Božo Frajman

A worldwide molecular phylogeny and classification of the leafy spurges, Euphorbia subgenus Esula (Euphorbiaceae)

The leafy spurges, Euphorbia subg. Esula, make up one of four main lineages in Euphorbia. The subgenus comprises about 480 species, most of which are annual or perennial herbs, but with a small number of dendroid shrubs and nearly leafless, pencil-stemmed succulents as well. The subgenus constitutes the primary northern temperate radiation in Euphorbia. While the subgenus is most diverse from central Asia to the Mediterranean region, members of the group also occur in Africa, in the Indo-Pacific region, and in the New World. We have assembled the largest worldwide sampling of the group to date (273 spp.), representing most of the taxonomic and geographic breadth of the subgenus. We performed phylogenetic analyses of sequence data from the nuclear ribosomal ITS and plastid ndhF regions. Our individual and combined analyses produced well-resolved phylogenies that confirm many of the previously recognized clades and also establish a number of novel groupings and place- ments of previously enigmatic species. Euphorbia subg. Esula has a clear Eurasian center of diversity, and we provide evidence for four independent arrivals to the New World and three separate colonizations of tropical and southern Africa. One of the latter groups further extends to Madagascar and New Zealand, and to more isolated islands such as Reunion and Samoa. Our results confirm that the dendroid shrub and stem-succulent growth forms are derived conditions in E. subg. Esula. Stem- succulents arose twice in the subgenus and dendroid shrubs three times. Based on the molecular phylogeny, we propose a new classification for E. subg. Esula that recognizes 21 sections (four of them newly described and two elevated from subsectional rank), and we place over 95% of the accepted species in the subgenus into this new classification.

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.

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 regions 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 areas 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.

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

BackgroundPolyploidisation 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.ResultsGenome 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.ConclusionsWe 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.

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.

How many taxa? Spatiotemporal evolution and taxonomy of Amphoricarpos (Asteraceae, Carduoideae) on the Balkan Peninsula

Amphoricarpos Vis. is an early diverging genus within tribe Cardueae (Carduoideae, Asteraceae), which is disjunctly distributed in the Balkan Peninsula, Anatolia and the Caucasus; the Anatolian and Caucasian taxa are sometimes treated as separate genus Alboviodoxa. We focus on the monophyletic Balkan populations, which have been treated very inconsistently in previous taxonomic accounts (one polymorphic species with or without varying sets of intraspecific taxa vs. two species, one of them with two subspecies). In order to disentangle relationships among populations across the entire distribution area of Amphoricarpos on the Balkan Peninsula, we employed amplified fragment length polymorphisms (AFLPs) as well as nuclear and plastid DNA sequences (ITS and rps16–trnK) to a dense sampling of populations. ITS was also used to reconstruct the genus’ spatiotemporal evolution. In addition, we contrasted the genetic results with morphological data to provide a sound taxonomic revision of Amphoricarpos on the Balkan Peninsula. The split between the Balkan populations and the Anatolian A. exsul took place in the late Miocene or early Pliocene, whereas diversification within the Balkan lineage is much younger and likely started in the Pleistocene. The deepest splits seen in AFLPs and/or ITS separate the geographically disjunct northern- and southern-most populations. Divergence within the continuous distribution area in the centre is shallower, but allowed recognition of three largely allopatric clusters. Morphometric data, however, were neither in line with previous multi-taxon treatments nor with patterns of genetic divergence. We therefore refrain from recognising any of the genetic groups as a distinct taxonomic entity and rather suggest treating all Balkan populations as a single, genetically, morphologically and ecologically variable species, Amphoricarpos neumayerianus (Vis.) Greuter, without intraspecific taxa.

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

Summary 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.

Does hybridization with a widespread congener threaten the long-term persistence of the Eastern Alpine rare local endemic Knautia carinthiaca?

Abstract Interspecific hybridization, especially when regularly followed by backcrossing (i.e., introgressive hybridization), conveys a substantial risk for many endangered organisms. This is particularly true for narrow endemics occurring within distributional ranges of widespread congeners. An excellent example is provided by the plant genus Knautia (Caprifoliaceae): Locally endemic K. carinthiaca is reported from two isolated populations in southern Austria situated within an area predominantly occupied by widespread K. arvensis. While K. carinthiaca usually inhabits low‐competition communities on rocky outcrops, K. arvensis occurs mainly in dry to mesic managed grasslands, yet both species can coexist in marginal environments and were suspected to hybridize. Flow cytometry revealed that diploid K. carinthiaca only occurs at its locus classicus, whereas the second locality is inhabited by the morphologically similar but tetraploid K. norica. In the, therefore, single population of K. carinthiaca, flow cytometry and AFLP fingerprinting showed signs of introgressive hybridization with diploid K. arvensis. Hybridization patterns were also reflected in intermediate habitat preferences and morphology of the hybrids. Environmental barriers to gene flow seem to prevent genetic erosion of K. carinthiaca individuals from the core ecological niches, restricting most introgressed individuals to peripheral habitats. Efficient conservation of K. carinthiaca will require strict protection of its habitat and ban on forest clear cuts in a buffer zone to prevent invasion of K. arvensis. We demonstrate the large potential of multidisciplinary approaches combining molecular, cytometric, and ecological tools for a reliable inventory and threat assessment of rare species.

Leaf anatomy of two reciprocally non-monophyletic mountain plants (Heliosperma spp.): does heritable adaptation to divergent growing sites accompany the onset of speciation?

Evolution is driven by natural selection, favouring individuals adapted in phenotypic traits to the environmental conditions at their growing site. To shed light on ecological and (epi-) genetically based differentiation between Heliosperma pusillum and Heliosperma veselskyi, two reciprocally non-monophyletic, but morphologically and ecologically divergent species from the south-eastern Alps, we studied various leaf anatomical traits and investigated chloroplast ultrastructure in leaves of the two species grown either in their natural habitat or in a common garden. The alpine H. pusillum occurs in open, wet rock habitats, whereas its close relative H. veselskyi is restricted to dry, shady habitats below overhanging rocks in the montane belt. H. pusillum exhibited higher thickness of leaves and palisade layers as adjustments and/or adaptations to higher irradiance and a higher stomatal area index reflecting better water availability. Traits were adjusted plastically, but differed between species grown in a common garden, suggesting that the differentiation between the two species is not solely based on phenotypic plasticity but also has a genetic basis. Our study thus supports the hypothesis that differentiation between the highly interfertile species is likely driven by natural selection.