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.