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.

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

BACKGROUND: Sleep 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. RESULTS: We 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. CONCLUSIONS: Taken 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.

The population genomics of repeated evolution in the blind cavefish Astyanax mexicanus.

Distinct populations of Astyanax mexicanus cavefish offer striking examples of repeatable convergence or parallelism in their independent evolutions from surface to cave phenotypes. However, the extent to which the repeatability of evolution occurred at the genetic level remains poorly understood. To address this, we first characterized the genetic diversity of 518 single-nucleotide polymorphisms (SNPs), obtained through RAD tag sequencing and distributed throughout the genome, in seven cave and three groups of surface populations. The cave populations represented two distinct lineages (old and new). Thirty-one SNPs were significantly differentiated between surface and old cave populations, two SNPs were differentiated between surface and new cave populations, and 44 SNPs were significantly differentiated in both old and new cave populations. In addition, we determined whether these SNPs map to the same locations of previously described quantitative trait loci (QTL) between surface and cave populations. A total of 25 differentiated SNPs co-map with several QTL, such as one containing a fibroblast growth factor gene (Fgf8) involved in eye development and lens size. Further, the identity of many SNPs that co-mapped with QTL was the same in independently derived cave populations. These conclusions were further confirmed by haplotype analyses of SNPs within QTL regions. Our findings indicate that the repeatability of evolution at the genetic level is substantial, suggesting that ancestral standing genetic variation significantly contributed to the population genetic variability used in adaptation to the cave environment.

Gene flow and population structure in the Mexican blind cavefish complex (Astyanax mexicanus).

BACKGROUND: Cave animals converge evolutionarily on a suite of troglomorphic traits, the best known of which are eyelessness and depigmentation. We studied 11 cave and 10 surface populations of Astyanax mexicanus in order to better understand the evolutionary origins of the cave forms, the basic genetic structuring of both cave and surface populations, and the degree to which present day migration among them affects their genetic divergence. RESULTS: To assess the genetic structure within populations and the relationships among them we genotyped individuals at 26 microsatellite loci. We found that surface populations are similar to one another, despite their relatively large geographic separation, whereas the cave populations are better differentiated. The cave populations we studied span the full range of the cave forms in three separate geographic regions and have at least five separate evolutionary origins. Cave populations had lower genetic diversity than surface populations, correlated with their smaller effective population sizes, probably the result of food and space limitations. Some of the cave populations receive migrants from the surface and exchange migrants with one another, especially when geographically close. This admixture results in significant heterozygote deficiencies at numerous loci due to Wahlund effects. Cave populations receiving migrants from the surface contain small numbers of individuals that are intermediate in both phenotype and genotype, affirming at least limited gene flow from the surface. CONCLUSIONS: Cave populations of this species are derived from two different surface stocks denoted "old" and "new." The old stock colonized caves at least three times independently while the new stock colonized caves at least twice independently. Thus, the similar cave phenotypes found in these caves are the result of repeated convergences. These phenotypic convergences have occurred in spite of gene flow from surface populations suggesting either strong natural or sexual selection for alleles responsible for the cave phenotype in the cave environment.

ß-adrenergic signaling regulates evolutionarily derived sleep loss in the Mexican cavefish.

Sleep is a fundamental behavior exhibited almost universally throughout the animal kingdom. The required amount and circadian timing of sleep differs greatly between species in accordance with habitats and evolutionary history. The Mexican blind cavefish, Astyanax mexicanus, is a model organism for the study of adaptive morphological and behavioral traits. In addition to loss of eyes and pigmentation, cave populations of A. mexicanus exhibit evolutionarily derived sleep loss and increased vibration attraction behavior, presumably to cope with a nutrient-poor environment. Understanding the neural mechanisms of evolutionarily derived sleep loss in this system may reveal critical insights into the regulation of sleep in vertebrates. Here we report that blockade of ß-adrenergic receptors with propranolol rescues the decreased-sleep phenotype of cavefish. This effect was not seen with α-adrenergic antagonists. Treatment with selective ß1-, ß2-, and ß3-antagonists revealed that the increased sleep observed with propranolol could partially be explained via the ß1-adrenergic system. Morphological analysis of catecholamine circuitry revealed conservation of gross catecholaminergic neuroanatomy between surface and cave morphs. Taken together, these findings suggest that evolutionarily derived changes in adrenergic signaling underlie the reduced sleep of cave populations.

Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish.

In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting.

Altered rest-activity patterns evolve via circadian independent mechanisms in cave adapted balitorid loaches.

Circadian rhythms and rest homeostasis are independent processes, each regulating important components of rest-activity patterns. Evolutionarily, the two are distinct from one another; total rest time is maintained unaffected even when circadian pacemaker cells are ablated. Throughout the animal kingdom, there exists a huge variation in rest-activity patterns, yet it is unclear how these behaviors have evolved. Here we show that four species of balitorid cavefish have greatly reduced rest times in comparison to rest times of their surface relatives. All four cave species retained biological rhythmicity, and in three of the four there is a pronounced 24-hour rhythm; in the fourth there is an altered rhythmicity of 38-40 hours. Thus, consistent changes in total rest have evolved in these species independent of circadian rhythmicity. Taken together, our data suggest that consistent reduction in total rest times were accomplished evolutionarily through alterations in rest homeostasis.

Evolutionary convergence on sleep loss in cavefish populations.

Patterns of sleep vary widely among species, but the functional and evolutionary principles responsible for this diversity remain unknown. The characin fish, Astyanax mexicanus, has eyed surface and numerous blind cave populations. The cave populations are largely independent in their origins, and the species is ideal for studying the genetic bases of convergent evolution. Here we show that this system is also uniquely valuable for the investigation of variability in patterns of sleep. We find that a clearly defined change in ecological conditions, from surface to cave, is correlated with a dramatic reduction in sleep in three independently derived cave populations of A. mexicanus. Analyses of surface × cave hybrids show that the alleles for reduced sleep in the Pachón and Tinaja cave populations are dominant in effect to the surface alleles. Genetic analysis of hybrids between surface and Pachón cavefish suggests that only a small number of loci with dominant effects are involved. Our results demonstrate that sleep is an evolutionarily labile phenotype, highly responsive to changes in ecological conditions. To our knowledge, this is the first example of a single species with a convergence on sleep loss exhibited by several independently evolved populations correlated with population-specific ecologies.