This poster,  presenting selected preliminary findings of an ongoing Cichla population genetics study,
                                                    was presented at the Brazilian Ichthyology Society meeting, January, 2005
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Different Population Genetics Patterns in two Species of Peacock Bass
(Cichla:Perciformes) of Tributaries of the Rio Negro
Diferentes padrões genético-populacionais em duas espécies de tucunarés
(Cichla:Perciformes) de tributários do rio Negro.
Vasconcelos, W. R. (1);  Nunes, M. S (1);  Reiss, P. (2);  Farias, I. P. (1).
(1)Laboratory of Evolution and Animal Genetics – L.E.G.AL - UFAM.
  (2) Acute Angling

rangel@inpa.gov.br
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Introduçtion
The rivers Unini and Xeriuini are tributaries of the Rio Negro, itself the largest tributary of the Amazon River. They harbor a great diversity of aquatic vertebrates.  To determine if the Rio Negro functions as a barrier to the genetic flow  between populations of non-migratory fish, four populations of peacock bass of the species Cichla temensis and Cichla orinocensis were sampled along the above cited affluents.  We used AMOVA and F statistics to test the hypothesis of population fragmentation, with the objective of identifying the patterns of distribution of intra-specific genetic variation.  This information can furnish data about the evolutionary dynamics of these two economically important species, not previously studied in depth at the population level.
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Materials and Methods 
Tissue Samples were collected in the locations indicated on the map (Fig. 1) and were preserved in 95% ethanol.  The DNA was extracted following the protocol for extraction by phenol-chloroform of Sambrook et al. (1989).  The region of the gene for ATPase was amplified via PCR, with the use of primers specifically developed by Sivasundar et al., (2001).  The sequencing reactions were performed in accordance with the recommendations of the manufacturers utilizing the Terminator Cycle Sequencing Kit (Amersham Bioscience), and analized in the MegaBACE 1000 automatic sequencer.
Intra and Inter-populational Analyses
The genetic structure of the populations was analyzed using AMOVA (Analysis of Molecular Variance) (Excoffier et al., 1992), and pairwise FsT (Cockerham & Weir, 1993), implemented by the program Harlequin 2.0 (Schneider et al., 2000).  Indices of genetic diversity and tests of selective neutrality of mutations were accessed using the program Harlequin 2.0.  The haplotype network was constructed with the program TCS versão 1.18 (Clement et al., 2000).
Fig. 2a- Example of C. orinocensis of the Rio Unini 
Fig. 2b- Example of C. temensis of the Rio Unini 
Results and Discussions

Based on the analyses of 605 base pairs and 46 gene copies, we found 11 haplotypes in C. temensis, with 1 most frequent and 9 singularly occuring.  While in the two populations of C. orinocensis, we analyzed 25 gene copies, we found 10 haplotypes, 3 being more frequent in relation to the rest.  Values of genetic diversity in general were more elevated in the populations of C. orinocensis than in C. temensis (Tab. 1).  The neutral presumption of mutations was tested through the index of selective neutrality  Fs of Fu and indicated genetic disequilibrium in the population of C. orinocensis and C. temensis of the rio Unini (Tab. 1).  Meanwhile D of Tajima indicated a probable population growth only in C. temensis.  Significant values (P<0,05) of the parameter Fst were observed between the populations of C. orinocensis, even with the correction of Bonferroni (Rice 1989).
Fig. 3a -Haplotype network showing the geneological relationships between the ten haplotypes detected and identified in C. orinocensis.
The color black indicates haplotypes found on the Rio Unini and yellow indicates those of the Rio Xeriuini. 		 Fig. 3b -Haplotype network showing the geneological relationships between the ten haplotypes detected and identified in C. temensis.
The color black indicates haplotypes found on the Rio Unini and yellow indicates those of the Rio Xeriuini. 

Tab. 1– Principal measures of intra-specific genetic polymorphism and tests of selective neutrality.  P< 0.05. 
Note: RU=Rio Unini and RX=Rio Xeriuini
Population
No.  of
samples
No.  of Poly-
morphic sites
Genetic Diversity
Nucleotide Diversity
D of Tajima
Fsof Fu
RU–C. orinocensis
11
10
0.9333 +/- 0.0773 
0.005657 +/- 0.003569 
-0.14123 
-3.11607 *
RX-C. orinocensis
14
10
0.8901 +/- 0.0603 
0.005657 +/- 0.003569 
-0.68577 
-2.09245 
RU-C. temensis
32
4
0.3377 +/- 0.1278 
0.000601 +/- 0.000672 
-1.87763 *
-3.81699 *
RX-C. temensis
14
3
0.2949 +/- 0.1558 
0.000763 +/- 0.000802 
-1.65231 *
-0.68877 
Table 2a –Values of Pairwise Fst below the line and  Effective number of migrants (Nm) above the line for C. orinocensis.
Nota: RU=Rio Unini and RX=Rio Xeriuini. 
Poipulation
RU–C. orinocensis
RX-C. orinocensis
RU– C. orinocensis
0.84186
RX-C. orinocensis
0.23878 *
 		 Table 2b –Values of Pairwise Fst below the line and  Effective number of migrants (Nm) above the line for C. orinocensis. C.  temensis.
Nota: RU=Rio Unini and RX=Rio Xeriuini.
Population
RU– C.  temensis
RX- C.  temensis
RU– C.  temensis
RX- C.  temensis
0
 
No indication of genetic difference  was demonstrated in the two populations of C. temensis, an indication of elevated genetic flux. () (Tab. 3b). 

The results of AMOVA also show this pattern. In the populations of C. orinocensis, 37,26% of the genetic variation occurred between the two populations (Fst=0,372; P=0). While in C. temensis, the variation between the two populations was  –0,97% (Fst=-0,0096; P=0.7575). Fst and AMOVA indicate population fragmentation in C. orinocensis.

These results demonstrate that a distance of ~100 Km between tributaries of the Rio Negro can function as a barrier to the dispersion of some species of fish as seen in C. orinocensis, but not in others as in C. temensis. Species of peacock bass , for example, can exhibit different patterns of migration, reflectining different life strategies.
Bibliography and References

Clement, M.; Posada, D. and Crandall, K. A.  (2000) TCS: a computer program to estimate gene 6 genealogies. Mol. Ecol., 9, 1657-1659. 
Cockerham, C. C. and Weir B. S. (1993) Estimation of gene flow from F-statistics. Evolution, 47, 855-863. 
Excoffier, L.; Smouse, P. E. and Quattro, J. M. (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics, 131, 479-491. 
Rice. 1989. Analysing table of statistical tests. Evolution 43: 223-225
Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, second edition Vol. 2. Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY. 
Schneider, S.; Roessli, D. and Excoffier, L. (2000). Arlequin ver. 2000: A software for population genetic data analysis. Genetics and Biometry Laboratory, University of Geneva. Geneva, Switzerland.
Sivasundar, A.; Bermigham, E.  and Orti, G. 2001. Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers. Mol. Ecol. 10 (2): 407-417. 
Xia, X. and Xie. Z. 2001 DAMBE: Data analysis in molecular biology and evolution. Journal of Heredity 92:371-373.
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Peacock Bass DNA Study Results - Portuguese

The 5 species of Peacock Bass

Gamefish of the Amazon Basin
 
Funding
International
Foundation
for Science
Acute Angling
Sport Fishing
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