Long-wavelength sensitive opsin (LWS) gene variability in Neotropical cichlids (Teleostei: Cichlidae)

THOMAZ M.C. FABRIN SONIA MARIA A.P. PRIOLI ALBERTO JOSÉ PRIOLI About the authors

ABSTRACT

Cichlid fishes are an important group in evolutionary biology due to their fast speciation. This group depends widely of vision for feeding and reproduction. During the evolutionary process it plays a significant role in interspecific and intraspecific recognition and in its ecology. The molecular basis of vision is formed by the interaction of the protein opsin and retinal chromophore. Long-wavelength sensitive opsin (LWS) gene is the most variable among the opsin genes and it has an ecological significance. Current assay identifies interspecific variation of Neotropical cichlids that would modify the spectral properties of the LWS opsin protein and codons selected. Neotropical species present more variable sites for LWS gene than those of the African lakes species. The LWS opsin gene in Crenicichla britskii has a higher amino acid similarity when compared to that in the African species, but the variable regions do not overlap. Neotropical cichlids accumulate larger amounts of variable sites for LWS opsin gene, probably because they are spread over a wider area and submitted to a wider range of selective pressures by inhabiting mainly lotic environments. Furthermore, the codons under selection are different when compared to those of the African cichlids.

Key words:
convergence; ecology; evolution; visual system.

INTRODUCTION

The Cichlid family is an important freshwater group of fish that presents a varied color pattern and widely uses the visual system (Nelson 2006NELSON J. 2006, Fishes of the World. 4th ed., New Jersey: Wiley, 601 p.). They are also considered a model in evolutionary studies due to their fast radiation and speciation in several African lakes (Seehausen et al. 1999SEEHAUSEN O , MAYHEW P AND ALPHEN J. 1999. Evolution of colour patterns in East African cichlid fish. J Evol Biol 12: 514-534., Smith et al. 2011SMITH AR , D'ANNUNZIO L, SMITH AE, SHARMA A, HOFMANN CM , MARSHALL NJ AND CARLETON K L. 2011. Intraspecific cone opsin expression variation in the cichlids of Lake Malawi. Mol Ecol 20: 299-310.). In addition, Neotropical cichlids exhibit a diversity of ecological niches, behavioral and morphological adaptations (Maan and Sefc 2013MAAN ME AND SEFC KM. 2013. Colour variation in cichlid fish: developmental mechanisms, selective pressures and evolutionary consequences. Semin Cell Dev Biol 24: 516-528.), besides being a very diversified group (López-Fernández et al. 2010LÓPEZ-FERNÁNDEZ H, WINEMILLER KO AND ­HONEYCUTT RL. 2010. Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Mol Phylogenet Evol 55: 1070-1086.).

The genus Cichla has been used for phylogenetic and population structure studies (Oliveira et al. 2006OLIVEIRA AV, PRIOLI AJ , PRIOLI SMAP, BIGNOTTO TS, JÚLIO JR HF, CARRER H, AGOSTINHO CS AND PRIOLI LM. 2006. Genetic diversity of invasive and native Cichla (Pisces: Perciformes) populations in Brazil with evidence of interspecific hybridization. J Fish Biol 69: 260-277., Willis et al. 2007WILLIS SC, NUNES MS, MONTAÑA CG, FARIAS IP AND LOVEJOY NR. 2007. Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae). Mol Phylogenet Evol 44: 291-307., 2012WILLIS SC, MACRANDER J, FARIAS IP AND ORTÍ G. 2012. Simultaneous delimitation of species and quantification of interspecific hybridization in Amazonian peacock cichlids (genus Cichla) using multi-locus data. BMC Evol Biol 12: 96.) since it is one of the most basal (Poletto and Ferreira 2010POLETTO A, FERREIRA I, CABRAL-DE-MELO DC, NAKAJIMA RT, MAZZUCHELLI J, RIBEIRO HB, VENERE PC, NIRCHIO M, KOCHER TD AND MARTINS C. 2010. Chromosome differentiation patterns during cichlid fish evolution. BMC Genet 11: 50.) which forms a monophyletic group when compared to other Neotropical cichlids (López-Fernández et al. 2010LÓPEZ-FERNÁNDEZ H, WINEMILLER KO AND ­HONEYCUTT RL. 2010. Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Mol Phylogenet Evol 55: 1070-1086.). The body shape is a characteristic in Cichla and species are identified mainly on coloration (Kullander and Ferreira 2006KULLANDER SO AND FERREIRA EJG. 2006. A review of the South American cichlid genus Cichla, with descriptions of nine new species (Teleostei: Cichlidae). Ichthyol Explor Freshwaters 17: 289-398.). A breaking down of reproductive isolation was detected between these two species in the Paraná River basin through molecular evidence (Oliveira et al. 2006OLIVEIRA AV, PRIOLI AJ , PRIOLI SMAP, BIGNOTTO TS, JÚLIO JR HF, CARRER H, AGOSTINHO CS AND PRIOLI LM. 2006. Genetic diversity of invasive and native Cichla (Pisces: Perciformes) populations in Brazil with evidence of interspecific hybridization. J Fish Biol 69: 260-277., Almeida-Ferreira et al. 2011ALMEIDA-FERREIRA GC, OLIVEIRA AV DE, PRIOLI AJ AND PRIOLI SMAP. 2011. Spar genetic analysis of two invasive species of Cichla (Tucunaré) (Perciformes: Cichlidae) in the Paraná river basin. Acta Sci Biol Sci 33: 79-85.), namely, Cichla kelberi and Cichla piquiti. However, there is no evidence of natural hybrids between the two species in the Tocantins-Araguaia basin where the two species coexist (Willis et al. 2007WILLIS SC, NUNES MS, MONTAÑA CG, FARIAS IP AND LOVEJOY NR. 2007. Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae). Mol Phylogenet Evol 44: 291-307., 2012WILLIS SC, MACRANDER J, FARIAS IP AND ORTÍ G. 2012. Simultaneous delimitation of species and quantification of interspecific hybridization in Amazonian peacock cichlids (genus Cichla) using multi-locus data. BMC Evol Biol 12: 96.).

A modification of the isolation mechanisms that had existed between them where they were native may have occurred, but would not be functioning in the new environment. Thus, the color that differentiates the two species may also function as a pre-zygotic isolation and become a maintainer mechanism of sympatric species which are closely related, preventing gene flow and hybridization process (Miyagi et al. 2012MIYAGI R, TERAI Y, AIBARA M, SUGAWARA T, IMAI H, TACHIDA H, MZIGHANI SI, OKITSU T, WADA A AND OKADA N. 2012. Correlation between nuptial colors and visual sensitivities tuned by opsins leads to species richness in sympatric Lake Victoria cichlid fishes. Mol Biol Evol 29: 3281-3296.).

The way coloring is perceived by aquatic organisms should be considered since the light wavelength reaching the environment and limnological characteristics act as a background for vision (Gray and McKinnon 2007GRAY SM AND MCKINNON JS. 2007. Linking color polymorphism maintenance and speciation. Trends Ecol Evol 22: 71-79.). In this case, vision would play an important role among species that use coloring as a mechanism of recognition (Terai et al. 2002TERAI Y, MAYER WE, KLEIN J, TICHY H AND OKADA N. 2002. The effect of selection on a long wavelength-sensitive (LWS) opsin gene of Lake Victoria cichlid fishes. PNAS 99: 15501-15506.).

Visual sensitiveness is determined by the visual pigments formed from the interaction between opsin proteins and retinal chromophores (Schwanzara 1967SCHWANZARA S. 1967. The visual pigments of freshwater fishes. Vision Res 7: 121-148.), enabling the perception of light wavelengths according to opsin proteins which are expressed by opsin genes and, due to their plasticity, may also vary according to environment and life stage of fish (Spady et al. 2006SPADY TC, PARRY JWL, ROBINSON PR, HUNT DM, BOWMAKER JK AND CARLETON K L. 2006. Evolution of the cichlid visual palette through ontogenetic subfunctionalization of the opsin gene arrays. Mol Biol Evol 23: 1538-1547., Yokoyama 2008YOKOYAMA S. 2008. Evolution of dim-light and color vision pigments. Annu Rev Genomics Hum Genet 9: 259-282., Hofmann et al. 2009HOFMANN CM , O'QUIN KE, MARSHALL NJ , CRONIN TW, SEEHAUSEN O AND CARLETON K L. 2009. The eyes have it: regulatory and structural changes both underlie cichlid visual pigment diversity. PLoS Biol 7: e1000266., Hofmann and Carleton 2009CARLETON K. 2009. Cichlid fish visual systems: mechanisms of spectral tuning. Integr Zool 4: 75-86., O'Quin et al. 2010O'QUIN KE , HOFMANN CM , HOFMANN HÁ AND CARLETON K L. 2010. Parallel evolution of opsin gene expression in African cichlid fishes. Mol Biol Evol 27: 2839-2854., Smith et al. 2011SMITH AR , D'ANNUNZIO L, SMITH AE, SHARMA A, HOFMANN CM , MARSHALL NJ AND CARLETON K L. 2011. Intraspecific cone opsin expression variation in the cichlids of Lake Malawi. Mol Ecol 20: 299-310.).

In the case of studies on these genes in Neotropical cichlids, Weadick et al. (2012WEADICK CJ, LOEW ER , RODD FH AND CHANG BSW. 2012. Visual pigment molecular evolution in the Trinidadian pike cichlid (Crenicichla frenata): a less colorful world for neotropical cichlids? Mol Biol Evol 29: 3045-3060.) investigated the molecular evolution of visual pigments in Crenicichla frenata, a species native to the Caribbean island of Trinidad, while Schott et al. (2014SCHOTT RK, REFVIK SP, HAUSER FE, LÓPEZ-FERNÁNDEZ H AND CHANG BSW. 2014. Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Mol Biol Evol 31: 1149-1165.) studied the molecular evolution of the opsin gene RH1 from 32 species of Neotropical cichlids and compared them with those of African cichlids.

African cichlids have seven cone opsin genes which express the opsin proteins sensitive to short-wavelength - SWS1, SWS2B, SWS2A; medium-wavelength - RH2B, RH2Aβ, RH2Aα; and long-wavelength - LWS. There is also a rod opsin gene - RH1, responsible for scotopic vision (Spady et al. 2005SPADY TC , SEEHAUSEN O , LOEW ER, JORDAN RC, KOCHER TD AND CARLETON K L. 2005. Adaptive molecular evolution in the opsin genes of rapidly speciating cichlid species. Mol Biol Evol 22: 1412-1422., Carleton et al. 2010CARLETON K L, HOFMANN CM, KLISZ C, PATEL Z, CHIRCUS LM, SIMENAUER LH, SOODOO N, ALBERTSON RC AND SER JR. 2010. Genetic basis of differential opsin gene expression in cichlid fishes. J Evol Biol 23: 840-853.). However, LWS presents the highest variability (Terai et al. 2002TERAI Y, MAYER WE, KLEIN J, TICHY H AND OKADA N. 2002. The effect of selection on a long wavelength-sensitive (LWS) opsin gene of Lake Victoria cichlid fishes. PNAS 99: 15501-15506.) and is related to ecological adaptation and sexual selection (Terai et al. 2006TERAI Y ET AL. 2006. Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids. PLoS Biol 4: e433.).

Variable sites of these sequences may cause small changes in the absorbed light wavelength due to the replacement of amino acids as long as they occur in specific regions of exons (Yokoyama 2008YOKOYAMA S. 2008. Evolution of dim-light and color vision pigments. Annu Rev Genomics Hum Genet 9: 259-282., Carleton 2009CARLETON K. 2009. Cichlid fish visual systems: mechanisms of spectral tuning. Integr Zool 4: 75-86.). Since water is an environment that requires adaptations in the visual system, the heterogeneity of light would represent a strong selection pressure on these genes. Any eventual variation may be associated with differences in sensitivity to light absorption spectra, similar to patterns shown by other studies (Yokoyama 2008YOKOYAMA S. 2008. Evolution of dim-light and color vision pigments. Annu Rev Genomics Hum Genet 9: 259-282., Seehausen et al. 2008SEEHAUSEN O ET AL. 2008. Speciation through sensory drive in cichlid fish. Nature 455: 620-626.).

Two hypotheses may be raised: (1) the occurrence of interspecific variation related to the visual sensitivity of the Neotropical species under analysis, and (2) different codons would be under selection when the studied groups of African and Neotropical cichlids are compared.

Amino acid and nucleotide sequences of coding regions of the LWS gene were analyzed and compared in ten species of Neotropical cichlids belonging to five genera. Subsequently, codon selection tests were performed to compare which codons were under selection between the African and Neotropical cichlids.

MATERIALS AND METHODS

STUDIED SPECIES

Current assay comprised ten Neotropical species and nineteen African species. The Neotropical species were Cichla kelberi (n=9), Cichla piquiti (n=5), Cichla monoculus (n=5), Crenicichla britskii (n=4), Crenicichla haroldoi (n=4), Crenicichla jupiaensis (n=3), Astronotus crassipinnis (n=4) Geophagus proximus (n=4), Satanoperca pappaterra (n=3) (Figure 1). The LWS gene sequence of Crenicichla frenata was obtained from GenBank (JN990732).

Figure 1
Sampling locations of Cichla populations native from the Tocantins-Araguaia (TO) and Amazonas river (AM) basins and from Neotropical cichlids species from upper Paraná river (aPR). TO-PA: Tocantins river, near to Pedro Afonso city (08° 58´ S; 48° 10´ W); TO-PN: Lajeado's resevoir, in the Tocantins river, near to Porto Nacional city (09°45´ S; 48°22´ W); TO-PX: Tocantins river, near to Peixe city - TO (11° 52´ S; 48° 35´ W); AM-MN: Amazonas river (03° 07´ S; 59° 55´ W, near to Manaus city- AM; aPR-PL: Upper Paraná river floodplain, close to Porto Rico city - PR (22°47´ S; 53°19´ W).

The African species sequences were obtained from the GenBank: one species from the Nile River, Oreochromis niloticus (AF247128); four species from Lake Tanganyika, Astatotilapia burtoni (AY660540), Opthalmotilapia ventralis (AY780512), Neolamprologus brichardi (AY780513), Tropheus duboisi (AY780516); two species from Lake Victoria, Pundamilia nyererei (AY673688), Pundamilia pundamilia (AY673689); and eleven species from Lake Malawi, Aulonocara heuseri (AY780517), Labeotropheus fuelleborni (AF247127), Metriaclima zebra (AF247126), Melanochromis auratus (AY780518), Lethrinops parvidens (AY780519), Tyrannochromis maculatus (AY780520), Cynotilapia afra (AY780521), Mylochromis lateristriga (AY780522), Labiochromis chisumulae (AY780515), Copadochromis borleyi (AY780514), Stigmatochromis modestus (AY780523). The Nile River and Lake Victoria are characterized as turbid environments, whereas lakes Malawi and Tanganyika are clear water environments (Spady et al. 2005SPADY TC , SEEHAUSEN O , LOEW ER, JORDAN RC, KOCHER TD AND CARLETON K L. 2005. Adaptive molecular evolution in the opsin genes of rapidly speciating cichlid species. Mol Biol Evol 22: 1412-1422.). Twenty-eight species were used in current analysis.

DNA EXTRACTION AND PCR

Muscle tissue samples, preserved in 96% ethanol and stored at -20 °C, were used for DNA extraction of species from the genus Cichla. Samples were provided by the tissue bank of the Genetics Laboratory of the Nucleus for Research in Limnology, Ichthyology and Aquaculture (Nupelia), Universidade Estadual de Maringá, Maringá, PR, Brazil. The other Neotropical species, collected within the Long Term Ecological Research Project (Projeto de Pesquisa Ecológica de Longa Duração, PELD), were also preserved in 96% ethanol and stored at -20 °C.

Genomic DNA was extracted with Promega kit (Wizard Genomic DNA Purification A1125) following manufacturer's instructions.

Primers described by Weadick et al. (2012WEADICK CJ, LOEW ER , RODD FH AND CHANG BSW. 2012. Visual pigment molecular evolution in the Trinidadian pike cichlid (Crenicichla frenata): a less colorful world for neotropical cichlids? Mol Biol Evol 29: 3045-3060.) were used for PCR. The amplicons were purified (Rosenthal et al. 1993ROSENTHAL A, COUTELLE O AND CRAXTON M. 1993. Large-scale production of DNA sequencing templates by microtitre format PCR. Nucleic Acids Res 21: 173-174.) and the sequencing reaction was performed with Big Dye Terminator kit with the ABI3730 automated DNA sequencer.

SEQUENCE ANALYSIS

The sequences were manually edited with the program BioEdit (Hall 1999HALL T. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.). Sequences alignment data using the Clustal W algorithm (Thompson et al. 1994THOMPSON JD, HIGGINS DG AND GIBSON TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680.) and the construction of phylogenetic trees inferred from the Neighbor-Joining method were performed with MEGA 6 software (Tamura et al. 2013TAMURA K, STECHER G, PETERSON D, FILIPSKI A AND KUMAR S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725-2729.). The selected codons were estimated with the FUBAR (Murrell et al. 2013MURRELL B, MOOLA S, MABONA A, WEIGHILL T, SHEWARD D, KOSAKOVSKY POND SL AND SCHEFFLER K. 2013. FUBAR: a fast, unconstrained bayesian approximation for inferring selection. Mol Biol Evol 30: 1196-1205.) and REL (Pond and Muse 2005POND SLK, FROST SDW AND MUSE SV. 2005. HyPhy: hypothesis testing using phylogenies. Bioinformatics 21: 676-679.) methods, available in the HyPhy software package (Pond et al. 2005POND SK AND MUSE SV. 2005. Site-to-site variation of synonymous substitution rates. Mol Biol Evol 22: 2375-2385.) available in the Datamonkey web server (Delport et al. 2010DELPORT W, POON AFY, FROST SDW AND POND SLK. 2010. Datamonkey 2010: A suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26: 2455-2457.), and CODEml, available in the PAML software (Yang 2007YANG Z. 2007. PAML 4: Phylogenetic analysis by maximum likelihood. Mol Biol Evol 24: 1586-1591., Xu and Yang 2013XU B AND YANG Z. 2013. PamlX: A graphical user interface for PAML. Mol Biol Evol 30: 2723-2724.).

So that variable sites and their relation to the spectral properties of the opsin protein could be identified, the sequence was aligned according to bovine rhodopsin (Bowmaker 1995BOWMAKER J. 1995. The visual pigments of fish. Prog Retin Eye Res 15., Hofmann et al. 2009HOFMANN CM , O'QUIN KE, MARSHALL NJ , CRONIN TW, SEEHAUSEN O AND CARLETON K L. 2009. The eyes have it: regulatory and structural changes both underlie cichlid visual pigment diversity. PLoS Biol 7: e1000266., Smith and Carleton 2010SMITH AR AND CARLETON K L. 2010. Allelic variation in Malawi cichlid opsins: a tale of two genera. J Mol Evol 70: 593-604., Schott et al. 2014SCHOTT RK, REFVIK SP, HAUSER FE, LÓPEZ-FERNÁNDEZ H AND CHANG BSW. 2014. Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Mol Biol Evol 31: 1149-1165.).

RESULTS

VARIABLE SITES IN NEOTROPICAL CICHLIDS

Fragments comprised exons 2-4 (633 bp) of the LWS gene. Whereas the Neotropical species presented 64 nucleotide and 27 amino acid variable sites, the African cichlids exhibited 45 nucleotide and 24 amino acidic variable sites (Table I). Exon 2 was the most variable in the two groups.

TABLE I
Number of variable sites in LWS gene of Neotropical and African cichlids. E2 = exon 2; E3 = exon 3; E4 = exon 4; N = nucleotide variable sites; Aa = amino acidic variable sites; S = synonymous substitutions; nS = nonsynonymous substitutions.

The amino acid and nucleotide genetic distances were calculated according to the Jones-Taylor-Thornton (Jones et al. 1992JONES DT, TAYLOR WR AND THORNTON JM.1992. The rapid generation of mutation data matrices from protein sequences. Bioinformatics 8: 275-282.) and Kimura-2-parameters (Kimura 1980KIMURA M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111-120.) models, respectively, among the Neotropical and African cichlids (Table SI - Supplementary Material). Figure 2 presents the phylogenetic trees based on the Neighbor-Joining method.

Figure 2
Phylogenetic tree from fragment of the coding regions of the opsin gene LWS (633 bp) of Neotropical and African cichlids, according to (a) nucleotide substitution model Kimura-2-parameter with 1000 bootstrap resampling and (b) JTT + G substitution model with 1000 bootstrap resampling and statistical neighbor-joining method.

Two species of the genus Crenicichla, C. britskii and C. frenata, evidenced evolutionary convergence in one amino acidic site (site 119), and they rated a smaller genetic distance when compared to some African species rather than to species of the genus Cichla, which is a basal group for Neotropical cichlids (López-Fernández et al. 2010LÓPEZ-FERNÁNDEZ H, WINEMILLER KO AND ­HONEYCUTT RL. 2010. Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Mol Phylogenet Evol 55: 1070-1086.) (Table SII). Eight out of the 10 Neotropical species had a substitution at amino acid 164 which results in tuning in the maximum wave spectrum absorbed by LWS opsin protein (Yokoyama 2008YOKOYAMA S. 2008. Evolution of dim-light and color vision pigments. Annu Rev Genomics Hum Genet 9: 259-282.).

CODON SELECTION

Selected codons were estimated by FUBAR, REL and M8 BEB model (available in CODEml) tests and were different between Neotropical and African cichlids, considering posterior probability above 80% to both models (Tables SIII to SVI). In the case of the REL evolution model, an evolution tree was built by the same model, taking into consideration REV substitution model and the neighbor-joining statistical method. There were nine different coding sequences among the ten Neotropical species selected, and twelve different coding sequences among the seventeen African species. The analyzed fragments comprised the codon 22-232 of the LWS gene.

REL model estimated eleven codons as positive selection, whereas the FUBAR and M8 models estimated nine and seven codons, respectively, for the Neotropical cichlids. All codons estimated by FUBAR and M8 models were also estimated by REL. In the case of the African lakes cichlids, REL test estimated eight codons under selection, FUBAR and M8 model estimated seven codons under selection. Only one codon under selection overlapped the two groups (site 119). Figure 3 shows posterior probabilities estimated by FUBAR model.

Figure 3
Posterior probability of codons under selection estimated by FUBAR model (a = Neotropical cichlids; b = African cichlids).

The position of the opsin protein codons LWS has been established according to the crystal structure of bovine rhodopsin (Stenkamp et al. 2002STENKAMP RE, TELLER DC AND PALCZEWSKI K. 2002. Crystal Structure of Rhodopsin : A G-Protein-Coupled Receptor. Chem BioChem 3: 963-967.) (Table II).

TABLE II
Codons under positive selection and its position in the structure of the LWS opsin protein according to FUBAR (F), REL (R) and M8 BEB model (M).

DISCUSSION

DIVERGENT SELECTION OF LWS GENE IN NEOTROPICAL AND AFRICAN CICHLIDS

LWS presents the greatest variability among opsin genes (Terai et al. 2002TERAI Y, MAYER WE, KLEIN J, TICHY H AND OKADA N. 2002. The effect of selection on a long wavelength-sensitive (LWS) opsin gene of Lake Victoria cichlid fishes. PNAS 99: 15501-15506., Spady et al. 2005SPADY TC , SEEHAUSEN O , LOEW ER, JORDAN RC, KOCHER TD AND CARLETON K L. 2005. Adaptive molecular evolution in the opsin genes of rapidly speciating cichlid species. Mol Biol Evol 22: 1412-1422., Miyagi et al. 2012MIYAGI R, TERAI Y, AIBARA M, SUGAWARA T, IMAI H, TACHIDA H, MZIGHANI SI, OKITSU T, WADA A AND OKADA N. 2012. Correlation between nuptial colors and visual sensitivities tuned by opsins leads to species richness in sympatric Lake Victoria cichlid fishes. Mol Biol Evol 29: 3281-3296.). Fragments of exon 2-5 (872 bp) were used in other research work (Terai et al. 2006TERAI Y ET AL. 2006. Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids. PLoS Biol 4: e433., Seehausen et al. 2008SEEHAUSEN O ET AL. 2008. Speciation through sensory drive in cichlid fish. Nature 455: 620-626.) since they encoded the transmembrane domains of the protein. The changes in specific sites of the opsin gene sequences which provide the longest (LWS) and shortest (SWS1) wavelength appear to be more critical (Carleton 2009CARLETON K. 2009. Cichlid fish visual systems: mechanisms of spectral tuning. Integr Zool 4: 75-86., Carleton et al. 2010CARLETON K L, HOFMANN CM, KLISZ C, PATEL Z, CHIRCUS LM, SIMENAUER LH, SOODOO N, ALBERTSON RC AND SER JR. 2010. Genetic basis of differential opsin gene expression in cichlid fishes. J Evol Biol 23: 840-853.), especially those that occur between exon 1-3 (Terai et al. 2006TERAI Y ET AL. 2006. Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids. PLoS Biol 4: e433.). Further, the opsin genes seem to accumulate the highest amount of variation (Hofmann et al. 2012HOFMANN CM , MARSHALL NJ, ABDILLEH K, PATEL Z , SIEBECK EU AND CARLETON K L. 2012. Opsin evolution in damselfish: convergence, reversal, and parallel evolution across tuning sites. J Mol Evol 75: 79-91.).

Papers on the gene in African cichlid species, comprising sequences of exons 2-5, reported ten variable sites in four species of three distinct genera inhabiting the same lake and thirteen variable sites in two sympatric species of the same genus (Terai et al. 2006TERAI Y ET AL. 2006. Divergent selection on opsins drives incipient speciation in Lake Victoria cichlids. PLoS Biol 4: e433.), with most leads towards non-synonymous changes. Nine out of the ten studied species of Neotropical cichlids comprised new species that had fragments of their LWS opsin gene sequenced (exons 2-4) and five genera with a total of 64 nucleotide variable sites.

Neotropical cichlids presented a higher amount of variable sites for this gene when compared to African cichlids. Moreover, variation sites do not overlap, even though some relationship involving environment, nutrition and species behavior seems to exist. The species under analysis are very distinct from each other. Furthermore, it seems that the period since speciation among Neotropical cichlids is superior (López-Fernández et al. 2010LÓPEZ-FERNÁNDEZ H, WINEMILLER KO AND ­HONEYCUTT RL. 2010. Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Mol Phylogenet Evol 55: 1070-1086.) than the time elapsed since speciation of cichlids from African lakes, due to their adaptive radiation (Seehausen et al. 1999SEEHAUSEN O , MAYHEW P AND ALPHEN J. 1999. Evolution of colour patterns in East African cichlid fish. J Evol Biol 12: 514-534.). Thus, the Neotropical species would accumulate more substitutions over time.

However, a recent study has shown a positive selection on the rhodopsin gene RH1 in Neotropical cichlids where selective pressure would be divergent between lotic and lentic environment and the encoding sites of the protein under selection (Schott et al. 2014SCHOTT RK, REFVIK SP, HAUSER FE, LÓPEZ-FERNÁNDEZ H AND CHANG BSW. 2014. Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Mol Biol Evol 31: 1149-1165.). Current analysis corroborated the above. Among the codons under selection, estimated by FUBAR and REL models, only one codon overlapped and the others codons were divergent. Since Neotropical cichlids presented a greater number of codons under selection, this fact showed that the gene in this group would be under different evolutionary pressures.

Only one variable site was found when compared to sequences of LWS opsin gene between species from the African cichlids of Lake Malawi. However, when compared to Oreochromis niloticus, an African cichlid from lotic environment, 22 amino acids differed (Carleton and Kocher 2001CARLETON K AND KOCHER T. 2001. Cone opsin genes of African cichlid fishes: tuning spectral sensitivity by differential gene expression. Mol Biol Evol 18: 1540-1550.). These genes seem to be under an intense selection pressure due to the meaning of their ecological and behavior (Bowmaker 1995BOWMAKER J. 1995. The visual pigments of fish. Prog Retin Eye Res 15., Hofmann et al. 2012HOFMANN CM , MARSHALL NJ, ABDILLEH K, PATEL Z , SIEBECK EU AND CARLETON K L. 2012. Opsin evolution in damselfish: convergence, reversal, and parallel evolution across tuning sites. J Mol Evol 75: 79-91.). Moreover, they differ from the analyzed nucleotide sequence for the coding regions, in which African and Neotropical cichlids form two monophyletic groups.

Although the great African lakes comprise a wide and extensive region, the area in which the Neotropical cichlids are distributed is probably wider and more dynamic, since it is made of various types of environments, for example, floodplain environments (Thomaz et al. 1997THOMAZ SM, ROBERTO MC AND BINI LM. 1997. Caracterização limnológica dos ambientes aquáticos e influência dos níveis fluviométricos. In: VAZZOLER AE ET AL. (Eds), A planície inundaçao do alto rio Paraná - Aspectos físicos, biológicos e socioeconômicos. Maringá: EDUEM , 460 p.). Therefore, these species would be under more types of selective pressures which would, consequently, lead to a greater divergence, especially among such diverse genera as the ones studied here (Graça and Pavanelli 2007GRAÇA WJ AND PAVANELLI CS. 2007. Peixes da planície de inundação do alto rio Paraná e áreas adjacentes. Maringá: EDUEM, 241 p.).

AN EVOLUTIONARY CONVERGENCE EVIDENCE?

Due to the Cichla basal characteristics (López-Fernández et al. 2010) and the Crenicichla derived character, the LWS opsin gene shows a region (site 119) that would converge evolutionarily as the Crenicichla genus. Although they show a greater divergence than that in African species, the coding sequence of the LWS opsin protein is closer to that of some species of African lakes than Cichla are. For example, amino acidic divergence between Cichla piquiti and Cynotilapia afra is 11.91%, while between Crenicichla britskii and C. afra is 9.23%.

Taking into consideration the convergent evolution hypothesis due to the distance between the groups (Arendt and Reznick 2008ARENDT J AND REZNICK D. 2008. Convergence and parallelism reconsidered: what have we learned about the genetics of adaptation? Trends Ecol Evol 23: 26-32.), a strong evidence of pressure by natural selection mechanism is presumed (Nagai et al. 2011NAGAI H, TERAI Y, SUGAWARA T, IMAI H, NISHIHARA H, HORI M AND OKADA N. 2011. Reverse evolution in RH1 for adaptation of cichlids to water depth in Lake Tanganyika. Mol Biol Evol 28: 1769-1776. ). It is actually a viable hypothesis (1) from the point of view of the two groups: African and Neotropical cichlids form monophyletic branches in which a group is mostly limited to lentic environments and the other is subjected to a greater diversity of environments, respectively. Both groups would be under different kinds of pressure; (2) the two genera Cichla and Crenicichla are very different: a great divergence of LWS opsin gene is expected since it is a gene of ecological and behavioral relevance. However, despite higher amino acid similarity between some African species and Crenicichla britskii and C. frenata, many variable sites do not overlap.

CONCLUSIONS

Neotropical cichlids seem to accumulate more LWS opsin gene variability than African cichlids from lentic environments, probably due to the differences between the African lakes and Neotropical regions. The latter factor would exert different selective pressures (Schott et al. 2014SCHOTT RK, REFVIK SP, HAUSER FE, LÓPEZ-FERNÁNDEZ H AND CHANG BSW. 2014. Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Mol Biol Evol 31: 1149-1165.), considering the divergence of the estimated codons to be under selection and the speciation time of both groups. Therefore, studies involving other opsin genes and more species of the Neotropical cichlids group should be performed. Moreover, given the variability of this gene in Neotropical cichlids, studies analyzing the possibility of its use as a marker for species identification and assistance in phylogenetic studies should be encouraged.

Research involving opsin genes and species of Neotropical cichlids is just starting (Weadick et al. 2012WEADICK CJ, LOEW ER , RODD FH AND CHANG BSW. 2012. Visual pigment molecular evolution in the Trinidadian pike cichlid (Crenicichla frenata): a less colorful world for neotropical cichlids? Mol Biol Evol 29: 3045-3060., Schott et al. 2014SCHOTT RK, REFVIK SP, HAUSER FE, LÓPEZ-FERNÁNDEZ H AND CHANG BSW. 2014. Divergent positive selection in rhodopsin from lake and riverine cichlid fishes. Mol Biol Evol 31: 1149-1165.) and seems to be highly promising. Further studies would contribute towards the understanding of the species' sensory system and their relationship with the dynamics of Neotropical environments.

ACKNOWLEDGMENTS

The authors would like to thank the Programa de Ecologia de Ambientes Aquáticos Continentais (PEA), the Núcleo de Pesquisa em Limnologia, Ictiologia e Aquicultura (Nupelia) and the Universidade Estadual de Maringá (UEM) for the infrastructure in current research. Thanks are also due to the Programa Ecológico de Longa Duração (PELD-CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for funding the project and the grant of a research scholarship. The authors are also grateful to Rodrigo Júnio da Graça and to Luciano Seraphim Gasques for their contributions and comments on this paper.

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Publication Dates

  • Publication in this collection
    Mar 2017

History

  • Received
    30 Sept 2015
  • Accepted
    22 Dec 2016
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