1 | Introduction
Ecotypic divergence in plants often occurs in alpine (including sub-alpine) habitats, where environmental conditions are more stressful than those in lower elevations (Konečná et al. 2019). Plants in alpine habitats suffer from stresses including freezing and snowfall, low and fluctuating temperature, strong wind, drought, low nutrient, and high UV radiation. Local adaptation to these stressful environments often leads to diversification in morphological and physiological traits, which results in divergence of alpine ecotypes (Wos et al. 2022). In cases that reproductive barriers arise between ecotypes in alpine and mountain zones due to not only temporal and spatial isolations in mating but also natural selection from local environments, genetic divergence occurs between ecotypes and proceeds toward speciation (Hirao et al. 2019). Because alpine habitats are geographically separated between different mountain ranges, two evolutionary processes can be applied to genetic divergence of an alpine ecotype across multiple mountain ranges (Holliday et al. 2016). One is parallel evolution of the alpine ecotype that occurred independently in different mountain ranges, where genetically-differentiated populations of the mountain ecotype are distributed (Trucchi et al. 2017; Szukala et al. 2023) (Figure S1a in Supporting Information). The other is stepping-stone colonization of different mountain summits by a single lineage of the alpine ecotype that had already diverged from the mountain ecotype (Bettin et al. 2007) (Figure S1b). Recent studies have demonstrated that the two evolutionary processes are responsible for the genetic divergence of alpine ecotypes in various plant taxa (Knotek et al. 2020; Bohutínská et al. 2021).
The Japanese Archipelago has mountain chains with alpine zones ranging north and south along the islands (Ohsawa and Ide 2011). Most of alpine plants in Japan are characterized by genetic divergence between central Honshu and northern Japan, the latter of which includes northern Honshu and/or Hokkaido (Fujii and Senni 2006). This genetic divergence are thought to result from multiple colonization of the Japanese Archipelago by arctic plants during glacial cycles in the Pleistocene (Ikeda 2022). These plants migrated southward and colonized central Honshu in glacial periods. In subsequent post-glacial periods, the colonized populations were isolated in alpine zones of central Honshu and diverged from the populations that retreated northward. On the other hand, temperate plants in mountain zones in the Japanese Archipelago show various phylogeographic patterns (Ohsawa and Ide 2011). In temperate trees in mountain zones, populations in southern Japan tend to have higher genetic diversity, which reflects multiple refugia in glacial periods, than populations in northern Japan with relatively homogeneous genetic structure, which reflects expansion from the refugia in post-glacial periods (Tomaru et al. 2022). As a result, latitudinal genetic divergence is often found in mountain trees, while genetic boarders of the divergence are located in various places. In addition to the latitudinal divergence, genetic divergence between northeastern and southwestern coastal sides of the Japanese Archipelago sometimes occurs due to contrasting climate conditions on the opposite sides of the islands (Tsumura 2006; Tsumura 2022). Thus, plants in both alpine and mountain zones often show genetic differentiation within species among mountain ranges along the Japanese Archipelago.
A white oak (Section Quercus ) species, Quercus crispulaBlume (Qc ), is common in cool-temperate forests in mountain zones in the Japanese Archipelago (Figure 1a–c). This species name is a synonym of Q. mongolica var. crispula (Blume) H. Ohashi, which is used in a taxonomic system widely accepted (Ohashi 1988; Aizawa et al. 2021). In this species, a sub-alpine variety, Q. crispulavar. horikawae H. Ohba (Qch ), is recognized (Ohba 1989) (Figure 1d–f). Because there is no combination of taxonomic naming under Q. mongolica Fisch. ex Ledeb., we follow nomenclature of Ohba (1989) in this study. Qch is usually found in sub-alpine zones or steep mountain slopes with heavy snowfall and is characterized by shrubby habit, bent trunk often decumbent near the ground, small leaf size, and dense hairs on the abaxial leaf surface (Ohba 2006). Because these phenotypes of Qch are discontinuous from those of Qcin a mountain range (Mt. Makihata, 37.0˚N, 139.0˚E, 1967 m) and are likely to be adaptive to sub-alpine environments (Noshiro 1984),Qc and Qch are regarded as different ecotypes. Genetic variation in nuclear-encoded allozymes in two Qc and twoQch populations did not indicate clear divergence betweenQc and Qch (Tanimoto et al. 1992).
Genetic structure of Qc has been investigated using different genetic markers. In chloroplast DNA haplotypes, higher haplotype diversity was found in southern Japan, while a few haplotypes were dominated in northern Japan (Kanno et al. 2004; Okaura et al. 2007; Liu and Harada 2014). The southern boarders of the northern haplotypes were located in central Honshu (Liu and Harada 2014; Onosato et al. 2021). In genotypes of nuclear microsatellite (simple sequence repeat, SSR), higher genetic diversity in southern populations and gradual genetic divergence between northern and southern populations were found, supporting post-glacial northward colonization from southern refugia (Ohsawa et al. 2011). In single nucleotide polymorphism (SNP) in some nuclear genes, however, northern populations harbored high nucleotide diversity and fast decay of linkage equilibrium, which were comparable to southern populations (Quang et al. 2008). These findings suggest that genetic variation was maintained in some genes during northward colonization. Therefore, the ecotypic divergence of Qch should be considered in the demographic history of post-glacial northward colonization of Qc .
Here, we proposed three hypotheses: (1) parallel evolution of ecotypic divergence between Qc and Qch that occurred independently in genetically-differentiated populations in different mountain ranges (Figure S1a), (2) stepping-stone colonization of different mountain summits by Qch populations that belong to a single lineage divergent from Qc (Figure S1b), and (3) another case of genetic differentiation of populations of the two ecotypes in different mountain ranges (Figure S1c). To test the three hypotheses, we selected pairs ofQc and Qch populations in multiple mountain ranges along central to northern Honshu and measured climatic conditions, leaf characters, and genetic variation in chloroplast and nuclear genomes.