CAM evolution is associated with gene family expansion in an explosive bromeliad radiation.

The subgenus Tillandsia (Bromeliaceae) belongs to one of the fastest radiating clades in the plant kingdom and is characterised by the repeated evolution of Crassulacean acid metabolism (CAM). Despite its complex genetic basis, this water-conserving trait has evolved independently across many plant families and is regarded as a key innovation trait and driver of ecological diversification in Bromeliaceae. By producing high-quality genome assemblies of a Tillandsia species pair displaying divergent photosynthetic phenotypes, and combining genome-wide investigations of synteny, transposable element (TE) dynamics, sequence evolution, gene family evolution and temporal differential expression, we were able to pinpoint the genomic drivers of CAM evolution in Tillandsia. Several large-scale rearrangements associated with karyotype changes between the two genomes and a highly dynamic TE landscape shaped the genomes of Tillandsia. However, our analyses show that rewiring of photosynthetic metabolism is mainly obtained through regulatory evolution rather than coding sequence evolution, as CAM-related genes are differentially expressed across a 24-hour cycle between the two species but are not candidates of positive selection. Gene orthology analyses reveal that CAM-related gene families manifesting differential expression underwent accelerated gene family expansion in the constitutive CAM species, further supporting the view of gene family evolution as a driver of CAM evolution.

Homoplastic versus xenoplastic evolution: exploring the emergence of key intrinsic and extrinsic traits in the montane genus Soldanella (Primulaceae).

Specific ecological conditions in the high mountain environment exert a selective pressure that often leads to convergent trait evolution. Reticulations induced by incomplete lineage sorting and introgression can lead to discordant trait patterns among gene and species trees (hemiplasy/xenoplasy), providing a false illusion that the traits under study are homoplastic. Using phylogenetic species networks, we explored the effect of gene exchange on trait evolution in Soldanella, a genus profoundly influenced by historical introgression. At least three features evolved independently multiple times: the single-flowered dwarf phenotype, dysploid cytotype, and ecological generalism. The present analyses also indicated that the recurring occurrence of stoloniferous growth might have been prompted by an introgression event between an ancestral lineage and a still extant species, although its emergence via convergent evolution cannot be completely ruled out. Phylogenetic regression suggested that the independent evolution of larger genomes in snowbells is most likely a result of the interplay between hybridization events of dysploid and euploid taxa and hostile environments at the range margins of the genus. The emergence of key intrinsic and extrinsic traits in snowbells has been significantly impacted not only by convergent evolution but also by historical and recent introgression events.

Ice holes microrefugia harbor genetically and functionally distinct populations of Vaccinium vitis-idaea (Ericaceae).

In the mountain terrain, ice holes are little depressions between rock boulders that are characterized by the exit of cold air able to cool down the rock surface even in summer. This cold air creates cold microrefugia in warmer surroundings that preserve plant species probably over thousands of years under extra-zonal climatic conditions. We hypothesized that ice hole populations of the model species Vaccinium vitis-idaea (Ericaceae) show genetic differentiation from nearby zonal subalpine populations, and high functional trait distinctiveness, in agreement with genetic patterns. We genotyped almost 30,000 single nucleotide polymorphisms using restriction site-associated DNA sequencing and measured eight functional traits indicative of individual performance and ecological strategies. Genetic results showed high differentiation among the six populations suggesting isolation. On siliceous bedrock, ice hole individuals exhibited higher levels of admixture than those from subalpine populations which could have experienced more bottlenecks during demographic fluctuations related to glacial cycles. Ice hole and subalpine calcareous populations clearly separated from siliceous populations, indicating a possible effect of bedrock in shaping genetic patterns. Trait analysis reflected the bedrock effect on populations' differentiation. The significant correlation between trait and genetic distances suggests the genetic contribution in shaping intraspecific functional differentiation. In conclusion, extra-zonal populations reveal a prominent genetic and phenotypic differentiation determined by history and ecological contingency. Therefore, microrefugia populations can contribute to the overall variability of the species and lead to intraspecific-driven responses to upcoming environmental changes.

Recurrent allopolyploidizations diversify ecophysiological traits in marsh orchids (Dactylorhiza majalis s.l.).

Whole-genome duplication has shaped the evolution of angiosperms and other organisms, and is important for many crops. Structural reorganization of chromosomes and repatterning of gene expression are frequently observed in allopolyploids, with physiological and ecological consequences. Recurrent origins from different parental populations are widespread among polyploids, resulting in an array of lineages that provide excellent models to uncover mechanisms of adaptation to divergent environments in early phases of polyploid evolution. We integrate here transcriptomic and ecophysiological comparative studies to show that sibling allopolyploid marsh orchid species (Dactylorhiza, Orchidaceae) occur in different habitats (low nutrient fens vs. meadows with mesic soils) and are characterized by a complex suite of intertwined, pronounced ecophysiological differences between them. We uncover distinct features in leaf elemental chemistry, light-harvesting, photoprotection, nutrient transport and stomata activity of the two sibling allopolyploids, which appear to match their specific ecologies, in particular soil chemistry differences at their native sites. We argue that the phenotypic divergence between the sibling allopolyploids has a clear genetic basis, generating ecological barriers that maintain distinct, independent lineages, despite pervasive interspecific gene flow. This suggests that recurrent origins of polyploids bring about a long-term potential to trigger and maintain functional and ecological diversity in marsh orchids and other groups.

Parallel adaptation to lower altitudes is associated with enhanced plasticity in Heliosperma pusillum (Caryophyllaceae).

High levels of phenotypic plasticity are thought to be inherently costly in stable or extreme environments, but enhanced plasticity may evolve as a response to new environments and foster novel phenotypes. Heliosperma pusillum forms glabrous alpine and pubescent montane ecotypes that diverged recurrently and polytopically (parallel evolution) and can serve as evolutionary replicates. The specific alpine and montane localities are characterized by distinct temperature conditions, available moisture, and light. Noteworthy, the ecotypes show a home-site fitness advantage in reciprocal transplantations. To disentangle the relative contribution of constitutive versus plastic gene expression to altitudinal divergence, we analyze the transcriptomic profiles of two parallely evolved ecotype pairs, grown in reciprocal transplantations at native altitudinal sites. In this incipient stage of divergence, only a minor proportion of genes appear constitutively differentially expressed between the ecotypes in both pairs, regardless of the growing environment. Both derived, montane populations bear comparatively higher plasticity of gene expression than the alpine populations. Genes that change expression plastically or constitutively underlie similar ecologically relevant pathways, related to response to drought and trichome formation. Other relevant processes, such as photosynthesis, rely mainly on plastic changes. The enhanced plasticity consistently observed in the montane ecotype likely evolved as a response to the newly colonized, drier, and warmer niche. We report a striking parallelism of directional changes in gene expression plasticity. Thus, plasticity appears to be a key mechanism shaping the initial stages of phenotypic evolution, likely fostering adaptation to novel environments.

Drivers of genomic landscapes of differentiation across a Populus divergence gradient.

Speciation, the continuous process by which new species form, is often investigated by looking at the variation of nucleotide diversity and differentiation across the genome (hereafter genomic landscapes). A key challenge lies in how to determine the main evolutionary forces at play shaping these patterns. One promising strategy, albeit little used to date, is to comparatively investigate these genomic landscapes as progression through time by using a series of species pairs along a divergence gradient. Here, we resequenced 201 whole-genomes from eight closely related Populus species, with pairs of species at different stages along the divergence gradient to learn more about speciation processes. Using population structure and ancestry analyses, we document extensive introgression between some species pairs, especially those with parapatric distributions. We further investigate genomic landscapes, focusing on within-species (i.e. nucleotide diversity and recombination rate) and among-species (i.e. relative and absolute divergence) summary statistics of diversity and divergence. We observe relatively conserved patterns of genomic divergence across species pairs. Independent of the stage across the divergence gradient, we find support for signatures of linked selection (i.e. the interaction between natural selection and genetic linkage) in shaping these genomic landscapes, along with gene flow and standing genetic variation. We highlight the importance of investigating genomic patterns on multiple species across a divergence gradient and discuss prospects to better understand the evolutionary forces shaping the genomic landscapes of diversity and differentiation.

Pervasive Introgression During Rapid Diversification of the European Mountain Genus Soldanella (L.) (Primulaceae).

Hybridization is a key mechanism involved in lineage diversification and speciation, especially in ecosystems that experienced repeated environmental oscillations. Recently radiated plant groups, which have evolved in mountain ecosystems impacted by historical climate change provide an excellent model system for studying the impact of gene flow on speciation. We combined organellar (whole-plastome) and nuclear genomic data (RAD-seq) with a cytogenetic approach (rDNA FISH) to investigate the effects of hybridization and introgression on evolution and speciation in the genus Soldanella (snowbells, Primulaceae). Pervasive introgression has already occurred among ancestral lineages of snowbells and has persisted throughout the entire evolutionary history of the genus, regardless of the ecology, cytotype, or distribution range size of the affected species. The highest extent of introgression has been detected in the Carpathian species, which is also reflected in their extensive karyotype variation. Introgression occurred even between species with dysploid and euploid cytotypes, which were considered to be reproductively isolated. The magnitude of introgression detected in snowbells is unprecedented in other mountain genera of the European Alpine System investigated hitherto. Our study stresses the prominent evolutionary role of hybridization in facilitating speciation and diversification on the one hand, but also enriching previously isolated genetic pools. [chloroplast capture; diversification; dysploidy; European Alpine system; introgression; nuclear-cytoplasmic discordance; ribosomal DNA.].

Polygenic routes lead to parallel altitudinal adaptation in Heliosperma pusillum (Caryophyllaceae).

Understanding how organisms adapt to the environment is a major goal of modern biology. Parallel evolution-the independent evolution of similar phenotypes in different populations-provides a powerful framework to investigate the evolutionary potential of populations, the constraints of evolution, its repeatability and therefore its predictability. Here, we quantified the degree of gene expression and functional parallelism across replicated ecotype formation in Heliosperma pusillum (Caryophyllaceae), and gained insights into the architecture of adaptive traits. Population structure analyses and demographic modelling support a previously formulated hypothesis of parallel polytopic divergence of montane and alpine ecotypes. We detect a large proportion of differentially expressed genes (DEGs) underlying divergence within each replicate ecotype pair, with a strikingly low number of shared DEGs across pairs. Functional enrichment of DEGs reveals that the traits affected by significant expression divergence are largely consistent across ecotype pairs, in strong contrast to the nonshared genetic basis. The remarkable redundancy of differential gene expression indicates a polygenic architecture for the diverged adaptive traits. We conclude that polygenic traits appear key to opening multiple routes for adaptation, widening the adaptive potential of organisms.

Down, then up: non-parallel genome size changes and a descending chromosome series in a recent radiation of the Australian allotetraploid plant species, Nicotiana section Suaveolentes (Solanaceae).

BACKGROUND AND AIMS: The extent to which genome size and chromosome numbers evolve in concert is little understood, particularly after polyploidy (whole-genome duplication), when a genome returns to a diploid-like condition (diploidization). We study this phenomenon in 46 species of allotetraploid Nicotiana section Suaveolentes (Solanaceae), which formed <6 million years ago and radiated in the arid centre of Australia. METHODS: We analysed newly assessed genome sizes and chromosome numbers within the context of a restriction site-associated nuclear DNA (RADseq) phylogenetic framework. KEY RESULTS: RADseq generated a well-supported phylogenetic tree, in which multiple accessions from each species formed unique genetic clusters. Chromosome numbers and genome sizes vary from n = 2x = 15 to 24 and 2.7 to 5.8 pg/1C nucleus, respectively. Decreases in both genome size and chromosome number occur, although neither consistently nor in parallel. Species with the lowest chromosome numbers (n = 15-18) do not possess the smallest genome sizes and, although N. heterantha has retained the ancestral chromosome complement, n = 2x = 24, it nonetheless has the smallest genome size, even smaller than that of the modern representatives of ancestral diploids. CONCLUSIONS: The results indicate that decreases in genome size and chromosome number occur in parallel down to a chromosome number threshold, n = 20, below which genome size increases, a phenomenon potentially explained by decreasing rates of recombination over fewer chromosomes. We hypothesize that, more generally in plants, major decreases in genome size post-polyploidization take place while chromosome numbers are still high because in these stages elimination of retrotransposons and other repetitive elements is more efficient. Once such major genome size change has been accomplished, then dysploid chromosome reductions take place to reorganize these smaller genomes, producing species with small genomes and low chromosome numbers such as those observed in many annual angiosperms, including Arabidopsis.

Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale.

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Repeat Dynamics across Timescales: A Perspective from Sibling Allotetraploid Marsh Orchids (Dactylorhiza majalis s.l.).

To provide insights into the fate of transposable elements (TEs) across timescales in a post-polyploidization context, we comparatively investigate five sibling Dactylorhiza allotetraploids (Orchidaceae) formed independently and sequentially between 500 and 100K generations ago by unidirectional hybridization between diploids D. fuchsii and D. incarnata. Our results first reveal that the paternal D. incarnata genome shows a marked increased content of LTR retrotransposons compared to the maternal species, reflected in its larger genome size and consistent with a previously hypothesized bottleneck. With regard to the allopolyploids, in the youngest D. purpurella both genome size and TE composition appear to be largely additive with respect to parents, whereas for polyploids of intermediate ages we uncover rampant genome expansion on a magnitude of multiple entire genomes of some plants such as Arabidopsis. The oldest allopolyploids in the series are not larger than the intermediate ones. A putative tandem repeat, potentially derived from a non-autonomous miniature inverted-repeat TE (MITE) drives much of the genome dynamics in the allopolyploids. The highly dynamic MITE-like element is found in higher proportions in the maternal diploid, D. fuchsii, but is observed to increase in copy number in both subgenomes of the allopolyploids. Altogether, the fate of repeats appears strongly regulated and therefore predictable across multiple independent allopolyploidization events in this system. Apart from the MITE-like element, we consistently document a mild genomic shock following the allopolyploidizations investigated here, which may be linked to their relatively large genome sizes, possibly associated with strong selection against further genome expansions.

Genomic insights into recent species divergence in Nicotiana benthamiana and natural variation in Rdr1 gene controlling viral susceptibility.

One of the most commonly encountered and frequently cited laboratory organisms worldwide is classified taxonomically as Nicotiana benthamiana (Solanaceae), an accession of which, typically referred to as LAB, is renowned for its unique susceptibility to a wide range of plant viruses and hence capacity to be transformed using a variety of methods. This susceptibility is the result of an insertion and consequent loss of function in the RNA-dependent RNA polymerase 1 (Rdr1) gene. However, the origin and age of LAB and the evolution of N. benthamiana across its wide distribution in Australia remain relatively underexplored. Here, we have used multispecies coalescent methods on genome-wide single nucleotide polymorphisms (SNPs) to assess species limits, phylogenetic relationships and divergence times within N. benthamiana. Our results show that the previous taxonomic concept of this species in fact comprises five geographically, morphologically and genetically distinct species, one of which includes LAB. We provide clear evidence that LAB is closely related to accessions collected further north in the Northern Territory; this species split much earlier, c. 1.1 million years ago, from their common ancestor than the other four in this clade and is morphologically the most distinctive. We also found that the Rdr1 gene insertion is variable among accessions from the northern portions of the Northern Territory. Furthermore, this long-isolated species typically grows in sheltered sites in subtropical/tropical monsoon areas of northern Australia, contradicting the previously advanced hypothesis that this species is an extremophile that has traded viral resistance for precocious development.

Population structure in Neotropical plants: Integrating pollination biology, topography and climatic niches.

Animal pollinators mediate gene flow among plant populations, but in contrast to well-studied topographic and (Pleistocene) environmental isolating barriers, their impact on population genetic differentiation remains largely unexplored. Comparing how these multifarious factors drive microevolutionary histories is, however, crucial for better resolving macroevolutionary patterns of plant diversification. Here we combined genomic analyses with landscape genetics and niche modelling across six related Neotropical plant species (424 individuals across 33 localities) differing in pollination strategy to test the hypothesis that highly mobile (vertebrate) pollinators more effectively link isolated localities than less mobile (bee) pollinators. We found consistently higher genetic differentiation (FST ) among localities of bee- than vertebrate-pollinated species with increasing geographical distance, topographic barriers and historical climatic instability. High admixture among montane populations further suggested relative climatic stability of Neotropical montane forests during the Pleistocene. Overall, our results indicate that pollinators may differentially impact the potential for allopatric speciation, thereby critically influencing diversification histories at macroevolutionary scales.

Taxon-specific or universal? Using target capture to study the evolutionary history of rapid radiations.

Target capture has emerged as an important tool for phylogenetics and population genetics in nonmodel taxa. Whereas developing taxon-specific capture probes requires sustained efforts, available universal kits may have a lower power to reconstruct relationships at shallow phylogenetic scales and within rapidly radiating clades. We present here a newly developed target capture set for Bromeliaceae, a large and ecologically diverse plant family with highly variable diversification rates. The set targets 1776 coding regions, including genes putatively involved in key innovations, with the aim to empower testing of a wide range of evolutionary hypotheses. We compare the relative power of this taxon-specific set, Bromeliad1776, to the universal Angiosperms353 kit. The taxon-specific set results in higher enrichment success across the entire family; however, the overall performance of both kits to reconstruct phylogenetic trees is relatively comparable, highlighting the vast potential of universal kits for resolving evolutionary relationships. For more detailed phylogenetic or population genetic analyses, for example the exploration of gene tree concordance, nucleotide diversity or population structure, the taxon-specific capture set presents clear benefits. We discuss the potential lessons that this comparative study provides for future phylogenetic and population genetic investigations, in particular for the study of evolutionary radiations.

La captura selectiva de secuencias de ADN ha surgido como una herramienta importante para la filogenética y la genética de poblaciones en taxones no-modelo. Mientras que el desarrollo de sondas de captura específicas para cada taxón requiere un esfuerzo sostenido, las colecciones de sondas universales disponibles pueden tener una potencia disminuida para la reconstrucción de relaciones filogenéticas poco profundas o de radiaciones rápidas. Presentamos aquí un conjunto de sondas para la captura selectiva desarrollado recientemente para Bromeliaceae, una familia de plantas extensa, ecológicamente diversa y con tasas de diversificación muy variables. El conjunto de sondas se centra en 1776 regiones de codificación, incluyendo genes supuestamente implicados en rasgos de innovación clave, con el objetivo de potenciar la comprobación de una amplia gama de hipótesis evolutivas. Comparamos la potencia relativa de este conjunto de sondas diseñado para un taxón específico, Bromeliad1776, con la colección universal Angiosperms353. El conjunto específico da lugar a un mayor éxito de captura en toda la familia. Sin embargo, el rendimiento global de ambos kits para reconstruir árboles filogenéticos es relativamente comparable, lo que pone de manifiesto el gran potencial de los kits universales para resolver las relaciones evolutivas. Para análisis filogenéticos o de genética de poblaciones más detallados, como por ejemplo la exploración de la congruencia de los árboles de genes, la diversidad de nucleótidos o la estructura de la población, el conjunto de captura específico para Bromeliaceae presenta claras ventajas. Discutimos las lecciones potenciales que este estudio comparativo proporciona para futuras investigaciones filogenéticas y de genética de poblaciones, en particular para el estudio de las radiaciones evolutivas.

The Evolutionary History of New Zealand Deschampsia Is Marked by Long-Distance Dispersal, Endemism, and Hybridization.

The contrasting evolutionary histories of endemic versus related cosmopolitan species provide avenues to understand the spatial drivers and limitations of biodiversity. Here, we investigated the evolutionary history of three New Zealand endemic Deschampsia species, and how they are related to cosmopolitan D. cespitosa. We used RADseq to test species delimitations, infer a dated species tree, and investigate gene flow patterns between the New Zealand endemics and the D. cespitosa populations of New Zealand, Australia and Korea. Whole plastid DNA analysis was performed on a larger worldwide sampling. Morphometrics of selected characters were applied to New Zealand sampling. Our RADseq review of over 55 Mbp showed the endemics as genetically well-defined from each other. Their last common ancestor with D. cespitosa lived during the last ten MY. The New Zealand D. cespitosa appears in a clade with Australian and Korean samples. Whole plastid DNA analysis revealed the endemics as members of a southern hemisphere clade, excluding the extant D. cespitosa of New Zealand. Both data provided strong evidence for hybridization between D. cespitosa and D. chapmanii. Our findings provide evidence for at least two migration events of the genus Deschampsia to New Zealand and hybridization between D. cespitosa and endemic taxa.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb.

Background and Aims: Quantifying genetic variation is fundamental to understand a species' demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies. Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables. Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria. Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Long-term isolation of European steppe outposts boosts the biome's conservation value.

The European steppes and their biota have been hypothesized to be either young remnants of the Pleistocene steppe belt or, alternatively, to represent relicts of long-term persisting populations; both scenarios directly bear on nature conservation priorities. Here, we evaluate the conservation value of threatened disjunct steppic grassland habitats in Europe in the context of the Eurasian steppe biome. We use genomic data and ecological niche modelling to assess pre-defined, biome-specific criteria for three plant and three arthropod species. We show that the evolutionary history of Eurasian steppe biota is strikingly congruent across species. The biota of European steppe outposts were long-term isolated from the Asian steppes, and European steppes emerged as disproportionally conservation relevant, harbouring regionally endemic genetic lineages, large genetic diversity, and a mosaic of stable refugia. We emphasize that conserving what is left of Europe's steppes is crucial for conserving the biological diversity of the entire Eurasian steppe biome.

Phylogenomic Relationships of Diploids and the Origins of Allotetraploids in Dactylorhiza (Orchidaceae).

Disentangling phylogenetic relationships proves challenging for groups that have evolved recently, especially if there is ongoing reticulation. Although they are in most cases immediately isolated from diploid relatives, sets of sibling allopolyploids often hybridize with each other, thereby increasing the complexity of an already challenging situation. Dactylorhiza (Orchidaceae: Orchidinae) is a genus much affected by allopolyploid speciation and reticulate phylogenetic relationships. Here, we use genetic variation at tens of thousands of genomic positions to unravel the convoluted evolutionary history of Dactylorhiza. We first investigate circumscription and relationships of diploid species in the genus using coalescent and maximum likelihood methods, and then group 16 allotetraploids by maximum affiliation to their putative parental diploids, implementing a method based on genotype likelihoods. The direction of hybrid crosses is inferred for each allotetraploid using information from maternally inherited plastid RADseq loci. Starting from age estimates of parental taxa, the relative ages of these allotetraploid entities are inferred by quantifying their genetic similarity to the diploids and numbers of private alleles compared with sibling allotetraploids. Whereas northwestern Europe is dominated by young allotetraploids of postglacial origins, comparatively older allotetraploids are distributed further south, where climatic conditions remained relatively stable during the Pleistocene glaciations. Our bioinformatics approach should prove effective for the study of other naturally occurring, nonmodel, polyploid plant complexes.

Early diversification and permeable species boundaries in the Mediterranean firs.

BACKGROUND AND AIMS: Inferring the evolutionary relationships of species and their boundaries is critical in order to understand patterns of diversification and their historical drivers. Despite Abies (Pinaceae) being the second most diverse group of conifers, the evolutionary history of Circum-Mediterranean firs (CMFs) remains under debate. METHODS: We used restriction site-associated DNA sequencing (RAD-seq) on all proposed CMF taxa to investigate their phylogenetic relationships and taxonomic status. KEY RESULTS: Based on thousands of genome-wide single nucleotide polymorphisms (SNPs), we present here the first formal test of species delimitation, and the first fully resolved, complete species tree for CMFs. We discovered that all previously recognized taxa in the Mediterranean should be treated as independent species, with the exception of Abies tazaotana and Abies marocana. An unexpectedly early pulse of speciation in the Oligocene-Miocene boundary is here documented for the group, pre-dating previous hypotheses by millions of years, revealing a complex evolutionary history encompassing both ancient and recent gene flow between distant lineages. CONCLUSIONS: Our phylogenomic results contribute to shed light on conifers' diversification. Our efforts to resolve the CMF phylogenetic relationships help refine their taxonomy and our knowledge of their evolution.