The enigmatic tropical alpine flora on the African sky islands is young, disturbed, and unsaturated.

Tropical alpine floras are renowned for high endemism, spectacular giant rosette plants testifying to convergent adaptation to harsh climates with nightly frosts, and recruitment dominated by long-distance dispersal from remote areas. In contrast to the larger, more recent (late Miocene onward) and contiguous expanses of tropical alpine habitat in South America, the tropical alpine flora in Africa is extremely fragmented across small patches on distant mountains of variable age (Oligocene onward). How this has affected the colonization and diversification history of the highly endemic but species-poor afroalpine flora is not well known. Here we infer phylogenetic relationships of ∼20% of its species using novel genome skimming data and published matrices and infer a timeframe for species origins in the afroalpine region using fossil-calibrated molecular clocks. Although some of the mountains are old, and although stem node ages may substantially predate colonization, most lineages appear to have colonized the afroalpine during the last 5 or 10 My. The accumulation of species increased exponentially toward the present. Taken together with recent reports of extremely low intrapopulation genetic diversity and recent intermountain population divergence, this points to a young, unsaturated, and dynamic island scenario. Habitat disturbance caused by the Pleistocene climate oscillations likely induced cycles of colonization, speciation, extinction, and recolonization. This study contributes to our understanding of differences in the histories of recruitment on different tropical sky islands and on oceanic islands, providing insight into the general processes shaping their remarkable floras.

Afro-alpine flagships revisited: Parallel adaptation, intermountain admixture and shallow genetic structuring in the giant senecios (Dendrosenecio).

Distantly related lineages of the enigmatic giant rosette plants of tropical alpine environments provide classical examples of convergent adaptation. For the giant senecios (Dendrosenecio), the endemic landmarks of the East African sky islands, it has also been suggested that parallel adaptation has been important for within-lineage differentiation. To test this hypothesis and to address potential gene flow and hybridization among the isolated sky islands, we organized field expeditions to all major mountains. We sampled all currently accepted species and all but one subspecies and genotyped 460 plants representing 109 populations. We tested whether genetic structuring corresponds to geography, as predicted by a parallel adaptation hypothesis, or to altitudinal belt and habitat rather than mountains, as predicted by a hypothesis of a single origin of adaptations. Bayesian and Neighbor-Net analyses showed that the main genetic structure is shallow and largely corresponds to geography, supporting a hypothesis of recent, rapid radiation via parallel altitude/habitat adaptation on different mountains. We also found evidence for intermountain admixture, suggesting several long-distance dispersals by wind across vast areas of unsuitable habitat. The combination of parallel adaptation, secondary contact, and hybridization may explain the complex patterns of morphological variation and the contradicting taxonomic treatments of these rare enigmatic giants, supporting the use of wide taxonomic concepts. Notably, the within-population genetic diversity was very low and calls for increased conservation efforts.

Sweet vernal grasses (Anthoxanthum) colonized African mountains along two fronts in the Late Pliocene, followed by secondary contact, polyploidization and local extinction in the Pleistocene.

High tropical mountains harbour remarkable and fragmented biodiversity thought to a large degree to have been shaped by multiple dispersals of cold-adapted lineages from remote areas. Few dated phylogenetic/phylogeographic analyses are however available. Here, we address the hypotheses that the sub-Saharan African sweet vernal grasses have a dual colonization history and that lineages of independent origins have established secondary contact. We carried out rangewide sampling across the eastern African high mountains, inferred dated phylogenies from nuclear ribosomal and plastid DNA using Bayesian methods, and performed flow cytometry and AFLP (amplified fragment length polymorphism) analyses. We inferred a single Late Pliocene western Eurasian origin of the eastern African taxa, whose high-ploid populations in one mountain group formed a distinct phylogeographic group and carried plastids that diverged from those of the currently allopatric southern African lineage in the Mid- to Late Pleistocene. We show that Anthoxanthum has an intriguing history in sub-Saharan Africa, including Late Pliocene colonization from southeast and north, followed by secondary contact, hybridization, allopolyploidization and local extinction during one of the last glacial cycles. Our results add to a growing body of evidence showing that isolated tropical high mountain habitats have a dynamic recent history involving niche conservatism and recruitment from remote sources, repeated dispersals, diversification, hybridization and local extinction.

DNA Metabarcoding Reveals Diet Overlap between the Endangered Walia Ibex and Domestic Goats - Implications for Conservation.

Human population expansion and associated degradation of the habitat of many wildlife species cause loss of biodiversity and species extinctions. The small Simen Mountains National Park in Ethiopia is one of the last strongholds for the preservation of a number of afro-alpine mammals, plants and birds, and it is home to the rare endemic Walia ibex, Capra walie. The narrow distribution range of this species as well as potential competition for resources with livestock, especially with domestic goat, Capra hircus, may compromise its future survival. Based on a curated afro-alpine taxonomic reference library constructed for plant taxon identification, we investigated the diet of the Walia ibex and addressed the dietary overlap with domestic goat using DNA metabarcoding of faecal samples. Faeces of both species were collected from different localities in the National Park. We show that both species are browsers, with forbs, shrubs and trees comprising the largest proportion of their diet, supplemented by grasses. There was a considerable overlap in dietary preferences. Several of the preferred diet items of the Walia ibex (Alchemilla sp., Hypericum revolutum, Erica arborea and Rumex sp.) were also among the most preferred diet items of the domestic goat. These results indicate that there is potential for competition between the two species, especially during the dry season, when resources are limited. Our findings, in combination with the expected increase in domestic herbivores, suggest that management plans should consider the potential threat posed by domestic goats to ensure future survival of the endangered Walia ibex.

Colonization and diversification in the African 'sky islands': insights from fossil-calibrated molecular dating of Lychnis (Caryophyllaceae).

The flora on the isolated high African mountains or 'sky islands' is remarkable for its peculiar adaptations, local endemism and striking biogeographical connections to remote parts of the world. Ages of the plant lineages and the timing of their radiations have frequently been debated but remain contentious as there are few estimates based on explicit models and fossil-calibrated molecular clocks. We used the plastid region maturaseK (matK) and a Caryophylloflora paleogenica fossil to infer the age of the genus Lychnis, and constructed a data set of three plastid (matK; a ribosomal protein S16 (rps16); and an intergenic spacer (psbE-petL)) and two nuclear (internal transcribed spacer (ITS) and a region spanning exon 18-24 in the second largest subunit of RNA polymerase II (RPB2)) loci for joint estimation of the species tree and divergence time of the African representatives. The time of divergence of the African high-altitude Lychnis was placed in the late Miocene to early Pliocene. A single speciation event was inferred in the early Pliocene; subsequent speciation took place sporadically from the late Pliocene to the middle Pleistocene. We provide further support for a Eurasian origin of the African 'sky islands' floral elements, which seem to have been recruited via dispersals at different times: some old, as in Lychnis, and others very recent. We show that dispersal and diversification within Africa play an important role in shaping these isolated plant communities.

RAD-seq data point to a northern origin of the arctic-alpine genus Cassiope (Ericaceae).

Many arctic-alpine plants display a highly disjunct distribution between the Arctic/Boreal regions and the southern Asian mountains. Two main hypotheses have been proposed to explain the origin of this biogeographic pattern: (1) south-to-north migration in the late Pliocene/early Pleistocene, and (2) north-to-south migration during the Miocene. The genus Cassiope is disjunctly distributed between the Arctic/Boreal regions and the Himalayan-Hengduan Mountains (HHM) and was selected to test these hypotheses. We constructed a fossil-calibrated phylogeny of Ericaceae using two plastid regions to estimate the crown group age of Cassiope, and used sequence data from thousands of loci produced by restriction site associated DNA sequencing (RAD-seq) to reconstruct the phylogeny of Cassiope. We also performed Bayesian divergence time analysis and biogeographic analysis. The Cassiope crown group was estimated to have originated in the Miocene, which predates the onset of Northern hemisphere glaciation. All HHM species formed a clade together with one eastern Siberian species, and this clade was sister to all other Arctic/Boreal species. This topology implies a northern origin of Cassiope, which is confirmed by our biogeographic analysis. Our results thus suggest that the ancient north-to-south migration hypothesis is most consistent with the origin of Cassiope.

Thousands of RAD-seq Loci Fully Resolve the Phylogeny of the Highly Disjunct Arctic-Alpine Genus Diapensia (Diapensiaceae).

Restriction-site associated DNA sequencing (RAD-seq) has recently become an important method to generate genome-wide molecular data for species delimitation, phylogeography, and population genetic studies. However, very few empirical studies have so far tested its applicability in phylogenetic reconstruction. The alpine-arctic genus Diapensia was selected to study the origin of the disjunction between the Arctic and the Himalayan-Hengduan Mountains (HHM). However, a previous phylogenetic analysis based on one nuclear and four plastid DNA regions failed to resolve the oldest divergences in Diapensia as well as the relationship between the two HHM species. Here we reconstruct a fully resolved phylogeny of Diapensia and address the conflict between the currently accepted taxonomy and the gene trees in the HHM species using RAD-seq. Based on a data set containing 2,650 loci selected to maximize the number of parsimony informative sites and allowing for a high level of missing data (51%), the phylogeny of Diapensia was fully resolved and each of the four species was reciprocally monophyletic. Whereas the arctic D. lapponica was inferred as sister to the HHM clade in the previous study, the RAD-seq data resolved the two arctic species as sisters to the HHM clade. Similar relationships were inferred from a differently filtered data set with far fewer loci (114) and less missing data (21%), but with lower support and with one of the two HHM species as non-monophyletic. Bayesian concordance analysis and Patterson's D-statistic tests suggested that admixture has occurred between the two HHM species.

The evolutionary history of Afrocanarian blue tits inferred from genomewide SNPs.

A common challenge in phylogenetic reconstruction is to find enough suitable genomic markers to reliably trace splitting events with short internodes. Here, we present phylogenetic analyses based on genomewide single-nucleotide polymorphisms (SNPs) of an enigmatic avian radiation, the subspecies complex of Afrocanarian blue tits (Cyanistes teneriffae). The two sister species, the Eurasian blue tit (Cyanistes caeruleus) and the azure tit (Cyanistes cyanus), constituted the out-group. We generated a large data set of SNPs for analysis of population structure and phylogeny. We also adapted our protocol to utilize degraded DNA from old museum skins from Libya. We found strong population structuring that largely confirmed subspecies monophyly and constructed a coalescent-based phylogeny with full support at all major nodes. The results are consistent with a recent hypothesis that La Palma and Libya are relic populations of an ancient Afrocanarian blue tit, although a small data set for Libya could not resolve its position relative to La Palma. The birds on the eastern islands of Fuerteventura and Lanzarote are similar to those in Morocco. Together they constitute the sister group to the clade containing the other Canary Islands (except La Palma), in which El Hierro is sister to the three central islands. Hence, extant Canary Islands populations seem to originate from multiple independent colonization events. We also found population divergences in a key reproductive trait, viz. sperm length, which may constitute reproductive barriers between certain populations. We recommend a taxonomic revision of this polytypic species, where several subspecies should qualify for species rank.

Use of ancient sedimentary DNA as a novel conservation tool for high-altitude tropical biodiversity.

Conservation of biodiversity may in the future increasingly depend upon the availability of scientific information to set suitable restoration targets. In traditional paleoecology, sediment-based pollen provides a means to define preanthropogenic impact conditions, but problems in establishing the exact provenance and ecologically meaningful levels of taxonomic resolution of the evidence are limiting. We explored the extent to which the use of sedimentary ancient DNA (sedaDNA) may complement pollen data in reconstructing past alpine environments in the tropics. We constructed a record of afro-alpine plants retrieved from DNA preserved in sediment cores from 2 volcanic crater sites in the Albertine Rift, eastern Africa. The record extended well beyond the onset of substantial anthropogenic effects on tropical mountains. To ensure high-quality taxonomic inference from the sedaDNA sequences, we built an extensive DNA reference library covering the majority of the afro-alpine flora, by sequencing DNA from taxonomically verified specimens. Comparisons with pollen records from the same sediment cores showed that plant diversity recovered with sedaDNA improved vegetation reconstructions based on pollen records by revealing both additional taxa and providing increased taxonomic resolution. Furthermore, combining the 2 measures assisted in distinguishing vegetation change at different geographic scales; sedaDNA almost exclusively reflects local vegetation, whereas pollen can potentially originate from a wide area that in highlands in particular can span several ecozones. Our results suggest that sedaDNA may provide information on restoration targets and the nature and magnitude of human-induced environmental changes, including in high conservation priority, biodiversity hotspots, where understanding of preanthropogenic impact (or reference) conditions is highly limited.

Hybridization and long-distance colonization at different time scales: towards resolution of long-term controversies in the sweet vernal grasses (Anthoxanthum).

BACKGROUND AND AIMS: Repeated hybridization and/or polyploidization confound classification and phylogenetic inference, and multiple colonizations at different time scales complicate biogeographical reconstructions. This study investigates whether such processes can explain long-term controversies in Anthoxanthum, and in particular its debated relationship to the genus Hierochloë, the evolution of its conspicuously diverse floral morphology, and the origins of its strikingly disjunct occurrences. A hypothesis for recurrent polyploid formation is proposed. METHODS: Three plastid (trnH-psbA, trnT-L and trnL-F) and two nuclear (ITS, ETS) DNA regions were sequenced in 57 accessions of 17 taxa (including 161 ETS clones) and Bayesian phylogenetic analyses were conducted. Divergence times were inferred in *BEAST using a strict molecular clock. KEY RESULTS: Anthoxanthum was inferred as monophyletic and sister to one species of Hierochloë based on the plastid data, whereas the nuclear data suggested that one section (Anthoxanthum section Anthoxanthum) is sister to a clade including the other section (Anthoxanthum section Ataxia) as sister to the genus Hierochloë. This could explain the variation in floral morphology; the aberrant characters in Ataxia seem to result from a Miocene hybridization event between one lineage with one fertile and two sterile florets (the Anthoxanthum lineage) and one which probably had three fertile florets as in extant Hierochloë. The distinct diploid A. gracile lineage originated in the Miocene; all other speciation events, many of them involving polyploidy, were dated to the Late Pliocene to Late Pleistocene. Africa was apparently colonized twice in the Late Pliocene (from the north to afro-alpine eastern Africa, and from south-east Asia to southern Africa), whereas Macaronesia was colonized much later (Late Pleistocene) by a diploid Mediterranean lineage. The widespread European tetraploid A. odoratum originated at least twice. CONCLUSIONS: Many of the controversies in Anthoxanthum can be explained by recurring hybridization and/or polyploidization on time scales ranging from the Miocene to the Late Pleistocene. All but one of the extant species shared most recent common ancestors in the Late Pliocene to the Late Pleistocene. The disjunct occurrences in Africa originated in the Late Pliocene via independent immigrations, whereas Macaronesia was colonized in the Late Pleistocene.

A single Mid-Pleistocene long-distance dispersal by a bird can explain the extreme bipolar disjunction in crowberries (Empetrum).

The proposed age of the striking biogeographic disjunction between the Arctic and southernmost South America varies from more than 65 million to a few thousand years, but no estimates based on explicit models and molecular data are available. Here we address the origin of bipolarity in crowberries (Empetrum), which are heath-forming dwarf shrubs with animal-dispersed fruits. We apply a fossil-calibrated relaxed molecular clock to model sequence evolution in two nuclear low-copy and two plastid DNA regions from 41 individual plants (420 clones for the nuclear regions) representing the entire geographic distribution of crowberries. The plastid region matK and four fossil calibration points were used to infer the ages of the crowberry stem and crown groups. All analyses resolved three major crowberry clades (A-C). Clade A contained sequences from the eastern Canadian pink-fruited crowberry (E. eamesii) as sister to clades B and C, which both contained sequences from the black-fruited northern hemisphere crowberry (E. nigrum). Clade B also contained a subclade with all sequences from the red-fruited southern hemisphere crowberry, which is often referred to as a distinct species, E. rubrum. Its closest relatives were consistently identified as black-fruited plants from northwestern North America. The median time to the most recent common ancestor for northern and southern hemisphere crowberries was estimated to 0.56-0.93 Ma, and 0.26-0.59 Ma for the southern plants only. We conclude that a single dispersal by a bird from northwestern North America to southernmost South America, taking place in the Mid-Pleistocene, is sufficient to explain the disjunction in crowberries.

Glacial survival may matter after all: nunatak signatures in the rare European populations of two west-arctic species.

Biogeographers claimed for more than a century that arctic plants survived glaciations in ice-free refugia within the limits of the North European ice sheets. Molecular studies have, however, provided overwhelming support for postglacial immigration into northern Europe, even from the west across the Atlantic. For the first time we can here present molecular evidence strongly favouring in situ glacial persistence of two species, the rare arctic-alpine pioneer species Sagina caespitosa and Arenaria humifusa. Both belong to the 'west-arctic element' of amphi-Atlantic disjuncts, having their few and only European occurrences well within the limits of the last glaciation. Sequencing of non-coding regions of chloroplast DNA revealed only limited variation. However, two very distinct and partly diverse genetic groups, one East and one West Atlantic, were detected in each species based on amplified fragment length polymorphisms (AFLPs), excluding postglacial dispersal from North America as explanation for their European occurrences. Patterns of genetic diversity and distinctiveness indicate that glacial populations existed in East Greenland and/or Svalbard (A. humifusa) and in southern Scandinavia (S. caespitosa). Despite their presumed lack of long-distance dispersal adaptations, intermixed populations in several regions indicate postglacial contact zones. Both species are declining in Nordic countries, probably due to climate change-induced habitat loss. Little or no current connectivity between their highly fragmented and partly distinct populations call for conservation of several populations in each geographic region.

Molecular phylogeny and biogeography of the bipolar Euphrasia (Orobanchaceae): recent radiations in an old genus.

Euphrasia includes perennial or annual green parasitic plants, and has a disjunct bipolar distribution except for one transtropical connection across the high mountains of Oceania. The disjunction is coupled with strikingly contrasting patterns of morphological diversity between the southern and northern hemispheres, making it an exciting model to study processes of evolutionary diversification which shaped present floras. We inferred the relationships among 51 species representing 14 of the 15 sections of the genus based on nrDNA ITS and cpDNA trnL intron, trnL-trnF and atpB-rbcL intergenic spacers. Maximum parsimony and Bayesian inference support monophyly of the genus and of several intrageneric groups characterized by morphology, ploidy level, and geographic range. Molecular phylogenetic dating using Bayesian "relaxed" clock methods suggests that the earliest Euphrasia radiations occurred minimum 11-8 Mya with bipolarity being achieved 7-5 Mya. Biogeographic analyses using explicit model-based approach inferred Eurasia as an ancestral area for the genus. The most parsimonious reconstruction found by a dispersal-vicariance analysis requires 17 dispersals to account for the current biogeographic pattern and supports Eurasian origin for Euphrasia. Both long-distance dispersal and across land vicariance can be invoked to explain the diversification in the genus, which experienced rapid radiations driven by new ecological opportunities of the late Pliocene and Pleistocene but also retained a set of local endemic or relict species of an earlier origin.

Circumpolar phylogeography of Juncus biglumis (Juncaceae) inferred from AFLP fingerprints, cpDNA sequences, nuclear DNA content and chromosome numbers.

We explored the circumpolar phylogeographic history of the arctic-alpine Juncus biglumis using amplified fragment length polymorphisms (AFLPs), sequences of cpDNA, relative nuclear DNA content and chromosome numbers. The analyses of the AFLP and cpDNA data gave congruent results and revealed three distinct clades. One of them, represented by a single population from the Taymyr peninsula in northern Siberia, had approximately fourfold larger genome size than the other samples and produced an AFLP pattern that was too aberrant to be analysed together with the rest of the data set. The two other clades represented different ploidy levels (2n = 60 and 120) as judged from chromosome counts of selected populations but differed only in c. 6% relative DNA content. Based on the AFLP and partly also on the cpDNA data, each of the two main clades was further subdivided into two well-supported subgroups. Three of the subgroups were widespread and exhibited largely overlapping distribution patterns. The fourth subgroup seems to be absent from the North Atlantic region and from western Siberia. We suggest that the four subgroups diverged during isolation in different glacial refugia during the Quaternary. Interestingly, individuals of both main clades were encountered in geographically close populations in eastern Greenland and even within a single population from Svalbard, indicating that both areas were colonised at least twice. The different genome sizes and ploidy levels strongly suggest that the three main clades represent distinct gene pools and act as cryptic species.

Origin and evolution of North American polyploid Silene (Caryophyllaceae).

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.

The origin and number of introductions of the Hawaiian endemic Silene species (Caryophyllaceae).

The Hawaiian endemic Silene are a small group of woody or semiwoody representatives from a large, predominantly herbaceous, species-rich genus. We here investigated the origin and number of introductions of the endemic Hawaiian Silene based on phylogenetic relationships inferred from DNA sequences from both the plastid (the rps16 intron) and the nuclear (ribosomal internal transcribed sequences, ITS, and intron 23 of the RPB2 gene) genomes. Silene antirrhina, a widespread weedy American annual, is strongly supported as sister to a monophyletic group consisting of the Hawaiian Silene, indicating a single colonization event. There are no obvious morphological similarities between S. antirrhina and any of the species of Hawaiian Silene. Our results suggest an American origin for the Hawaiian endemics because that would require only a single trans-ocean dispersal. Two of the Hawaiian endemics (S. struthioloides and S. hawaiiensis) that form a subclade in the analyses have evolved woodiness after introduction to the Hawaiian Islands. Our results contribute to other recent results based on molecular phylogenetics that emphasize the American continent as a source area for the Hawaiian flora and support a striking morphological radiation and evolution of woodiness from a single introduction to the archipelago.

Polyploid origins in a circumpolar complex in Draba (Brassicaceae) inferred from cloned nuclear DNA sequences and fingerprints.

Polyploid evolution has been of major importance in the arctic flora, but rarely addressed on the full circumpolar scale. Herein we study the allopolyploid Draba lactea and its close allies, which form a taxonomically intricate arctic-alpine complex including diploids, tetraploids, and hexaploids. Based on samples from the entire circumpolar area, we inferred the origins of polyploids in this complex using cloned DNA sequences from two nuclear regions (one intron from a gene encoding a second largest subunit in the RNA polymerase family, RPD2, and the ribosomal internal transcribed spacer region, ITS) and DNA fingerprints (random amplified polymorphic DNAs, RAPDs). Although D. lactea and all other polyploids examined in Draba are genetic alloploids showing fixed heterozygosity, the data obtained in the present study suggest that each of the polyploids analyzed here may have originated from a single diploid lineage: hexaploid D. lactea via tetraploid D. lactea from the D. palanderiana lineage (not from the D. fladnizensis and D. nivalis lineages as previously hypothesized), the tetraploid D. turczaninovii from the D. fladnizensis lineage, the tetraploid D. porsildii from the D. lonchocarpa lineage, and a tetraploid here named Draba spB from the D. nivalis lineage. Draba lactea has probably originated several times in the Beringian area, and it is not necessary to invoke complex origins based on a combination of different species lineages as previously suggested.

Evolution of a RNA polymerase gene family in Silene (Caryophyllaceae)-incomplete concerted evolution and topological congruence among paralogues.

Four low-copy nuclear DNA intron regions from the second largest subunits of the RNA polymerase gene family (RPA2, RPB2, RPD2a, and RPD2b), the internal transcribed spacers (ITSs) from the nuclear ribosomal regions, and the rps16 intron from the chloroplast were sequenced and used in a phylogenetic analysis of 29 species from the tribe Sileneae (Caryophyllaceae). We used a low stringency nested polymerase chain reaction (PCR) approach to overcome the difficulties of constructing specific primers for amplification of the low copy nuclear DNA regions. Maximum parsimony analyses resulted in largely congruent phylogenetic trees for all regions. We tested overall model congruence in a likelihood context using the software PLATO and found that ITSs, RPA2, and RPB2 deviated from the maximum likelihood model for the combined data. The topology parameter was then isolated and topological congruence assessed by nonparametric bootstrapping. No strong topological incongruence was found. The analysis of the combined data sets resolves previously poorly known major relationships within Sileneae. Two paralogues of RPD2 were found, and several independent losses and incomplete concerted evolution were inferred. The among-site rate variation was significantly lower in the RNA polymerase introns than in the rps16 intron and ITSs, a property that is attractive in phylogenetic analyses.