III. A Clade Exclusive to Northeast Asia
Our analysis verifies the existence of a clade exclusive to Northeast
Asia (Clade H), previously suggested by other studies (Fu et al., 2005;
Hu et al., 2007; Borzée et al., 2017). Clade H was strongly supported
(bootstrap value=100) and genetically distinct from other clades
(Fig. 2 ). In previous genetic studies, B. gargarizans was
treated as the B. gargarizans complex composed of several clades
across a large area without differentiation according to region or
altitude (Hu et al., 2007; Zhan & Fu, 2011; Borzée et al., 2017). These
features made it difficult to understand the evolutionary history ofB. gargarizans .
The existence of the Clade H suggests new interpretations of the
differentiation process of B. gargarizans in Northeast Asia. We
estimate the divergence time estimate of Clade H to be 2.25 Ma (0.5
Ma–4.33 Ma; Fig. 5 ), in the Pliocene and early Pleistocene.
During the early Pleistocene (Gelasian age), there were major geological
events (landification due to fluctuations in sea level) (He et al.,
2015) and dramatic climate change (glacial range expansion). Our
multilocus haplotype network infers that the ancestor of Clade H was in
southeastern China, as indicated by the mixed membership of Group 7 (SE
and NE regions) (Fig. 3 ).
Previous studies suggested that faunal exchange between China and the
Korean Peninsula occurred through the Yellow Sea land bridge at times of
low sea levels (Zhang et al., 2016; Du et al., 2019), including B.
gargarizans (Borzée et al., 2017). After dispersal into Northeast Asia,
the subsequent rise of sea level and the expansion of glaciers would
have isolated Clade H in a glacial refugium on the Korean Peninsula. A
similar pattern of a glacial refugium on the Korean Peninsula was found
in other organisms (Lee et al., 2008; Yoshikawa et al., 2008; Zhang et
al., 2008; Kim et al., 2013; Borzée et al., 2017; Fong et al., 2020).
Although we had limited sampling from North Korea, Heilongjiang Province
(China), and the Russian Far East, there is a preliminary pattern
indicating that South Korea is relatively diverse, which would support a
scenario of range contraction into South Korea during a glacial cycle,
followed by a range expansion northward during an interglacial cycle
(Fig. 4 ). To verify this hypothesis, additional samples are
needed from in the Northeast China, North Korea, and the Russian Far
East.