FIRST RECORD OF THE Saccostrea OYSTER IN BERTIOGA , SÃO PAULO , BRAZIL

1 Centro Avançado de Pesquisa Tecnológica do Agronegócio do Pescado Marinho, Instituto de Pesca, Santos, Brasil. E-mail: margalvao@pesca. sp.gov.br (corresponding author) 2 Universidade de Mogi das Cruzes, Núcleo Integrado de Biotecnologia, P.O. Box 411, 08701-970, Mogi das Cruzes, SP, Brasil. PRIMEIRO REGISTRO DA OSTRA Saccostrea EM BERTIOGA, SÃO PAULO, BRASIL ABSTRACT This study presents the first record of an alien species of oyster in Bertioga, São Paulo State (Southeast Brazilian coast). Alien oysters were found attached to mangrove roots, rock shores, stones and gravel in the riverbed, forming clusters of 10–20 individuals and cohabiting with native oyster species (Crassostrea mangle, C. brasiliana and Ostrea sp.). Results are presented based on molecular analysis of specimens collected in the Itaguaré River in June 2014. We used partial sequences of 16S and COI genes to assess the taxonomic identity. The Neighbor-joining method was used to analyze phenetic relationships among samples and the genetic diversity was calculated from the Kimura two-parameter (K2P) distances. The sequences in this work clustered with a sequence of “Saccostrea cucullata” from Madagascar for both genes (COI and 16S) and presented a genetic distance of 1.7 – 2.2% and 3.5 – 5.3% from other sequences of “S. cucullata group” for 16S and COI fragments, respectively. The genetic distances from others Saccostrea species (S. palmula, S. glomerata and S. mordax) ranged from 4.7 to 9.1% for 16S and from 13.8 to 19.0% for COI. The genetic distances from other oysters’ species sequences (genera Ostrea and Crassostrea) are over than 14.0% and 25.0% for 16S and COI, respectively. The record is discussed in the context of possible consequences on the environment and probable pathways of introduction. This is the first published record of a Saccostrea species in the southwestern Atlantic Ocean.


INTRODUCTION
Oysters play an important ecological role in nearshore ecosystems, and have economic significance for local fishing communities (GRABOWSKI et al., 2012).Among the native Brazilian bivalves, representatives of the Ostreidae family include the genera Ostrea Linnaeus, 1758, Crassostrea Sacco, 1897, and Dendostrea Swainson, 1835(RIOS, 2009).Oysters exhibit high degrees of morphological plasticity and wide geographic distribution.This has led to errors and confusion in their taxonomy because classification and identification of oysters is mainly based on morphological characters (LAM and MORTON, 2006;LIU et al., 2011;LOHAN et al., 2015).
In the last decade, the analysis of DNA sequence data has provided suitable molecular tools for species identification (REECE et al., 2008;SALVI et al., 2014;LOHAN et al., 2015).DNA barcoding based on a standard mitochondrial fragment cytochrome c oxidase subunit I (COI) is a valuable tool in identifying species with high levels of morphological plasticity (HEBERT et al., 2003;SEKINO and YAMASHITA, 2013).Studies involving mitochondrial and nuclear DNA have been conducted to elucidate the taxonomic status of Brazilian Crassostrea oysters (IGNACIO et al., 2000;LAPÈGUE et al., 2002;LAZOSKI et al., 2011), and to identify the presence of exotic species, such as Indo-Pacific Crassostrea species in northern and southeastern Brazil (VARELA et al., 2007;GALVÃO et al., 2013), and natural C. gigas banks on the south coast of Brazil (MELO et al., 2010).
In the present work, we used mitochondrial DNA sequences to assess the taxonomic identity of an alien oyster species found in an estuary located in São Paulo State, on the southeastern Brazilian coast.

METHODS
Oysters were collected from the bottom (2-6 m deep) of the Itaguaré River, Bertioga, São Paulo State, Brazil (45°57.9'W;23°46.7'S)(Figure 1), by divers in June 2014.The coastal region where the Itaguaré River flows 12.5 km into the Atlantic Ocean is surrounded by mangrove, Atlantic forest, floodplain forest, and resting vegetation.Biometric data, i.e., height (the dorsal-ventral axis), length (the anterior-posterior axis), and width (the lateral axis), were taken for each oyster with a calliper ± 0.01 mm accuracy (GALTSOFF, 1964).The material was deposited in the mollusk collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP) receiving the register number MZ 134246.
Samples of the adductor muscle from 15 specimens were placed in 95% ethanol and stocked in a freezer at −20°C.Genomic DNA of each oyster was individually extracted using the Illustra tissue and cells genomicPrep Mini Spin Kit (GE Healthcare, Life Sciences, Buckinghamshire, UK).Two partial mitochondrial fragments, COI and 16S rDNA, were amplified by polymerase chain reaction (PCR) using universal primers designed by FOLMER et al. (1994) for the COI gene (LCO1490 5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3'and HCO2198 5' -TAA ACT TCA GGG TGA CCA AAA AAT CA-3') and by PALUMBI (1996) for the 16S gene (16S.AR 5'-CGC CTG TTT ATC AAA AAC AT-3' and 16S.BR 5'-CCG GTC TGA ACT CAG ATC ACG T-3').PCR was performed in a final volume of 50 uL with 1× reaction buffer, 0.2-mM each dNTP, 1.5-mM MgCl 2 , 0.2-uM of each primer (forward and reverse), 1.0 U PLATINUM ® Taq DNA Polymerase High Fidelity (Invitrogen TM , Carlsbad, CA, USA), containing 0.5-2 ng DNA.The DNA amplification reaction was conducted in a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) with initial cycle of denaturation at 94°C for 4 min, followed by 32 cycles (94°C for 30 s, 56°C for 40 s, 72°C for 1 min), and a final extension at 72°C for 10 min.
The amplification products were purified with the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit and eluted in ultrapure water.The purified PCR products were sequenced bi-directionally using the BigDye ® Terminator Cycle Sequencing kit (Applied BiosystemsTM, Carlsbad, CA, USA) according to the manufacturer's protocols.Electrophoresis of the purified samples was performed in ABI PRISM ® 3100 DNA Sequencer (Applied Biosystems TM , Carlsbad, CA, USA).
Sequences were edited and aligned using CodonCode Aligner v3.5.6 (CodonCode Aligner Corp., Edham, MA, USA).The assembled sequences were deposited in GenBank with the accession numbers KT970455-KT970466.The in silico DNA sequences comparison analysis was performed in MegaBLAST (ZHANG et al., 2000;MORGULIS et al., 2008) to find highly similar sequences.The consensus sequences of each specimen and additional sequences obtained from GenBank were then aligned with ClustalW (THOMPSON et al., 1994) as implemented in MEGA v6 (TAMURA et al., 2013).The neighbor-joining method was used to analyze phenetic relationships among samples (SAITOU and NEI, 1987).Bootstrap re-sampling analysis was performed to assess support for individual nodes using 1,000 replicates with 10,000 random additions (NEI and KUMAR, 2000).The genetic diversity was calculated from the Kimura two-parameter (K2P) distances (KIMURA, 1980) using MEGA v.6 software (TAMURA et al., 2013).

RESULTS
Figure 2 shows the oysters collected for this work.The left valve is a cup shape, with a deep concavity in the umbonal region.Radial ripples can be seen from the umbo to the margins on the outer edge of the left valve.Projections are present along the margin.The right valve is smaller and flat, presenting small denticles along the inner edge, producing corresponding depressions on the left valve.The adductor muscle scar is reniform, and located in the central region.The 15 oysters collected were 56.3 ± 16.8 mm high, 39.9 ± 14.8 mm long, and 18.3 ± 6.9 mm wide (average ± standard deviation).The sizes ranged from 40 to 98 mm in height, from 22 to 65 mm in length, and from 9 to 30 mm in width.
The neighbor-joining dendrogram, obtained from the K2P distance, shows the genetic distances and clustering of related species (Figures 3 and 4).The bootstrap values indicate the branches' consistency index.The sequences in this work (ScB1, ScB2, ScB3, ScB4, ScB5 and ScB6 -GenBank accession numbers KT970455-KT970466) clustered with a sequence of Saccostrea cucullata from Madagascar (KP967577).Nevertheless, they presented a genetic distance of 1.7 -2.2% and 3.5 -5.3% from others sequences of "S.cucullata group" for 16S and COI fragments, respectively.The genetic distances from others Saccostrea species (S. palmula, S. glomerata and S. mordax) ranged from 4.7 to 9.1 % for 16S and from 13.8 to 19.0% for COI.The genetic distances from other oysters' species sequences (genera Ostrea and Crassostrea) are over than 14.0% and 25.0% for 16S and COI, respectively.

DISCUSSION
According to these results, the specimens collected on Itaguaré River belong to Saccostrea genus, but it is not possible precise which species it belongs.This is the first published record of a Saccostrea species in the southwestern Atlantic Ocean.
Species of the genus Saccostrea Dollfus & Dautzenberg, 1920, are common oysters that live on rocky shores in the Indo-Pacific Ocean (LAM and MORTON, 2006), but the taxonomy of this group is very controversial.A chaotic taxonomy has resulted from morphological ecotypes being given different names by different authors.According to LAM and MORTON (2006), in the Indo-West Pacific, Saccostrea cucullata (Born, 1778) sensu lato occupies a wide range of habitats, from mangroves to rocky shores; oyster populations experiencing different degrees of wave exposure and salinity have distinctive ecotypes.This species complex comprises at least seven species, with broadly overlapping geographic distributions (LAM and MORTON, 2006;HAMAGUCHI, 2014).Currently, there are only 10 valid taxonomic species among the dozens of species names for Saccostrea (HUBER, 2010).Many molecular analyses and phylogenetics studies have been carried out to elucidate the taxonomy of this group (LIU et al., 2011;HAMAGUCHI et al., 2014;SALVI et al., 2014;LOHAN et al., 2015).S. cucullata is believed to have been introduced into a number of locations where it has become established, including Hawaii (COLES et al., 1999), the Mediterranean Sea (ÇYNAR et al., 2011), and the Caribbean Sea (LOHAN et al., 2015).
Recent introductions of exotic bivalve species such as Isognomon bicolor, Mytilopsis leucophaeata, Crassostrea gigas, Myoforceps aristatus (TEIXEIRA et al., 2010), and Limnoperna fortunei (OLIVEIRA et al., 2006) have been reported along the Brazilian coast.Rising concern about the harmful impacts of nonnative species has resulted in substantial literature evaluating the risks of bivalve introductions (RUESINK et al., 2005).For instance, the golden mussel, a bioinvasive mollusk that colonizes the freshwater cooling systems of boats (OLIVEIRA et al., 2006) and power generation plants (DARRIGRAN and DRAGO, 2000), has resulted in economic losses in different watersheds in Brazil.Oyster introductions can also impact habitat structure, influencing trophic dynamics and water quality, and by introducing disease-causing organisms resulting in economic, ecological, and social damage (RUESINK et al., 2005;McKINDSEY et al., 2007).
Global shipping activity has been identified as the main source of species introductions in coastal estuarine and marine habitats.Ballast water and hull fouling are the main vectors associated with this invasion pathway (RUIZ et al., 1997).The area where Saccostrea oyster was found is close to the Port of Santos, the largest commercial harbor in Latin America.This area is also one of the most important petrochemical, chemical, and metallurgical industrial complexes in Brazil.This suggests the presence of Saccostrea oyster in the region may be the result of shipping activity in the harbor.LOHAN et al. (2015) also suggest that Saccostrea sp. have been introduced to the Caribbean Sea by either recreational or commercial vessels due the high connectivity of shipping and boating in the Panama Canal.
Another pathway is the introduction of species outside of their native range for aquaculture.This is one of the most common modes of introduction of exotic bivalves (McKINDSEY et al., 2007).One example is the Pacific oyster, Crassostrea gigas, one of the most commonly introduced oyster species for aquaculture in worldwide.This species was introduced in Brazil for aquaculture purpose and today it was found amongst the native species in oyster banks in Brazilian South Coast.However, we do not believe that aquaculture was responsible for the introduction of Saccostrea oyster in this particular case.
Previous studies have reported that Saccostrea species are well established in the Mediterranean (ÇINAR et al., 2011) andCaribbean Seas (LOHAN et al., 2015).Furthermore, S. cucullata and S. commercialis are listed in the Delivering Alien Invasive Species Inventories for Europe (DAISIE European Invasive Alien Species Gateway, 2008).These facts raise concerns about this species as a bioinvader, and the potential consequences of this occurrence in the southwest Atlantic warrants further investigation.Therefore, this first occurrence of the Saccostrea oyster and its eventual expansion along the Brazilian coast should be monitored in the future.

Figure 1 .
Figure 1 .Map of sampling site location (Elaborated by the authors)

Figure 4 .
Figure 4 .Neighbor-joining dendrogram of the COI sequences for oysters sampled in Itaguaré River, Bertioga, SP, Brazil.The bootstrap values are indicated on the branches