MOLECULAR GENETIC CHARACTERIZATION OF UKRAINIAN REPRESENTATIVES OF THE COMPLEX SPECIES ACONITUM ANTHORA BASED ON THE ITS1-5.8S- ITS2 REGION OF THE NUCLEAR GENOME

Abstract

Problem. The question of determining the boundaries of a species, in addition to the fundamental aspects, is of practical importance for nature conservation activities. One of the taxonomically problematic groups is the complex species A. anthora s. l. In the flora of Ukraine, four taxa with controversial status are distinguished within its borders, A. anthora L., A. eulophum Rchb., A. jacquinii Rchb. and A. pseudanthora Błocki ex Pacz. The last two species are included in the Red Book of Ukraine and the IUCN list. Determining the taxonomic position of these closely related forms requires the use of molecular genetic approaches. The ITS1-5.8S-ITS2 region of 35S rDNA is considered the most popular and informative marker for plant taxonomy and barcoding. Aim. To clarify the taxonomic status and phylogenetic relationships of representatives of A. anthora s. l. using the molecular marker ITS1-5.8S-ITS2 of 35S rDNA. Methods. Herbarium specimens of four representatives of the group A. anthora s. l. were collected on the territory of Western Ukraine. DNA was isolated by a modified CTAB method and then used for PCR amplification of the ITS1-5.8S-ITS2 region. PCR products were purified and Sanger sequenced. The sequences obtained were aligned with the sequences deposited in the GenBank database for representatives of two subgenera of the genus Aconitum, i.e. the nominative subgenus and the subgenus Lycoctonum. The resulting matrix was used for principal component analysis (PCA) and phylogenetic analysis using the Maximum Likelihood method. Main results. The sequences of ITS1-5.8S-ITS2 that we obtained for A. anthora, A. eulophum, A. jacquinii, and A. pseudanthora samples were found to be almost identical. They differed from each other in only five of the 641 alignment positions. Among them, four out of five positions appeared to be polymorphic even within individual genomes. According to the results of PCA analysis, all four samples of A. anthora s. l. formed a dense cluster separated by significant distances from the clusters formed by representatives of the subgenera Aconitum and Lycoctonum. In the generated phylogenetic tree, three main monophyletic clades are present, which correspond to the subgenera Aconitum and Lycoctonum and the A. anthora s. l. group. The Lycoctonum clade was sister to other members of the genus, and the Aconitum and A. anthora s. l. clades were sister to each other. Conclusions. The use of the ITS1-5.8S-ITS2 region of 35S rDNA for phylogenetic analysis confirms the need to grant the A. anthora s. l. group the status of a separate subgenus within the genus Aconitum. The high genetic affinity between samples of A. anthora s. l. from the territory of Ukraine indicates that the species names A. eulophum, A. jacquinii, and A. pseudanthora should be considered synonyms of A. anthora.