Altai Mountains - cradle of hybrids and introgressants: A case study in Veronica subg. Pseudolysimachium (Plantaginaceae) Author Khan, Gulzar Eike Mayland-Quellhorst, & Petr A. Kosachev, & Institute for Biology and Environmental Sciences, Carl von Ossietzky-University Oldenburg, Carl von Ossietzky-Str. & type. The GISH investigation in one case strongly supports homoploid hybridization (origin of V. × schmakovii from V. longifolia and V. porphyriana. Divergence times of Altai Veronica species are estimated to be within & - Author Mayland-Quellhorst, Eike Eike Mayland-Quellhorst, & Petr A. Kosachev, & Institute for Biology and Environmental Sciences, Carl von Ossietzky-University Oldenburg, Carl von Ossietzky-Str. & type. The GISH investigation in one case strongly supports homoploid hybridization (origin of V. × schmakovii from V. longifolia and V. porphyriana. Divergence times of Altai Veronica species are estimated to be within & - Author Kosachev, Petr A. Terezie Mandáková, & Faculty of Biology, Altai State University, Lenina 61, 656049 Barnaul, Russia & million years ago with high probability of gene flow over that time. Our results also demonstrate that the direction of gene flow is mainly from the locally endemic V. porphyriana. We hypothesize that the large Siberian plains and topographically diverse foreland of the Altai Mountains provide an ideal setting for hybridization with the potential for adaptive introgression of alleles conferring tolerance to cooler climates, to the lowland species migrating into the Altai Mountains. Author Mandáková, Terezie & Dirk C. Albach & Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Author Lysak, Martin A. & Dirk C. Albach & Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Author Albach, Dirk C. Eike Mayland-Quellhorst, & Petr A. Kosachev, & Institute for Biology and Environmental Sciences, Carl von Ossietzky-University Oldenburg, Carl von Ossietzky-Str. & type. The GISH investigation in one case strongly supports homoploid hybridization (origin of V. × schmakovii from V. longifolia and V. porphyriana. Divergence times of Altai Veronica species are estimated to be within & - text TAXON 2024 2024-04-30 73 2 530 546 http://dx.doi.org/10.1002/tax.13176 journal article 305150 10.1002/tax.13176 73548f05-40de-4973-a03f-cc7ca3fcce2a 1996-8175 14047615 ■ RESULTS Species boundaries and cohesion. — The assignment test in STRUCTURE with the dataset including all 233 individuals recovered K = 5 as optimal both by Evanno Δ K and likelihood scores (suppl. Fig. S3 ), which differs from the expected K = 10 based on species assignment ( Table 1 ). The result of three different groupings, K = 4, 5, 6 recovered more or less highly genetically cohesive groups including Veronica longifolia , V . pinnata , V. porphyriana and V . spicata ; however, the individuals from V . incana , in K4 showed admixture of V . longifolia and V. porphyriana , but were recovered as a separate group in K5 and K6 with high genetic portion from V . porphyriana ( Fig. 1 ). Removing the putative hybrids and including only putative purebreds (174 individuals, ~75% of total dataset), the STRUCTURE result of K = 5 again showed more or less the same results ( Fig. 2B , suppl. Fig. S3 ). The DAPC recovered 20 principal components as the best to group our data and clearly resolved all the individuals into K = 5 ( Fig. 2C ; suppl. Fig. S4 ). The DAPC results were supported by the unrooted trees inferred by IQ-TREE ( Fig. 2A ). The morphometric analysis (suppl. Appendix S1.) also supported the results of the genetic analyses in delimiting five main groups similar to those in Fig. 1 . Genetic differentiation among species based on AMOVA was F ST = 0.16 ( Table 2 ). The hierarchical STRUCTURE analysis retrieved five cohesive groups including Veronica incana , V. longifolia , V. pinnata , V. spicata and V. porphyriana . In addition to these five groups, V. arenosa merged with V. pinnata , while V. sajanensis , and V. spuria with V. spicata . However, V. reverdattoi and V. taigischensis did not fit to any clear group (suppl. Table S2 ). Chromosome localization of rDNA loci and classes of putative hybrids. — Chromosome numbers and rDNA loci were determined in six species. Veronica longifolia , V. pinnata , V. porphyriana , V. ×schmakovii , and V. spicata were found to have 2 n = 2 x = 34 chromosomes and one 5S rDNA-bearing chromosome pair, whereas V. incana had a tetraploid chromosome number (2 n = 4 x = 68) with a corresponding double number of 5S loci, i.e., two 5S-bearing chromosome pairs. In contrast to the conserved number of 5S rDNA, we found a large interspecific variation in the number of 35S rDNA: four NOR-bearing chromosomes in V. porphyriana , six in V. ×schmakovii and V. spicata , seven in V. pinnata , eight in V. longifolia , and at least nine in the tetraploid V. incana . In V. incana , additional very weak signals were observed ( Fig. 3 ). Each gDNA of V. longifolia and V. porphyriana hybridized to one half of the chromosome complement of V. ×schmakovii . Therefore, GISH strongly suggests a homoploid hybrid origin of V. ×schmakovii from parental genomes closely related to V. longifolia and V. porphyriana . This is also supported by the number of 35S rDNA-bearing chromosomes in V. ×schmakovii (six), an intermediate value between V. longifolia (eight) and V. porphyriana (four). The hypothesis of hybridization between and among species of Veronica in the Altai Mountains based on their morphotypes was mostly supported by STRUCTURE, and NewHybrids ( Fig. 4 ; suppl. Table S3). The first-level STRUCTURE analysis is a bit more inconclusive in that respect showing admixture of various levels involving both the biparental hybrids and potential triparental hybrid ( Fig. 2 ). The maximum likelihood tree based on all individuals also revealed that the putative hybrid individuals are mostly sister to one of the suggested parents (suppl. Fig. S5 ). The genetically admixed individuals were mostly the ones identified as hybrids a priori by morphological characteristics (suppl. Fig. S1 ); however, we also recovered admixture in individuals considered to be pure species, e.g., V . sajanensis , V . spuria , V . taigischensis , and V . reverdattoi . The STRUCTURE results showed that in most cases the putative hybrid individuals in all five scenarios had posterior probabilities ranging between 0.15 <Q <0.50 or 0.50 <Q <0.85, which suggests a hybrid swarm from F1 to later-generation hybrids ( Fig. 4 ; suppl. Table S3). The analysis implemented in NewHybrids to group the hybrid individuals supported the results of STRUCTURE with the exception of scenario 5 and recovered hybrids of different classes but dominated by F1 (suppl. Table S3). Fig. 2. Assignments of individuals to K clusters/populations based on Bayesian, multivariate and likelihood approaches as implemented in STRUCTURE, DAPC (in the adegenet package) and IQ-TREE programs respectively. A , Maximum likelihood unrooted tree reconstructed in IQ-TREE, calibrated with 1000 ultrafast bootstrap replicates. The numbers near the nodes represent bootstrap confidence. This analysis was based on 174 putative pure individuals. B , Probability of ancestry of each individual (horizontal axis; total 174 individuals) to each of K = 5 populations (vertical axis). The five populations correspond mostly to the morphotypes hypothesized for putative pure species. C , Discriminant analysis of principal components using the same dataset as in (B). D , Corresponding morphotypes of K = 5. — Photos: P.A. Kosachev. Fig. 2. Continued. Demographic history of pure species. — Using G-PhoCS to model the associated demographic history in the species of Veronica ( Fig. 5 ), we retrieved divergence times between the major clades and branches to be recent (1.14 Mya to 1.57 Mya) at 95% HPD (highest posterior density; see suppl. Table S4 for complete information). Even with the upper HPD at 95%, the divergence time was in the range of 1.35 Mya to 1.84 Mya. Among the five species, the earliest recently diverged species are V . porphyriana and V . incana (1.14 Mya), while the most divergent species is V . pinnata (1.84 Mya) at upper 95% HPD. Similarly, the lowest effective population size is that of V . pinnata (4280 individuals) followed by V . porphyriana (6150 individuals), while the largest one belongs to V. spicata (21,000 individuals) at average 95% HPD. The effective population sizes of their ancestors were also different in comparison with the extant effective population sizes of all the five species, e.g., the smallest one of V . porphyriana and V . spicata ancestors (1160 individuals), whereas the largest one for the ancestor population of all species (16,100 individuals) ( Fig. 5 ; suppl. Table S4). Our modeling of gene flow in both forward and reverse directions showed high probability of frequent gene flow from V . longifolia and V . pinnata to V . incana , V. porphyriana and V . spicata . Nevertheless, from V . incana and V . porphyriana to the other three species, the probability of gene flow was also comparatively high. Similarly, the gene flow among V . incana , V . porphyriana and V . spicata was high in both forward and reverse directions ( Fig. 5 ; suppl. Table S4).