Direct Evidence for Homologous Recombination in Mussel (Mytilus galloprovincialis) Mitochondrial DNA

Emmanuel D. Ladoukakis and Eleftherios Zouros

Department of Biology, University of Crete, Crete, Greece
Institute of Marine Biology of Crete, Crete, Greece


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
The assumption that animal mitochondrial DNA (mtDNA) does not undergo homologous recombination is based on indirect evidence, yet it has had an important influence on our understanding of mtDNA repair and mutation accumulation (and thus mitochondrial disease and aging) and on biohistorical inferences made from population data. Recently, several studies have suggested recombination in primate mtDNA on the basis of patterns of frequency distribution and linkage associations of mtDNA mutations in human populations, but others have failed to produce similar evidence. Here, we provide direct evidence for homologous mtDNA recombination in mussels, where heteroplasmy is the rule in males. Our results indicate a high rate of mtDNA recombination. Coupled with the observation that mammalian mitochondria contain the enzymes needed for the catalysis of homologous recombination, these findings suggest that animal mtDNA molecules may recombine regularly and that the extent to which this generates new haplotypes may depend only on the frequency of biparental inheritance of the mitochondrial genome. This generalization must, however, await evidence from animal species with typical maternal mtDNA inheritance.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Uniparental transmission is a common feature of mitochondrial genomes in all eukaryotes (Birky 1995Citation ). The gamete through which the mtDNA is inherited is the female, but exceptions occur, e.g., in the redwood Sequoia sempervirens (Neale, Marshall, and Sederoff 1989Citation ) and in the banana Musa acuminata (Faure et al. 1994Citation ), for which the mitochondrial genome is transmitted through the male gamete. A further exception is the mussel system, for which there exist two mtDNA lineages, one with maternal and one with paternal inheritance. These exceptions do not violate the rule of uniparental transmission (Hurst and Hoekstra 1994Citation ). One aspect, however, in which animal mtDNA is assumed to differ from that of plants and fungi is that it does not undergo homologous recombination. Direct evidence for homologous recombination exists for plants, fungi, and protists (for review, see Gray 1989Citation ). In animals, no physical evidence for homologous mtDNA recombination exists so far. On the other hand, several independent observations have led to the indirect conclusion that animal mtDNA does not recombine. These observations include failure to detect excision repair activity and crossover products in mammalian mitochondria (Clayton, Doda, and Friedberg 1975Citation ), failure to detect mtDNA recombination in somatic cell hybrids (Zuckerman et al. 1984Citation ), and sequestration of mtDNA molecules into clusters and, therefore, prevention of physical contact of unrelated molecules (Nass 1969Citation ; Satoh and Kuriowa 1991Citation ). Because recombination is considered to be an indispensable part of DNA replication and repair (Kowalczykowski 2000)Citation , the elevated mtDNA mutation rate in animals, compared with that of nuclear DNA (Wallace et al. 1987Citation ), was also taken as an indication of absence of homologous recombination in animal mtDNA (Howell 1997Citation ).

Other observations are, however, suggestive of the possibility of recombination in animal mtDNA. Thyagarajan, Padua, and Campbell (1996)Citation have demonstrated that human mitochondria contain the enzymes necessary for homologous recombination. Fusion of mitochondria is well demonstrated in Drosophila (Yaffe 1999Citation ). Nonhomologous recombination was proposed as the mechanism responsible for variation in the number of tandemly repeated motifs (variant number of tandem repeats [VNTR] polymorphism) observed in noncoding regions of mtDNA in a number of animal species (such as Drosophila [Solignac, Monnerot, and Mounolou 1986Citation ], scallops [Snyder et al. 1987Citation ], crickets [Rand and Harrison 1989Citation ], and sturgeons [Buroker et al. 1990Citation ]). Lunt and Hyman (1997)Citation have indeed provided direct evidence of this type of recombination in the nematode Meloidogyne javanica. Two population studies have also raised the possibility of recombination in primate mtDNA. One of these was based on a high frequency of homoplasies (Eyre-Walker, Smith, and Maynard Smith 1999Citation ), and the other was based on a negative correlation between linkage disequilibrium and physical distance along the molecule of mtDNA variants (Awadalla, Eyre-Walker, and Maynard Smith 1999Citation ). These studies have, however, been criticized on several grounds (Arctander 1999Citation ; Merriweather and Kaestle 1999Citation ; Kivisild et al. 2000Citation ). Moreover, two more recent studies failed to produce evidence for recombination in human mtDNA (Ingman et al. 2000Citation ; Elson et al. 2001)Citation . Here, we present direct evidence of homologous recombination in the mussel Mytilus galloprovincialis.

Mussels of the families Mytilidae (sea mussels) and Unionidae (freshwater mussels) possess two mtDNA lineages, one transmitted through the egg (the F lineage) and one transmitted through the sperm (the M lineage) (Skibinski, Gallagher, and Beynon 1994a, 1994bCitation ; Zouros et al. 1994a, 1994bCitation ; Liu, Mitton, and Wu 1996Citation ). Sperm mtDNA is delivered into the oocyte and eliminated within the first 24 h after fertilization in females (Sutherland et al. 1998Citation ) but is retained in males which develop into "homoplasmic mosaics," with their gonads dominated by the M molecule and their somatic tissues dominated by the F molecule (Garido-Ramos et al. 1998Citation ). The phenomenon has become known as doubly uniparental inheritance (DUI; Zouros et al. 1994aCitation ). M and F DNA sequences normally differ by >20% (Hoeh et al. 1997Citation ). This difference is considered prohibitive for homologous recombination because it necessitates a high activity of mismatch repair, which in turn suppresses homologous recombination (Rayssiguier, Thayler, and Radman 1989Citation ; Selva et al. 1995Citation ; de Wind et al. 1995Citation ). However, occasionally F genomes invade the M transmission route (Hoeh et al. 1997Citation ). This reversal of roles results in newly established M molecules (for which we here use the symbol Mf) whose sequences are initially similar to F but diverge rapidly owing to the high rate of mutation accumulation that characterizes male-transmitted lineages (Stewart et al. 1996Citation ). Males from natural populations that inherit an Mf from the father and an F from the mother are heteroplasmic for molecules that differ by about 4% (Hoeh et al. 1997Citation ), an amount not large enough to prevent homologous recombination and yet large enough to allow its detection if it has occurred. Our demonstration of homologous recombination was performed using this type of male.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
The experimental protocol consisted of the following steps:

  1. As part of a study of mtDNA variation in a population of M. galloprovincialis from the Black Sea, a number of male individuals were scored for restriction fragment length polymorphism (RFLP) at an 860-bp fragment of the COIII gene (Ladoukakis 1998Citation ). For this purpose, total DNA was extracted from gonadal and somatic tissues using the method of Miller, Dykes, and Polesky (1988)Citation as modified by Zouros et al. (1992)Citation . PCR amplification was carried out using the protocol of Saavedra, Reyero, and Zouros (1997)Citation with primers FOR1 (5'-TAT GTA CCA GGT CCA AGT CCG TG-3') and REV1 (5'-TGC TCT TCT TGA ATA TAA GCG TA-3'). These primers correspond to the nucleotide positions 460–482 and 1362–1301 of segment 5 of the Mytilus edulis F genome of Hoffman, Boore, and Brown (1992)Citation . The PCR reactions were carried out with 1 µl of source DNA, 0.5 mM of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, and 0.5 U Taq polymerase (Gibco-BRL) in the buffer supplied by the company in a total volume of 10 µl. The mixture was incubated at 94°C for 2 min for initial denaturation and then at 94°C for 1 min, 54°C for 30 s, and 72°C for 1 min for 35 cycles and a final extension at 72°C for 5 min. PCR products were digested with RsaI (Minotech) to determine the restriction type of the F and M mtDNA genomes of each male.
  2. Individual males were characterized as "typical" if their restriction profile was compatible with the presence of a typical F (maternal) and a typical M (paternal) mtDNA molecule, or as "atypical" if no M molecule could be detected from the restriction analysis. The restriction patterns of atypical males varied in complexity. Some patterns were compatible with the presence of only one F type molecule, and others were compatible with the presence of several F type molecules.
  3. PCR products from gonadal DNA of 10 randomly chosen atypical males were cloned on pGEM T vector (Promega). PCR products from somatic tissues of one of these males (male 68) were also cloned.
  4. Samples from several positive clones from each individual were subjected to PCR amplification with the same primers and conditions as above, and the PCR products were digested with RsaI (Minotech) and separated on a 1.5% agarose gel. The numbers of assayed clones varied among individuals according to the complexity of the restriction pattern seen in the original PCR product (step 2).
  5. Clones from the same individual were classified in groups of the same restriction pattern, and one randomly chosen clone from each group was subjected to double-strand sequencing on a PTC100 automated sequencer from MG Research. The primers used for sequencing were the primers of the vector SP6 (5'-ATT TAG GTG ACA CTA TAG-3') for one strand and T7 (5'-GTA ATA CGA CTC ACT ATA GGG C-3') for the other.

In contrast to gonadal tissue, which produced complex restriction profiles, somatic tissues always produced simple patterns, compatible with the presence of a single F molecule. As a result, the PCR product from the DNA of somatic tissues was directly sequenced. The resulting chromatograms were indeed consistent with a single molecule. An additional check of somatic tissue homoplasmy was made by examining a large number of clones from somatic DNA of individual 68, which contained the largest number of different sequences in the gonadal tissue (table 1 ). It is known that somatic tissues of males contain predominantly the maternal genome, whereas the gonad contains both the paternal and the maternal genomes (Garido-Ramos et al. 1998Citation ). This allows the identification of the maternal genome as the one found in somatic tissues. No such identification is possible for the paternal genome (or genomes) when more than two molecules are recovered from the gonad and when the possibility of recombination exists between maternal and paternal genomes.


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Table 1 Numbers of Clones Examined, Different Sequences Detected, and Numbers of Recombinant Sequences Found in the Gonads of 10 Individuals

 
A segment of 681 bp was unambiguously read from all sequences which corresponded to nucleotide positions 618–1299 of segment 5 of the M. edulis F genome of Hoffman, Boore, and Brown (1992)Citation . Sequences were aligned according to the ClustalX program (Thompson et al. 1997Citation ). No deletions or insertions were observed. Kimura's two-parameter distance model was used to construct an unweighted pair grouping method with arithmetic means (UPGMA) tree with the help of the MEGA package (Kumar, Tamura, and Nei 1993Citation ). The bootstrap values (1,000 replications) were calculated with the same program. Sequences were deposited in GenBank/EMBL with accession numbers AF214041AF214053.

The PCR assay for verification of the presence of recombinant molecules in extracts from gonadal tissues (see Results) was performed using primers RECF (5'-GCT TTA AGG CCC TCA-3') and RECR (5'-GCC TCA AAC CCA AAG-3'), an annealing temperature of 48°C, and a MgCl2 concentration of 1.5 mM.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Evidence for Recombination
The number of mtDNA clones from each individual subjected to restriction analysis is given in table 1 . All clones from three individuals (individuals 1, 12, and 13) produced the same profile, but two to five different profiles were recovered among clones from the other seven individuals. For each individual, clones with the same restriction profile were grouped together and one randomly chosen clone from each group was sequenced. A total of 13 different sequences for a 681-bp fragment of the COIII gene were recovered among the 24 clones that were sequenced. The sequences were clustered using the UPGMA method and produced two major phylads. The two sequences that were recovered more than once in the sample (sequences 2 and 5) were assigned to different phylads, which, for this reason, were named phylad 2 and phylad 5 (fig. 1 ). Restriction analysis of PCR products of the same COIII region from a larger sample of individuals (n = 66) from the same population estimated the frequency of sequence 2 at 0.74 and that of sequence 5 at 0.22 (Ladoukakis 1998Citation ); thus, these two sequences were the most common in the population as well. The complete sequence 2 is given in figure 2 . Figure 3 represents a convenient way of presenting the differences among sequences of the same or different phylads and demonstrating recombinant exchanges. The figure shows only the nucleotides at the 41 variable sites (sites in bold in fig. 2 ). The nucleotides of sequences 2 and 5 at the 41 variable sites are given in the middle. The sequences that belong to phylad 2 are listed under sequence 2. Likewise, the sequences that belong to phylad 5 are listed above sequence 5. Thus, the 13 complete sequences can be obtained from figures 2 and 3 .



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Fig. 1.—An unweighted pair grouping method with arithmetic means clustering of 13 different sequences recovered among clones from 10 individuals. The sequences form two distinct phylads, which were named after the two most common sequences in the population. Within phylads, topologies that are due to recombinational exchanges (fig. 3 ) have low bootstrap support because nucleotide differences are localized rather than random. Branches caused by dispersed mutational differences have good support

 


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Fig. 2.—Sequence 2 for a 681-bp fragment of the COIII mtDNA gene. Bold numbers indicate sites at which different nucleotides were encountered among the 13 sequences reported

 


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Fig. 3.—Differences among 13 nucleotide sequences of a 681-bp segment of the COIII mtDNA gene recovered from 10 males of Mytilus galloprovincialis. Sequences belonging to two phylads (fig. 1 ) are separated by thick lines. Numbers between lines refer to the 41 variable sites. The nucleotides for all 41 sites are given for sequences 2 and 5. Within-phylad differences are shown as differences from sequence 5 (upper part) or sequence 2 (lower part). Site numbers in bold indicate change of amino acid. Recombinant fragments are shown in gray boxes, with their lengths given in the right part of the figure

 
Figure 3 contains all the essential information with regard to homologous recombination. First, it must be observed that three nucleotides are singletons (site 142, sequence 11; site 450, sequence 7; site 483, sequence 10). In all likelihood, these are PCR errors that occurred during the production of the copies that were subsequently cloned. Errors of this type are common (Paabo, Irwin, and Wilson 1990Citation ; McPherson, Quirke, and Taylor 1991Citation , pp. 237–242; see also below), and they are identified by the fact that they are unique to the clone in which they are detected. When these substitutions are removed, sequences 7 and 11 become identical to sequences 2 and 5, respectively, and the total number of sequences is reduced to 11.

In phylad 5, sequences 12 and 3 can be seen as results of simple recombination exchange between sequences 5 and 2. For sequence 12, the beginning of the segment that is derived from phylad 2 is between the diagnostic sites 519 and 639, and the end is between sites 663 and 672; thus, the recombinant fragment has a length between 24 and 153 nt. The substitution at site 171 of this sequence can be either a PCR error or a homoplasy (given that it also occurs in phylad 2). For sequence 3, the beginning of recombination is between sites 507 and 516, and the recombinant fragment apparently extends beyond the cloned region.

In phylad 2 we may recognize three subgroups of sequences. One consists of sequence 2 and sequences 4, 13, and 8, all of which can be derived from simple recombination exchanges between sequences 2 and 5. The second subgroup consists of sequences 9 and 10. Sequence 9 differs from sequence 2 at seven sites (sites 42, 54, 324, 375, 418, 467, and 672) and must represent a mutational derivative of sequence 2. Sequence 10 shares the same differences as sequence 9 from sequence 2 but, in addition, contains a stretch of sequence 5 and therefore can be seen as a recombination result between sequences 5 and 9. The third subgroup consists of sequences 6 and 1, which share six nucleotide differences from sequence 2 (at sites 111, 342, 366, 467, 672, and 681). In total, after ignoring single-nucleotide differences, we are left with six unambiguous recombinant sequences, of which five (sequences 3, 12, 4, 13, and 8) are products of recombination between sequences 2 and 5 and one (sequence 10) is a product of recombination between sequences 9 and 5. For sequences for which both ends of the recombinant fragment lie within the sequenced region, the length of the fragment varies from 24 to 255 bp. In sequences 4 and 3, the length may be longer (fig. 3 ).

Amino acid substitutions have been observed in 3 of the 41 variable sites (sites 25, 142, and 467; fig. 3 ). The singleton substitution at site 142 in sequence 11 is, as noted, most likely the result of PCR error. At site 25, all sequences of phylad 2 have leucine and all sequences of phylad 5 have phenylalanine. At site 467, all sequences of phylad 5 and sequences 9, 10, 6, and 1 of phylad 2 have methionine, while all other sequences have threonine. The important observation is that both of these sites lie in regions that have not recombined. As a result, none of the recombination exchanges we observed resulted in any amino acid replacement. At present, we cannot say whether this is simply an accidental observation or that products of recombination with amino acid changes do not persist.

Elimination of the Possibility of Artifactual PCR Recombination
A previous study (Paabo, Irwin, and Wilson 1990Citation ) has shown that when the template DNA of the PCR reaction contains a mixture of DNA molecules, Taq DNA polymerase may "jump" from one molecule to another, thus producing hybrid products. In our protocol, the first PCR reaction was carried out on DNA extracts from gonad tissue, and the products were subsequently cloned and sequenced. Theoretically, there is a possibility that in the presence of more than one mtDNA molecule in the original DNA extract, PCR jumping could produce recombinant molecules. The original study by Paabo, Irwin, and Wilson (1990)Citation was designed to facilitate PCR jumping. The source DNA contained highly homologous sequences digested with restriction enzymes to produce fragments with single-strand protruding ends. These conditions did not apply to our DNA source. Nevertheless, we performed two experiments to eliminate the possibility that the recombinant sequences we observed were caused by PCR jumping.

The first experiment consisted of mixing DNA from two clones of known sequence, using the mixture as the PCR template, cloning the PCR products, and assaying the cloned sequences by restriction analysis following steps 1, 3, and 4 from Materials and Methods. In such an experiment, any restriction pattern other than that of the two source sequences would be due to PCR error. This experiment was performed twice. In the first experiment, we used a pair of clones, one containing sequence 2 and one containing sequence 5. These were the sequences which upon recombination would have produced sequences 4, 13, 8, 3, and 12 in our experimental animals. Forty clones from the PCR product were digested with RsaI. Fifteen clones produced the restriction pattern of sequence 2, and 25 produced the pattern of sequence 5. In the second experiment, we used a pair of clones, one containing sequence 9 and one containing sequence 5, the sequences which upon recombination would have produced sequence 10. Thirty-seven clones were assayed as above, of which 27 had the restriction pattern of sequence 9, 9 had the pattern of sequence 5, and 1 had a novel restriction pattern. The clone with the novel restriction pattern was sequenced and found to differ from sequence 5 by a single substitution: it had C in position 443 instead of T. Thus, the experiment failed to produce recombinant sequences by PCR jumping but demonstrated that PCR errors of single-nucleotide substitution occur with low frequency.

The purpose of the second experiment was to recover the recombinant sequences we observed from the original DNA extracts. The rationale was that if we designed a PCR reaction which would specifically amplify a recombinant sequence, the sequence would be recovered if it was present in the original extract (and thus in the animal itself), but it would not be recovered if in the original experiment it was produced by PCR jumping. We used the primers RECF (5'-GCT TTA AGG CCC TCA-3') and RECR (5'-GCC TCA AAC CCA AAG-3'), which corresponded to sites 130–144 and 516–530 of figure 2 , respectively. The first primer anneals on sequence 5, and the second anneals on sequence 2. Before they were used on the source DNA, the primers were used on a mixture of two clones, one containing sequence 2 and one containing sequence 5. The same was done with another pair of clones, one containing sequence 5 and one containing sequence 9. In both cases, we failed to produce a PCR product. The primers were also tried against a clone containing sequence 8 and against a clone containing sequence 13 (both of which consisted of recombinant sequences) and produced the expected PCR product. After these pilot trials, the primers were used directly on the DNA that was extracted from the gonads of individuals 59 and 35. The PCR product from animal 59 was sequenced and found to be identical to the recombinant sequence 13. The product from animal 35 was identical to the recombinant sequence 8 except at position 411, where the new product had T instead C, which must be a PCR point error.

How Recent Is the Recombination?
How recent might the recombination events that generated the observed sequences be? This question is important because different sequences found in an individual may be the results of recombination that happened in the individual itself, or they can be parts of a long-standing polymorphism generated by past recombination events. Four individuals (individuals 20, 59, 35, and 68) carry recombinant sequences and can be used to examine this question. The other individuals are either homoplasmic (individuals 1, 12, and 13) or heteroplasmic (individuals 23, 61, and 81) for the two common sequences (sequences 2 and 5) and thus are assumed to carry no recombinant sequences (table 1 ).

In individual 68, sequence 10 can be seen as a recombination product between sequences 9 and 5 (fig. 4 ), both of which were found in this individual (as mentioned earlier, the difference at position 483 is either a PCR error or a postrecombination mutation). Given the rarity of sequence 9 in the population as a whole (Ladoukakis 1998Citation ), its presence in the same individual with its recombination product implies a recent recombination event. In individual 20, sequences 3 and 4 are products of recombination between sequences 2 and 5 (fig. 4 ). Sequence 2 was recovered in individual 20, but sequence 5 was not. Unlike nuclear recombination, there is no cellular mechanism that would prevent loss upon cytokinesis of one or more mtDNA sequences. Indeed, loss of either a parental or a recombinant sequence is more probable than retention of the full complement of sequences (Koehler et al. 1991Citation ). Thus, sequences 3 and 4 are most likely products of a recombination event between sequences 2 and 5 that happened either in individual 20 itself or in a recent ancestral individual from which they were cotransmitted to individual 20. This transmission is unlikely to have occurred through the egg because these sequences were not found in the somatic tissues. Transmission of more than one type of mtDNA genome through the sperm is a possibility for which we have no evidence one way or the other. Whatever the case, there is evidence from two of the four informative animals that the events of recombination are recent and may in fact have occurred in the individuals in which they were observed. No such information can be extracted from animals 35 and 59, but this is compatible with stochastic loss of mitotypes.



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Fig. 4.—Recombination events that may account for the sequences found in individuals 68 (upper part) and 20 (lower part). The number of variable sites in each recombined fragment is given. Also identified are the variable sites that define the interval of sequence exchange

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
We present here firm evidence for homologous recombination in males of the Mediterranean mussel M. galloprovincialis. This is a different type of recombination from that shown to occur in the nematode M. javanica (Lunt and Hyman 1997Citation ) and is postulated to be responsible for the variable number of repeats seen in the mtDNA of various animal species (Solignac, Monnerot, and Mounolou 1986Citation ; Snyder et al. 1987Citation ; Rand and Harrison 1989Citation ; Buroker et al. 1990Citation ; Ludwig et al. 2000Citation ). The latter type of recombination is mediated by a mechanism of unequal crossing over and results in products of unequal lengths. It is restricted to parts of the genome where tandem repeats occur. For the majority of animal mtDNA, such arrays of repeats occur in the replication control region. Our observations refer to a typical coding part of the genome and imply that homologous recombination may occur in any part of the mussel mitochondrial genome. The recombination we saw was anticipated by Thyagarajan, Padua, and Campbell (1996)Citation , who observed that mitochondria in cultured human cells contained the enzymes that catalyze nuclear recombination. The implication, therefore, is that these enzymes are imported in the mitochondrion as well as in the nucleus and that the molecular mechanism of mtDNA recombination is similar to nuclear recombination.

We performed two experiments in order to eliminate the possibility that the recombination we observed was an artifact of the PCR reaction. Paabo, Irwin, and Wilson (1990)Citation have demonstrated that under certain conditions, Taq DNA polymerase may move from one template molecule to another, with the result that the PCR product may contain stretches of DNA from two different molecules. In the first experiment, the PCR polymerase was offered two types of DNA molecules, but we failed to detect any hybrid molecule among the products. The second experiment was designed to recover the observed recombinant sequences from the original DNA extract. The specific primers that were used for this experiment failed to produce a product when used against a mixture of sequences that could produce the recombinants by PCR jumping. When the same primers were used on the original DNA source, they produced the expected recombinant sequences. We conclude that the recombinant sequences we observed were present in the gonads of the individuals and cannot be attributed to PCR error.

The demonstration of recombination in mussel mtDNA raises several questions about the mussel mtDNA system itself and about animal mtDNA in general. One question is how common homologous recombination might be in mussel mtDNA. Our results suggest that it may be very common. Of the 24 sequences we obtained from the gonads (table 1 ), 13 were different, and of these, 6 were recombinant. Unless this region happened to be a recombination hot spot, we have to conclude that recombinant fragments would possibly be found in all molecules if longer stretches of DNA were examined. Thus, we have to assume that in the male gonad of the mussel, homologous mtDNA recombination is common, and it may be limited only by the degree of sequence divergence among the molecules that are contributed by the egg and the sperm.

Another question is to what extent do the results from the mussel mtDNA apply to the animal mtDNA as a whole? MtDNA transmission in mussels, now known as DUI, differs from that of standard maternal inheritance in several important ways (see Saavedra, Reyero, and Zouros 1997Citation ). For the question at hand, the issue is whether the homologous recombination we observed was an integral part of the DUI mechanism or a consequence. Whereas we have at present no evidence for the former, we can cite two arguments in favor of the latter. One is the claim for homologous recombination in primate mtDNA from population data (Awadalla, Eyre-Walker, and Maynard Smith 1999Citation ; Eyre-Walker, Smith, and Maynard Smith 1999Citation ). Even though these studies have been criticized (Arctander 1999Citation ; Merriweather and Kaestle 1999Citation ; Kivisild et al. 2000Citation ) and were not corroborated by two more recent studies (Ingman et al. 2000Citation ; Elson et al. 2001)Citation , they remain suggestive of the possibility of rare recombination in primate mtDNA. The second argument arises from the demonstration that mammalian mitochondria house the enzyme apparatus that is necessary for recombination (Thyagarajan, Padua, and Campbell 1996Citation ). Rather than assume that this apparatus is somehow inoperative in the animal mitochondria, it would be more inductive to further research on the issue of animal mtDNA recombination if one assumes that it is active and that the success in detecting recombination might simply be proportional to the degree of differentiation of the recombining genomes. In the face of rare heteroplasmy or even relatively common heteroplasmy for molecules with low degrees of molecular divergence, recombination will be difficult to detect either in present or in historical time. Transmission of paternal mtDNA has been reported in a number of species that cover a wide spectrum of the animal kingdom (Drosophila [Kondo et al.1990Citation ], mice [Gyllensten et al. 1991Citation ; Kaneda et al. 1995Citation ; Shitara et al. 1998Citation ], and anchovies [Magoulas and Zouros 1993Citation ]). For mice, the "leakage" of paternal mtDNA was estimated at about 10-4 of an individual's mtDNA pool. These observations suggest that incidental paternal mtDNA transmission may be the rule in animals, despite the presence of mechanisms for sperm mtDNA elimination in the fertilized ovum (Shitara et al. 1998Citation ; Sutovsky et al. 1999Citation ). In this study, the degree of differentiation between a genuine F genome inherited from the mother and an F-like genome inherited from the father has made the detection of recombination relatively easy.

If homologous recombination occurs in animal mtDNA, it will have an important effect on our understanding of mtDNA mutation and repair mechanisms and rates of mutation accumulation. Homologous recombination is essential for DNA repair in yeast (Ling et al. 1995Citation ) and is expected to play a similar role in animal mtDNA (Thyagarajan, Padua, and Campbell 1996Citation ; Howell 1997Citation ). Recombination errors may lead to unequal crossing over and deletions between short direct repeats of the type associated with mitochondrial diseases (Holt, Harding, and Morgan-Hughes 1988Citation ). It must be emphasized that the implications of homologous recombination of mtDNA for mitochondrial disease, of which there is a growing list (Chinnery and Turnbull 1999Citation ), aging (Ozawa 1999Citation ), and certain methods of cloning (Evans et al. 1999Citation ; Steinborn et al. 2000Citation ) do not depend on initial heteroplasmy or on whether or not it generates new haplotypes in the population.

The implications are equally serious for the use of mtDNA in studies of phylogeny, phylogeography, and dating where the genome is assumed to be clonally transmitted. Recent simulation studies by Schierup and Hein (2000a, 2000b)Citation suggest that assuming no recombination when recombination does occur leads to longer basal branches and shorter terminal branches than the real situation might and also to apparent violation of the molecular clock. To this we can suggest two other complications that could be of special importance in the reconstruction of phylogenies of closely related but reproductively isolated taxa. One is the possibility of concerted evolution in mtDNA. Concerted evolution was suggested in the A/T-rich region of Drosophila (Solignac, Monnerot, and Mounolou 1986Citation ) but is now a possibility for the whole mitochondrial genome. The second complication may result from variation (either among individuals or among populations) in the recombinational activity of the mitochondrion, which must be controlled by nuclear genes. All of these will have the effect of adding a heavy layer of noise around the phylogenetic signal. At the limit, mtDNA would be reduced to just another molecule with anastomosing lineages (Hey 2000)Citation .


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
We have demonstrated homologous recombination in mussel mtDNA. It is a matter of future research to establish how frequent and pervasive this recombination might be and whether recombinant molecules have the same chance to survive in the population as do nonrecombinant molecules. It also remains to be established whether the recombination we observed is an integral part of the exceptional transmission system of mussel mtDNA, in which case its importance would be restricted to species with DUI, or a consequence of heteroplasmy, in which case its importance would extend to animal mtDNA in general.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
We thank Prof. C. Louis and his associates for help with the sequencing of DNA, and Drs. C. Delidakis, A. Magoulas, and C. Saavedra for assistance at various stages of the work.


    Footnotes
 
Adam Eyre-Walker, Reviewing Editor

1 Keywords: mtDNA recombination doubly uniparental inheritance Mytilus COIII Back

2 Address for correspondence and reprints: Eleftherios Zouros, Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio Crete, Greece. E-mail: zouros{at}imbc.gr . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 

    Arctander P., Mitochondrial recombination? 1999 Science 284:2090–2091

    Awadalla P., A. Eyre-Walker, J. Maynard Smith, 1999 Linkage disequilibrium and recombination in hominid mitochondrial DNA Science 286:2524-2525[Abstract/Free Full Text]

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    Chinnery P. F., D. M. Turnbull, 1999 Mitochondrial DNA and disease Lancet 354:(Suppl.)17-21

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Accepted for publication March 7, 2001.