Computational protocol: Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea)

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[…] Samples of Crassostrea gasar and C. rhizophorae, previously identified by means of rRNA 16S DNA sequences (), were compared to those published by in the GenBank (AJ312937 and AJ312938, respectively), and to the sequence of C. brasiliana (= C. gasar) deposited in the GenBank (DQ839413) by . In order to verify the existence of Crassostrea paraibanensis, the rRNA 16S gene of five individuals from the Paraíba river estuary (Paraíba state, Brazil) were sequenced. The results showed that these sequences were identical to those of C. rhizophorae published by . Therefore, COI genes of oysters from the Paraíba river estuary were not sequenced.The samples of C. gasar (n = 215) and C. rhizophorae (n = 67) included in our analyses were collected from nine and five localities, respectively (, ). Oysters were also collected at Vila Lauro Sodré (00°51'11.2” S, 47°53'24.7” W; municipality of Curuçá, Pará state). Preliminary studies (rRNA16S) revealed these to be C. gasar (unpublished data). Young specimens (n = 10; < 2 months old) of a third unidentified Crassostrea sp. (n = 10), were obtained from plastic spat collectors at two sites on Canela Island, in the municipality of Bragança (00°47'02” S, 46°43'32.9” W), as well as from a wooden bridge on the Furo do Café tidal channel (00°50'43” S, 46°38'50” W). The scientific names and GenBank sequence accession numbers of oysters compared in the present study are described in Tables and . Crassostrea sp. collected from Bragança is referred to as Crassostrea sp. Canela, the first site where this oyster was found by the authors.DNA was extracted from the adductor muscle, according to the protocol of . Crassostrea rhizophorae COI sequences were obtained by direct sequencing of PCR amplified fragments, using the primers described by . Samples of Crassostrea gasar and Crassostrea sp. Canela could only be amplified with a pair of primers designed by C. H. Tagliaro (LCOC-CG-1490 5'- TGTCAACAAATCATT TAGACATTGG-3' and HCOC-CG-2190 5'- TACTTGA CCAAAAACATAAGACATGA-3'), based on the mitochondrial genome sequence of Crassostrea gigas (GenBank: NC_001276). The reaction protocol for the samples consisted of initial denaturing at 95 °C for 3 min; 35 cycles of 1 min at 95 °C, 1 min at 45 °C (C. gasar), 45.5 °C (C. rhizophorae) or 41.4 °C (Crassostrea sp. Canela), and 90 s at 72 °C, followed by a final extension at 72 °C for 7 min. The PCR products were purified using ExoSAP-IT® (Pharmacia). DNA sequences were obtained on both strands using dye terminator cycle sequencing reactions (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction, Applied Biosystems), that were subsequently loaded onto an automatic sequencer (Applied Biosystems model 377), according to manufacturer's protocols.Sequence alignment was carried out with the BioEdit 7 () and Clustal X 1.82 () programs. Only distinct COI sequences of C. gasar, C. rhizophorae and Crassostrea sp. Canela were aligned with sequences of the other species obtained from the GenBank. Nucleotide frequencies and transition/transversion ratios were obtained by means of Mega 4.0.2 software (). A saturation test was carried out with the DAMBE 4.2.13 program (). A set of aligned sequences is considered to be phylogenetically informative if the observed substitution saturation index (Iss) is significantly lower than the critical value of Iss (). Phylogenetic analyses were undertaken with PAUP* 4.0b10 (), using neighbor-joining (NJ) and maximum parsimony (MP), and with PHYML version 3 () using maximum likelihood (ML) methods. MODELTEST 3.07 () was used for choosing the best model for use in NJ and ML analyses through Hierarchical Likelihood Ratio Tests (HLRTs). Heuristic search was applied in MP and NJ analyses. The robustness of phylogenetic hypotheses obtained, were tested by bootstrapping () with 1000 pseudo-replicates for ML and 2000 for NJ and MP. The criterion adopted to evaluate robustness was to consider bootstrap values equal or superior to 90% as being informative.Two sets of sequence alignments were carried out. For the first set (Analysis I), use was made of only one partial COI sequence of C. rhizophorae, one of C. gasar, two of Crassostrea sp. Canela, and sequences collected from the GenBank: 10 different species of Crassostrea, five of Ostrea, one of Ostreola and one of Saccostrea, besides two Lophinae as outgroups (). For the second set (Analysis II), use was made of 52 different sequences of COI from three Brazilian species of oysters (), and sequences collected from the GenBank: 10 species of Crassostrea, one sequence of Saccostrea cucullata (AY038076), with sequences of Ostrea chilensis (AF112286) and Ostrea edulis (AF120651) as outgroups (). Analysis I was undertaken to verify whether Crassostrea was a monophyletic group, and Analysis II to avoid any influence from saturation that might be caused by Lophinae sequences in the analysis of Crassostrea species, as well as to reduce the number of sequences in the analysis itself. […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA, DAMBE, PhyML, ModelTest-NG
Application Phylogenetics
Organisms Crassostrea virginica, Crassostrea gasar