Callicarpa subpubescens, endemic to the Ogasawara Islands, is suggested to have multiple ecotypes in the Hahajima Islands, specifically in the central part of the Ogasawara Islands. In this study, associations between genetic groups and spatial distribution, habitat, leaf morphology, size structure, and flowering time of each genetic group were investigated on Hahajima and the satellite Imoutojima Islands. Genetic groups were identified using EST-SSR markers, revealing four ecotypes named based on morphological features: Dwarf (D), Glabrescent (G), Tall (T), and Middle (M), with M being a result of the hybridization of G and T. Ecotype D, adapted to dry environments, is characterized by small tree size, dense thick leaves with abundant hairs, and is distributed in dry scrub. Ecotype G, adapted to understory of mesic forests, lacks leaf hairs. Ecotype T, adapted to the canopy of mesic forests, has hairy leaves and is tall in tree height. Ecotype M, adapted to the canopy of mesic scrub or edges of mesic forests, has hairy leaves but with a shorter tree height than ecotype T. Flowering peaks differed among all ecotype pairs except G and M, but the flowering times more or less overlapped among all ecotypes, suggesting that pre-mating isolation among ecotypes is not perfect. Post-mating isolation is considered absent, as there were no differences in the results, germination, and survival rates of one-year seedlings among inter- and intra-ecotype crossings. The existence of such ecotypes provides valuable insights into the ongoing speciation processes adapting to the oceanic island environments.

Genotypes of each sample were characterized by the 17 EST-SSR markers, which were developed for Callicarpa subpubescens (Setsuko et al. 2018). PCR was carried out in 6 µl reaction mixtures containing ca. 1 ng genomic DNA, 2.5 µl Type-it Multiplex PCR Master Mix (Qiagen, Hilden, Germany), and 0.2 µM of each primer. PCR conditions were as follows: 95°C for 5 min, then 35 or 38 cycles of 94°C for 30 s, 55°C or 60°C for 90 s, 72°C for 90 s, followed by final extension at 60°C for 30 min. PCR fragments were then separated using a 3130 Genetic Analyzer (Applied Biosystems, CA, USA) and genotyped using GeneMarker software (SoftGenetics, PA, USA).