Cryptic invasion suggested by a cytogeographic analysis of the halophytic Puccinellia distans complex (Poaceae) in Central Europe.

Introduction: Despite the wealth of studies dealing with the invasions of alien plants, invasions of alien genotypes of native species (cryptic invasions) have been vastly neglected. The impact of cryptic invasions on the biodiversity of plant communities can, however, be significant. Inland saline habitats and halophytes (i.e., salt-tolerant plant species) are especially threatened by this phenomenon as they inhabit fragmented remnants of largely destroyed habitats, but at the same time some of these halophytic species are rapidly spreading along salt-treated roads. To study potential cryptic invasion of halophytes, the patterns of genome size and ploidy variation in the Puccinellia distans complex (Poaceae), the most rapidly spreading roadside halophyte in Central Europe, were investigated. Methods: DNA flow cytometry with confirmatory chromosome counts were employed to assess ploidy levels of 1414 individuals from 133 populations of the P. distans complex. In addition, climatic niche modelling was used to predict the distributions of selected cytotypes. Results: Eight groups differing in ploidy level and/or genome size were discovered, one diploid (2x; 2n = 14), two tetraploid (4xA, 4xB; 2n = 28), one pentaploid (5x; 2n = 35), three hexaploid (6xA, 6xB, 6xC; 2n = 42), and one heptaploid (7x; 2n = 49). The hexaploids (mostly the 6xC cytotype) were widespread through the study area, spreading intensively in both anthropogenic and natural habitats and probably hybridizing with the natural habitat dwelling tetraploids. In contrast, the non-hexaploid cytotypes rarely spread and were predominantly confined to natural habitats. Discussion: The extensive spread of the hexaploid cytotypes along roadsides has most likely facilitated their incursion into natural habitats. The colonization of new natural habitats by the hexaploids may pose a threat to the indigenous Puccinellia populations by compromising their genetic integrity and/or by outcompeting them.

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.

Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

The Rubiaceae tribe Rubieae has a world-wide distribution with up to 1,000 species. These collectively exhibit an enormous ecological and morphological diversity, making Rubieae an excellent group for macro- and microevolutionary studies. Previous molecular phylogenetic analyses used only a limited sampling within the tribe or missed lineages crucial for understanding character evolution in this group. Here, we analyze sequences from two plastid spacer regions as well as morphological and biogeographic data from an extensive and evenly distributed sampling to establish a sound phylogenetic framework. This framework serves as a basis for our investigation of the evolution of important morphological characters and the biogeographic history of the Rubieae. The tribe includes three major clades, the Kelloggiinae Clade (Kelloggia), the Rubiinae Clade (Didymaea, Rubia) and the most species-rich Galiinae Clade (Asperula, Callipeltis, Crucianella, Cruciata, Galium, Mericarpaea, Phuopsis, Sherardia, Valantia). Within the Galiinae Clade, the largest genera Galium and Asperula are para- and polyphyletic, respectively. Smaller clades, however, usually correspond to currently recognized taxa (small genera or sections within genera), which may be used as starting points for a refined classification in this clade. Life-form (perennial versus annual), flower shape (long versus short corolla tube) and fruit characters (dry versus fleshy, with or without uncinate hairs) are highly homoplasious and have changed multiple times independently. Inference on the evolution of leaf whorls, a characteristic feature of the tribe, is sensitive to model choice. Multi-parted leaf whorls appear to have originated from opposite leaves with two small interpetiolar stipules that are subsequently enlarged and increased in number. Early diversification of Rubieae probably started during the Miocene in western Eurasia. Disjunctions between the Old and the New World possibly are due to connections via a North Atlantic land bridge. Diversification of the Galiineae Clade started later in the Miocene, probably in the Mediterranean, from where lineages reached, often multiple times, Africa, eastern Asia and further on the Americas and Australia.

Dating the Species Network: Allopolyploidy and Repetitive DNA Evolution in American Daisies (Melampodium sect. Melampodium, Asteraceae).

Allopolyploidy has played an important role in the evolution of the flowering plants. Genome mergers are often accompanied by significant and rapid alterations of genome size and structure via chromosomal rearrangements and altered dynamics of tandem and dispersed repetitive DNA families. Recent developments in sequencing technologies and bioinformatic methods allow for a comprehensive investigation of the repetitive component of plant genomes. Interpretation of evolutionary dynamics following allopolyploidization requires both the knowledge of parentage and the age of origin of an allopolyploid. Whereas parentage is typically inferred from cytogenetic and phylogenetic data, age inference is hampered by the reticulate nature of the phylogenetic relationships. Treating subgenomes of allopolyploids as if they belonged to different species (i.e., no recombination among subgenomes) and applying cross-bracing (i.e., putting a constraint on the age difference of nodes pertaining to the same event), we can infer the age of allopolyploids within the framework of the multispecies coalescent within BEAST2. Together with a comprehensive characterization of the repetitive DNA fraction using the RepeatExplorer pipeline, we apply the dating approach in a group of closely related allopolyploids and their progenitor species in the plant genus Melampodium (Asteraceae). We dated the origin of both the allotetraploid, Melampodium strigosum, and its two allohexaploid derivatives, Melampodium pringlei and Melampodium sericeum, which share both parentage and the direction of the cross, to the Pleistocene ($<$1.4 Ma). Thus, Pleistocene climatic fluctuations may have triggered formation of allopolyploids possibly in short intervals, contributing to difficulties in inferring the precise temporal order of allopolyploid species divergence of M. sericeum and M. pringlei. The relatively recent origin of the allopolyploids likely played a role in the near-absence of major changes in the repetitive fraction of the polyploids' genomes. The repetitive elements most affected by the postpolyploidization changes represented retrotransposons of the Ty1-copia lineage Maximus and, to a lesser extent, also Athila elements of Ty3-gypsy family.

A novel method to infer the origin of polyploids from Amplified Fragment Length Polymorphism data reveals that the alpine polyploid complex of Senecio carniolicus (Asteraceae) evolved mainly via autopolyploidy.

Despite its evolutionary and ecological relevance, the mode of polyploid origin has been notoriously difficult to be reconstructed from molecular data. Here, we present a method to identify the putative parents of polyploids and thus to infer the mode of their origin (auto- vs. allopolyploidy) from Amplified Fragment Length Polymorphism (AFLP) data. To this end, we use Cohen's d of distances between in silico polyploids, generated within a priori defined scenarios of origin from a priori delimited putative parental entities (e.g. taxa, genetic lineages), and natural polyploids. Simulations show that the discriminatory power of the proposed method increases mainly with increasing divergence between the lower-ploid putative ancestors and less so with increasing delay of polyploidization relative to the time of divergence. We apply the new method to the Senecio carniolicus aggregate, distributed in the European Alps and comprising two diploid, one tetraploid and one hexaploid species. In the eastern part of its distribution, the S. carniolicus aggregate was inferred to comprise an autopolyploid series, whereas for western populations of the tetraploid species, an allopolyploid origin involving the two diploid species was the most likely scenario. Although this suggests that the tetraploid species has two independent origins, other evidence (ribotype distribution, morphology) is consistent with the hypothesis of an autopolyploid origin with subsequent introgression by the second diploid species. Altogether, identifying the best among alternative scenarios using Cohen's d can be straightforward, but particular scenarios, such as allopolyploid origin vs. autopolyploid origin with subsequent introgression, remain difficult to be distinguished.

rDNA Loci Evolution in the Genus Glechoma (Lamiaceae).

Glechoma L. (Lamiaceae) is distributed in eastern Asia and Europe. Understanding chromosome evolution in Glechoma has been strongly hampered by its small chromosomes, constant karyotype and polyploidy. Here phylogenetic patterns and chromosomal variation in Glechoma species are considered, using genome sizes, chromosome mapping of 5S and 35S rDNAs by fluorescence in situ hybridisation (FISH), and phylogenetic analyses of internal transcribed spacers (nrITS) of 35S rDNA and 5S rDNA NTS sequences. Species and populations of Glechoma are tetraploid (2n = 36) with base chromosome number of x = 9. Four chromosomes carry pericentric 5S rDNA sites in their short arms in all the species. Two to four of these chromosomes also carry 35S rDNA in subterminal regions of the same arms. Two to four other chromosomes have 35S rDNA sites, all located subterminally within short arms; one individual possessed additional weak pericentric 35S rDNA signals on three other chromosomes. Five types of rDNA locus distribution have been defined on the basis of 35S rDNA variation, but none is species-specific, and most species have more than one type. Glechoma hederacea has four types. Genome size in Glechoma ranges from 0.80 to 0.94 pg (1C), with low levels of intrapopulational variation in all species. Phylogenetic analyses of ITS and NTS sequences distinguish three main clades coinciding with geographical distribution: European (G. hederacea-G. hirsuta), Chinese and Korean (G. longituba), and Japanese (G. grandis). The paper presents the first comparative cytogenetic analyses of Glechoma species including karyotype structure, rDNA location and number, and genome size interpreted in a phylogenetic context. The observed variation suggests that the genus is still in genomic flux. Genome size, but not rDNA loci number and distribution, provides a character for species delimitation which allows better inferences of interspecific relationships to be made, in the absence of well-defined morphological differentiation.

The Impact of Reconstruction Methods, Phylogenetic Uncertainty and Branch Lengths on Inference of Chromosome Number Evolution in American Daisies (Melampodium, Asteraceae).

Chromosome number change (polyploidy and dysploidy) plays an important role in plant diversification and speciation. Investigating chromosome number evolution commonly entails ancestral state reconstruction performed within a phylogenetic framework, which is, however, prone to uncertainty, whose effects on evolutionary inferences are insufficiently understood. Using the chromosomally diverse plant genus Melampodium (Asteraceae) as model group, we assess the impact of reconstruction method (maximum parsimony, maximum likelihood, Bayesian methods), branch length model (phylograms versus chronograms) and phylogenetic uncertainty (topological and branch length uncertainty) on the inference of chromosome number evolution. We also address the suitability of the maximum clade credibility (MCC) tree as single representative topology for chromosome number reconstruction. Each of the listed factors causes considerable incongruence among chromosome number reconstructions. Discrepancies between inferences on the MCC tree from those made by integrating over a set of trees are moderate for ancestral chromosome numbers, but severe for the difference of chromosome gains and losses, a measure of the directionality of dysploidy. Therefore, reliance on single trees, such as the MCC tree, is strongly discouraged and model averaging, taking both phylogenetic and model uncertainty into account, is recommended. For studying chromosome number evolution, dedicated models implemented in the program ChromEvol and ordered maximum parsimony may be most appropriate. Chromosome number evolution in Melampodium follows a pattern of bidirectional dysploidy (starting from x = 11 to x = 9 and x = 14, respectively) with no prevailing direction.

Ecological differentiation of diploid and polyploid cytotypes of Senecio carniolicus sensu lato (Asteraceae) is stronger in areas of sympatry.

BACKGROUND AND AIMS: Ecological differentiation is recognized as an important factor for polyploid speciation, but little is known regarding whether the ecological niches of cytotypes differ between areas of sympatry and areas where single cytotypes occur (i.e. niche displacement). METHODS: Ecological niches of four groups of Senecio carniolicus sensu lato (s.l.) (western and eastern diploid lineages, tetraploids and hexaploids) were characterized via Landolt indicator values of the accompanying vascular plant species and tested using multivariate and univariate statistics. KEY RESULTS: The four groups of S. carniolicus s.l. were ecologically differentiated mainly with respect to temperature, light and soil (humus content, nutrients, moisture variability). Niche breadths did not differ significantly. In areas of sympatry hexaploids shifted towards sites with higher temperature, less light and higher soil humus content as compared with homoploid sites, whereas diploids and tetraploids shifted in the opposite direction. In heteroploid sites of tetraploids and the western diploid lineage the latter shifted towards sites with lower humus content but higher aeration. CONCLUSIONS: Niche displacement can facilitate the formation of stable contact zones upon secondary contact of polyploids and their lower-ploid ancestors and/or lead to convergence of the cytotypes' niches after they have attained non-overlapping ranges. Niche displacement is essential for understanding ecological consequences of polyploidy.

Underestimated diversity in one of the world's best studied mountain ranges: The polyploid complex of Senecio carniolicus (Asteraceae) contains four species in the European Alps.

Senecio carniolicus (Asteraceae) is an intricate polyploid complex distributed in the European Alps (di-, tetra- and hexaploids) and Carpathians (hexaploids only). Molecular genetic, ecological, and crossing data allowed four evolutionary groups within S. carniolicus to be identified. Here, we establish that these four groups (two vicariant diploid lineages, tetraploids and hexaploids) are also morphologically differentiated. As a consequence, we draw taxonomic conclusions by characterizing four species, including the more narrowly circumscribed S. carniolicus (lectotypified here), the taxonomically elevated S. insubricus comb. nov. (lectotypified here), and the two newly described species S. disjunctus and S. noricus.

Ecological differentiation, lack of hybrids involving diploids, and asymmetric gene flow between polyploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea carniolica, Asteraceae).

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long-term integrity of lineages in contact zones. Here, we assessed fine-scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di-, tetra-, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra- and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra- to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.

Polyploidisation and geographic differentiation drive diversification in a European High Mountain Plant Group (Doronicum clusii Aggregate, Asteraceae).

Range shifts (especially during the Pleistocene), polyploidisation and hybridization are major factors affecting high-mountain biodiversity. A good system to study their role in the European high mountains is the Doronicum clusii aggregate (Asteraceae), whose four taxa (D. clusii s.s., D. stiriacum, D. glaciale subsp. glaciale and D. glaciale subsp. calcareum) are differentiated geographically, ecologically (basiphilous versus silicicolous) and/or via their ploidy levels (diploid versus tetraploid). Here, we use DNA sequences (three plastid and one nuclear spacer) and AFLP fingerprinting data generated for 58 populations to infer phylogenetic relationships, origin of polyploids-whose ploidy level was confirmed by chromosomally calibrated DNA ploidy level estimates-and phylogeographic history. Taxonomic conclusions were informed, among others, by a Gaussian clustering method for species delimitation using dominant multilocus data. Based on molecular data we identified three lineages: (i) silicicolous diploid D. clusii s.s. in the Alps, (ii) silicicolous tetraploid D. stiriacum in the eastern Alps (outside the range of D. clusii s.s.) and the Carpathians and (iii) the basiphilous diploids D. glaciale subsp. glaciale (eastern Alps) and D. glaciale subsp. calcareum (northeastern Alps); each taxon was identified as distinct by the Gaussian clustering, but the separation of D. glaciale subsp. calcareum and D. glaciale subsp. glaciale was not stable, supporting their taxonomic treatment as subspecies. Carpathian and Alpine populations of D. stiriacum were genetically differentiated suggesting phases of vicariance, probably during the Pleistocene. The origin (autopolyploid versus allopolyploid) of D. stiriacum remained unclear. Doronicum glaciale subsp. calcareum was genetically and morphologically weakly separated from D. glaciale subsp. glaciale but exhibited significantly higher genetic diversity and rarity. This suggests that the more widespread D. glaciale subsp. glaciale originated from D. glaciale subsp. calcareum, which is restricted to a prominent Pleistocene refugium previously identified in other alpine plant species.

Testing the efficiency of nested barriers to dispersal in the Mediterranean high mountain plant Edraianthus graminifolius (Campanulaceae).

Due to strong spatial heterogeneity and limited Pleistocene glaciation, the Balkan Peninsula is a major European biodiversity hot spot. Surprisingly little, however, is known about patterns and processes of intraspecific diversification of its biota in general and of high-altitude species in particular. A well-suited system to test hypotheses with respect to various isolating factors acting at different geographic scales and to explore full-range phylogeographic patterns on the Balkan Peninsula is Edraianthus graminifolius (Campanulaceae), distributed in the western Balkan mountain systems, the southwestern Carpathians and the Apennine Peninsula. To this end, we used a dense population sampling and employed amplified fragment length polymorphism (AFLP) markers and plastid DNA sequences supplemented by ecological niche modelling. The strongest splits were inferred to separate southern and northern Balkan populations from the central ones, from where range extension occurred to the Carpathians and, in more recent times, once or twice to the Apennine Peninsula. The three genetic groups in the western Balkan Peninsula were remarkably congruent among molecular markers, suggesting that the barriers to gene flow acted over long time periods facilitating allopatric differentiation. Each main group of Balkan populations contained genetically and geographically distinct subgroups, which likely are the result of local refugia during warmer periods. Evidently, the topographically highly complex and during the Last Glacial Maximum only locally glaciated Balkan Peninsula is a hot spot of species richness and endemism as well as a sanctuary of intraspecific genetic diversity, even if the underlying causes remain insufficiently understood.

Parental ploidy strongly affects offspring fitness in heteroploid crosses among three cytotypes of autopolyploid Jacobaea carniolica (Asteraceae).

Reproductive interactions among cytotypes in their contact zones determine whether these cytotypes can co-exist and form stable contact zones or not. In autopolyploids, heteroploid cross-compatibilities might depend on parental ploidy, but tests of this hypothesis in autopolyploid systems with more than two ploidies are lacking. Here, we study Jacobaea carniolica, which comprises diploid, tetraploid, and hexaploid individuals regularly forming contact zones. Seeds obtained from in situ cross-pollinations within and among cytotypes were subjected to DNA flow cytometry and greenhouse germination experiments. Hybrid fitness and parental effects on hybrid fitness were tested with regression models comparing fitness parameters of early life stages. Irrespective of the direction of crosses, seed viability and seedling survival in diploid-polyploid crosses were substantially lower than in tetraploid-hexaploid crosses. In contrast, seedling growth traits indicated neither transgressive character expression nor any selection against hybrid offspring. Congruent with a model of genome dosage effects, these traits differed between reciprocal crosses, especially of diploids and tetraploids, where trait values resembled those of the maternal parent. The strong effect of parental ploidy on offspring fitness in heteroploid crosses may cause contact zones involving exclusively polyploid cytotypes to be less stable over longer terms than those involving diploids and polyploids.

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.

Phylogenetic relationships in Brassicaceae tribe Alysseae inferred from nuclear ribosomal and chloroplast DNA sequence data.

Numerous molecular systematic studies within Brassicaceae have resulted in a strongly improved classification of the family, as morphologically defined units at and above the generic level were often found to poorly reflect phylogenetic relationships. Here, we focus on tribe Alysseae, which despite its size (accounting for about 7% of all species) has only received limited coverage in previous phylogenetic studies. Specifically, we want to test phylogenetic hypotheses implied by current tribal and generic circumscriptions and to put diversification within tribe Alysseae into a temporal context. To this end, sequence data from the nrDNA ITS and two plastid regions (ndhF gene, trnL-F intergenic spacer) were obtained for 176 accessions, representing 16 out of 17 currently recognized genera of the tribe, and were phylogenetically analysed, among others, using a relaxed molecular clock. Due to large discrepancies with respect to published ages of Brassicaceae, age estimates concerning Alysseae are, however, burdened with considerable uncertainty. The tribe is monophyletic and contains four strongly supported major clades and Alyssum homalocarpum, whose relationships among each other remain uncertain due to incongruences between nuclear and plastid DNA markers. The largest genus of the tribe, Alyssum, is not monophyletic and contains, apart from A. homalocarpum, two distinct lineages, corresponding to sections Alyssum, Psilonema, Gamosepalum and to sections Odontarrhena and Meniocus, respectively. Clypeola, whose monophyly is supported only by the plastid data, is very closely related to and possibly nested within the second Alyssum lineage. Species of the genus Fibigia intermingle with those of Alyssoides, Clastopus, Degenia, and Physoptychis, rendering Fibigia polyphyletic. The monotypic genera Leptoplax and Physocardamum are embedded in Bornmuellera.

Next-generation sequencing reveals the impact of repetitive DNA across phylogenetically closely related genomes of Orobanchaceae.

We used next-generation sequencing to characterize the genomes of nine species of Orobanchaceae of known phylogenetic relationships, different life forms, and including a polyploid species. The study species are the autotrophic, nonparasitic Lindenbergia philippensis, the hemiparasitic Schwalbea americana, and seven nonphotosynthetic parasitic species of Orobanche (Orobanche crenata, Orobanche cumana, Orobanche gracilis (tetraploid), and Orobanche pancicii) and Phelipanche (Phelipanche lavandulacea, Phelipanche purpurea, and Phelipanche ramosa). Ty3/Gypsy elements comprise 1.93%-28.34% of the nine genomes and Ty1/Copia elements comprise 8.09%-22.83%. When compared with L. philippensis and S. americana, the nonphotosynthetic species contain higher proportions of repetitive DNA sequences, perhaps reflecting relaxed selection on genome size in parasitic organisms. Among the parasitic species, those in the genus Orobanche have smaller genomes but higher proportions of repetitive DNA than those in Phelipanche, mostly due to a diversification of repeats and an accumulation of Ty3/Gypsy elements. Genome downsizing in the tetraploid O. gracilis probably led to sequence loss across most repeat types.

The evolutionary history of the white-rayed species of Melampodium (Asteraceae) involved multiple cycles of hybridization and polyploidization.

PREMISE OF THE STUDY: Polyploidy plays an important role in race differentiation and eventually speciation. Underlying mechanisms include chromosomal and genomic changes facilitating reproductive isolation and/or stabilization of hybrids. A prerequisite for studying these processes is a sound knowledge on the origin of polyploids. A well-suited group for studying polyploid evolution consists of the three species of Melampodium ser. Leucantha (Asteraceae): M. argophyllum, M. cinereum, and M. leucanthum. METHODS: The origin of polyploids was inferred using network and tree-based phylogenetic analyses of several plastid and nuclear DNA sequences and of fingerprint data (AFLP). Genome evolution was assessed via genome size measurements, karyotype analysis, and in situ hybridization of ribosomal DNA. KEY RESULTS: Tetraploid cytotypes of the phylogenetically distinct M. cinereum and M. leucanthum had, compared to the diploid cytotypes, doubled genome sizes and no evidence of gross chromosomal rearrangements. Hexaploid M. argophyllum constituted a separate lineage with limited intermixing with the other species, except in analyses from nuclear ITS. Its genome size was lower than expected if M. cinereum and/or M. leucanthum were involved in its origin, and no chromosomal rearrangements were evident. CONCLUSIONS: Polyploids in M. cinereum and M. leucanthum are of recent autopolyploid origin in line with the lack of significant genomic changes. Hexaploid M. argophyllum also appears to be of autopolyploid origin against the previous hypothesis of an allopolyploid origin involving the other two species, but some gene flow with the other species in early phases of differentiation cannot be excluded.

The promiscuous and the chaste: frequent allopolyploid speciation and its genomic consequences in American daisies (Melampodium sect. Melampodium; Asteraceae).

Polyploidy, an important factor in eukaryotic evolution, is especially abundant in angiosperms, where it often acts in concert with hybridization to produce allopolyploids. The application of molecular phylogenetic techniques has identified the origins of numerous allopolyploids, but little is known on genomic and chromosomal consequences of allopolyploidization, despite their important role in conferring divergence of allopolyploids from their parental species. Here, using several plastid and nuclear sequence markers, we clarify the origin of tetra- and hexaploids in a group of American daisies, allowing characterization of genome dynamics in polyploids compared to their diploid ancestors. All polyploid species are allopolyploids. Among the four diploid gene pools, the propensity for allopolyploidization is unevenly distributed phylogenetically with a few species apparently more prone to participate, but the underlying causes remain unclear. Polyploid genomes are characterized by differential loss of ribosomal DNA loci (5S and 35S rDNA), known hotspots of chromosomal evolution, but show genome size additivity, suggesting limited changes beyond those affecting rDNA loci or the presence of processes counterbalancing genome reduction. Patterns of rDNA sequence conversion and provenance of the lost loci are highly idiosyncratic and differ even between allopolyploids of identical parentage, indicating that allopolyploids deriving from the same lower-ploid parental species can follow different evolutionary trajectories.

No evidence for a role of competitive capabilities of adults in causing habitat segregation of diploid and hexaploid Senecio carniolicus (Asteracaeae).

Hexaploid individuals of Senecio carniolicus (Asteraceae) predominantly occur in dense swards while diploids prevail in open vegetation. We test whether this habitat segregation is due to differential responses to competition. Linear regression models were used to relate biomass and maximum leaf length of adults to vegetation cover within radii of 20 cm around target individuals. Biomass differed between ploidy levels, but was independent from vegetation cover in both cytotypes. Maximum leaf length of diploids increased with vegetation cover, but remained constant in hexaploids. This suggests that at the adult stage diploids respond to increasing competition by changes in plant architecture rather than changes in resource utilization, while hexaploids are unaffected by competition. Consequently, other factors, such as competitive interactions at earlier life stages, likely are responsible for habitat segregation of diploid and hexaploid S. carniolicus.

Distribution and habitat segregation on different spatial scales among diploid, tetraploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the Eastern Alps.

BACKGROUND AND AIMS: The spatial distribution of cytotypes can provide valuable insights into evolutionary patterns of polyploid complexes. In a previous study the macro-scale distribution of the three main cytotypes in Senecio carniolicus (Asteraceae) within the Eastern Alps was characterized. Employing a roughly 12-fold extended sampling, the present study focuses on unravelling patterns of cytotype distribution on the meso- and microscale and on correlating those with ecological properties of the growing sites. METHODS: DAPI flow cytometry of dried samples was used to determine DNA ploidy level in 5033 individuals from 100 populations spread over the entire Eastern Alpine distribution area of S. carniolicus. Descriptors of microhabitats as well as spatial data were recorded in the field, and analysed with a mixed-effects ANOVA. KEY RESULTS: Extensive variation in DNA ploidy levels (2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x) was detected. Of the main cytotypes, diploids and hexaploids were widespread and had strongly overlapping distributions resulting in the frequent occurrence of cytotype mixtures (half of the investigated populations), whereas tetraploids were disjunctly distributed and occurred in the south-west and the east of the species' distribution area. In spite of the frequent co-occurrence of cytotypes, only 1 % of the samples belonged to secondary cytotypes (3x, 5x, 7x, 8x, 9x). Diploids, tetraploids and hexaploids were altitudinally segregated, but with broad overlap. Similarly, highly significant differences in vegetation and rock cover as well as microhabitat exposure were found between the main cytotypes. CONCLUSIONS: Senecio carniolicus shows a remarkable diversity of cytotypes. The distribution of the three main cytotypes (2x, 4x, 6x) has been shaped by Pleistocene glaciations to different extents. Whereas tetraploids are nearly entirely restricted to refugia, hexaploids colonized areas that were extensively glaciated. Diploid and hexaploid individuals often co-occur in mixed populations, where they are spatially and ecologically segregated at both the meso-scale (altitudinal differentiation, exposure of the growing site) and the micro-scale (cover of vegetation and bare rock). With regard to the ecological parameters investigated, the tetraploid cytotype occupies an intermediate position. The rareness of secondary cytotypes suggests the presence of strong pre- or post-zygotic mating barriers.