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The Journal of Experimental Biology 213, 3586-3592 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.045914

Identification and characterization of visual pigments in (Amphibia: ), an order of limbless with rudimentary eyes

S. M. Mohun1,2, W. L. Davies1,3, J. K. Bowmaker1, D. Pisani4, W. Himstedt5, D. J. Gower2, D. M. Hunt1,6 and M. Wilkinson2,* 1UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK, 2Department of Zoology, Natural History Museum, London, Cromwell Road, London SW7 5BD, UK, 3Nuffield Laboratory of Ophthalmology, University of Oxford, Levels 5-6, West Wing, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK, 4Department of Biology, National University of Ireland Maynooth, County Kildare, Ireland, 5Department of Biology, Darmstadt University of Technology, Schnittspahnstrasse 3-5, D-64287, Germany and 6School of Biology, University of Western , 35 Stirling Highway, Perth, WA 6009, Australia *Author for correspondence ([email protected])

Accepted 13 July 2010

SUMMARY In comparison with the other orders, the Anura () and Urodela (), knowledge of the visual system of the -like Gymnophiona (caecilians) is relatively sparse. Most caecilians are fossorial with, as far as is known any surface activity occurring mainly at night. They have relatively small, poorly developed eyes and might be expected to possess detectable changes in the spectral sensitivity of their visual pigments. Microspectrophotometry was used to determine the spectral sensitivities of the photoreceptors in three species of , bivittatum, seraphini and natans. Only rod opsin visual pigment, which may be associated with scotopic (dim light) vision when accompanied by other ‘rod- specific’ components of the phototransduction cascade, was found to be present. Opsin sequences were obtained from the eyes of two species of caecilian, cf. kohtaoensis and T. natans. These rod opsins were regenerated in vitro with 11-cis retinal to give pigments with spectral sensitivity peaks close to 500nm. No evidence for cone photoreception, associated with diurnal and colour vision, was detected using molecular and physiological methods. Additionally, visual pigments are short- wavelength shifted in terms of the maximum absorption of light when compared with other amphibian lineages. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/213/20/3586/DC1 Key words: opsin, visual pigments, evolution, spectral tuning, caecilian, Gymnophiona.

INTRODUCTION (Crescitelli et al., 1972; Yokoyama, 2000; Bowmaker and Hunt, The Gymnophiona, commonly known as caecilians, are one of the 2006; Hart and Hunt, 2007). Rods, which generally express the rh1 three orders of extant Amphibia. They are readily distinguished from opsin gene and rod-specific components of the phototransduction frogs (Anura) and salamanders (Urodela) by their annulated, cascade, are generally responsible for scotopic or dim light vision. limbless bodies, and are distinct in many other characters, including By contrast, up to four classes of cone opsin genes exist in rudimentary eyes (e.g. Taylor, 1968; Wilkinson and Nussbaum, vertebrates that are associated with colour and photopic vision (Hunt 2006). Reduction in the visual system of the Gymnophiona, et al., 2001a; Bowmaker and Hunt, 2006). The encoded proteins of associated with a mainly fossorial lifestyle, has long been recognized these genes have lmax values (based on a vitamin A1 chromophore) (see Norris and Hughes, 1918; Walls, 1942) but the extent to which between about 360–440nm (‘ultraviolet’ or ‘violet’, short the caecilian eye is functional remains uncertain (Wake, 1980; Wake, wavelength-sensitive; the sws1 gene) 440–470nm (‘blue’, short 1985; Himstedt, 1995; Himstedt, 1996). Adult caecilian eyes are wavelength-sensitive; the sws2 gene), 470–520nm (‘green’, relatively small, covered with skin and sometimes bone, lack middle wavelength-sensitive; the rh2 gene) and 500–570nm (‘red’, features expected of well-developed eyes and have long wavelength-sensitive; the lws gene). several features apparently co-opted to novel sensory structures and Electroretinography has detected a single class of visual pigment functions (e.g. Himstedt and Simon, 1995). Relaxed molecular clock in the caecilian Ichthyophis cf. kohtaoensis (Himstedt, 1995), and analysis suggests that the last common ancestor of extant caecilians this was presumed to be a rod pigment based on its lmax value at occurred in the or (Roelants et al., 2007; San Mauro, 502nm. Consistent with this, fragments of rh1 coding sequence have 2010) and that caecilians may therefore have possessed a reduced been isolated from four species of morphologically and visual system for over 150 million . phylogenetically diverse caecilians (Frost et al., 2006; Venkatesh It is widely assumed that variations in the peak spectral et al., 2001), suggesting that this class of visual pigment may be sensitivities (lmax) of the visual system represent adaptations to present throughout the Gymnophiona. Immunohistochemistry has specific visual needs associated with particular habitats or lifestyles also been performed on a similarly diverse group of caecilians using (Lythgoe, 1979; Peichl, 2005; Davies et al., 2009). Spectral antibodies raised to rod opsin, transducin, and cone opsin (Nguyen, sensitivity is determined either by changes in the tertiary structure 2003). Strong reactions were observed with anti-rod antibodies and of the opsin protein via amino acid substitutions and/or by the use weaker reactions with one anti-cone antibody, although the latter of alternate chromophores derived from vitamin A1 or A2 reaction is most probably the result of cross-reactivity with the rod

THE JOURNAL OF EXPERIMENTAL BIOLOGY Visual pigments in caecilians 3587 pigment (Nguyen, 2003; Foster et al., 1993). An anti-bovine Polymerase chain reaction transducin antibody also reacted positively, suggesting that the rod Fragments of opsin coding sequence were amplified from cDNA phototransduction pathway is intact in the retina of caecilians. Thus, using degenerate primers designed from an alignment of vertebrate a functional scotopic visual pathway would appear to be present in opsins (Davies et al., 2007a; Davies et al., 2007b). Polymerase chain caecilians, even in species with rudimentary eyes concealed under reaction (PCR) amplifications using genomic DNA from the same bone, without any evidence for the presence of photopic vision. caecilian species and amphibian cone class-specific and degenerate The aim of this study was to identify and characterize the visual primers were also performed (supplementary material TableS1). A pigments expressed in the retina within ecologically and nested protocol was used to detect sequences from exon 1 to exon phylogenetically diverse caecilian species and to determine the lmax 4. From the sequences obtained using standard nested PCR, specific of such pigments using in vivo and in vitro methods. primers (supplementary material TableS1) were designed for 5Ј- and 3Ј-RACE to isolate the untranslated regions (UTRs) of the rod MATERIALS AND METHODS opsin mRNA transcript. The reaction mixture typically contained –1 –1 –1 Tissue sources 10mmoll of each dNTP, 2mmoll MgCl2, 0.1mmoll of each Adult were used for all experiments as follows: one forward and reverse primer, 2.5i.u. Biotaq DNA polymerase specimen of (Guérin-Méneville 1829) (Bioline, London, UK) and 20–100 ng of cDNA. Cycling conditions (voucher number MW 2390) was used for microspectrophotometry were, an initial denaturing step at 94°C for 5min, then 40 cycles of (MSP); six specimens of (Fischer 1880) (MW denaturing at 94°C for 30s, annealing at 55°C for 1min and 7346–MW 7351), with one specimen being used for MSP and five extension at 72°C for 1.5min, with a final extension at 72°C for specimens for mRNA extraction; one specimen of Geotrypetes 10min. Negative controls were run in parallel containing the same seraphini (Duméril 1859) (MW 3854) was used for MSP; two reagents but without the addition of cDNA. A second round specimens of Ichthyophis cf. kohtaoensis (Taylor, 1960) (MW 4764 amplification used 1/10 dilution of the first round PCR products, and MW 4765) for mRNA extraction. All caecilian species were under identical conditions to the first round except for an annealing obtained by fieldwork or from licensed commercial sources as temperature of 60°C. approved by the local Animal Ethics Committee and were killed The 5Ј and 3Ј ends of the visual pigment cDNA were amplified using an approved procedure. All vouchers will be accessioned into using the 5Ј/3Ј-RACE kit (Roche), following the manufacturer’s the collections of the Natural History Museum, London. instructions. PCR products were cloned into pGEM T-Easy vector (Promega, Southampton, UK) prior to sequencing using either T7 Microspectrophotometry or SP6 primers and the Big Dye Terminator v3.1 Cycle Sequencing Physiological data were determined using MSP. A modified kit on an ABI 3730 sequencer (Applied Biosystems, Warrington, Liebman dual beam microspectrophotometer under computer control UK). was used to determine the spectral sensitivities of the photoreceptors. With the help of an infrared converter, the measuring beam In vitro regeneration of visual pigments (normally 2mm square cross section) was aligned to pass transversely The full-length coding sequences of T. natans and I. cf. kohtaoensis through the photoreceptor outer segments or transparent pieces of rh1 were amplified using a proofreading KOD XL Taq polymerase skin covering the eye, while the reference beam passed through a (Novagen, Nottingham, UK) (supplementary material Fig. S1). The clear space adjacent to the object examined. Spectra were scanned primer pairs were designed for the 5Ј and 3Ј ends with restriction from 750 to 350nm in 2nm increments and back from 251 to 749nm sites for EcoRI and SalI. The digested PCR products were at the interleaved wavelengths. Only one absorption spectrum was directionally cloned into a derivative of the mammalian expression usually obtained from a given outer segment to minimize the effects vector pMT4 carrying the sequence of bovine rod opsin 1D4 epitope of bleaching. To verify the presence of a photolabile pigment, (including the stop codon) at the 3Ј end of the coding sequence putative outer segments were bleached by exposure to white light (Franke et al., 1988). HEK293T cells were transfected using a from the monochromator passing through measuring beams of the calcium phosphate precipitation technique with vector containing microspectrophotometer. A standardized computer program was the opsin insert. Cells were harvested after 48h and washed with used to estimate the lmax for each outer segment. The spectra 1ϫ phosphate-buffered saline (PBS) buffer. Pigments were obtained from the outer segments were averaged to obtain a mean reconstituted by resuspending the harvested cells in PBS buffer and curve. The curve was then fitted to a standard template curve in incubating with 20mmoll–1 11-cis retinal in the dark. The pigments order to obtain an estimate of lmax (Govardovskii et al., 2000). The were isolated by incubating with 1% (w/v) n-dodecyl-b-D-maltoside standard template curve used was the Dartnall standard curve for (DDM) and 20mgml–1 phenylmethylsulphonylfluoride (PMSF) rhodopsin placed with its lmax at 502nm and expressed on an before passage over a cyanogen-bromide-activated (CNBr) abscissal scale of log frequency. Sepharose binding column coupled to an anti-1D4 monoclonal antibody. Messenger RNA extraction and complementary DNA The dark and the bleached (photobleached with broad spectrum synthesis white light for at least 30min) absorption spectra were measured Complementary DNA (cDNA) was generated from mRNA extracted using a dual path spectrophotometer (Spectronic Unicam, from the eyes of two species of caecilian, I. cf. kohtaoensis and T. Cambridge, UK). The lmax was determined from the difference natans, using three and ten eyes, respectively. The QuickPrepTM spectra (dark minus light bleached) by fitting a Govardovskii A1 micro mRNA purification kit (GE Healthcare, Little Chalfont, UK) template (Govardovskii et al., 2000) to the data using an Excel was used to purify polyadenylated mRNA. First strand cDNA was program. synthesized in a reaction using 500ng of oligo(dT) or for 3Ј-RACE primer from the 5Ј-/3Ј-RACE Kit (Roche, Burgess Hill, UK) and Phylogenetic analysis 1–2mg of mRNA, 1i.u. Superscript III reverse transcriptase An alignment of coding region amino acids was done manually in (Invitrogen, Paisley, UK), following the manufacturer’s instructions. MacClade 4.08 (Maddison and Maddison, 2000), using the

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3588 S. M. Mohun and others translated caecilian sequences and 68 vertebrate visual opsin 0.04 0.03 sequences downloaded from the NCBI database (alignment and A Dark B 488.5 nm GenBank accession numbers are given in supplementary material 0.03 0.02 Fig.S1), representing the five classes of visual opsins and including Bleached all amphibian representatives (typically up to three where available 0.01 and including any with rudimentary eyes) of major vertebrate 0.02 lineages. Bayesian inferences used Markov Chain Monte Carlo 0 (MCMC) implemented in MRBAYES 3.1.2 (Huelsenbeck and 0.01 Ronquist, 2001; Ronquist and Huelsenbeck, 2003) sampling every –0.01 1000 generations from two parallel runs (each of four chains) 0 of 1,000,000 generations. Convergence was established by 0.06 Dark comparing the standard deviations of split frequencies and burnins C 0.04 D 487.5 nm determined by visual inspection of likelihoods using TRACER 1.4 (Rambaut and Drummond, 2007) (Tracer v1.4, Available from 0.04 Bleached 0.02 http://beast.bio.ed.ac.uk/Tracer). Model jumping was used within MRBAYES 3.1.2 to estimate the fixed-rate model for amino acid 0.02 data during the tree-search, which chose the BLOSUM62 (BLOck A bs or ba nce 0 SUbstitution Matrix) model of amino acid substitution that was produced from multiple alignments of evolutionarily divergent 0 proteins (Henikoff and Henikoff, 1992). The analysis was rooted Dark 0.03 using LWS opsin protein sequences as outgroups. The sequences E F 486.6 nm were identified using BLASTP 2.2.20 and BLASTN (Altschul et 0.03 0.02 al., 1997) against published opsin sequences from the NCBI Bleached database. 0.02 0.01 RESULTS 0 Microspectrophotometry 0.01 Three species from ecologically diverse and phylogenetically –0.01 distinct caecilian lineages were investigated. Rhinatrema bivittatum 0 (a species that retains many ancestral features) and Geotrypetes 400 500 600 400 500 600 Wavelength (nm) seraphini, are terrestrial as adults, whereas Typhlonectes natans is aquatic. All have relatively well-developed eyes for caecilians, but Fig.1. MSP absorbance spectra for three caecilian species, Typhlonectes those of T. natans and G. seraphini are possibly secondarily well natans (A,B), Rhinatrema bivittatum (C, D) and developed. In all three species, only a single class of rod (E,F). (A,C,E) Dark and bleached spectra, (B,D,F) difference spectra. For photoreceptors was found, and microspectrophotometry (MSP) each species, the absorbance peak shifts to around 380nm after bleaching. This represents the oxime (stabilized form of the bleached demonstrated the presence in each species of a single visual pigment photopigment chromophore) of the liberated retinal. The fitted spectra to with similar lmax values of 488.5±0.9nm for T. natans (N31), both the dark and difference spectra are shown as the solid lines. 487.5±1.0nm for R. bivittatum (N13), and 486.6±2.2nm for G. seraphini (N8; Fig.1). The light transmission of the skin immediately above the eye of amino acid identity between T. natans and I. cf. kohtaoensis T. natans was determined by MSP. Fig.2 shows that this skin is sequences. almost transparent with only a small amount of absorbance attenuation of about 10% at wavelengths <500nm. Expression of visual pigments In order to confirm that the cloned gene sequences encoded the Opsin sequence analysis and phylogenetics pigments identified in situ in photoreceptors by MSP, the T. natans Of the three species used for the MSP analysis, only eye tissue samples pigment was generated in vitro by isolation of recombinant opsin from T. natans were available for molecular studies. A fourth species, I. cf. kohtaoensis, was therefore added to the study at this stage. Opsin sequences were PCR amplified from cDNA; in all cases, the sequence 100 of the amplified fragments corresponded to the rh1 gene. This was confirmed by phylogenetic analysis, with both caecilian sequences falling, as expected, within the amphibian rh1 group as a sister group 75 of (Fig.3). The aligned sequences used are shown in

supplementary material Fig.S1. No cone opsin sequences were 50

ion ss mi s n obtained by PCR amplifications from genomic DNA or cDNA using a

either gene specific or degenerate primers. Tr % 25 The full rh1 coding and deduced amino acid sequences for T. natans and I. cf. kohtaoensis are shown in supplementary material FigsS1 and S2. The sequences show an 80–82% nucleotide and 400 500 600 700 Wavelength (nm) a 87–89% amino acid identity for T. natans and a 79–81% nucleotide and a 86–90% amino acid identity for I. cf. kohtaoensis Fig.2. MSP transmission spectrum through the skin covering the eye of to the rh1 orthologues of other amphibian species. There is a 96% Typhlonectes natans.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Visual pigments in caecilians 3589

Fig.3. Phylogenetic results of Bayesian analyses of vertebrate amino acid sequence analysis using Blosum62. Numbers are posterior probabilities. Branches without numbers are maximally supported and branches with posterior probability support values below 0.6 were collapsed. See legend to supplementary material Fig.S2 for opsin sequence accession numbers.

from mammalian cells transfected with an expression vector DISCUSSION containing the opsin coding sequence. After pigment reconstitution Only a single spectral class of photoreceptor was found in three by the addition of 11-cis-retinal, the dark and bleached absorption caecilian species, Rhinatrema bivittatum, Typhlonectes natans and spectra were measured by conventional UV–visible Geotrypetes seraphini, with mean absorbance peaks between spectrophotometry. The difference spectrum yielded a lmax at 487–489nm. Consistent with this, the only expressed opsin detected 493nm (Fig.4), which is sufficiently similar to the photoreceptor in the eye of two caecilian species, T. natans and I. cf. kohtaoensis, peak at 488.5±0.9nm obtained in situ by MSP to confirm the identity belongs to the rod or Rh1 class of opsins that is expressed in rod of the photoreceptor pigment as rod opsin. The opsin coding photoreceptors throughout the vertebrate kingdom. No other classes sequence of I. cf. kohtaoensis was also expressed and regenerated of photoreceptors or expressed opsins were found and it is concluded in vitro to give an identical lmax to that of T. natans at 493nm. that these caecilian species possess a rod-only retina, which agrees Compared with the rod pigments of other Amphibia, which peak with previous research (Wake, 1985; Himstedt, 1995). Because cone around 502–506nm (Partridge et al., 1992; Chen et al., 1996; opsins homologous with those of other vertebrates are found in Fyhrquist et al., 1998; Ala-Laurila et al., 2002), the caecilian members of the Urodela (salamanders) and Anura (frogs) (Rohlich pigments measured in situ are short wavelength-shifted by between and Szel, 2000; Takahashi et al., 2001; Sakikabara et al., 2002), it 13 and 19nm. This shift must be the result of particular amino acid would appear that the loss of cone opsins, and cone photoreceptors, differences in the caecilian rh1 opsin pigments compared with other is a synapomorphy of Gymnophiona. amphibian rh1 opsins. Key amino acid sites 83, 122, 207, 211, 265, The peak sensitivities of rod visual pigments in Amphibia 292, 295, which are known to be important in the spectral tuning typically lie between 502–506nm (Liebman and Entine, 1968; of rh1 pigments (Davies et al., 2007a) are invariant across all the Harosi, 1975; Partridge et al., 1992; Chen et al., 1996; Fyhrquist et amphibian pigments (Fig.5). However, the two caecilian pigments al., 1998; Ala-Laurila et al., 2002); peak sensitivities of caecilian show consistent differences from other at sites 107, 158, rod pigments are therefore short wavelength-shifted by between 13 159, 213, 260, 289, 337 and 352 (Table1) which may influence the and 19nm. These shifts are comparable to those seen in a number spectral tuning of caecilian visual pigments. of deep-water vertebrates (Hunt et al., 2001b; Bowmaker et al., 1994;

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3590 S. M. Mohun and others

0.15 Table1. Conserved amino acid residues of the protein encoded by AB493 nm the caecilian rh1 genes compared with the same position in the 0.02 493 nm protein from other amphibians 0.10 Residue Domain Other amphibia 107 ECD1 Thr Glu, Ala, Val 158 TMD4 Leu Ala, Met –0.02 0.05 159 TMD4 Leu Phe, Met 213 TMD5 Ser Cys, Thr 260 TMD6 Gly Phe, Ala 281 ECD3 Ala Ser, Thr 0 –0.06 337 C-terminus Gly Ala 352 C-terminus Ala Ser A bs or ba nce difference Sequence numbering is based on bovine rod opsin. For sequences and –0.05 alignments used see supplementary material Fig. S1. ECD, extracellular –0.10 domain; TMD, transmembrane domain.

–0.10 400 450 500 550 600 400 450 500 550 600 Wavelength (nm) differs from the situation in I. cf. kohtaoensis where absorbance of the skin covering the eye approaches 100% at 410nm, reducing to Fig.4. In vitro difference absorbance spectra for (A) Ichthyophis cf. around 40% at 500nm and to almost 0% at 550nm (Himstedt, 1995). kohtaoensis and (B) Typhlonectes natans rod opsin pigments. The effect of this is to shift the peak sensitivity of the eye to around 520nm, and this remains the case even when the true peak of 493nm obtained from the present data is substituted for the peak of 502nm Hope et al., 1997) and marine mammals (Fasick et al., 1998; Fasick assumed by Himstedt (Himstedt, 1995). and Robinson, 2000) where they are thought to be an adaptation to The cave Proteus anguinus shows a similar the narrow bandwidth of down-welling light centred around 485nm morphological diversity and rudimentation of the eye to that in that penetrates deep water (Hunt et al., 2001b). These shifts involve caecilians (Kos et al., 2001). One population from the Otovec doline different combinations of substitutions at a number of amino acid in south-west Slovenia appears to parallel caecilians in the loss of sites that include 83, 261, 292 and 299 (Hunt et al., 1996; Fasick cone opsins while retaining the rod pigment. The eyes of and Robinson, 2000; Hunt et al., 2001b). None of these sites are, salamanders from this population have regressed and photoreceptors however, substituted in the caecilian pigments when compared with have no discernable outer segments (Kos et al., 2001). This contrasts the rod pigments of other amphibians. Differences are, however, with another population from the Planina cave which have retained found at sites 107, 158, 159, 213, 260, 281, 337 and 352; these are both a long wavelength-sensitive (red) cone opsin and a rod opsin, therefore potential spectral tuning sites, although none has yet been as determined by immunocytochemistry. The estimated time of implicated in the tuning of visual pigments. Thus, at present, it is divergence of surface and cave-dwelling salamanders is 2–5million not possible to conclude which of these sites is responsible for the years (Trontelj et al., 2007). Therefore, loss of cone pigments in short wavelength shift of caecilian rod pigments. cave-dwelling species has occurred over a relatively short The skin above the eye of T. natans shows only minimal evolutionary time and their loss in caecilians, which diverged from absorption of light across the spectrum from 400 to 750nm. other amphibians at a much earlier time, is unsurprising. Absorbance by the skin would not be expected therefore to alter Caecilians are found in both aquatic (swamps, rivers) and the peak spectral sensitivity of the eye of this caecilian species. This terrestrial (forest, open bush and agricultural landscapes) habitats

Fig.5. Potential spectral tuning sites in rod opsin S 100 140 S pigments expressed in the retinas of caecilians. The + schematic representation of the three-dimensional ECD1 ICD2 structure of a visual pigment showing the seven 134 + 185 transmembrane domains (TMD 1–7), three extracellular 20 ICD1 TMD2 122 187 domains (ECD 1–3), three intracellular domains (ICD 93 + 89 TMD4 181 86 1–3), the amino- and carboxy-termini (N and C), and the TMD3 180 120 118 40 retinal chromophore. Amino acids are represented by 15 83 90 164 113 52 114 160 circles. Grey shading indicates residues that are crucial Retinal Glycosylation sites ECD2 60 + for maintaining protein structural integrity; black, those N N 2 TMD1 207 49 H known to influence the spectral sensitivity of vertebrate 265 280 261 211 pigments and those with a square inside are sites that + TMD5 + 296 269 220 46 affect Rh1 and Rh2 visual pigments (Yokoyama, 2000; 300 292 ECD3 T4MD Davies et al., 2007b). Non-conservative amino acid 200 changes in caecilians compared with other amphibians T7MD 260 + 295 are shown with a cross. Modified from Davies et al. Palmitoylation sites ICD3 (Davies et al., 2007a).

323 240 322 320319 C 340 + + 338 340 336 342 343 334 335

Phosphorylation sites

THE JOURNAL OF EXPERIMENTAL BIOLOGY Visual pigments in caecilians 3591 in the wet tropics (e.g. Gower et al., 2004; Jones et al., 2006). Fasick, J. I. and Robinson, P. R. (2000). Spectral-tuning mechanisms of marine mammal rhodopsins and correlations with foraging depth. Vis. Neurosci. 17, 781- Terrestrial species live mostly in soil but may also exploit epigeic 788. microhabitats such as under leaf litter and rotting vegetation (e.g. Fasick, J. I., Cronin, T. W., Hunt, D. M. and Robinson, P. R. (1998). The visual pigments of the bottlenose dolphin (Tursiops truncatus). Vis. Neurosci. 15, 643- Burger et al., 2007). Caecilians (at least those species that are 651. partially surface active) are mostly considered nocturnal (Himstedt, Foster, R. G., Garcia Fernandez, J. M., Provencio, I. and Degrip, W. J. (1993). 1995; Kupfer et al., 2004; Burger et al., 2007), so it is likely that Opsin localisation and chromophore retinoids identified within the basal brain of the Anolis carolinensis. J. Comp. Physiol. A 172, 33-45. the peak absorbance of caecilian rod photoreceptor pigments are Franke, R. R., Sakmar, T. P., Oprian, D. D. and Khorana, H. G. (1988). A single spectrally tuned to maximize the absorbance of light and the amino acid substitution in rhodopsin (Lys248Leu) prevents activation of transducin. J. Biol. Chem. 263, 2119-2122. wavelengths available in either a forest floor, soil or night Frost, D. R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C. F. B., De environment. Tropical forests are heterogeneous in the spectral Sa, R. O., Channing, A., Wilkinson, M., Donnellan, S. C. et al. (2006). The amphibian tree of life. Bull. Am. Mus. Nat. Hist. 297, 1-370. composition of ambient light (Endler, 1993), where the light varies Fyhrquist, N., Donner, K., Hargrave, P. A., Mcdowell, J. H., Popp, M. P. and from white in large gaps between vegetation to yellowish-red in Smith, W. C. (1998). Rhodopsins from three and toad species: sequences and functional comparisons. Exp. Eye Res. 66, 295-305. small gaps. In forest shade, essentially all of the light has been Govardovskii, V. I., Fyhrquist, N., Reuter, T., Kuzmin, D. G. and Donner, K. (2000). transmitted through or reflected from leaves to give a range of In search of the visual pigment template. Vis. Neurosci. 17, 509-528. Gower, D. J., Loader, S. P., Wilkinson, M. and Moncreiff, C. B. (2004). Niche wavelengths of 470–490 nm (Endler, 1993), similar therefore to the separation and comparative abundance of boulengeri and maximum absorbance of caecilian rod visual pigments. By contrast, (Amphibia: Gymnophiona) in East Usambara forest, at night the wavelengths of light are long wavelength-shifted, but Tanzania. Afr. J. Herpetol. 53, 183-190. Harosi, F. (1975). Absorption spectra and linear dichroism of some amphibian there is no evidence for a shift towards longer wavelengths in any photoreceptors J. Gen. Physiol. 66, 357-382. nocturnal animals (Peichl, 2005), including caecilians. The light at Hart, N. S. and Hunt, D. M. (2007). Avian visual pigments: characteristics, spectral tuning, and evolution. Am. Nat. 169, S7-S26. twilight is predominately around 480nm, which is similar to the Henikoff, S. and Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proc. Nalt. Acad. Sci. 89, 10915-10919. lmax of caecilian pigments, and after the sun sinks below the horizon, Himstedt, W. (1995). Structure and function of the eyes in the caecilian Ichthyophis the loss of middle wavelengths becomes more pronounced (Endler, kohtaoensis (Amphibia, Gymnophiona) Zoo. Anal. Comp. Syst. 99, 81-94. 1993). Therefore, it is possible that the main role of the rod visual Himstedt, W. (1996). Die Blindwuhlen. Neue Brehm-Bucherei, Vol. 630. Magdeburg/Heidelberg: Westarp/Spektrum. pigment in caecilians is to detect twilight, a critical period during Himstedt, W. and Simon, D. (1995). Sensory basis of foraging behaviour in caecilians which diurnal and nocturnal species change behaviour patterns and (Amphibia, Gymnophiona) Herp. J. 5, 266-270. Hope, A. J., Partridge, J. C., Dulai, K. S. and Hunt, D. M. (1997). Mechanisms of locations and when prey detection and predator avoidance becomes wavelength tuning in the rod opsins of deep-sea fishes Proc. R. Soc. Lond. B. Biol. highly significant (Munz and Mcfarland, 1977). Sci. 264, 155-163. Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754-755. ACKNOWLEDGEMENTS Hunt, D. M., Fitzgibbon, J., Slobodyanyuk, S. J. and Bowmaker, J. K. (1996). We are grateful to Dr Rosalie Crouch for the generous gift of 11-cis retinal. We Spectral tuning and molecular evolution of rod visual pigments in the species flock of thank Alex Kupfer (Universität Jena), Jeannot and Odette (Camp Patawa), Guy cottoid fish in Lake Baikal. Vision Res. 36, 1217-1224. Tiego (Direction regionale de l’environment Guyane), Céline Dupuy (Direction des Hunt, D. M., Wilkie, S. E., Bowmaker, J. K. and Poopalasundaram, S. (2001a). Services, Vétérinaires de la Guyane), and especially Philippe Gaucher (Centre Vision in the ultraviolet Cell. Mol. Life Sci. 58, 1583-1598. Hunt, D. M., Dulai, K. S., Partridge, J. C., Cottrill, P. and Bowmaker, J. K. (2001b). National de la Recherche Scientifique) for facilitating our collection of Rhinatrema The molecular basis for spectral tuning of rod visual pigments in deep-sea fish. J. bivittatum in French Guiana. We would also like to thank Dr Livia Carvalho, Dr Exp. Biol. 204, 3333-3344. Susan Wilkie and Dr Jill Cowing for technical assistence with the in vitro Jones, D. T., Loader, S. P. and Gower, D. J. (2006). Trophic ecology of East African regeneration of caecilian visual pigments. This work was supported by a UK caecilians (Amphibia: Gymnophiona), and their impact on forest soil invertebrates J. 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