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

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

Phylogenetic relationships, diversification and expansion of chili peppers (Capsicum, Solanaceae).

BACKGROUND AND AIMS: Capsicum (Solanaceae), native to the tropical and temperate Americas, comprises the well-known sweet and hot chili peppers and several wild species. So far, only partial taxonomic and phylogenetic analyses have been done for the genus. Here, the phylogenetic relationships between nearly all taxa of Capsicum were explored to test the monophyly of the genus and to obtain a better knowledge of species relationships, diversification and expansion. METHODS: Thirty-four of approximately 35 Capsicum species were sampled. Maximum parsimony and Bayesian inference analyses were performed using two plastid markers (matK and psbA-trnH) and one single-copy nuclear gene (waxy). The evolutionary changes of nine key features were reconstructed following the parsimony ancestral states method. Ancestral areas were reconstructed through a Bayesian Markov chain Monte Carlo analysis. KEY RESULTS: Capsicum forms a monophyletic clade, with Lycianthes as a sister group, following both phylogenetic approaches. Eleven well-supported clades (four of them monotypic) can be recognized within Capsicum, although some interspecific relationships need further analysis. A few features are useful to characterize different clades (e.g. fruit anatomy, chromosome base number), whereas some others are highly homoplastic (e.g. seed colour). The origin of Capsicum is postulated in an area along the Andes of western to north-western South America. The expansion of the genus has followed a clockwise direction around the Amazon basin, towards central and south-eastern Brazil, then back to western South America, and finally northwards to Central America. CONCLUSIONS: New insights are provided regarding interspecific relationships, character evolution, and geographical origin and expansion of Capsicum A clearly distinct early-diverging clade can be distinguished, centred in western-north-western South America. Subsequent rapid speciation has led to the origin of the remaining clades. The diversification of Capsicum has culminated in the origin of the main cultivated species in several regions of South to Central America.

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

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

The origin of unique diversity in deglaciated areas: traces of Pleistocene processes in north-European endemics from the Galium pusillum polyploid complex (Rubiaceae).

The role of glacial oscillations in shaping plant diversity has been only rarely addressed in endemics of formerly glaciated areas. The Galium pusillum group represents a rare example of an ecologically diverse and ploidy-variable species complex that exhibits substantial diversity in deglaciated northern Europe. Using AFLP and plastid and nuclear DNA sequences of 67 populations from northern, central, and western Europe with known ecological preferences, we elucidate the evolutionary history of lineages restricted to deglaciated areas and identify the eco-geographic partitioning of their genetic variation. We reveal three distinct endemic northern lineages: (i) diploids from southern Sweden + the British Isles, (ii) tetraploids from southern Scandinavia and the British Isles that show signs of ancient hybridization between the first lineage and populations from unglaciated central Europe, and (iii) tetraploids from Iceland + central Norway. Available evidence supports a stepwise differentiation of these three lineages that started at least before the last glacial maximum by processes of genome duplication, interlineage hybridization and/or allopatric evolution in distinct periglacial refugia. We reject the hypothesis of more recent postglacial speciation. Ecological characteristics of the populations under study only partly reflect genetic variation and suggest broad niches of postglacial colonizers. Despite their largely allopatric modern distributions, the north-European lineages of the G. pusillum group do not show signs of rapid postglacial divergence, in contrast to most other northern endemics. Our study suggests that plants inhabiting deglaciated areas outside the Arctic may exhibit very different evolutionary histories compared with their more thoroughly investigated high-arctic counterparts.

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

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

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.

Nuclear and plastid haplotypes suggest rapid diploid and polyploid speciation in the N Hemisphere Achillea millefolium complex (Asteraceae).

BACKGROUND: Species complexes or aggregates consist of a set of closely related species often of different ploidy levels, whose relationships are difficult to reconstruct. The N Hemisphere Achillea millefolium aggregate exhibits complex morphological and genetic variation and a broad ecological amplitude. To understand its evolutionary history, we study sequence variation at two nuclear genes and three plastid loci across the natural distribution of this species complex and compare the patterns of such variations to the species tree inferred earlier from AFLP data. RESULTS: Among the diploid species of A. millefolium agg., gene trees of the two nuclear loci, ncpGS and SBP, and the combined plastid fragments are incongruent with each other and with the AFLP tree likely due to incomplete lineage sorting or secondary introgression. In spite of the large distributional range, no isolation by distance is found. Furthermore, there is evidence for intragenic recombination in the ncpGS gene. An analysis using a probabilistic model for population demographic history indicates large ancestral effective population sizes and short intervals between speciation events. Such a scenario explains the incongruence of the gene trees and species tree we observe. The relationships are particularly complex in the polyploid members of A. millefolium agg. CONCLUSIONS: The present study indicates that the diploid members of A. millefolium agg. share a large part of their molecular genetic variation. The findings of little lineage sorting and lack of isolation by distance is likely due to short intervals between speciation events and close proximity of ancestral populations. While previous AFLP data provide species trees congruent with earlier morphological classification and phylogeographic considerations, the present sequence data are not suited to recover the relationships of diploid species in A. millefolium agg. For the polyploid taxa many hybrid links and introgression from the diploids are suggested.

Allopolyploid speciation and ongoing backcrossing between diploid progenitor and tetraploid progeny lineages in the Achillea millefolium species complex: analyses of single-copy nuclear genes and genomic AFLP.

BACKGROUND: In the flowering plants, many polyploid species complexes display evolutionary radiation. This could be facilitated by gene flow between otherwise separate evolutionary lineages in contact zones. Achillea collina is a widespread tetraploid species within the Achillea millefolium polyploid complex (Asteraceae-Anthemideae). It is morphologically intermediate between the relic diploids, A. setacea-2x in xeric and A. asplenifolia-2x in humid habitats, and often grows in close contact with either of them. By analyzing DNA sequences of two single-copy nuclear genes and the genomic AFLP data, we assess the allopolyploid origin of A. collina-4x from ancestors corresponding to A. setacea-2x and A. asplenifolia-2x, and the ongoing backcross introgression between these diploid progenitor and tetraploid progeny lineages. RESULTS: In both the ncpGS and the PgiC gene tree, haplotype sequences of the diploid A. setacea-2x and A. asplenifolia-2x group into two clades corresponding to the two species, though lineage sorting seems incomplete for the PgiC gene. In contrast, A. collina-4x and its suspected backcross plants show homeologous gene copies: sequences from the same tetraploid individual plant are placed in both diploid clades. Semi-congruent splits of an AFLP Neighbor Net link not only A. collina-4x to both diploid species, but some 4x individuals in a polymorphic population with mixed ploidy levels to A. setacea-2x on one hand and to A. collina-4x on the other, indicating allopolyploid speciation as well as hybridization across ploidal levels. CONCLUSIONS: The findings of this study clearly demonstrate the hybrid origin of Achillea collina-4x, the ongoing backcrossing between the diploid progenitor and their tetraploid progeny lineages. Such repeated hybridizations are likely the cause of the great genetic and phenotypic variation and ecological differentiation of the polyploid taxa in Achillea millefolium agg.

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

[This corrects the article DOI: 10.1371/journal.pone.0207615.].

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.

Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic.

The plastid matK gene, trnL/F spacer, and nuclear rDNA ITS were sequenced for 36 species of Leontodon and 29 taxa of related genera of tribe Lactuceae. Phylogenetic relationships inferred from the independent and combined data are largely congruent and reveal that Leontodon sensu lato (s.l.) as presently defined is diphyletic: L. subgenus Leontodon forms a clade with Helminthotheca, Picris and Hypochaeris as sister genera, whereas L. subgenus Oporinia appears as a separate clade with strong bootstrap support and is thus better treated as a separate genus. Previous sectional classifications of Leontodon s.l. are considered in the light of DNA and additional morphological and karyological data. Support is presented for a core group of Hypochaeridinae sensu stricto (s.s.) with the two clades of Leontodon s.l., Helminthotheca, Picris, and Hypochaeris, whereas Urospermum, Hyoseris, Aposeris, and Rhagadiolus appear to be positioned more distantly.

AFLP analyses demonstrate genetic divergence, hybridization, and multiple polyploidization in the evolution of Achillea (Asteraceae-Anthemideae).

Achillea, a temperate genus of herbaceous allogamous perennials, is a model for evolutionary radiation through hybridization and polyploidization. AFLP analyses were performed on 300 individuals of 66 populations and 27 taxa/cytotypes, mainly from the polyploid A. millefolium aggregate and its suspected hybrid links with other clades of the genus. The mosaic genetic structure of hybrids and polyploids is revealed by specific AFLP bands shared with their assumed parents. In E Asia, A. alpina-4x and A. wilsoniana-4x are allotetraploids between A. acuminata-2x (sect. Ptarmica) and A. asiatica-2x (sect. Achillea-A. millefolium agg.). A. virescens-4x is a hybrid species linking A. nobilis agg. and A. millefolium agg. in S Europe. The hybrid swarm A. clypeolata-2x yen A. collina-4x recently formed in Bulgaria shows no AFLP bands additive to its parents; by contrast, other more ancient allopolyploids exhibit genetic innovations. Relationships within A. millefolium agg. are complex. Five 2x-taxa, mostly well separated and regressive, are limited to Eurasia; seven 4x- and 6x-taxa are intimately linked by hybridization, are expansive, and through A. asiatica-2x/4x have formed the N American polyploids. All these results from AFLPs correspond well to other evidence, and indicate a long history of reticulate evolution in Achillea.

Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry.

Flow cytometric measurements of nuclear DNA content were performed using ethidium bromide as the DNA stain (internal standard, Hordeum vulgare 'Ditta', 1C = 5.063 pg) in 25 samples belonging to nine diploid species and four varieties of Capsicum: C. chacoense, C. parvifolium, C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum var. baccatum, C. baccatum var. pendulum, C. baccatum var. umbilicatum, C. eximium and C. pubescens, all with 2n = 24, and C. campylopodium with 2n = 26. In addition, one sample each of C. annuum var. annuum and C. pubescens were also analysed using Feulgen densitometry (standard, Allium cepa 'Stuttgarter Riesen', 1C = 16.75 pg). Both staining methods resulted in very similar relative values. Genome size displays significant variation between but not within species (except in C. campylopodium), and contributes to their taxonomic grouping. 1C-values range from 3.34-3.43 pg (3273-3361 Mbp) in C. chacoense and the C. annuum complex to 4.53-5.77 pg (4439-5655 Mbp) in C. campylopodium and C. parvifolium. The data obtained support conclusions on phylogenetic relationships in the genus derived from karyotype analyses using chromosome banding approaches. In Capsicum, constitutive heterochromatin amount is correlated with genome size, except in C. parvifolium, and is regarded as an additive genomic component.