The genus Tlaloc belongs to the family Profundulidae, a group of small freshwater fishes found nowhere else on Earth except southern Mexico and Central America. This restricted distribution makes profundulids one of the very few freshwater fish families endemic to the Mesoamerican region. The fish are modest in size, reaching a maximum standard length of roughly 120 millimeters, and they often constitute the only fish species inhabiting the upper reaches of the river basins they occupy, a fact that underscores how isolated and vulnerable many of their populations may be. Currently, Tlaloc comprises just four valid species, all confined to the upper Grijalva–Usumacinta basin in Mexico and Guatemala and to the Ulúa River in Honduras.

The two species at the center of the new research are Tlaloc labialis, first described in 1866, and Tlaloc candalarius, described in 1924. These two are hypothesized sister species, meaning they are thought to be each other’s closest relatives, and their taxonomic validity has been repeatedly questioned. Previous genetic work revealed remarkably low variation within this species complex, and the two fish are so similar in their external appearance that some researchers have suggested they should be synonymized, effectively collapsing them into a single species. In a group where genera and species remain poorly defined and osteological knowledge is sparse, the need for new characters that can sharpen species boundaries has become increasingly urgent.

The urohyal bone offered a promising place to look. Long considered a synapomorphy, a shared derived feature, of teleost fishes, the urohyal is a single bony structure formed by the ossification of an unpaired tendon of the sternohyoideus muscle. Decades of research have shown that its shape carries genuine taxonomic signal: features of the urohyal have been used to discriminate fish families, genera, and even species, including species of the cyprinodontiform genus Aphanius. Osteology more broadly has been central to building hypotheses of phylogenetic relationships among cyprinodontiform fishes and among profundulids themselves, which is precisely why the authors of the new study hypothesized that the urohyal might contain enough information to resolve the Tlaloc question.

To examine the bone, the researchers turned to museum specimens held in the Fish Collection of the Zoology Museum of the University of Sciences and Arts of Chiapas. All specimens had been fixed in 10% formalin and preserved in 70% ethanol, and the material came from the Grijalva–Usumacinta basin in Chiapas, Mexico, and along the Mexico–Guatemala border. The team selected adult specimens without separating the sexes, owing to the limited number of individuals available, and analyzed eight individuals of each species. Standard lengths ranged from 52.12 to 84.18 millimeters in T. labialis and from 40.87 to 71.56 millimeters in T. candalarius, with urohyal total lengths between 3.0 and 5.5 millimeters and 3.0 and 4.5 millimeters respectively.

The technical centerpiece of the study was a classic preparation method known as clearing and double-staining. Following the technique described by Taylor and Van Dyke in 1985, the specimens were treated so that bone stained red with alizarin while cartilage stained blue with alcian blue, rendering the delicate skeletal elements visible through the surrounding tissues. Each urohyal bone was photographed from the left side using a digital camera attached to a Zeiss Stemi DV4 stereomicroscope, and morphometric measurements were taken with an ocular micrometer connected to the same instrument. Crucially, all measurements were expressed as percentages of the total length of the urohyal, a standardization designed to limit the distorting influence of allometric growth, in which body proportions change as an animal grows. Differences between species were then tested statistically using the non-parametric Mann–Whitney U test, with significance set at an alpha level of 0.05.

The results were striking. In both species, the urohyal proved to be an enlarged, laterally compressed, triangular bone in lateral view, with a convex posterior portion, consistent with earlier descriptions of the structure in other cyprinodontiforms and in a third Tlaloc species, T. hildebrandi. The bone consists of a basibranchial attachment, a hypohyal attachment, and a condyle on its anterior margin, along with four distinguishable edges and two plates. But the decisive character was the neck of the urohyal, the narrow region of the bone. In T. candalarius, the neck height measured between 6.66% and 11.11% of the total urohyal length, with a mean of 8.75%, while in T. labialis it ranged from 13.15% to 25%, with a mean of 19.04%. The difference was highly significant, with a Mann–Whitney U test yielding Z = 3.360 and P < 0.001, and, notably, the ranges for the two species showed no overlap whatsoever.

A second, qualitative difference reinforced the morphometric one. In T. candalarius, the dorsal extension of the urohyal makes contact with the basal portion of the basibranchial attachment, whereas in T. labialis the dorsal extension reaches the upper end of that attachment. The authors suggest that quantifying such shape differences more rigorously would likely require geometric morphometric methods, which analyze shape mathematically across landmark points, but even at the descriptive level the contrast between the two species was clear. Taken together, the neck width and the configuration of the dorsal extension provide a reliable pair of characters for telling these fish apart.

The implications extend beyond a single species pair. The study’s authors conclude that T. candalarius should be maintained as a distinct taxon, since the urohyal characters allow it to be discriminated from its presumed sister species. That conclusion independently corroborates recent work published in 2025 by Domínguez-Cisneros and colleagues, who reached the same verdict using characters of the caudal complex and counts of vertebrae. Two independent lines of skeletal evidence now converge on the same answer, considerably strengthening the case that T. candalarius is valid. Morcillo and colleagues had previously suggested that T. candalarius may be the product of a very recent speciation event, which would explain the low genetic divergence that once fueled doubts about its status, and the new findings fit neatly with that scenario: recently separated species can look nearly identical on the outside while retaining diagnosable differences in their skeletons.

There is also a practical lesson in methodology here. Clearing and double-staining is operationally simple and fast, in sharp contrast to X-ray radiography or high-resolution two-dimensional X-ray imaging, which are highly specialized and expensive to acquire. For laboratories and museums working with limited budgets in the very regions where these endemic fishes live, a technique that requires little more than staining chemistry, a stereomicroscope, and an ocular micrometer opens the door to rigorous species delimitation. The authors caution that further research is needed, particularly with larger sample sizes and more localities, and they note that the urohyal’s potential in taxonomic and systematic studies of profundulids is only beginning to be explored. Still, the message of the study is clear: in the effort to catalog and protect Mesoamerica’s unique freshwater fish heritage, some of the most decisive evidence may come from structures no larger than a few millimeters, hidden in the throat of a fish small enough to fit in a palm.

Subject of Research: Comparative osteology of the urohyal bone for species delimitation in the Mesoamerican fish genus Tlaloc

Article Title: Comparative morphology of the urohyal bone between two species of the genus Tlaloc ( Actinopterygii , Cyprinodontiformes , Profundulidae )

Article References: Padilla-Sánchez, A., Domínguez-Cisneros, S. E., Velázquez-Velázquez, E., & Kobelkowsky, A. (2025). Comparative morphology of the urohyal bone between two species of the genus Tlaloc (Actinopterygii, Cyprinodontiformes, Profundulidae). Acta Ichthyologica et Piscatoria, 55, 133-137. https://doi.org/10.3897/aiep.55.151622

Image Credits: AI Generated

DOI: 10.3897/aiep.55.151622

Keywords: Tlaloc, Profundulidae, urohyal bone, taxonomy, osteology, Mesoamerica, freshwater fishes, Cyprinodontiformes, clearing and double-staining, species delimitation, morphometrics, Grijalva–Usumacinta basin