Phylogenetic challenges in a recently diversified and polyploid-rich Alyssum (Brassicaceae) lineage: low divergence, reticulation, and parallel polyploid speciation.

Elucidating the evolution of recently diverged and polyploid-rich plant lineages may be challenging even with high-throughput sequencing, both for biological reasons and bioinformatic difficulties. Here, we apply target enrichment with genome skimming (Hyb-Seq) to unravel the evolutionary history of the Alyssum montanum-A. repens species complex. Reconstruction of phylogenetic relationships in diploids supported recent and rapid diversification accompanied by reticulation events. Of the 4 main clades identified among the diploids, 3 clades included species from the Alps, Apennine, and Balkan peninsulas, indicating close biogeographic links between these regions. We further focused on the clade distributed from the Western Alps to the Iberian Peninsula, which comprises numerous polyploids as opposed to a few diploids. Using a recently developed PhyloSD (phylogenomic subgenome detection) pipeline, we successfully tracked the ancestry of all polyploids. We inferred multiple polyploidization events that involved 2 closely related diploid progenitors, resulting into several sibling polyploids: 2 autopolyploids and 6 allopolyploids. The skewed proportions of major homeolog-types and the occurrence of some minor homeolog-types, both exhibiting geographic patterns, suggest introgression with the progenitors and other related diploids. Our study highlights a unique case of parallel polyploid speciation that was enhanced by ecological and geographic separation and provides an excellent resource for future studies of polyploid evolution.

Allele Sorting as a Novel Approach to Resolving the Origin of Allotetraploids Using Hyb-Seq Data: A Case Study of the Balkan Mountain Endemic Cardamine barbaraeoides.

Mountains of the Balkan Peninsula are significant biodiversity hotspots with great species richness and a large proportion of narrow endemics. Processes that have driven the evolution of the rich Balkan mountain flora, however, are still insufficiently explored and understood. Here we focus on a group of Cardamine (Brassicaceae) perennials growing in wet, mainly mountainous habitats. It comprises several Mediterranean endemics, including those restricted to the Balkan Peninsula. We used target enrichment with genome skimming (Hyb-Seq) to infer their phylogenetic relationships, and, along with genomic in situ hybridization (GISH), to resolve the origin of tetraploid Cardamine barbaraeoides endemic to the Southern Pindos Mts. (Greece). We also explored the challenges of phylogenomic analyses of polyploid species and developed a new approach of allele sorting into homeologs that allows identifying subgenomes inherited from different progenitors. We obtained a robust phylogenetic reconstruction for diploids based on 1,168 low-copy nuclear genes, which suggested both allopatric and ecological speciation events. In addition, cases of plastid-nuclear discordance, in agreement with divergent nuclear ribosomal DNA (nrDNA) copy variants in some species, indicated traces of interspecific gene flow. Our results also support biogeographic links between the Balkan and Anatolian-Caucasus regions and illustrate the contribution of the latter region to high Balkan biodiversity. An allopolyploid origin was inferred for C. barbaraeoides, which highlights the role of mountains in the Balkan Peninsula both as refugia and melting pots favoring species contacts and polyploid evolution in response to Pleistocene climate-induced range dynamics. Overall, our study demonstrates the importance of a thorough phylogenomic approach when studying the evolution of recently diverged species complexes affected by reticulation events at both diploid and polyploid levels. We emphasize the significance of retrieving allelic and homeologous variation from nuclear genes, as well as multiple nrDNA copy variants from genome skim data.

So Closely Related and Yet So Different: Strong Contrasts Between the Evolutionary Histories of Species of the Cardamine pratensis Polyploid Complex in Central Europe.

Recurrent polyploid formation and weak reproductive barriers between independent polyploid lineages generate intricate species complexes with high diversity and reticulate evolutionary history. Uncovering the evolutionary processes that formed their present-day cytotypic and genetic structure is a challenging task. We studied the species complex of Cardamine pratensis, composed of diploid endemics in the European Mediterranean and diploid-polyploid lineages more widely distributed across Europe, focusing on the poorly understood variation in Central Europe. To elucidate the evolution of Central European populations we analyzed ploidy level and genome size variation, genetic patterns inferred from microsatellite markers and target enrichment of low-copy nuclear genes (Hyb-Seq), and environmental niche differentiation. We observed almost continuous variation in chromosome numbers and genome size in C. pratensis s.str., which is caused by the co-occurrence of euploid and dysploid cytotypes, along with aneuploids, and is likely accompanied by inter-cytotype mating. We inferred that the polyploid cytotypes of C. pratensis s.str. are both of single and multiple, spatially and temporally recurrent origins. The tetraploid Cardamine majovskyi evolved at least twice in different regions by autopolyploidy from diploid Cardamine matthioli. The extensive genome size and genetic variation of Cardamine rivularis reflects differentiation induced by the geographic isolation of disjunct populations, establishment of triploids of different origins, and hybridization with sympatric C. matthioli. Geographically structured genetic lineages identified in the species under study, which are also ecologically divergent, are interpreted as descendants from different source populations in multiple glacial refugia. The postglacial range expansion was accompanied by substantial genetic admixture between the lineages of C. pratensis s.str., which is reflected by diffuse borders in their contact zones. In conclusion, we identified an interplay of diverse processes that have driven the evolution of the species studied, including allopatric and ecological divergence, hybridization, multiple polyploid origins, and genetic reshuffling caused by Pleistocene climate-induced range dynamics.