* Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimioupolis, Athens, Greece; and Department of Biology, University of Crete, Heraklion, Crete, Greece
Correspondence: E-mail: grodakis{at}biol.uoa.gr.
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Abstract |
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Key Words: Mytilus mitochondrial genome maternally or paternally inherited mtDNA
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Introduction |
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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?
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Materials and Methods |
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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 50100 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 5001,200 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 1060210669 and 1067610741, and AB065374, regions 92759340 and 93399409, 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.
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Results and Discussion |
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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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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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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 88808944) 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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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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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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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.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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References |
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