Prospecting molecular markers to distinguish Cichla kelberi , C . monoculus and C . piquiti

Peacock bass, a fish of the genus Cichla, is an exotic species from the upper river Paraná floodplain in which the species Cichla kelberi and C. piquiti have been confirmed, coupled to the specie C. monoculus upstream in the Capivara and Taquaruçu dams. The introduction of this genus has caused negative impacts on the diversity of native species. Current research prospects DNA sequences capable of distinguishing the three species and provide molecular data for the taxonomic characterization of the species in the upper Paraná River basin. Sequencing of nuclear (tmo4c4, dlx2 and bmp4) and mitochondrial (cox1, cytb) loci were done from fish of the three species of the genus Cichla reported in the literature of the upper Paraná River basin. Sequence analysis provided molecular differentiation for the species through the usage of loci cytb, dlx2 and cox1. Since the latter only distinguished C. piquiti from the other Cichla species, the loci bmp4 and tmo4c4 were not adequate to accomplish our aim.


Introduction
Fish of the genus Cichla belong to the order Perciformes and the family Cichlidae, popularly known in Brazil as peacock bass (Tucunaré).The family is one of the main models of speciation in evolutionary biology due to its diversification on the Great Lakes of Africa, which led to the formation of species with different ecological adaptations in a relatively short period of geological time (JOYCE et al., 2011).Thus, it has been focused upon by many phylogenetic researches (LÓPEZ-FERNÁNDEZ et al., 2010).
The genus Cichla, widely distributed in neotropical regions, derives from a monophyletic lineage and is considered the baseline genus for a divergence of neotropical cichlids (FARIAS et al., 2001).The genus has 15 described species which are native to Tocantins, Amazon and Orinoco river basins (KULLANDER;FERREIRA, 2006).Since genetic analysis has shown genetic introgression among some of the species, the validity of this classification has been questioned (WILLIS et al., 2012).
The introduction of species in the upper Paraná River basin, developed by hydroelectric companies Acta Scientiarum.Biological Sciences Maringá, v. 37, n. 4, p. 455-462, Oct.-Dec., 2015 (ESPÍNOLA et al., 2010), is a strategy to increase fish stocks which started in the 1990s (AGOSTINHO et al., 2007).However, the origin of this genus in the Paraná river basin is officially unknown, except for some reports of accidental tanks leaks involving Cichla monoculus (Block & Schneider, 1801) during the January 1997 flood in the Paranapanema and Tibagi rivers (ORSI; AGOSTINHO, 1999).
Other relevant aspects related to the illegal introduction of the species are related to its characteristics as sports fishing, due to its aggressiveness when caught with artificial baits, and to the commercial acceptance of its meat (WINEMILLER, 2001, AGOSTINHO et al., 2007;PELICICE;AGOSTINHO, 2009).
The capture of a great number of juveniles of the genus in the above-mentioned environment has shown a high degree of the species´ reproduction success in the Upper Paraná River floodplain (ESPÍNOLA et al., 2010).The hybridization between C. kelberi and C. piquiti recorded by genetic researches is another important event that has occurred in the floodplain (ALMEIDA-FERREIRA et al., 2011;OLIVEIRA et al., 2008OLIVEIRA et al., , 2006)).
Combination analyses of molecular markers that identify regions which display different evolutionary rates are necessary to understand the complex evolutionary process in neotropical fish (FABRIN et al., 2014;BEHEREGARAY, 2008) and its hybridization dynamics (MIMS et al., 2010;STREELMAN et al., 2004).In fact, genetic studies are important to provide data that may be used in taxonomic characterization, supporting ecological studies aiming at the management of hybrid populations.
Current research prospects DNA sequences capable of distinguishing the three species C. kelberi, C. piquiti and C. monoculus.et al. (2006).Figure 1 demonstrates the sites from where the specimens were collected.Temperature conditions and time used in the amplification of the respective regions were appropriate and specified in Table 1.The amplified genes were sequenced individually in both directions using the primers already described (Table 2) with Big Dye Terminator kit.The nucleotide sequence determination was conducted in MegaBace automated sequencer (Amersham), according to the manufacturer's instructions.

Sequences analysis
Sequences were edited with BioEdit software while alignment (Clustal W) and distance p were determined with MEGA 6 software (TAMURA et al., 2013).Distance matrices were provided in the supplementary material.
Alignments were previously evaluated by the best-fit model test in MEGA 6 software to elaborate the phylogenetic reconstructions.Each was conducted through its bases substitution model by the maximum likelihood method.Each phylogeny used as a parameter 1000 bootstrap resamplings with the presence of an outgroup (preferably of the same family and with closer phylogenetic analysis, considering the GenBank availability) and also conducted in MEGA 6.The phylogenetic analysis elucidated the clades, whilst bootstraps rates determined the validity of clades distinction.Table 3 provides the GenBank accession number of the sequences obtained in current study.

Results
The regions cox1 and cytb were individually evaluated for alignment.The number of variable sites was checked and evaluated according to the best evolutionary model.Sequences obtained from the region cox1 (544 bp) revealed 57 variable sites (10.47%).The Hasegawa-Kishino-Yano model with gamma distribution for the reconstruction of the mitochondrial phylogeny of the three species of Cichla from cox1 region was employed.Figure 2 provided the results of the phylogenetic reconstruction by the maximum likelihood method.
The second prospected mitochondrial region was related to cytochrome b (cytb) (464 bp) gene.Due to the low number of sequences obtained from the surveyed specimens, three sequences from GenBank (gi294471571, gi294471583 and gi294471565), one from each species, were included for comparison.Figure 2 shows the phylogenetic reconstruction of this region and was based on Hasegawa-Kishino-Yano model, performed by the maximum likelihood algorithm with 1000 bootstrap re-samplings.
Further, tmo4c4, dlx2 and bmp4 were the evaluated nuclear genome regions and the best evolution model, the number of variable sites and size of alignment were calculated for each region.After calculating the best evolution model for each region, the phylogenies were reconstructed with the maximum likelihood method, taking into consideration 1000 bootstrap re-samplings.Figure 3 demonstrates results for each region.Among the smaller interspecific variation with less than 1%, the loci tmo4c4 and bmp4 were highlighted for all evaluated species.The accumulated variations at these loci do not appear to be enough for a sharp distinction since rates are close to the intraspecific variation ones.
Although the region with the highest intraspecific variation was cox1 (3.9%) for C. kelberi, it was less than or equal to interspecific rates discovered in this region for the combination with the other two species, perhaps due to the fact that the sequence of bases of specimen Ck12 differed a lot when compared to other specimens.The above may be observed in this group by the high standard deviation of the intrapopulational distance p (2.5%).
The lower intraspecific variations occurred in the species C. piquiti, in the cytb and tmo4c4 sequences (0 and 0.1%, respectively); tmo4c4 (0.1%) had the lowest rates for C. kelberi; whereas the greatest intraspecific distance rates (all greater than 4%) were from cox1 and cytb.
When interspecific and intraspecific average rates from the different prospected regions were compared, three loci showed lower internal species variations than the rate reported among the groups.Consequently, the three species could be differentiated.Although the mentioned regions were cytb, cox1 and dlx2, only cytb presented a significant difference among the maximum distance p values.cytb had the interspecific variation at least 2.15 times higher than the intraspecific variation rates.Differences between the rates of the maximum distance p were lower for regions cox1 and dlx2, with 1.07 and 1.125 times respectively.
However, other loci may be useful to distinguish the species and to evaluate the distances p between pairs of species.Species C. monoculus and C. piquiti could be segregated in the region cox1.The variation between the intra-and inter-specific distances p from these species is at least 2.12 times.However, it is still not possible to clearly distinguish C. kelberi from the others in this region since the intra-and inter-specific variations are very close to each other.
Among the nuclear loci evaluated, dlx2 seems to be the most promising when it comes to species differentiation.The variation of the groups´ internal distance p was 0.7% for C. monoculus, 0.5% for C. kelberi and 1.6% for C. piquiti.When the genetic distances among the groups were compared, it could be perceived that they were at least 1.12 times different, if the smallest change that occurred in C. monoculus was considered with the other species.Table 4 shows the interspecific polymorphic sites between C. monoculus, C. kelberi and C. piquiti.
In the case of the nuclear region tmo4c4, low intraspecific variation is extant for C. piquiti, with low interspecific variation rates, and thus low variability in this region.The above indicates that the locus may not be suitable for the differentiation of these species.The evaluation of region bmp4 points in the same direction as the tmo4c4 analysis.Although the distance p variation for the region bmp4 indicates a distinction between C. piquiti and C. kelberi, a more precise evaluation shows the overlap of specimens of these species, which invalidates the use of this marker in the distinction.

Discussion
Although the cox1 region has been widely used to identify species and has even been used as the base sequence in the 'Barcodes of Life Data System Project' (BOLD) (RATNASINGHAM; HERBERT, 2003), the three species C. kelberi, C. piquiti and C. monoculus could not be identified by this region because the sequences failed to have enough variance to differentiate Cichla monoculus from Cichla kelberi.A similar result occurred for the cox1 region in the case of another cichlid genus (Oreochromis), in which the divergence was not significant to differentiate all the evaluated species (WU; YANG, 2012).Nevertheless, the region analysis could distinguish Cichla piquiti from the others.
The phylogeny based on the cytochrome b gene region (cytb) indicated the possibility of distinguishing specimens of the three species.Further, the spreading of specimens showed welldefined groupings for the three species.Since there is a clear divergence of species, the region´s polymorphism seems to characterize them clearly.
The analysis of cytb region has been used in phylogenetic studies since it contains low and high variable regions, that is, more preserved regions and regions of wider domain.It may be thus considered a good marker for the study of molecular phylogeny in monophyletic families (FARIAS et al., 1999(FARIAS et al., , 2001;;GENNER et al., 2007;MUSILOVÁ et al., 2008;PUEBLA, 2009;SMITH et al., 2008).This region has also been used in biogeographical studies of the Cichla genus and demonstrated its potential for this purpose (WILLIS et al., 2007).
Although there is the distinction for the three species with locus dlx2, the region would be safer to distinguish between C. piquiti and C. kelberi.The gene is part of the group of 'dlx homeobox' gene family and is involved in the embryonic formation of the brain, jaws and teeth of vertebrates (PANGANIBAN; RUBESTEIN, 2002).In Lake Malawi´s cichlids, the analysis of these sequences was able to distinguish two species with introgressive hybridization (MIMS et al., 2010).Further, it has been used for cichlids phylogeny in other research works (HULSEY et al., 2010;SMITH et al., 2008).
The nuclear locus tmo4c4 showed random and very low variability, preventing the species distinction process.The locus is known because of its low similarity to TINTIN proteins becoming an immunoglobulin domain.The region does not seem to be associated with positive selection (STREELMAN et al., 1998); for closely related species, the intraspecific diversity may be equal to interspecific differences.
The impossibility of 'bone morphogenic protein 4' (bmp4) gene usage may be due to its morphological differences among different The gene has been effective when used as a parameter for the study of the genetic relationships in other fish groups, such as the genus Scarus (SMITH et al., 2008).When East African cichlids were evaluated, the bmp4 gene could be found mainly associated with species that have undergone a rapid speciation and a high replacement rate (PUEBLA, 2009).This feature does not appear to be associated with the case of the genus Cichla, perhaps because the morphological characteristics of this group are not as diverse as African cichlids.
Phylogeny results show a greater proximity between C. kelberi and C. monoculus, which may be evidenced by the phylogenies presented to these loci.Results corroborate those reported by Willis et al. (2010) who, using the mitochondrial DNA controlling region, posited similarly the two species within the same cladze, whereas they placed C. Piquiti in another one.
When the mitochondrial regions were evaluated, although the positioning of the species evaluated for mitochondrial cox1 region was the same discovered for nuclear loci, it was possible to perceive an interesting difference in assessing the phylogeny of cytb locus.A greater proximity between C. monoculus and C. piquiti than C. monoculus and C. kelberi could be observed.The fact that there are discrepancies between the reconstructed phylogenies based on nuclear loci and mitochondrial is common (TOEWS; BRESFORD, 2012).However, in current study, the mitochondrial locus cox1 points at the same nuclear direction, while cytb points towards a discrepant one.Species closeness is another aspect that should be underscored.

Conclusion
Since the effectiveness of different loci in differentiating Cichla monoculus, C. kelberi and C. piquiti species is tested, the analysis foregrounds distinction between C. monoculus and C. kelberi from C. piquiti by cytb and dlx2 loci sequences, and the difference between C. piquiti from the other two species of the genus Cichla present in the upper Paraná River basin by cox1.However, data rejected the use of bmp4 and tmo4c4 loci to identify these species.
Tissues preserved in alcohol 70% from the tissue bank of the Genetics Laboratory of The Research Center of Limnology Ichthyology and Aquaculture (NUPELIA), State University of Maringá, were used for DNA extraction.Cichla monoculus specimens from the Paranapanema River (n = 7) and Taquaruçu reservoir (Upper Parana River basin), used by Briñez et al. (2013), were retrieved, coupled to Cichla kelberi (n = 6) and C. piquiti (n = 6) from Tocantins-Araguaia basin, used by Oliveira

Figure 1 .
Figure 1.Collection sites of Cichla specimens.1 = C. monoculus was retrieved from the river Paranapanema (22°54'S; 52°00'W); 2 = C. kelberi and C. piquiti were retrieved from the Tocantins-Araguaia basin (09°45´S; 48°22´W).DNA amplification and sequencing DNA extraction followed method by Prioli et al. (2002) and the Polymerase Chain Reactions (PCRs) were done in a total volume of 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 volume.The nuclear locus tmo4c4 was amplified according to Schelly et al. (2006); primers described by Streelman et al. (1998) were used for locus tmo4c4; and primers described by Smith et al. (2008) were used for loci bmp4 and dlx2.The mitochondrial cytochrome oxidase I sequences (cox1) were amplified as described by Steinke et al. (2009) and the cytochrome b (cytb) region as described by Kocher et al. (1989).Temperature conditions and time used in the amplification of the respective regions were appropriate and specified in Table1.The amplified genes were sequenced individually in both directions using the primers already described (Table2) with Big Dye Terminator kit.The nucleotide sequence determination was conducted in MegaBace automated sequencer (Amersham), according to the manufacturer's instructions.

Figure 2 .
Figure 2. a) Phylogenetic reconstruction of the cox1 (544 bp) gene from specimens of the species Cichla monoculus (Cm), C. kelberi (Ck) and C. piquiti (Cp) by the maximum likelihood method (1000 bootstrap re-samplings) with the Hasegawa-Kishino-Yano model with gamma distribution.Astronotus ocellatus was used as outgroup.b) Phylogenetic reconstruction of cytb (464 bp) gene from specimens of the species C. monoculus (Cm), C. kelberi (Ck) and C. piquiti (Cp) by the maximum likelihood method (1000 re-samplings of bootstrap) with the Hasegawa-Kishino-Yano model.Astronotus ocellatus was the outgroup.

Table 1 .
PCR reaction conditions for the different regions of mitochondrial and nuclear Cichla DNA.

Table 2 .
Primers used in the amplifications for the different regions of mitochondrial and nuclear Cichla DNA.

Table 3 .
GenBank accession number of specimens sequenced in current study.