The genomic basis of adaptation to calcareous and siliceous soils in Arabidopsis lyrata.

Edaphic conditions are important determinants of plant fitness. While much has been learnt in recent years about plant adaptation to heavy metal contaminated soils, the genomic basis underlying adaptation to calcareous and siliceous substrates remains largely unknown. We performed a reciprocal germination experiment and whole-genome resequencing in natural calcareous and siliceous populations of diploid Arabidopsis lyrata to test for edaphic adaptation and detect signatures of selection at loci associated with soil-mediated divergence. In parallel, genome scans on respective diploid ecotypes from the Arabidopsis arenosa species complex were undertaken, to search for shared patterns of adaptive genetic divergence. Soil ecotypes of A. lyrata display significant genotype-by-treatment responses for seed germination. Sequence (SNPs) and copy-number variants (CNVs) point towards loci involved in ion transport as the main targets of adaptive genetic divergence. Two genes exhibiting high differentiation among soil types in A. lyrata further share trans-specific single nucleotide polymorphisms with A. arenosa. This work applies experimental and genomic approaches to study edaphic adaptation in A. lyrata and suggests that physiological response to elemental toxicity and deficiency underlies the evolution of calcareous and siliceous ecotypes. The discovery of shared adaptive variation between sister species indicates that ancient polymorphisms contribute to adaptive evolution.

Genomic Resources Notes accepted 1 April 2015 - 31 May 2015.

This article documents the public availability of transcriptomic resources for (i) the stellate sturgeon Acipenser stellatus, (ii) the flowering plant Campanula gentilis and (iii) two endemic Iberian fish, Squalius carolitertii and Squalius torgalensis.

Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean.

At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin - a hotspot of biodiversity - provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low-copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid-Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.

Phylogeny of Campanuloideae (Campanulaceae) with emphasis on the utility of nuclear pentatricopeptide repeat (PPR) genes.

BACKGROUND: The Campanuloideae (Campanulaceae) are a highly diverse clade of angiosperms found mostly in the Northern Hemisphere, with the highest diversity in temperate areas of the Old World. Chloroplast markers have greatly improved our understanding of this clade but many relationships remain unclear primarily due to low levels of molecular evolution and recent and rapid divergence. Furthermore, focusing solely on maternally inherited markers such as those from the chloroplast genome may obscure processes such as hybridization. In this study we explore the phylogenetic utility of two low-copy nuclear loci from the pentatricopeptide repeat gene family (PPR). Rapidly evolving nuclear loci may provide increased phylogenetic resolution in clades containing recently diverged or closely related taxa. We present results based on both chloroplast and low-copy nuclear loci and discuss the utility of such markers to resolve evolutionary relationships and infer hybridization events within the Campanuloideae clade. RESULTS: The inclusion of low-copy nuclear genes into the analyses provides increased phylogenetic resolution in two species-rich clades containing recently diverged taxa. We also obtain support for the placement of two early diverging lineages (Jasione and Musschia-Gadellia clades) that have previously been unresolved. Furthermore, phylogenetic analyses of PPR loci revealed potential hybridization events for a number of taxa (e.g., Campanula pelviformis and Legousia species). These loci offer greater overall topological support than obtained with plastid DNA alone. CONCLUSION: This study represents the first inclusion of low-copy nuclear genes for phylogenetic reconstruction in Campanuloideae. The two PPR loci were easy to sequence, required no cloning, and the sequence alignments were straightforward across the entire Campanuloideae clade. Although potentially complicated by incomplete lineage sorting, these markers proved useful for understanding the processes of reticulate evolution and resolving relationships at a wide range of phylogenetic levels. Our results stress the importance of including multiple, independent loci in phylogenetic analyses.

How to handle speciose clades? Mass taxon-sampling as a strategy towards illuminating the natural history of Campanula (Campanuloideae).

BACKGROUND: Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions. METHODOLOGY/PRINCIPAL FINDINGS: Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from "classification-guided" (D088) and "phylogeny-guided sampling" (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates. CONCLUSIONS/SIGNIFICANCE: A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.

Genetic diversity in widespread species is not congruent with species richness in alpine plant communities.

The Convention on Biological Diversity (CBD) aims at the conservation of all three levels of biodiversity, that is, ecosystems, species and genes. Genetic diversity represents evolutionary potential and is important for ecosystem functioning. Unfortunately, genetic diversity in natural populations is hardly considered in conservation strategies because it is difficult to measure and has been hypothesised to co-vary with species richness. This means that species richness is taken as a surrogate of genetic diversity in conservation planning, though their relationship has not been properly evaluated. We tested whether the genetic and species levels of biodiversity co-vary, using a large-scale and multi-species approach. We chose the high-mountain flora of the Alps and the Carpathians as study systems and demonstrate that species richness and genetic diversity are not correlated. Species richness thus cannot act as a surrogate for genetic diversity. Our results have important consequences for implementing the CBD when designing conservation strategies.

Towards a species level tree of the globally diverse genus Chenopodium (Chenopodiaceae).

Chenopodium is a large and morphologically variable genus of annual and perennial herbs with an almost global distribution. All subgenera and most sections of Chenopodium were sampled along with other genera of Chenopodieae, Atripliceae and Axyrideae across the subfamily Chenopodioideae (Chenopodiaceae), totalling to 140 taxa. Using Maximum parsimony and Bayesian analyses of the non-coding trnL-F (cpDNA) and nuclear ITS regions, we provide a comprehensive picture of relationships of Chenopodium sensu lato. The genus as broadly classified is highly paraphyletic within Chenopodioideae, consisting of five major clades. Compared to previous studies, the tribe Dysphanieae with three genera Dysphania, Teloxys and Suckleya (comprising the aromatic species of Chenopodium s.l.) is now shown to form one of the early branches in the tree of Chenopodioideae. We further recognize the tribe Spinacieae to include Spinacia, several species of Chenopodium, and the genera Monolepis and Scleroblitum. The Chenopodium rubrum and the Ch. murale-clades were newly discovered as distinct major lineages but their relationships within Chenopodioideae will need further evaluation. Based on our results, we suggest the delimitation of Chenopodium to include Einadia and Rhagodia because these are part of the crown group composed of species of subg. Chenopodium that appear sister to the Atripliceae. The tetraploid crops such as Ch. berlandieri subsp. nuttalliae and Ch. quinoa also belong to Chenopodium sensu stricto. Trees derived from trnL-F and ITS were incongruent within this shallow crown group clade. Possible biological causes are discussed, including allopolyploidization.

The polyphyletic genus Sebaea (Gentianaceae): a step forward in understanding the morphological and karyological evolution of the Exaceae.

Within the Gentianaceae-Exaceae, the most species-rich genus Sebaea has received very little attention in terms of phylogenetic or karyological investigations. As a result, the exact number of species remains vague and the relationships with the other members of the Exaceae poorly understood. In this paper, we provide the first comprehensive phylogeny of the Exaceae including most Sebaea species known so far based on four cpDNA sequence regions. In addition, morphological and karyological characters were mapped on the inferred phylogenetic trees to detect possible non-molecular synapomorphies. Our results reveal the paraphyly of Sebaea and highlight new generic relationships within the Exaceae. Sebaea pusilla (lineage S1--Lagenias) forms a highly supported and early diverging clade with Sebaeas.str. (clade S2 -Sebaea). A third clade of the former Sebaea s.l. (clade S3--Exochaenium) contains exclusively tropical African species, and is sister with a large clade containing all the remaining genera of Exaceae. Within the latter, the proposed sister relationships between the recently described Klackenbergia and Ornichia are highly supported. Optimization of several morphological characters onto the inferred phylogenetic trees reveals several synapomorphies for most highly supported clades. In particular, lineage S1 (Lagenias) is supported by medifixed anthers that are inserted at the base of the corolla tube and cubical seeds with polygonal testa cells; clade S2 (Sebaea) is supported by both the presence of secondary stigmas along the style and ridged seeds with rectangular testa cells arranged in row; clade S3 (Exochaenium) is supported by its particular gynoecium (stylar polymorphism and clavate, papillose stigma). Finally, karyological reconstructions suggest a basal number of x=7 for the Exaceae and several episodes of dysploidy leading to x=8 and 9.

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

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

Disentangling reticulate evolution in an arctic-alpine polyploid complex.

Although polyploidy plays a fundamental role in plant evolution, the elucidation of polyploid origins is fraught with methodological challenges. For example, allopolyploid species may confound phylogenetic reconstruction because commonly used methods are designed to trace divergent, rather than reticulate patterns. Recently developed techniques of phylogenetic network estimation allow for a more effective identification of incongruence among trees. However, incongruence can also be caused by incomplete lineage sorting, paralogy, concerted evolution, and recombination. Thus, initial hypotheses of hybridization need to be examined via additional sources of evidence, including the partitioning of infraspecific genetic polymorphisms, morphological characteristics, chromosome numbers, crossing experiments, and distributional patterns. Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) represents an ideal case study to examine reticulation because specific hypotheses have been derived from morphology, karyology, interfertility, and distribution to explain the observed variation of ploidy levels, ranging from diploidy to 14-ploidy. Sequences from 5 chloroplast and 1 nuclear ribosomal DNA (nrDNA) markers were analyzed to generate the respective phylogenies and consensus networks. Furthermore, extensive cloning of the nrDNA marker allowed for the identification of shared nucleotides at polymorphic sites, investigation of infraspecific genetic polymorphisms via principal coordinate analyses PCoAs, and detection of recombination between putative progenitor sequences. The results suggest that most surveyed polyploids originated via hybridization and that 2 taxonomic species formed recurrently from different progenitors, findings that are congruent with the expectations of speciation via secondary contact. Overall, the study highlights the importance of using multiple experimental and analytical approaches to disentangle complex patterns of reticulation.

Phylogenetic analysis informed by geological history supports multiple, sequential invasions of the Mediterranean Basin by the angiosperm family Araceae.

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin. We focused on the origin of four species, Ambrosina bassii, Biarum dispar, Helicodiceros muscivorus, Arum pictum, subendemic or endemic to Corsica, Sardinia, and the Balearic Archipelago. The results support two main invasions of the Mediterranean Basin by the Araceae, one from an area connecting North America and Eurasia in the Late Cretaceous and one from the Anatolian microplate in western Asia during the Late Eocene, thus confirming the proposed heterogeneous origins of the Mediterranean flora. The subendemic Ambrosina bassii and Biarum dispar likely diverged sympatrically from their widespread Mediterranean sister clades in the Early-Middle Eocene and Early-Middle Miocene, respectively. Combined evidence corroborates a relictual origin for the endemic Helicodiceros muscivorus and Arum pictum, the former apparently representing the first documented case of vicariance driven by the initial splitting of the Hercynian belt in the Early Oligocene. A recurrent theme emerging from our analyses is that land connections and interruptions, caused by repeated cycles of marine transgressions-regressions between the Tethys and Paratethys, favored geodispersalist expansion of biotic ranges from western Asia into the western Mediterranean Basin and subsequent allopatric speciation at different points in time from the Late Eocene to the Late Oligocene.

Evolution of biogeographic patterns, ploidy levels, and breeding systems in a diploid-polyploid species complex of Primula.

Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) includes diploid, self-incompatible heterostyles and polyploid, self-compatible homostyles, the latter generally occurring at higher latitudes than the former. This study develops a phylogenetic hypothesis for Aleuritia to elucidate the interactions between Pleistocene glacial cycles, biogeographic patterns, ploidy levels and breeding systems. Sequences from five chloroplast DNA loci were analyzed with parsimony to reconstruct a phylogeny, haplotype network, and ancestral states for ploidy levels and breeding systems.The results supported the monophyly of Aleuritia and four major biogeographic lineages: an amphi-Pacific, a South American, an amphi-Atlantic and a European/North American lineage. At least four independent switches to homostyly and five to polyploidy were inferred. An Asian ancestor probably gave origin to an amphi-Pacific clade and to a lineage that diversified on the European and American continents. Switches to homostyly occurred exclusively in polyploid lineages, which mainly occupy previously glaciated areas. The higher success of the autogamous polyploid species at recolonizing habitats freed by glacial retreat might be explained in terms of selection for reproductive assurance.

Generic delimitation and phylogenetic relationships within the subtribe Chironiinae (Chironieae: Gentianaceae), with special reference to Centaurium: evidence from nrDNA and cpDNA sequences.

To better understand the evolutionary history of the genus Centaurium and its relationship to other genera of the subtribe Chironiinae (Gentianaceae: Chironieae), molecular analyses were performed using 80 nuclear ribosomal ITS and 76 chloroplast trnLF (both the trnL UAA intron and the trnL-F spacer) sequences. In addition, morphological, palynological, and phytochemical characters were included to a combined data matrix to detect possible non-molecular synapomorphies. Phylogenetic reconstructions support the monophyly of the Chironiinae and an age estimate of ca. 22 million years for the subtribe. Conversely, both molecular data sets reveal a polyphyletic Centaurium, with four well-supported main clades hereafter treated as separate genera. The primarily Mediterranean Centaurium s.s. is closely related to southern African endemics Chironia and Orphium, and to the Chilean species Centaurium cachanlahuen. The resurrected Mexican and Central American genus Gyrandra is closely related to Sabatia (from eastern North America). Lastly, the monospecific genus Exaculum (Mediterranean) forms a monophyletic group together with the two new genera: Schenkia (Mediterranean and Australian species) and Zeltnera (all other indigenous American centauries). Several biogeographical patterns can be inferred for this group, supporting a Mediterranean origin followed by dispersals to (1) North America, Central America, and South America, (2) southern Africa (including the Cape region), and (3) Australia and Pacific Islands.

Phylogenetic relationships within the New World endemic Zeltnera (Gentianaceae-Chironiinae) inferred from molecular and karyological data.

The New World endemic genus Zeltnera consists of 25 species mainly distributed in the western part of the United States and Mexico. Chromosome counts performed on 113 populations (24 species) reveal extensive congruence between chromosomal groups and the assemblages obtained from analyses of nuclear ribosomal DNA (ITS) and chloroplast DNA (trnL intron and trnL-F intergenic spacer) sequences. Karyological and molecular data sets support three main biogeographic groups for Zeltnera. A first and mainly unresolved cluster (n = 17 and n = 20) occurs in California, whereas two other clades are centered in the Texas region (n = 20 and n = 21) and in Mexico (n = 21 and n = 22). Under the assumption of a molecular clock, and using both dispersal and vicariance explanations for the current distribution of the respective species, the genus is thought to have a North American origin with considerable diversification in the early Pliocene (ca. 5 million years ago). Geological events, such as desert formation and mountain orogenies, have created insuperable barriers that today separate the three major and likely vicariant groups.

Monophyly and relationships of the tribe Exaceae (Gentianaceae) inferred from nuclear ribosomal and chloroplast DNA sequences.

Both chloroplast trnL (UAA) intron and nuclear ribosomal ITS sequences highly confirmed the monophyly of the tribes of the Gentianaceae defined by the recent classification, and revealed the tribe Exaceae as a basal clade just next to the basal-most lineage, the tribe Saccifolieae. Within the tribe Exaceae, Sebaea (except Sebaea madagascariensis) appeared as the most basal clade as the sister group to the rest of the tribe. The Madagascan endemic genera Gentianothamnus and Tachiadenus were very closely related to each other, together standing as sister to a clade comprising Sebaea madagascariensis, Ornichia, and Exacum. The saprophytic genus Cotylanthera nested deeply inside Exacum. Sebaea madagascariensis was shown closer to the Madagascan endemic genus Ornichia than to any other sampled Sebaea species. Exacum appeared as the most derived taxon within this tribe. The topology of the phylogenetic trees conform with the Gondwana vicariance hypothesis regarding the biogeography of Exaceae. However, no evidence for matching the older relationships within the family to the tectonic history could be corroborated with various divergence time analyses. Divergence dating estimated a post-Gondwana diverging of the Gentianaceae about 50 million years ago (MYA), and the tribe Exaceae as about 40 MYA. The Mozambique Channel land-bridge could have played an important role in the biogeographic history of the tribe Exaceae.