Genetic variation among the five species
To assess genetic diversity within each species, we calculated
nucleotide polymorphism, haplotype polymorphism (Hd),
nucleotide diversity (π/kb),
polymorphic sites (θ/kb), and Tajima’s D (Table 2). Except for S.
torqueola , four of the species have comparable π values, ranging from
2.73-4.52(/kb). I. mcclellandii , A. hueti and P.
pectoralis have significantly high θ values (5.68 – 8.51/kb) compared
to L. lutea (1.22/kb) and S. torqueola (1.8/kb). Hd values
are similar in all five species (0.83 to 0.972). Tajima’s D is
significantly negative in I. mcclellandii and L. lutea ,
suggesting substantial recent population expansion or a selective sweep
in these species (Tajima 1989). Population expansion is also supported
by their radiating haplotype networks (Figure 2). S. torqueola ,
despite wide regional sampling and a relatively large number of
individuals (n = 39), appears to differ substantially from the other
species in variation. It has a relatively low number of SNPs (single
versus double digits) and low values of π and θ. Its Tajima’s D is close
to zero, signaling little recent population expansion. As a resident of
moderate elevation (700-1500m), S. torqueola also presents a
highly homogenous population composition suggestive of substantial gene
flow (Figure 2E).
Except for S. torqueola , the species generally display comparable
haplotype diversity, complexity, and patterns of recent population
expansion. Most of their substitutions are synonymous, and their
networks contain conspicuous patterns of haplotype radiation from highly
diverse ancestral clusters (e.g., Figure 2A-D). In all 5 species’
networks, divergence among haplotypes of 2-5 bp is common between
populations, strongly indicating rapid substitution combined with
homogenous haplotype distribution in populations following isolation.
Some of the species included a conspicuous core haplotype distinct from
derived haplotypes, e.g., the blue haplotypes in I. mcclellandiiand L. lutea (Figure 2A and 2C). This pattern is less notable in
other circumstances, e.g., the green cluster in L. lutea (Figure
2C). Under the conventional substitution rate of
10-8/site/year (Nguyen and Ho 2016), this pattern
suggests divergence times of 0.1~0.3 Ma among diverged
haplotypes in all species.
Interestingly, the haplotype network of L. lutea suggests this
species went through a strong bottleneck, followed by rapid radiation of
each of its haplotype groups (Figure 2C). The two star-like clusters are
separated by a 2 bp substitution. One of these substitutions (at bp
1033) is non-synonymous, causing an amino acid change from Isoleucine
(assumed to be the ancestral state by reference to the outgroup,L. argentauris , in the green cluster) to Valine (in the blue
cluster). The high haplotype diversity in the ancestral cluster and the
radiating haplotype network in the derived cluster suggest recent
expansion of the new non-synonymous substitution, which is homogenously
distributed among the sampled (blue) populations.
Genetic clustering returned different results for the 5 species.
Overall, all species were confidently clustered by the gmyc(Supporting information). I. mcclellandii split into 3 maximum
likelihood clusters, while A. hueti split into 4 clusters,L. leiothrix split into 6 clusters, P. pectoralis into 15
clusters, and S. torqueola into 5 clusters. None of the clusters
show strong alias to populations except for P. pectoralis . InP. pectoralis , the clustering divided populations into smaller
closely related groups (Supporting information). Gmyc also
inferred the threshold time of the split. The threshold time of I.
mcclellandii, A. hueti, L. lutea and S. torqueola are in the
approximate range of 0.2 MYA, while the inferred split time of P.
pectoralis is later than 23 000 and 12 000 years ago (Supporting
information). The clustering result is supported with significant
likelihood ratio test. Homogeneous ancestry is an indication of
simultaneous demographic changes. While for P. pectoralis, the
short threshold time and fine arranged population clusters indicate that
they probably were dispersed recently, e.g., in the last glacial cycle.