Cave Populations Research Articles

Incipient regressive evolution of the circadian rhythms of a cave amphipod

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.

Response to conspecific chemical cues in surface- and cave-dwelling populations of Atlantic mollies, Poecilia mexicana

Cavefishes typically evolve sensory adaptations to compensate for the loss of visual orientation and communication in their naturally dark habitats. We compared the response to chemical cues from conspecifics between surface- and cave-dwelling populations of Atlantic mollies (Poecilia mexicana) using dichotomous association preference tests. In one of the two described cave populations, females spent significantly more time in the preference zone containing chemical cues of conspecifics compared to the preference zone receiving control water, while no discrimination was detected in the other cave population and in two surface populations. Our findings suggest chemo-sensory adaptations in only one of two cave populations and independent evolutionary trajectories in both cave populations.

Trophic plasticity among spring vs. cave populations of Gammarus minus: examining functional niches using stable isotopes and C/N ratios

AbstractIn some environments, species may exhibit trophic plasticity, which allows them to extend beyond their assigned functional group. For Gammarus minus, a freshwater amphipod classified as a shredder or detritivore, cave populations have been observed consuming heterotrophs as well as shredding leaves, and therefore may be exhibiting trophic plasticity. To test this possibility, we examined the C and N stable isotope and C/N ratios for cave and spring populations of G. minus. A 15‐day feeding experiment using leaves and G. minus from a spring population established that the diet‐tissue discrimination factor was 3.2 ‰ for δ15N. Cave G. minus were 8 ‰ higher in δ15N relative to cave leaves, indicating they did not derive nitrogen from leaves, whereas field collected spring populations were 2–3 ‰ higher than spring leaves, indicating that they did. Cave G. minus were 2.6 ‰ higher in δ15N than the cave isopod, Caecidotea holsingeri. Relative to spring populations, Organ Cave G. minus were 15N enriched by 6 ‰, suggesting they occupied a different trophic level, or incorporated an isotopically distinct N source. While stable isotopes cannot tell what the cave G. minus are eating, the isotopes certainly show that G. minus are not eating leaves and are trophically distinct form the surface populations. Differences in C/N ratios were observed, but reflect the size of the G. minus examined and not feeding group or habitat. The isotope data strongly support the hypothesis that cave populations of G. minus have become generalist or omnivorous by including animal protein in their diet.

Influence of Mining Pollution on Metal Bioaccumulation and Biomarker Responses in Cave Dwelling Fish, Clarias gariepinus.

Cave ecosystems remain largely unstudied and risk being severely degraded as a result of anthropogenic activities. The Wonderfontein Cave, situated in the extensive gold mining region of the Witwatersrand Basin, is one such system that hosts a population of Clarias gariepinus, which is exposed to the influx of polluted mine water from the Wonderfontein Spruit River. The aim of this study was to investigate the bioaccumulation of metals, as well as relevant biomarkers, in C. gariepinus specimens sampled from the Wonderfontein Cave during high (April 2013) and low (September 2013) flow surveys. Results were also compared to a surface population associated with the Wonderfontein Spruit River. There were temporal differences in metal bioaccumulation patterns and this was attributed to the lack of dilution during the low flow period. Metals associated with acid mine drainage, i.e. Co, Mn and Zn were significantly higher in the Wonderfontein Cave population and were reflected in an increase in oxidative stress biomarkers (catalase, protein carbonyls and superoxide dismutase) and the induction of metallothionein, a biomarker of metal exposure. The surface population was exposed to metals associated with geological weathering processes, i.e. Fe and Al.

Influence of geomorphology and surface features on the genetic structure of an important trogloxene, the secret cave cricket (Ceuthophilus secretus)

Cave ecosystems supporting a variety of endemics depend on the carbon, nitrogen, and nutrients brought into caves by trogloxenic species, such as the secret cave cricket (Ceuthophilus secretus). Surface movements of trogloxenes may comprise the strongest ecological connections among caves. Our objective was to better understand dispersal patterns in C. secretus in order to inform management of this species and the cave endemics that depend upon them. We used microsatellite loci to estimate gene flow and genetic diversity among 42 karst features supporting C. secretus on the Fort Hood Military Reserve, Texas, USA. This sampling was used to assess the influences of karst topography and other landscape features on genetic diversity and population structure. Cave populations did not exhibit evidence of recent bottlenecks and genetic diversity was similar among sites, with the exception of one sample from an isolated cave. Samples exhibited a strong pattern of isolation by distance, but karst topology was also influential, with genetic differentiation being much higher between samples from separate ridges than among those on the same ridge. It appears that co-location on a ridge was an important factor facilitating dispersal among karst features. There was little evidence that other surface features such as forest cover, roads or streams influenced gene flow and genetic differentiation. The low genetic connectivity among ridges suggests that isolated caves on ridges where cricket habitat is uncommon or degraded might not be easily recolonized after extinction events, with potentially negative consequences for associated cave communities.

Genetics and hybridization in surface and caveAstyanax(Teleostei): a comparison of regressive and constructive traits

Change in ecological conditions, as seen in surface and cave populations of Astyanax (Teleostei), has caused the divergent evolution of a large number of traits like eyes, coloration, taste, lateral line, and different kinds of behaviour like schooling, sleep or feeding posture. Because of the interfertility of surface and cave forms these fish are an exceptional object to study the morphological and genetic basis of the evolution of such complex regressive and constructive traits. Classical crossing analyses and genomic studies are contributing to growing understanding. Both kinds of traits mostly rely on multiple genetic bases and the phenotypic manifestation in the various crosses is similar. The gene effect underlying the phenotypic manifestation may exhibit an exponential increase at differing amounts in the various traits and crosses. Missing or presence of such genetic interaction helps determine whether the variability of eyes or pigmentation exhibited by Astyanax cave fish populations like Micos, is due to a more recent origin or to secondary hybridization with the surface fish. Neither crossing analysis nor QTL mapping revealed that eye reduction is pleiotropically antagonistically related to the increase of taste buds or lateral line sense. Independent inheritance of traits suggests that Astyanax cave fish are subjected to mosaic evolution.

Alterations in Mc1r gene expression are associated with regressive pigmentation in Astyanax cavefish.

Diverse changes in coloration across distant taxa are mediated through alterations in certain highly conserved pigmentation genes. Among these genes, Mc1r is a frequent target for mutation, and many documented alterations involve coding sequence changes. We investigated whether regulatory mutations in Mc1r may also contribute to pigmentation loss in the blind Mexican cavefish, Astyanax mexicanus. This species comprises multiple independent cave populations that have evolved reduced (or absent) melanic pigmentation as a consequence of living in darkness for millions of generations. Among the most salient cave-associated traits, complete absence (albinism) or reduced levels of pigmentation (brown) have long been the focus of degenerative pigmentation research in Astyanax. These two Mendelian traits have been linked to specific coding mutations in Oca2 (albinism) and Mc1r (brown). However, four of the seven caves harboring the brown phenotype exhibit unaffected coding sequences compared to surface fish. Thus, diverse genetic changes involving the same genes likely impact reduced pigmentation among cavefish populations. Using both sequence and expression analyses, we show that certain cave-dwelling populations harboring the brown mutation have substantial alterations to the putative Mc1r cis-regulatory region. Several of these sequence mutations in the Mc1r 5' region were present across multiple, independent cave populations. This study suggests that pigmentation reduction in Astyanax cavefish evolves through a combination of both coding and cis-regulatory mutations. Moreover, this study represents one of the first attempts to identify regulatory alterations linked to regressive changes in cave-dwelling populations of A. mexicanus.

Melanocortin 4 receptor mutations contribute to the adaptation of cavefish to nutrient-poor conditions

Despite recent advances in the understanding of morphological evolution, the genetic underpinnings of behavioral and physiological evolution remain largely unknown. Here, we study the metabolic changes that evolved in independently derived populations of the Mexican cavefish, Astyanax mexicanus. A hallmark of cave environments is scarcity of food. Cavefish populations rely almost entirely on sporadic food input from outside of the caves. To survive under these conditions, cavefish have evolved a range of adaptations, including starvation resistance and binge eating when food becomes available. The use of these adaptive strategies differs among independently derived cave populations. Although all cavefish populations tested lose weight more slowly than their surface conspecifics during restricted rations, only a subset of cavefish populations consume more food than their surface counterparts. A candidate gene-based screen led to the identification of coding mutations in conserved residues of the melanocortin 4 receptor (MC4R) gene, contributing to the insatiable appetite found in some populations of cavefish. Intriguingly, one of the mutated residues has been shown to be linked to obesity in humans. We demonstrate that the allele results in both reduced maximal response and reduced basal activity of the receptor in vitro. We further validate in vivo that the mutated allele contributes to elevated appetite, growth, and starvation resistance. The allele appears to be fixed in cave populations in which the overeating phenotype is present. The presence of the same allele in multiple caves appears to be due to selection from standing genetic variation present in surface populations.

Trophic ecology of groundwater species reveals specialization in a low‐productivity environment

Summary Identifying feeding strategies at lower bounds of habitat productivity is fundamental to understand the relationship between energy availability and trophic specialization. Low productivity is expected to severely constrain trophic specialization because organisms may no longer be able to fulfil their energy requirements by feeding on a reduced set of resources. However, species living in low‐productivity habitats often exhibit particular biological traits such as low metabolic rates and high food‐finding abilities, which may release constraints on trophic specialization. In the present study, we used carbon (C) and nitrogen (N) stable isotopes to measure the degree of trophic specialization in two species of isopods (Proasellus valdensis and Proasellus cavaticus) living in groundwater, one of the most energy‐limited environments on earth. Fundamental specialization was obtained from a 13C/15N‐labelling experiment in the laboratory: we measured separately the carbon and nitrogen assimilation rates of the two species across the three food sources encountered in their natural cave habitats (fine and coarse particulate organic matter and sedimentary biofilm). Then, for each species, we tested for variation in diet composition among individuals and populations by quantifying the relative contribution of the three food sources to the diet of multiple individuals within 5 cave populations. The labelling experiment showed that both species assimilated about 10 times more carbon and at least 4 times more nitrogen from sedimentary biofilm than from both forms of particulate organic matter. Field samplings showed that sedimentary biofilm made up, on average, 83% of the diet of isopods. Moreover, we found almost no variation in diet among individuals of a cave population as well as among cave populations within species. This study provides the first evidence of a high degree of trophic specialization in a low‐productivity cave environment. Both species exhibited a strong fundamental specialization on sedimentary biofilm and most probably fed selectively on this food source in their natural environment. Our findings challenge the prediction that species would adopt generalist feeding strategies at lower bounds of habitat productivity.

Potential rapid evolution of foot morphology in Italian plethodontid salamanders (Hydromantes strinatii) following the colonization of an artificial cave.

How organisms respond to environmental change is a long-standing question in evolutionary biology. Species invading novel habitats provide an opportunity to examine contemporary evolution in action and decipher the pace of evolutionary change over short timescales. Here, we characterized phenotypic evolution in the Italian plethodontid salamander, Hydromantes strinatii, following the recent colonization of an artificial cave by a forest floor population. When compared with a nearby and genetically related population in the natural forest floor and a nearby cave population, the artificial cave population displayed significant differences in overall foot shape, with more interdigital webbing relative to the other populations. Further, this population evolved significantly larger feet, which corresponded more closely to those found in other cave populations than to forest floor populations to which the cave population is closely related. Finally, we quantified the rate of evolution for both foot shape and foot area, and found that both traits displayed large and significant evolutionary rates, at levels corresponding to other classic cases of rapid evolution in vertebrates. Together, these findings reveal that the response to novel environmental pressures can be large and rapid and that the anatomical shifts observed in the artificial cave population of H. strinatii may represent a case of rapid evolution in response to novel environmental pressures.

Role of density and resource competition in determining aggressive behaviour in salamanders

AbstractIntraspecific aggression is an important aspect of social behaviour that can significantly affect individual survivorship and population dynamics. Aggressiveness may be particularly important in food‐deprived habitats, like caves, but few studies have investigated behavioural and ecological determinants of aggressiveness and cannibalism in cave vertebrates. In this study, we evaluated the role of density, competition for food, starvation and rearing history under cave‐like conditions in promoting aggression. We performed a behavioural experiment on larvae collected from multiple underground springs and neighbouring streams, and we reared them under two different settings: total darkness and outdoor conditions. Intraspecific attacks were very scarce in hatchlings, and increased after 1 month. Starvation periods and high density clearly increased aggressiveness. Furthermore, aggressiveness strongly increased under competition conditions (presence of other larvae feeding). Aggressiveness increased with age more quickly in larvae from caves than in those from streams. Environmental features typically experienced by cave populations, such as starvation and high density, increase intraspecific attacks and might promote cannibalism. Strong plasticity for aggression may enhance the possibility to successfully colonize and exploit food‐deprived environments where no other predators can survive, such as underground environments.

Distinct genetic architecture underlies the emergence of sleep loss and prey-seeking behavior in the Mexican cavefish.

BackgroundSleep is characterized by extended periods of quiescence and reduced responsiveness to sensory stimuli. Animals ranging from insects to mammals adapt to environments with limited food by suppressing sleep and enhancing their response to food cues, yet little is known about the genetic and evolutionary relationship between these processes. The blind Mexican cavefish, Astyanax mexicanus is a powerful model for elucidating the genetic mechanisms underlying behavioral evolution. A. mexicanus comprises an extant ancestral-type surface dwelling morph and at least five independently evolved cave populations. Evolutionary convergence on sleep loss and vibration attraction behavior, which is involved in prey seeking, have been documented in cavefish raising the possibility that enhanced sensory responsiveness underlies changes in sleep.ResultsWe established a system to study sleep and vibration attraction behavior in adult A. mexicanus and used high coverage quantitative trait loci (QTL) mapping to investigate the functional and evolutionary relationship between these traits. Analysis of surface-cave F2 hybrid fish and an outbred cave population indicates that independent genetic factors underlie changes in sleep/locomotor activity and vibration attraction behavior. High-coverage QTL mapping with genotyping-by-sequencing technology identify two novel QTL intervals that associate with locomotor activity and include the narcolepsy-associated tp53 regulating kinase. These QTLs represent the first genomic localization of locomotor activity in cavefish and are distinct from two QTLs previously identified as associating with vibration attraction behavior.ConclusionsTaken together, these results localize genomic regions underlying sleep/locomotor and sensory changes in cavefish populations and provide evidence that sleep loss evolved independently from enhanced sensory responsiveness.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0119-3) contains supplementary material, which is available to authorized users.

Cave dwelling Onychophora from a Lava Tube in the Galapagos

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.

Inferring demographic structure with moccasin size data from the Promontory Caves, Utah

The moccasin assemblage Julian Steward recovered from the Promontory caves in 1930-31 provides a novel example in which material culture can be used to understand the structure of an AD thirteenth century population. Several studies shed light on the relationship between shoe size, foot size, and stature. We develop an anthropometric model for understanding the composition of the Promontory Cave population by using moccasin size as a proxy for foot size. We then predict the stature of the individual who would have worn a moccasin. Stature is closely related to age for children, subadults and adult males. Although there are predictable sex and age factors biasing moccasin discard practices, moccasin dimensions suggest a relatively large proportion of children and subadults occupied the Promontory caves. This bison and antelope hunting population appears to have thrived during its stay on Promontory Point.

Characterization of 19 new microsatellite loci for the Omani barb Garra barreimiae from 454 sequences.

BackgroundGarra barreimiae is a cyprinid fish from the southeastern Arabian Peninsula, which inhabits regularly desiccating wadis and survives in isolated ponds or underground. In 1984 a cave-dwelling population was found in the Al Hoota cave system and previous genetic analyses revealed some differentiation with limited gene flow between the surface populations and the cave population. Since no suitable markers are available for evaluation of gene flow between the cave population and the adjacent surface populations, we focused on designing and establishing novel microsatellite markers from next generation sequencing data.Findings19 microsatellite markers containing di- and tetranucleotide simple sequence repeats were developed from 454 sequences. Forty-four individuals from two surface populations (Wadi Al Falahi and Misfat Al Abriyeen) of G. barreimiae (sampling permission number 13/2012, export permission number 29/2012) were used for analyses and characterization of the loci. On average, the number of alleles per locus is 7.6 (range: 2–20). Two markers displayed indication of linkage disequilibrium in both populations (DL6X, 9XNC). Significant deviation from Hardy-Weinberg equilibrium was observed at four loci in the Misfat Al Abriyeen population (2PUM, 88CM, 1EHE, 3Z7M) and at two loci in the Wadi Al Falahi population (QLIM, 3 N43). Three of the microsatellite loci were significant for null alleles in one of the two populations (Misfat Al Abriyeen: CJHG; Wadi Al Falahi: PH8A, 3ROZ). Expected and observed heterozygosities ranged from 0 to 95.0% respectively from 0 to 95.8% (Wadi Al Falahi) and from 0 to 89.1% respectively from 0 to 95.0% (Misfat Al Abriyeen). Fourteen of these markers were successfully cross-amplified in G. rufa.ConclusionThis 19 microsatellite loci provide a useful tool to understand the structure and genetic differences of populations. Moreover, these markers will help to evaluate species delimitation in G. barreimiae and potentially even in related species.

Next generation phylogeography of cave and surface Astyanax mexicanus

The loss of traits is a commonly observed evolutionary pattern in cave organisms, but due to extensive morphological convergence, inferring relationships between cave and surface populations can be difficult. For instance, Astyanax mexicanus (the blind Mexican cavefish) is thought to have repeatedly lost its eyes following colonization of cave environments, but the number of evolutionarily independent invasions of this species into caves remains unclear. Because of these repeated losses, it has become a model organism for studying the genetic basis of phenotypic trait loss. Here we reconstruct a high-resolution phylogeography for A. mexicanus inferred from both mitochondrial DNA and several thousand single nucleotide polymorphisms. We provide novel insight into the origin of cave populations from the Sabinos and Río Subterráneo caves and present evidence that the Sabinos cave population is part of a unique cave lineage unrelated to other A. mexicanus cave populations. Our results indicate A. mexicanus cave populations have at least four independent origins.

A spider species complex revealed high cryptic diversity in South China caves

Cryptic species, which are an important component of biodiversity, have rarely been studied in South China karst. We investigated cryptic diversity in the cave species complex Telema cucurbitina, which has a narrow niche but widespread distribution among multiple caves. We sampled another 15 populations (caves) in addition to the population from the type locality. Phylogenetic results indicated that individuals from the same cave constituted well-supported clades. Species diversity within this species complex was assessed in a coalescent framework, first with a Bayesian extension of the general mixed Yule coalescent (bGMYC) model and a Bayesian species delimitation method (BPP). Both species delimitation methods identified each cave population as a separate species. We propose that each cave population within this species complex was a separate evolving lineage and therefore 16 OTUs were recovered based on our molecular data despite their high morphological similarities. We also propose that the unrecognized organism’s diversity within South China caves might be extremely large considering our case. Furthermore, our work reveals that species discovery of cave organisms by morphological data has a high probability of underestimating hidden diversity. Our work also highlights the need for conservation strategies to protect this largely neglected diversity of cave organisms.

Circadian rhythms in Mexican blind cavefish Astyanax mexicanus in the lab and in the field

Biological clocks have evolved as an adaptation to life on a rhythmic planet, synchronising physiological processes to the environmental light-dark cycle. Here we examine circadian clock function in Mexican blind cavefish Astyanax mexicanus and its surface counterpart. In the lab, adult surface fish show robust circadian rhythms in per1, which are retained in cave populations, but with substantial alterations. These changes may be due to increased levels of light-inducible genes in cavefish, including clock repressor per2. From a molecular standpoint, cavefish appear as if they experience 'constant light' rather than perpetual darkness. Micos River samples show similar per1 oscillations to those in the lab. However, data from Chica Cave shows complete repression of clock function, while expression of several light-responsive genes is raised, including DNA repair genes. We propose that altered expression of light-inducible genes provides a selective advantage to cavefish at the expense of a damped circadian oscillator.

Convergence in feeding posture occurs through different genetic loci in independently evolved cave populations of Astyanax mexicanus

When an organism colonizes a new environment, it needs to adapt both morphologically and behaviorally to survive and thrive. Although recent progress has been made in understanding the genetic architecture underlying morphological evolution, behavioral evolution is poorly understood. Here, we use the Mexican cavefish, Astyanax mexicanus, to study the genetic basis for convergent evolution of feeding posture. When river-dwelling surface fish became entrapped in the caves, they were confronted with dramatic changes in the availability and type of food source and in their ability to perceive it. In this setting, multiple independent populations of cavefish exhibit an altered feeding posture compared with their ancestral surface forms. We determined that this behavioral change in feeding posture is not due to changes in cranial facial morphology, body depth, or to take advantage of the expansion in the number of taste buds. Quantitative genetic analysis demonstrates that two different cave populations have evolved similar feeding postures through a small number of genetic changes, some of which appear to be distinct. This work indicates that independently evolved populations of cavefish can evolve the same behavioral traits to adapt to similar environmental challenges by modifying different sets of genes.

Loss of Schooling Behavior in Cavefish through Sight-Dependent and Sight-Independent Mechanisms

Surface populations of Astyanax mexicanus, living in rivers like their common ancestors, school, while several, independently derived cave populations of the same species have lost schooling behavior. We quantify schooling behavior in individual A.mexicanus and identify quantitative trait loci (QTL) for this trait. We find that the evolutionary modulation of schooling has both vision-dependent and -independent components. We also quantify differences in the lateral line and vision between cavefish and surface fish and relate these differences to the evolutionary loss of schooling behavior. We provide evidence that a monoamine neurotransmitter may have played a role in the evolution of schooling behavior. We find that vision is essential for schooling tendency in A.mexicanus, while the lateral line has a small effect on this behavior. Schooling behavior in A.mexicanus has evolved both through changes in sensory systems and through changes in genetic loci that likely act downstream of sensory inputs.