DOI: 10.5061/dryad.69p8cz9d3
title: Dual expansion routes likely underlie the present-day population structure in a Parnassius butterfly across the Japanese Archipelago

description: The Japanese Archipelago consists of a series of isolated yet
 interconnected islands off the Eurasian continent. The linear topography
 of the archipelago presents a unique biogeographic context for the
 dispersal of organisms from the continent. In this study, mitochondrial
 DNA (mtDNA) and single-nucleotide polymorphism (SNP) variation were
 employed to elucidate the dispersal history of the Japanese clouded
 butterfly (Parnassius glacialis) across the Japanese Archipelago,
 including the northern island (Hokkaido), the main island (Honshu), and
 Shikoku Island. Network analysis of 1192 bp of mtDNA (cytochrome oxidase I
 and II) regions revealed 49 haplotypes and three distinct haplotype
 groups, which correspond geographically to Eastern Japan, Western Japan,
 and Chugoku–Shikoku. The Chugoku–Shikoku group is the most ancient
 lineage. Divergence time estimates using whole-genome sequencing of mtDNA
 suggest that the Japanese lineage diverged from the continental P.
 glacialis approximately 3.08 million years ago (Ma). Subsequently, the
 Eastern Japan and Western Japan lineages diverged from the Chugoku–Shikoku
 lineage around 1.06 Ma, with subsequent divergence of the Eastern and
 Western Japan lineages at approximately 0.62 Ma. P. glacialis is estimated
 to have expanded its distribution via a land bridge that once connected
 China and the Japanese Archipelago. Population structure analysis based on
 3067 SNP genotypes revealed five distinct genetic structures within the
 Japanese Archipelago, indicating geographical differentiation. Through
 mtDNA and SNP variation analyses, four primary genetic barriers were
 identified: between Hokkaido and Honshu, between Eastern and Central
 Japan, within the Kansai region, and in the Chugoku region. The first
 three barriers correspond to notable geographical features, the Blakiston
 Line, a line parallel to the Itoigawa–Shizuoka Tectonic Line, and a
 boundary crossing Lake Biwa. These findings suggest that Japanese P.
 glacialis diverged from the continental P. glacialis and expanded its
 range across the Japanese Archipelago via western routes, leading to its
 current distribution.
description: SNPs were detected from the extracted DNA using the genotyping by
 random amplicon sequencing, direct (GRAS-Di) method designed by Enoki and
 Takeuchi (2018) and described by Hosoya et al. (2019); this method offers
 simple library construction and the capacity to detect many SNPs. Briefly,
 libraries were constructed through two sequential PCR steps, similar to
 multiplexed inter-simple sequence repeat genotyping by sequencing (Suyama
 and Matsuki 2015). The first PCR primers consisted of 10 bases of Illumina
 Nextera adaptor 3-end sequences plus 3-base random oligomers (13 bases).
 The final PCR product was purified using columns or magnetic beads without
 size selection and then employed for sequencing on an Illumina platform
 (Illumina, San Diego, CA, USA). Cyclized DNA was prepared using the
 produced library and the MGIEasy universal library conversion kit (App-A,
 MGI Tech) according to the manufacturer’s instructions. DNBs were prepared
 using the DNBSEQ DNB Rapid Make reagent kit (MGI Tech) and high-throughput
 pair-end sequencing primer kit (App-D, MGI Tech) as recommended in the
 instructions. Sequencing was performed using DNBSEQ-T7RS high-throughput
 sequencing kit (MGI Tech) using the supplied primers (App-D) and the
 DNBSEQ-T7 (MGI Tech) platform under 2 × 150 bp conditions. To remove
 primer sequences, the first three bases of each read were removed using
 Cutadapt v4.0 (Martin, 2011). Paired reads with QS &lt;30 or fewer
 than 75 bases were filtered out using Sickle v.1.33 (Joshi and Fass,
 2011). Sequences &gt; 75 nucleotides were removed to standardize read
 length for data analysis. The isolated DNA was
 resuspended in Tris-HCl buffer (pH 7.5) and stored at 4°C. Polymerase
 chain reaction (PCR) was conducted to amplify two gene regions: the
 contiguous mitochondrial COI region (721 bp) and COII region (470 bp). The
 primers used to amplify the COI region were JNfwd
 (5’-GCAGGAACTGGATGAACAG-3’) and Krev (5’-GAGTATCGTCGAGGTATCC-3’) (Dechaine
 and Martin, 2004), and those used to amplify the COII region PierreIII
 (5’-AGAGAGTTTCACCTTTAATAGAACA-3’; Nazari et al., 2007) and Eva
 (5’-GAGAACATTACTTGCTTTCAGTCATCT-3’; Bogdanowicz et al., 1993). Both
 fragments were amplified using TaKaRa Ex Taq Hot Start Version (TaKaRa,
 Tokyo, Japan). The PCR conditions for the COI region were 94°C for 5 min,
 followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min.
 For the COII region, the amplification conditions were 95°C for 5 min,
 followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min,
 and finally 72°C for 10 min. Sequencing reactions were performed using
 BigDye Terminator v3.1 (Applied Biosystems, Carlsbad, USA). All samples
 were sequenced using an ABI PRISM 3100-Avant Genetic Analyzer (Applied
 Biosystems, Carlsbad, CA, USA).
description: # Dual expansion routes likely underlie the present-day population
 structure in a Parnassius butterfly across the Japanese Archipelago
 [https://doi.org/10.5061/dryad.69p8cz9d3](https://doi.org/10.5061/dryad.69p8cz9d3) ## Description of the data and file structure Each sample’s raw fastq sequence data for SNP and haplotype of mtDNA ### Files and variables #### File: Supporting\_Material\_PG.xls **Description:** Each sample’s raw fastq sequence data and haplotype ##### Variables * Sheet 1: an overview of each sheet. * Sheet 2: raw sequence data for each  SNP detected with the Grass-Di method. * Sheet 3: haplotype number for each collected individual (collected year, individual code, collection site, Prefecture, Prefecture number, haplotype number)