MOLECULAR IDENTIFICATION OF NATIVE OYSTERS ON THE COAST OF MARANHÃO , BRAZIL *

Oysters are found along the whole coast of Brazil. The phenotypes of the species vary considerably according to the characteristics of the habitat. The present study investigated the existence of different oyster species of the genus Crassostrea on the coast of Maranhão, using the Multiplex PCR technique and DNA Barcoding. The results of the Multiplex PCR revealed two distinct bands characteristic of the species C. gasar and C. rhizophorae in a total of 135 samples analyzed. The sequencing of the COI gene of 98 samples produced a 695 bp fragment and 15 haplotypes for C. gasar and 640 bp and eight haplotypes for C. rhizophorae. The haplotype tree divided the two species clearly into different clades with 100% bootstrap support. Intraspecific genetic divergence was 0.2% in both species, while interspecific divergence was 23.6%. The similarity between the sequences generated and those available in BoldSystems ranged from 97.01% to 98.37% for C. rhizophorae and from 97.55% to 99.84% for both C. gasar and C. brasiliana, reinforcing the taxonomic problems in this group, which supports the synonymization of these species. The DNA barcoding permitted the reliable identification of the samples and confirmed the existence of two species of oyster in the study area.


INTRODUCTION
The oysters of the genus Crassostrea (SACCO, 1897) are widely distributed and are found attached to substrates such as roots and rocks in the intertidal zone along the whole coast of Brazil (RIOS, 1994).The considerable morphological similarities of the different taxa hamper the reliable identification of these organisms, and contribute to widespread taxonomic uncertainties (RIOS, 1994;IGNACIO et al., 2000;BONDIOLI et al., 2017).
The phenotypic plasticity of the oysters makes the identification of native species extremely difficult, although molecular techniques have been developed to resolve the taxonomic and zoogeographic problems of the species of the genus Crassostrea (IGNACIO et al., 2000;LAPEGUE et al., 2002;PIE et al., 2006;VARELA et al., 2007;REECE et al., 2008;MELO et al., 2010;GALVÃO et al., 2013).The oysters of this genus are the most cultivated, worldwide, given the well-established husbandry technology and their economic importance (WAKAMATSU, 1973;GALVÃO et al., 2017).In Brazil, there have been a number of attempts to develop the commercial farming of native oysters, although most have been unsuccessful, due primarily to the difficulties of obtaining and identifying the seed of species with potential for farming.
Multiplex PCR techniques have been developed specifically for the identification of oysters of the genus Crassostrea (LUDWIG et al., 2011;MELO et al., 2013), with the objective of optimizing the taxonomic identification of these organisms, providing an important tool for aquaculture and fishery operations.In addition to this approach, a fragment of approximately 650 base pairs of the 5' extremity of the mitochondrial Cytochrome oxidase subunit I (COI) gene has been adopted as a universal identification system for animal species, including the oysters of the Atlantic Ocean.Each species is normally represented by a unique sequence or a closely similar group of sequences of this gene fragment, which is known as the DNA barcode (HEBERT et al., 2003).
In the area of the present study on the coast of Maranhão, oysters are found throughout the littoral, in natural beds, in both estuarine environments, where they can be found attached to the roots of mangrove trees, and on open beaches, where they are typically attached to rocky outcrops.Previous molecular studies that included samples from this coast (MELO et al., 2010;LAZOSKI et al., 2011;CAVALEIRO et al., 2013) recorded the presence of only a single oyster species, Crassostrea gasar.However, the lack of more detailed studies on the native species and their distribution in the different habitats of the coast of Maranhão represents a drawback for the development of oyster farming in the region and the understanding of the actual status of the local stocks of this important fishery resource.
The principal objective of the present study was to identify the native oyster species found on the coast of Maranhão, based on the banding pattern generated by the multiplex PCR and the sequences of the mitochondrial DNA barcoding fragment.

METHODS Samples
The adult specimens were captured in different habitats of the intertidal and subtidal zones, where they were found attached to substrates such as mangrove roots and rocks.The study area encompasses seven sampling points on the coast of Maranhão (Figure 1).Two of the points (Carutapera and Cururupu) are located in the western extreme of Maranhão state, two (Primeira Cruz and Tutóia) are located in the eastern extreme, and three in the center of the state, with samples being collected in 2014 and 2015 (Table 1).The samples were frozen at -20 o C and deposited in the tissue and DNA collection of the fauna of Maranhão (COFAUMA) at the Maranhão State University (UEMA) before being transferred to the UEMA Genetics and Pathology Laboratory, where the adductor muscle was removed and preserved in 100% ethanol.
The total DNA was isolated from the tissue of the adductor muscle using MEDRANO et al. (1990)  amplification was based on the protocol and primers described by MELO et al. (2013).
The results of the PCR were visualized by horizontal electrophoresis for 80 minutes at 50 V in a 1.5% agarose gel stained with Ethidium bromide.A 1 Kb molecular ladder (1kb Plus DNA Ladder, Life Technologies, Rockville, MD, USA) was used for each migration and the gel was photodocumented for the analysis of the banding pattern and the identification of the Crassostrea oysters, as in MELO et al. (2013).
The Polymerase Chain Reaction (PCR) was used to isolate and amplify the mitochondrial COI region, using the primers described by FOLMER et al. (1994) and MELO et al. (2010).The amplification protocol was the same as that used by MELO et al. (2010).The PCR products were visualized in 1% agarose gel and purified with ExoSAP-IT following the maker's protocol.The sequencing reaction was based on the SANGER et al. (1977) method, using a Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems).The samples were precipitated in EDTA-Ethanol Sodium Acetate and then sequenced in an ABI 3500 automatic DNA sequencer (Life Technologies) following the manufacturer's recommendations.

Data analysis
The sequences were edited and aligned using the ClustalW tool (THOMPSON et al., 1994) in the BIOEDIT program, version 7.0.5.2, (HALL, 1999).The data matrix, mean genetic distances, and phylogenetic analyses were run in MEGA 6.0 (TAMURA et al., 2013), using the neighboring-joining approach, with the Kimura 2-parameter model (SAITOU and NEI, 1987).A bootstrap analysis (FELSENSTEIN, 1985) was used to verify the significance of the groupings.The DnaSP program, version 5 (LIBRADO and ROZAS, 2009) was used to analyze haplotype and nucleotide diversity.Molecular identification using the COI gene was based on comparisons of the sequences obtained in the present study with those available on the BOLDSYSTEMS (Barcode of Life Data Systems) platform (HEBERT et al., 2003).A sequence of the COI gene of Crassostrea sp.Canela (HM003525) obtained from the region of Bragança, in Pará, northern Brazil, was incorporated into the database as the outgroup.

Multiplex PCR
A total of 135 individuals were analyzed by multiplex PCR.The bands revealed by the amplification in agarose gel corresponded to those of the species C. gasar and C. rhizophorae (Figure 2).The bands obtained from the samples from the municipalities of Carutapera, Cururupu, Primeira Cruz and Tutóia were all of 718 bp, corresponding to the ITS 1 region, indicating that C. gasar is present at these localities.In the samples from the municipalities of São José de Ribamar, Paço do Lumiar and Raposa, however, two banding patterns were obtained.One was the single band characteristic of C. gasar, while the other was a double band, with a 377 bp sequence for the COI region, and 718 bp corresponding to the ITS 1, confirming the presence of C. rhizophorae at all three sampling points.

Sequencing of the COI gene
A total of 98 sequences were obtained from the samples of Crassostrea oysters, of which, 78 corresponded to the species C. gasar, with a fragment of 695 bp, and 20 were consistent with C. rhizophorae, with a fragment of 640 bp.Eight haplotypes were recovered from the C. rhizophorae samples, with haplotype diversity (h) of 0.795 and nucleotide diversity (π) of 0.002.In C. gasar, a total of 15 haplotypes were identified, with haplotype diversity (h) of 0.428 and nucleotide diversity (π) of 0.002.
The haplotype tree based on the Neighbor Joining (NJ) approach and the Kimura 2-parameter (K2P) model presented two strongly-supported clades.All the C. rhizophorae haplotypes were grouped in a basal clade (100% bootstrap support), while the C. gasar haplotypes constituted a second, clade with equally strong support (Figure 3).
The clear separation of C rhizophorae and C. gasar in distinct groups confirms the occurrence of two Crassostrea species in the study area on the coast of Maranhão, corroborated by the high genetic divergence values (23.9-25.9%)recorded between the haplotypes of the two taxa.A mean intraspecific genetic divergence of 0.20% was recorded for both the oyster species, whereas the divergence recorded between C. gasar and C. rhizophorae was 23.6% (Table 2).The existence of distinct C. rhizophorae and C. gasar populations is thus further supported by the high genetic divergence value recorded between these two taxa.

Molecular identification
The COI sequences were submitted to the BOLDSYSTEMS platform for the identification of the samples through comparisons with those deposited in this system.Sequences identified as C. rhizophorae were 97.01% to 98.37% similar to the sequences of this species available in BOLDSYSTEMS (Figure 4).In the case of the samples identified as C. gasar, similar percentages of similarity, varying from 97.55% to 99.84%, were found in relation to the BOLDSYSTEMS sequences of both C. gasar and C. brasiliana, raising a taxonomic conundrum (Figure 5).These  results are corroborated by the haplotype tree generated using the GenBank sequences of C. brasiliana, which groups with C. gasar with 100% bootstrap support, indicating the formation of a single clade (Figure 3).

DISCUSSION
The results of the Multiplex PCR confirmed the occurrence of C. rhizophorae in the study area, on the Maranhão coast, where it had not been recorded previously.Up to now, only C. gasar had been recorded in Maranhão (MELO et al., 2010;LAZOSKI et al., 2011).The two species were found coexisting sympatrically on the same stretch of coast in the municipalities of São José de Ribamar (on rocky substrates) and Paço do Lumiar (in mangrove habitats).Sympatry between two or more native species of oyster has been recorded in some other Brazilian estuaries (PIE et al., 2006;GARDUNHO et al., 2012).
The presence of more than one native oyster species on the coast of Maranhão reinforces the need for more detailed studies in reproductive biology and the evaluation of the stocks of both species.As two species are now known to occur in the region, previous evaluations of the status of this fishery resource would be incomplete, given that they considered the presence of only a single species in the area.
In the study region, oysters are typically harvested in a rudimentary way by the traditional local communities, with no concern for the management of the resource or its sustainability.Scientific studies that provide reliable data on the status of stocks provide the potential for the implementation of effective management measures by these local communities.Sustainable techniques, such as oyster farming (CAVALLI and FERREIRA, 2010), represent an economically viable alternative source of subsistence for the populations that depend on this natural resource.However, the correct identification of the native species found in a given area is a fundamental first step in the development of effective oyster farming operations.
Even though all the native species of Crassostrea oysters can be farmed commercially, C. gasar has been shown to perform best, in terms of captive growth rates, indicating that the cultivation of this species may be the most lucrative in most cases (CHRISTO and ABSHER, 2006).In this case, the mapping of the distribution of the two native species found in the study area will be of fundamental importance for the effective recruitment of oyster seed.There are a number of points on the coast of Maranhão -Carutapera, Primeira Cruz, Cururupu and Tutoia -that have considerable potential for the recruitment of oyster seed, given that only C. gasar was found in these areas.However, new studies need to focus on the genetic identification of specimens and the recruitment of oysters to confirm this conclusion.
The findings of the present study have extremely important implications for the development of oyster farming and harvesting operations on the Maranhão coast, given the general lack of data from this region.While this region has an enormous potential for oyster farming, there is no captive production whatsoever, which reinforces the need for the optimization of the points for the recruitment of native oyster seed.
While the Multiplex PCR was regulated for the identification of up to four oyster species, one (C.gigas) was not expected for the study area.This is because C. gigas, an exotic species from the Indo-Pacific region, would not be expected to tolerate the high temperatures typical of the area of the present study (MELO et al., 2010).The Multiplex PCR proved effective for the identification of the Crassostrea species.The efficiency of this approach indicates that it may be a practical alternative for the monitoring of the distribution of oyster species, as well as being an accessible tool for the oyster farmer.

Figure 1 .
Figure 1.Map of the sampling points on the coast of Maranhão, Brazil.

Figure 3 .
Figure 3. Haplotype tree produced by the neighbor joining (NJ) method using the K2P model based on the sequences of the COI gene in oysters of the genus Crassostrea in this study and GenBank sequences.The numbers at the nodes represent the bootstrap values (1000 replicates).CAR = Carutapera; CUR = Cururupu; PC = Primeira Cruz and TUT = Tutóia; SJR = São José de Ribamar; and PL = Paço do Lumiar; CSP = Crassostrea sp.

Table 2 .Figure 4 .
Figure 4. Percentage genetic similarity of the C. rhizophorae haplotypes obtained by molecular identification in the BOLDSYSTEMS platform.

Figure 5 .
Figure 5. Percentage genetic similarity of the C. gasar haplotypes obtained by molecular identification in the BOLDSYSTEMS platform.

Table 1 .
Number of oyster samples collected per collecting point on the coast of Maranhão, Brazil.