Abstract:

Aims: Integrative taxonomy, which combines multiple lines of evidence, offers an effective approach for resolving species boundaries. The cannabina Clade of the genus Urtica is widely distributed across the “Third Pole”, a region characterized by exceptional biodiversity and distinctive ecological environments. Despite their wide distribution, species boundaries within this clade have been challenging due to complex morphological traits and complicated evolutionary history. In this study, we conducted an integrative taxonomic assessment of four species of cannabina Clade (Urtica cannabina; U. triangularis; U. dioica; U. hyperborea) from the Third Pole by integrating genomic data with analyses of geographic distribution and morphological traits, using U. mairei, U. membranifolia and several related genera as outgroups. 

Methods: Geographic boundaries were delineated using ArcGIS based on occurrence records from GBIF, PPBC, NPSRC, iNaturalist, and records collected from the field. Morphological traits differentiation was assessed via Principal Coordinate Analysis (PCoA) of 16 morphological traits. Phylogenetic relationships and population genetic structure were reconstructed based on plastid genome data and the Angiosperms353 nuclear gene set from 50 individuals of the cannabina Clade. 

Results: Our analyses revealed the following key findings: (1)Urtica hyperborea is a well-supported monophyletic clade, exhibiting distinct morphological and geographic range. Although it overlaps in distribution with U. dioica in the Himalaya and Hengduan Mountains, they typically occupy different altitudinal gradients; (2)Urtica dioica subsp. afghanica and U. dioica subsp. gansuensis display significant divergence in distribution, morphology, and genomic from U. dioica subsp. dioica, suggesting the possible presence of cryptic species. However, their taxonomic status requires further investigation; (3) Urtica triangularis, U. cannabina, and U. dioica show geographical overlapping and little clustering difference in both morphological and genetic data. The observed cyto-nuclear discordance indicates potential genetic introgression among these taxa. Morphological convergence among species in high-altitude environments might contribute to the difficulty in distinguishing between them based on their traits. Furthermore, our study demonstrates that the integration of plastome data with the Angiosperm353 nuclear gene dataset shows great promise in delimiting closely related species. 

Conclusion: Through the integration of multiple lines of evidences, this study comprehensively delineates species boundaries within the cannabina Clade and offers new insights into plant evolutionary processes in the Third Pole. These findings not only enhance our understanding of interspecific relationships and distribution patterns within the genus Urtica, but also offer a robust methodological framework for exploring the integrated taxonomy in other plant groups across the Third Pole region.

Key words: Urtica, closely related species, Third Pole, Angiosperms 353 genes, plastid genome, integrative taxonomy, species boundary