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Anostomidae is a widely distributed group that includes the species rich Leporinus. This genus is widespread in South America, including its south portion where they are mainly distributed in the São Francisco and La Plata basins. Here we obtained the genetic similarities observed among species does not corroborate the division of species of Leporinus in groups according to color patterns. Additionally, our results show that the species of Leporinus from the La Plata and São Francisco are not more similar among themselves than to species of other drainages which could be explained by the occurrence of recent fauna exchanges between these two basins and adjacent basins. Keywords: Phylogeny, Molecular Sequencing, Mitochondrial DNA, Fish. systematics, suggesting a possible local differentiation of these species. An interesting finding of our study is that both in number of species and number of individuals in the rivers where they occur [1,3]; because of this, several attempts were made trying to divide the species of Leporinus in smaller groups. Fowler  created the subgenus Myocharax, based on the dentition presented by Leporinus desmotes, which shows a very long pair of symphyseal teeth. Borodin  attempted to divide the genus Leporinus in two subgenera and created the subgenus Hypomasticus that included species with a lower mouth. Another subgenus, Leporinops, was created by Géry , based on tooth and cranial differences. This subgenus was based on Leporinus moralesi . All these proposals were not accept by Garavello & Britski  in the catalog of species of the family but Hyposmastictus was recognized as valid by Sidlauskas & Vari . Garavello & Britski , in a paper describing Leporinus amblyrhynchus and L. paranensis from the upper Paraná Basin, showed that most species of the genus may be placed into three artificial groups based on the color pattern of the trunk: 1) barred species, 2) striped species and 3) spotted species. The number of teeth is also a diagnostic feature for the species of the genus and is often used in identification keys. With the exception of the recently described species Leporinus venerei , which *Corresponding author: Gleisy S. Avelino, Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Caixa Postal 510, Rubião Júnior, s/n, 18618-970, Botucatu, SP, Brazil, E-mail: email@example.com Fausto Foresti, Claudio Oliveira, Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Caixa Postal 510, Rubião Júnior, s/n, 18618-970, Botucatu, SP, Brazil Heraldo A. Britski, Museu de Zoologia da Universidade de São Paulo, Caixa Postal 42494, 04299-970, São Paulo, SP, Brazil Garavello & Britski  recognized nine species of Leporinus in the La Plata Basin, four species in the São has the dental formula 4/3, unique among species of the genus, all species are artificially placed into three groups according to the dental formula (number of tooth in premaxillary/dentary in each half of the jaw): 3/3, 3/4 or 4/4 . © 2015 Gleisy S. Avelino et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Francisco River Basin, and two species were reported for both basins. Britski et al.  described a new species for the La Plata Basin (L. piavussu), recognized that L. elongatus is endemic to the Rio Jequitinhonha and Rio Pardo (coastal rivers) and recognize L. aguapeiensis and L. silvestrii as junior synonyms of L. obtusidens. Considering the previous hypothesis of closer relationships between the fish fauna of the São Francisco and La Plata basins (including the Parana, Paraguay and Uruguay rivers) , the widespread distribution of Leporinus in these basins [1,10] and in the south portion of South America and the economical importance of species of this genus, in the present study, partial sequences of the mitochondrial genes 16S rRNA, Cytochrome B (CytB) and Cytochrome c Oxidase I (COI) and of the nuclear gene -Tropomyosin (Trop) from 19 species of Leporinus were used to test the hypothesis that the current morphological recognized species in south portion of South America basins can be correctly identified using DNA markers and to test the similarities between species from São Francisco and La Plata basins. 2 Material and Methods Eighteen species of Leporinus (73 samples) were used, out of which 40 samples were from the Parana River Basin, 9 samples from the Paraguay River Basin, 1 sample from the Uruguay River Basin, 12 samples from the São Francisco River Basin and 8 samples from the coastal Brazilian rivers. Additionally 1 sample from the Amazonas River Basin and 2 samples from the Orinoco River Basin were analyzed as comparative material. Species, collecting sites, and museum collection numbers are shown in Table 1. Figure 1 shows the collecting sites of the samples obtained from the south part of South America. One species, from rio Araguaia, could not be positively identified and was named Leporinus sp. and one species was identified as L. aff. paranensis because it differs from L. paranensis in the number of series of peduncule scales (12 vs. 16). Tissue samples were taken from all specimens investigated for DNA isolation (preserved in 95% ethanol) and the specimens were fixed in 10% formaldehyde and preserved in 70% ethanol. The animals studied are Table 1. Specimens sequenced in the present study and Genbank sequence numbers. Voucher specimens are deposited in the fish collection of Laboratório de Biologia e Genética de Peixes (LBP), Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil. Species Leporinus amblyrhynchus LBP 3505 Locality Rio Itararé/Rio Paranapanema/ Fartura/SP/S 23°24'44.9'' W 49°34'15.4'' Reservatório de Chavantes/Rio Paranapanema/Chavantes/SP/S 23°08'01.1'' W 49°40'34.4'' Rio Capivari/Rui Barbosa/BA/S 12°10'45''S 40°24'16''W Lagoa Feia/Rio Paraíba do Sul/ Leste/Campos dos Goytacazes/ RJ/S 22°00' W 41°20' Rio Orinoco/Rio Orinoco/Caicara del Orinoco/Bolívar/Venezuela/N 07°38'11.6'' W 66°19'04.2'' Região de Rombado, tributary of the rio Pirai/Poconé/MT/S 16°25'40.8'' W 56°25'08.58'' Reservatório de Jurumirim/Rio Paranapanema/Itatinga/SP/S 23°20' W 48°34' Rio Tietê/Rio do Peixe/Rio Paraná/Bofete/SP/S 22°46'29.9'' W 48°08'43.5'' Região de Rombado, tributary of rio Pirai/Poconé/MT/S 16°25'40.8' W 56°25'08.6'' Code PA2 Specimen Genes number 20121 16S EU181625 Cyt b EU183041 COI EU185541 Trop EU181641 Leporinus bahiensis Leporinus copelandii Leporinus cf. fasciatus Leporinus friderici PA2 PA2 PA2 BA EC2 EU181626 EU181627 EU181628 EU183046 EU183045 EU185542 EU185543 EU185544 EU181642 EU181644 EU181643 EU181599 EU183015 EU185593 EU181688 OR OR PY1 EU181613 EU181614 EU181574 EU183027 EU182993 EU185598 EU185599 EU185563 EU181659 PA2 EU181602 EU183019 EU185551 EU181677 Leporinus lacustris PA3 PA3 PY1 EU181638 EU181598 EU181573 EU183013 EU183014 EU182992 EU185566 EU185562 EU181658 contiuned Table 1. Specimens sequenced in the present study and Genbank sequence numbers. Voucher specimens are deposited in the fish collection of Laboratório de Biologia e Genética de Peixes (LBP), Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil. LBP 1422 Locality Rio Taquari/Rio Paraguai/Coxim/ MS S 18°25'42.5'' W 54°50'02.8'' Rio Montividiu/Rio Paraná/ Montividiu/GO/S 17°26'25.8'' W 51°10'26.7'' Rio São Francisco/Três Marias/MG/S 18°11'28.5'' W 45°14'51.42'' Rio São Francisco/Três Marias/ MG/S 18°11'21.0' W 45°15'10.3'' Code PY3 PY3 PY3 PA5 Specimen Genes 12505 19492 19493 16370 EU181590 EU181612 EU181634 EU183005 EU183039 EU183040 EU183016 EU185582 EU185583 EU185584 EU185585 EU181664 EU181662 EU181663 - Species Leporinus macrocephalus Leporinus 2491 microphthalmus Leporinus obtusidens 250 SF1 SF1 SF1 EU181567 EU181568 EU182984 EU182985 EU185545 EU185546 EU181648 EU181646 SF1 Rio Tietê/Rio Paraná/Botucatu/ PA3 SP/S 22°37'55.7'' W 48°10'30.2'' Rio Tietê/Rio Paraná/Botucatu/ PA3 SP/S 22°37'55.7'' W 48°10'30.2'' PA3 PA3 PA3 Rio Paraná/Rio Paraguai/Ayolas/ PA1 Misiones/Paraguai/S 27°23' W PA1 56°53' Lagoas Marginais Rio Ibicuí/ UY Rio Uruguai/Uruguaiana/RS/S 29°24'00'' W 56°42'00'' Reservatório de Jurumirim/Rio PA2 Paranapanema/Itatinga/SP/S 23°20' W 48°34' Lagoa dos Patos/Porto Alegre/ EC1 RS/S 29°59'11''S 51°16'17''W EC1 EC1 EC1 Rio Paraná/Rio Paraguai/Ayolas/ PA1 Misiones/Paraguai/S 27°23' W PA1 56°53' PA1 PA1 PA1 Reservatório de Jurumirim/Rio PA2 Paranapanema/Itatinga/SP/S 23°20' W 48°34' Rio Cuiabá/Rio Paraguai/Santo PY2 Antônio do Leverger/MT/S PY2 15°52'07.7' W 56°06'14.6'' PY2 PY2 Rio da Prata/Entre Rios/ PA7 Argentina/S 33°43'49.4'' W 59°25'42.4'' PA7 PA7 PA7 PA7 EU181596 EU181611 EU181620 EU181622 EU181624 EU181582 EU181588 EU181606 EU183011 EU183026 EU183034 EU183036 EU183038 EU182997 EU183003 EU183022 EU185561 EU185556 EU185558 EU185559 EU185560 EU185548 EU185549 - EU181652 EU181650 EU181654 EU181649 EU181647 EU181657 EU181655 - EU181601 EU183018 EU185550 EU181651 EU181583 EU181584 EU181585 EU181587 EU181589 EU181579 EU182998 EU182999 EU183000 EU183002 EU183004 EU183049 EU185579 EU185580 EU185572 EU185581 EU185574 EU185569 EU181675 EU181670 - EU181607 EU181608 EU181609 EU181610 EU183023 EU183024 EU183025 EU185552 EU185553 EU185554 EU185555 EU181685 EU181656 EU181686 EU181687 contiuned Table 1. Specimens sequenced in the present study and Genbank sequence numbers. Voucher specimens are deposited in the fish collection of Laboratório de Biologia e Genética de Peixes (LBP), Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil. Species Leporinus piavussu LBP 2706 3303 Locality Code Specimen Genes 15587 19851 19854 19848 19827 19828 21936 EU181597 EU181621 EU181623 EU181619 EU181615 EU181616 EU181635 EU183012 EU183035 EU183037 EU183033 EU183028 EU183029 EU183042 EU185575 EU185577 EU185578 EU185557 EU185586 EU185587 EU185588 EU181666 EU181665 EU181653 EU181676 EU181681 Rio Tietê/Rio Paraná/Botucatu/ PA3 SP/S 22°37'55.7'' W 48°10'30.2'' Rio Tietê/Rio Paraná/Botucatu/ PA3 SP/S 22°37'55.7'' W 48°10'30.2'' PA3 PA3 PA2 PA2 PA2 Leporinus octofasciatus Leporinus aff. paranensis 17722 Rio Tietê/Rio Paraná/Botucatu/ SP/S 22°37'55.7'' W 48°10'30.2'' 3939 Rio Paranapanema/Rio Paraná/ Chavantes/SP/S 23°08'01.1'' W 49°40'34.9 3808 Rio Novo/ Rio Paranapanema/ Avaré/SP/S 23°01'26.2' W 48°49'32.6'' Leporinus paranensis Leporinus piau Rio Formoso/Mineiros/GO/S 18°15'40.3' W 52°53'00.1'' Represa de Três Marias/Três Marias/MG/S 18°13'39.61'' W 45°14'51.4'' Rio Picão/Rio São Francisco/ Abaeté/MG/S 19°35,361' W 45°18,006' Represa de Três Marias/Três Marias/MG/S 18°13'39.61'' W 45°14'51.4'' Rio Mucuri/Carlos Chagas/MG/S 17°41'42.4' W 40°46'11.3'' Rio Taquari - Pesqueiro Recanto Alegre/Rio Paraguai/Coxim/MS S 18°25'42.5'' W 54°50'02.8'' Reservatório de Jurumirim/ Paranapanema/SP/S 23°20' W 48°34' Tributary of córrego Fogaça/Rio Araguaia/Barra do Garças/MT/ S 15°40'53.9'' W 52°13'21.4'' Represa de Três Marias/Três Marias/MG/S 18°13'39.61'' W 45°14'51.4'' Ribeirão Santo Inácio/Rio São Francisco/Moema/MG S 19°52'39.2'' W 45°26'04.62'' PA2 PA2 PA6 PA6 SF1 SF1 SF2 EU181629 EU181630 EU183043 EU183044 EU185589 - EU181678 EU181679 EU181563 EU181564 HM015214 EU182981 EU182980 HM015216 EU185590 HM015215 EU181682 HM015217 Leporinus reinhardti Leporinus steindachneri Leporinus striatus SF1 SF1 EC3 EC3 PY3 EU181562 EU181565 EU182979 EU182982 EU185592 EU185591 EU181684 EU181683 EU181591 EU183006 EU185595 EU181691 3180 2736 Leporinus sp. 1805 PA2 PA2 AM EU181600 EU181636 EU181594 EU183017 EU183020 EU183008 EU185596 EU185594 EU185600 EU181690 EU181689 EU181692 Leporinus taeniatus SF1 EU181566 EU182983 EU185597 EU181693 SF3 SF3 EU181631 EU181632 EU182986 EU182989 EU181694 EU181695 1- Animal deposited in the Museu de Zoologia da USP (MZUSP 88618). 2 Animal deposited in the Museu de Ciências da PUC (MCP 28917). deposited in the fish collection of the Laboratory of Biology and Genetics of Fish (LBP), Institute of Biosciences, Paulista State University, Botucatu, São Paulo, Brazil or in the Museu de Ciências da Pontifícia Universidade Católica, Porto Alegre, Rio Grande do Sul, Brazil. DNA was extracted with the Phenol technique based on the protocol of Sambrook & Russell  and with the extraction buffer technique based on the protocol of Aljanabi & Martinez . Partial sequences of the mitochondrial 16S rRNA genes, Cytochrome B, Cytochrome Oxidase I and nuclear Fig. 1. Map showing the collection points in the Southern area of South America. Paraná River (blue spots): Ayolas (PA1), Paranapanema (PA2), Tiete (PA3), Mogi-Guaçu (PA4), Montividiu (PA5), Mineiros (PA6) and Entre Ríos (PA7); Paraguay River (orange spots): Poconé (PY1), Cuiabá (PY2) and Taquari (PY3); Uruguay River (yellow spot - UY1); São Francisco River (red spots): Três Marias dam (SF1) and Picão (SF2); Weastern Coastal Region: Jacuí (EC1 - pink spot), Paraiba do Sul (EC2 - light green spot) and Carlos Chagas (EC3 - dark green spot). -Tropomyosin gene were amplified by polymerase chain reactions (PCR). The primers used are listed in Table 2. The amplification was performed in a total volume of 25.0 µL of a solution containing 16.5 µL of distilled water, 2.5 µL dNTP (8 mm), 2.5 µL buffer 10x, 1.2 µL of each primer (10 M) and 0.1 µL DNA Polymerase (1 unit), 1.0 µL of DNA sample and from 0.5 to 1.0 µL MgCl2, when required. The reaction conditions for PCR primers were: 16S (30 s at 95°C, 45 s at 50°C, 45 s at 68°C and 420 s at 68°C), CytB (30 s at 95°C, 45 s at 48-50°C, 120 s at 68°C and 420 s at 68°C), COI (30 s at 95°C, 45 s at 48-50°C, 45 s at 68 and 72°C and 420 s at 68 and 72°C) and Trop (30 s at 95°C, 45 s at 55°C, 90 s at 72°C and 420 s at 72°C), all the conditions with 30 cycles. The PCR products were identified on 1% agarose gel. The DNA was purified using the PCR Kit GFXTM Gel Band The sequences were aligned using the ClustalW program  implemented in DAMBE version 5.2.14  and BioEdit  programs. The range and pattern of nucleotide substitution and genetic distance were examined using the MEGA 5.05 . The saturation of nucleotides was analyzed by plotting the absolute number of transitions (Ti) and transversions (Tv) Purification (Amersham Biosciences) and a salt extraction method based on the protocol used by Travis Glenn  and with the ExoSAP-IT kit. The sequencing reaction was performed with the Amershan Bioscience, DYEnamic Terminator kit. The sequences were determined in an automated ABI PRISMTM 377 DNA Sequencer (PerkingElmer). All sequences were read at least twice (forward and reverse). Table 2. Primers used for PCR and sequencing. Gene 16S F 16S R Cyt b L 14841 Cyt b H 15915 Fish F1 Fish R1 TROP F TROP R Sequences 5´ - ACG CCT GTT TAT CAA AAA CAT - 3´ 5´ - CCG GTC TGA ACT CAG ATC ACG T - 3´ 5´ - AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA - 3´ 5´ - AAC TGC CAG TCA TCT CCG GTT TAC AAG AC - 3´ 5´ - TCA ACC AAC CAC AAA GAC ATT GGC AC - 3´ 5´ - TAG ACT TCT GGG TGG CCA AAG GAA TCA - 3´ 5´ - CCA CTG CCC TGC AGA AGC TGG AGG A - 3´ 5´ - CTC CTC AGT ACG CTC CAG CTC ACC CTC A - 3´ References Kocher et al. (1989)  Kocher et al. (1989)  Ward et al. (2005)  Calcagnotto et al. (2005)  Present study versus the values of genetic distance using the program DAMBE version 5.2.14 . The program Modeltest  was used to select the nucleotide substitution model that best fits the data obtained. RAxML  using the web servers RAxML BlackBox  was used for all maximum likelihood analyses using a mixed partition model. Random starting trees were used for each independent ML tree search and all other parameters were set on default values. All analyses were conducted under GTR+G since RAxML only applies this model . Topological robustness was investigated using 1000 non-parametric bootstrap replicates. A set of four partitioning schemes ranging from 1 to 8 partitions was tested following the procedures outlined by Li et al.  under the AIC and BIC criteria. In the first, test we included all the dataset (1 partition), in the second test, all genes were analyzed separately (4 partitions: 16S, CytB, COI, Trop), in the third test, the 16S and Trop were separated and the first, second, and third position of CytB and COI were analyzed together (5 partitions), and in the fourth test, the 16S, Trop and the first, second, and third position of CytB and COI were analyzed separately (8 partitions). Maximum parsimony (MP) analyses were conducted with PAUP* 4.0b10 . Heuristic searches were performed using 1000 random addition replicates and TBR branch swapping. All characters were unordered, all character transformations were equally weighted, and branches with maximum length of zero were collapsed. Gaps were treated as missing data. Clade robustness was assessed using 1000 bootstrap pseudoreplicates  with the same parameters as above. 3 Results The sequences obtained in this study were deposited in the GenBank (Table 1). We obtained sequences of the mitochondrial 16S rRNA gene of 73 specimens (100%), Cytochrome B of 65 specimens (89%), Cytochrome c Oxidase I of 73 specimens (100%), and the gene -Tropomyosin of 54 specimens (72%). After the alignment procedure and manual correction of alignment, we obtained a matrix with 2582 characters, out of which 1578 were conserved, 992 variable, and 728 informative for parsimony analysis. The proportion transition/transversion (Ti/Tv) rate was 2.68. The average composition, in percentage of bases was adenine (A) 26.9%, cytosine (C) 26.4%, guanine (G) 19.4%, and thymine (T) 27.3%. A Chi-square test of heterogeneity of nucleotide frequencies among OTUs with Yates correction (performed in DAMBE) showed no significant values. Base composition was computed for all taxa in the concatenated alignment excluding constant sites to gauge the effect of possible base compositional bias on the resulting phylogeny. A graphical analysis of the relationship between transitions (Ti) and transversions (Tv) and genetic distance estimated by the GTR distance model indicates that there is no saturation of these nucleotides (data not shown). The overall average distance between sequences was d = 0.090 ± 0.004, according the Kimura-2-parameter substitution model . Among species with more than one specimen available the genetic distance ranged from d = 0.000 ± 0.000 in Leporinus paranensis to d = 0.041 ± 0.002 in L. obtusidens (Table 3). Genetic distance among species pairs ranged from d = 0.008 ± 0.002 from L. octofasciatus and L. paranensis to d = 0.151 ± 0.009 between L. copelandii and L. cf. fasciatus (Table 3). Four different partitioning schemes ranging from one to 8 partitions were tested to establish the optimal number of data partitions (following Li et al. ) for the final analysis. The results showed that the 8 partition model was the best choice (data not shown); however, ML analysis conducted with the other partitioning schemes resulted in the same final topology, with minor differences in branch length and support values (not shown). Throughout the text and in the figures, measures of support are indicated as a series of two numbers on selected internal branches of the trees, starting with Table 3. K2P genetic distance obtained among the species of Leporinus. Mean below diagonal and standard error above diagonal. In the diagonal (in bold) the mean value and standard error for each species. n/c = not computed. 3 0.004 0.007 n/c 0.008 0.030 ± 0.009 n/c 0.007 0.001 ± 0.004 0.012 ± 0.008 0.010 ± 0.009 n/c 0.008 0.041 ± 0.008 0.014 ± 0.003 0.026 0.072 0.128 0.113 0.101 0.133 0.128 0.030 0.080 0.110 0.043 0.130 0.119 0.107 0.146 0.061 0.113 0.112 0.111 0.046 0.097 0.123 0.081 0.083 0.123 0.128 0.103 0.103 0.101 0.122 0.131 0.124 0.118 0.133 0.140 0.115 0.118 0.107 0.129 0.123 0.113 0.128 0.122 0.110 0.110 0.123 0.024 0.044 0.125 0.119 0.117 0.045 0.072 0.112 0.125 0.109 0.107 0.097 0.105 0.042 0.042 0.124 0.081 0.092 0.008 0.068 0.128 0.113 0.095 0.125 0.125 0.043 0.000 ± 0.000 0.052 0.092 0.094 0.072 0.098 0.104 0.022 0.011 ± 0.002 0.122 0.107 0.093 0.120 0.115 0.054 0.028 ± 0.003 0.084 0.115 0.132 0.116 0.116 0.038 ± 0.005 0.112 0.130 0.109 0.104 n/c 0.112 0.109 0.087 0.007 0.002 ± 0.001 0.114 0.107 0.024 ± 0.003 0.100 0.005 ± 0.001 0.007 0.007 0.007 0.007 0.008 0.008 0.008 0.007 0.007 0.005 0.007 0.008 0.007 0.008 0.008 0.007 0.007 0.007 0.007 0.005 0.007 0.009 0.010 0.010 0.010 0.010 0.004 0.002 0.047 0.110 0.125 0.057 0.060 0.138 0.112 0.121 0.002 0.005 0.009 0.007 0.007 0.008 0.008 0.005 0.009 0.008 0.009 0.125 0.008 0.007 0.006 0.008 0.012 0.008 0.002 0.106 0.125 0.128 0.131 0.110 0.113 0.103 0.118 0.139 0.104 0.102 0.142 0.008 0.003 0.008 0.005 0.008 0.007 0.008 0.007 0.009 0.007 0.008 0.007 0.006 0.007 0.007 0.005 0.006 0.004 0.007 0.003 0.143 0.145 0.139 0.121 0.126 0.007 0.008 0.008 0.008 0.008 0.008 0.007 0.007 0.005 0.006 0.003 0.008 0.001 0.059 0.092 0.117 0.035 0.007 0.006 0.007 0.007 0.004 0.007 0.007 0.008 0.009 0.008 0.009 0.011 0.008 0.008 0.008 0.116 0.007 0.006 0.007 0.007 0.010 0.009 0.008 0.008 0.010 0.008 0.008 0.009 0.008 0.005 0.135 0.059 0.100 0.151 0.006 0.007 0.007 0.007 0.008 0.004 0.009 0.005 0.005 0.009 0.008 0.008 0.005 0.005 0.006 0.009 0.008 0.007 0.103 0.009 0.009 0.008 0.008 0.006 0.006 0.007 0.007 0.006 0.007 0.009 0.006 0.007 0.007 0.007 0.007 0.007 0.008 0.009 0.008 0.008 0.004 0.005 0.008 0.008 0.007 0.004 0.004 0.005 0.008 0.007 0.006 0.008 0.007 `14 1 Leporinus aff 0.003 ± paranensis 2 Leporinus 0.014 ± amblyrhynchus 3 Leporinus bahiensis 4 Leporinus cf fasciatus 5 Leporinus copelandii 0.118 6 Leporinus friderici 7 Leporinus lacustris 8 Leporinus macrocephalus 9 Leporinus microphthalmus 10 Leporinus obtusidens 11 Leporinus octofasciatus 12 Leporinus paranensis 13 Leporinus piau 14 Leporinus piavussu 15 Leporinus reinhardti 16 Leporinus sp. 17 Leporinus steindachneri 18 Leporinus striatus 19 Leporinus taeniatus 0.029 non-parametric bootstrap percentages from ML and MP analyses, respectively (e.g. 100/100, see Fig. 2). The general tree topology observed in all analyses was very similar, although statistical support was not strong for some nodes. The final topology obtained by ML analysis will be used to discuss relationships among taxa (Figs. 2-3), but important differences with results obtained by ML and MP analyses will be discussed in the text. A general analysis of the genetic distance among specimens among species with more than one specimen available showed that the genetic distance was low (less than 2%) in ten species (Leporinus paranensis, L. friderici, L. aff. paranensis, L. taeniatus, L. macrocephalus, L. piau, L. lacustris, L. octofasciatus, L. steindachneri and L. amblyrhynchus - Table 3) and high (more than 2%) in five species (L. striatus, L. piavussu, L. cf. fasciatus, L. Fig. 2. Partial maximum likelihood (ML) tree showing relationships among some species of Leporinus obtained by a partitioned analysis of the concatenated dataset. A series of two numbers (e.g., 100/100) at each of the main nodes represents the percentage of bootstrap support (1000 bootstrap replicates) obtained by ML and maximum parsimony (MP) analysis, respectively. Asterisks represents a node that was not obtained by MP analyses. Blue lines represent the Parana River samples, orange lines represent the Paraguay River samples, red lines represent the São Francisco River samples, black lines represent the Amazon River and Orinoco River samples, and the pink line represents the Western Coastal River sample. Fig. 3. Partial maximum likelihood (ML) tree showing relationships among some species of Leporinus obtained by a partitioned analysis of the concatenated dataset (please check Fig. 2). A series of two numbers (e.g., 100/100) at each of the main nodes represents the percentage of bootstrap support (1000 bootstrap replicates) obtained by ML and maximum parsimony (MP) analysis, respectively. Asterisks represents a node that was not obtained by MP analyses. Blue lines represent the Parana River samples, orange lines represent the Paraguay River samples, red lines represent the São Francisco River samples, black lines represent the Amazon River and Orinoco River samples, and the pink line represents the Western Coastal River sample. reinhardti and L. obtusidens -Table 3). This difference may be explained, in part by the sample distribution since some samples of L. striatus and L. obtusidens were collect in far points in their distribution area (Table 1). However, the samples of L. piavussu, L. cf. fasciatus and L. reinhardti were collected in the same geographical area and the observed genetic difference may be an intrinsic characteristic of these species. Studies involving molecular identification of species have reported several cases of deep intra-specific divergence [26-31] and in many of them this diversity has been associated to the existence of cryptic species. Carefully studies involving other genetic markers and populational sampling should be done in the future to better check this genetic variability. Genetic distance was computed among 153 species pairs and only in two cases the values observed were less than 2%: between L. paranensis and L. octofasciatus (d = 0.008 ± 0.002) and between L. paranensis and L. aff. paranensis (d = 0.018 ± 0.004) (Table 3). The comparison of the sequences of L. paranensis and L. octofasciatus shows that only four position are different (diagnostic), all in the COI gene. The comparison of the sequences of L. paranensis and L. aff. paranensis shows that 23 position were different (diagnostic), 18 in the COI gene and 5 in the 16S gene. Several molecular identification studies have suggested the use of a nucleotide diagnostic approach as an alternative to distance approach to identify species [30-34]. Thus, considering the morphological differences among the above cited species we believe that the small genetic differences among them may be result of recent speciation. The general distance between L. obtusidens and L. piavussu was d = 0.045 ± 0.003 (Table 3). However, in all the trees obtained the species L. piavussu was found among the specimens of L. obtusidens (Fig. 3). A close analyses of Figure 3 shows that the samples of L. obtusidens can be separated in four groups, identified here as Group A to Group D. Two of these groups are geographically distinct: the Group B with specimens from São Francisco, and the Group C with specimens from Rio Cuiabá (Table 1, Fig. 1). The other two are more widely distributed: the Group A with specimens in the Paraná, Uruguay and Lagoa dos Patos and Group D with specimens in the Paraná and Paraguay rivers (Table 1, Fig. 1). The genetic distance among these groups ranges from d = 0.033 ± 0.003 between Group A and Group B to d = 0.061 ± 0.004 between Group A and Group C (Table 4). As discussed above this high genetic diversity may be associated to the existence of cryptic species and thus additional studies (involving different markers as genetic and molecular markers) should be conducted in this species. 4 Discussion Analyzing the genetic similarities among species studied we found that the samples from the Amazon Basin (Leporinus sp.) and one species from the Orinoco River Basin (Leporinus cf. fasciatus) are more similar between themselves than with other species (Figs. 2). It should be emphasized that Leporinus sp. showed a great morphological and meristic similarity with L. obtusidens, but could not be identified at species level. The species L. lacustris, L. friderici, and L. piau formed a well supported group being L. friderici more similar to L. piau (Fig. 2). All these three species fit the color pattern 3 of Garavello & Britski , and have four teeth in both jaws. A second group close related to the cited above is composed of L. bahiensis, L. taeniatus, L. octofasciatus, L. paranensis and L. aff. paranensis (Fig. 2). The species L. bahiensis was included in the present study due its deep morphological similarity with L. aff. paranensis (Britski, personal observation). The results show that these two last species are not particularly genetically similar and L. bahiensis is more similar to L. taeniatus (Fig. 2). Although all species in this group have the same number of teeth in both jaws, three teeth in the premaxillary and four teeth in the dentary, they have a different color pattern; L. taeniatus has a color pattern 2, L. octofasciatus has a color pattern 1, and L. paranensis, L. aff. paranensis and L. bahiensis have a color pattern 3, according to Garavello & Britski  classification. Leporinus microphthalmus appears genetically more similar to L. amblyrhynchus (Fig. 2). L. microphthalmus has three teeth in the premaxilla and four in the dentary and L. amblyrhynchus has three teeth in both bones. Moreover, L. amblyrhynchus has an intermediate color pattern between Table 4. K2P genetic distance obtained among the groups of Leporinus obtusidens and L. piavussu. Mean below diagonal and standard error above diagonal. In the diagonal (in bold) the mean value and standard error for each species. 1 1 L. obtusidens A 2 L. obtusidens B 3 L. obtusidens C 4 L. obtusidens D 5 L. piavussu 0.017 ± 0.001 0.033 0.061 0.051 0.051 2 0.003 0.007 ± 0.001 0.058 0.050 0.051 3 0.004 0.004 0.041 ± 0.003 0.055 0.059 4 0.004 0.004 0.004 0.018 ± 0.002 0.029 5 0.004 0.004 0.004 0.003 0.028 ± 0.003 the barred and striped pattern, an uncommon pattern among species of Leporinus . A species resembling L. amblyrhynchus, in color pattern, is L. taeniatus, a species that occurs in the basin of the São Francisco, however, this species differs from L. amblyrhynchus for having 16 circumpeduncular scale series (12 in L. amblyrhynchus) and four teeth in each dentary (three in L. amblyrhynchus) . In the tree obtained in this work, L. amblyrhynchus and L. taeniatus do not stick together, so the grouping by color pattern was not confirmed in this case. Leporinus steindachneri, L. striatus, L. macrocephalus, and L. reinhardti appear successively as genetically more similar to L. obtusidens + L. piavussu (Figs. 2 - 3). L. steindachneri has four teeth in the premaxilla and in the dentary and has a color pattern 3; L. striatus has three teeth in the premaxillary and four teeth in the dentary and has a color pattern 2; L. macrocephalus, L. reinhardti, L. obtusidens and L. piavussu have three teeth in both jaws and a color pattern 3 according to Garavello & Britski  and Britski et al. . An interesting finding of our study is that the genetic similarities observed among species do not corroborate the division of species of Leporinus in groups according to color patterns. For example, L. striatus, a species that fits the color pattern 2 of Garavello & Britski  is closely related to L. steindachneri, a species that fits the color pattern 3 of the same authors. Additionally, regarding the number of teeth, L striatus has three teeth in the premaxillary and four in the dentary and L. steindachneri has four teeth in both bones, reinforcing the hypothesis that these characters may be also not phylogenetically informative. Another example are the species L. octofasciatus and Leporinus cf. fasciatus which, despite having different teeth (L. octofasciatus with three teeth in the premaxillary and four in the dentary and Leporinus cf. fasciatus with four teeth in both jaws), fall into the same color pattern 1, but are not together in the tree (Fig. 2). On the other hand, an example of clustering in the phylogeny, which is in accordance with the division by groups with similar color pattern and dentition, is the group formed by the species L. lacustris, L. friderici, and L. piau. They all fit in the color pattern 3 and have four teeth in both jaws . The most important result obtained in our analysis is that the species of Leporinus from the La Plata and São Francisco are not more similar among themselves than to species of other drainages in the south part of South America. Two main hypotheses could explain this result: (1) the genus could be very old, and speciation could have precluded these basins formation; (2) the occurrence of recent fauna exchanges between these two basins could give origin to recent `species-pairs' in these basins. Considering that the main formation of these basins is a very ancient event dating back to the Jurassic or Cretaceous ages , before or close to the separation between Africa and South America, the first hypotheses does not seem probable, since we do not have any sign of the presence of these fishes in Africa. On the other hand, the second hypothesis is sustainable by strong evidences of recent reactivation of Precambrian shear zones from the Miocene to the present day [36,37]. A particularly clear example of this type of exchange was documented by Ribeiro et al.  in the Upper Rio Guaratuba, which originally flowed toward the Paraná River and became a coastal river due to the Quaternary activity of NW-trending faults. The occurrence of several `species-pairs' in our tree, such as L. taeniatus (São Francisco) and L. octofasciatus and L. paranensis (La Plata); L. piau (São Francisco) and L. friderici (La Plata); and L. reinhardti (São Francisco) and L. obtusidens (La Plata, São Francisco and other South America drainages) may represent a good example of the recent connection between the La Plata and São Francisco basins. Based on these data we strongly suggest that these species-pairs should be deeply analyzed to confirm their taxonomic status (one or two species). Acknowledgements: We thank all the individuals who assisted us in the collection and identification of the specimens that served as the basis for this study and to Museu de Ciências e Tecnologia da Pontifícia Universidade Católica do Rio Grande do Sul and Museu de Zoologia of Universidade de São Paulo for the donation of fish tissues. Fundação de Apoio à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil provided funds to the development of the present study. Conflict if interest: Dr Avelino delcares nothing to disclose.
DNA Barcodes – de Gruyter
Published: Jan 1, 2015
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