Unveiling the Genome-Wide Consequences of Range Expansion and Mating System Transitions in Primula vulgaris.

Genetic diversity is heterogeneously distributed among populations of the same species, due to the joint effects of multiple demographic processes, including range contractions and expansions, and mating systems shifts. Here, we ask how both processes shape genomic diversity in space and time in the classical Primula vulgaris model. This perennial herb originated in the Caucasus region and was hypothesized to have expanded westward following glacial retreat in the Quaternary. Moreover, this species is a long-standing model for mating system transitions, exemplified by shifts from heterostyly to homostyly. Leveraging a high-quality reference genome of the closely related Primula veris and whole-genome resequencing data from both heterostylous and homostylous individuals from populations encompassing a wide distribution of P. vulgaris, we reconstructed the demographic history of P. vulgaris. Results are compatible with the previously proposed hypothesis of range expansion from the Caucasus region approximately 79,000 years ago and suggest later shifts to homostyly following rather than preceding postglacial colonization of England. Furthermore, in accordance with population genetic theoretical predictions, both processes are associated with reduced genetic diversity, increased linkage disequilibrium, and reduced efficacy of purifying selection. A novel result concerns the contrasting effects of range expansion versus shift to homostyly on transposable elements, for the former, process is associated with changes in transposable element genomic content, while the latter is not. Jointly, our results elucidate how the interactions among range expansion, transitions to selfing, and Quaternary climatic oscillations shape plant evolution.

Phylogeography and ecological niche modeling implicate multiple microrefugia of Swertia tetraptera during quaternary glaciations.

BACKGROUND: Climate fluctuations during the Pleistocene and mountain uplift are vital driving forces affecting geographic distribution. Here, we ask how an annual plant responded to the Pleistocene glacial cycles. METHODS: In this study, we analyzed the population demographic history of the annual herb Swertia tetraptera Maxim (Gentianaceae) endemic to Qinghai-Tibetan Plateau (QTP). A total of 301 individuals from 35 populations of S. tetraptera were analyzed based on two maternally inherited chloroplast fragments (trnL-trnF and trnS-trnG). Phylogeographic analysis was combined with species distribution modeling to detect the genetic variations in S. tetraptera. RESULTS: The genetic diversity of S. tetraptera was high, likely due to its wide natural range, high proportion of endemic haplotypes and evolutionary history. Fifty-four haplotypes were identified in S. tetraptera. Only a few haplotypes were widespread (Hap_4, Hap_1, Hap_3), which were dispersed throughout the present geographical range of S. tetraptera, while many haplotypes were confined to single populations. The cpDNA dataset showed that phylogeographic structuring was lacking across the distribution range of S. tetraptera. Analyses of molecular variance showed that most genetic variation was found within populations (70.51%). In addition, the relationships of the haplotypes were almost completely unresolved by phylogenetic reconstruction. Both mismatch distribution analysis and neutrality tests showed a recent expansion across the distribution range of S. tetraptera. The MaxEnt analysis showed that S. tetraptera had a narrow distribution range during the Last Glacial Maximum (LGM) and a wide distribution range during the current time, with predictions into the future showing the distribution range of S. tetraptera expanding. CONCLUSION: Our study implies that the current geographic and genetic distribution of S. tetraptera is likely to have been shaped by Quaternary periods. Multiple microrefugia of S. tetraptera existed during Quaternary glaciations. Rapid intraspecific diversification and hybridization and/or introgression may have played a vital role in shaping the current distribution patterns of S. tetraptera. The distribution range of S. tetraptera appeared to have experienced contraction during the LGM; in the future, when the global climate becomes warmer with rising carbon dioxide levels, the distribution of S. tetraptera will expand.

The European Pine Marten Martes martes (Linnaeus, 1758) Is Autochthonous in Sicily and Constitutes a Well-Characterised Major Phylogroup within the Species (Carnivora, Mustelidae).

No molecular data are currently available for the Sicilian populations of the European pine marten Martes martes, thus preventing any sound inference about its native or non-native status on the island, as well as the local phylogeography of the species. In order to investigate these issues, we sequenced two mtDNA markers in road-killed specimens collected in Sicily. Both markers consistently demonstrated the existence of a well-characterised Sicilian clade of the species, which is endemic to the island and constitutes the sister group of a clade including the Mediterranean and Central-North European major phylogroups of the European pine marten. Such evidence supports the autochthony of Martes martes in Sicily and points to a natural Pleistocene colonisation of the island followed by isolation. The occurrence of a, to date undetected, major phylogroup of the species in Sicily calls for the dedicated monitoring of the Sicilian populations of the species in order to preserve this evolutionarily significant unit.

A DNA barcode library for the butterflies of North America.

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.

Phylogeographic analyses of poplar revealed potential glacial refugia and allopatric divergence in southwest China.

The distribution pattern and genetic structure of plant species have been profoundly influenced by climate oscillations. Phylogeographic analyses have been numerously conducted in biodiversity hotspot regions and some general conclusions have been documented. However, other regions have received less attentions and these places may serve as potential glacial refugia for plant species to survive the Quaternary glaciation. Here, we used six nuclear and three cpDNA markers to estimate the phylogeographic pattern of Populus davidiana, a widespread species distributed in southwest China. As a widely distributed species in southwest China, the nucleotide diversity of P. davidiana was relatively high (Na = 6.28, HO = 0.534, and HE = 0.658). Genetic differentiation (FST) between the two main distribution regions, Yunnan and Guizhou provinces, was 0.21221. According to the composition of chloroplast haplotypes and the result of structure in these populations, we clearly distinguished two distantly sublineages corresponding to two distribution regions. Results of the Mantel test showed that there was a significant correlation between genetic distance and geographical distance (R2 = 0.8252, p<.05). The topographically heterogeneous regions and the low dispersal ability of seed and pollen may lead to high genetic differentiation between these two regions. A potential glacial refugia for P. davidiana located in adjacent regions to the Hengduan range was revealed and allopatric divergence in separated glacial refugia may directly lead to the present phylogeographic pattern of this species.

Last Glacial Maximum led to community-wide population expansion in a montane songbird radiation in highland Papua New Guinea.

BACKGROUND: Quaternary climate fluctuations are an engine of biotic diversification. Global cooling cycles, such as the Last Glacial Maximum (LGM), are known to have fragmented the ranges of higher-latitude fauna and flora into smaller refugia, dramatically reducing species ranges. However, relatively less is known about the effects of cooling cycles on tropical biota. RESULTS: We analyzed thousands of genome-wide DNA markers across an assemblage of three closely related understorey-inhabiting scrubwrens (Sericornis and Aethomyias; Aves) from montane forest along an elevational gradient on Mt. Wilhelm, the highest mountain of Papua New Guinea. Despite species-specific differences in elevational preference, we found limited differentiation within each scrubwren species, but detected a strong genomic signature of simultaneous population expansions at 27-29 ka, coinciding with the onset of the LGM. CONCLUSION: The remarkable synchronous timing of population expansions of all three species demonstrates the importance of global cooling cycles in expanding highland habitat. Global cooling cycles have likely had strongly different impacts on tropical montane areas versus boreal and temperate latitudes, leading to population expansions in the former and serious fragmentation in the latter.

Environmental differences are correlated with the distribution pattern of cytotypes in Veronica subsection Pentasepalae at a broad scale.

BACKGROUND AND AIMS: The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider approx. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and their relationship to environmental variables. METHODS: A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained principal coordinates analysis) analyses. KEY RESULTS: Four ploidy levels (2x, 4x, 6x and 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south to north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. CONCLUSIONS: The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.

Phylogeography of the Tibetan hamster Cricetulus kamensis in response to uplift and environmental change in the Qinghai-Tibet Plateau.

AIM: The evolutionary process of an organism provides valuable data toward an understanding of the Earth evolution history. To investigate the relationship between the uplift of the Qinghai-Tibet Plateau (QTP) and mammalian evolution since the late Cenozoic, the geographic distribution of genetic variations in the Tibetan hamster Cricetulus kamensis was investigated using phylogeographical methods. In particular, population divergence, demographic history, genetic variation, and the prediction of species distribution area were investigated. LOCATION: The Qinghai-Tibet Plateau. METHODS: A total of 53 specimens, representing 13 geographic populations, were collected from the QTP. The phylogeographical pattern and demographic history of C. kamensis were analyzed, and the probable factors in the QTP uplift and the Quaternary glacial periods were inferred from one nuclear and four mitochondrial genes. Furthermore, the species distribution model (SDM) was used to predict changes in potentially suitable habitats since the last Interglacial. RESULTS: Phylogenetic analysis demonstrated that two major genetic differentiations of the C. kamensis population occurred during the Early Pleistocene that were influenced by the Qing-Zang tectonic movement from the Middle Pliocene to the Early Pleistocene. Genetic distance between two major clades indicated low genetic divergence. Demographic history analysis showed that the C. kamensis population was affected by the Quaternary glacial period. SDM analysis indicated that C. kamensis was endemic to the QTP and the suitable habitat was affected by climate change, especially during the Last Glacial Maximum (LGM). MAIN CONCLUSION: Our results indicated that the QTP uplift led to the population divergence of C. kamensis, and vicariance well accounted for the geographic distribution of genetic variation in C. kamensis as a result of genetic divergence and lack of gene flow. The genetic distance shows that C. alticola may be a subspecies of C. kamensis. Demographic history analysis suggests that the QTP was affected by the last glacial period. SDM analysis supports that almost the entire QTP is covered by a huge ice sheet during the LGM.

Influence of the Quaternary Glacial Cycles and the Mountains on the Reticulations in the Subsection Willkommia of the Genus Centaurea.

Late Neogene and Quaternary climatic oscillations have greatly shaped the genetic structure of the Mediterranean Basin flora, with mountain plant species tracking warm interglacials/cold glacials by means of altitudinal shifts instead of broad latitudinal ones. Such dynamics may have enhanced population divergence but also secondary contacts. In this paper, we use a case example of subsection Willkommia of Centaurea (comprising three narrowly distributed endemic species, Centaurea gadorensis, C. pulvinata, and C. sagredoi) to test for reticulate evolution and recurrent hybridizations between nearby populations. For this, we combine analyses of genetic diversity and structuring, gene flow and spatial correlation, and ecological niche modeling. Our results support the contention that the current genetic structure of the three species is the result of historical gene flow at sites of secondary contact during the glacial periods, followed by isolation after the retraction of populations to the middle-upper areas of the mountains during the interglacial periods. The extent and direction of the gene flow was determined largely by the location of the populations on mountainsides oriented toward the same valley or toward different valleys, suggesting the intermountain valleys as the areas where secondary contacts occurred.

Rapid Intraspecific Diversification of the Alpine Species Saxifraga sinomontana (Saxifragaceae) in the Qinghai-Tibetan Plateau and Himalayas.

An increasing number of phylogeographic studies have been conducted for plant species in the Qinghai-Tibetan Plateau (QTP) and its flanking mountains. However, these studies have mainly focused on the determination of glacial refugia and routes of inter-/post-glacial expansions. Rapid intraspecific diversification of plants in this region have not been thoroughly discussed. Herein, we investigate the effects of the Quaternary climate changes on population genetic structure and diversifications of a herbaceous alpine species, Saxifraga sinomontana, which may have an evolutionary time scale <5 million years in the QTP and Himalayan regions. Using a total of 350 individuals from 29 populations, we studied the evolutionary history of S. sinomontana by analyzing cpDNA trnL-trnF, rpl16 and nrDNA ITS sequences. A total of 89 haplotypes and 158 genotypes were detected for cpDNA and ITS sequences, respectively. Only a few haplotypes/genotypes were widespread, while an extremely large number of haplotypes/genotypes were restricted to single populations, which were scattered throughout the current geographical range of S. sinomontana. This suggests the existence of microrefugia of this species during the Quaternary glaciations. In addition, the relationships of the haplotypes/genotypes were almost completely not resolved by phylogenetic reconstruction. Combining characteristics in terms of high haplotype richness, large proportion of private haplotypes, and shallow haplotype divergence, we speculate that recent intraspecific diversification has occurred in S. sinomontana. Molecular clock analysis estimated that the onset diversification within S. sinomontana to be 1.09 Ma (95% HPD = 0.80-1.45), coinciding with the extensive Quaternary glaciations on the QTP which started ca. 1.17 Ma. The Quaternary climatic oscillations may have triggered rapid intraspecific diversification in this QTP-Himalayan species. However, large niche breadth, as well as introgression/hybridization between the studied species and its closely related sympatric saxifrages, may also played a role to some extent on the current genetic structure of S. sinomontana, which need to be further studied.

Cold-blooded in the Ice Age: "refugia within refugia", inter-and intraspecific biogeographic diversification of European whipsnakes (Squamata, Colubridae, Hierophis).

In this work, we performed a biogeographic analysis with Bayesian binary MCMC (BBM) statistical dispersal-vicariance analysis (S-DIVA) and species distribution models (SDM) on three phylogenetically closely related Mediterranean whipsnakes (Hierophis gemonensis, H. carbonarius, H. viridiflavus), to investigate the pathways of their geographical diversification and locate putative refugial areas in the last glacial maximum (LGM). Our analysis suggests that the diversification processes between the studied species overall followed an east-west route, from eastern Greece to the Iberian Peninsula and continental France, highlighting a significant role of dispersal and vicariance processes at both inter- and intraspecific levels. In particular, the main lineage-splitting events between H. gemonensis, H. carbonarius and H. viridiflavus coincide with two events of vicariance, involving respectively eastern Greece and eastern Italy, and eastern Italy and western Italy, Iberian Peninsula and continental France. SDM analyses highlight the occurrence of multiple putative glacial refugia in the Balkans, Italy and southern France, which represent well the occurrence of distinct "refugia within refugia" in the main Mediterranean peninsulas. Furthermore, our results suggest how these refugia may have played an important role during the Quaternary climatic oscillations in shaping the current haplotype distribution of European whipsnakes.

Much late onset of Quaternary glaciations on the Tibetan Plateau: determining the age of the Shishapangma Glaciation using cosmogenic 26Al and 10Be dating.

The onset of Quaternary glaciations is a critical event in the climate and tectonic history of the Tibetan Plateau. The Shishapangma Glaciation, defined based on the till deposit from the northern slopes of Mt. Shishapangma, has been identified as the oldest glaciation on the Tibetan Plateau. However, the timing of this glaciation has not been constrained. We measured 10Be and 26Al concentrations of a set of boulders on top of this till and simulated their complex exposure-burial histories. The simulated results indicate that the formation age of this till is likely around 835.2 ±â€¯241.0 ka, representing the minimum timing of glacial onset on the Tibetan Plateau. The Shishapangma Glaciation is apparently much younger than the glacial onset in many other areas of the world, such as Europe and North America, and was likely driven by the coupled effect between tectonic uplift and climate cooling during the early-middle Pleistocene transition.

Phylogeographic analyses of a widely distributed Populus davidiana: Further evidence for the existence of glacial refugia of cool-temperate deciduous trees in northern East Asia.

Despite several phylogeographic studies had provided evidence to support the existence of glacial refugia of cool-temperate deciduous trees in northeast China, the species used in these studies were limited by the species ranges, which could not exclude the possibility that northern populations were the colonists from southern refugial populations during the last glacial maximum (LGM). Here, we estimated the nucleotide variation in Populus davidiana, a widespread species distributed in Eurasia. Three groups in northeast, central, and southwest China were constructed according to the simulation results from SAMOVA, composition of chloroplast haplotypes and structure results. We revealed that the northeast China had endemic haplotypes, the haplotypes and nucleotide diversity in northern regions were not lower than that in southern China, and this species has not experienced population expansion base on the estimation of Bayesian skyline plots. Ecological niche modeling (ENM) indicated that the northeast China had a high suitability score during the last glacial maximum. The combined evidence clearly demonstrated that northeastern and southwestern refugia were maintained across the current distributional range of P. davidiana during the LGM. The genetic differentiation between these two refugia might be mainly caused by differences of climate among these areas. The phylogeographic analyses of a widely distributed P. davidiana provided robust evidence to clarify the issue of refugia in northeast China, and these results are of great importance for understanding the influence of Quaternary glaciations on the distribution and evolution of species in East Asia.

Pleistocene range shifts, refugia and the origin of widespread species in western Palaearctic water beetles.

Quaternary glacial cycles drove major shifts in both the extent and location of the geographical ranges of many organisms. During glacial maxima, large areas of central and northern Europe were inhospitable to temperate species, and these areas are generally assumed to have been recolonized during interglacials by range expansions from Mediterranean refugia. An alternative is that this recolonization was from non-Mediterranean refugia, in central Europe or western Asia, but data on the origin of widespread central and north European species remain fragmentary, especially for insects. We studied three widely distributed lineages of freshwater beetles (the Platambus maculatus complex, the Hydraena gracilis complex, and the genus Oreodytes), all restricted to running waters and including both narrowly distributed southern endemics and widespread European species, some with distributions spanning the Palearctic. Our main goal was to determine the role of the Pleistocene glaciations in shaping the diversification and current distribution of these lineages. We sequenced four mitochondrial and two nuclear genes in populations drawn from across the ranges of these taxa, and used Bayesian probabilities and Maximum Likelihood to reconstruct their phylogenetic relationships, age and geographical origin. Our results suggest that all extant species in these groups are of Pleistocene origin. In the H. gracilis complex, the widespread European H. gracilis has experienced a rapid, recent range expansion from northern Anatolia, to occupy almost the whole of Europe. However, in the other two groups widespread central and northern European taxa appear to originate from central Asia, rather than the Mediterranean. These widespread species of eastern origin typically have peripherally isolated forms in the southern Mediterranean peninsulas, which may be remnants of earlier expansion-diversification cycles or result from incipient isolation of populations during the most recent Holocene expansion. The accumulation of narrow endemics of such lineages in the Mediterranean may result from successive cycles of range expansion, with subsequent speciation (and local extinction in glaciated areas) through multiple Pleistocene climatic cycles.

Phylogeography reveals an ancient cryptic radiation in East-Asian tree frogs (Hyla japonica group) and complex relationships between continental and island lineages.

BACKGROUND: In contrast to the Western Palearctic and Nearctic biogeographic regions, the phylogeography of Eastern-Palearctic terrestrial vertebrates has received relatively little attention. In East Asia, tectonic events, along with Pleistocene climatic conditions, likely affected species distribution and diversity, especially through their impact on sea levels and the consequent opening and closing of land-bridges between Eurasia and the Japanese Archipelago. To better understand these effects, we sequenced mitochondrial and nuclear markers to determine phylogeographic patterns in East-Asian tree frogs, with a particular focus on the widespread H. japonica. RESULTS: We document several cryptic lineages within the currently recognized H. japonica populations, including two main clades of Late Miocene divergence (~5 Mya). One occurs on the northeastern Japanese Archipelago (Honshu and Hokkaido) and the Russian Far-East islands (Kunashir and Sakhalin), and the second one inhabits the remaining range, comprising southwestern Japan, the Korean Peninsula, Transiberian China, Russia and Mongolia. Each clade further features strong allopatric Plio-Pleistocene subdivisions (~2-3 Mya), especially among continental and southwestern Japanese tree frog populations. Combined with paleo-climate-based distribution models, the molecular data allowed the identification of Pleistocene glacial refugia and continental routes of postglacial recolonization. Phylogenetic reconstructions further supported genetic homogeneity between the Korean H. suweonensis and Chinese H. immaculata, suggesting the former to be a relic population of the latter that arose when the Yellow Sea formed, at the end of the last glaciation. CONCLUSIONS: Patterns of divergence and diversity were likely triggered by Miocene tectonic activities and Quaternary climatic fluctuations (including glaciations), causing the formation and disappearance of land-bridges between the Japanese islands and the continent. Overall, this resulted in a ring-like diversification of H. japonica around the Sea of Japan. Our findings urge for important taxonomic revisions in East-Asian tree frogs. First, they support the synonymy of H. suweonensis (Kuramoto, 1980) and H. immaculata (Boettger, 1888). Second, the nominal H. japonica (Günther, 1859) represents at least two species: an eastern (new taxon A) on the northern Japanese and Russian Far East islands, and a southwestern species (n. t. B) on southern Japanese islands and possibly also forming continental populations. Third, these continental tree frogs may also represent an additional entity, previously described as H. stepheni Boulenger, 1888 (senior synonym of H. ussuriensis Nikolskii, 1918). A complete revision of this group requires further taxonomic and nomenclatural analyses, especially since it remains unclear to which taxon the species-epitheton japonica corresponds to.

Phylogeography of postglacial range expansion in Juglans mandshurica (Juglandaceae) reveals no evidence of bottleneck, loss of genetic diversity, or isolation by distance in the leading-edge populations.

The past studies of postglacial recolonization patterns in high latitude regions have revealed a significant role of dispersal capacity in shaping the genetic diversity and population structure of temperate trees. However, most of these studies have focused on species with long-distance dispersal followed by exponential population growth and were therefore unable to reveal the patterns in the case of a gradual expansion. Here we studied the impacts of postglacial range expansions on the distribution of genetic diversity in the Manchurian walnut (Juglans mandshurica), a common tree of East Asian cool-temperate deciduous forests that apparently lacks long-distance seed dispersal ability. The genetic diversity and structure of 19 natural walnut populations in Northeast China and the Korean Peninsula were examined using 17 nuclear simple sequence repeat (SSR) loci. Potential habitats under current and past climatic conditions were predicted using the ecological niche modelling (ENM) method. Bayesian clustering analysis revealed three groups, which were inferred to have diverged through multiple glacial-interglacial cycles in multiple refugia during the Quaternary Period. ENM estimated a southward range shift at the LGM, but high suitability scores still occurred in the western parts of the Changbai Mountains (Northeast China), the Korean peninsula and the exposed seafloor of the Yellow Sea. In contrast to most other cool-temperate trees co-occurring in the same region, the Manchurian walnut did not show any evidence of a population bottleneck, loss of genetic diversity or isolation by distance during the postglacial expansion. Our study clearly indicates that current northern populations originated from one glacial lineage and recolonization via a gradually advancing front due to the lack of a long-distance seed dispersal mechanism led to no latitudinal decrease in genetic diversity.

Evolutionary melting pots: a biodiversity hotspot shaped by ring diversifications around the Black Sea in the Eastern tree frog (Hyla orientalis).

Hotspots of intraspecific genetic diversity, which are of primary importance for the conservation of species, have been associated with glacial refugia, that is areas where species survived the Quaternary climatic oscillations. However, the proximate mechanisms generating these hotspots remain an open issue. Hotspots may reflect the long-term persistence of large refugial populations; alternatively, they may result from allopatric differentiation between small and isolated populations, that later admixed. Here, we test these two scenarios in a widely distributed species of tree frog, Hyla orientalis, which inhabits Asia Minor and southeastern Europe. We apply a fine-scale phylogeographic survey, combining fast-evolving mitochondrial and nuclear markers, with a dense sampling throughout the range, as well as ecological niche modelling, to understand what shaped the genetic variation of this species. We documented an important diversity centre around the Black Sea, composed of multiple allopatric and/or parapatric diversifications, likely driven by a combination of Pleistocene climatic fluctuations and complex regional topography. Remarkably, this diversification forms a ring around the Black Sea, from the Caucasus through Anatolia and eastern Europe, with terminal forms coming into contact and partially admixing in Crimea. Our results support the view that glacial refugia generate rather than host genetic diversity and can also function as evolutionary melting pots of biodiversity. Moreover, we report a new case of ring diversification, triggered by a large, yet cohesive dispersal barrier, a very rare situation in nature. Finally, we emphasize the Black Sea region as an important centre of intraspecific diversity in the Palearctic with implications for conservation.

Multiple glacial refugia for cool-temperate deciduous trees in northern East Asia: the Mongolian oak as a case study.

In East Asia, temperate forests are predicted to have retracted southward to c. 30° N during the last glacial maximum (LGM) based on fossil pollen data, whereas phylogeographic studies have often suggested glacial in situ survival of cool-temperate deciduous trees in their modern northern ranges. Here we report a study of the genetic diversity and structure of 29 natural Mongolian oak (Quercus mongolica) populations using 19 nuclear simple sequence repeat (nSSR) loci and four chloroplast DNA fragments. Bayesian clustering analysis with nSSRs revealed five groups, which were inferred by approximate Bayesian computation (ABC) to have diverged in multiple refugia through multiple glacial-interglacial cycles. Analysis of chloroplast DNA variation revealed four lineages that were largely but incompletely geographically disjunct. Ecological niche modelling (ENMs) indicated a southward range shift of the oak's distribution at the LGM, although high suitability scores were also evident in the Changbai Mts. (Northeast China), the Korean Peninsula, areas surrounding the Bohai Sea, and along the coast of the Russian Far East. In addition, endemic chloroplast DNA haplotypes and nuclear lineages occurred in high-latitude northern areas where the ENM predicted no suitable habitat. The combined evidence from nuclear and chloroplast DNA, and the results of the ENM clearly demonstrate that multiple northern refugia, including cryptic ones, were maintained across the current distributional range of the Mongolian oak during the LGM or earlier glacial periods. Though spatially limited, postglacial expansions from these refugia have led to a pattern of decreased genetic diversity with increasing latitude.

The intraspecific genetic variability of siliceous and calcareous Gentiana species is shaped by contrasting demographic and re-colonization processes.

The Ciminalis section of Gentiana comprises seven species, two of them growing on siliceous substrates (G. alpina and G. acaulis), the other ones being calcareous taxa (G. clusii, G. angustifolia, G. ligustica, G. occidentalis and G. dinarica). A total of 515 individuals from 183 populations over the entire Ciminalis distribution range was analyzed using four chloroplast loci (trnH-psbA, matK, rpoB and rpoC1) and the nuclear ribosomal marker ITS2. The siliceous species display only two chloroplast haplotypes each and are both characterized by patterns of range expansions all over the Alps. Conversely, the calcareous species are on average more diverse (two to 13 haplotypes per species) with strong patterns of local structuring. We suggest that the occurrence of many calcareous refugia at the periphery of the Alps must have led to local adaptation and morphological diversification, and helped preserving intraspecific diversities during the last glaciations for the associated taxa. ITS2 was more efficient in delineating species boundaries than the chloroplast markers for which several haplotypes are shared among species. This might be either due to chloroplast capture among species and/or to recent divergence. Species adapted to the same substrate are generally only distantly related when they co-occur in the same place. For both types of markers, G. clusii is found genetically distant from all other species.