Supplementary Materials for Article Whole-genome sequencing of 84 Japanese eels reveals evidence against panmixia and support for sympatric speciation Yoji Igarashi 1, Hong Zhang1 , Engkong Tan 1, Masashi Sekino2 , Kazutoshi Yoshitake 1, Shigeharu Kinoshita 1, Susumu Mitsuyama 1, Tatsuki Yoshinaga 3, Seinen Chow 2, Hiroaki Kurogi 4, Akira Shinoda 5, Yu-San Han 6, Ryoshiro Wakiya 7, Noritaka Mochioka 7, Toshihiro Yamamoto 4, Hiroshi Kuwada 8, Yoshitsugu Kaji 9, Yutaka Suzuki 10, Takashi Gojobori 11, Takanori Kobayashi 2, Kenji Saitoh 2, Shugo Watabe 3 and Shuichi Asakawa 1,* 1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan; [email protected] (Y.I.); [email protected] (H.Z.); [email protected] (E.T.); [email protected] (K.Y.); [email protected] (S.K.); [email protected] (S.M.) 2 National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, Yokohama, Kanagawa 236-8648, Japan; [email protected] (M.S.); [email protected] (S.C.); [email protected] (T.K.) 3 School of Marine Biosciences, Kitasato University, Sagamihara, Kanagawa 252-0373, Japan; [email protected] (T.Y.); [email protected] (S.W.) 4 Yokosuka Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Yokosuka, Kanagawa 238-0316, Japan; [email protected] (H.K.); [email protected] (T.Y.) 5 Department of Biology, Tokyo Medical University, Tokyo 160-8402, Japan; [email protected] 6 Institute of Fishery Science, College of Life Science, National Taiwan University, Taipei 10617, Taiwan; [email protected] 7 Bioresource Sciences, Faculty of Agriculture, Kyushu University, Fukuoka 812-0053, Japan; [email protected] (R.W.); [email protected] (N.M.) 8 Minami-Izu Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Kamo, Shizuoka 415-0156, Japan 9 Wakayama Prefectural Museum of Natural History, Kainan, Wakayama 642-0001, Japan; [email protected] 10 Department of Medical Genome Science, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8561, Japan; [email protected] 11 Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan; [email protected] † Present address: National Association for the Promotion of Productive Seas, Kodenmacho Matsumura Bldg, Nihonbashi Kodenmacho, Chuo, Tokyo 103-0001, Japan; [email protected] ‡ Present address: Tohoku National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Shiogama, Miyagi 985-0001, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-3-5841-5296 Genes 2018, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/genes Genes 2018, 9, x FOR PEER REVIEW 2 of 34 Figure S1. Molecular phylogenetic tree constructed by the maximum likelihood method on the whole mitochondrial DNA sequences from the 84 Japanese eels Anguilla japonica assembled in this study and 19 Anguilla species reported previously. Common Japanese conger (Conger myriaster, AB038381), Kaup’s arrowtooth eel (Synaphobranchus kaupii, AP002977), and sawtooth eel (Serrivomer sector, AP007250) were used as outgroups. The bootstrap probabilities from a 1,000 replicate analyses are given as percentages at the nodes. The results confirmed that all of the 84 eels used in this study are A. japonica. Genes 2018, 9, x FOR PEER REVIEW 3 of 34 Figure S2. Plots of the ratio of average depth of coverage at SNP sites regarded as homozygous and heterozygous against the sequence depth of samples. To distinguish heterozygous SNPs from homozygous SNPs and determine the genotype exactly, sequences with greater depth are more accurate. If the depth is sufficient and the genotyping is accurate, the ratio is expected to converge to 1. Genes 2018, 9, x FOR PEER REVIEW 4 of 34 Figure S3. Cluster analysis using half of the raw data of 9 representative samples together with the 84 samples. Each half data of each sample (the depth of coverage are from 6.9 to 9.7) was independently processed. For all of 9 samples, each-half data and original full data formed a cluster with a closer exclusive relationship, indicating that sequences with a depth of 6.9 or more are sufficient. Genes 2018, 9, x FOR PEER REVIEW 5 of 34 (a) TW15 MR01 TC11 TC12 TC05 MR02 TC08 TC09 TC07 TW03 MR05 TW05 TM01 TC10 TK03 MR06 SG10 TW13 KM13 TC06 TC13 MR03 SG01 TW08 SG02 KM01 TW22 SG07 TW10 SG05 SG04 SG03 SG20 SG13 TW02 SG06 TW19 KM16 SG19 TK02 SG16 SG11 TW06 TW23 SG17 SG09 MR04 TW24 TC03 TC04 SG08 TW16 TW11 TW21 TK01 TW20 TW04 MR07 Depth: 800-900 SG12 SG14 SG15 TW12 TW17 TW18 SG18 KM03 TW07 TW14 KM09 KM08 KM10 TC01 TC02 KM07 10,305,454 SNPs KM11 KM12 Height KM05 KM06 KM14 KM15 KM02 KM04 1200 1300 1400 1500 TW01 TW09 TW15 MR01 TC12 TC05 MR02 TC08 MR05 KM01 KM13 TC09 TC07 TM01 TW03 TK03 MR06 MR03 TW05 TC13 TC06 TW13 SG10 TC10 TC11 TW22 KM16 SG02 SG07 SG20 SG05 SG04 KM03 TW08 SG06 SG01 SG03 SG13 TW19 TW02 TW10 SG19 TK02 SG09 SG11 TW06 SG16 TW04 MR04 TW24 SG08 TK01 TW23 SG17 TW21 TW20 SG14 SG12 TW16 TW12 TW11 MR07 TW17 SG15 TC03 TC04 TW18 SG18 TW07 TW14 KM09 KM08 KM10 Depth: 900-1000 KM07 KM11 KM12 TC01 TC02 KM02 KM04 KM05 KM06 11,764,634 SNPs KM14 KM15 Height 1200 1300 1400 1500 TW01 TW09 MR01 TW15 KM01 TC12 TC05 MR02 KM13 MR05 TC08 TC09 TM01 TC07 KM03 TK03 MR03 TC13 TC06 TW03 MR06 SG10 KM16 TW13 SG02 SG20 SG05 SG07 SG06 TW22 TW05 SG13 SG04 SG03 TK02 SG01 TW02 TW19 SG19 TK01 TW08 SG08 TC10 TC11 SG09 MR04 SG16 SG11 TW06 TW23 SG12 SG14 TW10 TW04 SG17 MR07 TW24 TW20 TW21 SG15 TW17 KM09 TW12 TW16 KM08 KM10 TW11 SG18 TW18 TC03 TC04 TW14 TW07 Depth: 1000-1100 KM11 KM12 KM07 TC01 TC02 KM02 KM04 10,469,990 SNPs KM05 KM06 KM14 KM15 Height 1150 1250 1350 1450 Kuma River estuary eels TW01 TW09 (b) ●● ●● ●●●● ● ●●●● ● ● ●●●●●● ● ● ● ● ●●●●● ● ● ●● ● ●●●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● 200 0 200 0 200 0 − − − Depth: 1000-1100 400 400 Depth: 800-900 Depth: 900-1000 − − 400 − PC2 10,305,454 SNPs PC2 11,764,634 SNPs PC2 10,469,990 SNPs 600 600 − − 600 ● Mariana Ridge ● Mariana Ridge − ● Mariana Ridge ● Midori River ● Midori River ● Midori River ● Sagami River ● Sagami River ● Sagami River ● 800 ● ● 800 Taiwan Ta iw a n Taiwan ● − − ● Takase River ● Ta ka se R iv e r ● Takase Riv er ● Tam a River ● Ta ma Riv er ● 800 ● Tama River − ● ● Tsuchi River ● ● Tsuchi River ● ● Tsuchi River ● −500 −400 −300 −200 −100 0 100 −500 −400 −300 −200 −100 0 100 −400 −300 −200 −100 0 100 PC1 PC1 PC1 Figure S4. Genetic population analyses at 3 different sequence depths for the Japanese eel Anguilla japonica collected from different areas. (a) Cluster analysis and (b) PCA. The depth ranges of 800 to 900, 900 to 1000, and 1000 to 1100 include 10,305,454, 11,764,634, and 10,469,990 SNP sites, respectively. Genes 2018, 9, x FOR PEER REVIEW 6 of 34 (a) TW15 MR01 MR02 TC12 MR05 TC05 TC01 TC11 MR06 TM01 TW03 TC08 TC09 MR03 TC07 TW05 SG10 TC10 TK03 TW13 TW08 SG05 SG04 TW22 SG07 SG01 SG02 TC03 SG06 TW02 SG20 TW19 SG03 SG13 TC13 TC06 TW10 SG19 MR07 SG09 SG11 MR04 SG16 SG08 SG17 TW21 SG14 SG12 TW06 TK02 TW23 TW20 TW04 TW12 SG15 TW16 TW24 TC04 TW18 SG18 TK01 TW11 TW07 TW17 TW14 TC02 KM16 KM13 Height KM01 Kuma River estuary eels KM08 KM04 KM07 KM14 KM15 KM05 KM10 KM06 KM09 KM02 KM03 KM11 KM12 1300 1400 1500 1600 1700 TW01 TW09 (b) ● Mariana Ridge ● ● Midori River ● Sagami River ● Taiwan ● Takase River ● Tama River ● Tsuchi River ● PC2 0 200 400● 600 800● 1000 ● ●●●● ● ● ● ● ●●●●●●●● ● ● ● ● ● ●●●●●●●●●● ● ● ● ● ●●●● ● ● −500 −400 −300 −200 −100 0 100 PC1 Figure S5. Genetic population analysis was based on 4,557,695 major SNPs that were present in 15 to 66 of the 84 samples. (a) Cluster analysis and (d) PCA were performed using the SNPs. This result confirmed that it was not just certain minor SNPs characterizing the samples. Genes 2018, 9, x FOR PEER REVIEW 7 of 34 Figure S6. Molecular phylogenetic tree based on calculated pairwise genetic distance. Molecular phylogenetic tree constructed by the unweighted pair group method using MEGA6 software (a), the neighbor-joining method using MEGA6 software (b), the group average method using R (c), and the ward method using R (d) based on a pairwise genetic distance of 30 Japanese eel Anguilla japonica individuals. Genes 2018, 9, x FOR PEER REVIEW 8 of 34 Figure S7. Genetic analysis of the 84 sampled Japanese eels Anguilla japonica based on SNP sites at different linkage groups of A. japonica ranged from 13,825 for LG17 (minimum) to 161,375 for LG9 (maximum). Results of the Cluster analyses and PCA using the SNP sites on each linkage group are shown. Genes 2018, 9, x FOR PEER REVIEW 9 of 34 Figure S7. continued. Genes 2018, 9, x FOR PEER REVIEW 10 of 34 Figure S7. continued. Genes 2018, 9, x FOR PEER REVIEW 11 of 34 Figure S7. continued. Genes 2018, 9, x FOR PEER REVIEW 12 of 34 Figure S7. continued. Genes 2018, 9, x FOR PEER REVIEW 13 of 34 LG1 LG4 KM09 KM02 KM09 KM04 KM12 KM06 KM10 KM14 KM13 KM05 KM06 KM10 KM04 KM15 KM16 KM08 KM16 KM08 KM11 KM02 KM11 KM03KM07 KM01 KM14 KM05 KM03 KM07 KM12 KM01 KM15 KM13 KM05 KM03 KM08 KM01 KM02 KM13 KM06 KM09 KM15 KM07 KM16 KM11 KM10 KM04 KM12 KM14 LG2 LG5 KM11 KM12 KM02 KM10 KM08 KM14 KM07 KM04 KM06 KM03 KM13 KM01 KM15 KM05 KM09 KM16 KM05 KM06 KM08 KM09 KM01 KM15 KM13 KM02 KM07 KM04 KM14 KM16 KM10 KM03 KM12 KM11 LG3 LG6 KM11 KM10 KM08 KM03 KM12 KM15 KM16 KM06 KM01 KM09 KM14 KM05 KM07 KM13 KM02 KM04 Figure S7.