Seven new karyotypes reveal hidden chromosome diversity in microteiid lizards

Deep in the leaf litter of South America’s forests lives one of the continent’s most species-rich yet least-studied lizard families. The Gymnophthalmidae, commonly known as microteiids, comprise 297 recognized Neotropical species across 56 genera, most of them small enough to slip unnoticed through the forest floor. Despite decades of molecular work that has reshaped their classification, the chromosomes of these animals remained almost entirely unmapped. Now, a team of Brazilian researchers has published seven new karyotypes for the subfamily Cercosaurinae, tripling the amount of cytogenetic data available for two of its major tribes and exposing a striking degree of chromosomal diversity that had gone undocumented for more than a decade.

The study, published in the journal Comparative Cytogenetics, focused on the tribes Bachiini and Cercosaurini, which together contain roughly 204 species. Before this work, only three of them had ever had their karyotypes described. Using standard cytogenetic techniques including Giemsa staining, Ag-NOR staining to locate nucleolus organizer regions, C-banding to reveal constitutive heterochromatin, and RBG-banding for precise chromosome pairing, the researchers characterized seven species: Bachia bresslaui, Bachia dorbignyi, Cercosaura olivacea, Cercosaura schreibersii albostrigatus, Neusticurus bicarinatus, Placosoma cordylinum, and Placosoma glabellum. The specimens came from localities across Brazil, from Goiás and Mato Grosso to Rio de Janeiro, São Paulo, and Amazonas, and were deposited in the Museu de Zoologia da Universidade de São Paulo.

The diploid numbers uncovered ranged dramatically, from 2n = 32 in Bachia dorbignyi to 2n = 58 in Placosoma glabellum. That 26-chromosome spread within a single sample of closely related lizards underscores how dynamically their genomes have evolved. Almost all species displayed karyotypes composed of both macrochromosomes and microchromosomes, a hallmark of the family, but the number, morphology, and relative sizes of these elements varied considerably among species. Within the genus Bachia alone, B. dorbignyi carries 2n = 32 while B. bresslaui carries 2n = 46, a difference that immediately sets these species apart cytogenetically. Similarly, within Placosoma, P. cordylinum has 2n = 44 with a clear macro-microchromosome distinction, while P. glabellum has 2n = 58 with chromosomes decreasing gradually in size, a fundamentally different karyotype architecture.

One of the most intriguing findings concerns sex chromosomes. In Bachia dorbignyi, the researchers observed a dot-like microchromosome exclusively in male specimens, absent from the female. This pattern suggests a putative XX:XY sex determination system, with the dot-like element representing the Y chromosome. It is the first such mechanism with a dot-like Y described in Gymnophthalmidae. The researchers could not obtain meiotic spreads to confirm a heteromorphic bivalent, but they note that such heteromorphism is often undetectable in microchromosomes due to their very small size, as seen in other lizard species. Based on the known phylogenetic distribution of sex determination systems in squamates, the absence of heteromorphic sex chromosomes in the other six species studied may indicate that sex chromosomes in those species are homomorphic, meaning the X and Y are morphologically indistinguishable. Further analyses using cytogenomic techniques will be needed to clarify whether male heterogamety evolved once in the family or independently in different lineages.

Ag-NOR staining revealed notable variability in the number and position of nucleolus organizer regions, the chromosomal sites of ribosomal RNA genes. In Bachia bresslaui, the researchers found between two and six Ag-NORs per metaphase, located at telomeric regions of macrochromosomes and, in the female, also on a single microchromosome in at least 42 percent of cells analyzed. This intra- and interindividual polymorphism may reflect genomic plasticity in rDNA locus activation or positioning, potentially linked to differential gene expression. In contrast, Neusticurus bicarinatus and Placosoma glabellum showed a more conserved pattern, with a single pair of macrochromosomes bearing the NORs. Only in B. bresslaui and C. olivacea were multiple Ag-NORs detected on both macro- and microchromosomes, a pattern also seen in other microteiid species such as Nothobachia ablephara and Micrablepharus atticolus.

C-banding patterns, obtained for four of the seven species, revealed differences in both the amount and distribution of constitutive heterochromatin. Bachia bresslaui displayed conspicuous heterochromatic blocks at pericentromeric regions of all macrochromosomes, a pattern otherwise known only in the distantly related genus Procellosaurinus. Cercosaura olivacea showed small C-bands in both pericentromeric and telomeric regions of its macrochromosomes. In Neusticurus bicarinatus and Placosoma glabellum, heterochromatin was predominantly telomeric, with P. glabellum exhibiting lightly stained telomeric C-bands on most chromosomes, except for the short arm of a subtelocentric pair that was almost completely heterochromatic. These faint telomeric signals are consistent with patterns reported for other gymnophthalmid species, in which heterochromatin frequently serves structural or protective roles at chromosome ends.

RBG-banding, which uses bromodeoxyuridine incorporation to produce replication banding patterns, was successfully applied only to Cercosaura olivacea. The technique allowed the nine pairs of macrochromosomes to be precisely paired, proving its utility as a cytogenetic tool. The researchers note the difficulty in obtaining high-quality metaphase spreads for this technique in microteiids, whose extremely small body size, averaging just 35 millimeters snout-vent length, makes chromosome preparation from intestinal epithelial cells particularly challenging. Meiotic analyses confirmed the karyotypes, revealing 9 macrobivalents and 12 microbivalents in diakinesis cells of C. olivacea, 10 macrobivalents and 12 microbivalents in C. schreibersii albostrigatus, N. bicarinatus, and P. cordylinum, and 29 bivalents in P. glabellum.

Beyond the new karyotypes, the study provides a comprehensive compilation of all chromosomal data available for Gymnophthalmidae, synthesizing decades of scattered cytogenetic literature. The family now shows a diploid number range from 2n = 34 in Gymnophthalmus pleii to 2n = 62 in Nothobachia ablephara, with two distinct karyotype patterns: one with a clear size distinction between macro- and microchromosomes, and another with high diploid numbers and chromosomes decreasing gradually in size. Two forms of male heterogamety have been documented, a standard XX:XY system and an X₁X₁X₂X₂:X₁X₂Y multiple sex chromosome system. Supernumerary chromosomes, or B chromosomes, have also been described in the family, as have diploid and triploid parthenogenetic lineages in Gymnophthalmus and Loxopholis. A recent probabilistic analysis for the superfamily Gymnophthalmoidea, using data from 69 of its 502 species, inferred an ancestral diploid number of 2n = 42, implying that chromosomal fissions and fusions played a significant role in karyotype evolution.

From a taxonomic perspective, the new data highlight the potential utility of chromosomal features as diagnostic characters. The distinct karyotype of Bachia dorbignyi, with its low diploid number and putative sex chromosome system, reinforces the cytogenetic distinctiveness of this species relative to B. bresslaui and may aid in the delimitation of cryptic taxa. Similarly, chromosomal data for Cercosaura schreibersii albostrigatus may contribute to the ongoing debate over the validity of subspecies within C. schreibersii, supporting future integrative taxonomic revisions. The researchers emphasize that, despite the challenges of working with these tiny lizards, the high degree of chromosomal diversity uncovered here corroborates the dynamic nature of genome evolution in this clade and opens new avenues for research on speciation, sex determination, and genomic architecture in Neotropical lizards. Incorporating cytogenomic techniques and broader taxonomic sampling will be essential to fully elucidate the evolutionary mechanisms shaping karyotypes in this enigmatic group.

Subject of Research: Cytogenetics of Neotropical microteiid lizards

Article Title: Seven new karyotypes for subfamily Cercosaurinae ( Squamata , Gymnophthalmidae ) with a synthesis of chromosomal data

Article References: Rachid, L. S. F., Yonenaga-Yassuda, Y., Rodrigues, M. T., & Pellegrino, K. C. M. (2026). Seven new karyotypes for subfamily Cercosaurinae (Squamata, Gymnophthalmidae) with a synthesis of chromosomal data. Comparative Cytogenetics, 20, 21-42. https://doi.org/10.3897/compcytogen.20.170539

Image Credits: AI Generated

DOI: 10.3897/compcytogen.20.170539

Keywords: Gymnophthalmidae, karyotype, cytogenetics, lizards, chromosomes, Cercosaurinae, Seven, karyotypes, subfamily, Squamata, synthesis, chromosomal