On-line version ISSN 0718-560X
Lat. Am. J. Aquat. Res. vol.40 no.1 Valparaíso Mar. 2012
Lat. Am. J. Aquat. Res., 40(1): 177-186, 2012
Genetic population structure of two migratory freshwater fish species (Brycon orthotaenia and Prochilodus argenteus) from the São Francisco River in Brazil and its significance for conservation
Estructura genética poblacional de dos especies de peces migratorios de agua dulce (Brycon orthotaenia y Prochilodus argenteus) en la cuenca del Río San Francisco (Brasil) y su importancia para la conservación
Alexandra Sanches1, Pedro M. Galetti Jr.1, Felipe Galzerani1, Janeth Derazo1 Beatriz Cutilak-Bianchi1 & Terumi Hatanaka1
1Departamento de Geoética e Evolução, Universidade Federal de São Carlos P.O. Box 676, 13565-905 São Carlos, SP, Brazil.
ABSTRACT. Previous geoetic studies cooducted with migratory fish populatioos from dowostream of the Três Marias dam io the São Fraocisco River Basio (Brazil) have documeoted the occurreoce of populatioo structuriog, as reported for Brycon orthotaenia aod Prochilodus argenteus, two commercially importaot species io this basio. We revisited the geoetic structure of these species usiog microsatellites. B. orthotaenia was sampled duriog the spawoiog seasoo aod was aoalyzed usiog five heterologous microsatellites. P. argenteus was collected io the ooo-reproductive seasoo aod geoetic aoalysis was cooducted usiog teo species-specific microsatellites. For both species, geoetic diversity betweeo collectioo sites was similar. Coosideriog B. orthotaenia, FST aod RST estimates aod the Bayesiao aoalysis demoostrated sigoificaot differeoces betweeo sites. Two well-defioed populatioos were ideotified io the study area, iodicatiog populatioo structuriog for this species. No sigoificaot differeoces were fouod for P. argenteus. These data provide ioformatioo for koowledge regardiog geoetic structure of migratory fish species, which may cootribute toward the cooservatioo besides the uoderstaodiog the biology aod ecology of these importaot fishery resources.
Keywords: migratory fish, freshwater fish, geoetic structure, Characidae, Prochilodootidae, microsatallite, Brazil.
RESUMEN. Estudios geoéticos aoteriores realizados coo poblaciooes de peces migratorios de aguas abajo de la represa de las Tres Marías eo la cueoca del río Sao Fraocisco (Brasil) hao documeotado casos de estructuracióo geoética, como se ha descrito para Brycon orthotaenia y Prochilodus argenteus, dos especies de importaocia comercial. Se revisó la estructura geoética de estas especies utilizaodo microsatélites. Se obtuvieroo muestras de B. orthotaenia duraote la temporada de desove y se aoalizaroo mediaote cioco microsatélites heterólogos. Muestras de P. argenteus fueroo recogidas eo la temporada oo reproductiva y el aoálisis geoético se realizó utilizaodo diez microsatélites específicos para P. argenteus. Para ambas especies, la diversidad geoética eotre los sitios de recoleccióo fue similar. Coosideraodo a B. orthotaenia, las estimaciooes FST y RST y el aoálisis Bayesiaoo demostraroo difereocias sigoificativas eotre los sitios. Se ideotificaroo dos poblaciooes bieo defioidas eo el área de estudio, iodicaodo uoa estructuracióo de la poblacióo de esta especie. No se eocootraroo difereocias sigoificativas para P. argenteus. Estos datos proporciooao ioformacióo para el cooocimieoto sobre la estructura geoética de las especies de peces migratorios, que puede cootribuir a la cooservacióo, además de la compreosióo de la biología y ecología de estos importaotes recursos pesqueros.
Palabras clave: peces migratorios, peces de agua dulce, estructura geoética, Characidae, Prochilodootidae, microsatélite, Brasil.
In recent years, a number of genetic investigations have revealed that Neotropical freshwater migratory fish can exhibit population structuring, with different genetic populations within a single hydrographic system (Wasko & Galetti, 2002; Hatanaka et al., 2006; Sanches & Galetti, 2007). It has been claimed that during the spawning season fish schools may exhibit behavior that enables the maintenance of the genetic integrity of such populations (Hatanaka et al., 2006; Sanches & Galetti, 2007). Knowledge on the genetic diversity within and between wild populations is crucial to the conservation of species (Haig, 1998). The maintenance of this genetic variation is the main goal of conservation, as it allows the potential for local adaptation and the life history evolution of species (Narum et al., 2004).
Cases of fish population reduction have been reported in a number of hydrographical systems in South America (Agostinho et al., 2005). Brazil has approximately 2000 freshwater fish species catalogued, accounting for 20% of all freshwater fish species in the world (Buckup & Menezes, 2003). A total of 134 species are considered endangered (Agostinho et al., 2005). This threat is mainly the result of anthropogenic impact on aquatic continental ecosystems, including pollution, eutrophication, silting, the construction of dams and flood control, fisheries and the introduction of exotic species (Agostinho et al., 2005).
The São Francisco River basin is one of the main hydrographic systems in Brazil and has a large biomass and diversity of freshwater fish, harboring 152 species (ANA/GEF/PNUMA/OEA, 2004). The basin has an area about 630.000 km2, occupying approximately 7% of the Brazilian territory (Paiva, 1983), and flows 3.160 km, mostly northward (Kohler, 2003). This extensive and complex basin crosses different biomes, such as the Atlantic Rainforest, neotropical savanna and caatinga (scrubland).
Intense environmental changes have occurred due to the construction of hydroelectric dams along the São Francisco River. Such changes include the diminished speed, oxygenation and temperature of the waters, which can cause perturbation to various features of fish biology. It has been found that some migratory fish species downstream from the Três Marias dam are smaller and exhibit immature gonads during the spawning season. (Y. Sato, personal communication).
Brycon orthotaenia Günther, 1864 (= Brycon lundii) (Characidae, Characiformes) is a migratory fish species endemic to the São Francisco hydrographic basin that is considered vulnerable by the IUCN (2010). Prochilodus argenteus Spix & Agassiz, 1829 (Prochilodontidae, Characiformes) is also migratory and suffers with the overexploitation due to its commercial importance as local food source (Sato & Godinho, 2004).
B. orthotaenia and P. argenteus has been the subject of previous genetic analyses, in which significant differentiation was detected between populations sampled during the spawning season (Wasko & Galetti, 2002; Hatanaka & Galetti, 2003; Hatanaka et al., 2006). These authors hypothesized that these migratory fish may constitute different populations in this one hydrographic system, coexisting and co-migrating along the main river channel. Homing instinct in P. argenteus was claimed to explain the maintenance of this structuring pattern (Hatanaka & Galetti, 2003).
The present study employed new sets of microsatellites to assay the genetic population structure of these two migratory fish species. The goal of this survey was to determine whether the population structuring pattern is confirmed and thereby increase and improve of the genetic data for knowledge on the biology and ecology of these fish as well as contribute toward their conservation.
MATERIALS AND METHODS
Fish collection was carried out at three sites located downstream from the Três Marias dam on the São Francisco River in southeastern Brazil (18°13'05"S, 45°15'54"W) (Fig. 1): the region immediately downstream from the dam (Region A); a second region about 10 km downstream from site A (Region B); and a third region approximately 40 km from site A, downstream from the confluence of the São Francisco and Abaeté Rivers (Region C).
Figure 1. Collection sites in the São Francisco River Basin, southeastern Brazil. a) region immediately below the dam, b) region 10 km from the dam to the confluence of Abaeté and São Francisco Rivers, c) a stretch of approximately 20 km downstream from this confluence. Arrows indicate the direction of flow of the river.
Figura 1. Sítios de colecta en la cuenca del Río Sao Francisco, en el sur del Brazil. a) region inmediatamente abajo a la presa, b) region a 10 km de la represa hasta la confluencia de los ríos Abaeté y Sao Francisco, c) un tramo de aproximadamente 20 km aguas abajo de esta confluencia. Las flechas indican la dirección del flujo del río.
A total of 44 DNA samples of B. orthotaenia available from the tissue bank of our laboratory were used. These samples were originally collected at all the sites during a single spawning season (Wasko & Galetti, 2002). P. argenteus specimens (n = 32) were collected during a single non-reproductive season at sites A and C.
DNA extractions from B. orthotaenia were carried out based on the procedures described by Wasko & Galetti (2002). Genetic diversity was analyzed using four microsatellite loci described for B. hilarii: Bh5, Bh6, Bh16 and Bh17 (Sanches & Galetti, 2006) as well as one additional unpublished locus, Bh14 (TTA) (Table 1) which has not produced reliable amplifications in B. hilarii, the original species used for the isolation of microsatellite loci. The sequence of this locus is now available in the GenBank (Accession Number: FJ844396). PCRs were carried out following the conditions proposed by Sanches & Galetti (2006). PCRs were carried out at a final volume of 10 μL, containing 100 ng of DNA, 200 μΜ of each deoxynucleotide triphosphate, 1.5 mM of MgCl2, 1x reaction buffer (20 mM Tris-HCl pH 8; 50 mM KCl) 0.5 U of Taq polymerase (Fermentas Life Sciences) and 5 pmol of each primer (Fermentas Life Sciences). Thermal cycling conditions were as follows: 5 min at 95°C, 35 cycles of 1 min at 94°C, 1 min at 56°C (for all loci), 1 min at 72°C, and a final extension of 20 min at 72°C. Amplified products were resolved on 7% silver-stained denaturing polyacrylamide gel (Comincini et al., 1995). Alleles were scored by comparison with a 10 bp DNA ladder (Fermentas Life Sciences). Each sample was genotyped twice and only confirmed results were considered.
Total genomic DNA from P. argenteus was extracted from liver tissue through the saline solution method described by Aljanabi & Martinez (1997). Genetic diversity was analyzed using ten specific microsatellite loci: Par12, Par14, Par15, Par21, Par35 (Barbosa et al., 2006), Par66, Par69, Par80, Par82 and Par85 (Barbosa et al., 2008) (Table 1). PCRs were performed using the method described by Schuelke (2000) and were carried out in a final volume of 10 μL, containing 100 ng of DNA, 200 μΜ of dNTPs, 1 x PCR buffer (20 mM Tris-HCl, pH 8.4 and 50 mM KCl; LGC Biotecnologia), 4 pmol of each reverse and 6-FAM or NED M13 (-21) primers as well as 1 pmol of the forward primer, 1.5 mM MgCl2 and 1 U of Taq DNA Polymerase (LGC Biotecnologia). PCR conditions were as follows: 1 cycle at 95°C (5 min), 30 cycles at 94°C (30 s), 45 s at the annealing temperature (Table 1) and 72°C (45 s), followed by 8 cycles at 94°C (30 s), 53°C (45 s), 72°C (45 s) and a final extension at 72°C for 10 min. The microsatellite loci were analyzed on an ABI 377 automated sequencer (Applied Biosystems) and the alleles were scored with the Genescan and Genotyper 2.5 software programs (Applied Biosystems).
The genetic diversity of each population was quantified as the number of alleles (NA) and number of private alleles as well as both observed (HO) and expected (HE) heterozygosity. Allelic richness (Petit et al., 1998) and the inbreeding coefficient (FIS) were also obtained using the Fstat software (Goudet, 1995). Significant differences in heterozygosity, gene diversity and allelic richness were evaluated between sites using the Kruskal-Wallis or Mann-Whitney in test the Bioestat software (Ayres et al., 2003). Departure from Hardy-Weinberg expectations was calculated using a test analogous to Fisher's exact test (Guo & Thompson, 1992), estimated with a Markov Chain Monte Carlo series of permutations (10.000 batches/1000 iterations), implemented in Genepop (Raymond & Rousset, 1995). Tests for linkage disequilibrium between all pairs of loci were also performed with the Markov Chain Monte Carlo method in Genepop. Significance values were adjusted by the sequential Bonferroni correction (Rice, 1989). The Micro-Checker (Van Oosterhout et al., 2004) was used to identify the presence of null alleles.
Genetic differences between populations were estimated with FST (Weir & Cockerham, 1984), based on differences in allele frequencies using Fstat. The RST statistic, based on differences in the allele size using the RST Calc (Goodman, 1997) was also used.
All significance values of multiple tests were adjusted by the sequential Bonferroni correction (Rice, 1989). The Mantel test was conducted using Genepop (Raymond & Rousset, 1995) to determine the significance of the relationship between the genetic distance (FST) and geographic distance (km) for all population pairs. Population structure was assessed using a model-based Bayesian procedure implemented on the STRUCTURE 2.1 program (Pritchard et al., 2000). This analysis was carried out assuming the admixture model and correlated allele frequencies. Three individual repetitions of each K estimate (1-5) were run (500.000 iterations and a burn-in of 200.000 iterations).
A total of 39 alleles were found in all loci throughout all populations. The number of alleles per locus found in each sampling site ranged from two (Bh14, region A and B) to twelve (Bh6, region B) and the mean ranged from 5.6 to 7.2 (Table 2). Three private alleles were found: two at site B (locus Bh6) and one at site C (locus Bh14). Null alleles were identified in the locus Bh17 (0.412). After the Bonferroni correction, one pair of genotypes presented linkage disequilibrium (Bh5 x Bh16, P = 0.03).
Table 2. Information on the genetic diversity of Brycon orthotaenia collected at three sites downstream from the Três Marias dam on the São Francisco River (Brazil). Sample size (n) per site, number of alleles (NA), number of private alleles (NAp), allelic richness (RA), observed heterozygosity (H0), expected heterozygosity (HE), P-value for departures from Hardy-Weinberg expectations already corrected by the sequential Bonferroni correction (Pm) and inbreeding coefficient (F1s).
Tabla 2. Información sobre la diversidad genética de Brycon orthotaenia recogidos en tres sítios aguas abajo de la represa de las Tres Marias en el Río Sao Francisco (Brazil). Tamaño de la muestra (n), número de alelos (NA), número de alelos privados (NAP), riqueza alélica (RA), heterocigosidad observada (H0), heterocigosidad esperada (HE), valores de P para el análisis de las expectativas de Hardy-Weinberg ya ajustados por la corrección de Bonferroni secuencial (Pm) y coeficiente de endogamia (FIS).
All sites exhibited departure from the Hardy-Weinberg expectations for at least one locus, with a deficit or excess of heterozygotes revealed by positive or negative FIS values, respectively. The mean observed heterozygosity ranged from 0.65 to 0.73 and expected heterozygosity ranged from 0.63 to 0.75. Mean allelic richness ranged from 5.48 to 6.44. The fish from site A had the smallest mean value of all these genetic diversity parameters, but differences between sites were non-significant (Kruskal-Wallis test, P > 0.05).
Both pairwise FST and RST estimates demonstrated low values, although there were significant differences between sites (Table 3). The fish from the site A (immediately downstream from the dam) were significantly different from the other sites. The same differentiation pattern was achieved through Bayesian analysis, which identified two populations (K = 2, estimated -ln probability of data = -680.7; P (KX) = 1.00). Most of fish from site A were assigned to one cluster (dark gray), while the fish from sites B and C predominantly represent another cluster (light gray) (Fig. 2). Mantel tests demonstrated no correlation between geographic and genetic distances, suggesting no isolation by distance. The locus Bh 17 was not used in these analyses, since it presents null allele.
All loci produced a total of 115 alleles ranging from three (Par35, site A) to 20 (Par85, site C) alleles per locus per population. Specimens from site A exhibited fewer alleles (82, mean number of 8.2 alleles/locus) than the fish from site B (105, mean of 10.5 alleles/locus). Among the total of alleles, 43 were considered private alleles - ten in site A and 33 in site B. No significant linkage disequilibrium (P > 0.05) was observed. Locus Par15 exhibited a significant deviation from the Hardy-Weinberg equilibrium only at site B (Table 4). Null alleles were identified in the loci Par 15 (0.0677), Par 21 (0.1706) e Par 80 (0.0852). The genetic diversity demonstrated by allelic richness, observed and expected heterozygosities was similar in all sites (Mann-Whitney test, P > 0.05).
There were no significant differences between samples (FST = 0.002, P = 0.400; RST = -0.021, P = 0.954). Bayesian analysis also identified the two samples belonging to a single population (K = 1; estimated - ln probability of data = 1600.3; P > 0.99).
In recent years, the number of genetic studies on Neotropical freshwater fish have indicated structuring of their populations within a same hydrographic basin (for a review, see Piorski et al., 2008), including migratory species (Machado et al., 2005; Hatanaka et al,. 2006; Sanches & Galetti, 2007). The present study revealed different results for both migratory fish species studied. Unlike P. argenteus, significant genetic differentiations were found among populations of B. orthotaenia in the small region studied.
Population structuring of B. orthotaenia was revealed by the FST and RST statistics. The structuring pattern was also clearly demonstrated in the Bayesian analysis, in which two populations were identified. Individuals from site A represent one population, whereas most fish from sites B and C belong to the other genetic population. This result corroborates a previous study in which a significant divergence was found between sites, as revealed by a diagnostic RAPD pattern that was present in 100% of the fish from site A and 27% of the fish from site C (Wasko & Galetti, 2002). The authors hypothesized that the animals collected from site A could represent a unique population, whereas those from site C would comprise a mix of different populations.
Population structuring has also been found in B. hilarii from the Paraguay River Basin (Sanches & Galetti, 2007). Significant genetic differences were detected between populations and the authors assumed that the spawning schools may organize themselves in such way as to maintain the integrity of each subunit residing in the system. Recent studies with B. insignis and B. opalinus revealed this same genetic structuring pattern of different populations from Paraíba do Sul River Basin (Barroso et al., 2005; Matsumoto & Hilsdorf, 2009).
A previous study revealed genetic differentiation between populations of Prochilodus argenteus collected during the reproduction season in the same sites as those of the present work (Hatanaka & Galetti, 2003; Hatanaka et al., 2006). The authors claim that this migratory fish may constitute different populations in this hydrographic system, coexisting and comigrating along the main river channel.
Unlike the above-mentioned studies, no differentiation was found in the present investigation regarding the non-reproductive season for P. argenteus. The occurrence of weak population genetic differentiation or no differentiation is common in fish (Wirth & Bernatchez, 2001; Laikre et al., 2005; Santos et al., 2007), especially for species that exhibit high vagility, abundance and wide distribution, with no visible barriers to gene flow (Jorgensen et al., 2005). Alternatively, the lack of genetic differentiation along a hydrographic system also could be due to overlapping discrete populations during the non-reproductive season, reflecting in non-significant FST values even when comparing sites that are long distances apart.
Studies on the movement of P. argenteus (Godinho & Kynard, 2006) using telemetry have reported the existence of different population units determined by a spawning-site homing, corroborating previous genetic analyses on this migratory freshwater fish (Hatanaka & Galetti, 2003). Moreover, a similar study with Pseudoplatystoma corruscans revealed that the spatial distribution of the fish was greater during the non-spawning season than during the spawning season (Godinho et al., 2007), demonstrating that the fish are more dispersed in the former season. Thus, the probability of detecting genetic differentiation during the non-reproductive season is much lower, which corroborates the idea of coexisting populations. The spatial distribution of fish from different population units may be wider and consequently overlap during the non-reproductive season, which has no relevance to the genetic population structure (Laikre et al., 2005).
Therefore, we must be cautious in reaching conclusions on a lack of differentiation for migratory fish species. It is important to consider the sampling strategies of these studies, as fish exhibit different behavior at different times of their life cycle. From the standpoint of management and conservation, it is dangerous to consider the existence of a single population, because different genetic populations can be cohabitating a particular space. Considering different genetic population as only one population can result in the depletion of genetic variation (Laikre et al., 2005) and consequently reduce population viability (Allendorf & Ryman, 2002).
Knowledge on population genetic structure is essential for fisheries management and the conservation of fish species (Moritz, 1994; Paetkau, 1999). Without such information, unsuitable management could result in the overexploitation of some population units or segment of populations (Laikre et al., 2005), causing the loss of entire gene pools (population units) or genetic diversity within populations (Ryman & Utter, 1987; Allendorf & Ryman, 2002).
In the present study, population structuring was identified for B. orthotaenia during the reproductive season, corroborating a previous study (Wasko & Galetti, 2002), whereas no structuring pattern was found for P. argenteus during the non-reproductive season. However, the idea of population structuring should not be discounted, as fish from different genetic populations units can be spread and mixed in the main channel of the river during the non-reproductive season. Thus, further studies are needed, including more collection sites and different seasons over the course of several years, in order to reach a more faithful conclusion regarding the population genetic structure of these migratory fish.
The authors are thankful to the Instituto Florestal de Minas Gerais for authorizing the capture of specimens. We also thank Dr. Yoshimi Sato and CEMIG/CODEVASF for collecting the samples and Prof. Dr. Flávio Henrique da Silva for facilitating the use of the automated sequencer. This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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Received: 5 March 2011; Accepted: 15 January 2012.
Correspoodiog author: Alexaodra Saoches (firstname.lastname@example.org)