Department of Biology, University of Crete, Greece; and Institute of Marine Biology of Crete, Iraklio Crete, Greece
The question of whether animal mitochondrial DNA (mtDNA) undergoes recombination has recently been the subject of intense debate. In a recent paper (Ladoukakis and Zouros 2001)
, we presented evidence for mtDNA recombination from direct recovery of recombinant molecules in the gonads of several males of the mussel Mytilus galloprovincialis. Because in this species there exist two lineages of mitochondrial genomes, each with a different mode of transmission (a phenomenon known as doubly uniparental inheritance [DUI] of mtDNA; Zouros et al. 1994
), the possibility existed that mtDNA recombination was a peculiarity of DUI. Here, we present evidence for recombination in three different species with standard maternal mtDNA inheritance: a crustacean, an amphibian, and a mammal.
Our initial objective was to apply the method of Awadalla, Eyre-Walker, and Maynard Smith (1999)
to cases drawn from the literature. We used the study by Meyran, Gielly, and Taberlet (1998)
of a segment of cytochrome oxidase I in Gammarus fossarum as one such case. In the process of analyzing these data, we realized that the signature of recombination was clearly preserved in the amino acid sequences. As a result, we turned our search to studies that reported coding mtDNA sequences with substantial differences at the amino acid level, even if the sequences were drawn from different species of a genus. We confined the search to within volume 16 (year 2000) of Molecular Phylogenetics and Evolution, from which we selected the study by Martin et al. (2000)
on rodents of the genus Apodemus and the study by Sumida, Ogata, and Nishioka (2000)
on frogs of the genus Rana. These two studies report only parts of the known sequences of the cytochrome b gene, but our analysis was based on the full DNA sequences, which we retrieved from GenBank (accession numbers and references in fig. 1
).
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The Rana comparison involved a segment of 1,143 bp of the cytochrome b gene. The 14 available sequences were reduced to eight for our analysis. Twenty-two of the 381 amino acid sites were variable, and three domains were identified: one of nine, one of four, and one of six amino acid sites (fig. 1 ). Replacements within consensus groups were rare, as in the case of Gammarus. Exchanges were seen among all three domains (first with second, second with third, third with first). For DNA comparison, we chose sequences Rr2, Rr3, and Rc6. The recombinant fragment was defined as the region between nucleotide sites 636 and 863 (length 228 bp), with a corresponding postrecombination divergence of 0.053. No other continuous or random assemblage of nucleotide sites (steps 5 and 6 of the protocol) could produce a smaller value. For the homogeneity test, the sequences were divided into lengths of 50 nt. All three comparisons produced the expected results (for Rr2 vs. Rc6, F(2,21), P = 0.241; for Rr2 vs. Rr3, P = 0.000; for Rr3 vs. Rc6, P = 0.005). The divergence between Rr2 and Rc6 is 0.225. Given that the postrecombination divergence is 0.053, the time of the recombination event must be a quarter as recent as the time since the divergence of Rr2 and Rc6.
The Apodemus comparison involved a segment of 1,140 bp of the cytochrome b gene. All 10 original sequences were retained for the analysis. Twenty-three of the 380 amino acid sites were variable. We could identify two domains, one of five and one of six amino acid sites. One difference from Gammarus and Rana was that no domain could be identified in the last part of eight amino acid sites (or more than a third of the DNA sequence). Several combinations of three sequences can be used to trace the recombination at the DNA level. We used sequences Ag3, Ap5, and As26 as a triad that illustrates the recombination most clearly. The recombination fragment was defined as the region between sites 688 and 1105. Again, there was no continuous part of the DNA sequence that could produce a smaller postrecombination divergence, and only one out of 1,000 random combinations of nucleotide sites produced a lower value than the postulated recombination. As in the case of Rana, the sequences were divided into lengths of 50 nt for the homogeneity test. Again, all three comparisons produced the expected results (for Ap5 vs. Ap3, F(2,21), P = 0.244; for Ap5 vs. As26, P = 0.001; for As26 vs. Ap3, P = 0.040). The divergence between Ag3 and Ap5 is 0.184, and the postrecombination divergence is 0.149, which suggests that the recombination is 80% as old as the separation of the parental sequences.
The observation that recognizable amino acid domains with alternative consensus sequences occur in different combinations in a collection of sequences is in itself strong evidence for recombination. One could invoke some kind of within-domain convergent evolution to account for the observations, for example, that the first amino acid replacement in a domain may act as an agent for selective replacements in other sites. This type of selection, of interest in itself, cannot account for the nucleotide similarities. In particular, it cannot explain the parallel changes in amino acid similarities and similarities in synonymous sites (fig. 2B ). To reinforce this point and to make alternative hypotheses such as bias of codon usage less likely, we removed from each triad of sequences the codons responsible for variable amino acids and calculated nucleotide divergence on the truncated sequences. In all cases but one, the results were similar to those shown in the upper part of figure 2B ; i.e., for both the first and the second flanking regions, the smallest divergence was between sequences A and B, and for the middle region the smallest divergence was between sequences B and C. The exception was in the third region of the Apodemus triad, where the largest divergence occurred between As26 and Ap5. This exception is most likely due to the old age of the recombination event, which allowed for the accumulation of a large degree of divergence between shared sequence parts.
We conclude that amino acids simply provide stronger signals than nucleotides themselves of a process that causes regional homogenization of divergent DNA sequences. This process must be reciprocal homologous recombination, and not gene conversion alone. Evidence for this hypothesis comes from the case of Rana and Apodemus, in which all four combinations of consensus sequences for the first two amino acid domains can be found in the collection of sequences of figure 1
(e.g., Rd5, Rd4, Rr2, and Rr3 in Rana and As24, Ap5, As6, and Ag3 in Apodemus). The difference between the postrecombination divergence and the two nonrecombinant sequences (sequence A vs. sequence C; fig. 2A
) provides an estimate of the degree of divergence between sequences at the time of recombination. This degree is 0.13 for Gammarus, 0.03 for Apodemus, and 0.17 for Rana. Recombination between sequences with a higher degree of divergence would probably be unlikely to occur (Rayssiguier, Thayler, and Radman 1989
), whereas recombination between sequences with a lower degree would be difficult to detect.
The direct evidence of homologous recombination we have reported in mussels (Ladoukakis and Zouros 2001)
and the three cases of historical recombination we report here in three more species, each from a different major division of the animal kingdom, leave little doubt that recombination occurs in animal mtDNA. That we were able to come across three cases of recombination in the literature with not much searching effort suggests that many mtDNA lineages may contain evidence for recombination. This does not imply that it would be easy to estimate the frequency of mtDNA recombination. Extant recombinant sequences must represent only a small fraction of sequences generated by recombination. Yet, our observations help to reinforce the point that mtDNA genomes may recombine freely. This rarely results in new haplotypes owing to the limited frequency of heteroplasmy, which in turn is due to the rarity of biparental inheritance of animal mtDNA. The rate of paternal mtDNA "leakage" in animals has not been studied in a systematic way. The only crude estimates we have at present are 10-4 from laboratory crosses in mice (Gyllensten et al. 1991
) and 10-3 per fertilization in Drosophila hybrids (Kondo et al. 1990
; Kondo, Matsuura, and Chigusa 1992
). Finally, our results provide support for the claim of recombination in hominid mtDNA (Awadalla, Eyre-Walker, and Maynard Smith 1999
; Eyre-Walker, Smith, and Maynard Smith 1999
), with whatever consequences this may have on biohistorical inferences based on this genome.
Acknowledgements
We thank Drs. I. Karakassis and N. Primikirios for help.
Footnotes
Fumio Tajima, Reviewing Editor
1 Keywords: mtDNA recombination
COI
cyt b
Gammarus
Rana
Apodemus
2 Address for correspondence and reprints: Eleftherios Zouros, Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio Crete, Greece. zouros{at}imbc.gr
.
References
Awadalla P., A. Eyre-Walker, J. Maynard Smith, 1999 Linkage disequilibrium and recombination in hominid mitochondrial DNA Science 286:2524-2525
Eyre-Walker A., N. H. Smith, J. Maynard Smith, 1999 How clonal are human mitochondria? Proc. R. Soc. Lond. B Biol. Sci 266:477-483[ISI][Medline]
Gyllensten U., D. Wharton, A. Josefsson, A. C. Wilson, 1991 Paternal inheritance of mitochondrial DNA in mice Nature 352:255-257[ISI][Medline]
Jansa S. A., S. M. Goodman, P. K. Tucker, 1999 Molecular phylogeny and biogeography of Madagascar's native rodents (Muridae: Nesomyinae): a test of the single origin hypothesis Cladistics 15:253-270[ISI]
Jukes T. H., C. R. Cantor, 1969 Evolution of protein molecules Pp 21132 in H. N. Munro, ed. Mammalian protein metabolism. Academic Press, New York
Kim Y.-R., D.-E. Yang, H. Lee, J.-E. Lee, H.-I. Lee, S.-Y. Yang, H.-Y. Lee, 1999 Genetic differentiation of mitochondrial cytochrome b gene of the Korean Rana dybowskii (Amphibia: Ranidae) Korean J. Biol. Sci 3:199-205
Kimura M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]
Kondo R., E. T. Matsuura, S. I. Chigusa, 1992 Further observation of paternal transmission of Drosophila mitochondrial DNA by PCR selective amplification method Genet. Res 59:81-84[ISI][Medline]
Kondo R., Y. Satta, E. T. Matsuura, H. Ishiwa, N. Takahata, S. I. Chigusa, 1990 Incomplete maternal transmission of mitochondrial DNA in Drosophila Genetics 126:657-663
Ladoukakis E. D., E. Zouros, 2001 Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA Mol. Biol. Evol 18:1168-1175
Lee H.-I., D.-E. Yang, Y.-R. Kim, H. Lee, J.-E. Lee, S.-Y. Yang, H.-Y. Lee, 1999 Genetic variation of the mitochondrial cytochrome b sequence in Korean Rana rugosa (Amphibia; Ranidae) Korean J. Biol. Sci 3:89-96
Lee J.-E., D.-E. Yang, Y.-R. Kim, H. Lee, H.-I. Lee, S.-Y. Yang, H.-Y. Lee, 1999 Genetic relationships of Rana amurensis based on mitochondrial cytochrome b gene sequences Korean J. Biol. Sci 3:303-309
Martin Y., G. Gerlach, C. Schlotterer, A. Meyer, 2000 Molecular phylogeny of European muroid rodents based on complete cytochrome b sequences Mol. Phylogenet. Evol 16:37-47[ISI][Medline]
Meyran J. C., L. Gielly, P. Taberlet, 1998 Environmental calcium and mitochondrial DNA polymorphism among local populations of Gammarus fossarum (Crustacea, Amphipoda) Mol. Ecol 7:1391-1400[ISI]
Rayssiguier C., D. S. Thayler, M. Radman, 1989 The barrier to recombination between Escherichia coli and Salmonella typhymurium disrupted in mismatch-repair mutants Nature 342:396-401[ISI][Medline]
Serizawa K., H. Suzuki, K. Tsuchiya, 2000 A phylogenetic view on species radiation in Apodemus inferred from variation of nuclear and mitochondrial genes Biochem. Genet 38:27-40[ISI][Medline]
Sumida S., M. Ogata, M. Nishioka, 2000 Molecular phylogenetic relationships of pond frogs distributed in the Palearctic region inferred from DNA sequences of mitochondrial 12S ribosomal RNA and cytochrome b genes Mol. Phylogenet. Evol 16:278-285[ISI][Medline]
Suzuki H., K. Tsuchiya, N. Takezaki, 2000 A molecular phylogenetic framework for the Ryukyu endemic rodents Tokudaia osimensis and Diplothrix legata Mol. Phylogenet. Evol 15:15-24[ISI][Medline]
Zouros E., A. O. Ball, C. Saavedra, K. R. Freeman, 1994 Mitochondrial DNA inheritance Nature 368:818[ISI][Medline]