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The Astyanax fish exhibits two morphs: an eyed, pigmented surface morph and an eyeless, depigmented cave morph. Previous studies have shown that blind morphs swim nearly parallel to the wall and can sense detailed information about objects by gliding alongside them and sensing changes in the flow field around their body using their lateral line sensory system. Hence, cavefish can build hydrodynamic images of their surroundings. Field observations showed that one of their presumptive prey, mysid shrimp, is predominately found not on the floor, but crawling on the walls. In our study, the angle of the body axis with respect to a vertical wall was measured while fish swam in a tank. Results show that when swimming by a wall, cavefish incline the vertical axis of their body away from the wall. But most significantly, this angle is different when the right side or the left side of their body is oriented towards the wall. Intriguingly, cavefish have a leftward-biased dorso-cranial bend, where the convex side of the head is towards their right side. Other studies have shown behavioral “handedness”. When exhibiting Vibration Attraction Behavior (VAB), cavefish in the field show laterality on the preponderant side they circle to explore a vibrating stimulus. Likewise in larval prey capture (LPC) behavior, larvae strike towards prey preferentially located on one side. Our results support that cavefish also express behavioral lateralization during passive swimming by walls and/or when searching for food that is perched on the walls, such as mysid shrimp.

A new genus and species of troglobiotic nicoletiid (Insecta, Zygentoma, Nicoletiidae) is described from northern Alabama, USA. The type species was collected from three caves in the Highland Rim section of the Interior Low Plateau physiographic province on the northern side of the Tennessee River Valley. Morphological and genetic analysis using the mitochondrial 16S rRNA locus show that Spinanycta alabamensis sp. nov. is quite distinct from related nicoletiids in North America. The species differs from members of other genera by its urosternum I, which in males is modified with a central pointy extension. The new species significantly extends the distribution of cave-dwelling members of the family into the southeastern United States and suggests that additional nicoletiid diversity remains to be discovered from karst regions of the eastern United States.

Abstract Background Several studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish’s most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly. Results Using the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles. Conclusions We confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment. The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome.

The most studied cavefish in the world is Astyanax mexicanus, and the most frequently used specimens in research come from the Pachón cave in Northeast Mexico. A recent study using the capture–mark–recapture technique revealed that the Pachón population size is only in the hundreds of individuals, not in the thousands. This result raises serious concerns about their long-term survival and highlights the need to accurately estimate the population size of other Astyanax caves. In our study, we used a well-established, non-invasive method for estimating population size: the transect sampling method, which counts the abundance of organisms along a line at regular intervals. We used the 310 m long Sabinos Cave’s Pool #1 for measurements and estimated a population of 1142 individuals. Most importantly, we found that cavefish are not distributed uniformly: 68% of the individuals were clustered under a bat roost. To corroborate this observation, we used drones to explore submerged galleries inaccessible to scientists. Results from the Toro/Calera Cave system confirmed that most galleries were essentially void of cavefish, except for in those few areas with high amounts of nutrients. Scientists visiting the most studied Astyanax localities (Pachón, Sabinos, Tinaja, and Chica) experience what appear to be high-density populations of about 10 fish/m2, possibly because the pool they visit happens to be under bat roosts. Our study shows that one cannot extrapolate the density at these localities to the rest of the habitat, where densities might be two orders of magnitude lower. Future studies should consider that the apparent high density at visited sites is overrepresenting the total population size when calculating the number of fish specimens to be captured.

Pachón cave in the Sierra de El Abra, in Northeast Mexico, stands out as hosting the world’s most widely studied cavefish population – with over 500 scholarly articles published about the population. Refugio Cave was recently discovered in the El Abra region. This cave hosts the mysid cave shrimp Spelaeomysis quinterensis and the blind cave tetra fish, Astyanax mexicanus. This study aims to understand how the aquatic community of Refugio Cave es related to other cave populations in the area. For this purpose, the Histone H3 gene of mysid shrimps and the OCA2 gene that confers albinism in Astyanax fish was sequenced. Results support that the Refugio and Pachón aquatic communities, which are only 4.5 km away apart, are closely related. Thus, the Refugio Cave population may contribute to better understand the evolutionary history of such an important population and, perhaps, help with Pachon’s cavefish conservation.

In zebrafish larvae, the first response when detecting prey is an oculomotor behavior; eye convergence. Eye convergence increases the overlap between the visual fields of the left and right eyes to prepare for tracking prey. A high vergence angle is maintained throughout the prey-tracking and capture swim phases, enhancing binocular depth. Since the discovery of eye convergence, hundreds of articles reporting on this behavior in zebrafish have been published. In this study, we found that the larvae of blind tetra cavefish, Astyanax mexicanus, despite being adapted to the absence of visual stimuli due to the lack of light in the cave, have retained the oculomotor behavior of eye convergence in their vestigial eyes. In Astyanax, eye convergence responses can be triggered singlehandedly by vibrations elicited with a glass rod at frequencies similar to those generated by its prey (10–35 Hz). The blind cave tetra offers an intriguing combination of regression of the eye structure, while retaining several of the physiological functions and actions performed in the eye, including light-entrained retinomotor rhythms and eye convergence.

The karstic cave, la Cueva de Los Sabinos, located in the Sierra de El Abra in the state of San Luis Potosí, Mexico, is mostly known for hosting a population of blind, depigmented Astyanax mexicanus cavefish. Herein, we report the discovery of a non-pigmented sponge (Porifera) in the final sump of this cave. No genus or species name could be attributed because we did not collect any specimen. Up to now, the sponge distribution seems restricted to a single pool in la Cueva de Los Sabinos, but further careful exploration of other pools of the cave as well as closely related cavities is warranted. To our knowledge, this observation constitutes the fourth report of a freshwater, white, cave-adapted sponge in the world and the first for Mexico and North America. It is also the eleventh troglobite species encountered in Los Sabinos. Our discovery confirms the exceptionally rich biodiversity of this cave ecosystem.

The Astyanax genus represents an extraordinary example of phenotypic evolution, being their most extreme examples the blind and depigmented morphs, which have evolved from independent surface-dwelling lineages. Among cave organisms, Astyanax cavefish is a prominent model system to study regressive evolution. Before this study, 34 cave populations were known for the Astyanax genus to be inhabited by the cave morph. The majority of those cave populations are distributed in Northeast México, at the Sierra Madre Oriental (32 cavefish), in three main areas: Sierra de Guatemala, Sierra de El Abra, and Micos, and two in the Balsas basin in the state of Guerrero, Mexico. In the present study, we describe a new cave population found 4.5 km Southward of Pachón cave, the most northern cave population known for the Sierra de El Abra limestone. El Refugio cave is a resurgence with a mixed population of fish with different levels of troglomorphism, and surface fish, resembling other hybrid populations within the Sierra de El Abra. Based on a mitochondrial DNA characterization of the 16S ribosomal DNA sequence, we could identify the mitochondrial lineage of this population, which was placed closely related to the “New Lineage”, sharing haplotypes with the surface (i.e. Arroyo Lagartos) and Pachón populations, instead of with the cave populations from Central Sierra de El Abra (e.g. Tinaja cave). El Refugio cave population gives additional evidence of the intricate history of this system, where migration, drift, and selection have shaped the evolution of the cave morphs through the independent episodes of the Astyanax mexicanus history.

The lens plays a central role in the development of the optic cup. In fish, regression of the eye early in development affects the development of the craniofacial skeleton, the size of the olfactory pits, the optic nerve, and the tectum. Lens removal further affects olfaction, prey capture, and aggression. The similarity of the fish eye to other vertebrates is the basis for its use as an excellent animal model of human defects. Questions regarding the effects of eye regression are specifically well-suited to be addressed by using fish from the genus Astyanax. The species has two morphs; an eyeless cave morph and an eyed, surface morph. In the cavefish, a lens initially develops in embryos, but then degenerates by apoptosis. The cavefish retina is subsequently disorganized, degenerates, and retinal growth is arrested. The same effect is observed in surface fish when the lens is removed or exchanged for a cavefish lens. While studies can greatly benefit from a control group of surface fish with regressed eyes brought through lensectomies, few studies include them because of technical difficulties and the low survivorship of embryos that undergo this procedure. Here we describe a technique with significant modification for improvement for conducting lensectomy in one-day-old Astyanax and other fish, including zebrafish. Yields of up to 30 live embryos were obtained using this technique from a single spawn, thus enabling studies that require large sample sizes.

The evolution of foraging behaviors is key to colonizing challenging habitats such as a cave’s dark environment. Vibration attraction behavior (VAB) gives fish the ability to swim in the darkness toward a vibration stimulus produced by many prey crustaceans and insects. VAB evolved in the blind Mexican cave tetra, Astyanax mexicanus. VAB is regulated by an increased number of mechanosensory neuromasts, particularly in the eye orbital region. However, VAB in Astyanax is only correlated with the number of neuromasts on the left side. Astyanax also have a bent skull preferentially to the left and a QTL signal for the right-side number of neuromasts. We conducted field studies in five different cave populations for four years. Results support that all cave populations can express behavioral lateralization or preponderance of side to examine a vibrating object. The percentage of individuals favoring one side may change among pools and years. In one cave population (Pachón), for one year, this “handedness” was expressed by preferentially using the right side of their face. On the contrary, in most years and pools, Tinaja, Sabinos, Molino, and Toro cave populations explored preferentially using their left side. This suggests that if there is an adaptative effect, it selects for asymmetry on itself, and not necessarily for which side is the one to be specialized. Results also showed that the laterality varied depending on how responsive an individual fish was, perhaps due to its nutritional, motivational state, or mode of stimuli most relevant at the time for the fish.

The surface morph of the Mexican tetra fish (Astyanax mexicanus) exhibits strong territoriality behavior and high levels of aggression. In contrast, the eyeless cave-adapted morph from Sierra de El Abra, México, rarely are aggressive and have totally lost the territorial behavior. These behaviors are part of what has been called the cavefish behavioral syndrome. Here, we report that several Astyanax cave populations of Sierra de Guatemala, unlike those reported for the Sierra de El Abra cave populations, display significant territoriality and aggression when confined into a reduced space. We discuss divergent evolutionary trajectories in terms of agonistic behavior for cavefish populations inhabiting different mountain ranges.

The Pennsylvania grotto sculpin is known from just two caves of the Nippenose Valley in central Pennsylvania, USA. They exhibit emergent troglobitic morphological traits and are the second northern-most cave adapted fish in the world. Two mitochondrial (16S rRNA and D-loop gene) and one nuclear (S7 ribosomal protein gene intron) gene in both cave and epigean populations were sequenced. For the three markers, a large proportion of cave specimens possess unique haplotypes not found in their local surface counterparts, suggesting a vicariance in their evolutionary history. The cave population also has haplotypes from two separate lineages of surface sculpins of the Cottus cognatus/bairdii species complex. Since morphology, nuclear, and mitochondrial markers are not correlated among cave individuals, hybridization with introgression is suggested.

Cave organisms are often characterized by reduced pigmentation, eyesight, and enhanced mechanosensory functions. The stygobitic catfish Astroblepus pholeter is found within some subterranean drainages in Ecuador. The species was first described in 1962 with specimens that were all highly depigmented and troglomorphic. The next observations in the field occurred until 2011, 2015 and 2018. At such dates, specimens examined progressively displayed more surface-like appearance. Appendages in these individuals were progressively shorter and pigmentation levels are now as high as some surface Astroblepus. Based on sampled specimens, it would appear that since 1962, the population has been progressively composed of less troglomorphic individuals. One possibility is that the population has undergone introgressive hybridization in recent years as surface Astroblepus are known to enter the caves and cohabitate with the troglomorphic Astroblepus. Lastly, we report that Individuals are able to detect and respond to light. Histological analyses show that A. pholeter’s eyes have all of the major ocular structures (lens, optic nerve, and all retinal layers).

The Sierra de El Abra is a long (120 km) and narrow (10 km) karstic area in northeastern Mexico. Some studies have suggested independent evolutionary histories for the multiple populations of blind cavefish Astyanax mexicanus that inhabit this mountain range, despite the hydrological connections that may exist across the Sierra. Barriers between caves could have prevented stygobitic populations to migrate across caves, creating evolutionary significant units localized in discrete biogeographical areas of the Sierra de El Abra. The goal of the present study was to evaluate if there is a correspondence in phylogeographical patterns between Astyanax cavefish and the stygobitic mysid shrimp Spelaeomysis quinterensis. Astyanax mtDNA and mysid histone H3 DNA sequences showed that in both species, cave populations in central El Abra, such as Tinaja cave, are broadly different from other cave populations. This phylogeographical convergence supports the notion that the central Sierra de El Abra is a biogeographical zone with effective barriers for either cave to cave or surface to cave gene flow, which have modulated the evolutionary history across species of its aquatic stygobitic community.

The characiform fish Astyanax mexicanus comes in two forms, a surface-dwelling morph which lives in the rivers of North and Central America and a blind, depigmented cave-dwelling morph which inhabits caves in Mexico. In recent years, this species has arguably become among the most influential model system for the study of evolutionary development and genomics in cave biology. While recent articles have analysed in great detail Astyanax genetics, development, physiology, phylogeny and behaviour, there have been comparatively few recent studies concerning its ecology and in particular its biogeography. Mitchell et al. (1977) reported the species inhabiting 29 caves in the Sierra de El Abra region. Despite the elapsing of over 40 years and the latest surge of interest in the model, not a single new cave locality had been described for the species. We describe here a new and 30th cave locality, Chiquitita Cave, inhabited by troglomorphic A. mexicanus. Their morphology, eye histology, 16S rRNA DNA sequence, and smelling capabilities are analysed. This cave represents the southernmost extension for the cave morph’s habitat within the Sierra de El Abra. Its name, Chiquitita Cave (Tiny Cave), was chosen in reference to a potential hydrologic connection to “Chica Cave” (small cave), which is among the most studied populations of Astyanax.

Abstract Background Life in the darkness of caves is accompanied, throughout phyla, by striking phenotypic changes including the loss or severe reduction in eyes and pigmentation. On the other hand, cave animals have undergone constructive changes, thought to be adaptive, to survive in this extreme environment. The present study addresses the question of the evolution of growth in caves, taking advantage of the comparison between the river-dwelling and the cave-dwelling morphs of the Mexican tetra, Astyanax mexicanus. Results A sclerochronology approach was undertaken to document the growth of the species in these two very distinct habitats. Scales from 158 wild Astyanax mexicanus specimens were analyzed from three caves (Pachón, Tinaja and Subterráneo) and two rivers (Rio Gallinas and Arroyo Lagarto) in San Luis Potosi and Tamaulipas, Mexico. A 10–13% reduction in scales size was observed in the cave morphs compared to the surface morphs. Age could be reliably inferred from annual growth increments on the scales from the two morphs of the species. Further comparisons with growth curves in laboratory conditions, obtained using the von Bertalanffy growth model, were also performed. In the wild and in the laboratory, cavefish originating from the Pachón cave reached smaller sizes than surface fish from three different locations: Rio Gallinas and Arroyo Lagarto (wild sampling) and Texas (laboratory population), respectively. Wild Pachón cavefish also seemed to grow to smaller sizes than the two other wild cavefish populations studied, Tinaja and Subterráneo. Finally, growth in the laboratory was faster than in the wild, particularly in the two first years of life. Conclusions These data suggest that cavefish originating from the Pachón cave are subjected to an intrinsic limitation of their final size, which is at least in part independent from energy/food availability. This growth limitation may be an advantageous way of limiting energy expenditure and food needs in the cave environment. Moreover, growth regulation evolved differently in independently evolved cave populations. These results are discussed with regard to the sources of energy or general ecological conditions present in caves, and to the differences in behavior or feeding skills known in cavefish.

The subterranean environment is often described as “extreme” and food poor. Laboratory experiments have shown that blind Mexican tetra Astyanax mexicanus (De Filippi, 1853) cavefish are better at finding food in the dark than surface fish. Several morphological and behavioural attributes that could foster this obvious adaptive response to cave environments have been described. Nonetheless, it is currently unknown what young cavefish actually eat in their natural cave environment. Our results from the Pachón cave in México during the dry and rainy season show that fry are efficient predators in their natural cave environment. Their primary food item is aquatic crustaceans. The guts of post-larval, pre-juvenile stage individuals (n=9) contained an average of 17.9 water fleas (Cladocera), copepods, ostracods, and isopods. Thus, the fry in this cave are well-fed. The Pachón cave environment does not appear to be “food poor” for juvenile cavefish. Food regimes change between post-larval and adult stages to become more dependent on partially decomposed material, guano, or detritus from the mud. We discuss the data with regards to our current developmental and genetic understanding of cavefish morphological and behavioural evolution, particularly regarding its enhanced Vibration Attraction Behaviour (VAB).

The habitat of cave-adapted organisms is characterized by complete darkness and in some instances, an apparent lack of environmental distinction between day and night. It is unclear if cave-adapted organisms retain circadian rhythms that can be light-entrained. Stygobromus allegheniensis (Allegheny Cave Amphipod) is an eyeless troglobitic crustacean found in caves located in the Northeastern region of the United States. Two cave populations were examined for evidence of light-entrained circadian rhythms. The first population inhabits a small tectonic cave (Ice Caves, Sam’s Point Preserve, NY) and the second (Clarksville Cave, Clarksville, NY) inhabits a long cave system in limestone rock. Experiments conducted in both the field and the laboratory suggest that the capacity to exhibit motor rhythms has been conserved in at least some individuals of both populations. Nonetheless, their motor activity rhythms have high variability of period length between individuals and do not appear to be light-entrainable. It is thus proposed that in this species, light-entrainable circadian rhythms controlling motor activity have undergone incipient regressive evolution.

The Astyanax species complex has two morphs: a blind, depigmented morph which inhabits caves in México and an eyed, pigmented surface-dwelling morph. The eyed morph can also be found in a few caves, sometimes hybridizing with the cave morph. This species complex has arguably become the most prominent model system among cave organisms for the study of evolutionary development and genomics. Before this study, 32 caves were known to be inhabited by the cave morph, 30 of them within the El Abra region. The purpose of this study was to conduct new surveys of the area and to assess some unconfirmed reports of caves presumably inhabited by troglomorphic fish. We describe two new localities, Sótano del Toro #2 and Sótano de La Calera. These two caves comprise a single hydrologic system together with the previously described cave of Sótano del Toro. The system is inhabited by a mixed population of troglomorphic, epigeomorphic, and presumably hybrid fish. Furthermore, Astyanax cavefish and the mysid shrimp Spelaeomysis quinterensis show a phylogeographic convergence that supports the notion that the central Sierra de El Abra is a biogeographical region that has influenced the evolutionary history of its aquatic community across species. The presumptive location of the boundaries of this biogeographical region are identified.

Stygobromus allegheniensis Holsinger, 1967 (Allegheny Cave Amphipod) is a troglobiotic crustacean commonly found in caves of the Northeast United States. We describe several new populations from the unique tectonic Ice Caves found in the Shawangunk Ridge in New York, USA. Results also show that despite being an eyeless species, it can detect particular wavelengths of light and individuals display scotophilia, a preference for darkness. Finally, the Ice Caves pose a challenge to any aquatic troglobiont; in the winter months, the Ice Caves freeze and the floor and walls become covered in solid ice. Our results show that S. allegheniensis may seek warmer waters within the cave, but can also survive being frozen in solid ice.

A new population of velvet worms (Onychophora) inhabiting a lava tube cave in the island of Santa Cruz, Galapagos, is reported here. The population size is large, suggesting that they may be troglophilic. Its members are darkly pigmented, with no obvious troglomorphic features. Their 16S rRNA sequence showed no differences when compared to an unidentified species of surface velvet worm from the same island, thus supporting cave and surface populations belong to the same species. Based on the 16S rRNA data, the Galapagos velvet worms derived from an Ecuadorian/Colombian clade, as would be expected of ease of dispersal from the nearest mainland to the Galapagos Islands.

The “thysanuran” (Zygentoma: Nicoletiidae) Speleonycta ozarkensis is the only troglobiotic nicoletiid from the Ozark Highlands. It was originally described with only four specimens from four different cave systems in Arkansas and Oklahoma. The scarcity of available specimens has made it difficult to determine whether morphological variation among populations represents intraspecific or interspecific variation. We examined molecular (16S rRNA) variation among populations and found no evidence that they represent a species complex. Because of its limited distribution and lack of ecological and life history data, S. ozarkensis may be a species of conservation concern. We therefore conducted a conservation status assessment. We bioinventoried 44 caves in Arkansas and Oklahoma to determine the distribution of S. ozarkensis. A new locality in Adair Co., Oklahoma, was discovered and new specimens were collected to better assess morphological variation among populations. Data on ecology and life history was gathered. We determined the conservation status of the species and identified potential threats to existing populations. Despite being known from a few localities, S. ozarkensis has a broad distribution approaching 10,000 km2. Molecular data suggest S. ozarkensis is capable of considerable dispersal and is primarily an epikarstic species, perhaps explaining why it has been infrequently collected from caves. Conservation assessments revealed that S. ozarkensis is at a slight risk of extinction. We identified seven threats impacting populations that vary in scope and severity, but only recreational caving (three caves) and development associated with urbanization (one cave) have the greatest potential to immediately impact populations.

The blind Mexican tetra fish, Astyanax mexicanus, has become the most influential model for research of cave adapted organisms. Many authors assume that the Sierra de Guatemala populations and the Sierra de El Abra populations are derived from two independent colonizations. This assumption arises in part from biogeography. The 100 m high, 100 m wide Servilleta Canyon of the Boquillas River separates both mountain ranges and is an apparent barrier for troglobite dispersion. Anelpistina quinterensis (Nicoletiidae, Zygentoma, Insecta) is one of the most troglomorphic nicoletiid silverfish insects ever described. 16S rRNA sequences support that this species migrated underground to reach both mountain ranges within less than 12,000 years. Furthermore, literature shows a plethora of aquatic and terrestrial cave restricted species that inhabit both mountain ranges. Thus, the Servilleta canyon has not been an effective biological barrier that prevented underground migration of troglobites between the Sierra de Guatemala and the Sierra de El Abra. The Boquillas River has changed its course throughout time. Caves that in the past connected the two Sierras were only recently geologically truncated by the erosion of the new river course. It is likely that, with the geological changes of the area and throughout the 2-8 million years of evolutionary history of cave Astyanax, there have been opportunities to migrate across the Servilleta canyon.

Anelpistina taninuli sp. nov. (Insecta: Zygentoma: Nicoletiidae), a species from Taninul, Sierra de El Abra, San Luis Potosi, Mexico, is described. We also report the 16S rRNA sequence of this new species.