Parallelism of amino acid changes at the RH1 affecting spectral sensitivity among deep-water from Lakes Tanganyika and Malawi

Tohru Sugawara*, Yohey Terai*, Hiroo Imai†, George F. Turner‡, Stephan Koblmu¨ ller§, Christian Sturmbauer§, Yoshinori Shichida†, and Norihiro Okada*¶ʈ

*Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; †Department of Biophysics, Graduate School of Science, Kyoto University and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kyoto 606-8502, Japan; ‡Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom; §Department of Zoology, Karl Franzens University of Graz, Universitatsplatz 2, A-8010 Graz, Austria; and ¶Division of Cell Fusion, National Institute of Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan

Edited by Tomoko Ohta, National Institute of Genetics, Mishima, Japan, and approved March 7, 2005 (received for review July 27, 2004) Many examples of the appearance of similar traits in different focus on one of those genes, a rhodopsin, RH1, which is an lineages are known during the evolution of organisms. However, excellent candidate to test for evolutionary parallelism, because the underlying genetic mechanisms have been elucidated in very repeated amino acid replacements occurring at multiple posi- few cases. Here, we provide a clear example of evolutionary tions were suggested to have arisen convergently as functional parallelism, involving changes in the same genetic pathway, pro- adaptation (22). viding functional adaptation of RH1 pigments to deep-water hab- Visual pigment is a seven-transmembrane ␣-helical protein itats during the adaptive radiation of East African fishes. We that contains a light-absorbing chromophore, retinal (23, 24). determined the RH1 sequences from 233 individual cichlids. The Vertebrate visual pigments are classified into five groups in the reconstruction of cichlid RH1 pigments with 11-cis-retinal from 28 phylogenetic tree (25), and one of the groups is called Rh or sequences showed that the absorption spectra of the pigments of RH1, to which rhodopsins, visual pigments in rod photoreceptor nine species were shifted toward blue, tuned by two particular cells responsible for scotopic (twilight) vision, belong (25, 26). amino acid replacements. These blue-shifted RH1 pigments might The absorption spectrum of a visual pigment can be altered by have evolved as adaptations to the deep-water photic environ- amino acid substitutions within the protein moiety of the visual ment. Phylogenetic evidence indicates that one of the replace- pigment, and key replacement positions are located close to the ments, A292S, has evolved several times independently, inducing retinal-binding pocket (26–28). The chromophore is 11-cis al- similar functional change. The parallel evolution of the same dehyde of either vitamin A1 (retinaldehyde) or vitamin A2 mutation at the same amino acid position suggests that the (3,4-didehydroretinaldehyde) (23). Red-shifts of 10–40 nm can number of genetic changes underlying the appearance of similar be achieved by changing chromophores from A1 to A2 (29). traits in cichlid diversification may be fewer than previously The evolution of visual pigments serves as a prime model for expected. the study of molecular adaptation in vertebrates (26, 28), and several such cases have been reported in primates (30), birds rhodopsin ͉ adaptation (26), and fishes (31, 32). For example, color vision in primates shows a correlation between molecular adaptation and ecolog- orphologically and functionally similar characters often ical specialization (33–35) in that spectral sensitivity of eyes Mevolve more than once because of the operation of match the ’s environment, allowing for the processing of similar selective pressures in different lineages of organisms. greater amounts of information. The visual system of the Co- One of the fundamental questions concerning character sim- moran coelacanth (32) and deep-water Cottoid fishes in Lake ilarity is whether the genetic mechanisms underlying similar Baikal (31) have rod cells in which the peak sensitivity of visual pigments is blue-shifted to a range of 470–490 nm, because evolutionary changes are the same or are different. Similar longer and short wavelengths are more rapidly attenuated with evolutionary changes caused by the same genetic mechanism increasing water depth, and blue-green light (470–490 nm) are the result of parallelism, whereas those changes produced penetrates to the greatest depth in clear-water habitats (36). by different mechanisms are because of convergence (1). Aquatic environments provide a great diversity of photic con- Although a few cases of both parallelism and convergence in ditions, varying in aspects such as turbidity, color, and brightness Drosophila pigmentation have been recently observed (2–4), (28). The purpose of our study was to investigate the evolution the genetic bases of the appearance of similar characters of the RH1 gene in cichlid fishes of the East African Great Lakes largely remain to be elucidated. in the course of their adaptation to various lacustrine habitats. In East Africa, Lakes Victoria and Malawi harbor endemic Ϸ cichlid fish communities comprising 450–700 species, whereas Materials and Methods holds Ϸ250 species (5). These communities are DNA Sequencing and Sequence Analysis. Determination of the ecologically and morphologically highly diverse but encompass cichlid RH1 gene was as described (22). Nucleotide sequences relatively low genetic diversity, particularly in Malawi and Vic- were deposited in GenBank under accession nos. AB084924– toria (6–11). Cichlids are becoming a model system for under- AB084947, AB185213–AB185242, AB185390, and AB196147. standing the genetic basis of vertebrate speciation (12, 13). Parallel appearances of similar feeding morphologies (8, 14) and nuptial coloration (15) in different lakes or lineages have been This paper was submitted directly (Track II) to the PNAS office. documented. The repeated evolution of similarly adapted cichlid Data deposition: The sequences reported in this paper have been deposited in the GenBank species in various lineages in the same lake or even in different database (accession nos.AB084924–AB084947, AB185213–AB185242, AB185390, and lakes presents an opportunity to explore the issue of convergence AB196147). and parallelism. Recently, several studies of genes underlying the ʈTo whom correspondence should be addressed. E-mail: [email protected]. diversity of cichlids have been reported (12, 16–21). Here, we © 2005 by The National Academy of Sciences of the USA

5448–5453 ͉ PNAS ͉ April 12, 2005 ͉ vol. 102 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405302102 Downloaded by guest on September 27, 2021 Table 1. The informative positions of amino acid replacements among 28 cichlid RH1 genes, with ␭max values

Read vertically, the top three lines specify the amino acid positions in the cichlid RH1 pigment. A dot indicates an amino acid identical to the RH1 consensus. Open boxes at the top indicate the positions of the seven transmembrane regions. Shaded columns indicate the amino acid replacements that line the EVOLUTION chromophore-binding pockets or are in close proximity to the chromophore. The position of residues in each helix is based on the model of Baldwin (61) and on the crystal structure of bovine rhodopsin (62).

Comparison of Absorption Spectra of Cichlid RH1 Pigments. RH1 Results coding sequences were amplified by means of PCR using the Sequence Analysis of Cichlid RH1 Genes. We sequenced the RH1 cichlid genome as template with a pair of PCR primers desig- partial coding region (924 bp corresponding to amino acids nated RH1HindIIIF and RH1KpnIR (see Supporting Materials 13–320 among 354 of the full-length of the cichlid RH1 and Methods, which is published as supporting information on protein) of 56 species (Fig. 4, which is published as supporting the PNAS web site). PCR products were digested with KpnI and information on the PNAS web site). Among all sequences, 56 HindIII, and fragments were cloned into a pFLAG-CMV-5a amino acid positions were variable, 42 positions were within (Sigma-Aldrich, St. Louis) expression vector. Their expression the region of transmembrane helices, and 7 positions were and characterization was performed according to the method of close to the retinal-binding pocket. The other 14 variable Ueyama et al. (ref. 37, and for procedural details, see Supporting positions were within the region of extracellular loops, and 2 Materials and Methods). positions were close to the retinal-binding pocket (Table 1).

Chromophore Use. Adult cichlids were dark-adapted for 12 h, then Absorption Spectra. To test the relationships between the absorp- decapitated and enucleated. Retinal was extracted from crude tion spectra and ecology, we reconstructed RH1 pigments and eye-cup homogenates or isolated retina as an oxime, as described measured the absorption spectra for 28 cichlid species from (refs. 38–40, and for procedural details, see Supporting Materials African rivers and the three Great Lakes. First, we examined the and Methods). chromophore usage in laboratory-bred individuals of a Victorian ‘‘Haplochromis sp.’’ and the Malawian Dimidiochromis compres- Construction of RH1 Mutants. In vitro mutagenesis of the RH1 was siceps. The ratio of A1 to A2 was revealed to be Ϸ10:1 (data not performed by extension of DNA synthesis of overlapped oligo- shown), indicating that the predominant chromophore type of nucleotide primers using PCR (41). visual pigments in these cichlids is A1-derived. This result is

Sugawara et al. PNAS ͉ April 12, 2005 ͉ vol. 102 ͉ no. 15 ͉ 5449 Downloaded by guest on September 27, 2021 Fig. 2. Frequency distributions of ␭max of cichlid RH1 pigments. ␭max values were grouped according to whether the species occupy shallow- or deep- water habitats. Each square represents 1 species (total 28). S, 292S; N, 83N; within the square.

Among the variable amino acids, those at positions 83 and 292 were expected to be located in the transmembrane region, close to the retinal-binding pocket. At these positions, we constructed mutated cichlid RH1 pigments, such as S292A (amino acid change from serine to alanine at the position 292), A292S, D83N, and N83D. RH1 sequences came from the Tanganyikan cichlids Baileychromis centropomoides, frontosa, Limnochromis staneri, and Trematocara macrostoma, the Malaw- ian Diplotaxodon macrops, Pallidochromis tokolosh, and the widely distributed O. niloticus. Fig. 1 shows representative results of absorption spectra with ␭max values of wild-type and mutated RH1. In the RH1 of C. frontosa (Fig. 1a), the S292A mutation led to a shift in the peak sensitivity (␭max) from 487 Ϯ 2nmto 498 Ϯ 2 nm, an 11-nm shift toward the red end of the spectrum. Similarly, in T. macrostoma, a 12-nm red-shift, from 488 Ϯ 1nm to 500 Ϯ 1 nm, was observed in the S292A mutant. The reverse A292S mutation in the RH1 pigment of O. niloticus caused the ␭max value to shift 14 nm toward blue from 505 Ϯ 1nmto491Ϯ 2 nm (Fig. 1b). In the RH1 pigment of B. centropomoides, the N83D mutation caused an 8-nm shift of the ␭max value toward Fig. 1. Absorption spectra of the wild-type (wt) and mutated RH1 pigments red from 492 Ϯ 1nmto500Ϯ 2 nm (Fig. 1c). However, N83D evaluated by the dark–light difference spectra. When the pigment was not ␭ Ϯ bleached, RH1 pigments have ␭max values at Ϸ500 nm. When the regenerated caused only slight shifts of the max values from 500 1nmto pigments were exposed to light, new absorption peaks at Ϸ360 nm were 503 Ϯ 1nminP. tokolosh (Fig. 1d), and from 500 Ϯ 1nmto502Ϯ observed, indicating that 11-cis-retinal in the pigment was isomerized by light, 2nminD. macrops (data not shown) pigments. In the RH1 and all-transretinal oxime was released. In the different spectra, positive pigment of L. staneri, the reverse mutation D83N also caused the peaks at wavelengths of Ϸ500 nm correspond to the visual pigment; negative slight shift of ␭max value from 498 Ϯ 3nmto494Ϯ 1 nm (data peaks at 360 nm are derived from release of retinal oxime. Red and blue lines not shown). indicate the absorption spectra of wild-type and mutated RH1 pigments from C. frontosa (a), O. niloticus (b), B. centropomoides (c), and P. tokolosh (d), Discussion respectively. Spectral Tuning for Adaptation to Deep-Water Habitats. The wide range of absorption spectra of the RH1 pigments observed consistent with the previous data of cichlid visual pigments (21, in cichlids (Table 1) raises the issue of its evolutionary signifi- 42). We thus reconstructed all studied cichlid RH1 pigments cance. With the exception of a chameleon, the ␭max values of with A1-derived retinal. terrestrial vertebrate RH1 pigments have been found to range Absorption spectra were represented by their peak spectral from 495 to 508 nm (26, 43). For 19 of the surveyed cichlid sensitivity (␭max) values. In the 28 cichlid species tested, the species, the ␭max values of RH1 pigments fell within this ␭max values of the RH1 pigments varied from 484 Ϯ 1nmin ‘‘normal’’ range (Table 1; 495 Ϯ 1nminAulonocara ‘‘copper’’ to 505 Ϯ 1nminOreochromis to 505 Ϯ 1nminO. niloticus). Peak sensitivities of RH1 lay niloticus (Table 1). From repeated measurements of 35 samples, outside this range and shifted toward blue in nine of the species we determined that the mean measurement error (with 95% analyzed (Table 1; 484 Ϯ 1nminT. unimaculatum to 492 Ϯ 1 confidence intervals) was 1.4 Ϯ 0.25 nm. nm in B. centropomoides). For the species sampled from Lakes Malawi and Tanganyika, we have examined data on their ecology Amino Acids Responsible for the Differences in ␭max Values. To and habitat preferences (refs. 44–46, Table 2, and Supporting identify the residues responsible for spectral tuning of these References, which are published as supporting information on the pigments, we focused on amino acids near the chromophore. PNAS web site). The ␭max values of the RH1 pigments from all

5450 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405302102 Sugawara et al. Downloaded by guest on September 27, 2021 shallow-water cichlid species in our study fell within the range of However, many deep-water species did not show a blue shift, terrestrial vertebrate RH1 pigments. All of the blue-shifted including the Tanganyikan Bathybates fasciatus, Hemibates ste- species live in relatively deep waters in Lake Tanganyika, which nosoma, Gnathochromis permaxillaris, and L. staneri, and the has clear water, except near some river estuaries. Thus, the blue Malawian D. macrops and P. tokolosh. Perhaps such species have shift of their ␭max values appears to result from adaptation to other adaptations to the deep-water visual environment, such as deep-water environments (Fig. 2). having large eyes, as have D. macrops (47), P. tokolosh (48), and EVOLUTION

Fig. 3. Phylogenetic relationships among East African cichlid fishes, demonstrating the multiple origin of specific adaptations to deep-water habitat by means of amino acid substitutions from D to N at position 83 (‘‘Malawi͞Baileychromis-type;’’ bold gray branches) and from A to S at position 292 (‘‘Tanganyika-type;’’ bold black branches). Arrows indicate the occurrence of amino acid replacements of A292S and D83N at the branches. The deep-water species are underlined. (a) Strict consensus tree based on 924 bp of the RH1 gene summarizing the results of maximum parsimony (equal weights, heuristic search, random addition of taxa, 10 replicates; tree length, 270 mutations; CI excluding uninformative characters, 0.46), neighbor-joining (TrNϩIϩ⌫ model) and Bayesian inference (2,000,000 generations, no chains, trees sampled every 100 generations, and burn-in of 10,000). Bootstrap values for parsimony (above) and neighbor joining (center), and Bayesian posterior probabilities (below) are shown, when Ͼ50. Asterisks indicate where other taxa not shown (see Supporting Materials and Methods) had identical sequences. (b) RH1 gene tree obtained by Bayesian inference. (c) The phylogenetic tree based on SINEs (55, 56). (d) Strict consensus based on the NADH2 gene obtained by neighbor-joining (substitution model HKYϩIϩG), weighted maximum parsimony [search parameters and weights are as in Salzburger et al. (63), 127 most parsimonious trees] and Bayesian analysis (10 chains, 2 Mio generations, 1 tree saved per 100 generations, and burnin factor of 1 Mio). In this tree, we used existing sequences from some haplochromine species closely related to those used in the RH1 and SINE trees. These taxon substitutions would be unlikely to influence the structure of the tree in relation to the nodes where RH1 transitions are believed to occur.

Sugawara et al. PNAS ͉ April 12, 2005 ͉ vol. 102 ͉ no. 15 ͉ 5451 Downloaded by guest on September 27, 2021 H. stenosoma (49). Other deep-water species may not constantly independent origins of S at position 292. The A292S mutation is live in the light-limited depth water zone: L. staneri, H. steno- clearly functional. In most cases, the deep-water species with soma, and B. fasciatus have broad depth ranges, and some species A292S had close relatives found in shallow water for at least part are believed to migrate to the surface at night (44). of their range and in which position 292 was A and the RH1 pigment was not shifted toward blue (see Supporting Materials Effect of D83N and A292S in Cichlid RH1 Pigments. Several mutation and Methods). analysis studies targeting positions 83 and 292 have been conducted. Thus, it seems likely that fixation of spectral shift mutations at S292A in RH1 pigments in coelacanth or a replacement at the the same base position, A292S, occurred multiple times in corresponding position in mammalian red and green pigments deep-water-dwelling Lake Tanganyikan cichlids. This finding shifts the ␭max values 8–28 nm toward red (32, 50, 51), although the may be the result of independent mutational events at position reverse mutation in human pigment did not cause any change (52). 292 in different lineages. Another possibility is that the A292S It was also reported that D83N in bovine RH1 pigment shifts the transition might have arisen once early in the history of the ␭max values 6 nm toward blue (53). In the present study, we Tanganyikan radiation, but many of the descendent species demonstrated that A292S and S292A were responsible for signif- maintained A͞S polymorphism, with fixation for S occurring icant spectral shifts of Ͼ10 nm in cichlid RH1 pigments. We found independently in deep-water lineages, and fixation for A in other that D83N caused an 8-nm spectral shift toward blue in B. centro- species, including all those remaining in shallow water. Persistent pomoides RH1 pigments, but smaller spectral shifts in two Malaw- polymorphism in a functional gene, however, seems implausible ian cichlids (2–3 nm) and in L. staneri RH1 pigments (4 nm), which (it is not an obvious case to propose frequency-dependent is close to the measurement error of the ␭max values in this study selection), and we did not find any instances of A͞S polymor- amounting to Ϯ 1.4 nm. It is likely that substitutions at other sites phism among any of the taxa investigated (466 alleles tested, see affect the spectral tuning of RH1 pigments, in addition to those that Supporting Materials and Methods). we have studied. This assumption might explain the observations Introgressive hybridization, reported to have occurred in African that D83N seemed to cause minimal spectral shift in some cases, cichlid radiations (57, 58), might also cause the appearance of and might also explain the substantial differences in peak sensitiv- parallel evolution of a trait. In this case, the genetic basis of the trait ities of O. niloticus (␭max ϭ 505 Ϯ 1 nm) and A.‘‘copper’’ (␭max ϭ may have arisen less often that it appears from a phylogeny 495 Ϯ 1 nm), despite their having the same amino acids at both estimate, and have been transmitted horizontally between lineages positions 83 and 292 (Table 1). However, the independent fixation by introgression. Fixation may then have occurred, particularly of the D83N mutation in deep-water species in both lakes, and its when introgression was followed by strong directional selection for absence from shallow water and riverine species, suggests that it adaptive traits, such as appropriate variants of a functional gene like may have functional significance in adaptation to deep water, even RH1. The broad compatibility of the phylogeny of the major though our estimates of the resulting shifts in peak sensitivity were lineages estimates from mitochondrial NADH2 sequences and less than the range of measurement error in the present study. from the mainly SINE insertions provides no evidence for intro- gression. In phylogenies based on each of these methods, several Parallel Adaptations to Deep-Water Habitat by the Same Nucleotide independent A292S changes could be observed. Although it seems Changes. Trees based on RH1 sequences, mitochondrial ND2 unlikely, the possibility remains that the similarity of the RH1 tree sequences, and on SINE insertions all indicated an independent to other estimates of the species tree may be the result of recom- origin of the D83N substitution (a nucleotide substitution G247A) bination of introgressed RH1 sequences with ancestral RH1 se- in Lake Tanganyika Baileychromis and the Lake Malawi deep-water quences with eventual repeated independent fixation of an RH1 taxa (Fig. 3). Assuming a single gain of D83N at the base of the sequence that largely retains the character of the ancestral sequence Tanganyika radiation increases tree length by minimally 14 muta- of the particular lineage, but which has gained an S at position 292 from introgression. tion steps, based on an analysis using the software package MAC- It is believed that the replacement A292S resulting in blue CLADE (54). However, we did not obtain strong evidence that D83N necessarily results in a significant change in ␭max. Further exam- shifts of RH1 pigments has occurred several times during the 400 ination of RH1 functions may be illuminating. million years of vertebrate evolution (26, 59, 60). In the present From the relatively poorly resolved strict consensus RH1 gene study, however, we have demonstrated that the same replace- tree (Fig. 3a), A292S (a nucleotide substitution G874T) ap- ment has occurred in cichlids over a very short evolutionary peared to evolve independently between one and three times, period of a few million years, although several amino acid positions are known to shift the ␭max of RH1 pigment (26). The according to a reconstruction of ancestral states by using MAC- repeated fixation of the same substitution at the same position CLADE (54). By using a two-tailed Kishino–Hasegawa test (op- may indicate that the number of genes and͞or amino acid tions: full optimization and 1,000 bootstrap replicates, see Sup- positions responsible for the functional diversity of cichlid fishes porting Materials and Methods) to test a number of trees derived in the African Great Lakes might be fewer than previously by various methods, including the consensus tree, the best expected by assuming convergence. likelihood ratio score was assigned to a tree obtained by Bayesian inference (Fig. 3b), followed by a neighbor-joining tree (data not We thank Dr. T. Sato for helpful discussions and Ms. K. Sakai for shown). Both indicate three independent A292S transitions. technical assistance. This work was supported by the Ministry of Estimates of the species phylogeny derived from SINE insertions Education, Culture, Sports, Science, and Technology of Japan (N.O.), (55, 56) across the genome (Fig. 3c) and from mitochondrial the Austrian Academy of Sciences (S.K.), the Austrian Science Foun- NADH2 sequences (Fig. 3d) were also consistent with multiple dation (C.S.), and the Natural Environment Council U.K. (G.F.T.).

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