Originally published as Genetics Published Articles Ahead of Print on May 27, 2008.

Genetics, Vol. 179, 1029-1032, June 2008, Copyright © 2008
doi:10.1534/genetics.107.085274

Delimiting the Frequency of Paternal Leakage of Mitochondrial DNA in Chinook Salmon

Jonci N. Wolff*,1, Sandra Gandre{dagger}, Aleksander Kalinin* and Neil J. Gemmell*

* School of Biological Sciences, University of Canterbury, Christchurch 8041, New Zealand and {dagger} Department of Biotechnology, University of Applied Sciences, Mannheim 68163, Germany

1 Corresponding author: School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8001, New Zealand.
E-mail: jonci.wolff{at}pg.canterbury.ac.nz

Manuscript received December 2, 2007. Accepted for publication April 14, 2008.

ABSTRACT
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 >ABSTRACT
ACKNOWLEDGEMENTS
 LITERATURE CITED

We analyzed embryos of a wild-return hatchery population of chinook salmon for the presence of paternal mtDNA. None of the 10,082 offspring examined revealed paternally transmitted DNA, delimiting the maximum frequency of paternal leakage in this system to 0.03% (power of 0.95) and 0.05% (power of 0.99).

THE absence of both paternal transmission (paternal leakage) and heterologous recombination of animal mitochondrial DNA (mtDNA) are believed to be cornerstones of mtDNA inheritance (BIRKY 1995; BARR et al. 2005). These features combined with its small size (generally 15–20 kb), high copy number, and higher mutation rate (compared to nuclear genes) have greatly facilitated the investigation of complex genetic ancestries and phylogeographic or phylogenetic patterns (BIRKY et al. 1983; AVISE et al. 1987; MORITZ et al. 1987; BIRKY 2001; SLATE and GEMMELL 2004).

In recent years, however, there has been increasing evidence for paternal leakage and recombination of mtDNA in a wide range of animal species. Paternal leakage has been documented in at least 15 species (summarized in FONTAINE et al. 2007; see also ZOUROS et al. 1992; GUO et al. 2006; BRETON et al. 2007; THEOLOGIDIS et al. 2007) and recombination in at least 11 species (LUNT and HYMAN 1997; LADOUKAKIS and ZOUROS 2001; HOARAU et al. 2002; KRAYTSBERG et al. 2004; PIGANEAU and EYRE-WALKER 2004; GANTENBEIN et al. 2005; TSAOUSIS et al. 2005; GUO et al. 2006; ARMSTRONG et al. 2007; CIBOROWSKI et al. 2007; UJVARI et al. 2007), spanning highly divergent taxa including mammals, mollusks, reptiles, birds, fish, flatworms, and arthropods. Although the detected cases of either paternal leakage or recombination are assumed to be exceptions to the general rule, the increasing number of these events clearly questions our current understanding of mitochondrial inheritance and the frequency of paternal leakage in particular.

The occurrence of both paternal leakage and recombination of mtDNA in the animal kingdom has potentially substantial implications for traditional phylogenetic analysis (SCHIERUP and HEIN 2000). For example, assuming a molecular clock based on a linear rate of accumulating mutations over evolutionary time would lead to erroneous estimates if analyzed mitochondrial data sets contained sequences influenced by either event by increasing the number of potential mutations and haplotypes. Ignoring undetected recombination in genealogies can lead to underestimates of times of divergence and overestimates of the number of mutations and population size (EYRE-WALKER 2000; SCHIERUP and HEIN 2000; SLATE and GEMMELL 2004). It is therefore vital to determine at what frequency these events may occur, so that models of mtDNA evolution can be improved to estimate better evolutionary relationships and times of divergence.

Previous studies estimating the frequency of paternal leakage were greatly influenced by inbreeding and backcrossing (KONDO et al. 1990; GYLLENSTEN et al. 1991; SHITARA et al. 1998; SHERENGUL et al. 2006), crossing regimes that are assumed to promote paternal leakage (KANEDA et al. 1995; SUTOVSKY et al. 2000; SHERENGUL et al. 2006). Other studies detected paternal leakage but neglected to estimate how frequently this might occur (MEUSEL and MORITZ 1993; KVIST et al. 2003; GANTENBEIN et al. 2005; FONTAINE et al. 2007). Additionally, samples of at least 300 progeny with no detected paternal mtDNA are required to correctly delimit its frequency to 1% (MILLIGAN 1992) and failures to detect paternal leakage in previous studies were probably attributable to the use of low sample sizes. Here, we report the first large-scale study to systematically estimate the frequency of paternal leakage within a species under semiwild conditions, with potentially confounding factors such as inbreeding, backcrossing, and hybridization eliminated. To achieve this, we analyzed 10,082 embryos of a wild-return hatchery population of chinook salmon, generated through artificial fertilization, for the presence of paternal mtDNA. Previous work on this hatchery population revealed the presence of 36 single nucleotide polymorphisms (SNPs) within the mitochondrial genes mt-nd1–mt-nd5 (our unpublished data). Each of the five haplogroups present in this population contains at least one SNP that is unique for the corresponding haplogroup (diagnostic SNP). Nucleotide positions of diagnostic SNPs examined are 3957, 5842, 10,650, and 10,725 (hereafter single SNPs are referred to as nt3957, nt5842, nt10650, and nt10725) of the chinook mitochondrial genome (NCBI: NC_002980). The presence of diagnostic SNPs in all haplogroups allows for unambiguous determination of mtDNA origin in offspring (i.e., paternally or maternally derived) enabling us to readily investigate patterns of mitochondrial inheritance in this system. Furthermore, as both paternal leakage and recombination have been detected in a related teleost, the Atlantic salmon (CIBOROWSKI et al. 2007), this system appeared to be suitable for the detection of paternal leakage.

The experimental approach for the detection of paternal mtDNA at low copy numbers was specifically developed for this study to ensure highest possible sensitivity combined with the ability to process large sample sizes (WOLFF and GEMMELL 2008). The use of conventional allele-specific primers was not applicable due to minimal sequence differences (1 bp) between paternal and maternal alleles. Experiments with allele-specific primers designed for each diagnostic SNP prior to this study resulted repeatedly in crossannealing and amplification of maternal mtDNA with male-specific primers (data not shown).

Twelve independent fertilization experiments between individuals of different haplogroups were performed and each experiment generated on average 840 progeny, with 357 as the smallest and 1677 the largest number of offspring generated in a single fertilization experiment (Table 1). Because only a limited number of fish return to the hatchery each year, individuals for these experiments were chosen according to their availability and an even representation of all haplogroups could not be accounted for, introducing potential biases toward the overrepresentation of single haplogroups. A total of 10,082 samples have been genotyped, of which 7188 samples were examined with a detection limit of 1:64 and 2895 samples were analyzed with a detection limit of 1:2048. The difference in detection limit resulted from SNP-specific deviations of the genotyping experiments (WOLFF and GEMMELL 2008). In none of the 10,082 samples examined paternally derived mtDNA was detected.


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TABLE 1

Estimated maximum frequency of paternal leakage per cross

 
On the basis of the genotyping results, we calculated the maximum frequency at which paternal leakage can be excluded to occur (Table 1). Table 1 shows a summary of all genotyping experiments and the maximum frequencies of paternal leakage in relation to sample sizes of single fertilization experiments and the power of the test. The highest frequencies estimated in this study where 0.84% (power of 0.95) and 1.28% (power of 0.99) for a sample size of 357 and the lowest frequencies were 0.18% (power of 0.95) and 0.27% (power of 0.99) for a sample size of 1677. If sample sizes of single experiments are combined, the maximum frequencies at which paternal leakage can be excluded to occur according to a sample size of 10,082 offspring are 0.03% (power of 0.95) and 0.05% (power of 0.99).

The failure to detect paternally derived mtDNA in the samples investigated does not invariably exclude the potential presence of paternal mtDNA. First, the presence of paternally derived mtDNA cannot be excluded at ratios beyond the detection limits of 1:64 and 1:2048. Second, as the inheritance of paternal mtDNA can be tissue specific (SCHWARTZ and VISSING 2002), the detection of paternal mtDNA (if present) in tissues other than those investigated here might remain undetected. Nevertheless, as DNA extractions investigated here were made from tail tips (which contain tissues from all three germ layers) and with the exception of several bivalves (BRETON et al. 2007) the inheritance of paternal mtDNA does not appear to be tissue specific, paternal mtDNA was expected to be detected if present in somatic tissue.

The cases of paternal leakage documented in the literature cover highly divergent taxa, indicating that this phenomenon is not limited to single taxa or species. The occasional occurrence of paternal leakage is assumed to be due to a failure of those mechanisms that prevent paternal leakage. Interestingly, 12 of the 15 documented cases of paternal leakage were observed in hybrid zones, an environment where these mechanisms are assumed to be more relaxed and less stringent (ROKAS et al. 2003). In fact, fertilization experiments in fruit flies and cattle showed that paternal leakage occurs at a significantly higher frequency in hybridization experiments compared to intraspecific crosses (SUTOVSKY et al. 2000; SHERENGUL et al. 2006). SUTOVSKY et al. (2000) demonstrated that the ubiquitination preceding the proteolytic degradation of mammalian sperm mitochondria upon fertilization is not detectable in bovine hybrids whereas this process is reliable in intraspecific experiments (SUTOVSKY et al. 2000). The absence of this process in hybrids might be due to interspecific sequence differences and therefore to differences in the amino acid sequence of proteins catalyzing this process, disabling, for example, the recognition of sperm mitochondria (SUTOVSKY et al. 2000).

However, the remaining 3 of 15 documented cases of paternal leakage occurred either in intraspecific fertilization experiments or in natural populations (MAGOULAS and ZOUROS 1993; SCHWARTZ and VISSING 2002; SHERENGUL et al. 2006) demonstrating that this phenomenon is not exclusively limited to hybrid zones. Why mechanisms to prevent paternal leakage fail here can only be the subject of speculation. The occurrence of paternal leakage might be simply a quantitative phenomenon, occurring accidentally in relation to time and number of fertilization events. The increasing number of revealed incidents in recent years is potentially due to advances in technology, such as (i) increased sensitivity for minor allele contributions, (ii) the capability of single techniques to process large sample sizes, (iii) increased generation of mtDNA sequence data, and therefore (iv) increased gene coverage. In combination, such advances may allow for the detection of even rare phenomena, such as paternal leakage.

We did not detect any incidence of parental leakage of mtDNA in our study and have been able to define an upper limit for the frequency of such an event to 0.03–0.05%. However, while our result was negative, the increasing reports of paternal leakage substantiate its occurrence beyond any doubt and it now should be considered as an ongoing part of animal mitochondrial inheritance. The potential incidence of this phenomenon (with particular attention paid to hybrid zones) and its influence on subsequent analyses must be considered if mtDNA is applied as a molecular marker (EYRE-WALKER 2000; SCHIERUP and HEIN 2000; SLATE and GEMMELL 2004). The question that needs to be answered now is no longer about the general existence of paternal leakage but about its frequency in the population and species of interest, and if these are frequent enough, such leakage events may need to be explicitly considered in models of mtDNA evolution.


ACKNOWLEDGEMENTS
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 ABSTRACT
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LITERATURE CITED
We thank the NIWA Silverstream Hatchery team for their support, advice, and use of their facilities and present and past members of our lab for field assistance, in particular, Victoria Metcalf, Thorsten Horn, and Patrice Rosengrave. We also thank Dan White and Jawad Abdelkrim for valuable comments on the manuscript. This study was funded by the Royal Society of New Zealand, Marsden Fund grant UOC402. Approval of our work was obtained from all appropriate ethical and regulatory bodies, principally the Environmental Risk Management Authority (GMD00066), Ministry of Agriculture and Forestry (2003018889), our local animal ethics board, and the Fish and Game Council of New Zealand. J.N.W. was supported by the University of Canterbury Scholarships Board.


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Communicating editor: H. G. SPENCER