4 | Discussion
Our results from ncEST-SSR genotypes do not support parallel evolution
of ecotypic divergence between Qc and Qch in different
mountain ranges (Figure S1a) but do suggest colonization of different
mountain summits by Qch populations belonging to a single lineage
that had already diverged from Qc (Figure S1b). First, the NJ
tree indicated that the genetic group of Qch (eight Qchpopulations) and the genetic group of Qc (13 Qcpopulations and three Qch populations) and were separated in the
tree topology as shown in Figure S1b. Second, the PCA demonstrated that
the genetic groups of Qc and Qch were located at different
positions in the coordinates of PC1 and PC2, irrespective of their
geographic locations. Although the genetic groups of Qc andQch are genetically divergent, their genetic differentiation
(F ST = 0.046) was lower than that between
Japanese white oak species, Qc and Q. dentata Thunberg
(F ST = 0.133), Qc and Q. serrataMurray (F ST = 0.153), and Q. dentata andQ. serrata (F ST = 0.211) using ncEST-SSR
loci (Nagamitsu et al. 2019). This low genetic differentiation between
the genetic groups of Qc and Qch suggests recent
divergence and/or gene flow between them, which is consistent with
intermediate ancestry proportions of the Qc and Qchclusters frequently found in individuals of both genetic groups. Thus,
the taxonomic treatment of Qch as the variety of speciesQc seems appropriate (Ohba 1989), and the genetic groups ofQc and Qch can be recognized as ecotypes (Lowry 2012).
The genetic diversity in the genetic groups of Qc and Qchdid not differ. This result suggests that the Qch ecotype has
maintained its genetic variation in spite of restricted areas of its
habitats in sub-alpine zones. The latitudinal cline in allelic richness
implies northward colonization from southern refugia after the last
glacial period. This cline is common in white oak species, Q.
aliena Blume and Q. serrata (San Jose-Maldia et al. 2017) andQc (Ohsawa et al. 2011) in Japan. The geographic distributions of
cpDNA haplotypes in the Qc and Qch populations are
consistent with the previous knowledge in the Japanese white oak species
(Kanno et al. 2004; Okaura et al. 2007; Liu and Harada 2014; San
Jose-Maldia et al. 2017; Onosato et al. 2021). These findings suggest
that the Qch ecotype shares the post-glacial migration history
through seed dispersal with the white oak species and has colonized
sub-alpine zones toward northern mountain ranges. The genetic
sub-structure within the genetic groups of Qch shown in the
Bayesian clustering at K = 4 and the slightly higher genetic
differentiation within Qch (F ST = 0.039)
than within Qc (F ST = 0.019) may reflect
stepping-stone colonization of sub-alpine zones during northward
migration associated with founder effects and genetic drift.
Climatic conditions in the habitats of populations differed between the
genetic groups of Qc and Qch . The climatic conditions of
the genetic group of Qch were characterized by low temperature
and heavy snow. This correspondence between climatic and genetic
variations suggests that the Qch ecotype is isolated by temporal
and spatial reproductive barriers and/or adapted to climatic environment
in sub-alpine zones. In Moriyoshi (location 4), the Qc andQch populations had different cpDNA haplotypes, suggesting seed
dispersal barriers between mountain and sub-alpine zones in this
mountain range. Although phenological shift of reproductive events and
spatial separation in mountain topography potentially occur among
elevations, pollen and seed dispersal is feasible, because wind
dispersal of pollen ranges over long distances when tree populations are
fragmented (Ortego et al. 2014), and seed dispersers move to search
acorns among elevations (Gomez 2003; Bekku et al. 2019). Biotic and
abiotic factors that differ between mountain and sub-alpine zones can
affect survival and growth of plants (Wos et al. 2022), although
selective drivers responsible for local adaptation of the Qchecotype are unclear. The balance between gene flow and natural selection
may result in the weak genetic differentiation between the Qc andQch ecotypes.
The three populations 05Hx, 06Hx, and 07Hx were identified as Qchbased on phenotypes but were grouped to Qc based on ncEST-SSR
genotypes. Leaf sizes observed in the three Qch populations were
similar to those in five Qch populations of the genetic group ofQch and were smaller than those in four Qc populations of
the genetic group of Qc . This unexpected result implies that
environmental factors in the habitats of the three populations can
induce the Qch phenotypes in spite of their genetic background ofQc . Because the climatic conditions in these habitats are not
sub-alpine conditions as shown in the climatic PCA, specific factors,
such as strong wind, drought, and low nutrient, may facilitate
expression of the Qch phenotypes through morphological and
physiological responses to these factors (Nagamitsu et al. 2019;
Solé-Medina et al. 2022). In Oga (location 5) near the coast, for
example, strong wind from the sea may lead to small leaves and shrubby
habit. This phenotypic plasticity prevents us from defining diagnostic
morphology to identify the taxon Qch as the sub-alpine ecotype
recognized by ncEST-SSR genotypes. Common garden experiment in multiple
environments is useful to clarify phenotypic plasticity and local
adaptation mentioned above, which may help us to find the diagnostic
morphology and to treat the taxon Qch properly.
We do not know the origin of the genetic group of Qch . The most
plausible scenario is that the Qch ecotype derived from Qcin the Japanese Archipelago after the divergence between Qc andQ. mongolica , the latter of which is distributed in the
continental northeastern Asia (Ohashi 1988). Reconstruction of
geographic distributions in the last glacial period using species
distribution modeling indicated that the past potential habitats ofQc existed in northern Honshu in addition to southwestern parts
of the Japanese Archipelago (Onosato et al. 2021). Thus, marginal
populations in this northern refugium could diverge from main
populations in the southwestern refugia and colonize sub-alpine zones in
mountain ranges in central and northern Honshu after the last glacial
period.
In white oaks, most taxa are interfertile, and hybridization is involved
in speciation and ecotypic divergence (Hipp et al. 2020). In northern
Hokkaido, a coastal ecotype of Qc is derived from hybridization
with Q. dentata and is treated as a hybrid taxon Q×angustilepidota Nakai (Nagamitsu et al. 2019; Nagamitsu et al.
2020). In central Honshu, Q. mongolica var. mongolicoides(H. Ohba) M. Aizawa is thought to originate from ancient hybridization
between Qc and Q. mongolica , the latter of which had
probably colonized the Japanese Archipelago during glacial cycles
(Aizawa et al. 2018; Aizawa et al. 2021). In the continental
northwestern Asia, Q. mongolica var. liaotungensis(Koidz.) Nakai (syn.: var. undulatifolia (H. Lev.) Kitam. & T.
Hiroki), which is often treated as a separate species Q.
liaotungensis Koidz. (syn.: Q. wutaishanica Mayr.), has shrubby
habit (Aizawa et al. 2021). Thus, there is a possibility that Q.
liaotungensis is involved in the origin of the Qch ecotype
through ancient hybridization (Yang et al. 2016; Yang et al. 2018). The
origin of the sub-alpine Qch lineage and the history of its
ecotypic divergence should be investigated in future genomic studies
including Asian white oak taxa.