Characteristics of microsatellite loci isolated from
We isolated 41 and characterized 17 microsatellite loci for evaluating the genetic structure of the Amazonian fish Hypophthalmus marginatus, from the Tocantins and Araguaia River in the Eastern Amazonia. Of the 17 selected microsatellite sequences, 15 were dinucleotide repeats, 9 of which were perfect (5–31 repetitions) and 6 were composite motifs. Among these 17 microsatellites, only two were polymorphic. The average number of alleles (Na) observed in the five examined populations ranged from 3.5 to 4.5, while the average observed heterozygosity (Ho) ranged from 0.3 to 0.6. The allelic frequency was less homogeneous at the locus Hm 5 than that for the Hm 13. Genetic diversity was measured in three upstream and two downstream populations under the influence of the Tucuruí Hydroelectric Dam. Our findings provide evidence for low levels of genetic diversity in H. marginatus of the Tocantis basin possibility related to the Dam construction. The Fst and Rst analysis fits well with migratory characteristics of H. marginatus, suggesting the existence of a gene flow mainly in the upstream or downstream directions. To test the hypothesis that the Dam was responsible for the detected reduction on this species genetic diversity, a large number of genetic markers are recommended, covering geographic distribution range of the fish species.
- hydroelectric dam influence
- migratory fishes
- population genetic structure
Migratory freshwater fishes are vulnerable to a variety of anthropogenic impacts, including harvesting, pollution, and other types of habitat disturbance. The impact of dams is not only limited to the transformation of habitats from lentic to lotic but they also isolate the population from their places of spawning to feeding grounds [1–3]. Therefore, a survival and reproduction of migratory fish can be directly affected by changing thermal and hydrodynamic conditions in their habitat . In the Brazilian Amazonia, over 10 million hectares (ha) of forests are expected to become permanently flooded after the construction of new‐planned dams . Long‐term monitoring of fish populations is available only for Tucuruí Dam in the Tocantins River [6–10], where alterations following impoundment reduced the fish diversity on the reservoir resulting in the increase of predators such as
2. Materials and methods
The Tocantins is largely a plateau river, flowing for most of its length within an enclosed valley, draining an area of 343,000 km2. Over the past three decades, its basin has suffered from huge anthropogenic pressures, including widespread deforestation, mining, and the construction of the Tucuruí between 1984 and 1985. This dam is one of the world's largest hydroelectric dams, which has a reservoir of 2840 km2, most of which was originally covered with primary
Eighty‐two samples (14–19 per site) obtained from muscle or liver tissue of
2.2. Microsatellite development
Molecular tools such as microsatellites markers can give us a picture of the distribution of the genetic variability of the natural populations of Amazonian fish [15, 16]. To assess the genetic parameters of the
A total of 96 clones were sequenced, and 17 of these were selected for primer design using the software Primer3 . In all primer pairs, the forward primers had an M13(‐21) tail added to its 5ʹ end . An optimal annealing temperature was inferred using a gradient PCR with temperatures set between 52.3 and 70.5°C (Table 1).
|Primer sequences (5ʹ–3ʹ)||Repeat motif (5ʹ–3ʹ)||T (°C)||Size range|
Genotyping reactions were carried out in the Biocycler Thermal Cycler MJ96+/MJ96G (Applied Biosystems), in a final volume of 10 μL. Each reaction contained 3.8 μL of MilliQ water, 1.2 μL of 50 mM MgCl2, 1.0 μL of 10 mM dNTPs, 1.0 μL of PCR buffer (100 mM Tris‐HCl, pH 8.5, 500 mM KCl), 0.4 μL of 2 μM tailed forward primer, 0.4 μL of 2 μM fluorescently labeled primer, 0.8 μL of 2 μM reverse primer, 0.3 μL of 2.5 U Taq DNA polymerase, and 1 μL of DNA (50–100 ng/μL). Reactions were submitted to the following cycling profile: hot start at 94°C for 60 s followed by 25 cycles of denaturing at 94°C for 30 s, annealing for 30 s at locus specific temperatures (Table 1), and extension at 68°C for 30 s; labeling step consisted of 20 cycles of denaturing at 94°C for 20 s, annealing at 52°C for 30 s, and extension at 72°C for 60 s; final extension was performed at 72°C for 30 min. The fragments present in 1 μL of PCR product were separated in 8% polyacrylamide denaturing gel in an ALFexpressTM II (Amersham Biosciences, Freiburg, Germany) automatic DNA sequencer. Amplicon size was estimated by the Allele Locator 1.03 software (Amersham Biosciences, India), based on the internal and external standards provided by the manufacturer.
2.3. Data analysis
To measure the genetic variability within each population, the number of alleles per locus (A), effective number of alleles per locus (Ae), the observed heterozygosity (Ho), and expected (He) under Hardy‐Weinberg equilibrium (HWE) for each locus and their averages were calculated using Popgene 32 software .
The Genepop 3.1b program  was used to test whether the data obtained are significant deviations from Hardy‐Weinberg equilibrium and the occurrence of connection imbalance between the loci analyzed. This program uses a method of Markov chain to get an unbiased estimate of Fisher's exact test to detect a significant deficiency or excess of heterozygotes .
The differentiation between the populations was evaluated by comparing peer‐to‐peer two ways: 1. Estimates FST , using the Arlequin 3.5 program  and 2. Estimates RST , by program RSTCalc .
RST is an analog of Wright FST, which is based on the stepwise mutation model (SMM) for microsatellite loci and has the particularity of not displaying associated bias to differences in the size of the alleles in the samples of populations and / or differences the variance between loci. Some authors argue that the estimates of FST show lower when compared to RST estimates in same analysis .
The existence of linkage disequilibrium between the loci was evaluated by Genepop 3.4 program .
Among a total of 98 sequenced recombinant clones, 61 clones presented microsatellites. After sequence analysis, it was found that 41 clones showed more than four repeats of microsatellites and we have designed and purchased primers for 17 of SSRs.
Among the 17 selected microsatellite sequences were a hexanucleotide, a tetranucleotide, and 15 dinucleotide repeats. Of 15 dinucleotide repeats, nine were perfect (5–31 repetitions) and six were composite dinucleotide repeat type (Table 1). Of the 17 loci, only two (Hm 5 and Hm 13) were polymorphic among studied samples.
The average number of alleles (A) observed in five populations examined ranged from 3.5 to 4.5, while the average observed heterozygosity (Ho) ranged from 0.3 to 0.6 (Table 2). The allelic frequency was less homogeneous to the Hm 5 locus than for the Hm 13; its most frequent allele was the same for all populations (Table 3).
|Hm 5||Hm 13|
|Size (bps)||Relative frequencies||Size (bps)||Relative frequencies|
|Population (N)||A||Ae||Ho||He||P – EHW|
|Abaetetuba (14)||Hm 5||4.0||4.0||0.3||0.4||0.3|
|Cametá (19)||Hm 5||6.0||5.5||0.6||0.7||0.2|
|Itupiranga (15)||Hm 5||5.0||5.0||0.7||0.8||0.1|
|Tucuruí (19)||Hm 5||5.0||4.9||0.6||0.7||0.4|
|Conceição (15)||Hm 5||5.0||4.9||0.5||0.7||0.4|
Significant values of Fst were observed in all comparisons including Abaetetuba and the comparison of Tucuruí × Conceição (Table 4) gave negative Rst values representing interactions where the variance within a population exceeds the variance between populations.
|Conceição||****||0.28 (0.000)||0.05 (0.05)||0.04 (0.09)||0.10 (0.001)|
|Abaetetuba||0.71 (0.0000)||****||0.24 (0.00)||0.28 (0.00)||0.27 (0.000)|
|Itupiranga||0.35 (0.0006)||0.26 (0.0061)||****||0.005 (0.41)||0.006 (0.350)|
|Cametá||0.20 (0.0100)||0.47 (0.0002)||0.04 (0.19)||****||0.008 (0.350)|
|Tucuruí||0.41 (0.0000)||0.42 (0.0000)||-0.02 (0.63)||0.02 (0.223)||****|
Although tested just by two polymorphic SSRs, genetic variability of
Some populations of
A comparison made elsewhere of
The heterozygosity values provided by two microsatellite loci in
There is no much information on populations of
Laroche and Durand  studied the genetic structure of the Percidae
5. Preliminary conclusions
If low differentiation is prevalent, the geographic distribution range of large samples would be recommended for genetic analyses. It would also be useful to assess the levels of genetic variability within and among populations from different basins for a better understanding of population dynamics of these species. Although our conclusions were made by using only two microsatellite loci analyses, results are consistent with data from mitochondrial markers.
This study was supported by CAPES, and CNPq. We are also grateful to IBAMA, for providing the authorization 013‐2007 to capture, collect, and transport the biological material to the Centrais Elétricas do Norte do Brasil S.A. (ELETRONORTE S.A.) as well as for logistic support at Tucuruí. We thank Cassio Andrade (EMATER/PA) and Comunidade Jaraqüera Grande for providing logistic support at Cametá; Aparecida, Jhonis, and Leo for providing logistic support at Conceição de Araguaia. Special thanks to Soraya Andrade, Silvanira Ribeiro Barbosa for helping in laboratory. EJHR thanks Mauricio Papa de Arruda for collaboration with ALFexpress management.