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Mitonuclear linkage disequilibrium in
human populations
Daniel B. Sloan1, Peter D. Fields2 and Justin C. Havird1
1
Research
Cite this article: Sloan DB, Fields PD, Havird
JC. 2015 Mitonuclear linkage disequilibrium in
human populations. Proc. R. Soc. B 282:
20151704.
http://dx.doi.org/10.1098/rspb.2015.1704
Received: 16 July 2015
Accepted: 27 August 2015
Subject Areas:
genetics
Keywords:
assisted reproductive technologies, coevolution,
cytonuclear interactions, mitochondrial
genome, mtDNA, sex chromosomes
2
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
Zoological Institute, University of Basel, Vesalgasse 1, Basel, 4051, Switzerland
There is extensive evidence from model systems that disrupting associations
between co-adapted mitochondrial and nuclear genotypes can lead to deleterious and even lethal consequences. While it is tempting to extrapolate
from these observations and make inferences about the human-health effects
of altering mitonuclear associations, the importance of such associations
may vary greatly among species, depending on population genetics, demographic history and other factors. Remarkably, despite the extensive study of
human population genetics, the statistical associations between nuclear and
mitochondrial alleles remain largely uninvestigated. We analysed published
population genomic data to test for signatures of historical selection to maintain mitonuclear associations, particularly those involving nuclear genes that
encode mitochondrial-localized proteins (N-mt genes). We found that significant mitonuclear linkage disequilibrium (LD) exists throughout the human
genome, but these associations were generally weak, which is consistent
with the paucity of population genetic structure in humans. Although mitonuclear LD varied among genomic regions (with especially high levels on
the X chromosome), N-mt genes were statistically indistinguishable from
background levels, suggesting that selection on mitonuclear epistasis has not
preferentially maintained associations involving this set of loci at a specieswide level. We discuss these findings in the context of the ongoing debate
over mitochondrial replacement therapy.
1. Introduction
Author for correspondence:
Daniel B. Sloan
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.1704 or
via http://rspb.royalsocietypublishing.org.
The biology of eukaryotic cells depends upon highly coevolved interactions
between nuclear and mitochondrial genomes [1,2]. There is a growing body of
evidence that disrupting these interactions has harmful consequences for organismal fitness [3–5]. In fact, incompatibilities between interacting nuclear and
mitochondrial genes from diverging populations may represent one of the primary mechanisms responsible for reproductive isolation and the origin of
species [6,7]. With increasing awareness about the biomedical importance of mitochondrial DNA [8–10], there is also greater interest in how epistatic interactions
between mitochondrial variants and the nucleus may affect human health
[11,12]. There are numerous examples in which the severity of mitochondrial genetic diseases is dependent upon the nuclear background or vice versa [13–24], and
the use of human cell lines to generate cytoplasmic hybrids (cybrids) has provided
additional evidence for mitonuclear interactions [25–29]. To our knowledge,
however, there are no documented cases within humans of true mitonuclear mismatch, in which mitochondrial haplotypes have been shown to be superior on
their own ‘native’ nuclear backgrounds and compromised when found on foreign
nuclear backgrounds (i.e. sign epistasis; figure 1).
Interest in the effects of mitonuclear interactions on human health has
become particularly acute in recent years because of the controversy surrounding the approval of mitochondrial replacement (MR) therapy as an assisted
reproductive technology in the UK [30]. MR therapy aims to prevent the transmission of diseases resulting from mitochondrial mutations by transferring the
nuclear genetic material from an affected egg, polar body or fertilized zygote
into a healthy donor’s cytoplasm [31–35]. Much of the controversy about
these techniques has centred on ethical concerns and the potential side effects
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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fitness/health outcome
mitochondrial
haplotype 2
Figure 1. Illustration of sign epistasis in mitonuclear interactions. The
coloured organelles reflect the concept of matched and mismatched pairings
between mitochondria and nuclei.
associated with physical manipulation of reproductive cells,
but an additional concern has emerged from the field of evolutionary biology—specifically that MR could produce genetic
incompatibilities associated with novel pairings of nuclear
and mitochondrial genotypes [36 –38].
The idea that MR causes increased risk of genetic incompatibilities is supported by genetic crossing and cybrid studies in
model systems, including yeast, fruit flies, copepods and
rodents [39–47]. In these and other eukaryotic lineages, mitochondrial-targeted proteins such as OXPHOS complex
subunits [48–50], ribosomal subunits [51,52], tRNA synthetases [44] and RNA polymerases [53] have been found to
exhibit incompatibilities with certain mitochondrial backgrounds and/or accelerated evolution indicative of positive
selection and compensatory mitonuclear coevolution. One of
the more comprehensive bodies of work has established the
copepod Tigriopus californicus as a model for studying mitonuclear incompatibilities [54] and left little doubt that performing
MR as an assisted reproductive technique in T. californicus
could cause more health problems for the copepods than it
would fix. Of course, humans are not copepods and, unfortunately, questions about the health effects of disrupting
mitonuclear interactions in humans are less clear-cut. There is
difficulty in generalizing the results from model systems
because the extent of mitonuclear co-adaptation is likely to
be highly species-specific and depend on population genetic
factors such as mitochondrial and nuclear mutation rates, effective population size, mating system, generation time and
population subdivision [55–59]. There is some reason to
think that mitonuclear co-adaptation within human populations may be less extensive because of recent demographic
expansion and a relatively small amount of genetic structure
[60–62], but the controversy over MR has thus far received
little scrutiny through the lens of human population genetics.
A history of selection favouring specific combinations of
mitochondrial and nuclear alleles should leave detectable signatures in the genetic diversity of a species [63–65]. For
example, we would expect N-mt genes to be subject to more
intense selection for efficient mitonuclear interactions than
other nuclear genes and, therefore, to exhibit stronger statistical
associations with the mitochondrial genome (mitonuclear linkage disequilibrium, LD). Such population genetic information
2. Material and methods
(a) Human population genomic data
Genotypic data were obtained from the Human Genome Diversity Project (HGDP) [60]. This dataset comprises 660 918 single
nucleotide polymorphism (SNP) genotypes for each of 1043 individuals, including 163 SNPs from the mitochondrial genome.
The sampled individuals are broadly representative of human
genetic and geographical diversity, encompassing 51 different
populations [75]. We restricted our analysis to 585 300 nuclear
SNPs with a minor allele frequency of at least 5% to avoid
biases associated with measures of LD between loci with rare
alleles (electronic supplementary material, figure S1) [76].
The chromosome and nucleotide position for each SNP were
extracted from the SNP142 database obtained from the UCSC
human genome downloads site [77]. SNPs found in coding
sequences, introns, untranslated regions, or 50 and 30 flanking
regions were associated with corresponding Entrez Gene IDs
using SNPnexus [78] with the GRCh37/h19 human genome
assembly. N-mt genes were identified based on the human MitoCarta classification [79]. In addition, we manually selected a
smaller set of N-mt genes which code for proteins that form
multi-subunit complexes with mitochondrial-encoded gene products or directly interact with mitochondrial DNA or RNAs.
This subset included genes that encode OXPHOS subunits, ribosomal proteins, tRNA synthetases, and DNA and RNA polymerases
(electronic supplementary material, table S1). In total, 237 156
SNPs were assigned to genes in the MitoCarta dataset. Of those,
7677 were assigned to the entire set of MitoCarta N-mt genes,
and 754 were found in the more targeted subset.
(b) Determination of mitochondrial haplogroups
Illumina SNP genotyping probe sequences were obtained from the
NCBI Probe Database and used to identify nucleotide positions for
each mitochondrial SNP, which were then converted to the position numbering for the revised Cambridge Reference Sequence
(rCRS) [80]. The resulting SNP data were analysed with HAPLOGREP v. 2.0 Beta [81] with build 16 of PHYLOTREE [82] to assign a
mitochondrial haplogroup for each individual (electronic supplementary material, table S2). Haplogroups with fewer than
20 individuals were combined with closely related haplogroup(s)
following the mitochondrial genealogy described by van Oven &
Kayser [82] (electronic supplementary material, table S3). The L5
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Proc. R. Soc. B 282: 20151704
mitochondrial
haplotype 1
could offer a valuable complement to phenotypic and biomedical data. It has been proposed that human mitonuclear
incompatibilities are often cryptic, with poorly matched mitonuclear combinations being eliminated during oogenesis [38].
While such a mechanism would preclude directly observing
the phenotypic consequences of mitonuclear mismatch, the
elimination of specific mitonuclear genotypes from the population by natural selection would still alter the statistical
associations between mitochondrial and nuclear loci and potentially leave detectable footprints in patterns of mitonuclear LD.
The recent flood of human population genomic data has
been accompanied by detailed analysis of LD throughout the
nuclear genome [60,66–70] as well as phylogeographic and
functional analysis of variation in the mitochondrial genome
[71–74]. Remarkably, however, genome-wide patterns of mitonuclear LD remain almost entirely unexplored. Here, we take
advantage of existing human population genomic data to analyse patterns of mitonuclear LD and to test hypotheses about
the history of selection on mitonuclear interactions—a pertinent topic in light of the current controversy surrounding MR.
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nuclear background 1
nuclear background 2
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(d) Analysis of mitonuclear linkage disequilibrium
A custom Perl script was used to calculate LD between each
nuclear SNP locus and the mitochondrial genome (electronic supplementary material, file S1). Because there are more than two
mitochondrial alleles (haplogroups), we calculated multi-allelic
LD using the following measures as defined by Zhao et al. [76]:
D0 , r 2, D*, x 2df and x 20 . We accounted for differences in ploidy
between autosomes, sex chromosomes and the mitochondrial
genome by randomly sampling one allele per individual for
each diploid nuclear locus. D0 was only moderately correlated
with each of the other measures of LD (r , 0.5), reflecting
known differences in how low-frequency genotype combinations
differentially affect these measures [76]. The other four LD
measures were highly correlated with each other (r . 0.9). Therefore, for subsequent analyses, we considered D0 and only one of
the four other measures (r 2). D0 is the standard LD coefficient
(D) scaled to be between 0 and 1 based on the maximum possible
absolute value of D for the given allele frequencies [85]. By contrast, r 2 is the square of the correlation coefficient between a
pair of loci and represents the amount of predictive information
that one locus can provide about another [67,76,86].
For each nuclear SNP, the significance of the observed association between mitochondrial haplogroups and nuclear alleles was
assessed with a Fisher’s exact test, which was implemented in R
v. 3.1.2 [87], using the fisher.test function with p-values calculated by Monte Carlo simulations with 105 replicates. The false
discovery rate (FDR) for identifying non-random mitonuclear
associations was calculated with a Benjamini–Hochberg step-up
procedure [88]. Mitonuclear LD values were calculated for each
gene by averaging D0 and r 2 estimates across all SNPs associated
with that gene. ANOVAs were implemented in R to test whether
chromosomes maintain significantly different levels of mitonuclear LD based on mean values from all genes containing a
minimum of five SNPs. These values were log-transformed to
meet normality assumptions. We also performed Fisher’s exact
tests to determine whether chromosomes differed significantly
in the proportion of genes that were found in the upper 5% tail
of mitonuclear LD values across the entire genome. Because of
the small number of Y-linked SNPs in the HGDP dataset, the
Y chromosome was excluded from these analyses.
(e) Functional enrichment analysis
To test the hypothesis that selection on co-adapted mitonuclear
interactions has preferentially maintained LD between N-mt
genes and the mitochondrial genome, we used t-tests
implemented in R to compare log-transformed levels of mitonuclear LD (D0 and r 2) between N-mt genes and all other genes. We
also performed Fisher’s exact tests to determine whether N-mt
genes were overrepresented in the 5% tail of the distribution of
mitonuclear LD values. These tests were performed with the
entire set of N-mt genes and with the targeted subset of N-mt
0.05
0
J
HV*/V
H
−0.05
−0.05
0
0.05
nuclear PCA 1
0.10
Figure 2. Principal component analysis (PCA) of SNPs in N-mt genes. Individuals are colour-coded based on their mitochondrial haplogroups. The
N group consists of haplogroups N, N*, I, W, Y, A, S, X and O.
genes (electronic supplementary material, table S1) that were
chosen based on their more intimate molecular interactions with
mitochondrial-encoded gene products or the mitochondrial
genome itself. To identify any functional characteristics that are
over-represented in genes with high levels of mitonuclear LD,
we performed a gene ontology (GO) enrichment analysis with
the DAVID 6.7 web server [89]. We used the list of genes with the
top 5% of r2-values as the test set for this analysis. We restricted
each of the above analyses to genes with a minimum of five SNPs.
3. Results
SNP genotypes from 1041 geographically and genetically
diverse individuals revealed that mitonuclear LD is prevalent
throughout the human genome. The vast majority (89%) of
nuclear SNPs exhibited a significantly non-random association with mitochondrial haplogroups at an FDR threshold
of 0.05, and genome-wide measures of genetic diversity
tended to cluster with mitochondrial haplogroups (figure 2).
Although significant, the observed levels of mitonuclear LD
were generally low. The median and mean values of D0
were 0.2360 and 0.2541, respectively. Because the D0 statistic
is known to yield inflated values when certain genotypic combinations are rare or unobserved [76], r 2 is probably the more
informative measure of LD in this case. The median and mean
values of r 2 were only 0.0033 and 0.0041, respectively, with a
maximum observed value of 0.0304 (SNP rs8179271).
The magnitude of mitonuclear LD varied across different
regions of the genome (figure 3). There were significant differences among chromosomes for gene-averaged values of both
D0 and r 2 (p 0:001 for both measures; ANOVA). Most notably,
levels of mitonuclear LD were substantially higher in X-linked
genes (mean D0 ¼ 0.31; mean r 2 ¼ 0.0062) than autosomal
genes (figures 3 and 4) (mean D0 ¼ 0.26; mean r 2 ¼ 0.0041).
There were also significant (albeit smaller) differences among
autosomes for both D0 and r 2 (p 0:001 for both measures;
ANOVA) (electronic supplementary material, table S4). On
average, genes on chromosome 15 exhibited the strongest
mitonuclear LD (mean D0 ¼ 0.272; mean r 2 ¼ 0.0048), while
genes on chromosome 19 (mean D0 ¼ 0.250; mean r 2 ¼ 0.0040)
and chromosome 14 (mean D0 ¼ 0.252; mean r 2 ¼ 0.0039) had
the weakest associations in terms of D0 and r 2, respectively.
We did not find evidence that levels of mitonuclear LD
were significantly different for N-mt genes than for the rest
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Proc. R. Soc. B 282: 20151704
To provide a visual clustering of nuclear genetic diversity to
compare with mitochondrial haplotype variation, a principal
component analysis of all nuclear SNPs was performed using
SmartPCA, which is part of the EIGENSOFT v. 6.0.1 package [83].
We employed default settings for the number of eigenvectors calculated and for the removal of outlier individuals. Formatting for
the HGDP data for input into SmartPCA was performed with a
custom bash script and PLINK v. 1.07 [84].
nuclear PCA 2
(c) Principal component analysis
L0
L1
L2
L3*
M/M9/E/G
M*
C/M8/Z
D
Q
N
R*/P
B
F/R9
K/U8
U*
U
T
0.10
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haplogroup was excluded from subsequent analyses, because it
contained only two individuals in the HGDP dataset, and its position within the human mitochondrial genealogy precludes
merging it with any other haplogroups.
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4
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mitonuclear LD (r 2)
0.03
0.02
0.01
16
17
18
19
20
21
22
X
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
chromosome position
Figure 3. Variation in mitonuclear LD across the nuclear genome. Only SNPs associated with annotated genes are plotted, with those in N-mt genes shown in red.
0.006
r 2 0.005
autosomes
0.004
frequency (N-mt genes)
X chromosome
6000
70 000
SNPs in N-mt genes
SNPs in other genes
60 000
5000
50 000
4000
40 000
3000
30 000
2000
20 000
1000
10 000
0
0.0001
frequency (other genes)
7000
0.007
0
0.001
0.01
0.1
r2
0.003
0.20
0.25
0.30
D¢
0.35
0.40
Figure 4. Mitonuclear LD averaged by chromosome. Each point represents a
chromosome, and LD statistics (D0 and r 2) are calculated based on the mean
of all genes with at least five SNPs.
of the genome. Epistatic selection on mitonuclear interactions
is expected to preferentially involve nuclear-encoded proteins
that are targeted to mitochondria, yielding the prediction that
N-mt genes would exhibit elevated levels of mitonuclear LD.
However, this was not observed when LD was measured
across the global sample of individuals that make up the
HGDP dataset. Gene-averaged levels of mitonuclear LD
were nearly identical between N-mt genes (mean D0 ¼ 0.260;
mean r 2 ¼ 0.00419) and the rest of the genes in the genome
(mean D0 ¼ 0.258; mean r 2 ¼ 0.00419), resulting in non-significant differences for both D0 and r 2 ( p ¼ 0.83 and 0.54,
respectively; ANOVA). N-mt genes were slightly overrepresented in the set of ‘outlier’ genes with the highest levels of
mitonuclear LD, with 5.8 and 5.3% of N-mt genes being
found in the 5% upper tail of the mitonuclear LD distribution
based on D0 or r 2, respectively, but these values did not deviate significantly from the random expectation ( p ¼ 0.48 and
0.72 for D0 and r 2, respectively). Overall, N-mt genes were
statistically indistinguishable from the rest of the genome
with respect to their mitonuclear LD distributions (figures 3
and 5). Restricting these analyses to N-mt genes that are
expected to be involved in the most direct interactions with
the mitochondrial genome and its gene products (electronic
supplementary material, table S1) also yielded non-significant
Figure 5. Frequency distribution of mitonuclear LD (r 2) for SNPs in N-mt
genes and other genes that are not targeted to the mitochondria.
results (electronic supplementary material, table S5). There is
a potential for bias in these analyses because mitonuclear
LD levels differ substantially between the X chromosome
and the autosomes (figures 3 and 4), and it has been shown
that N-mt genes are under-represented on the mammalian X
chromosome [90]. To account for this bias, we repeated the
analysis of N-mt genes after excluding the X chromosome
and found no qualitative change in the results.
In the absence of a detectable effect associated with N-mt
genes, we considered the possibility that other functional
characteristics could be responsible for variation in levels of mitonuclear LD among genes. However, the subset of genes with the
top 5% highest levels of mitonuclear LD was not significantly
enriched for terms in any of the three GO categories (biological
processes, cellular components or molecular functions).
4. Discussion
(a) Human mitonuclear linkage disequilibrium
For mitochondrial and nuclear loci (or any loci that are not
physically linked), the maintenance of LD requires either
epistatic selection for certain combinations of alleles or nonrandom mating. In the absence of these forces, existing LD
would decay rapidly (50% loss each generation) [91]. By
itself, selection for specific combinations of alleles would
have to be very strong to counteract this decay and maintain
significant LD. Although it is conceivable that loci across the
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0
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The development and approval of MR as a clinical treatment
has reaffirmed the importance of understanding mitonuclear
interactions in humans [30–35]. One source of controversy surrounding this technology is its potential to create maladapted
pairings of mitochondrial and nuclear alleles, which is
5
Proc. R. Soc. B 282: 20151704
(b) Mitonuclear interactions and the risks of
mitochondrial replacement therapy
viewed as a serious concern by some researchers [36–38] but
has been dismissed by others based on the limited evidence
for such incompatibilities in humans and the fact that the genetic shuffling resulting from MR is similar (though not identical
[38]) to the normal results of biparental sexual reproduction
[62,97,98]. We propose that taking a species’ population genetic
history into account is important when assessing the potential
for mitonuclear interactions and incompatibilities. Our analysis
revealed significant non-random associations between mitochondrial haplotypes and nuclear alleles throughout the
human genome. In principle, these findings lend credibility to
the argument that MR has the potential to disrupt co-adapted
mitonuclear interactions. However, as noted above, the statistical associations were generally weak as would be expected
given the limited amounts of population genetic structure
found in humans [60,61] and the fact that mitochondrial haplotypes are only partially informative in predicting continental
ancestry [93] or controlling for population stratification in
association studies [99,100].
To further assess the importance of mitonuclear coevolution in human populations, we hypothesized that the effects
of epistatic selection (specifically mitonuclear LD) would
vary across the genome and be most pronounced in N-mt
genes. The logic behind this prediction is that selection for efficient mitonuclear interactions would: (i) disproportionately
affect genes which encode proteins that are targeted to the
mitochondria and involved in direct mitonuclear interactions,
(ii) lead to faster sorting of ancestral genetic diversity among
isolated populations at these loci, and (iii) preferentially
restrict introgression at these loci during periods of genetic
admixture. This line of logic is consistent with the observation
that N-mt genes can exhibit steep clines in hybrid zones that
mirror the distribution of mitochondrial genetic variation
[101,102] and that mitonuclear interactions play a disproportionate role in generating reproductive isolation between
species [6,7].
We did not detect elevated levels of mitonuclear LD for
N-mt genes, indicating that selection has not systematically
maintained stronger mitonuclear associations involving
these loci when assessed at the global scale of human genetic
diversity. However, there are reasons that this test may be
conservative, which point to important avenues for future
investigation. First, our separation of nuclear genes into two
categories (N-mt and ‘other’) is surely an oversimplification
that may mask more subtle variation among genes. Given
the extent to which mitochondria are integrated into the
broader metabolic, signalling and regulatory pathways
within the eukaryotic cell, it is possible that gene products
that are not physically targeted to the mitochondria could
still create epistatic fitness effects that depend on the mitochondrial background, thereby reducing any signal that
differentiates N-mt genes from the rest of the genome.
Second, major mitonuclear incompatibilities can result from
individual substitutions in a single N-mt gene. For example,
Meiklejohn et al. [44] identified mitonuclear incompatibility
with major fitness consequences in Drosophila melanogaster
that is based on a SNP in a mitochondrial-encoded tRNA
gene and a single amino acid polymorphism in the corresponding N-mt tRNA synthetase gene. Such effects that are
localized to a single gene may get lost in the noise when
pooled with hundreds of other N-mt genes in the genome.
Therefore, even though we did not detect greater mitonuclear
LD in N-mt genes, the genes exhibiting the highest levels
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entire nuclear genome are all involved in epistatic interactions
with the mitochondrial genome that produce such large fitness effects, we consider this very unlikely. Instead, the
prevalence of significant mitonuclear LD throughout the
entire human genome almost certainly reflects non-random
mating resulting from the history of isolation between
human populations [60,61].
There is obvious genetic differentiation among human
populations, but levels of population structure are generally
low [60,61]. In an analysis of the same HGDP dataset, Li et al.
[60] found that the among-population component represents
only 9% of total human genetic diversity. Population structure
is more pronounced for genetic variation in the mitochondrial
genome. Because of its asexual and effectively haploid mode of
inheritance, the mitochondrial genome has a lower effective
population size (Ne) and, therefore, should experience higher
rates of genetic divergence between populations as a result of
drift [92]. Accordingly, large geographical regions such as
Europe and East Asia are highly enriched for a small fraction
of mitochondrial haplotype diversity, reflecting the history of
repeated bottlenecks as human populations migrated out of
Africa [10,82,93]. The geographically structured distribution
of mitochondrial genetic variation may also have been
shaped by local adaptation to environmental conditions such
as climate [72,74,94]. Therefore, the significant but generally
weak statistical associations between nuclear and mitochondrial polymorphisms that are prevalent throughout the
human genome are consistent with our understanding of
human population genetics and demographic history.
The elevated level of mitonuclear LD that we observed for
X-linked genes is probably caused by the fact that the X
chromosome has a lower Ne than autosomes, resulting in
faster rates of population divergence by drift [60,95]. Estimates
of the among-population component of total diversity in the
HGDP sample are significantly higher for the X chromosome
(13%) than autosomes (9%) [60]. As both mitochondrial and
X-linked loci accumulate differences between populations at
a high rate, they will more rapidly build up associations with
each other. Another relevant issue is that two-thirds of X
chromosomes in a population are found in females and are,
therefore, co-transmitted with mtDNA, whereas autosomes
are shared equally by males and females [96]. As a result, in
populations with a recent history of admixture, there may be
a slower rate of decay of mitonuclear LD that involves
X-linked genes. It has also been proposed that higher rates of
co-transmission should facilitate co-adaptations between
X-linked genes and the mitochondrial genome [90,96]. Therefore, it is also conceivable that selection against disrupting coadapted mitonuclear genotypes may be partially responsible
for the higher levels of mitonuclear LD on the X chromosome.
Teasing apart the relative contributions of Ne, inheritance, and
selection is an important motivation for additional empirical
and theoretical work on the generation and maintenance of
mitonuclear LD on autosomes and sex chromosomes.
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Data accessibility. The SNP genotype data from the HGDP were previously published [60], and the data are available via the HGDP
website: http://www.hagsc.org/hgdp. The code used to analyse
these data and the resulting data tables are available as electronic
supplementary material accompanying this paper.
Authors’ contributions. D.B.S. conceived of and designed the study, performed analyses and drafted the manuscript. P.D.F. and J.C.H.
designed the study and performed analyses. All authors critically
revised the manuscript and gave final approval for publication.
Competing interests. We declare we have no competing interests.
Funding. Our research on the evolution of mitonuclear interactions is
supported by the National Science Foundation (MCB-1412260) and
Colorado State University. J.C.H. is supported by a National Institutes of Health Postdoctoral Fellowship (F32GM116361).
Acknowledgements. We thank John Brookfield, Adam Eyre-Walker and
an anonymous reviewer for insightful comments that helped us
improve an earlier version of the manuscript.
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Proc. R. Soc. B 282: 20151704
being observed, because they are selectively eliminated during
oogenesis [38].
Overall, our analysis did not find compelling evidence for
a history of strong selection to conserve co-adapted mitonuclear interactions in humans, which is in line with the
arguments that mitonuclear incompatibilities may be less
prevalent in humans than many other eukaryotic lineages
because of human demographic history [62]. Perhaps our
most striking observation, however, is how little we currently
know about mitonuclear LD in humans. It is clear that this
gap in our understanding of the population genetics of our
own species needs to be addressed. As researchers and clinicians evaluate MR techniques, this will help assess the risks
associated with mitonuclear incompatibilities alongside
other concerns about the ethics of genetic engineering, the
technical risks associated with manipulating reproductive
cells and the efficient allocation of biomedical resources. We
suggest that taking advantage of the ever-increasing availability of human population genomic data to analyse
patterns of mitonuclear LD will offer a novel approach to
identify loci that may have been historical targets of selection
for efficient mitonuclear interactions and to determine the
epistatic component of human phenotypes and diseases
with a complex genetic basis.
rspb.royalsocietypublishing.org
of mitonuclear LD represent candidates for involvement in
mitonuclear interactions and incompatibilities (electronic supplementary material, table S6). Third, because the published
data used in our analyses were derived from SNP genotyping
arrays, they are biased towards high-frequency polymorphisms (which we exacerbated by filtering based on minor
allele frequency). This bias is relevant because alleles that
are involved in epistatic interaction with highly deleterious
effects are less likely to rise to high frequencies. Therefore,
datasets based on whole genome or exome re-sequencing
could provide a valuable complement to this analysis.
Future analysis of mitonuclear LD in humans may benefit
from a targeted approach that focuses on specific N-mt genes
and specific populations with mitochondrial haplotypes that
have been implicated in mitonuclear epistasis [13–24]. Analysis of human cybrid cell lines is also a promising method for
manipulating mitonuclear genotypes and identifying loci
responsible for deleterious mismatched mitonuclear interactions [26–28]. To date, however, cybrid studies in humans
have each been performed on a single nuclear background,
which does not allow for distinguishing any mitonuclear
interaction effects from the additive effects of mitochondrial
genotype. Generating a full matrix of pairwise combinations
of nuclear and mitochondrial genotypes as has been done in
yeast [45] could be used to test for mitonuclear sign epistasis
(figure 1) in human cell lines and eventually to identify the
specific loci responsible for any epistatic effects.
Another important approach for expanding our understanding of mitonuclear interactions will be to measure LD
within relatively panmictic populations. Incorporating data
from human populations with a recent history of admixture
would be particularly valuable. One of the expected effects of
selection on mitonuclear interactions is to slow the decay of
LD at loci responsible for mitonuclear incompatibilities when
populations interbreed. This would be difficult to observe in
more isolated populations because alleles associated with
strong mitonuclear incompatibilities are likely to have already
been purged. Examining recently admixed populations will
be especially valuable for further testing the hypothesis that
mitonuclear incompatibilities commonly occur without ever
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