Fine-scale spatial genetic structure and gene dispersal in Silene latifolia.

Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global F(ST)=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5-30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal.

The effect of host plant and isolation on the genetic structure of phytophagous insects: A preliminary study on a bruchid beetle

Genetic differentiation is a consequence of the combination of drift and restriction in gene flow between populations due to barriers to dispersal, or selection against individuals resulting from inter-population matings In phytophagous insects, local adaptation to different kinds of host plants can sometimes lead to reproductive isolation and thus to genetic structuring, or even to speciation Acanthoscelides. obtectus Say is a bean bruchid specialized on beans of the Phaseolus vulgaris group, attacking both wild and domesticated forms of P vulgaris., and P coccineus This study reveals that the genetic structure of populations of this bruchid is explained mainly by their geographical location and is not related to a particular kind (wild or domesticated) of bean In contrast, the species of bean might have led, to some extent, to genetic structuring in these bruchids, although our sampling is too limited to address such process unambiguously. If confirmed, it would corroborate preliminary results found for the parasitoid species that attack Acanthoscelides species, which might show a genetic structure depending on the species of host plant

Comparative phylogeography of mutualists and the effect of the host on the genetic structure of its partners.

Whether or not species participating in specialized and obligate interactions display similar and simultaneous demographic variations at the intraspecific level remains an open question in phylogeography. In the present study, we used the mutualistic nursery pollination occurring between the European globeflower Trollius europaeus and its specialized pollinators in the genus Chiastocheta as a case study. Explicitly, we investigated if the phylogeographies of the pollinating flies are significantly different from the expectation under a scenario of plant-insect congruence. Based on a large-scale sampling, we first used mitochondrial data to infer the phylogeographical histories of each fly species. Then, we defined phylogeographical scenarios of congruence with the plant history, and used maximum likelihood and Bayesian approaches to test for plant-insect phylogeographical congruence for the three Chiastocheta species. We show that the phylogeographical histories of the three fly species differ. Only Chiastocheta lophota and Chiastocheta dentifera display strong spatial genetic structures, which do not appear to be statistically different from those expected under scenarios of phylogeographical congruence with the plant. The results of the present study indicate that the fly species responded in independent and different ways to shared evolutionary forces, displaying varying levels of congruence with the plant genetic structure

Predicting present and future intra-specific genetic structure through niche hindcasting across 24 millennia.

Paleoclimatic reconstructions coupled with species distribution models and identification of extant spatial genetic structure have the potential to provide insights into the demographic events that shape the distribution of intra-specific genetic variation across time. Using the globeflower Trollius europaeus as a case-study, we combined (1) Amplified Fragment Length Polymorphisms, (2) suites of 1000-years stepwise hindcasted species distributions and (3) a model of diffusion through time over the last 24,000 years, to trace the spatial dynamics that most likely fits the species' current genetic structure. We show that the globeflower comprises four gene pools in Europe which, from the dry period preceding the Last Glacial Maximum, dispersed while tracking the conditions fitting its climatic niche. Among these four gene pools, two are predicted to experience drastic range retraction in the near future. Our interdisciplinary approach, applicable to virtually any taxon, is an advance in inferring how climate change impacts species' genetic structures.

Comparative population genetic structure in a plant-pollinator/seed predator system.

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co-adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short-lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.

Forecasting changes in population genetic structure of alpine plants in response to global warming.

Species range shifts in response to climate and land use change are commonly forecasted with species distribution models based on species occurrence or abundance data. Although appealing, these models ignore the genetic structure of species, and the fact that different populations might respond in different ways because of adaptation to their environment. Here, we introduced ancestry distribution models, that is, statistical models of the spatial distribution of ancestry proportions, for forecasting intra-specific changes based on genetic admixture instead of species occurrence data. Using multi-locus genotypes and extensive geographic coverage of distribution data across the European Alps, we applied this approach to 20 alpine plant species considering a global increase in temperature from 0.25 to 4 °C. We forecasted the magnitudes of displacement of contact zones between plant populations potentially adapted to warmer environments and other populations. While a global trend of movement in a north-east direction was predicted, the magnitude of displacement was species-specific. For a temperature increase of 2 °C, contact zones were predicted to move by 92 km on average (minimum of 5 km, maximum of 212 km) and by 188 km for an increase of 4 °C (minimum of 11 km, maximum of 393 km). Intra-specific turnover-measuring the extent of change in global population genetic structure-was generally found to be moderate for 2 °C of temperature warming. For 4 °C of warming, however, the models indicated substantial intra-specific turnover for ten species. These results illustrate that, in spite of unavoidable simplifications, ancestry distribution models open new perspectives to forecast population genetic changes within species and complement more traditional distribution-based approaches.

Fine-scale genetic structure and marginal processes in an expanding population of Biscutella laevigata L. (Brassicaceae).

Evolutionary processes acting at the expanding margins of a species' range are still poorly understood. Genetic drift is considered prevalent in marginal populations, and the maintenance of genetic diversity during recolonization might seem puzzling. To investigate such processes, a fine-scale investigation of 219 individuals was performed within a population of Biscutella laevigata (Brassicaceae), located at the leading edge of its range. The survey used amplified fragment length polymorphisms (AFLPs). As commonly reported across the whole species distribution range, individual density and genetic diversity decreased along the local axis of recolonization of this expanding population, highlighting the enduring effect of the historical colonization on present-day diversity. The self-incompatibility system of the plant may have prevented local inbreeding in newly found patches and sustained genetic diversity by ensuring gene flow from established populations. Within the more continuously populated region, spatial analysis of genetic structure revealed restricted gene flow among individuals. The distribution of genotypes formed a mosaic of relatively homogenous patches within the continuous population. This pattern could be explained by a history of expansion by long-distance dispersal followed by fine-scale diffusion (that is, a stratified dispersal combination). The secondary contact among expanding patches apparently led to admixture among differentiated genotypes where they met (that is, a reshuffling effect). This type of dynamics could explain the maintenance of genetic diversity during recolonization.

Genetic structure of a linear population of Beta vulgaris ssp maritima (sea beet) revealed by isozyme and RFLP analysis

Knowledge of the genetic structure of plant populations is necessary for the understanding of the dynamics of major ecological processes. It also has applications in conservation biology and risk assessment for genetically modified crops. This paper reports the genetic structure of a linear population of sea beet, Beta vulgaris ssp. maritima (the wild relative of sugar beet), on Furzey Island, Poole Harbour. The relative spatial positions of the plants were accurately mapped and the plants were scored for variation at isozyme and RFLP loci. Structure was analysed by repeated subdivision of the population to find the average size of a randomly mating group. Estimates of F-ST between randomly mating units were then made, and gave patterns consistent with the structure of the population being determined largely by founder effects. The implications of these results for the monitoring of transgene spread in wild sea beet populations are discussed.

Chromosomal rearrangement and genetic structure in the shrews of the Sorex araneus group

ABSTRACT The role of chromosomal rearrangements in the speciation process is much debated and many theoretical models have been developed. The shrews of the Sorex araneus group offer extraordinary opportunities to study the relationship between chromosomal variation and speciation. Indeed, this group of morphologically very similar species received a great deal of attention due to its karyotypic variability, which is mainly attributed to Robertsonian fusions. To explore the impact of karyotypic changes on genetic differentiation, we first studied the relationship between genetic and karyotypic structure among Alpine species and among chromosome races of the S. araneus group using Bayesian admixture analyses. The results of these analyses confirmed the taxonomic status of the studied species even though introgression can still be detected between species. Moreover, the strong spatial sub-structure highlighted the role of historical factors (e.g. geographical isolation) on genetic structure. Next, we studied gene flow at the chromosome level to address the question of the impact of chromosomal rearrangements on genetic differentiation. We used flow sorted chromosomes from three different karyotypic taxa of the S. araneus group to map microsatellite markers at the chromosóme arm level. We have been able to map 24 markers and to show that the karyotypic organisation of these taxa is well conserved, which suggests that these markers can be used for further inter-taxa studies. A general prediction of chromosomal speciation models is that genetic differentiation between two taxa should be larger across rearranged chromosomes than across chromosomes common to both taxa. We combined two approaches using mapped microsatellites to test this prediction. First, we studied the genetic differentiation among five shrew taxa placed at different evolutionary levels (i.e. within and among species). In this large scale study, we detected an overall significant difference in genetic structure between rearranged vs. common chromosomes. Moreover, this effect varied among pairwise comparisons, which allowed us to differentiate the role of the karyotypic complexity of hybrids and of the evolutionary divergence between taxa. Secondly, we compared the levels of gene flow measured across common vs. rearranged chromosomes in two karyotypically different hybrid zones (strong vs. low complexity of hybrids), which show similar levels of genetic structure. We detected a significantly stronger genetic structure across rearranged chromosomes in the hybrid zone showing the highest level of hybrid complexity. The large variance observed among loci suggested that other factors, such as the position of markers within the chromosome, also certainly affects genetic structure. In conclusion, our results strongly support the role of chromosomal rearrangements in the reproductive barrier and suggest their importance in speciation process of the S. araneus group. RESUME Le rôle des réarrangements chromosomiques dans les processus de spéciation est fortement débattu et de nombreux modèles théoriques ont été développés sur le sujet. Les musaraignes du groupe Sorex araneus présentent de nombreuses opportunités pour étudier les relations entre les variations chromosomiques et la spéciation. En effet, ce groupe d'espèces morphologiquement très proches a attiré l'attention des chercheurs en raison de sa variabilité caryotypique principalement attribuée à des fusions Robertsoniennes. Pour explorer l'impact des changements caryotypiques sur la différenciation génétique, nous avons tout d'abord étudié les relations entre la structure génétique et caryotypique de races chromosomiques et d'espèces alpine du groupe S. araneus en utilisant des analyses Bayesiennes d' « admixture ». Les résultats de ces analyses ont confirmé le statut taxonomique des espèces étudiées bien que nous ayons détecté de l'introgression entre espèces. L'observation d'une sous structure spatiale relativement forte souligne l'importance des facteurs historiques (telle que l'isolation géographique) sur la structure génétique de ce groupe. Ensuite, nous avons étudié le flux de gène au niveau des chromosomes pour aborder de manière directe la question de l'impact des réarrangements chromosomiques sur la différenciation génétique. En conséquence, nous avons utilisé des tris de chromosomes de trois taxons du groupe S. araneus pour localiser des marqueurs microsatellites au niveau du bras chromosomique. Au cours de cette étude, nous avons pu localiser 24 marqueurs et montrer une forte conservation dans l'organisation du caryotype de ces taxa. Ce résultat suggère que leur utilisation est appropriée pour des études entre taxa. Une prédiction générale à tous les modèles de spéciation chromosomique correspond à la plus grande différenciation génétique des chromosomes réarrangés que des chromosomes communs. Nous avons combiné deux approches utilisant des microsatellites localisés au niveau du bras chromosomique pour tester cette prédiction. Premièrement, nous avons étudié la différenciation génétique entre cinq taxa du groupe S. araneus se trouvant à des niveaux évolutifs différents (i.e. à l'intérieur et entre espèce). Au cours de cette étude, nous avons détecté une différenciation globale significativement plus élevée sur les chromosomes réarrangés. Cet effet varie entre les comparaisons, ce qui nous a permis de souligner le rôle de la complexité caryotypique des hybrides et du niveau de divergence évolutive entre taxa. Deuxièmement, nous avons comparé le flux de gènes des chromosomes communs et réarrangés dans deux zones d'hybridation caryotypiquement différentes (forte vs. Faible complexité des hybrides) mais présentant un niveau de différenciation génétique similaire. Ceci nous a permis de détecter une structure génétique significativement plus élevée sur les chromosomes réarrangés au centre de la zone d'hybridation présentant la plus grande complexité caryotypic. La forte variance observée entre loci souligne en outre le fait que d'autres facteurs, tel que la position du marqueur sur le chromosome, affectent probablement aussi la structure génétique mesurée. En conclusion, nos résultats supportent fortement le rôle des réarrangements chromosomiques dans la barrière reproductive entre espèces ainsi que leur importance dans les processus de spéciation des musaraignes du groupe S. araneus.

Spatio-temporal population genetic structure of the parasitic mite Spinturnix bechsteini is shaped by its own demography and the social system of its bat host.

Information about the population genetic structures of parasites is important for an understanding of parasite transmission pathways and ultimately the co-evolution with their hosts. If parasites cannot disperse independently of their hosts, a parasite's population structure will depend upon the host's spatial distribution. Geographical barriers affecting host dispersal can therefore lead to structured parasite populations. However, how the host's social system affects the genetic structure of parasite populations is largely unknown. We used mitochondrial DNA (mtDNA) to describe the spatio-temporal population structure of a contact-transmitted parasitic wing mite (Spinturnix bechsteini) and compared it to that of its social host, the Bechstein's bat (Myotis bechsteinii). We observed no genetic differentiation between mites living on different bats within a colony. This suggests that mites can move freely among bats of the same colony. As expected in case of restricted inter-colony dispersal, we observed a strong genetic differentiation of mites among demographically isolated bat colonies. In contrast, we found a strong genetic turnover between years when we investigated the temporal variation of mite haplotypes within colonies. This can be explained with mite dispersal occuring between colonies and bottlenecks of mite populations within colonies. The observed absence of isolation by distance could be the result from genetic drift and/or from mites dispersing even between remote bat colonies, whose members may meet at mating sites in autumn or in hibernacula in winter. Our data show that the population structure of this parasitic wing mite is influenced by its own demography and the peculiar social system of its bat host.

The evolution of unicoloniality in ant societies : recognition, dispersal and population genetic structure

Introduction Societies of ants, bees, wasps and termites dominate many terrestrial ecosystems (Wilson 1971). Their evolutionary and ecological success is based upon the regulation of internal conflicts (e.g. Ratnieks et al. 2006), control of diseases (e.g. Schmid-Hempel 1998) and individual skills and collective intelligence in resource acquisition, nest building and defence (e.g. Camazine 2001). Individuals in social species can pass on their genes not only directly trough their own offspring, but also indirectly by favouring the reproduction of relatives. The inclusive fitness theory of Hamilton (1963; 1964) provides a powerful explanation for the evolution of reproductive altruism and cooperation in groups with related individuals. The same theory also led to the realization that insect societies are subject to internal conflicts over reproduction. Relatedness of less-than-one is not sufficient to eliminate all incentive for individual selfishness. This would indeed require a relatedness of one, as found among cells of an organism (Hardin 1968; Keller 1999). The challenge for evolutionary biology is to understand how groups can prevent or reduce the selfish exploitation of resources by group members, and how societies with low relatedness are maintained. In social insects the evolutionary shift from single- to multiple queens colonies modified the relatedness structure, the dispersal, and the mode of colony founding (e.g. (Crozier & Pamilo 1996). In ants, the most common, and presumably ancestral mode of reproduction is the emission of winged males and females, which found a new colony independently after mating and dispersal flights (Hölldobler & Wilson 1990). The alternative reproductive tactic for ant queens in multiple-queen colonies (polygyne) is to seek to be re-accepted in their natal colonies, where they may remain as additional reproductives or subsequently disperse on foot with part of the colony (budding) (Bourke & Franks 1995; Crozier & Pamilo 1996; Hölldobler & Wilson 1990). Such ant colonies can contain up to several hundred reproductive queens with an even more numerous workforce (Cherix 1980; Cherix 1983). As a consequence in polygynous ants the relatedness among nestmates is very low, and workers raise brood of queens to which they are only distantly related (Crozier & Pamilo 1996; Queller & Strassmann 1998). Therefore workers could increase their inclusive fitness by preferentially caring for their closest relatives and discriminate against less related or foreign individuals (Keller 1997; Queller & Strassmann 2002; Tarpy et al. 2004). However, the bulk of the evidence suggests that social insects do not behave nepotistically, probably because of the costs entailed by decreased colony efficiency or discrimination errors (Keller 1997). Recently, the consensus that nepotistic behaviour does not occur in insect colonies was challenged by a study in the ant Formica fusca (Hannonen & Sundström 2003b) showing that the reproductive share of queens more closely related to workers increases during brood development. However, this pattern can be explained either by nepotism with workers preferentially rearing the brood of more closely related queens or intrinsic differences in the viability of eggs laid by queens. In the first chapter, we designed an experiment to disentangle nepotism and differences in brood viability. We tested if workers prefer to rear their kin when given the choice between highly related and unrelated brood in the ant F. exsecta. We also looked for differences in egg viability among queens and simulated if such differences in egg viability may mistakenly lead to the conclusion that workers behave nepotistically. The acceptance of queens in polygnous ants raises the question whether the varying degree of relatedness affects their share in reproduction. In such colonies workers should favour nestmate queens over foreign queens. Numerous studies have investigated reproductive skew and partitioning of reproduction among queens (Bourke et al. 1997; Fournier et al. 2004; Fournier & Keller 2001; Hammond et al. 2006; Hannonen & Sundström 2003a; Heinze et al. 2001; Kümmerli & Keller 2007; Langer et al. 2004; Pamilo & Seppä 1994; Ross 1988; Ross 1993; Rüppell et al. 2002), yet almost no information is available on whether differences among queens in their relatedness to other colony members affects their share in reproduction. Such data are necessary to compare the relative reproductive success of dispersing and non-dispersing individuals. Moreover, information on whether there is a difference in reproductive success between resident and dispersing queens is also important for our understanding of the genetic structure of ant colonies and the dynamics of within group conflicts. In chapter two, we created single-queen colonies and then introduced a foreign queens originating from another colony kept under similar conditions in order to estimate the rate of queen acceptance into foreign established colonies, and to quantify the reproductive share of resident and introduced queens. An increasing number of studies have investigated the discrimination ability between ant workers (e.g. Holzer et al. 2006; Pedersen et al. 2006), but few have addressed the recognition and discrimination behaviour of workers towards reproductive individuals entering colonies (Bennett 1988; Brown et al. 2003; Evans 1996; Fortelius et al. 1993; Kikuchi et al. 2007; Rosengren & Pamilo 1986; Stuart et al. 1993; Sundström 1997; Vásquez & Silverman in press). These studies are important, because accepting new queens will generally have a large impact on colony kin structure and inclusive fitness of workers (Heinze & Keller 2000). In chapter three, we examined whether resident workers reject young foreign queens that enter into their nest. We introduced mated queens into their natal nest, a foreign-female producing nest, or a foreign male-producing nest and measured their survival. In addition, we also introduced young virgin and mated queens into their natal nest to examine whether the mating status of the queens influences their survival and acceptance by workers. On top of polgyny, some ant species have evolved an extraordinary social organization called 'unicoloniality' (Hölldobler & Wilson 1977; Pedersen et al. 2006). In unicolonial ants, intercolony borders are absent and workers and queens mix among the physically separated nests, such that nests form one large supercolony. Super-colonies can become very large, so that direct cooperative interactions are impossible between individuals of distant nests. Unicoloniality is an evolutionary paradox and a potential problem for kin selection theory because the mixing of queens and workers between nests leads to extremely low relatedness among nestmates (Bourke & Franks 1995; Crozier & Pamilo 1996; Keller 1995). A better understanding of the evolution and maintenance of unicoloniality requests detailed information on the discrimination behavior, dispersal, population structure, and the scale of competition. Cryptic genetic population structure may provide important information on the relevant scale to be considered when measuring relatedness and the role of kin selection. Theoretical studies have shown that relatedness should be measured at the level of the `economic neighborhood', which is the scale at which intraspecific competition generally takes place (Griffin & West 2002; Kelly 1994; Queller 1994; Taylor 1992). In chapter four, we conducted alarge-scale study to determine whether the unicolonial ant Formica paralugubris forms populations that are organised in discrete supercolonies or whether there is a continuous gradation in the level of aggression that may correlate with genetic isolation by distance and/or spatial distance between nests. In chapter five, we investigated the fine-scale population structure in three populations of F. paralugubris. We have developed mitochondria) markers, which together with the nuclear markers allowed us to detect cryptic genetic clusters of nests, to obtain more precise information on the genetic differentiation within populations, and to separate male and female gene flow. These new data provide important information on the scale to be considered when measuring relatedness in native unicolonial populations.

The effect of host social system on parasite population genetic structure: comparative population genetics of two ectoparasitic mites and their bat hosts.

BACKGROUND: The population genetic structure of a parasite, and consequently its ability to adapt to a given host, is strongly linked to its own life history as well as the life history of its host. While the effects of parasite life history on their population genetic structure have received some attention, the effect of host social system has remained largely unstudied. In this study, we investigated the population genetic structure of two closely related parasitic mite species (Spinturnix myoti and Spinturnix bechsteini) with very similar life histories. Their respective hosts, the greater mouse-eared bat (Myotis myotis) and the Bechstein's bat (Myotis bechsteinii) have social systems that differ in several substantial features, such as group size, mating system and dispersal patterns. RESULTS: We found that the two mite species have strongly differing population genetic structures. In S. myoti we found high levels of genetic diversity and very little pairwise differentiation, whereas in S. bechsteini we observed much less diversity, strongly differentiated populations and strong temporal turnover. These differences are likely to be the result of the differences in genetic drift and dispersal opportunities afforded to the two parasites by the different social systems of their hosts. CONCLUSIONS: Our results suggest that host social system can strongly influence parasite population structure. As a result, the evolutionary potential of these two parasites with very similar life histories also differs, thereby affecting the risk and evolutionary pressure exerted by each parasite on its host.

Genetic structure of Gymnures (genus Hylomys; Erinaceidae) on continental islands of Southeast Asia: Historical effects of fragmentation

Eustatic sea level changes during Pleistocene climatic fluctuations produced several cycles of connection-isolation among continental islands of the Sunda shelf. To explore the potential effects of these fluctuations, we reconstructed a model of the vicariant events that separated these islands, based on bathymetric information. Among many possible scenarios, two opposite phylogenetic patterns of evolution were predicted for terrestrial organisms living in this region: one is based on the classical allopatric speciation mode of evolution, while the other is the outcome of a sequential dispersal colonization of the archipelago. We tested the applicability of these predictions with an analysis of sequence variation of the cytochrome b gene from several taxa of Hylomys. They were sampled throughout SE-Asia and the Sunda islands. High levels of haplotype differentiation characterize the different island taxa. Such levels of differentiation support the existence of several allopatric species, as was suggested by previous allozyme and morphological data. Also in accordance with previous results, the occurrence of two sympatric species from Sumatra is suggested by their strongly divergent haplotypes. One species, Hylomys suillus maxi, is found both on Sumatra and in Peninsular Malaysia, while the other, H. parvus, is endemic to Sumatra. Its closest relative is H. suillus dorsalis from Borneo. Phylogenetic reconstructions also demonstrate the existence of a Sundaic clade composed of all island taxa, as opposed to those from the continent. Although there is no statistical support for either proposed biogeographic model of evolution, we argue that the sequential dispersal scenario is more appropriate to describe the genetic variation found among the Hylomys taxa. However, despite strong differentiation among island haplotypes, the cladistic relationships between some island taxa could not be resolved. We argue that this is evidence of a rapid radiation, suggesting that the separation of the islands may have been perceived as a simultaneous event rather than as a succession of vicariant events. Furthermore, the estimates of divergence times between the haplotypes of these taxa suggest that this radiation may actually have predated the climatic fluctuations of the Pleistocene. Further refinement of the initial palaeogeographic models of evolution are therefore needed to account for these results.

Morphological, ecological and genetic aspects associated with endemism in the Fly Orchid group.

The European genus Ophrys (Orchidaceae) is famous for its insect-like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives.

Genetic structure of the genus Leptospira by multilocus enzyme electrophoresis

Thirty strains from the 11 species of the genus Leptospira were studied by multilocus enzyme electrophoresis at 12 enzyme loci, all of which were polymorphic. The mean number of alleles per locus was 6.5. Twenty-five electrophoretic types were distinguished. Grouping of the strains by cluster analysis was in general agreement with species delineation as determined by DNA-DNA hybridization, except for the strains of Leptospira meyeri and Leptospira inadai, which were scattered throughout the genus, reflecting previously recognized taxonomic uncertainties. Analysis of the clonality within Leptospira interrogans sensu stricto indicated that this population was relatively heterogeneous and a lack of gene linkage disequilibrium could not be excluded. There was a genetic discrimination between the pathogenic species and the saprophytic ones. The phenotypically intermediate species (L. inadai and Leptospira fainei) were also genetically separated and were probably closer to the saprophytes than to the pathogens.