EVIDENCE FOR REPEATED LOSS OF SELECTIVE CONSTRAINT IN RHODOPSIN OF AMBLYOPSID CAVEFISHES (TELEOSTEI: AMBLYOPSIDAE)

The genetic mechanisms underlying regressive evolution—the degeneration or loss of a derived trait—are largely unknown, particularly for complex structures such as eyes in cave organisms. In several eyeless animals, the visual photoreceptor rhodopsin appears to have retained functional amino acid sequences. Hypotheses to explain apparent maintenance of function include weak selection for retention of light‐sensing abilities and its pleiotropic roles in circadian rhythms and thermotaxis. In contrast, we show that there has been repeated loss of functional constraint of rhodopsin in amblyopsid cavefishes, as at least three cave lineages have independently accumulated unique loss‐of‐function mutations over the last 10.3 Mya. Although several cave lineages still possess functional rhodopsin, they exhibit increased rates of nonsynonymous mutations that have greater effect on the structure and function of rhodopsin compared to those in surface lineages. These results indicate that functionality of rhodopsin has been repeatedly lost in amblyopsid cavefishes. The presence of a functional copy of rhodopsin in some cave lineages is likely explained by stochastic accumulation of mutations following recent subterranean colonization.     

Cave-adapted evolution in the North American amblyopsid fishes inferred using phylogenomics and geometric morphometrics

Cave adaptation has evolved repeatedly across the Tree of Life, famously leading to pigmentation and eye degeneration and loss, yet its macroevolutionary implications remain poorly understood. We use the North American amblyopsid fishes, a family spanning a wide degree of cave adaptation, to examine the impact of cave specialization on the modes and tempo of evolution. We reconstruct evolutionary relationships using ultraconserved element loci, estimate the ancestral histories of eye-state, and examine the impact of cave adaptation on body shape evolution. Our phylogenomic analyses provide a well-supported hypothesis for amblyopsid evolutionary relationships. The obligate blind cavefishes form a clade and the cave-facultative eyed spring cavefishes are nested within the obligate cavefishes. Using ancestral state reconstruction, we find support for at least two independent subterranean colonization events within the Amblyopsidae. Eyed and blind fishes have different body shapes, but not different rates of body shape evolution. North American amblyopsids highlight the complex nature of cave-adaptive evolution and the necessity to include multiple lines of evidence to uncover the underlying processes involved in the loss of complex traits.     

The molecular mechanisms of OPA1-mediated optic atrophy in Drosophila model and prospects for antioxidant treatment

Mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA). The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479), a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens and pigment deposition) eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS) production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition.     

Colour vision variation in leaf‐nosed bats (Phyllostomidae): Links to cave roosting and dietary specialization

Bats are a diverse radiation of mammals of enduring interest for understanding the evolution of sensory specialization. Colour vision variation among species has previously been linked to roosting preferences and echolocation form in the suborder Yinpterochiroptera, yet questions remain about the roles of diet and habitat in shaping bat visual ecology. We sequenced OPN1SW and OPN1LW opsin genes for 20 species of leaf‐nosed bats (family Phyllostomidae; suborder Yangochiroptera) with diverse roosting and dietary ecologies, along with one vespertilionid species (Myotis lavali). OPN1LW genes appear intact for all species, and predicted spectral tuning of long‐wavelength opsins varied among lineages. OPN1SW genes appear intact and under purifying selection for Myotis lavali and most phyllostomid bats, with two exceptions: (a) We found evidence of ancient OPN1SW pseudogenization in the vampire bat lineage, and loss‐of‐function mutations in all three species of extant vampire bats; (b) we additionally found a recent, independently derived OPN1SW pseudogene in Lonchophylla mordax, a cave‐roosting species. These mutations in leaf‐nosed bats are independent of the OPN1SW pseudogenization events previously reported in Yinpterochiropterans. Therefore, the evolution of monochromacy (complete colour blindness) has occurred in both suborders of bats and under various evolutionary drivers; we find independent support for the hypothesis that obligate cave roosting drives colour vision loss. We additionally suggest that haematophagous dietary specialization and corresponding selection on nonvisual senses led to loss of colour vision through evolutionary sensory trade‐off. Our results underscore the evolutionary plasticity of opsins among nocturnal mammals.     

Chromosomal localization of three mouse diacylglycerol kinase (DAGK) genes: genes sharing sequence homology to the Drosophila retinal degeneration A (rdgA) gene

There is growing evidence to support some form of light-activated phosphoinositide signal transduction pathway in the mammalian retina. Although this pathway plays no obvious role in mammalian phototransduction, mutations in this pathway cause retinal degenerations in Drosophila. These include the retinal degeneration A mutant, which is caused by an alteration in an eye-specific diacylglycerol kinase (DAGK) gene. In our efforts to consider genes mutated in Drosophila as candidates for mammalian eye disease, we have initially determined the map position of three DAGK genes in the mouse.     

Retinal determination genes as targets and possible effectors of extracellular signals

Retinal determination genes are sufficient to specify eyes in ectopic locations, raising the question of how these master regulatory genes define an eye developmental field. Genetic mosaic studies establish that expression of the retinal determination genes eyeless, teashirt, homothorax, eyes absent, sine oculis, and dachshund are each regulated by combinations of Dpp, Hh, N, Wg, and Ras signals in Drosophila. Dpp and Hh control eyeless, teashirt, sine oculis, and dachshund expression, Dpp and Ras control homothorax, and all the signaling pathways affect eyes absent expression. These results suggest that eye-specific development uses retinal determination gene expression to relay positional information to eye target genes, because the distinct, overlapping patterns of retinal determination gene expression reflect the activities of the extracellular signaling pathways.     

Genetic Perturbation of the Maize Methylome

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana.     

Relaxed purifying selection of rhodopsin gene within a Chinese endemic cavefish genus <Emphasis Type="Italic">Sinocyclocheilus</Emphasis> (Pisces: Cypriniformes)

The cyprinid fish genus Sinocyclocheilus, as the most cavefish rich genus, includes many species showing striking adaptation to caves and convergent reduction or even loss of eyes and pigmentation. RH1 is responsible for dim vision. In order to explore the evolution of RH1 gene in this genus, we sequenced the complete gene from 28 individuals of 16 representative species of Sinocyclocheilus, with cave and surface species included. Phylogenetic analyses supported the monophyly of Sinocyclocheilus and polyphyly of the cave species. Codon models implemented in PAML were used to infer the evolution of RH1. We found that Sinocyclocheilus had a significantly higher evolutionary rate for amino acids than other cyprinid fishes compared, which might be the result of relaxation of purifying selection and could be ascribed to cave habit of this genus. In contrast to previous hypotheses, both cave and surface lineages exhibited a similar rate of molecular evolution, so the RH1 of cave species may still be functional, although these species were highly adapted to cave environment. Two amino acid substitutions (D83G and E122V) that were not reported before were found, which may be useful for site-directed mutagenesis in the future. Handling editor: Christian Sturmbauer     

Standing genetic variation as a potential mechanism of novel cave phenotype evolution in the freshwater isopod,Asellus aquaticus

Novel phenotypes can come about through a variety of mechanisms including standing genetic variation from a founding population. Cave animals are an excellent system in which to study the evolution of novel phenotypes such as loss of pigmentation and eyes. Asellus aquaticus is a freshwater isopod crustacean found in Europe and has both a surface and a cave ecomorph which vary in multiple phenotypic traits. An orange eye phenotype was previously revealed by F₂ crosses and backcrosses to the cave parent within two examined Slovenian cave populations. Complete loss of pigmentation, both in eye and body, is epistatic to the orange eye phenotype and therefore the orange eye phenotype is hidden within the cave populations. Our goal was to investigate the origin of the orange eye alleles within the Slovenian cave populations by examining A. aquaticus individuals from Slovenian and Romanian surface populations and Asellus aquaticus infernus individuals from a Romanian cave population. We found orange eye individuals present in lab raised surface populations of A. aquaticus from both Slovenia and Romania. Using a mapping approach with crosses between individuals of two surface populations, we found that the region known to be responsible for the orange eye phenotype within the two previously examined Slovenian cave populations was also responsible within both the Slovenian and the Romanian surface populations. Complementation crosses between orange eye Slovenian and orange eye Romanian surface individuals suggest that the same gene is responsible for the orange eye phenotype in both surface populations. Additionally, we observed a low frequency phenotype of eye loss in crosses generated between the two surface populations and also in the Romanian surface population. Finally, in a cave population from Romania, A. aquaticus infernus, we found that the same region is also responsible for the orange eye phenotype as the Slovenian cave populations and the Slovenian and Romanian surface populations. Therefore, we present evidence that variation present in the cave populations could originate from standing variation present in the surface populations and/or transgressive hybridization of different surface phylogenetic lineages rather than de novo mutations.     

Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation

More than 20 populations of the cave-dwelling characid Astyanax occur within a restricted karst area in Mexico. The fish possess reduced eyes without lenses and visual cells. It is still an open question as to whether this condition evolved convergently after multiple entries of the surface ancestor into the different caves or whether a single cave ancestor, already characterized by reduced eyes, spread secondarily within them. In the crosses between specific populations, specimens appear that deviate considerably from those of the parents. They possess larger and better-developed eyes with histologically intact lenses and visual cells; they thus have the structural potential for vision. This indicates that in different cave populations, different mutations in the eye gene system have occurred. In cases where these non-functional rudimentary genes are recombined in hybrid specimens, gene expression may be restored. This is the result of separate evolution of several Astyanax cave populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 545−554.     

Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele

The Mexican tetra, Astyanax mexicanus, comprises 29 populations of cave-adapted fish distributed across a vast karst region in northeastern Mexico. These populations have a complex evolutionary history, having descended from ‘old'' and ‘young'' ancestral surface-dwelling stocks that invaded the region ∼6.7 and ∼2.8 MYa, respectively. This study investigates a set of captive, pigmented Astyanax cavefish collected from the Micos cave locality in 1970, in which albinism appeared over the past two decades. We combined novel coloration analyses, coding sequence comparisons and mRNA expression level studies to investigate the origin of albinism in captive-bred Micos cavefish. We discovered that albino Micos cavefish harbor two copies of a loss-of-function ocular and cutaneous albinism type II (Oca2) allele previously identified in the geographically distant Pachón cave population. This result suggests that phylogenetically young Micos cavefish and phylogenetically old Pachón cave fish inherited this Oca2 allele from the ancestral surface-dwelling taxon. This likely resulted from the presence of the loss-of-function Oca2 haplotype in the ‘young'' ancestral surface-dwelling stock that colonized the Micos cave and also introgressed into the ancient Pachón cave population. The appearance of albinism in captive Micos cavefish, caused by the same loss-of-function allele present in Pachón cavefish, implies that geographically and phylogenetically distinct cave populations can evolve the same troglomorphic phenotype from standing genetic variation present in the ancestral taxon.     

Do Chinese cavefish show intraspecific variability in morphological traits?

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One‐third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies.     

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.