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.

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.

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.

Meeting in Liguria: Hybridisation between Apennine endemic Euphorbia barrelieri and western Mediterranean E. nicaeensis led to the allopolyploid origin of E. ligustica.

The Mediterranean Basin is renowned for its extremely rich biota and is considered as one of the 25 Global Biodiversity Hotspots, but its diversity is not homogeneously distributed. Outstanding in the number of (endemic) species are the Ligurian Alps (Italy). At the foot of the Ligurian Alps, little above the Mediterranean Sea, a disjunct occurrence of Italian endemic Euphorbia barrelieri was reported. Using an array of integrative methods ranging from cytogenetic (chromosome number and relative genome size estimation), over phylogenetic approaches (plastid, ITS and RAD sequencing) to multivariate morphometrics we disentangled the origin of these populations that were shown to be tetraploid. We performed phylogenetic analyses of the nuclear ITS and plastid regions of a broad taxonomic sampling of Euphorbia sect. Pithyusa to identify possible species involved in the origin of the tetraploid populations and then applied various analyses of RADseq data to identify the putative parental species. Our results have shown that the Ligurian populations of E. barrelieri are of allotetraploid origin that involved E. barrelieri and western Mediterranean E. nicaeensis as parental species. We thus describe a new species, E. ligustica, and hypothesise that its adaptation to similar environments in which E. barrelieri occurs, triggered development of similar morphology, whereas its genetic composition appears to be closer to that of E. nicaeensis. Our study emphasises the importance of polyploidisation for plant diversification, highlights the value of the Ligurian Alps as a hotspot of biodiversity and endemism and underlines the importance of integrative taxonomic approaches in uncovering cryptic diversity.

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.

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.

Genomic analyses suggest parallel ecological divergence in Heliosperma pusillum (Caryophyllaceae).

The mosaic distribution of interbreeding taxa with contrasting ecology and morphology offers an opportunity to study microevolutionary dynamics during ecological divergence. We investigate here the evolutionary history of an alpine and a montane ecotype of Heliosperma pusillum (Caryophyllaceae) in the south-eastern Alps. From six pairs of geographically close populations of the two ecotypes (120 individuals) we obtained a high-coverage restriction site associated DNA sequencing (RADseq) dataset that was used for demographic inference to test the hypothesis of parallel evolution of the two ecotypes. The data are consistent with repeated ecological divergence in H. pusillum, uncovering up to five polytopic origins of one ecotype from the other. A complex evolutionary history is evidenced, with local isolation-with-migration in two population pairs and intra-ecotype migration in two others. In all cases, the time of divergence or secondary contact was inferred as postglacial. A metagenomic analysis on exogenous contaminant RAD sequences suggests divergent microbial communities between the ecotypes. The lack of shared genomic regions of high divergence across population pairs illustrates the action of drift and/or local selection in shaping genetic divergence across repeated cases of ecological divergence.

Patterns of rapid diversification in heteroploid Knautia sect. Trichera (Caprifoliaceae, Dipsacoideae), one of the most intricate taxa of the European flora.

BACKGROUND: Polyploidy is one of the most important evolutionary pathways in flowering plants and has significantly contributed to their diversification and radiation. Due to the prevalence of reticulate evolution spanning three ploidy levels, Knautia is considered one of the taxonomically most intricate groups in the European flora. On the basis of ITS and plastid DNA sequences as well as AFLP fingerprints obtained from 381 populations of almost all species of the genus we asked the following questions. (1) Where and when did the initial diversification in Knautia take place, and how did it proceed further? (2) Did Knautia undergo a similarly recent (Pliocene/Pleistocene) rapid radiation as other genera with similar ecology and overlapping distribution? (3) Did polyploids evolve within the previously recognised diploid groups or rather from hybridisation between groups? RESULTS: The diversification of Knautia was centred in the Eastern Mediterranean. According to our genetic data, the genus originated in the Early Miocene and started to diversify in the Middle Miocene, whereas the onset of radiation of sect. Trichera was in central parts of the Balkan Peninsula, roughly 4 Ma. Extensive spread out of the Balkans started in the Pleistocene about 1.5 Ma. Diversification of sect. Trichera was strongly fostered by polyploidisation, which occurred independently many times. Tetraploids are observed in almost all evolutionary lineages whereas hexaploids are rarer and restricted to a few phylogenetic groups. Whether polyploids originated via autopolyploidy or allopolyploidy is unclear due to the weak genetic separation among species. In spite of the complexity of sect. Trichera, we present nine AFLP-characterised informal species groups, which coincide only partly with former traditional groups. CONCLUSIONS: Knautia sect. Trichera is a prime example for rapid diversification, mostly taking place during Pliocene and Pleistocene. Numerous cycles of habitat fragmentation and subsequent reconnections likely promoted hybridisation and polyploidisation. Extensive haplotype sharing and unresolved phylogenetic relationships suggest that these processes occurred rapidly and extensively. Thus, the dynamic polyploid evolution, the lack of crossing barriers within ploidy levels supported by conserved floral morphology, the highly variable leaf morphology and unstable indumentum composition prevent establishing a well-founded taxonomic framework.

Heteroploid Knautia drymeia includes K. gussonei and cannot be separated into diagnosable subspecies.

PREMISE OF THE STUDY: Knautia drymeia is a morphologically variable, diploid and tetraploid temperate forest understory species distributed in southeastern Europe and adjacent areas. The species is an excellent system to explore the influence of polypoidy on taxonomic delineations, the role of hybridization among genetically distant populations in polyploid evolution, and the impact of glacial refugia on the evolution of polyploids. METHODS: Amplified fragment length polymorphism fingerprinting and multivariate analyses of morphological characters were performed on 57 populations spanning the distribution area of K. drymeia. K-means clustering, comparison of in-silico tetraploids and observed tetraploids, and a phylogeographic analysis using relaxed random walks were used to explore the genetic structure within the diploids, to infer the origin of the tetraploids and to reconstruct range expansion through time. Further, we contrasted the morphology and genetic groups with current taxonomy and evaluated the status of the tetraploid Apennine endemic K. gussonei and the intraspecific taxa of K. drymeia. KEY RESULTS: The genetic structure was strongly geographically correlated and yielded four genetic groups; K. gussonei was inseparable from K. drymeia. Distributions of diploid lineages are suggestive of glacial refugia in the northwesternmost and southeastern Balkan Peninsula. Polyploids originated at least two times, as autopolyploids and probably additionally also as allopolyploids. Morphological divergence corresponded with neither genetic groups nor current taxonomy. CONCLUSIONS: Genetic and morphometric data confirmed neither divergence of K. gussonei nor recognition of subspecies within K. drymeia. We therefore propose treating K. drymeia as a morphologically and genetically variable species without infraspecific taxa.

Cytotype diversity and genome size variation in Knautia (Caprifoliaceae, Dipsacoideae).

BACKGROUND: Polyploidisation is one of the most important mechanisms in the evolution of angiosperms. As in many other genera, formation of polyploids has significantly contributed to diversification and radiation of Knautia (Caprifoliaceae, Dipsacoideae). Comprehensive studies of fine- and broad-scale patterns of ploidy and genome size (GS) variation are, however, still limited to relatively few genera and little is known about the geographic distribution of ploidy levels within these genera. Here, we explore ploidy and GS variation in Knautia based on a near-complete taxonomic and comprehensive geographic sampling. RESULTS: Genome size is a reliable indicator of ploidy level in Knautia, even if monoploid genome downsizing is observed in the polyploid cytotypes. Twenty-four species studied are diploid, 16 tetraploid and two hexaploid, whereas ten species possess two, and two species possess three ploidy levels. Di- and tetraploids are distributed across most of the distribution area of Knautia, while hexaploids were sampled in the Balkan and Iberian Peninsulas and the Alps. CONCLUSIONS: We show that the frequency of polyploidisation is unevenly distributed in Knautia both in a geographic and phylogenetic context. Monoploid GS varies considerably among three evolutionary lineages (sections) of Knautia, but also within sections Trichera and Tricheroides, as well as within some of the species. Although the exact causes of this variation remain elusive, we demonstrate that monoploid GS increases significantly towards the limits of the genus' distribution.

Escaping to the summits: phylogeography and predicted range dynamics of Cerastium dinaricum, an endangered high mountain plant endemic to the western Balkan Peninsula.

The Balkans are a major European biodiversity hotspot, however, almost nothing is known about processes of intraspecific diversification of the region's high-altitude biota and their reaction to the predicted global warming. To fill this gap, genome size measurements, AFLP fingerprints, plastid and nuclear sequences were employed to explore the phylogeography of Cerastium dinaricum. Range size changes under future climatic conditions were predicted by niche-based modeling. Likely the most cold-adapted plant endemic to the Dinaric Mountains in the western Balkan Peninsula, the species has conservation priority in the European Union as its highly fragmented distribution range includes only few small populations. A deep phylogeographic split paralleled by divergent genome size separates the populations into two vicariant groups. Substructure is pronounced within the southeastern group, corresponding to the area's higher geographic complexity. Cerastium dinaricum likely responded to past climatic oscillations with altitudinal range shifts, which, coupled with high topographic complexity of the region and warmer climate in the Holocene, sculptured its present fragmented distribution. Field observations revealed that the species is rarer than previously assumed and, as shown by modeling, severely endangered by global warming as viable habitat was predicted to be reduced by more than 70% by the year 2080.

Disentangling relationships among the diploid members of the intricate genus Knautia (Caprifoliaceae, Dipsacoideae).

The genus Knautia (Caprifoliaceae, Dipsacoideae) encompasses 40-60 species mainly distributed in western Eurasia, with highest species diversity in the Alps and the Balkan Peninsula. It is traditionally regarded as one of the taxonomically most challenging European genera due to the widespread occurrence of polyploidy, the high incidence of hybridisation and the maintenance of morphologically intermediate forms. A prerequisite for assessing the complex spatiotemporal diversification of a polyploid group is a comprehensive hypothesis of the phylogenetic relationships among its diploid members. To this end, DNA sequence data (nrDNA ITS and plastid petN(ycf6)-psbM) combined with AFLP fingerprinting were performed on 148 diploid populations belonging to 35 taxa. Phylogenies obtained by maximum parsimony and Bayesian analyses were used to test the monophyly of the genus and its three sections Trichera, Tricheroides and Knautia, to provide insights into its evolutionary history and to test previous hypotheses of inter- and intrasectional classification. Both nuclear and chloroplast datasets support the monophyly of Knautia and its three sections, with ambiguous placement of K. cf. degenii. The majority of species belong to the nearly exclusively perennial section Trichera (x=10). Within section Trichera all markers revealed largely unresolved phylogenetic relationships suggesting rapid radiation and recent range expansion. In addition, extensive sharing of plastid haplotypes across taxa and wide geographic ranges of plastid haplotypes and ribotype groups were observed. The molecular data are partly at odds with the traditional informal grouping of taxa within section Trichera. Whereas the traditional groups of K. dinarica, K. drymeia and K. montana can be maintained, the new, smaller and well supported Midzorensis and Pancicii Groups as well as the SW European Group are separated from the heterogeneous traditional K. longifolia group. The former groups of K. arvensis, K. dalmatica, K. fleischmannii and K. velutina are clearly polyphyletic. Their diploid members have to be rearranged into the Xerophytic Group, the Carinthiaca Group, and the Northern and Southern Arvensis Groups. The annual sections Tricheroides (x=10) and Knautia (x=8) with only a few taxa are resolved in the ITS and plastid trees on long branches as early diverging lineages within the genus.

Phylogenetic data reveal a surprising origin of Euphorbia orphanidis (Euphorbiaceae) and environmental modeling suggests that microtopology limits its distribution to small patches in Mt. Parnassus (Greece).

The Mediterranean Basin is one of the most biodiverse areas in the world, harboring 25,000 plant species, of which 60% are endemic. Some of them have narrow distributions, such as Euphorbia orphanidis, which is only known from alpine screes on Mt. Parnassos in Greece. Its exact distribution in this mountain was, however, poorly known, and its phylogenetic origin was also unclear. We performed extensive field work in Mt. Parnassos and could register E. orphanidis only in five patches of limestone screes in the eastern part of this mountain range, emphasizing its very narrow distribution, which is likely limited by topography influencing water availability as indicated by environmental modeling. We also registered 31 accompanying species and thus characterized its habitat. Using nuclear ribosomal internal transcribed spacer and plastid ndhF-trnL and trnT-trnF sequences, we show that it belongs to E. sect. Patellares, despite not having connate raylet leaves typical for this section, and not to E. sect. Pithyusa as previously suggested. The relationships among the species of E. sect. Patellares are poorly resolved, suggesting their simultaneous divergence that dated to the late Pliocene, which coincided with the establishment of the Mediterranean climate. The relative genome size of E. orphanidis is in the range of that for the other members of E. sect. Patellares, suggesting that it is diploid. Finally, we performed multivariate morphological analyses to generate a comprehensive description of E. orphanidis. Based on its narrow distribution and the anticipated negative impact of global warming, we consider this species endangered. Our study demonstrates how microrelief can limit the distribution of plants in topographically heterogeneous mountain environments and likely plays an important, yet neglected, role in shaping the distribution patterns of plants in the Mediterranean Basin.

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.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

From Western Asia to the Mediterranean Basin: Diversification of the Widespread Euphorbia nicaeensis Alliance (Euphorbiaceae).

The Mediterranean Basin is an important biodiversity hotspot and one of the richest areas in the world in terms of plant diversity. Its flora parallels in several aspects that of the Eurasian steppes and the adjacent Irano-Turanian floristic region. The Euphorbia nicaeensis alliance spans this immense area from the western Mediterranean to Central Asia. Using an array of complementary methods, ranging from phylogenomic and phylogenetic data through relative genome size (RGS) estimation to morphometry, we explored relationships and biogeographic connections among taxa of this group. We identified the main evolutionary lineages, which mostly correspond to described taxa. However, despite the use of highly resolving Restriction Site Associated DNA (RAD) sequencing data, relationships among the main lineages remain ambiguous. This is likely due to hybridisation, lineage sorting triggered by rapid range expansion, and polyploidisation. The phylogenomic data identified cryptic diversity in the Mediterranean, which is also correlated with RGS and, partly, also, morphological divergence, rendering the description of a new species necessary. Biogeographic analyses suggest that Western Asia is the source area for the colonisation of the Mediterranean by this plant group and highlight the important contribution of the Irano-Turanian region to the high diversity in the Mediterranean Basin. The diversification of the E. nicaeensis alliance in the Mediterranean was triggered by vicariance in isolated Pleistocene refugia, morphological adaptation to divergent ecological conditions, and, to a lesser extent, by polyploidisation.

Genome Size Variation in Dianthus sylvestris Wulfen sensu lato (Caryophyllaceae).

Genome size (GS) is an important characteristic that may be helpful in delimitation of taxa, and multiple studies have shown correlations between intraspecific GS variation and morphological or environmental factors, as well as its geographical segregation. We estimated a relative GS (RGS) of 707 individuals from 162 populations of Dianthus sylvestris with a geographic focus on the Balkan Peninsula, but also including several populations from the European Alps. Dianthus sylvestris is morphologically variable species thriving in various habitats and six subspecies have been recognized from the Balkan Peninsula. Our RGS data backed-up with chromosome counts revealed that the majority of populations were diploid (2n = 30), but ten tetraploid populations have been recorded in D. sylvestris subsp. sylvestris from Istria (Croatia, Italy). Their monoploid RGS is significantly lower than that of the diploids, indicating genome downsizing. In addition, the tetraploids significantly differ from their diploid counterparts in an array of morphological and environmental characteristics. Within the diploid populations, the RGS is geographically and only partly taxonomically correlated, with the highest RGS inferred in the southern Balkan Peninsula and the Alps. We demonstrate greater RGS variation among the Balkan populations compared to the Alps, which is likely a result of more pronounced evolutionary differentiation within the Balkan Peninsula. In addition, a deep RGS divergence within the Alps likely points to persistence of the alpine populations in different Pleistocene refugia.

Stability in the South, Turbulence Toward the North: Evolutionary History of Aurinia saxatilis (Brassicaceae) Revealed by Phylogenomic and Climatic Modelling Data.

The Balkan Peninsula played an important role in the evolution of many Mediterranean plants and served as a major source for post-Pleistocene colonisation of central and northern Europe. Its complex geo-climatic history and environmental heterogeneity significantly influenced spatiotemporal diversification and resulted in intricate phylogeographic patterns. To explore the evolutionary dynamics and phylogeographic patterns within the widespread eastern Mediterranean and central European species Aurinia saxatilis, we used a combination of phylogenomic (restriction-site associated DNA sequencing, RADseq) and phylogenetic (sequences of the plastid marker ndhF) data as well as species distribution models generated for the present and the Last Glacial Maximum (LGM). The inferred phylogenies retrieved three main geographically distinct lineages. The southern lineage is restricted to the eastern Mediterranean, where it is distributed throughout the Aegean area, the southern Balkan Peninsula, and the southern Apennine Peninsula, and corresponds to the species main distribution area during the LGM. The eastern lineage extends from the eastern Balkan Peninsula over the Carpathians to central Europe, while the central lineage occupies the central Balkan Peninsula. Molecular dating places the divergence among all the three lineages to the early to middle Pleistocene, indicating their long-term independent evolutionary trajectories. Our data revealed an early divergence and stable in situ persistence of the southernmost, eastern Mediterranean lineage, whereas the mainland, south-east European lineages experienced more complex and turbulent evolutionary dynamics triggered by Pleistocene climatic oscillations. Our data also support the existence of multiple glacial refugia in southeast Europe and highlight the central Balkan Peninsula not only as a cradle of lineage diversifications but also as a source of lineage dispersal. Finally, the extant genetic variation within A. saxatilis is congruent with the taxonomic separation of peripatric A. saxatilis subsp. saxatilis and A. saxatilis subsp. orientalis, whereas the taxonomic status of A. saxatilis subsp. megalocarpa remains doubtful.

Giants and dwarfs: molecular phylogenies reveal multiple origins of annual spurges within Euphorbia subg. Esula.

Euphorbia (Euphorbiaceae) comprises over 2150 species and is thus the second-largest genus of flowering plants. In Europe, it is represented by more than 100 species with highest diversity in the Mediterranean area; the majority of taxa belong to subgenus Esula Pers., including about 500 taxa. The few available phylogenetic studies yielded contrasting results regarding the monophyly of subg. Esula, and the phylogenetic relationships among its constituents remain poorly understood. We have sampled DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid trnT-trnF region from about 100, predominantly European taxa of subg. Esula in order to infer its phylogenetic history. The plastid data support monophyly of subg. Esula whereas the ITS phylogeny, which is generally less resolved, is indecisive in this respect. Although some major clades have partly incongruent positions in the ITS and plastid phylogenies, the taxonomic content of the major terminal clades is congruent in both trees. As traditional sectional delimitations are largely not corroborated, an improved classification is proposed. Character state reconstruction illustrates that the annual life form developed independently several times in different clades of subgenus Esula from perennial ancestors, and that several morphological traits used in previous classifications of Euphorbia developed in parallel in different lineages.