Introduction:
Speciation occurs when populations accumulate genetic differences that
cause reproductive incompatibilities in hybrids, leading to maladaptive
phenotypes such as inviability, sterility, or to reduced fitness
relative to parentals. Bateson (Bateson 1909), Dobzhansky (Dobzhansky
1937), and Muller (Muller 1942) have proposed that such
incompatibilities would involve two or more loci, so that substitutions
that are adaptive or neutral in their own genomic background can be
functionally incompatible with alleles that are present in a foreign
genomic background; the so-called Bateson-Dobzhansky-Muller
incompatibilities or BDMIs. Although this model has guided research on
the genomic basis of speciation for more than 70 years, only within the
last decade have genomic regions (and in few cases genes) causing full
sterility or inviability been identified in taxa as diverse as yeast,
flies, mice and plants (Presgraves 2010). Such genes are found to evolve
rapidly, as expected for genes caught up in open-ended molecular
evolutionary arms race, and are often involved in co-evolution between
host and pathogen, or between selfish genes and suppressors (Presgraves
2010). Particularly in studies with highly divergent species pairs,
BDMIs seem to have a complex genomic architecture that involves tens of
genes spread throughout the genome (Tang & Presgraves 2009; Schumeret al. 2018). Yet, full sterility or inviability evolve later
during the speciation process, and thus it is unclear whether those
genes reflect initial barriers that arise between diverging populations
or whether they reflect other evolutionary forces that arose after
reproductive isolation was established. Therefore, understanding the
genetic architecture of incompatibilities causing partial reproductive
isolation, such as reduced fertility, remains an important task in
evolutionary biology (Corbett-Detig et al. 2013; Turner & Harr
2014; Rafati et al. 2018).
Recent studies in taxa as diverse as arthropods, nematodes, vertebrates,
yeast, and angiosperms (reviewed in Burton et al. 2013; Sloanet al. 2017; Hill et al. 2018) have shown that the
co-evolution between the mitochondrial and the nuclear genome often
results in partially reduced fitness in hybrids between closely related
taxa (i.e. species, subspecies, or even populations). This observation
has led several authors to suggest that mitonuclear BDMIs play a
disproportional role at early stages of reproductive isolation relative
to the more widely studied nuclear-nuclear BDMIs (Burton & Barreto
2012; Hill 2016). To understand why such a general pattern would arise
across species, one needs to consider the origin and evolution of the
mitochondrion in eukaryotes. Some 1.5 billion years ago, when a
proteobacterial heterotroph (proto-mitochondrion; Martijn et al.2018) became the obligatory endosymbiont of an archaebacterial
methanogen (proto-nucleus), this symbiosis resulted in a strong
functional partition (Rivera et al. 1998), where the
proto-mitochondrion became responsible for metabolic functions and the
proto-nucleus for transcription and translation. Because intracellular
clonal replication favors smaller genomes (Taylor et al. 2002;
Clark et al. 2012), a race for replication of the
proto-mitochondrion lead to the reduction in the endosymbiont genome
size, both through the loss of redundant genes and through gene transfer
to the host genome (Selosse et al. 2001; Timmis et al.2004). Such selective pressure is still observed today in the genome
evolution of intracellular endosymbionts in insects (Wernegreen 2002),
in the recent transposition of mitochondrial genes into the nuclear
genome (Hazkani-Covo et al. 2010), or even in the deletion of
essential mitochondrial genes with significant fitness costs for the
organism (Taylor et al. 2002). Currently, in most metazoans, over
1,000 proteins are housed in the mitochondrion (Calvo & Mootha 2010),
but only 13 of those remain coded in the mitochondrial genome. Hence,
mitochondrial function is dependent upon nuclear-encoded proteins, many
of which interact closely with mtDNA encoded proteins, RNAs or
DNA-binding sites. This mitonuclear cooperation sets the stage for a
tight evolutionary arms race between the nuclear and mitochondrial
genomes to preserve functionality within eukaryotic evolutionary
lineages in essential cell functions, such as respiration and
mitochondrial protein synthesis, that have large impacts on organismal
fitness. Such mitonuclear coadaptation becomes exposed in F2 hybrids,
where independently evolving mitochondrial and nuclear alleles are
forced to interact, often being functionally incompatible, disrupting
organelle function and, consequently, causing hybrid breakdown (Burtonet al. 2013).
Contrary to nuclear-nuclear BDMIs, mitonuclear incompatibilities are
expected to be highly asymmetric for a variety reasons, as described in
the model of compensatory coevolution (Rand et al. 2004). First,
because of the mode of replication of its circular genome, mutation rate
(µ ) is higher in the mitochondrial relative to the nuclear
genome. Second, due to its lack of recombination and matrilineal
inheritance, mitochondrial genes have an effective population size
(Ne ) that is 4 times smaller than that of nuclear genes,
resulting in higher rates of fixation via genetic drift and conversely a
reduced efficiency of selection. These two processes result in the
consistent observation of faster evolution rates of mitochondrial
relative to nuclear genes, which are 2-fold in drosophilids, 20-fold in
ungulates and up to 40-fold in primates (Osada & Akashi 2012). Even
though most de novo mutations are partially deleterious, the relatively
higher levels of genetic drift allow the fixation of these new
mutations. This asymmetry of µ and Ne leads to a higher
fixation rate for weakly deleterious mutations in the mitochondrial
genomes relative to the nuclear genome; a process known as “Muller’s
ratchet” (Lynch & Blanchard 1998). This accumulation of deleterious
mutations in the mitochondrial genome elicits compensatory mutations in
the interacting nuclear genes (Osada & Akashi 2012; Barreto & Burton
2013a; Sloan et al. 2014) but not in the rest of the nuclear
genome, maintaining the stability and function of mitonuclear protein
complexes. Although such mitochondrial deleterious mutations are
effectively silenced within a population, they become exposed in
interpopulation crosses where coadapted mitonuclear complexes become
mismatched in hybrids, contributing for the establishment of genetic
barriers between recently diverged taxa (Sloan et al. 2017). The
magnitude of such early barriers to gene flow directly depends on the
genetic architecture of mitonuclear BDMIs, which remains unknown across
species.
Although mitonuclear incompatibilities are now recognized to play a
general role in establishing reproductive isolation between emerging
species (Reinhardt et al. 2013), their effect is most visible in
taxa presenting exceptionally high mitochondrial evolution rates, such
as the copepod Tigriopus californicus (Willett 2012). Allopatric
divergence between populations of this species resulted in parallel
patterns of genomic divergence that are consistent with mitonuclear
coevolution happening within independent populations. Protein coding
genes from the mitochondria evolve 2 to 14 times faster than those from
the nuclear genome (Pereira et al. 2016), suggesting that
mitochondrial genes drive intragenomic coevolution. Nuclear encoded
proteins that functionally interact with the mitochondria evolve more
rapidly than non-interacting nuclear encoded proteins (Barreto et
al. 2018), suggesting that selection favoring compensatory mutations
targets specific nuclear genes. Finally, experimental interpopulation F2
hybrids show that fitness breakdown in multiple life history traits
(such as fecundity, survivorship and developmental time) scales with
mitochondrial divergence (Burton 1990a; Edmands 1999). Notably, F2
fitness breakdown is rescued in maternal backcrosses (Ellison & Burton
2008b), where mitochondrial and nuclear coevolving units are rematched,
demonstrating that fitness breakdown is caused by interactions between
the mitochondria and unknown nuclear loci. Despite such an evident
parallelism in mitonuclear coevolution within populations, evolutionary
processes conditioned by local habitat are non-parallel. For example,
ecological trade-offs along latitudinal gradients have resulted in
differential adaptation to temperature (Willett 2010; Hong & Shurin
2015; Pereira et al. 2017) and to salinity (Leong et al.2017). Moreover, smaller Ne at southern populations has resulted
in stronger genetic drift relative to northern populations (Pereiraet al. 2016), and potentially in a faster accumulation of
deleterious mutations genome wide.
Recent studies with interpopulation hybrids of T. californicushave established that mitonuclear incompatibilities result in fitness
breakdown at various organizational levels. While heterozygous F1s are
vigorous, mismatched mitonuclear complexes in F2 and inbred lines
results in: reduced mitochondrial function (Ellison & Burton 2008a),
reduced ATP production (Ellison & Burton 2006), elevated oxidative
damage to DNA (Barreto & Burton 2013b), upregulation of pathways
involved in physiologic stress (Barreto et al. 2015), and
breakdown at multiple life history traits (fecundity, survivorship and
developmental time; Burton 1990a; Edmands 1999). It is still unclear
whether such generalized fitness breakdown cascade from mitonuclear
BDMIs involving few nuclear genes with generalize effect, or involving
multiple regions spread throughout the nuclear genome. It is worth
noting that this species lacks sex chromosomes, and instead has
polygenic sex determination (Voordouw & Anholt 2002; Alexander et
al. 2015). Therefore, factors leading to the disproportionate role of
sex chromosomes in the evolution of BDMIs reported in many species
(Presgraves 2018), does not apply in this case.
Previous efforts to understand the genetic architecture of mitonuclear
BDMIs in T. californicus have focused on deviations from the
expected Mendelian inheritance in F2 hybrids surviving to adulthood
(Foley et al. 2013; Lima & Willett 2018; Lima et al.2019), in hybrid swarms with recovered fitness (Pritchard & Edmands
2012) or among high- and low-fitness hybrids within an F2 cohort (Healy
& Burton 2020). Although those generally confirm a prominent role of
mitonuclear incompatibilities relative to nuclear-nuclear BDMIs (but see
Pritchard et al. 2011), they strongly differ in the estimated
number, location and effect size of nuclear loci interacting with the
mitochondria (ranging from few loci to all 12 chromosomes).
Understanding the genomic architecture of mitonuclear BDMIs requires a
whole-genome approach, over multiple generations of hybridization and
selection, and under divergent selection associated to alternative
mitochondrial backgrounds. To address this, we employed an experimental
evolution approach initiated with replicate populations of low-fitness
F2 interpopulation hybrids. Over the course of the experiment, parental
nuclear alleles competed in alternate mitochondrial backgrounds (i.e.
treatment) established by reciprocal crosses, for about nine generations
(Fig. 1). To assess the efficiency of natural selection in small
experimental populations we tested for increase in population size and
for fitness recovery in female fecundity and nauplii survivorship, which
are known to be associated with mitonuclear incompatibilities. Next, to
find regions likely responding to selection (uniform or divergent
between treatments), we identified genomic regions with significant
allelic frequency change during the experiment. Finally, to tease apart
nuclear genomic regions involved in mitonuclear incompatibilities we
identified regions that differ among alternative mitochondrial
backgrounds. We found genomic regions likely involved in mitonuclear
BDMIs on multiple chromosomes and that these regions differ between
mitochondrial backgrounds, suggesting that mitonuclear incompatibilities
have a complex and asymmetric genetic architecture.