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Hirudinaria manillensis (Lesson, 1842), commonly known as the buffalo leech, shows a polymorphism of two ventral colorations. The green color morph has a plain green ventral surface and the red color morph has a brick-red ventral surface with two black submarginal stripes. Based on molecular and morphological evidence in the present study, these two color morphs were revealed as two different species. The red color morph fits well with the description of H. manillensis, while the green color morph showed some distinctions, and therefore is described herein as Hirudinaria thailandica Jeratthitikul & Panha, sp. nov. The new species can be distinguished from its congeners by the dark greenish or dark olive ventral surface and a round atrium with ventral insertion of ejaculatory ducts in the male reproductive organ. A phylogenetic tree based on concatenated data of COI and 28S genes supported the new species and further indicated it as a sister species to H. bpling Phillips, 2012.

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 Hirudinaria manillensis, 78.2% of all collected specimens and at all 17 locations, Hirudinaria javanica 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 Hirudinaria manillensis, Hirudinaria javanica and Hirudinaria sp., respectively. This revealed different chromosome numbers among all three species, with Hirudinaria javanica having n = 13, 2n = 26, Hirudinaria manillensis 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 Hirudinaria manillensis 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 Hirudinaria javanica, whilst it was on pairs 1, 2, 3 and 5 in Hirudinaria manillensis, and on pairs 3 and 5 for the unknown Hirudinaria sp.