Evidence from the primary structures of dermal antimicrobial peptides that Rana tagoi okiensis and Rana tagoi tagoi (Ranidae) are not conspecific subspecies

Morphological evidence and data from comparisons of nucleotide sequences of mitochondrial genes demonstrate considerable intraspecies variation among populations of the Japanese brown frog Rana tagoi Okada 1928 (Tago's brown frog). Five peptides with antimicrobial activity were isolated from an extract of the skins of specimens of Rana tagoi okiensis collected on the Oki Islands, Japan. Determination of their primary structures demonstrated that two peptides belong to the ranatuerin-2 family, two peptides to the temporin family, and one peptide to the brevinin-1 family. Ranatuerin-2 peptides were not previously identified in the skin of specimens of R. t. tagoi collected in Chiba Prefecture, Japan and the structures of the temporin peptides from R. t. okiensis (temporin-TOa: FLPILGKLLSGFL.NH2 and temporin-TOb: FLPILGKLLSGLL.NH2) are different from temporin-TGa (FLPILGKLLSGIL.NH2) isolated from R. t. tagoi. Similarly, the acyclic C-terminally α-amidated brevinin-1 peptide from R. t. okiensis (Brevinin-1TOa, GIGSILGVIAKGLPTLISWIKNR.NH2) shows three amino acid substitutions (Gly1 → Ala, Val8 → Ala, Ile9 → Leu) compared to the ortholog from R. t. tagoi. In addition, bradykinin, identical to the mammalian peptide, is present in high concentration in the skin of R. t. okiensis but not R. t. tagoi. The data provide evidence to support the proposal that R. t. tagoi and R. t. okiensis should be regarded as separate species (R. tagoi and R. okiensis) rather than conspecific subspecies.