Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

BACKGROUND: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species. RESULTS: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype. CONCLUSIONS: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

On Intraspecific and Interspecific Variation in Teleost Scleral Ossification.

Scleral ossicles are bony elements found along the eyes of many fishes, amphibians, and reptiles. These bones provide a superficial layer of support to the eye and may facilitate visual acuity. Previous research has shown that scleral ossicle diversity is generally limited among teleosts, but that scleral ossicles have been lost numerous times among teleosts inhabiting benthopelagic habitats (Franz-Odendaal. Anat Rec 291 (2008) 161-168). In this study, we further investigate these patterns of intraspecific and interspecific variation by examining eyes from multiple individuals of 10 riverine teleosts native to Kentucky as well as one population of the Mexican blind cavefish, Astyanax mexicanus, and by re-analyzing a quantitative database of scleral ossicle number and depth preference from over 100 teleosts using newly resolved teleost phylogenies. Consistent with the limited diversity of most teleost families, we find that intraspecific variation in scleral ossicle number and size is virtually nonexistent among the species sampled, although we do find evidence of additional interspecific variation among the Cyprinodontiformes, as well as dramatic intrapopulation variation among cavefish from Chica Cave. Although our data replicates the negative relationship between scleral ossicle number and the depth preference previously found among teleosts (Franz-Odendaal. Anat Rec 291 (2008) 161-168), even when accounting for phylogenetic relationships, our results further reveal that this relationship is relatively weak. We conclude that further sampling may reveal additional interspecific and even intraspecific variation among some groups of teleosts, and that depth could serve as a proxy for other life history traits that more directly influence teleost scleral ossicle diversity such as prey-capture strategies. Anat Rec, 302:1238-1249, 2019. © 2019 Wiley Periodicals, Inc.

Two - three loci control scleral ossicle formation via epistasis in the cavefish Astyanax mexicanus.

The sclera is the protective outer layer of the eye. In fishes, birds, and reptiles, the sclera may be reinforced with additional bony elements called scleral ossicles. Teleost fish vary in the number and size of scleral ossicles; however, the genetic mechanisms responsible for this variation remain poorly understood. In this study, we examine the inheritance of scleral ossicles in the Mexican tetra, Astyanax mexicanus, which exhibits both a cave morph and a surface fish morph. As these morphs and their hybrids collectively exhibit zero, one, and two scleral ossicles, they represent a microcosm of teleost scleral ossicle diversity. Our previous research in F2 hybrids of cavefish from Pachón cave and surface fish from Texas suggested that three genes likely influence the formation of scleral ossicles in this group through an epistatic threshold model of inheritance, though our sample size was small. In this study, we expand our sample size using additional hybrids of Pachón cavefish and Mexican surface fish to (1) confirm the threshold model of inheritance, (2) refine the number of genes responsible for scleral ossicle formation, and (3) increase our power to detect quantitative trait loci (QTL) for this trait. To answer these three questions, we scored surface fish and cavefish F2 hybrids for the presence of zero, one, or two scleral ossicles. We then analyzed their distribution among the F2 hybrids using a chi-square (χ2) test, and used a genetic linkage map of over 100 microsatellite markers to identify QTL responsible for scleral ossicle number. We found that inheritance of scleral ossicles follows an epistatic threshold model of inheritance controlled by two genes, which contrasts the three-locus model estimated from our previous study. Finally, the combined analysis of hybrids from both crosses identified two strong QTL for scleral ossicle number on linkage groups 4.2 and 21, and a weaker QTL on linkage group 4.1. Scleral ossification remains a complex trait with limited knowledge of its genetic basis. This study provides new insight into the number and location of genes controlling the formation of scleral ossicles in a teleost fish species.

Complex Evolutionary and Genetic Patterns Characterize the Loss of Scleral Ossification in the Blind Cavefish Astyanax mexicanus.

The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra (Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F1 hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F2 hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common-but not ubiquitous-among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F1 hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F2 hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.

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 cavefish genome reveals candidate genes for eye loss.

Natural populations subjected to strong environmental selection pressures offer a window into the genetic underpinnings of evolutionary change. Cavefish populations, Astyanax mexicanus (Teleostei: Characiphysi), exhibit repeated, independent evolution for a variety of traits including eye degeneration, pigment loss, increased size and number of taste buds and mechanosensory organs, and shifts in many behavioural traits. Surface and cave forms are interfertile making this system amenable to genetic interrogation; however, lack of a reference genome has hampered efforts to identify genes responsible for changes in cave forms of A. mexicanus. Here we present the first de novo genome assembly for Astyanax mexicanus cavefish, contrast repeat elements to other teleost genomes, identify candidate genes underlying quantitative trait loci (QTL), and assay these candidate genes for potential functional and expression differences. We expect the cavefish genome to advance understanding of the evolutionary process, as well as, analogous human disease including retinal dysfunction.

Interspecific variation in Rx1 expression controls opsin expression and causes visual system diversity in African cichlid fishes.

The mechanisms underlying natural phenotypic diversity are key to understanding evolution and speciation. Cichlid fishes are among the most speciose vertebrates and an ideal model for identifying genes controlling species differences. Cichlids have diverse visual sensitivities that result from species expressing subsets of seven cichlid cone opsin genes. We previously identified a quantitative trait locus (QTL) that tunes visual sensitivity by varying SWS2A (short wavelength sensitive 2A) opsin expression in a genetic cross between two Lake Malawi cichlid species. Here, we identify Rx1 (retinal and anterior neural fold homeobox) as the causative gene for the QTL using fine mapping and RNAseq in retinal transcriptomes. Rx1 is differentially expressed between the parental species and correlated with SWS2A expression in the F2 progeny. Expression of Rx1 and SWS2A is also correlated in a panel of 16 Lake Malawi cichlid species. Association mapping in this panel identified a 413-bp deletion located 2.5-kb upstream of the Rx1 translation start site that is correlated with decreased Rx1 expression. This deletion explains 62% of the variance in SWS2A expression across 53 cichlid species in 29 genera. The deletion occurs in both the sand and rock-dwelling cichlid clades, suggesting that it is an ancestral polymorphism. Our finding supports the hypothesis that mixing and matching of ancestral polymorphisms can explain the diversity of present day cichlid phenotypes.

QTL clustering as a mechanism for rapid multi-trait evolution.

Cave-dwelling animals exhibit remarkable convergence in multiple cave-related traits, yet the genetic mechanisms responsible for the evolution and integration of many such traits remain unclear. Astyanax mexicanus is a model cave-dwelling fish with sighted surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. Using a genetic cross between these morphs, we discovered significant correlations among several cave-related traits, including reduced eyes, increased superficial neuromast receptors located within the eye orbit (EO SN) and a vibration-attraction behavior (VAB) that facilitates foraging in darkness. Furthermore, we discovered that the quantitative trait loci (QTL) underlying these traits are clustered within the Astyanax genome. Following an ablation experiment that demonstrated that the EO SN contribute to VAB, we concluded that the adaptive evolution of VAB and EO SN has likely contributed to eye loss in cavefish. In this addendum, we further discuss the possible role of multi-trait QTL clustering in facilitating rapid adaptation.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish: response to Borowsky (2013).

Vibration attraction behavior (VAB) is the swimming of fish toward an oscillating object, a behavior that is likely adaptive because it increases foraging efficiency in darkness. VAB is seen in a small proportion of Astyanax surface-dwelling populations (surface fish) but is pronounced in cave-dwelling populations (cavefish). In a recent study, we identified two quantitative trait loci for VAB on Astyanax linkage groups 2 and 17. We also demonstrated that a small population of superficial neuromast sensors located within the eye orbit (EO SN) facilitate VAB, and two quantitative trait loci (QTL) were identified for EO SN that were congruent with those for VAB. Finally, we showed that both VAB and EO SN are negatively correlated with eye size, and that two (of several) QTL for eye size overlap VAB and EO SN QTLs. From these results, we concluded that the adaptive evolution of VAB and EO SN has contributed to the indirect loss of eyes in cavefish, either as a result of pleiotropy or tight physical linkage of the mutations underlying these traits. In a subsequent commentary, Borowsky argues that there is poor experimental support for our conclusions. Specifically, Borowsky states that: (1) linkage groups (LGs) 2 and 17 harbor QTL for many traits and, therefore, no evidence exists for an exclusive interaction among the overlapping VAB, EO SN and eye size QTL; (2) some of the QTL we identified are too broad (>20 cM) to support the hypothesis of correlated evolution due to pleiotropy or hitchhiking; and (3) VAB is unnecessary to explain the indirect evolution of eye-loss since the negative polarity of numerous eye QTL is consistent with direct selection against eyes. Borowsky further argues that (4) it is difficult to envision an evolutionary scenario whereby VAB and EO SN drive eye loss, since the eyes must first be reduced in order to increase the number of EO SN and, therefore, VAB. In this response, we explain why the evidence of one trait influencing eye reduction is stronger for VAB than other traits, and provide further support for a scenario whereby elaboration of VAB in surface fish may precede complete eye-loss.

Quantitative genetic analysis of retinal degeneration in the blind cavefish Astyanax mexicanus.

The retina is the light-sensitive tissue of the eye that facilitates vision. Mutations within genes affecting eye development and retinal function cause a host of degenerative visual diseases, including retinitis pigmentosa and anophthalmia/microphthalmia. The characin fish Astyanax mexicanus includes both eyed (surface fish) and eyeless (cavefish) morphs that initially develop eyes with normal retina; however, early in development, the eyes of cavefish degenerate. Since both surface and cave morphs are members of the same species, they serve as excellent evolutionary mutant models with which to identify genes causing retinal degeneration. In this study, we crossed the eyed and eyeless forms of A. mexicanus and quantified the thickness of individual retinal layers among 115 F(2) hybrid progeny. We used next generation sequencing (RAD-seq) and microsatellite mapping to construct a dense genetic map of the Astyanax genome, scan for quantitative trait loci (QTL) affecting retinal thickness, and identify candidate genes within these QTL regions. The map we constructed for Astyanax includes nearly 700 markers assembled into 25 linkage groups. Based on our scans with this map, we identified four QTL, one each associated with the thickness of the ganglion, inner nuclear, outer plexiform, and outer nuclear layers of the retina. For all but one QTL, cavefish alleles resulted in a clear reduction in the thickness of the affected layer. Comparative mapping of genetic markers within each QTL revealed that each QTL corresponds to an approximately 35 Mb region of the zebrafish genome. Within each region, we identified several candidate genes associated with the function of each affected retinal layer. Our study is the first to examine Astyanax retinal degeneration in the context of QTL mapping. The regions we identify serve as a starting point for future studies on the genetics of retinal degeneration and eye disease using the evolutionary mutant model Astyanax.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish.

BACKGROUND: How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression. RESULTS: Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN. CONCLUSIONS: We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

Evolution of cichlid vision via trans-regulatory divergence.

BACKGROUND: Phenotypic evolution may occur through mutations that affect either the structure or expression of protein-coding genes. Although the evolution of color vision has historically been attributed to structural mutations within the opsin genes, recent research has shown that opsin regulatory mutations can also tune photoreceptor sensitivity and color vision. Visual sensitivity in African cichlid fishes varies as a result of the differential expression of seven opsin genes. We crossed cichlid species that express different opsin gene sets and scanned their genome for expression Quantitative Trait Loci (eQTL) responsible for these differences. Our results shed light on the role that different structural, cis-, and trans-regulatory mutations play in the evolution of color vision. RESULTS: We identified 11 eQTL that contribute to the divergent expression of five opsin genes. On three linkage groups, several eQTL formed regulatory "hotspots" associated with the expression of multiple opsins. Importantly, however, the majority of the eQTL we identified (8/11 or 73%) occur on linkage groups located trans to the opsin genes, suggesting that cichlid color vision has evolved primarily via trans-regulatory divergence. By modeling the impact of just two of these trans-regulatory eQTL, we show that opsin regulatory mutations can alter cichlid photoreceptor sensitivity and color vision at least as much as opsin structural mutations can. CONCLUSIONS: Combined with previous work, we demonstrate that the evolution of cichlid color vision results from the interplay of structural, cis-, and especially trans-regulatory loci. Although there are numerous examples of structural and cis-regulatory mutations that contribute to phenotypic evolution, our results suggest that trans-regulatory mutations could contribute to phenotypic divergence more commonly than previously expected, especially in systems like color vision, where compensatory changes in the expression of multiple genes are required in order to produce functional phenotypes.

Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African cichlid fishes.

BACKGROUND: Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression. RESULTS: We identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover. We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression. CONCLUSIONS: This study is the first to systematically search the opsins of cichlids for putative cis-regulatory sequences. Although many putative regulatory regions are highly conserved across a large number of phenotypically diverse cichlids, we found at least nine divergent sequences that could contribute to opsin expression differences in cis and stand out as candidates for future functional analyses.

New evidence for the role of heterochrony in the repeated evolution of cichlid opsin expression.

SUMMARY Lake Malawi (LM) cichlids have undergone heterochronic shifts in the expression of their cone opsin genes, the genes responsible for color vision. These shifts have generated species with short-, middle-, and long-wavelength-sensitive cone photoreceptors and visual systems. However, it is unclear when during the evolution of African cichlids these shifts occurred, or whether they could account for similar short- and middle-wavelength-sensitive profiles among unrelated cichlids in Lake Tanganyika (LT). To address these questions, we surveyed opsin expression in developing fry of two African cichlids, Astatotilapia burtoni from LT and Melanochromis auratus from LM. We found that A. burtoni expresses a series of three different single-cone opsins over the course of development, while M. auratus exhibits variation in the expression of only two. Neither A. burtoni nor M. auratus exhibits much variation in the expression of its double-cone opsins. These patterns reveal that A. burtoni exhibits progressive development in the sensitivity of its single-cone photoreceptors, but direct development in the sensitivity of its double-cone photoreceptors. M. auratus exhibits neotenic development in the sensitivity of both photoreceptor sets. Given the intermediate phylogenetic placement of A. burtoni between cichlids from LT and LM, our results suggest that the ancestor of LM's cichlids exhibited a progressive developmental pattern of opsin expression. These results indicate that the heterochronic shifts which produced the short- and middle-wavelength-sensitive profiles of LM's cichlids occurred recently, and suggest that the presence of similar profiles among LT's cichlids are due to parallel heterochronic shifts.

Parallel evolution of opsin gene expression in African cichlid fishes.

Phenotypic evolution may occur either through alterations to the structure of protein-coding genes or their expression. Evidence for which of these two mechanisms more commonly contribute to the evolution of a phenotype can be garnered from examples of parallel and convergent evolution. The visual system of East African cichlid fishes is an excellent system with which to address this question. Cichlid fishes from Lakes Malawi (LM) and Victoria together exhibit three diverse palettes of coexpressed opsins and several important protein-coding mutations that both shift spectral sensitivity. Here we assess both opsin expression and protein-coding diversity among cichlids from a third rift lake, Lake Tanganyika (LT). We found that Tanganyikan cichlids exhibit three palettes of coexpressed opsins that largely overlap the short-, middle-, and long-wavelength-sensitive palettes of LM cichlids. Bayesian phenotypic clustering and ancestral state reconstructions both support the parallel evolution of the short- and middle-wavelength palettes among cichlids from LT and LM. In each case, these transitions occurred from different ancestors that expressed the same long-wavelength palette. We also identified similar but distinct patterns of correlated evolution between opsin expression, diet, and lens transmittance among cichlids from LT and LM as well. In contrast to regulatory changes, we identified few functional or potentially functional mutations in the protein-coding sequences of three variable opsins, with the possible exception of the SWS1 (ultraviolet) opsin. These results underscore the important contribution that gene regulation can make to rapid phenotypic evolution and adaptation.

Plasticity of opsin gene expression in cichlids from Lake Malawi.

Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild-caught fish to lab-reared F(1) that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild-caught fish were also expressed in lab-reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the SWS2B (violet) opsin, which was always expressed at higher levels in lab-reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi.

The relationship between lens transmission and opsin gene expression in cichlids from Lake Malawi.

The lens plays an important role in regulating the wavelengths of light that reach the retina. However, the evolutionary relationship between lens transmission and retinal sensitivity remains cloudy at best. We examined the relationship between lens transmission and opsin gene expression in a group of rapidly radiating cichlids from East Africa. Lens transmission was bimodal, either cutting off around 360 or 400 nm, and appeared to be quite labile evolutionarily. We found a strong correlation between lens transmission and SWS1 (UV) opsin gene expression, suggesting that UV transmitting lenses are adaptive in cichlids. Species which expressed high levels of SWS2B (violet) opsin varied in their lens transmission while most species that expressed high levels of SWS2A (blue) opsin had UV blocking lenses. In no instance did lens transmission appear to limit retinal sensitivity. Finally, the strong correlation that we observe between SWS1 expression and lens transmission suggests that these two traits might be coupled genetically.

The eyes have it: regulatory and structural changes both underlie cichlid visual pigment diversity.

A major goal of evolutionary biology is to unravel the molecular genetic mechanisms that underlie functional diversification and adaptation. We investigated how changes in gene regulation and coding sequence contribute to sensory diversification in two replicate radiations of cichlid fishes. In the clear waters of Lake Malawi, differential opsin expression generates diverse visual systems, with sensitivities extending from the ultraviolet to the red regions of the spectrum. These sensitivities fall into three distinct clusters and are correlated with foraging habits. In the turbid waters of Lake Victoria, visual sensitivity is constrained to longer wavelengths, and opsin expression is correlated with ambient light. In addition to regulatory changes, we found that the opsins coding for the shortest- and longest-wavelength visual pigments have elevated numbers of potentially functional substitutions. Thus, we present a model of sensory evolution in which both molecular genetic mechanisms work in concert. Changes in gene expression generate large shifts in visual pigment sensitivity across the collective opsin spectral range, but changes in coding sequence appear to fine-tune visual pigment sensitivity at the short- and long-wavelength ends of this range, where differential opsin expression can no longer extend visual pigment sensitivity.