Molecular analysis of threatened species of the freshwater snail genus Potamolithus (Prosobranchia: Truncatelloidea: Tateidae) in the Río de la Plata, Argentina

摘要

Molecular analyses were initiated from specimens of Potamolithus collected in January 2018 on Martín Garcia Island (Province of Buenos Aires). The specimens were first separated into five morphs according to shell shape and coloration, from which a foot fragment fixed in absolute alcohol was prepared from each specimen. The total genomic DNA was extracted, by the CTAB method for 15 specimens and by commercial kit for 14 specimens. The partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified by primers following Folmer et al. (1994). The amplification by Polymerase Chain Reaction (PCR) was performed in a final volume of 50 μl. The PCR products were purified using a Pure DNA-Clean Up kit (PB-L Bio-Logic Products) and the two DNA strands of the gene were directly sequenced in Macrogen Inc., Seoul, Korea. The resulting sequences were edited using the Chromas Lite and Bioedit software. They were then trimmed to eliminate primers and consensus sequences were compared with reference sequences deposited in GenBank. Sequence alignment was performed with Clustal X 2.0.12 software, optimized by visual inspection, and edited with a word processor. The genetic distance was then analyzed using the Neighbor-Joining algorithm, through the MEGA X program, using the Hasegawa-Kishino-Yano model. The maximum likelihood analysis was performed using the MEGA X program using the Hasegawa-Kishino-Yano model. In total, 23 COI sequences were obtained, which would correspond to 5 entities: (1) entity 1 and 2 correspond to P. agapetus (8 sequences) and P. buschii (4 sequences), both species previously cited for Martín Garcia Island and with registration of the COI gene in Genbank; (2) entity 3 corresponds anatomically with P. bisinuatus (7 sequences) -already mentioned for Martín Garcia, the first sequences obtained for this species; (3) entity 4 may correspond to P.