Together, the results support a shared evolutionary history for the two aquatic communities. Refugio Cave is notable because it contains a mixed population of Astyanax. Some fish are fully pigmented and have large eyes, resembling surface forms; others show intermediate reductions in pigmentation or eyes; and the most troglomorphic individuals lack pigmentation and retain only small external remnants of their eyes.

The population is described as including fully epigeomorphic fish, hybrids with varying degrees of eye and pigment regression, and specimens that appear as troglomorphic as Pachón cavefish. The cave entrance is at 229 meters above sea level, the same base level as a nearby ephemeral surface stream that flows only during the rainy season. The study was designed to distinguish two possible biogeographic positions for Refugio.

One hypothesis placed it in the northern Sierra de El Abra alongside Pachón, implying that the two nearby populations and their aquatic associates shared genetic and evolutionary affinities. The alternative hypothesis assigned Refugio to the central Sierra de El Abra, which would make the deep canyon between the caves a significant barrier to exchange. Refugio and Pachón are 4.5 kilometers apart, and the remnant fossil canyon between them is nearly 100 meters deep.

The canyon is now used in part by Highway 85 toward Ciudad Mante. Regional geological reconstructions indicate that the Río Comandante cut the canyon roughly one to four million years ago and later shifted northward as uplift and erosion continued. Fossil canyons in the Sierra de El Abra have elsewhere been shown to restrict the dispersal of aquatic organisms.

For the fish, the researchers tested a 66-base-pair fragment of exon 13 in the Oca2 gene using 11 A. mexicanus specimens from Refugio Cave. Pachón cavefish carry a single-nucleotide change at position 1252, with guanine replacing adenine. This mutation is associated with the independently evolved albinism of the Pachón population and had not previously been reported in other Sierra de El Abra cavefish populations, making it informative for testing whether Refugio fish shared ancestry or evolutionary influence with Pachón.

Among the 11 Refugio specimens, the most epigeomorphic fish carried the adenine found in surface fish, six fish with intermediate pigmentation or eye traits were heterozygous for adenine and guanine, and the four most troglomorphic fish were homozygous for the guanine cave allele. The genotypes broadly corresponded with the visible combinations of pigmentation and eye reduction reported in the fish. The presence of the Pachón-associated Oca2 allele in some Refugio fish indicates an evolutionary connection between the populations, although the marker alone cannot establish the age or route of that connection.

The result is also consistent with mitochondrial DNA results reported for Refugio A. mexicanus: those fish carried the same haplotype found in Pachón and surface populations, rather than the haplotype found in central Sierra de El Abra populations. The nuclear-marker and mitochondrial evidence therefore support assigning Refugio to the northern Sierra de El Abra biogeographic zone. The mixed phenotypes and heterozygous genotypes also show that surface and cave-associated genetic lineages meet within Refugio, consistent with the seasonal access of surface fish to the cave entrance.

For the shrimp, the researchers amplified and sequenced a 328-base-pair fragment of the Histone H3 gene. They collected three S. quinterensis specimens from Refugio Cave and compared their sequences with available data from caves representing the Sierra de Guatemala, northern, central and southern Sierra de El Abra, as well as two additional specimens from Piedras Cave in the central zone. Refugio shrimp sequences differed from Pachón by two base pairs, or 0.6 percent.

They differed from Sierra de Guatemala populations by seven base pairs, or 2.1 percent, and from the central Sierra de El Abra populations of Sabinos, Tinaja and Piedras by 34 base pairs, or 10.3 percent. Sabinos, Tinaja and Piedras had identical sequences in the comparison. The shrimp result provides an independent line of evidence for the northern placement of Refugio.

Earlier regional work had delineated northern, central and southern Sierra de El Abra biogeographic zones using underground hydrogeology, shrimp Histone H3 sequences and mitochondrial A. mexicanus DNA, with additional support from nuclear markers in the fish. Refugio now appears to contain the second known cavefish population in the northern zone, after Pachón. Its shrimp are much more similar to those of Pachón than to mysids from the central zone, while the fish carry a marker previously found only in Pachón among Sierra de El Abra cave populations.

These findings suggest that the affinity applies to both sampled aquatic species rather than to the cavefish alone. The combined genetic evidence also implies that the nearly 100-meter-deep fossil canyon between Refugio and Pachón did not create a complete barrier between the northern populations. At the same time, the study does not identify the mechanism that allowed organisms to move or exchange genetic material across or around the canyon.

The authors note that the caves are now independent springs and do not share a common aquifer, so any connection would have to occur at their headwaters, where the 4.5-kilometer separation may constitute a substantial barrier. Earlier hydrogeographic observations indicate that caves within the broader El Abra zones share hydrologic connections, but the present marker comparison does not reconstruct the detailed route or timing of gene flow between Refugio and Pachón. Refugio may be especially useful because Pachón has been one of the most intensively studied cavefish populations and its population has been the subject of intensive biological research.

Recent assessments cited by the study suggest that the Pachón population may be declining and may contain only a few hundred individuals. Refugio appears to be the closest and most closely related cavefish population to Pachón in the El Abra region, making it a potential alternative site for examining the evolutionary forces acting on northern Astyanax and for investigating the evolutionary history of that lineage. It might also reduce repeated sampling pressure on Pachón.

These are potential applications of the present result: the study did not test conservation outcomes, demonstrate that sampling at Refugio would protect Pachón, or establish population trends at either cave. Refugio Cave therefore adds a geographically close and genetically informative counterpart to one of evolutionary biology’s best-known cavefish populations. The study’s central conclusion rests on two species and two marker systems: a distinctive nuclear Oca2 allele in A. mexicanus and closely matching Histone H3 sequences in S. quinterensis.

Their agreement places the cave’s aquatic community within the northern Sierra de El Abra and supports a closer relationship between Refugio and Pachón than between either locality and the central-zone populations.

Subject of Research: Genetic biogeographic affinities of the aquatic community in Refugio Cave, Mexico

Article Title: Biogeographical affinities of the aquatic community of Refugio Cave, a newly discovered Astyanax cave

Article References: Espinasa, L., & Pech, A. (2023). Biogeographical affinities of the aquatic community of Refugio Cave, a newly discovered Astyanax cave. Subterranean Biology, 46, 77-86. https://doi.org/10.3897/subtbiol.46.102043

Image Credits: AI Generated

DOI: 10.3897/subtbiol.46.102043

Keywords: Refugio Cave, Astyanax mexicanus, cavefish, Spelaeomysis quinterensis, mysid shrimp, Oca2 gene, Histone H3, biogeography, El Abra, Pachón Cave, Biogeographical, affinities