Morphological and molecular identification of leech species of Lankaran-Astara (Azerbaijan) region

Leeches are a class within Annelida and there are more than 700 identified species. Although the majority of leeches live in fresh waters, 15% of them have adapted to the seas and a few of them have adapted to terrestrial life. This research was carried out between 2019 and 2022 to determine the morphological and molecular features of Hirudinea fauna living in the Lankaran-Astara region of the Republic of Azerbaijan. In total 62 wetlands covering 6 provinces as Astara, Jalilabad, Masally, Lankaran, Lerik, and Yardimly in the Lankaran-Astara region were observed and among these wetlands in 33 areas, leeches determined. The leeches collected from the wetlands were transported with special containers in 70% alcohol to Fırat University, Fisheries Faculty, Fish Diseases Laboratory. In order to determine the leech species, first of all, classification was made by dissection under the microscope for morphological identification. Afterward, sequencing of the COX1 mtDNA gene region of the classified samples was carried out. At the result of the study, 6 leech species belonging to 3 families, Erpobdellidae (Dina lineata, Trocheta n.sp.), Glossiphoniidae (Helobdella stagnalis, Hemiclepsis marginata, Placobdella costata) and Hirudinidae (Hirudo orientalis) were identified. Phylogenetic trees were obtained by using Maximum Likelihood (ML), Maximum Parsimony (MP) and Neighbour-Joining (MJ) methods using a total of 28 DNA fragments consisting of 15 mtDNA sequences of these leeches and 14 comparison sequences obtained from GenBank. By comparing the phylogenetic trees obtained by these different methods, the definitive diagnosis and identification of the Azerbaijan leeches were reached. Although Placobdella costata and Hemiclepsis marginata species also were determined morphologically in the region, no sequences obtained for the COX1 gene region in DNA analysis. In the study, Trocheta n. sp. was determined to be a new species because the mtDNA COX1 gene region differed by 8-14% from other species of this genus. DNA sequences of Azerbaijan leech species were uploaded to GenBank database and their registration numbers were obtained. By using the 15 mtDNA sequences obtained in this study, the haplotype status and network of leech species were prepared for Azerbaijan. As a result of these analyzes, H. orientalis, Trocheta n. sp. and H. stagnalis species were determined to be gathered under a single haplotype roof. However, D. lineata species belonging to the Erpobdella family were found to be grouped under 5 different haplotypes.