Evolution of the Sabulina verna group (Caryophyllaceae) in Europe: A deep split, followed by secondary contacts, multiple allopolyploidization and colonization of challenging substrates.

One of the major goals of contemporary evolutionary biology is to elucidate the relative roles of allopatric and ecological differentiation and polyploidy in speciation. In this study, we address the taxonomically intricate Sabulina verna group, which has a disjunct Arctic-alpine postglacial range in Europe and occupies a broad range of ecological niches, including substrates toxic to plants. Using genome-wide ddRAD sequencing combined with morphometric analyses based on extensive sampling of 111 natural populations, we aimed to disentangle internal evolutionary relationships and examine their correspondence with the pronounced edaphic and ploidy diversity within the group. We identified two spatially distinct groups of diploids: a widespread Arctic-alpine group and a spatially restricted yet diverse Balkan group. Most tetraploids exhibited a considerably admixed ancestry derived from both these groups, suggesting their allopolyploid origin. Four genetic clusters in congruence with geography and mostly supported by morphological traits were recognized in the diploid Arctic-alpine group. Tetraploids are split into two distinct and geographically vicariant groups, indicating their repeated polytopic origin. Furthermore, our results also revealed at least five-fold parallel colonization of toxic substrates (serpentine and metalliferous), altogether demonstrating a complex interaction between geography, challenging substrates and polyploidy in the evolution of the group. Finally, we propose a new taxonomic treatment of this complex.

A Multigene Phylogeny of Native American Hawkweeds (Hieracium Subgen. Chionoracium, Cichorieae, Asteraceae): Origin, Speciation Patterns, and Migration Routes.

Native American hawkweeds are mainly mountainous species that are distributed all over the New World. They are severely understudied with respect to their origin, colonization of the vast distribution area, and species relationships. Here, we attempt to reconstruct the evolutionary history of the group by applying seven molecular markers (plastid, nuclear ribosomal and low-copy genes). Phylogenetic analyses revealed that Chionoracium is a subgenus of the mainly Eurasian genus Hieracium, which originated from eastern European hawkweeds about 1.58-2.24 million years ago. Plastid DNA suggested a single origin of all Chionoracium species. They colonized the New World via Beringia and formed several distinct lineages in North America. Via one Central American lineage, the group colonized South America and radiated into more than a hundred species within about 0.8 million years, long after the closure of the Isthmus of Panama and the most recent uplift of the Andes. Despite some incongruences shown by different markers, most of them revealed the same crown groups of closely related taxa, which were, however, largely in conflict with traditional sectional classifications. We provide a basic framework for further elucidation of speciation patterns. A thorough taxonomic revision of Hieracium subgen. Chionoracium is recommended.

Molecular Evolution and Organization of Ribosomal DNA in the Hawkweed Tribe Hieraciinae (Cichorieae, Asteraceae).

Molecular evolution of ribosomal DNA can be highly dynamic. Hundreds to thousands of copies in the genome are subject to concerted evolution, which homogenizes sequence variants to different degrees. If well homogenized, sequences are suitable for phylogeny reconstruction; if not, sequence polymorphism has to be handled appropriately. Here we investigate non-coding rDNA sequences (ITS/ETS, 5S-NTS) along with the chromosomal organization of their respective loci (45S and 5S rDNA) in diploids of the Hieraciinae. The subtribe consists of genera Hieracium, Pilosella, Andryala, and Hispidella and has a complex evolutionary history characterized by ancient intergeneric hybridization, allele sharing among species, and incomplete lineage sorting. Direct or cloned Sanger sequences and phased alleles derived from Illumina genome sequencing were subjected to phylogenetic analyses. Patterns of homogenization and tree topologies based on the three regions were compared. In contrast to most other plant groups, 5S-NTS sequences were generally better homogenized than ITS and ETS sequences. A novel case of ancient intergeneric hybridization between Hispidella and Hieracium was inferred, and some further incongruences between the trees were found, suggesting independent evolution of these regions. In some species, homogenization of ITS/ETS and 5S-NTS sequences proceeded in different directions although the 5S rDNA locus always occurred on the same chromosome with one 45S rDNA locus. The ancestral rDNA organization in the Hieraciinae comprised 4 loci of 45S rDNA in terminal positions and 2 loci of 5S rDNA in interstitial positions per diploid genome. In Hieracium, some deviations from this general pattern were found (3, 6, or 7 loci of 45S rDNA; three loci of 5S rDNA). Some of these deviations concerned intraspecific variation, and most of them occurred at the tips of the tree or independently in different lineages. This indicates that the organization of rDNA loci is more dynamic than the evolution of sequences contained in them and that locus number is therefore largely unsuitable to inform about species relationships in Hieracium. No consistent differences in the degree of sequence homogenization and the number of 45S rDNA loci were found, suggesting interlocus concerted evolution.

Evolutionary history and genetic diversity of apomictic allopolyploids in Hieracium s.str.: morphological versus genomic features.

PREMISE: The origin of allopolyploids is believed to shape their evolutionary potential, ecology, and geographical ranges. Morphologically distinct apomictic types sharing the same parental species belong to the most challenging groups of polyploids. We evaluated the origins and variation of two triploid taxa (Hieracium pallidiflorum, H. picroides) presumably derived from the same diploid parental pair (H. intybaceum, H. prenanthoides). METHODS: We used a suite of approaches ranging from morphological, phylogenetic (three unlinked molecular markers), and cytogenetic analyses (in situ hybridization) to genome size screening and genome skimming. RESULTS: Genotyping proved the expected parentage of all analyzed accessions of H. pallidiflorum and H. picroides and revealed that nearly all of them originated independently. Genome sizes and genome dosage largely corresponded to morphology, whereas the maternal origin of the allopolyploids had no discernable effect. Polyploid accessions of both parental species usually contained genetic material from other species. Given the phylogenetic distance of the parents, their chromosomes appeared only weakly differentiated in genomic in situ hybridization (GISH), as well as in overall comparisons of the repetitive fraction of their genomes. Furthermore, the repeatome of a phylogenetically more closely related species (H. umbellatum) differed significantly more. CONCLUSIONS: We proved (1) multiple origins of hybridogeneous apomicts from the same diploid parental taxa, and (2) allopolyploid origins of polyploid accessions of the parental species. We also showed that the evolutionary dynamics of very fast evolving markers such as satellite DNA or transposable elements does not necessarily follow patterns of speciation.

Polyploid evolution: The ultimate way to grasp the nettle.

Polyploidy is one of the major forces of plant evolution and widespread mixed-ploidy species offer an opportunity to evaluate its significance. We therefore selected the cosmopolitan species Urtica dioica (stinging nettle), examined its cytogeography and pattern of absolute genome size, and assessed correlations with bioclimatic and ecogeographic data (latitude, longitude, elevation). We evaluated variation in ploidy level using an extensive dataset of 7012 samples from 1317 populations covering most of the species' distribution area. The widespread tetraploid cytotype (87%) was strongly prevalent over diploids (13%). A subsequent analysis of absolute genome size proved a uniform Cx-value of core U. dioica (except for U. d. subsp. cypria) whereas other closely related species, namely U. bianorii, U. kioviensis and U. simensis, differed significantly. We detected a positive correlation between relative genome size and longitude and latitude in the complete dataset of European populations and a positive correlation between relative genome size and longitude in a reduced dataset of diploid accessions (the complete dataset of diploids excluding U. d. subsp. kurdistanica). In addition, our data indicate an affinity of most diploids to natural and near-natural habitats and that the tetraploid cytotype and a small part of diploids (population from the Po river basin in northern Italy) tend to inhabit synanthropic sites. To sum up, the pattern of ploidy variation revealed by our study is in many aspects unique to the stinging nettle, being most likely first of all driven by the greater ecological plasticity and invasiveness of the tetraploid cytotype.

Cytotype coexistence in the field cannot be explained by inter-cytotype hybridization alone: linking experiments and computer simulations in the sexual species Pilosella echioides (Asteraceae).

BACKGROUND: Processes driving ploidal diversity at the population level are virtually unknown. Their identification should use a combination of large-scale screening of ploidy levels in the field, pairwise crossing experiments and mathematical modelling linking these two types of data. We applied this approach to determine the drivers of frequencies of coexisting cytotypes in mixed-ploidy field populations of the fully sexual plant species Pilosella echioides. We examined fecundity and ploidal diversity in seeds from all possible pairwise crosses among 2x, 3x and 4x plants. Using these data, we simulated the dynamics of theoretical panmictic populations of individuals whose progeny structure is identical to that determined by the hybridization experiment. RESULTS: The seed set differed significantly between the crossing treatments, being highest in crosses between diploids and tetraploids and lowest in triploid-triploid crosses. The number of progeny classes (with respect to embryo and endosperm ploidy) ranged from three in the 2x-2x cross to eleven in the 3x-3x cross. Our simulations demonstrate that, provided there is no difference in clonal growth and/or survival between cytotypes, it is a clear case of minority cytotype exclusion depending on the initial conditions with two stable states, neither of which corresponds to the ploidal structure in the field: (i) with prevalent diploids and lower proportions of other ploidies, and (ii) with prevalent tetraploids and 9% of hexaploids. By contrast, if clonal growth differs between cytotypes, minority cytotype exclusion occurs only if the role of sexual reproduction is high; otherwise differences in clonal growth are sufficient to maintain triploid prevalence (as observed in the field) independently of initial conditions. CONCLUSIONS: The projections of our model suggest that the ploidal structure observed in the field can only be reached via a relatively high capacity for clonal growth (and proportionally lower sexual reproduction) in all cytotypes combined with higher clonal growth in the prevailing cytotype (3x).

CLO-PLA: a database of clonal and bud-bank traits of the Central European flora.

This dataset presents comprehensive and easy-to-use information on 29 functional traits of clonal growth, bud banks, and lifespan of members of the Central European flora. The source data were compiled from a number of published sources (see the reference file) and the authors' own observations or studies. In total, 2,909 species are included (2,745 herbs and 164 woody species), out of which 1,532 (i.e., 52.7% of total) are classified as possessing clonal growth organs (1,480, i.e., 53.9%, if woody plants are excluded). This provides a unique, and largely unexplored, set of traits of clonal growth that can be used in studies on comparative plant ecology, plant evolution, community assembly, and ecosystem functioning across the large flora of Central Europe. It can be directly imported into a number of programs and packages that perform trait-based and phylogenetic analyses aimed to answer a variety of open and pressing ecological questions.

Stress-induced memory alters growth of clonal offspring of white clover (Trifolium repens).

PREMISE OF THE STUDY: The phenotype of an individual can be modified by the environment experienced by its predecessors, a phenomenon called transgenerational or maternal effects. These effects are studied mostly across sexual generations and are thought to be mediated also by epigenetic variation. However, we do not know how important transgenerational effects are across asexual generations of clonal plants. METHODS: We investigated the role of different drought intensities and durations experienced by parental plants of Trifolium repens on the growth of offspring ramets after transplantation of clonal cuttings to control conditions. We also treated half of the plants with 5-azacytidine, which is a demethylating agent, to test the potential role of DNA methylation on transgenerational effects. KEY RESULTS: Transgenerational effects were manifested as increased biomass of offspring ramets if parental plants experienced medium drought applied for a short period and decreased biomass of offspring ramets if parental plants experienced intense drought for a short period. These transgenerational effects were not observed for offspring of parents from the same treatments if these were treated with 5-azacytidine, whose application significantly decreased the amount of 5-methyl-2'-deoxycytidine in plants. CONCLUSIONS: Transgenerational effects might play an important role in the clonal plant Trifolium repens and are probably mediated by epigenetic variation. The growth and behavior of clonal plants might be affected not only by the ambient environment but also by environments that are no longer present at the time of clonal reproduction. This phenomenon can have yet unacknowledged ecological and evolutionary implications for clonal plants.

Diversity and endemism in deglaciated areas: ploidy, relative genome size and niche differentiation in the Galium pusillum complex (Rubiaceae) in Northern and Central Europe.

BACKGROUND AND AIMS: Plants endemic to areas covered by ice sheets during the last glaciation represent paradigmatic examples of rapid speciation in changing environments, yet very few systems outside the harsh arctic zone have been comprehensively investigated so far. The Galium pusillum aggregate (Rubiaceae) is a challenging species complex that exhibits a marked differentiation in boreal parts of Northern Europe. As a first step towards understanding its evolutionary history in deglaciated regions, this study assesses cytological variation and ecological preferences of the northern endemics and compares the results with corresponding data for species occurring in neighbouring unglaciated parts of Central and Western Europe. METHODS: DNA flow cytometry was used together with confirmatory chromosome counts to determine ploidy levels and relative genome sizes in 1158 individuals from 181 populations. A formalized analysis of habitat preferences was applied to explore niche differentiation among species and ploidy levels. KEY RESULTS: The G. pusillum complex evolved at diploid and tetraploid levels in Northern Europe, in contrast to the high-polyploid evolution of most other northern endemics. A high level of eco-geographic segregation was observed between different species (particularly along gradients of soil pH and competition) which is unusual for plants in deglaciated areas and most probably contributes to maintaining species integrity. Relative monoploid DNA contents of the species from previously glaciated regions were significantly lower than those of their counterparts from mostly unglaciated Central Europe, suggesting independent evolutionary histories. CONCLUSIONS: The aggregate of G. pusillum in Northern Europe represents an exceptional case with a geographically vicariant and ecologically distinct diploid/tetraploid species endemic to formerly glaciated areas. The high level of interspecific differentiation substantially widens our perception of the evolutionary dynamics and speciation rates in the dramatically changing environments of Northern Europe.

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes.

PREMISE OF THE STUDY: The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. METHODS AND RESULTS: Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. CONCLUSIONS: The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.

Bridging global and microregional scales: ploidy distribution in Pilosella echioides (Asteraceae) in central Europe.

BACKGROUND AND AIMS: A detailed knowledge of cytotype distribution can provide important insights into the evolutionary history of polyploid systems. This study aims to explore the spatial distribution of different cytotypes in Pilosella echioides at various spatial scales (from the whole distributional range to the population level) and to outline possible evolutionary scenarios for the observed geographic pattern. METHODS: DNA-ploidy levels were estimated using DAPI flow cytometry in 4410 individuals of P. echioides from 46 populations spread over the entire distribution range in central Europe. Special attention was paid to the cytotype structure in the most ploidy-diverse population in south-west Moravia. KEY RESULTS: Five different cytotypes (2x, 3x, 4x, 5x and 6x) were found, the last being recorded for the first time. Although ploidy-uniform (di- or tetraploid) sites clearly prevailed, nearly one-quarter of the populations investigated harboured more (up to all five) cytotypes. Whereas penta- and hexaploids constituted only a minority of the samples, a striking predominance of the triploid cytotype was observed in several populations. CONCLUSIONS: The representative sampling confirmed previous data on cytotype distribution, i.e. the spatial aggregation of mixed-ploidy populations in south-west Moravia and Lower Austria and the predominance of ploidy-uniform populations in other parts of the area investigated. Recurrent origin of polyploids from diploid progenitors via unreduced gametes and their successful establishment are considered the key factors promoting intrapopulational ploidy mixture ('primary hybrid zones'). As an alternative to the generally accepted theory of cytotype co-existence based on the development of different means of inter-ploidy reproductive isolation, it is suggested that a long-term ploidy mixture can also be maintained in free-mating populations provided that the polyploids originate with a sufficient frequency. In addition, the prevalence (or subdominance) of the triploid cytotype in several mixed-ploidy populations represents the first evidence of such a phenomenon in plant systems with exclusively sexual reproduction.

Intra-individual polymorphism in diploid and apomictic polyploid hawkweeds (Hieracium, Lactuceae, Asteraceae): disentangling phylogenetic signal, reticulation, and noise.

BACKGROUND: Hieracium s.str. is a complex species-rich group of perennial herbs composed of few sexual diploids and numerous apomictic polyploids. The existence of reticulation and the near-continuity of morphological characters across taxa seriously affect species determination, making Hieracium one of the best examples of a 'botanist's nightmare'. Consequently, its species relationships have not previously been addressed by molecular methods. Concentrating on the supposed major evolutionary units, we used nuclear ribosomal (ETS) and chloroplast (trnT-trnL) sequences in order to disentangle the phylogenetic relationships and to infer the origins of the polyploids. RESULTS: Despite relatively low interspecific variation, the nuclear data revealed the existence of two major groups roughly corresponding to species with a Western or Eastern European origin. Extensive reticulation was mainly inferred from the character additivity of parental ETS variants. Surprisingly, many diploid species were of hybrid origin whilst several polyploid taxa showed no evidence of reticulation. Intra-individual ETS sequence polymorphism generally exceeded interspecific variation and was either independent of, or additional to, additive patterns accounted for by hybrid origin. Several ETS ribotypes occurred in different hybrid taxa, but never as the only variant in any species analyzed. CONCLUSION: The high level of intra-individual ETS polymorphism prevented straightforward phylogenetic analysis. Characterization of this variation as additive, shared informative, homoplasious, or unique made it possible to uncover the phylogenetic signal and to reveal the hybrid origin of 29 out of 60 accessions. Contrary to expectation, diploid sexuals and polyploid apomicts did not differ in their molecular patterns. The basic division of the genus into two major clades had not previously been intimated on morphological grounds. Both major groups are thought to have survived in different glacial refugia and to have hybridized as a result of secondary contact. Several lines of evidence suggest the data is best explained by the presence of an extinct range of variation and a larger diversity of ancestral diploids in former times rather than by unsampled variation. Extinct diversity and extensive reticulation are thought to have largely obscured the species relationships. Our study illustrates how multigene sequences can be used to disentangle the evolutionary history of agamic complexes or similarly difficult datasets.

Genome size in Hieracium subgenus Hieracium (Asteraceae) is strongly correlated with major phylogenetic groups.

BACKGROUND AND AIMS: Hieracium subgenus Hieracium is one of the taxonomically most intricate groups of vascular plants, due to polyploidy and a diversity of breeeding systems (sexuality vs. apomixis). The aim of the present study was to analyse nuclear genome size in a phylogenetic framework and to assess relationships between genome size and ploidy, breeding system and selected ecogeographic features. METHODS: Holoploid and monoploid genome sizes (C- and Cx-values) of 215 cultivated plants from 89 field populations of 42 so-called 'basic' Hieracium species were determined using propidium iodide flow cytometry. Chromosome counts were available for all analysed plants, and all plants were tested experimentally for their mode of reproduction (sexuality vs. apomixis). For constructing molecular phylogenetic trees, the external transcribed spacer region of nuclear ribosomal DNA was used. KEY RESULTS: The mean 2C values differed up to 2.37-fold among different species (from 7.03 pg in diploid to 16.67 in tetraploid accessions). The 1Cx values varied 1.22-fold (between 3.51 and 4.34 pg). Variation in 1Cx values between conspecific (species in a broad sense) accessions ranged from 0.24% to 7.2%. Little variation (not exceeding the approximate measurement inaccurracy threshold of 3.5%) was found in 33 species, whereas variation higher than 3.5% was detected in seven species. Most of the latter may have a polytopic origin. Mean 1Cx values of the three cytotypes (2n, 3n and 4n) differed significantly (average of 3.93 pg in diploids, 3.82 pg in triploids and 3.78 pg in tetraploids) indicating downsizing of genomes in polyploids. The pattern of genome size variation correlated well with two major phylogenetic clades which were composed of species with western or eastern European origin. The monoploid genome size in the 'western' species was significantly lower than in the 'eastern' ones. Correlation of genome size with latitude, altitude and selected ecological characters (light and temperature) was not significant. A longitudinal component was only apparent for the whole data set, but absent within the major lineages. CONCLUSIONS: Phylogeny was the most important factor explaining the pattern of genome size variation in Hieracium sensu stricto, species of western European origin having significantly lower genome size in comparison with those of eastern European origin. Any correlation with ecogeographic variables, including longitude, was outweighed by the divergence of the genus into two major phylogenetic lineages.

Incongruent plastid and nuclear DNA phylogenies reveal ancient intergeneric hybridization in Pilosella hawkweeds (Hieracium, Cichorieae, Asteraceae).

Phylogenetic relationships for Hieracium subgen. Pilosella were inferred from chloroplast (trnT-trnL, matK) and nuclear (ITS) sequence data. Chloroplast markers revealed the existence of two divergent haplotype groups within the subgenus that did not correspond to presumed relationships. Furthermore, chloroplast haplotypes of the genera Hispidella and Andryala nested each within one of these groups. In contrast, ITS data were generally in accord with morphology and other evidence and were therefore assumed to reflect the true phylogeny. They revealed a sister relationship between Pilosella and Hispidella and a joint clade of Hieracium subgenera Hieracium and Chionoracium (Stenotheca) while genus Andryala represented a third major lineage of the final ingroup cluster. Detailed analysis of trnT-trnL character state evolution along the ITS tree suggested two intergeneric hybridization events between ancestral lineages that resulted in cytoplasmic transfer (from Hieracium/Chionoracium to Pilosella, and from the introgressed Pilosella lineage to Andryala). These chloroplast capture events, the first of which involved a now extinct haplotype, are the most likely explanation for the observed incongruencies between plastid and nuclear DNA markers.

Loss of genetic variation in geographically marginal populations of Atriplex tatarica (Chenopodiaceae).

BACKGROUND AND AIMS: Genetic variability was estimated for Atriplex tatarica from 25 populations in the Czech Republic. Since its north-western range margin is in central Europe, a relationship between marginality and low within-population genetic diversity was tested in accordance with the Central-Marginal Model. METHODS: Population genetic diversity was expressed by assessing patterns of variation at 13 putatively neutral allozyme loci (comprising 30 putative alleles) within and between 25 natural populations of A. tatarica along a north-west-south-east transect in the Czech Republic. KEY RESULTS: Atriplex tatarica is a species of human-made habitats with a mixed mating system and wide geographic distribution. Overall, A. tatarica displayed moderate levels of genetic diversity in comparison with other herbaceous plants. The percentage of loci that were polymorphic was 47.1%, with average values of 1.55, 0.151 and 0.155 for the average number of alleles per polymorphic locus (A), observed heterozygosity (Ho) and expected heterozygosity (He), respectively. There was only weak evidence of inbreeding within populations (FIS=0.031) and significant population differentiation (FST=0.214). Analysis of the data provides no evidence for isolation-by-distance for the whole study area. However, Mantel tests were highly significant for the marginal Bohemian region and non-significant for the central Moravian region. While northern populations of A. tatarica showed significantly lower allelic richness (A=1.462) than populations from the southern part of the study area (A=1.615), they did not differ in observed heterozygosity (Ho), gene diversity (HS), inbreeding within populations (FIS) or population differentiation (FST), despite generally lower values of particular genetic measurements in the marginal region. CONCLUSIONS: Genetic diversity, with the exception of allelic richness, was not significantly lower at the margins of the species' range. This, therefore, provides only weak support for the predictions of the Central-Marginal Model.