Systematics of the freshwater leech genus Hirudinaria Whitman, 1886 (Arhynchobdellida, Hirudinidae) from northeastern Thailand

In total, 435 specimens of the Southeast Asian freshwater leech species within the Hirudinidae family were collected from 17 locations of various types of aquatic habitats in northeastern Thailand. They were all morphologically placed within the genus Hirudinaria Whitman, 1886 and there were three distinct species: the common Hirudinariamanillensis, 78.2% of all collected specimens and at all 17 locations, Hirudinariajavanica at 20.3% of collected samples and from five locations and a rarer unidentified morphospecies (Hirudinaria sp.) with six samples from only two locations. The karyotypes of these three species were examined across their range in this study area for 38, 11 and 6 adult specimens of Hirudinariamanillensis, Hirudinariajavanica and Hirudinaria sp., respectively. This revealed different chromosome numbers among all three species, with Hirudinariajavanica having n = 13, 2n = 26, Hirudinariamanillensis lacked one small chromosome pair with n = 12, 2n = 24, and the unknown Hirudinaria sp. differed from any known Hirudinaria karyotypes in exhibiting a higher chromosome number (n = 14, 2n = 28) and a gradual change in size from large to small chromosomes. This suggests that the unknown Hirudinaria sp. is a new biological species. However, phylogenetic analysis based upon a 658 bp fragment of the cytochrome oxidase subunit I gene placed this unknown morphospecies within the Hirudinariamanillensis clade, perhaps then suggesting a recent sympatric speciation, although this requires further confirmation. Regardless, the chromosomes of all three species were asymmetric, most with telocentric elements. A distinct bi-armed chromosome marker was present on the first chromosome pair in Hirudinariajavanica, whilst it was on pairs 1, 2, 3 and 5 in Hirudinariamanillensis, and on pairs 3 and 5 for the unknown Hirudinaria sp.