* Department of Biology, University of Oulu, Oulu, Finland
Institute of Zoology, Johannes Gutenberg University, Mainz, Germany
Institute of Biology and Soil Science, Far East Branch; Vladivostok, Russia
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Abstract |
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Key Words: heteroplasmy hybrid zone mitochondrial control region Parus major
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Introduction |
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Another possibility is that heteroplasmy may arise through paternal leakage, which is to say that the paternal mitochondria are not always eliminated during fertilization of an egg. Paternal leakage has been reported to occur from time to time in Drosophila, mice, mussels, and humans (Kondo et al. 1990; Gyllensten et al. 1991; Zouros et al. 1992; Schwartz and Vissing 2002, respectively). One explanation proposed to account for the rare detection of biparental inheritance of mitochondria is that the two haplotypes should be dissimilar enough to be detected during usual screening, e.g., for population genetic studies. Studies of hybrid zones, where populations harboring relatively genetically distant mitochondrial haplotypes meet, provide an ideal opportunity to search for paternal mitochondrial leakage. Here we present evidence that introgression and paternal leakage of mitochondrial haplotypes occurs in a hybrid zone where two subspecies groups of the great tit, Parus major, meet. To our knowledge this is the first report of paternal leakage in birds.
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Materials and Methods |
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We extracted DNA from blood samples using the standard phenol-chloroform procedure from a total of 27 great tits originating from four sampling sites in the middle Amur Valley. Of these birds, 15 were phenotypically major and 12 were minor birds or individuals close to those phenotypes. Amplification of the mitochondrial control region was performed with primers L16700 (5'ATCATAAATTCTCGCCGGGACTCT3') and H636 (5'GAGATGAGGAGTATTCAACCGAC3'). The amplified region covered all of the first domain and part of the second domain of the control region. Polymerase chain reaction (PCR) was performed in 50 µl volume containing about 250 ng of template DNA, 1.0 µM of each primer, 0.2 mM of each dNTP, 5 µl of 10x PCR buffer (2.5 mM MgCl2), and 1.0 unit of Dynazyme (Finnzymes). The amplification profile was 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min, and a final extension in 72°C for 5 min. Sequencing reactions were performed with the primer H636 with Big Dye Terminator Cycle Sequencing Kit version 2.0 and run with the ABI 377 automatic sequencer.
The sequence from one phenotypically major individual was repeatedly a mixture of minor and major haplotypes, making the sequence unreadable after a region where there were two indels (the one of a single and the other of two base pairs) between the different haplotypes. Therefore, new primer pairs were designed to amplify only the minor (L16700 + H328minor 5'GGGACATTA-TTCGTATACTGG3' and L288minor 5'CGTACAT-ACAAACTCCACCAG3' + H636) or major (L16700 + H351major 5'CTTTAGGAGGTGGGCTTCATGC3' and L288major 5'ACAAACTCCACTCTAGTATACGGA3' + H636) haplotypes. The PCR conditions were the same as described above.
Sequencing reactions were performed with primers H328minor (5' end of the minor control region), H351major (5' end of the major control region), or H636 (central part of major or minor control region). These primers produced pure major (GenBank accession number AF537976) and minor (GenBank accession number AF537975) sequences from this individual, from which both haplotypes were sequenced (altogether 578 bp) four times from independent PCRs. A maximum likelihood tree of the 28 sequences (GenBank accession numbers AF537962AF537989) was constructed using the program fastDNAml (Olsen et al. 1994), with a transition/transversion ratio of 11, empirical base frequencies, and 100 bootstraps.
In addition, to rule out a possible mixing of two samples in one, six polymorphic microsatellite loci (http://www.shef.ac.uk/misc/groups/molecol/Passerineprimers.xls) from the heteroplasmic bird were screened: Pdo5 (Griffith et al. 1999), Pocc6 (Bensch, Price, and Kohn 1997), Esc6 (Hanotte et al. 1994), PK12 (GenBank accession number AF041466), Ppi2 (Martinez et al. 1999), and Pca8 (Dawson et al. 2000). One specimen of a minor genotype and one of a major genotype were screened for comparison. Pdo5, Esc6, and Ppi2 were amplified in a 10 µl PCR containing 50 ng of template DNA, 0.4 µM of each primer, 0.1 mM of each dNTP, 1 µl of 10x PCR buffer (2.5 mM MgCl2), and 0.16 units of Dynazyme (Finnzymes) using the following profile: 94 for 2 min followed by 35 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for 45 s; and a final extension in 72°C for 2 min. Pocc6, Pca8, and PK12 were amplified similarly, except the annealing temperature was 55°C, and MgCl2 was 3.0 mM for Pca8 and 1.5 mM for PK12.
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Results and Discussion |
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Obviously, introgression of mitochondrial haplotypes must occur from one group to another as a result of hybridization. Finding a heteroplasmic bird having the two very distinct haplotypes of minor and major was, however, a surprise, because this kind of heteroplasmy very likely occurred by paternal leakage, not somatic mutations. Heteroplasmic conditions thus far reported from other bird species may have been generated by two distinct somatic mutational processes. Variable numbers of heteroplasmic tandem repeats, likely produced through slipped strand mispairing (Densmore, Wright, and Brown 1985), have been documented at least in the shrike (Lanius ludovicianus: Mundy, Winchell, and Woodruff 1996), in some auks, gulls, and a wader (family Laridae and Calidris maritima: Berg, Moum, and Johansen 1995), and in other gulls and some terns (genera Larus and Sterna: Crochet and Desmarais 2000). To our knowledge, the only bird species which has been shown to be heteroplasmic due to a single-site base substitution is the razorbill (Alca torda: Moum and Bakke 2001). In the case reported here, the difference between the two haplotypes of the heteroplasmic great tit is too large to be explained by somatic mutations within an individual, and it would be highly unlikely that such somatic mutations would result in a haplotype of a sympatric subspecies group.
Whether the mitochondrial DNA has leaked from the father of the bird, or whether such leakage occurred in previous generations and was followed by maternal transmission of the heteroplasmy, cannot be determined, even though the fact that the phenotype is of a major type supports an older introgression from minor to major. Some authors have reported that the proportion of different mitochondrial haplotypes estimated from all the offspring is about the same as the proportion in their heteroplasmic mother, but the level of heteroplasmy varies among the offspring (Chinnery et al. 2000 and references therein). Often, heteroplasmic conditions are resolved within one or few generations through a bottleneck during oogenesis, a process analogous to strong genetic drift (e.g., Ashley, Laipis, and Hauswirth 1989; Gocke, Benko, and Rogan 1998). The level of heteroplasmy also varies in different tissues, as seen, for example, in humans, where clinical features in the affected tissues differ, depending on the relative amounts of pathogenic mitochondria and normal mitochondria (Chinnery 2002; Schwartz and Vissing 2002). Unfortunately, there are no data from which to examine the transmission of mitochondria from parents to offspring in birds or to study the possible proportional differences of mitochondrial haplotypes in different tissues. In mice, there is some experimental evidence for persistent transmission of the leaked haplotype to subsequent generations (Gyllensten et al. 1991) and other evidence against ongoing transmission (Shitara et al. 1998). The proportion of leaked mitochondria in mice has been estimated from intraspecific crosses to be about 0.01% (Gyllensten et al. 1991). From crossing experiments of Drosophila simulans x D. mauritiana, the proportion of leaked paternal mtDNA per fertilization was estimated to be about 0.1%. In three of 331 lines, the maternal type was completely replaced by the paternal type of mitochondria, whereas a fourth line was heteroplasmic (Kondo et al. 1990).
Paternal leakage of mitochondria seems to be a widespread phenomenon among the animal phyla, being present at least in molluscs, insects, and vertebrates (mammals and birds). Further studies of hybrid zones could give more insight into the extent of paternal leakage in the animal kingdom, but estimation of the amount and persistence of leaked mitochondria would need controlled laboratory experiments. Paternal leakage of relatively distinct mtDNA also composes a framework for detection of possible mitochondrial recombination, a phenomenon which has recently been the subject of strong debate (Eyre-Walker and Awadalla 2001 and references therein).
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Acknowledgements |
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Footnotes |
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Adam Eyre-Walker, Associate Editor
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