Molecular Identification of Leporinus from the South Portion of South America

DNA Barcodes 2015; Volume 3: 98–109

Research Article Open Access

Gleisy S. Avelino*, Heraldo A. Britski, Fausto Foresti, Claudio Oliveira Molecular identification of Leporinus from the south portion of South America

DOI 10.1515/dna-2015-0013 Received January 30, 2015; accepted July 6, 2015 1 Introduction

Abstract: Anostomidae is a widely distributed group Anostomidae is a family of freshwater fishes from South that includes the species rich Leporinus. This genus America that ranges from the Atrato River, western is widespread in South America, including its south Colombia, down south to central portions of Argentina portion where they are mainly distributed in the São [1]. A hypothesis of phylogenetic relationships within Francisco and La Plata basins. Here we obtained the Anostomidae was recently proposed by Sidlauskas & sequences of mitochondrial and nuclear genes of 19 Vari [2], who recognize that the family includes fourteen species of Leporinus with the objective of testing our genera. Leporinus is the largest genus of Anostomidae methods of molecular species identification and to both in number of species and number of individuals test the similarities between species of Leporinus of in the rivers where they occur [1,3]; because of this, the São Francisco and La Plata basins. Results show several attempts were made trying to divide the species that two species pair have a genetic distance below of Leporinus in smaller groups. Fowler [4] created the 2% (L. paranensis x L. obtusidens and L. paranensis x subgenus Myocharax, based on the dentition presented L. aff. paranensis). Our results also show that a high by Leporinus desmotes, which shows a very long pair genetic distance occurs in L. obtusidens and L. striatus of symphyseal teeth. Borodin [5] attempted to divide suggesting a possible local differentiation of these the genus Leporinus in two subgenera and created species. An interesting finding of our study is that the subgenus Hypomasticus that included species the genetic similarities observed among species does with a lower mouth. Another subgenus, Leporinops, not corroborate the division of species of Leporinus in was created by Géry [6], based on tooth and cranial groups according to color patterns. Additionally, our differences. This subgenus was based on Leporinus results show that the species of Leporinus from the La moralesi [7]. All these proposals were not accept by Plata and São Francisco are not more similar among Garavello & Britski [1] in the catalog of species of the themselves than to species of other drainages which family but Hyposmastictus was recognized as valid by could be explained by the occurrence of recent fauna Sidlauskas & Vari [2]. exchanges between these two basins and adjacent Garavello & Britski [8], in a paper describing Leporinus basins. amblyrhynchus and L. paranensis from the upper Paraná Basin, showed that most species of the genus may be Keywords: Phylogeny, Molecular systematics, placed into three artificial groups based on the color Sequencing, Mitochondrial DNA, Fish. 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 *Corresponding author: Gleisy S. Avelino, Departamento de used in identification keys. With the exception of the Morfologia, Instituto de Biociências, Universidade Estadual Paulista recently described species Leporinus venerei [9], which “Júlio de Mesquita Filho” (UNESP), Caixa Postal 510, Rubião Júnior, has the dental formula 4/3, unique among species of s/n, 18618-970, Botucatu, SP, Brazil, E-mail: [email protected] the genus, all species are artificially placed into three Fausto Foresti, Claudio Oliveira, Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista “Júlio de groups according to the dental formula (number of Mesquita Filho” (UNESP), Caixa Postal 510, Rubião Júnior, s/n, tooth in premaxillary/dentary in each half of the jaw): 18618-970, Botucatu, SP, Brazil 3/3, 3/4 or 4/4 [9]. Heraldo A. Britski, Museu de Zoologia da Universidade de São Pau- Garavello & Britski [1] recognized nine species of lo, Caixa Postal 42494, 04299-970, São Paulo, SP, Brazil Leporinus in the La Plata Basin, four species in the São

© 2015 Gleisy S. Avelino et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. - 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 99

Francisco River Basin, and two species were reported for 2 Material and Methods both basins. Britski et al. [10] described a new species for the La Plata Basin (L. piavussu), recognized that L. Eighteen species of Leporinus (73 samples) were used, out elongatus is endemic to the Rio Jequitinhonha and Rio of which 40 samples were from the Parana River Basin, 9 Pardo (coastal rivers) and recognize L. aguapeiensis and L. samples from the Paraguay River Basin, 1 sample from the silvestrii as junior synonyms of L. obtusidens. Uruguay River Basin, 12 samples from the São Francisco Considering the previous hypothesis of closer River Basin and 8 samples from the coastal Brazilian rivers. relationships between the fish fauna of the São Francisco Additionally 1 sample from the Amazonas River Basin and and La Plata basins (including the Parana, Paraguay 2 samples from the Orinoco River Basin were analyzed and Uruguay rivers) [11], the widespread distribution of as comparative material. Species, collecting sites, and Leporinus in these basins [1,10] and in the south portion of museum collection numbers are shown in Table 1. Figure South America and the economical importance of species 1 shows the collecting sites of the samples obtained from of this genus, in the present study, partial sequences of the south part of South America. One species, from rio the mitochondrial genes 16S rRNA, Cytochrome B (CytB) Araguaia, could not be positively identified and was and Cytochrome c Oxidase I (COI) and of the nuclear gene named Leporinus sp. and one species was identified as α-Tropomyosin (Trop) from 19 species of Leporinus were L. aff. paranensis because it differs from L. paranensis in used to test the hypothesis that the current morphological the number of series of peduncule scales (12 vs. 16). recognized species in south portion of South America Tissue samples were taken from all specimens basins can be correctly identified using DNA markers and investigated for DNA isolation (preserved in 95% ethanol) to test the similarities between species from São Francisco and the specimens were fixed in 10% formaldehyde and La Plata basins. 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 LBP Locality Code Specimen Genes

number 16S Cyt b COI Trop Leporinus 3505 Rio Itararé/Rio Paranapanema/ PA2 20121 EU181625 EU183041 EU185541 EU181641 amblyrhynchus Fartura/SP/S 23°24’44.9’’ W 49°34’15.4’’ 3917 Reservatório de Chavantes/Rio PA2 21828 EU181626 EU183046 EU185542 EU181642 Paranapanema/Chavantes/SP/S PA2 21829 EU181627 - EU185543 EU181644 23°08’01.1’’ W 49°40’34.4’’ PA2 21830 EU181628 EU183045 EU185544 EU181643 Leporinus 45851 Rio Capivari/Rui Barbosa/BA/S BA bahiensis 12°10’45’’S 40°24’16’’W Leporinus 2380 Lagoa Feia/Rio Paraíba do Sul/ EC2 16074 EU181599 EU183015 EU185593 EU181688 copelandii Leste/Campos dos Goytacazes/ RJ/S 22°00’ W 41°20’ Leporinus cf. 3077 Rio Orinoco/Rio Orinoco/Caicara OR 19698 EU181613 EU183027 EU185598 - fasciatus del Orinoco/Bolívar/Venezuela/N OR 19699 EU181614 - EU185599 - 07°38’11.6’’ W 66°19’04.2’’ Leporinus 655 Região de Rombado, tributary PY1 8087 EU181574 EU182993 EU185563 EU181659 friderici of the rio Pirai/Poconé/MT/S 16°25’40.8’’ W 56°25’08.58’’ 3179 Reservatório de Jurumirim/Rio PA2 16873 EU181602 EU183019 EU185551 EU181677 Paranapanema/Itatinga/SP/S 23°20’ W 48°34’ Leporinus 2340 Rio Tietê/Rio do Peixe/Rio PA3 15940 EU181638 EU183013 - - lacustris Paraná/Bofete/SP/S 22°46’29.9’’ PA3 15941 EU181598 EU183014 EU185566 - W 48°08’43.5’’ 4058 Região de Rombado, tributary PY1 8086 EU181573 EU182992 EU185562 EU181658 of rio Pirai/Poconé/MT/S 16°25’40.8’ W 56°25’08.6’’

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access 100 G.S. Avelino, et al.

contiunedTable 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 LBP Locality Code Specimen Genes

Leporinus 1422 Rio Taquari/Rio Paraguai/Coxim/ PY3 12505 EU181590 EU183005 EU185582 EU181664 macrocephalus MS PY3 19492 - EU183039 EU185583 EU181662 S 18°25’42.5’’ W 54°50’02.8’’ PY3 19493 EU181612 EU183040 EU185584 EU181663 Leporinus 2491 Rio Montividiu/Rio Paraná/ PA5 16370 EU181634 EU183016 EU185585 - microphthalmus Montividiu/GO/S 17°26’25.8’’ W 51°10’26.7’’ Leporinus 250 Rio São Francisco/Três SF1 4211 EU181567 EU182984 EU185545 EU181648 obtusidens Marias/MG/S 18°11’28.5’’ W SF1 4212 EU181568 EU182985 EU185546 EU181646 45°14’51.42’’ 8091 Rio São Francisco/Três Marias/ SF1 37504 MG/S 18°11’21.0’ W 45°15’10.3’’ SF1 37505 2199 Rio Tietê/Rio Paraná/Botucatu/ PA3 15586 EU181596 EU183011 EU185561 EU181652 SP/S 22°37’55.7’’ W 48°10’30.2’’ 3304 Rio Tietê/Rio Paraná/Botucatu/ PA3 19470 EU181611 EU183026 EU185556 EU181650 SP/S 22°37’55.7’’ W 48°10’30.2’’ PA3 19849 EU181620 EU183034 EU185558 EU181654 PA3 19853 EU181622 EU183036 EU185559 EU181649 PA3 19855 EU181624 EU183038 EU185560 EU181647 2509 Rio Paraná/Rio Paraguai/Ayolas/ PA1 9917 EU181582 EU182997 EU185548 EU181657 Misiones/Paraguai/S 27°23’ W PA1 9939 EU181588 EU183003 EU185549 EU181655 56°53’ 28392 Lagoas Marginais Rio Ibicuí/ UY 17677 EU181606 EU183022 - - Rio Uruguai/Uruguaiana/RS/S 29°24’00’’ W 56°42’00’’ 3912 Reservatório de Jurumirim/Rio PA2 16872 EU181601 EU183018 EU185550 EU181651 Paranapanema/Itatinga/SP/S 23°20’ W 48°34’ 4715 Lagoa dos Patos/Porto Alegre/ EC1 24906 RS/S 29°59’11’’S 51°16’17’’W EC1 24908 EC1 24909 EC1 24910 2509 Rio Paraná/Rio Paraguai/Ayolas/ PA1 9923 EU181583 EU182998 EU185579 EU181675 Misiones/Paraguai/S 27°23’ W PA1 9932 EU181584 EU182999 EU185580 - 56°53’ PA1 9935 EU181585 EU183000 EU185572 - PA1 9938 EU181587 EU183002 EU185581 - PA1 9945 EU181589 EU183004 EU185574 EU181670 2506 Reservatório de Jurumirim/Rio PA2 9232 EU181579 EU183049 EU185569 - Paranapanema/Itatinga/SP/S 23°20’ W 48°34’ 2745 Rio Cuiabá/Rio Paraguai/Santo PY2 18008 EU181607 EU183023 EU185552 EU181685 Antônio do Leverger/MT/S PY2 18009 EU181608 EU183024 EU185553 EU181656 15°52’07.7’ W 56°06’14.6’’ PY2 18011 EU181609 - EU185554 EU181686 PY2 18012 EU181610 EU183025 EU185555 EU181687 5669 Rio da Prata/Entre Rios/ PA7 27701 Argentina/S 33°43’49.4’’ W 59°25’42.4’’ PA7 27702 PA7 27703 PA7 27704 PA7 27705

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 101

Species LBP Locality Code Specimen Genes

Leporinus 2706 Rio Tietê/Rio Paraná/Botucatu/ PA3 15587 EU181597 EU183012 EU185575 - piavussu SP/S 22°37’55.7’’ W 48°10’30.2’’ 3303 Rio Tietê/Rio Paraná/Botucatu/ PA3 19851 EU181621 EU183035 EU185577 EU181666 SP/S 22°37’55.7’’ W 48°10’30.2’’ PA3 19854 EU181623 EU183037 EU185578 EU181665 17722 Rio Tietê/Rio Paraná/Botucatu/ PA3 19848 EU181619 EU183033 EU185557 EU181653 SP/S 22°37’55.7’’ W 48°10’30.2’’ Leporinus 3939 Rio Paranapanema/Rio Paraná/ PA2 19827 EU181615 EU183028 EU185586 - octofasciatus Chavantes/SP/S 23°08’01.1’’ W PA2 19828 EU181616 EU183029 EU185587 EU181676 49°40’34.9 Leporinus aff. 3808 Rio Novo/ Rio Paranapanema/ PA2 21936 EU181635 EU183042 EU185588 EU181681 paranensis Avaré/SP/S 23°01’26.2’ W 48°49’32.6’’ PA2 21937 EU181629 EU183043 EU185589 EU181678 PA2 21939 EU181630 EU183044 - EU181679 Leporinus 9573 Rio Formoso/Mineiros/GO/S PA6 44902 paranensis 18°15’40.3’ W 52°53’00.1’’ PA6 44903 Leporinus piau 260 Represa de Três Marias/Três SF1 4163 EU181563 EU182981 - - Marias/MG/S 18°13’39.61’’ W SF1 4162 EU181564 EU182980 EU185590 EU181682 45°14’51.4’’ 340 Rio Picão/Rio São Francisco/ SF2 4309 HM015214 HM015216 HM015215 HM015217 Abaeté/MG/S 19°35,361’ W 45°18,006’ Leporinus 259 Represa de Três Marias/Três SF1 4156 EU181562 EU182979 EU185592 EU181684 reinhardti Marias/MG/S 18°13’39.61’’ W SF1 4163 EU181565 EU182982 EU185591 EU181683 45°14’51.4’’ Leporinus 8098 Rio Mucuri/Carlos Chagas/MG/S EC3 37533 steindachneri 17°41’42.4’ W 40°46’11.3’’ EC3 37536 Leporinus 1482 Rio Taquari - Pesqueiro Recanto PY3 12708 EU181591 EU183006 EU185595 EU181691 striatus Alegre/Rio Paraguai/Coxim/MS S 18°25’42.5’’ W 54°50’02.8’’ 3180 Reservatório de Jurumirim/ PA2 16871 EU181600 EU183017 EU185596 EU181690 2736 Paranapanema/SP/S 23°20’ W PA2 17483 EU181636 EU183020 EU185594 EU181689 48°34’ Leporinus sp. 1805 Tributary of córrego Fogaça/Rio AM 13060 EU181594 EU183008 EU185600 EU181692 Araguaia/Barra do Garças/MT/ S 15°40’53.9’’ W 52°13’21.4’’ Leporinus 261 Represa de Três Marias/Três SF1 4174 EU181566 EU182983 EU185597 EU181693 taeniatus Marias/MG/S 18°13’39.61’’ W 45°14’51.4’’ 329 Ribeirão Santo Inácio/Rio São SF3 4250 EU181631 EU182986 - EU181694 Francisco/Moema/MG SF3 4281 EU181632 EU182989 - EU181695 S 19°52’39.2’’ W 45°26’04.62’’

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 extracted with the Phenol technique based on the protocol and Genetics of Fish (LBP), Institute of Biosciences, of Sambrook & Russell [12] and with the extraction buffer Paulista State University, Botucatu, São Paulo, Brazil technique based on the protocol of Aljanabi & Martinez or in the Museu de Ciências da Pontifícia Universidade [13]. Partial sequences of the mitochondrial 16S rRNA Católica, Porto Alegre, Rio Grande do Sul, Brazil. DNA was genes, Cytochrome B, Cytochrome Oxidase I and nuclear

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access 102 G.S. Avelino, et al.

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).

α-Tropomyosin gene were amplified by polymerase chain Purification (Amersham Biosciences) and a salt extraction reactions (PCR). The primers used are listed in Table 2. method based on the protocol used by Travis Glenn [14] The amplification was performed in a total volume of 25.0 and with the ExoSAP-IT kit. The sequencing reaction was µL of a solution containing 16.5 µL of distilled water, 2.5 performed with the Amershan Bioscience, DYEnamic µL dNTP (8 mm), 2.5 µL buffer 10x, 1.2 µL of each primer Terminator kit. The sequences were determined in an (10 μM) and 0.1 µL DNA Polymerase (1 unit), 1.0 µL of DNA automated ABI PRISM™ 377 DNA Sequencer (Perking- sample and from 0.5 to 1.0 µL MgCl2, when required. The Elmer). All sequences were read at least twice (forward reaction conditions for PCR primers were: 16S (30 s at 95°C, and reverse). 45 s at 50°C, 45 s at 68°C and 420 s at 68°C), CytB (30 s at The sequences were aligned using the ClustalW 95°C, 45 s at 48-50°C, 120 s at 68°C and 420 s at 68°C), COI program [15] implemented in DAMBE version 5.2.14 (30 s at 95°C, 45 s at 48-50°C, 45 s at 68 and 72°C and 420 s [16] and BioEdit [17] programs. The range and pattern at 68 and 72°C) and Trop (30 s at 95°C, 45 s at 55°C, 90 s at of nucleotide substitution and genetic distance were 72°C and 420 s at 72°C), all the conditions with 30 cycles. examined using the MEGA 5.05 [18]. The saturation The PCR products were identified on 1% agarose gel. The of nucleotides was analyzed by plotting the absolute DNA was purified using the PCR Kit GFXTM Gel Band number of transitions (Ti) and transversions (Tv)

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 103

Table 2. Primers used for PCR and sequencing.

Gene Sequences References

16S F 5´ - ACG CCT GTT TAT CAA AAA CAT - 3´ Kocher et al. (1989) [39] 16S R 5´ - CCG GTC TGA ACT CAG ATC ACG T - 3´ Cyt b L 14841 5´ - AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA - 3´ Kocher et al. (1989) [39] Cyt b H 15915 5´ - AAC TGC CAG TCA TCT CCG GTT TAC AAG AC - 3´ Fish F1 5´ - TCA ACC AAC CAC AAA GAC ATT GGC AC - 3´ Ward et al. (2005) [40] Fish R1 5´ - TAG ACT TCT GGG TGG CCA AAG GAA TCA - 3´ TROP F 5´ - CCA CTG CCC TGC AGA AGC TGG AGG A - 3´ Calcagnotto et al. (2005) [41] TROP R 5´ - CTC CTC AGT ACG CTC CAG CTC ACC CTC A - 3´ Present study

versus the values of genetic distance using the program I of 73 specimens (100%), and the gene α-Tropomyosin of DAMBE version 5.2.14 [16]. The program Modeltest [19] 54 specimens (72%). After the alignment procedure and was used to select the nucleotide substitution model manual correction of alignment, we obtained a matrix that best fits the data obtained. with 2582 characters, out of which 1578 were conserved, RAxML [20] using the web servers RAxML BlackBox 992 variable, and 728 informative for parsimony analysis. [21] was used for all maximum likelihood analyses using The proportion transition/transversion (Ti/Tv) rate was a mixed partition model. Random starting trees were 2.68. The average composition, in percentage of bases was used for each independent ML tree search and all other adenine (A) 26.9%, cytosine (C) 26.4%, guanine (G) 19.4%, parameters were set on default values. All analyses were and thymine (T) 27.3%. A Chi-square test of heterogeneity conducted under GTR+G since RAxML only applies this of nucleotide frequencies among OTUs with Yates model [20]. Topological robustness was investigated using correction (performed in DAMBE) showed no significant 1000 non-parametric bootstrap replicates. A set of four values. Base composition was computed for all taxa in partitioning schemes ranging from 1 to 8 partitions was the concatenated alignment excluding constant sites tested following the procedures outlined by Li et al. [22] to gauge the effect of possible base compositional bias under the AIC and BIC criteria. In the first, test we included on the resulting phylogeny. A graphical analysis of the all the dataset (1 partition), in the second test, all genes relationship between transitions (Ti) and transversions were analyzed separately (4 partitions: 16S, CytB, COI, (Tv) and genetic distance estimated by the GTR distance Trop), in the third test, the 16S and Trop were separated model indicates that there is no saturation of these and the first, second, and third position of CytB and COI nucleotides (data not shown). were analyzed together (5 partitions), and in the fourth The overall average distance between sequences was test, the 16S, Trop and the first, second, and third position d = 0.090 ± 0.004, according the Kimura-2-parameter of CytB and COI were analyzed separately (8 partitions). substitution model [25]. Among species with more than Maximum parsimony (MP) analyses were conducted one specimen available the genetic distance ranged with PAUP* 4.0b10 [23]. Heuristic searches were performed from d = 0.000 ± 0.000 in Leporinus paranensis to d = using 1000 random addition replicates and TBR branch 0.041 ± 0.002 in L. obtusidens (Table 3). Genetic distance swapping. All characters were unordered, all character among species pairs ranged from d = 0.008 ± 0.002 from transformations were equally weighted, and branches L. octofasciatus and L. paranensis to d = 0.151 ± 0.009 with maximum length of zero were collapsed. Gaps were between L. copelandii and L. cf. fasciatus (Table 3). treated as missing data. Clade robustness was assessed Four different partitioning schemes ranging from using 1000 bootstrap pseudoreplicates [24] with the same one to 8 partitions were tested to establish the optimal parameters as above. number of data partitions (following Li et al. [22]) for the final analysis. The results showed that the 8 partition model was the best choice (data not shown); however, ML 3 Results analysis conducted with the other partitioning schemes resulted in the same final topology, with minor differences The sequences obtained in this study were deposited in in branch length and support values (not shown). the GenBank (Table 1). We obtained sequences of the Throughout the text and in the figures, measures mitochondrial 16S rRNA gene of 73 specimens (100%), of support are indicated as a series of two numbers on Cytochrome B of 65 specimens (89%), Cytochrome c Oxidase selected internal branches of the trees, starting with

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access 104 G.S. Avelino, et al. 0.008 0.007 0.007 0.008 0.005 0.004 0.005 0.007 0.008 0.008 0.004 0.004 0.008 0.007 0.004 0.006 0.005 ± 0.001 0.003 0.007 19 0.007 0.007 0.007 0.007 0.007 0.010 0.008 0.007 0.009 0.008 0.007 0.007 0.007 0.009 0.008 0.007 0.100 0.007 0.024 ± 0.003 18 0.002 ± 0.001 0.007 0.008 0.008 0.007 0.010 0.008 0.007 0.008 0.008 0.007 0.007 0.006 0.009 0.009 0.007 0.107 0.008 0.114 17 0.112 n/c 0.008 0.007 0.007 0.010 0.007 0.007 0.008 0.008 0.006 0.006 0.007 0.008 0.007 0.007 0.087 0.006 0.109 16 0.130 0.112 0.038 ± 0.005 0.005 0.007 0.010 0.007 0.005 0.008 0.007 0.007 0.007 0.008 0.009 0.008 0.007 0.104 0.007 0.109 15 0.132 0.115 0.084 0.028 ± 0.003 0.008 0.009 0.009 0.003 0.008 0.006 0.007 0.008 0.008 0.008 0.009 0.007 0.116 0.008 0.116 `14 0.120 0.093 0.107 0.122 0.011 ± 0.002 0.007 0.005 0.007 0.008 0.008 0.004 0.003 0.008 0.008 0.006 0.006 0.054 0.005 0.115 13 0.098 0.072 0.094 0.092 0.052 0.000 ± 0.000 0.002 0.009 0.009 0.012 0.006 0.006 0.011 0.010 0.005 0.009 0.022 0.004 0.104 12 0.125 0.095 0.113 0.128 0.068 0.008 0.014 ± 0.003 0.008 0.008 0.008 0.005 0.005 0.009 0.008 0.005 0.007 0.043 0.004 0.125 11 0.125 0.112 0.072 0.045 0.117 0.092 0.121 0.041 ± 0.002 0.008 0.006 0.007 0.007 0.008 0.008 0.008 0.006 0.107 0.007 0.109 10 0.128 0.113 0.123 0.129 0.119 0.081 0.112 0.125 n/c 0.009 0.007 0.007 0.009 0.009 0.008 0.007 0.110 0.008 0.122 9 0.131 0.122 0.101 0.107 0.125 0.124 0.138 0.103 0.142 0.010 ± 0.002 0.008 0.008 0.008 0.010 0.009 0.007 0.118 0.008 0.124 8 0.112 0.083 0.103 0.118 0.044 0.042 0.060 0.113 0.102 0.126 0.012 ± 0.003 0.004 0.007 0.007 0.005 0.006 0.046 0.005 0.111 7 0.113 0.081 0.103 0.115 0.024 0.042 0.057 0.110 0.104 0.121 0.035 0.001 ± 0.001 0.007 0.007 0.005 0.006 0.043 0.004 0.107 6 0.061 0.123 0.128 0.140 0.123 0.105 0.125 0.131 0.139 0.139 0.117 0.116 n/c 0.009 0.009 0.008 0.110 0.008 0.119 5 0.146 0.097 0.123 0.133 0.110 0.097 0.110 0.128 0.118 0.145 0.092 0.100 0.151 0.030 ± 0.005 0.008 0.008 0.080 0.008 0.130 4 0.133 0.101 0.113 0.128 0.072 0.026 0.047 0.125 0.106 0.143 0.059 0.059 0.135 0.103 n/c 0.007 0.030 0.004 0.128 3 0.124 0.101 0.114 0.117 0.098 0.094 0.111 0.113 0.092 0.121 0.095 0.091 0.127 0.117 0.112 0.014 ± 0.002 0.090 0.006 0.111 2 0.113 0.082 0.107 0.118 0.057 0.018 0.039 0.110 0.112 0.118 0.049 0.045 0.118 0.097 0.040 0.090 0.029 1 0.003 ± 0.001 0.108 Leporinus 17 Leporinus steindachneri sp. 16 Leporinus Leporinus 15 Leporinus reinhardti Leporinus piavussu 14 Leporinus Leporinus piau 13 Leporinus Leporinus 12 Leporinus paranensis Leporinus 11 Leporinus octofasciatus Leporinus 10 Leporinus obtusidens Leporinus 9 Leporinus microphthalmus Leporinus 8 Leporinus macrocephalus Leporinus lacustris 7 Leporinus Leporinus friderici 6 Leporinus Leporinus copelandii 5 Leporinus Leporinus cf 4 Leporinus fasciatus Leporinus bahiensis 3 Leporinus Leporinus 2 Leporinus amblyrhynchus taeniatus 19 Leporinus Leporinus aff aff 1 Leporinus paranensis striatus 18 Leporinus . Mean below diagonal and standard error above diagonal. In the diagonal (in bold) the mean value and standard error for for error standard and value the mean (in bold) In the diagonal diagonal. above error standard and diagonal below . Mean Leporinus of among the species obtained distance 3. K2P genetic Table computed. = not n/c species. each

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 105 non-parametric bootstrap percentages from ML and A general analysis of the genetic distance among MP analyses, respectively (e.g. 100/100, see Fig. 2). The specimens among species with more than one specimen general tree topology observed in all analyses was very available showed that the genetic distance was low (less similar, although statistical support was not strong for than 2%) in ten species (Leporinus paranensis, L. friderici, some nodes. The final topology obtained by ML analysis L. aff. paranensis, L. taeniatus, L. macrocephalus, L. will be used to discuss relationships among taxa (Figs. piau, L. lacustris, L. octofasciatus, L. steindachneri and 2-3), but important differences with results obtained by L. amblyrhynchus - Table 3) and high (more than 2%) in ML and MP analyses will be discussed in the text. 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.

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access 106 G.S. Avelino, et al.

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 However, the samples of L. piavussu, L. cf. fasciatus and may be explained, in part by the sample distribution L. reinhardti were collected in the same geographical since some samples of L. striatus and L. obtusidens were area and the observed genetic difference may be an collect in far points in their distribution area (Table 1). intrinsic characteristic of these species. Studies involving

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 107 molecular identification of species have reported several above this high genetic diversity may be associated to the cases of deep intra-specific divergence [26-31] and in many existence of cryptic species and thus additional studies of them this diversity has been associated to the existence (involving different markers as genetic and molecular of cryptic species. Carefully studies involving other genetic markers) should be conducted in this species. markers and populational sampling should be done in the future to better check this genetic variability. Genetic distance was computed among 153 species 4 Discussion pairs and only in two cases the values observed were Analyzing the genetic similarities among species less than 2%: between L. paranensis and L. octofasciatus studied we found that the samples from the Amazon (d = 0.008 ± 0.002) and between L. paranensis and L. aff. Basin (Leporinus sp.) and one species from the Orinoco paranensis (d = 0.018 ± 0.004) (Table 3). The comparison River Basin (Leporinus cf. fasciatus) are more similar of the sequences of L. paranensis and L. octofasciatus between themselves than with other species (Figs. 2). It shows that only four position are different (diagnostic), should be emphasized that Leporinus sp. showed a great all in the COI gene. The comparison of the sequences of morphological and meristic similarity with L. obtusidens, L. paranensis and L. aff. paranensis shows that 23 position but could not be identified at species level. were different (diagnostic), 18 in the COI gene and 5 in the The species L. lacustris, L. friderici, and L. piau formed 16S gene. Several molecular identification studies have a well supported group being L. friderici more similar to suggested the use of a nucleotide diagnostic approach L. piau (Fig. 2). All these three species fit the color pattern as an alternative to distance approach to identify species 3 of Garavello & Britski [8], and have four teeth in both [30-34]. Thus, considering the morphological differences jaws. A second group close related to the cited above is among the above cited species we believe that the small composed of L. bahiensis, L. taeniatus, L. octofasciatus, L. genetic differences among them may be result of recent paranensis and L. aff. paranensis (Fig. 2). The species L. speciation. bahiensis was included in the present study due its deep The general distance between L. obtusidens and L. morphological similarity with L. aff. paranensis (Britski, piavussu was d = 0.045 ± 0.003 (Table 3). However, in all the personal observation). The results show that these two trees obtained the species L. piavussu was found among last species are not particularly genetically similar and L. the specimens of L. obtusidens (Fig. 3). A close analyses bahiensis is more similar to L. taeniatus (Fig. 2). Although of Figure 3 shows that the samples of L. obtusidens can all species in this group have the same number of teeth be separated in four groups, identified here as Group in both jaws, three teeth in the premaxillary and four A to Group D. Two of these groups are geographically teeth in the dentary, they have a different color pattern; distinct: the Group B with specimens from São Francisco, L. taeniatus has a color pattern 2, L. octofasciatus has a and the Group C with specimens from Rio Cuiabá (Table color pattern 1, and L. paranensis, L. aff. paranensis and L. 1, Fig. 1). The other two are more widely distributed: the bahiensis have a color pattern 3, according to Garavello & Group A with specimens in the Paraná, Uruguay and Britski [8] classification. Lagoa dos Patos and Group D with specimens in the Leporinus microphthalmus appears genetically more Paraná and Paraguay rivers (Table 1, Fig. 1). The genetic similar to L. amblyrhynchus (Fig. 2). L. microphthalmus has distance among these groups ranges from d = 0.033 ± three teeth in the premaxilla and four in the dentary and L. 0.003 between Group A and Group B to d = 0.061 ± 0.004 amblyrhynchus has three teeth in both bones. Moreover, L. between Group A and Group C (Table 4). As discussed 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 2 3 4 5

1 L. obtusidens A 0.017 ± 0.001 0.003 0.004 0.004 0.004

2 L. obtusidens B 0.033 0.007 ± 0.001 0.004 0.004 0.004

3 L. obtusidens C 0.061 0.058 0.041 ± 0.003 0.004 0.004

4 L. obtusidens D 0.051 0.050 0.055 0.018 ± 0.002 0.003

5 L. piavussu 0.051 0.051 0.059 0.029 0.028 ± 0.003

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access 108 G.S. Avelino, et al. the barred and striped pattern, an uncommon pattern basins could give origin to recent ‘species-pairs’ in these among species of Leporinus [8]. A species resembling L. basins. Considering that the main formation of these amblyrhynchus, in color pattern, is L. taeniatus, a species basins is a very ancient event dating back to the Jurassic that occurs in the basin of the São Francisco, however, or Cretaceous ages [35], before or close to the separation this species differs from L. amblyrhynchus for having 16 between Africa and South America, the first hypotheses circumpeduncular scale series (12 in L. amblyrhynchus) does not seem probable, since we do not have any sign of and four teeth in each dentary (three in L. amblyrhynchus) the presence of these fishes in Africa. On the other hand, [8]. In the tree obtained in this work, L. amblyrhynchus the second hypothesis is sustainable by strong evidences and L. taeniatus do not stick together, so the grouping by of recent reactivation of Precambrian shear zones from color pattern was not confirmed in this case. the Miocene to the present day [36,37]. A particularly Leporinus steindachneri, L. striatus, L. macrocephalus, clear example of this type of exchange was documented and L. reinhardti appear successively as genetically by Ribeiro et al. [38] in the Upper Rio Guaratuba, which more similar to L. obtusidens + L. piavussu (Figs. 2 - 3). originally flowed toward the Paraná River and became a L. steindachneri has four teeth in the premaxilla and in coastal river due to the Quaternary activity of NW-trending the dentary and has a color pattern 3; L. striatus has three faults. The occurrence of several ‘species-pairs’ in our tree, teeth in the premaxillary and four teeth in the dentary such as L. taeniatus (São Francisco) and L. octofasciatus and has a color pattern 2; L. macrocephalus, L. reinhardti, and L. paranensis (La Plata); L. piau (São Francisco) and L. obtusidens and L. piavussu have three teeth in both jaws L. friderici (La Plata); and L. reinhardti (São Francisco) and and a color pattern 3 according to Garavello & Britski [8] L. obtusidens (La Plata, São Francisco and other South and Britski et al. [10]. America drainages) may represent a good example of the An interesting finding of our study is that the genetic recent connection between the La Plata and São Francisco similarities observed among species do not corroborate basins. Based on these data we strongly suggest that these the division of species of Leporinus in groups according species-pairs should be deeply analyzed to confirm their to color patterns. For example, L. striatus, a species that taxonomic status (one or two species). fits the color pattern 2 of Garavello & Britski [8] is closely related to L. steindachneri, a species that fits the color Acknowledgements: We thank all the individuals who pattern 3 of the same authors. Additionally, regarding assisted us in the collection and identification of the the number of teeth, L striatus has three teeth in the specimens that served as the basis for this study and to premaxillary and four in the dentary and L. steindachneri Museu de Ciências e Tecnologia da Pontifícia Universidade has four teeth in both bones, reinforcing the hypothesis Católica do Rio Grande do Sul and Museu de Zoologia of that these characters may be also not phylogenetically Universidade de São Paulo for the donation of fish tissues. informative. Another example are the species L. Fundação de Apoio à Pesquisa do Estado de São Paulo octofasciatus and Leporinus cf. fasciatus which, despite and Conselho Nacional de Desenvolvimento Científico e having different teeth (L. octofasciatus with three teeth in Tecnológico do Brasil provided funds to the development the premaxillary and four in the dentary and Leporinus cf. of the present study. fasciatus with four teeth in both jaws), fall into the same color pattern 1, but are not together in the tree (Fig. 2). Conflict if interest: Dr Avelino delcares nothing to On the other hand, an example of clustering in the disclose. 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. References piau. They all fit in the color pattern 3 and have four teeth [1] Garavello J.C., Britski H.A., Family Anostomidae (Headstanders), in both jaws [8]. In: Reis R.E., Kullander S.O., Ferraris Jr., Carl J. (Eds), Cheklist of The most important result obtained in our analysis the freshwater fishes of South and Central America, Porto Alegre, is that the species of Leporinus from the La Plata and EDIPUCRS, 2003 São Francisco are not more similar among themselves [2] Sidlauskas B.L., Vari R.P., Phylogenetic relationships within the South American fish family Anostomidae (Teleostei, Ostariophysi, than to species of other drainages in the south part of Characiformes), Zool. J. Linn. Soc., 2008, 154, 70-210 South America. Two main hypotheses could explain this [3] Froese R., Pauly D., FishBase, www.fishbase.org, 2011, version result: (1) the genus could be very old, and speciation (09/2011) could have precluded these basins formation; (2) the [4] Fowler H.W., Fishes from the Rupununi: River, British Guiana, Proc. occurrence of recent fauna exchanges between these two Acad. Nat. Sci, Philadelphia., 1914, 66, 229-284

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access Molecular identification of Leporinus from the south portion of South America 109

[5] Borodin N.A., Notes on some species and subspecies of the [24] Felsenstein J., Confidence limits on phylogenies: an approach genus Leporinus Spix, Mem. Mus. Com. Zool. Cambridge, using the bootstrap, Evolution, 1985, 39, 783-791 Massachusetts, 1929, 50, 269-290 [25] Nei M., Kumar S., Molecular evolution and phylogenetics, New [6] Géry J., Contributions a 1’etude des poisons characoides (n °8). Un York, Oxford University Press, 2000 nouveau sous-genre de Leporinus (Erythrinidae, Anostomidae): [26] Handfield D., Handfield L., A new species of Plusia (Lepidoptera: Leporinops, type L. moralesi Fowler, Bull. Mus. Hist. Nat., Paris, Noctuidae) from North America, Can. Entomol., 2006, 138, 1960, 32 (4), 308-313 (in French) 853–859 [7] Fowler H.W., Los Peces del Peru, Bol. Mus. Hist. Nat. Universidad [27] Smith M.A., Woodley N.E., Janzen D.H., Hallwachs W., Hebert de San Marcos, 1942, 22-23, 352-381 (in Spanish) P.D.N., DNA barcodes reveal cryptic host-specificity within the [8] Garavello J.C., Britski H.A., Duas novas espécies do gênero presumed polyphagus members of a genus of parasitoid flies Leporinus Spix, 1829, da bacia do alto Paraná (Teleostei, (Diptera: Tachinidae), PNAS, 2006, 103, 3657–3662 Anostomidae), Comun. Mus. Cienc. da PUCRS, Porto Alegre, 1987, [28] Ward R.D., Holmes B.H., Yearsley G.K., DNA barcoding reveals 44, 153-165 (in Portugese) a likely second species of Asin sea bass (barramundi) (Lates [9] Britski H.A., Birindelli J.L.O., Description of a new species of the calcarifer), J. Fish. Biol. 2008, 72, 458–463 genus Leporinus Spix (Characiformes: Anostomidae) from the Rio [29] Pereira L.H.G., Pazian M.F., Hanner R., Foresti F., Oliveira C., DNA Araguaia, Brazil, with comments on the taxonomy and distribution barcoding reveals hidden diversity in the Neotropical freshwater of L. parae and L. lacustris, Neotrop. Ichthyol., 2008, 6 (1), 45-51 fish Piabina argentea (Characiformes: Characidae) from the Upper [10] Britski H.A., Birindelli J.L.O., A new species of Leporinus Agassiz, Paraná Basin of Brazil, MDN, 2011, 22 (Suppl 1), 87–96 1829 (Characiformes: Anostomidae) from the Rio Tocantins, Brazil, [30] April J., Mayden R.L., Hanner R., Bernatchez L., Genetic calibration Neotrop. Ichthyol., 2013, 11 (1), 25-32 of species diversity among North America´s freshwater fishes, [11] Menezes N.A., Distribuição e origem da fauna de peixes de água PNAS, 2011, 108 (26), 10602–10607 doce das grandes bacias fluviais do Brasil, In: Comissão Intere- [31] Pereira L.H.G., Hanner R., Foresti F., Oliveira C., Can DNA stadual da Bacia do Paraná-Paraguai. Poluição e Piscicultura. São barcoding accurately discriminate megadiverse Neotropical Paulo: USP. Faculdade de Saúde Pública, 1972 (in Portugese) freshwater fish fauna?, BMC Genet., 2013, 14, 20 [12] Sambrook J., Russel D.W., Molecular Cloning - a Laboratory [32] Desalle R., Egan M.G., Siddall M., The unholy trinity: taxonomy, Mannual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold species delimitation and DNA barcoding, Philos. Trans. R. Soc. Spring Harbor, 2001 Lond. B. Biol. Sci., 2005, 360, 1905–1916 [13] Aljanabi S.M., Martinez I., Universal and rapid salt-extraction of [33] Wong E.H.K., Shivji M.S., Hanner R.H., Identifying sharks with high quality genomic DNA for PCR-based techniques, Nucleic. DNA barcodes: assessing the utility of a nucleotide diagnostic Acids. Res., 1997, 25, 4692-4693 approach, Mol. Ecol. Res., 2009, 9 (1), 243–256 [14] Travis Glenn (text available at http://www.uga.edu/srel/DNA_Lab/ [34] Birstein V.J., Desalle R., Doukakis P., Hanner R., Ruban G.I., Wong PEG_Precip’00.rtf) E., Testing taxonomic boundaries and the limit of DNA barcoding [15] Thompson J.D., Higgins D.G., Gibson T.J., CLUSTAL W: improving in the Siberian sturgeon, Acipenser baerii, MDN, 2009, 20 (5–6), the sensitivity of progressive multiple sequence alignment 110–118 through sequence weighting, positions-specific gap penalties and [35] Potter P.E., The Mesozoic and Cenozoic paleodrainage of South weight matrix choice, Nucleic. Acids Res., 1994, 22, 4673-4680 America: a natural history, J. S. Am. Earth. Sci., 1997, 10, 331-344 [16] Xia X., Xie Z., DAMBE: Data analysis in molecular biology and [36] Riccomini C., Asumpção M., Quaternary tectonics in Brazil, evolution, J. Hered., 2001, 92, 371-373 Episodes, 1999, 22, 221-225 [17] Hall T.A., BioEdit: a user-friendly biological sequence alignment [37] Riccomini C., Sant’Anna L.G., Ferrari A.L., Evolução geológica do editor and analysis program for Windows 95/98/NT, Nucleic Acids Rift Continental do Sudeste do Brasil, Pp. 383-405, In: Mantesso- Symposium Series, 1999, 41, 95-98 Neto V., Bartorelli A., Carneiro C.D.R., Brito-Neves B.B., (Eds.), [18] Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S., Geologia do continente Sul-Americano: evolução da obra de MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Fernando Flávio Marques de Almeida, Editora Beca, São Paulo, Likelihood, Evolutionary Distance, and Maximum Parsimony 2004 Methods, Mol. Biol. Evol., 2011, 28, 2731-2739 [38] Ribeiro A.C., Lima F.C., Riccomini C., Menezes N.A., Fishes of the [19] Posada D., Crandall K.A., Modeltest: testing the model of DNA Atlantic Rainforest of Boracéia: testimonies of the Quaternary substitution, Bioinformatics, 1998, 14, 817-818 fault reactivation within a Neoproterozoic tectonic province in [20] Stamatakis A., RAxML-VI-HPC: Maximum likelihood-based Southeastern Brazil, Ichthyol. Explor. Fres., 2006, 17, 157-164 phylogenetic analyses with thousands of taxa and mixed models, [39] Kocher T.D., Thomas W.K., Meyer A., Edwards S.V., Pääbo S., Bioinformatics, 2006, 22, 2688-2690 Villablanca F.X., Wilson A., Dynamics of mitochondrial DNA [21] Stamatakis A., Hoover P., Rougemont J., A rapid bootstrap evolution in animals: ampliWcation and sequencing with algorithm for the RAxML web servers, Syst. Biol., 2008, 57, conserved primers, Proc. Natl. Acad. Sci., 1989, 86, 6196–6200. 758-771 [40] Ward R. D., Zemlak T. S., Innes B. H., Last P. R., Hebert P. D. N., DNA [22] Li C., Ortí G., Zhang G., Lu G., A practical approach to barcoding Australia’s fish species, Phil. Trans. R. Soc. B, 2005, phylogenomics: The phylogeny of ray-finned fish (Actinopterygii) 360, 1847–1857. as a case study, BMC Evol. Biol., 2007, 7, 44-55 [41] Calcagnotto D., Schaefer S.A., DeSalle R., Relationships among [23] Swofford D.L., PAUP* - Phylogenetic analysis using parsimony characiform fishes inferred from analysis of nuclear and (*and other methods), Version 4.0b10, Sinauer Associates, mitochondrial gene sequences, Mol. Phylogenet. Evol., 2005, 36, Sunderland, Massachusetts, 2001 135–153.

- 10.1515/dna-2015-0013 Downloaded from De Gruyter Online at 09/13/2016 04:12:13PM via free access