UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de botanique et de biologie végétale Prof. R. Spichiger Ecology and genetics of the rare plant Aster amellus L. in a fragmented landscape Thèse présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie par Romain Mayor de Genève (GE) Genève 2008 Contents Chapter 1 General Introduction 5 Chapter 2 Identification and characterization of eight 19 microsatellite loci in Aster amellus L. (Asteraceae) Chapter 3 Inbreeding and inbreeding depression in the rare perennial 27 Aster amellus L. in natural conditions with biotic and ecological interactions Chapter 4 Effects of distance, density and population size on 53 gene flow in the rare Aster Amellus L. in a fragmented landscape Chapter 5 Effects of ecology and genetic among fragmented 79 populations of the long-lived Aster amellus L. in experimental conditions with biotic and abiotic treatment Chapter 6 Synthesis 103 Résumé 108 Chapter 1 General Introduction 5 CHAPTER 1 This thesis deals with the effects of fragmentation on a rare plant species. We will first introduce the theoretical background and present general aspects of the study, thus giving the reader the option to go directly to the synthesis in the final chapter. The corpus of the thesis (Chapters 2 to 5) itself presents important information including graphics and details of procedures. Complementary information, provided in the introduction, can help to understand the corpus, but care has been taken to avoid overlap of contents. From Sadi Carnot to fragmentation In the last 20 years, there has been an increasing interest in the population biology of wild species to assess the threats induced by human activity. Indeed, since the thermodynamic treatises of Sadi Carnot ( 1824), it has expanded from a rural lifestyle to an urban and industrialised one. Extensive farming has been replaced by intensive farming: traditional, fragmented, agricultural land has been grouped to form extended areas without natural habitats, and semi-natural habitats have been abandoned. This has had three evident effects. The first is the destruction of habitat leading a given population directly to its death. The second is the reduction of habitat size. The third is the fragmentation of remnant populations. We will focus on the effects of fragmentation which, as we will see, are intimately bound to the first two effects. Other consequences of the industrial revolution, such as pollution, are not considered in this study. Fragmentation Fragmentation is the process whereby one unified entity is divided into smaller entities. For a group of individuals called a population, living in a given landscape and participating together in reproduction, fragmentation of the landscape has two major impacts. First, one big population breaks up into several small populations which form a metapopulation; second, 6 GENERAL INTRODUCTION individuals previously “connected” in the reproduction process become isolated, impeding in this way the maintenance of panmixia. Small and isolated populations are more susceptible to perish through stochastic processes such as environmental perturbation, demographic change, and genetic drift (Shaffer 1987). They have to face a diminution of gene flow and therefore an increase of inbreeding and the perpetuation of genetic drift without possible rescue. In order to tackle the fragmentation issue, we studied a rare plant whose populations are like islands surrounded by intensive farming and urban areas. Inbreeding Inbreeding is caused either by autofecondation or by non-random association of conspecifics in the reproduction process (Keller & Waller 2002). As a general consequence, an increasing number of individuals in the subsequent generations become homozygote at different loci. From generation to generation, the number of homozygote loci increases at the individual level and thus at the population level. In order to assess inbreeding in small populations, we analysed the mean observed heterozygosity of the populations with 7 microsatellite markers, and related it to census sizes of the populations. Microsatellites are small regions in the DNA where base pairs are repeated in sequences of 1bp to 4bp. They are neutral codominant markers, thus ideal for studying inbreeding, drift and gene flow (Estoup et al. 1998; Ouborg et al. 1999; Ross et al. 1999). Drift Drift is a stochastic process in which the set of alleles of a population at generation t0 is progressively lost in subsequent generations (Ellstrand & Elam 1993). The reason for this is that in the reproduction process, one individual gives only half of its alleles to one offspring. 7 CHAPTER 1 Drift is less visible in large populations where the initial diversity of alleles at t0 is nearly preserved because the possibility for an individual to give more than half of its alleles is increased by the number of fecundation events. Moreover if a certain allele is rare in a given population (large or small), its chance of being transmitted to the next generation is enhanced if it is carried by a larger number of individuals, i.e. in larger populations. This leads us to the concept of purge (see section on purge). In order to assess genetic drift in small populations, we analysed the average gene diversity of populations with seven microsatellite markers and related it to census population sizes. Inbreeding depression and drift depression The threat of inbreeding is inbreeding depression. Three cases of inbreeding depression are postulated: the lethal recessive case, ovedominance, and partial dominance. The lethal recessive case is characterised by a strong selection on inbred genotypes which give no chance of survival. Overdominance is the case where homozygotes are less fit than heterozygotes; this permits survival of inbred individuals, but with a genetic load implying a constant pressure on the individual’s fitness. Partial dominance is the case where slightly or mildly deleterious recessive alleles are partially masked by dominant alleles (Charlesworth & Charlesworth 1987; Leberg & Firmin 2008). Drift depression occurs when the variability of alleles is diminished through genetic drift, thus decreasing the capacity of a population to adapt itself to changing conditions (Barrett & Kohn 1991) (see section on purge). Moreover, loss of alleles could lead to inbreeding by drift (Jacquard 1968; Glémin 2003) resulting in cases similar to those of inbreeding. 8 GENERAL INTRODUCTION In order to detect inbreeding depression in natural conditions and in controlled ex situ conditions, we measured fitness traits of plants and related them to the level of inbreeding measured with microsatellite markers. Purge A purge is the possibility of a given population to eliminate deleterious alleles. It may simply happen by genetic drift (case were drift has a positive effect) or by inbreeding (Glémin 2003). In both cases, small populations have a greater chance to experience a purge. The reason for this is that deleterious alleles are better conserved in large populations, either through lack of drift or through maintenance of deleterious alleles among heterozygous individuals. Purge may be viewed as a possible rescue for small populations, but it should not be thought of as having a generally positive effect. A good example of this is the maintenance of high polymorphism of melanin genes in insect species (True 2003); the debate about the role played here by selection is ongoing, but variability seems to be important in this case although a morph is not always advantageous. In our study, we did not test whether a purge occurred, but we introduced this term because a purge may lead to failure in the detection of inbreeding effects in small populations. Moreover we were faced with a confusing pattern between purge and maternal effects in ex situ experiments (see Chapter 5). Gene flow Gene flow is the process whereby genes “travel” throughout an entire population or among the populations of a metapopulation. In plants, gene flow is mediated by pollen dispersal, seed dispersal (Levin & Kerster 1974) and clonal propagation (Johansson 1993; Johansson & Nilsson 1993). A panmixia situation is produced by gene flow between individuals of a population. Any interruption in gene flow causes a rift in panmixia. The longer the 9 CHAPTER 1 interruption, the more we loose panmixia. One current rule says that one migrant per generation is sufficient to maintain panmixia in a metapopulation (Wright 1931). However simulations showed that 5 to 20 migrants are not sufficient to reach a Hardy-Weinberg equilibrium between populations (Lacy 1987) . Rupture of gene flow increases situations of genetic drift and in this way prevents rescue by heterosis. In this study, we analyse how gene flow is influenced by fragmentation parameters: isolation by density in terms of number of surrounding individuals around one individual and number of surrounding populations around one population, isolation by size of populations in terms of the number of counted individuals in a given site and isolation by distance. Ecological parameters We have seen how genetic parameters can influence the fate of populations; however fate of populations can also be driven by physical abiotic or biotic pressures. This is what we call environmental stochasticity (see section on fragmentation). In plant species, ecological parameters are extremely important because fate lies there: to stay in place alive or to die. Playing an evident