Survival in nunatak and peripheral glacial refugia of three alpine plant species is partly predicted by altitudinal segregation.

Mountain biota survived the Quaternary cold stages most probably in peripheral refugia and/or ice-free peaks within ice-sheets (nunataks). While survival in peripheral refugia has been broadly demonstrated, evidence for nunatak refugia is still scarce. We generated RADseq data from three mountain plant species occurring at different elevations in the southeastern European Alps to investigate the role of different glacial refugia during the Last Glacial Maximum (LGM). We tested the following hypotheses. (i) The deep Piave Valley forms the deepest genetic split in the species distributed across it, delimiting two peripheral refugia. (ii) The montane to alpine species Campanula morettiana and Primula tyrolensis survived the LGM in peripheral refugia, while high-alpine to subnival Saxifraga facchinii likely survived in several nunatak refugia. (iii) The lower elevation species suffered a strong population decline during the LGM. By contrast, the higher elevation species shows long-term stability of population sizes due to survival on permanently ice-free peaks and small population sizes at present. We found peripheral refugia on both sides of the Piave Valley, which acted as a major genetic barrier. Demographic modelling confirmed nunatak survival not only for S. facchinii but also for montane to alpine C. morettiana. Altitudinal segregation influenced the species' demographic fluctuations, with the lower elevation species showing a significant population increase at the end of the LGM, and the higher elevation species either showing decrease towards the present or stable population sizes with a short bottleneck. Our results highlight the role of nunatak survival and species ecology in the demographic history of mountain species.

Evolutionary dynamics of Euphorbia carniolica suggest a complex Plio-Pleistocene history of understorey species of deciduous forest in southeastern Europe.

Deciduous forests form the dominant natural vegetation of Europe today, but were restricted to small refugia during Pleistocene cold stages, implying an evolutionary past shaped by recurrent range contractions and expansions. Cold-stage forest refugia were probably widespread in southern and central Europe, with the northwestern Balkan Peninsula being of particular importance. However, the actual number and location of deciduous forest refugia, as well as the connections between them, remain disputed. Here, we address the evolutionary dynamics of the deciduous forest understorey species Euphorbia carniolica as a proxy for past forest dynamics. To do so, we obtained genomic and morphometric data from populations representing the species' entire range, investigated phylogenetic position and intraspecific genetic variation, tested explicit demographic scenarios and applied species distribution models. Our data support two disjoint groups linked to separate refugia on the northwestern and central Balkan Peninsula. We find that genetic differentiation between groups started in the early Pleistocene via vicariance, suggesting a larger distribution in the past. Both refugia acted as sources for founder events to the southeastern Alps and the Carpathians; the latter were likely colonised before the last cold stage. In line with traditional views on the pre-Pleistocene origin of many southeastern European deciduous forest species, the origin of E. carniolica was dated to the late Pliocene. The fact that E. carniolica evolved at a time when a period of continuous forestation was ending in much of Eurasia provides an interesting biogeographical perspective on the past links between Eurasian deciduous forests and their biota.

Parallel adaptation to lower altitudes is associated with enhanced plasticity in Heliosperma pusillum (Caryophyllaceae).

High levels of phenotypic plasticity are thought to be inherently costly in stable or extreme environments, but enhanced plasticity may evolve as a response to new environments and foster novel phenotypes. Heliosperma pusillum forms glabrous alpine and pubescent montane ecotypes that diverged recurrently and polytopically (parallel evolution) and can serve as evolutionary replicates. The specific alpine and montane localities are characterized by distinct temperature conditions, available moisture, and light. Noteworthy, the ecotypes show a home-site fitness advantage in reciprocal transplantations. To disentangle the relative contribution of constitutive versus plastic gene expression to altitudinal divergence, we analyze the transcriptomic profiles of two parallely evolved ecotype pairs, grown in reciprocal transplantations at native altitudinal sites. In this incipient stage of divergence, only a minor proportion of genes appear constitutively differentially expressed between the ecotypes in both pairs, regardless of the growing environment. Both derived, montane populations bear comparatively higher plasticity of gene expression than the alpine populations. Genes that change expression plastically or constitutively underlie similar ecologically relevant pathways, related to response to drought and trichome formation. Other relevant processes, such as photosynthesis, rely mainly on plastic changes. The enhanced plasticity consistently observed in the montane ecotype likely evolved as a response to the newly colonized, drier, and warmer niche. We report a striking parallelism of directional changes in gene expression plasticity. Thus, plasticity appears to be a key mechanism shaping the initial stages of phenotypic evolution, likely fostering adaptation to novel environments.

Apomictic Mountain Whitebeam (Sorbus austriaca, Rosaceae) Comprises Several Genetically and Morphologically Divergent Lineages.

The interplay of polyploidisation, hybridization, and apomixis contributed to the exceptional diversity of Sorbus (Rosaceae), giving rise to a mosaic of genetic and morphological entities. The Sorbus austriaca species complex from the mountains of Central and South-eastern Europe represents an allopolyploid apomictic system of populations that originated following hybridisation between S. aria and S. aucuparia. However, the mode and frequency of such allopolyploidisations and the relationships among different, morphologically more or less similar populations that have often been described as different taxa remain largely unexplored. We used amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequencing, and analyses of nuclear microsatellites, along with multivariate morphometrics and ploidy data, to disentangle the relationships among populations within this intricate complex. Our results revealed a mosaic of genetic lineages-many of which have not been taxonomically recognised-that originated via multiple allopolyploidisations. The clonal structure within and among populations was then maintained via apomixis. Our results thus support previous findings that hybridisation, polyploidization, and apomixis are the main drivers of Sorbus diversification in Europe.

Disentangling Relationships among the Alpine Species of Luzula Sect. Luzula (Juncaceae) in the Eastern Alps.

Polyploidisation, agmatoploidy and symploidy have driven the diversification of Luzula sect. Luzula. Several morphologically very similar species with different karyotypes have evolved, but their evolutionary origins and relationships are unknown. In this study, we used a combination of relative genome size and karyotype estimations as well amplified fragment length polymorphism (AFLP) fingerprinting to investigate the relationships among predominately (sub)alpine Luzula alpina, L. exspectata, L multiflora and L. sudetica in the Eastern Alps, including also some samples of L. campestris and L. taurica as outgroup. Our study revealed common co-occurrence of two or three different ploidies (di-, tetra- and hexaploids) at the same localities, and thus also common co-occurrence of different species, of which L. sudetica was morphologically, ecologically and genetically most divergent. Whereas agmatoploid L. exspectata likely originated only once from the Balkan L. taurica, and hexaploid L. multiflora once from tetraploid L. multiflora, the AFLP data suggest multiple origins of tetraploid L. multiflora, from which partly agmatoploid individuals of L. alpina likely originated recurrently by partial fragmentation of the chromosomes. In contrast to common recurrent formation of polyploids in flowering plants, populations of agmatoploids resulting by fission of complete chromosome sets appear to have single origins, whereas partial agmatoploids are formed recurrently. Whether this is a general pattern in Luzula sect. Luzula, and whether segregation of ecological niches supports the frequent co-occurrence of closely related cytotypes in mixed populations, remains the subject of ongoing research.

Homoploid hybrids are common but evolutionary dead ends, whereas polyploidy is not linked to hybridization in a group of Pyrenean saxifrages.

Hybridization and polyploidy are major forces in plant evolution. Homoploid hybridization can generate new species via hybrid speciation, or modify extant evolutionary lineages through introgression. Polyploidy enables instantaneous reproductive isolation from the parental lineage(s) and is often coupled with evolutionary innovations, especially when linked to hybridization. While allopolyploidy is a well-known and common mechanism of plant speciation, the evolutionary role of autopolyploidy might have been underestimated. Here, we studied the saxifrages of Saxifraga subsection Saxifraga in the Pyrenees, which easily hybridise and include polyploid populations of uncertain origin, as a model to unravel evolutionary consequences and origin of hybridization and polyploidy. Additionally, we investigate the phylogenetic relationship between the two subspecies of the endemic S. pubescens to ascertain whether they should rather be treated as different species. For these purposes, we combined ploidy-informed restriction associated DNA analyses, plastid DNA sequences and morphological data on a comprehensive population sample of seven species. Our results unravel multiple homoploid hybridization events at the diploid level between different species pairs, but with limited evolutionary impact. The ploidy-informed analyses reveal that all tetraploid populations detected in the present study belong to the widespread alpine species S. moschata. Although of autopolyploid origin, they are to some extent morphologically differentiated and underwent a different evolutionary pathway than their diploid parent. However, the high plastid DNA diversity and the internal structure within eastern and western population groups suggest multiple origins of the polyploids. Finally, our phylogenetic analyses show that S. pubescens and S. iratiana are clearly not sister lineages, and should consequently be considered as independent species.

Polygenic routes lead to parallel altitudinal adaptation in Heliosperma pusillum (Caryophyllaceae).

Understanding how organisms adapt to the environment is a major goal of modern biology. Parallel evolution-the independent evolution of similar phenotypes in different populations-provides a powerful framework to investigate the evolutionary potential of populations, the constraints of evolution, its repeatability and therefore its predictability. Here, we quantified the degree of gene expression and functional parallelism across replicated ecotype formation in Heliosperma pusillum (Caryophyllaceae), and gained insights into the architecture of adaptive traits. Population structure analyses and demographic modelling support a previously formulated hypothesis of parallel polytopic divergence of montane and alpine ecotypes. We detect a large proportion of differentially expressed genes (DEGs) underlying divergence within each replicate ecotype pair, with a strikingly low number of shared DEGs across pairs. Functional enrichment of DEGs reveals that the traits affected by significant expression divergence are largely consistent across ecotype pairs, in strong contrast to the nonshared genetic basis. The remarkable redundancy of differential gene expression indicates a polygenic architecture for the diverged adaptive traits. We conclude that polygenic traits appear key to opening multiple routes for adaptation, widening the adaptive potential of organisms.

Post-glacial range formation of temperate forest understorey herbs - Insights from a spatio-temporally explicit modelling approach.

Aim: Our knowledge of Pleistocene refugia and post-glacial recolonization routes of forest understorey plants is still very limited. The geographical ranges of these species are often rather narrow and show highly idiosyncratic, often fragmented patterns indicating either narrow and species-specific ecological tolerances or strong dispersal limitations. However, the relative roles of these factors are inherently difficult to disentangle. Location: Central and south-eastern Europe. Time period: 17,100 BP - present. Major taxa studied: Five understorey herbs of European beech forests: Aposeris foetida, Cardamine trifolia, Euphorbia carniolica, Hacquetia epipactis and Helleborus niger. Methods: We used spatio-temporally explicit modelling to reconstruct the post-glacial range dynamics of the five forest understorey herbs. We varied niche requirements, demographic rates and dispersal abilities across plausible ranges and simulated the spread of species from potential Pleistocene refugia identified by phylogeographical analyses. Then we identified the parameter settings allowing for the most accurate reconstruction of their current geographical ranges. Results: We found a largely homogenous pattern of optimal parameter settings among species. Broad ecological niches had to be combined with very low but non-zero rates of long-distance dispersal via chance events and low rates of seed dispersal over moderate distances by standard dispersal vectors. However, long-distance dispersal events, although rare, led to high variation among replicated simulation runs. Main conclusions: Small and fragmented ranges of many forest understorey species are best explained by a combination of broad ecological niches and rare medium- and long-distance dispersal events. Stochasticity is thus an important determinant of current species ranges, explaining the idiosyncratic distribution patterns of the study species despite strong similarities in refugia, ecological tolerances and dispersal abilities.

Postglacial range expansion of high-elevation plants is restricted by dispersal ability and habitat specialization.

Aim: Species' ecological traits influence their spatial genetic patterns. Bedrock preference strongly shapes the phylogeography of alpine plants, but its interactions with other ecological traits have rarely been disentangled. Here, we explore whether dispersal ability and degree of habitat specialization account for divergent postglacial expansion patterns of high-elevation plants in spite of similar bedrock preference. Location: The Pyrenees, southwestern Europe. Taxon: Cirsium glabrum (Asteraceae), Salix pyrenaica (Salicaceae) and Silene borderei (Caryophyllaceae). Methods: Phylogenetic, genetic structure and demographic modelling analyses based on restriction-site-associated DNA sequencing (RADseq) data from a range-wide populational sampling were conducted. Occurrence data and environmental variables were used to construct species distribution models, which were projected under current and Last Glacial Maximum conditions, and were combined with RADseq data to reconstruct the postglacial history of the study species. The degree of habitat specialization of each species was estimated based on the plant communities within which they occur, and their climatic niche breadth. Results: Salix pyrenaica, which occupies a broad range of habitats, shows a high level of range filling, a blurred genetic structure and an admixture cline between the two main genetic groups, congruent with rapid postglacial expansion. The microsite specialists C. glabrum and S. borderei exhibit a strong genetic structure and low levels of range filling, indicative of slow postglacial expansion. The good disperser C. glabrum shows higher levels of admixture between genetic groups and weaker population differentiation than the poor disperser S. borderei. Main Conclusions: Factors other than bedrock preference have a strong impact on the postglacial range dynamics of high-elevation species. Habitat specialization plays an important role, allowing species occupying a broad range of habitats to more rapidly expand their ranges after environmental change. The effect of dispersal ability is lower than expected for the study species.

Congruent evolutionary responses of European steppe biota to late Quaternary climate change.

Quaternary climatic oscillations had a large impact on European biogeography. Alternation of cold and warm stages caused recurrent glaciations, massive vegetation shifts, and large-scale range alterations in many species. The Eurasian steppe biome and its grasslands are a noteworthy example; they underwent climate-driven, large-scale contractions during warm stages and expansions during cold stages. Here, we evaluate the impact of these range alterations on the late Quaternary demography of several phylogenetically distant plant and insect species, typical of the Eurasian steppes. We compare three explicit demographic hypotheses by applying an approach combining convolutional neural networks with approximate Bayesian computation. We identified congruent demographic responses of cold stage expansion and warm stage contraction across all species, but also species-specific effects. The demographic history of the Eurasian steppe biota reflects major paleoecological turning points in the late Quaternary and emphasizes the role of climate as a driving force underlying patterns of genetic variance on the biome level.

Evidence for Glacial Refugia of the Forest Understorey Species Helleborus niger (Ranunculaceae) in the Southern as Well as in the Northern Limestone Alps.

Glacial refugia of alpine and subnival biota have been intensively studied in the European Alps but the fate of forests and their understory species in that area remains largely unclear. In order to fill this gap, we aimed at disentangling the spatiotemporal diversification of disjunctly distributed black hellebore Helleborus niger (Ranunculaceae). We applied a set of phylogeographic analyses based on restriction-site associated DNA sequencing (RADseq) data and plastid DNA sequences to a range-wide sampling of populations. These analyses were supplemented with species distribution models generated for the present and the Last Glacial Maximum (LGM). We used exploratory analyses to delimit genomically coherent groups and then employed demographic modeling to reconstruct the history of these groups. We uncovered a deep split between two major genetic groups with western and eastern distribution within the Southern Limestone Alps, likely reflecting divergent evolution since the mid-Pleistocene in two glacial refugia situated along the unglaciated southern margin of the Alps. Long-term presence in the Southern Limestone Alps is also supported by high numbers of private alleles, elevated levels of nucleotide diversity and the species' modeled distribution at the LGM. The deep genetic divergence, however, is not reflected in leaf shape variation, suggesting that the morphological discrimination of genetically divergent entities within H. niger is questionable. At a shallower level, populations from the Northern Limestone Alps are differentiated from those in the Southern Limestone Alps in both RADseq and plastid DNA data sets, reflecting the North-South disjunction within the Eastern Alps. The underlying split was dated to ca. 0.1 mya, which is well before the LGM. In the same line, explicit tests of demographic models consistently rejected the hypothesis that the partial distribution area in the Northern Limestone Alps is the result of postglacial colonization. Taken together, our results strongly support that forest understory species such as H. niger have survived the LGM in refugia situated along the southern, but also along the northern or northeastern periphery of the Alps. Being a slow migrator, the species has likely survived repeated glacial-interglacial circles in distributional stasis while the composition of the tree canopy changed in the meanwhile.

Performance comparison of two reduced-representation based genome-wide marker-discovery strategies in a multi-taxon phylogeographic framework.

Multi-locus genetic data are pivotal in phylogenetics. Today, high-throughput sequencing (HTS) allows scientists to generate an unprecedented amount of such data from any organism. However, HTS is resource intense and may not be accessible to wide parts of the scientific community. In phylogeography, the use of HTS has concentrated on a few taxonomic groups, and the amount of data used to resolve a phylogeographic pattern often seems arbitrary. We explore the performance of two genetic marker sampling strategies and the effect of marker quantity in a comparative phylogeographic framework focusing on six species (arthropods and plants). The same analyses were applied to data inferred from amplified fragment length polymorphism fingerprinting (AFLP), a cheap, non-HTS based technique that is able to straightforwardly produce several hundred markers, and from restriction site associated DNA sequencing (RADseq), a more expensive, HTS-based technique that produces thousands of single nucleotide polymorphisms. We show that in four of six study species, AFLP leads to results comparable with those of RADseq. While we do not aim to contest the advantages of HTS techniques, we also show that AFLP is a robust technique to delimit evolutionary entities in both plants and animals. The demonstrated similarity of results from the two techniques also strengthens biological conclusions that were based on AFLP data in the past, an important finding given the wide utilization of AFLP over the last decades. We emphasize that whenever the delimitation of evolutionary entities is the central goal, as it is in many fields of biodiversity research, AFLP is still an adequate technique.

Long-term isolation of European steppe outposts boosts the biome's conservation value.

The European steppes and their biota have been hypothesized to be either young remnants of the Pleistocene steppe belt or, alternatively, to represent relicts of long-term persisting populations; both scenarios directly bear on nature conservation priorities. Here, we evaluate the conservation value of threatened disjunct steppic grassland habitats in Europe in the context of the Eurasian steppe biome. We use genomic data and ecological niche modelling to assess pre-defined, biome-specific criteria for three plant and three arthropod species. We show that the evolutionary history of Eurasian steppe biota is strikingly congruent across species. The biota of European steppe outposts were long-term isolated from the Asian steppes, and European steppes emerged as disproportionally conservation relevant, harbouring regionally endemic genetic lineages, large genetic diversity, and a mosaic of stable refugia. We emphasize that conserving what is left of Europe's steppes is crucial for conserving the biological diversity of the entire Eurasian steppe biome.

Multiple auto- and allopolyploidisations marked the Pleistocene history of the widespread Eurasian steppe plant Astragalus onobrychis (Fabaceae).

The Eurasian steppes occupy a significant portion of the worldwide land surface and their biota have been affected by specific past range dynamics driven by ice ages-related climatic fluctuations. The dynamic alterations in conditions during the Pleistocene often triggered reticulate evolution and whole genome duplication events. Employing genomic, genetic and cytogenetic tools as well as morphometry we investigate the intricate evolution of Astragalus onobrychis, a widespread Eurasian steppe plant with diploid, tetraploid and octoploid cytotypes. To analyse the heteroploid RADseq dataset we employ both genotype-based and genotype-free methods that result in highly consistent results, and complement our inference with information from the plastid ycf1 region. We uncover a complex and reticulate evolutionary history, including at least one auto-tetraploidization event and two allo-octoploidization events; one of them involved also genetic contributions from other species, most likely A. goktschaicus. The present genetic structure points to the existence of four main clades within A. onobrychis, which only partly correspond to different ploidies. Time-calibrated diffusion models suggest that diversification within A. onobrychis was associated with ice age-related climatic fluctuations during the last million years. We finally argue for the usefulness of uniparentally inherited plastid markers, even in the genomic era, especially when investigating heteroploid systems.

Is the incidence of survival in interior Pleistocene refugia (nunataks) underestimated? Phylogeography of the high mountain plant Androsace alpina (Primulaceae) in the European Alps revisited.

Temperate mountain ranges such as the European Alps have been strongly affected by the Pleistocene glaciations. Glacial advances forced biota into refugia, which were situated either at the periphery of mountain ranges or in their interior. Whereas in the Alps peripheral refugia have been repeatedly and congruently identified, support for the latter scenario, termed "nunatak hypothesis," is still limited and no general pattern is recognizable yet. Here, we test the hypothesis of nunatak survival for species growing in the high alpine to subnival zones on siliceous substrate using the cushion plant Androsace alpina (Primulaceae), endemic to the European Alps, as our model species. To this end, we analyzed AFLP and plastid DNA sequence data obtained from a dense and range-wide sampling. Both AFLPs and plastid sequence data identified the southwestern-most population as the most divergent one. AFLP data did not allow for discrimination of interior and peripheral populations, but rather identified two to three longitudinally separated major gene pools. In contrast, in the eastern half of the Alps several plastid haplotypes of regional or local distribution in interior ranges-the Alpine periphery mostly harbored a widespread haplotype-were indicative for the presence of interior refugia. Together with evidence from other Alpine plant species, this study shows that in the eastern Alps silicicolous species of open habitats in the alpine and subnival zone survived, also or exclusively so, in interior refugia. As the corresponding genetic structure may be lost in mostly nuclear-derived, rapidly homogenizing marker systems such as AFLPs or RAD sequencing tags, markers not prone to homogenization, as is the case for plastid sequences (Sanger-sequenced or extracted from an NGS data set) will continue to be important for detecting older, biogeographically relevant patterns.

Ancestral remnants or peripheral segregates? Phylogenetic relationships of two narrowly endemic Euphrasia species (Orobanchaceae) from the eastern European Alps.

Endemism in mountain ranges is considered to be the result of a number of factors, including restriction to refugia during Pleistocene climate fluctuations. However, isolation in glacial refugia cannot explain the origin of narrowly endemic taxa restricted to formerly heavily glaciated areas. Here, we investigate the phylogeny of two narrowly endemic species, Euphrasia inopinata and E. sinuata (Orobanchaceae), found exclusively in formerly heavily glaciated areas of the eastern European Alps. As both species are diploid and very similar to the widespread (allo)polyploid E. minima, we test whether the restricted distributions of E. inopinata and E. sinuata are relictual, i.e. the two species are ancestral diploid remnants of a polyploid complex, or whether they are derived, i.e. the two species are peripheral segregates of a more widespread diploid. Based on internal transcribed spacer (ITS) sequence and amplified fragment length polymorphism (AFLP) fingerprint data it is shown that E. inopinata and E. sinuata, whose diploid ploidy level is confirmed for all analysed individuals via flow cytometry, are phylogenetically closely related to diploid E. alpina s. l. (series Alpinae) instead of E. minima (series Parviflorae). In addition, there is no evidence that these two diploid species participated in the formation of allotetraploid E. minima. Thus, E. inopinata and E. sinuata are interpreted as peripheral segregates of the widespread E. alpina s. l. Shifts in pollination system from allogamy in E. alpina s. l. to autogamy in E. inopinata and E. sinuata, genetic drift in small populations and geographic isolation at the periphery of the range of E. alpina s. str. probably contributed to the morphological and ecological differentiation of E. inopinata and E. sinuata.

Contrasting evolutionary origins of two mountain endemics: Saxifraga wahlenbergii (Western Carpathians) and S. styriaca (Eastern Alps).

BACKGROUND: The Carpathians and the Alps are the largest mountain ranges of the European Alpine System and important centres of endemism. Among the distinctive endemic species of this area is Saxifraga wahlenbergii, a Western Carpathians member of the speciose genus Saxifraga. It was frequently considered a taxonomically isolated Tertiary palaeopolyploid and palaeoendemic, for which the closest relatives could not yet be traced. A recently described narrow endemic of the Eastern Alps, S. styriaca, was hypothesized to be closely related to S. wahlenbergii based on shared presence of peculiar glandular hairs. To elucidate the origin and phylogenetic relationships of both species we studied nuclear and plastid DNA markers based on multiple accessions and analysed the data in a wide taxonomic context. We applied Sanger sequencing, followed by targeted next-generation sequencing (NGS) for a refined analysis of nrITS variants to detect signatures of ancient hybridization. The ITS data were used to estimate divergence times of different lineages using a relaxed molecular clock. RESULTS: We demonstrate divergent evolutionary histories for the two mountain endemics. For S. wahlenbergii we revealed a complicated hybrid origin. Its maternal parent belongs to a Western Eurasian lineage of high mountain taxa grouped in subsect. Androsaceae and is most likely the widespread S. androsacea. The putative second parent was most likely S. adscendens, which belongs to the distantly related subsect. Tridactylites. While Sanger sequencing of nrITS only showed S. adscendens-related variants in S. wahlenbergii, our NGS screening revealed presence of sequences from both lineages with clear predominance of the paternal over the maternal lineage. CONCLUSIONS: Saxifraga styriaca was unambiguously assigned to subsect. Androsaceae and is not the sister taxon of S. wahlenbergii. Accordingly, the similarity of the glandular hairs observed in both taxa rests on parallelism and both species do not constitute an example of a close evolutionary link between the floras of the Western Carpathians and Eastern Alps. With the origin of its paternal, S. adscendens-like ITS DNA estimated to ca. 4.7 Ma, S. wahlenbergii is not a relict of the mid-Tertiary climate optimum. Its hybrid origin is much younger and most likely took place in the Pleistocene.

Integrating phylogenomics, phylogenetics, morphometrics, relative genome size and ecological niche modelling disentangles the diversification of Eurasian Euphorbia seguieriana s. l. (Euphorbiaceae).

Next generation sequencing has revolutionised biology. Restriction-associated DNA sequencing (RADseq) has primarily been used to study infraspecific relationships but has also been applied in multi-species phylogenomic analyses. In this study, we used a combination of phylogenomic (with RADseq data) and phylogenetic (with sequences of the nuclear internal transcribed spacer, ITS) methods to explore relationships within the taxonomically intricate Euphorbia seguieriana s. l., one of the most widespread Euphorbia taxa inhabiting zonal and extrazonal steppes from Iberia to Central Asia. In the inferred phylogenies the southeastern Balkan and Anatolian populations were clearly separated, supporting the distinction of E. niciciana from E. seguieriana at the species level. Within E. seguieriana, the populations from the Caucasus, Iran, and easternmost Anatolia were sister to all other populations based on RADseq, making necessary the description of a new, morphologically divergent subspecies, E. seguieriana subsp. armeniaca. Conversely, additional studies are needed to understand the status of E. seguieriana subsp. hohenackeri, which is sympatric with E. seguieriana subsp. armeniaca. Niche analyses indicated that differences in the climatic niche between E. niciciana and E. seguieriana are relatively small compared with the climatic differences between the regions over which they are distributed. Contrary to previous believes, E. niciciana and E. seguieriana are allopatric and have likely diverged during the Pleistocene in two different glacial refugia as suggested by distribution modelling. Euphorbia niciciana nowadays has a submediterranean distribution, occupying habitats that are slightly warmer, moister, and less seasonal in temperature but more seasonal in precipitation than E. seguieriana, a characteristic species of continental steppes. Using flow cytometry, we demonstrate that the relative genome sizes of E. niciciana and E. seguieriana differ significantly. Additionally, multivariate morphometric analyses of 56 morphological characters indicated clear morphological divergence of the two species. Importantly, we also provide a revised taxonomic treatment including formal nomenclatural changes, an identification key and species descriptions. Our study demonstrates that an integrative approach, combining modern phylogenomic methods with traditional phylogenetic, cytogenetic, environmental and morphological analyses can result in satisfactorily resolved relationships in intricate groups of closely related species. Finally, phylogenetic inference using ITS sequences is still a useful tool for resolving relationships among the taxa at the species level, but the phylogenomic approach based on RADseq data certainly provides better resolution both among and within species.

Phylogeography of western Mediterranean Cymbalaria (Plantaginaceae) reveals two independent long-distance dispersals and entails new taxonomic circumscriptions.

The Balearic Islands, Corsica and Sardinia (BCS) constitute biodiversity hotspots in the western Mediterranean Basin. Oligocene connections and long distance dispersal events have been suggested to cause presence of BCS shared endemic species. One of them is Cymbalaria aequitriloba, which, together with three additional species, constitute a polyploid clade endemic to BCS. Combining amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequences and morphometrics, we inferred the phylogeography of the group and evaluated the species' current taxonomic circumscriptions. Based on morphometric and AFLP data we propose a new circumscription for C. fragilis to additionally comprise a group of populations with intermediate morphological characters previously included in C. aequitriloba. Consequently, we suggest to change the IUCN category of C. fragilis from critically endangered (CR) to near threatened (NT). Both morphology and AFLP data support the current taxonomy of the single island endemics C. hepaticifolia and C. muelleri. The four species had a common origin in Corsica-Sardinia, and two long-distance dispersal events to the Balearic Islands were inferred. Finally, plastid DNA data suggest that interspecific gene flow took place where two species co-occur.

Origin and Diversification of South American Polyploid Silene Sect. Physolychnis (Caryophyllaceae) in the Andes and Patagonia.

The Andes are an important biogeographic region in South America extending for about 8000 km from Venezuela to Argentina. They are - along with the Patagonian steppes - the main distribution area of ca. 18 polyploid species of Silene sect. Physolychnis. Using nuclear ITS and plastid psbE-petG and matK sequences, flow cytometric ploidy level estimations and chromosome counts, and including 13 South American species, we explored the origin and diversification of this group. Our data suggest a single, late Pliocene or early Pleistocene migration of the North American S. verecunda lineage to South America, which was followed by dispersal and diversification of this tetraploid lineage in the Andes, other Argentinian mountain ranges and the Patagonian steppes. Later in the Pleistocene South American populations hybridized with the S. uralensis lineage, which led to allopolyploidisation and origin of decaploid S. chilensis and S. echegarayi occurring at high elevations. Additionally, we show that the morphological differentiation in leaf shape correlated with divergent habitats (high elevation Andes vs. lower elevation Patagonian steppes) is also supported phylogenetically, especially in the ITS tree. Lastly, the species boundaries among the narrow-leaved Patagonian steppe species are poorly resolved and need more thorough taxonomic revision.