Allele frequency change is consistent with asymmetric
mitonuclear incompatibilities
By measuring allelic frequency change from the initial F2 hybrids to the
replicated hybrid lines that evolved under each mitochondrial
background, we can scan the genome for regions likely under selection,
which should be consistent across replicates, from regions under genetic
drift, which should show no or inconsistent change across replicates.
Although the choice of sequenced lines is debatable, we assume that the
three chosen replicates were adequate for testing our hypothesis because
they all show increased in population size, and survivorship has
recovered to parental fitness for multiple generations (Fig. 2D). In
agreement with selection, we found some consistency among replicate
populations of each reciprocal cross (Fig. S2). This was particularly
pronounced on the SC lines, where we found more extreme P-values
rejecting the null hypothesis of genetic drift (Fig. 3); also reflected
by the higher lineage-specific thresholds based on a z-score of 2
(-log10 (P-value) of 4.17 for SC and 1.73 for SD). Together with our
observation that F2 breakdown in survivorship is stronger in the SC
background (Fig. 2C), this suggests that the strength of selection
acting against mitonuclear incompatibilities was stronger in this
mitochondrial background relative to SD. Such asymmetries in the
strength of mitonuclear incompatibilities is also supported by
reciprocal crosses of multiple populations of T. californicus(Ganz & Burton 1995; Peterson et al. 2013).
By comparing the genomic regions responding to selection in lines
evolving under alternative mitochondrial backgrounds, we can distinguish
regions responding to uniform selection (i.e. where the same nuclear
allele dominates), from regions responding to divergent selection (i.e.
where different alleles dominate). Consistent with uniform selection in
6.5% of the genome (Table 1), we find that the SC allele dominates in
much of chromosome 7 and a small segment of chromosome 8. Uniform
selection could be explained by BDMIs between 2 or more nuclear factors
(nuclear-nuclear instead of mitonuclear incompatibilities), purging of
slightly deleterious alleles that have been fixed in one of the parental
populations, or by adaptation to laboratorial conditions favoring
alleles of one of the populations. These possibilities are also not
mutually exclusive, and previous work in this species lend support to
any of these. Studies with reciprocal F2 hybrids from multiple
population crosses have found skews towards the same allele in one or
two chromosomes (Foley et al. 2013; Lima et al. 2019),
suggesting a modest role of nuclear-nuclear incompatibilities inT. californicus . Previous genomic studies of natural populations
found that effective population size in SD is less than half that of SC
(Pereira et al. 2016), and thus this population is expected to
accumulate deleterious mutations twice as fast. Moreover, experimental
studies have revealed that these populations are differentially adapted
to temperature (Willett 2010; Hong & Shurin 2015; Pereira et al.2017) and to salinity (Leong et al. 2017), and that rearing
temperature used in this study favors SC alleles, relative to SD
(Willett 2010). While these three selective regimes are impossible to
distinguish under our experimental design, future
evolve-and-resequencing experiments varying environmental factors (such
as in Griffiths et al. (2020), but with reciprocal mitochondrial
backgrounds) will shed light on the relative role of ecological
adaptation driving this signature of uniform selection.
Consistent with divergent selection, we find that allele frequencies
differ in 33% of the genome, depending on the mitochondrial background.
Yet, concerning the targets of divergent selection, there is no region
where opposite alleles dominate, as would be expected if the same
genomic region was under divergent selection in both mitochondrial
backgrounds, i.e. if mitonuclear incompatibilities had a symmetric
genetic architecture. Instead, the location of genomic regions
consistent with divergent selection strongly differs among mitochondrial
backgrounds. For example, the SC mitochondria seems to interact strongly
with regions in chromosomes 2, 3, 8, 9, 11 and 12, while the SD
mitochondria seems to interact with regions in chromosomes 4, 6 and 9
(Fig. 3). In agreement, previous studies using F2 hybrids from
reciprocal crosses have reported allele frequency changes during a
single generation in multiple chromosomes, both in response selection
for rapid development (5 chromosomes, in Healy & Burton 2020) or simply
as a consequence of differential survivorship during development (4 to 5
chromosomes, in Lima et al (2019)). In contrast to those studies,
where skews consistent with divergent selection were small in magnitude
(up to ~15%) and were consistent across entire
chromosomes, by evolving hybrids over multiple generations, we were able
to identify genomic regions that span between half to small portions of
the chromosomes (62 Kbp – 6.2 Mbp; Fig. 3). Nevertheless, our regions
under divergent selection still contain hundreds or thousands of genes,
and thus prevent identifying the causal genes underlying mitonuclear
incompatibilities. Future “evolve-and-resequence” studies increasing
effective recombination rate, either by increasing the size of
experimental populations or the number of generations under experimental
evolution, will provide higher resolution into the asymmetric and
polygenic genomic architecture of mitonuclear incompatibilities
described here.
Consistent with our hypothesis, the direction of divergent selection
favoring mitonuclear adaptation is strongly supported, as nuclear
alleles matched the mitochondrial background in 88.6 and 87% of the
cases (Table 1). Some of the remaining windows with skews towards the
mismatched allele can be explained by non-biological reasons. For
example, in SD lines, all windows showing skews towards the mismatched
SC allele are physical linked to a large region of chromosome 7 where
the SC allele is favored in both mitochondrial backgrounds, and thus can
be explained by the stochasticity of recombination events during this
experiment. In contrast, in SC lines, windows with the mismatched allele
can only be explained by antagonistic selection, as these are located in
half of chromosome 11, which shows no change in the reciprocal cross.
Although this pattern is usually not expected for mitonuclear
incompatibilities, empirical studies in F2 hybrids show that it is not
uncommon. In studies with F2 hybrids, Lima et al 2019 found
patterns consistent with antagonistic selection in one to three
chromosomes, depending on the population cross, and Healy & Burton
(2020) found such pattern in F2 hybrids between SD and SC also in
chromosome 11.
Taken together, our results offer new insights into the genomic
architecture of mitonuclear incompatibilities. We find that mitonuclear
incompatibilities are highly asymmetric, both in the magnitude of
selection and in the location of targeted nuclear regions, supporting
theoretical predictions of BDMIs involving uniparentally inherited genes
(Turelli & Moyle 2007). Despite the small size and limited gene content
of the mitochondrial genome, we find that it exerts significant
divergent selection in many genomic regions located in multiples
chromosomes of the nuclear genome. Our finding that mitonuclear
interactions predominantly favor coadapted gene complexes imply that
such early BDMIs can result in barriers to gene flow that are widespread
across the genome. The rapid evolution of the mitochondria and
coevolving nuclear genes during periods of geographic isolation, may
thus constitute important reproductive barriers that persist after taxa
establish secondary contact or sympatry.