Primary hybrid zone formation in Tephroseris helenitis (Asteraceae), following postglacial range expansion along the central Northern Alps.

Distinguishing between secondary versus primary hybrid zone formation remains a challenging task as, for instance, the time window in which these historical (vicariant) versus contemporary (environmental-selective) processes are distinguishable may be relatively narrow. Here, we examine the origin and structure of a transition zone between two subspecies of Tephroseris helenitis along the central Northern Alps, using molecular (AFLP) and morphological (achene type) data in combination with ecological niche models (ENMs) to hindcast ranges at the Last Glacial Maximum (LGM) and mid-Holocene. Samples were collected over a c. 350 km long transect, largely covered by ice during the LGM. Genetically nonadmixed individuals of subspp. helenitis versus salisburgensis dominated the westernmost versus eastern transect areas, with admixed individuals occurring in between. Clines for achene morphology and outlier loci potentially under climate-driven selection were steep, largely noncoincidental, and displaced to the east of the cline centre for neutral AFLPs. During the LGM, ssp. helenitis should have been able to persist in a refugium southwest of the transect, while suitable habitat for ssp. salisburgensis was apparently absent at this time. Together with patterns of genetic and clinal variation, our ENM data are suggestive of a primary hybrid zone that originated after the species' postglacial, eastward expansion. The observed clinal changes may thus reflect random/nonadaptive processes during expansion and selection on particular loci, and possibly achene type, in response to a long-term, west-to-east climate gradient in the direction of more stressful (e.g., wetter/cooler) conditions. Overall, this study adds to the vast hybrid zone literature a rare example of a hybrid zone caused by primary differentiation within a plant species, underlaid by historical range expansion.

Occurrence of apomictic conspecifics and ecological preferences rather than colonization history govern the geographic distribution of sexual Potentilla puberula.

The geographic distribution of sexual-apomictic taxa (i.e., comprising individuals usually reproducing either sexually or asexually via seeds) is traditionally thought to be driven by their ecological preferences and colonization histories. Where sexuals and apomicts get into contact with each other, competitive and reproductive interactions can interfere with these factors, an aspect which hitherto received little attention in biogeographic studies. We disentangled and quantified the relative effects of the three factors on the distribution of tetraploid sexuals in Potentilla puberula in a latitudinal transect through the Eastern European Alps, in which they are codistributed with penta-, hepta-, and octoploid apomictic conspecifics. Effects were explored by means of binomial generalized linear regression models combining a single with a multiple predictor approach. Postglacial colonization history was inferred from population genetic variation (AFLPs and cpDNA) and quantified using a cost distance metric. The study was based on 235 populations, which were purely sexual, purely apomictic, or of mixed reproductive mode. The occurrence of apomicts explained most of the variation in the distribution of sexuals (31%). Specifically, the presence of sexual tetraploids was negatively related to the presence of each of the three apomictic cytotypes. Effects of ecological preferences were substantial too (7% and 12% of the total variation explained by ecological preferences alone, or jointly with apomicts' occurrence, respectively). In contrast, colonization history had negligible effects on the occurrence of sexuals. Taken together, our results highlight the potentially high impact of reproductive interactions on the geographic distribution of sexual and apomictic conspecifics and that resultant mutual exclusion interrelates to ecological differentiation, a situation potentially promoting their local coexistence.

Genome-wide nuclear data confirm two species in the Alpine endemic land snail Noricella oreinos s.l. (Gastropoda, Hygromiidae).

The Austrian endemic land snail species Noricella oreinos (formerly Trochulus oreinos) occurs in the Northeastern Calcareous Alps at high elevations. Two morphologically highly similar subspecies N. o. oreinos and N. o. scheerpeltzi have been described. First analyses of mitochondrial and nuclear marker sequences indicated a high genetic divergence between them. In the present study, we aimed to assess gene flow between the two subspecies which should help to re-evaluate their taxonomic status. Sequence data and amplified fragment length polymorphism (AFLP) markers of 255 Noricella specimens covering the whole distribution range were analyzed. A clear geographic separation was found within the potential contact zone, the Haller Mauern mountain range. Samples of all western sites were part of the clade representing N. o. scheerpeltzi and almost all samples from the eastern sites clustered with N. o. oreinos. However, within two sampling sites of the eastern Haller Mauern, a few individuals possessed a COI sequence matching the N. o. oreinos clade whereas at the ITS2 locus they were heterozygous possessing the alleles of both taxa. Contrary to the ITS2 results indicating historical and/or ongoing hybridization, AFLP analyses of 202 individuals confirmed a clear separation of the two taxa congruent with the mitochondrial data. Although they occur on the same mountain range without any physical barrier, no indication of ongoing gene flow between the two taxa was found. Thus, we conclude that the two taxa are separate species N. oreinos and N. scheerpeltzi.

Difference in reproductive mode rather than ploidy explains niche differentiation in sympatric sexual and apomictic populations of Potentilla puberula.

Apomicts tend to have larger geographical distributional ranges and to occur in ecologically more extreme environments than their sexual progenitors. However, the expression of apomixis is typically linked to polyploidy. Thus, it is a priori not clear whether intrinsic effects related to the change in the reproductive mode or rather in the ploidy drive ecological differentiation. We used sympatric sexual and apomictic populations of Potentilla puberula to test for ecological differentiation. To distinguish the effects of reproductive mode and ploidy on the ecology of cytotypes, we compared the niches (a) of sexuals (tetraploids) and autopolyploid apomicts (penta-, hepta-, and octoploids) and (b) of the three apomictic cytotypes. We based comparisons on a ploidy screen of 238 populations along a latitudinal transect through the Eastern European Alps and associated bioclimatic, and soil and topographic data. Sexual tetraploids preferred primary habitats at drier, steeper, more south-oriented slopes, while apomicts mostly occurred in human-made habitats with higher water availability. Contrariwise, we found no or only marginal ecological differentiation among the apomictic higher ploids. Based on the pronounced ecological differences found between sexuals and apomicts, in addition to the lack of niche differentiation among cytotypes of the same reproductive mode, we conclude that reproductive mode rather than ploidy is the main driver of the observed differences. Moreover, we compared our system with others from the literature, to stress the importance of identifying alternative confounding effects (such as hybrid origin). Finally, we underline the relevance of studying ecological parthenogenesis in sympatry, to minimize the effects of differential migration abilities.

Diversification in continental island archipelagos: new evidence on the roles of fragmentation, colonization and gene flow on the genetic divergence of Aegean Nigella (Ranunculaceae).

Background and Aims: Disentangling the relative roles of past fragmentation (vicariance), colonization (dispersal) and post-divergence gene flow in the genetic divergence of continental island organisms remains a formidable challenge. Amplified fragment length polymorphisms (AFLPs) were used to (1) gain further insights into the biogeographical processes underlying the Pleistocene diversification of the Aegean Nigella arvensis complex; (2) evaluate the role of potential key factors driving patterns of population genetic variability (mating system, geographical isolation and historical contingencies); and (3) test the robustness of conclusions previously drawn from chloroplast (cp) DNA. Methods: Genetic diversity was analysed for 235 AFLP markers from 48 populations (497 individuals) representing 11 taxa of the complex using population genetic methods and Bayesian assignment tests. Key Results: Most designated taxa are identifiable as genetically distinct units. Both fragmentation and dispersal-driven diversification processes occurred at different geological time scales, from Early to Late Pleistocene, specifically (1) sea barrier-induced vicariant speciation in the Cyclades, the Western Cretan Strait and Ikaria; and (2) bi-regional colonizations of the 'Southern Aegean Island Arc' from the Western vs. Eastern Aegean mainland, followed by allopatric divergences in Crete vs. Rhodos and Karpathos/Kasos. Outcrossing island taxa experienced drift-related demographic processes that are magnified in the two insular selfing species. Population genetic differentiation on the mainland seems largely driven by dispersal limitation, while in the Central Aegean it may still be influenced by historical events (island fragmentation and sporadic long-distance colonization). Conclusions: The biogeographical history of Aegean Nigella is more complex than expected for a strictly allopatric vicariant model of divergence. Nonetheless, the major phylogeographical boundaries of this radiation are largely congruent with the geography and history of islands, with little evidence for ongoing gene exchange between divergent taxa. The present results emphasize the need to investigate further biological and landscape features and contemporary vs. historical processes in driving population divergence and taxon diversification in Aegean plant radiations.

Sexual intraspecific recombination but not de novo origin governs the genesis of new apomictic genotypes in Potentilla puberula (Rosaceae).

Apomixis - asexual reproduction via seeds - might arise de novo following polyploidisation events, or via reproductive transfer of apomixis. Both processes can be obtained within species or via hybridisation. We aimed to determine the origin of apomictic genotypes in Potentilla puberula, a rosaceous species showing reproductive differentiation with ploidy: sexual tetraploids and apomictic penta- to octoploids, which regularly co-occur in sympatry. The study is based on 726 individuals, comprising all cytotypes, collected from 138 populations in the Eastern European Alps. We established relationships of cytotypes based on AFLP fingerprinting and cpDNA sequencing to test (1) whether the apomicts are of recurrent allopolyploid origin or originated from within the species via autopolyploidy, and (2) whether there are indications for reproductive transfer versus de novo origin of apomixis. Three principal pathways were identified which explain the origin of new apomictic genotypes, all involving at least one apomictic parent and thus compatible with the idea of reproductive transfer of the apomictic trait to the progeny: (1) self-fertilisation of unreduced egg cells in apomicts; (2) cross-fertilisation among apomicts; and (3) occasionally, heteroploid crosses among sexuals and apomicts. Autopolyploids derived from tetraploid sexuals were repeatedly observed, but did not express apomixis. Finally, our results suggest no role of other species in the origin of extant apomictic genotypes of P. puberula, although local hybrids with P. crantzii were identified. In conclusion, our results show that the formation of new apomictic genotypes required a genetic contribution from at least one apomictic parent. This finding is in accordance with the idea that apomixis is inheritable in P. puberula. On the contrary, lack of apomixis in penta- and hexaploids derived from sexual backgrounds did not support the hypothesis of a de novo origin of apomixis. Relatively high frequency of remnant sexuality in the apomicts involving different cytological pathways of seed formation can explain their high cytological and genotypic diversity. Finally, lack of global introgression from a third taxon is in support of P. puberula as a concise, although highly diverse, species.

Long-distance plant dispersal to North Atlantic islands: colonization routes and founder effect.

Long-distance dispersal (LDD) processes influence the founder effect on islands. We use genetic data for 25 Atlantic species and similarities among regional floras to analyse colonization, and test whether the genetic founder effect on five islands is associated with dispersal distance, island size and species traits. Most species colonized postglacially via multiple dispersal events from several source regions situated 280 to >3000 km away, and often not from the closest ones. A strong founder effect was observed for insect-pollinated mixed maters, and it increased with dispersal distance and decreased with island size in accordance with the theory of island biogeography. Only a minor founder effect was observed for wind-pollinated outcrossing species. Colonization patterns were largely congruent, indicating that despite the importance of stochasticity, LDD is mainly determined by common factors, probably dispersal vectors. Our findings caution against a priori assuming a single, close source region in biogeographic analyses.

Molecular phylogenetic analyses identify Alpine differentiation and dysploid chromosome number changes as major forces for the evolution of the European endemic Phyteuma (Campanulaceae).

Phyteuma is a chromosomally and ecologically diverse vascular plant genus and constitutes an excellent system for studying both the role of chromosomal change for species diversification and the evolution of high-mountain biota. This kind of research is, however, hampered by the lack of a sound phylogenetic framework exacerbated by the notoriously low predictive power of traditional taxonomy with respect to phylogenetic relationships in Campanulaceae. Based on a comprehensive taxon sampling and analyses of nuclear and plastid sequence and AFLP fingerprint data, Phyteuma is confirmed as a monophyletic group sister to the monotypic Physoplexis, which is in line with their peculiar flower morphologies. Within Phyteuma two clades, largely corresponding to previously recognized sections, are consistently found. The traditional circumscription of taxonomic series is largely rejected. Whereas distinctness of the currently recognized species is mostly corroborated, some interspecific relationships remain ambiguous due to incongruences between nuclear and plastid data. Major forces for diversification and evolution of Phyteuma are descending dysploidy (i.e., a decrease in chromosome base number) as well as allopatric and ecological differentiation within the Alps, the genus' center of species diversity.

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.

Tales of the unexpected: phylogeography of the arctic-alpine model plant Saxifraga oppositifolia (Saxifragaceae) revisited.

Arctic-alpine biota occupy enormous areas in the Arctic and the northern hemisphere mountain ranges and have undergone major range shifts during their comparatively short history. The origins of individual arctic-alpine species remain largely unknown. In the case of the Purple saxifrage, Saxifraga oppositifolia, an important model for arctic-alpine plants, phylogeographic studies have remained inconclusive about early stages of the species' spatiotemporal diversification but have provided evidence for long-range colonization out of a presumed Beringian origin to cover today's circumpolar range. We re-evaluated the species' large-scale range dynamics based on a geographically extended sampling including crucial areas such as Central Asia and the (south-)eastern European mountain ranges and employing up-to-date phylogeographic analyses of a plastid sequence data set and a more restricted AFLP data set. In accordance with previous studies, we detected two major plastid DNA lineages also reflected in AFLP divergence, suggesting a long and independent vicariant history. Although we were unable to determine the species' area of origin, our results point to Europe (probably the Alps) and Central Asia, respectively, as the likely ancestral areas of the two main lineages. AFLP data suggested that contact areas between the two clades in the Carpathians, Northern Siberia and western Greenland were secondary. In marked contrast to high levels of diversity revealed in previous studies, populations from the major arctic refugium Beringia did not exhibit any plastid sequence polymorphism. Our study shows that adequate sampling of the southern, refugial populations is crucial for understanding the range dynamics of arctic-alpine species.

Genetic consequences of climate change for northern plants.

Climate change will lead to loss of range for many species, and thus to loss of genetic diversity crucial for their long-term persistence. We analysed range-wide genetic diversity (amplified fragment length polymorphisms) in 9581 samples from 1200 populations of 27 northern plant species, to assess genetic consequences of range reduction and potential association with species traits. We used species distribution modelling (SDM, eight techniques, two global circulation models and two emission scenarios) to predict loss of range and genetic diversity by 2080. Loss of genetic diversity varied considerably among species, and this variation could be explained by dispersal adaptation (up to 57%) and by genetic differentiation among populations (F(ST); up to 61%). Herbs lacking adaptations for long-distance dispersal were estimated to lose genetic diversity at higher rate than dwarf shrubs adapted to long-distance dispersal. The expected range reduction in these 27 northern species was larger than reported for temperate plants, and all were predicted to lose genetic diversity according to at least one scenario. SDM combined with F(ST) estimates and/or with species trait information thus allows the prediction of species' vulnerability to climate change, aiding rational prioritization of conservation efforts.

Allopolyploid origins of the Galeopsis tetraploids--revisiting Müntzing's classical textbook example using molecular tools.

Whole-genome duplication coupled with hybridization is of prime importance in plant evolution. Here we reinvestigate Müntzing's classical example of allopolyploid speciation; the first report of experimental synthesis of a naturally occurring allopolyploid species, Galeopsis tetrahit. Various molecular markers (cpDNA, NRPA2, amplified fragment length polymorphisms (AFLPs)) and flow cytometry were surveyed in population samples of subgenus Galeopsis, including two allopolyploid species and their potential diploid parents. The presence of two divergent copies of single-copy NRPA2 confirms the allopolyploid origins of G. tetrahit and Galeopsis bifida. However, the two allopolyploids do not share the same maternal genome, as originally suggested by Müntzing. The results support independent origins, but not recurrent formation, of the two allotetraploids. Data further indicate frequent gene flow and introgression within ploidy levels, but less so between ploidy levels. Our results confirm and elaborate on Müntzing's classical conclusion about allopolyploid origins of G. tetrahit and G. bifida. We address questions of general interest within polyploidy research, such as recurrent formation, gene flow and introgression within and between ploidy levels.

Pleistocene distribution range shifts were accompanied by breeding system divergence within Hornungia alpina (Brassicaceae) in the Alps.

Impact of glacial history on the phylogeography of silicate-dwelling plants of the European Alps has been particularly well studied, whereas virtually no data are available for species growing on different bedrock types, as for Hornungia alpina. Bayesian clustering of AFLP data only partly support the distinction of three subspecies as morphologically defined. Whereas the phylogeographical N-group corresponds to subsp. alpina, the congruence of the SW-group and SE-group with subsp. brevicaulis, and subsp. austroalpina, respectively, is limited. High levels of rarity and genetic diversity in the N-group suggest Pleistocene survival along the outer margin of the Alpine arc. For subsp. brevicaulis we suggest a single origin from a refugium in the Southwestern Alps, whereas subsp. austroalpina might have originated twice in the Southern and Southeastern Alps. Different levels of genetic diversity and partitioning of genetic variation indicate a divergence in breeding system, which is corroborated by pollinator exclusion experiments revealing self-incompatibility in the N-group and autonomous selfing in the SE-group.

History or ecology? Substrate type as a major driver of spatial genetic structure in Alpine plants.

Climatic history and ecology are considered the most important factors moulding the spatial pattern of genetic diversity. With the advent of molecular markers, species' historical fates have been widely explored. However, it has remained speculative what role ecological factors have played in shaping spatial genetic structures within species. With an unprecedented, dense large-scale sampling and genome-screening, we tested how ecological factors have influenced the spatial genetic structures in Alpine plants. Here, we show that species growing on similar substrate types, largely determined by the nature of bedrock, displayed highly congruent spatial genetic structures. As the heterogeneous and disjunctive distribution of bedrock types in the Alps, decisive for refugial survival during the ice ages, is temporally stable, concerted post-glacial migration routes emerged. Our multispecies study demonstrates the relevance of particular ecological factors in shaping genetic patterns, which should be considered when modelling species projective distributions under climate change scenarios.

Plant speciation in continental island floras as exemplified by Nigella in the Aegean Archipelago.

Continental shelf island systems, created by rising sea levels, provide a premier setting for studying the effects of geographical isolation on non-adaptive radiation and allopatric speciation brought about by genetic drift. The Aegean Archipelago forms a highly fragmented complex of mostly continental shelf islands that have become disconnected from each other and the mainland in relatively recent geological times (ca <5.2 Ma). These ecologically fairly homogenous islands thus provide a suitable biogeographic context for assessing the relative influences of past range fragmentation, colonization, gene flow and drift on taxon diversification. Indeed, recent molecular biogeographic studies on the Aegean Nigella arvensis complex, combining phylogenetic, phylogeographic and population level approaches, exemplify the importance of allopatry and genetic drift coupled with restricted gene flow in driving plant speciation in this continental archipelago at different temporal and spatial scales. While the recent (Late Pleistocene) radiation of Aegean Nigella, as well as possible instances of incipient speciation (in the Cyclades), is shown to be strongly conditioned by (palaeo)geographic factors (including changes in sea level), shifts in breeding system (selfing) and associated isolating mechanisms have also contributed to this radiation. By contrast, founder event speciation has probably played only a minor role, perhaps reflecting a migratory situation typical for continental archipelagos characterized by niche pre-emption because of a long established resident flora. Overall, surveys of neutral molecular markers in Aegean Nigella have so far revealed population genetic processes that conform remarkably well to predictions raised by genetic drift theory. The challenge is now to gain more direct insights into the relative importance of the role of genetic drift, as opposed to natural selection, in the phenotypic and reproductive divergence among these Aegean plant species.

Complex distribution patterns of di-, tetra-, and hexaploid cytotypes in the European high mountain plant Senecio carniolicus (Asteraceae).

DNA ploidy levels were estimated using DAPI-flow cytometry of silica-dried specimens of the European mountain plant Senecio carniolicus (Asteraceae), covering its entire distribution area in the Eastern Alps (77 populations, 380 individuals) and the Carpathians (five populations, 22 individuals). A complex pattern of ploidy level variation (2x, 4x, 5x, 6x, and 7x cytotypes) was found in this species, which has been considered uniformly hexaploid. Hexaploids predominated in the Eastern Alps and was the only cytotype found in the Carpathians, while odd ploidy levels (5x, 7x) constituted a small fraction of the samples (<1.3%). Tetraploids occurred in two disjunct areas, which correspond with putative Pleistocene refugia for silicicolous alpine plants. Diploids occurred in large portions of the Alps but were absent from areas most extensively glaciated in the past. Intrapopulational cytotype mixture was detected in 22 populations-the majority involving diploids and hexaploids-with intermediate ploidy levels mostly lacking, suggesting limited gene flow and the evolution of reproductive isolation. Significant and reproducible intracytotype variation in nuclear DNA content was observed. Higher genome size in western diploids might be due to ancient introgression with the closely related S. incanus or to different evolutionary pathways in the geographically separated diploids.

Comparative phylogeography of the Veronica alpina complex in Europe and North America.

The Veronica alpina complex comprises eight species of alpine habitats over a wide range of mountain systems in the Northern Hemisphere. The occurrence of sympatric species in the European and North American mountain systems allowed us not only to investigate the effect of the ice ages on intraspecific phylogeographical patterns and genetic diversity in different continents of the Northern Hemisphere, but also to compare these patterns in closely related species. Plastid DNA trnL-F sequences and AFLP (amplified fragment length polymorphism) fingerprints were used to infer the phylogenetic history of the group and phylogeographical patterns within species. Hybrid origin of tetraploid eastern North American V. wormskjoldii from western North American V. nutans (= V. wormskjoldii s.l.) and Eurasian V. alpina is suggested. A number of phylogeographical groups have been found both in V. alpina from Europe and in V. nutans from western North America. Phylogeographical substructuring in the Alps is inferred for V. alpina but not for V. bellidioides, which is moreover characterized by an overall very low genetic diversity. Western North American V. cusickii is much more genetically diverse than its sympatric relative, V. nutans, an effect that is likely due to differences in the breeding system. Populations of V. nutans are differentiated into three groups, those from the Cascades and from the southern and the northern Rocky Mountains. Genetic diversity seems to be higher in the North American V. nutans than in the morphologically and ecologically similar European V. alpina. A possible scenario to explain this pattern is suggested.

Saponaria pumila (caryophyllaceae) and the ice age in the European alps.

The polymerase chain reaction (PCR)-based amplified fragment length polymorphism (AFLP) technique was applied to elucidate the glacial history of the alpine cushion plant Saponaria pumila in the European Alps. Special emphasis was given to a dense sampling of populations. Our data support a survival of S. pumila during the last ice age in at least three refugia, which are characterized by unique marker sets. Patterns of genetic diversity and divergence can be explained by survival in peripheral refugia and additional in situ survival within the ice sheet on peripheral nunataks. A nunatak survival in interior parts of the Alps needs not be postulated to explain our results. The level of genetic diversity is dramatically different between populations (Shannon's diversity index: 0.87-19.86). Some peripheral populations are characterized by a high number of rare fragments indicating long isolation, but not necessarily by a high level of genetic diversity. Parts of the present distributional area were recolonized via recent long-distance dispersal, leading to severely bottlenecked populations lacking private or rare fragments. The combination of our data with palaeogeological and palaeoclimatological evidence allows us to confine Pleistocene refugia to certain regions and to draw a detailed scenario of the glacial and postglacial history of S. pumila.