The likelihood of each partition model was compared using differences in AIC. The partitions that best fit the data are indicated. Additionally, we searched for the presence of more than one sws1 variant by sequencing the exon 1 from genomic DNA of single individuals of the Helicops species: H.
In all Helicops species, we consistently found both residues Phe86 and Val86 supplementary fig. S4 , Supplementary Material online , indicating that the presence of two short-wavelength sensitive opsins, a UV and a violet, within individuals is widespread in the genus.
In all other species non- Helicops investigated, we found the presence of residue Phe86, only, indicating a single UV-sensitive SWS1 opsin. Furthermore, we analyzed intronic sequences of single individuals from six Helicops species. We amplified by PCR and sequenced the intron 1 of H.
The amplified fragments were isolated from agarose gel supplementary fig. S5 , Supplementary Material online and sequenced in both directions. All species had an intron 1 with approximately bp, except H. In individuals of two species, H. S5 , Supplementary Material online. This finding strongly indicates that the two sws1 variants represent distinct paralogous genes. Moreover, based on the electrophoresis images supplementary fig.
S5 , Supplementary Material online and preliminary sequencing analyses data not shown , we identified signals of the presence of three distinct intron 1s with different lengths in H. We searched for distinct signatures of selection acting on the sws1 opsin genes of Helicops snakes fig. Using CmC models, we also tested partitions isolating as foregrounds, respectively: 1 the aquatic lineage Hydropsini snakes represented here by Helicops species and Hydrops caesurus , and 2 the Helicops clade.
S6 and table S2 , Supplementary Material online. M0 , as expected for a protein-coding gene under strong purifying selection. M1a, M8 vs. M7; supplementary table S3 , Supplementary Material online. We did not find evidence for positive selected sites in specific clades of interest aquatic snakes, Helicops, sws1a , and sws1b using branch-site models supplementary table S4 , Supplementary Material online.
We used microspectrophotometry MSP of retinas on two wild-caught H. In the H. S7 and table S5 , Supplementary Material online. The contralateral eye of this second individual was used for the genetic analysis revealing the expression of a UV Phe86 sws1a opsin gene.
The combined MSP and genetic results for this individual of H. Four visual opsins are simultaneously expressed in retinas of Helicops snakes. C Lens and spectacle normalized transmittance of H.
D Effect of spectacle transmittance on spectral sensitivity in H. This amino acid combination and spectral peak were described in diurnal colubroids Schott et al. Analysis of ocular media transmittance of two individuals of H. We calculated the impact of the ocular media transmittance in visual sensitivity and the amount of incoming light available to the visual system by quantifying the effect of the spectacle transmittance on photoreceptors spectral sensitivity curves of H.
We investigated the spectral phenotype of H. Sws1a and sws1b of H. The expressed proteins were purified by immunoaffinity and reconstituted with cis -retinal chromophore in the dark. Spectroscopy of the purified proteins revealed that both visual pigments are functional and have distinct absorption peaks.
Molecular mechanisms underlying the spectral tuning of UV and violet opsins of Helicops : UV-visible dark absorption spectra of H.
WT, wildtype. We explored the functional role of the single amino acid substitution Phe86Val using site-directed mutagenesis and in vitro expression of mutant opsins. These results show experimentally, for the first time in snakes, that residue 86 is responsible for a major shift between UV and violet opsins, whereas other residues might have minor additional effects on spectral tuning. The substitution Val86Phe in a violet opsin background generated a complete shift toward the UV-band, indicating that Phe86 might cause the loss of electrostatic protonation of the chromophore Schiff base Cowing et al.
However, the spectral peak of the mutated UV opsin, Phe86Val, did not cause the complete shift toward the violet band, indicating that other residues present in the violet opsin SWS1B might be necessary for fully stabilizing the protonation of the Schiff base Fahmy and Sakmar resulting in the difference in the degree of spectral shift in the forward and backward mutations.
We investigated the photoreceptor types, densities, and distribution in retinas of Helicops snakes, using immunohistochemistry. Additionally, the absence of typical rods with long and slender outer segment is characteristic of primarily diurnal species Walls ; Underwood ; Wong ; Sillman et al.
Nocturnal colubroids, on the other hand, have duplex retinas with a high amount of typical rods and a thick outer nuclear layer Hauzman et al. Using different combinations of specific anti-opsin antibodies double-labeling , previously used in snake retinas Schott et al. The retinas are dominated by the LWS cone class with two subpopulations, large single cones and double cones fig.
Small single SWS1 cones represent a small population of photoreceptors, and a fourth group of photoreceptors contain the rhodopsin RH1 photopigment and were classified as cone-like rods fig. The retinal morphology of Helicops. Confocal images of retinal sections of H. No opsin colocalization was observed in any cone. We analyzed the total density and distribution of the photoreceptors and of the SWS1 cones in three whole-mounted retinas of H.
The topographic maps showed higher densities of total photoreceptor and of the SWS1 cones in the ventral retina, with anisotropic area centralis fig. This type of specialization is likely to provide higher visual acuity in the upper visual field and suggest a higher absorption of short-wavelength photons from above. The retinal topographic maps of Helicops modestus. Total photoreceptors and SWS1 cones with anisotropic area centralis in the ventral region.
The optic nerve head is depicted as a white circle. In this study, we showed that two distinct SWS1 opsins with UV- or violet sensitivity can be expressed simultaneously in the retina of the aquatic dipsadid snake Helicops and that a single amino acid substitution is responsible for the UV-to-violet shift between the two SWS1 opsins.
The novel violet-sensitive SWS1B might compensate, at least in part, for the ancestral loss of blue opsins in snakes. The presence of two SWS1 provides a potential substrate for trichromatic or even tetrachromatic vision in this lineage. Finally, we find that the unique expansion of the opsin toolbox in Helicops was accompanied by morphological specializations of the retina such as a higher density of photoreceptors and short-wavelength sensitive cones in the ventral retina, possibly reflecting the freshwater habitats of this lineage.
Microspectrophotometry showed that both UV and violet single cones together with long-wavelength cones and rods can be simultaneously present in Helicops retina. The spectral peaks of the UV-sensitive and the violet-sensitive SWS1 cones measured by MSP matched the theoretical predictions based on tuning sites and directly measured by in vitro expression of the reconstructed SWS1 opsins.
Genome mining suggested that sws1 duplications might have independently occurred in some fish lineages Minamoto and Shimizu ; Lagman et al. In Helicops snakes, the two distinct SWS1 opsins might have evolved as a secondary adaptation to freshwater aquatic habitats, reducing the spectral gap between the ancestral UV-sensitive cones and the yellow-red sensitive LWS cones.
An intriguing possibility is that the pathways beginning with UV and violet cone excitation might lead to postreceptoral opponent responses allowing color discrimination in the very short waveband, an exciting venue for future behavioral and physiological studies. Alternatively, the two SWS1 opsins might have evolved completely distinct roles not involved in color vision, but specialized in other functions such as polarization vision, as proposed for UV cones in other vertebrates Bennett and Cuthill ; Hawryshyn The authors speculated that heterozygous individuals might express both alleles and gain an advantage over homozygous ones in a similar fashion to that enjoyed by heterozygote females of New World monkeys.
However, evidence that two SWS1 opsins are simultaneously expressed in these snakes is still lacking. In addition, the proposed fitness advantage of heterozygotes would only be present if both alleles were expressed not only simultaneously but in codominant fashion in distinct photoreceptors. Mammal ancestors passed through a nocturnal bottleneck that resulted in the loss of visual structures and two cone opsins sensitive to the core region of the visible spectral, SWS2 and RH2 Davies et al.
Some primates are unique among mammals in displaying trichromatic color vision based on SWS1, MWS, and LWS opsins, a gain achieved through two distinct mechanisms in different lineages Carvalho et al. In Old World monkeys, an lws duplication followed by substitutions at spectral tuning sites resulted in spectrally distinct opsins MWS and LWS conferring trichromacy in both males and females Nathans et al.
Worth noting, substitutions at the same residues are responsible for the spectral shift of the MWS and LWS opsins in both primate lineages.
In snakes, the fact that the same opsin site 86 was involved in SWS1 tuning in two independent lineages might represent a remarkable example of convergent evolution in which distinct lineages independently evolved similar phenotypes through the recruitment of substitutions at the same residue. The invasion of aquatic environments by a terrestrial vertebrate is probably at the origin of convergent visual phenotypes in these two independent snake lineages but the presence of mutations at the same site underlying such convergence points to mutational biases or genetic constraints in this opsin Losos ; Stern , an exciting avenue for future research.
These predictions were confirmed by our in vitro expression of the sws1a and sws1b genes of H. Moreover, using site-directed mutagenesis, we confirmed for the first time the functional role of the single amino acid substitution at residue 86 in generating the wide spectral shift of UV and violet photopigments in this lineage of snakes.
In the majority of snakes investigated so far, residue 86 is thought to predict well the spectral peak of SWS1 opsins Davies et al. The SWS1 is the only vertebrate visual opsin class that can reach a spectral sensitivity peak in the UV range, the consequence of the absence of protonation in the chromophore Schiff base in the dark state Fahmy and Sakmar In the other visual opsins, including violet SWS1 photopigments, the Schiff base of the cis -retinal chromophore is protonated, with a highly conserved glutamate counterion E acting on the proton stabilization Nathans Site-directed mutagenesis assays demonstrated that substitutions at site 86 from various amino acids Tyr, Ser, Leu, Val characteristic of violet-sensitive SWS1 opsins in mammals, and now snakes, to Phe86, cause the loss of electrostatic stabilization of the protonation, shifting the absorption peak of the photopigment to the UV range Cowing et al.
On the other hand, the nonpolar residues Val86, Met86, and Leu86, despite being naturally found in violet opsins of guinea pig, primates, and amphibians, respectively, were not able to generate any spectral change in UV opsins of fish Cowing et al. This is probably achieved by the interaction of residue Val86 with other spectral tuning sites, such as the nonpolar residue Met93 Shi et al.
However, we observed considerable noise in the mutant Phe86Val opsin curve possibly caused by a low yield due to reduced stability of the generated opsin Hauser et al. The resulting curve did not optimally fit a typical opsin template which led to only an approximate estimate of the spectral peak, around nm. We detected a remarkable difference in the evolutionary rates of the two Helicops sws1 opsin genes.
After this transient stage establishing the new paralog in the population, purifying selection will act ubiquitously on this gene as in the ancestral copy, resulting in a gradual return to preduplication evolutionary rates. Pegueroles et al. In their comprehensive study, the observed acceleration was limited to a relatively short period of time following the duplication event, gradually decreasing and being completely erased after approximately Although our data do not allow reliable estimates of divergence time for the two sws1 paralogs, species trees indicate that the genus Helicops diverged about This places the origin of the sws1 paralogs in a time window consistent with Pegueroles et al.
The fate of a duplicated gene is largely determined by its potential to evolve new functions Ohno ; Walsh ; Gojobori and Innan In fish, where opsin gene duplications are not uncommon, paralogs of opsins sensitive to the central portion of the spectrum i.
Duplications of the sws1 gene are rare and, to the best of our knowledge, have been suggested only in few fish species Minamoto and Shimizu ; Musilova et al. However, no study has demonstrated the simultaneous expression of more than one sws1 variant in a retina of any vertebrate.
In Helicops snakes, the gain of a new violet opsin in addition to the UV opsin might have been selected upon to partially compensate for the ancestral loss of the SWS2 opsin.
The additional violet opsin of Helicops snakes fills a spectral gap between the UV and LWS opsins, enabling a broader coverage of the visible light spectrum available. Generally, freshwater environments have less light at short wavelengths, especially in the UV range, due to suspended organic matter and rapid absorption of the shortest wavelengths with depth Loew and Lythgoe ; Lythgoe and Partridge The life of a Helicops snake is at the interface of two drastically different visual worlds, terrestrial and aquatic.
The unique adaptations of its visual system reflect this duality: underwater, a violet pigment may enable efficient photon capture at short wavelengths and, together with LWS cones, provides the potential for color vision. Above water, UV light is abundant and the snake can take full advantage of the broader light space available by employing the additional UV photoreceptor, and potentially gaining color discrimination at the shortest wavelengths.
However, unexpectedly, their retinal structure is similar to that of diurnal snakes, with a low density of photoreceptors, absence of typical rods Walls ; Underwood ; Wong ; Sillman et al.
The low density of photoreceptors points to lower light sensitivity compared with nocturnal snakes with duplex retinas and high density of typical rods Walls ; Sillman et al. A similar apparent incongruence was described in the nocturnal dipsadid Thamnodynastes with a typical diurnal retina Hauzman It was suggested that a shift to nocturnal activity in Thamnodynastes is associated with hunting anurans Torello-Viera and Marques , whereas the diurnal retinal pattern may be due to phylogenetic inertia, so that the change in the circadian rhythm might have not been accompanied yet by changes in retinal morphology.
More work on the diel activity patterns of Helicops is needed to evaluate whether similar selective forces are at the origin of its typically diurnal retina. So far, only a handful of studies have described retinal topography in snakes Wong ; Hart et al. In dipsadid snakes, a visual streak was found in the arboreal Philodryas olfersii Hauzman et al. On the other hand, in the closely related terrestrial species, Philodryas patagoniensis , a ventral anisotropic area centralis was described, which would enable detection of predators from above Hauzman et al.
In Helicops modestus , we observed higher density of photoreceptors and SWS cones in the ventral retina, as in P. Behavioral studies revealed chromatic discrimination in the superior visual field of the murine Jacobs et al. In Helicops , it would be valuable to investigate whether similar selective pressures led to the observed retinal organization, enhancing the ability to perceive dark objects against a brighter background such as aerial predators and allowing a fast escape dive underwater.
Indeed, a wide variety of birds prey on aquatic and terrestrial snakes Guthrie ; Mushinsky and Miller , including Helicops Franz et al. However, one must exert caution when interpreting retinal organization at the photoreceptor level given the complexity of visual processing pathways within the retina and beyond Baden et al.
This could be achieved through higher acuity or improved chromatic discrimination in the upper visual field, facilitating the detection of anurans on aquatic plants above the snake. Further investigations on retinal circuitry, including photoreceptor connectivity to bipolar and ganglion cells, and visually guided behaviors will be required to fully understand the adaptive link between the retinal organization, visual function, and the life history of Helicops.
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