Snake venomics of Bitis species reveals large intragenus venom toxin composition variation:: Application to taxonomy of congeneric taxa

The protein composition of the venoms of the West African Gaboon viper (Bitis gabonica rhinoceros), the rhinoceros viper (Bitis nasicornis), and the horned puff adder (Bitis caudalis) were analyzed by RP-HPLC, N-terminal sequencing, SDS-PAGE, MALDI-TOF peptide mass fingerprinting, and CID-MS/MS. In line with previous proteomic and transcriptomic analyses showing that snake venom proteins belong to only a few major protein families, the venom proteomes of Bitis gabonica rhinoceros, Bitis nasicornis, and Bitis caudalis comprise, respectively, toxins from 11, 9, and 8 toxin families. Dimeric disintegrins, PLA(2) molecules, serine proteinases, a CRISP, C-type lectin-like proteins, L-amino acid oxidases, and snake venom metalloproteases are present in the three Bitis snake venoms, though they depart from each other in the composition and the relative abundance of their toxins. The venom composition appears to keep information on the evolutionary history of congeneric taxa. Protein similarity coefficients used to estimate the similarity of venom proteins of the Bitis taxa sampled here and in previous studies (eg. Bitis arietans and Bitis gabonica gabonica) support the monophyly of the three West African taxa (B.g. gabonica, B.g. rhinoceros, and B. nasicornis) based on genetic distance reconstructions, the lack of alliances between B. arietans and any other Bitis species, and are consistent with the taxonomic association of Bitis caudalis within the differentiated group of small Bitis species. The low level of venom toxin composition similarity between the two conventionally recognized subspecies of Bitis gabonica, B. g. gabonica and B. g. rhinoceros, supports the consideration by some authors of B. g. rhinoceros as a separate species, Bitis rhinoceros. Moreover, our proteomic data fit better to a weighted phylogram based on overall genetic distances than to an unweighted maximum-parsimony tree.