Dedicated ichthyological surveys in four active karstic caves in the Mexican state of Chiapas (Grijalva River drainage basin) resulted in the discovery of the same number of hypogean populations of Rhamdia catfishes assignable to two different species: R. laticauda and R. guatemalensis. The taxonomic identity of these populations was initially determined based on morphological traits and subsequently corroborated with molecular data in a phylogenetic framework. For the most part, these newly discovered populations exhibit partial and variable troglomorphism (vs. fixed), a pattern that has been observed in most other cave-dwelling species/populations of Mexican Rhamdia, and possibly caused by gene flow with and/or incipient speciation from epigean lineages. Since most hypogean forms of Mexican Rhamdia derive from/are part of a larger R. laticauda clade, our discovery of cave-dwelling populations assignable to R. guatemalensis is noteworthy and includes the very first record of a R. guatemalensis population with pronounced and widespread troglomorphism. Our discovery of hitherto unrecorded populations of hypogean Rhamdia highlights the continued importance of exploration in the process of documenting subterranean biodiversity, particularly in regions of the world rich with cave systems. Our findings corroborate the notion that, among Neotropical fishes, the catfish genus Rhamdia is one of the most prone and effective at colonizing subterranean habitats and establishing viable hypogean populations.

Cavefishes, hypogean populations, troglobitic fishes, troglomorphism

Catfishes of the genus Rhamdia Bleeker, 1858 are a clade of Neotropical freshwater fishes widely distributed throughout the continent–from Mexico to Argentina–and with considerable taxonomic diversity (27 currently valid species) (Silfvergrip 1996; Perdices et al. 2002; Hernández et al. 2015; Angrizani and Malabarba 2020; Fricke et al. 2020). Remarkably, Rhamdia catfishes have a propensity to colonize subterranean habitats, particularly in the form of active karst caves, as evidenced by the fact that about a quarter of their overall species diversity, plus several populations lacking definite species adscription, are stygobitic (living exclusively in groundwater) (Arroyave and De La Cruz Fernández 2021a, b).

The six valid species of hypogean (cave-dwelling) and troglobitic (displaying phenotypic adaptations to cave life) Rhamdia are: the Brazilian Rhamdia enfurnada (Bichuette and Trajano 2005), the Venezuelan Rhamdia guasarensis (DoNascimiento et al. 2004), and the Mexican Rhamdia reddelli (Miller 1984), Rhamdia zongolicensis (Wilkens 1993), Rhamdia macuspanensis (Weber and Wilkens 1998), and Rhamdia laluchensis (Weber et al. 2003). Notably, all of them are characterized by being fully troglomorphic and microendemic to their respective type-locality caves, at least based on their original descriptions. Meanwhile, cave-dwelling Rhamdia populations of dubious taxonomic designation and with varying degree of troglomorphism have so far only been reported for Mexico (Robertson 1983; Mosier 1984; Arroyave and De La Cruz Fernández 2021a, b).

As evidenced by the above, the bulk of the known diversity of hypogean Rhamdia resides in Mexico, which is rather unsurprising, given that roughly 20% of Mexican territory is karstic landscape (Bautista 2023). In Mexico, Rhamdia catfishes have colonized karst formations in the southeast, with most populations (at least five taxonomically unassigned, although probably R. laticauda) and species (R. zongolicensis and R. reddelli) reported from the mountainous karst region of the Sierra de Zongolica, state of Veracruz, bordering with the state of Oaxaca, Papaloapan River basin (Arroyave and De La Cruz Fernández 2021a, b). Only after ~350 km southeast of Zongolica do other cave Rhamdia species occur, in the state of Chiapas (R. laluchensis) and further east in Tabasco (R. macuspanensis), in active caves that are part of the Grijalva-Usumascinta River basin (Arroyave and De La Cruz Fernández 2021b).

Besides cave-dwelling forms, Mexican Rhamdia include the surface species Rhamdia laticauda, Rhamdia guatemalensis, and Rhamdia parryi (Miller 2005). Existing research on the evolutionary history of Rhamdia in Middle America suggests that the diversity of cave-dwelling lineages in the region derives from the epigean and more widespread species R. laticauda, with the possibility that all Mexican cave-dwelling forms (including all four valid species) are in fact cave-adapted populations of R. laticauda (Perdices et al. 2002; Arroyave and De La Cruz Fernández 2021a, b). To date, the only known exception to this pattern (R. laticauda being the “ancestor” of all cave Rhamdia in Mexico) would be a non-troglomorphic cave-dwelling population of Rhamdia from the Grutas de Coconá (Tabasco), assignable to the surface species R. guatemalensis (Arroyave and De La Cruz Fernández 2021b).

While a renewed interest and recent research including field surveys and exploration has resulted in improved understanding of the taxonomic diversity and evolutionary history of Mexican cave Rhamdia (Arroyave and De La Cruz Fernández 2021a, b), vast extensions of karstic landscape remain to be surveyed for the presence of hypogean populations. The state of Chiapas, with its rich karstic topography that includes the Sierra Madre de Chiapas, the Chiapas Highlands, and the Chiapas Depression, offers a promising target for the discovery and documentation of previously unknown and/or understudied hypogean populations of Rhamdia. Besides the formally described troglobitic species Rhamdia laluchensis (Weber et al. 2003), existing records of hypogean catfishes in caves of Chiapas are limited to a few reports from historical international speleological expeditions in the region and from anecdotical accounts from local spelunkers, namely: Sistema Pecho Blanco (Cintalapa de Figueroa) (Sbordoni et al. 1986; Sbordoni and Lucarelli 1989), Cueva de los dos Hermanos (Berriozabal) (Sbordoni et al. 2004), an unnamed cave in the Selva del Mercadito (Sbordoni and Lucarelli 1989), El Chorro Grande cave (Río Suchiapa) (http://www.oztotl.com/ps/reports/El%20Chorro%20Grande.pdf, http://eksa.free.fr/chiapas2008/chiapas2008.pdf), Paso Burro cave (Berriozabal), and Los Bordos and El Encanto caves (Río La Venta). None of these gray-literature and anecdotical reports, however, has been previously investigated and documented from a taxonomic standpoint. Consequently, we embarked on an ichthyological expedition devoted to survey and sample some of these caves. This study presents our findings, documenting hitherto unknown cave-dwelling populations of Rhamdia catfishes from the state of Chiapas, and shedding light on their taxonomic nature and evolutionary history based on analyses of morphological and genetic comparative data.

Our ichthyological surveys in four cave systems in the state of Chiapas, Mexico, resulted in the discovery of four hitherto undocumented hypogean populations of catfishes of the genus Rhamdia, three of which with individuals only partially (vs. completely) troglomorphic, and to a varying degree (Fig. 3). Despite its cave-dwelling nature, the only specimen collected at El Encanto cave displayed normal (non-troglomorphic) phenotype (Fig. 3). Specimens from these four newly discovered populations were initially ascribed, based on external morphology, to the epigean species R. guatemalensis (Los Bordos and El Encanto caves) and R. laticauda (Paso Burro and El Chorro Grande caves), a result that was further corroborated by comparative molecular data in a phylogenetic context (Fig. 4). Morphological and meristic data from the specimens collected at the surveyed caves are presented in Tables 2, 3. The phylogeny presented in Fig. 4 clearly shows that the newly discovered cave-dwelling populations of Rhamdia documented herein (colored in bold red) are well nested within two major species-level clades that correspond to the epigean R. guatemalensis (Los Bordos and El Encanto caves) and R. laticauda (Paso Burro and El Chorro Grande caves), respectively. The phylogeny also shows that samples from Los Bordos and El Encanto are closely associated with and almost genetically indistinguishable from epigean samples from the Río La Venta (colored in blue) collected at El Aguacero and La Conchuda waterfalls. Within the R. guatemalensis clade, the only other known hypogean population is found in the Grutas de Coconá cave system, in the state of Tabasco. As shown in the phylogeny, the bulk of the diversity of hypogean Middle American Rhamdia is however represented by populations of R. laticauda, including two documented for the first time in this study: El Chorro Grande and Paso Burro caves. Unsurprisingly, the most closely related sample to those from Paso Burro cave is from an epigean population found in the river flowing out of the cave (Río Sabinal). While there is strong nodal support for the clade consisting of El Chorro Grande, Paso Burro, and Sabinal samples, its relationship to other components of the R. laticauda radiation is rather uncertain (due to the low support of the nodes involved). Despite this fact, it appears that this clade is more closely related to R. laticauda Central American lineages (including R. nicaraguensis) than to those occurring further north in Mexico, including cave-dwelling species and populations found in Veracruz (R. zongolicensis and hypogean R. laticauda), Oaxaca (R. reddelli), Chiapas (R. laluchensis), and Tabasco (R. macuspanensis).

Figure 3. 

Specimens of newly discovered populations of hypogean Rhamdia from Chiapas, showing observable external morphological features, including variation in the degree of troglomorphism (eye reduction and depigmentation) a preserved specimens of R. laticauda from Paso Burro cave b preserved specimens of R. laticauda from El Chorro Grande cave c preserved specimens of R. guatemalensis from Los Bordos cave d preserved specimen of R. guatemalensis from El Encanto cave e fresh specimen (prior to euthanasia and formalin fixation of R. laticauda from Paso Burro cave in lateral (body, both sides) and dorsal (head) views, displaying partial troglomorphism characterized by depigmentation and complete absence of the right eye f fresh specimens of R. guatemalensis from Los Bordos cave, displaying a wide spectrum of variation in the degree of troglomorphism, including complete loss of eyes and pigmentation.

Figure 4. 

Phylogenetic tree of Middle American Rhamdia based on comparative COI sequence data, highlighting the phylogenetic position of individuals from the newly discovered hypogean populations as well as from epigean populations near the surveyed caves. Cave-dwelling species/populations in red (newfound in bold). Epigean species/populations in black, except for localities surveyed herein, in blue. Terminals corresponding to COI sequences obtained from GenBank (www.ncbi.nlm.nih.gov/Genbank), BOLD (www.barcodinglife.org), or from unpublished data previously generated by the senior author (JA), include the corresponding accession or voucher/catalog number in the label (between the species name and the country of origin). Colored circles on nodes indicate degree of clade support as determined by bootstrap values (B).

The catfish genus Rhamdia is unarguably one of the most successful groups of freshwater fishes at colonizing hypogean habitats, particularly in Mexico, where most cave-dwelling species/populations are found, mainly in the Sierra de Zongolica, a karstic mountainous region that drains tributaries of the Papaloapan River basin in the state of Veracruz (Arroyave and De La Cruz Fernández 2021a). Prior to this study, the only taxonomically authoritative record of a cave-dwelling form of Rhamdia from the state of Chiapas corresponded to the species R. laluchensis, microendemic from the Sótano de La Lucha, a pit cave on the Grijalva River basin (Weber et al. 2003). The few grey-literature and anecdotical records of hypogean Rhamdia from Chiapas had neither been corroborated nor further investigated to establish the taxonomic nature of these populations. Our ichthyological surveys of four of these purportedly cavefish-harboring caves resulted in the rather unsurprising discovery of what appear to be well-established populations of hypogean Rhamdia, thus bringing the number of taxonomically verified (including catalogued vouchers) cave-dwelling populations of Rhamdia for the state of Chiapas up to five.

A first notable discovery from this study is that the Rhamdia specimens collected inside the surveyed caves do not represent undescribed species but populations of more widespread epigean species, specifically of R. laticauda and R. guatemalensis, and that each cave only harbors a single catfish species. Both morphological and molecular evidence strongly support this conclusion (Figs 3, 4). Whereas morphological identification of these populations was relatively straightforward (primarily based on pectoral spine serration pattern), characters traditionally used to distinguish between R. laticauda and R. guatemalensis, such as interdorsal space and adipose fin length, were not as clear-cut as expected (R. laticauda normally having a shorter adipose fin and correspondingly longer interdorsal space) (Table 3). Regardless of this potentially confounding morphological observation, phylogenetic analysis of comparative COI sequence data unequivocally resolved the populations from Paso Burro and El Chorro Grande as R. laticauda and those from Los Bordos and El Encanto as R. guatemalensis (Fig. 4). Furthermore, COI pairwise genetic distances between the newly discovered hypogean populations and their respective epigean species never exceeded the traditionally employed ~3% sequence divergence heuristic threshold for conspecifics (Hebert et al. 2003), providing additional support for the abovementioned species-level designation. Although our phylogenetic results also expose the non-monophyly of R. laticauda samples with respect to R. nicaraguensis, this pattern has been previously reported and discussed (Perdices et al. 2002; Arroyave and De La Cruz Fernández 2021b), and while deserving of further scrutiny, it is not the topic of our present study. This is, however, the subject of ongoing research by the senior author (JA) and collaborators, who are using genome-wide molecular markers and denser taxonomic and geographic sampling to address the systematics of the genus in Middle America.

The resulting phylogeny (Fig. 4) also makes absolute sense biogeographically, with samples from Paso Burro closely related–and almost genetically identical–to the epigean sample form Río Sabinal (river flowing out of the cave), and these samples altogether forming a clade sister to the sample from El Chorro Grande cave. Similarly, samples from Los Bordos and El Encanto caves were mainly resolved well nested within epigean samples from El Aguacero and La Conchuda waterfalls, an unsurprising result given that all four localities are part of the Río La Venta basin and in close proximity (no more than 10 km apart from each other). Furthermore, El Aguacero and La Conchuda waterfalls are located almost immediately outside of El Encanto and Los Bordos caves, respectively. Notably, the branch subtending the clade consisting of Paso Burro and El Chorro Grande samples (inclusive of the Río Sabinal sample) is particularly long, even longer than that of any of the troglobitic species currently recognized as distinct and valid (although nested within the R. laticauda radiation). While the causes behind such large genetic divergence are unclear at this point, this pattern certainly deserves attention and future scrutiny.

Another noteworthy finding of our study has to do with patterns of troglomorphism in cave Rhamdia. Except for El Encanto, for which no troglomorphic individuals were sampled (likely because of the small sample size, N = 1), all surveyed caves contained fishes with varying degree of troglomorphism (Fig. 3). This finding offers further support to the notion that hypogean fishes will eventually evolve a troglobitic phenotype characterized by eye reduction/loss and depigmentation, likely as adaptation to life in aphotic subterranean environments (Wilkens and Strecker 2017). Notably, complete troglomorphism (complete eye loss and depigmentation) is not fixed in any of the newly discovered hypogean populations. Instead, this condition is variable and only partial in most individuals sampled (Fig. 3). Partial troglomorphism in these populations is characterized by a varying degree of depigmentation and eye reduction (from normal to completely absent eyes). Remarkably, a specimen of hypogean R. laticauda from Paso Burro cave was found to display asymmetrical troglomorphism, lacking the right eye while still having a seemingly normal left one (Fig. 3e). The herein observed pattern of partial and variable troglomorphism conforms with recent research on Mexican cave-dwelling Rhamdia (Arroyave and De La Cruz Fernández 2021b), which uncovered that species believed to be completely troglomorphic, as per their original descriptions (Miller 1984; Wilkens 1993; Weber and Wilkens 1998; Weber et al. 2003), in reality display a spectrum of variation in the degree of troglomorphism. Such variable troglomorphism was also explicitly reported in the original description of the Brazilian R. enfurnada (Bichuette and Trajano 2005). Similarly, hypogean populations of R. laticauda from the Sierra de Zongolica have also been shown to exhibit clinical morphological variation in regressive troglomorphic traits (Arroyave and De La Cruz Fernández 2021a). Altogether, these findings suggest that varying (vs. fixed) troglomorphism might be the norm rather than the exception in hypogean Rhamdia. Although the exact causes behind this pattern have yet to be uncovered, time since cave colonization (incipient speciation) and crossings between hypogean and epigean individuals (gene flow) might be the main drivers constraining fixation of complete troglomorphism in these populations, a hypothesis recently advanced by previous authors (Arroyave and De La Cruz Fernández 2021b).

Admittedly, the discovery of hypogean populations of R. guatemalensis was contrary to our expectations, given that almost all known lineages of Mexican cave-dwelling Rhamdia derive from/are part of the R. laticauda radiation (Arroyave and De La Cruz Fernández 2021a, b). To our knowledge, the only prior record of a fully subterranean population of R. guatemalensis corresponds to the Grutas de Coconá cave system in the state of Tabasco (CNPE-IBUNAM 23815), with all three sampled specimens displaying a normal, non-troglomorphic phenotype (Arroyave and De La Cruz Fernández 2021b). As in the case of El Encanto cave, given this small sample size (N = 3), the existence of troglomorphic individuals in the population from Grutas de Coconá cannot be completely discounted. In any case, our discovery of two novel cave-dwelling populations of R. guatemalensis is noteworthy, particularly the one from Los Bordos cave, which effectively constitutes the very first record of a hypogean population of R. guatemalensis with pronounced and widespread troglomorphism (Fig. 3c, f). While some populations of R. guatemalensis from cenotes (karstic sinkholes) in the Yucatán Peninsula aquifer are considered somewhat hypogean, to the extent that they have been assigned subspecies status due to their incipient troglomorphism (i.e., R. g. decolor and R. g. stygaea) (Hubbs 1936), these populations are, strictly speaking, not troglobitic but trogloxenes, for the cenote is in most cases not a subterranean but an epigean habitat, and these fishes appear to only rarely venture into the cavern and cave zones of the aquifer (Arroyave et al. 2021).

We believe that the existence of hypogean fish populations, regardless of their taxonomic/phylogenetic distinctiveness, should eventually result in conservation efforts aimed at protecting them and their habitats. While the taxonomic rank of subspecies has been proposed to be of conservation utility (Phillimore and Owens 2006), it is our view that subspecies should be recognized on the basis of evidence beyond ecological/environmental singularity (e.g., cave-dwelling nature), such as discontinuities in the geographical distribution of phenotypic traits and some degree of phylogenetic distinctiveness and phylogeographic substructure. For this reason, as tempting as it is to propose subspecies names for each of the hypogean populations documented herein (with the ensuing conservation implications), we refrain from this course of action, at least until future research results in conclusive evidence in support of not only ecological but also evolutionary uniqueness. Notwithstanding this taxonomic resolve, we urge environmental policy-makers to acknowledge the ecological uniqueness of subterranean populations as diversity worth preserving.

Our discovery and documentation of hitherto taxonomically non-verified populations of hypogean Rhamdia highlights the continued importance of exploration in the process of documenting subterranean biodiversity, particularly in regions of the world rich with cave systems. Our findings corroborate the notion that, among Neotropical fishes, Rhamdia catfishes are one of the most prone and effective genera at colonizing subterranean habitats and establishing viable hypogean populations. One implication of this being that the diversity of cave-dwelling Rhamdia is most likely grossly underestimated, not only in Mexico but also throughout the distribution range of the genus. Consequently, much more exploration and taxonomically sound documentation work are needed to reach a more accurate picture of its hypogean diversity and ultimately about its evolutionary history.

This research was financially supported by the Universidad Nacional Autónoma de México (UNAM) through a “Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT)” grant (IN214922) to JA. Special thanks to Jorge Paz Tenorio for providing us with information that was instrumental in allowing us to efficiently survey El Chorro Grande cave. Additional thanks to Dr. Eloisa Torres, Collection Manager at CNPE-IBUNAM, for her assistance with cataloging the specimens used in this research.