Cytogenetic and DNA barcoding reveals high divergence within the trahira , Hoplias malabaricus ( Characiformes : Erythrinidae ) from the lower Amazon River

Molecular and cytogenetic data have provided evidence of cryptic speciation in the widespread South American trahira, Hoplias malabaricus. In the present study, karyotypes and DNA barcode sequences of specimens from seven populations inhabiting the lower Amazon River were analyzed in order to characterize the levels of genetic divergence within a single karyomorph. All the specimens presented karyotypes with 2n = 40 chromosomes (20m+20sm) that were consistent with the species’ C karyomorph. The DNA barcodes revealed six haplogroups, with clear divergence between populations from Brazil and Argentina. The results support the species complex hypothesis and indicate that a single karyomorph of H. malabaricus may harbor more than one species.

The trahira Hoplias malabaricus is a common species found in lentic habitats in the most of the river basins of South America (Nelson, 2006).This species has been studied intensively using a cytogenetic approach, which revealed a karyotypic polymorphism among populations from distinct river basins (Bertollo et al., 2000;Rosa et al., 2009;Santos et al., 2009;Blanco et al., 2010).Seven karyomorphs (denominated A-G) have been identified based on diploid number and chromosome morphology, and are currently recognized as distinct independent evolutionary units (Bertollo et al., 2000).A new karyomorph from the Araguaia River was recently reported by Vitorino et al. (2011).
The A, C, E, F, and G karyomorphs have been collected in the Amazon basin, although the A, C, and F karyomorphs occur outside this basin in eastern and southern Brazil, the G and E karyomorphs seem to be restricted to a small number of Amazonian localities (Bertollo et al., 2000).
The lack of records of hybrids from areas in which distinct karyomorphs are sympatric has been considered to be strong evidence for the existence of a species complex (Scavone et al., 1994;Lopes et al., 1998;Bertollo et al., 2000;Pazza & Júlio, 2003;Rosa et al., 2009.).This conclusion was further reinforced by morphological (Rosa et al., 2009;Piorsky, 2010) and molecular data (Dergam et al., 1998;2002;Santos et al., 2009;Piorsky, 2010;Pereira et al., 2013).Thus, the available chromosomal, morphological and DNA evidence provide strong support for the existence of a complex of cryptic species within the typical Hoplias malabaricus morphotype, which requires careful taxonomic revision.
The accurate delimitation of species is a classic problem of the biological sciences.Recently, DNA barcoding has become an increasingly popular approach to the delimitation of species (Hebert et al., 2003;Blaxter, 2004;Ward et al., 2005Ward et al., , 2009;;Kerr et al., 2007Kerr et al., , 2009;;Steinke et al., 2009;Casiraghi et al., 2010).In this approach, a partial sequence of the cytochrome c oxidase subunit I gene (COI) has been standardized as a DNA barcode for the analysis of animal groups, including cryptic species complexes (Hebert et al., 2004a(Hebert et al., , 2004b;;Hubert et al., 2008;Lara et al., 2010;Allcock et al., 2011).
In order to contribute to a better understanding of the evolutionary history and diversification of the Hoplias malabaricus species complex, we analyzed DNA barcodes and karyotypes of specimens representing karyomorph C from the Amazon basin.To assess the geographical structure we compared DNA barcodes of specimens from Amazon Basin and São Francisco River basin (Brazil) and Pampa plain region (Argentina).Our principal objective was to test the hypothesis that a given karyomorph could represent a single species.

Sampling and karyotypes
A total of 35 specimens of Hoplias malabaricus were collected from seven sites located along a 350 km stretch of the main channel of lower Amazon River in the Brazilian state of Pará (Table 1, Fig. 1).Specimen collection was authorized by the Instituto Chico Mendes, Brazil, through special license number 24384-1.All the specimens were identified as H. malabaricus based on their external morphology, using the diagnostic characters provided by Oyakawa & Mattox (2009).Voucher specimens (UFOPA-I 301 -335) were photographed, fixed in 10% formalin and stored in 70% ethanol, and deposited in the fish collection of the Zoology Museum at the Universidade Federal do Oeste do Pará (UFOPA), Brazil.
For comparative purposes with further studies, morphological measurements of selected specimens are given in Table 2.Such variables were examined taken point-to-point along the left side of the specimen and were taken with digital callipers with a precision of 0.01 mm following Rosa et al. (2009).
Karyotypes were prepared from kidney cells following the protocol of Bertollo et al. (1978).Cytogenetic parameters such as the diploid number (2n), karyotypic formula and C-banding (Sumner, 1972) were used for the identification of karyomorphs.For the purposes of the present study, each karyomorph that was defined using these criteria was assumed to represent a distinct species.

DNA extraction, PCRs and sequencing
Fragments of the heart tissue of each specimen were collected and fixed in ethanol 95%.Genomic DNA extraction was carried out using the standard phenol-chlorophorm protocol (Sambrook et al., 1989).
The PCR cycle was as follows: initial denaturing at 94ºC for 3 minutes, followed by 35 cycles of denaturing at 94 o C for 30 seconds, annealing at 59 o C for 1 minute, extension at 72 o C for 2 minutes, and a final extension at 72 o C for 7 minutes.The products were checked on a 1% Agarose gel stained with GelRed and the positive reactions were purified using Exosap IT Kit (GE Healthcare) according to the manufacturer's instructions.Barcode sequences were obtained using the ABI Prism Big Dye terminator sequencing kit V. with the primer M-13.Electrophoresis was conducted in an ABI 3500 automated DNA analyzer (Applied Biosystems).
A preliminary phylogenetic reconstruction of the mitochondrial haplotypes was based on the Neighbor-Joining (NJ) method (Saitou & Nei, 1987) using the Kimura 2 Parameters (K2P) substitution model implemented in MEGA v.5 (Tamura et al., 2011) following the routine analytical procedure established in previous DNA barcoding studies (www.boldsystems.org).The initial NJ analysis was complemented with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods.Either complementary analysis was processed with MEGA v.5 and statistical support for the nodes was provided by a Bootstrap test with 1000 pseudoreplicates (Felsenstein, 1985).For the ML inference the best substitution model to explain the variation in the data was chosen by KAKUSAN v.4 (Tanabe, 2007), based on the Akaike Information Criteria (AIC).

Results
All the specimens analyzed presented 2n = 40 chromosomes and a karyotypic formula with 20 metacentrics and 20 submetacentrics.The C-banded karyotype revealed heterochromatic blocks in the centromeres of all the chromosomes and telomeres in some of the pairs.A conspicuous and heteromorphic C-band was detected in the proximal region of chromosome 14 (Fig. 2).
The molecular dataset was based on partial 652-bp sequences of the mitochondrial COI gene of 44 specimens of Hoplias malabaricus and one of H. intermedius.No evidence of substitution saturation was found in the analysis of transition-transversions versus divergence.The fragment had 150 variable sites, 92 of which were informative for parsimony analysis.The sequences produced 35 haplotypes, which were used for phylogenetic analyses (Table 3).Haplotype 1 (Hap_1) represents the species H. intermedius, which was included as outgroup.The best evolutionary model fitted to the data was the HKY85_gamma model.
The phylogenetic analyses yielded gene trees with three major clades with strong statistical support.A closer examination of the tree topologies was concordant to highlight six haplogroups (Gp) and revealed slight differences in the positions of the groups 3 and 4. Furthermore, haplotype 29 was joined with Gp_4 by NJ analysis; however, such link has not been corroborated with ML and MP methods (Fig. 3, Table 3).
DNA barcodes successfully separated Hoplias malabaricus populations from Brazil (Gp 2-6) and Argentina (Gp1).In the other hand, Brazilian populations from distinct hydrographic regions, São Francisco and Amazonas River Basins, remained close together (Fig. 3).A common pattern visualized in the Amazonian clades is the contribution of several sample sites to build each haplogroup node.
Due to Hap_29 not showed clear phylogenetic relationship with other clades we chosen remove it from the distance analysis among the haplogroups.The distances varied from 9% in Gp1 x Gp2 to 1.8% in Gp5 x Gp6 (Table 4).The largest distance within the group was found in Gp4 (2%) and a comparison between Hoplias malabaricus and the outgroup H. intermedius revealed 16.4% of genetic divergence.We observed a mean distance of 8.6% when compared populations from Brazil (Gp2-6) to Argentina (Gp1).

Discussion
Cytogenetic markers have provided important criteria for the delimitation of H. malabaricus populations and the analysis of the evolutionary pathways of karyotypic diversification in this group (Bertollo et al., 2000;Cioffi et al., 2009;Jacobina et al., 2011;Vitorino et al., 2011).The chromosomal morphology and C-banding patterns of the H. malabaricus specimens analyzed in the present study were consistent with the C karyomorph lineage.This identification is supported by the diploid number (2n = 40), the presence of 20 meta-and 20 submetacentrics in both males and females, and the C-banding pattern of the heteromorphic pair 14, which may be interpreted as homologous to pair 11 of the C karyomorph from Bento Gomes River described by Cioffi et al. (2009) and Cioffi & Bertollo (2010).The heteromorphism of pair 14 appears to represent a signature of the nascent XX/XY sex system indicated by karyomorph C (Cioffi & Bertollo, 2010).However, the populations from the lower Amazon and Bento Gomes rivers diverged in their karyotypic formulae.In fact, a certain degree of intra-karyomorph variation would be expected, considering that the H. malabaricus complex is widely distributed in the river basins of South America and is known to contain high levels of karyotype polymorphism.Indeed, Blanco et al. (2010) recently reported extensive karyotypic variation in karyomorph A.
The DNA barcode analysis of Hoplias malabaricus revealed the presence of six haplogroups with a clear distinction between Brazilian and Argentinean populations.Additionally, similar levels of high genetic divergence could be observed between the Brazilian group Gp2 and the other populations.Recently, Rosso et al. (2012) argued for the strong geographic structure revealed by COI sequences of H. malabaricus from distant hydrographic basins in South America, and highlighted for the possibility of undiscovered species in the Pampa Plain region, Argentina.Our results corroborate the singularity of H. malabaricus from Argentina and show that deep genetic divergence also occurs in a smaller geographic scale as observed in populations from Amazon Basin.
Deep divergence in DNA barcode sequences thus appears to be a characteristic of Hoplias lineages.Previous studies have found high levels of divergence in Hoplias  malabaricus karyomorphs A and F from the São Francisco basin using the mitochondrial ATPase 6 marker.In this case, molecular divergence was accompanied by marked chromosomal variation, given that the A karyomorph is 2n = 42 while F is 2n = 40 (Santos et al., 2009).If we assume that all the specimens collected in the present study (35 specimens) are representative of karyomorph C, then this cytogenetic categorization should work with a range of genetic distance as high as the observed to discriminate distinct karyomorphs (A and F) and possibly distinct species.Such profound divergence in the DNA barcode within species indicates cryptic speciation or phylogeographic structuring.When it occurs between congener species, it may be explained by a long evolutionary history of diversification (De Carvalho et al., 2011).
Originally, Hebert et al. (2003) proposed that a divergence of 2-3% in the DNA barcode represents the threshold for the definition of species status in a lot of taxonomic groups.Several studies with fish groups have documented a larger range of genetic divergence (8-10%) between congeneric species (Ward et al., 2005(Ward et al., , 2009;;Hubert et al., 2008;Lara et al., 2010;De Carvalho et al., 2011;Pereira et al., 2011).Similar levels of divergence were found herein among Hoplias malabaricus haplogroups, what can be interpreted as indicative of speciation event.
The effectiveness of DNA barcodes for the assessment of biodiversity and resolution of taxonomic problems, such as the identification of species, is currently under debate, although this approach has proven to be successful for delimiting species in a number of different animal groups (Hebert et al., 2004a, b;Clare et al., 2006;Ward et al., 2009).The results of the present study indicate that species-level genetic divergence within Hoplias malabaricus may not be restricted to populations with distinct karyomorphs, but even among individuals that present the same karyomorph, which indicates the possibility that different chromosomal races may also encompass species complexes.If the speciation process in H. malabaricus taken place without gross modification in the karyotypes, as seems to be the case with the karyomorph C, then the cytogenetic categorization that discriminate seven karyomorphs (A-G) should be carefully examined when interpreted as indicative of independent evolutionary units.It is clear that further studies based on the integrative approaches with genetic (molecular and  cytogenetic markers) and morphological data will be necessary for a more definitive understanding of the evolutionary relationships within the H. malabaricus complex.

Fig. 3 .
Fig. 3. Phylogenetic trees of Hoplias malabaricus haplotypes based on COI mitochondrial gene sequences.a) Neighbor-Joining; b) Maximum likelihood and c) Maximum Parsimony.Values in the nodes indicate the statistical support from bootstrap test.

Table 1 .
3 (Perkin Elmer) Samples, collect sites and collection numbers of Hoplias malabaricus from Amazon basin.