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Simple Summary: The wetland violets of Central and Northern Europe (Viola epipsila Ledeb., V. palustris L.) are endangered because the ranges of both species are drastically decreased due to global climatic changes. Their disappearance might also be the result of the formation of interspecific hybrids which can replace the parent species. The study of such species is particularly important because they might be considered as indicators of anthropogenic changes occurring in peatlands and their disappearances. The taxonomic situation of studied species is intricate, and the presence of interspecific hybrids and putative introgressants [V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta)] makes it even more complicated. The main goal of our study was to reconstruct the origin of V. pubifolia and its genetic relatedness to both putative parental species—V. palustris and V. epipsila—using advanced molecular methods. The taxonomic problem has been finally solved. We found no basis to separate V. pubifolia as a species in its own right because its morphological characters as well as genetic variation fall well within the range of variability of V. palustris. We have also concluded that the low genetic differentiation and heterozygosity of V. epipsila in Europe might be a cause of the reduced tolerance of this species to changing environmental conditions and can possibly lead to its extinction. In Europe, the V. epipsila—V. palustris group comprises V. epipsila Ledeb., V. palustris L., V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta), interspecific hybrids, and putative introgressants. The genetic affinity of V. pubifolia to V. palustris, and their shared origin via hybridization followed by polyploidization, were confirmed using inter simple sequence repeat (ISSR) markers, restriction site-associated DNA sequencing (RAD-Seq), and a low-copy nuclear gene, GPI, which encodes glucose-6-phosphate isomerase. The other taxa of subsect. Stolonosae were not identified as putative parents of V. pubifolia by GPI. Our analyses indicated that V. pubifolia can be included in the morphological and genetic variation of V. palustris. The ISSR, RAD-Seq, and genome size value separated well V. palustris from V. epipsila and hybrids. The results also reopen the discussion on intraspecific variation in the context of taxa ranks and species concepts. The reduced tolerance of V. epipsila in Europe to changing environmental conditions might result from low genetic differentiation and heterozygosity, as well as the increased number of interspecific hybrids (V. epipsila × V. palustris), and eventually can possibly lead to its extinction. The disappearance of populations/individuals of this species may indicate anthropogenic changes occurring in peatlands. [ABSTRACT FROM AUTHOR]

The Viola epipsila-V. palustris complex is a highly taxonomically complicated group of species in its entire circumboreal range of distribution. Habitat loss, forest flooding, and hybridization could lead to the extinction of V. epipsila. A hybrid index and principal component analysis (PCA) were used to select qualitative and quantitative morphological features to distinguish parent species and hybrids, inter simple sequence repeat (ISSR) markers to determine the genetic diversity of the populations, flow cytometry to estimate the genome size (GS), and non-coding chloroplast DNA (cpDNA) regions to indicate the directions of crosses. All taxa are very morphologically variable, and their features can change within a season. The most stable feature is the distance of the bracts on the pedicel from the rhizome. The genetic diversity of all taxa populations is low and highly influenced by selfing and vegetative propagation. The population structure is differentiated: populations of V. epipsila or V. palustris, mixed populations with both parent species, F1 hybrids and populations with introgressive forms occur in different regions. The interspecific GS variation corresponds to the ploidy level (4x = 2.52 pg, 8x = 4.26 pg, 6x = 3.42 pg). Viola epipsila is the mother plant of the hybrids. Research has shown the risk of V. epipsila extinction in Central Europe and the importance of local populations in studying the role of hybridization in reducing/maintaining/increasing biodiversity. [ABSTRACT FROM AUTHOR]