Study species and sampling
Callicarpa parvifolia grows in sunny dry dwarf scrub on rocky ground in the Chichijima Islands (Toyoda, 2003), with a flowering peak in July (Table 1), whereas C. glabra grows in the understory of dry scrub in the Chichijima Islands (Toyoda, 2014) and has its flowering peak in August. These species are classified as “endangered” and “critically endangered” in the Red List of Threatened Species of Japan (Ministry of the Environment Government of Japan, 2020), respectively. In contrast, C. subpubescens is not listed as a threatened species and is widely distributed in the Bonin and Volcano Islands.Callicarpa subpubescens exhibits different ecotypes, each with distinct habitats and some with different flowering peaks. For example, the Chichijima Islands’ ecotype (S) inhabits the forest edge of mesic forests, with peak flowering in June. The Hahajima Islands have four ecotypes: the glabrescent ecotype (SG), the tall ecotype (ST), the dwarf ecotype (SD), and the hybrid ecotype (SH; previously called the intermediate ecotype M; Setsuko et al. 2023). The ecotype SG inhabits the understory of mesic forests, with a flowering peak in July. The ecotype ST forms the canopy of tall mesic forests, with a flowering peak in October. The ecotype SD forms the canopy of dry scrub, with two flowering peaks in August and November. The ecotype SH forms the canopy of mesic scrub (cloud forests) or inhabits the forest edge of mesic forests, with a flowering peak in July (Setsuko et al., 2023; Sugai et al., 2019). The Mukojima Islands have two ecotypes (STm and Sm), which are genetically close to the ecotype ST in the Hahajima Islands and the ecotype S in the Chichijima Islands (Sugai et al. 2019; also refer to the Results section). However, substantial forest areas have been lost due to feral goats (Shimizu, 2003), making it challenging to determine their original habitats. Flowering surveys on the Mukojima Islands were conducted only once in July 2010, as the islands are currently uninhabited, with no ocean liner services, and distant from inhabited Chichijima Island.
To cover species and ecotypes of each island in the Bonin Islands, leaf samples were collected from 94 individuals across 14 populations for DNA extraction (Table 1; Fig. S1). These samples included two populations in the Mukojima Islands, six populations from the Chichijima Islands, and six populations from the Hahajima Islands. As outgroups, one individual each of Callicarpa japonica and Callicarpa mollis , which grow in Kyoto Prefecture, Japan, was also collected (Fig. S1). Silica gel was used to immediately dry leaf samples used for DNA extraction.
Leaves were also sampled for phenotypic measurements from the same individuals used for DNA extraction. As leaf morphology varies within individuals depending on sunlight exposure, leaves were collected from the sunlit upper canopy. However, owing to time constraints, sunlit leaves could not be collected from the SGi population.