Genotyping-by-sequencing reveals range expansion of Adonis vernalis (Ranunculaceae) from Southeastern Europe into the zonal Euro-Siberian steppe.

The Euro-Siberian steppe flora consists of warm- and cold-adapted species, which may have responded differently to Pleistocene glacials and interglacials. Genotyping-by-sequencing individuals from across the distribution range of the pheasant's eye (Adonis vernalis), we aimed to gain insight into steppe florogenesis based on the species' evolutionary history. Although the primary area of origin of the species group comprising A. vernalis, A. villosa and A. volgensis is in Asia, our results indicate that recent populations of A. vernalis are not of Asian origin but evolved in the southern part of Europe during the Pleistocene, with Spanish populations clearly genetically distinct from the Southeastern European populations. We inferred that A. vernalis migrated eastwards from the sub-Mediterranean forest-steppes of Southeastern Europe into the continental forest-steppe zone. Eastern European populations had the highest private allelic richness, indicating long-term large population sizes in this region. As a thermophilic species, A. vernalis seems unlikely to have survived in the cold deserts of the Last Glacial Maximum in Western Siberia, so this region was likely (re)colonized postglacially. Overall, our results reinforce the importance of identifying the area of origin and the corresponding ecological requirements of steppe plants in order to understand the composition of today's steppe flora.

Pleistocene dynamics of the Eurasian steppe as a driving force of evolution: Phylogenetic history of the genus Capsella (Brassicaceae).

Capsella is a model plant genus of the Brassicaceae closely related to Arabidopsis. To disentangle its biogeographical history and intrageneric phylogenetic relationships, 282 individuals of all five currently recognized Capsella species were genotyped using a restriction digest-based next-generation sequencing method. Our analysis retrieved two main lineages within Capsella that split c. one million years ago, with western C. grandiflora and C. rubella forming a sister lineage to the eastern lineage consisting of C. orientalis. The split was attributed to continuous latitudinal displacements of the Eurasian steppe belt to the south during Early Pleistocene glacial cycles. During the interglacial cycles of the Late Pleistocene, hybridization of the two lineages took place in the southwestern East European Plain, leading to the allotetraploid C. bursa-pastoris. Extant genetic variation within C. orientalis postdated any extensive glacial events. Ecological niche modeling showed that suitable habitat for C. orientalis existed during the Last Glacial Maximum around the north coast of the Black Sea and in southern Kazakhstan. Such a scenario is also supported by population genomic data that uncovered the highest genetic diversity in the south Kazakhstan cluster, suggesting that C. orientalis originated in continental Asia and migrated north- and possibly eastwards after the last ice age. Post-glacial hybridization events between C. bursa-pastoris and C. grandiflora/rubella in the southwestern East European Plain and the Mediterranean gave rise to C. thracica. Introgression of C. grandiflora/rubella into C. bursa-pastoris resulted in a new Mediterranean cluster within the already existing Eurasian C. bursa-pastoris cluster. This study shows that the continuous displacement and disruption of the Eurasian steppe belt during the Pleistocene was the driving force in the evolution of Capsella.

The phylogeographic history of Krascheninnikovia reflects the development of dry steppes and semi-deserts in Eurasia.

Constituting one of Earth's major biomes, steppes are characterised by naturally treeless extra-tropical vegetation. The formation of the Eurasian steppe belt, the largest steppe region in the world, began in Central Asia during the Neogene. In the glacial stages of the Pleistocene, steppe displaced forest vegetation, which in turn recolonised the area during the warmer interglacial periods, thus affecting the distribution of plants adapted to these habitats. Krascheninnikovia ceratoides (Chenopodiaceae) is a plant characteristic of dry steppe and semi-desert formations. Earlier studies showed that the ancestor of this autochthonous steppe element originated in Central Asia during the Miocene/Pliocene, i.e., in the same region and at the same time as the first appearance of steppe vegetation. However, as the extant lineages of Krascheninnikovia ceratoides diversified only 2.2 ± 0.9 Mya, it may represent a modern element of current dry steppe and semi-desert formations, rather than a component of the first steppe precursors of the Miocene. As such, it may have capitalised on the climatic conditions of the cold stages of the Quaternary to expand its range and colonise suitable habitats outside of its area of origin. To test this hypothesis, phylogeographic methods were applied to high-resolution genotyping-by-sequencing data. Our results indicate that Krascheninnikovia originated in western Central Asia and the Russian Altai, then spread to Europe in the West, and reached North America in the East. The populations of eastern Central Asia and North America belong to the same clade and are genetically clearly distinct from the Euro-Siberian populations. Among the populations west of the Altai Mountains, the European populations are genetically distinct from all others, which could be the result of the separation of populations east and west of the Urals caused by the Pleistocene transgressions of the Caspian Sea.

Extremely low levels of chloroplast genome sequence variability in Astelia pumila (Asteliaceae, Asparagales).

Astelia pumila (G.Forst.) Gaudich. (Asteliaceae, Asparagales) is a major element of West Patagonian cushion peat bog vegetation. With the aim to identify appropriate chloroplast markers for the use in a phylogeographic study, the complete chloroplast genomes of five A. pumila accessions from almost the entire geographical range of the species were assembled and screened for variable positions. The chloroplast genome sequence was obtained via a mapping approach, using Eustrephus latifolius (Asparagaceae) as a reference. The chloroplast genome of A. pumila varies in length from 158,215 bp to 158,221 bp, containing a large single copy region of 85,981-85,983 bp, a small single copy region of 18,182-18,186 bp and two inverted repeats of 27,026 bp. Genome annotation predicted a total of 113 genes, including 30 tRNA and four rRNA genes. Sequence comparisons revealed a very low degree of intraspecific genetic variability, as only 37 variable sites (18 indels, 18 single nucleotide polymorphisms, one 3-bp mutation)-most of them autapomorphies-were found among the five assembled chloroplast genomes. A Maximum Likelihood analysis, based on whole chloroplast genome sequences of several Asparagales accessions representing six of the currently recognized 14 families (sensu APG IV), confirmed the phylogenetic position of A. pumila. The chloroplast genome of A. pumila is the first to be reported for a member of the astelioid clade (14 genera with c. 215 species), a basally branching group within Asparagales.

SSR-seq: Genotyping of microsatellites using next-generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring.

Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next-generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom-designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single-nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%-63.5% (mean over all loci) in the three study species. Thus, the information obtained by next-generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.

Tabula rasa in the Patagonian Channels? The phylogeography of Oreobolus obtusangulus (Cyperaceae).

The extent of the Pleistocene glaciations in the Patagonian Channel region (southwesternmost South America) and their impact on the vegetation there are largely unknown. Whether the regional flora was wiped out completely (tabula rasa) or survived in ice-free pockets (in situ survival) is still an open question. The molecular imprint of either scenario should still be visible in extant populations. Therefore, DNA sequence data of Oreobolus obtusangulus Gaudich. (Cyperaceae) were analysed. This species is an abundant constituent of Patagonian cushion peat bogs, one of the Patagonian Channel region's major vegetation types. Three hundred and eighty-four individuals from 48 populations were sequenced for two chloroplast (ycf3-psaA and trnQUUG -psbK intergenic spacers) and 14 nuclear loci containing simple sequence repeats (SSRs; microsatellites). Phylogenetic reconstructions and the geographic distribution of genetic diversity revealed that the species was split into three main lineages whose general distributions comprise three separate major regions, that is, south-central Chile, Fuego-Patagonia and the East Patagonian Andes, which probably constitute glacial refugia. Postglacial migration fronts formed a suture zone with high levels of genetic diversity in the Northwest Patagonian Andes, where remnants of a supposedly ancestral lineage were also found to be locally restricted to a single population (Huinay). The heavily glaciated Patagonian Channels were likely recolonized from the northwest, and partly from the south. Although the westernmost Patagonian Channel population (Estero Bachem) harboured private SSR alleles (singletons) and showed slightly elevated genetic diversity, it remained unclear whether this population actually survived in situ. This study helps fill a major gap in reconstructing the Pleistocene vegetation history of West and Andean Patagonia.