The Complete Maternal and Paternal Mitochondrial Genomes of the Mediterranean Mussel Mytilus galloprovincialis: Implications for the Doubly Uniparental Inheritance Mode of mtDNA

Athanasia Mizi*, Eleftherios Zouros{dagger}, Nicholas Moschonas{dagger} and George C. Rodakis*

* Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimioupolis, Athens, Greece; and {dagger} Department of Biology, University of Crete, Heraklion, Crete, Greece

Correspondence: E-mail: grodakis{at}biol.uoa.gr.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
The maternal (F) and paternal (M) mitochondrial genomes of the mussel Mytilus galloprovincialis have diverged by about 20% in nucleotide sequence but retained identical gene content and gene arrangement and similar nucleotide composition and codon usage bias. Both lack the ATPase8 subunit gene, have two tRNAs for methionine and a longer open-reading frame for cox3 than seen in other mollusks. Between the F and M genomes, tRNAs are most conserved followed by rRNAs and protein-coding genes, even though the degree of divergence varies considerably among the latter. Divergence at nad3 is exceptionally low most likely because this gene includes the origin of transcription of the lagging strand (OL). Noncoding regions are the least conserved with the notable exception of the central domain of the main control region and a segment of another noncoding region immediately following nad3. The amino acid divergence (14%) of the two genomes is smaller than in two other pairs of conspecific genomes that are available in GenBank, that of the clam Venerupis philippinarum (34%) and of the fresh water mussel Inversidens japanensis (50%), suggesting that doubly uniparental inheritance of mtDNA emerged at different times in the three species or that there has been a relatively recent replacement of the male genome by the female in the Mytilus line. The latter hypothesis is supported from phylogenetic and population studies of Mytilidae. That the M genome contains a full complement of genes with no premature termination codons argues against it being a selfish element that rides with the sperm. It is shorter than the F by 118 bp, which apparently cannot account for the postulated replicative advantage of this genome over the F in male gonads. The high similarity of the two genomes explains why the F genome may assume the role of the M genome, but it does not exclude the possibility that for this to happen some M-specific sequences must be transferred on to the F genome by means of recombination. If such sequences exist they would most likely be located in noncoding regions.

Key Words: Mytilus • mitochondrial genome • maternally or paternally inherited mtDNA


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Several bivalve species are known to have two highly differentiated mitochondrial genomes (Skibinski, Gallagher, and Beynon 1994a, 1994b; Zouros et al. 1994a, 1994b; Liu, Mitton, and Wu 1996; Hoeh et al. 1997; Passamonti and Skali 2001; Curole and Kocher 2002; Hoeh, Stewart, and Guttman 2002; Serb and Lydeard 2003) one of which follows the standard maternal inheritance (and is known as type F for female-transmitted) and the other is transmitted through the sperm (and is known as M for male-transmitted). Thus, each genome obeys the rule of uniparental transmission. The phenomenon, known as doubly uniparental inheritance (DUI, Zouros et al. 1994a), has been studied more extensively in the blue mussel genus Mytilus, where it was first noted (Skibinski, Gallagher, and Beynon 1994a, 1994b; Zouros et al. 1994a, 1994b).

Even before the discovery of DUI, it was known from the work of Hoffmann, Boore, and Brown (1992) that the Mytilus mtDNA possesses characteristics not commonly found among metazoan mitochondrial genomes. It lacks the ATPase8 gene, has two tRNAs for methionine, and most notably, its gene arrangement is very different from other known animal mtDNAs. It was subsequently determined that the genome examined by Hoffmann, Boore, and Brown (1992) was maternally transmitted (Skibinski, Gallagher, and Beynon 1994b; Rawson and Hilbish 1995; Stewart et al. 1995). Comparison of partial sequences from both genomes suggested that the M genome evolves faster than the F (Skibinski, Gallagher, and Beynon 1994b; Rawson and Hilbish 1995; Stewart et al. 1995; Quesada, Skibinski, and Skibinski 1996; Hoeh et al. 1997; Quesada, Warren, and Skibinski 1998). Further studies of DUI in mussels suggested that the F genome may occasionally invade the sperm-transmission route and assume the role of the M genome (Hoeh et al. 1997; Saavedra, Reyero, and Zouros 1997; Quesada, Wenne, and Skibinski 1999). Also it has been observed that F and M genomes may recombine in the male gonad, where they are found together (Ladoukakis and Zouros 2001).

For a better understanding of the mechanism and evolution of DUI we need the complete sequences of the two genomes. At present, the complete sequences of two F genomes have been published, those of the blue Mytilus edulis (Hoffmann, Boore, and Brown 1992; Boore, Medina, and Rosenberg 2004) and of the fresh water mussel Lampsilis ornata (Serb and Lydeard 2003). The sequences of the F and M genomes of the venerid clam Venerupis philippinarum and the fresh water mussel Inversidens japanensis are available in GenBank (accession numbers AB065375, AB065374, AB055625, AB055624, respectively; M. Okazaki and R. Ueshima, personal communication). This paper is the first to present together the complete sequence of F and M genomes of a species, namely, the Mediterranean mussel Mytilus galloprovincialis. In particular, the M. galloprovincialis M genome is the first paternally transmitted mitochondrial genome whose complete sequence is presented and discussed.

In addition to presenting these sequences, we use them to obtain answers to the following specific questions. (1) Do the two genomes differ in gene content and gene arrangement? Such differences might be expected from two genomes that have different patterns of transmission and distribution in male and female tissues and whose sequences have diverged, on evidence from partial sequences, by more than 20%. (2) Do different parts of the genome diverge at different rates? (3) Are there common patterns of divergence among F and M pairs from different species with DUI?


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Adult mussels were collected from the port of Heraklion in Crete and from Nea Peramos in Saronicos Gulf, Greece. Mussels were sexed by microscopic examination for presence of eggs or sperm in gonad tissue. For the F genome, total DNA was extracted from the gonad tissue of females. The M genome is present predominantly in the gonads of males, whose somatic cells contain the F genome. In order to minimize the contamination of the preparation of the M genome by the F, we used the male's sperm after induction of spawning.

PROMEGA Taq polymerase was used in all polymerase chain reaction (PCR) reactions. All primer pairs used are available as Supplementary Material online. To obtain the F genome, the mtDNA of a female individual was amplified by long PCR in two fragments. Long PCR amplifications were carried out in 50-µl reaction volumes containing 50–100 ng of template DNA, 0.3 mM of each primer, 0.5 mM deoxynucleoside triphosphate, 3.5 mM MgCl2, and 0.75 µl of enzyme mix (Roche "Expand Long Template PCR System") in buffer 2 supplied by the company. Reaction conditions were in accordance to supplier's recommendations. The first pair of primers, Lola1 and LCOIIIr, amplified a fragment of 6.7 kb while the second, LCOIIIf and Lola2, a fragment of 10 kb. These two large fragments served as templates in PCR reactions to obtain products of 500–1,200 bp. Given that the majority of the F mitochondrial sequence was known for M. edulis (Hoffmann, Boore, and Brown 1992), several sets of primers were designed using this sequence.

The M genome of one male individual was amplified by long PCR in two fragments using the same reaction conditions and two sets of primers. The first, 16M-Sl-f (with a recognition site for SalI) and C3M-Sc-r (with a recognition site for SacI), amplified a fragment of 6.7 kb. The second, C3M-f-X and 16M-r-X (both with sites for XbaI), amplified a fragment of 11 kb. The product of the first reaction was digested with SacI and SalI and three fragments of length 1.2, 2, and 3.5 kb were obtained. The digestion of the product of the second reaction with SacI and XbaI gave four fragments of length 1.1, 1.2, 1.7, and 7 kb. The 7-kb fragment was subsequently used as template in PCR reactions to obtain three smaller fragments of 1.7, 2, and 3.5 kb length. All fragments were cloned into plasmid vector pBluesript II KS (Stratagene) in Escherichia coli DH5a cells. Conjunctive fragments between sequential clones were amplified by PCR reactions.

Sequencing was performed by an ABI 377 automated sequencer with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems), in addition to the standard dideoxynucleotide chain termination method (Sanger, Nicklen, and Coulson 1977) or the use of a commercial outlet (MWG Company, Germany). Primer walking was applied when cloned fragments were greater than ~1.2 kb.

Sequences were characterized after alignment and comparison with data from the National Center for Biotechnology Information databank, using the Blast network service (Altschul et al. 1990), or from cited publications. Alignment of DNA and protein sequences was performed by ClustalX version 1.83 (Thompson et al. 1997) after selection of optimal parameters (opening and extension gap penalties). Corrections were made manually aiming at maximizing sequence similarity. tRNA genes were identified by examining regions known to code for tRNA genes in the genomes of M. edulis (Hoffmann, Boore, and Brown 1992) and of Mytilus californianus (Beagley, Okimoto, and Wolstenholme 1999). Sequences that have the potential to form the characteristic mitochondrial tRNA cloverleaf structure were found only in these regions of the M. galloprovincialis genome. To examine whether nucleotide differences or indels between the F and M genomes accumulate at different rates in different regions of the tRNA secondary structure (e.g., stems versus loops), nucleotide differences at the same region of the tRNA structure were counted over all tRNAs and compared among regions with a chi-square test. The two methionine tRNAs in each of the two mitochondrial genomes of M. galloprovincialis (this study) and of V. philippinarum (GenBank accession numbers AB065375, regions 10602–10669 and 10676–10741, and AB065374, regions 9275–9340 and 9339–9409, respectively; M. Okazaki and R. Ueshima, personal communication) were aligned using the program ClustalX version 1.83 (Thompson et al. 1997) with equal "pairwise" and "multiple" alignment parameter values (gap opening and extension penalties = 3.00), and compared phylogenetically by a neighbor-joining tree constructed using the program MEGA version 2.1 (Kumar et al. 2001) with Jukes-Cantor correction. Potential secondary structures near or at the 5'-end of protein genes have been produced by the RNA "mfold" program 3.1 (Zuker 2003). Sequences of the main control region (CR) were aligned and divided in domains according to Cao et al. (2004).

Amino acid sequences for protein-coding genes were obtained using the genetic code of Drosophila mtDNA (Hoffmann, Boore, and Brown 1992), and nucleotide alignments were subsequently adjusted to correspond to amino acid alignments. Zerofold-, twofold-, and fourfold-degenerated positions were identified using software DnaSP version 3.53 (J. Rozas and R. Rozas 1999). This program was also used to estimate codon usage and to construct sliding window plots. Estimation of genetic distances (K) was based on Kimura's two-parameter model (1980) using the software MEGA version 2.1 (Kumar et al. 2001). The divergence of protein genes in synonymous (Ks) and nonsynonymous (Ka) sites was calculated by the modified Nei-Gojobori method with Jukes-Cantor correction, and the p distance at the amino acid level was calculated using the computer program MEGA version 2.1 (Kumar et al. 2001). The four-cluster analysis method under minimum evolution (Rzhetsky, Kumar, and Nei 1995) was used to examine whether the F and M mitochondrial genomes from different species cluster according to their mode of transmission or according to species of origin. For each tetrad of compared sequences, the analysis produces a "complement probability" (CP) which denotes how much more probable the produced topology is over any other alternative topology. CP values larger than 0.95 indicate that the topology is significantly better than any other topology. For this analysis the amino acid sequences of any given protein gene from the maternal and the paternal mitochondrial genomes of three species that are known to have DUI (M. galloprovincialis, this study; V. philippinarum and I. japanensis, GenBank accession numbers AB065375, AB065374, AB055625, AB055624, respectively, M. Okazaki and R. Ueshima, personal communication) were aligned by the program ClustalX version 1.83 (Thompson et al. 1997) using the default parameters, and all alternative topologies were tested using the program PHYLTEST version 2.0 (Kumar 1996). This was done separately for each protein gene and for the concatenated alignments of all protein genes.

Sequence data from this article have been deposited to GenBank under the accession numbers AY497292, AY363687, AY496974, and AY496975.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Genome Size, Nucleotide Composition, and Gene Number and Order
The F genome of M. galloprovincialis was found to be virtually identical to the F genome of M. edulis, as presented in the incomplete form by Hoffmann, Boore, and Brown (1992) and in the complete form by Boore, Medina, and Rosenberg (2004). Mytilus edulis and M. galloprovincialis are members of the M. edulis species complex (which also includes Mytilus trossulus) they hybridize in the areas of sympatry, and their taxonomic status as distinct species is debatable (Gossling 1992). The sequence of the F genome of M. galloprovincialis we report here is 16,744 bp, only four nucleotides longer from the M. edulis F genome assembled by Boore, Medina, and Rosenberg (2004). Its gene order (fig. 1) is also identical to that of the M. edulis F genome and the overall nucleotide divergence, K, is 0.009 (standard error, SE = 0.001), which is within the range expected for conspecific mtDNA genomes. For comparison, the overall mean divergence of four human mitochondrial genomes (Cambridge Reference Sequence [NC_001807], a Swedish [X93334], an African [D38112], and a Japanese [AB055387]) is 0.005 (SE = 0.000) and the mean divergence for the 26 complete genomes of Drosophila simulans (Ballard 2004) is 0.016 (SE = 0.001).



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FIG. 1.— Gene maps of the mitochondrial genomes of Mytilus galloprovincialis. All genes are transcribed clockwise. The one-letter amino acid code is used for tRNA designation. L1, L2, M1, M2, S1, and S2 designate tRNAs recognizing codons CUN, UUR, AUA, AUG, AGN, and UCN, respectively. Black areas indicate noncoding regions; CR, putative CR; UR1–UR6, unassigned regions 1 to 6. Noncoding sequences of less than 10 bp are not shown. atp6, ATP synthase subunit 6; cox13, cytochrome c oxidase subunits I, II, and III; cob, cytochrome b apoenzyme; nad16 and nad4L, nicotinamide adenine dinucleotide dehydrogenase subunits 1–6 and 4L; rrnaS and rrnaL, small and large subunits of ribosomal RNA. The dotted line shows the position of a 1,045-bp insertion that was found in the sequenced M genome. N{Delta}, corresponds to the first 11 bp of the normal trnN; {Delta}CR, corresponds to bases 77–902 of the CR; Y, is a full copy of the trnY; cob{Delta}, corresponds to the first 88 bp of cob; {Delta}G, corresponds to the last 56 bp of trnG. Numbers inside the circle indicate the size of the genome.

 
The M genome of M. galloprovincialis has the same gene order as the F genome including noncoding regions (fig. 1). The most prominent difference of the M genome from the F is the presence of a 1,045-bp insertion, which consists of the first 11 bp of trnN and the last 56 bp of trnG separated by a continuous length of 978 bp that contains part of the main CR, a full copy of the trnY and the first 88 bp of cob. Examination of 19 M genomes, each extracted from a different individual, failed to identify the presence of this segment. In all these genomes the PCR product obtained after using primers 12S-f-X and 16M-r-X (see Supplementary Material online), was approximately 1 kb shorter than the product from the sequenced genome. Two of the short products were reamplified using the internal primers SMALL-f-X and LARGE-r-X and found to be ~800 bp, instead of 1,853 bp which was the size of the product from the sequenced genome. Part of the two 800 bp products was sequenced and found to match the corresponding part of the sequenced genome after the excision of the 1,045-bp segment (GenBank accession numbers AY496974 and AY496975). This suggests that this segment represents an insertion that is present in the specific genome we have sequenced and is not typical of the M. galloprovincialis M genome. A detailed model of how this insertion was produced will be published elsewhere. The net size of the typical M genome is 16,626 bp, shorter by 118 bp from the F genome. The difference is accounted mostly by the main CR and specifically its first and third domain (Cao et al. 2004).

The gene arrangement of both M. galloprovincialis genomes and the M. edulis F genome are identical to each other, but is remarkably different from that known in other fully sequenced metazoan mtDNAs (Hoffmann, Boore, and Brown 1992; Boore 1999). No overlapping genes are observed. Both genomes of M. galloprovincialis contain the full complement of genes of the metazoan mtDNA and an extra tRNA for methionine, but lack the ATPase8 subunit gene. There are seven noncoding sequences larger than 10 bp. Of these the largest (shown as CR in fig. 1) is apparently the main CR for replication and transcription (Cao et al. 2004).

As noted by Hoffmann, Boore, and Brown (1992), all genes of the mussel mtDNA are coded by the same strand. Coding of all genes by the same DNA strand is a feature of all marine bivalves whose full mtDNA sequence is known (Crassostrea gigas, NC_001276, S.-H. Kim, E.-Y. Je, and D.-W. Park, personal communication; V. philippinarum, AB065374 and AB065375, M. Okazaki and R. Ueshima, personal communication) but not in the two freshwater species L. ornata (Serb and Lydeard 2003) and I. japanensis (AB055624, AB055625, M. Okazaki and R. Ueshima, personal communication). In other mollusks a relatively small number of mitochondrial genes are transcribed from the second strand. The scaphopods Graptacme eborea and Siphonodentalium lobatum are an exception, with about an equal number of genes encoded by each strand (Boore, Medina, and Rosenberg 2004; Dreyer and Steiner 2004). The occurrence of all genes in the same strand is a relatively rare phenomenon in metazoans and, in addition to bivalves, has been reported in some annelids (Lumbricus terrestris, Boore and Brown 1995; Platynereis dumerilii, Boore and Brown 2000) and brachiopods (Terebratulina retusa, Stechmann and Schlegel 1999; Terebratalia transversa, Helfenbein, Brown, and Boore 2001; Laqueus rubellus, Noguchi et al. 2000).

The nucleotide compositions of the two genomes are summarized in table 1. The G + T content of the F and M coding strand is 58.1% and 56.8%, respectively, and thus the sense strand can be considered as the heavy (H) strand of the molecule. The A + T content of the H strand is also relatively high (61.8%, F; 63.0%, M). Similar values have been reported in L. ornata (62%, Serb and Lydeard 2003), Pupa strigosa (61.1%, Kurabayashi and Ueshima 2000), and Cepaea nemoralis (59.8%, Yamazaki et al. 1997), but in other mollusks the A + T content is much higher (Albinaria coerulea, 70.7%, Hatzoglou, Rodakis, and Lecanidou 1995; Katharina tunicata, 69.0%, Boore and Brown 1994; G. eborea, 74.1%, Boore, Medina, and Rosenberg 2004). This variation in A + T content is among the highest observed within a phylum and reflects the high heterogeneity of molluscan mtDNA (Boore 1999).


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Table 1 Length, Base Composition, and Sequence Divergence of F and M Genes and Noncoding Regions

 
There is a marked bias in favor of T against C, which is not restricted to any particular class of genes and does not differ between the two genomes. The GC and AT asymmetry between the two mitochondrial DNA strands can be expressed in terms of GC skew and AT skew calculated according to Perna and Kocher (1995): GC skew = (G – C)/(G + C) and AT skew = (A – T)/(A + T), where G, C, A, and T are the occurrences of the four bases in the H strand. In M. galloprovincialis F and M mitochondrial genomes, the GC skew and the AT skew are F: +0.24 and –0.17, and M: +0.28 and –0.16, respectively. Similar calculations for fourfold synonymous sites produce a remarkably high value for GC skew (+0.45, F; +0.42, M), indicating strong bias against codons ending in C. It is common for animal mitochondrial genomes to deviate from random usage of nucleotides. The deviation has been attributed to several factors: unequal presentation of specific nucleotides in the nucleotide pool; preference of mitochondrial gamma DNA polymerase for specific nucleotides; a higher incidence of mutation due to longer exposure of the leading strand during replication (Sueoka 1962; Asakawa et al. 1991; Jermiin and Crozier 1994; Jermiin et al. 1994, 1996). L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis, and E. Zouros (unpublished data) have obtained evidence for the involvement of the last factor.

The amount of divergence varies along the genome (fig. 2). For most of the length of the genome the divergence remains similar to the average of 0.20 (table 1). There are three regions of high and two regions of low divergence. The first region of high divergence maps at the major CR and specifically its first domain (variable domain 1, VD1). It is followed by a region of low divergence, which maps in the second domain (conserved domain, CD) of the major CR. The high divergence of VD1 and the conservation of CD have been discussed by Cao et al. (2004). The second region of high divergence corresponds to another noncoding region (UR1, fig. 1). The second conserved region contains the nad3 and the adjacent segment of UR4 and most likely corresponds to OL, the origin of the transcription of the lagging strand (L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis, and E. Zouros, unpublished data). This conserved region is followed immediately by the third highly divergent point, which corresponds to the remaining part of UR4.



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FIG. 2.— Degree of divergence between F and M genomes. The genome was linearized at the first nucleotide position of CR (fig. 1) and scanned with a sliding window of 150 bp moved in steps of 50 bp. The two highly conserved regions (horizontal arrows) correspond to: (A) central domain (CD) of the putative CR; and (B) nad3 plus the adjacent 100 bp of UR4. The three highly diverged regions (downward arrows) correspond to: (C) first variable domain (VD1) of CR; (D) UR2; and (E) the remaining part of UR4.

 
Ribosomal RNA Genes
The definition of boundaries of rrnaS was based on the assumption that it occupies all the space between trnF and trnG. For rrnaL we assumed that the first base at the 5'-end comes immediately after trnD, but the 3'-end of the gene cannot be decided objectively because it is followed by a noncoding sequence (CR). For consistency, we adopted the endpoint suggested by Hoffmann, Boore, and Brown (1992). The location, the length, and the nucleotide content of the two ribosomal RNA genes do not differ in the two M. galloprovincialis genomes. The divergence between the genomes is the same for the two rRNA genes and smaller than the average for the genome (fig. 1 and table 1).

Transfer RNA Genes
The identification of tRNA genes was based on their potential to form cloverleaf structures (fig. 3). All tRNA genes exist in the F and M genomes of M. galloprovincialis. Beagley, Okimoto, and Wolstenholme (1999) have shown that the M. californianus F genome possesses a trnS2 gene which does not contain a dihydrouridine arm (DHU arm) and is located between cox3 and trnM1. Absence of a DHU arm is not unusual in mitochondrial serine tRNAs, whether they recognize the UCN or the AGN codon (Tomita et al. 2002; Serb and Lydeard 2003). We have found that the trnS2 gene of both the F and M genomes of M. galloprovincialis is also located between cox3 and trnM1 (fig. 1). Hoffmann, Boore, and Brown (1992) suggested that trnS2 was located between nad3 and cox1 in the M. edulis F genome, but Beagley, Okimoto, and Wolstenholme (1999) presented direct evidence that the transcript of the region containing this sequence remains linked to the transcript of cox1 and is in fact a pseudogene. The sequence proposed by Hoffmann, Boore, and Brown (1992) as trnS2 was also found in the UR4 of the F genome of M. galloprovincialis (nucleotide positions 8880–8944) and can be folded into a typical tRNA secondary structure, but with a CGA rather than a TGA anticodon, which is the anticodon in all molluscan mtDNAs. In the M genome of M. galloprovincialis, this sequence (region UR4, nucleotide positions from 8614 to 8678) cannot form a tRNA-like structure with a serine anticodon. Further evidence that this sequence is not a functional tRNA comes from the sequence's divergence between the two genomes. The Kimura distance is 0.455 (SE = 0.116) compared to 0.088 (SE = 0.041) for trnS2 that is located between cox3 and trnM1. The 0.455 value is outside the range for homologous tRNAs, which varies from 0.067 to 0.169.



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FIG. 3.— Putative cloverleaf structures for the 23 tRNA genes of the F Mytilus galloprovincialis genome. Circled nucleotides indicate substitutions in the M genome. Arrows indicate nucleotides that are present in the M genome but missing from the F. Watson-Crick pairing is shown by solid lines and G-T pairs by dots.

 
Both F and M genomes contain 23 tRNA genes, one more than is typical for the metazoan mtDNA. The additional tRNA has the anticodon TAT, thus the extra tRNA codes for methionine. Nine out of the 12 protein genes of the Mytilus genome start with the ATG codon and two with the ATA (table 2). From this and also from the fact that in all known metazoan mtDNAs the most common initiation codon is ATG, we may conclude that the methionine tRNA with the CAT anticodon represents the ancestral form and that, as suggested by Hoffmann, Boore, and Brown (1992), the second methionine tRNA has arisen by duplication. The F and M genomes of the venerid V. philippinarum also have two tRNA genes for methionine, but both have the "ancestral" CAT anticodon. The F genome of L. ornata, both F and M genomes of I. japanensis, and the genome of the oyster Crassostrea gigas, a species that apparently does not have DUI (Curole and Kocher 2002), contain only one tRNA for methionine, again with the CAT anticodon. Therefore, presence of two methionine tRNA genes is not a feature of bivalves in general or of bivalve species with DUI in particular.


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Table 2 Number of Amino Acids and Initiation and Termination Codons in Completely Sequenced Molluscan mtDNAs

 
The Mytilidae and Ostreidae belong to subclass Pteriomorphia, while the Veneridae belong to subclass Heteroconchia (Carter, Campbell, and Campbell 2000). This and the fact that the four methionine tRNAs of M. galloprovincialis and the four tRNAs of V. philippinarum form two distinct clusters in phylogenetic trees (fig. 4) make the hypothesis of two independent duplication events more likely than the alternative hypothesis before the separation of Mytilidae and Veneridae. The presence of the duplication in both F and M genomes of mytilids M. edulisM. galloprovincialis and of the venerid V. philippinarum may suggest that the duplication predates the split of the two genomes and thus the emergence of DUI in these species. But this explanation would be inconsistent with the absence of the duplication from the two unionid species, L. ornata and I. japanensis which have DUI, unless we assumed that DUI emerged independently in each of the three families (Mytilidae, Unionidae, and Veneridae) in which it is known to occur. A more likely explanation is that separate duplication events occurred in an F genome of the M. edulis/galloprovincialis line and in an F genome of the V. philippinarum line and that these F genomes replaced their respective M genomes through the phenomenon of "mtDNA masculinization" or "sex reversal." One feature of DUI in mytilids is that occasionally an F genome may ride with the sperm and become maternally inherited from that point onward (Zouros et al. 1994b; Hoeh et al. 1996, 1997; Saavedra, Reyero, and Zouros 1997; Quesada, Wenne, and Skibinski 1999). Such "masculinized" F genomes are found in high frequencies in several populations together with the standard M genome (Ladoukakis et al. 2002) and, on evidence from phylogenetic trees, have replaced the original M lineage and reset the F/M divergence to zero in several taxa (Hoeh et al. 1997; Quesada, Wenne, and Skibinski 1999). To explain the presence of the TAT anticodon in one of the duplicates in the M. edulisM. galloprovincialis genomes, one would need to make the further assumption that a C-to-T mutation had occurred in the anticodon of one of the duplicate genes of the F genome before its masculinization. This hypothesis is fully consistent with the nucleotide divergences of the four methionine tRNA genes (fig. 4) which join the genes according to their anticodon rather than according to the genome in which they are found.



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FIG. 4.— Neighbor joining tree of the methionine tRNAs of Mytilus galloprovincialis and Venerupis philippinarum mitochondrial genomes. F and M denote gender-specific genomes. Numbers 1 and 2 denote orthology. trnM1 of M. galloprovincialis are located between trnS2 and nad2 and have the TAT anticodon, whereas trnM2 are located between genes trnK and trnL1 and have the CAT anticodon. trnM(1) and trnM(2) of V. philippinarum are found in tandem between trnY and trnD in both genomes and both have the CAT anticodon. Numbers indicate percentage of bootstrap support from 1,000 replicates.

 
The tRNAs are the most conserved genes between the two M. galloprovincialis mitochondrial genomes (table 1). Substitutions are unequally distributed among the various structural parts of the tRNA. Even though substitutions are more common in loops than in stems, there are large differences among the four types of stems (P = 0.001, from the chi-square test) and among the three types of loops (P = 6.5 x 10–11, from the chi-square test). Excepting the two bases before DHU which are fully conserved, we may recognize four classes according to the degree of conservation (table 3). One class consists of the DHU stem, the anticodon stem, and the anticodon loop all of which are equally and highly conserved (P = 0.129, from the chi-square test) with an average of 3% substitution rate. The amino acid acceptor stem, the extra arm, the T{Psi}C stem, and the discriminator base form the second class with a mean substitution rate of 10% (P = 0.879, from the chi-square test). The DHU loop at 23% and the T{Psi}C loop at 40% form the two most variable classes. In total, only 8.2% of nucleotide differences between the two genomes are in sites that affect base pairing; 59.1% of the substitutions are located in loops. Another 30.4% are located in stem positions but have not affected base pairing. The remaining 2.3% of substitutions are located in positions where no pairing is observed. The overall small degree of divergence of tRNAs can therefore be attributed to the small number of sites that do not affect tRNA function. Yet, even nucleotides that are located in loops cannot be considered as fully neutral because several of them participate in the formation of the tertiary structure of the molecule (Dirheimaer et al. 1995).


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Table 3 Number of Nucleotide Substitutions (percent in parenthesis) in Different Regions of the tRNA Secondary Structure Summed Over All tRNA Genes

 
Secondary Structures at Junctions Between Protein-Coding Genes
Ojala, Montoya, and Attardi (1981) suggested that the secondary structure of a tRNA gene between a pair of protein genes is responsible for the precise cleavage of the polycistronic primary transcript. In the absence of an intervening tRNA, this role can be played by a stem-loop structure, the 5'-end part of the gene itself, or a combination of the two. Potential hairpin structures at protein-protein gene junctions with no intervening tRNA have been reported in several studies (e.g., Bibb et al. 1981; Clary and Wolstenholme 1985; Okimoto et al. 1992; Boore and Brown 1994). Nineteen of the 23 tRNA genes of the M. galloprovincialis genome are clustered in four groups of two to seven members (fig. 1). Of the remaining four, one (trnY) lies immediately before the 5'-end of cob and the other three (trnV, trnT, trnF) occupy positions between protein-coding genes. Thus, 5 of the 12 protein-coding genes (cob, nad4, nad2, nad3, and nad4L) have a tRNA preceding their 5'-end. Four other genes (cox2, nad1, cox1, and nad5) have a noncoding sequence preceding their 5'-end that is capable of forming a stem and loop structure (fig. 5). In cox1 this structure corresponds to the pseudo-trnS2, which Beagley, Okimoto, and Wolstenholme (1999) have shown is included in the cox1 mRNA (discussed above). This leaves only three protein-coding genes (cox3, atp6, and nad6) without a tRNA or a noncoding sequence at the 5' end. In all these three genes, a putative stem-and-loop structure that includes the translation initiation codon can be formed downstream from the 5'-end part of the gene (fig. 5).



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FIG. 5.— Putative secondary structures preceding or included in the 5'-end of protein-coding genes that are not preceded by a tRNA. UR1, UR2, and UR4 precede cox2, nad1, and cox1, respectively. For cox3, atp6, nad5, and nad6 the putative structure includes the initiation codon (enclosed). Numbers indicate nucleotide positions or length in bp between two structures. Solid lines indicate Watson-Crick pairing and dots G-T pairs.

 
Protein Genes
The ATPase8 subunit gene, that is normally present in the metazoan mitochondrial genome, is missing from the mussel mtDNA, thus reducing the protein genes to 12. Atp8 is also missing from the mtDNAs of other bivalves (Crassostrea, Venerupis, and Inversidens), nematodes (Okimoto et al. 1992), platyhelminths (Le et al. 2000), and chaetognaths (unpublished data, cited in Boore, Medina, and Rosenberg 2004) but, interestingly, is present in the mitochondrial F genome of the unionid bivalve L. ornata (Serb and Lydeard 2003).

The protein-encoding genes are of the same length in the two genomes, except for cox1, cob, and nad5 in which the length difference is 4, 5, and 6 amino acids, respectively (table 2). An ambiguity arises concerning the termination point of cox3. The size of this gene is relatively conserved in metazoans (normally below 300 codons; see examples in table 2), but in both M. galloprovincialis genomes this gene has a length of 311 codons. Hoffmann, Boore, and Brown (1992) were the first to note the increased number of codons in the F genome of M. edulis and suggested that the polypeptide may consist of 311 amino acids or that transcription is terminated before the end of the open-reading frame. Based on the sequence similarity to other known cox3 genes, Boore, Medina, and Rosenberg (2004) suggested that the gene in M. edulis may have an incomplete transcription termination codon resulting in a transcript of 264 codons. This hypothesis is supported by the fact that the additional 47 amino acid sequence bears no resemblance to any known cox3 genes. Alternatively, one may use the conventional rule that preference for the end of the coding part of a gene must be given to a complete termination codon, as long as it appears before the first nucleotide of the next gene. According to this convention, the gene must be assumed to consist of 311 amino acids. Our comparison of the two genomes is based on the assumption that the extra length of 47 codons belongs to the protein-coding part of the genome.

Synonymous (Ks) and non-synonymous (Ka) values between the two genomes vary among protein genes (table 1). Ka is particularly low for cox1, but Ks is not, suggesting that this gene is under selective constraint. The conservation of cox1 is common in animal mtDNA (Pesole et al. 1999; Saccone et al. 1999). Ks and Ka deviate from average in nad6, but in opposite ways. In nad3 both K values are lower than average. Similar comparisons can be made for the V. philippinarum and I. japanensis genomes. In both pairs the cox1 has the lowest divergence and the nad6 divergence is among the largest, but in neither pair is the nad3 divergence particularly low (fig. 6). The low rate of evolution of nad3 relative to other protein genes seems to be a characteristic property of the M. edulis species complex. A broad survey of sequenced animal mitochondrial genomes (table 4) shows that a rate of evolution at the nad3 gene as low as in Mytilidae is found in Petromyzoniformes, but this low rate is shared by all protein-coding genes of Petromyzoniformes (see "ratio(b)/(a)" in table 4). The explanation for the conservation of the nad3 gene in the M. edulis species complex may lie in the fact that nad3 contains the origin of the replication of the lagging stand (L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis, and E. Zouros, unpublished data).



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FIG. 6.— Amino acid divergence (p distance) of three pairs of conspecific genomes for the 12 protein genes. Black bars: Mytilus galloprovincialis; gray bars: Venerupis philippinarum; blank bars: Inversidens japanensis.

 

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Table 4 Average Intrataxon Diversity (p distances) of Mitochondrial Protein Genes of Completely Sequenced Metazoan mtDNAs

 
Codon Usage
All codons occur in both M. galloprovincialis mitochondrial genomes (table 5). TTT (phenylalanine) is the most frequent codon followed by TTA (leucine). TTT is also the most frequent codon in L. ornata (Serb and Lydeard 2003) and in C. nemoralis (Terrett, Miles, and Thomas 1996), whereas TTA is most common in A. coerulea (Hatzoglou, Rodakis, and Lecanidou 1995), P. strigosa (Kurabayashi and Ueshima 2000), Roboastra europaea (Grande et al. 2002), G. eborea (Boore, Medina, and Rosenberg 2004), and K. tunicata (Boore and Brown 1994). These two codons are the most frequently used codons in other invertebrate mtDNAs (Garesse 1988; Cantatore et al. 1989; Okimoto et al. 1992; Asakawa et al. 1995; De Giorgi et al. 1996; Helfenbein, Brown, and Boore 2001). TTT is also very frequent in primitive chordates (like amphioxus, Branchiostoma lanceolatum, Spruyt et al. 1998), but not in most of vertebrates, where CTA (e.g., Cyprinus, Chang, Huang, and Lo 1994; Homo sapiens, Ingman et al. 2000) or ATT (e.g., Xenopus laevis, Roe et al. 1985; Danio rerio, Broughton, Milam, and Roe 2001) are the most frequently used codons. The four least used codons in M. galloprovincialis mtDNA are TCC, CGC, ACC, and ACG. Of these, CGC is also among the least common in the mtDNA of other mollusks.


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Table 5 Codon Usage in F and M Genomes

 
Synonymous codons, whether fourfold (4FD) or twofold (2FD) degenerate, are recognized by the same tRNA (table 5), with the exception of the methionine codons which are recognized by a different tRNA (see above). In nine 2FD and seven 4FD families the most frequently used codon does not match the tRNA's anticodon. This has been observed in other metazoan mtDNA as well (Roe et al. 1985; Wolstenholme 1992, Rand and Kann 1998; Crease 1999; Broughton, Milam, and Roe 2001) and suggests that strict codon-anticodon complementarity has not affected the codon composition of the genome.

Deviations from equal frequency of the four nucleotides in 4FD sites are common in the animal mtDNA and have been attributed to several factors, such as unequal presence of the four nucleotides in the nucleotide pool, preference of the mitochondrial gamma DNA polymerase for specific nucleotides, or asymmetrical mutation rate owing to different duration of exposure of the lagging strand during replication (Sueoka 1962; Asakawa et al. 1991; Jermiin and Crozier 1994; Jermiin et al. 1994, 1996). L. Cao, E. Kenchington, A. Mizi, G. C. Rodakis, and E. Zouros (unpublished data) have noted that 4FD sites with longer exposure have a higher probability to be occupied by T and a lower probability to be occupied by G. This observation implies that the frequency with which the four codons of an amino acid are used depends on the distribution of the amino acid's residues along the genome. Yet, these correlations account for less than a third of the bias in codon usage and cannot explain the bias in 2FD codon families. Thus, other factors must also affect codon bias. One among these may be the degree with which tRNAs recognize their different synonymous codons. If this degree varies among codons, the tRNA itself may act as a selection factor for synonymous mutations and may thus determine the frequency of synonymous codons in the genome.

Implications for DUI of mtDNA
The two mitochondrial genomes of M. galloprovincialis are remarkably similar in gene content, gene arrangement, nucleotide composition, and codon usage in spite the fact that their primary DNA sequence has diverged by 20% and, more importantly, that their mode of transmission and distribution among female and male tissues is dramatically different. This is not true for the F and M genomes of two other species (V. philippinarum and I. japanensis) whose complete sequences are available in GenBank (accession numbers, AB065375, AB065374, AB055625, AB055624, respectively; M. Okazaki and R. Ueshima, personal communication). Figure 6 illustrates the amino acid divergence at all 12 protein genes between the F and M genomes of M. galloprovincialis, V. philippinarum, and I. japanensis. In all 12 comparisons the divergence is smallest in M. galloprovincialis and largest in I. japanensis, with the exception of cox2 and nad4L, where the divergence in V. philippinarum is higher than in I. japanensis. In order to examine the phylogenetic status of the F and M mitochondrial genomes among the three species, we applied the four-cluster analysis to all combinations of four sequences drawn from two different species. Given the large phylogenetic divergence among the three families, only amino acid sequences were used for this analysis. When genes were used individually, the sequences paired according to their species rather than according to their mode of transmission for all 12 genes. Figure 7 shows the result of the four-cluster analysis based on the concatenated alignment of all protein genes. The CP for any combination is one, suggesting that the topology joining the sequences according to species is much better than any alternative topology.



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FIG. 7.— A treelike presentation of the results of four-cluster analysis based on the concatenated amino acid sequence of all 12 protein-coding genes of the F and M genomes of Mytilus galloprovincialis, Venerupis philippinarum, and Inversidens japanensis. CP: complement probability. The p distance for the concatenated amino acid sequences between the F and M genomes of M. galloprovincialis, V. philippinarum and I. japanensis is 0.139 (SE = 0.006), 0.343 (SE = 0.008) and 0.506 (SE = 0.008), respectively. Branch length is proportional to p distance.

 
One explanation for the result of figure 7 is that DUI emerged independently in the lines leading to the three species. The question of multiple origins of DUI was first addressed by Hoeh et al. (1996, 1997) on the basis of partial sequences of F and M genomes from different species of Mytilidae and Unionidae families. These authors rejected the hypothesis of multiple origins of DUI in favor of the hypothesis of "masculinization" or "role reversal" of the F genome and subsequent replacement of the "old" M with the newly masculinized F. This phenomenon has been observed in pair-matings of M. galloprovincialis (Saavedra, Reyero, and Zouros 1997) and in natural populations of M. trossulus (Stewart et al. 1995) and M. galloprovincialis (Ladoukakis et al. 2002) where males were found with two types of F genomes, one in the soma and the other in the gonad. Role reversal also provides the most likely explanation for the F and M genomes of M. trossulus in the Baltic Sea (Quesada, Wenne, and Skibinski 1999). This explanation suggests that the history of species with DUI is punctuated with waves of invasion and replacement of the M line by the F. It remains a matter of speculation whether the replacement is stochastic or driven by selection. If we assumed a relatively constant rate, the whole-genome data comparison we present here indicates that the last wave in M. edulisM. galloprovincialis occurred much more recently than in V. philippinarum or I. japanensis.

Mitochondrial gene arrangement in mollusks is known to be very different from typical metazoan mtDNAs and to vary extensively among molluscan species (Boore 1999). Serb and Lydeard (2003) have discussed this feature in bivalves from the point of its phylogenetic utility. Concentrating on the three F/M pairs, we see again a marked difference between M. galloprovincialis, where the gene arrangement is identical, and I. japanensis, where there must have been at least two gene-order inversions in the light strand and one in the heavy strand. Also two tRNAs (trnD and trnV) are coded by opposite stands. Interestingly, the gene order of the M genome of I. japanensis is more similar to the gene order of the L. ornata F genome published by Serb and Lydeard (2003) than to its conspecific F genome, an observation that raises the question of whether gene arrangement can be a reliable taxonomic character for species with DUI. In V. philippinarum the differences are confined to a gene duplication of cox2 in the F genome and an extra trnM in the M genome.

The large divergence between the F and M genomes of I. japanensis is typical of all unionid species that have been examined so far. Hoeh, Stewart, and Guttman (2002) analyzed that cox1 sequences form a collection of unionid species from seven different genera. All species appeared to possess the DUI mode of inheritance and, remarkably, F and M types formed two distinct clusters, suggesting that no masculinization event has occurred in unionids for the last 200 Myr, the estimated time of divergence between the most distantly related species in the collection. Curole and Kocher (2002) observed that in the unionids Lampsilis teres, Quadrula quadrula, and Quadrula refulgens, cox2 is longer in the M genome than in the F and speculated that this difference may render the F genome unsuitable to function as M. A similar argument can be based on differences in gene arrangement between F and M genomes if the large differences observed in I. japanensis, the only unionid species whose complete sequences of both genomes are available, turned out to be typical for unionids.

The small number of mitochondria carried by the sperm and the preponderance of the M genome in the male gonad of Mytilidae forced early studies of DUI to suggest that the M genome enjoys a replication advantage over the F during male gametogenesis (Skibinski, Gallagher, and Beynon 1994b; Zouros et al. 1994b; Saavedra, Reyero, and Zouros 1997). It is known that replication is faster in mitochondrial genomes with large deletions (e.g., Diaz et al. 2002 and references therein). As noted, the M genome of M. galloprovincialis is smaller than the F by 118 bp. Assuming a linear relation between molecule length and rate of replication, it would mean that the relative replicative advantage of M will be about 1%, hardly sufficient to explain this genome's domination in the male gonad. A more likely hypothesis is that male primordial cells start with a mitochondrial population in which egg and sperm mitochondria are not in the same proportion to the frequencies in the fertilized egg (Cao, Kenchington, and Zouros 2004) or that egg mitochondria are actively eliminated from the developing gonad.

The M genome occurs almost exclusively in the male gonad and sperm where the F genome is absent or occurs in hardly detectable amounts (Garrido-Ramos et al. 1998). This in combination with this genome's faster rate of evolution prompted the speculation that the M genome may simply be a selfish element whose only function is to secure a ride with the sperm (Hurst and Hoekstra 1994). An opposite suggestion has also been made, i.e., that far from being nonfunctional the M genome may evolve under pressure to meet the specific needs of sperm (Skibinski, Gallagher, and Beynon 1994b). Our results do not address the second hypothesis but argue against the first. That no premature termination codons occur in any protein gene of the M genome is unexpected from a molecule devoid of normal function for 5 Myr, which is the time of split of the M from the F genome in the M. edulis species group suggested by Rawson and Hilbish (1995) on the basis of rrnaS divergence. An even stronger argument can be made from the F and M genomes of V. philippinarum and I. japanensis whose F/M split must be much older (fig. 7) and where also no premature stop codons occur.

The remarkable similarity between the two genomes of M. galloprovincialis may explain the relatively high frequency of masculinization in the M. edulis species group, but also makes more difficult to answer the question of whether F and M genomes contain sequences that are responsible for their different mode of transmission. The most notable difference between the two M. galloprovincialis genomes is in the first domain of the large unassigned region, which most likely corresponds to the major CR of the genome (Cao et al. 2004). This domain, which is referred to as the first variable region (VR1), is about 150 bp shorter in the M genome (25% of its length), and its divergence between the two genomes is almost 50% (Cao et al. 2004). Similar differences have been found between F and M genomes of M. trossulus from the Baltic Sea (Burzynski et al. 2003). As suggested by Burzynski et al. (2003) and Cao et al. (2004) the CR, and more specifically its VR1 domain, is the most probable region of the mussel mitochondrial genome to house "transmission-specific" sequences, i.e., sequences that may be part of the mechanism that determines whether the genome will be transmitted through the egg or the sperm. If M- and F-specific indels and nucleotide sequence differences in the CR do actually have a transmission-specific role, then an F genome cannot function as M unless it acquires M-specific sequences at this part of the molecule. This acquirement might be possible through recombination. Ladoukakis and Zouros (2001) and Burzynski et al. (2003) have shown that recombination does indeed occur in the Mytilus mtDNA. The question can be settled only if several typical F, typical M, and masculinized F genomes are sequenced. The possible involvement of recombination in masculinization suggests still another explanation why masculinization is presently rare or absent in unionids. Given the large differences in gene arrangement between conspecific F and M genomes, recombination in these species will likely generate nonfunctional molecules and, thus, will prevent the transfer of critical "masculinizing" sequence domains from the M genome into the F.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
Table of oligonucleotides used in PCR reactions and for sequencing of F- and M-type genomes.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 
This work was supported by the Greek General Secretariat for Research and Technology (grant PENED-01ED42 to A. M., E. Z. and G.C.R.) and by the University of Athens (to G.C.R.). We are grateful to the two anonymous reviewers and particularly to one of them who made extensive editorial suggestions on the first draft of the paper.


    Footnotes
 
Richard Thomas, Associate Editor


    References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Supplementary Material
 Acknowledgements
 References
 

    Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.[CrossRef][ISI][Medline]

    Asakawa, S., H. Himeno, K. Miura, and K. Watanabe. 1995. Nucleotide sequence and gene organization of the starfish Asterina pectinifera mitochondrial genome. Genetics 140:1047–1060.[Abstract/Free Full Text]

    Asakawa, S., Y. Kumazawa, T. Araki, H. Himeno, K. Miura, and K. Watanabe. 1991. Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes. J. Mol. Evol. 32:511–520.[ISI][Medline]

    Ballard, J. W. 2004. Sequential evolution of a symbiont inferred from the host: Wolbachia and Drosophila simulans. Mol. Biol. Evol. 21:428–442.[Abstract/Free Full Text]

    Beagley, C. T., R. Okimoto, and D. R. Wolstenholme. 1999. Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene. Genetics 152:641–652.[Abstract/Free Full Text]

    Bibb, M. J., R. A. Van Etten, C. T. Wright, M. W. Walberg, and D. A. Clayton. 1981. Sequence and gene organization of mouse mitochondrial DNA. Cell 26:167–180.[CrossRef][ISI][Medline]

    Boore, J. L. 1999. Animal mitochondrial genomes. Nucleic Acids Res. 27:1767–1780.[Abstract/Free Full Text]

    Boore, J. L., and W. M. Brown. 1994. Complete DNA sequence of the mitochondrial genome of the black chiton Katharina tunicata. Genetics 138:423–443.[Abstract/Free Full Text]

    ———. 1995. Complete sequence of the mitochondrial DNA of the annelid worm Lumbricus terrestris. Genetics 141:305–319.[Abstract/Free Full Text]

    ———. 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17:87–106.[Abstract/Free Full Text]

    Boore, J. L., M. Medina, and L. A. Rosenberg. 2004. Complete sequences of the highly rearranged molluscan mitochondrial genomes of the scaphopod Graptacme eborea and the bivalve Mytilus edulis. Mol. Biol. Evol. 21:1492–1503.[Abstract/Free Full Text]

    Broughton, R. E., J. E. Milam, and B. A. Roe. 2001. The complete sequence of the zebrafish (Danio rerio) mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA. Genome Res. 11:1958–1967.[Abstract/Free Full Text]

    Burzynski, A., M. Zbawicka, D. O. F. Skibinski, and R. Wenne. 2003. Evidence for recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic. Mol. Biol. Evol. 20:388–392.[Abstract/Free Full Text]

    Cantatore, P., M. Roberti, G. Rainaldi, M. N. Gadaleta, and C. Saccone. 1989. The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus. J. Biol. Chem. 264:10965–10975.[Abstract/Free Full Text]

    Cao, L., E. Kenchington, and E. Zouros. 2004. Differential segregation patterns of sperm mitochondria in embryos of the blue mussel (Mytilus edulis). Genetics 166:883–894.[Abstract/Free Full Text]

    Cao, L., E. Kenchington, E. Zouros, and G. C. Rodakis. 2004. Evidence that the large noncoding sequence is the main control region of maternally and paternally transmitted mitochondrial genomes of the marine mussel (Mytilus spp.). Genetics 167:835–850.[Abstract/Free Full Text]

    Carter, J. G., Campbell, D. C., and M. R. Campbell. 2000. Cladistic perspectives on early bivalve evolution. Pp. 47–79 In E. M. Harper, J. D. Taylor, and J. A. Crame, eds. The evolutionary biology of the bivalvia. Special Publications 177. Geological Society, London.

    Chang, Y. S., F. L. Huang, and T. B. Lo. 1994. The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome. J. Mol. Evol. 38:138–155.[ISI][Medline]

    Clary, D. O., and D. R. Wolstenholme. 1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J. Mol. Evol. 22:252–271.[ISI][Medline]

    Crease, T. J. 1999. The complete sequence of the mitochondrial genome of Daphnia pulex (Cladocera: Crustacea). Gene 233:89–99.[CrossRef][ISI][Medline]

    Curole, J. P., and T. D. Kocher. 2002. Ancient sex-specific extension of the cytochrome c oxidase II gene in bivalves and the fidelity of doubly-uniparental inheritance. Mol. Biol. Evol. 19:1323–1328.[Abstract/Free Full Text]

    De Giorgi, C., A. Martiradonna, C. Lanave, and C. Saccone. 1996. Complete sequence of the mitochondrial DNA in the sea urchin Arbacia lixula: conserved features of the echinoid mitochondrial genome. Mol. Phylogenet. Evol. 5:323–332.[CrossRef][ISI][Medline]

    DeJong, R. J., A. M. Emery, and C. M. Adema. 2004. The mitochondrial genome of Biomphalaria glabrata (gastropoda, basommatophora), intermediate host of Schistosoma mansoni. J. Parasitol. 90:991–997.[Medline]

    Diaz, F., M. P. Bayona-Bafaluy, M. Rana, M. Mora, H. Hao, and C. T. Moraes. 2002. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 30:4626–4633.[Abstract/Free Full Text]

    Dirheimaer, G., G. Keith, P. Dumas, and E. Westhof. 1995. Primary, secondary, and tertiary structures of tRNAs. Pp. 93–126 in D. Söll and U. L. RajBahandary, eds. tRNA: structure, biosynthesis and function. ASM Press, Washington, DC.

    Dreyer, H., and G. Steiner. 2004. The complete sequence and gene organization of the mitochondrial genome of the gadilid scaphopod Siphonodentalium lobatum (Mollusca). Mol. Phylogenet. Evol. 31:605–617.[CrossRef][ISI][Medline]

    Garesse, R. 1988. Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations. Genetics 118:649–663.[Abstract/Free Full Text]

    Garrido-Ramos, M. A., D. T. Stewart, B. W. Sutherland, and E. Zouros. 1998. The distribution of male-transmitted and female-transmitted mitochondrial DNA types in somatic tissues of blue mussels: implications for the operation of doubly uniparental inheritance of mitochondrial DNA. Genome 41:818–824.[CrossRef][ISI]

    Gossling, E. 1992. Systematics and geographic distribution of Mytilus. Pp. 1–20 in E. Gossling, ed. The mussel Mytilus: ecology, physiology, genetics and aquaculture. Elsevier, Amsterdam.

    Grande, C., J. Templado, J. L. Cervera, and R. Zardoya. 2002. The complete mitochondrial genome of the nudibranch Roboastra europaea (Mollusca: Gastropoda) supports the monophyly of opisthobranchs. Mol. Biol. Evol. 10:1672–1685.

    Hatzoglou, E., G. C. Rodakis, and R. Lecanidou. 1995. Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea. Genetics 140:1353–1366.[Abstract/Free Full Text]

    Helfenbein, K. G., W. M. Brown, and J. L. Boore. 2001. The complete mitochondrial genome of the articulate brachiopod Terebratalia transversa. Mol. Biol. Evol. 18:1734–1744.[Abstract/Free Full Text]

    Hoeh, W. R., D. T. Stewart, and S. I. Guttman. 2002. High fidelity of mitochondrial genome transmission under the doubly uniparental mode of inheritance in freshwater mussels (Bivalvia: Unionoidea). Evol. Int. J. Org. Evol. 56:2252–2261.

    Hoeh, W. R., D. T. Stewart, C. Saavedra, B. W. Sutherland, and E. Zouros. 1997. Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae). Mol. Biol. Evol. 14:959–967.[Abstract]

    Hoeh, W. R., D. T. Stewart, B. W. Sutherland, and E. Zouros. 1996. Cytochrome c oxidase sequence comparisons suggest an unusually high rate of mitochondrial DNA evolution in Mytilus (Mollusca: Bivalvia). Mol. Biol. Evol. 13:418–421.[Free Full Text]

    Hoffmann, R. J., J. L. Boore, and W. M. Brown. 1992. A novel mitochondrial genome organization for the blue mussel Mytilus edulis. Genetics 131:397–412.[Abstract/Free Full Text]

    Hurst, L. D., and R. F. Hoekstra. 1994. Evolutionary genetics. Shellfish genes kept in line. Nature 368:811–812.[CrossRef][ISI][Medline]

    Ingman, M., H. Kaessmann, S. Pääbo, and U. Gyllensten. 2000. Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713.[CrossRef][ISI][Medline]

    Jermiin, L. S., and R. H. Crozier. 1994. The cytochrome b region in the mitochondrial DNA of the ant Tetraponera rufoniger: sequence divergence in Hymenoptera may be associated with nucleotide content. J. Mol. Evol. 38:282–294.[CrossRef][ISI][Medline]

    Jermiin, L. S., P. G. Foster, D. Graur, R. M. Lowe, and R. H. Crozier. 1996. Unbiased estimation of symmetrical directional mutation pressure from protein-coding DNA. J. Mol. Evol. 42:476–480.[ISI][Medline]

    Jermiin, L. S., D. Graur, R. M. Lowe, and R. H. Crozier. 1994. Analysis of directional mutation pressure and nucleotide content in mitochondrial cytochrome b genes. J. Mol. Evol. 39:160–173.[ISI][Medline]

    Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120.[ISI][Medline]

    Kumar, S. 1996. PHYLTEST: a program for testing phylogenetic hypotheses. Pennsylvania State University, University Park.

    Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245.[Abstract/Free Full Text]

    Kurabayashi, A., and R. Ueshima. 2000. Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organization. Mol. Biol. Evol. 17:266–277.[Abstract/Free Full Text]

    Ladoukakis, E. D., C. Saavedra, A. Magoulas, and E. Zouros. 2002. Mitochondrial DNA variation in a species with two mitochondrial genomes: the case of Mytilus galloprovincialis from the Atlantic, the Mediterranean and the Black Sea. Mol. Ecol. 11:755–769.[CrossRef][ISI][Medline]

    Ladoukakis, E. D., and E. Zouros. 2001. Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA. Mol. Biol. Evol. 18:1168–1175.[Abstract/Free Full Text]

    Le, T. H., D. Blair, T. Agatsuma et al. (14 co-authors). 2000. Phylogenies inferred from mitochondrial gene orders—a cautionary tale from the parasitic flatworms. Mol. Biol. Evol. 17:1123–1125.[Free Full Text]

    Liu, H.-P., J. B. Mitton, and S. K. Wu. 1996. Paternal mitochondrial DNA differentiation far exceeds maternal DNA and allozyme differentiation in the fresh water mussel Anodonda grantis grantis. Evolution 50:952–957.[ISI]

    Noguchi, Y., K. Endo, F. Tajima, and R. Ueshima. 2000. The mitochondrial genome of the brachiopod Laqueus rubellus. Genetics 155:245–259.[Abstract/Free Full Text]

    Ojala, D., J. Montoya, and G. Attardi. 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470–474.[CrossRef][ISI][Medline]

    Okimoto, R., J. L. Macfarlane, D. O. Clary, and D. R. Wolstenholme. 1992. The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. Genetics 130:471–498.[Abstract/Free Full Text]

    Passamonti, M., and V. Skali. 2001. Gender-associated mitochondrial DNA heteroplasmy in the venerid clam Tapes philippinarum (Mollusca: Bivalvia). Curr. Genet. 39:117–124.[CrossRef][ISI][Medline]

    Perna, N. T., and T. D. Kocher. 1995. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J. Mol. Evol. 41:353–358.[ISI][Medline]

    Pesole, G., C. Gissi, A. De Chirico, and C. Saccone. 1999. Nucleotide substitution rate of mammalian mitochondrial genomes. J. Mol. Evol. 48:427–434.[ISI][Medline]

    Quesada, H., D. A. Skibinski, and D. O. Skibinski. 1996. Sex-biased heteroplasmy and mitochondrial DNA inheritance in the mussel Mytilus galloprovincialis Lmk. Curr. Genet. 29:423–426.[ISI][Medline]

    Quesada, H., M. Warren, and D. O. Skibinski. 1998. Nonneutral evolution and differential mutation rate of gender-associated mitochondrial DNA lineages in the marine mussel Mytilus. Genetics 149:1511–1526.[Abstract/Free Full Text]

    Quesada, H., R. Wenne, and D. O. Skibinski. 1999. Interspecies transfer of female mitochondrial DNA is coupled with role-reversals and departure from neutrality in the mussel Mytilus trossulus. Mol. Biol. Evol. 16:655–665.[Abstract]

    Rand, D. M., and L. M. Kann. 1998. Mutation and selection at silent and replacement sites in the evolution of animal mitochondrial DNA. Genetica 102/103:393–407.[CrossRef]

    Rawson, P. D., and T. J. Hilbish. 1995. Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex. Mol. Biol. Evol. 12:893–901.[Abstract]

    Roe, B. A., D. P. Ma, R. K. Wilson, and J. F. Wong. 1985. The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260:9759–9774.[Abstract/Free Full Text]

    Rozas, J., and R. Rozas. 1999. DnaSP version3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174–175.[Abstract/Free Full Text]

    Rzhetsky A., Kumar S., and M. Nei. 1995. Four-cluster analysis: a simple method to test phylogenetic hypotheses. Mol. Biol. Evol. 12:163–167.[Abstract]

    Saavedra, C., M. I. Reyero, and E. Zouros. 1997. Male-dependent doubly uniparental inheritance of mitochondrial DNA and female-dependent sex-ratio in the mussel Mytilus galloprovincialis. Genetics 145:1073–1082.[Abstract/Free Full Text]

    Saccone, C., C. De Giorgi, C. Gissi, G. Pesole, and A. Reyes. 1999. Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system. Gene 238:195–209.[CrossRef][ISI][Medline]

    Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.[Abstract]

    Serb, J. M., and C. Lydeard. 2003. Complete mtDNA sequence of the North American freshwater mussel, Lampsilis ornata (Unionidae): an examination of the evolution and phylogenetic utility of mitochondrial genome organization in Bivalvia (Mollusca). Mol. Biol. Evol. 20:1854–1866.[Abstract/Free Full Text]

    Skibinski, D. O. F., C. Gallagher, and C. M. Beynon. 1994a. Mitochondrial DNA inheritance. Nature 368:817–818.[ISI][Medline]

    ———. 1994b. Sex limited mitochondrial DNA transmission in the marine mussel Mytilus edulis. Genetics 138:801–809.[Abstract/Free Full Text]

    Spruyt, N., C. Delarbre, G. Gachelin, and V. Laudet. 1998. Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates. Nucleic Acids Res. 26:3279–3285.[Abstract/Free Full Text]

    Stechmann, A., and M. Schlegel. 1999. Analysis of the complete mitochondrial DNA sequence of the brachiopod Terebratulina retusa places Brachiopoda within the protostomes. Proc. R. Soc. Lond. B Biol. Sci. 266:2043–2052.[CrossRef][ISI][Medline]

    Stewart, D. T., E. R. Kenchington, R. K. Singh, and E. Zouros. 1996. Degree of selective constraint as an explanation of the different rates of evolution of gender-specific mitochondrial DNA lineages in the mussel Mytilus. Genetics 143:1349–1357.[Abstract/Free Full Text]

    Stewart, D. T., C. Saavedra, R. R. Stanwood, A. O. Ball, and E. Zouros. 1995. Male and female mitochondrial lineages in the blue mussel (Mytilus edulis) species group. Mol. Biol. Evol. 12:735–747.[Abstract]

    Sueoka, N. 1962. On the genetic basis of variation and heterogeneity of DNA base composition. Proc. Natl. Acad. Sci. USA 48:582–592.[ISI][Medline]

    Terrett, J. A., S. Miles, and R. H. Thomas. 1996. Complete DNA sequence of the mitochondrial genome of Cepaea nemoralis (Gastropoda: Pulmonata). J. Mol. Evol. 42:160–168.[ISI][Medline]

    Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876–4882.[CrossRef]

    Tomita, K., S. Yokobori, T. Oshima, T. Ueda, and K. Watanabe. 2002. The cephalopod Loligo bleekeri mitochondrial genome: multiplied noncoding regions and transposition of tRNA genes. J. Mol. Evol. 54:486–500.[CrossRef][ISI][Medline]

    Wolstenholme, D. R. 1992. Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141:173–216.[ISI][Medline]

    Yamazaki, N., R. Ueshima, J. A. Terrett et al. (12 co-authors).1997. Evolution of pulmonate gastropod mitochondrial genomes: comparisons of gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures. Genetics 145:749–758.[Abstract/Free Full Text]

    Zouros, E., A. O. Ball, C. Saavedra, and K. R. Freeman. 1994a. Mitochondrial DNA inheritance. Nature 368:818.[CrossRef][ISI][Medline]

    ———. 1994b. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc. Natl. Sci. USA 91:7463–7467.[Abstract/Free Full Text]

    Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31:3406–3415.[Abstract/Free Full Text]

Accepted for publication January 3, 2005.