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.