BACKGROUND
mitochondrial  dna is widely used for phylogenetic analysis because of its unique architecture, inheritance and small size.
metazoan mtdna is nearly always a circular molecule except for some cnidarians  <cit> .
it contains the same  <dig> genes, specifying  <dig> proteins of the respiratory chain ,  <dig> ribosomal rnas and  <dig> transfer rnas.
although there are exceptions, most mtdnas range in size from  <dig> to  <dig> kb.
typically, there are few intergenic nucleotides except for a single large non-coding region generally thought to contain elements that control the initiation of replication and transcription  <cit> .
size variation in mtdna is usually due to the different length of the non-coding regions.
gene order is generally conserved in most metazoan taxa although some groups show considerable variation.
variations in both mtdna sequence and gene order have been used for phylogenetic analysis.
because mtdna is fast evolving and nucleotide mutations may return to an early state, mtdna sequences may not allow deep phylogenetic reconstruction.
gene order, on the other hand, has very small probability of back-mutation and may be particularly useful for high level phylogenetic analysis.
although the mechanism of mtdna rearrangement is poorly understood, mt gene order has been increasingly used for phylogenetic studies  <cit> .
as the second species-rich phylum of the animal kingdom after arthropoda, mollusca exhibits tremendous variation in their mt genomes.
seven bivalve lineages  have been found to have an unusual mode of inheritance for mtdna, termed doubly uniparental inheritance   <cit> .
some pulmonate gastropods have unusual trnas lacking the t-stem or the d-stem, similar to nematode mt trnas  <cit> .
the atp <dig> and atp <dig> genes are separated in the scaphopods and two groups of gastropods .
in addition, several mt genes are duplicated in cephalopods watasenia scintillans and todarodes pacificus  <cit> .
unlike the general conservation in gene order in most other metazoan groups, most molluscan mt genomes reported so far contain considerable rearrangements especially in bivalvia and scaphopoda  <cit> .
at the same time, the phylogeny of molluscs is poorly studied.
phylogenetic relationships among major molluscan groups are not well understood.
the species identity and classification of some most common molluscs remain questionable.
oysters are bivalve molluscs widely distributed in world oceans.
they are benthic, sessile filter-feeders with important roles in estuary ecology.
some species support major fishery and aquaculture industries worldwide.
despite the abundance, ecological and economic significance of oysters, we know little about their species diversity and evolutionary history.
classification of oysters remains a challenge partly due to the lack of well-defined morphological characters.
shell morphology, the main character used in oyster classification, is known to be plastic and subject to environmental variation.
much of the oyster classification to date is based on shell characteristics and has resulted in considerable errors and confusion.
the difficulty of oyster classification is particularly pronounced in china and other parts of asia where a large number of species are sympatric  <cit> .
about thirty species have been recorded along the coast of china  <cit> .
the presence of a large number of oyster species has led many to believe that asia pacific is the epicenter of oyster speciation, but the inability to reliably identifying them has hindered our understanding of oyster evolution.
among the extent oysters, five crassostrea oysters, namely c. gigas, c. angulata, c. sikamea, c. hongkongensis and c. ariakensis are commonly found in china and other parts of asia and yet, they have been difficult to identify by shell morphology alone.
recently, some confusion in the oyster identification have been resolved using dna sequence data  <cit> , and the five crassostrea oysters can be reliably identified  <cit> .
still, we know little about the evolutionary history of the crassostrea species and how they relate to each other and the other oysters.
for example, it is not clear whether c. angulata should be considered as an independent species and which species is the closest relative of the newly described c. hongkongensis.
most of the phylogenetic analyses so far are based on short dna fragments, yielding variable results  <cit> .
the ability to sequence and compare whole mt genomes provides a new impetus for phylogenetic analysis of oysters and other molluscs.
complete mt genome sequences have been obtained for two crassostrea oysters from asia  and one species from the atlantic   <cit> .
comparative analysis shows that the two asian species share the same gene order that is very different from the atlantic species  <cit> .
to determine if gene order is conserved in other asian species and to understand the evolution of crassostrea oysters, we sequenced the mt genomes of four crassostrea oysters from asia plus one that was previously published  <cit> , obtained newly released mt genomes of two asian oysters , and compared them with other molluscan genomes.
here we report the first estimates of divergence time among crassostrea species based on complete mt sequences and the complete conservation in gene order among the six asian crassostrea species.
the complete conservation of gene order is highly unusual considering the tremendous rearrangement of mtdna in most marine bivalves.
it provides strong evidence for recent emergence of the six crassostrea species in asia.
our analysis also suggests that rearrangement of the mt genome may not be strictly a function of time but subject to some other constraints that are presently not well understood.
RESULTS
genome composition
genome composition and organization of the six asian crassostrea oysters is summarized in figure  <dig> and additional file 1: table s <dig>  organization of the american oyster c. virginica, a sister species from the atlantic ocean, is listed for comparative analysis .
the complete mt genomes of c. gigas, c. angulata, c. sikamea, c. hongkongensis and c. ariakensis are  <dig>  bp,  <dig>  bp,  <dig>  bp,  <dig>  bp and  <dig>  bp in length, respectively  <cit> .
these sequences have been deposited in genbank under the accession number eu <dig>  nc_012648-nc_ <dig>  nc_ <dig>  the american oyster c. virginica mtdna is  <dig>  bp in length.
the mt genome of s. mordax is  <dig>  bp in length.
however, the mt genome of c. iredalei,  <dig>  bp in length, is obviously longer than all other oysters  <cit> .
the size of molluscan mt genomes varies dramatically, ranging from  <dig>  bp in the snail biomphalaria glabrata to  <dig>  bp in the sea scallop placopecten magellanicus  <cit> .
the six asian crassostrea oyster genomes encode  <dig> genes including  <dig> protein-coding genes,  <dig> rrnas and  <dig> trnas on the same strand .
in contrast to the typical animal mt genomes, they lack one protein-coding gene atp <dig> and have duplications of three trnas: trnm, trnk and trnq.
another unique character is that rrnl gene is split into two segments  and rrns has a nearly identical duplication, which has never been reported in other animal mt genomes  <cit> .
among the seven crassostrea oysters, the lowest a+t content is  <dig> % in c. virginica, while the highest a+t content is  <dig> % in c. hongkongensis.
in the other five species, the a+t content varies from  <dig> % in c. ariakensis to  <dig> % in c. iredalei  <cit>  .
the highest a+t content of c. hongkongensis is mainly caused by the increased total length and high a+t content of the non-coding region.
the length of coding regions in the asian-pacific oyster genomes is similar and larger than that in c. virginica, because of duplications of two trnas  and one rrns.
rrnl gene
rrns gene
gene arrangement
molluscs, especially bivalves, display an extraordinary amount of variation in gene arrangement.
gene order of selected molluscs was presented and compared with that of oysters in figure  <dig>  the black chiton katharina tunicata is the only sequenced representative of the polyplacophora, an early diverged class of the mollusca  <cit> .
its gene order may represent the pleisomorphic gene arrangement in mollusca.
at high taxonomic levels, the gene order tends to be conserved across polyplacophoran, cephalopods and gastropods.
the gene order of k. tunicata differs from that of the common octopus octopus vulgaris by merely the inversion of trnp and translocation of trnd.
gene order of other cephalopods resembles that of o. vulgaris with some translocations of trna or the switch of large gene blocks  <cit> .
additionally, the gene arrangement of k. tunicata differs from that of the blacklip abalone haliotis rubra merely by the inversion of trnp plus transposition of four trnas.
in comparison, however, mt genomes of bivalvia show tremendous gene rearrangements.
to date, all bivalves whose mt genomes are available belong to three subclasses: palaeoheterodonta, heterodonta and pteriomorphia.
gene order in freshwater mussel lampsilis ornata  is nearly identical to that of it sister species inversidens japanensis except for the translocation of several trnas, and protein-coding genes nad <dig> and nad <dig>   <cit> .
in addition, l. ornata genome contains the atp <dig> gene which is absent in i. japanensis .
the remaining bivalves whose mt genomes have been sequenced are marine species, where the gene order is dramatically rearranged.
comparing the gene arrangement between meretrix petechialis and venerupis philippinarum , they share four identical gene blocks: two large blocks cox1-l1-nad1-nad2-nad4l-i and cox2-p-cob-rrnl-nad4-h-e-s2-atp6-nad3-nad <dig>  and two small blocks only containing trna genes.
they share only one gene block cob-rrnl-nad4-atp <dig> with acanchocardia tuberculata of the same order.
meanwhile, a. tuberculata and hiatella arctica share only two small gene blocks trnm-w and nad2-cox <dig>  species sequenced in pteriomorphia belong to three orders: mytiloida, ostreoida and pectinoida.
few gene blocks are shared between any two pairs.
among the four scallops of the family pectinidae , gene arrangement of mizuhopecten yessoensis closely resembles that of chlamys farreri.
they share three large gene blocks nad4l-nad6-l2-cob, cox3-k-f-q-e-atp6-cox2-nad2-t-p-i-l1-m-nad3-nad <dig> and n-nad1-r-rrnl-m. but their gene order is very different from that of the other two scallops, despite being members of the same family.
the two genomes of c. farreri and argopecten irradians only show three small shared gene blocks nad6-l2-cob, nad3-nad <dig> and nad1-r-rrnl, while a. irradians and p. magellanicus only share one gene block l2-cob-cox <dig>
on the other hand, the mt gene order in the six asian crassostrea oysters is completely identical to each other .
this is highly unusual considering the tremendous rearrangement observed in other groups of bivalvia.
even within the same genus, comparison between the asian crassostrea oysters of the pacific and the american oyster c. virginica of the atlantic ocean show that gene order has been drastically rearranged.
gene order of the protein-coding genes  is the same, while several trnas has translocated after the divergence between the asian and american species.
the six asian crassostrea oysters have duplicated gene trnk, trnq and rrns compared with the american oyster.
they differ from the atlantic sister species by as many as nine trna changes , and furthermore, they differ significantly from s. mordax in pcgs.
if the trna is not considered, they share three pcgs blocks cox1-cox3-cob-cox <dig>  nad3-nad1-nad4l, nad5-nad <dig> and atp6-nad <dig>  it is amazing that crassostrea sister species from the two oceans have so much difference in gene order and close relatives even have more difference in gene order, while gene order is completely conserved in the six crassostrea species from asia.
this raises the questions what causes rearrangements of mt genomes and how do they evolve over time.
additional studies of mt genomes of other oyster species would be interesting to see if this pattern should preserve, which may further our understanding of mt genome evolution.
protein-coding genes
of the  <dig> typical pcgs , twelve genes were determined and the atp <dig> gene was absent in all oyster genomes.
all pcgs are encoded on and transcribed from the same strand.
these features have been observed in all other marine bivalve genomes published so far except for h. arctica where the atp <dig> gene has been reported, and for two species of palaeoheterodonta  where genes are coded on both strands .
thus, coding genes on the same strand and missing atp <dig> gene are the most distinct features of marine bivalve mt genomes, though from a recent publication from breton et al  <cit>  this might only hold true for ostreoida and a few other bivalves.
the two palaeoheterodonta species that use both strands for coding are the only known members of bivalvia showing significant conservation in gene order with other molluscan classes that also use both strands for coding.
we suspect that coding on both strands may be inhibitory to mt genome rearrangement, and marine bivalves show a tremendous amount of mt genome rearrangement because they only use one strand for coding.
rearranging a genome with dual-strand coding may be more complicated and cause more harm than rearranging a genome that codes on one strand.
mt genomes often use a variety of non-standard initiation codons  <cit> .
in some cases, identification of the very clear initiation codon is difficult when several alternatives are inferred representing the start of coding sequence in a region.
most of pcgs initiate with the standard start codon atg.
standard start codon ata is used for cox <dig> in c. virginica, nad <dig> in c. hongkongensis and nad4l in c. sikamea and in c. ariakensis.
non-standard initiation codon gtg is used for nad <dig> in c. ariakensis, and att is used for nad <dig> in c. hongkongensis .
there are no obvious patterns in termination codon usage; the usage frequency of stop codon tag is similar to that of taa.
incomplete termination codons t and ta were also used.
termination codon taa is used in  <dig> pcgs of c. virginica; if the incomplete termination codon is considered, the number of taa is up to eleven.
the pcgs with identical termination codon in the six genomes are nad <dig> and nad4l, which ended with taa and incomplete stop codon t, respectively .
cgi
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*rrnl <dig> and rrnl <dig> indicate the two parts of 5' half and 3'half rrnl, respectively; rrns <dig> and rrns <dig> indicate the duplication of rrns.
the number of amino acids coded by each of the mt genomes is approximately equal.
excluding the stop codons, the c. virginica mtdna encodes the least amino acids , while the c. gigas mtdna encodes the most amino acids .
oysters with the lowest  and highest  a+t compositions of protein-coding region are c. virginica and c. hongkongensis, respectively.
similarly, oysters showing the lowest and highest a+t content of the first and the third positions are also c. virginica and c. hongkongensis:  <dig> % and  <dig> % for the first position, and  <dig> % and  <dig> % for the third position, respectively.
however, the a+t content of the second position is approximately the same in the six genomes ranging from  <dig> % in c. hongkongensis to  <dig> % in c. virginica.
it is obvious that the a+t content of the third codon position is higher than that of the first and the second positions.
the genomic features of  <dig> bivalve sequences are presented in table  <dig>  the statistics of a+t content, start and stop codon, and amino acid number of pcgs in c. iredalei is already described in wu et al's paper  <cit> .
nonsynonymous and synonymous substitutions
the estimation of nonsynonymous  and synonymous  substitution rates is of great significance in understanding evolutionary dynamics of protein-coding sequences across closely related species  <cit> .
to detect the influence of selection pressure in crassostrea species, the numbers of ka and ks were calculated and their ratios were plotted for all pairwise comparisons among the seven oysters .
the ratio of ka/ks in all  <dig> protein-coding genes varied from  <dig>  for cox <dig> in c. angulata_c.
sikamea and c. gigas_c.
sikamea to  <dig>  for nad <dig> in c. angulata_c.
ariakensis, which supports the existence of different mutation constraints among genes.
most of the amino acid substitutions are localized in the nadh complex genes.
it suggests a relaxation of purifying selection in the nad <dig>  nad <dig>  nad <dig>  nad <dig> genes compared with the more conservative genes such as cox <dig>  cox <dig> and atp <dig>
transfer and ribosomal rna genes
the mt genomes of the six asian crassostrea oysters encode  <dig> trna genes.
in addition to the  <dig> typical trnas, there are three duplications of trnm, trnk and trnq.
in comparison, however, there is no duplication of trnk and trnq in c. virginica.
the trna structure of c. gigas is referred to as the standard when the nucleotide variation in other four asian oysters is examined .
nucleotide variations including transition/transversion and insertion/deletion mostly occur at dhu and tΨc loops .
duplication of trna is common in molluscs.
a second trnm is also presented in the mussel mytilus edulis, m. galloprovincialis and m. trossulus  <cit> , and the clam v. philippinarum, h. arctica and a. tuberculata  <cit> .
in p. magellanicus, there are up to ten copies of trnm  <cit> .
in m. trossulus mtdna, an additional copy of trnq has also been reported  <cit> .
the anticodon usage of oyster genomes is congruent to the corresponding trna of other molluscs with one exception.
the anticodon of two trnms in the oysters is cau while that of two trnms is cau and uau in mytilus.
the difference in anticodon corresponds to the third wobble position.
the boundaries of both the small and the large ribosomal genes were determined by blast with the revised annotation of c. gigas genome  <cit> .
the rrnl gene is split into two segments, one segment of 5' end is distributed between trnq <dig> and trnn, and the other segment of 3' end is located between cox <dig> and cox <dig>  the split rrnl is first reported in c. virginica, but so far not reported in other metazoan mt genomes.
the mt genome of s. mordax also has the split rrnl, however, it does not have the duplicated rrns.
the duplication of rrns is only found in the six asian crassostrea oysters and the similarity between intraspecies and interspecies is high  in the conserved  <dig> nucleotides at the center of rrns sequences.
the size of rrnl in the asian crassostrea oysters is nearly equal, and smaller than that in c. virginica .
the length of rrns in c. virginica is  <dig> bp, while total length of rrns in the asian crassostrea oysters varies from  <dig>  bp  to  <dig>  bp .
non-coding regions
as in most bivalves, the oyster mtdnas contain a large number of unassigned nucleotides.
there are more than  <dig> non-coding regions throughout the seven crassostrea genomes.
the unassigned nucleotides vary from  <dig>  bp in c. gigas to  <dig>  bp in c. iredalei.
the proportion of the unassigned nucleotides in the whole genome varies from  <dig> % in c. gigas to  <dig> % in c. iredalei.
the largest non-coding region in the asian oysters is located between gene trng and trnv, while it is between gene trnp and trng in c. virginica.
the sequence of each corresponding non-coding region from the six asian crassostrea oysters was listed separately and regions large than  <dig> nucleotides were aligned using clustalx  <dig> .
there are  <dig> non-coding regions aligned .
the alignment shows that there are sequence conservation in the non-coding region, especially, higher sequence conservation in the regions between trns <dig> and trnp, trnv and nad <dig>  the sequence similarity among species in the non-coding region is positively correlated with their relatedness.
phylogenetic analysis and divergence time estimation
phylogenetic analysis using all mt coding sequences provides clear evolutionary relationships among the seven crassostrea and one saccostrea oyster species .
c. gigas is first clustered with c. angulata and then united with c. sikamea, meanwhile c. hongkongensis and c. ariakensis formed a clade.
finally, c. iredalei with other five crassostrea oysters form a large asian clade.
together with asian crassostrea oysters, the c. virginica formed the crassostrea clade.
s. mordax diverged early from crassostrea oysters and was positioned at the base of the large oyster clade.
all the phylogenetic relationships are supported with high values.
the close relationships between c. gigas and c. angulata, and c. hongkongensis and c. ariakensis are clearly demonstrated on the phylogenetic trees , which has been the subject of debate for some time  <cit> .
the complete mt genomes of c. gigas and c. angulata differed by 3%, providing strong support for their status as two independent species.
the first appearance of gastropoda and bivalvia in fossil record is approximately  <dig> myr ago  <cit> , providing the calibration constraint for divergence estimation.
divergence time estimates between species and/or clades are given in table  <dig> and figure  <dig>  our estimation shows that c. virginica and the asian oysters diverged about  <dig>  myr ago.
the six asian crassostrea oysters started their divergence about  <dig>  myr ago with the separation of c. iredalei from other species first.
other two large clades c. hongkongensis and c. ariakensis, and c. sikamea, c. gigas and c. angulata diverged  <dig>  myr ago.
c. hongkongensis and c. ariakensis diverged  <dig>  myr ago.
in the other clade, c. sikamea diverged from c. gigas and c. angulata  <dig>  myr ago.
the two most closely related species, c. angulata and c. gigas, diverged about  <dig>  myr ago.
these estimates indicate that, relative to the divergence with c. virginica about  <dig> myr ago, the speciation of the six asian crassostrea species is rather recent or 3- <dig> myr ago.
still, it is difficult to reconcile molecular divergence with genome rearrangement data observed here.
assuming the divergence time estimates are correct, it is intriguing that the mt genomes of c. virginica and the asian species diverged with nine structure rearrangements  in about  <dig> myr, while there was not a single rearrangement event among the six asian crassostrea species in the past  <dig> myr.
during the same time frame, numerous rearrangement events occurred among scallop species .
thus, the complete conservation of gene order among the asian crassostrea oysters in the past  <dig> myr is highly unusual, given the amazing rate of mt genome rearrangements in marine bivalves in general and within the genus crassostrea itself.
while this finding provides strong evidence for recent speciation of the six asian crassostrea species, it also indicates that mt genome rearrangement may not be strictly a function of time, but constrained by other factors.
detailed mechanisms of mt genome rearrangement are unknown.
we suspect that coding on both strands may be one of the factors inhibiting mt genome rearrangement.
this hypothesis is supported by the fact that the only two bivalve molluscs  with dual-strand coding have relatively fewer rearrangements of gene order compared with other molluscan classes .
all marine bivalves use one strand for coding and show tremendous rearrangements.
the extensive rearrangement of bivalve mt genomes may be a consequence of coding on one strand.
furthermore, the fact that massive rearrangements in marine bivalves have not disrupted single-strand coding or changed the direction of transcription suggests that the rearrangements are not caused by inversion or reverse transposition.
thus, transposition or "tandem repeat-random loss" <cit>  may be important for mt genome rearrangement  <cit> .
dual-strand coding may not be the only factor limiting mt genome rearrangement, as it cannot explain the absence of rearrangements in the six asian oysters over  <dig> myr.
we speculate that some other unknown features in mt genomes of the six asia species may also limit genome rearrangements.
the most noticeable structure change is the duplication of rrns in the six asian species, though it is difficult to determine if it has any effects on genome rearrangement at current stage.
further studies on the mt genomes of other oysters and marine bivalves may shed light on the origin and evolution of genome rearrangements.
gene order data have been shown to be valuable in phylogenetic analysis  <cit> .
while our analysis demonstrates the power of rearrangement data, it also argues for a better understanding of mt genome rearrangement before using them to infer divergence time.
CONCLUSIONS
asia pacific has long been suspected to be the center of oyster speciation.
our analysis of complete mt genomes provides strong evidence that crassostrea oysters have diversified in asia and the divergence was rather recent or within the last 3- <dig> myr.
the complete conservation of gene order in the six asian crassostrea species over a period of  <dig> myr is highly unusual given the remarkable rate of rearrangements in their sister species and other bivalves during the same time frame.
it provides strong evidence, in addition to sequence data, for the recent speciation of crassostrea oysters in asia.
it also indicates that changes in mt gene order may not be strictly a function of time but subject to other constraints that are presently not well understood.

 mitochondrial dna is widely used for phylogenetic analysis because of its unique architecture, inheritance and small size. metazoan mtdna is nearly always a circular molecule except for some cnidarians. it contains the same genes, specifying proteins of the respiratory chain, ribosomal rnas and transfer rnas. although there are exceptions, most mtdnas range in size from to kb. typically, there are few intergenic nucleotides except for a single large non-coding region generally thought to contain elements that control the initiation of replication and transcription. size variation in mtdna is usually due to the different length of the non-coding regions. gene order is generally conserved in most metazoan taxa although some groups show considerable variation. variations in both mtdna sequence and gene order have been used for phylogenetic analysis. because mtdna is fast evolving and nucleotide mutations may return to an early state, mtdna sequences may not allow deep phylogenetic reconstruction. gene order, on the other hand, has very small probability of back-mutation and may be particularly useful for high level phylogenetic analysis. although the mechanism of mtdna rearrangement is poorly understood, mt gene order has been increasingly used for phylogenetic studies. as the second species-rich phylum of the animal kingdom after arthropoda, mollusca exhibits tremendous variation in their mt genomes. seven bivalve lineages have been found to have an unusual mode of inheritance for mtdna, termed doubly uniparental inheritance. some pulmonate gastropods have unusual trnas lacking the t-stem or the d-stem, similar to nematode mt trnas. the atp and atp genes are separated in the scaphopods and two groups of gastropods. in addition, several mt genes are duplicated in cephalopods watasenia scintillans and todarodes pacificus. unlike the general conservation in gene order in most other metazoan groups, most molluscan mt genomes reported so far contain considerable rearrangements especially in bivalvia and scaphopoda. at the same time, the phylogeny of molluscs is poorly studied. phylogenetic relationships among major molluscan groups are not well understood. the species identity and classification of some most common molluscs remain questionable. oysters are bivalve molluscs widely distributed in world oceans. they are benthic, sessile filter-feeders with important roles in estuary ecology. some species support major fishery and aquaculture industries worldwide. despite the abundance, ecological and economic significance of oysters, we know little about their species diversity and evolutionary history. classification of oysters remains a challenge partly due to the lack of well-defined morphological characters. shell morphology, the main character used in oyster classification, is known to be plastic and subject to environmental variation. much of the oyster classification to date is based on shell characteristics and has resulted in considerable errors and confusion. the difficulty of oyster classification is particularly pronounced in china and other parts of asia where a large number of species are sympatric. about thirty species have been recorded along the coast of china. the presence of a large number of oyster species has led many to believe that asia pacific is the epicenter of oyster speciation, but the inability to reliably identifying them has hindered our understanding of oyster evolution. among the extent oysters, five crassostrea oysters, namely c. gigas, c. angulata, c. sikamea, c. hongkongensis and c. ariakensis are commonly found in china and other parts of asia and yet, they have been difficult to identify by shell morphology alone. recently, some confusion in the oyster identification have been resolved using dna sequence data, and the five crassostrea oysters can be reliably identified. still, we know little about the evolutionary history of the crassostrea species and how they relate to each other and the other oysters. for example, it is not clear whether c. angulata should be considered as an independent species and which species is the closest relative of the newly described c. hongkongensis. most of the phylogenetic analyses so far are based on short dna fragments, yielding variable results. the ability to sequence and compare whole mt genomes provides a new impetus for phylogenetic analysis of oysters and other molluscs. complete mt genome sequences have been obtained for two crassostrea oysters from asia and one species from the atlantic. comparative analysis shows that the two asian species share the same gene order that is very different from the atlantic species. to determine if gene order is conserved in other asian species and to understand the evolution of crassostrea oysters, we sequenced the mt genomes of four crassostrea oysters from asia plus one that was previously published, obtained newly released mt genomes of two asian oysters, and compared them with other molluscan genomes. here we report the first estimates of divergence time among crassostrea species based on complete mt sequences and the complete conservation in gene order among the six asian crassostrea species. the complete conservation of gene order is highly unusual considering the tremendous rearrangement of mtdna in most marine bivalves. it provides strong evidence for recent emergence of the six crassostrea species in asia. our analysis also suggests that rearrangement of the mt genome may not be strictly a function of time but subject to some other constraints that are presently not well understood.  genome composition genome composition and organization of the six asian crassostrea oysters is summarized in figure and additional file 1: table s organization of the american oyster c. virginica, a sister species from the atlantic ocean, is listed for comparative analysis. the complete mt genomes of c. gigas, c. angulata, c. sikamea, c. hongkongensis and c. ariakensis are bp, bp, bp, bp and bp in length, respectively. these sequences have been deposited in genbank under the accession number eu nc_012648-nc_ nc_ the american oyster c. virginica mtdna is bp in length. the mt genome of s. mordax is bp in length. however, the mt genome of c. iredalei, bp in length, is obviously longer than all other oysters. the size of molluscan mt genomes varies dramatically, ranging from bp in the snail biomphalaria glabrata to bp in the sea scallop placopecten magellanicus. the six asian crassostrea oyster genomes encode genes including protein-coding genes, rrnas and trnas on the same strand. in contrast to the typical animal mt genomes, they lack one protein-coding gene atp and have duplications of three trnas: trnm, trnk and trnq. another unique character is that rrnl gene is split into two segments and rrns has a nearly identical duplication, which has never been reported in other animal mt genomes. among the seven crassostrea oysters, the lowest a+t content is % in c. virginica, while the highest a+t content is % in c. hongkongensis. in the other five species, the a+t content varies from % in c. ariakensis to % in c. iredalei. the highest a+t content of c. hongkongensis is mainly caused by the increased total length and high a+t content of the non-coding region. the length of coding regions in the asian-pacific oyster genomes is similar and larger than that in c. virginica, because of duplications of two trnas and one rrns. rrnl gene rrns gene gene arrangement molluscs, especially bivalves, display an extraordinary amount of variation in gene arrangement. gene order of selected molluscs was presented and compared with that of oysters in figure the black chiton katharina tunicata is the only sequenced representative of the polyplacophora, an early diverged class of the mollusca. its gene order may represent the pleisomorphic gene arrangement in mollusca. at high taxonomic levels, the gene order tends to be conserved across polyplacophoran, cephalopods and gastropods. the gene order of k. tunicata differs from that of the common octopus octopus vulgaris by merely the inversion of trnp and translocation of trnd. gene order of other cephalopods resembles that of o. vulgaris with some translocations of trna or the switch of large gene blocks. additionally, the gene arrangement of k. tunicata differs from that of the blacklip abalone haliotis rubra merely by the inversion of trnp plus transposition of four trnas. in comparison, however, mt genomes of bivalvia show tremendous gene rearrangements. to date, all bivalves whose mt genomes are available belong to three subclasses: palaeoheterodonta, heterodonta and pteriomorphia. gene order in freshwater mussel lampsilis ornata is nearly identical to that of it sister species inversidens japanensis except for the translocation of several trnas, and protein-coding genes nad and nad. in addition, l. ornata genome contains the atp gene which is absent in i. japanensis. the remaining bivalves whose mt genomes have been sequenced are marine species, where the gene order is dramatically rearranged. comparing the gene arrangement between meretrix petechialis and venerupis philippinarum, they share four identical gene blocks: two large blocks cox1-l1-nad1-nad2-nad4l-i and cox2-p-cob-rrnl-nad4-h-e-s2-atp6-nad3-nad and two small blocks only containing trna genes. they share only one gene block cob-rrnl-nad4-atp with acanchocardia tuberculata of the same order. meanwhile, a. tuberculata and hiatella arctica share only two small gene blocks trnm-w and nad2-cox species sequenced in pteriomorphia belong to three orders: mytiloida, ostreoida and pectinoida. few gene blocks are shared between any two pairs. among the four scallops of the family pectinidae, gene arrangement of mizuhopecten yessoensis closely resembles that of chlamys farreri. they share three large gene blocks nad4l-nad6-l2-cob, cox3-k-f-q-e-atp6-cox2-nad2-t-p-i-l1-m-nad3-nad and n-nad1-r-rrnl-m. but their gene order is very different from that of the other two scallops, despite being members of the same family. the two genomes of c. farreri and argopecten irradians only show three small shared gene blocks nad6-l2-cob, nad3-nad and nad1-r-rrnl, while a. irradians and p. magellanicus only share one gene block l2-cob-cox on the other hand, the mt gene order in the six asian crassostrea oysters is completely identical to each other. this is highly unusual considering the tremendous rearrangement observed in other groups of bivalvia. even within the same genus, comparison between the asian crassostrea oysters of the pacific and the american oyster c. virginica of the atlantic ocean show that gene order has been drastically rearranged. gene order of the protein-coding genes is the same, while several trnas has translocated after the divergence between the asian and american species. the six asian crassostrea oysters have duplicated gene trnk, trnq and rrns compared with the american oyster. they differ from the atlantic sister species by as many as nine trna changes, and furthermore, they differ significantly from s. mordax in pcgs. if the trna is not considered, they share three pcgs blocks cox1-cox3-cob-cox nad3-nad1-nad4l, nad5-nad and atp6-nad it is amazing that crassostrea sister species from the two oceans have so much difference in gene order and close relatives even have more difference in gene order, while gene order is completely conserved in the six crassostrea species from asia. this raises the questions what causes rearrangements of mt genomes and how do they evolve over time. additional studies of mt genomes of other oyster species would be interesting to see if this pattern should preserve, which may further our understanding of mt genome evolution. protein-coding genes of the typical pcgs, twelve genes were determined and the atp gene was absent in all oyster genomes. all pcgs are encoded on and transcribed from the same strand. these features have been observed in all other marine bivalve genomes published so far except for h. arctica where the atp gene has been reported, and for two species of palaeoheterodonta where genes are coded on both strands. thus, coding genes on the same strand and missing atp gene are the most distinct features of marine bivalve mt genomes, though from a recent publication from breton et al this might only hold true for ostreoida and a few other bivalves. the two palaeoheterodonta species that use both strands for coding are the only known members of bivalvia showing significant conservation in gene order with other molluscan classes that also use both strands for coding. we suspect that coding on both strands may be inhibitory to mt genome rearrangement, and marine bivalves show a tremendous amount of mt genome rearrangement because they only use one strand for coding. rearranging a genome with dual-strand coding may be more complicated and cause more harm than rearranging a genome that codes on one strand. mt genomes often use a variety of non-standard initiation codons. in some cases, identification of the very clear initiation codon is difficult when several alternatives are inferred representing the start of coding sequence in a region. most of pcgs initiate with the standard start codon atg. standard start codon ata is used for cox in c. virginica, nad in c. hongkongensis and nad4l in c. sikamea and in c. ariakensis. non-standard initiation codon gtg is used for nad in c. ariakensis, and att is used for nad in c. hongkongensis. there are no obvious patterns in termination codon usage; the usage frequency of stop codon tag is similar to that of taa. incomplete termination codons t and ta were also used. termination codon taa is used in pcgs of c. virginica; if the incomplete termination codon is considered, the number of taa is up to eleven. the pcgs with identical termination codon in the six genomes are nad and nad4l, which ended with taa and incomplete stop codon t, respectively. cgi can csi cho car cvi cgi can csi cho car cvi cgi can csi cho cai cvi cox1 cox2 cox3 nad1 nad2 nad3 nad4 nad4l nad5 nad6 cob atp6 *rrnl and rrnl indicate the two parts of 5' half and 3'half rrnl, respectively; rrns and rrns indicate the duplication of rrns. the number of amino acids coded by each of the mt genomes is approximately equal. excluding the stop codons, the c. virginica mtdna encodes the least amino acids, while the c. gigas mtdna encodes the most amino acids. oysters with the lowest and highest a+t compositions of protein-coding region are c. virginica and c. hongkongensis, respectively. similarly, oysters showing the lowest and highest a+t content of the first and the third positions are also c. virginica and c. hongkongensis: % and % for the first position, and % and % for the third position, respectively. however, the a+t content of the second position is approximately the same in the six genomes ranging from % in c. hongkongensis to % in c. virginica. it is obvious that the a+t content of the third codon position is higher than that of the first and the second positions. the genomic features of bivalve sequences are presented in table the statistics of a+t content, start and stop codon, and amino acid number of pcgs in c. iredalei is already described in wu et al's paper. nonsynonymous and synonymous substitutions the estimation of nonsynonymous and synonymous substitution rates is of great significance in understanding evolutionary dynamics of protein-coding sequences across closely related species. to detect the influence of selection pressure in crassostrea species, the numbers of ka and ks were calculated and their ratios were plotted for all pairwise comparisons among the seven oysters. the ratio of ka/ks in all protein-coding genes varied from for cox in c. angulata_c. sikamea and c. gigas_c. sikamea to for nad in c. angulata_c. ariakensis, which supports the existence of different mutation constraints among genes. most of the amino acid substitutions are localized in the nadh complex genes. it suggests a relaxation of purifying selection in the nad nad nad nad genes compared with the more conservative genes such as cox cox and atp transfer and ribosomal rna genes the mt genomes of the six asian crassostrea oysters encode trna genes. in addition to the typical trnas, there are three duplications of trnm, trnk and trnq. in comparison, however, there is no duplication of trnk and trnq in c. virginica. the trna structure of c. gigas is referred to as the standard when the nucleotide variation in other four asian oysters is examined. nucleotide variations including transition/transversion and insertion/deletion mostly occur at dhu and tΨc loops. duplication of trna is common in molluscs. a second trnm is also presented in the mussel mytilus edulis, m. galloprovincialis and m. trossulus, and the clam v. philippinarum, h. arctica and a. tuberculata. in p. magellanicus, there are up to ten copies of trnm. in m. trossulus mtdna, an additional copy of trnq has also been reported. the anticodon usage of oyster genomes is congruent to the corresponding trna of other molluscs with one exception. the anticodon of two trnms in the oysters is cau while that of two trnms is cau and uau in mytilus. the difference in anticodon corresponds to the third wobble position. the boundaries of both the small and the large ribosomal genes were determined by blast with the revised annotation of c. gigas genome. the rrnl gene is split into two segments, one segment of 5' end is distributed between trnq and trnn, and the other segment of 3' end is located between cox and cox the split rrnl is first reported in c. virginica, but so far not reported in other metazoan mt genomes. the mt genome of s. mordax also has the split rrnl, however, it does not have the duplicated rrns. the duplication of rrns is only found in the six asian crassostrea oysters and the similarity between intraspecies and interspecies is high in the conserved nucleotides at the center of rrns sequences. the size of rrnl in the asian crassostrea oysters is nearly equal, and smaller than that in c. virginica. the length of rrns in c. virginica is bp, while total length of rrns in the asian crassostrea oysters varies from bp to bp. non-coding regions as in most bivalves, the oyster mtdnas contain a large number of unassigned nucleotides. there are more than non-coding regions throughout the seven crassostrea genomes. the unassigned nucleotides vary from bp in c. gigas to bp in c. iredalei. the proportion of the unassigned nucleotides in the whole genome varies from % in c. gigas to % in c. iredalei. the largest non-coding region in the asian oysters is located between gene trng and trnv, while it is between gene trnp and trng in c. virginica. the sequence of each corresponding non-coding region from the six asian crassostrea oysters was listed separately and regions large than nucleotides were aligned using clustalx. there are non-coding regions aligned. the alignment shows that there are sequence conservation in the non-coding region, especially, higher sequence conservation in the regions between trns and trnp, trnv and nad the sequence similarity among species in the non-coding region is positively correlated with their relatedness. phylogenetic analysis and divergence time estimation phylogenetic analysis using all mt coding sequences provides clear evolutionary relationships among the seven crassostrea and one saccostrea oyster species. c. gigas is first clustered with c. angulata and then united with c. sikamea, meanwhile c. hongkongensis and c. ariakensis formed a clade. finally, c. iredalei with other five crassostrea oysters form a large asian clade. together with asian crassostrea oysters, the c. virginica formed the crassostrea clade. s. mordax diverged early from crassostrea oysters and was positioned at the base of the large oyster clade. all the phylogenetic relationships are supported with high values. the close relationships between c. gigas and c. angulata, and c. hongkongensis and c. ariakensis are clearly demonstrated on the phylogenetic trees, which has been the subject of debate for some time. the complete mt genomes of c. gigas and c. angulata differed by 3%, providing strong support for their status as two independent species. the first appearance of gastropoda and bivalvia in fossil record is approximately myr ago, providing the calibration constraint for divergence estimation. divergence time estimates between species and/or clades are given in table and figure our estimation shows that c. virginica and the asian oysters diverged about myr ago. the six asian crassostrea oysters started their divergence about myr ago with the separation of c. iredalei from other species first. other two large clades c. hongkongensis and c. ariakensis, and c. sikamea, c. gigas and c. angulata diverged myr ago. c. hongkongensis and c. ariakensis diverged myr ago. in the other clade, c. sikamea diverged from c. gigas and c. angulata myr ago. the two most closely related species, c. angulata and c. gigas, diverged about myr ago. these estimates indicate that, relative to the divergence with c. virginica about myr ago, the speciation of the six asian crassostrea species is rather recent or 3- myr ago. still, it is difficult to reconcile molecular divergence with genome rearrangement data observed here. assuming the divergence time estimates are correct, it is intriguing that the mt genomes of c. virginica and the asian species diverged with nine structure rearrangements in about myr, while there was not a single rearrangement event among the six asian crassostrea species in the past myr. during the same time frame, numerous rearrangement events occurred among scallop species. thus, the complete conservation of gene order among the asian crassostrea oysters in the past myr is highly unusual, given the amazing rate of mt genome rearrangements in marine bivalves in general and within the genus crassostrea itself. while this finding provides strong evidence for recent speciation of the six asian crassostrea species, it also indicates that mt genome rearrangement may not be strictly a function of time, but constrained by other factors. detailed mechanisms of mt genome rearrangement are unknown. we suspect that coding on both strands may be one of the factors inhibiting mt genome rearrangement. this hypothesis is supported by the fact that the only two bivalve molluscs with dual-strand coding have relatively fewer rearrangements of gene order compared with other molluscan classes. all marine bivalves use one strand for coding and show tremendous rearrangements. the extensive rearrangement of bivalve mt genomes may be a consequence of coding on one strand. furthermore, the fact that massive rearrangements in marine bivalves have not disrupted single-strand coding or changed the direction of transcription suggests that the rearrangements are not caused by inversion or reverse transposition. thus, transposition or "tandem repeat-random loss" may be important for mt genome rearrangement. dual-strand coding may not be the only factor limiting mt genome rearrangement, as it cannot explain the absence of rearrangements in the six asian oysters over myr. we speculate that some other unknown features in mt genomes of the six asia species may also limit genome rearrangements. the most noticeable structure change is the duplication of rrns in the six asian species, though it is difficult to determine if it has any effects on genome rearrangement at current stage. further studies on the mt genomes of other oysters and marine bivalves may shed light on the origin and evolution of genome rearrangements. gene order data have been shown to be valuable in phylogenetic analysis. while our analysis demonstrates the power of rearrangement data, it also argues for a better understanding of mt genome rearrangement before using them to infer divergence time.  asia pacific has long been suspected to be the center of oyster speciation. our analysis of complete mt genomes provides strong evidence that crassostrea oysters have diversified in asia and the divergence was rather recent or within the last 3- myr. the complete conservation of gene order in the six asian crassostrea species over a period of myr is highly unusual given the remarkable rate of rearrangements in their sister species and other bivalves during the same time frame. it provides strong evidence, in addition to sequence data, for the recent speciation of crassostrea oysters in asia. it also indicates that changes in mt gene order may not be strictly a function of time but subject to other constraints that are presently not well understood. 
they shared the same gene order that differed from an atlantic sister species by as many as nine trna changes and even differed significantly from s. mordax in protein-coding genes. to understand the species diversity and its evolutionary history, we collected five crassostrea species from asia and sequenced their complete mitochondrial genomes in addition to two newly released asian oysters for a comprehensive analysis. the complete conservation of gene order in the six asian crassostrea species over myr is highly unusual given the remarkable rate of rearrangements in their sister species and other bivalves. it provides strong evidence for the recent speciation of the six crassostrea species in asia. phylogenetic analysis indicates that the six asian crassostrea species emerged between and myr ago, while the atlantic species evolved myr ago. it further indicates that changes in mt gene order may not be strictly a function of time but subject to other constraints that are presently not well understood. it is long been suspected, based on the extraordinary species diversity observed, that asia pacific is the epicenter of oyster speciation. their genomes contained a split of the rrnl gene and duplication of trnm, trnk and trnq genes.
 oysters are morphologically plastic and hence difficult subjects for taxonomic and evolutionary studies. it is long been suspected, based on the extraordinary species diversity observed, that asia pacific is the epicenter of oyster speciation. to understand the species diversity and its evolutionary history, we collected five crassostrea species from asia and sequenced their complete mitochondrial genomes in addition to two newly released asian oysters for a comprehensive analysis.  the six asian crassostrea mt genomes ranged from to bp in size, and all coded for genes on the same strand. their genomes contained a split of the rrnl gene and duplication of trnm, trnk and trnq genes. they shared the same gene order that differed from an atlantic sister species by as many as nine trna changes and even differed significantly from s. mordax in protein-coding genes. phylogenetic analysis indicates that the six asian crassostrea species emerged between and myr ago, while the atlantic species evolved myr ago.  the complete conservation of gene order in the six asian crassostrea species over myr is highly unusual given the remarkable rate of rearrangements in their sister species and other bivalves. it provides strong evidence for the recent speciation of the six crassostrea species in asia. it further indicates that changes in mt gene order may not be strictly a function of time but subject to other constraints that are presently not well understood. 