Molecular Phylogenetics and Evolution 69 (2013) 1209–1214

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Molecular Phylogenetics and Evolution

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Short Communication

Multilocus molecular phylogeny of Gasteropelecidae (Ostariophysi:Characiformes) reveals the existence of an unsuspected diversity

1055-7903/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ympev.2013.07.005

⇑ Corresponding author. Address: Departamento de Morfologia, Instituto deBiociências, UNESP, Univ. Estadual Paulista, 18618-970 Botucatu, São Paulo, Brazil.Tel.: +55 1438800464.

E-mail address: claudio@ibb.unesp.br (C. Oliveira).

Kelly T. Abe a, Tatiane C. Mariguela a, Gleisy S. Avelino a, Ricardo M.C. Castro b, Claudio Oliveira a,⇑a Laboratório de Biologia e Genética de Peixes, Departamento de Morfologia, Instituto de Biociências, Univ. Estadual Paulista, UNESP, Botucatu, São Paulo, Brazilb Laboratório de Ictiologia de Ribeirão Preto (LIRP), Departamento de Biologia da FFCLRP, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 May 2013Revised 3 July 2013Accepted 4 July 2013Available online 18 July 2013

Keywords:CharaciformsHatchetfishesEvolutionPhylogenySystematics

The characiform family Gasteropelecidae, the so-called freshwater hatchetfishes, is comprised of threegenera and nine species found in Panama and all South American countries except Chile. Our goal wasto investigate the molecular characteristics, phylogenetic relationships among the species and generaof Gasteropelecidae and phylogenetic relationships between the Gasteropelecidae family with otherCharaciformes. DNA fragments from two mitochondrial (16S rRNA and Cytochrome B) and three nucleargenes (Rag1, Rag2 and Myh6) were sequenced. Our results corroborate the morphology-based hypothe-sized monophyly of the Gasteropelecidae family and most of the relationships among its genera. How-ever, the genus Gasteropelecus is polyphyletic because G. maculatus is placed as the sister group to allother gasteropelecids, whereas G. sternicla is more closely related to species of Carnegiella. Similarly,the species Carnegiella strigata is not monophyletic, which suggests that the family needs a taxonomicreview. Moreover, the species Thoracocharax stellatus was composed by four distinct lineages suggestingthe this species may represents a species complex.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Characiformes comprises a large freshwater fish order withapproximately 2000 species distributed in 23 families, 19 of whichare exclusively Neotropical and four are exclusively African(Oliveira et al., 2011; Eschmeyer and Fong, 2013). Several characi-forms are commercially important as food or ornamental fishes.Among the ornamental characiforms, the species of Gasteropeleci-dae, which are collectively known in the aquarium trade ashatchetfishes, are popular aquarium fishes. These fishes aredistributed in Panama and all South American countries exceptChile and occur in the drainage basins of the Amazon, Orinoco,Paraguay, and Marowini (Maroni) Rivers (Weitzman and Palmer,2003).

The Gasteropelecidae (Fig. 1) is comprised of three genera(Gasteropelecus, Carnegiella and Thoracocharax) and nine valid spe-cies (Weitzman and Palmer, 2003) and are easily recognized bytheir relatively small size and extremely developed and sharp ante-ro-ventral keel (hence the ‘‘hatch’’ in their English popular name)formed by their greatly enlarged, strongly convex muscular pector-al girdle region, which consists of greatly expanded coracoid bonesfused to a single fan-shaped and corrugated median bone. This

antero-ventral keel is functionally connected to a pair of elongateand developed pectoral fins, which allows gasteropelecins to jumprelatively long or high distances. The entire skull and pectoral gir-dle of the three genera is highly modified for jumping and feeding,mainly on insects, at the surface of water (Weitzman and Palmer,2003). The Gasteropelecus and Thoracocharax species occur in theopen waters of larger rivers, streams and lakes, whereas Carnegiellaare found in small creeks and streams.

Although the first species of Gasteropelecidae to be described,Gasteropelecus sternicla (Linnaeus, 1758), was described more than250 years ago, the relationships of the family with the remainingCharaciformes has been poorly studied until now. Weitzman(1962) formally defined the Gasteropelecinae (=Gasteropelecidae)and concluded that ‘‘this subfamily has been evolving for a longtime, and the Gasteropelecinae may well have been derived fromsome relatively primitive characid with five branchiostegal rays’’.In addition, because Thoracocharax has a ‘‘somewhat more primi-tive morphology with respect to other members of the Gasterope-lecinae (=Gasteropelecidae)’’, which arises from a commonancestor with Gasteropelecus, and Carnegiella ‘‘seems to be aneotenic form of Gasteropelecus and directly derived from it’’,Thoracocharax was placed alone in the Thoracocharacini tribe,and Carnegiella and Gasteropelecus were placed together in theGasteropelecini tribe.

Mirande (2010) presented a cladistic analysis for the Characidaebased on 360 morphological characteristics scored for 160 charac-iform species. Although this study is the most wide study to date

Fig. 1. Preserved representative specimens of three recognized genera of Gasteropelecidae: (A) Carnegiella strigata; (B) Gasteropelecus sternicla; (C) Thoracocharax stellatus(Photos A. Datovo) and in D) Geographical distribution of the Gasteropelecidae samples. 1, Río Iguesia and Río Pirre, Panama (Gasteropelecus maculatus); 2, General ManuelCedeño, Venezuela (Thoracocharax stellatus); 3, Barcelos, Amazonas (Carnegiella marthae and C. strigata); 4, Rio Preto da Eva and Careiro da Várzea, Amazonas (C. Strigata and T.stellatus); 5, Cruzeiro do Sul and Mâncio Lima, Acre (C. strigata, G. sternichla, and T. stellatus); 6, Cuiabá, Mato Grosso (T. stellatus); 7, Barra do Garça, Mato Grosso (T. stellatus).See Table 1 for species identities and detailed information of collection localities.

1210 K.T. Abe et al. / Molecular Phylogenetics and Evolution 69 (2013) 1209–1214

employing morphological data, it did not include representativesof many genera of the Characidae and or representatives fromthe characiform families Alestidae, Chilodontidae, Citharinidae,Ctenoluciidae and Hepsetidae. In this study, Gasteropelecidae(represented by the genera Thoracocharax and Carnegiella) wasfound with Engraulisoma taeniatum as a sister group of allremaining Characoidea, including Serrasalmidae, Alestidae andCharacidae.

The first molecular studies on characiform phylogenies wereperformed by Orti and Meyer (1996, 1997) using partial sequencesof 12S and 16S rRNA mitochondrial genes and by analyzing partialsequences of the nuclear gene ependymin. In these studies, mito-chondrial data show Gasteropelecidae (represented by Carnegiellaand Gasteropelecus) as a sister group of Anostomidae, Chilodonti-dae and Crenuchidae, whereas nuclear data display the Gasterope-lecidae (represented by Gasteropelecus) as a sister group of thegenera Gymnocorymbus and Paracheirodon (Characidae). Javonilloet al. (2010) proposed a phylogenetic hypothesis for the group theyrecognized as Characidae; however, the poor out-group represen-tatives (only Chalceus macrolepidotus) did not permit the testingof the real monophyly of the studied groups. However, three gen-era of Gasteropelecidae appeared as a monophyletic group in alarge polytomy that involved most of the taxa studied. In a molec-ular phylogenetic study of Characidae in which representatives ofall families of Characiformes were included, Oliveira et al. (2011)found Gasteropelecidae as a sister group of Bryconidae and Triport-heidae, which were newly defined by the authors.

In view of the currently unstudied phylogenetic relationshipamong gasteropelecid species, two mitochondrial and three nucle-ar genes of representatives of the Gasteropelecidae genera wereanalyzed, and the data were employed to formulate a hypothesiscovering the relationships among the genera and species ofGasteropelecidae.

2. Material and methods

2.1. Taxa sampled and molecular data collection

Our in-group was composed of tissue samples obtained fromspecimens belonging to five of nine currently recognized speciesof Gasteropelecidae (Fig. 1) from 12 known and 1 unknown(aquarium trade specimens) localities (Fig. 1, Table 1). The

sampling was focused in the acquisition of representatives fromall three gasteropelecid genera and specimens from a wide repre-sentative distribution area (Fig. 1). To obtain a representativeout-group, we used the data of the families Bryconidae, Chalceidaeand Triportheidae published by Oliveira et al. (2011) in a broadstudy of Characidae.

Total DNA was extracted from ethanol-preserved muscle sam-ples using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA,USA) following the manufacturer’s instructions. Partial sequencesof the 16S rRNA, Cytochrome b (Cyt b), recombination activatinggene 1 (Rag1), recombination activating gene 2 (Rag2), and Myosinheavy chain 6 from cardiac muscle alpha (Myh6) genes wereamplified by polymerase chain reaction (PCR) with the primersdescribed by Kocher et al. (1989), Palumbi (1996), Lovejoy andCollette (2001), Irwing et al. (1991), Li et al. (2007), Li and Ortí(2007) and Oliveira et al. (2011). Amplifications were performedin a total volume of 25 ll with 2.5 ll of 10X buffer (10 mMTris–HCl + 15 mM MgCl2 buffer), 0.5 ll MgCl2, 0.5 ll each primer(5 lM), 0.4 ll dNTPs (200 nM of each), 0.2 ll Taq Platinumpolymerase (Invitrogen), 1 ll template DNA (10–50 ng) and19.4 ll ddH2O. The thermo-cycler profile used for the fragments16S rRNA and Cyt b contained 35 cycles and an annealing temper-ature of 50–55 �C. Nested-PCR was used to amplify the nucleargenes Rag1, Rag2, and Myh6. Conditions for amplification of thesegenes for both rounds of PCR used 15 cycles with an annealingtemperature of 56 �C followed by 15 cycles with an annealing tem-perature at 54 �C. PCR products were purified using ExoSap-IT�

(USB Corporation), sequenced using the ‘‘Big DyeTM Terminator v3.1 Cycle Sequencing Ready Reaction Kit’’ (Applied Biosystems),purified by ethanol precipitation and loaded on an automaticsequencer 3130-Genetic Analyzer (Applied Biosystems) at Institutode Biociências, Universidade Estadual Paulista, Botucatu, São Paulo,Brazil. All sequences obtained through this study have been depos-ited in GenBank (Additional file 1).

2.2. Alignment and phylogenetic analyses

Sequences of each gene were independently aligned using theMuscle algorithm with default parameters (Edgar, 2004), and thealignments were inspected by eye for any obvious misalignments,which were corrected. A quality control step was included in ourworkflow, as described in Oliveira et al. (2011). Genetic distances

Table 1Species of Gasteropelecidae analyzed in the present phylogenetic study.

Species Voucher Specimen Locality Geographic position Position inFig. 1

Carnegiella marthae LBP 4199 23601 23793 Brazil, Amazonas, Barcelos, Igarapé Puxirituba 00�53018.600 S 62�40036.100

W3

Carnegiella strigata LBP 2512 17027 17030 Brazil, Aquarium trade specimens – –Carnegiella strigata LBP 4200 23602 23798 Brazil, Amazonas, Barcelos, Igarapé Puxirituba 00�53018.600 S 62�40036.100

W3

Carnegiella strigata LBP 3167 19318 Brazil, Amazonas, Rio Preto da Eva, Rio Preto da Eva, 02�45016.600 S 59�37030.900

W4

Carnegiella strigata LBP 4177 22802 Brazil, Acre, Cruzeiro do Sul, Igarapé Preto 07�35033.40 S 72�45017.700 W 5Gasteropelecus

maculatusSTRI3506 19869 Panama, Rio Iglesia 08�25023.000 N 78�00005.000

W1

Gasteropelecusmaculatus

STRIAM208 19868 Panama, Rio Pirre 08�07059.900 N 77�43059.900

W1

Gasteropelecus sternicla LBP 4070 22974 2297822975

Brazil, Acre, Mâncio Lima, Rio Japiim 07�34028.80 S 72�55024.900 W 5

Thoracocharax stellatus LBP 3942 22595 22596 Venezuela, Bolívar, General Manuel Cedeño, RioOrinoco

07�38030.300 N 66�18038.000

W2

Thoracocharax stellatus LBP 1694 12769 Brazil, Amazonas, Careiro da Várzea, Lago do Vanico 03�09017.300 S 59�53012.300

W4

Thoracocharax stellatus LBP 4179 22801 Brazil, Acre, Mâncio Lima, Rio Moa 07�26035.500 S 73�03033.500

W5

Thoracocharax stellatus LBP 7534 35343 Brazil, Mato Grosso, Cuiabá, Rio Cuiabá 15�39009.900 S 56�04008.600

W6

Thoracocharax stellatus LBP 1588 11714 11716 Brazil, Mato Grosso, Barra do Garças, Rio das Garças 15�54018.100 S 52�19024.200

W7

K.T. Abe et al. / Molecular Phylogenetics and Evolution 69 (2013) 1209–1214 1211

among sequences were calculated in Mega 5.04 (Tamura et al.,2011). To evaluate the occurrence of substitution saturation, weestimated the index of substitution saturation (Iss) in DAMBE5.3.31 (Xia, 2013).

Six reasonable partitioning schemes, ranging from 1 to 13 par-titions (Additional file 1), were tested following the proceduresoutlined by Li et al. (2008) under the AIC and BIC criteria. Thebest-fit model of nucleotide substitution was calculated in Mega5.04 (Tamura et al., 2011) with default parameters using theAkaike information criterion (Posada and Buckley, 2004).

Maximum parsimony (MP) analyses were conducted usingPAUP* 4.0b10 (Swofford, 2003). Heuristic searches were performedwith a minimum of 1000 random addition replicates and TBRbranch swapping. All characteristics were unordered, all character-istic transformations were equally weighted, and branches with amaximum length of zero were collapsed. Gaps were treated asmissing data. Clade robustness was assessed using 1000 bootstrappseudo-replicates (Felsenstein, 1985) with the same parameters asabove.

RAxML which uses the web servers RAxML-HPC2 on TG(Stamatakis et al., 2008; Miller et al., 2010), was used for all

Table 2Information content and characteristics of each gene partition.

Gene

16S Cyt b

Number of sequences 35 (100%) 34 (97bp After alignment 606 992Number of variable sites 230 496Number of informative characteristics under parsimony 187 459% Informative characteristics under parsimony 30.9 46.2PA 31.4 26.9PC 22.9 27.5PG 22.8 14.6PT 22.9 31.0Overall mean genetic distance (p-Distance) 0.118 ± 0.008 0.210Nucleotide substitution model GTR HKYa (shape) parameter of C distribution 0.26 1.06Proportion of invariant (I) sites – 0.45

maximum likelihood (ML) analyses using a mixed partition model.Random starting trees were used for each independent ML treesearch and all other parameters were set to the default values.All ML analyses were conducted following the 13 partition scheme,which was suggested by the AIC and BIC criteria (Table 2). Topolog-ical robustness was investigated using 1000 non-parametric boot-strap replicates.

Phylogenetic analyses using a partitioned Bayesian approach(B) were conducted in MrBayes 3.1.2 (Ronquist and Huelsenbeck,2003). A mixed model analysis was implemented, which allowedindividual models of nucleotide substitution to be estimated inde-pendently for each partition. As MrBayes 3.1.2 only implements 1,2, and 6 substitution rate models, it was often not possible toimplement the preferred model selected by the AIC. In these situ-ations, the nearest over-parameterized model was used to avoidnegative consequences of model violations or under parameteriza-tion (Li et al., 2008). Therefore, the model for all partitions was setas ‘‘lset nst = 6’’, ‘‘rates = invgamma’’ (G + I), and the commands‘‘unlink’’ and ‘‘prset ratepr = variable’’ were used to unlink modelparameters across data partitions and define a rate multiplier foreach partition. Two independent Bayesian analyses were

Final

Myh6 Rag1 Rag2 Matrix

%) 24 (69%) 24 (69%) 31 (80%) 35 (100%)752 1265 1031 4646229 410 361 1726179 285 247 135723.8 22.5 23.9 29.230.7 25.9 24.4 27.321.2 23.6 25.2 24.523.2 27.3 27.2 22.924.9 23.2 23.2 25.3

± 0.007 0.083 ± 0.005 0.087 ± 0.004 0.075 ± 0.004 0.127 ± 0.003T92 K2P K2P GTR0.96 0.39 0.45 0.59– – – 0.37

1212 K.T. Abe et al. / Molecular Phylogenetics and Evolution 69 (2013) 1209–1214

conducted. Four independent MCMC chains were run with15,000,000 replicates each, which sampled one tree every 1000steps. The distribution of log likelihood scores was examined todetermine stationarity for each search and to decide if extra runswere required to achieve convergence using the program Tracer1.4 (Rambaut and Drummond, 2004). Initial trees estimated priorto convergence were discarded as part of a burn-in procedure,and the remaining trees were used to construct a 50% majority ruleconsensus tree in Paup*.

Alternative phylogenetic hypotheses were compared using like-lihood-based tests implemented in the program Treefinder (Jobb,2008). These tests assess the statistical significance of differencesin likelihood scores between two or more hypotheses. Probabilitiesfor alternative hypotheses were obtained for the Shimodaira–Hasegawa (SH) and the approximately unbiased (AU) tests (Shimo-daira and Hasegawa, 1999; Shimodaira, 2002). Both testing proce-dures are adequate to compare hypotheses a posteriori based onthe same data set, but because the SH test is more conservative(Shimodaira, 2002), significance was determined when the P-val-ues obtained were P < 0.05 and P < 0.01 for SH and AU, respectively.Alternative hypotheses were constructed by performing treesearches under specific topological constraints to find the ML treethat satisfies the enforced branching pattern. The constraints fixedeither the topology or the composition for major clades, but in eachcase, multifurcations within these clades or elsewhere in the treewere resolved via tree searches. Searches were conducted underML using the program Treefinder with a 13-partition scheme anda GTR + G + I model optimized independently for each partition(the same approach used with RAxML). The results from each ofthese constrained tree searches were saved individually and subse-quently joined into a single hypothesis file to perform topologytests according to the Treefinder manual (Jobb, 2008).

3. Results

Partial sequences of two mitochondrial (16S rRNA and Cyt b)and three nuclear genes (Myh6, Rag1 and Rag2) were obtainedfor 35 specimens, of which 16 were sequenced for the presentstudy and 19 were obtained from Oliveira et al. (2011) (Additionalfile 1). The final matrix had 4646 bp and was deposited in TreeBase(http://treebase.org) under number 14439.

Missing data, because of problems with PCR experiments,sequencing, or missing data in GenBank, corresponded to 15.5% (Ta-ble 2). Data absence was more prevalent among nuclear (24.8%)than mitochondrial genes (1.5%), likely because of non-conservedpriming regions and an increased risk of cross-contamination inthe nested PCR procedure. For each matrix and gene, the numberand percentage of sequences obtained, including their size (bp),number of variable sites, base pair composition, mean pairwise ge-netic distances among taxa (p-distance), best substitution modelfor the gene, a (shape) parameter of C distribution, proportion ofinvariant (I) sites, number of informative characteristics under par-simony, and proportion of informative characteristics under parsi-mony, are presented in the Table 2. The mean pairwise geneticdistances among taxa observed was between 0.083 ± 0.005(Myh6) and 0.210 ± 0.007 (Cyt b), which suggests that the analyzedsequences have enough genetic variation for the phylogenetic stud-ies of species, genera and families. Each gene and codon positionpartition was tested further to investigate the occurrence of substi-tution saturation, and the results showed that there is little satura-tion for the Cyt b 3rd codon position in the symmetrical topologytest (results not shown); however, considering that the Iss.c valueis greater than the Iss value and that there is no significant satura-tion in the asymmetrical topology test, the information found inthis position can be used in phylogenetic analyses. The best-fitting

model of nucleotide substitution calculated for each gene wasGTR + I (16S), HKY + I + C (Cyt b), T92 + I (Myh6 and Rag1) andK2P + I (Rag2) (Table 2). The combined data set contains significantphylogenetic information given that most major lineages along thebackbone of the tree were supported by high bootstrap values.

Six different partitioning schemes, ranging from 1 to 13 parti-tions (Additional file 2), were tested to establish the optimal num-ber of data partitions (following Li et al., 2008) for the finalanalysis. The results showed that the 13-partition model was thebest choice; however, ML analysis conducted with other partition-ing schemes resulted in the same topology with minor differencesin branch length and support values (not shown).

Throughout the text and in the Fig. 2, measures of support areindicated as a series of three numbers on selected internalbranches of the trees subtending labeled clades, starting withposterior probabilities in Bayesian analysis and followed bynon-parametric bootstrap percentages from ML and MP analyses,respectively (e.g., 1/100/100, see Fig. 2); dashes represent valueslower than 0.5% (B) or 50% (ML and MP) and asterisks representnodes that have different topologies in different analytical meth-ods. Nodes without support values greater than 0.5% (B) and 50%(ML and MP) were collapsed. An ML tree that summarizes the phy-logenetic results is presented in Fig. 2. The general tree topologyobserved in all analyses was similar; however, statistical supportwas not strong at some nodes. Thus, we selected the ML topologyto discuss relationships among taxa.

3.1. Phylogenetic relationships of Gasteropelecidae

Fig. 2 shows that Gasteropelecidae is the sister group of Brycon-idae (0.94/*/53) and these two families are the sister group ofTriportheidae (1/100/100). In the ML analysis Gasteropelecidaewas resolved as sister group of Triportheidae in 51% of bootstrapreplicates. The monophyly of Gasteropelecidae was corroboratedin all analyses with high bootstrap values (1/100/97). Inside Gaste-ropelecidae, we found Gasteropelecus as a polyphyletic genus. G.maculatus is a sister group of all remaining Gasteropelecidae. Tho-racocharax is monophyletic (1/100/100) and is the sister group ofall samples of Carnegiella and Gasteropelecus sternicla (0.99/88/90). G. sternicla is the sister group of all Carnegiella specimensand this is a monophyletic genus (1/100/100). The species C. mart-hae is monophyletic (1/100/100) but C. strigata is not.

4. Discussion

4.1. Relationships among Gasteropelecidae and remainingcharaciforms

Gasteropelecidae, including Gasteropelecus, Carnegiella and Tho-racocharax, was resolved as monophyletic and as a sister group ofBryconidae and these two families are the sister group of Triport-heidae in the MP and B analysis (Fig. 2). However, in the ML anal-ysis Gasteropelecidae was placed as sister group of Triportheidaein 51% of the bootstraped trees. The first hypotheses is the sameas that presented by Oliveira et al. (2011), but the further additionof Bryconidae and Triportheidae data may result in a differenthypotheses of relationship among these families. In both cases,the present results refute the hypothesis of Mirande (2010), whichstated that Engraulisoma could belong to the family Gasteropelec-idae as a sister group of Thoracocharax plus Carnegiella.

4.2. Phylogenetic relationships among Gasteropelecidae genera andspecies

Among Gasteropelecidae, we found Gasteropelecus as polyphy-letic (Fig. 2). G. maculatus is the sister group of all remaining

Fig. 2. Best maximum likelihood tree (ln-28844.8) obtained in the partitioned analysis of the concatenated dataset and emphasizes the relationship among species ofGasteropelecidae (signaled). A series of three numbers (e.g., 1/100/93) at each of the main nodes represent the posterior probability for that split obtained in Bayesian analysis(B), percentage of bootstrap support obtained by ML, and percentage of bootstrap support obtained by MP analysis, respectively (1000 bootstrap replicates). Dashes representvalues lower than 0.5 (B) or 50% (ML and MP). Asterisks represent nodes that were not obtained by B, ML or MP analyses. Some branches are collapsed because of the lack ofresolution in our analyses. Clades labeled in red correspond to species collected in Panama, in green in Venezuela, in pink in the Araguaia basin, in light blue in the Paraguaybasin, and in dark blue in the Amazon.

K.T. Abe et al. / Molecular Phylogenetics and Evolution 69 (2013) 1209–1214 1213

Gasteropelecidae, whereas G. sternicla is a sister group of Carnegi-ella. As stated above, an important osteological characteristic foundin Gasteropelecus maculatus is the presence of the supraorbital bone(Géry, 1977). This characteristic is absent in all remaining Gaste-ropelecidae. However, a topology test (Additional file 2) resultsin non significant P-values in both the SH and AU tests suggestingthe Gasteropelecus may be a monophyletic genus. Additional datawill be necessary to better test the monophyly of this genus.

Thoracocharax stellatus is in a monophyletic lineage; however,four well-supported clades were found among our samples(Fig. 2) separated by relatively large genetic distances and follow-ing a well defined geographic orientation. The first one, the sistergroup of all other samples, was comprised of fishes from theOrinoco River. A second monophyletic clade was comprised ofsamples from the Paraguay River (a single sample), Araguaia Riverand Amazonas River. The genetic distance [computed consideringthe Kimura-2-parameter model (Kimura, 1980)] among the stud-ied samples ranged from 0.023 ± 0.002 between the Paraguayand Amazonas Rivers to 0.068 ± 0.004 between the Araguaia andOrinoco Rivers when considering all genes. Considering only theCyt b gene, the genetic distance among the studied samples rangedfrom 0.021 ± 0.004 between the Araguaia and Paraguay Rivers to0.103 ± 0.009 between the Araguaia and Orinoco Rivers. Wardet al. (2009) compared genetic distances among 546 fish species(1677 sequences) using sequences of the Cytochrome c Oxidase I

gene, a gene with a similar evolutionary rate as Cytochrome b,and found that the mean genetic distance within species was0.0035 ± 0.0001 and 0.0811 ± 0.0004 within genera. These datasuggest that T. stellatus may represent a species complex insteadof a single species. However, further revision of this species willbe necessary to understand this genetic variation correctly.

The genus Carnegiella is monophyletic (Fig. 2). However, as thetwo samples of C. marthae form a monophyletic lineage, the C.strigata samples do not comprise a monophyletic clade. Enforcingtopological constraints to test the monophyly of Carnegiella resultsin highly significant P-values in both, the SH and AU tests (Addi-tional file 2) rejecting this monophyly hypotheses.

The two samples collected in the Igarapé Puxirituba in Barcelos(Rio Negro drainage) appear unrelated in our phylogenetic analysis(Fig. 2). Géry (1977) extensively discussed what he called colorpolymorphisms observed in the different species of Carnegiellaand recognized several subspecies that were not recognized inthe last revision of the family (Weitzman and Palmer, 2003). Ourresults suggest that a thorough revision, including genetic data,should be performed in this genus, which should aim for more ro-bust species identification.

Further inclusion of samples of the remaining species of gaste-ropelecid Carnegiella myersi, C. schereri, Gasteropelecus levis, andThoracocharax securis as well as more local populations will be use-ful for a necessary revision of the whole family.

1214 K.T. Abe et al. / Molecular Phylogenetics and Evolution 69 (2013) 1209–1214

Acknowledgements

We would like to thank all of the people who helped us withspecimen collection and identification, especially Dr. Mauro Nir-chio from Universidad de Oriente for the donation of fish tissuesamples from Venezuela and Dr. Oris Sanjur from SmithsonianTropical Research Institute for the donation of tissue samples ofGasteropelecus maculatus, Dr. Ana Carla M.M. de Aquino and AléssioDatovo, both from LIRP, for helping with specimen preparation andpreparation of Fig. 1. This study is part of the FAPESP (Fundação deApoio à Pesquisa do Estado de São Paulo) Thematic Project ‘‘Phylo-genetic relationships in the Characidae (Ostariophysi: Characifor-mes) (FAPESP Grant Number 04/09219-6, R.M.C. Castro, PrincipalInvestigator). The fourth and fifth authors are CNPq (ConselhoNacional de Desenvolvimento Científico e Tecnológico do Brasil)researchers (CNPq Grant Numbers 309632/2007-2 and 303854/2009-0, respectively). The second and third authors are FAPESPPostdoctoral Researchers (FAPESP Grant Numbers 2010/17999-2and 2011/17746-0, respectively), and this research was developedprimarily as the first author’s Master of Science Dissertation.Financial support was also provided by CAPES (Coordenação deAperfeiçoamento de Pessoal de Nível Superior).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2013.07.005.

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