Molecular identification of Cichla ( Perciformes : Cichlidae ) introduced in reservoirs in Southern Brazil

Species of peacock bass were introduced in several watersheds in South America and worldwide, mainly due to its importance to sport fishing, by being a fighting fish. A recent revision of the genus Cichla showed that the species introduced in reservoirs of the South, Southeast and Northeast regions of Brazil are two new species, described as Cichla kelberi (yellow peacock bass) and Cichla piquiti (blue peacock bass), erroneously identified as C. monoculus and C. ocellaris. With the purpose to identify the populations of Cichla in Paranapanema and Paraná rivers, a total of 323 base pairs (bp) of the mtDNA control region were sequenced, obtained from 84 specimens of Cichla in six different localities (Tapajós river, Solimões river, Capivara, Taquaruçu and Rosana reservoirs in the Paranapanema river, and in the upper Paraná river floodplain). The analyses revealed the genetic diversity of Cichla monoculus, introduced into the Capivara reservoir, originally from the region of Manaus (Amazonas State), and spread in the reservoirs downstream (Taquaruçu and Rosana). The occurrence of the same haplotypes in the three reservoirs suggests one single introduction. This study confirmed the introduction of Cichla in the Capivara reservoir and showed the genetic diversity of Cichla in the Paranapanema river.


Introduction
The introduction of invasive species is the second leading cause of extinction of both animals and plants, being surpassed only by habitat changes, such as the construction of large dams and deforestation.Species of the genus Cichla (peacock bass) are restricted to the Neotropical region, and are the representatives of the family Cichlidae that reach the largest size, have considerable economic importance in the Amazon region, both in commercial and recreational fishing.Recently, Kullander and Ferreira (2006) reviewed the species of the genus Cichla and observed that the species introduced in the reservoirs of the South, Southeast and Northeast regions are two new species, described as Cichla kelberi (yellow peacock bass) and Cichla piquiti (blue peacock bass), mistakenly identified as C. monoculus and C. ocellaris.Afterwards Oliveira et al. (2008) reported the presence of C. monoculus in the Capivara reservoir and more recently Carvalho et al. (2009) showed the genetic origin of the invasive peacock bass populations in four major river basins of Minas Gerais State.
The species of peacock bass, mainly due to their sporting characteristics, have been introduced in many river basins of South America and worldwide.In the upper Paraná river floodplain, species of Cichla, such as C. kelberi (yellow peacock bass) and C. piquiti (blue peacock bass) were introduced in reservoirs of the rivers Grande, Tietê and Paranaíba.The capture of large numbers of immature specimens suggests a high reproductive success in these environments (ESPÍNOLA et al., 2010).In general, it is possible to find these two species and hybrids in the floodplain (OLIVEIRA et al., 2006).Dams constructed before the introduction of the species in the Paraná river prevented their spread to the Paranapanema river.As shown by Oliveira et al. (2006Oliveira et al. ( , 2008)), the population of the Capivara reservoir is characterized as Cichla monoculus from an independent introduction.The present study aimed to identify Cichla species introduced in the reservoirs of the Paranapanema river and upper Paraná river Basin.

Samples collection
This study encompassed three reservoirs of the Paranapanema river (Rosana, Taquaruçu and Capivara), arranged in sequence along the river; the Paraná river in the region of Porto Rico; and samples of the rivers Solimões and Tapajós (Table 1, Figure 1).Muscle tissue samples from eighty-four specimens of Cichla (peacock bass) were collected between March and December 2008, and preserved in absolute ethanol and kept at -20°C.The adult specimens of peacock bass were fixed in 40% formaldehyde and deposited in the Ichthyology Collection of Nupélia (Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura), Cod: Nupélia 6838.
Extraction and Amplification of DNA DNA was extracted according to Monesi et al. (1998) with some modifications (PRIOLI et al., 2002).The amplification of mtDNA fragments by PCR was based on Prioli et al. (2002).The fragment was amplified by the pair of primers L A21 mtDNA control region (5'-AGAGCGTCGGTCTTGT AAACC-3') (CRONIN et al., 1993) and H16498 (5'-CCTGAAGTAGGAACCAGATG-3') (MEYER et al., 1990), containing approximately 550 base pairs (bp).This is constituted of the sequence of the gene tRNA Pro , which encodes the tRNA of the amino acid proline, and the rest of the fragment contains the hyper variable sequence 5' of the control region of the mitochondrial DNA molecule.The PCR reaction volume was 25 μL, containing Tris-KCl (20 mM Tris-HCl pH 8.4 with 50 mM KCl), 1.5 mM MgCl 2 , 2.5 μM of each primer, 0.1 mM of each dNTP, 2.5 U of Taq DNA polymerase, 15 ng of DNA and Milli-Q water to complete the 25 μL volume.The mixture was placed in a thermocycler  and DNA denatured at 94 o C for 4 minutes.The amplification occurred during 40 cycles, with each one consisting of denaturation at 94 o C for 15 seconds, annealing at 59 o C for 30 seconds and 72°C for 2 minutes, followed by a final extension of 72 o C for 10 minutes.The DNA was amplified and quantified, and in order to eliminate the excess of primers and dNTPs that could interfere with the sequencing, a purification process was performed following the protocol of Rosenthal et al. (1993).

Sequencing
The obtained fragments were sequenced according to the manufacturer's instructions using the primer H16498.About 50 ng of DNA of the final product of each PCR was used as sample for the sequencing in the automatic sequencer MegaBace (Amersham) following the manufacturer's instructions.
The sequences were thus aligned using the program CLUSTAL W (THOMPSON et al., 1994) and manually edited with the program BIOEDIT (HALL, 1999).The p-distances and the numbers of polymorphic nucleotides were calculated using MEGA 4.0 (TAMURA et al., 2007).
The evolutionary model was selected using the programs PAUP 4.0 (SWOFFORD, 2002) and MODELTEST 3.7 (POSADA;CRANDALL, 1998).The corrected Akaike Information Criterion (AlCc) and Bayesian Information Criterion (BIC) were used to select the nucleotide substitution model.To construct the phylogenetic tree, we used the maximum likelihood method on the selected model, and bootstrap based on 10,000 resamplings.The posterior distribution of trees was approximated by using the Markov chain Monte Carlo as implemented in MrBayes (HUELSENBECK et al., 2001).The sequences of GenBank DQ841871 and DQ841865 were used as external groups for C. ocellaris and C. orinocensis, respectively.The haplotype diversity (h) and nucleotide diversity (π) were determined using the programs Arlequin 3.1 (EXCOFFIER et al., 2007) and DNAsp (LIBRADO;ROZAS, 2009).

Results
Sequences with a total of 323 base pairs (bp) of the mtDNA control region were obtained from eighty-four specimens of Cichla (GenBank accession numbers FJ872833 to FJ 872916) in six different locations (reservoirs Capivara, Taquaruçu and Rosana in the Paranapanema river, and rivers Tapajós, Solimões and Paraná in the region of Porto Rico) (Table 1).Forty-nine haplotypes were found in the samples of C. monoculus collected in Amazonas and four haplotypes were found in the samples collected in the Paranapanema river (Capivara, Taquaruçu and Rosana reservoirs) and Paraná river in the Porto Rico region.In the Paraná State, four haplotypes of C. kelberi were also found (region of Porto Rico and Taquaruçu reservoir).The number of mutations and haplotypes, and haplotype and nucleotide diversity presented in the studied Basins, are listed in the Table 2. Of the four haplotypes of C. monoculus observed in the Upper Paraná basin, the first haplotype was found in the reservoirs Capivara, Taquaruçu and Rosana, and also in samples of the Solimões river.But it was not observed in the samples of Porto Rico.The haplotype frequency was 31%.The second haplotype was found in all sampling sites (Capivara, Rosana, Taquaruçu and Porto Rico).Importantly, this haplotype is 99% similar to the haplotype of the Tapajós river collected in the region of Santarém, with a frequency of 64%.The third haplotype was found in Capivara, Taquaruçu and Porto Rico, and differs from the others by the deletion of two thymines, with a frequency of 2.4%.The fourth haplotype was found only in Porto Rico, with a single thymine nucleotide deletion compared to the second haplotype.The haplotype frequency was 2.4% (  The haplotype diversity and the nucleotide diversity were, as expected, higher in samples from the Amazonas basin than in the upper Paraná basin.The genetic diversity of the samples of each location is presented in Table 2.These parameters were not calculated for C. kelberi owing the bilateral hybridization between the two congeners introduced in this basin (GRAÇA; PAVANELLI, 2007;OLIVEIRA et al., 2006).
The corrected Akaike Information Criterion (AICC) and Bayesian Information Criterion  Within each of the six clades, there was a low genetic variation (Table 4); or no index computed (N/C) -since only one individual formed the clades used as external group.Clade four had the highest genetic diversity; composed of samples collected in Madeira, Orinoco and Negro rivers by Willis et al. (2007).The difference of 2.3% can be explained because the groups of the present study were made according to the analysis of Willis et al. (2007), which also found three groups of C. monoculus.The smallest genetic distance was observed within the group one of C. kelberi, which highlights the high degree of relatedness between specimens of this species.
The largest distance was found between the clades four of C. monoculus and C. kelberi (0.089) and the smallest distance between the clades three and four of C. monoculus (0.041) (Table 4).
The p-distance matrix was used to construct a scatterplot of the main coordinates of six groups of Cichla (Figure 3).C. kelberi formed an isolated group, detected along the axis one.C. monoculus formed two groups, but only detected by the dispersion in the axis two.Moreover, the species C. orinocensis and C. ocellaris showed no marked differences in relation to C. monoculus.

Discussion
The obtained results evidenced that four haplotypes of C. monoculus presented in the Paraná river have a high haplotype frequency, showing a similarity with the haplotypes of the samples collected in the rivers Tapajós and Solimões.The first haplotype was not found in Porto Rico.The third and fourth haplotypes differ by having a deletion of thymine regarding the first and the second haplotypes.The study performed by Oliveira et al. (2006) showed that in the samples gathered in Capivara reservoir, there was one haplotype shared with the native population of the Amazonas river.Then, Oliveira et al. (2008) analyzed sixty-five specimens of Cichla of the Upper Paraná river and Amazon basins by employing the non-transcribed region of 5S rDNA gene to obtain specific markers for related species, and observed that all specimens of C. monoculus of the Amazon river basin and the upper Paraná river basin presented the same amplified fragments.This confirms the results herein registered, i.e., the populations introduced in the Paranapanema river were possibly specimens from the Solimões and Tapajós rivers.
According to Almeida-Ferreira et al. (2011), Cichla monoculus Spix, 1831 was the first species identified in the Paraná river in 1986.However, according to Oliveira et al. (2006) The peacock bass is recorded for at least 35% of 71 Brazilian reservoirs reviewed by Agostinho et al. (2007).These authors emphasized that these species are present in these reservoirs for decades and have established viable populations, and in some, dominate the assemblies, both in number and biomass.The first introduction of the peacock bass in the Paranapanema river was in Capivara reservoir, in 1997 (ORSI;AGOSTINHO, 1999), and the first confirmed capture took place in 1999 (SHIBATTA et al., 2002).Currently, it is also found in Rosana and Taquaruçu reservoirs, located downstream.The peacock bass is present in practically all reservoirs located along the rivers Grande, Tietê and Paraná.In the reservoirs of the Paranapanema river, the percentage of occurrence was lower (25%), although samplings performed in the Capivara (HOFFMANN et al., 2005) and Taquaruçu reservoirs (BRITTO; CARVALHO, 2006) showed that the distribution in this basins has increased rapidly.In smaller sub-basins, usually with small reservoirs, the occurrence of this species is more restricted (AGOSTINHO et al., 2007).The data obtained showed a strong evidence that Cichla monoculus was introduced in the Capivara reservoir as described by Oliveira et al. (2006Oliveira et al. ( , 2008)), from the region of Manaus, and suggest the dispersion to the reservoirs located downstream (Taquaruçu and Rosana) by sequential occupation according to the models proposed by Heger and Trepl (2003), comprising the phases of establishment, population growth and dispersal of specimens (propagule pressure).This hypothesis is supported by the results found by Luiz et al. (2005) and Pelicice et al. (2005), since samplings undertaken in Rosana reservoir in 2000-2003 had not detected its presence.Moreover, the occurrence of the same haplotypes in the three reservoirs suggests the occurrence of a single introduction.

Conclusion
The present study proposed the genetic identification of the peacock bass and assisted the understanding of Neotropical invasions on the Parana river, Brazil.Additionally, an experimental study in genetics population would be useful to test the gene flow and the asymmetric patterns of migration in order to analyze and identify what is happening with dispersal of Cichla after the introduction into the Capivara reservoir.
(BIC) of ModelTest have indicated the HKY + G as the optimal evolutionary model for the D-loop sequences.The frequencies of the nucleotide bases were freqA = 0.4207, freqC = 0.1692, freqG = 0.0952, freqT = 0.3148 and of the replacing models Ti / Tv ratio = 5.0888; the parameter gamma distribution (G) was G = 0.3370 and the proportion of invariable sites (I) was I = 0.The transition/transversion ratios were K1 = 4.647 (purines) and K2 = 9.613 (pyrimidines) and the general transition/transversion ratio R= [A*G*k1 + T*C*k2]/[(A+G)*(T+C)] was R = 1.976.The heuristic inference (maximum likelihood) (Figure 2) generated by the PAUP, resulted in a tree with six major clades denominated groups.The topology was not affected by the inclusion of the two external groups.The phylogenetic reconstruction placed some species available in GenBank (C.cf.monoculus being C. kelberi.This is because the description of the latter species was made subsequently (KULLANDER; FERREIRA, 2006).The tree shows six main clades.The first clade contains C. kelberi from Araguaia (S.Willis, personal communication), Itaipu, Tocantins and the upper Paraná river basin in the region of Porto Rico (present study).The second clade contains C. monoculus from the reservoirs Taquaruçu, Rosana, Capivara and the upper Paraná river basin in the region of Porto Rico, Tapajós river in the region of Santarém (present study), and the rivers Tapajós, Amazonas and Xingu (WILLIS et al., 2007).The third clade contains C. monoculus from the Solimões river, reservoirs Taquaruçu, Capivara and Rosana (present study) and the rivers Solimões, Xingu and Tapajós reported by Willis et al. (2007) and Renno et al. (2006).The fourth clade includes C. monoculus from the rivers Madeira, Negro and Orinoco reported by Willis et al. (2007).The fifth clade is represented by C. orinocensis and the sixth by C. ocellaris (WILLIS et al., 2007).
, the genus Cichla is represented by two species (C.kelberi and C. piquiti) erroneously identified as C.monoculus and Cichla sp.Oliveira et al. (2008) identified C. kelberi and C. piquiti in the Paraná river, in accordance with the data reported by Kullander and Ferreira (2006), and identified haplotypes from the Solimões river in samples of C. monoculus collected in the Paranapanema river.In the present study, we recorded two species of Cichla (C.monoculus and C. kelberi) in the study areas.A small number of samples of C. kelberi were taken from the Taquaruçu reservoir, indicating the need for further sampling or that the species was recently introduced.Agostinho et al. (2007) stated that the species of peacock bass appear to have established in the basin of the Paraná river by other mechanisms other than the restocking programs developed by the electricity sector.

Table 1 .
Location and number of specimens of two species of Cichla examined in the present study.
*Specimens collected for this study; **Sequences available in GenBank.

Table 2 .
Haplotype and nucleotide diversity of the populations of C. monoculus.

Table 4 .
Genetic distance (P) within and between clades of Cichla haplotypes from invasive and native populations, based on the hypervariable sequence of the mtDNA control region.Scatterplot of the main coordinates of the six groups of Cichla.Group 1: C. kelberi from São Felix de Araguaia, Itaipu reservoir, region of Porto Rico, Taquaruçu reservoir and Tocantins basin; Group 2: C. monoculus from the reservoirs Rosana, Taquaruçu and Capivara in the Paranapanema river, Paraná river in the region of Porto Rico, Tapajós river and according to Willis et al. (2007) regions of Xingu, Amazonas and Tapajós; Group 3: C. monoculus from Solimões, Taquaruçu, Capivara, Rosana and according to Willis et al. (2007) from Solimões, Xingu and Tapajós; Group 4: C. monoculus according to Willis et al. (2007) from Madeira, Orinoco and Negro rivers; Group 5: C. orinocensis and Group 6: C. ocellaris.