: Isolation by distance for 19 Trollius europaeus populations in north-eastern Switzerland. Pairwise (FST / (1-FST)) and log-transformed geographic distances, based on IBD analysis with a correlation coefficient of 0.209 (P = 0.0598).

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Table 3: Analysis of raw harvest data of the greenhouse experiment of 19 Trollius europaeus populations grown with and without competition by Trysetum flavescens and using an ANCOVA type III. leaf area DF Sum Sq F value P intercept 1 117062 46.6722 <0.0001 initial leaf width 1 525108 209.3591 <0.0001 treatment 1 417489 133.7272 <0.0001 compartment 1 1132 0.5268 0.4773 population 18 200304 4.4367 <0.0001 treatment : population 18 56195 1.2547 0.2126 compartment : population 1 2149 0.8638 0.3510 residuals 536 1333690 average stalk length DF Sum Sq F value P intercept 1 36403 94.9148 <0.0001 initial leaf width 1 22005 57.3733 <0.0001 treatment 1 58420 11.7581 <0.0001 compartment 1 189.0000 0.0040 population 18 40678 5.8923 <0.0001 treatment : population 18 9937 1.4580 0.0997 compartment : population 1 2 0.0044 0.9473 residuals 542 205226 above ground biomass DF Sum Sq F value P intercept 1 2.882 47.7448 <0.0001 initial leaf width 1 11.656 193.1322 <0.0001 treatment 1 8.081 135.0585 <0.0001 compartment 1 0.026 2.0000 0.1781 population 18 3.84 3.5352 <0.0001 treatment : population 18 1.077 0.9896 0.4700 compartment : population 1 0.013 0.2190 0.6400 residuals 543 32.828 below ground biomass DF Sum Sq F value P intercept 1 9.358 81.9745 <0.0001 initial leaf width 1 37.746 330.6584 <0.0001 treatment 1 34.895 429.9179 <0.0001 compartment 1 0.327 0.3438 0.5575 population 18 8.577 4.1743 <0.0001 treatment : population 18 1.461 0.7136 0.7985 compartment : population 1 0.951 8.0447 0.0047 residuals 541 61.55

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Table 4: Linear mixed-effects model for analysis of the absolute severity of competition (ASC) of Trollius europaeus.

ASC random effects intercept residual leaf area 0.0000 0.2572 fixed effects estimate SE DF t value P intercept 0.2399 0.0625 265 3.8406 0.0002 asc (inital leaf width) 0.9878 0.1491 265 6.6265 <0.0001 population size1 -0.0072 0.0085 15 -0.8419 0.4141 population density 0.0028 0.0060 15 0.4753 0.6414 expected heterozygosity2 -0.9682 0.5179 15 -1.8695 0.0812 ASC random effects intercept residual average stalk 0.0321 0.1288 length fixed effects estimate SE DF t value P intercept -0.0914 0.0443 271 -2.1242 0.0346 asc (inital leaf width) 0.3438 0.0756 271 4.5476 <0.0001 population size1 -0.0007 0.0058 15 -0.1254 0.9019 population density -0.0056 0.0041 15 -1.3851 0.1863 expected heterozygosity2 -0.0839 0.3571 15 -0.2351 0.8173 ASC random effects intercept residual above ground 0.0296 0.2732 biomass fixed effects estimate SE DF t value P intercept 0.1982 0.0709 272 2.7924 0.0056 asc (inital leaf width) 1.0408 0.1584 272 6.5694 <0.0001 population size1 -0.0003 0.0096 15 -0.0361 0.9717 population density -0.0006 0.0067 15 -0.0938 0.9265 expected heterozygosity2 -0.4608 0.5903 15 -0.7807 0.4471 ASC random effects intercept residual below ground 0.0320 0.2339 biomass fixed effects estimate SE DF t value P intercept 0.3000 0.0642 270 4.6706 <0.0001 asc (inital leaf width) 1.1143 0.1375 270 8.1025 <0.0001 population size1 -0.0097 0.0087 15 -1.1082 0.2852 population density 0.0126 0.0061 15 2.0690 0.0562 expected heterozygosity2 -0.4098 0.5297 15 -0.7737 0.4512 1 log-transformed, 2 centred

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Discussion

Genetic diversity and differentiation of Trollius europaeus populations

For Trollius europaeus, we found a relatively low FST of 0.033 and populations contained similar levels of genetic diversity independent of their sizes. Given the apparent isolation of populations across the landscape and the specialized pollination system we expected a higher degree of genetic differentiation, which was not confirmed by our results. Nevertheless, the retention of genetic variation within populations seems to be a common pattern in outcrossing perennial plants (Nybom and Bartish, 2000). The degree of genetic differentiation often increases with the area under study (Nybom and Bartish, 2000). Indeed, we found much smaller differentiation than Després et al. (2002), whose study area covered the Pyrenees, Alps and Fennoscandia (0.033 vs. 0.152; same marker systems used). Isolation by distance was marginally significant for our study populations (P = 0.06), suggesting that gene flow can extend over several kilometres and/or accumulates over time. In contrast, an allozyme study of Chiastocheta in discrete populations of T. europaeus (minimum distance to the next T. europaeus patch of 1 km) in Denmark found local differentiation and low dispersal between the sampled insect populations (Johannesen and Loeschcke, 1996). This indicates that pollination by Chiastocheta occurs primarily on a local scale, as also suggested by Després (2003), most probably resulting in the isolation of plant populations. Seed dispersal as a source for gene flow is probably also limited. Globeflower seeds do not have any morphological characteristics that aid dispersal, and usually drop to the ground in the close vicinity of the mother plant. Therefore, historical gene flow in an only moderately fragmented landscape seems to be responsible for the observed genetic pattern in Trollius europaeus. We further cannot rule out the possibility that the assumed isolation of populations is counteracted by small undetected populations of T. europaeus distributed throughout the landscape, acting as stepping stones for Chiastocheta and facilitating pollen movement, even though it seems unlikely for our study region. In addition, larger Diptera and other insects very occasionally visited T. europaeus flowers (personal observation CK). Although such visits were rare, they might contribute disproportionately to long distance gene flow due to a greater foraging range. Species distribution range is likely influencing not only genetic differentiation but also diversity. Després et al. (2002) found significantly higher within-population genetic diversity of T. europaeus in the Alps than in the Pyrenees or Fennoscandia. This higher diversity was similar to the diversity levels found in our study (HE = 0.229 compared to HE = 50

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

0.309 in our study). Després et al. (2002) interpreted this to be caused by the proximity to suggested glacial refugia and suggested that the alpine populations represent moderately fragmented relics of a large southern ancestral distribution area. As our study populations are located within the same geographical range, it is plausible that the vicinity to potential refugia shaped within-population genetic diversity.

Despite an overall FIS of 0.117, only three within population inbreeding measures were significant. Even though, those populations contained all less than 5000 flowers, another eight populations of this size range had no indication for inbreeding, indicating that the loss of genetic diversity due to matings among relatives is of minor importance in small populations of T. europaeus.

Effects of population size, plant density and genetic diversity on plant performance Trollius europaeus plants in the greenhouse experiment did respond to competition with Trisetum flavescens with decreased leaf area and biomass, confirming a reduced performance under competition. The severities of competition in the different traits under study, however, were not affected by the population size, the population density or the expected heterozygosity. Likewise, the individual plant traits were not affected by population size and expected heterozygosity (details not shown). Leaf stalks reacted to competition by elongation, a likely response to competition for light, but this was independent of population size, population density or expected heterozygosity (details not shown). The similar levels of genetic diversity across populations might explain the lack of differences in plant performance. While many studies have found positive effects between population size, neutral genetic diversity and plant fitness (Gaudeul et al., 2000, Lienert et al., 2002, Galeuchet et al., 2005, Leimu et al., 2006, de Vere et al., 2009, Jacquemyn et al., 2009, Krauss et al., 2010), the absence of such a relationship, as in this study, has also been found for other species across a wide range of habitats (Schmidt and Jensen, 2000, Podolsky, 2001, Kuss et al., 2008, Brownlie et al., 2009, Qiu et al., 2009, Spigler et al., 2010). Furthermore, while reduced population size and decreased genetic variability often occur jointly (Oostermeijer et al., 1994, Fischer and Matthies, 1998, Lienert et al., 2002, Vergeer et al., 2003a, Vergeer et al., 2003b), other studies did not find such relationships (Ouborg and Vantreuren, 1995, Lammi et al., 1999, Fischer et al., 2000, Luijten et al., 2000, Pluess and Stöcklin, 2004, Dostalek et al., 2010), indicating more complex mechanisms regulating plant fitness and performance.

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Conclusions and implications for conservation Given our results, it seems most likely that the retention of historical genetic patterns or sufficient gene flow between populations are counteracting negative effects of changes in population sizes and habitat alteration. In Switzerland, major fragmentation and land conversion events have taken place in the last 60 years (Schulz and Dosch, 2005), the time period in which a decline of T. europaeus has been observed in our study region (Artendatenbank Canton Zurich, http://www.aln.zh.ch). Even though Trollius europaeus reaches reproductive maturity after ~2 years, its lifespan is thought to be considerably longer, thus probably only a few population turnovers (i.e. exchange of all individuals) took place. As populations often lose variability very slowly (Amos and Balmford, 2001) and perennial plants are assumed to have intermediate rates of population declines (Eriksson and Ehrlen, 2001), it could also be that we will see more pronounced effects in the future. Due to the approach used here, we cannot infer current gene flow among our study populations. The observed results might thus be considerably influenced by historic gene flow patterns preserved in the present day populations. As Trollius europaeus is becoming increasingly rare across Europe due to the loss of suitable, moist habitats, especially in areas of intense agriculture and dense settlement structures, a main aim of our study was to understand possible threats to this species triggered by habitat fragmentation, especially given the higher expected vulnerability due to its nursery pollination system. Here as well as in a parallel study of in situ reproductive success (Klank et al., 2010) we found no negative effects of small population size on plant performance. T. europaeus ability to persist in relatively small remnant habitats in combination with its easily recognizable characteristic globose flowers might make it an eligible candidate as a flagship species for the conservation of moist meadows (as also suggested by Lemke, 2011). Furthermore, the fact that for T. europaeus population size had no effect on population genetic diversity or performance suggests that management for its conservation should acknowledge that small populations retain considerable viability and therefore conservation value.

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CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

Appendix

- 55.4 55.4 65.9 71.92 71.92 35.93 58.17 55.26 34.81 58.33 66.52 34.97 58.72 63.21 58.15 66.02 69.36 60.58 37.01 65.22 Kriens

- , and * , and 0.8 0.8 4.6 7.63 7.63 4.92 31.2 8.57 2.45 1.55 37.65 37.65 30.37 16.72 10.22 32.56 11.55 10.48 11.67 30.94 Kafer 0.0123* 0.0123* SURV - F07 F07 28.6 28.6 31.7 37.92 37.92 15.42 21.16 20.81 18.04 19.73 23.37 33.75 15.13 23.09 32.03 22.03 35.49 23.95 0.0496* 0.0496* 0.0265*

- 3.6 3.6 F41 F41 5.12 5.12 9.35 6.57 7.35 5.14 9.58 27.22 27.22 29.18 31.99 16.47 29.77 13.22 12.07 15.67 0.0515* 0.0515* 0.0146* 0.0238*

- derived from AFLP from derived 3.5 3.5 F34 F34 4.02 4.02 7.04 3.91 3.91 40.33 40.33 34.11 20.79 14.71 36.12 16.15 12.22 34.98 13.12 14.94 14.94 ST 0.0152* 0.0152* 0.0739* 0.0331* 0.0474*

F - 11 11 F30 F30 8.36 8.36 4.81 9.21 3.14 1.41 37.73 37.73 31.15 17.14 33.34 12.25 31.99 11.07 0.0212* 0.0212* 0.0078* 0.0488* 0.0138* 0.0283*

- 3.1 3.1 F29 F29 4.25 4.25 2.15 8.09 7.02 7.02 5.77 14.86 14.86 26.51 11.95 30.38 30.38 25.89 28.63 0.0306* 0.0306* 0.0297* 0.0232* 0.0706* 0.0204* 0.0344*

- 8.86 8.86 3.42 9.99 3.87 39.11 39.11 30.79 18.26 11.22 32.88 12.84 31.65 F25_27 F25_27 0.0340* 0.0340* 0.0149* 0.0209* 0.0258* 0.0454* 0.0192* 0.0290* - F24 F24 28.7 28.7 8.01 9.11 5.24 6.92 6.13 36.13 36.13 14.52 31.02 29.51 0.014* 0.014* 0.0069* 0.0069* 0.0379* 0.0008* 0.0325* 0.0141* 0.0099* 0.0150*

- F23 F23 9.15 9.15 4.58 3.38 2.22 32.29 32.29 25.63 28.27 12.79 26.31 0.0380* 0.0380* 0.0296* 0.0231* 0.0382* 0.0323* 0.0290* 0.0530* 0.0276* 0.0203*

- F02 F02 0.97 0.97 29.5 2.95 50.74 50.74 22.07 23.43 25.26 31.84 0.0679* 0.0679* 0.0403* 0.0639* 0.0471* 0.0408* 0.0806* 0.0379* 0.0785* 0.0555* 0.0209*

populations.half-matrix Thepairwise lower shows - F15 F15 11.3 11.3 42.53 42.53 30.93 21.44 13.08 32.78 15.29 0.0482* 0.0482* 0.0474* 0.0156* 0.0176* 0.0294* 0.0205* 0.0455* 0.0147* 0.0529* 0.0251* 0.0215*

- F13 F13 3.19 3.19 4.01 34.51 34.51 24.52 11.27 27.05 0.0447* 0.0447* 0.0655* 0.0516* 0.0503* 0.0451* 0.0302* 0.0407* 0.0275* 0.0249* 0.0629* 0.0283* 0.0259*

Trollius europaeus - F11 F11 22.8 22.8 8.72 3.51 32.48 32.48 25.54

0.0470* 0.0470* 0.0369* 0.0490* 0.0387* 0.0108* 0.0354* 0.0333* 0.0200* 0.0603* 0.0149* 0.0189* 0.0113* 0.0152* - F01 F01 3.02 3.02 53.56 53.56 32.02 23.89 0.0401* 0.0401* 0.0085* 0.0487* 0.0675* 0.0728* 0.0412* 0.0576* 0.0531* 0.0456* 0.0638* 0.0395* 0.0340* 0.0320* 0.0369*

- B03 B03 35.91 35.91 21.34 12.18 0.0726* 0.0726* 0.0227* 0.0527* 0.0198* 0.0465* 0.0137* 0.0018* 0.0017* 0.0179* 0.0084* 0.0211* 0.0174* 0.0382* 0.0371* 0.0290*

- B15 B15 23.78 23.78 29.06

0.0097* 0.0097* 0.0439* 0.0368* 0.0204* 0.0216* 0.0540* 0.0140* 0.0188* 0.0074* 0.0024* 0.0205* 0.0224* 0.0113* 0.0491* 0.0232* 0.0308* and geographic distances for 19 19 for distances geographic and ST F -

B11 B11 50.55 50.55 0.0180* 0.0180* 0.0338* 0.0206* 0.0215* 0.0291* 0.0290* 0.0463* 0.0387* 0.0150* 0.0361* 0.0372* 0.0211* 0.0415* 0.0129* 0.0333* 0.0237* 0.0331*

- Pairwise Pairwise ARV 0.0233* 0.0233* 0.0270* 0.0508* 0.0133* 0.0418* 0.0016* 0.0308* 0.0564* 0.0521* 0.0435* 0.0413* 0.0357* 0.0299* 0.0300* 0.0255* 0.0595* 0.0176* 0.0073* A1:

ARV B11 B15 B03 F01 F11 F13 F15 F02 F23 F24 F25_27 F29 F30 F34 F41 F07 Kafer Kriens Table indicate a significant differentiation between populations. The upper half shows the geographic distances in km. km. in distances geographic the shows half upper The populations. between differentiation a significant indicate 58

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

(%) -46 -38.3 -38.3 -34.8 -28.4 -44.72 -21.51 -34.33 -25.86 -44.43 -43.91 -42.72 -40.98 -41.49 -25.99 -38.81 -29.73 -30.26 -19.65 -35.12 ∆ Trollius SE -0.1 -0.1 -0.2 -0.1 -0.2 -0.1 -0.1 -0.1 -0.2 -0.1 -0.2 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.2 -0.1 -0.1 none none of 19 19 of 1.3 1.3 0.68 0.68 1.21 1.16 1.18 0.93 0.91 0.82 0.99 1.39 1.42 0.73 1.08 0.83 1.28 1.04 1.39 1.06 0.69 mean -0 -0 SE -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 below ground biomass biomass ground below grass grass 0.38 0.38 0.95 0.71 0.78 0.69 0.51 0.46 0.53 0.79 0.84 0.43 0.96 0.66 0.59 0.83 0.72 1.12 0.76 0.45 mean

(%) -18 -9.87 -20.1 -28.09 -22.38 -33.94 -35.92 -13.71 -41.37 -33.96 -13.55 -24.09 -31.91 -40.19 -12.43 -36.67 -13.04 -28.68 -21.97 ∆ the plants grownplantsthe competition in compared -0 -0 SE -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 none none 0.32 0.32 0.69 0.73 0.84 0.57 0.83 0.53 0.44 0.72 0.72 0.56 0.62 0.69 0.46 0.71 0.71 0.76 0.73 0.41 mean -0 -0 -0 -0 -0 SE -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 -0.1 above ground biomass biomass ground above grass grass 0.5 0.5 0.4 0.23 0.23 0.53 0.48 0.54 0.49 0.35 0.38 0.54 0.49 0.33 0.55 0.44 0.58 0.51 0.68 0.57 0.32 mean

(%) 58.6 58.6 15.5 61.8 23.58 40.36 46.42 26.61 34.47 52.18 41.06 21.69 50.91 30.91 18.54 26.25 17.89 31.51 23.42 69.12 ∆ SE -3.5 -3.5 -5.4 -3.4 -4.4 -6.6 -6.8 -3.9 -3.9 -4.5 -3.9 -5.6 -3.4 -3.4 -6.3 -4.7 -4.4 -4.5 -6.8 -3.7 representspercentagedifference the for none none 69.5 69.5 (%) 49.89 76.23 73.16 82.54 81.83 84.28 63.68 55.96 62.06 80.32 69.09 70.91 70.55 70.64 71.88 72.97 72.35 50.05 mean ∆ -6 -6 SE -4.6 -4.6 -4.9 -6.5 -3.9 -6.5 -7.1 -4.2 -4.3 -3.2 -5.1 -5.5 -4.9 -6.3 -6.8 -6.5 -6.1 -5.7 -4.9 average stalk length length stalk average grass grass 79.13 94.21 102.7 120.9 103.6 97.34 85.63 85.16 87.54 97.74 104.3 90.98 84.06 89.07 114.3 84.74 95.96 89.29 84.65 mean and above and below ground biomass recorded at harvest in the greenhouse experiment experiment greenhouse the in harvest at recorded biomass ground below and above and (%) -36.6 -36.6 -28.87 -27.48 -32.09 -38.23 -13.11 -42.99 -28.19 -27.16 -12.48 -44.42 -16.54 -32.21 -14.42 -15.94 -25.51 -12.47 -27.04 -17.29 ∆ SE -17 -9.6 -9.6 -21.4 -21.4 -17.2 -19.4 -12.9 -24.5 -13.9 -19.4 -17.3 -20.1 -19.6 -13.5 -12.5 -13.7 -20.6 -17.9 -17.9 -10.2 none none 167 75.37 166.8 182.4 124.8 185.9 116.8 113.8 148.9 172.3 130.2 151.7 158.7 99.62 153.2 172.8 173.9 174.6 83.59 mean totalarea leaf SE -13 -7.71 -9.38 -13.7 -11.9 -11.5 -19.4 -8.55 -9.76 -13.8 -14.1 -9.72 -8.55 -9.27 -15.5 -13.7 -15.7 -16.3 -11.8 grass grass 106 82.9 82.9 53.61 121.1 113.3 112.7 108.4 83.88 130.3 109.3 72.39 126.6 107.6 85.26 128.8 128.8 152.2 127.4 69.14 mean

n 16 16 16 16 15 15 16 15 14 14 14 16 16 16 16 15 16 16 14 Means for leaf area, average stalk length stalk average for leaf area, Means populations. Standard errors are given in the SE column. column. SE the in given are errors Standard populations.

R opulation to the control. KRIENS KRIENS Table A2: p europaeus ARV B03 B11 B15 F01 F02 F07 F11 F13 F15 F23 F24 F25_27 F29 F30 F34 F41 KAFE

CHAPTER 3 – GENETIC VARIATION AND PLANT PERFORMANCE

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

CONSERVATION IMPLICATIONS BASED ON NEUTRAL AND

QUANTITATIVE GENETIC DIFFERENTIATION IN SWISS

TROLLIUS EUROPAEUS POPULATIONS

Charlotte Klank, Jaboury Ghazoul and Andrea R. Pluess

submitted

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Summary

While genetic diversity and reproductive ecology provide valuable information for evaluating population persistence and differentiation, understanding the processes causing population differentiation is an important requirement for conservation management. Populations might differ due to divergent selection or primarily due to random processes or might be similar due to extensive gene flow and/or unifying selection. These mechanisms affect the potential use of populations as sources for reinforcing populations under management regimes. Using the comparison of quantitative (QST) and neutral (FST) genetic differentiation we assessed the importance of selection versus genetic drift in Trollius europaeus. We contrasted four small with four large populations in Switzerland, where the habitat of the focal species has undergone substantial fragmentation.

Among the small populations, similar QST and FST estimates suggested that population differentiation is mainly driven by genetic drift, while population genetic variation indicated no loss of diversity in small compared with large populations and a slight isolation by distance effect. Among the large as well as all populations, QST > FST suggested diversifying selection. Excluding the population at highest elevation reduced the QST estimates to similar values as FST, i.e. the pattern can be explained by genetic drift alone, suggesting that elevation is most likely the factor shaping divergent selection. Thus the addition of genetic material from similar altitudes would be a suitable conservation strategy for maintaining Trollius europaeus in nature reserves, demonstrating how understanding processes which shape population differentiation can support decision makers for an informed conservation management.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Introduction

Conservation actions often focus on the preservation of rare and endangered plants, yet also of importance is the need to manage populations of species that are currently still common in many parts of Europe (see Galeuchet et al., 2005, Bowman et al., 2008, Honnay and Jacquemyn, 2007). Given the trend of increasing urban sprawl, agricultural intensification and landscape fragmentation (McKinney, 2006, Antrop, 1998, Schulz and Dosch, 2005), common species face increasing pressures due to the loss of suitable habitats (Lienert and Fischer, 2003, Stehlik et al., 2007, Stöcklin and Fischer, 1999). Thus the identification of conservation units (i.e. populations or groups of populations that merit conservation) in addition to knowledge of the factors affecting local population persistence is an important prerequisite for good conservation practice (Moritz, 1994). While information on the genetic diversity and reproductive ecology can provide information to evaluate population persistence and differentiation, a good understanding of the processes causing differentiation is also important as they might affect the potential use of populations as sources for reinforcing populations under management regimes. Population divergence can be attributed to two main processes: genetic drift and natural selection (e.g. Frankham, 2005, Lande, 1988), which can be counterbalanced by gene flow between populations, thus slowing down or halting divergence. Genetic drift is an arbitrary process whereby random changes in allele frequencies shape the genetic structure of a population, a process more pronounced in small than large populations. Natural selection, on the other hand, is driven by varying environmental pressures in a heterogeneous landscape favouring certain genotypes within a population. Both processes shape the variation of genotypes within a population, which is an important source for a species adaptive and evolutionary potential (Hamrick et al., 1991, Frankham, 1999). While neutral marker surveys such as AFLPs can be used to make inferences on the influence of genetic drift as the cause for population differentiation, detecting differentiation caused by natural selection is harder to achieve as phenotypic traits are commonly under polygenetic control (Podolsky and Holtsford, 1995, McKay and Latta, 2002, Houle, 1989). One approach to differentiate between these two processes is a comparison between quantitative genetic variation (QST), based on substantially heritable morphological traits, and genetic population differentiation (FST) derived from neutral molecular markers (Spitze, 1993, Merila and Crnokrak, 2001). With this approach the relative roles of natural selection

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION and genetic drift can be assessed and used to determine if plant populations should be treated as separate units or not (Leinonen et al., 2008).

The comparison of QST and FST leads to three principal results (Leinonen et al., 2008). First, if there is no difference between QST and FST, genetic drift and natural selection cannot be distinguished, i.e. the observed differentiation could be achieved by drift alone. Second, if

QST exceeds FST, population differentiation is caused by diversifying selection favouring different genotypes within different populations. Third, if QST is lower than FST, unifying selection is prevalent while neutral genetic variation will differentiate primarily due to genetic drift.

In our study we compared quantitative trait differentiation (QST) among small and large populations of globeflower (Trollius europaeus) with neutral genetic differentiation (FST) to evaluate whether populations are subject to diversifying or unifying selection or mainly determined by drift. Trollius europaeus was chosen as their population sizes and numbers have declined across Europe owing to a loss of wet meadows through drainage and due to elevated nutrient inputs from agriculture (Buhler and Schmid, 2001). Yet in Switzerland, T. europaeus is still relatively common, but populations are of highly different sizes and mostly located in nature protection areas. Therefore, different processes might shape the genetic structure of small and up to 40 times larger populations. First, we estimated the level of heritability of the different traits under study with the expectation that these estimates don’t differ among population subsets. Second, we estimated population differences with the expectation that the effect of genetic drift is more prevalent in small populations, resulting in similar QST and FST values, whereas large populations could either experience unifying or diversifying selection, depending on whether the population sites are experiencing the same selection forces or not. Such information could help in identifying appropriate management actions for small Trollius europaeus populations by providing information on the suitability of plant and seed material for transplants to reinforce populations.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Material & Methods

Study Species Characterized by its bright yellow, globose flower, Trollius europaeus L. (Ranunculaceae) is a perennial, hermaphroditic, self-incompatible herb occurring in moist habitats throughout northern and mid-Europe (Jaeger and Després, 1998, Lauber and Wagner, 2001, Pellmyr, 1992, Pellmyr, 1989). Up to six fly species of the genus Chiastocheta form a highly specialized nursery pollination system with T. europaeus (Pellmyr, 1989, Pellmyr, 1992, Jaeger and Després, 1998), where Chiastocheta are the sole pollinators of globeflowers (Pellmyr, 1989). Given the small body size of Chiastocheta (~5 mm), pollen dispersal between populations might be limited. Seed dispersal might be limited too, as seeds do not have any morphological dispersal aides.

Figure 1: Location of the eight Trollius europaeus populations in Switzerland. Large populations are symbolised by a filled circle, small populations by a filled triangle. The grey areas and lines are lakes and river system, respectively. Reproduced with the permission of swisstopo (JA100120).

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Study sites and plant material Seed and leaf material was collected from eight T. europaeus populations in eastern Switzerland (Figure 1, Table 1). Population sizes ranged between 140 and 820,690 flowers (see Klank et al., 2010 for a detailed description on population size). Four of them were classified as “small” with 140 to 1’120 flowers per population and the other four were classified as “large” populations with 43’360 to 820’700 flowers per population. The distances between populations were 1.57 – 37.64 km, and populations were located at 537 to 1250 m a.s.l. (Table 1, 2).

Genetic diversity and differentiation in neutral markers (FST) To determine population differentiation in neutral marker loci we used an Amplified Fragment Length Polymorphism (AFLP) survey in 15 – 22 individuals per population (Table 1). Using the three primer pairs Eco-ATC/Mse-CAG, Eco-ATC/Mse-CAT and Eco- AGA/Mse-CTC (Després et al., 2002) we obtained 76 polymorphic loci for analysis. A detailed description of the protocol and scoring procedure can be found in the supporting information.

The inbreeding coefficients (FIS) were calculated with FAFLPcalc (Dasmahapatra (2008).

Expected heterozygozity (HE) and overall (i.e. among all, among small and among large populations) as well as pair-wise population differentiations (FST) were calculated in

AFLPSURV (Vekemans et al., 2002). FST values were obtained using the Bayesian approach with non-uniform prior distributions and under consideration of the FIS estimates. The significance of the overall FST estimates were calculated with 1000 permutations of individuals among populations while the 95% confidence intervals (CI) of the estimates were calculated with a jackknife procedure over loci. Significance of the pairwise FST values were checked by calculating the 95% CI based on 4999 permutations The pairwise FST were then modified according to Rousset (1997) and tested for isolation by distance using IBD (Bohonak, 2002) and log-transformed geographic distances with 1000 randomizations.

2 Genetic differentiation in quantitative traits (QST) and narrow-sense heritability (h ) Seeds of 7-8 seed families per population were cold stratified on moist commercial germination soil at 4°C in the dark for four months and then placed in growth cabinets for 10 weeks for germination (MLR-351H, SANYO Electric Co. Ltd.; alternating 12 h light cycles at 15 °C, 50 % relative humidity and 14.4 klx). Seedlings were then moved into greenhouse compartments and 5-10 individuals of each seed family (Ntotal = 426) were transplanted after

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION two weeks into 0.5 L pots filled with commercial soil. Plants were then grown under addition of artificial lights (20 klx) from 8am to 6pm and 20/16 °C at day- and night-time, respectively. Pot positions were randomized every two weeks. After transplanting the seedlings, we recorded the number of leaves. At the end of the experiment (i.e. after 24 weeks), we recorded again the number of leaves, measured stalk lengths of all leaves, leaf area of all leaves and oven-dried above and below ground biomass. We further calculated the ratio above/below ground biomass and the relative growth rate (RGR) for the total number of leaves using log-transformed values. Each trait was analysed for differences between size class and populations using an ANOVA. We grouped populations within size class and seed families within populations. A Tukey HSD test was performed to evaluate differences between population pairs. Using a half-sibling design, the narrow sense heritability was calculated following Petit et al. 2 (2001) as h = 4VFAM / (4VFAM + VE). VFAM represents the seed family variance component and VE the residual variance. QST were calculated as QST = VPOP / (8VFAM + VPOP) where VPOP is the population variance component. Variance components were obtained using a fully randomized Linear Mixed Effects Model with the REML method and seed families nested 2 within populations as random factors. The 95% CIs for h and QST were obtained by applying a bootstrap across seed families with 5000 iterations using the statistical software R, version 2.10 (RDevelopmentCoreTeam, 2009).

To determine the relationship of QST and FST, we calculated the difference of each bootstrap

QST and a randomly chosen jackknifed FST, analogous to an equivalence test. The 95%CI of these difference measures (CIdelta) indicates if QST is bigger (CIdelta with a positive range), similar (CIdelta including ‘0’) or smaller (CIdelta in the negative range) than FST.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Results

Genetic diversity and differentiation in neutral markers (FST)

Expected heterozygosity (HE) ranged from 0.26 – 0.34 (Table 1) and did not differ between size classes (Wilcoxon test: W = 4, P = 0.343). The FST among all populations was 0.033 with a CI of [0.0300, 0.0337] based on a FIS of 0.109. The FST of the four small populations was similar (FST = 0.0329; CI: [0.0336, 0.0407], based on FIS = 0.144) while the estimate for the four large populations was smaller (FST = 0.0269; CI: [0.0241, 0.0285], based on FIS =

0.145). Population pair-wise FST ranged from 0.015 – 0.0807 (P < 0.05 for all but one comparison; Table 2). Pair-wise FSTs tended to increase with distance among all populations (Z = 1.209, r = 0.466, one-sided P = 0.066, Fig. 2).

Quantitative traits While small and large populations showed similar growth traits (P > 0.1 for all traits), all traits differed among populations and seed families (P < 0.03 for all tests; see Table S1 for trait averages and Table S2 for Anova results in the electronic supplemental material). For population pairwise trait comparisons, the Tukey HSD test revealed differences in 45.8% comparisons (P < 0.05 for those tests; Table S3 in the electronic supplemental material). Arvenbuel had the highest frequency of significant trait differences to all other studied populations (33 out of 42 comparisons; P < 0.02). For pairwise comparisons between small populations, 13 out of 36 comparisons differed (P < 0.05), while for large populations 21 out of 36 were different (P < 0.05).

Narrow-sense heritability and QST vs. FST 2 In the full dataset, h ranged from 0.18 to 0.35 and QST ranged from 0.09 to 0.59 for all traits (Table S4 in the electronic supplemental material). In accordance to the Anova results of no 2 differences between size classes, h and QST for the two size classes (small and large populations, respectively) were similar as indicated by the overlapping CIs of the respective traits despite of above ground biomass, where h2 was higher among the small than among the large populations (Table S4).

Using the delta criterion described above, we found that the majority of the QST estimates exceeded the FST estimates (12 out of 18 comparisons, Table 3). For the datasets using all populations as well as in the subset of large populations, QST > FST was found for all traits except for below ground biomass. QST of below ground biomass did not differ from FST in 68

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION both population subsets as well as when it was estimated over all populations. Analysing the subset of small populations, no differences between QST and FST were found in all traits but the ratio above/below ground biomass and RGR leaf number, where QST exceeded FST.

Table 1: Population characteristics of the Trollius europaeus study populations and sample sizes used in the experiments. quantitative trait neutral genetic analyses analyses no of H size (no of no of no of E size individuals (expected population elev. coordinates1 flowers per seed individuals class per seed hetero- population) families for AFLP family) zygosity) Kafer small 740 708'454/252'875 140 8 5 - 10 19 0.317 F2 small 745 682'861/235'022 230 8 6 - 8 22 0.279 F15 small 712 705'852/257'047 310 7 7 – 8 15 0.301 F2527 small 612 707'931/254'335 1120 8 5 – 9 22 0.285 F34 large 597 709'868/257'254 43360 7 6 - 8 15 0.300 B3 large 537 702'884/244'312 57780 8 5 - 7 21 0.261 F41 large 752 711'868/241'711 86460 8 6 - 8 21 0.323 Arvenbuel large 1250 732'336/223'768 820700 7 5 - 8 21 0.336 1 Coordinates in meters according to the Swiss topographical maps (Bundesamt für Landestopographie, Wabern, Switzerland)

Table 2: Geographic distances in km and pairwise FST for the eight Trollius europaeus populations. Pairwise FST were significantly different from zero except B3-F2527. Small populations Large populations Kafer F2 F15 F2527 F34 B3 F41 Arv. Kafer - 0.056 0.023 0.019 0.033 0.038 0.015 0.018 Small F2 31.23 - 0.046 0.065 0.081 0.046 0.038 0.056 pop. F15 4.93 31.89 - 0.015 0.043 0.017 0.012 0.029 F2527 1.57 31.69 3.42 - 0.020 0 0.026 0.042 F34 4.71 35.08 4.01 3.58 - 0.021 0.024 0.03 Large B3 10.3 21.76 13.01 11.27 14.84 - 0.017 0.051 pop. F41 11.71 29.75 16.53 13.28 15.83 9.86 - 0.025 Arv. 37.64 50.7 42.54 39.12 40.42 36.4 27.19 - Arv. is used as abbreviation for Arvenbuel.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

large - large large - small small - small ) ST F /1− ST F pairwise ( 0.00 0.02 0.04 0.06 0.08

51020304050

geographic distance [km] Figure 2: Isolation by distance graph relating pairwise neutral genetic differences (FST / (1-FST)) to log-scaled geographic distances [km] for the Trollius europaeus populations of different sizes. Filled circles indicate pairs of large – large populations, open circles combinations of large – small populations and filled triangles of small – small populations.

Table 3: Differences between Qst and Fst values (delta: Qst-Fst) including confidence intervals for six plant traits in small, large and all Trollius europaeus populations under study. Delta values with CIdelta including zero are printed in italic. delta: Qst-Fst all large small lower upper lower upper lower upper CIdelta CIdelta CIdelta CIdelta CIdelta CIdelta above ground biomass 0.1076 0.4461 0.1884 0.8340 -0.0396 0.2378 below ground biomass -0.0302 0.1361 -0.0162 0.3134 -0.0407 0.0056 ratio above/below 0.2186 0.7400 0.2358 0.9730 0.1789 0.8260 average stalk length 0.1074 0.4462 0.0933 0.8115 -0.0400 0.1489 total leaf area 0.1062 0.4656 0.1923 0.8554 -0.0395 0.3278 RGR leaf number 0.1148 0.4816 0.1163 0.6048 0.0407 0.9605

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Discussion

Across the eight Trollius europaeus populations, QST values were frequently larger than FST indicating diversifying selection. While diversifying selection was also prevalent in the large population subset, the small population subset had QST values similar to FST for four of the six traits. Therefore, genetic drift cannot be excluded as main driver of population differentiation in above- and below ground biomass as well as average stalk length and total leaf area in small populations. Small populations are less resistant to random changes in the allelic frequencies caused by genetic drift than large populations (Ellstrand and Elam, 1993). The results of our study suggest that this stochastic process acts among small populations of T. europaeus. The test for isolation by distance indicates that gene flow tended to decrease with geographic distance: while the small populations in less than five kilometre distance seemed to be well connected by gene exchange, F2, the population farthest away, differed from the other small populations to a higher degree. However, the trait values of F2 were similar to the others with only four out of eighteen population pair-wise trait comparisons being different from zero suggesting that F2 is not under divergent selection from the other small populations. Willi et al. (2007) found in Ranunculus reptans populations of different sizes not only an enhanced influence of drift but also an increased divergent selection in small populations. Besides a decrease of divergence with increasing population size, quantitative trait divergence also decreased but their estimates exceeded those of the neutral genetic differentiation. In T. europaeus, the QST values were not larger but also not smaller than the

FST values, indicating that diversifying and unifying selection can be ruled out. Among all as well as among large populations, the indication for diversifying selection is a conservative finding, because several effects which might bias QST estimates such as maternal effects, dominance, epistatis and non-additive components, lower the QST estimate relative to the FST (Lopez-Fanjul et al., 2003, Yang et al., 1996, Goudet and Buchi, 2006,

Whitlock, 1999). Our finding of a QST larger than FST is in accordance with other studies (see reviews by Leinonen et al., 2008, McKay and Latta, 2002, Merila and Crnokrak, 2001), indicating that diversifying selection is the predominant cause of plant population differentiation. However, the relatively low FST found in the AFLP survey suggests that while there may be some differentiation on the level of quantitative genetic traits, gene flow counteracted differentiation of neutral genes in T. europaeus to a large extent, a result which

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION has also been found in other species (e.g. Chun et al., 2011, Sanou et al., 2005, Sambatti and Rice, 2006, Volis and Zhang, 2010). Diversifying selection can be found along environmental gradients resulting into local adaptation (Leimu and Fischer, 2008). The main information on environmental differences among populations in our study is the altitudinal origin: the study sites were located between 540 and 749m a.s.l., despite of the population called Arvenbuel which was located at 1250m a.s.l.. Population pairs including Arvenbuel were similarly differentiated in neutral genetic markers as the other population pairs. In contrast, Arvenbuel showed the most differences in plant traits to all other populations analysed (see Table S3). For example, for below ground biomass, no significant pairwise comparisons were found except for pairs including Arvenbuel. This population had the lowest mean of all populations in this trait. Generally, individuals reared from seeds of Arvenbuel had the lowest mean values for almost all traits measured. As growth of plants from higher altitudes reared at lower altitudes can be reduced, as shown e.g. by Clausen et al. (1941) for Potentilla glandulosa and Achiella lanulosa, our results suggest that the greenhouse conditions were probably closer to the field conditions of the populations at lower altitudes. Excluding Arvenbuel from the analysis of quantitative genetic variation changed the result of the QST - FST comparison in the large population data set as well as for the whole data set: QST estimates were similar to FST estimates suggesting that genetic drift shaped the population differentiation (details not shown). This change in the QST estimates indicates that indeed, selective forces might change with altitude.

Generally, potential responses to selection are positively related to the heritability of the respective traits (Silvertown and Charlesworth, 2001). All traits measured in T. europaeus showed reasonable estimates of heritability. Also in the population subsets, the estimates were in all but one case above the median heritability value of ca. 0.15 found in a literature survey on 1214 plant traits (Geber and Griffen, 2003). The heritability estimates in T. europaeus suggests that the observed traits might be formed by selective forces, if these are strong enough. But observed estimates might also overestimate in situ values (Geber and Griffen 2003). Despite the altitude of population, we did not test for further potential environmental variation which might shape differentiation among populations. However, as quantitative trait differentiation did not exceed the differentiation in neutral traits in combination with the restricted area covered by our sampling approach, we conclude that no other major selective forces were present.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Conclusion for conservation of small populations Given that ecotypic variation can play an important role in conservation management when dealing with habitat restoration or reinforcing populations (McKay et al., 2005, Hufford and Mazer, 2003, Vergeer et al., 2004, Galen et al., 1991), information on the suitability of plants of non-local origin is important. To circumvent potential outbreeding depression through artificially combination of distinct populations (Edmands, 1999, Tallmon et al., 2004), populations with similar trait measures should be used. A further conservation concern is the level of genetic diversity (Leimu et al., 2010, Schemske et al., 1994, Ellstrand and Elam, 1993), which in our study was similar across the populations of different sizes, indicating that small populations are not genetically depleted and generally suitable as seed sources for management schemes. The diversifying selection found among all populations as well as the large population subset is thus most likely caused by the differences in elevation, highlighting that plants from higher altitudes are less suitable for use in reinforcing populations at lower elevations. Even though our results indicated that genetic drift might the main driver of population differentiation, the low neutral genetic differentiation and the finding of no divergent selection among all populations after excluding Arvenbuel suggest that the populations of similar altitude within our study form a potential conservation unit. We conclude that promoting the addition of genetic material to small populations would be a suitable conservation strategy for maintaining Trollius europaeus in small nature reserves, given that plant material originates from similar elevations.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

References

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Stöcklin, J. & Fischer, M. (1999) Plants with longer-lived seeds have lower local extinction rates in grassland remnants 1950-1985. Oecologia, 120, 539-543. Tallmon, D.A., Luikart, G. & Waples, R.S. (2004) The alluring simplicity and complex reality of genetic rescue. Trends in Ecology & Evolution, 19, 489-496. Vekemans, X., Beauwens, T., Lemaire, M. & Roldan-Ruiz, I. (2002) Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size. Molecular Ecology, 11, 139- 151. Vergeer, P., Sonderen, E. & Ouborg, N.J. (2004) Introduction strategies put to the test: Local adaptation versus heterosis. Conservation Biology, 18, 812-821. Volis, S. & Zhang, Y.H. (2010) Separating Effects of Gene Flow and Natural Selection along an Environmental Gradient. Evolutionary Biology, 37, 187-199. Whitlock, M.C. (1999) Neutral additive genetic variance in a metapopulation. Genetical Research, 74, 215-221. Whitlock, R., Hipperson, H., Mannarelli, M., Butlin, R.K. & Burke, T. (2008) An objective, rapid and reproducible method for scoring AFLP peak-height data that minimizes genotyping error. Molecular Ecology Resources, 8, 725-735. Willi, Y., Van Buskirk, J., Schmid, B. & Fischer, M. (2007) Genetic isolation of fragmented populations is exacerbated by drift and selection. Journal of Evolutionary Biology, 20, 534- 542. Yang, R.C., Yeh, F.C. & Yanchuk, A.D. (1996) A comparison of isozyme and quantitative genetic variation in Pinus contorta ssp latifolia by F-ST. Genetics, 142, 1045-1052.

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Appendix AFLP protocol Leaves from 22 randomly sampled individuals with a minimum distance of ~4 meters were collected from each population in 2006 and 2007. Leaf material was dried in silica gel immediately after collection. DNA extractions were done with the DNeasy Plant Maxi Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s protocol using 10 mg of dried plant material. DNA concentrations were measured with a Nanodrop 1000 (Thermo Scientific) and averaged at ~24 ng/µl. Preselective and selective AFLP primer pairs were Eco-ATC – Mse-CAG, Eco-ATC – Mse-CAT and Eco-AGA – Mse-CTC as used by Després et al. (Després, Loriot et al. 2002). For the digestion-ligation step, first 10 µl extracted DNA (~240 ng) were digested for 1 h at 37°C in a 27 µl restriction mix consisting of 2 U EcoR1, 2 U Mse1, 0.4 µl bovine serum albumin (10 mg/ml), and 4 µl NEB2 buffer (10x, BioLabs). Second, PCR adaptors were ligated to the DNA fragments for 3 h at room temperature by adding 14 µl ligation mix consisting of 15 pmol EcoR1 adaptor, 136.35 pmol Mse1 adaptor, 0,1 µl bovine serum albumin, 5 µl ATP (10 mM), 1 µl NEB2 (10x, BioLabs), and 20 U T4 Ligase (BioLabs). This step was finished by inactivating the ligase for 10 min at 65° C. For preselective amplification 1.5 µl restriction-ligation mix were added to 19 µl PCR reaction mix containing 157.5 ng of each preselective primer Eco-A and Mse-C, 0.3 µl dNTP mix (25 mM), 1.5 µl MgCl2 (25mM), 4 µl Promega GoTaq© reaction buffer (5x, no MgCl2) and 0.375 U GoTaq polymerase (Promega). PCR reactions cycling conditions were as follows: 1 cycle (94°C for 3 min), 20 cycles (94°C for 30 s, 60° for 30 sec., 72°C for 60 s) and 1 cycle (72°C for 1 min). This preselective PCR product was then 1:20 times diluted with 1x TE buffer. Selective amplifications were performed by adding 3 µl of the diluted preselective

PCR product to 11 µl selective PCR mix containing 1.5 µl MgCl2 (25 mM), 0.5 µl dNTPs

(25 mM), 3 µl GoTaq reaction buffer (5x, no MgCl2) and 0,375 U GoTaq polymerase. PCR cycling conditions were 1 cycle (94°C for 3 min), 10 cycles (94°C for 30 s, 65°C for 30 s with a reduction of the annealing temperature of -1°C from 65° to 56°), then 25 cycles (94°C for 30 s, 54°C for 30 s, 72°C for 1 min) and 1 cycle (72°C for 60 seconds). All PCRs were carried out using a Bio-Rad dyad cycler. Fragments were separated on an ABI3720xl (GeneScanTM 500 LIZ® sizing standard) and Genemapper 3.5 software (Applied Biosystems) used for fragment reading. We used the following procedure to generate the fragment absence/presence matrix: Preliminary peak positions were selected manually for each primer pair in Genemapper 3.5 (ABI) and peak height data was recorded. Using AFLPScore 1.4 (Whitlock et al., 2008) we retained those loci that had an error rate of < 5%

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION using 28 duplicated samples, resulting in a total of 101 loci. As recommended by Lynch and Milligan (1994), we retained only loci with a frequency above 3/n, leading to a set 76 loci for analysis.

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Table A1: Means and standard errors of each trait of the greenhouse experiment calculated for each Trollius europaeus population and population size class.

Kafer F2 F15 F2527 B3 F34 F41 Arvenbuel mean 0.419 0.533 0.744 0.443 0.560 0.332 0.618 0.201 std. error 0.030 0.040 0.070 0.035 0.068 0.024 0.048 0.024 above ground small large biomass [g] mean 0.532 0.437 std. error 0.024 0.025 mean 2.193 2.206 2.139 2.014 1.670 1.974 2.111 1.335 std. error 0.109 0.135 0.144 0.120 0.128 0.111 0.132 0.112 below ground small large biomass [g] mean 2.140 1.799 std. error 0.063 0.064 mean 0.185 0.238 0.341 0.211 0.334 0.162 0.296 0.148 ratio std. error 0.005 0.008 0.021 0.009 0.027 0.005 0.014 0.013 above/below small large ground biomass mean 0.242 0.237 std. error 0.007 0.010 mean 49.18 53.86 61.84 50.12 51.69 48.14 55.68 38.52 std. error 2.15 2.13 2.29 1.63 2.01 1.65 1.87 1.62 average stalk small large length [mm] mean 53.61 48.96 std. error 1.08 1.00 mean 81.48 100.25 125.06 84.31 93.91 69.17 109.92 34.57 std. error 4.99 6.99 9.88 5.80 9.49 4.60 7.86 4.33 total leaf area small large [cm2] mean 97.36 78.77 std. error 3.68 4.00 mean 0.046 0.059 0.060 0.048 0.062 0.038 0.055 0.031 RGR leaf std. error 0.002 0.002 0.002 0.002 0.003 0.002 0.002 0.003 number small large -1 [N d ] mean 0.053 0.047 std. error 0.002 0.001

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Table A2: ANOVA results for each trait of Trollius europaeus measured in the greenhouse experiment with size class, population and seed family as explanatory variables. Traits were log or square root transformed when necessary to obtain a normal error distribution. above ground biomass Df SumSQ MeanSQ F value P size class 1 9.82 9.815 1.141 0.3265 population [size class] 6 51.61 8.602 12.282 0.0000 seed family [population] 53 37.1 0.700 1.665 0.0039 Residuals 365 153.55 0.421 below ground biomass Df SumSQ MeanSQ F value P size class 1 0.019 0.019 0.059 0.8157 population [size class] 6 1.903 0.317 26.906 0.0000 seed family [population] 53 0.625 0.012 1.437 0.0305 Residuals 365 2.993 0.008 ratio above/below ground biomass Df SumSQ MeanSQ F value P size class 1 0.804 0.804 0.853 0.3913 population [size class] 6 5.652 0.942 6.789 0.0000 seed family [population] 53 7.354 0.139 1.870 0.0005 Residuals 365 27.083 0.074 average stalk length Df SumSQ MeanSQ F value P size class 1 4.666 4.666 3.711 0.1023 population [size class] 6 7.544 1.257 2.824 0.0187 seed family [population] 53 23.598 0.445 1.926 0.0003 Residuals 365 84.373 0.231 total leaf area Df SumSQ MeanSQ F value P size class 1 145.91 145.909 1.2139 0.3128 population [size class] 6 721.18 120.197 12.3072 0.0000 seed family [population] 53 517.62 9.766 1.5198 0.0151 Residuals 365 2345.51 6.426 RGR leaf number Df SumSQ MeanSQ F value P size class 1 0.004 0.004 0.691 0.4375 population [size class] 6 0.038 0.006 13.788 0.0000 seed family [population] 53 0.024 0.0005 1.677 0.0038 Residuals 351 0.096 0.0003

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

Table A3: Tukey HSD P-values between Trollius europaeus population pairs. Values printed in bold are smaller than 0.05. above ground biomass Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.6900 x F15 0.0034 0.3475 x F2527 1.0000 0.7174 0.0044 x B3 0.0245 0.6908 0.9998 0.0293 x F34 0.9999 0.8885 0.0150 0.9999 0.0757 x F41 0.0000 0.0037 0.7860 0.0000 0.5296 0.0000 x Arv 0.0002 0.0000 0.0000 0.0002 0.0000 0.0001 0.0000 x below ground biomass Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.9998 x F15 0.9985 0.9999 x F2527 0.9525 0.9974 0.9998 x B3 0.8760 0.9821 0.9959 0.9999 x F34 0.9831 0.8713 0.7968 0.4794 0.3515 x F41 0.8130 0.5235 0.4301 0.1654 0.1083 0.9995 x Arv 0.0065 0.0253 0.0545 0.1634 0.3375 0.0003 0.0000 x ratio above/below ground biomass Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.0190 x F15 0.0000 0.0000 x F2527 0.7717 0.6543 0.0000 x B3 0.0000 0.0000 1.0000 0.0000 x F34 0.9914 0.0013 0.0000 0.2751 0.0000 x F41 0.0000 0.0027 0.9426 0.0000 0.9577 0.0000 x Arv 0.2928 0.0000 0.0000 0.0051 0.0000 0.8212 0.0000 x average stalk length Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.5308 x F15 0.0000 0.0841 x F2527 0.9927 0.9576 0.0031 x B3 0.0791 0.9629 0.7140 0.4268 x F34 0.6847 0.9999 0.0689 0.9861 0.9332 x F41 0.0002 0.1307 0.9999 0.0055 0.8228 0.1075 x Arv 0.1494 0.0003 0.0000 0.0198 0.0000 0.0010 0.0000 x total leaf area Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.5713 x F15 0.0017 0.3425 x F2527 0.9999 0.7003 0.0039 x B3 0.0503 0.9030 0.9907 0.0856 x F34 0.9857 0.9847 0.0509 0.9961 0.4076 x F41 0.0000 0.0117 0.9265 0.0000 0.4613 0.0006 x Arv 0.0007 0.0000 0.0000 0.0005 0.0000 0.0000 0.0000 x

CHAPTER 4 – NEUTRAL AND QUANTITATIVE GENETIC DIFFERENTIATION

RGR leaf number Kafer F2 F15 F2527 B3 F34 F41 Arv Kafer x F2 0.0007 x F15 0.0006 0.9999 x F2527 0.9996 0.0114 0.0088 x B3 0.0000 0.0844 0.1478 0.0000 x F34 0.9999 0.0002 0.0002 0.9896 0.0000 x F41 0.0000 0.9957 0.9993 0.0009 0.3899 0.0000 x Arv 0.1409 0.0000 0.0000 0.0521 0.0000 0.3055 0.0000 x

2 Table A4: QST and h with 95 % confidence intervals for each trait calculated for subsets (four small, four large) as well as all Trollius europaeus populations under study. 2 QST h 2 QST lower CI upper CI h lower CI upper CI all 0.3421 0.1337 0.4804 0.2767 0.2573 0.3276 above ground biomass large 0.5127 0.0416 0.4830 0.2193 0.1934 0.2791 small 0.0853 0.0000 0.2675 0.3425 0.3074 0.4139 all 0.0929 0.0040 0.1649 0.3455 0.2901 0.5601 below ground -9 -9 biomass large 0.93e 0.65e 0.0484 0.3341 0.1822 0.5984 small 0.1457 0.18e-8 0.1922 0.3182 0.2113 0.5364 ratio all 0.5912 0.2503 0.7695 0.1798 0.1529 0.2406 above/below ground large 0.6863 0.2578 1.0000 0.1544 0.0000 0.5494 biomass small 0.5354 0.2212 0.8550 0.2030 0.1616 0.2941 all 0.1810 0.0403 0.2624 0.3282 0.3057 0.3831 average stalk length large 0.4380 0.1257 0.8652 0.1878 0.0351 0.5143 small 0.0603 0.0000 0.1924 0.4151 0.2951 0.6353 all 0.3833 0.1482 0.4927 0.2277 0.1648 0.4794 total leaf area large 0.5255 0.2131 0.8733 0.2129 0.0448 0.4823 small 0.1268 0.0000 0.3611 0.2451 0.0947 0.5551 all 0.3509 0.1480 0.5041 0.2783 0.1838 0.5302 RGR number of leaves large 0.3408 0.1412 0.6126 0.3668 0.1623 0.6181 small 0.4243 0.0788 1.0000 0.1243 0.0000 0.4670

CHAPTER 5 - CONCLUSIONS

CONCLUSIONS

CHAPTER 5 - CONCLUSIONS

Trollius europaeus in the Swiss Plateau

In our study we sought to understand the effects of habitat fragmentation on the population persistence of the globeflower Trollius europaeus as a case study in Central Europe using a field study, greenhouse experiments and a neutral genetic marker survey. Based on the results presented in the chapters 2 - 4, we found no clear indication that the small populations in our study performed worse than the large populations. Neither reproductive success, genetic diversity nor competitive ability were related to population size. For reproductive success we found that Chiastocheta abundance and plant population density, rather than population size, was the main driver given the intricate nursery pollination mechanism. Population density had a mitigating effect on seed predation, which was confirmed by the decreasing effect of local flower density on Chiastocheta abundance. Mean overall population differentiation was low (FST of 0.033), indicating that the populations have not undergone substantial differentiation in neutral markers. Using the QST vs FST comparison, we found indications that natural selection is acting within T. europaeus populations, with diversifying selection prevalent in large populations, while small populations had non-significant QSTs, indicating unifying selection.

Overall, our results show that the 19 Trollius europaeus populations studied in the Swiss lowlands are able to persist. We found no negative effects of small population size on the reproductive success, genetic diversity or competitive ability, indicating that these populations are not yet affected by the ongoing land conversion. The main driver was Chiastocheta abundance, a pollinator that also acts as a seed predator, making plant density an important characteristic determining reproductive success. The fact that Chiastocheta abundance, and not plant population size, determined reproductive success shows that the intricate nursery pollination system is still intact in these populations, even though pollinator interactions are often disturbed through land use modifications and habitat fragmentation (Potts et al., 2010, Kolb, 2008, Steffan-Dewenter and Westphal, 2008). Chiastocheta abundance significantly decreased reproductive success and was locally related to flower density, explaining how plant population density had a positive effect on reproductive success by providing a “safety in numbers” environment for individual globeflowers.

In Europe, Trollius europaeus is becoming locally extinct in those areas where land use change has destroyed suitable habitats. In Germany, globeflowers are listed as endangered 84

CHAPTER 5 - CONCLUSIONS due to drainage and over-fertilization of habitats (Ludwig and Schnittler, 1996), and listed as endangered, vulnerable or rare in other European countries (Muncaciu et al., 2010). In Switzerland as a whole the species is classified as of “least concern”, but as “vulnerable” in the Swiss plateau where our study populations are located (Moser et al., 2002). Our results indicate that globeflower populations are able to persist under intensifying land conversion and agriculture, making the protection of the remaining populations of the emblematic globeflower a worthwhile cause. Regarding conservation management, it is important to recognise that other factors than population size, which is often considered to be the main factor, are determining reproductive success and thus population persistence. In our study species, population density was shown to have a mitigating effect on seed predation, and thus needs to be taken into consideration when making management decisions. Another option of population management could be reinforcing globeflower populations through seed transfers from other natural populations. The results from chapter 3 and 4 showed that genetic differentiation between populations was low, though diversifying selection was acting in some of the populations. Using seed material from populations located on similar elevations and with similar surrounding landscape matrixes could be a viable option to ensure population survival in cases where population sizes have become very low.

In general, the Trollius europaeus populations are doing well within the fragmented landscape of the Swiss plateau. Still, we need to consider the possibility that we did not detect negative effects of small population size due to time lag effects. Extinction debts or time lag effects following environmental change have been described in theory (Krauss et al., 2010, Kuussaari et al., 2009, Jackson and Sax, 2010, Tilman et al., 1994, Helm et al., 2006, Hahs et al., 2009). Negative effects of habitat change might come into effect only with a considerable time lag effect, especially in species with longer lifespans. If mortality only slightly exceeds the establishment of seedlings, it might take decades before long-lived species show first signs of detrimental consequences of habitat change. In Switzerland, most of the drastic changes in the landscape took place within the last 100 years, with the majority of land use change taking place in the post-war period due to increased human population growth, often associated with biotic homogenisation and habitat destruction (e.g. McKinney, 2006, Foley et al., 2005, Thompson and Jones, 1999) and the intensification of agriculture (Tilman et al., 2002, Tilman et al., 2001). As Trollius europaeus is a perennial, herbaceous plant, it is difficult to judge whether some of the

CHAPTER 5 - CONCLUSIONS observed results might be a result of plants being relicts of previously larger populations. This is especially the case for the assessment of neutral genetic diversity using AFLPs, as this technique does not allow us to assess contemporary gene flow. It is thus possible that the missing link between genetic diversity and population size and the low FST could be relicts of historical gene flow which have been preserved so far due to long life spans of individual plants. Furthermore, no comprehensive and reliable data exists on the former distribution of Trollius europaeus in our study area. While we can presume that many populations have been lost or reduced in size in the past decades (personal communication with local land owners), it is possible that the studied populations are historically small which may be reflected in the low level of genetic variation (McKay et al., 2005, Hamrick and Godt, 1996).

General implications for nature conservation

With now approximately 7 billion people living on earth, almost no ecosystem remains untouched by human activities (Bloom, 2011, Vitousek et al., 1997). Land use changes related to agriculture or urban developments are a common cause for habitat fragmentation across the globe and are occurring with increasing speed as demands for resources increase, frequently causing losses in biodiversity (Tilman et al., 2001, McKinney, 2006, Thompson and Jones, 1999). As biodiversity represents one of humankind most valuable resource, understanding the consequences of habitat fragmentation on species persistence provides a needed basis for managing and protecting the remaining intact ecosystems. Biodiversity can act as an insurance mechanism when species become locally extinct, providing a buffer against environmental fluctuations (Loreau et al., 2001, Yachi and Loreau, 1999). It is thus important to gain a thorough understanding of processes driving population persistence not only in already rare plants, but also in species that are still common and/or characterized by non-generalist pollinators. Our study highlights the importance to carefully assess what determines the quality of reproductive success – in our case rather plant density than pure plant number – and shows that a good understanding of the pollination system and population dynamics is needed to make sound management decision.

CHAPTER 5 - CONCLUSIONS

References

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CHAPTER 5 - CONCLUSIONS

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ACKNOWLEDGEMENTS

I would like to thank Jaboury Ghazoul and Andrea Pluess for their guidance throughout my

PhD, Jaboury Ghazoul for providing the opportunity to write this thesis as well as the Swiss

Federal Institute of Technology Zurich for funding this study, the department of nature conservation of the canton Zurich for their permission to study populations located on nature protection areas as well as the private land owners for allowing access and the Eschikon research facility staff for their assistance.

I would also like to thank Jaboury Ghazoul, Andrea Pluess , Prof. Widmer and Prof. Kunin for their time and effort taken to examine my thesis.

My special thanks to:

ADRIEN – ALINE – ANNE – BERNT – CHRISTOPH – CONSTANZE – ERNEST – ESTHER – FINE –

FLORIAN – HOLGER – JULIA – PHIL – SMITHA – THOMAS – THE FUNGHI PEOPLE - THE ITES

CURRICULUM VITAE

Personal data

Name: Charlotte Klank born: 03.09.1979, Marburg / Germany nationality: German

Education 11/2011 Graduation Dr. sc. (ETH Zurich) 04/2006 – 11/2011 PhD student ETH Zurich, Ecosystem Management Thesis Title: “Trollius europaeus in a fragmented landscape: Reproductive success, genetic diversity and trait differentiation in a nursery pollinated plant” 11/2005 Graduation as Dipl.-Biologin („Sehr Gut”), University of Goettingen Diploma thesis on Diplomarbeit im Bereich der botanischen OEkologie 05/2004 – 09/2004 field work for the diploma thesis, Sulawesi / Indonesia 02/2002 – 12/2002 Study abroad Biology, University of of Queensland, Australien courses in Entomology and zoological Ecology 10/1999 – 11/2005 Studies of Biology, University of Goettingen Majors: Zoology (Entomology), Botany (Ecology) and Geology 04/1999 – 10/1999 Chemistry, University of Goettingen 10/1998 – 04/1999 Mineralogy, University of Hamburg 06/1998 Abitur, IGS Goettingen (Grade 1,3)

Work experience 02/2011 – dato iForest.ch / iGarten.ch freelance: translation of iPhone applications (german – English) 07/2011 – 08/2011 University of Freiburg, Geobotany field assistant, pathogen survey on tree species in EU project BACCARA 05/2011 – 06/2011 Biodiversity and climate research centre (BiK-F), Frankfurt lab assistant: Establishment of genetic marker system 04/2005 – 05/2005 Plant Ecology and Ecosystem Research, University of Goettingen Supervision of advanced practical in Ecology

CURRICULUM VITAE

Internships 12/2005 – 02/2006 Museum of the geological faculty, University of Goettingen Preparation of the amber collection 08/2000 – 09/2000 ARCHELON (Sea Turtle Protection Society of Greece), Griechenland Protection of nesting sites and public awareness campaign 02/2000 – 04/2000 Flamingo Primary School, Namibia Teaching in German, Englisch, Biology and Sports 01/1999 – 04/1999 Max-Planck-Institute of biophysical Chemistry, Goettingen Introduction to molecular biology techniques

Courses 07/2009 Introduction to Access 2007 (ETH Zurich) 09/2008 Life Cycle Assessments and Ecobalance (University of Zurich) 06/2008 Business Tools: Effective communication and negotiation (ETH Zurich) 09/2006 Project management (University of Zurich)

Languages German: native Englisch: fluent French: good knowledge

Computer skills Very good MS Office skills (Word, Excel, PowerPoint) Basic knowledge of Access 2007 Good knowledge of the statistical software R Good knowledge of the various genetic programs