1 Introduction. DNA Barcodes 2015; 3: DOI /dna Received February 21, 2015; accepted July 6, 2015

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1 DNA Barcodes 2015; 3: Research Article Open Access Camila da Silva de Souza, Claudio Oliveira, Luiz Henrique Garcia Pereira* Knodus moenkhausii (Characiformes: Characidae): one fish species, three hydrographic basinsa natural or anthropogenic phenomenon? DOI /dna Received February 21, 2015; accepted July 6, 2015 Abstract: We used the DNA barcoding technique (COI and CytB markers) combined with GMYC analysis to characterize the genetics of the widely distributed Neotropical fish species Knodus moenkhausii from three different isolated hydrographic basins. Despite the fact that most of the Neotropical hydrographic basins have been isolated for millions of years, species could be shared between basins due to natural events (stream capture) or anthropogenic activities. Recent surveys, however, have shown that many widely distributed species are actually species complexes divided into previously unrecognized cryptic species. In this study, we tested the hypothesis that K. moenkhausii from three hydrographic basins represent a single panmictic species and discuss the most likely explanation of its present geographical distribution. The GMYC analysis revealed that all specimens of K. moenkhausii represent a single species: the intra- and intergroup minimum K2P genetic distances for both genes were zero and haplotypes were shared among the three hydrographic basins. This suggests there has been recent interchange of K. moenkhausii throughout the three hydrographic basins. It is likely that this is due to recent human activities, either the transposition of natural barriers or intentional introduction or accidental escape due to the ornamental fish trade. Keywords: Neotropical, COI, CytB, Upper Paraná river, São Francisco river, Paraíba do Sul river, Human activities, species introduction, GMYC, ornamental fishes. *Corresponding author: Luiz Henrique Garcia Pereira, Centro de Ciências da Vida e da Natureza, Universidade Federal da Integração Latino-Americana UNILA, Foz do Iguaçu, Paraná, Brazil, , luiz.pereira@unila.edu.br Camila da Silva de Souza, Claudio Oliveira, Departamento de Morfologia, Universidade Estadual Paulista UNESP, Botucatu, São Paulo, Brazil, Introduction The Neotropical region comprises 78 freshwater ecoregions (hydrographic basins), of which 28 occur in Brazil, including some of the major river basins of the world, such as the Amazonas, Paraná and Uruguay Rivers [1]. This region contains the most diversified freshwater ichthyofauna of the world, with about 6,000 species to date [2]. The geographical distribution of this fauna is highly complex, with some species occurring in restricted areas, sometimes only known from the type locality (e.g. Trichomycterus maracaya, Characidium xanthopterum), while others have broad distributions and occur even in multiple hydrographic basins (e.g. Hoplias malabaricus, Astyanax paranae, and Knodus moenkhausii) [2,3]. The latter phenomenon is especially interesting, since most hydrographic basins have been isolated for millions of years [4,5]. Nevertheless, shared species among Neotropical hydrographic basins are a frequent occurrence, such as between Paraíba do Sul and the Upper Paraná River basins with 21 shared species and between the Upper Paraná and São Francisco River basins with 47 shared species [6,7,8]. The occurrence of shared species among hydrographic basins could be attributed either to natural phenomena such as headwater stream capture or to anthropogenic activities such as the accidental or intentional introduction of nonnative species. Headwater stream capture (also called stream capture or stream piracy) occurs when the whole or a part of a stream/river is shifted to the drainage of a neighboring basin due to geomorphological processes, which then allows the dispersal of species to the new basin [5,9]. Headwater stream capture has been used to explain the occurrence of many shared species between neighboring basins, such as in the case of Mimagoniates microleps (Characidae) between the Iguaçu River and the Coastal Eastern basins [10] and Cnesterodon brevirostratus (Poeciliidae) between the Uruguai River and the South Coastal drainages [4]. On the other hand, the introduction 2015 Camila da Silva de Souza et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

2 130 C. da Silva de Souza, et al. of species by anthropogenic activities has become an increasingly common event [11,12,13]. The number of species found outside of their native region has doubled in the last three decades [14]. In Brazil, the number of introduced species is uncertain, due mostly to the lack of surveys and because many regions of Brazilian watersheds are poorly explored [3,6,15]. Nevertheless, the few surveys show an extensive number of introduced species (either exotic or allochthonous), such as in the Alto Paraná and Paraíba do Sul River basins which contain 74 (23.9%) and 64 (49%) introduced species respectively [6,16,17]. An alternative explanation for apparently shared species between basins is that these species represent unrecognized cryptic species, which would only be revealed by molecular studies. Indeed, some recent studies have shown that many widely distributed species are actually cases of cryptic species complexes which had gone unrecognized [18-23]. In this study, we assess the genetic status of populations of the freshwater fish species Knodus moenkhausii which occurs in three isolated hydrographic basins (of the Upper Paraná, São Francisco, and Paraíba do Sul Rivers). Knodus moenkhausii (Eigenmann & Kennedy, 1903) is a small fish (~5 cm) belonging to the family Characidae. This species mainly inhabits small streams with structurally simple habitats of sandy bottom, mild currents, shallow water (~45 cm), and without cover and marginal vegetation; often K. moenkhausii is the most abundant species in this habitat [24,25]. Identifying this species can be difficult due to its similarity to other species of the genus and the related genera Creagrutus, Piabina, and Bryconamericus. The species was first described from the Paraguay River basin (Eigenmann & Kennedy, 1903), and later reported from the Upper Paraná [6,24-27] and São Francisco River basins [28]. Recently, our research group collected specimens of K. moenkhausii in the Paraíba do Sul River basin as well. These findings raise the question whether K. moenkhausii represents a single panmictic species or distinct genetic populations native to each hydrographic basin in which it occurs. To resolve this question, we used DNA barcoding combined with GMYC analysis (Generalized Mixed Yule Coalescent) for delimiting species. DNA barcoding was proposed specifically to characterize species using a standard, short (~650 pb), and universal sequence fragment of the Cytochrome c Oxidase subunit I (COI) gene [29]. The COI sequence can be considered a genetic barcode, which ideally would be unique to each species, since some divergence would be expected to develop in this region during the evolutionary history of different taxa. At present, the barcoding database (BOLD) contains about 10,800 species of fishes ( org), and in some studies the successful identification rate can exceed 90% [30,31]. The GYMC analysis [32] uses maximum-likelihood statistics and a time-calibrated gene tree to delimit species using sequences from a single locus. The method combine a speciation model (Yule) with a population model (coalescent) to delimit species by characterizing the transition between intra-specific (population) and interspecific (species) events [32]. In general, this method is more robust and less subjective than traditional barcoding analysis methods. In this study, we analyzed specimens of K. moenkhausii from the Upper Paraná, São Francisco and Paraíba do Sul River basins to test the hypothesis that these specimens belong to a single panmictic species. Furthermore, we discuss the likely determinants of the current geographical distribution of this species. 2 Material and Methods We analyzed 36 K. moenkhausii specimens from three hydrographic basins (Upper Paraná, São Francisco and Paraíba do Sul Rivers) (Fig. 1), available in the tissue collection of the Laboratório de Biologia e Genética de Peixes (LBP), São Paulo, Brazil (Supplementary table 1). A single specimen each of Knodus borki, Knodus chapadae, Knodus victoriae, and Bryconamericus sp., were used for comparison (Supplementary table 1). 2.1 Extraction, PCR and Sequencing Total genomic DNA was isolated from fin or muscle tissue of each specimen using the DNeasy Blood and Tissue Kit (Qiagen California USA) according to the manufacturer s instructions. The partial mitochondrial COI gene, was amplified by the PCR using the primers: C_FishF1t1/C_FishR1t1, 5 GTA AAA CGA CGG CCA GTC AAC CAA CCA CAA AGA CAT TGG CAC-3, 5 -TGT AAA ACG ACG GCC AGT CGA CTA ATC ATA AAG ATA TCG GCA C-3, 5 -CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA A-3, 5 -CAG GAA ACA GCT ATG ACA CCT CAG GGT GTC CGA ARA AYC ARA A-3 [33]. We also amplified the mitochondrial gene Cytochrome B (CytB), amplified by PCR using the primers LNF (5 -GAC TTGA AAA ACC AYC GTT GT) and H08R2 (5 -GCT TTG GGA GTT AGD GGT GGG AGT TAG AAT C) [34]. PCR was carried out on a thermocycler Veriti 96-well Fast (ABI-Applied Biosystems California - USA), with a final volume of 10.0 µl containing 5.0 µl Buffer 2X, 3.3 µl ultrapure water, 1.0 µl each primer (10µM ), 0.2 µl enzyme Phire Hot Start II DNA polymerase (Life Technologies

3 One fish species, three hydrographic basins: natural or anthropogenic? 131 Figure 1: South America Hydrographic Map: Hydrographic map showing the three hydrographic basins with K. moenkhausii sampled. Blue = Upper Paraná River basin; green = São Francisco River basin; red = Paraíba do Sul River basin. Red dots = K. moenkhausii sample sites; yellow dots = K. chapadae sample sites; green dots = K. victoriae sample sites; grey squares = Bryconamericus sp. sample sites; dark line = Itaipu Dam. California USA) (5U) and 0.5 µl of DNA template (~50 ng). The thermocycler conditions to amplify the COI gene were initial denaturation at 98 C for 5 min followed by 30 cycles denaturation at 98 C for 5 s, annealing at 56 C for 20 s and extension at 72 C for 30 s, followed by a final extension step at 72 C for 5 min. The thermocycler conditions to amplify the CytB gene were initial denaturation at 98 C for 5 min followed by 30 cycles of denaturation at 98 for 5 s, annealing at 50 C for 15 s and extension at 72 C for 45 s, followed by a final extension step at 72 C for 5 min. Amplified products were checked on 1% agarose gels. The PCR products were purified with ExoSap-IT (USB Corporation, Cleveland, OH, USA), and the purified PCR product was used as template to sequencing both DNA strands. Sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Ready Reaction (Applied Biosystems California - USA) and sequencing was performed on the automatic sequencer ABI 3130 DNA Analyzer (Applied Biosystems California - USA). 2.2 Data analysis The sequences were edited using the software programs ATGC (Genetyx Corporation Tokio - Japan) and Bioedit [35] to obtain the consensus sequences. For verification of contaminants (exogenous DNA), the sequences were submitted to the software BLAST available on the NCBI site. The sequences were aligned by the editor ClustalW [36] coupled with Dambe software [37]. The genetic distances were calculated using the Kimura-2- parameter (K2P) distance model [38] by the program MEGA v.6.06 [39]. The K2P model was chosen due its widespread application to calculate genetic distances in barcoding surveys. To determine species boundaries, we used GMYC analysis with single threshold as described in Pons et al [32]. For the analysis we generated a uncalibrated ultrametric tree with the Beast 1.5 program [40]. This tree was obtained with the General Time Reversible (GTR) model and a relaxed clock (uncorrelated lognormal). We used the speciation birth-death process model. We started the search with a UPGMA tree and then a Markov Chain Monte Carlo (MCMC) model with 30 x10 6 generations. One tree was sampled every 1000 generations. The tree with the highest probability was selected in the TreeAnnotator V1.5.3 [41] and used as an ultrametric tree for the GMYC analysis. The GMYC analysis was carried out on the GMYC web server available at gmyc/.

4 132 C. da Silva de Souza, et al. 3 Results We obtained 36 COI gene sequences (~554 bp) and 24 CytB gene sequences (~850 bp) from our K. moenkhausii samples as well as single COI and CytB sequences for the congeneric species K. borki, K. chapadae, K. victoriae and the related species Bryconamericus sp.. No sequences showed insertions, deletions, or stop-codons, and the analysis with the BLAST tool did not show contaminants. The GMYC analysis suggest the existence of five ML entities (independent coalescent group) for both genes (confidence interval = 5-5 and 5-6 and, threshold time = and for COI and CytB, respectively), corresponding to Bryconamericus sp., K. borki, K. chapadae, K. victoriae and K. moenkhausii (Fig. 2). All specimens of K. moenkhausii from the three different hydrographic basins grouped in a single ML cluster (Fig. 2). To calculate the genetic divergence values, K. moenkhausii sequences were divided into three different groups corresponding to each hydrographic basin (Upper Paraná, São Francisco, and Paraíba do Sul Rivers) for both genes. No genetic divergence was observed for the COI sequences among and within K. moenkhausii groups, except for the Upper Paraná group that showed a mean intraspecific genetic divergence of 0.1% (Table 1). The COI sequences of K. moenkhausii consist of four haplotypes, with the most frequent haplotype (Hap1 86.1%) shared across the three hydrographic basins. The three remaining haplotypes (Hap2, Hap3 and Hap4) were exclusive to the Upper Paraná River basin (Table 2). The genetic distance between K. moenkhausii and the three other congeneric species ranged from 8.6% to 11.0%, and from 14.8% to Table 1: K2P genetic divergences for COI (below diagonal) and CytB (above diagonal) between K. moenkhausii groups and related species. Red = K2P genetic divergences within groups; Blue = among three K. moenkhausii groups (groups = separate hydrographic basin samples) Upper Paraná river basin 0.1/ Paraíba do Sul river basin 0 0/ São Francisco river basin 0 0 0/ Knodus victoriae / Knodus chapadae / Knodus borki / Bryconamericus sp /- Table 2: Haplotypes of COI and CytB genes for K. moenkhausii. UP = Upper Paraná River basin; SF = São Francisco River basin; PS = Paraíba do Sul River basin. COI haplotypes Nucleotide position Absolute number Frequency Occurrence Hap1 C T G % UP, SF, PS Hap2 C C G 3 8.3% UP Hap3 C C A 1 2.8% UP Hap4 A T G 1 2.8% UP CytB haplotypes Hap1 G C T T A T % UP, SF, PS Hap2 G T C C G T 2 8.3% UP Hap3 G C T T A C 1 4.2% UP Hap4 A C T T A T 1 4.2% SF

5 Figure 2: Generalized Mixed Yule Coalescent species delimitation tree showing the five independent coalescence groups (ML entities) found for both genes (COI and CytB). Blue = Upper Paraná River basin, green = São Francisco River basin, red = Paraíba do Sul River basin. One fish species, three hydrographic basins: natural or anthropogenic? 133

6 134 C. da Silva de Souza, et al. 15.1% between K. moenkhausii and Bryconamericus sp. (Table 1). For the CytB sequence, the genetic distance ranged from 0 to 0.1% and 0 to 0.1% among and within the three groups, respectively (Table 1). The CytB sequences of K. moenkhausii comprise four haplotypes, with the most frequent haplotype (Hap1 83.3%) shared across the three hydrographic basins. The Hap2 and Hap3 haplotypes were exclusive to the Upper Paraná River basin and Hap4 was exclusive to the São Francisco River basin (Table 2). The genetic distance in CytB between K. moenkhausii and its three congeneric species ranged from 9.3% to 11.0% and from 13.8% to 14.0% from Bryconamericus sp (Table 1). 4 Discussion The results obtained in the present study indicate that the K. moenkhausii specimens from the Upper Paraná, São Francisco and Paraíba do Sul River basins represent a panmictic genetic unit, likely representing a single species. This finding is inconsistent with many studies that suggest a pattern of limited dispersal in small Neotropical freshwater fishes [42,43]. This limited dispersal ability should restrict the geographical distribution of these fishes facilitating the subdivision of populations and leading to speciation by geographic isolation (allopatry) [43]. Additionally, several surveys have shown that many species with wide geographic distribution actually represent complexes of distinct cryptic species. For example, a recent review of Pseudoplatystoma using morphological characters showed that the widely distributed species P. fasciatum actually represents a complex of five species, one in each hydrographic basin that they occupy [44]. Another survey of different populations of Gymnotus pantherinus from southeast Brazil showed that each population was genetically distinct with few shared and many exclusive haplotypes [45]. The authors suggested that the development of geographic barriers resulting from the formation of the Serra do Mar, resulted in a pattern of allopatric speciation [45]. Two surveys analyzing Piabina argentea from the Upper Paraná and São Francisco River basins with molecular and cytogenetic tools showed that the nominal species comprises a species complex with six distinct putative species [18,19]. Interestingly, this species shows a similar geographic distribution to K. moenkhausii and is a similar small Neotropical fish [46]. In contrast to the above cases, we found that K. moenkhausii presents a very different genetic profile. The GMYC analyses conducted with both mitochondrial genes (COI and CytB) showed that all samples of K. moenkhausii form a single genetic unit. There was no genetic divergence among the three hydrographic basins and the haplotypes of both genes were shared among the three hydrographic basins revealing a complete lack of population structure. The genetic distance from congeners ranged from 8.6% to 11.0 and 9.3% to 11.0% for COI and CytB genes, respectively: values consistent with those found in other DNA barcoding surveys that showed average genetic distances between congeners ranging from 7% to 11% in more than 90% of comparisons [28,30,31,47,48]. Similarly, these other studies report genetic divergences within species of about 0.5%, a similar value to that found in the present survey. Ward [30] analyzed the COI sequences of 1088 fish species available in BOLD, and found a minimum interspecific distance of 2% to be a useful threshold to delimiting species, usually with an order of magnitude lower variation within species. These findings generally support the hypothesis that K. moenkhausii represents a single panmictic species. 4.1 Geographical distribution of K. moenkhausii Headwater stream capture could explain the occurrence of K. moenkhausii in the three different hydrographic basins analyzed. However, the absence of genetic divergence among the three groups for both genes and broadly shared haplotypes indicates completely unstructured populations. Considering that the three hydrographic basins have been isolated for millions of years [4,5], one would expect, at least, some degree of population structure, considering that K. moenkahusii has a relatively limited dispersal ability [42,43]. Based on this, we believe headwater stream capture may be insufficient to explain the genetic profile we found in the current geographical distribution of K. moenkhausii. On the other hand, the introduction of species by human activities may better explain the pattern we found. The numerous shared haplotypes among the three hydrographic basins suggest a recent event. The main anthropogenic pathways introducing nonnative fish species across hydrographic basin boundaries include the transposition of natural barriers through the construction of canals, allowing communication of water between isolated rivers or the inundation of areas due the construction of hydroelectric power dams. In addition, aquaculture includes the intentional introduction of fishes using exotic or allochthonous species (stocking strategy), fishing techniques using live bait introduce nonnative fishes, and the trade in ornamental fishes includes transferring species between locations [11,12,49]. In fact, the number of recognized introduced species (either

7 One fish species, three hydrographic basins: natural or anthropogenic? 135 exotic and allochthonous) to the Upper Paraná, Paraíba do Sul, and São Francisco River basins are extensive, with 74 (23.9%), 64 (49%), and 24 (9.8%) introduced species, respectively [6,16,50]. Additonal evidence that anthropogenic activities are responsible for the present distribution pattern of K. moenkhausii is provided by the species list of Upper Paraná River basin [6]. K. moenkhausii was described from the Paraguay River, in the drainage of the Lower Paraná River basin (Eigenmann and Kennedy 1903). K. moenkhausii was recorded in the Upper Paraná River basin only after the construction of the Itaipu hydroelectric power dam in 1982 [6]. The Itaipu Reservoir inundated an area of about 1350 km 2 [51] that included the Sete Quedas Falls, a natural and effective barrier that was the limit of the hydrographic basins of the Lower and Upper Paraná Rivers [6,52]. The inundation of Sete Quedas falls moved the barrier between these two hydrographic basins to 150 Km below the Itaipu Dam, allowing the mixing of species from the two basins. At present, at least 33 species initially limited to the Lower Paraná River basin have successfully colonized the Upper Paraná River basin, including K. moenkhausii [52]. The lists of introduced species in the Paraíba do Sul and São Francisco River basins do not include K. moenkhausii [16,50]. The occurrence of K. moenhkausii in the São Francisco river basin was first documented in 2011 by Carvalho [28] and its occurrence in the Paraíba do Sul River is first reported here. We propose that this recent spread is due to construction of hydroelectric power dams or canals as occurred in the Upper Paraná River basin. These three watersheds are located in the most urbanized and exploited area of Brazil. Brazil has 1175 hydroelectric plants in operation (476 small, 498 medium and 201 large), most of them (45%) located along these three hydrographic basins [53]. There are also more 49 hydroelectric plants under construction and 173 planned [53]. It is likely that flooding behind dams has connected isolated streams or rivers from different hydrographic basins, such as in the aforementioned case of the Itaipu Dam. A similar well-recorded case occurred when the Piumhi River (with its 22 tributaries) which belonged to the Upper Paraná River basin was entirely transferred to São Francisco River basin after construction of the Furnas hydroelectric power dam [54]. This transposition introduced at list nine species from Upper Paraná River basin to São Francisco River basin [55,56,57,58,59]. However, in general, the impact of transpositions of rivers due to hydroelectric power dams are rarely recorded. The Paraíba do Sul River, the most anthropogenically impacted river of Brazil [8], is a well-documented case of the transposition of water by construction of canals [54,60], however, there are no studies about its impact on the fauna of the river basins. An alternative or additional explanation is the artificial spread of K. moenkhausii by the ornamental fish trade. The ornamental fish trade was valued at about US$330 million in 2009, selling approximately 2 billion specimens of almost 2,000 species [61,62]. It is likely that there are extensive intentional introductions or accidental escapes of fishes into non-native basins [64]. It is a plausible explanation for K. moenkhausii which is traded as an ornamental fish due its small size and attractive coloration in aquariums, although it is traded in relatively low numbers (data from Ministry of Development, Industry and Foreign Trade of Brazil MDIC ). The ornamental fish trade has been reported as the cause of many species introductions, for example in the Upper Paraná River basin where eleven fishes species were introduced [6,65] and the São Francisco river basin, where seven species were introduced [7]. The Paraíba do Sul River basin is documented to have 56 introduced species [66]. This high level reflects the fact that it is the major ornamental fish-farming center of Brazil with about 350 farms with many documented intentional and accidental escapes of fishes to neighboring streams and rivers [66,67]. Since K. moenkhausii is traded as an ornamental fish, it is likely that K. moenkhausii species has also been introduced into the São Francisco and Paraíba do Sul Rivers basins in this way, especially since the species is highly opportunistic, facilitating its adaptation to new habitats [25] and accounting for its rapid spread into different hydrographic basins. 5 Conclusions The analysis of mitochondrial COI and CytB sequences showed that K. moenkhausii represents a single panmictic species occupying the three hydrographic basins we studied (Upper Paraná, Paraíba do Sul, and São Francisco Rivers). The results are most consistent with the hypothesis that the presence of K. moenkhausii in the three hydrographic basins is due its introduction by human activities, probably due to either or both the transposition of natural barriers to dispersal or to intentional or accidental escapes caused by the trade in ornamental fishes. Our results confirm the efficacy and utility of the combination of DNA barcoding and GYMC analysis for helping to delimit and confirm species status and exploring the determinants of the geographic distribution of Neotropical freshwater fishes.

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