DISCUSSION

The type specimen comes from the Astrakhan region (Ledebour et al., 1833), and Geltman (1986) even treated this taxon as endemic to the Volga river delta. By detailed morphological examination of the items (obtained from D. Geltman, St. Petersburg), the individuals fall into a well‑separated cluster of U. d. subsp. pubescens in the morphometrics analysis (CS II). Unfortunately, it was not possible to verify the ploidy level (other plants from the region were proved to be diploid, Geltman pers. comm.) or obtain any molecular data from the items. Moreover, when investigating the original locations in the Volga river delta, no individuals were found. As a result, it was impossible to provide an acceptable explanation for this disjunction and prevalence of diploids in the Po Plain.

Origin of tetraploid cytotype

In the cytogeographical study, the predominance of tetraploid cytotype (mostly referred to as U. d. subsp. dioica) has been confirmed, which widely occurs in various synanthropic habitats and has a cosmopolitan distribution (Meusel et al., 1965; POWO, 2022; CS I). Subsequent molecular analyses (Hyb‑Seq approach) and extraction of SNPs showed a clear differentiation of Middle‑Eastern tetraploid individuals. That also corresponded to the splitting of tetraploids into two separate branches of the species tree, comprising European individuals, which merged with all diploids, and plants from Southwest Asia (Anatolia, Iran, and Georgia; CS II). The molecular data did not indicate that Asian tetraploids originated from the partly co‑occurring diploids assigned to U. d. subsp. kurdistanica. One possible explanation is that sampling density is low in Asia compared to Europe, which could lead to omitting any ancestral diploids. Another possible explanation is that tetraploids originated from other sympatric diploids (e.g., mentioned diploid subspecies from Central Asia – U. d. subsp. afghanica and U. d. subsp. gansuensis; Chen et al., 2003) outside the study area and migrated into the area covered by the study. Finally, the least likely is that hybridization occurred at the tetraploid level.

The examples of the possible spontaneous formation of tetraploids are the tetraploid individuals collected in mixed‑ploidy populations with diploids assigned to U. d. subsp.

pubescens in the Po Plain. The absence of any other diploid taxa within the distance of 200 km suggests that the individuals are likely of autopolyploid origin (CS II). This hypothesis is supported by the morphometric analysis, where the tetraploid individuals from mixed‑ploidy populations either fell into the well‑separated diploid cluster of U. d. subsp. pubescens or formed a separate cluster on the edge of other tetraploids assigned to U. d. subsp. dioica (CS II).

The morphometric evaluation of both ploidy levels shows the overlap between

the tetraploids (U. d. subsp. dioica) and diploids assigned to U. d. subsp. subinermis and

U. d. subsp. sondenii, which might indicate the participation of these plants in the origin

of tetraploids. However, it cannot be excluded the possibility of the formation of

tetraploids from a different mixture of allo‑ and autopolyploid lineages (hybridization

among different diploids followed by polyploidization), which hybridize further at 4x

8. DISCUSSION |

level, which might lead to a complicated origin that is difficult to uncover. For European samples outside of the Po Plain, the molecular data do not provide any plausible evidence for the origin of tetraploid individuals due to the lack of variability at both diploid and tetraploid level.

Therefore, it was impossible to elucidate the evolutionary history of these tetraploids, which also reflected the results from the morphometric evaluation and previous complicated taxonomy of U. d. subsp. dioica in the latest phylogenies (Henning et al., 2014; Grosse‑Veldmann et al., 2016b; CS II).

Taxonomic consequences

Although the different sets of molecular markers were used and all plants were checked by flow cytometry, no structure within Urtica dioica (CS II) has been found, as well as in the case of previous phylogenetic studies (Henning et al., 2014; Grosse‑Veldmann et al., 2016b). On the other hand, the results have contributed to delimitation within the related species to U. dioica. The position of related species U. kioviensis and U. gracilis was clearly delimited, corresponding to Henning et al. (2014), who obtained almost the same topology using ITS (internal transcribed spacer) and chloroplast DNA regions (trnS–G, trnH–psbA and trnL–F). The results have also contributed to clarifying the position of endemic U. cypria from Cyprus, which had been considered a subspecies of U. dioica (Weigend, 2006). However, the molecular data supported by chromosome counts (Table 1, CS II, Appendix III), and nuclear genome size estimation (Table 1, CS I) suggest separation to the species rank, as previously proposed by Hand (2019). Together, these support their assignment to other Mediterranean endemic species (Urtica atrovirens, U. bianorii, U. rupestris), and confirming the position outside of U. dioica s.s. clade. In the first study (CS I), the 1Cx‑value for U. cypria has been estimated incorrectly, because it was expected it to be triploid, as suggested by its genome size compared to other subspecies of U. dioica. Besides that, other included species are well‑recognizable based on their genome size (Table 1; except U. atrovirens, which has the same genome size as the mutually indistinguishable diploid subspecies of U. dioica). Genome size can aid in the detection of taxa and may be indicative of genetic distance. One example is U. bianorii, which was previously classified as a variety or subspecies of U. atrovirens and was often mistakenly identified, while genome size can differentiate it reliably (Paiva, 1993; similar examples in other taxa e.g., Loureiro et al., 2010; Vít et al., 2016; Yan et al., 2016). Additionally, species from the Urtica genus can be determined by the differing morphology of their achenes, although this is not applicable at subspecies level (Chrtek, 1979; Wolters et al., 2005;

Appendix IV).