Hidden Chromosome Clues Reveal Evolutionary Secrets of a Cryptic Frog Group

Deep in the Atlantic Forest and the cerrado landscapes of South America, a group of small, burrowing frogs has been quietly hiding a chromosomal puzzle that has intrigued evolutionary biologists for decades. The genus Physalaemus, which belongs to the family Leptodactylidae and comprises roughly 50 species distributed from southern Venezuela to Uruguay and much of Brazil, is notorious for its cryptic species—animals that look nearly identical on the outside but differ profoundly at the genetic and chromosomal level. A new study published in the journal Comparative Cytogenetics has now filled in a crucial missing piece of this puzzle by describing, for the first time, the complete karyotype of Physalaemus evangelistai, one of only two species in the P. gracilis group whose chromosomes had never been characterized.

The research team, led by scientists from Brazilian and Argentine institutions, applied a combination of classical cytogenetic techniques and modern molecular mapping methods to five species within the P. gracilis group. Their goal was ambitious: to test a long-standing hypothesis about chromosome homology across these closely related frogs, and to determine whether the nucleolus organizer regions (NORs)—the chromosomal sites where ribosomal RNA genes cluster—occupy the same evolutionary position in different species, or whether they have shifted over time through structural rearrangements. The answer, as it turns out, is more nuanced than anyone expected.

Physalaemus evangelistai proved to have a diploid number of 22 chromosomes and a fundamental number of 44, which is entirely consistent with the karyotypic configuration seen across the entire genus. Chromosome pairs 1, 5, 6, and 10 were metacentric, while pairs 2 through 4 and 7 through 11 were submetacentric. C-banding revealed small blocks of constitutive heterochromatin in the centromeric regions of all chromosomes, a pattern that is remarkably conserved across the group. But the real surprise came when the researchers examined chromosome 8 in detail.

In every specimen analyzed, chromosome 8 carried an interstitial NOR on its long arm, coinciding with a secondary constriction visible under standard Giemsa staining. Yet the NOR was not uniform in size across individuals. The team identified two distinct morphotypes: chromosome 8a, bearing a small NOR, and chromosome 8b, carrying a much larger one. Three of the four males examined were heterozygous for this trait, while both females analyzed were homozygous for the smaller variant. More intriguingly, both females carried an additional NOR on one homologue of chromosome pair 9—a feature entirely absent from all four males and from every other karyotyped species in the group.

This sex-specific pattern immediately raised the possibility that P. evangelistai possesses a ZZ/ZW sex determination system, with the NOR serving as a cytogenetic marker distinguishing the sex chromosomes. Such a mechanism has been documented in other anuran species, including Physalaemus ephippifer, Gastrotheca riobambae, Buergeria buergeri, and Hyla femoralis, where NORs have been identified as the sole cytogenetic difference between sex chromosomes. However, the researchers are careful to note that NOR polymorphisms are relatively common among frogs in general, and the limited sample size—particularly the small number of females—prevents any definitive conclusion. Whether this heteromorphism is truly sex-linked or represents an autosomal polymorphism remains an open question that will require larger sampling efforts to resolve.

Beyond the novel karyotype description, the study made a significant contribution to the ongoing debate about chromosome homology within the P. gracilis group. Four of the six species had been karyotyped previously, and their NORs occupy seemingly different positions: interstitial on chromosome 8 in P. carrizorum and P. lisei, terminal on chromosome 8 in P. gracilis, and terminal on chromosome 10 in P. barrioi. Because chromosomes 8 through 11 in these karyotypes are very similar in size and morphology, determining whether the NOR-bearing chromosome of P. barrioi is truly homologous to chromosome 8 of the other species has been extremely challenging. The new study found a subtle but consistent clue: an interstitial DAPI-positive C-band on the short arm of the NOR-bearing chromosomes in P. evangelistai, P. carrizorum from Palmas, and P. barrioi. This shared heterochromatic feature, though faint and easily missed in some metaphase preparations, provides the first independent cytogenetic evidence supporting the hypothesis that these chromosomes are indeed homologous across the group.

The researchers also mapped two repetitive DNA sequences—PcP190 satellite DNA and the U2 small nuclear RNA gene—across the karyotypes of the analyzed species. PcP190 is a satellite DNA family widely distributed among hyloid anurans, having been reported in seven families to date. In the P. gracilis group, it produces a small number of chromosomal clusters arranged in apparently species-specific patterns. The new mapping revealed that P. carrizorum from Palmas, Paraná, carries PcP190 clusters on chromosomes 1 and 3, while P. evangelistai has a cluster only on chromosome 1, and P. barrioi shares the chromosome 1 and 3 pattern. This distribution is phylogenetically informative: the species carrying the chromosome 1 cluster (P. evangelistai, P. carrizorum, and P. barrioi) form one clade, while P. lisei and P. gracilis, which lack it, form another. The most parsimonious explanation is that the chromosome 1 cluster was present in the ancestor of the group and subsequently lost in the lineage leading to P. lisei and P. gracilis.

Perhaps the most striking finding of the entire study concerns P. carrizorum itself. Specimens from Palmas in Paraná, Brazil, differ markedly from those previously described from Misiones, Argentina—localities separated by approximately 242 kilometers. The Palmas population shows a terminal NOR on chromosome 8 and PcP190 clusters on chromosomes 1 and 3, whereas the Misiones population has an interstitial/pericentromeric NOR on chromosome 8 and only a single PcP190 cluster on chromosome 3. Additional differences in heterochromatin distribution further distinguish the two populations. These cytogenetic divergences could reflect intraspecific variation, but they could equally signal the presence of cryptic species hidden within what is currently recognized as a single taxon. The authors emphasize that further taxonomic analyses, integrating morphological, molecular, and bioacoustic data, will be essential to determine which explanation is correct.

The U2 snRNA gene mapping provided a contrasting picture of evolutionary stasis. In all four analyzed species of the P. gracilis group, U2 snRNA gene clusters are restricted to the terminal region of the short arm of chromosome 6. This same condition has been observed in other species of Physalaemus and in the related genus Leptodactylus, supporting the hypothesis that chromosome 6 is homologous across these lineages. While the authors caution that mapping this sequence in additional leptodactylid genera is needed before drawing definitive conclusions, the consistency of this marker across such a broad taxonomic range makes it one of the most reliable chromosomal landmarks available for comparative cytogenetics in the family.

Taken together, these findings paint a picture of a frog group whose genomes are simultaneously conserved and dynamic. The fundamental karyotype—22 chromosomes with a fundamental number of 44—has remained stable across the genus, yet the positions of NORs, the distribution of satellite DNA clusters, and even the number of heterochromatic bands vary considerably between species and even between populations of the same species. This combination of stability and lability makes the P. gracilis group an excellent natural laboratory for studying how chromosomal rearrangements contribute to speciation, particularly in a lineage already known for its high frequency of cryptic diversity. As the authors note, much work remains to be done, but the characterization of P. evangelistai‘s karyotype and the identification of shared heterochromatic markers on NOR-bearing chromosomes represent important steps toward resolving the evolutionary history of this enigmatic group of Neotropical frogs.

Subject of Research: Comparative cytogenetics and karyotype characterization of frog species in the Physalaemus gracilis group

Article Title: Comparative cytogenetics of the Physalaemus gracilis group ( Anura , Leptodactylidae ) with characterization of the karyotype of Physalaemus evangelistai Bokermann, 1967

Article References: Mosquini, P. H. P., Souza, L. H. B., Ferro, J. M., & Lourenço, L. B. (2025). Comparative cytogenetics of the Physalaemus gracilis group (Anura, Leptodactylidae) with characterization of the karyotype of Physalaemus evangelistai Bokermann, 1967. Comparative Cytogenetics, 19, 171-188. https://doi.org/10.3897/compcytogen.19.171637

Image Credits: AI Generated

DOI: 10.3897/compcytogen.19.171637

Keywords: Physalaemus, cytogenetics, karyotype, NOR, satellite DNA, FISH, anurans, chromosome evolution, Leptodactylidae, heterochromatin, cryptic species, U2 snRNA