Delimiting Evolutionarily Significant Units of the Fish, Piaractus brachypomus

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1 Journal of Heredity, 2015, doi: /jhered/esv047 Symposium Article Symposium Article Delimiting Evolutionarily Significant Units of the Fish, Piaractus brachypomus (Characiformes: Serrasalmidae), from the Orinoco and Amazon River Basins with Insight on Routes of Historical Connectivity Maria Doris Escobar L., Juana Andrade-López, Izeni P. Farias, and Tomas Hrbek From the Laboratório de Evolução e Genética Animal, Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. Gen. Rodrigo Octávio Jordão Ramos, 3000, Campus Universitário, Bairro Coroado I, Manaus, AM, Brasil (Escobar, Farias, and Hrbek); Laboratorio Aplicado de Biogeografia y Bioacústica, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Bogotá, DF, Colombia (Escobar); Laboratório de Sistemática de Peces, Instituto de Zoología Tropical, Universidad Central de Venezuela, Caracas, Venezuela (Andrade-López); and Facultad de Ciencias Agrarias. Fundación Universitaria Juan de Castellanos, Boyacá, Colombia (Andrade-López). Address correspondence to Tomas Hrbek at the address above, or hrbek@evoamazon.net. Received August 5, 2014; First decision November 28, 2014; Accepted June 24, Corresponding Editor: Kathryn Rodriguez-Clark Abstract The freshwater fish Piaractus brachypomus is an economically important for human consumption both in commercial fisheries and aquaculture in all South American countries where it occurs. In recent years the species has decreased in abundance due to heavy fishing pressure. The species occurs in the Amazon and Orinoco basins, but lack of meristic differences between fishes from the 2 basins, and extensive migration associated with reproduction, have resulted in P. brachypomus being considered a single panmictic species. Analysis of 7 nuclear microsatellites, mitochondrial DNA sequences (D-loop and COI), and body shape variables demonstrated that each river basin is populated by a distinct evolutionarily significant unit (ESU); the 2 groups had an average COI divergence of 3.5% and differed in body depth and relative head length. Historical connection between the 2 basins most probably occurred via the Rupununi portal rather than via the Casiquiare canal. The 2 ESUs will require independent fishery management, and translocation of fisheries stocks between basins should be avoided to prevent loss of local adaptations or extinction associated with outbreeding depression. Introductions of fishes from the Orinoco basin into the Putumayo River basin, an Amazon basin drainage, and evidence of hybridization between the 2 ESUs have already been detected. Resumen Piaractus brachypomus, es una especie comercialmente importante para el consumo proveniente de las pesquerías y acuicultura en todos los país donde ocurre en Suramérica. En los últimos años se ha reportado la disminución de la abundancia de la especie debido a la presión de pesca. The American Genetic Association All rights reserved. For permissions, please journals.permissions@oup.com 428

2 Journal of Heredity, 2015, Vol. 106, Special Issue 429 Esta especie se encuentra distribuida en la cuenca del Amazonas así como en el Orinoco, pero la falta de diferencias merísticas entre los peces de las dos cuencas, asociado a la extensa migración reproductiva ha llevado a considerar a P. brachypomus como una especie panmítica. El análisis de siete microsatélites nucleares, secuencias de DNAm (D-loop y COI) y variables de la forma del cuerpo han demostrado que cada cuenca hidrográfica esta poblada por un grupo biológico distinto. Los dos grupos muestran una divergencia promedio de 3.5% con COI y diferencias en la profundidad del cuerpo y longitud de la cabeza, representando dos Unidades Evolutivas Significativas (ESU). Se ha considerado que las conexiones históricas entre las dos cuencas para esta especie es más probable que ocurra vía el portal del Rupununi que por el canal del Casiquiare. Los dos ESU requieren de manejo pesquero independiente y levitar la translocación de peces entre las dos cuencas. Por primera vez, se ha detectado la introducción de peces de la cuenca del Orinoco al río Putumayo un afluente del río Amazonas y evidencia de hibridación entre los ESUs. Subject areas: Conservation genetics and biodiversity; Population structure and phylogeography Key words: Casiquiare canal, geometric morphometrics, hybridization, microsatellites, mitochondrial DNA, Putumayo River, Rupununi portal An important goal of conservation genetic studies is to identify populations and thereby delineate management units that can be effectively monitored for the effects of anthropogenic activities (Olsen et al. 2014). Modern fishery management also aims to conserve genetic diversity within stocks as the basis for the administration and conservation of fishery resources (Allendorf and Ryman 1987; Dudgeon et al. 2012). The key to successful application of molecular methods in natural resource management is the evaluation of population differentiation to determine whether separate management strategies are needed and justified (Reiss et al. 2009). Molecularly assisted monitoring techniques have thus become one of the main tools for fishery managers to help guarantee sustainability of fish harvests and avoid fish resource depletion (Reiss et al. 2009). Evolutionarily significant units (ESUs) sensu Waples (1991) and Crandall et al. (2000) are one such category of differentiated populations. The principal criteria for delimiting ESUs are reproductive isolation (no gene flow), adaptive differentiation, reciprocal monophyly, and concordance among different data sets (genetics, morphology, behavior, life history, and geography). Among these, isolation and adaptive differentiation have been considered to be the most important to differentiate among units that merit conservation and separate management strategies (Palsbøll et al. 2007). ESUs are often viewed as species or as evolutionary lineages in incipient stages of speciation (Hey 2001; De Queiroz 2007). The Orinoco and Amazon basins share a considerable number of fish species (Reis et al. 2003), in spite of geological evidence suggesting that the 2 basins became separated during the early Miocene (Cooper et al. 1995; Lundberg et al. 1998). Piaractus brachypomus (Cuvier 1818) is one such shared species. Distributed widely in the Orinoco and Amazon River basins (Machado-Allison 1982; Jegú 2003), this large migratory fish is commercially important in Colombia, Peru, Bolivia, Venezuela, and Brazil (Goulding 1981; Loubens and Panfili 2001; Novoa 2002; Lasso et al. 2011a). As a seed consuming herbivore, it is also ecologically important for its role in seed dispersal (Machado-Allison 1982; Goulding 1989). Fishery studies in the Colombian Orinoco and Amazon River basins report that populations of P. brachypomus have drastically fallen in recent years (Ramírez-Gil and Ajiaco-Martínez 2001; Lasso et al. 2011b). A drastic reduction in harvested individuals from overfishing was also observed much earlier by Goulding (1979) for the upper Madeira River, a southern tributary of the Amazon River. Although management strategies have been drawn up in a number of the countries where this species occurs, they largely represent generic solutions in the absence of relevant data, broadly encompass other commercial species, and have not led to the restoration of specific target populations, calling into doubt their effectiveness for species conservation and management (Lasso et al. 2011b). Here, we report the results of the first study to take into consideration the entire geographic distribution of P. brachypomus in the Orinoco and Amazon River basins. The principal objective of our study was to test for population structuring between the 2 basins using an integrated molecular and morphological approach. We also evaluated potential routes of connectivity between the 2 basins and examined the practical implications of our results for the conservation and management of the species within each basin. Materials and Methods Sample Collection Tissue samples from P. brachypomus were collected from 5 localities in the Orinoco basin and 6 localities in the Amazon basin (Figure 1) totaling 215 individuals, 95 from the Orinoco and 120 from the Amazon basin. The samples were preserved in 95% ethanol and deposited in the CTGA-UFAM tissue collection maintained by the Laboratory of Evolution and Animal Genetics (LEGAL) at the Federal University of Amazonas (UFAM), Manaus, Brazil. Specimens were photographed, and 2 specimens from each basin were preserved in 10% formaldehyde and deposited in the ichthyological collections of the National Institute of Amazonian Research (INPA 39118), Manaus, Brazil, and the Guyana Hydrobiological Research Station of La Fundación La Salle, San Felix, Venezuela (CRI-EDIHG ). Additional specimens deposited at the Ichthyological Collection Center in the Museum of Biology, Central University of Venezuela (MBUCV V , MBUCV V , and MBUCV V ) and the National Institute of Amazonian Research, Manaus, Brazil (INPA 22329, INPA 20122, INPA 5790, and INPA 1098) were used for morphometric analyses. Molecular Data Collection DNA extraction was performed using CTAB 2% (Doyle and Doyle 1987) and phenol chloroform (Sambrook et al. 1989) methods.

3 430 Journal of Heredity, 2015, Vol. 106, Special Issue Figure 1. Collection localities and the geographic range of Piaractus brachypomus. N = sample size. Yellow shading (light gray) indicates distribution in the Orinoco River basin: (1) San José del Guaviare (N = 20), (2) Puerto López (N = 20), (3) Puerto Carreño (N = 20), (4) San Fernando de Apure (N = 20), (5) San Felix/ Ciudad Guayana (N = 15). Red shading (dark gray) indicates distribution in the Amazon Basin: (6) Boa Vista (N = 20), (7) Santarém (N = 21), (8) Janauacá/Manaus (N = 19), (9) Tefé (N = 20), (10) Letícia/Tabatinga (N = 20), (11) Puerto Asis (N = 20). Distribution in the Essequibo basin is in gray representing our uncertainty as to which group of P. brachypomus occurs there. Fish for morphometric analyses were collected in San Fernando de Apure, Orinoco, and Janauacá, Amazonas. Names in capital letters are principal basins. Oval maked by A is the Casiquiare canal; oval marked by B is the Rupununi portal. The red (gray) arrow indicates a potential connection between the Orinoco and Essequibo rivers resulting from the capture of the Cuyuni River by the Essequibo River. Black arrows indicate a potential connection between the Orinoco and Essequibo rivers via a coastal route. Quality of the extraction was evaluated on a 0.8% agarose gel stained with GelRed (Biotium). Quantification of DNA was determined spectrophotometrically by Nanodrop 2000 Thermo-Scientific and diluted to a final concentration of 50 ng/ml. Given that microsatellite markers have not been developed for P. brachypomus, we tested 16 microsatellite loci developed for the species Piaractus mesopotamicus (Calcagnotto et al. 2001) and the sister of the Piaractus clade, Colossoma macropomum (Santos et al. 2009). Seven of the 16 microsatellites tested were chosen for final analyses based on polymorphism and absence of null alleles (see below; see Supplementary Material 1 online). The microsatellites were amplified in a 10 μl polymerase chain reaction (PCR) using the following reaction conditions: 2.3 μl of ddh2o, 1.0 μl of dntp (2.5 mm), 1.0 μl of MgCl (25 mm), 1.0 μl of 10x buffer (750 mm Tris HCl ph 8.8 at 25 C, 200 mm (NH4)2SO4, 0.1% (v/v) Tween 20), 0.7 μl of primer forward (2 mm), 1.0 μl of primer reverse (2 mm), 0.3 μl of FAM labeled M13 primer, 0.5 μl Taq (1 U/μL), and 1.0 μl of DNA (50 ng). The PCR conditions were as follows: first cycle of 30 repetitions (10 s at 94 C, 30 s at primerspecific annealing temperature (Table 1), and extension at 68 C for 40 s), a second cycle of 20 repetitions (10 s at 94 C, 30 s at 53 C, and 30 s at 68 C), and a final extension at 72 C for 30 min. The amplified product was verified by electrophoresis on a 1% agarose gel. Genotyping was performed on the ABI 3130xl automated sequencer (Applied Biosystems) using 1.0 μl diluted PCR product (10 40 depending on the primer), 8.0 μl of formamide, and 1.0 μl of ROX size standard (DeWoody et al. 2004). Microsatellite alleles were sized using the software GeneMapper version 4.0 (Applied Biosystems). For the mitochondrial D-loop (Sivasundar et al. 2001) and COI (Ivanova et al. 2007) genes, PCR amplification was performed in a total volume of 15 μl with the following reaction conditions: 6.2 μl of ddh2o, 1.5 μl of dntp (2.5 mm), 1.5 μl MgCl (25 mm), 1.5 μl of 10x buffer (750 mm Tris HCl ph 8.8 at 25 C, 200 mm (NH4)2SO4, 0.1% (v/v) Tween 20), 1.5 μl of each primer (2 mm), 0.3 μl Taq (1.0 U/μL), and 1 μl of DNA (50 ng). The PCR reaction consisted of 35 cycles (60 s at 92 C, s at 48 C, and s at 68 C) and a final extension of 7 min at 72 C. The product was purified using ExoSAP-IT (USB Corporation) following the manufacturer s instructions. The purified PCR product was sequenced using Big Dye Terminator Kit V3.1 (Applied Biosystems) according to manufacturer s instructions, precipitated with EtOH/ EDTA and resolved in the 3130xl ABI automated sequencer (Applied Biosystems). The sequences were edited in the program BioEdit (Hall 1999) and aligned in Clustal W (Thompson et al. 1996). In analyses involving the COI gene we also included sequences of P. brachypomus (HQ420838, FJ978042), P. mesopotamicus (HQ420833, GU701416), C. macropomum (HQ420847), and Mylossoma duriventre (HM453212) obtained from GenBank. Morphometric Data Collection Thirty-four standardized digital photos from the left side of individuals of each basin were taken, representing 11 specimens from the Orinoco and 12 from the Amazon rivers (Figure 1). Standard length varied from 100 to 220 mm. On each image coordinates of 14 homologous points were recorded (see Supplementary Material 2 online) using TPS Dig software (Rohlf 2013).

4 Journal of Heredity, 2015, Vol. 106, Special Issue 431 Molecular Analyses Microsatellite data were analyzed for the presence of null alleles and their frequencies using the programs Micro-Checker (Van Oosterhout et al. 2004) and FreeNA (Chapuis and Estoup 2007). Extent of linkage disequilibrium between pairs of loci was evaluated in the program Arlequin 3.5 (Excoffier and Lischer 2010) using permutations; significance was estimated using sequential Bonferroni correction (Rice 1989). The number of biological populations in the dataset was estimated in the program Structure (Pritchard et al. 2000). Analyses were performed using the admixture and correlated allelic frequencies model. We generated 1 million topologies using the Markov Monte Carlo chain (MCMC), sampling every 1000th topology and discarding the first hundred 1000 topologies as burnin. Convergence was assessed via examination of α values. We ran analyses for K = 1 7, each with 20 independent replicates. The most likely number of biological populations was selected using the method of Evanno et al. (2005) implemented in the software Structure Harvester (Earl and VonHoldt 2012). For the most likely value of K, the proportion of allocation (Q) of the localities sampled within detected groups, and individual allocation ratio q (proportion of ancestral genome of each individual in the group) were estimated. Additionally, the differentiation (F ST ) between the biological groups was estimated using Arlequin 3.5 (Excoffier and Lischer 2010). The evolutionary relationships of individuals in the population were determined from the control region (D-loop). Maximum likelihood analysis (ML), generated in the Treefinder program (Jobb et al. 2004), was used to estimate the evolutionary model that best fit the data. The resulting topology was visualized in the program Haploviewer (Salzburger et al. 2011). The cohesion of the populations was inferred from control region sequence data by applying a coalescence analysis in the IMa2 software (Hey and Nielsen 2007). This analysis allowed us to estimate the size (θ), rate of immigration (m), and time since separation of the descendant populations (t). The analysis consisted of 25 independent MCMC runs, sampling 5 hundred 1000 genealogies after a burnin of genealogies. In the calculations a generation time of 3 years (age of reproductive maturity in females) was assumed (Novoa 2002; Landines-Parra and Mojica-Benítez 2005) and the mean substitution rate of nucleotides per site per year estimated from divergence of trans-isthmenian actinopterygian species pairs (Lessios 2008). The most likely phylogenetic relationship and divergence among COI haplotypes was estimated using the maximum likelihood framework implemented in the program Treefinder (Jobb et al. 2004), using a model of molecular evolution best fitting the data, also estimated in Treefinder. Phylogenetic support was assessed via 5000 bootstrap replicates. Colossoma macropomum and M. duriventre were used as outgroups (Ortí et al. 2008), and divergent time estimation was based on the mean substitution rate of nucleotides per site per year (Lessios 2008). Phylogenetic analyses were replicated in the software BEAST v (Drummond and Rambaut 2007) resulting in an identical topology. Morphometric Analyses The 14 homologous points were subjected to Procrustes transformation (Rohlf and Slice 1990) to translate, rotate, and scale each image, thus eliminating differences in position and size of each specimen (Zelditch et al. 2004). Transformed points were used to generate 25 linear variables representing the shape and contour of each individual fish using the Euclidean distance matrix analysis (EDMA). Linear variables were then used in a principal component analysis. All analyses were conducted in the program PAST 1.70 (Hammer et al. 2001). We directly tested for differences in shape between fish from the Orinoco versus Amazon basins using the Hotelling T 2 statistic (Dryden and Mardia 1998). Additionally, we carried out a generalized Procrustes transformation followed by a visualization on a thin plate spline grid of the transformation of the mean shape in each basin to the mean shape in the other basin. These analyses were carried out in the shapes statistical package (Dryden 2012) in R (R Development Core Team 2011). Data Archiving In fulfillment of data archiving guidelines (Baker 2013), we have deposited the primary genotype and morphometric data underlying these analyses with Dryad. All sequence data were deposited in GenBank under accession numbers KP to KP and KP to KP Two specimens from each basin were deposited in the ichthyological collections of the National Institute of Amazonian Research (INPA 39118), Manaus, Brazil, and the Guyana Hydrobiological Research Station of La Fundación La Salle, San Felix, Venezuela (CRI-EDIHG ). Results A total of 215 individuals, 95 from the Orinoco and 120 from the Amazon basin were analyzed using 7 polymorphic microsatellite loci. Analysis of the data in Micro-Checker suggested an excess of homozygotes, probably due to the presence of null alleles in the loci Pme4, Pme5, Pme20, and Cm1B8 in the Orinoco basin and Pme2, Pme4, Pme21, Cm1G7, and Cm1B6 in the Amazon basin. However, FreeNA analyses suggested no significant levels of null alleles. Uncorrected data and data corrected for the possible presence of null alleles using the method of Chapuis and Estoup (2007) resulted in similar F ST values: (95% CI: ) versus (95% CI: ), respectively. The difference was not statistically significant (Wilcoxon signed-rank test; W = 6; P > 0.05), so we applied no correction to the data. The data also showed no evidence of linkage disequilibrium after the Bonferroni correction for multiple comparisons (Rice 1989), leading us to believe that the loci were inherited independently. In the 7 polymorphic microsatellite loci analyzed, fish from the Amazon and Orinoco basins had a comparable average number of alleles per locus, but both observed and expected heterozygosities were lower in the Orinoco basin (Amazon basin: A = 6.3, H O = 0.68, H E = 0.72 vs. Orinoco basin: A = 6.1, H O = 0.55, H E = 0.66). Bayesian inference of population structuring suggested the existence of 2 biological populations, one in the Orinoco and another in the Amazon basin (K = 2; mean LnProb = ) (Figure 2). The value of attribution (Q) showed that each biological unit was composed of individuals with a high proportion of differentiated ancestral genome (Q > 0.99), except for the individuals sampled from Boa Vista and Puerto Asis (Figure 1, see Supplementary Material 3 online). On average, individuals from Boa Vista (N = 20) shared 7.4% of their genome with individuals from the Orinoco basin. Genome sharing as high as 34% was observed in 1 individual, with 3 individuals sharing more than 10% of their genome, and 7 sharing more than 5% of their genome. The ancestry of all individuals from the Puerto Asis (Putumayo River, Amazon basin) was divided approximately equally between the biological groups occupying the Amazon and Orinoco basins, suggesting that these individuals were F1 generation hybrids; all individuals also had mtdna haplotypes of fishes from the Orinoco basin (see below). We obtained 103 D-loop sequences of 733 bp length from 60 individuals from the Orinoco basin and 43 individuals from the

5 432 Journal of Heredity, 2015, Vol. 106, Special Issue Amazon basin. The evolutionary model that best fit the data was HKY85 + gamma. The maximum likelihood phylogenetic network indicated 2 groups separated by 35 mutational steps (Figure 3). One group contained all individuals from the Orinoco basin while the other group contained only individuals from the Amazon basin (Figure 3). The individuals from Puerto Asis, despite being geographically located within the Amazon basin, grouped with individuals from the Orinoco basin. All individuals from Boa Vista grouped together with the other Amazonian samples. Furthermore, application of the isolation-with-migration model to both the D-loop and the microsatellite data, excluding the Puerto Asis samples, supported a hypothesis of no gene flow between the 2 basins. The estimated time of divergence of the 2 populations under the isolation-withmigration model was 438 ky (95% HPD ky). Phylogenetic analyses of the COI gene showed a pattern of reciprocal monophyly for the sequences sampled from individuals from the Orinoco and Amazon basins ( ln likelihood = ), with the exception of the Puerto Asis samples, which again grouped with the Orinoco samples. Individuals from Boa Vista also again clustered together with other Amazonian samples. Sequences of P. brachypomus and P. mesopotamicus deposited in GenBank (HQ420838, FJ and HQ420833, GU701416) were phylogenetically nested within the Amazon basin clade (Figure 4). Mean Kimura-2-parameter divergence between the Orinoco and Amazon basin clades was 3.5% while the mean divergence of Piaractus spp. from C. macropomum was 22.8% and from M. duriventre was 25.6%. Mean divergence of P. mesopotamicus and P. brachypomus from the Amazon basin was 0%. Based on upper and lower estimates of COI divergence rates (1.03% per million years and 1.77% per million years, respectively) estimated from the comparison of actinopterygian sister species occurring on either side of the Isthmus of Panama (Lessios 2008), we estimated the divergence of the Orinoco and Amazon clades of P. brachypomus to have occurred between 1.95 and 3.36 million years ago with a mean divergence of 2.54 million years ago. The Orinoco and Amazon basin clades also had significant differences in body shape (Hotelling T 2 = , P < ). Individuals from the Amazon basin were relatively deeper in the mid body, and had a relatively short head and snout in the anterior to posterior direction, with a less upturned mouth (Figure 5). When shape was summarized via principal components (see Supplementary Material 4 online), the first principal component (PC1) had contributions primarily from measurements describing body depth, while the second principal component (PC2) had contributions from measurements describing relative length of the head and mouth. Additionally, specimens from the Amazon basin had larger eye diameter, longer anal fins, and greater distances between the posterior margin of the dorsal fin and the origin of the adipose fin compared with the Orinoco basin individuals. Amazon basin individuals generally varied more in shape than Orinoco basin individuals. Discussion Population Structure The Orinoco and the Amazon basins arose as independent drainages with the rise of the Vaupes arch during the early Miocene (Cooper et al. 1995; Lundberg et al. 1998). In spite of this separation, they Figure 2. Results of structure analysis. Yellow/light gray symbolizes the genome of Piaractus brachypomus from the Orinoco basin. Red/dark gray symbolizes the genome of P. brachypomus from the Amazon basin. Figure 3. Phylogenetic network of Piaractus brachypomus D-loop haplotypes sampled from the Orinoco basin (yellow/light gray) and the Amazon basin (red/ dark gray).

6 Journal of Heredity, 2015, Vol. 106, Special Issue Putumayo_1 Putumayo_2 SanFelix_1 Meta_25 Meta_24 Guaviare_16 Guaviare_15 Apure_3 Apure_1 SanFelix_3 P. brachypomus_cepta007 Janauaca_2 Branco_5 P. mesopotamicus_lbp23804 P. brachypomus_n192 P. mesopotamicus_cepta001 Branco_3 LaPedrera_2 LaPedrera_1 Janauaca_1 Itaituba_2 Itaituba_1 GuajaraMirim_2 GuajaraMirim_1 Santarem_10 Santarem_ Colossoma macropomum Mylossoma duriventre P. brachypomus Orinoco basin P. brachypomus P. mespotamicus Amazon basin Paraguay basin Figure 4. Phylogenetic tree of Piaractus brachypomus based on the mitochondrial COI gene. Note that Piaractus mesopotamicus is a phylogenetic member of the P. brachypomus clade from the Amazon basin. Number above branches are bootstrap values. are currently connected via the Casiquiare canal (Winemiller and Willis 2011) and alternate corridors have been proposed between the upper Orinoco and Negro Rivers (Winemiller and Willis 2011) or between the delta of the Orinoco and the Branco River (Lovejoy and Araújo 2000; Hubert and Renno 2006). Due to this shared history and current connectivity, a number of aquatic species are found in both basins, many of them in the upper Orinoco and the upper Negro Rivers. Piaractus brachypomus is one of these, but it is one of the few species that has a basin-wide distribution in both the Amazon and the Orinoco basins. Additionally, both P. brachypomus and P. mesopotamicus are long distance migrants, and very important and effective seed dispersers (Galetti et al. 2008; Anderson et al. 2009). In spite of theoretical connectivity between the populations, evidence from both morphological and molecular data indicated that P. brachypomus forms independent evolutionary units in the Orinoco and Amazon basins. Fish from the Amazon basin were more robust, and had shorter snouts (Figure 5), relatively larger eyes, and a larger distance separating the dorsal and adipose fins. The 2 groups are independent biological entities significantly differentiated from each other at nuclear loci (F ST = ; P < 0.001), with individuals in each group having over 99% probability of belonging to just one biological group (Figure 2). The only 2 exceptions were individuals from Boa Vista (Q = 94%), suggesting recent connectivity between or population divergence of the Orinoco and Amazon groups in the region of Boa Vista; and the individuals from Puerto Asis (Q = 46%), suggesting hybridization. Fishes from the 2 basins were also reciprocally monophyletic (Figures 3 and 4) the exception being the Puerto Asis sample with an average between-basin pairwise divergence of 3.5%. This pairwise divergence corresponds to the split of the Orinoco and Amazon clades of P. brachypomus to have occurred between 1.95 and 3.36 million years ago with a mean divergence of 2.54 million years ago. Surprisingly, P. mesopotamicus was phylogenetically nested within the Amazon group of P. brachypomus (Figure 4). All the sequences deposited in Genbank resulted in the same inference. The P. mesopotamicus sequences were generated in 4 independent studies, and thus likely characterize the species. This result is, however, not entirely unexpected. In a study published by Ortí et al. (2008), P. brachypomus and P. mesopotamicus are also not reciprocally monophyletic. Potential Routes of Connectivity Differentiation between the Orinoco and Amazon groups of P. brachypomus postdated the rise of the Vaupes arch and the separation of the 2 basins (~2.5 million years ago vs. ~10.5 million years ago Hoorn et al. 1995). The rise of the Vaupes arch is thought to have played a major role in divergence of the faunas of the 2 basins (Lundberg et al. 1998). The 2 basins and their faunas are connected via the Casiquiare canal, but the canal also acts as an ecological filter (Winemiller et al. 2008) due to strong gradients in many physicochemical factors, and thus limits the distribution of species. This region also seems to act as an ecological filter for P. brachypomus. Museum records for P. brachypomus in the upper Negro and Orinoco rivers are nonexistent and the inability to collect specimens as well as our informal interviews with artisanal fishermen suggest that the species does not occur in this region. Life history and ecological data suggest that Piaractus is mostly restricted to clear and white waters, exceptions being the black waters of lower Negro and Caroni Rivers, Venezuela, and thus it is unlikely that P. brachypomus would use the Casiquiare canal or shallow floodplain connections between the black water headwaters of the Negro and Orinoco rivers (Winemiller and Willis 2011), to move between the 2 basins. The observed divergence is best explained by an alternative connection between the Orinoco and Amazon basins via the Essequibo River and the Rupununi portal (Lovejoy and Araújo 2000). Based on surveys of fish fauna, there is evidence that the Cuyuni River, in the past part of the Caroni-Orinoco River drainage, was captured by the Mazaruni River which drains into the Essequibo River (Lasso et al. 1990). The Essequibo itself is connected via the flooded savannahs of the Rupununi River and the Takutu River to the Branco River which drains into the lower Negro River. Until about 1 million years ago, the upper Branco River drained into the Essequibo River, and only in the mid Pleistocene was it captured by the Negro River to become part of the Amazon basin (Schaefer and Vale Júnior 1997). The connection via the Rupununi portal and the Essequibo River seems plausible. Piaractus brachypomus is found in the clear or lightly

7 434 Journal of Heredity, 2015, Vol. 106, Special Issue a Amazonas to Orinoco transformation relative body depth relative body length b Orinoco to Amazonas transformation relative body depth relative body length Figure 5. Thin plate spline grid visualizing the transformation of the mean shape of Orinoco individuals to the mean shape of Amazon individuals (a), and vice versa (b). turbid waters of the Paragua River, an affluent of the Caroni-Orinoco system (Samudio et al. 2008), and the Essequibo River (Lowe- McConnell 1962) including its Mazaruni and Cuyuni tributaries (Watkins et al. 2005); and it is the Cuyuni drainage that was apparently captured from the Paragua-Caroni drainage of the Orinoco system (Lujan and Armbruster 2011). Such an alternate connection via the Essequibo River would explain the presence of P. brachypomus in the Branco River system, and its apparent absence in the central and upper Negro River (Figure 1). The Essequibo connection would also explain the presence of a small portion, on average 7.4% but with 1 individual sharing at least 36%, of the genome of the Orinoco clade in the fishes of Boa Vista, Branco River drainage. Instead of stream capture, Hubert and Renno (2006) proposed dispersal via a coastal route. The authors propose that during the Pleistocene the deltas of the Orinoco, Essequibo, and Amazon rivers were in closer proximity, and the greater quantity of water discharged would have formed a freshwater or brackish corridor between the 3 deltas. Such a corridor would have allowed the interchange of freshwater faunas, and according to Hubert and Renno (2006) explains the large number of shared characid taxa between the Orinoco and Amazon rivers and intervening Guianan drainages. Piaractus brachypomus is apparently not found in the Amazon delta, but is common and abundant in the Orinoco delta. Additionally, it is capable of tolerating 18% salinity (Novoa 2002; Lasso and Sánchez-Duarte 2011), and has been reported in freshwater plumes which form in the Atlantic Ocean past the Orinoco delta during the rainy season (Mees 1974). Therefore, P. brachypomus potentially could have dispersed via the coastal route between the Orinoco and the Essequibo

8 Journal of Heredity, 2015, Vol. 106, Special Issue 435 deltas, and then between the Essequibo and Branch Rivers via the floodplains of the Rupununi savannah. Whether via drainage capture or via dispersal along the coast, the connection and separation of P. brachypomus of the Amazon and Orinoco basins seems more likely to have occurred via the Essequibo and the Rupununi portal rather than the connection between the upper Orinoco and Negro rivers. In addition to this study, only 3 published studies have provided evidence for the Essequibo and the Rupununi portal route. In the study of the needlefishes of the genus Potamorrhaphis where Lovejoy and Araújo (2000) proposed connection between the 2 basins via the Rupununi portal, the authors observed a 3.5% pairwise sequence divergence between localities from the Orinoco and lower Amazon basins. Using the mean substitution rate of nucleotides per site per year estimated from divergence of trans-isthmenian actinopterygian species pairs (Lessios 2008), we arrive at 2.50 million years divergence between the 2 groups. Similarly, Turner et al. (2004) observed 2.2% sequence divergence between Prochilodus clades from the Orinoco and Negro + Essequibo basins, suggesting a divergence of 1.57 million years. Lastly, Hrbek et al. (2005) observed sister taxon relationship between the species Austrofundulus transilis from the Venezuelan llanos and the Unare River basin, and the species Austrofundulus rupununensis from the Rupununi savannah. The Rupununi savannah is drained by the Rupununi (Essequibo drainage) and the Takutu (Branco drainage) rivers, and regularly floods, thus connecting the 2 basins. Although Hrbek et al. (2005) did not estimate the time of this divergence, a calibration based on a rate of substitutions per site per year (from Hrbek and Larson 1999) suggests a divergence of 2.95 million years ago. Thus, the divergences observed in Austrofundulus spp., Potamorrhaphis spp., and Prochilodus spp. shared between the 2 basins are comparable with the 2.54 million years divergence observed in P. brachypomus in this study. These, as well as the current study cast doubt on the preeminent role of the uplift of the Vaupes arch at approximately 10.5 million years ago (Cooper et al. 1995) in driving vicariant diversification of the aquatic faunas of the Orinoco and Amazon basins. Conservation Recommendations and a Case Study from the Colombian Amazon The Orinoco and Amazon basin populations of P. brachypomus are differentiated morphologically, and molecularly in both the mitochondrial and nuclear genomes. Therefore, based on the multiple criteria proposed by Waples (1991), Moritz (1994) and Crandall et al. (2000), the 2 groups are sufficiently differentiated to be considered ESUs. Independent studies of population parameters, reproductive seasonality, and other life-history characteristics are the next step in order to manage these ESUs as independent stocks and produce scientifically founded fisheries regulations that address such factors as minimum size of capture, fishing season limits, and repopulation and reintroduction strategies tailored for each ESU/basin. This recommendation for management of the populations as separate ESUs cannot be stressed enough, as the taxonomic impediment in conservation and management policies is enormous (Mace 2004). In spite of finding 2 clear ESUs, we also found clear evidence for hybridization (Figure 2); this may be the result of aquaculture programs that are also highly relevant to conservation efforts. Following a decades-long conflict between the Colombian government and various rebel groups which controlled large rural areas, peace is beginning to return to Colombia. Given that much of the conflict has been rooted in poverty, uneven distribution of wealth and resources, and inaccessibility of government to principally rural sections of the society (Arnson et al. 2004), the government has therefore taken up an ambitious program of social inclusion and socioeconomic reconstruction. One such socioeconomic rural development initiative is centered on aquaculture programs and the restoration of wild fish stocks (Agudelo Cordoba et al. 2006; Gutiérrez et al. 2012). However, even at this early stage of its implementation, it is clear that problems with this program have emerged. The aquaculture and restoration program focuses on a mix of native and nonnative species (Cevallos Ruiz 2006), with a number of the nonnative species, such as tilapia (species of the African genus Oreochromis), known to be highly invasive (Zengeya et al. 2013). Of the native species, the majority are restricted to a single basin; however, 2 species, P. brachypomus and C. macropomum, are shared between the Orinoco and Amazon basins. For historical reasons, aquaculture stocks are derived from fishes from the Orinoco basin, which in the case of P. brachypomus represents a different ESU than that which occurs in the Amazon basin. (For the other species, we do not know if the Orinoco and Amazon basins contain different ESUs.) As aquaculture expands into the Colombian Amazon, the Orinoco ESU is being introduced into the Amazon basin under the assumption that P. brachypomus of the Orinoco and Amazon basins are one and the same. As with any nonlocal aquaculture species, the species may escape from aquaculture stations or be intentionally released, and may become locally established with ecological as well as genetic consequences for the local species or ESU (e.g., Naylor et al. 2005; Zengeya et al. 2013). We have evidence of such events from Puerto Asis in the upper Putumayo River, Amazon River basin. The animals sampled from this region are of hybrid origin with an average of 54% of their genome being from the Orinoco ESU and 46% from the Amazon ESU. These individuals were most likely first-generation hybrids between females of the Orinoco ESU and males of the Amazon ESU. The fertility of these probable hybrids is still unclear, that is, if postzygotic isolating mechanisms exist since clearly prezygotic isolating mechanisms do not. Depending on the number of individuals of the Orinoco ESU being introduced, the existence of postzygotic isolating mechanisms could lead to severe reduction of numbers or even local extinction of the Amazon ESU in the upper Putumayo River basin. Whether the Orinoco ESU individuals are escaped or intentionally released aquaculture individuals is unclear; however, intentional release is probably more likely, since 1 of the 2 main objectives of the socioeconomic rural development initiative is the restoration of wild fish stocks via introductions (Agudelo Cordoba et al. 2006). The upper Putumayo is a petroleum-producing region, and as such repopulation with native fishes is carried out within the scope of environmental compensation initiatives. It is unlikely that there is current or recent historical connectivity between the 2 basins via their headwater regions. The Putumayo River is at least 200 km south of the Guaviare River, the southernmost distribution of P. brachypomus in the Orinoco basin. Between these 2 drainages lie the Caqueta and Apaporis Rivers. Piaractus brachypomus does not occur in the Apaporis, or upstream of the Araracuara rapids in the middle Caqueta; and in the lower reaches of the Caqueta where P. brachypomus occurs, we found no evidence of hybridization between the 2 ESUs. Despite uncertainties about how the Orinoco ESU was introduced into the Putumayo River ecosystem, and whether it is also present in other Amazonian drainages of Colombia, this example clearly points out the lack of basic taxonomic knowledge for even economically important South American fishes. The knowledge gap is enormous with an estimated 50% of South American fish species yet to be described (Reis et al. 2003) and is not confined to small, cryptic, or commercially irrelevant species. Large and commercially

9 436 Journal of Heredity, 2015, Vol. 106, Special Issue important fishes such as the multiple species of catfishes of the genus Pseudoplatystoma (Buitrago-Suárez and Burr 2007), and the peacock basses of the genus Cichla (Kullander and Ferreira 2006) have only recently been described. Similar taxonomic deficit affects other groups, including the supposedly well-known South American mammalian megafauna (e.g., Cozzuol et al. 2013; Hrbek et al. 2014). The taxonomic impediment can have serious practical consequences, with outbreeding depression derailing well-meaning socioeconomic programs, and undermining conservation and management policies (Mace 2004). Restocking efforts with different source populations can often lead to outbreeding depression through the disruption of co-adapted gene complexes thus rather than rescuing, further endangering the local population (Huff et al. 2011). Outbreeding depression also brings important detrimental consequences to aquaculture and management practices (Gilk et al. 2004). In the case when hybridization occurs between highly divergent lineages, outbreeding depression can lead to extinction (Templeton 1986). Therefore, we strongly advocate investment in scientific studies that will characterize population structure and identify ESUs and principally locally adapted populations (Funk et al. 2012) of species that are of commercial importance and areas likely to be intensively managed or cultured. The importance of these studies cannot be overemphasized especially for those situations where the same species is shared across different basins. Supplementary Material Supplementary material can be found at Funding Conselho Nacional de Desenvolvimento Científico e Técnológico (CNPq / to T.H.; CNPq/PPG / and CNPq/CT-Amazon / to I.P.F.), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) PhD fellowship to M.D.E.L., and CNPq research fellowship to I.P.F. (306804/2013-3) and T.H. (303646/2010-1). Acknowledgments We especially thank Antonio Machado-Allison from the Tropical Zoology Institute of the Central University of Venezuela for the assistance in the morphological analyses, and Nirson Gonzalez, Aniello Barbarino, Rosa Elena Martinez Ajiaco, Luis Francisco Cubillos, Luz Marina Rodriguez, Henry Elias Yucuna Kubeo, Maria da Conceição Pires, and Adam Leão for collaborating with field sampling. We also thank SISBIO/Brazil, INDECOR/Colombia, and INSOPESCA/Venezuela for providing collecting permits. Finally, we thank Kathryn Rodriguez-Clark for giving us the opportunity to contribute to this special issue. This study is part of M.D.E.L. s Doctoral thesis in the Biological Diversity graduate program of UFAM. References Agudelo Cordoba E, Alonso JC, Moya Ibañez LA Perspectivas para el ordenamiento de la pesca y la acuicultura en el área de investigación fronteriza Colombo Peruana del río Putumayo. Bogotá (Colombia): Instituto Amazónico de Investigaciones Científicas SINCHI & Instituto Nacional de Dessarrollo del Perú INADE. Allendorf FW, Ryman N Genetic management of hatchery stocks. In: Ryman N, Utter F, editors. Population genetic and fishery management. Seattle (WA): University of Washington Press. p Anderson JT, Rojas SJ, Flecker AS High-quality seed dispersal by fruiteating fishes in Amazonian floodplain habitats. Oecologia. 161: doi: /s Arnson CJ, Chueca Mora A, Gómez Buendía H, Schaerer JP The social and economic dimensions of conflict and peace in Colombia. Washington (DC): The Wilson Center. Baker CS Journal of heredity adopts joint data archiving policy. J Hered. 104:1. doi: /jhered/ess137 Buitrago-Suárez UA, Burr BM Taxonomy of the catfish genus Pseudoplatystoma Bleeker (Siluriformes: Pimelodidae) with recognition of eight species. Zootaxa. 1512:1 38. Calcagnotto D, Russello MA, DeSalle R Isolation and characterization of microsatellite loci from Piaractus mesopotamicus and their applicability in other Serrasalminae fish. Mol Ecol Notes. 1: Cevallos Ruiz L Estado actual de la piscicultura en el su de la Amazonia Colombiana. Rev Electrónica Ingeneiría en Prod Acuícola. 2:1 5. Available from: Chapuis M-P, Estoup A Microsatellite null alleles and estimation of population differentiation. Mol Biol Evol. 24: doi: /molbev/msl191 Cooper MA, Addison FT, Alvarez R, Coral M, Graham RH, Hayward SH, Martinez J, Naar J, Peñas R, Pulham AJ, et al Basin development and tectonic history of the Llanos Basin, Eastern Cordillera, and Middle Magdalena Valley, Colombia. Am Assoc Pet Geol Bull. 79: Cozzuol MA, Clozato CL, Holanda EC, Rodrigues FHG, Nienow S, de Thoisy B, Redondo RAF, Santos FR A new species of tapir from the Amazon. J Mammal. 94: doi: /12-mamm-a Crandall KA, Bininda-Emonds ORP, Mace GM, Wayne RK Considering evolutionary processes in conservation biology. Trends Ecol Evol. 15: De Queiroz K Species concepts and species delimitation. Syst Biol. 56: doi: / DeWoody JA, Schupp J, Kenefic L, Busch J, Murfitt L, Keim P Universal method for producing ROX-labeled size standards suitable for automated genotyping. Biotechniques. 37: Doyle JJ, Doyle JL A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 19: Drummond AJ, Rambaut A BEAST: Bayesian Evolutionary Analysis by Sampling Trees. BMC Evol Biol. 7:214. doi: / Dryden IL shapes package. 42. Available from: Dryden IL, Mardia KV Statistical shape analysis. Chichester (UK): John Wiley and Sons. Dudgeon CL, Blower DC, Broderick D, Giles JL, Holmes BJ, Kashiwagi T, Kruck NC, Morgan JAT, Tillet BJ, Ovenden JR A review of the application of molecular genetics for fisheries management and conservation of sharks and rays. J Fish Biol. 80: doi: /j x Earl DA, VonHoldt BM STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour. 4: doi: /s Evanno G, Regnaut S, Goudet J Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 14: doi: /j x x Excoffier L, Lischer HEL Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 10: doi: /j x Funk WC, McKay JK, Hohenlohe PA, Allendorf FW Harnessing genomics for delineating conservation units. Trends Ecol Evol. 27: doi: /j.tree Galetti M, Donatti CI, Pizo MA, Giacomini HC Big fish are the best: Seed dispersal of Bactris glaucescens by the pacu fish (Piaractus mesopotamicus) in the Pantanal, Brazil. Biotropica. 40: doi: / j x Gilk SE, Wang IA, Hoover CL, Smoker WW, Taylor SG, Gray AK, Gharrett AJ Outbreeding depression in hybrids between spatially separated pink salmon, Oncorhynchus gorbuscha, populations: marine survival, homing ability, and variability in family size. Environ Biol Fishes. 69: doi: /b:ebfi c1 Goulding M Ecologia de Pesca do Rio Madeira. Manaus (Brazil): INPA. Goulding M Man and fisheries on an Amazon frontier. The Hague (Belgium): Dr. W. Junk Publishers.

10 Journal of Heredity, 2015, Vol. 106, Special Issue 437 Goulding M Amazon: the flooded forest. London (UK): BBC Books. Gutiérrez F de P, Sánchez-Duarte P, Lasso CA Piaractus brachypomus (Cuvier 1818). In: Gutiérrez F de P, Lasso CA, Baptite MP, Sánchez-Duarte P, Díaz AM, editors. VI. Catálogo de la biodiversidad acuática exótica y trasplantada en Colombia: moluscos, crustáceos, peces, anfibios, reptiles y ave. Serie Editorial Recursos Hidrobiológico y Pesqueros Continentales de Colombia. Bogotá (Colombia): Instituto de Investigación de los Recursos Biológicos Alexander von Humboldt (IAvH). p Hall T BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 41: Available from: programs/bioedit/bioedit.html Hammer Ø, Harper DAT, Ryan PD PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron. 4:1 9. Hey J Genes, categories and species: the evolutionary and cognitive causes of the species problem. New York (NY): Oxford University Press. Hey J, Nielsen R Integration with the Felsenstein equation for improved Markov chain Monte Carlo methods in population genetics. Proc Natl Acad Sci U S A. 104: doi: /pnas Hoorn C, Guerrero J, Sarmiento GA, Lorente MA Andean tectonics as a cause for changing drainage patterns in Miocene northern South America. Geology. 23: doi: / (1995)023<0237:ata ACF>2.3.CO;2 Hrbek T, da Silva VMF, Dutra N, Gravena W, Martin AR, Farias IP A new species of river dolphin from Brazil or: how little do we know our biodiversity. PLoS One. 9:e doi: /journal.pone Hrbek T, Larson A The evolution of diapause in the killifish family Rivulidae (Atherinomorpha, Cyprinodontiformes): a molecular phylogenetic and biogeographic perspective. Evolution (N Y). 53: doi: / Hrbek T, Taphorn DC, Thomerson JE Molecular phylogeny of Austrofundulus Myers (Cyprinodontiformes: Rivulidae), with revision of the genus and the description of four new species. Zootaxa. 825:1 39. Hubert N, Renno J-F Historical biogeography of South American freshwater fishes. J Biogeogr. 33: doi: /j x Huff DD, Miller LM, Chizinski CJ, Vondracek B Mixed-source reintroductions lead to outbreeding depression in second-generation descendents of a native North American fish. Mol Ecol. 20: doi: / j x x Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes. 7: doi: /j x Jegú M Subfamily Serrasalminae. In: Reis RE, Kullander SO, Ferraris CJ, editors. Check list of the freshwater fishes of South and Central America. Porto Alegre (Brazil): EDIPUCRS. p Jobb G, Haeseler A von, Strimmer K TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics. BMC Evol Biol. 4:18. doi: / Kullander SO, Ferreira EJG A review of the South American cichlid genus Cichla, with descriptions of nine new species (Teleostei: Cichlidae). Ichthyol Explor Freshwaters. 17: Landines-Parra MA, Mojica-Benítez HO Manejo y reproducción de carácidos. In: Daza PV, Landines-Parra MA, Sanabria-Ochoa AI, editors. Reproducción de Peces en el Trópico. Bogotá (Colombia: INCODER). p Lasso CA, Agudelo Cordoba E, Jiménez L, Ramirez-Gil H, Morales-Betancourt MA, Ajiaco-Martínez RE, Paula-Gutiérrez F, Usma-Oviedo JS, Muñoz S, Sanabria AI. 2011a. Catalogo de los recursos pesqueros continentales de Colombia. Bogotá (Colombia): Instituto Alexander von Humboldt. Lasso CA, Gutiérrez F de P, Morales-Betancourt MA, Agudelo Cordoba E, Ramírez-Gil H, Ajiaco-Martínez RE. 2011b. Serie Editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia II. Pesquerías Continentales de Colombia: Cuencas del Magdalena-Cauca, Sinú, Canalete, Atrato, Orinoco, Amazonas y Vertiente del Pacífico. Bogotá (Colombia): Instituto Alexander von Humboldt. Lasso CA, Machado-Allison A, Hernandez RP Consideraciones zoogeograficas de los peces de La Gran Sabana (Alto Caroni) Venezuela, y sus relaciones con las cuencas vecinas. Mem Soc Ciencias Nat La Salle. 20: Lasso CA, Sánchez-Duarte P Los peces del delta del Orinoco. Diversidad, bioecología, uso y conservación. Caracas (Venezuela): Fundación La Salle de Ciencias Naturales y Chevron C.A. Lessios HA The Great American Schism: divergence of marine organisms after the rise of the Central American Isthmus. Annu Rev Ecol Evol Syst. 39: doi: /annurev.ecolsys Loubens G, Panfili J Biologie de Piaractus brachypomus (Teleostei: Serrasalmidae) dans le bassin du Mamoré (Amazonie bolivienne). Ichtyology Explor Freshwaters. 12: Lovejoy NR, De Araújo ML Molecular systematics, biogeography and population structure of neotropical freshwater needlefishes of the genus Potamorrhaphis. Mol Ecol. 9: Lowe-McConnell RH Note on the fishes in Georgetown fish markets and their seasonal fluctuations. Fish Bull Dep Agric Georg Guyana. 4:1 31. Lujan NK, Armbruster JW The Guiana Shield. In: Albert JS, Reis RE, editors. Historical biogeography of neotropical freshwater fishes. Berkeley (CA): University of California Press. p Lundberg JG, Marshall LG, Guerrero J, Horton B, Malabarba MCSL, Wesselingh FP The stage for Neotropical fish diversification: a history of tropical South American rivers. In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS. editors. Phylogeny and classification of neotropical fishes. Porto Alegre (Brazil): EDIPUCRS. p Mace GM The role of taxonomy in species conservation. Philos Trans R Soc London B Biol Sci. 359: doi: /rstb Machado-Allison A Estudios sobre la subfamilia Serrasalminae (Teleostei, Characidae). Parte 1. Estudio Comparado de los Juveniles de las Cachamas de Venezuela (Genero: Colossoma y Piaractus). Acta Biol Venez. 11: Mees GF The Auchenipteridae and Pimelodidae of Suriname (Pisces, Nematognathi). Zool Verh. 132: Moritz C Defining evolutionarily significant units for conservation. Trends Ecol Evol. 9: Naylor R, Hindar K, Fleming IA, Goldburg RJ, Williams S, Volpe J, Whoriskey F, Eagle J, Kelso D, Mangel M Fugitive salmon: assessing the risks of escaped fish from net-pen aquaculture. Bioscience. 55: doi: / (2005)055[0427:fsatro]2.0.co;2 Novoa D Los recursos pesqueros del eje fluvial Orinoco Apure: presente y futuro. Caracas (Venezuela): INAPESCA. p Olsen MT, Andersen LW, Dietz R, Teilmann J, Härkönen T, Siegismund HR Integrating genetic data and population viability analyses for the identification of harbour seal (Phoca vitulina) populations and management units. Mol Ecol. 23: doi: /mec Ortí G, Sivasundar A, Dietz K, Jégu M Phylogeny of the Serrasalmidae (Characiformes) based on mitochondrial DNA sequences. Genet Mol Biol. 31: doi: /s Palsbøll PJ, Bérubé M, Allendorf FW Identification of management units using population genetic data. Trends Ecol Evol. 22: doi: /j. tree Pritchard JK, Stephens M, Donnelly P Inference of population structure using multilocus genotype data. Genetics. 155: R Development Core Team R: a language and environment for statistical computing. Vienna (Austria): R Foundation for Statistical Computing. Ramírez-Gil H, Ajiaco-Martínez RH La pesca en la Baja Orinoquia colombiana: pasado, presente y futuro. Boletín Científico Inpa. 7: Reis RE, Kullander SO, Ferraris CJ Check list of the freshwater fishes of South and Central America. Porto Alegre (Brazil): EDIPUCRS. p Reiss H, Hoarau G, Dickey-Collas M, Wolff WJ Genetic population structure of marine fish: mismatch between biological and fisheries management units. Fish Fish. 10: doi: /j x Rice WR Analyzing tables of statistical tests. Evolution (N Y). 43: doi: / Rohlf FJ tpsdig version Stony Brook (NY): Department of Ecology and Evolution, State University of New York at Stony Brook.