Abstract 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.

A eritropoese e a atividade de transcrição de DNA de hemoglobina foram analisadas em Odontophrynus americanus diplóides e tetraplóides. Dados de conteúdo celular relativo de DNA, RNA e mitocôndrias obtidos por citometria de fluxo mostraram uma atividade gênica aumentada em ambos os animais durante a eritropoese in vivo estimulada por tratamento com fenilhidrazina, com valores maiores em eritrócitos tetraplóides imaturos. No décimo dia de recuperação da anemia foram encontradas grandes quantidades de RNA mensageiro em ambos espécimens. Com base no conteúdo mitocondrial, as células tetraplóides apresentaram um metabolismo energético mais intenso. O. americanus diplóides apresentaram três vezes mais células eritróides que os animais tetraplóides, caracterizando um mecanismo regulador da produção de células eritróides. Determinações hematológicas mostraram que as células tetraplóides contêm 30% mais hemoglobina que as células diplóides. Uma vez que 25-30% mais ribossomos estão presentes nas células tetraplóides, parece que um mecanismo regulador da síntese de hemoglobina esteja relacionado ao nível transcricional. Como as inclusões citoplasmáticas semelhantes a corpos de Heinz, porém associadas com RNA ou RNP, são encontradas em ambos os espécimens, é possível que outro mecanismo regulador esteja presente, similar ao das "hidden breaks" descritas em peixes poliplóides, acumulando rRNA transcrito em excesso e não traduzido. Estas diferenças em níveis fisiológicos e moleculares, detectadas durante a eritropoese, reforçam que divergências em nível de especiação estão em curso entre os O. americanus diplóides e tetraplóides.

Erythropoietic and hemoglobin DNA transcriptional activities were analyzed in the diploid and the tetraploid Odontophrynus americanus. Flow cytometric analyses of DNA, RNA and mitochondrial contents showed increased genic activity in both diploid and tetraploid animals during erythropoiesis in vivo elicited by pretreatment phenylhydrazine. Generally, higher values were seen in immature tetraploid erythroid cells. On the 10th day of recovery from anemia, large amounts of messenger RNA were found in both specimens. Based on the mitochondrial content, the tetraploid cells had more intense energy metabolism than the diploid cells. Diploid O. americanus had about three times more erythroid cells than tetraploid specimens, indicating that there were differences in the regulatory mechanisms of erythroid cells. Hematological parameters showed that tetraploid cells had 30% more hemoglobin than the diploid, suggesting a regulatory mechanism of hemoglobin synthesis at the transcriptional level. Cytoplasmic inclusions resembling Heinz bodies were found in both types of cells. In the tetraploid cells they were previously found associated with RNA or RNP, suggesting that other regulatory system which controls the accumulation of nontranslated RNA transcribed in excess must be present. These differences at the physiological and molecular levels during erythropoiesis reinforce the hypothesis that speciation is occurring between diploid and tetraploid O. americanus.