Discordance between mitochondrial and nuclear markers
The internal transcribed spacer (ITS1) of the ribosomal DNA has widely
been used as a molecular marker for phylogenetic analysis at species
level and for species identification of closely related molluscan taxa,
including mussel species (Santaclara et al., 2006; Wood, Apte, MacAvoy,
& Gardner, 2007). Nevertheless, unlike the deep divergence we uncovered
in mitochondrial sequences, nuclear DNA sequences (ITS1) were not highly
divergent (a maximum pairwise sequence divergence of 0.6%) and in the
phylogenetic tree, all ITS1 sequences clustered together, irrespective
of their geographic origin (Fig. 3A). The ITS1 network showed two
genotype groups (a single step away from each other), each forming a
star-like genealogy made of an admixture of northern and southern
mitochondrial cox1 members (Fig. 3B). This type of mito-nuclear
discordance is often found in biogeography studies and can be explained
by many possible hypotheses (reviewed in Toews and Brelsford 2012).
Further investigation will be needed to define the precise cause of
mito-nuclear discordance in this species, but we discussed two
hypotheses in the case of M. virgata : incomplete lineage sorting
and selection on mitochondrial genes.
The first hypothesis invokes the stochasticity of coalescent processes
at the two independent loci, resulting in divergent patterns. Incomplete
lineage sorting is often mentioned in studies that uncover mito-nuclear
discordance because nuclear markers have a larger effective population
size (i.e. slower coalescence) than mitochondrial DNA (Moore, 1995;
Pesole, Gissi, De Chirico, & Saccone, 1999). However, in the present
study the difference between the cox1 and ITS1 results are so
extreme (very deep divergence versus negligible divergence) that the
incomplete lineage sorting hypothesis is inadequate given the inferred
demographic history. Incomplete lineage sorting can only occur if two
subpopulations maintain large population sizes before they merge into a
common ancestral population; in contrast, our mtDNA cox1 and ITS1
genealogies both support very rapid and recent population growth
following a population bottleneck. This is indicated by the genealogies
with very short external branches only (a “star genealogy;” see Figs.
2A, 2B and Fig. 3), the significant negative values in Tajima’s Dand Fu’s FS test (Table 3), and the unimodal peak
skewed over the Y-axis in the pairwise mismatch distribution analysis
for the two mitochondrial lineages (Figs. 6A and 6B). Furthermore, the
coalescence time between northern and southern cox1 lineages is
estimated to be more than five times older than those of within-lineage
coalescences (Fig. 5). Given a small ancestral population(s) and/or a
very old splits of an ancestral population, incomplete lineage sorting
at ITS1 or a long waiting time prior to the coalescence of the mtDNA
lineages are unlikely to be the reason for mito-nuclear discordance.
Another hypothesis worth considering is that natural selection may have
produced long-term, balanced mtDNA polymorphism. Although it is a
long-held assumption that most of the sequence variation in mtDNA is
selectively neutral (Ballard & Whitlock 2004; Meiklejohn, Montooth, &
Rand, 2007), it is also well documented that balancing selection can
occur under spatial heterogeneity (Hedrick, 2006; Scott et al., 2011).
We note that the spatial distribution of the northern and southerncox1 lineages strongly correlates with surface seawater
temperature (Fig. 1). Therefore, we suggest that these two divergentcox1 lineages are a proxy for two mitochondrial genotypes adapted
to different surface water temperature zones, possibly due to divergence
in the energy-metabolic functions in mitochondria. To test for divergent
natural selection on the mt genome, we compared the whole mitochondrial
genome sequences of two individuals representing the northern and
southern mitochondrial lineages. Calculating the ratio of nonsynonymous
(dN ) over synonymous (dS ) substitutions for the 12
mitochondrial protein-coding genes of the complete mitochondrial genomes
revealed that none of the 12 protein-coding genes (includingcox1 , the genetic marker used in this study) showed a significant
excess of dN/dS > 1 (Supplementary Table 2). These results
do not provide supporting evidence for the selection on the
mitochondrial genome; however, we cannot rule out the possibility that
the two divergent lineages might still be maintained by balancing
selection. The functional difference between two lineages might result
from a small number of amino acid substitutions (not readily detectable
in this case) or gene regulatory sequence differences. Additional
mitochondrial genome sequencing that can cover the genetic diversity for
the two mitochondrial lineages is further required to address whether
deep divergence in mitochondrial genes is related to adaptation to
different surface water temperature zones in the NWP.