Phylogenomics and the rise of the angiosperms.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5-7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Strategies of diaspore dispersal investment in Compositae: the case of the Andean highlands.

BACKGROUND AND AIMS: Understanding diaspore morphology and how much a species invests on dispersal appendages is key for improving our knowledge of dispersal in fragmented habitats. We investigate diaspore morphological traits in high-Andean Compositae and their main abiotic and biotic drivers and test whether they play a role in species distribution patterns across the naturally fragmented high-Andean grasslands. METHODS: We collected diaspore trait data for 125 Compositae species across 47 tropical high-Andean summits, focusing on achene length and pappus-to-achene length ratio, with the latter as a proxy of dispersal investment. We analysed the role of abiotic (temperature, elevation and latitude) and biotic factors (phylogenetic signal and differences between tribes) on diaspore traits and whether they are related to distribution patterns across the Andes, using phylogenomics, distribution modelling and community ecology analyses. KEY RESULTS: Seventy-five percent of the studied species show small achenes (length <3.3 mm) and 67% have high dispersal investment (pappus length at least two times the achene length). Dispersal investment increases with elevation, possibly to compensate for lower air density, and achene length increases towards the equator, where non-seasonal climate prevails. Diaspore traits show significant phylogenetic signal, and higher dispersal investment is observed in Gnaphalieae, Astereae and Senecioneae, which together represent 72% of our species. High-Andean-restricted species found across the tropical Andes have, on average, the pappus four times longer than the achene, a significantly higher dispersal investment than species present only in the northern Andes or only in the central Andes. CONCLUSIONS: Small achenes and high diaspore dispersal investment dominate among high-Andean Compositae, traits typical of mostly three tribes of African origin; but traits are also correlated with the environmental gradients within the high-Andean grasslands. Our results also suggest that diaspore dispersal investment is likely to shape species distribution patterns in naturally fragmented habitats.

A Bioinformatic Pipeline to Estimate Ploidy Level from Target Capture Sequence Data Obtained from Herbarium Specimens.

Whole genome duplications (WGD) are frequent in many plant lineages; however, ploidy level variation is unknown in most species. The most widely used methods to estimate ploidy levels in plants are chromosome counts, which require living specimens, and flow cytometry estimates, which necessitate living or relatively recently collected samples. Newly described bioinformatic methods have been developed to estimate ploidy levels using high-throughput sequencing data, and these have been optimized in plants by calculating allelic ratio values from target capture data. This method relies on the maintenance of allelic ratios from the genome to the sequence data. For example, diploid organisms will generate allelic data in a 1:1 proportion, with an increasing number of possible allelic ratio combinations occurring in individuals with higher ploidy levels. In this chapter, we explain step-by-step this bioinformatic approach for the estimation of ploidy level.

Effective population size in a partially clonal plant is not predicted by the number of genetic individuals.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

PREMISE: Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome. METHODS: We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω). RESULTS: Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK. CONCLUSIONS: We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.

Multiple pre- and postzygotic components of reproductive isolation between two co-occurring Lysimachia species.

Genetic divergence between species depends on reproductive isolation (RI) due to traits that reduce interspecific mating (prezygotic isolation) or are due to reduced hybrid fitness (postzygotic isolation). Previous research found that prezygotic barriers tend to be stronger than postzygotic barriers, but most studies are based on the evaluation of F1 hybrid fitness in early life cycle stages. We combined field and experimental data to determine the strength of 17 prezygotic and postzygotic reproductive barriers between two Lysimachia species that often co-occur and share pollinators. We assessed postzygotic barriers up to F2 hybrids and backcrosses. The two species showed near complete RI due to the cumulative effect of multiple barriers, with an uneven and asymmetric contribution to isolation. In allopatry, prezygotic barriers contributed more to reduce gene flow than postzygotic barriers, but their contributions were more similar in sympatry. The strength of postzygotic RI was up to three times lower for F1 progeny than for F2 or backcrossed progenies, and RI was only complete when late F1 stages and either F2 or backcrosses were accounted for. Our results thus suggest that the relative strength of postzygotic RI may be underestimated when its effects on late stages of the life cycle are disregarded.

Genomic, spatial and morphometric data for discrimination of four species in the Mediterranean Tamus clade of yams (Dioscorea, Dioscoreaceae).

BACKGROUND AND AIMS: Among the numerous pantropical species of the yam genus, Dioscorea, only a small group occurs in the Mediterranean basin, including two narrow Pyrenean endemics (Borderea clade) and two Mediterranean-wide species (D. communis and D. orientalis, Tamus clade). However, several currently unrecognized species and infraspecific taxa have been described in the Tamus clade due to significant morphological variation associated with D. communis. Our overarching aim was to investigate taxon delimitation in the Tamus clade using an integrative approach combining phylogenomic, spatial and morphological data. METHODS: We analysed 76 herbarium samples using Hyb-Seq genomic capture to sequence 260 low-copy nuclear genes and plastomes, together with morphometric and environmental modelling approaches. KEY RESULTS: Phylogenomic reconstructions confirmed that the two previously accepted species of the Tamus clade, D. communis and D. orientalis, are monophyletic and form sister clades. Three subclades showing distinctive geographic patterns were identified within D. communis. These subclades were also identifiable from morphometric and climatic data, and introgression patterns were inferred between subclades in the eastern part of the distribution of D. communis. CONCLUSIONS: We propose a taxonomy that maintains D. orientalis, endemic to the eastern Mediterranean region, and splits D. communis sensu lato into three species: D. edulis, endemic to Macaronesia (Canary Islands and Madeira); D. cretica, endemic to the eastern Mediterranean region; and D. communis sensu stricto, widespread across western and central Europe. Introgression inferred between D. communis s.s. and D. cretica is likely to be explained by their relatively recent speciation at the end of the Miocene, disjunct isolation in eastern and western Mediterranean glacial refugia and a subsequent westward recolonization of D. communis s.s. Our study shows that the use of integrated genomic, spatial and morphological approaches allows a more robust definition of species boundaries and the identification of species that previous systematic studies failed to uncover.

Genome-wide footprints in the carob tree (Ceratonia siliqua) unveil a new domestication pattern of a fruit tree in the Mediterranean.

Intense research efforts over the last two decades have renewed our understanding of plant phylogeography and domestication in the Mediterranean basin. Here we aim to investigate the evolutionary history and the origin of domestication of the carob tree (Ceratonia siliqua), which has been cultivated for millennia for food and fodder. We used >1000 microsatellite genotypes to delimit seven carob evolutionary units (CEUs). We investigated genome-wide diversity and evolutionary patterns of the CEUs with 3557 single nucleotide polymorphisms generated by restriction-site associated DNA sequencing (RADseq). To address the complex wild vs. cultivated status of sampled trees, we classified 56 sampled populations across the Mediterranean basin as wild, seminatural or cultivated. Nuclear and cytoplasmic loci were identified from RADseq data and separated for analyses. Phylogenetic analyses of these genomic-wide data allowed us to resolve west-to-east expansions from a single long-term refugium probably located in the foothills of the High Atlas Mountains near the Atlantic coast. Our findings support multiple origins of domestication with a low impact on the genetic diversity at range-wide level. The carob was mostly domesticated from locally selected wild genotypes and scattered long-distance westward dispersals of domesticated varieties by humans, concomitant with major historical migrations by Romans, Greeks and Arabs. Ex situ efforts to preserve carob genetic resources should prioritize accessions from both western and eastern populations, with emphasis on the most differentiated CEUs situated in southwest Morocco, south Spain and eastern Mediterranean. Our study highlights the relevance of wild and seminatural habitats in the conservation of genetic resources for cultivated trees.

Genome Insights into Autopolyploid Evolution: A Case Study in Senecio doronicum (Asteraceae) from the Southern Alps.

Polyploidy is a widespread phenomenon across angiosperms, and one of the main drivers of diversification. Whilst it frequently involves hybridisation, autopolyploidy is also an important feature of plant evolution. Minority cytotypes are frequently overlooked due to their lower frequency in populations, but the development of techniques such as flow cytometry, which enable the rapid screening of cytotype diversity across large numbers of individuals, is now providing a more comprehensive understanding of cytotype diversity within species. Senecio doronicum is a relatively common daisy found throughout European mountain grasslands from subalpine to almost nival elevations. We have carried out a population-level cytotype screening of 500 individuals from Tête Grosse (Alpes-de-Haute-Provence, France), confirming the coexistence of tetraploid (28.2%) and octoploid cytotypes (71.2%), but also uncovering a small number of hexaploid individuals (0.6%). The analysis of repetitive elements from short-read genome-skimming data combined with nuclear (ITS) and whole plastid DNA sequences support an autopolyploid origin of the polyploid S. doronicum individuals and provide molecular evidence regarding the sole contribution of tetraploids in the formation of hexaploid individuals. The evolutionary impact and resilience of the new cytotype have yet to be determined, although the coexistence of different cytotypes may indicate nascent speciation.

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.

A customised target capture sequencing tool for molecular identification of Aloe vera and relatives.

Plant molecular identification studies have, until recently, been limited to the use of highly conserved markers from plastid and other organellar genomes, compromising resolution in highly diverse plant clades. Due to their higher evolutionary rates and reduced paralogy, low-copy nuclear genes overcome this limitation but are difficult to sequence with conventional methods and require high-quality input DNA. Aloe vera and its relatives in the Alooideae clade (Asphodelaceae, subfamily Asphodeloideae) are of economic interest for food and health products and have horticultural value. However, pressing conservation issues are increasing the need for a molecular identification tool to regulate the trade. With > 600 species and an origin of ± 15 million years ago, this predominantly African succulent plant clade is a diverse and taxonomically complex group for which low-copy nuclear genes would be desirable for accurate species discrimination. Unfortunately, with an average genome size of 16.76 pg, obtaining high coverage sequencing data for these genes would be prohibitively costly and computationally demanding. We used newly generated transcriptome data to design a customised RNA-bait panel targeting 189 low-copy nuclear genes in Alooideae. We demonstrate its efficacy in obtaining high-coverage sequence data for the target loci on Illumina sequencing platforms, including degraded DNA samples from museum specimens, with considerably improved phylogenetic resolution. This customised target capture sequencing protocol has the potential to confidently indicate phylogenetic relationships of Aloe vera and related species, as well as aid molecular identification applications.

Microsatellites and petal morphology reveal new patterns of admixture in Orchis hybrid zones.

PREMISE: The genetic structure of hybrid zones provides insight into the potential for gene flow to occur between plant taxa. Four closely related European orchid species (Orchis anthropophora, O. militaris, O. purpurea, and O. simia) hybridize when they co-occur. We aimed to characterize patterns of hybridization in O. militaris-O. purpurea, O. purpurea-O. simia, and O. anthropophora-O. simia hybrid zones using molecular and morphological data. METHODS: We used 11 newly isolated nuclear microsatellites to genotype 695 individuals collected from seven hybrid zones and six allopatric parental populations in France. Geometric morphometric analysis was conducted using 15 labellum landmarks to capture the main aspects of petal shape. RESULTS: Backcrossing was asymmetric toward O. militaris in multiple O. militaris-O. purpurea hybrid zones. Hybrids in O. purpurea-O. simia and O. anthropophora-O. simia hybrid zones were largely limited to F1 and F2 generations, but further admixture had occurred. These patterns were reflected in labellum geometric morphometric data, which correlated strongly with nuclear microsatellite data in all three species combinations. CONCLUSIONS: The coexistence of parental and admixed individuals in these Orchis hybrid zones implies they are likely to be tension zones being maintained by a balance between gene flow into the hybrid zone and selection acting against admixed individuals. The pattern of admixture in the three species combinations suggests intrinsic selection acting on the hybrids is weaker in more closely related taxa.

Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing.

Leaves of the wild yam species Dioscorea sansibarensis display prominent forerunner or "drip" tips filled with extracellular bacteria of the species Orrella dioscoreae.1 This species of yam is native to Madagascar and tropical Africa and reproduces mainly asexually through aerial bulbils and underground tubers, which also contain a small population of O. dioscoreae.2,3 Despite apparent vertical transmission, the genome of O. dioscoreae does not show any of the hallmarks of genome erosion often found in hereditary symbionts (e.g., small genome size and accumulation of pseudogenes).4-6 We investigated here the range and distribution of leaf symbiosis between D. sansibarensis and O. dioscoreae using preserved leaf samples from herbarium collections that were originally collected from various locations in Africa. We recovered DNA from the extracellular symbiont in all samples, showing that the symbiosis is widespread throughout continental Africa and Madagascar. Despite the degraded nature of this DNA, we constructed 17 symbiont genomes using de novo methods without relying on a reference. Phylogenetic and genomic analyses revealed that horizontal transmission of symbionts and horizontal gene transfer have shaped the evolution of the symbiont. These mechanisms could help explain lack of signs of reductive genome evolution despite an obligate host-associated lifestyle. Furthermore, phylogenetic analysis of D. sansibarensis based on plastid genomes revealed a strong geographical clustering of samples and provided evidence that the symbiosis originated at least 13 mya, earlier than previously estimated.3.

Biogeography and genome size evolution of the oldest extant vascular plant genus, Equisetum (Equisetaceae).

BACKGROUND AND AIMS: Extant plant groups with a long fossil history are key elements in understanding vascular plant evolution. Horsetails (Equisetum, Equisetaceae) have a nearly continuous fossil record dating back to the Carboniferous, but their phylogenetic and biogeographic patterns are still poorly understood. We use here the most extensive phylogenetic analysis to date as a framework to evaluate their age, biogeography and genome size evolution. METHODS: DNA sequences of four plastid loci were used to estimate divergence times and investigate the biogeographic history of all extant species of Equisetum. Flow cytometry was used to study genome size evolution against the framework of phylogenetic relationships in Equisetum. KEY RESULTS: On a well-supported phylogenetic tree including all extant Equisetum species, a molecular clock calibrated with multiple fossils places the node at which the outgroup and Equisetum diverged at 343 Mya (Early Carboniferous), with the first major split among extant species occurring 170 Mya (Middle Jurassic). These dates are older than those reported in some other recent molecular clock studies but are largely in agreement with a timeline established by fossil appearance in the geological record. Representatives of evergreen subgenus Hippochaete have much larger genome sizes than those of deciduous subgenus Equisetum, despite their shared conserved chromosome number. Subgenus Paramochaete has an intermediate genome size and maintains the same number of chromosomes. CONCLUSIONS: The first divergences among extant members of the genus coincided with the break-up of Pangaea and the resulting more humid, warmer climate. Subsequent tectonic activity most likely involved vicariance events that led to species divergences combined with some more recent, long-distance dispersal events. We hypothesize that differences in genome size between subgenera may be related to the number of sperm flagellae.

Corrigendum: Museomics Unveil the Phylogeny and Biogeography of the Neglected Juan Fernandez Archipelago Megalachne and Podophorus Endemic Grasses and Their Connection With Relict Pampean-Ventanian Fescues.

[This corrects the article DOI: 10.3389/fpls.2020.00819.].

Phenolic Compounds Content and Genetic Diversity at Population Level across the Natural Distribution Range of Bearberry (Arctostaphylos uva-ursi, Ericaceae) in the Iberian Peninsula.

Bearberry (Arctostaphylos uva-ursi) is a medicinal plant traditionally employed for the treatment of urinary tract infections due to high contents of arbutin (hydroquinone ß-D-glucoside), which is now mainly used as a natural skin-whitening agent in cosmetics. Bearberry has also been proposed as a natural antioxidant additive due to the high contents of phenolic compounds in leaves. We studied the variation on phenolic compounds in 42 wild populations of bearberry, aiming to elucidate if intrinsic biological, climatic, and/or geographic factors affect phenolic contents across its natural distribution in the Iberian Peninsula. Bearberry leaves were collected during autumn over a three-year period (2014-2016) in populations across a latitude and altitude gradient. Methanolic extracts showed a wide range of variation in total phenols content, and different phenolic profiles regarding arbutin (levels of this major constituent varied from 87 to 232 mg/g dr wt), but also catechin and myricetin contents, which were affected by geographic and climatic factors. Moderate levels of variation on genome size-assessed by flow cytometry-and on two plastid DNA regions were also detected among populations. Genetic and cytogenetic differentiation of populations was weakly but significantly associated to phytochemical diversity. Elite bearberry genotypes with higher antioxidant capacity were subsequently identified.

Toward Unifying Global Hotspots of Wild and Domesticated Biodiversity.

Global biodiversity hotspots are areas containing high levels of species richness, endemism and threat. Similarly, regions of agriculturally relevant diversity have been identified where many domesticated plants and animals originated, and co-occurred with their wild ancestors and relatives. The agro-biodiversity in these regions has, likewise, often been considered threatened. Biodiversity and agro-biodiversity hotspots partly overlap, but their geographic intricacies have rarely been investigated together. Here we review the history of these two concepts and explore their geographic relationship by analysing global distribution and human use data for all plants, and for major crops and associated wild relatives. We highlight a geographic continuum between agro-biodiversity hotspots that contain high richness in species that are intensively used and well known by humanity (i.e., major crops and most viewed species on Wikipedia) and biodiversity hotspots encompassing species that are less heavily used and documented (i.e., crop wild relatives and species lacking information on Wikipedia). Our contribution highlights the key considerations needed for further developing a unifying concept of agro-biodiversity hotspots that encompasses multiple facets of diversity (including genetic and phylogenetic) and the linkage with overall biodiversity. This integration will ultimately enhance our understanding of the geography of human-plant interactions and help guide the preservation of nature and its contributions to people.

Museomics Unveil the Phylogeny and Biogeography of the Neglected Juan Fernandez Archipelago Megalachne and Podophorus Endemic Grasses and Their Connection With Relict Pampean-Ventanian Fescues.

Oceanic islands constitute natural laboratories to study plant speciation and biogeographic patterns of island endemics. Juan Fernandez is a southern Pacific archipelago consisting of three small oceanic islands located 600-700 km west of the Chilean coastline. Exposed to current cold seasonal oceanic climate, these 5.8-1 Ma old islands harbor a remarkable endemic flora. All known Fernandezian endemic grass species belong to two genera, Megalachne and Podophorus, of uncertain taxonomic adscription. Classical and modern classifications have placed them either in Bromeae (Bromus), Duthieinae, Aveneae/Poeae, or Loliinae (fine-leaved Festuca); however, none of them have clarified their evolutionary relationships with respect to their closest Festuca relatives. Megalachne includes four species, which are endemic to Masatierra (Robinson Crusoe island) (M. berteroniana and M. robinsoniana) and to Masafuera (Alejandro Selkirk island) (M. masafuerana and M. dantonii). The monotypic Podophorus bromoides is a rare endemic species to Masatierra which is only known from its type locality and is currently considered extinct. We have used museomic approaches to uncover the challenging evolutionary history of these endemic grasses and to infer the divergence and dispersal patterns from their ancestors. Genome skimming data were produced from herbarium samples of M. berteroniana and M. masafuerana, and the 164 years old type specimen of P. bromoides, as well as for a collection of 33 species representing the main broad- and fine-leaved Loliinae lineages. Paired-end reads were successfully mapped to plastomes and nuclear ribosomal cistrons of reference Festuca species and used to reconstruct phylogenetic trees. Filtered ITS and trnTLF sequences from these genomes were further combined with our large Loliinae data sets for accurate biogeographic reconstruction. Nuclear and plastome data recovered a strongly supported fine-leaved Fernandezian clade where Podophorus was resolved as sister to Megalachne. Bayesian divergence dating and dispersal-extinction-cladogenesis range evolution analyses estimated the split of the Fernandezian clade from its ancestral southern American Pampas-Ventanian Loliinae lineage in the Miocene-Pliocene transition, following a long distance dispersal from the continent to the uplifted volcanic palaeo-island of Santa Clara-Masatierra. Consecutive Pliocene-Pleistocene splits and a Masatierra-to-Masafuera dispersal paved the way for in situ speciation of Podophorus and Megalachne taxa.

Microsatellite marker development in the crop wild relative Linum bienne using genome skimming.

PREMISE: Nuclear microsatellite markers were developed for Linum bienne, the sister species of the crop L. usitatissimum, to provide molecular genetic tools for the investigation of L. bienne genetic diversity and structure. METHODS AND RESULTS: Fifty microsatellite loci were identified in L. bienne by means of genome skimming, and 44 loci successfully amplified. Of these, 16 loci evenly spread across the L. usitatissimum reference nuclear genome were used for genotyping six L. bienne populations. Excluding one monomorphic locus, the number of alleles per locus ranged from two to 12. Four out of six populations harbored private alleles. The levels of expected and observed heterozygosity were 0.076 to 0.667 and 0.000 to 1.000, respectively. All 16 loci successfully cross-amplified in L. usitatissimum. CONCLUSIONS: The 16 microsatellite loci developed here can be used for population genetic studies in L. bienne, and 28 additional loci that successfully amplified are available for further testing.