Phylogenetic analysis of the order Pleuronectiformes (Teleostei) based on sequences of 12S and 16S mitochondrial genes

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The fish order Pleuronectiformes, composed of 14 families, has two suborders: Psettodoidei (with one family) and Pleuronectoidei (with thirteen families). The relationships among families of Pleuronectoidei and among the genera of their families have
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  Phylogenetic analysis of the order Pleuronectiformes (Teleostei)based on sequences of 12S and 16S mitochondrial genes Marisa F.C. Azevedo 1* , Claudio Oliveira 1 , Belén G. Pardo 2 , Paulino Martínez 2 and Fausto Foresti 1 1  Laboratório de Biologia e Genética de Peixes, Instituto de Biociências,Universidade Estadual Paulista, Botucatu, SP, Brazil. 2  Laboratorio de Genética, Faculdade de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain. Abstract The fish order Pleuronectiformes, composed of 14 families, has two suborders: Psettodoidei (with one family) andPleuronectoidei (with thirteen families). The relationships among families of Pleuronectoidei and among the generaoftheirfamilieshaveextensivelybeendebatedandaconsensushasnotyetbeenreached.Inthepresentstudy,par-tial sequences of the 12S and 16S mitochondrial rRNA genes were obtained from 19 species belonging to the fami-lies Achiridae, Bothidae, Cynoglossidae, Paralichthyidae, Pleuronectidae, Scophthalmidae, and Soleidae.Additionalsequencesof42pleuronectiformspecieswereobtainedfromGenBank.Phylogeneticanalyseswerecon-ducted by the methods of maximum-parsimony, maximum-likelihood and Bayesian inference. Our results corrobo-rate the monophyletic status of all families, excluding Paralichthyidae. In the family Achiridae, the genus Catathyridium  (freshwater)wasthesistergroupof Trinectes  (saltwater),and Hypoclinemus  (freshwater)wasthesis-tergroupof Achirus  (saltwater).Assumingthattheputativeancestorofachiridslivedinsaltwater,itissuggestedthatthe freshwater habitats in South America were colonized independently by different achirid lineages.  Key words:  phylogeny, molecular systematics, mitochondrial DNA, fish evolution, flatfishes.Received: September 11, 2006; Accepted: May 14, 2007. Introduction Flatfishes of the Pleuronectiformes order are easilyrecognized because the adults are not bilaterally symmetri-cal (Nelson, 2006). Young flatfishes are bilaterally sym-metrical and swim upright, but early in their developmentoneeyemigratesacrossthetopoftheskulltolieadjacenttothe eye on the other side (Ahlstrom  et al. , 1984). Most spe-cies have both eyes on the right side and lie on the left side(dextral) but some suffer an opposite development (si-nistral) (Nelson, 2006). In some species, such as  Platichthys stellatus , both dextral and sinistral individualsmay occur and this difference appears to be largely under geneticcontrol(Policansky,1982).However,thereappearsto be no convincing arguments for a direct adaptive advan-tage for being sinistral or dextral (Nelson, 2006). Manyflatfish species have great economical importance, mainlydue to the quality of their meat, and have been extensivelyexploited (Cerqueira  et al. , 1997).Regan (1910, cited by Ramos, 1998) proposed thefirst morphological classification for the order then namedHeterostomata. The author considered the group as mono- phyletic and divided it in two putative monophyletic subor-ders, Psettodoidei and Pleuronectoidei. Currently, the mainclassification of the order is still based on morphologicalcharacters. However, the monophyletic nature of severalfamilies has been extensively debated (Chapleau, 1993;Chapleau and Keast, 1988; Chanet, 1997; Fukui, 1997;Hensley, 1997; Cooper and Chapleau, 1998; Berendzen and Dimmick, 2002; Pardo  et al. , 2005).The most recent and extensive phylogenetic study of Pleuronectiformes using morphological data was done byChapleau (1993). According to this author, Psettodidae isthe most primitive family, while Cynoglossidae andSoleidaearethemostderived.Citharidae,Scophthalmidae,Paralichthyidae, Bothidae, Pleuronectidae, and Achiridaeare considered intermediary families. According to theclassification proposed by Nelson (2006), mainly based onthe studies of  Chapleau (1993), Cooper and Chapleau(1998) and Hoshino (2001), the order has 14 families.Psettodidae is the only family of the suborder Psettodoideiand the remaining families belong to the suborder Pleuronectoidei. Genetics and Molecular Biology , 31, 1 (suppl), 284-292 (2008)Copyright © 2008, Sociedade Brasileira de Genética. Printed in Brazilwww.sbg.org.br  Send correspondence to Marisa Fagundes Carvalho de Azevedo. Nú-cleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé,Universidade Federal do Rio de Janeiro, NUPEM/UFRJ, Caixa Postal119331,27910-970Macaé,RJ,Brazil.E-mail:marisafca@yahoo.com.*Present address: Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé, Universidade Federal do Rio de Janeiro, Ma-caé, RJ, Brazil. Research Article  The first molecular study conducted on Pleuronec-tiformes by Tinti  et al.  (1999) reinforced the previously proposed relationships based on morphological characters, butonlyninespecieswereinvestigated.Pardo etal. (2005),analyzed fragments of about 650 base pairs (bp) of the 16SmtDNA gene of 30 species, and Berendzen and Dimmick (2002) analyzed DNA sequences of about 1200 bp of the12S and 16S mtDNA genes of 44 species. These analysesresulted in well-resolved phylogenies at species and genuslevels. However, the relationship among families remainedunresolved.Mostspeciesofflatfishesliveinsaltwaterorbrackishwater, but some are also found in freshwater (Nelson,2006). Among the few freshwater species those of the fam-ily Achiridae are encountered in Brazil. Achirids aremainly shore fishes restricted to both sides of Americas(Ramos, 2003a). According to Ramos (1998), the most primitive achirid species are freshwater species of the gen-era  Hypoclinemus  and  Catathyridium . In the present study, partial sequences of the mitochondrial genes 12S and 16Sof mtDNA of 19 species belonging to seven families of Pleuronectiformesweredeterminedwiththeaimofinvesti-gating the monophyletic nature of the recognized families,mainly that of the South America achirids. Materials and Methods Tissue samples Tissue samples were obtained from specimens of 19species of Pleuronectiformes belonging to the familiesAchiridae, Bothidae, Cynoglossidae, Paralichthyidae,Pleuronectidae, Scophthalmidae, and Soleidae. Speciesnames and sampling sites are shown in Table 1. Additionalsequences of 42 pleuronectiform species were retrievedfrom GenBank (Table 1). The single species of Psettodidae(  Psettodes erumei ) was used as outgroup, following the re-sults by Chapleau (1993) who demonstrated that this is themost primitive family among Pleuronectiformes. Azevedo  et al.  285 Table1 -SpecimensanalyzedinthepresentstudyandrespectiveGenBankaccessnumbers.FW=freshwater;BW=brackishwater;SW=saltwater.As-terisks show the species sequenced in the present study.Family Species Source 12S rRNA 16S rRNAAchiridae  Achirus declivis*  BW, Pará, Brazil  AY998041 AY998029Catathyridium jenynsi*  FW, Paraná, Brazil  AY998034 AY998022 Hypoclinemus mentalis*  FW, Acre, Brazil  AY998035 AY998023Trinectes paulistanus*  SW, São Paulo, Brazil  AY998036 AY998024Trinectes maculatus  GenBank   AF488496 AF488446  Bothidae  Arnoglossus imperialis*  SW, Spain  AY141358 AY359651 Arnoglossus thori  GenBank   AF542208 AY157329 Arnoglossus laterna  GenBank   AF542210 AY359653 Bothus lunatus  GenBank   AF488508 AF488458 Bothus ocellatus*  SW, São Paulo, Brazil  AY998038 AY998026  Bothus podas  GenBank   AF542221 AY157326  Bothus robinsi  GenBank   AF488509 AF488459Crossorhombus kobensis  GenBank   AF488506 AF488456  Laeops kitaharae  GenBank   AF488511 AF488461 Citharidae  Citharoides macrolepis  GenBank   AF488513 AF488463Citharus linguatula  GenBank   AF542220 AY157325 Cynoglossidae  Symphurus plagusia*  SW, São Paulo, Brazil  AF488497 DQ017374Symphurus tesselatus*  SW, São Paulo, Brazil  AY998037 AY998025 Paralichthyidae  Citharichthys xanthostigma  GenBank   AF488499 AF488449Cyclopsetta chittenden*i  SW, São Paulo, Brazil  AY998031 AY998019 Etropus crossotus*  SW, São Paulo, Brazil  AY998032 AY998020 Etropus longimanus*  SW, São Paulo, Brazil  AY998033 AY998021 Etropus microstomus  GenBank   AF488502 AF488452 Paralichthys dentatus  GenBank   AF488501 AF488451 Paralichthys patagonicus*  SW, Santa Catarina, Brazil  AY998040 AY998028 Pseudorhombus pentophthalmus  GenBank   AF488505 AF488455Syacium papillosum*  SW, São Paulo, Brazil  AY998039 AY998027 Tarphops oligolepis  GenBank   AF488507 AF488457  Xystreurys liolepis  GenBank   AF488504 AF488454  DNA extraction and sequencing Total DNA was extracted from ethanol-preservedliver or muscle tissue with the Wizard Genomic DNA Puri-fication Kit (Promega). Partial sequences of the mitochon-drial genes 12S and 16S rRNA were amplified by the polymerase chain reaction (PCR) with the following prim-ers: L1091 and H1478 (Kocher   et al. , 1989) for the gene12S rRNA, and 16Sa-L and 16Sb-H (Palumbi  et al. , 1991)for the gene 16S rRNA. Primer concentrations were 5  µ M.Amplifications were performed in a total volume of 25  µ Lfor 35 cycles (30 s at 95 °C, 60 s at 50-60 °C, and 120 s at72 °C). The PCR products were visualized on a 1% agarosegel. The amplified segments were extracted from the gelwith the kit GFX PCR DNA and Gel Purification (GEHealthcare).Sequencingreactionswereperformedwiththekit Big Dye Terminator v3.1 Cycle Sequencing Ready Re-action (Applied Biosystems) and analyzed in an automatedsequencer ABI Prism 377 DNA Sequencer (Applied Bio-systems). Some sequencing reactions were done with thekit Thermo Sequenase fluorescent labeled primer cycle se-quencing kit with 7-deaza-dGTP (Amersham Biosciences)and analyzed in an automated sequencer model ALF Ex- press II (Amersham Biosciences). All sequences were readat least two times (forward and reverse). Sequence analyses and phylogenetic approaches Individual sequences of each species were initiallyanalyzed with the software DAMBE (Xia and Xie, 2001)and a consensus sequence was obtained for each DNA seg-mentofeachspecies.Afterthat,allsequenceswerealigned 286 Molecular phylogeny of flatfishes Table 1 (cont.) Family Species Source 12S rRNA 16S rRNAPleuronectidae  Eopsetta jordani  GenBank   AF488476 AF488426 Glyptocephalus zachirus  GenBank   AF488486 AF488436  Hippoglossus stenolepis  GenBank   AF488483 AF488433 Isopsetta isolepis  GenBank   AF488481 AF488431 Lepidopsetta bilineata  GenBank   AF488479 AF488429 Limanda aspera  GenBank   AF488491 AF488441 Lyopsetta exilis  GenBank   AF488484 AF488434 Microstomus bathybius  GenBank   AF488490 AF488440 Microstomus pacificus  GenBank   AF488480 AF488430 Parophrys vetula  GenBank   AF488488 AF488438 Platichthys flesus*  SW, Spain  AF542206 AY359670 Platichthys stellatus  GenBank   AF488482 AF488432 Pleuronectes platessa  GenBank   AF542207 AY157328 Pleuronichthys guttulatus  GenBank   AF488487 AF488437  Pleuronichthys verticalis  GenBank   AF488489 AF488439 Psettichthys melanostictus  GenBank   AF488485 AF488435 Pseudopleuronectes americanus  GenBank   AF488478 AF488428 Psettodidae  Psettodes erumei  GenBank   AF488518 AF488468 Samaridae  Samariscus xenicus  GenBank   AF488517 AF488467  Plagiopsetta glossa  GenBank   AF488516 AF488466  Scophthalmidae  Lepidorhombus wiffiagonis*  SW, Spain  AY998042 AY998030Scophthalmus aquosus  GenBank   AF488512 AF420449Scophthalmus maximus*  SW, Spain  AY998043 AY998019Scophthalmus rhombus*  SW, Spain  AY998044 AY998020 Soleidae  Aseraggodes kobensis  GenBank   AF488493 AF488443 Dicologlossa cuneata*  SW, Spain  AF542211 AY359660 Heteromycteris japonicus  GenBank   AF488494 AF488444 Microchirus azevia  GenBank   AF542216 AY157318 Microchirus ocellatus  GenBank   AF542218 AF112850 Microchirus variegatus  GenBank   AY141359 AF112851 Monochirus hispidus  GenBank   AF542219 AF112852Solea solea  GenBank   AF488492 AF112845Solea sonegalensis*  SW, Spain  AF542205 AY359661  withthesoftwareClustalW,implementedinDAMBE(Xiaand Xie, 2001). Nucleotide variation, substitution patternsand genetic distances were examined using MEGA 2.1(Kumar   et al. , 2001). Nucleotide saturation was analyzed by plotting the absolute number of transitions (Ti) andtransversions (Tv) against genetic distance values in thesoftware DAMBE (Xia and Xie, 2001).Maximum-parsimony (MP) based phylogenetic anal-yses were performed using the software PAUP* beta ver-sion 4.0b10 (Swofford, 2002) with heuristic searches usingrandom addition of sequences and the tree bisection andreconnection (TBR) algorithm. In all analyses, the charac-ter-state optimization method employed was the acceler-ated transformation (ACCTRAN). Parsimony trees weregenerated using Ti/Tv ratios of 1:1 and 1:2, consideringgapsasmissingdataorasafifthbase.Bootstrapresampling(Felsenstein, 1985) was applied to assess support for indi-vidual nodes using 1000 replicates with random additionsand TBR branch swapping.Geneticdistancesamongsequenceswereestimatedbythe Hasegawa-Kishino-Yano (HKY85) nucleotide substitu-tion model (Hasegawa  et al. , 1985), incorporating the pro- portion of invariant sites and among-site rate heterogeneity based on a hierarchical hypothesis test of alternative modelsimplemented in the program Modeltest 3.7 (Posada andCrandall, 1998). The Bayesian-inference method of phylo-geneticanalysis(Huelsenbeck  etal. ,2001)wasusedtoeval-uate alternative tree topologies through the estimation of  posterior probabilities using MrBayes v. 3.0 (Ronquist andHuelsenbeck, 2003). The MrBayes analysis ran four chainssimultaneously, each for 1 million generations. Every 100 th generation was sampled and the asymptote of the likelihoodscore was detected with the SUMP command. All sampledtopologies beneath the asymptote were discarded from the population of trees considered in the subsequent major-ity-rule consensus. The frequency with which a particular clade appeared in the population of retained topologies wasinterpretedasitsposteriorprobability.Posteriorprobabilitieswereinterpretedasameasureofhowlikelythecladeappearsin the optimal topology, rather than accuracy of the nodewith respect to species relationships or clade stability. Con-sensus trees were produced with the software TreeExplorer implemented in MEGA 2.1 (Kumar   et al. , 2001).Maximum likelihood (ML) phylogenetic analyseswere performed using the software PHYML (Guindon andGascuel,2003)initswebsiteversion(Guindon etal. ,2005)using the Hasegawa-Kishino-Yano (HKY85) nucleotidesubstitution model (Hasegawa  et al. , 1985). Clade stabilitywas estimated by non-parametric bootstrapping in 500 rep-licates with PHYML. Results Sequences obtained for this study have been depos-ited in GenBank (accession numbers AY998019 -AY998044) (Table 1).Thecombinedsequencedataofthe61flatfishtaxare-sulted in 1428 bp, of which 314 were conserved sites and981 were parsimony informative. The transition/transver-sion (Ti/Tv) ratio observed was 0.9. Percent base composi-tion for sequenced regions of the L-strand was determinedas follows: adenine (A) 29.4; cytosine (C) 26.1; guanine(G) 22.3; and thymine (T) 22.2. The analysis of these dataclearly shows that the base composition of the L-strand issomewhatA-rich,similartothatdescribedforseveralmito-chondrial genes of fishes (Alves-Gomes  et al. , 1995; Shi-mabukuro-Dias  et al. , 2004), lizards (Reeder, 1995), and snakes (Parkinson, 1999). The results from the analysis of  plotting transitions and transversions against genetic dis-tance suggest the occurrence of low nucleotide saturation(Figure 1).Initially, a total of eight MP heuristic searches wereconducted employing all data or excluding 179 characters belonging to six segments with alignment problems, in-cluding or excluding gaps, and considering the Ti/Tv ratiosof 1:1 or 1:2. The resultant phylogenies were largely con-gruent. The best resolved MP consensus tree obtained fromthe analysis of 1000 bootstrap replicates including all posi-tions, treating gaps as missing data, and the Ti/Tv ratio of 1:1 is presented in Figure 2. For this tree, the results ob-tained were: tree length = 4332, consistency index(CI) = 0.4084, homoplasy index (HI) = 0.5916 and reten-tion index (RI) = 0.6259.Phylogenetic analyses with the Bayesian method re-sulted in a similar tree with the nodes supported by valuesusually higher than those found in MP analysis (Figure 3).The phylogeny obtained with the ML method was similar to that obtained with the MP method (Figure 4) but somefamilies were differently positioned. Discussion Considering Psettodidae as sister group of all other  pleuronectiforms (Chapleau, 1993), all remaining familiesinvestigated in our study appeared as monophyletic (withthe exception of Paralichthyidae) and were supported by Azevedo  et al.  287 Figure 1  - Graphic showing the frequency of observed transitions andtransversions versus genetic distance (Kimura, 1980) of 12S and 16SrRNAgenes.Transitionsareblacksquares,transversionsareopencircles.  high bootstrap and posterior probability values. However,the data obtained did not resolve the relationship amongseveral families, as already observed in previous studiesemploying 12S and 16S rRNA sequences (Berendzen andDimmick, 2002; Pardo  et al. , 2005).Paralichthyidae with about 16 genera and 105 species(Nelson, 2006), has been recognized as a paraphyleticgroup (Hensley and Ahlstrom, 1984; Chapleau, 1993; Par-do  et al. , 2005; Berendzen and Dimmick, 2002). In the present study, the analysis of specimens from eight generashowed that they belong to two or three independent lin-eages. The group identified as Paralichthyidae 1 was re-lated to Bothidae in all analyses (Figures 2 to 4). The groupidentified as Paralichthyidae 2 in the MP and ML analysesappeared as polyphyletic (Figures 2 and 4), as also ob-served in the Bayesian analysis (Figure 3). The first group(Paralichthyidae 1), composed by the genera  Cyclopsetta , Syacium , Citharichthys ,and  Etropus waspreviouslyrecog-nized by Chapleau (1993) as a natural group (named Cyclopsetta  group) sharing a urinary papilla oriented to-wards the blind size, an ocular pelvic fin based on themid-ventrallineofthebody,ablindsizepelvic-finbasean- 288 Molecular phylogeny of flatfishes Figure 2  - Pleuronectiforms consensus maximum-parsimony tree pro-duced when gaps were treated as missing data and the Ti/Tv ratio of 1:1(TL = 4332, CI = 0.4084, HI = 0.5916, RI = 0.6259). Numbers above branchesarebootstrapvaluesbasedon1000replicates.Valuesbelow50%are not shown. Figure3 -PleuronectiformesconsensustreeproducedbyaBayesiananal-ysis. Numbers above nodes are posterior probabilities recovered by theBayesian analysis. Values below 50% are not shown.
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