Even for a frog expert, it is practically impossible to differentiate the species just by looking Physalaemus cuvieri e Physalaemus ephippifer Both can be found in flooded savannas and in mirrors of water formed in the middle of pastures, from the Amazon to Rio Grande do Sul. Roughly speaking, the specimens from the northern region were called P. ephippifer and the others from P. cuvieri or, popularly, dog-frog.
Now, new studies funded by FAPESP suggest that this group of amphibians may harbor not just two, but between four and seven species, which can only be accurately differentiated through advanced molecular biology techniques.
So far, no morphological characteristics have been sufficient to discriminate these animals with brown head and back, dark and irregular lateral stripes and reddish spots on the inner thighs and groin. Also, no specificity was observed in the song intoned by males to attract females. However, scientists found cytogenetic and molecular differencesrobust enough to allow subdivision.
The work was published in the journal Frontiers in Genetics - Evolutionary and Population Genetics by researchers from the State University of Campinas (Unicamp), Paulista State University (Unesp), Institute of Scientific and Technological Research of the State of Amapá (IEPA), Federal University of Maranhão (UFMA) and institutions from Argentina and the United States.
"In theory, this group of amphibian frogs is composed of two species, but a few years ago we did a study of the karyotypes of specimens of this group and began to suspect that there were other species besides the two already described," said Luciana Bolsoni Lourenço, a professor at the Institute of Biology (IB) at Unicamp andcoordinator of the study.
The work mentioned by the researcher was conducted as part of a project funded by FAPESP. To investigate the hypothesis raised, the group collected frogs of these two supposed species in nine locations in western Pará and one location in Roraima. In addition, the researchers increased the amount of molecular markers - DNA sequences capable of revealing differences between individuals- used to classify species.
The researcher did part of the work during an internship at the University of Tennessee, in the United States, with the support of FAPESP. Lourenço had the collaboration of Benjamin Minault Fitzpatrick, one of the co-authors of the article.
The work also integrates a Thematic Project and a Research Grant - Partnership for Technological Innovation (PITE), both coordinated by Célio Fernando Baptista Haddad, professor of the Institute of Biosciences at Unesp, in Rio Claro, who also signs the article.
Despite the molecular differences, which became even more evident when the so-called RAD-seq (restriction site-associated DNA sequencing) markers were included, the formal description of new species still demands more classical knowledge.
To be able to classify species as cryptic, that is, whose differences can only be perceived at the molecular level, researchers need to make sure that there are, in fact, no morphological or acoustic characteristics that discriminate them. In addition, more individuals from outside the Amazon need to be analyzed to have a larger and more representative sample.
The fact that the animals are so similar physically raises the question that there could be mating between the species. Data not yet published by the group bring evidence of some contact zones between the different genetic lineages. Therefore, the researchers are now interested in assessing whether differences in karyotypes may play a role in the isolation of these lineages.
One feature that differs P. ephippifer In this species, females present heteromorphic sex chromosomes, that is, easily distinguishable from each other. In the others, sex chromosomes are homomorphic (equal in males and females).
"One hypothesis is that relative differences in sex chromosomes entail a reproductive barrier, which may have aided in the isolation of some of the strains," said the researcher.
The discovery that in this group previously composed of two species may actually exist between four and seven has an impact on conservation actions. currently, P. cuvieri e P. ephippifer are in the "least concern" category of the International Union for Conservation of Nature (IUCN) red list of threatened species.
It is not known whether this status will prevail for new members of the group, who may be in areas more susceptible to human action, for example.
"When there are two species distributed over a large number of localities, the concern for conservation policies may be quite different from that in a scenario with several species occurring in more restricted areas," Lourenço said.