DOI: 10.5061/dryad.zkh1893bw
title: History of the terrestrial isopod genus Ligidium in Japan based on phylogeographic analysis

description: Background Phylogeographical approaches explain the genetic diversity of
 local organisms in the context of their geological and geographic
 environments. Thus, genetic diversity can be a proxy for geological
 history. Here we propose a genus of woodland isopod, Ligidium, as a marker
 of geological history in relation to orogeny and the Quaternary glacial
 cycle. Results Mitochondrial analysis of 721 individuals from 97 sites
 across Japan revealed phylogenetic divergence between the northeastern and
 southwestern Japan arcs from 7 to 3.5 million years ago. It also showed
 repeated population expansions in northeastern Japan in response to
 Quaternary glacial and interglacial cycles. Genome-wide analysis of 83
 selected individuals revealed multiple genetic nuclear clusters. The
 genomic groupings were consistent with the local geographic distribution,
 indicating that the Ligidium phylogeny reflects its migration history.
 Conclusion Ligidium DNA sequence analysis can provide insight into the
 geological, geographical, and paleoenvironmental history of the studied
 region.
description: Sample
 collection We surveyed <em>Ligidium</em>
 populations in Japan. We collected 828 <em>Ligidium</em>
 specimens from 97 sites and sequenced 721 samples (Fig. 1, Table 1).
 Samples were preserved in 70–99.5% ethanol in 2-mL microtubes at 4°C or room
 temperature. DNA
 extraction, amplification, and sequencing
 Genomic DNA was isolated from
 the muscles of the abdomen and legs with a DNA Mini Kit (Qiagen,
 Germantown, MD, USA). PCR amplification was conducted using the primer
 pair LCO-1490 and HCO-2198. Amplification and cycling conditions were as
 in a previous work.
 Details of the experimental conditions are provided in the Supplemental
 Data. Sequencing was conducted with an ABI 3130 Genetic Analyzer (Applied
 Biosystems, Waltham, MA, USA). Sequences were checked and assembled using
 MEGA7. Mitochondrial gene
 locus (CO1) sequences for 721 individuals were determined and aligned using
 ClustalW. RAD-seq We performed
 RAD-seq analysis to search for SNPs in individuals from Niigata and
 Hokkaido obtained in previous studies, two regions in northern Japan (Aomori and
 Sendai), and two regions in western Japan (Shizuoka and
 Sendai). Tables 1 and S1
 list the samples used, and the sampling sites are shown in Fig. 1c.
 Genomic DNA was isolated from almost whole-body tissues with a DNA Mini
 Kit (Qiagen). Libraries for RAD-seq were prepared with EcoRI and BglII
 restriction enzymes. The
 library was sequenced with 150 + 150 bp paired-end reads in one lane of an
 Illumina HiSeqX instrument (Illumina, San Diego, CA, USA) by Macrogen
 (Seoul, South Korea). Raw reads were trimmed using
 Trimmomatic-0.39 with the
 following parameters: ILLUMINACLIP: adapter.fasta:2:30:10:keepBothReads,
 SLIDINGWINDOW: 4:15, CROP: 132, HEADCROP: 2, and MINLEN: 130. Sequences
 are available at the DNA Data Bank of Japan (DDBJ) Sequence Read Archive
 (DRA014204). We used two pipeline programs to call the SNPs: denovo_map.pl
 provided by Stacks and
 ipyrad. Following a
 previous work, we varied
 the combinations of the denovo_map.pl parameters as follows: (n, M) = (2,
 1), (3, 2), (4, 3), (5, 6), and selected (n, M) = (2, 1), which called the
 most SNPs. We used Stacks’ populations program to analyze populations of
 individual samples, calculate population genetics statistics, and export
 data in various output formats for analysis. PLINK
 v1.90b6.18 was used for
 data handling. Alleles with a frequency of &lt; 1% and sites with
 &gt; 50% heterozygosity were removed. Only SNPs shared by ~80% of the
 individuals were retained. With ipyrad, loci with frequencies of &gt;
 50% heterozygosity were removed, and SNPs shared by ~70% of the local
 populations were retained. We retained SNPs shared by at least two
 individuals and filtered out individuals that did not have 80% of all SNPs
 using TASSEL 5. After
 filtering with TASSEL 5, we used PGDSpider to convert the VCF files for
 other analyses. Genetic
 structure analysis We tested the
 ability of Structure v. 2.3.4 to determine the genetic structure of
 populations using Bayesian cluster analysis. Ten simulations were run,
 with the burn-in period and Markov chain Monte Carlo iterations set to
 10<sup>5</sup> and 10<sup>6</sup>, respectively.
 The maximum value of K was determined based on the mtDNA results and
 geographical distribution. For the Structure analysis, one SNP was
 randomly sampled from each locus to avoid the effect of linkage
 disequilibrium. The python script vcf_single_snp.py (radseq/vcf_single_snp.py at master · pimbongaerts/radseq · GitHub) was used to obtain the one SNP datum from ipyrad, and drawings were created using the R package pophelper. In addition, PCA was performed to visualize the genetic differences among populations using the adegenet package in R. We obtained pairwise Fst values for the RAD-seq dataset using Arlequin 3.5.1.2. Fst values were used to test population structure, supported by cluster analysis, and statistical significance was based on 1000 restored extractions.
description: This data repository contains the following files:
 RAD-seq_trimmed.zip<br>See the details in
 Materials and Methods "RAD-seq" and README files in each folder.
 This folder contains SNPs VCF files after calling by two pipeline
 programs, denovo_map.pl provided by Stacks  (Catchen et al., 2013) and
 ipyrad (Eaton & Overcast, 2020). Folders are divided in groups.
 See Supplemental Information for grouping information Fig. S3.
 Northeastern_Japan and Western_Japan folder contains Structure analyses
 results in the article. The files are summarized by Structure Harvester
 (Earl et al., 2012).  Bioinformatic_scripts.docx:
 Bioinformatic scripts included in “History of the terrestrial isopod
 genus <em>Ligidium</em> in Japan based on phylogeographic
 analysis“.<br>It contains the scripts of TreeAnnotator Analysis,
 Stacks Analysis, ipyrad Analysis, and the Python script for random
 sampling one SNPs, R codes for plotting structure results by pophelper,
 and R codes for plotting PCA results by adgenet package in R. 
 Supplemental Information: Supplemental Information of “History
 of the terrestrial isopod genus <em>Ligidium</em> in Japan
 based on phylogeographic analysis“.