Scholarly article on topic 'Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification'

Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification Academic research paper on "Biological sciences"

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Academic research paper on topic "Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification"

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RESEARCH ARTICLE Open Access

Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification

Gwenaelle Danic-Tchaleu, Serge Heurtebise, Benjamin Morga and Sylvie Lapegue*

Abstract

Background: Because of its typical architecture, inheritance and small size, mitochondrial (mt) DNA is widely used for phylogenetic studies. Gene order is generally conserved in most taxa although some groups show considerable variation. This is particularly true in the phylum Mollusca, especially in the Bivalvia. During the last few years, there have been significant increases in the number of complete mitochondrial sequences available. For bivalves, 35 complete mitochondrial genomes are now available in GenBank, a number that has more than doubled in the last three years, representing 6 families and 23 genera. In the current study, we determined the complete mtDNA sequence of O. edulis, the European flat oyster. We present an analysis of features of its gene content and genome organization in comparison with other Ostrea, Saccostrea and Crassostrea species.

Results: The Ostrea edulis mt genome is 16 320 bp in length and codes for 37 genes (12 protein-coding genes, 2 rRNAs and 23 tRNAs) on the same strand. As in other Ostreidae, O. edulis mt genome contains a split of the rrnL gene and a duplication of trnM. The tRNA gene set of O. edulis, Ostrea denselamellosa and Crassostrea virginica are identical in having 23 tRNA genes, in contrast to Asian oysters, which have 25 tRNA genes (except for C. ariakensis with 24). O. edulis and O. denselamellosa share the same gene order, but differ from other Ostreidae and are closer to Crassostrea than to Saccostrea. Phylogenetic analyses reinforce the taxonomic classification of the 3 families Ostreidae, Mytilidae and Pectinidae. Within the Ostreidae family the results also reveal a closer relationship between Ostrea and Saccostrea than between Ostrea and Crassostrea.

Conclusions: Ostrea edulis mitogenomic analyses show a high level of conservation within the genus Ostrea, whereas they show a high level of variation within the Ostreidae family. These features provide useful information for further evolutionary analysis of oyster mitogenomes.

Background

Because of its typical architecture, inheritance and small size, animal mitochondrial (mt) DNA is widely used for phylogenetic studies. Combined with these characteristics, its typically maternal inheritance contributes to a fast rate of evolution. Nucleotide changes combined with gene order and rearrangement data can provide valuable information on major evolutionary changes at different taxonomic levels. Typically, animal mtDNA is a compact molecule (14 to 17 kb), though some mtDNA can be vastly larger (e.g., Plactopecten magella-nicus [1]), and usually encodes 13 proteins, 22 transfer

* Correspondence: slapegue@ifremer.fr

IFREMER, Laboratoire de Génétique et Pathologie, F-17390 La Tremblade, France

BioMed Central

RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) [2]. There are often few intergenic nucleotides except for a single large non-coding region generally thought to contain elements that control the initiation of replication and transcription [3]. Size variation in mtDNA is usually due to the different length of the non-coding regions. Gene order is generally conserved in most taxa, although some groups show considerable variation. This is particularly so in the Mollusca phylum, especially in Bivalvia and Scaphopoda [4]. In addition to the fact that phylogenetic relationships among major molluscan groups are not well understood, the species classification of some of the most common mollusks remains difficult.

A case in point is oysters, for which a plastic growth pattern is a major feature, resulting in a wide range of

© 2011 Lapegue et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

overlapping ecophenotypic variants [5,6]. Oysters are bivalve molluscs that are widely distributed in the world's oceans. As benthic, sessile filter-feeders, they play an important role in estuarine ecosystems. Moreover, some species are of economic importance, like the Pacific cupped oyster, which is grown in 27 countries and is the most highly produced mollusc species in the world. Oysters have been introduced all over the world for culture and many species are sympatric. Numerous species (30-40 according to the classifications) of oysters of the genus Ostrea have been described. Their geographical range is particularly wide in warm and temperate waters of all oceans, although they have a predominantly tropical distribution [6,7]. In Europe, along the Atlantic and Mediterranean coasts, the European flat oyster, Ostrea edulis, is an important economic marine resource: in 2009 almost 3000 tons were produced in the world, mainly (91%) in Europe (Spain, France, Ireland ...) [8].

During the last few years, there have been significant increases in the number of complete mitochondrial sequences available for all species. The number has more than doubled for molluscs in the last three years [9], so that 98 complete mollusk mitochondrial genomes are now available in GenBank, mainly from gastropods (43), bivalves (35) and cephalopods (14). Among bivalves, the sequenced genomes represent 6 families and 23 genera. In the Ostreidae, the genus Crassostrea has been thoroughly studied, with 7 representatives (6 Asian oysters and 1 American oyster) [10]. In contrast, there is only one representative of the genus Saccostrea (Saccostrea mordax), and one of the genus Ostrea (Ostrea denselamellosa). Recent studies have provided a more comprehensive picture of the cupped oyster genome, showing an unusually high conservation of mito-chondrial gene order in Asian Crassostrea species [11]. Even though molecular tools, such as mitochondrial or microsatellite markers, already exist for the European flat oyster and allow population genetics [12] or quantitative genetics [13] studies, the complete characterization of its mtDNA will allow a better study to be made of phylogenetic relationships among members of the genus, especially between the closely-related species O. edulis and O. angasi [14], to improve classification of the Ostreidae family within the Bivalvia.

Results and discussion

Genome composition

The complete mitochondrial genome of Ostrea edulis [GenBank: JF274008] is 16 320 nt in length and encodes 37 genes, including 12 protein-coding genes (PCGs), 2 rRNAs and 23 tRNAs on the same strand (Figure 1 and Table 1). This size is very close to that of O. denselamel-losa (16 277 bp), shorter than that of other Ostreidae

(16 532 bp for S. mordax to 22 446 bp for C. iredalei), and is within the size range of the Pteriomorphia mt genomes published to date: from 16 211 nt for Argopec-ten irradians [15] to 32 115 nt for Placopecten magella-nicus [1].

In the mt genome of O. edulis, a total of 965 bp of non-coding nucleotides is spread over 21 intergenic regions (each over 5 bp) including a major non-coding region (MNR) of 695 bp. A large non-coding region suggests a putative control region based on its AT content of 74.4% [16]. In contrast to typical animal mito-chondrial genomes, the O. edulis genome may lack the protein-coding gene atp8, although some recent studies have found a good candidate for atp8 gene in Mytilidae and possibly in some Ostreidae [17]. Furthermore, O. edulis genome also has duplications of three tRNAs: trnM, trnS and trnL. The rrnL gene is split into 2 fragments, a phenomenon previously observed in the Ostrei-dae [11]. The rrnS is not duplicated (also in O. denselamellosa, S. mordax and C. virginica), in contrast to Asian Crassostrea.

The molecule has an overall A+T composition value of 64.9% and the size of the coding region is 15 379 nt in length, accounting for 94.2% of the whole genome. The AT content is slightly higher than those of Pectinidae (55.3 to 59.6% [18]) or Mytilidae (61.5 to 61.8%). The AT composition of O. edulis is, therefore, within the AT content range of the Ostreidae: the lowest known AT content is 60.7% in O. denselamellosa, while the highest is 65.3% in C. hongkongensis [19]. In S. mordax, the AT content is 64.4% which is very similar to O. edulis.

As observed in O. denselamellosa (16 277 bp), S. mor-dax (16,532 bp) and C. virginica (17 244 bp), the lack of duplicated rrnS in O. edulis, added to the lack of 2 tRNAs (not duplicated trn-K and trn-Q) compared to Asian Crassostrea may account for the difference in length compared with other Crassostrea (C. gigas 18225 bp, C. hongkongensis 18 622 bp).

The genome composition of O. edulis is, thus, identical to O. denselamellosa (except for AT composition) and close to S. mordax in terms of complete genome size, AT content and the non-duplicated rrnS. More mitochondrial genome sequences from Ostrea and Sac-costrea will be needed to assess relationships between the Ostrea, Crassostrea and Saccostrea genera.

Gene arrangement

Animal mt gene order is relatively stable within major groups and generally variable among groups [2]. Bivalve species show variability in terms of mt genome size, gene arrangement and tRNA number [20]. As observed in four Pectinidae (A. irradians, M. yessoensis, C. farreri and P. magellanicus), gene arrangement can be very different despite species being members of the same family

D MNR cokI

nad4 v

Figure 1 Mitochondrial genome map of Ostrea edulis. Genes for proteins (green) and rRNAs (blue) are abbreviated with standard abbreviations. The one letter amino acid code is used for tRNA (red) designation. The major non-coding region (MNR) is shown in grey.

[15]. In contrast, in Mytilus congeners M. edulis, M. trossulus and M. galloprovincialis, PCGs are arranged in an identical order, but tRNA, rRNA and control regions are also almost the same [21]. The mt gene order of O. edulis is identical to that of O. denselamellosa (Figure 2). The arrangement of PCGs in O. edulis is cox1, cox3, cytb, cox2, nad2, atp6, nad4, nad5, nad6, nad3, nadl, nad4L and is nearly identical to that of Crassostrea, except for the inversion of two PCGs nad2 and atp6. Among the six Asian Crassostrea and their Atlantic sister species C. virginica, only protein-coding gene order is identical [10]. Besides, gene order of Crassostrea differs significantly from S. mordax. Ostreidae share three

PCG blocks cox1-cox3-cytb-cox2, nad5-nad6 and nad3-nad1-nad4L. Moreover, the nad2-atp6-nad4 block of O. edulis is inverted in S. mordax (Figure 2), but the remaining genes are extensively rearranged. The major non-coding region (MNR) is located after trnD (Figure 1), while this region is found between trnG and trnV near atp6 and nad2 in Crassostrea. If tRNAs and rRNAs are considered, there are six blocks conserved within the Ostreidae: cox3-trnI, cox2-trnM, trnM-trnS, trnY-atp6, trnH-nad4 and trnF-trnA-nad1-nad4L. Between O. edu-lis and Asian Crassostrea, seven blocks are shared: cox3-trnI-trnT-trnE-cytb, cox2-trnM-trnS, trnM-trnS, trnY-atp6, trnH-nad4, nad5-nad6-trnQ-nad3 and trnL-trnF-

Table 1 Features of Ostrea edulis mitochondrial genome

Feature Sequence location Size Start codon Stop codon Intergenic region*

cox1 1-1566 1566 ATG TAA 6

trnG 1573-1639 67 95

cox3 1735-2622 888 ATA TAG -1

trnl 2622-2687 66 10

trnT 2698-2761 64 7

trnE 2769-2836 68 -6

cytb 2831-3997 1167 CTA TAA 1

cox2 3999-4691 693 ATG TAA 3

trnM1 4695-4759 65 7

trnS1 4767-4836 70 151

trnM2 4988-505- 64 51

trnS2 5103-5172 70 1

trnL1 5173-5239 67 1

trnP 5241-5304 64 17

rrnL 5'end 5322-5896 575 25

nad2 5922-6929 1008 ATT TAA 73

trnC 7003-7066 64 3

trnY 7070-7134 65 6

atp6 7141-7809 669 ATA TAG 3

trnN 7813-7883 71 14

trnR 7898-7964 67 4

trnV 7969-8035 67 27

trnH 8063-8126 64 -15

nad4 8112-9476 1365 ATA TAG 0

rrnS 9477-10411 935 81

rrnL 3'end 10493-11200 708 172

nad5 11373-12920 1548 ATG TAA 7

nad6 12928-13395 468 ATA TAA 9

trnQ 13405-13470 66 1

nad3 13472-13825 354 ATG TAG -1

trnK 13825-13891 67 3

trnL2 13895-13960 66 1

trnF 13962-14028 67 13

trnA 14042-14106 65 79

nad1 14186-15118 933 ATG TAA 1

nad4L 15120-15401 282 ATG TAA 60

trnW 15462-15524 63 33

trnD 15558-15625 68 0

MNR 15626-16320 695 0

^Negative numbers indicate overlapping nucleotides between adjacent genes

trnA-nad1-nad4L-trnW; while between O. edulis and S. mordax, seven blocks are also shared but these are different: trnG-cox3-tmI, trnE-cytb-cox2-tmM, trnM-trnS-trnL-trnP-rrnL(5'end), trnF-trnA-nad1-nad4L, rrnL (3'end)-nad5-nad6, trnH-nad4 and trnY-atp6. It should be noted that rrnL is in one piece in Saccostrea but not within Crassostrea.

In terms of gene arrangement, it is thus clear that O. edulis is more similar to Crassostrea than to S. mordax when comparing PCGs. As shown in Figure 2, the

complete genome arrangement of O. edulis is similar to that of Asian Crassostrea while it appears completely reorganized from trnY to the end of mt genome when compared with that of S. mordax.

Protein-coding genes

All PCGs are encoded on and transcribed from the same strand. Twelve open reading frames (ORFs) were detected for the thirteen typical PCGs (cox1-cox3, cytb, nad1-nad6, nad4L, atp6 and atp8). Although we

Figure 2 The gene arrangement map of Ostreidae mitochondrial genomes. The bars show identicalgene blocks. Allgenes are transcribed from left-to-right.

carefully looked for candidate regions for atp8 gene, we could not identify any, as in all Pteriomorphia complete genomes already published. However, a recent publication [17] suggests that a putative ORF represents a good candidate to start an atp8 gene in most bivalve mt genomes. Within the invertebrate mt code there are three standard initiation codons (M-AUG, M-AUA, and I-AUU), but mt genomes often use a variety of non conventional start codons [22]. In this study, most of PCGs use conventional initiation codons: ATA is used for cox3, nad4, nad6 and atp6; ATG is used for cox1, cox2, nad1, nad3, nad4L and nad5; ATT is used for nad2, but cytb uses the alternative start codon CTA (as in C. gigas and C. angulata [10]). Eight protein-coding genes were terminated by a stop codon (TAA and TAG).

Transfer and ribosomal RNA genes

In total, 23 tRNA coding genes were identified in the size range of 63 to 71 nucleotides, based on typical secondary structure (Additional file 1). An additional trnM was detected as found in C. gigas, C. hongkongensis [9], C. virginica [16] and Mytilus [23]. Two serine and two leucine tRNA genes were also differentiated in O. edulis by their anticodons (UCA Ser1, AGA Ser2, and CUA Leu1, UUA Leu2) as found in O. denselamellosa [24], Crassostrea and some other species (M. edulis, M. gallo-provincialis and Argopecten irradians). The anticodon usage of O. edulis was congruent with the corresponding tRNA genes of other molluscan mtDNAs.

Identification of both the small and the large riboso-mal RNA genes in O. edulis was accomplished by BLAST comparison with other published ribosomal RNA genes, especially O. denselamellosa [GenBank: HM015199], S. mordax [GenBank:FJ841968] and C. gigas [GenBank:EU672831]. Although putative gene boundaries for the two rRNA genes have been found, these cannot be precisely determined until transcript

mapping is carried out. Besides rrnS of O. edulis is 935 bp in length and flanked by nad4 and rrnL 3'end.

The rrnL gene is split into two segments: one segment, of the 5' end (matches with rrnL 5'end from O. denselamellosa and Saccostrea), is 575 bp long and positioned between trnP and nad2; and the other segment, of the 3' end, is 708 bp and located between rrnS and nad5. The length of the rrnS is similar to that of most bivalves, but smaller than that of O. denselamellosa (1017 bp) and that of Crassostrea (946 to 1207 bp) [10]. The size of rrnL (1283 bp in all) is similar to that of O. denselamellosa (1299 bp), but smaller than that of other bivalves. This bias may be due to the method (BLAST) used to compare the rRNA sequences because this method only checks the identity between a few sequences and because it's easier to compare sequences from same species as they show higher identity.

Non-coding regions

As in most bivalves, O. edulis mtDNA contains a large number of unassigned nucleotides. There are as many as 21 non-coding regions (> 5 bp) up to 965 nucleotides found throughout the O. edulis mitochondrial genome. Eight of these non-coding regions are more than 50 bp in length. Among these regions, the major non-coding region (MNR) has been identified and located, that remains the most promising region in which to find regulatory and/or gender-specific sequences [25]. The O. edulis mtDNA MNR is positioned between trnD and cox1 and is 695 bp in length, similar to that of O. dense-lamellosa (689 bp), making it the longest MNR within the Ostreidae apart from C. virginica (832 bp) and C. ariakensis (716 bp). It has an A+T content of 74.4% which is higher than the remainder of the mt genome (64.4%), as it includes several (A)n and (T)n homopolymer tracts, features which are typically used for

identification of the mitochondrial control region and thought to contain the replication origin [2].

Phylogenetic analysis

In recent years there have been many phylogenetic studies on the taxonomy and evolution of the Ostreidae based on molecular data, especially mitochondrial DNA [26-30]. However, most of these previous studies have been based on partial sequences and incomplete molecular information. Recently, Ren et al. [11] have compared 7 complete mt genomes from Asian oysters.

In the present study's aa-based tree built with twelve concatened PCGs from 19 mitochondrial genomes in Pteromorphia (Figure 3), we can observe that, at the Ostreidae level, O. edulis is first clustered with O. dense-lamellosa as congeneric species. Then this group of species falls into a highly supported clade with S. mordax. Ostrea and Saccostrea are then clustered with the Cras-sostrea species group. In this latest clade, the single American oyster C. virginica falls at the base of a nested clade that contains the Asian oysters. Very similar results were obtained with a nucleotide phylogenetic

tree with low differences of bootstrap values. In Figure 4, more Ostreidae species are included as more numerous cox1 sequences are available in Genbank. The same phylogenetic relationship between Ostrea, Saccostrea, and Crassostrea is observed, especially the first grouping of Ostrea and Saccostrea, but not between Ostrea and Crassostrea, with however far less robust nodes. This same result was observed when considering the evolution of the tRNA anticodons in marine bivalve mito-chondrial genomes, where the relationship presented are also based on concatenated nucleotide sequences of 12 protein-coding genes by Bayesian inference analysis [24]. However a recent study [31], based on cox1 and 16S sequences, showed a closer relationship between Ostrea and Crassostrea, than with Ostrea and Saccostrea. However, for the cox1 analysis, only one Ostrea sequence was included, and for the 16S analysis, much more Ostrea sequences were included but the bootstrap value was between 50 and 80%. Those comparisons seem to indicate that phylogenetic analyses are more powerful when including several sequences as the 12 concatened PCGs.

100 r~ Crassostrea angulata L- Crassostrea gigas

-Crassostrea sikamea

-Crassostrea ariakensis

95 I-Crassostrea hongkongensis

Crassostrea iredalei

-Crassostrea virginica

Saccostrea mordax -Ostrea denselamellosa

Ostrea edulis

Ostreidae

■ Argopecten irradians

-Placopecten magellanicus

-Mimachlamys nobilis

-Mizuhopecten yessoensis

Chlamys farreri

Pectinidae

■ Musculista senhousia — Mytilus trossulus r Mytilus galloprovincialis

100 L Mytilus edulis

Mytilidae

Haliotis rubra ] Outgroup

Figure 3 Phylogenetic tree based on twelve concatenated PCGs from 19 mitochondrial genomes.

- Ostrea edulis cox1 Ostrea angasi cox1 Ostrea chilensis cox1

Ostrea denselamellosa cox1

Ostreola conchaphila cox1 — Ostrea puelchana cox1 -Ostrea aupouria cox1

Ostrea

Saccostrea mordax cox1 - Saccostrea cucullata cox1

-Saccostrea glomerata cox1

-Saccostrea kegaki cox1

-Crassostrea virginica cox1

Crassostrea iredalei cox1

-Crassostrea ariakensis cox1

-Crassostrea hongkongensis cox1

-Crassostrea sikamea cox1

Saccostrea

— Crassostrea gigas cox1 Crassostrea angulata cox1 -Haliotis rubra cox1 ] Outgroup

Crassostrea

Figure 4 Phylogenetic tree based on cox1 from all published Ostrea, Ostreola, Saccostrea and Crassostrea.

Finally, the phylogenetic tree presented in Figure 3, which includes mt genomes from all published Pterio-morphia, reinforces the taxonomic classification of the 3 families Ostreidae, Mytilidae and Pectinidae [32,11].

Conclusion

In conclusion, the complete mitochondrial genome of O. edulis is 16 320 bp in length. A common phenomenon is that mitogenomes of most bivalves contain two trnM genes and most metazoan mitochondria have a set of 22 tRNA, including two trnL and two trnS. However the tRNA gene sets of O. edulis, O. denselamellosa and C. virginica are identical in having 23 tRNA genes. Another important characteristic is that the rrnS gene is not duplicated in O. edulis, a feature shared with O. densela-mellosa, S. mordax and C. virginica and which contrasts with Asian Crassostrea.

The phylogenetic analyses confirm the relationships between each family (Ostreidae, Mytilidae and Pectini-dae), but also within each genus (Ostrea, Saccostrea and

Crassostrea). Within the Ostreidae, phylogenetic analyses show that Ostrea are closer to Saccostrea than Crassostrea, although gene arrangement may show a closer relationship between Ostrea and Crassostrea, indicating that several types of information are needed to infer relationships between genome species as evolution is acting at different levels of the genomes. As many questions remain unanswered on the phylogeny of Ostreidae, especially between Ostrea and Saccostrea, it would be desirable to increase the resolution by adding samples of more taxa in order to extend molecular information among the major lineages of the Ostreidae and within the Pteriomorphia as a whole.

Methods

PCR amplification and DNA sequencing

Adductor muscle from three O. edulis collected in Quiberon Bay (Bretagne, France) was used in this study. Total genomic DNA was extracted using a Wizard®DNA Clean-up System (Promega). The

mitochondrial genome was amplified in 4 overlapping fragments using species-specific primers (Additional file 2). PCR was performed in 25 ^l reaction volumes in a thermocycler (Applied Biosystems). Each reaction contained 13.3 ^l dH2O, 5.0 ^l buffer 5x (Promega), 2.0 ^l MgCl2 (25 mM), 2.5 ^l dNTP (2 mM), 0.5 ^l of each primer (20 ^M), and 0.2 ^l GoTaq®DNA polymerase (5U/^l, Promega). PCR cycling conditions were 94 °C for 2 min; then 30 cycles of 94 °C for 30 sec, 57 °C for 30 sec and 72 °C for 2 min; and finally a step of 72 °C for 10 min. PCR products were verified by electrophoresis (1% agarose gel) and purified using Monta-ge®PCR Centrifugal Filter Devices (Millipore). Purified products were then used directly as templates in cycle sequencing reactions with dyelabeled terminators (Big Dye 3.1, Applied Biosystems). Specific primers were designed and used for primer walking sequencing, which was performed for both strands of each sample on an ABI 3130XL/Genetic Analyser (ABI).

Sequence analysis and gene annotation

During the processing of large fragments and those from primer walking sequencing, regular and manual examinations were used to ensure there was reliable overlapping and correct genome assembly.

Protein-coding and ribosomal RNA genes were firstly identified using BLAST [33] searches at GenBank, and then by alignment with previously published mt genomes from species of Crassostrea, Saccostrea and other closely-related molluscs. Amino-acid sequences of protein-coding genes were inferred with ORF Finder [34] using invertebrate mitochondrial genetic code. Transfer RNAs were identified using DOGMA [35]http://dogma. ccbb.utexas.edu/, and tRNAscan-SE [36]http://selab.jane-lia.org/tRNAscan-SE/ using mito/chloroplast genetic code and default search mode, or setting the cove cutoff score to 1 when necessary. Assembly of the genome and gene map of the mitochondrial genome of Ostrea edulis was performed using CLC Main Workbench (CLC bio).

Phylogenetic analysis

To date, 20 Pteriomorphia mt genomes are available in GenBank [37] and we used 19 of these (excluding Argo-pecten irradians irradians that is very close to Argopec-ten irradians: 99% similarity) in our phylogenetic analysis, together with O. edulis mt genome obtained in this study (Table 2). The blacklip abalone Haliotis rubra (Gastropoda) was used as the outgroup. The nucleotide and amino-acid sequences from all 12 PCGs (protein-coding genes) were concatenated for each genome and

Table 2 List of complete mitogenomes used in this study

Tax on Classification GenBank Accession Number Size

Mollusca

Bivalvia

Pteriomorphia

Mytilus edulis Mytiloida; Mytiloidea; Mytilidae AY484747 16,740 nt

Mytilus galloprovincialis Mytiloida; Mytiloidea; Mytilidae AY497292 16,744 nt

Mytilus trossulus Mytiloida; Mytiloidea; Mytilidae AY823625 18,652 nt

Musculista senhousia Mytiloida; Mytiloidea; Mytilidae GU001954 20,612 nt

Crassostrea angulata Ostreoida; Ostreoidea Ostre dae EU672832 18,225 nt

Crassostrea ariakensis Ostreoida; Ostreoidea; Ostre dae EU672835 18,414 nt

Crassostrea gigas Ostreoida; Ostreoidea; Ostre dae EU67283' 18,225 nt

Crassostrea hongkongensis Ostreoida; Ostreoidea; Ostre dae EU672834 18,622 nt

Crassostrea iredalei Ostreoida; Ostreoidea; Ostre dae FJ841967 22,446 nt

Crassostrea sikamea Ostreoida; Ostreoidea; Ostre dae EU672833 18,243 nt

Crassostrea virginica Ostreoida; Ostreoidea; Ostre dae Y905542 17,244 nt

Saccostrea mordax Ostreoida; Ostreoidea; Ostre dae FJ841968 16,532 nt

Ostrea denselamellosa Ostreoida; Ostreoidea; Ostre dae HM015199 16,277 nt

Ostrea edulis Ostreoida; Ostreoidea; Ostre dae JF274008 16,320 nt

Argopecten irradians Pectinoida; Pectinoidae; Pectinidae EU023915 16,22' nt

Chlamys farreri Pectinoida; Pectinoidae; Pectinidae EU715252 21,695 nt

Mizuhopecten yessoensis Pectinoida; Pectinoidae; Pectinidae AB271769 20,414 nt

Placopecten magellanicus Pectinoida; Pectinoidae; Pectinidae DQ088274 32,115 nt

Mimachlamys nobilis Pectinoida; Pectinoidea; Pectinidae FJ415225 17,963 nt

Gastropoda

Vetigastropoda

Haliotis rubra Haliotoidea; Haliotidae NC_005940 16,907 nt

aligned using MUSCLE [38]. The size of the concatenated alignment nucleotides varied from 10 411 bp for M. yessoensis to 11 240 bp for P. magellanicus). To the alignments, we applied a Maximum Likelihood (ML) phylogenetic reconstruction approach using 100 bootstraps with MEGA5 [39]. A second phylogenetic analysis was performed using 5 additional cox1 genes from Ostrea (Ostrea angasi [GenBank:AF112287.1], Ostrea aupouria [GenBank:AF112288.1], Ostrea chilensis [Gen-Bank:AF112289.1], Ostrea puelchana [GenBank: DQ226521.1], and Ostreola conchaphila [GenBank: DQ464125.1]) and 3 from Saccostrea (Saccostrea cucul-lata [GenBank:AY038076.1], Saccostrea glomerata [Gen-Bank:EU007483.1] and Saccostrea kegaki [GenBank: AB076910.1]).

Additional material

Additional file 1: The potential secondary structures of 22 tRNAs of Ostrea edulis. The duplication of methionine is named Ml and M2 respectively. Codons recognized are shown for the pairs of leucine (L1 and L2) and serine (S1 and S2).

Additional file 2: Primers used for amplification of 4 large fragments in mitochondrial genome of Ostrea edulis.

Acknowledgements

The authors thank Aimé Langlade who provided biological material, Denis Saulnier who gave valuable advice, Nicole Faury for her technicalassistance and Helen McCombie for English editing. We also thank two anonymous reviewers for their usefulcomments on the manuscript.

Authors' contributions

GDT, SH and BM obtained the sequences. GDT and SL performed analyses and wrote the first draft of the publication. Allthe co-authors finalised the manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 9 June 2011 Accepted: 12 October 2011 Published: 12 October 2011

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doi:10.1186/1756-0500-4-400

Cite this article as: Danic-Tchaleu et al.: Complete mitochondrial DNA sequence of the European flat oyster Ostrea edulis confirms Ostreidae classification. BMC Research Notes 2011 4:400.

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