Hemoglobin protein profile as a parameter for taxonomic analysis in Brazilian Testudinidae

The Brazilian Testudinidae family is widespread across South America. It includes Chelonoidis denticulatus, the largest tortoise in South America and Chelonoidis carbonarius, found mostly in the north and northwestern part of the continent. Using hemoglobin to identify species is cheaper than other methods such as DNA sequencing and can offer useful information, since the hemoglobin molecule is a well-preserved protein chain during the evolution of species. Thus, in order to establish a hemoglobin profile for the Brazilian Testudinidae C. denticulatus, C. carbonarius and morphotype 1, hemoglobin electrophoresis was performed at acid pH in phosphate agar and at alkaline pH in cellulose acetate, in order to visualize the specific fractions of each species. High performance liquid chromatography was used for the quantification of fractions. For an in-depth analysis and better detailing of the hemoglobin profile of the species, polypeptide chain electrophoresis was performed at acid and alkaline pH. We observed differences in the hemoglobin profiles of C. denticulatus in relation to C. carbonarius and morphotype 1, which suggests that this methodology, not common in taxonomic studies, can help determine relationships between species, since hemoglobins are proteins with well-preserved genes. We found differences in hemoglobin mobility between C. denticulatus, C. carbonarius and morphotype 1 in electrophoresis at alkaline pH, however, the behavior of globin chains was similar between the three groups. High performance liquid chromatography showed different retention times in the globin fractions of C. denticulatus and C. carbonarius, but not between C. carbonarius and morphotype 1, indicating that, possibly, the divergence time between C. carbonarius and morphotype 1 is more recent than the divergence between C. denticulatus and C. carbonarius, due to the highly conserved character of this functional protein. Thus, considering the high degree of conservation of hemoglobins in vertebrates, and the differences observed in electrophoresis at alkaline pH and HPLC, we infer that C. carbonarius and morphotype 1 present a common branch.