Adaptive radiation of the Callicarpa genus in the Bonin Islands revealed through double-digest restriction site–associated DNA sequencing analysis

Suzuki SETSUKO¹, Satoshi NARITA², Ichiro TAMAKI³, Kyoko SUGAI⁴, Atsushi J NAGANO^5,6, Tokuko UJINO-IHARA¹, Hidetoshi KATO⁷, Yuji ISAGI²

¹Department of Forest Molecular Genetics and Biotechnology, Forestry and Forest Products Research Institute, Forest Research and Management Organization, 1 Matsunosato, Tsukuba, Ibaraki, 305-8786, Japan

²Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan,

³Gifu Academy of Forest Science and Culture, 88 Sodai, Mino, Gifu, 501-3714, Japan

⁴Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

⁵Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga, 520-2194, Japan

⁶Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihouji, Tsuruoka, Yamagata, 997-0017, Japan

⁷Makino Herbarium, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo, 192-0397, Japan

Running title: Adaptive radiation in Callicarpa

*Correspondence; Suzuki Setsuko, E-mail:setsukos@affrc.go.jp

ORCID Suzuki Setsuko, 0000-0002-0612-1853; Kyoko Sugai, 0000-0003-2426-6156; Ichiro Tamaki, 0000-0003-2315-243X; Atsushi J. Nagano, 0000-0001-7891-5049; Tokuko Ujino-Ihara, 0000-0002-9243-1638; Yuji Isagi, 0000-0002-9777-076X

ABSTRACT

The Bonin Islands, comprising of the Mukojima, Chichijima, and Hahajima Islands, are known for their isolated and distinctive habitats, hosting a diverse array of endemic flora and fauna. In these islands, adaptive radiation has played a remarkable role in speciation, particularly evident in the Callicarpa genus that is represented by three species: Callicarpa parvifolia and Callicarpa glabraexclusive to the Chichijima Islands, and Callicarpa subpubescens , distributed across the entire Bonin Islands. Notably, C. subpubescens exhibits multiple ecotypes, differing in leaf hair density, flowering time, and tree size. In this study, we used double-digest restriction site–associated DNA sequencing to analyze species, ecotypes and geographical variations within Callicarpain the Bonin Islands. We aimed to determine detailed phylogenetic relationships, investigate species and ecotype diversification patterns, estimate divergence times, and explore cryptic species using genetic and phenotypic data. Genetic analysis revealed that C. parvifolia andC. glabra formed a single, distinct genetic groups. Conversely,C. subpubescens showed seven genetic groups corresponding to different ecotypes and regions, with one ecotype derived from the hybridization of two others. Phylogenetic and population demography analyses, focusing on six Chichijima and Hahajima Islands–based species/ecotypes, indicated the divergence of an ecotype adapted to tall mesic forests approximately 170 kya, whereas the other five species/ecotypes diverged nearly simultaneously around 73–77 kya. Environmental changes during the glacial period likely contributed to this process of adaptive radiation. Moreover, leaf morphology, flowering time, and genetic analyses suggested the presence of two cryptic species within C. subpubescens .

Key words: ddRAD-Seq, Lamiaceae, Oceanic islands, Quaternary, Ogasawara Islands

INTRODUCTION

Oceanic islands are geographically isolated—never been connected to large landmasses. Owing to their distinctive characteristics and limited colonization opportunities, the biotas of oceanic islands exhibit a greater degree of endemic flora and fauna, often resulting from frequent ecological speciation and adaptive radiation (Gillespie et al., 2001; Givnish, 1997). Adaptive radiation refers to the rapid diversification of lineages into multiple taxa, each adapted to distinct ecological niches (Rundle & Nosil, 2005; Schluter, 2000). Some examples include Darwin’s finches in the Galapagos Islands (Grant, 1986; Grant, 1998; Grant & Grant, 2007) and silverswords and Hawaiian lobeliads in the Hawaiian Islands (Carlquist et al., 2003; Givnish et al., 2009).

The Bonin Islands, comprising of the Mukojima, Chichijima, and Hahajima Islands, are oceanic islands located approximately 1,000 km south of Tokyo, Japan (Fig. S1). Despite their smaller land area (70 km²) and lower altitude (463 m) compared with other oceanic islands, such as the Canary Islands (area, 7,490 km²; elevation, 3,718 m) and the Galapagos Islands (area, 7,870 km²; 1707 m), the Bonin Islands exhibit a high proportion of endemic vascular flora, with ~43% of species being endemic (Ono et al., 1986), exceeding 26% and 43% endemism in the Canary Islands (Aedo et al., 2013) and the Galapagos Islands (Porter, 1978), respectively. Diverse habitats, including forests, grasslands, and coastal areas, providing varied microclimates and supporting multifarious plant species, coupled with the islands’ isolation, have facilitated the evolution of endemic species adapted to specific ecological conditions in the Bonin Islands. The role of adaptive radiation in driving the notable endemism rate of the Bonin Islands is underscored by various genera, such as Crepidiastrum(Ito & Ono, 1990), Symplocos (Soejima et al., 1994), andPittosporum (Ito et al., 1997), which have experienced diversification yielding three to four species each.

In the Bonin Islands, the genus Callicarpa is another example of adaptive radiation (Ono, 1991; Shimizu & Tabata, 1991). Comprising three endemic species, Callicarpa parvifolia , Callicarpa glabra , and Callicarpa subpubescens . Callicarpa parvifolia and C. glabra are restricted to the Chichijima Islands, whereas C. subpubescens exhibits a wider distribution across the Mukojima and Hahajima Islands (Fig. S1). Using microsatellite markers, Sugai et al. (2019) demonstrated genetic differentiation among the three Callicarpa species in the Chichijima Islands, as well as multiple genetic groups within C. subpubescens in the Mukojima and Hahajima Islands. Furthermore, Setsuko et al. (2023) identified four ecotypes in the Hahajima Islands, one derived from hybridization of two other ecotypes, with these ecotypes found to be locally adapted to soil moisture and light conditions. However, detailed phylogenetic relationships, as well as species/ecotype diversification timing and order remain unclear. By examining differentiation patterns and divergence time, it may be possible to infer how plants in the oceanic islands diversified with geohistorical and/or environmental changes (Kadereit & Abbott, 2021).

In this study, we aimed to elucidate detailed phylogenetic relationships within the Callicarpa genus in the Bonin Islands, determine species and ecotype diversification patterns, estimate divergence times, and investigate cryptic species. To achieve these objectives, we employed double-digest restriction site–associated DNA sequencing (ddRAD-Seq), a genomic approach for detecting a large number of single nucleotide polymorphisms (SNPs) throughout the genome. Known for high resolution, ddRAD-Seq is suitable for investigating intricate phylogenetic relationships within closely related taxa (Wagner et al., 2013). The results of this study will offer fundamental insights into the evolutionary history of Callicarpa in the Bonin Islands, contribute essential knowledge on factors influencing species diversification in island ecosystems, and enhance our understanding of speciation processes. Moreover, the identification of potential cryptic species highlights the importance of comprehensive genetic analyses in biodiversity studies.

MATERIALS AND METHODS