DOI: 10.5061/dryad.05qfttfc1
title: Data from: Adaptive radiation of the Callicarpa genus in the Bonin Islands revealed through double-digest restriction site–associated DNA sequencing analysis

description: The Bonin Islands, comprised 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 C. glabra exclusive 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 aimed to
 investigate species and ecotype diversification patterns, estimate
 divergence times, and explore cryptic species within Callicarpa in the
 Bonin Islands using phenotypic and genetic data (double-digest restriction
 site–associated DNA sequencing). Genetic analysis revealed that C.
 parvifolia and C. glabra both formed single, distinct genetic groups.
 Conversely, C. subpubescens consisted of six genetic groups corresponding
 to different ecotypes and regions, and a hybrid group resulting from the
 hybridization between two of these genetic groups. Population demography
 analysis focusing on six Chichijima and Hahajima Islands–based
 species/ecotypes indicated that all species and ecotypes except one
 ecotype diverged simultaneously around 73–77 kya. The star-shaped
 neighbor-net tree also suggests the simultaneous divergence of species and
 ecotypes. The species and ecotypes that simultaneously diverged adapted to
 dry environments and understory forests, suggesting that aridification may
 have contributed to this process of adaptive radiation. Moreover, leaf
 morphology, flowering time, and genetic analyses suggested the presence of
 two cryptic species and one hybrid species within C. subpubescens.
description: <strong>Study species and sampling</strong>
 <em>Callicarpa</em><em> parvifolia</em>
 grows in sunny dry dwarf scrub on rocky ground in the Chichijima Islands,
 whereas <em>C. glabra</em> grows in the understory of dry
 scrub in the Chichijima Islands. In contrast, <em>C.
 subpubescens</em> is not listed as a threatened species and is
 widely distributed in the Bonin and Volcano Islands, situated
 approximately 150 km southwest of the Bonin Islands.
 <em>Callicarpa</em><em> subpubescens</em> 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).
 The ecotype SG inhabits the understory of mesic forests. The ecotype ST
 forms the canopy of tall mesic forests. The ecotype SD forms the canopy of
 dry scrub. The ecotype SH forms the canopy of mesic scrub (cloud forests)
 or inhabits the forest edge of mesic forests. 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,
 respectively. 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 (ecotypes STm and Sm) in the
 Mukojima Islands, six populations　from the Chichijima Islands,
 representing three species and ecotypes (P, G, S) from two islands,
 Anijima and Chichijima Islands. Additionally, six populations from the
 Hahajima comprised two ecotypes (SG and SD) from the two islands, Hahajima
 and Imoutojima Islands, and ecotypes ST and SH from Hahajima Island. As
 outgroups, one individual each of <em>Callicarpa japonica</em>
 and <em>Callicarpa mollis</em>, both of which grow in Kyoto
 Prefecture, mainland Japan, was also collected (Fig. S1). Silica gel was
 used to immediately dry leaf samples used for DNA extraction.
 <strong>ddRAD genotyping and SNP
 filtering</strong> DNA was extracted using a
 modified CTAB method (Milligan, 1992). The DNA samples were quantified
 using a Qubit 2.0 Fluorometer (Invitrogen, MA, USA) and adjusted to 12.6
 ng/μl through dilution with TE buffer. Sequencing libraries were prepared
 following a modified version of Peterson’s protocol for ddRAD-seq
 (Peterson et al., 2012). For detailed library preparation methods, refer
 to appendix 1. The libraries were sequenced using a HiSeq2000 platform
 (Illumina, CA, USA) with 51 bp single-end reads at BGI Japan (Kobe,
 Japan). To ensure appropriate data resolution and
 accuracy for each specific analysis, three datasets were created: denovo,
 referenced, and demography datasets. SNPs were detected using dDocent
 (Puritz et al., 2014) and Stacks version 2.60 (Catchen et al., 2013;
 Catchen et al., 2011). The detection conditions and number of SNPs used in
 each analysis are summarized in Table S1. In all data sets, we excluded
 five individuals with low individual-level genotyping rates from SNP
 detection. In the referenced and denovo datasets, SNPs were detected using
 dDocent, following its tutorial. When creating the denovo dataset, the
 reference genome of <em>C. subpubescens</em> was not used as a
 reference sequence and the two outgroup individuals were not included.
 This dataset is optimal for detecting population genetic structure without
 reference bias. In contrast, when creating the referenced dataset, the
 reference genome and two outgroup individuals were used. This dataset
 provides more accurate SNP calling for phylogenetic analysis. Total raw
 SNPs generated via dDocent were filtered using vcftools -0.1.14 to meet
 the conditions outlined in Table S1. For the demography
 dataset, SNPs were re-extracted from the .bam files created for the
 referenced dataset using dDocent. First, gstacks from Stacks was used to
 generate catalogs of variable sites (Rochette et al., 2019). Subsequently,
 populations from Stacks were employed to extract SNPs with the following
 options: -r 0.8 -p X --min-mac 1 --max-obs-het 0.5 --vcf (where X
 represents the number of species/ecotypes in each dataset). The pairwise
 two-dimensional minor allele site frequency spectrum (2D-mSFS) was
 calculated from the .vcf file using the R script 2D-msfs-R
 (https://github.com/garageit46/2D-msfs-R). Missing data were addressed
 through bootstrapping within the same ecotype. This dataset includes
 non-variable sites and low-frequency SNPs, making it suitable for
 inferring evolutionary processes and population history.
description: # Data from: Adaptive radiation of the Callicarpa genus in the Bonin
 Islands revealed through double-digest restriction site–associated DNA
 sequencing analysis
 [https://doi.org/10.5061/dryad.05qfttfc1](https://doi.org/10.5061/dryad.05qfttfc1) This study investigated species and ecotype diversification patterns, estimated divergence times, and explored cryptic species within *Callicarpa* in the Bonin Islands using phenotypic and dagenetic data, revealing simultaneous divergence driven by aridification and identifying two cryptic species and one hybrid species within *C. subpubescens*. This data includes SNPs of 96 individuals characterized by ddRAD-Seq. ## Description of the data and file structure MS Excel sheet **Individual_information.xlsx** contains "Individual ID: Column A", "Population ID: Column B", and "Species/ecotype: Column C" of 96 individuals used in this study. The abbreviations for the species and ecotypes are as follows: P: *C. parvifolia*; G: *C. glabra*; S: ecotype of *C. subpubescens* in the Chichijima Islands; SG: glabrescent ecotype of *C. subpubescens* in the Hahajima Islands; ST: tall ecotype of *C. subpubescens* in the Hahajima Islands; SD: dwarf ecotype of *C. subpubescens* in the Hahajima Islands; SH: hybrid ecotype of *C. subpubescens* in the Hahajima Islands; STm: ecotype of *C. subpubescens* similar to ecotype ST in the Mukojima Islands; Sm: *C. subpubescens* similar to ecotype S in the Mukojima Islands.　 **Denovo_ADMIXTURE.vcf** is the SNP dataset used in the ADMIXTURE analysis with the denovo dataset. The VCF file contains SNP information for each individual and each locus, with individual IDs listed in the Individual_information.xlsx **Referenced_SplitsTree_with_SH.vcf** and **Referenced_SplitsTree_without_SH.vcf** are SNP datasets used in SplitsTree, based on the referenced dataset. The former includes the SH ecotype, while the latter excludes it. The VCF file contains SNP information for each individual and each locus, with individual IDs listed in the Individual_information.xlsx **Referenced_30_RAxML-ng_without_SH.vcf**, **Referenced_50_RAxML-ng_without_SH.vcf**, and **Referenced_80_RAxML-ng_without_SH.vcf** are SNP datasets used in RaxML-ng analysis, each filtered based on the referenced dataset with genotyping rates of 30%, 50%, and 80%, respectively. The VCF file contains SNP information for each individual and each locus, with individual IDs listed in the Individual_information.xlsx **Demography_model_a.vcf**, **Demography_model_b.vcf**, **Demography_model_c.vcf**, **Demography_model_d.vcf**, **Demography_model_e.vcf**, **Demography_model_f.vcf**, and **Demography_model_g.vcf** contain SNP datasets used in demographic analyses for models a, b, c, d, e, f, and g, respectively, with fastsimcoal2. The VCF file contains SNP information for each individual and each locus, with individual IDs listed in the Individual_information.xlsx ### Usage Notes Excel can be used to access the .xlsx file. All .vcf files can be viewed using TASSEL or a standard text editor. ## Sharing/access Information Links to publications that cite or use the data: Setsuko S, Narita S, Tamaki I, Sugai K, Nagano AJ, Ihara-Udino T, Kato H, Isagi Y (2024) Adaptive radiation of the *Callicarpa* genus in the Bonin Islands revealed through double-digest restriction site–associated DNA sequencing analysis. Ecology and Evolution Links to other publicly accessible locations of the data: No Links/relationships to ancillary data sets: No Was data derived from another source? No If yes, list source(s): NA Recommended citation for this dataset: Setsuko S, Narita S, Tamaki I, Sugai K, Nagano AJ, Ihara-Udino T, Kato H, Isagi Y (2024) Adaptive radiation of the *Callicarpa* genus in the Bonin Islands revealed through double-digest restriction site–associated DNA sequencing analysis. Dryad Digital Repository.