MOLECULAR PHYLOGENETIC ANALYSIS OF Crotalus durissus (VIPERIDAE: CROTALINAE) SUBSPECIES. INFERENCE FROM MITOCHONDRIAL DNA

R. T. Yassaka(1), Rádis-Baptista G. (1), Callefo, M.E.V.(2), Furtado, M.F.(2), Yamane, T. (1)

(1)Laboratory of Molecular Toxinology, Instituto Butantan, (2)Laboratory of Herpetology, Instituto Butantan, Avenida Vital Brazil, 1500, SãoPaulo, 05503-900.

The present work aims at, using sequences of conserved genes, to assist the systematic of serpents of the Crotalus genus. For this study, were collected, in the limits of the Southeastern region of Brazil, 20 individuals of the Crotalus durissus terrificus subspecies, 3 individuals of the Crotalus durissus colillineatus subspecies and 4 individuals of theCrotalus genus - whose morphologic classification was not possible. The blood was taken from the esofagic vein. Mitochondrial DNA was purified by SDS and proteinase K method. Oligonucleotide primers, specific for conserved domains of cytochrome b and subunit 4 of the NADH dehydrogenase - both of the mitochondrial DNA – were used in PCR reactions. The amplified products that correspond to the segments of cytochrome b gene (758 bp) and gene of subunit 4 of the NADH (900 bp) were cloned, propagated in E. coli cells and sequenced. At the present moment, the sequences are being analyzed using different programs, among them, the EditSeq and the SeqMan in the package of DNA Star software (Lasergene, Madison, WI). The alignment of the sequences is based on the Clustal method. Both nucleotide and amino acid sequences are used for the cladistic analysis and for construction of phylogenetic tree. The consistence of those relationships is proved by maximum parsimony and maximum likelihood methods, based on reliable coefficients (Bootstrap, Half Jack-Knife).

The fragments of mitochondrial DNA genes can supply information to complement the morphological analysis and establish the systematic of these taxa, Crotalus durissus subspecies. Molecular data from Crotalus durissus ruruima is being added, to corroborate this analysis. Comparison of the sampling loci and the monophilyletism of the taxa, contributes to elucidate the history and the taxonomic classification of this group, emphasizing differences in this subspecies level or the polymorphic aspects.

Financial support: FAPESP, FUNDAP

CORRESPONDENCE TO:

R. T. Yassaka, Laboratory of Molecular Toxinology, Instituto Butantan, Avenida Vital Brazil, 1500, São Paulo, 05503-900, Brasil, Email: ryassaka@hotmail.com