Genetic divergence among invasive and native populations of the yellow peacock cichlid Cichla kelberi a

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1 Journal of Fish Biology (2016) 89, doi: /jfb.13144, available online at wileyonlinelibrary.com Genetic divergence among invasive and native populations of the yellow peacock cichlid Cichla kelberi a A. C. P. B. Marques*, A. C. S. Franco*, F. Salgueiro, E. García-Berthou* and L. N. Santos* *Laboratory of Theoretical and Applied Ichthyology, Federal University of Rio de Janeiro State, Rio de Janeiro, Av. Pasteur, 458 R314A, , Rio de Janeiro, RJ, Brazil, Graduate Course in Ecology, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho, 373, , Rio de Janeiro, RJ, Brazil, Grupo de Pesquisa em Biodiversidade Molecular Vegetal, Federal University of Rio de Janeiro State, Rio de Janeiro, Av. Pasteur, 458 R512, , Rio de Janeiro, RJ, Braziland GRECO, Institute of Aquatic Ecology, University of Girona, E-17071, Girona, Catalonia, Spain This study used the hypervariable domain of the mitochondrial DNA (mtdna) control region (CR) to assess the genetic divergence among native and invasive populations of Cichla kelberi, which is considered the first peacock cichlid introduced and established throughout Brazil and is among the most invasive populations of this genus worldwide. The maximum likelihood tree based on 53 CR sequences with strong bootstrap support revealed that C. kelberi forms a monophyletic clade, confirming that all 30 C. kelberi studied belong to this morphotype. Additionally, the haplotype analysis of the C. kelberi sequences from 11 sampling sites revealed that invasive populations are much less diverse than native ones and largely dominated by a single haplotype that prevailed in reservoirs at the Paraíba do Sul River basin. Two haplotypes were recorded exclusively in an invasive population at Porto Rico, southern Brazil, and one private haplotype was detected in two reservoirs from Paraíba do Sul (Pereira Passos and Paracambi), suggesting more than one introduction event and that native populations should be better evaluated to encompass the entire genetic diversity of native C. kelberi. The possible route and pathways of C. kelberi introduction are also briefly discussed The Fisheries Society of the British Isles Key words: genetic diversity; invasive species; mitochondrial DNA; Paraíba do Sul River; peacock bass. INTRODUCTION During the past century, rates of species introduction by human activities have increased, owing to the accelerated worldwide movement of people and products (García-Berthou, 2007; Essl et al., 2015). Invasive species are recognized as one of the major threats to native biodiversity and ecosystems functioning by the Convention on Biological Diversity (SCBD, 2000; Clavero & García-Berthou, 2005), of which Brazil and many other countries are signatories. In the Neotropics, a great number of exotic Author to whom correspondence should be addressed. Tel.: ; luciano.santos@unirio.br a This paper was presented at the FSBI Symposium, Bangor, in July Its content may not follow the usual style and format of the Journal of Fish Biology The Fisheries Society of the British Isles

2 2596 A. C. P. B. MARQUES ET AL. fish species have been introduced in almost all hydrographic basins, despite this region harbouring the highest fish biodiversity in the world. In Brazil, several freshwater fish species were brought from other countries or translocated among Brazilian basins, with only a few river basins of the south-east and north-east not having introduced species nowadays (Agostinho et al., 2007). In recent decades, these exotic fish species were introduced in order to increase reservoir fish production for sport fishing and also by escaping from fish farms (Chellappa et al., 2003; Agostinho et al., 2007). Naturally found in lakes and rivers of Amazon, Orinoco and Tocantins-Araguaia river basins, peacock cichlids, genus Cichla Bloch & Schneider 1801, have been introduced in several Brazilian basins, especially in the north-east, south-east and south, as well as in other regions of the world, such as in Central and North America and Hawai i (Zaret & Paine, 1973; Shafland, 1996; Elredge, 2000; Agostinho et al., 2007; Santos et al., 2011). Cichla spp. are visual predators that thrive in lentic environments such as reservoirs and can cause significant deleterious effects on native biodiversity (Zaret & Paine, 1973; Latini & Petrere, 2004; Menezes et al., 2012; Pelicice et al., 2015). Due to its excellent taste and high importance in sport fishing (Gomiero & Braga, 2003), these fishes are still being introduced, with Cichla spp. invasive populations being established in several fluvial lakes, rivers and reservoirs, especially in south-eastern Brazil (Agostinho et al., 2007). Therefore, they raise serious concerns for the conservation of aquatic biodiversity, due to their strong predation on smaller species (Latini & Petrere, 2004). Although recognized as the most invasive Cichla spp., the yellow peacock cichlid Cichla kelberi Kullander & Ferreira, 2006 is still misidentified, since morphometric analyses support the existence of five species in the native range (C. kelberi, Cichla monoculus Spix & Agassiz 1801, Cichla ocellaris Bloch & Schneider 1801, Cichla nigromaculata Jardine & Schomburgk 1843 and Cichla pleiozona Kullander & Ferreira, 2006; Kullander & Ferreira, 2006), whereas a single species (Cichla ocellaris sensu lato) is recognized through molecular techniques (Willis et al., 2012). Therefore, further studies of the native and invasive populations of C. kelberi is warranted, including taxonomic and genetic studies that might help to trace the origin of introductions and assist management (Fitzpatrick et al., 2012). Molecular markers have been successfully used for intraspecific genetic divergence studies with invasive species, especially the mitochondrial DNA (mtdna) control region (CR) (Prioli et al., 2002; Oliveira et al., 2006; Fitzpatrick et al., 2012; Panarari-Antunes et al., 2012; Willis et al., 2012; Gasques et al., 2015). The mtdna CR is much more variable than nuclear DNA loci (e.g. 16S, Xscr, Mitf, tmo4c4, dlx2 and bmp4) and even mtdna loci (e.g. cox, cytb, and mtatpase) (Willis et al., 2007, 2012; Carvalho et al., 2009; Gasques et al., 2015), thereby providing important information about the genetic diversity and taxonomy of species. The objective of this study was to assess the genetic divergence among native and invasive populations of C. kelberi in Brazil, using the hypervariable domain of mtdna CR. MATERIALS AND METHODS FISH SAMPLING AND MORPHOLOGICAL IDENTIFICATION Samples of introduced C. kelberi were obtained during its post-reproductive period (autumn; from April to June 2014 and 2015) in six reservoirs from Paraíba do Sul River basin (from up to downstream): Funil (FUN), Lajes (LAJ), Santana (SAN), Vigário (VIG), Pereira Passos

3 GENETIC DIVERGENCE IN C. KELBERI POPULATIONS 2597 Fig. 1. Photograph of an adult male of Cichla kelberi (L T 312 mm) caught in Funil reservoir, showing the major features used for species identification (Kullander & Ferreira, 2006): three dark vertical bars, light spots on the pelvic, anal and caudal fins, and irregular dark blotches on abdominal side. (PPA) and Paracambi (PAR). FUN was the only reservoir located in main stem of the Paraíba do Sul River, whereas SAN and VIG were built through water diversions from this river. LAJ was filled through deviation and impoundment of the River Piraí, a tributary of the River Paraíba do Sul, while PPA and PAR reservoirs receive a mixture of pumped waters from LAJ and VIG. Fishes were caught by hook and line using artificial baits during a week of angling effort in each reservoir. A total of 30 C. kelberi were used for DNA analyses and the number of fish per reservoir (n = 5) was standardized according to the system (e.g. SAN) that provided the least number of C. kelberi. Whenever possible, fish from different length classes ( mm total length (L T ), corresponding to 0 2 year-old fish) were used in order to maximize the genetic variability within populations. The present study provided thus 30 additional CR sequences for C. kelberi, which means an increase of 45% in relation to all sequences available for this species in GenBank (n = 36). Theindividuals ofc. kelberi were identified according to Kullander & Ferreira (2006), which morphologically described this species with three dark vertical bars and light spots on the pelvic and anal fins and also on the lower lobe of the caudal fin. Cichla kelberi also had an absence of black or ocellated stripes or spots on the head or on the gill (Fig. 1). Some individuals exhibited some irregular dark blotches on the anterior abdominal sides. Although C. kelberi and the other four morphotypes of yellow peacock cichlid are recognized as C. ocellaris sensu lato (Willis et al., 2012), C. kelberi was considered as a valid species in this study because most sequences available in GenBank follow the morphological classification proposed by Kullander & Ferreira (2006). DNA ISOLATION, AMPLIFICATION AND SEQUENCING Cichla kelberi specimens were collected and kept frozen ( 2 C) until DNA extraction. Total genomic DNA was extracted from muscle using the NucleoSpin Tissue kit (Macherey-Nagel GmbH & Co.; following the manufacturer s instructions andthenstoredat 20 C. The mtdna CR was amplified via polymerase chain reaction (PCR) using the pair of primers tpro2-5 (5 -ACCCTAACTCCCAAAGC-3 ) and HN-20-3 (5 -GTGTTATGCTTTAGTTAAGC-3 )(Leeet al., 1995; Palumbi, 1996). These primers were designed to amplify c. 900 bp of the mtdna CR. As the accurate sequencing of the 3 portion of this fragment downstream of a poly-t region is problematic (Willis et al., 2007), only c. 520 bp of the 5 end were sequenced and used in the present analysis. The amplification reaction mixture (25 μl) contained 1 unit (U) Taq DNA polymerase (Thermo Scientific Inc.; 1 reaction buffer with NH 4 SO 4,2 5mMMgCl 2,0 16 mm deoxynucleotide triphosphate (dntp) (Thermo Scientific), 8 pmol of each primer and 5 ηg of genomic DNA. The PCR cycling comprised an initial 5 min heating step at 94 C, followed by 40 cycles of 94 C for 30 s, 45 C for 30 s, 72 C for 1 min and a final extension at 72 Cfor5min.A 4 μl aliquot of each PCR reaction was checked by electrophoresis in 1% agarose gels stained

4 2598 A. C. P. B. MARQUES ET AL. with GelRed (Biotium Inc.; PCR products were purified and sequenced by Macrogen Inc. ( in both directions using the PCR primers. DATA ANALYSIS Sequence reads were manually checked and edited using the software BioEdit (Hall, 1999). Individual consensus sequences were aligned using the ClustalW algorithm implemented in MEGA 6.06 software (Tamura et al., 2013). The 30 sequences obtained from non-nativepopulations in the present study were analysed together with 23 other C. kelberi sequences retrieved from GenBank, totalling 53 sequences from 11 localities. Only localities with more than one sequence available in GenBank were included in the analysis. Localities with imprecise geographic coordinates were disregarded (Table I). First, the mtdna haplotypes, containing only C. kelberi sequences, were defined by analysing sequences with the DNASP 5 programme (Librado & Rozas, 2009). Then, to confirm the specimen identification, the sequences of the defined haplotypes were compared with sequences of the closely related species C. monoculus, C. ocellaris, C. pinima and C. pleiozona available in GenBank (Table SI, Supporting Information). Therefore, a maximum likelihood (ML) phylogenetic tree with 1000 bootstrap replications was constructed using the MEGA software. The jmodeltest2 software (Darriba et al., 2012) was used to select the best-fit nucleotide substitution model (HKY + I). A CR sequence of Astronotus ocellatus (Agassiz 1831) was used as outgroup. The genealogical relationships among the inferred haplotypes were determined using the median-joining algorithm implemented in NETWORK 5 (Bandelt et al., 1999). The haplotype (h) and nucleotide diversity (π) indices were calculated using the Arlequin (Excoffier & Lischer, 2010). A hierarchical analysis of molecular variance (AMOVA) was also conducted using the Arlequin software. Two groups were defined, one with the native populations and another with invasive populations. RESULTS Approximately 520 bp of the mtdna CR were sequenced from 30 individuals of C. kelberi from six reservoirs (FUN, LAJ, SAN, VIG, PPA and PAR), outside their native range, (five individuals from each reservoir) from the Paraíba do Sul river basin, south-eastern Brazil. These 30 sequences along with 23 other C. kelberi sequences available in GenBank [from Tucuruí reservoir (TUC), São Félix do Araguaia (SFA), Três Marias reservoir (TMA), Itumbiara (ITU) and Porto Rico (PRI)] were used to evaluate the levels of genetic diversity among invasive and native populations and to conduct a phylogeographic analysis of the C. kelberi. The alignment of these 53 sequences included 513 nucleotides. Taking into account the 53 C. kelberi CR sequences, nine haplotypes of C. kelberi were identified using the DNAsp software (Table II). The ML phylogenetic reconstruction (nucleotide substitution model: HKY + I) showed that C. kelberi haplotypes formed a monophyletic group with high bootstrap support (88%). The sequences of C. monoculus, C. ocellaris, C. pinima and C. pleiozona also formed monophyletic groups with high bootstrap values (Fig. 2). The haplotype H1 was present in the native SFA and TUC populations and was the most widely distributed among invasive populations, being present in 10 of 11 analysed populations (Table I and Fig. 3). Most of the invasive populations analysed (five of nine) are monomorphic and present this haplotype H1. Considering the invasive area, the ITU population is the only one to present the H6 haplotype, also found in the native area. Three C. kelberi populations presented private haplotypes. The native

5 GENETIC DIVERGENCE IN C. KELBERI POPULATIONS 2599 Table I. Population name, code, sample size, geographic co-ordinates and genetic diversity indexes for each Cichla spp. population studied Population Code Sample size Number of Latitude haplotypes o S Longitude o W (A) Number of private haplotypes (P) Haplotype diversity (h mean ± s.d.) Nucleotide diversity (π mean ± s.d.) Native populations S. F. Araguaia SFA ± ± Tucuruí TUC ± ± Mean Total ± ± Invasive populations Funil FUN ± ± Itumbiara ITU ± ± Lajes LAJ ± ± Paracambi PAR ± ± Porto Rico PRI ± ± Pereira Passos PPA ± ± Santana SAN ± ± Três Marias TMA ± ± Vigário VIG ± ± Mean Total ± ± All populations Mean Total ± ±

6 2600 A. C. P. B. MARQUES ET AL. Table II. Polymorphic sites that define the nine different mtdna haplotypes of Cichla kelberi Control region polymorphic sites Haplotypes H1 C T T G T T A A H2 * C * * * * * * H3 * * C * * C * * H4 T * C * * * * G H5 * * C * * * * * H6 * * C A * * * * H7 * * * * C * * * H8 * * * A * * G * H9 * * C A * * G * *, the same nucleotide of H1 haplotype. SFA and TUC populations exhibited three (H3, H4 and H5) and one (H7) private haplotypes, respectively. Among the invasive populations, Porto Rico (PRI) exhibited two private haplotypes (H8 and H9) (Table I). The haplotype network obtained through the median-joining analysis in the software NETWORK is presented in Fig. 3. Considering native populations, the total haplotype diversity (h) and nucleotide diversity (π) were (mean ± s.d.), h = 0 93 ± 0 08 and π = ± The native SFA and TUC populations presented equal levels of h and π diversity (Table I). The SFA population, however, exhibited five different haplotypes while three were observed in the TUC population. In invasive populations, the total h = 0 36 ± 0 09 and π = ± Among them, the PRI population was the most polymorphic, with three different haplotypes, h = 0 60 ± 0 13 and π = ± (Table I). The AMOVA analysis revealed that 60 15% of the genetic variation was found within populations, 24 10% among populations within groups and 15 75% among groups (native v. invasive populations) (Table III). DISCUSSION The mtdna CR shows one of the fastest mutation rates in the genome, providing unambiguous assessment of genealogical connection between individuals in order to estimate intraspecific gene flow (Willis et al., 2012). The hypervariable domain of the mtdna CR of C. kelberi demonstrated a higher genetic diversity in the native range in comparison with invasive populations. Although the total number of haplotypes was not very different between native and invasive C. kelberi populations (i.e. six v. five haplotypes), the haplotype and nucleotide diversity were three times greater in the native range. Founder events and population bottlenecks are considered responsible for the loss of diversity of many invasive species, due to the low number of introduced individuals (Allendorf & Lundquist, 2003). This low genetic diversity associated with founder effects was found for other populations of Cichla spp. and also other invasive fishes (Oliveira et al., 2006; Vidal et al., 2010; Panarari-Antunes et al., 2012; Briñez et al., 2013). The high dominance of a single or few haplotypes in invasive populations is another clue of the occurrence of founder effects. The genetic profile for most non-native C. kelberi populations showed a high dominance of haplotype 1 (H1), except

7 GENETIC DIVERGENCE IN C. KELBERI POPULATIONS DQ GU DQ C. monoculus GU GU JQ JQ JQ C. pleiozona JQ JQ JQ JQ GU C. ocellaris DQ JQ JQ JQ JQ C. pinima JQ JQ JQ /haplotype 1 KU /haplotype 2 JQ /haplotype 3 GU /haplotype 4 GU /haplotype 5 C. kelberi GU /haplotype 6 FJ /haplotype 7 FJ /haplotype 8 FJ /haplotype 9 NC_ Astronotus ocellatus Fig. 2. Maximum likelihood tree based on Cichla spp. control region mtdna sequences. Numbers at nodes represent bootstrap values after 1000 replications (cut off = 80%). Out group: Astronotus ocellatus. for ITU which was dominated by a single haplotype (H6). This dominance pattern of only few haplotypes seems to be common to invasive populations, which reflect only partially the genetic diversity of native populations (Lindholm et al., 2005; Grapputo et al., 2006; Vidal et al., 2010). The two haplotypes (H1 and H6) shared by both native and non-native C. kelberi populations corroborate the suggestions of Kullander & Ferreira (2006) and Willis et al. (2007) that invasive populations of this species originated from translocations of native individuals from Tocantins and Araguaia River basins. By contrast, Agostinho et al.

8 2602 A. C. P. B. MARQUES ET AL. (a) (b) TUC H8 56 SFA H9 194 TMA N ITU H6 114 PRI H3 136 H H FUN H2 H7 LAJ H1 km VIG SAN PPA PAR Fig. 3. (a) Map showing the distribution of mtdna haplotypes (, H1;, H2;, H3;, H4;, H5;, H6;, H7;, H8;, H9) at 10 sampling locations (FUN, Funil; LAJ, Lajes; SAN, Santana; VIG, Vigário; PPA, Pereira Passos; PAR, Paracambi; TUC, Tucuruí; SFA, São Félix do Araguaia; TMA, Três Marias; ITU, Itumbiara). (b) Median-joining network for Cichla kelberi native and invasive populations. The network circle size is proportional to the haplotype frequency when considering all populations and the distance between nodes is proportional to the number of mutational steps that separates each haplotype. (2007) suggested without the support of molecular analyses that invasive Cichla spp. populations in Paraíba do Sul, São Francisco, Doce and Paraná Rivers originated from translocations from the River Amazon basin. While molecular validation for the occurrence of other species of yellow peacock bass (i.e. C. monoculus) in non-native systems of south-eastern and southern Brazil is still in discussion (Oliveira et al., 2006; Briñez et al., 2013), the findings of the current study strongly confirm that C. kelberi is the main Cichla spp. that has invaded the reservoirs in the Paraíba do Sul River basin. Despite the dominance of the haplotype H1 in eight invasive populations of Cichla spp, three haplotypes not detected within the native distribution were also recorded (H2, H8 and H9). Polymorphisms in the mtdna sequences indicate the possibility of multiple C. kelberi introductions in reservoirs of the Paraíba do Sul River basin (H2 in PPA and PAR) and in PRI (H8 and H9). Multiple introductions generally increase within-population genetic variation of introduced populations relative to native populations and can be inferred if alleles that do not co-occur in native populations are found in introduced samples (Kolbe et al., 2004; Johnson et al., 2011; Fitzpatrick et al., 2012). This can be also indicative of increased propagule pressure, enhancing the probability of establishment because mutation and genetic drift among introduced populations are unlikely to generate intrinsic population differentiation over historical timescales (Frankham, 2005).

9 GENETIC DIVERGENCE IN C. KELBERI POPULATIONS 2603 Table III. Hierarchical analysis of molecular variance (AMOVA) based on 53 Cichla kelberi mtdna sequences; all results P < Samples were divided in two groups: native and invasive populations Source of variation d.f. Sum of squares Variance components Percentage of variation Among groups Among populations within groups Within populations Total Although all the pathways of C. kelberi introduction beyond its native range are still not fully understood, the findings of the current study provide novel and more accurate trails on the invasion routes and stocking events of this species in the south-eastern and southern Brazilian systems, especially when the patterns of haplotype distribution are addressed together with the first Cichla spp. records in these invaded sites. Taking into account the high dominance of the haplotype H1 together with the oldest record of C. kelberi introduction (c. 1950; Santos et al., 2001) among the invasive populations addressed here, there is a strong evidence that the first translocation of Cichla spp. to the south-eastern and southern Brazilian systems occurred in Lajes reservoir, one of the oldest, large ( 30 km 2 ) reservoirs in Brazil (Araújo & Santos, 2001). Cichla kelberi was apparently recorded for the first time between 1950 and 1960 at SAN and VIG reservoirs and although concurrent Cichla spp. introductions cannot be entirely discarded, these two reservoirs were probably invaded through transferred individuals from Lajes reservoir, because of the low distance (<10 km) among reservoirs and intentional introductions of local anglers (Santos et al., 2008). The first spread of C. kelberi to Pereira Passos and Paracambi reservoirs probably occurred in a more passive way, apparently through young fish that survived turbine passage from Lajes and VIG reservoirs, in the case of Pereira Passos colonisation (c. 1970) and similarly for Paracambi reservoir. Deliberate introductions by local sport fishing associations also seem to be the main explanation for C. kelberi invasion of Funil reservoir (probably during the 1970s), separated <100 km from Lajes reservoir (i.e. the two reservoirs have angling clubs, sharing some affiliates). Despite the dominance of the haplotype H1, it is unlikely that C. kelberi populations of TMA and Porto Rico floodplain came from individuals translocated from the reservoirs of the Paraíba do Sul River, which are separated >1000 km from those sites. This difference, together with the exclusive record of haplotype H6 for the population at ITU and the three haplotypes (H2, H8 and H9) not detected within the native area, support the occurrence of multiple introductions through the south-eastern and southern Brazilian systems. The occurrence of multiple C. kelberi introductions was previously detected by Oliveira et al. (2006) and Briñez et al. (2013) for the Porto Rico floodplain. Therefore, the polymorphisms indicating the occurrence of multiple C. kelberi introductions in the Pereira Passos and Paracambi reservoirs, two of the most downstream reservoirs in the Paraíba do Sul River that were studied, could be related to their proximity (c. 80 km) to the Rio de Janeiro metropolis, with 11 million people living in its environs, thereby increasing the chance of recurrent events of introduction. Although the extent of differentiation among introduced populations, however, appears to be explained more by introduction history (Marisco et al.,

10 2604 A. C. P. B. MARQUES ET AL. 2011; Fitzpatrick et al., 2012), it is possible that the record of three haplotypes found exclusively in invasive populations (i.e. Porto Rico, Pereira Passos and Paracambi; Fig. 1) can be explained also by the low sampling effort usually observed in previous studies directed to DNA analyses of native populations, which failed to encompass the entire genetic variability. Efforts to describe the genetic diversity of C. kelberi populations introduced in ponds and reservoirs of the Brazilian semi-arid region, where the first translocations beyond its native range were reported (Fontenele, 1948), are crucial to further disentangle the introduction history of this invasive species. We especially thank the Graduate Course in Neotropical Biodiversity (PPGBIO-UNIRIO) and people at Laboratório de Ictiologia Teórica e Aplicada for providing the logistic support. This work was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil (research grant to L.N.S., E /2012, E-26/ /2015) and Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), Brazil (graduate grant to A.C.S.F. and visiting professorship to E.G.B., ref / ). Supporting Information Supporting Information may be found in the online version of this paper: Table SI Samples for which defined haplotypes confirmed species identification for Cichla kelberi compared with sequences of the closely related species available in GenBank. Sampling site population codes: FUN, Funil; LAJ, Lajes; SAN, Santana; VIG, Vigário; PPA, Pereira Passos; PAR, Paracambi; TUC, Tucuruí; SFA, São Félix do Araguaia; TMA, Três Marias; ITU, Itumbiara. References Agostinho, A. A., Gomes, L. C. & Pelicice, F. (2007). Ecologia e Manejo dos Recursos Pesqueiros em Reservatórios do Brazil. Maringá: EDUEM. Allendorf, F. W. & Lundquist, L. L. (2003). Introduction: population biology, evolution and control of invasive species. Conservation Biology 17, Araújo, F. G. & Santos, L. N. (2001). Distribution of fish assemblages in Lajes reservoir, Rio de Janeiro, Brazil. Brazilian Journal of Biology 61, Bandelt, H. J., Forster, P. & Röhl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16, Briñez, B., Júlio, H. F. Jr., Prioli, S. M. A. P., Maniglia, T. C. & Prioli, A. J. (2013). Molecular identification of Cichla (Perciformes: Cichlidae) introduced in reservoirs in Southern Brazil. Acta Scientiarum Biological Sciences 35, Carvalho, D. C., Oliveira, D. A. A., Sampaio, I. & Beheregaray, L. B. (2009). Microsatellite markers for the Amazon peacock bass (Cichla piquiti). Molecular Ecology Resources 9, Chellappa, S., Câmara, M. R. & Chellappa, N. T. (2003). Ecology of Cichla monoculus (Osteichthyes: Cichlidae) from a reservoir in the semi-arid region of Brazil. Hydrobiologia 504, Clavero, M. & García-Berthou, E. (2005). Invasive species are a leading cause of animal extinctions. Trends in Ecology and Evolution 20, 110. Darriba, D., Taborda, G. L., Doallo, R. & Poasada, D. (2012). jmodeltest2: more models, new heuristics and parallel computing. Nature Methods 9, 772. Elredge, L. G. (2000). Non-indigenous freshwater fishes, amphibians and crustaceans of the Pacific and Hawaiian islands. In Invasive Species in the Pacific: A Technical Review and Draft Regional Strategy (Sherley, G., ed), pp Apia: South Pacific Regional Environment Programme.

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