ABSTRACT. We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The nonbanded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were Method for peripheral blood lymphocyte culture able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.
Two allopatric populations of Brazilian diploid and tetraploid
Odontophrynus americanus
species complex, both from São
Paulo state, had their blood hemoglobin biochemically analyzed. In addition,
these specimens were cytogenetically characterized. Biochemical characterization
of hemoglobin expression showed a distinct banding pattern between the
allopatric specimens. Besides this, two distinct phenotypes, not linked to
ploidy, sex, or age, were observed in adult animals of both populations.
Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw
with reduced interpupillary distance and shorter hind limbs. Phenotype B shows
yellowish-colored body with larger papillae, arch-shaped jaw with broader
interpupillary distance and longer hind limbs. Intermediate phenotypes were also
found. Considering the geographical isolation of both populations, differences
in chromosomal secondary constrictions and distinct hemoglobins banding
patterns, these data indicate that 2n and 4n populations represent cryptic
species in the
O. americanus
species complex. The observed
phenotypic diversity can be interpreted as population genetic variability.
Eventually future data may indicate a probable beginning of speciation in these
Brazilian frogs. Such inter- and intrapopulational differentiation/speciation
process indicates that
O. americanus
species complex taxonomy
deserves further evaluation by genomics and metabarcoding communities, also
considering the pattern of hemoglobin expression, in South American frogs.
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