Genetic diversity of invasive and native (Pisces ... · Non-native freshwater ﬁshes have been...

Genetic diversity of invasive and native Cichla(Pisces: Perciformes) populations in Brazil with

evidence of interspecific hybridization

A. V. OLIVEIRA*†, A. J. PRIOLI*‡§, S. M. A. P. PRIOLI*‡,T. S. BIGNOTTO*, H. F. JULIO JR*‡, H. CARRERk,

C. S. AGOSTINHO{ AND L. M. PRIOLI*#

*Nucleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupelia), UniversidadeEstadual de Maringa, Av. Colombo 5790, Bloco G-90, 87020-900 Maringa, PR, Brasil,‡Departamento de Biologia Celular e Genetica, Universidade Estadual de Maringa, Av.Colombo 5790, Bloco H-67, 87020-900 Maringa, PR, Brasil, kDepartamento de CienciasBiologicas, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo,

Av. Padua Dias 11, 13418-900 Piracicaba, SP, Brasil, {Universidade Federal doTocantins, Jardim dos Ipes, 77500-000 Porto Nacional, TO, Brasil and #Departamento de

Biologia, Universidade Estadual de Maringa, Av. Colombo 5790, Bloco H-78,87020-900 Maringa, PR, Brasil

(Received 7 July 2005, Accepted 1 August 2006)

Invasive and native populations of the Amazonian fishes ‘peacock bass’ Cichla monoculus and of

a not yet described species ‘blue tucunare’ here referred as Cichla sp. ‘Azul’ were analysed for

genetic diversity using the hypervariable domain of the mitochondrial DNA (mtDNA) control

region plus steady diagnostic random amplified polymorphic DNA loci. There is no detailed

historical record of the introduction of Cichla species into the Upper Parana River basin, where

they became invasive and a potential threat to local ichthyofauna. Genetic diversity among

invasive populations confirmed the hypothesis of multiple introductions in this hydrographic

basin. Moreover, a large and previously unknown population of natural fertile hybrids between

C. cf. monoculus and Cichla sp. ‘Azul’ was identified in the Itaipu hydroelectric reservoir and

in the floodplain of the Upper Parana River. Crossbred morphotypes were similar to C. cf.

monoculus, but their morphological identification was not unequivocal. This hybrid population

was characterized by high genetic diversity and it was composed of hybrids possessing

concurrently nuclear DNA fragments specific for C. cf. monoculus as well as fragments specific

for Cichla sp. ‘Azul’. The nuclear DNA markers indicated that reproductive isolation between

C. cf. monoculus and Cichla sp. ‘Azul’ has broken down in the new environment, and mtDNA

sequences revealed that both species can be the female donor in the interspecific crosses. The

data presented herein are potentially useful for future taxonomic, genetic and evolutionary

studies in the complex Cichla group, for monitoring of invasive populations, and for further

development of ecological guidelines. # 2006 The Authors

Journal compilation # 2006 The Fisheries Society of the British Isles

Key words: Cichla; Cichlid; D-loop; interspecific hybrids; peacock bass; tucunare.

§Author to whom correspondence should be addressed. Tel. and fax: þ55 44 3263 1424;

email: [email protected]

†Present address: Centro Universitario de Maringa (Cesumar), Av. Guedner 1610, 87050-390 Maringa,

PR, Brasil.

Journal of Fish Biology (2006) 69 (Supplement B), 260–277

doi:10.1111/j.1095-8649.2006.01291.x, available online at http://www.blackwell-synergy.com

260# 2006 The Authors

Journal compilation # 2006 The Fisheries Society of the British Isles

INTRODUCTION

Non-native freshwater fishes have been deliberately introduced in Neotropicalhydrographic basins even though these habitats are naturally abundant withnative fish species. During the last decades, Brazil has received the highestnumber of non-native fishes in spite of its currently >2100 catalogued fish spe-cies, which comprise c. 21% of the world list (Buckup & Menezes, 2003). In-troductions have included fishes from other countries and also transfersamong Brazilian hydrographic basins (Agostinho et al., 1994, 2003; Julio &Agostinho, 2003). As extensively reported, non-native introduced fishes maybecome invasive and have a serious impact on aquatic ecosystems. As discussedby Agostinho et al. (2005), in Brazilian inland waters, fish species introductionshave been recognized as one of the principal direct causes of biodiversity loss.Such introductions have been done mainly for aquaculture, fish stocking andrecreational fisheries, and generally without considering their potentialadverse impact on the environment and on the biodiversity of local aquaticecosystems.The Upper Parana River floodplain, a unique ecosystem with >250 reported

fish species, has been strongly affected by non-native introduced fishes. It com-prises an environmental protected area, as well as the only remaining runningwater stretch of the Parana River in Brazilian territory, which is not restrainedby hydroelectric dams. In 1982, when the Itaipu hydroelectric dam was closed,the floodplain received a massive introduction of at least 35 fish species fromthe Middle Parana River basin (Agostinho et al., 2003; Julio & Agostinho,2003). These fishes were introduced because the resulting Itaipu reservoirsubmerged the Guaıra Falls (Seven Falls), which had previously formed thenatural geographic barrier between these two ichthyological provinces. Asa consequence, c. 150 km of the Parana River downstream of the falls weremerged with the Upper Parana River. On top of that, during the past threedecades populations of both non-native and local fishes have been intentionallyintroduced in the Upper Parana River basin. Amazonian piscivores have beenthe most successful colonizers in this basin where they have spread out of res-ervoirs and are now affecting areas with high abundance of endemic species,including the floodplain (Agostinho et al., 2004, 2005).Fishes of the genus Cichla Schneider, 1801, are among the species that were

deliberately introduced in many hydrographic basins, including in the UpperParana (Agostinho et al., 1994, 2003, 2004; Shafland, 1996; Julio & Agostinho,2003). Most Cichla species are native to the Amazon and Orinoco basins.Morphological traits have been the basis of Cichla taxonomy, but this genusremains problematic. Although 15 different Cichla morphotypes have beenreported (Kullander, 1986; Kullander & Nijssen, 1989), presently only fivespecies are described: Cichla temensis Humboldt, 1821 (Orinoco, Negro andTapajos Rivers); Cichla monoculus Spix & Agassiz, 1831 (Amazon basin, includ-ing the Tocantins–Araguaia sub-basin); Cichla ocellaris Bloch & Schneider,1801 (Guyana rivers, from the Marowijne drainage in Suriname and FrenchGuyana to the Essequibo drainage in Guyana); Cichla orinocensis Humboldt,1821 (Orinoco and Negro rivers); Cichla intermedia Machado-Allison, 1971(Upper Negro River and Middle Orinoco River).

GENETIC DIVERSITY OF C I C H L A POPULATIONS 261

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Journal compilation # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 69 (Supplement B), 260–277

In 1985, a few specimens identified as C. monoculus species, which is popularlyknown as ‘tucunare’, ‘peacock bass’ and ‘pavon’, were found for the first time inthe Itaipu hydroelectric reservoir (Agostinho et al., 1994, 2004). Sometime later,a not yet described Cichla species (S. Kullander, pers. comm.), which is popularlyknown as ‘tucunare azul’ and ‘blue tucunare’ and native to the Tocantins–Araguaia sub-basin of the Amazon River basin, was found in the Itaipu reser-voir. Initially, both Cichla populations seemed to be present at low density,but they increased rapidly and spread from the reservoir into many rivers andstreams. Diverse morphotypes resembling C. monoculus, but of unclear identifica-tion as regards their morphological analysis, have also been found in the Itaipureservoir and in the floodplain (C. S. Pavanelli, pers. comm.). Taxonomy, originand introduction of Cichla in the Upper Parana River basin remain unclear. Ithas been assumed that multiple Cichla introductions might have occurred intothis basin, particularly in waters that are regulated by dams. Supposedly, Cichlawere introduced by sport fishing associations but incidental escapes from pisci-culture might also have occurred occasionally (Orsi & Agostinho, 1999). Morerecently, Cichla populations have also been found in other areas of the UpperParana basin, mainly in reservoirs, but as far as is known, there is no recordof their introductions. Cichla species are voracious predators feeding on a widerange of prey and displaying complex reproductive strategies (Fontanele & Peix-oto, 1979; Novaes et al., 2004). The introduction of Cichla populations in theUpper Parana basin has developed into a controversial issue because while theybecame the most important species for sport fishery, they also have becomehighly invasive and voracious piscivores, a menace to local fishes, includingendemic species (Fontenele & Peixoto, 1979; Agostinho et al., 2003, 2004).Knowledge of their genetic diversity and distinctive taxonomy is crucial for

monitoring introduced Cichla populations, particularly those that are nowinvasive and a steady part of the current fauna in many areas of the UpperParana River basin. Mitochondrial genome and nuclear DNA fragments haveproved useful in taxonomic and genetic studies. The hypervariable domain ofthe mitochondrial DNA (mtDNA) control region has been the main nucleotidesequence of choice for population and phylogenetic studies among closelyrelated species. Diagnostic nuclear DNA fragments such as steady randomamplified polymorphic DNA (RAPD) markers (Williams et al., 1990) havebeen helpful in taxonomic and genetic research, including studies of hybridiza-tion events. In native and non-native fish populations, these nuclear and mito-chondrial molecular markers have been informative (Bardakci & Skibinski,1994; Callejas & Ochando, 2001; Weiss et al., 2001; Oliveira et al., 2002; Prioliet al., 2002; Rubidge & Taylor, 2004).Genetic studies have not previously been reported on Cichla non-native pop-

ulations and little is known about their native populations. Evidence of hybrid-ization between C. monoculus and C. temensis (‘speckled pavon’) species hasbeen reported for natural populations native to Amazonian regions (Andradeet al., 2001; Brinn et al., 2004; Teixeira & Oliveira, 2005). These studies suggestthe possibility of interspecific crosses in regions where more than one Cichlaspecies have been introduced. Genetic studies of native and invasive Cichlapopulations will help to improve current knowledge of taxonomy and evolutionwithin the cichlid group (Farias et al., 1999).

262 A. V. OLIVEIRA E T A L .

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The objective of this work was to analyse the genetic diversity of native andinvasive populations, which have been identified as C. monoculus and Cichla sp.species, by using the hypervariable domain of the mtDNA control region plusdiagnostic nuclear RAPD loci. With the taxonomic and genetic characteriza-tion possible with these tools the aim was to improve the understanding ofCichla introduction in the Upper Parana basin, and to test the hypothesis ofhybridization between introduced species.

MATERIALS AND METHODS

FISH SAMPLING AND DNA EXTRACTION

Cichla populations were sampled in four locations of the Upper Parana Riverbasin and in three locations of the Amazon River basin (Fig. 1). On the basis ofmorphological traits, the specimens (voucher specimens: NUP1908; NUP3180;NUP1746; NUP3379; NUP3884) were initially identified as C. monoculus and C.temensis (Kullander, 1986), and a third species not yet described, which is popularlyknown as ‘blue tucunare’ and ‘tucunare azul’ (S. Kullander, pers. comm.). Giventhe unclear status of the taxonomy of Cichla species, the species included in thisstudy are referred to as Cichla cf. monoculus, Cichla sp. ‘Azul’ and Cichla cf. temensis.Cichla specimens were captured with gillnets, and muscle tissues were immediatelyfixed in ethanol and stored at �20° C. Samples of total DNA were extracted frommuscle tissues macerated in the presence of liquid nitrogen, according to Monesiet al. (1998) with few modifications. After phenol/chloroform extraction, DNA wasprecipitated with ethanol and resuspended in diluted TE buffer (Tris 1 mM, EDTA0�1 mM pH 8�0) containing RNAase (20 mg ml�1). DNA aliquots were usedfor quantification by comparison with known quantities of l phage DNA in 1%agarose gel.

AMPLIFICATION AND ANALYSIS OF RAPD LOCI

The eight RAPD primers OPW-04, OPW-09, OPW-17, OPW-19, OPA-06, OPE-09,OPX-05 and OPX-18 (Operon Technologies Inc., Alameda, CA, U.S.A.) were usedfor polymerase chain reaction (PCR) amplifications. The PCR reaction mix andDNA amplification were performed according to Williams et al. (1990) with minormodifications (Prioli et al., 2002). A sample without template DNA was included asa negative control in each experiment. In order to test reproducibility of PCR amplifi-cation products, reactions were performed at least twice and one to two samples of pre-viously amplified and analysed PCR products, with the same primer, were included ineach agarose gel. Electrophoresis profiles were visualized under UV radiation and pho-tographed with Kodak EDAS-290. Sizes of DNA fragments were estimated by compar-ison with standard 100 base pair (bp) ladder (Invitrogen Life Technologies�, Carlsbad,CA, U.S.A.).

RAPD electrophoresis profiles were analysed for polymorphism based on the pres-ence and absence of accurate steady DNA bands on agarose gel. Specimens were com-pared within and among populations. Indexes of molecular diversity were estimatedbased on the average gene diversity over all haplotype loci using Arlequin v. 3.0(Excoffier et al., 2005). Distance matrix between specimens, taken two by two, wasobtained by the arithmetic complement of Nei & Li (1979) similarity index, usingRAPDPLOT (Black, 1997). Because the genetic distance of Nei & Li (1979) is not met-rical, the Lingoes correction (Legendre & Anderson, 1999) was applied using theDistPCoA software (Legendre & Anderson, 1998).


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FIG. 1. Map of South America displaying the seven sampling locations in which Cichla populations were

surveyed. Locations 1 to 4 are situated in the Upper Parana River basin ( ) where Cichla monoculus

and Cichla sp. ‘Azul’ were introduced and became invasive. Locations 5 to 7 are situated in the

Amazon hydrographic basin ( ) including its Tocantins–Araguaia sub-basin ( ). Native species C.

monoculus is found in both and areas, C. temensis in and Cichla sp. ‘Azul’ in . Sampling

locations: 1, Upper Parana River floodplain (22°479 S; 53°199 W) near Porto Rico township: Cichla

sp. ‘Azul’, C. monoculus and interspecific hybrids; 2, Itaipu hydroelectric reservoir (between 24°059;25°339 S and 54°009; 54°379 W) in the Parana River, downstream the floodplain: Cichla sp. ‘Azul’

and interspecific hybrids; 3, Capivara hydroelectric reservoir (27°399 S; 51°219 W) in the Para-

napanema River: C. monoculus; 4, Promissao hydroelectric reservoir (21°189 S; 17°479W) in the Tiete

River, near Promissao township: Cichla sp. ‘Azul’; 5, Tocantins River (09°459 S; 48°229 W), Lajeado

reservoir, in the Amazon Tocantins–Araguaia sub-basin, near Porto Nacional city: Cichla sp. ‘Azul’

and C. monoculus; 6, Amazon River (03°079 S; 59°559 W) near the Solimoes River and Manaus city:

C. monoculus; 7, Fish farm near the Teles Pires River, Lucas do Rio Verde City, Mato Grosso State

(13°039 S; 55°549 W): C. temensis.


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SEQUENCING AND ANALYSIS OF MITOCHONDRIAL DNA

A mtDNA fragment of c. 460 bp was PCR amplified from DNA samples of four tosix specimens of each population. The primers D-loop L 59-AGAGCGTCGGTCTTG-TAAACC-39 (Cronin et al., 1993) and H16498 59-CCTGAAGTAGGAACCAGATG-39(Meyer et al., 1990) were used for PCR amplifications. The reaction mix consisted ofTris-KCl (20 mM Tris-HCl pH 8�4 and 50 mM KCl), 1�5 mM MgCl2, 2�5 mM of eachprimer, 0�1 mM of each dNTP, 2�5 U Taq DNA polymerase, 15 ng DNA and water toa total volume of 25 ml. PCR amplification started with one cycle of 94° C for 4 min,50° C for 30 s and 72° C for 2 min, followed by 40 cycles of 94° C for 15 s, 56° C for 30s, 72° C for 2 min and a final extension step at 72° C for 10 min. The mtDNA segmentfrom each specimen was amplified in two independent PCR reactions, as replicates, andthen bi-directionally sequenced.

PCR products were directly used as templates for sequencing in an automatedsequencer ABI-3100 (Perkin Elmer, Norwalk, CT, U.S.A.). Approximately 50 ng oftemplate DNA and 20 pmol of either primer H16498 or D-Loop L were added to eachsequencing reaction. The reaction mix was heated at 94° C for 4 min, and amplifica-tions were performed in 35 cycles of 30 s at 94° C, 30 s at 55° C and 1 min 30 s at60° C, followed by 5 min at 60° C and then kept at 4° C. Sequence data were submittedto quality check, assembly and alignment on the Vector NTI Suite 6.0 (Informax, Inc.,Invitrogen Life Technologies�). DNA sequences were aligned using the CLUSTALWand genetic analyses were conducted using MEGA 3.1 (Kumar et al., 2004). Matrix ofdistances among specimens and among haplotypes was obtained from estimates ofgenetic distances of Tamura & Nei (1993). Clustering was performed by the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analyses were based on 1000 replications.

RESULTS

Each of the selected RAPD primers amplified from eight to 11 intense DNAfragments, which ranged from c. 350 bp to 2�4 kb. Eighty-three most intense,defined and repeatable DNA fragments were chosen for analyses. Of those 83RAPD loci, 53 (63�86%) were polymorphic and 30 (36�14%) were monomor-phic. Electrophoresis profiles for the OPW-09 primer are illustrated in Fig. 2.All primers, except OPA-06, produced steady monomorphic DNA fragments,which were exclusive either to C. cf. monoculus or to Cichla sp. ‘Azul’. Mono-morphic and exclusive DNA bands were identified in numbers suitable to dis-tinguish these species and their studied populations, and they were used asdiagnostic nuclear markers.Specimens from the native C. cf. monoculus populations, two individuals

from the Tocantins River (Fig. 1, location 5) and three individuals from theAmazon River (Fig. 1, location 6), were clearly distinguished from each otherby exclusive monomorphic DNA fragments. In the native specimens from theTocantins River, 10 monomorphic DNA fragments were found to be exclusiveto Cichla sp. ‘Azul’ and 12 were exclusive to C. cf. monoculus. Cichla sp. ‘Azul’from the Tocantins River contained 12 exclusive monomorphic DNA frag-ments when compared to C. cf. monoculus from the Amazon River, while thelatter contained 14 exclusive monomorphic DNA fragments when comparedto Cichla sp. ‘Azul’ from the Tocantins River. All of the exclusive diagnosticDNA fragments were confirmed in the native and invasive populations. Theinvasive Cichla sp. ‘Azul’ populations sampled in the floodplain and in the Itaipuand Promissao reservoirs shared characteristic exclusive monomorphic nuclearmarkers with the native population from Tocantins River. The population


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invasive to Promissao reservoir, however, contained two monomorphic DNAfragments that were absent in the Tocantins, floodplain and Itaipu populations.On the other hand, these latter populations shared three monomorphic DNAfragments that were absent in the Promissao population. The non-native C. cf.monoculus population from Capivara reservoir (Fig. 1, location 3) shared twomonomorphic DNA fragments with the population native to the Amazon River(Fig. 1, location 6), which were absent in the Tocantins population (Fig. 1, loca-tion 5). The analysed C. cf. temensis specimens were characterized by five exclu-sive monomorphic RAPD fragments.Of the 68 Cichla specimens captured in the Upper Parana River floodplain

and in the Itaipu reservoir (Fig. 1, locations 1 and 2), a total of 52 specimenscontained simultaneously nuclear monomorphic DNA fragments exclusive toC. cf. monoculus and fragments exclusive to Cichla sp. ‘Azul’. They resembledC. cf. monoculus species based on morphological traits. Two of the C. cf.monoculus exclusive monomorphic fragments were specific to the populationnative to the Tocantins River and absent in the Amazon River population.Genetic variability within these 52 specimens was high, as indicated by42�17% of RAPD polymorphic loci. In contrast, the percentage of polymorphicloci was low in the C. cf. monoculus and in the Cichla sp. ‘Azul’ populations,varying from 1�2 to 14�46%. The haplotype molecular diversity index, as basedon the average gene diversity over all haplotype loci, was estimated as 0�143 forall populations analysed as a group. The haplotype molecular diversity indexfor those 52 specimens sampled the floodplain and Itaipu reservoir was esti-mated as 0�085. This genetic diversity estimate was high when compared tothe haplotype molecular diversity indexes varying from 0�012 to 0�040 withinall other populations studied.

FIG. 2. Electrophoresis profiles, obtained by using the RAPD primer OPW-09, of specimens from native

and invasive Cichla populations. Lanes 1–3, Cichla temensis native to the Amazon River basin. Lanes

4–6, Cichla sp. ‘Azul’ native to the Tocantins River. Lanes 7–8, Cichla monoculus native to the

Tocantins River. Lanes 9–11, C. monoculus invasive to the Capivara reservoir. Lanes 12–15, hybrids

(C. monoculus � Cichla sp. ‘Azul’) sampled in the Upper Parana River floodplain. Lanes 16–18,

hybrids (C. monoculus � Cichla sp. ‘Azul’) sampled in the Itaipu reservoir. Note the presence of

DNA fragments exclusive to C. monoculus (lanes 7–11) and Cichla sp. ‘Azul’ (lanes 4–6). Arrows in

lane 15 indicate DNA fragments exclusive to either C. monoculus (arrow at right) or Cichla sp. ‘Azul’

(arrow at left), which were inherited by this crossbred. L, Molecular mass markers (Ladder 100).


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The genetic differentiation pattern on the basis of Nei & Li (1979) similarityindex is represented by principal co-ordinates (Fig. 3), where the three distinctspecies C. cf. monoculus, Cichla sp. ‘Azul’ and C. cf. temensis were distinguishedfrom each other. Cichla cf. temensis was positioned closer to Cichla sp. ‘Azul’than to C. cf. monoculus. The 52 specimens from the Upper Parana River flood-plain and Itaipu reservoir, which shared C. cf. monoculus and Cichla sp. ‘Azul’nuclear DNA fragments, were arranged as a population in an intermediateposition between these two species. These results are evidence of crossbreedingbetween C. cf. monoculus and Cichla sp. ‘Azul’. The specific nuclear diagnosticDNA fragments were not equally distributed in the 52 specimens from theinvasive populations established in the floodplain and Itaipu reservoir, whichindicate that they could represent C. cf. monoculus v. Cichla sp. ‘Azul’ advancedprogenies.The mtDNA fragments (c. 460 bp) consisted of a partial sequence of the

tRNAThr gene, immediately followed by the complete sequence of the tRNAPro

gene, and then by c. 360 bp corresponding to the hypervariable domain ofthe control region (GenBank accession numbers AY836716 to AY836750).Nucleotide polymorphism was low among the tRNA sequences, therefore theywere not informative and were excluded from the analysis. The CLUSTALWalignment of the mtDNA control region sequence (c. 360 bp) from 35 Cichlaspecimens revealed a total of 97 polymorphic nucleotide sites, which were

FIG. 3. The first two axes in the principal co-ordinates analysis (PCO) of Cichla populations: Invasive to

the Upper Parana River basin: ( ) Upper Parana River floodplain composed of Cichla sp. ‘Azul’

(left) and ( ) interspecific hybrids (middle); ( ) Itaipu reservoir composed of Cichla sp. ‘Azul’ (left)

and ( ) interspecific hybrids (middle); ( ) Promissao Reservoir consisted of Cichla sp. ‘Azul’; ( )

Capivara reservoir composed of C. monoculus. Native to the Amazon River basin: ( ) Tocantins

River comprising C. monoculus and Cichla sp. ‘Azul’ ( ) Amazon River near Manaus composed of

C. monoculus; ( ) fish farm near the Teles Pires River, in Mato Grosso State, comprising C. temensis.

Analyses were corrected by the Lingoes method from the matrix of arithmetic complements of Nei

and Li’s (1979) similarity coefficients obtained from RAPD markers.


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distributed in 10 haplotypes (Table I). Almost all changes were single nucleo-tide substitutions, and transitions were the most frequent (average ti:tv ¼2�12) as is typical for the mtDNA control region.Genetic distances of Tamura & Nei (1993), as based on the mtDNA control

region sequences, are shown in Table II. Genetic distances were low amonghaplotypes found within the same species, varying from 0�006 to 0�057. Asto native populations, the C. cf. monoculus haplotypes (Hapl-Cmn-VI andHapl-Cmn-II) were discriminated from Cichla sp. ‘Azul’ (Hapl-Csp-I) by17�5–18�6% nucleotide divergence (63–67 sites), and from C. cf. temensis(Hapl-CtmI) by c. 18�5% divergence. As expected, the genetic distance esti-mates were proportional to the nucleotide diversity when C. cf. monoculuswas compared to Cichla sp. ‘Azul’ or to C. cf. temensis, varying from 0�177to 0�195. On the other side, the estimates indicated that Cichla sp. ‘Azul’ ismore closely related to C. cf. temensis (0�125) than to the C. cf. monoculus(0�178–0�195) populations. In addition, the analysed mtDNA control regionsequence was suitable to discriminate the populations of C. cf. monoculus nativeeither to the Tocantins River (Hapl-Cmn-II) or to the Amazon River (Hapl-Cmn-VI). They were differentiated by 19 nucleotide site polymorphisms(5�3%), with a genetic distance estimate of 0�054.The mtDNA control region sequences indicated the possible native popula-

tions that were involved in the Cichla introductions to the locations studied inUpper Parana River basin. Interestingly, the same C. cf. monoculus haplotype(Hapl-Cmn-VI) from the Amazon River native population (Fig. 1, location 6)was identified in the population introduced in the Capivara reservoir (Fig. 1,location 3). In addition, a second C. cf. monoculus haplotype (Hapl-Cmn-VII)was identified in the Capivara reservoir. As compared to the native C. cf.monoculus, the haplotype Hapl-Cmn-VII was more closely related to theHapl-Cmn-VI from the Amazon River specimens (3�9% divergence) than tothe Hapl-Cmn-II from Tocantins specimens (7�8% divergence). The haplotypesof Cichla sp. ‘Azul’ invasive to the floodplain and Itaipu reservoir were identi-cal to those of Cichla sp. ‘Azul’ native to the Tocantins River. The neighbour-joining dendrogram based on Tamura & Nei (1993) genetic distance matrixamong Cichla specimens is represented in Fig. 4. Three major haplotypegroups, corresponding to C. cf. monoculus, Cichla sp. ‘Azul’ and C. cf. temensis,were separated and supported by a 100% bootstrap rate. The species Cichla sp.‘Azul’ was more closely related to C. cf. temensis than to C. cf. monoculus(Table II and Fig. 4).As shown in Table I, the specimens identified as interspecific hybrids con-

tained the mitochondrial genome from either C. cf. monoculus or Cichla sp.‘Azul’. Nine of the 11 interspecific hybrids analysed contained C. cf. monoculushaplotypes, which were divergent by only one to three nucleotide sites from theHapl-Cmn2 haplotype from the population native to the Tocantins River. TheC. cf. monoculus haplotypes of populations invasive to the floodplain and Itaipureservoir diverged by c. 19 nucleotide changes (5�3%) when compared to thepopulation native to the Amazon River. As expected, these nine specimenswere clustered with specimens native to the Tocantins River in the neigh-bour-joining dendrogram based on Tamura & Nei (1993) genetic distances(Fig. 4). The remaining two interspecific hybrids carried a haplotype identical


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TABLEI.

Nucleotidepolymorphismsin

thehypervariablesequence

(c.360bp)ofthemtD

NAcontrolregion(D

-loop)from

Cic

hlainvasiveand

nativepopulations.Samplinglocationsare

indicatedbythefirstnumber

ineach

specim

enidentification:1,Upper

ParanaRiver

floodplain;2,

Itaipureservoir;3,Capivara

reservoir;5,TocantinsRiver;6,AmazonasRiver;7,fish

farm

nearTeles

Pires

River

(see

Fig.1).Haplotypes:

Hapl-Cmn,

C.

mo

no

culu

s;Hapl-Csp,

Cic

hla

sp.‘A

zul’;Hapl-Ctm

,C

.te

men

sis.Entire

sequencesatGenbank:AY836716to

AY836750

Specim

enHaplotypes

Identification

0112222233335566677777888889999111111111111111111111111111111111111111111222222222222222222333333

7451345856783506824789067890246111112222222333344455666667777788888899999000123333445577788223455

123450125679037917809245792345803578902458127070278265905814060316

2-TUC83

Hapl-Cmn-I

Hybrid

CCCATACAATTAATTTGGTCGTTTATGAACTTTAAC—GATTACCTGATTCATAT-TGTTAACTTAACAATTT-G

CA-C

CTCAATACGTCTTACGT

2-TUC85

Hapl-Cmn-I

Hybrid

....................................-

--.................-.................-...-

..................

2-TUC90

Hapl-Cmn-I

Hybrid

....................................-

--.................-.................-...-

..................

2-TUC62

Hapl-Cmn-I

Hybrid

....................................-

--.................-.................-...-

..................

1-TUC18

Hapl-Cmn-I

Hybrid

....................................-

--.................-.................-...-

..................

1-TUC26

Hapl-Cmn-I

Hybrid

....................................-

--.................-.................-...-

..................

5-TUC103

Hapl-Cmn-II

C.

monocu

lus*

...............................C

....-

--.................-.A

...............-...-

..................

5-TUC104

Hapl-Cmn-II

C.

monocu

lus*

...............................C

....-

--.................-.A

...............-...-

..................

5-TUC105

Hapl-Cmn-II

C.

monocu

lus*

...............................C

....-

--.................-.A

...............-...-

..................

1-TUC16

Hapl-Cmn-III

Hybrid

...............................C

....-

--.................-.A

...............-...-

.....G

............

1-TUC15

Hapl-Cmn-IV

Hybrid

....C

..........................C

....-

--.................-.A

...............-...-

.....G

............

1-TUC21

Hapl-Cmn-V

Hybrid

...G

..G

........................C

....-

--.................-.A

...............-...-

.....G

............

6-TUC114

Hapl-Cmn-V

IC

.m

onocu

lus*

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

6-TUC115

Hapl-Cmn-V

IC

.m

onocu

lus*

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

6-TUC116

Hapl-Cmn-V

IC

.m

onocu

lus*

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

6-TUC120

Hapl-Cmn-V

IC

.m

onocu

lus*

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

3-TUC42

Hapl-Cmn-V

IC

.m

onocu

lus

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

3-TUC55

Hapl-Cmn-V

IC

.m

onocu

lus

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

3-TUC77

Hapl-Cmn-V

IC

.m

onocu

lus

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.TT

.C..-...-...........

TC

....C

3-TUC80

Hapl-Cmn-V

IC

.m

onocu

lus

.T

...G

.................A

...T

....C

...-

--.......C

A.C

...A

..C

.A

.C

.....G

.T

T.C

..-...-...........T

C....C

3-TUC57

Hapl-Cmn-V

IIC

.m

onocu

lus

T....G

.......C

.C

...A

...C

..A

T........-

--........A

.C

.-.A

..C

.A

C....C

.G.TT.C

..-...-...........T

......

3-TUC78

Hapl-Cmn-V

IIC

.m

onocu

lus

T....G

.......C

.C

...A

...C

..A

T........-

--........A

.C

.-.A

..C

.A

C....C

.G.TT.C

..-...-...........T......

3-TUC79

Hapl-Cmn-V

IIC

.m

onocu

lus

T....G

.......C

.C

...A

...C

..A

T........-

--........A

.C

.-.A

..C

.A

C....C

.G.TT.C

..-...-...........T......


# 2006 The Authors


TABLEI.

Continued

Specim

enHaplotypes

Identification

0112222233335566677777888889999111111111111111111111111111111111111111111222222222222222222333333

7451345856783506824789067890246111112222222333344455666667777788888899999000123333445577788223455

123450125679037917809245792345803578902458127070278265905814060316

1-TUC5

Hapl-Csp-I

Hybrid

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C

TCCAAAATCTTATTG..AACT.A

TGAC

1-TUC19

Hapl-Csp-I

Hybrid

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

1-TUC68

Hapl-Csp-I

Cic

hla

sp.

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

1-TUC69

Hapl-Csp-I

Cic

hla

sp.

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

1-TUC70

Hapl-Csp-I

Cic

hla

sp.

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

2-TUC63

Hapl-Csp-I

Cic

hla

sp.

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

5-TUC98

Hapl-Csp-I

Cic

hla

sp.*

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

5-TUC96

Hapl-Csp-I

Cic

hla

sp.*

.....G

.GC...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATCAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

5-TUC95

Hapl-Csp-II

Cic

hla

sp.*

.....G

.GT...T

.-...A

TAC..TAATTAC..T.A

CATACC..ATAAT.A

.TAC.T

AA.C

-T..ATT.C


TGAC

7-TUC73

Hapl-Ctm

-IC

.te

men

sis*

..T..G.G

TAATT.-.A

AAT.C

C...A

T..C..CGTTATAC.C

CATCACCC.T

ATCA.A

..-.-.ATT.C

.CCGAATGCTTA..GCCAACTC.T

...

7-TUC74

Hapl-Ctm

-IC

.te

men

sis*

..T..G.G

TAATT.-.A

AAT.C

C...A

T..C..CGTTATAC.C

CATCACCC.T

ATCA.A

..-.-.ATT.C


...

7-TUC75

Hapl-Ctm

-IC

.te

men

sis*

..T..G.G

TAATT.-.A

AAT.C

C...A

T..C..CGTTATAC.C

CATCACCC.T

ATCA.A

..-.-.ATT.C


...

*Specim

ensfrom

nativepopulations.

270 A. V . OLIVEIRA E T A L .

# 2006 The Authors


TABLEII.Genetic

distancesofTamura

&Nei

(1993)among

Cic

hla

haplotypes

from

invasiveand

nativepopulations,

based

on

the

hypervariable

sequence

ofthemtD

NA

controlregion.Haplotypes:Hapl-Cmn,

C.

mo

no

culu

s;Hapl-Csp,

Cic

hla

sp.‘A

zul’;Hapl-Ctm

,C

.te

men

sis.Samplinglocations:1,Upper

ParanaRiver

floodplain;2,Itaipureservoir;3,Capivara

reservoir;5,TocantinsRiver;6,Amazonas

River;7,fish

farm

ingnearTeles

Pires

River

(see

Fig.1)

Haplotypes,locations

Hapl-Cmn-II

C.

mo

no

culu

sHapl-Cmn-V

IC

.m

on

ocu

lus

Hapl-Cmn-V

IIC

.m

onocu

lus

Hapl-Cmn-I

Hybrid

Hapl-Csp-I

Cic

hla

sp.*

Hapl-Csp-I

Hybrid

5*

6*and3

31and2

5*,1and2

1

Hapl-Cmn-V

I,C

.m

on

ocu

lus

0� 054

6*and3

Hapl-Cmn-V

II,

C.

mo

no

culu

s0� 057

0� 042

3 Hapl-Cmn-I,Hybrid

0� 006

0� 054

0� 057

1and2

Hapl-Csp-I,

Cic

hla

sp.*

0� 192

0� 178

0� 195

0� 192

5*,1and2

Hapl-Csp-I,Hybrid

0� 192

0� 178

0� 195

0� 192

0� 000

1 Hapl-Ctm

-IC

.te

men

sis

0� 192

0� 177

0� 187

0� 191

0� 125

0� 125

7 *Specim

ensfrom

nativepopulations.


# 2006 The Authors


to native Cichla sp. ‘Azul’, and they were grouped with this species populationnative to the Tocantins River. Therefore, the specimens positioned in the inter-mediate area of the graph of PCO factors I and II (Fig. 3) had maternal ances-tors from the C. cf. monoculus and Cichla sp. ‘Azul’ species.

DISCUSSION

The molecular genetic data described in this work confirm that the speciesC. cf. monoculus and the Cichla sp. ‘Azul’ (blue tucunare), a not yet describedspecies, were introduced into the southern part of the Upper Parana River

FIG. 4. Neighbour-joining dendrogram obtained from the hypervariable domain of the mtDNA control

region (D-loop) sequences of Cichla haplotypes from populations invasive to the Upper Parana

River basin and populations native to the Amazon hydrographic basin: ( ) Upper Parana River

floodplain; ( ) Itaipu reservoir; ( ) Capivara reservoir; ( ) Tocantins River; ( ) Amazon River

near Manaus; ( ) fish farm near the Teles Pires River, in Mato Grosso State. Genetic distances were

estimated by the Tamura & Nei (1993) method. Numbers in the dendrogram indicate bootstrap

probability as based on 1000 replicates. *, Specimens from native populations.


# 2006 The Authors


basin. Moreover, the data indicate that introduced populations of C. cf.monoculus and Cichla sp. ‘Azul’ interbred in the Itaipu reservoir and in thefloodplain of the Upper Parana River. Hybrids between C. cf. monoculus andCichla sp. ‘Azul’ have not been described in the Tocantins–Araguaia sub-basinwhere both species are native and sympatric.Polymorphisms in the mtDNA sequences and in nuclear DNA fragments

confirm the hypothesis of multiple Cichla introductions in the Upper ParanaRiver basin. Data revealed two subpopulations of C. cf. monoculus in theCapivara reservoir and indicated that they were derived from populationsnative to the Amazon River basin, which are diverse of the Tocantins–Araguaia sub-basin. In the Upper Parana floodplain and Itaipu reservoir,however, invasive populations might have been introduced at first from C.cf. monoculus and Cichla sp. ‘Azul’ native to the Tocantins–Araguaia sub-basin. Data also indicated that a diverse subpopulation of Cichla sp. ‘Azul’was introduced in the Promissao reservoir. Therefore, multiple intentional in-troductions of Cichla species from the Tocantins–Araguaia sub-basin and fromthe Amazon River are likely to have occurred into the Upper Parana Riverbasin, and they obviously involved diverse native subpopulations at the differentevents. In addition, Orsi & Agostinho (1999) reported recent Cichla introduc-tions into the Capivara reservoir probably were the result of fish escapes fromfish farming operations.The genetic differentiation pattern demonstrated 52 specimens genetically

intermediate between C. cf. monoculus and Cichla sp. ‘Azul’ (Fig. 3). The datapresented here provide strong indication for the breakdown of reproductiveisolation between Cichla species in a new environment, resulting in hybridiza-tion. The low frequencies of Cichla parental species in the Itaipu reservoirand in the floodplain of the Upper Parana basin reinforce the assumption oflocal hybridization. Because hybrids were prevalent (76�5%) in the establishedinvasive populations plus the evidence that currently they would representadvanced progenies, it could be hypothesized that the C. cf. monoculus v. Cichlasp. ‘Azul’ hybrids are fertile. The number and repeatability of the exclusivediagnostic nuclear DNA bands were sufficient for an unambiguous discrimina-tion of the three Cichla species and their populations. Diagnostic nuclear DNAfragments exclusive to the same studied Cichla species and populations havealso been confirmed using the inter-simple sequence repeat (ISSR) technique.In addition, the ISSR diagnostic markers corroborate the results reported inthis work both for the native and invasive Cichla populations and for the exis-tence of C. cf. monoculus � Cichla sp. ‘Azul’ hybrids (G. C. A. Almeida, pers.comm.). RAPD diagnostic loci have been used effectively for preliminaryassessments of genetic variability and for unequivocal detection of naturalinterspecific hybrids in other fishes (Bardakci & Skibinski, 1994; Callejas &Ochando, 2001; Weiss et al., 2001; Oliveira et al., 2002; Khrisanfova et al.,2004).The Cichla hybrids identified in the Upper Parana River basin inherited

mtDNA either from C. cf. monoculus or from Cichla sp. ‘Azul’, hence demon-strating that both parental species can act as the female donor in the interspe-cific crosses. Hybrid haplotypes were clustered with their corresponding nativespecies, which must have been the mother in the interspecific crosses, and


# 2006 The Authors


the clustering did not discriminate native and introduced Cichla specimens. Asbased on the mtDNA nucleotide similarity (Fig. 4 and Table I), a C. cf. mono-culus population from the Tocantins-Araguaia hydrographic basin rather thanthe Amazon River must have been involved in the interspecific crosses.Introgressive hybridization is a common feature between divergent lineages

of fishes, particularly when allopatric taxa are introduced into new habitats(Hubbs, 1955; Arthington, 1991; Pierce & Van Den Avyle, 1997; Avise et al.,2002; Rubidge & Taylor, 2004). In the Upper Parana River floodplain, forinstance, diagnostic RAPD markers revealed crossbreeding between theendemic Steindachnerina insculpta (Eigenmann & Eigenmann, 1889) andSteindachnerina brevipinna (Fernandez-Yepez, 1948), which was introducedfrom the Middle Parana River (Oliveira et al., 2002). Cichla species havea highly similar karyotype (2n ¼ 48) macrostructure (Alves, 1998; Nishiyama,1998), and the possibility of occasional natural hybridization between C. cf.monoculus and C. cf. temensis has been previously reported in their native re-gions, as inferred from karyological and esterase analyses (Andrade et al.,2001; Brinn et al., 2004; Teixeira & Oliveira, 2005). The three species studiedwere sharply separated in the neighbour-joining dendrogram (Fig. 4). ThemtDNA sequences indicated that C. cf. temensis population is more closelyrelated to Cichla sp. ‘Azul’ than to C. cf. monoculus, but there are no reportsof natural coexistence and hybridization between them.Crossbreeding between C. cf. monoculus and Cichla sp. ‘Azul’ in their native

region has not been reported. Hybridization between closely related fish specieshas been described in regions where the introduced species is genetically com-patible to either local or other introduced species (Hubbs, 1955; Arthington,1991; Pierce & Van Den Avyle, 1997; Oliveira et al., 2002). Displacement ofspecies outside their native region may disrupt isolation mechanisms (Scribneret al., 2001). Cichla species are known to have complex reproductive andbehaviour strategies such as nest building and parental care (Agostinho et al.,2003). It appears that the C. cf. monoculus and Cichla sp. ‘Azul’ populationshave not encountered major restrictions in the new habitat, since alreadyshortly after their introduction they were widespread in the lentic environmentsof the Upper Parana River basin. Moreover, the high frequency of interspecifichybrids in the Upper Parana River suggests that the new environment wasfavourable for hybridization between Cichla sp. ‘Azul’ and C. cf. monoculus.As discussed by Smith et al. (2003), hybridization among cichlids may be

more significant as an evolutionary impact than previously assumed. Moreover,interspecific hybridization can lead to local species extinction and can representa threat to the integrity of unique gene pools (Scribner et al., 2001; Perry et al.,2002). When hybridization between species results in fertile hybrids, well-adapted and vigorous strains may be created, which are potentially more com-petitive than the most aggressive variants of the parental species (Arthington,1991). Since the number of hybrid specimens in the Upper Parana basin wasrelatively large, they may have some competitive advantage over the parentalspecies. Continued surveillance of these populations, including studies on pop-ulation density and differentiation migration patterns, might be useful in deter-mining causes for hybridization and thus that will be of genetic and ecologicalinterest.


# 2006 The Authors


The data presented herein are potentially useful for the monitoring of theinvasive Cichla populations and for future taxonomic, genetic and evolutionarystudies within this genus. The detection of a large interspecific hybrid popula-tion calls attention to the imminent possibility of natural hybridization withunpredictable consequences when two or more Cichla species are concurrentlyintroduced. Therefore, the data are also of interest for the development offuture ecological guidelines.

The authors gratefully acknowledge A. A. Agostinho and M. Petrere Jr for valuablediscussions and suggestions, C. S. Pavanelli and W. J. Gracxa for assisting with speciesidentification, E. K. Okada for helping with fish sampling, C. T. Harms for revising themanuscript, and Nupelia-UEM for logistic support. Part of this research was supportedby grants from CNPq and CAPES.

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Electronic Reference

Buckup, P. A. & Menezes, A. (2003). Catalogo dos peixes marinhos e de agua doce doBrasil. Available from http://www.mnrj.ufrj.br/catalogo


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