*Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen;
Laboratoire Biologie Cellulaire 4, Université Paris-Sud
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Abstract |
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Introduction |
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Here, we provide the first direct evidence of recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus (Teleostei: Pleuronectiformes [flatfish]). In flounder, as in many other fishes (Nesbo, Arab, and Jakobsen 1998
, Ludwig et al. 2000
), the 5'-end of the mitochondrial control region is characterized by the presence of a variable number of tandem repeats (VNTRs) and a high level of length heteroplasmy for this region. The cause of variation in the repeat number is not fully understood but is variously attributed to slipped strand mispairing, illegitimate elongation, and termination-associated sequence (TAS)-based replication (Ludwig et al. 2000
). It may also be caused by mtDNA recombination, although this is more difficult to demonstrate when VNTRs consist of perfect repeats.
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Materials and Methods |
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Results and Discussion |
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Again from the same set of 18 individuals, 36 clones per individual were sequenced (total of 84) to characterize the array motifs themselves and their number. Two types of core repeats were detected, a "C" type and a "T" type, which differed by two point mutations (fig. 1 ). Among the 18 individuals, 13 contained only the pure "C" type array, 4 only the pure "T" type array, and 1 individual a compound array of "C" and "T" (fig. 1A, B, and C , respectively). The compound array is indicative of mtDNA recombination. It is highly unlikely that the compound array arose by mutation as the differences between the "C" type and the "T" type involve two independent mutations and neither seems to be associated with secondary structure such as hairpin (no palindrome in the sequence of the repeat unit). Moreover these two positions are highly conserved in the closely related species, Pleuronectes platessa (unpublished data).
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Further support for true recombination comes from the fact that the recombinant "CT" array was found in each of three independent DNA extractions, PCR, and sequencing reactions from the recombinant individual. In addition, the entire DNA extraction, amplification, cloning, and sequencing procedure was conducted in an independent laboratory where no studies on fish have ever been conducted. Twelve clones were sequenced and recombinant "CT" arrays were found in seven clones and a parental "T" was found in the five other clones (table 2
). Interestingly, only one of the "parental" types ("T") is found together with recombinant arrays. This suggests that recombination took place at least a generation ago and that the other "parental" type ("C") was lost by random drift during ontogenesis or gametogenesis (Chinnery et al. 2000
). Variation in the number of "C" or "T" repeats in recombinant arrays further suggests that other mechanisms, such as slipped strand mispairing, besides (or in addition to) recombination are involved in the evolution of the number of repeats.
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In principle, an alternative hypothesis to recombination could be that the compound array is the ancestral form and that the derived type of array we have observed evolved by a series of duplication-and-loss events. In this scenario, the two mutations (i.e., switching between the C to T type and vice versa) occurred in a duplicated region, such that both it and the ancestral form would be retained in the same individual. The different number of basic repeats could then be accounted for by a history of duplication and loss. Given the range in the number of repeat regions found (110), such duplications and losses might occur fairly regularly. However, we do not favor this alternative hypothesis because (1) if the "CT" arrays are indeed the ancestral form, then they should have been found more frequently in P. flesus and in closely related flatfish such as Pleuronectes platessa (plaice)plaice contains only one repeat, suggesting that the ancestral form was one repeat and (2) this hypothesis requires that the two mutations be retained in the same individual, whereas the recombination scenario requires only that the two types be present in the same population.
The mtDNA recombination we found between "C" and "T" type arrays in flounder has several implications for vertebrate mtDNA in general. Before such recombination is possible, the "C" and "T" type mitochondria must be present in the same cell and their DNA must be coupled. This implies paternal leakage followed by fusion of the mitochondria. Paternal leakage has been reported for mice (Gyllensten et al. 1991
) and anchovies (Magoulas and Zouros 1993
), whereas fusion of mitochondria has been demonstrated in Drosophila (Yaffe 1999
), and the enzymes necessary for recombination have been found in human mitochondria (Thyagarajan, Padua, and Campbell 1996
). It appears therefore that all these properties are present in flounder and in vertebrates more generally.
The importance of recombination in vertebrate mitochondria has broad implications across several fields, ranging from human mitochondrial diseases (Schon 2000
) to the compromise of phylogenetic and population studies that assume strict clonal inheritance of mtDNA (Schierup and Hein 2000
). In the case of human mitochondrial diseases, mtDNA recombination will greatly change the mode and patterns of inheritance, which in turn may affect current diagnostic methods. Recombination can also affect the accuracy of phylogenetic reconstruction (Posada and Crandall 2002
), inferences related to demographic history, and the application of molecular clocks (Schierup and Hein 2000
).
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Acknowledgements |
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Footnotes |
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Keywords: heteroplasmy
recombination
mitochondria
DLoop
VNTR
Platichthys flesus
Address for correspondence and reprints: Galice Hoarau, Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. E-mail: g.hoarau{at}biol.rug.nl
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