The origins and spread of domestic horses from the Western Eurasian steppes.

Domestication of horses fundamentally transformed long-range mobility and warfare1. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling2-4 at Botai, Central Asia around 3500 BC3. Other longstanding candidate regions for horse domestication, such as Iberia5 and Anatolia6, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 BC, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association7 between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 BC8,9 driving the spread of Indo-European languages10. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium BC Sintashta culture11,12.

Dynamic evolution of size and colour in the highly specialized Zodarion ant-eating spiders.

Ecological specialists constitute relevant case studies for understanding the mechanisms, potential and limitations of evolution. The species-rich and strictly myrmecophagous spiders of the genus Zodarion show diversified defence mechanisms, including myrmecomorphy of different ant species and nocturnality. Through Hybridization Capture Using RAD Probes (hyRAD), a phylogenomic technique designed for sequencing poorly preserved specimens, we reconstructed a phylogeny of Zodarion using 52 (approx. a third of the nominal) species that cover its phylogenetic and distributional diversity. We then estimated the evolution of body size and colour, traits that have diversified noticeably and are linked to defence mechanisms, across the group. Our genomic matrix of 300 loci led to a well-supported phylogenetic hypothesis that uncovered two main clades inside Zodarion. Ancestral state estimation revealed the highly dynamic evolution of body size and colour across the group, with multiple transitions and convergences in both traits, which we propose is likely indicative of multiple transitions in ant specialization across the genus. Our study will allow the informed targeted selection of Zodarion taxa of special interest for research into the group's remarkable adaptations to ant specialization. It also exemplifies the utility of hyRAD for phylogenetic studies using museum material.

The untapped potential of macrofossils in ancient plant DNA research.

The rapid development of ancient DNA analysis in the last decades has induced a paradigm shift in ecology and evolution. Driven by a combination of breakthroughs in DNA isolation techniques, high-throughput sequencing, and bioinformatics, ancient genome-scale data for a rapidly growing variety of taxa are now available, allowing researchers to directly observe demographic and evolutionary processes over time. However, the vast majority of paleogenomic studies still focus on human or animal remains. In this article, we make the case for a vast untapped resource of ancient plant material that is ideally suited for paleogenomic analyses: plant remains, such as needles, leaves, wood, seeds, or fruits, that are deposited in natural archives, such as lake sediments, permafrost, or even ice caves. Such plant remains are commonly found in large numbers and in stratigraphic sequence through time and have so far been used primarily to reconstruct past local species presences and abundances. However, they are also unique repositories of genetic information with the potential to revolutionize the fields of ecology and evolution by directly studying microevolutionary processes over time. Here, we give an overview of the current state-of-the-art, address important challenges, and highlight new research avenues to inspire future research.

Hybridization fuelled diversification in Spialia butterflies.

The importance of hybridization and introgression is well documented in the evolution of plants but, in insects, their role is not fully understood. Given the fact that insects are the most diverse group of organisms, assessing the impact of reticulation events on their evolution may be key to comprehend the emergence of such remarkable diversity. Here, we used an insect model, the Spialia butterflies, to gather genomic evidence of hybridization as a promoter of novel diversity. By using double-digest RADseq (ddRADseq), we explored the phylogenetic relationships between Spialia orbifer, S. rosae and S. sertorius, and documented two independent events of interspecific gene flow. Our data support that the Iberian endemism S. rosae probably received genetic material from S. orbifer in both mitochondrial and nuclear DNA, which could have contributed to a shift in the ecological preferences of S. rosae. We also show that admixture between S. sertorius and S. orbifer probably occurred in Italy. As a result, the admixed Sicilian populations of S. orbifer are differentiated from the rest of populations both genetically and morphologically, and display signatures of reproductive character displacement in the male genitalia. Additionally, our analyses indicated that genetic material from S. orbifer is present in S. sertorius along the Italian Peninsula. Our findings add to the view that hybridization is a pervasive phenomenon in nature and in butterflies in particular, with important consequences for evolution due to the emergence of novel phenotypes.

Poor performance of DNA barcoding and the impact of RAD loci filtering on the species delimitation of an Iberian ant-eating spider.

Genomic data provide unprecedented power for species delimitation. However, current implementations are still time and resource consuming. In addition, bioinformatic processing is contentious and its impact on downstream analyses is insufficiently understood. Here we employ ddRAD sequencing and a thorough sampling for species delimitation in Zodarion styliferum, a widespread Iberian ant-eating spider. We explore the influence of the loci filtering strategy on the downstream phylogenetic analyses, genomic clustering and coalescent species delimitation. We also assess the accuracy of one mitochondrial (COI) and one nuclear (ITS) barcode for fast and inexpensive species delineation in the group. Our genomic data strongly support two morphologically cryptic but ecologically divergent lineages, mainly restricted to the central-eastern and western parts of the Iberian Peninsula, respectively. Larger matrices with more missing data showed increased genomic diversity, supporting that bioinformatic strategies to maximize matrix completion disproportionately exclude loci with the highest mutation rates. Moderate loci filtering gave the best results across analyses: although larger matrices returned concatenated phylogenies with higher support, middle-sized matrices performed better in genetic structure analyses. COI displayed high diversity and a conspicuous barcode gap, revealing 13 mitochondrial lineages. Mitonuclear discordance is consistent with ancestral isolation in multiple groups, probably in glacial refugia, followed by range expansion and secondary contact that produced genomic homogenization. Several apparently (unidirectionally) introgressed specimens further challenge the accuracy of species identification through mitochondrial barcodes in the group. Conversely, ITS failed to separate both lineages of Z. styliferum. This study shows an extreme case of mitonuclear discordance that highlights the limitations of single molecular barcodes for species delimitation, even in presence of distinct barcode gaps, and brings new light on the effects of parameterization on shallow-divergence studies using RAD data.

Climate drives community-wide divergence within species over a limited spatial scale: evidence from an oceanic island.

Geographic isolation substantially contributes to species endemism on oceanic islands when speciation involves the colonisation of a new island. However, less is understood about the drivers of speciation within islands. What is lacking is a general understanding of the geographic scale of gene flow limitation within islands, and thus the spatial scale and drivers of geographical speciation within insular contexts. Using a community of beetle species, we show that when dispersal ability and climate tolerance are restricted, microclimatic variation over distances of only a few kilometres can maintain strong geographic isolation extending back several millions of years. Further to this, we demonstrate congruent diversification with gene flow across species, mediated by Quaternary climate oscillations that have facilitated a dynamic of isolation and secondary contact. The unprecedented scale of parallel species responses to a common environmental driver for evolutionary change has profound consequences for understanding past and future species responses to climate variation.

Rapid colour shift by reproductive character displacement in Cupido butterflies.

Reproductive character displacement occurs when competition for successful breeding imposes a divergent selection on the interacting species, causing a divergence of reproductive traits. Here, we show that a disputed butterfly taxon is actually a case of male wing colour shift, apparently produced by reproductive character displacement. Using double digest restriction-site associated DNA sequencing and mitochondrial DNA sequencing we studied four butterfly taxa of the subgenus Cupido (Lepidoptera: Lycaenidae): Cupido minimus and the taxon carswelli, both characterized by brown males and females, plus C. lorquinii and C. osiris, both with blue males and brown females. Unexpectedly, taxa carswelli and C. lorquinii were close to indistinguishable based on our genomic and mitochondrial data, despite displaying strikingly different male coloration. In addition, we report and analysed a brown male within the C. lorquinii range, which demonstrates that the brown morph occurs at very low frequency in C. lorquinii. Such evidence strongly suggests that carswelli is conspecific with C. lorquinii and represents populations with a fixed male brown colour morph. Considering that these brown populations occur in sympatry with or very close to the blue C. osiris, and that the blue C. lorquinii populations never do, we propose that the taxon carswelli could have lost the blue colour due to reproductive character displacement with C. osiris. Since male colour is important for conspecific recognition during courtship, we hypothesize that the observed colour shift may eventually trigger incipient speciation between blue and brown populations. Male colour seems to be an evolutionarily labile character in the Polyommatinae, and the mechanism described here might be at work in the wide diversification of this subfamily of butterflies.

A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris.

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double-digest restriction-site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex-biased asymmetries, NUMTs, natural selection, introgression or Wolbachia-mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.

Long-term in situ persistence of biodiversity in tropical sky islands revealed by landscape genomics.

Tropical mountains are areas of high species richness and endemism. Two historical phenomena may have contributed to this: (i) fragmentation and isolation of habitats may have promoted the genetic differentiation of populations and increased the possibility of allopatric divergence and speciation and (ii) the mountain areas may have allowed long-term population persistence during global climate fluctuations. These two phenomena have been studied using either species occurrence data or estimating species divergence times. However, only few studies have used intraspecific genetic data to analyse the mechanisms by which endemism may emerge at the microevolutionary scale. Here, we use landscape analysis of genomic SNP data sampled from two high-elevation plant species from an archipelago of tropical sky islands (the Trans-Mexican Volcanic Belt) to test for population genetic differentiation, synchronous demographic changes and habitat persistence. We show that genetic differentiation can be explained by the degree of glacial habitat connectivity among mountains and that mountains have facilitated the persistence of populations throughout glacial/interglacial cycles. Our results support the ongoing role of tropical mountains as cradles for biodiversity by uncovering cryptic differentiation and limits to gene flow.

Phylogeography and population genomics of a lotic water beetle across a complex tropical landscape.

The habitat template concept applied to a freshwater system indicates that lotic species, or those which occupy permanent habitats along stream courses, are less dispersive than lentic species, or those that occur in more ephemeral aquatic habitats. Thus, populations of lotic species will be more structured than those of lentic species. Stream courses include both flowing water and small, stagnant microhabitats that can provide refuge when streams are low. Many species occur in these microhabitats but remain poorly studied. Here, we present population genetic data for one such species, the tropical diving beetle Exocelina manokwariensis (Dytiscidae), sampled from six localities along a ~300 km transect across the Birds Head Peninsula of New Guinea. Molecular data from both mitochondrial (CO1 sequences) and nuclear (ddRAD loci) regions document fine-scale population structure across populations that are ~45 km apart. Our results are concordant with previous phylogenetic and macroecological studies that applied the habitat template concept to aquatic systems. This study also illustrates that these diverse but mostly overlooked microhabitats are promising study systems in freshwater ecology and evolutionary biology. With the advent of next-generation sequencing, fine-scale population genomic studies are feasible for small nonmodel organisms to help illuminate the effect of habitat stability on species' natural history, population structure and geographic distribution.

A tale of two forests: ongoing aridification drives population decline and genetic diversity loss at continental scale in Afro-Macaronesian evergreen-forest archipelago endemics.

Background and Aims: Various studies and conservationist reports have warned about the contraction of the last subtropical Afro-Macaronesian forests. These relict vegetation zones have been restricted to a few oceanic and continental islands around the edges of Africa, due to aridification. Previous studies on relict species have generally focused on glacial effects on narrow endemics; however, little is known about the effects of aridification on the fates of previously widespread subtropical lineages. Methods: Nuclear microsatellites and ecological niche modelling were used to understand observed patterns of genetic diversity in two emblematic species, widely distributed in these ecosystems: Canarina eminii (a palaeoendemic of the eastern Afromontane forests) and Canarina canariensis (a palaeoendemic of the Canarian laurel forests). The software DIYABC was used to test alternative demographic scenarios and an ensemble method was employed to model potential distributions of the selected plants from the end of the deglaciation to the present. Key Results: All the populations assessed experienced a strong and recent population decline, revealing that locally widespread endemisms may also be alarmingly threatened. Conclusions: The detected extinction debt, as well as the extinction spiral to which these populations are subjected, demands urgent conservation measures for the unique, biodiversity-rich ecosystems that they inhabit.

Clustering Genes of Common Evolutionary History.

Phylogenetic inference can potentially result in a more accurate tree using data from multiple loci. However, if the loci are incongruent-due to events such as incomplete lineage sorting or horizontal gene transfer-it can be misleading to infer a single tree. To address this, many previous contributions have taken a mechanistic approach, by modeling specific processes. Alternatively, one can cluster loci without assuming how these incongruencies might arise. Such "process-agnostic" approaches typically infer a tree for each locus and cluster these. There are, however, many possible combinations of tree distance and clustering methods; their comparative performance in the context of tree incongruence is largely unknown. Furthermore, because standard model selection criteria such as AIC cannot be applied to problems with a variable number of topologies, the issue of inferring the optimal number of clusters is poorly understood. Here, we perform a large-scale simulation study of phylogenetic distances and clustering methods to infer loci of common evolutionary history. We observe that the best-performing combinations are distances accounting for branch lengths followed by spectral clustering or Ward's method. We also introduce two statistical tests to infer the optimal number of clusters and show that they strongly outperform the silhouette criterion, a general-purpose heuristic. We illustrate the usefulness of the approach by 1) identifying errors in a previous phylogenetic analysis of yeast species and 2) identifying topological incongruence among newly sequenced loci of the globeflower fly genus Chiastocheta We release treeCl, a new program to cluster genes of common evolutionary history (http://git.io/treeCl).

Climatic niche evolution is faster in sympatric than allopatric lineages of the butterfly genus Pyrgus.

Understanding how speciation relates to ecological divergence has long fascinated biologists. It is assumed that ecological divergence is essential to sympatric speciation, as a mechanism to avoid competition and eventually lead to reproductive isolation, while divergence in allopatry is not necessarily associated with niche differentiation. The impact of the spatial context of divergence on the evolutionary rates of abiotic dimensions of the ecological niche has rarely been explored for an entire clade. Here, we compare the magnitude of climatic niche shifts between sympatric versus allopatric divergence of lineages in butterflies. By combining next-generation sequencing, parametric biogeography and ecological niche analyses applied to a genus-wide phylogeny of Palaearctic Pyrgus butterflies, we compare evolutionary rates along eight climatic dimensions across sister lineages that diverged in large-scale sympatry versus allopatry. In order to examine the possible effects of the spatial scale at which sympatry is defined, we considered three sets of biogeographic assignments, ranging from narrow to broad definition. Our findings suggest higher rates of niche evolution along all climatic dimensions for sister lineages that diverge in sympatry, when using a narrow delineation of biogeographic areas. This result contrasts with significantly lower rates of climatic niche evolution found in cases of allopatric speciation, despite the biogeographic regions defined here being characterized by significantly different climates. Higher rates in allopatry are retrieved when biogeographic areas are too widely defined-in such a case allopatric events may be recorded as sympatric. Our results reveal the macro-evolutionary significance of abiotic niche differentiation involved in speciation processes within biogeographic regions, and illustrate the importance of the spatial scale chosen to define areas when applying parametric biogeographic analyses.

Genetic consequences of Quaternary climatic oscillations in the Himalayas: Primula tibetica as a case study based on restriction site-associated DNA sequencing.

The effects of Quaternary climatic oscillations on the demography of organisms vary across regions and continents. In taxa distributed in Europe and North America, several paradigms regarding the distribution of refugia have been identified. By contrast, less is known about the processes that shaped the species' spatial genetic structure in areas such as the Himalayas, which is considered a biodiversity hotspot. Here, we investigated the phylogeographic structure and population dynamics of Primula tibetica by combining genomic phylogeography and species distribution models (SDMs). Genomic data were obtained for 293 samples of P. tibetica using restriction site-associated DNA sequencing (RADseq). Ensemble SDMs were carried out to predict potential present and past distribution ranges. Four distinct lineages were identified. Approximate Bayesian computation analyses showed that each of them have experienced both expansions and bottlenecks since their divergence, which occurred during or across the Quaternary glacial cycles. The two lineages at both edges of the distribution were found to be more vulnerable and responded in different ways to past climatic changes. These results illustrate how past climatic changes affected the demographic history of Himalayan organisms. Our findings highlight the significance of combining genomic approaches with environmental data when evaluating the effects of past climatic changes.

Assessing the potential of RAD-sequencing to resolve phylogenetic relationships within species radiations: The fly genus Chiastocheta (Diptera: Anthomyiidae) as a case study.

Determining phylogenetic relationships among recently diverged species has long been a challenge in evolutionary biology. Cytoplasmic DNA markers, which have been widely used, notably in the context of molecular barcoding, have not always proved successful in resolving such phylogenies. However, with the advent of next-generation-sequencing technologies and associated techniques of reduced genome representation, phylogenies of closely related species have been resolved at a much higher detail in the last couple of years. Here we examine the potential and limitations of one of such techniques-Restriction-site Associated DNA (RAD) sequencing, a method that produces thousands of (mostly) anonymous nuclear markers, in disentangling the phylogeny of the fly genus Chiastocheta (Diptera: Anthomyiidae). In Europe, this genus encompasses seven species of seed predators, which have been widely studied in the context of their ecological and evolutionary interactions with the plant Trollius europaeus (Ranunculaceae). So far, phylogenetic analyses using mitochondrial markers failed to resolve monophyly of most of the species from this recently diversified genus, suggesting that their taxonomy may need a revision. However, relying on a single, non-recombining marker and ignoring potential incongruences between mitochondrial and nuclear loci may provide an incomplete account of the lineage history. In this study, we applied both classical Sanger sequencing of three mtDNA regions and RAD-sequencing, for reconstructing the phylogeny of the genus. Contrasting with results based on mitochondrial markers, RAD-sequencing analyses retrieved the monophyly of all seven species, in agreement with the morphological species assignment. We found robust nuclear-based species assignment of individual samples, and low levels of estimated contemporary gene flow among them. However, despite recovering species' monophyly, interspecific relationships varied depending on the set of RAD loci considered, producing contradictory topologies. Moreover, coalescence-based phylogenetic analyses revealed low supports for most of the interspecific relationships. Our results indicate that despite the higher performance of RAD-sequencing in terms of species trees resolution compared to cytoplasmic markers, reconstructing inter-specific relationships among recently-diverged lineages may lie beyond the possibilities offered by large sets of RAD-sequencing markers in cases of strong gene tree incongruence.

Is hybridization driving the evolution of climatic niche in Alyssum montanum.

PREMISE OF THE STUDY: After decades of interest, the contribution of hybridization to ecological diversification remains unclear. Hybridization is a potent source of novelty, but nascent hybrid lineages must overcome reproductive and ecological competition from their parental species. Here, we assess whether hybrid speciation is advantageous over alternative modes of speciation, by comparing the geographical and ecological ranges and climatic niche evolutionary rates of stabilized allopolyploid vs. autopolyploids in the Alyssum montanum species complex. METHODS: We combined an extensive review of studies addressing the systematics and genetic diversity of A. montanum s.l., with flow cytometry and cloning of nuclear markers, to establish the ploidy level and putative hybrid nature of 205 populations. The respective geographic distribution and climatic niche evolution dynamics of the allo- and autopolyploids were investigated using multivariate analyses and comparative phylogenetic approaches. KEY RESULTS: As expected by theory, allopolyploids occur mainly along contact zones and are generally spatially overlapping with their diploid counterparts. However, they demonstrate higher rates of niche evolution and expand into different climatic conditions than those of their diploid congeners. In contrast, autopolyploids show lower rates of niche evolution, occupy ecological niches similar to their ancestors and are restricted to less competitive and peripheral geographic areas. CONCLUSIONS: Hybridization thus seems advantageous by promoting ecological niche evolution and more readily allowing escape from competitive exclusion.

Wheat alleles introgress into selfing wild relatives: empirical estimates from approximate Bayesian computation in Aegilops triuncialis.

Extensive gene flow between wheat (Triticum sp.) and several wild relatives of the genus Aegilops has recently been detected despite notoriously high levels of selfing in these species. Here, we assess and model the spread of wheat alleles into natural populations of the barbed goatgrass (Aegilops triuncialis), a wild wheat relative prevailing in the Mediterranean flora. Our sampling, based on an extensive survey of 31 Ae. triuncialis populations collected along a 60 km × 20 km area in southern Spain (Grazalema Mountain chain, Andalousia, totalling 458 specimens), is completed with 33 wheat cultivars representative of the European domesticated pool. All specimens were genotyped with amplified fragment length polymorphism with the aim of estimating wheat admixture levels in Ae. triuncialis populations. This survey first confirmed extensive hybridization and backcrossing of wheat into the wild species. We then used explicit modelling of populations and approximate Bayesian computation to estimate the selfing rate of Ae. triuncialis along with the magnitude, the tempo and the geographical distance over which wheat alleles introgress into Ae. triuncialis populations. These simulations confirmed that extensive introgression of wheat alleles (2.7 × 10(-4) wheat immigrants for each Ae. triuncialis resident, at each generation) into Ae. triuncialis occurs despite a high selfing rate (Fis ≈ 1 and selfing rate = 97%). These results are discussed in the light of risks associated with the release of genetically modified wheat cultivars in Mediterranean agrosystems.

A deep dig­­hindsight on Holocene vegetation composition from ancient environmental DNA.

Want a glimpse at past vegetation? Studying pollen and other plant remains, which are preserved for example in lake sediments or mires for thousands of years, allows us to document regional occurrences of plant species over radiocarbon-dated time series. Such vegetation reconstructions derived from optical analyses of fossil samples are inherently incomplete because they only comprise taxa that contribute sufficient amounts of pollen, spores, macrofossil or other evidences. To complement optical analyses for paleoecological inference, molecular markers applied to ancient DNA (aDNA) may help in disclosing information hitherto inaccessible to biologists. Parducci et al. (2013) targeted aDNA from sediment cores of two lakes in the Scandes Mountains with generic primers in a meta-barcoding approach. When compared to palynological records from the same cores, respective taxon lists show remarkable differences in their compositions, but also in quantitative representation and in taxonomic resolution similar to a previous study (Jørgensen et al. 2012). While not free of assumptions that need critical and robust testing, notably the question of possible contamination, this study provides thrilling prospects to improve our knowledge about past vegetation composition, but also other organismic groups, stored as a biological treasure in the ground.

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.

The abrupt climate change at the Eocene-Oligocene boundary and the emergence of South-East Asia triggered the spread of sapindaceous lineages.

BACKGROUND AND AIMS: Paleoclimatic data indicate that an abrupt climate change occurred at the Eocene-Oligocene (E-O) boundary affecting the distribution of tropical forests on Earth. The same period has seen the emergence of South-East (SE) Asia, caused by the collision of the Eurasian and Australian plates. How the combination of these climatic and geomorphological factors affected the spatio-temporal history of angiosperms is little known. This topic is investigated by using the worldwide sapindaceous clade as a case study. METHODS: Analyses of divergence time inference, diversification and biogeography (constrained by paleogeography) are applied to a combined plastid and nuclear DNA sequence data set. Biogeographical and diversification analyses are performed over a set of trees to take phylogenetic and dating uncertainty into account. Results are analysed in the context of past climatic fluctuations. KEY RESULTS: An increase in the number of dispersal events at the E-O boundary is recorded, which intensified during the Miocene. This pattern is associated with a higher rate in the emergence of new genera. These results are discussed in light of the geomorphological importance of SE Asia, which acted as a tropical bridge allowing multiple contacts between areas and additional speciation across landmasses derived from Laurasia and Gondwana. CONCLUSIONS: This study demonstrates the importance of the combined effect of geomorphological (the emergence of most islands in SE Asia approx. 30 million years ago) and climatic (the dramatic E-O climate change that shifted the tropical belt and reduced sea levels) factors in shaping species distribution within the sapindaceous clade.