Whole-genome analyses reveal the phylogeny and gene flow of Japanese and Ryukyu wild boars

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Within this archipelago, there are two subspecies: the Japanese wild boar (JWB, Sus scrofa leucomystax ) and the Ryukyu wild boar (RWB, Sus scrofa riukiuanus ), which exhibit differences in morphology and habitat. Until now, the whole genomes of JWB and RWB individuals with known origins had not been sequenced, leaving their genetic relationships with other populations unclear. In this study, we resequenced the whole genomes of five JWB individuals from Honshu (specifically Tochigi, Aichi, and Kyoto), one from Shikoku (Tokushima), and one from Kyushu (Saga). Additionally, we resequenced two RWB individuals from Okinawa and Ishigaki Islands. Results We analyzed the genomes to clarify the genetic relationships between JWB, RWB, and other lineages of the genus Sus . Phylogenetic trees based on genomic data indicate that the JWB and RWB populations form distinct clades, and these two clades form a monophyletic clade. The JWB/RWB clade diverged from a common ancestor with the Eastern Russian and Northern Chinese wild boars. Principal component analysis confirmed distinct genetic clusters for JWB and RWB. These results suggest that JWB and RWB represent independent boar lineages that evolved following isolation within the Japanese archipelago. However, the f4 statistic indicates gene flow between JWB and Eastern Russian/Northern Chinese boars, suggesting migration across Northern East Asia. Conclusion Overall, our results show that JWB and RWB diverged from East Asian boars through dispersal and isolation within the Japanese archipelago. Wild boar Whole genome sequencing introgression Japanese archipelago Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The wild boar ( Sus scrofa ) is a species found worldwide [ 1 ]. Phylogenetic studies indicate that its ancestral population originated in Southeast Asia and split into Eastern and Western Eurasian lineages approximately 200,000 years ago [ 2 ]. Wild boars have not only expanded to major continents but also to islands separated by seas, such as those in Southeast Asia [ 3 ]. They have a high potential for environmental adaptation, including high-altitude areas [ 4 ] and low-temperature regions [ 5 ]. Previous genomic studies have documented gene flow between European and Chinese domestic pig lineages [ 6 ] as well as between wild boars and domestic pigs in both Europe and China [ 7 ]. The Japanese Archipelago is home to two subspecies of wild boar: the Japanese wild boar (JWB, Sus scrofa leucomystax ) and the Ryukyu wild boar (RWB, Sus scrofa riukiuanus ). JWB is found on the islands of Honshu, Shikoku, and Kyushu, while RWB inhabits the Ryukyu Islands. The morphological differences between these two subspecies are well-documented. One significant distinction is their body size; JWB individuals are generally larger than RWB individuals, as studies on mandible and dental morphology indicate [ 8 ]. Furthermore, RWB has been reported to be the smallest wild boar in the world [ 9 ]. To clarify the genetic relationships among JWB/RWB, studies using mitochondrial DNA have been conducted [ 10 , 11 ]. Furthermore, a study using microsatellite DNA analysis has revealed the population structure of JWB across Honshu, Shikoku, and Kyushu [ 12 ]. In a study with higher sample resolution, 15 clusters were observed due to geographic barriers such as natural topography, human settlements, and man-made structures [ 13 ]. Currently, the whole genome sequence of only one individual of the Japanese wild boar has been reported (accession number: ERR173212) [ 7 ]. However, this study did not specify the geographic origin of this individual. The genome of this individual has been used in studies, such as Zhang, Traspov et al. [ 5 ], which demonstrated a sister relationship between this individual and the clade that includes Russian and Korean wild boars. However, it remains unclear which subspecies this individual belongs to. In this study, we resequenced the whole genomes of five Japanese wild boar (JWB) individuals from Honshu (Tochigi, Aichi, and Kyoto), as well as one from Shikoku (Tokushima) and one from Kyushu (Saga). Additionally, we resequenced two Ryukyu wild boar (RWB) individuals from Okinawa and Ishigaki Islands. Using our data, along with publicly available genomic sequences from wild boars (WBs) and domestic pigs (DPs), we analyzed the genomes to clarify the genetic relationships between JWB, RWB, and other lineages of the genus Sus . Methods Animal samples Tissue samples from 5 JWB were selected along 1,160 km from the main islands of Japan (Honshu, Kyushu and Shikoku) and 2 RWB from Ryukyu archipelago (Okinawa and Ishigaki Islands). We recorded the date, location, estimated age, and weight associated with each animal (Supplementary Table 2). Until processing, samples were stored at -20°C. A preliminary selection of individuals for whole genome sequencing (WGS) was conducted from a wider JWB and RWB collection of 1,062 individuals from National Agriculture and Food Organization (NARO), Tsukuba, Japan. JWB and RWB whole genome sequencing dataset construction The ear and muscle tissues were collected between 2014 and 2017. There is evidence that local domesticated pigs had recently introgressed into the wild boar population, which found hybrids between JWB and European Domestic pigs [14]. To accurately capture the evolutionary history and diversity of JWB and RWB, sample selection was conducted based on the following criteria: (i) Individuals were classified into different group based on the results from Sawai et al. 2023 [13], (ii) A subset of samples were selected from different islands, prefectures, and sounders to reduce closely related individuals based on spatial coordinates, (iii) (ii) was analyzed by mitochondrial DNA (mtDNA) D‑loop haplotype analysis to exclude hybrids, and then, consequently seven individuals from divergent branches of mtDNA Neighbor-Joining network [15] were taken to define the final sequencing dataset . DNA extraction and sequencing Genomic DNA extraction from the ear and tongue tissue samples was conducted using the QiAmp DNA mini kit (QIAGEN Ltd., Hilden, Germany) in accordance with the manufacturer’s instructions. Genomic DNA quantity and quality were assessed the Agilent 2200 using TapeStation (Agilent Technologies), which revealed high‑molecular‑weight genomic DNA ranging from 4,600 to 8,000 bp. Subsequently, DNA samples were re‑quantified and normalized to prepare whole‑genome sequencing libraries constructed with an Illumina DNA Prep kit and standard TruSeq adapters (Illumina, San Diego, CA) according to the manufacturer’s protocol. The sequenced paired‑end 150 bp reads were obtained in an Illumina NovaSeq X Plus. Data availability The data have been deposited with links to BioProject accession number PRJDB40278 in the DDBJ BioProject database. Extraction of single nucleotide polymorphisms (SNPs) and vcf file preparation We used sequencing data from 34 wild boars, 19 domestic pigs, and two outgroup species (Supplement Table 1). Sequence reads were processed using AdapterRemoval v2 with parameters --trimns --trimqualities --minquality 25 --minlength 25, which removed adaptor sequences, ambiguous bases (N), and terminal bases with Phred quality scores below 25. The trimmed reads were mapped to the Boar and Pig reference genome (GCF_000003025.6_Sscrofa11.1)[16] using BWA “BWA-MEM” algorithm. The mapping data were exported in BAM file format and sorted and indexed using SAMtools [17]. The duplicated reads in the BAM files were marked by the MarkDuplicates algorithm, implemented in GATK v4.6 (https://gatk.broadinstitute.org/hc/en-us). We performed haplotype calling on all individuals analyzed in this study using the HaplotypeCaller algorithm in GATK v4.6, and output as gvcf format (-ERC GVCF option). All gvcf files were combined into a single gvcf file by the CombineGVCFs algorithm in GATK v4.6. The combined gvcf file was genotyped by the GenotypeGVCFs algorithm in GATK v4.6. To maximize the number of SNPs for analyses, we prepared datasets from the genotyped vcf file for each analysis by following filtering using vcftools [18]. Dataset of Pig and Boar for principal components analysis (PCA) and phylogenetic tree construction We removed all sites with missing data (Max-missing 1). Then, we removed all indels, singleton, and doubleton sites to eliminate PCR and sequencing errors that may have occurred in one individual by minor allele count (mac) filtering (mac = 3). We extracted bi-allelic sites with coverage equal to or more than one in all individuals and with GQ values equal to or more than eight in all individuals, using VCFtools [19]. Next, to obtain approximately independent SNPs, we performed linkage disequilibrium (LD) pruning. Using PLINK ver. 1.90b6.21 [20] with an option “--indep-pairwise 50 10 0.1” to conduct the LD pruning. The final dataset consisted of 225,140 sites. For PCA, we removed outgroup species. Dataset of Pig and Boar for Admixtools We removed all sites with missing data (Max-missing 1). Then, we removed all indels, singleton, and doubleton sites to eliminate PCR and sequencing errors that may have occurred in one individual by minor allele count (mac) filtering (mac = 3). We extracted bi-allelic sites with coverage equal to or more than one in all individuals and with GQ values equal to or more than eight in all individuals, using VCFtools [19]. Phylogenetic analysis The SNP datasets for phylogenetic analysis were converted to PHYLIP format using TASSEL 5 [21]. 10 kb sequences from the 5’ end of the PHYLIP format file were extracted and a model for the Maximum Likelihood (ML) method was selected using MEGA ver. X [22]. A phylogenetic tree was constructed using the ML method using PhyML ver. 3.3.20220408 [23], using the GTR model (“-m GTR”) based on the best-fit model selected in MEGA, with 100 bootstrap replicates. PCA (and ADMIXTURE) We performed principal component analysis (PCA) using PLINK ver. 1.90b6.21 [24], and individual ancestry proportions were estimated using ADMIXTURE ver. 1.3.0 [25]. PCA and ADMIXTURE analysis utilized a dataset excluding the outgroup Outgroup-f3, and f4 statistics We used the Outgroup- f3 statistics to measure shared genetic drift between JWB or RWB and the other Sus populations, and the f4 statistics to test the genetic affinity between JWB/RWB and WB populations from Northern China, Eastern Russia, and European DP. Outgroup- f3 and f4 statistics implemented in ADMIXTOOLS ver. 7.0.2 [26] were used to evaluate the shared genetic drift among JWB/RWB and the Sus population from the other regions. Results Genetic relationship of JWB/RWB to the other Sus We resequenced the whole genomes of seven individuals of Japanese wild boars, comprising five JWB (Japanese Wild Boar) and two RWB (Ryukyu Wild Boar) (see Fig. 1, Table1). To clarify the phylogenetic relationships among JWB, RWB, and other Sus populations, we constructed a genome-wide SNP dataset comprising genomic information from WBs and DPs worldwide (refer to Fig. 1, Supplement Table 1 for details). Using this SNP dataset, we constructed an ML phylogenetic tree (see Fig. 2). The tree shows the first major split between European and East Asian clades, with the Northern Chinese and Eastern Russian clades diverging from the Southern Chinese WB, consistent with the previous study [5] (Fig. 2). JWB and RWB formed a monophyletic clade, within which each subspecies formed its own subclade. Notably, the JWB/RWB clade is a sister clade to the Eastern Russian/Northern Chinese WB. Within the JWB subclade, the individual from Kyushu (Saga) is basal, followed by the individual from Shikoku (Tokushima), and the three individuals from Honshu cluster (Kyoto, Aichi, and Tochigi) together. This branching pattern reflects their geographic distribution across the Japanese mainland (see Fig. 1). Note that an individual, namely ERR173212, which has been recognized as the JWB in the public database, is placed in the clade of RWB, indicating this individual originates from the RWB (Fig. 2). Table 1 Sample and sequencing information ID Population Location Mapped reads Depth A13 Japanese Wild Boar Tochigi 597,940,320 34.7 B43 Japanese Wild Boar Aichi 603,397,399 34.9 D28 Ryukyu Wild Boar Okinawa 623,072,951 36.2 E10 Japanese Wild Boar Saga 637,877,951 35.4 F22 Japanese Wild Boar Tokushima 632,351,129 35.1 G28 Ryukyu Wild Boar Ishigaki 638,476,155 35.5 H37 Japanese Wild Boar Kyoto 615,839,582 35.7 Next, we conducted PCA on the same dataset used for the ML tree (Fig. 3). The results indicated that the first principal component (PC1) separated European DPs and Western Russian WBs from Eastern Asian WBs and DPs, which included Chinese, Eastern Russian/Northern Chinese, RWB, and JWB populations. The second principal component (PC2) further differentiated the Southern Chinese WBs and Chinese DPs into distinct clusters: WBs in Eastern Russian/Northern Chinese, RWB, and JWB (Fig. 3). These findings indicate that JWB and RWB are genetically distinct from the other WB and DP populations. Furthermore, JWB and RWB are closely related yet exhibit genetic differentiation. In the ADMIXTURE analysis (Supplement Fig. 1-2), at K = 4, JWB and RWB shared a common ancestry component. However, at K = 5, JWB and RWB are assigned their own ancestry, respectively. Additionally, the previously reported JWB (ERR173212) showed the same ancestry component as RWB at K = 5, further confirming that this individual belongs to the RWB. We further investigated the genetic relationship among JWB, RWB, and other Sus individuals, using outgroup- f3 statistics [26] with the warthog ( Phacochoerus africanus ) as the outgroup (Fig. 4a, 4b, Supplement Fig. 3-8). Comparisons of f3 values for Okinawa RWB and other individuals showed that the highest f3 values were observed with ERR173212, followed by RWB (Ishigaki), JWB, and the WBs from Eastern Russia/Northern China (see Fig. 4a). Therefore, the closest population to RWB is JWB. When comparing f3 values between individuals of JWB and individuals from other populations, the highest f3 values were observed with other JWB individuals, followed by WBs from Eastern Russia/Northern China (see Fig. 4b, Supplementary Fig. 5-8). This result was inconsistent with the earlier findings that JWB is closest to RWB (see Fig. 2). This inconsistency raises the possibility of introgression between the JWB and Eastern Russian/Northern Chinese WBs. If introgression had occurred between JWB and Eastern Russian/Northern Chinese WBs, we would expect the f3 value between them to be slightly higher. Introgression between JWB and Eastern Russian and Northern Chinese WBs We investigated a potential introgression between the JWB/RWB populations and other populations using f 4 statistics [26]. Our analysis of introgression between the Eastern Russian (Fig. 5a) and Northern Chinese (Fig. 5b) WBs revealed that only individuals from the JWB population exhibited significant positive f 4 values, while individuals from the RWB population did not exhibit significant f 4 values. These results suggest that introgression events have occurred between the JWB and the Eastern Russian/Northern Chinese WBs. Additionally, we examined the possibility of introgression between the JWB/RWB populations and the European DP (Supplementary Fig. 9), since a European mtDNA haplotype was identified in one JWB individual [14, 27]. We tested gene flow between European DP and JWB/RWB using f 4 statistics. The results showed a weaker positive f 4 value between European DP and JWB than that shown between JWB and the Eastern Russian/Northern Chinese WBs. This result indicates the possibility of past hybridization between JWB and European DP, supporting the conclusions of previous studies [14, 27]. Discussion Geographic history of the Japanese Archipelago and JWB/RWB divergence Our phylogenetic analysis showed that a monophyletic JWB and RWB clade is placed within the East Asian clade, and that it diverged from the common ancestor with Eastern Russian/Northern Chinese WBs. This finding suggests that the ancestors of the JWB and RWB likely originated in East Asia before migrating to the Japanese archipelago, where they evolved under isolation. Zhang et al. 2022, 2025 [ 2 , 5 ] estimated divergence time using a mutation rate of 3.6 × 10⁻⁹ per generation and a generation time of 3 years. Their studies estimate that the divergence between European and East Asian wild boars occurred around 250,000 years ago, with southern and northern Chinese populations diverging approximately 30,000 years ago, and a more recent separation between the Northern Chinese and the Eastern Russian populations occurring around 10,000 years ago. Based on the estimated divergence times, the ancestral population of JWB/RWB separated from the Eastern Russian/Northern Chinese lineage approximately 30,000 to 10,000 years ago (Fig. 2 ). Our study did not intend to extrapolate the exact location on the mainland where the common ancestor of JWB/RWB migrated to the Japanese archipelago. In the future, given that genomic data from modern and ancient wild boars along the continental coast become available, the discovery of the origins of JWB/RWB will progress robustly. Since the Jomon period (160,000–3,000 years ago), numerous wild boar remains have been excavated on the Japanese mainland (Honshu, Shikoku, and Kyushu) [ 28 ]. Additionally, the earliest known occurrences of wild boars on Okinawa and Ishigaki Islands date back to the Late Pleistocene [ 29 ]. Archeological remains of wild boars from the Late Pleistocene have been found in the Shiraho Saonetabaru Cave on Okinawa Island, dating from around 24,000 to 16,000 years ago [ 30 ]. Similarly, WBs remains have been reported from the Sakitari Cave on Ishigaki Island, dating to approximately 20,000 years ago [ 31 ]. These archaeological evidences indicate that wild boars likely arrived in the Japanese archipelago between 10,000 and 30,000 years ago, which is concordant with this study. In the JWB clade, the Saga (Kyushu) individual is positioned as basal, followed by the Shikoku individual, and then the Honshu individuals. This tree topology corresponds with the geographical distribution of these individuals and supports previous research by microsatellite markers [ 12 , 13 ]. During the Last Glacial Maximum, Honshu, Shikoku, and Kyushu were connected by land due to lower sea levels. Sea level rose during the postglacial period, resulting in the separation of Honshu and Shikoku around 7,000 years ago and of Honshu and Kyushu around 5,000 years ago [ 32 ]. These geological events are likely responsible for the population divergence observed among the JWB. Similar patterns of population structure have been reported in other mammals, such as the red fox ( Vulpes vulpes ) [ 33 ]. In the case of RWB, individuals from Okinawa and Ishigaki formed distinct clusters. Notably, these two islands have never been connected by land, even during periods of low sea levels. It is likely that the ancestral RWB first colonized one island before being dispersed to others, where the isolated populations subsequently differentiated. Introgression event of the JWB Our analysis indicates that there is gene flow between JWB and WB populations in Eastern Russia and Northern China. However, it remains unclear in which direction this gene flow occurred: whether individuals from eastern Russia and northern China introgressed into JWB, or vice versa. Furthermore, the f 4 values between JWB and individuals from Eastern Russia and Northern China did not show significant differences (Fig. 5 a, 5 b), leaving us uncertain about which specific WB populations on the continent have experienced gene flow with JWB. Collecting more modern and ancient genomic data will help clarify the details of gene flow. Conclusion In this study, we newly determined the whole genome sequences of five Japanese wild boars and two Ryukyu wild boars and investigated their genetic relationships with other Sus populations. Our results showed that JWB and RWB form a monophyletic clade diverging from the common ancestor with the Eastern Russian/Northern Chinese wild boar lineage, and that JWB and RWB each form distinct clades. Furthermore, we detected gene flow between JWB and wild boars from Eastern Russia/Northern China, suggesting WB migration in Northern East Asia. This initial NGS-based genomic analysis of JWB and RWB establishes a fundamental baseline for addressing the still-uncertain origins of their ancestors, and dispersion across the islands. Further genomic data from modern and ancient WB populations will elucidate the unresolved aspects of the historical and geographical distribution of Sus Scrofa in Asia. Abbreviations JWB: Japanese wild boar DP: domestic pig PCA: principal component analysis RWB: Ryukyu wild boar SNP: single nucleotide polymorphism WB: wild boar Declarations Ethics approval and consent to participate Wild boar samples were purchased from a commercial source, with their origin information. No sampling of live animals was performed in this study, and therefore no ethical approval was required. Consent for publication Not applicable Availability of data and materials The nucleotide sequences in this study have been deposited in the DDBJ Sequenced Read Archive under accession number PRJDB40278. Competing interests The authors declare that they have no competing interests Funding This work was supported by JSPS KAKENHI Grant Number 24H01588 under the Grant-in-Aid for Transformative Research Areas (A) Integrative bioarchaeological studies on human prehistory in the Japanese archipelago to MT, and Japan Society for the Promotion of Science (JSPS) International Fellowships for Research in Japan Short-term (PE21054) to DG. Acknowledgements We thank Dr. Takehisa Yamamoto from National Institute of Animal Health, National Agriculture and Food Research Organization, for the provision of wild boar samples. Author Contributions RI performed population genetic analyses using the whole genome sequence data and wrote and edited manuscript. DG selected and purified genomic DNA samples based on the basic genetic analysis of Japanese and Ryukyu wild boar, obtained WGS data, and edited manuscript. YT edited manuscript and supported genomic data and population genetic analyses. 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Supplementary Files SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplemantFigure.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 11 May, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 11 Apr, 2026 Reviewers agreed at journal 11 Apr, 2026 Reviewers invited by journal 09 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Editor invited by journal 06 Apr, 2026 Submission checks completed at journal 06 Apr, 2026 First submitted to journal 06 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9264188","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621414886,"identity":"ab49971c-d4ea-430d-a9d9-bba0b9c0935d","order_by":0,"name":"Rikako Itoi","email":"","orcid":"","institution":"The Graduate University for Advanced Studies, SOKENDAI","correspondingAuthor":false,"prefix":"","firstName":"Rikako","middleName":"","lastName":"Itoi","suffix":""},{"id":621414887,"identity":"2d53b46e-63c6-462d-96a0-015edded7e41","order_by":1,"name":"David Gamarra","email":"","orcid":"","institution":"National Agriculture and Food Research Organization","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Gamarra","suffix":""},{"id":621414888,"identity":"8078e558-2ba5-4f16-ab49-efe6f5bbbede","order_by":2,"name":"Yohey Terai","email":"","orcid":"","institution":"The Graduate University for Advanced Studies, SOKENDAI","correspondingAuthor":false,"prefix":"","firstName":"Yohey","middleName":"","lastName":"Terai","suffix":""},{"id":621414889,"identity":"ddb5655d-2cd5-41d0-b057-079815bd24ad","order_by":3,"name":"Jun Gojobori","email":"","orcid":"","institution":"The Graduate University for Advanced Studies, SOKENDAI","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Gojobori","suffix":""},{"id":621414890,"identity":"27cfbc6c-4b67-4afb-acb5-43e9ad007808","order_by":4,"name":"Masaaki Taniguchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYNCCAxJy9sebD8D5BsRoMWY4cyyBJC0MiQw3cohQCAK67ccffvhxxiKBsefMtwc/99gwyLs3MBQX4NFidibHWLLnhkQeM3vvdsOeZ2kMhmcOMBjPwKflQA6DBM8HiWI2nrPbJHgOHGYwnJHAYMyDT8v5549//vkgkdgjkfNM8g9RWm4kmEnz3JBInCGRwyYNskVegqCWN2bWMmckjA14jplJyxxI4zHgOdiA3y/n0x/ffHOsTs6AvfmZ5JsDNnLy7c3HjPGFGAbgMTjA2GZMig4GBvkGBubHpGkZBaNgFIyCYQ4A4ZhRVTthyusAAAAASUVORK5CYII=","orcid":"","institution":"National Agriculture and Food Research Organization","correspondingAuthor":true,"prefix":"","firstName":"Masaaki","middleName":"","lastName":"Taniguchi","suffix":""}],"badges":[],"createdAt":"2026-03-30 08:24:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9264188/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9264188/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107183104,"identity":"29546b6a-f344-4828-b147-5e02b738ebff","added_by":"auto","created_at":"2026-04-17 18:04:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215379,"visible":true,"origin":"","legend":"\u003cp\u003eA map showing the origin of the genomic data used in this study.\u003c/p\u003e\n\u003cp\u003eTriangles indicate the newly sequenced genomic data for JWB and RWB.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/c120eaabab2e2ed8171cfed6.png"},{"id":107183107,"identity":"be2bb548-d149-4d9f-aa3f-e2e5a998811c","added_by":"auto","created_at":"2026-04-17 18:04:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":220417,"visible":true,"origin":"","legend":"\u003cp\u003eAn ML tree of boars based on 225,140 unlinked SNPs.\u003c/p\u003e\n\u003cp\u003eBootstrap values are shown on or under the nodes. Bootstrap values ≥ 90 are indicated with asterisks.\u003c/p\u003e\n\u003cp\u003eER and NC indicate individuals from the Eastern Russian and Northern Chinese populations, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/57fbd0897744f2bd672b1719.png"},{"id":107481858,"identity":"6c176fbf-0a73-438a-ae1e-66d1a6f07ad8","added_by":"auto","created_at":"2026-04-22 02:20:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131588,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Components Analysis (PC1 versus PC2) of WB and DP.\u003c/p\u003e\n\u003cp\u003eNewly sequenced JWB and RWB genome data are shown by triangles\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/e097bd1e451418c1f75dcff8.png"},{"id":107483288,"identity":"98a81edc-d99d-433a-bb23-54ebda745bce","added_by":"auto","created_at":"2026-04-22 02:27:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349955,"visible":true,"origin":"","legend":"\u003cp\u003eShared genetic drift between (a) Ishigaki or (b) Tochigi and all other WB/DP measured by outgroup f3 statistics. Each f3 statistical value is plotted in order of highest to lowest value from the top. The names of the WB/DP individuals are shown on the left side of the panel. Error bars represent standard errors.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/758e16db02ea7738be433631.png"},{"id":107485948,"identity":"b891c01a-d250-49df-9c40-e94b2a642729","added_by":"auto","created_at":"2026-04-22 02:36:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2178452,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/2b123c46-3b9b-4796-b9db-f4752916d54e.pdf"},{"id":107482467,"identity":"afda2cea-6049-4e06-9206-f4c4eb9bf8c8","added_by":"auto","created_at":"2026-04-22 02:23:38","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20861,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/1dae5a385c3222a57cf18412.xlsx"},{"id":107483285,"identity":"c71fc549-7ca2-4d63-9c58-85f21c215727","added_by":"auto","created_at":"2026-04-22 02:27:10","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18635,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/3c27c2489df8fb994be2a051.xlsx"},{"id":107183109,"identity":"869c91d1-a969-4369-b787-001cf54aaf9f","added_by":"auto","created_at":"2026-04-17 18:04:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":423295,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemantFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9264188/v1/fd08129da8cd880ffd5ca4e4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-genome analyses reveal the phylogeny and gene flow of Japanese and Ryukyu wild boars","fulltext":[{"header":"Background","content":"\u003cp\u003eThe wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) is a species found worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Phylogenetic studies indicate that its ancestral population originated in Southeast Asia and split into Eastern and Western Eurasian lineages approximately 200,000 years ago [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Wild boars have not only expanded to major continents but also to islands separated by seas, such as those in Southeast Asia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. They have a high potential for environmental adaptation, including high-altitude areas [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and low-temperature regions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous genomic studies have documented gene flow between European and Chinese domestic pig lineages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] as well as between wild boars and domestic pigs in both Europe and China [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Japanese Archipelago is home to two subspecies of wild boar: the Japanese wild boar (JWB, \u003cem\u003eSus scrofa leucomystax\u003c/em\u003e) and the Ryukyu wild boar (RWB, \u003cem\u003eSus scrofa riukiuanus\u003c/em\u003e). JWB is found on the islands of Honshu, Shikoku, and Kyushu, while RWB inhabits the Ryukyu Islands. The morphological differences between these two subspecies are well-documented. One significant distinction is their body size; JWB individuals are generally larger than RWB individuals, as studies on mandible and dental morphology indicate [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, RWB has been reported to be the smallest wild boar in the world [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo clarify the genetic relationships among JWB/RWB, studies using mitochondrial DNA have been conducted [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, a study using microsatellite DNA analysis has revealed the population structure of JWB across Honshu, Shikoku, and Kyushu [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In a study with higher sample resolution, 15 clusters were observed due to geographic barriers such as natural topography, human settlements, and man-made structures [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the whole genome sequence of only one individual of the Japanese wild boar has been reported (accession number: ERR173212) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, this study did not specify the geographic origin of this individual. The genome of this individual has been used in studies, such as Zhang, Traspov et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which demonstrated a sister relationship between this individual and the clade that includes Russian and Korean wild boars. However, it remains unclear which subspecies this individual belongs to.\u003c/p\u003e \u003cp\u003eIn this study, we resequenced the whole genomes of five Japanese wild boar (JWB) individuals from Honshu (Tochigi, Aichi, and Kyoto), as well as one from Shikoku (Tokushima) and one from Kyushu (Saga). Additionally, we resequenced two Ryukyu wild boar (RWB) individuals from Okinawa and Ishigaki Islands. Using our data, along with publicly available genomic sequences from wild boars (WBs) and domestic pigs (DPs), we analyzed the genomes to clarify the genetic relationships between JWB, RWB, and other lineages of the genus \u003cem\u003eSus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal samples\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples from 5 JWB were selected along 1,160 km from the main islands of Japan (Honshu, Kyushu and Shikoku) and 2 RWB from Ryukyu archipelago (Okinawa and Ishigaki Islands). We recorded the date, location, estimated age, and weight associated with each animal (Supplementary Table 2). Until processing, samples were stored at -20\u0026deg;C. A preliminary selection of individuals for whole genome sequencing (WGS) was conducted from a wider JWB and RWB collection of 1,062 individuals from National Agriculture and Food Organization (NARO), Tsukuba, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eJWB and RWB whole genome sequencing dataset construction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ear and muscle tissues were collected between 2014 and 2017. There is evidence that local domesticated pigs had recently introgressed into the wild boar population, which found hybrids between JWB and European Domestic pigs [14]. To accurately capture the evolutionary history and diversity of JWB and RWB, sample selection was conducted based on the following criteria: (i) Individuals were classified into different group based on the results from Sawai et\u0026nbsp;al. 2023 [13], (ii) A subset of samples were selected from different islands, prefectures, and sounders to reduce closely related individuals based on spatial coordinates, (iii) (ii) was analyzed by mitochondrial DNA (mtDNA) D‑loop haplotype analysis to exclude hybrids, and then, consequently seven individuals from divergent branches of mtDNA Neighbor-Joining network\u0026nbsp;[15]\u0026nbsp;were taken to define the final sequencing dataset\u003cs\u003e.\u003c/s\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA extraction and sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA extraction from the ear and tongue tissue samples was conducted using the QiAmp DNA mini kit (QIAGEN Ltd., Hilden, Germany) in accordance with the manufacturer\u0026rsquo;s instructions. Genomic DNA quantity and quality were assessed the Agilent 2200 using TapeStation (Agilent Technologies), which revealed high‑molecular‑weight genomic DNA ranging from 4,600 to 8,000 bp. Subsequently, DNA samples were re‑quantified and normalized to prepare whole‑genome sequencing libraries constructed with an Illumina DNA Prep kit and standard TruSeq adapters (Illumina, San Diego, CA) according to the manufacturer\u0026rsquo;s protocol. The sequenced paired‑end 150 bp reads were obtained in an Illumina NovaSeq X Plus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data have been deposited with links to BioProject accession number PRJDB40278 in the DDBJ BioProject database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExtraction of single nucleotide polymorphisms (SNPs) and vcf file preparation\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used sequencing data from 34 wild boars, 19 domestic pigs, and two outgroup species (Supplement Table 1). Sequence reads were processed using AdapterRemoval v2 with parameters --trimns --trimqualities --minquality 25 --minlength 25, which removed adaptor sequences, ambiguous bases (N), and terminal bases with Phred quality scores below 25. The trimmed reads were mapped to the Boar and Pig reference genome (GCF_000003025.6_Sscrofa11.1)[16] using BWA \u0026ldquo;BWA-MEM\u0026rdquo; algorithm.\u003c/p\u003e\n\u003cp\u003eThe mapping data were exported in BAM file format and sorted and indexed using SAMtools [17]. The duplicated reads in the BAM files were marked by the MarkDuplicates algorithm, implemented in GATK v4.6 (https://gatk.broadinstitute.org/hc/en-us). We performed haplotype calling on all individuals analyzed in this study using the HaplotypeCaller algorithm in GATK v4.6, and output as gvcf format (-ERC GVCF option). All gvcf files were combined into a single gvcf file by the CombineGVCFs algorithm in GATK v4.6. The combined gvcf file was genotyped by the GenotypeGVCFs algorithm in GATK v4.6. To maximize the number of SNPs for analyses, we prepared datasets from the genotyped vcf file for each analysis by following filtering using vcftools [18].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDataset of Pig and Boar for principal components analysis (PCA) and phylogenetic tree construction\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe removed all sites with missing data (Max-missing 1). Then, we removed all indels, singleton, and doubleton sites to eliminate PCR and sequencing errors that may have occurred in one individual by minor allele count (mac) filtering (mac = 3). We extracted bi-allelic sites with coverage equal to or more than one in all individuals and with GQ values equal to or more than eight in all individuals, using VCFtools [19].\u003c/p\u003e\n\u003cp\u003eNext, to obtain approximately independent SNPs, we performed linkage disequilibrium (LD) pruning. Using PLINK ver. 1.90b6.21 [20] with an option \u0026ldquo;--indep-pairwise 50 10 0.1\u0026rdquo; to conduct the LD pruning. The final dataset consisted of 225,140 sites. For PCA, we removed outgroup species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDataset of Pig and Boar for Admixtools\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe removed all sites with missing data (Max-missing 1). Then, we removed all indels, singleton, and doubleton sites to eliminate PCR and sequencing errors that may have occurred in one individual by minor allele count (mac) filtering (mac = 3). We extracted bi-allelic sites with coverage equal to or more than one in all individuals and with GQ values equal to or more than eight in all individuals, using VCFtools [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhylogenetic analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SNP datasets for phylogenetic analysis were converted to PHYLIP format\u0026nbsp;using TASSEL 5 [21]. 10 kb sequences from the 5\u0026rsquo; end of the PHYLIP format file were extracted and a model for the Maximum Likelihood (ML) method was selected using MEGA ver. X [22].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A phylogenetic tree was constructed using the ML method using PhyML ver. 3.3.20220408 [23], using the GTR model (\u0026ldquo;-m GTR\u0026rdquo;) based on the best-fit model selected in MEGA, with 100 bootstrap replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCA (and ADMIXTURE)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed principal component analysis (PCA) using PLINK ver. 1.90b6.21 [24], and individual ancestry proportions were estimated using ADMIXTURE ver. 1.3.0 [25]. PCA and ADMIXTURE analysis utilized a dataset excluding the outgroup\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOutgroup-f3, and f4 statistics\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used the Outgroup-\u003cem\u003ef3\u003c/em\u003e statistics\u0026nbsp;to measure shared genetic drift between JWB or RWB and the other \u003cem\u003eSus\u003c/em\u003e populations, and the \u003cem\u003ef4\u003c/em\u003e statistics to test the genetic affinity between JWB/RWB and WB populations from Northern China, Eastern Russia, and European DP. Outgroup-\u003cem\u003ef3\u003c/em\u003e and \u003cem\u003ef4\u003c/em\u003e statistics implemented in ADMIXTOOLS ver. 7.0.2 [26] were used to evaluate the shared genetic drift among JWB/RWB and the \u003cem\u003eSus\u003c/em\u003e population from the other regions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenetic relationship of JWB/RWB to the other Sus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe resequenced the whole genomes of seven individuals of Japanese wild boars, comprising five JWB (Japanese Wild Boar) and two RWB (Ryukyu Wild Boar) (see Fig. 1, Table1). To clarify the phylogenetic relationships among JWB, RWB, and other \u003cem\u003eSus\u003c/em\u003e populations, we constructed a genome-wide SNP dataset comprising genomic information from WBs and DPs worldwide (refer to Fig. 1, Supplement Table 1 for details). Using this SNP dataset, we constructed an ML phylogenetic tree (see Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tree shows the first major split between European and East Asian clades, with the Northern Chinese and Eastern Russian clades diverging from the Southern Chinese WB, consistent with the previous study [5] (Fig. 2).\u0026nbsp;JWB and RWB formed a monophyletic clade, within which each subspecies formed its own subclade. Notably, the JWB/RWB clade is a sister clade to the Eastern Russian/Northern Chinese WB. Within\u0026nbsp;the JWB subclade, the individual from Kyushu (Saga) is basal, followed by the individual from Shikoku (Tokushima), and the three individuals from Honshu cluster (Kyoto, Aichi, and Tochigi) together. This branching pattern reflects their geographic distribution across the Japanese mainland (see Fig. 1). Note that an individual, namely ERR173212, which has been recognized as the JWB in the public database, is placed in the clade of RWB, indicating this individual originates from the RWB (Fig. 2).\u003c/p\u003e\n\u003cp\u003eTable 1 \u0026nbsp; Sample and sequencing information\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.67742%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26.3441%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePopulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8925%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMapped reads\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDepth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eA13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eJapanese Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eTochigi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e597,940,320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e34.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eB43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eJapanese Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eAichi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e603,397,399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e34.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eD28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eRyukyu Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eOkinawa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e623,072,951\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e36.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eE10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eJapanese Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eSaga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e637,877,951\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e35.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eF22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eJapanese Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eTokushima\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e632,351,129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e35.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eG28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eRyukyu Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eIshigaki\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e638,476,155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.67742%;\"\u003e\n \u003cp\u003eH37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 26.3441%;\"\u003e\n \u003cp\u003eJapanese Wild Boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.8925%;\"\u003e\n \u003cp\u003eKyoto\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e615,839,582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.043%;\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNext, we conducted PCA on the same dataset used for the ML tree (Fig. 3). The results indicated that the first principal component (PC1) separated European DPs and Western Russian WBs from Eastern Asian WBs and DPs, which included Chinese, Eastern Russian/Northern Chinese, RWB, and JWB populations. The second principal component (PC2) further differentiated the Southern Chinese WBs and Chinese DPs into distinct clusters: WBs in Eastern Russian/Northern Chinese, RWB, and JWB (Fig. 3). These findings indicate that JWB and RWB are genetically distinct from the other WB and DP populations. Furthermore, JWB and RWB are closely related yet exhibit genetic differentiation.\u003c/p\u003e\n\u003cp\u003eIn the ADMIXTURE analysis (Supplement Fig. 1-2), at K = 4, JWB and RWB shared a common ancestry component. However, at K = 5, JWB and RWB are assigned their own ancestry, respectively. Additionally, the previously reported JWB (ERR173212) showed the same ancestry component as RWB at K = 5, further confirming that this individual belongs to the RWB.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We further investigated the genetic relationship among JWB, RWB, and other \u003cem\u003eSus\u003c/em\u003e individuals, using outgroup-\u003cem\u003ef3\u003c/em\u003e statistics [26] with the warthog (\u003cem\u003ePhacochoerus africanus\u003c/em\u003e) as the outgroup (Fig. 4a, 4b, Supplement Fig. 3-8). Comparisons of \u003cem\u003ef3\u003c/em\u003e values for Okinawa RWB and other individuals showed that the highest \u003cem\u003ef3\u003c/em\u003e values were observed with ERR173212, followed by RWB (Ishigaki), JWB, and the WBs from Eastern Russia/Northern China (see Fig. 4a). Therefore, the closest population to RWB is JWB.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;When comparing \u003cem\u003ef3\u003c/em\u003e values between individuals of JWB and individuals from other populations, the highest \u003cem\u003ef3\u003c/em\u003e values were observed with other JWB individuals, followed by WBs from Eastern Russia/Northern China (see Fig. 4b, Supplementary Fig. 5-8). This result was inconsistent with the earlier findings that JWB is closest to RWB (see Fig. 2). This inconsistency raises the possibility of introgression between the JWB and Eastern Russian/Northern Chinese WBs. If introgression had occurred between JWB and Eastern Russian/Northern Chinese WBs, we would expect the \u003cem\u003ef3\u003c/em\u003e value between them to be slightly higher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIntrogression between JWB and Eastern Russian and Northern Chinese WBs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated a potential introgression between the JWB/RWB populations and other populations using \u003cem\u003ef\u003c/em\u003e4 statistics [26]. Our analysis of introgression between the Eastern Russian (Fig. 5a) and Northern Chinese (Fig. 5b) WBs revealed that only individuals from the JWB population exhibited significant positive \u003cem\u003ef\u003c/em\u003e4 values, while individuals from the RWB population did not exhibit significant \u003cem\u003ef\u003c/em\u003e4 values. These results suggest that introgression events have occurred between the JWB and the Eastern Russian/Northern Chinese WBs.\u003c/p\u003e\n\u003cp\u003eAdditionally, we examined the possibility of introgression between the JWB/RWB populations and the European DP (Supplementary Fig. 9), since a European mtDNA haplotype was identified in one JWB individual [14, 27]. We tested gene flow between European DP and JWB/RWB using \u003cem\u003ef\u003c/em\u003e4 statistics. The results showed a weaker positive \u003cem\u003ef\u003c/em\u003e4 value between European DP and JWB than that shown between JWB and the Eastern Russian/Northern Chinese WBs. This result indicates the possibility of past hybridization between JWB and European DP, supporting the conclusions of previous studies [14, 27].\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeographic history of the Japanese Archipelago and JWB/RWB divergence\u003c/h2\u003e \u003cp\u003eOur phylogenetic analysis showed that a monophyletic JWB and RWB clade is placed within the East Asian clade, and that it diverged from the common ancestor with Eastern Russian/Northern Chinese WBs. This finding suggests that the ancestors of the JWB and RWB likely originated in East Asia before migrating to the Japanese archipelago, where they evolved under isolation.\u003c/p\u003e \u003cp\u003eZhang et al. 2022, 2025 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] estimated divergence time using a mutation rate of 3.6 \u0026times; 10⁻⁹ per generation and a generation time of 3 years. Their studies estimate that the divergence between European and East Asian wild boars occurred around 250,000 years ago, with southern and northern Chinese populations diverging approximately 30,000 years ago, and a more recent separation between the Northern Chinese and the Eastern Russian populations occurring around 10,000 years ago. Based on the estimated divergence times, the ancestral population of JWB/RWB separated from the Eastern Russian/Northern Chinese lineage approximately 30,000 to 10,000 years ago (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur study did not intend to extrapolate the exact location on the mainland where the common ancestor of JWB/RWB migrated to the Japanese archipelago. In the future, given that genomic data from modern and ancient wild boars along the continental coast become available, the discovery of the origins of JWB/RWB will progress robustly.\u003c/p\u003e \u003cp\u003eSince the Jomon period (160,000\u0026ndash;3,000 years ago), numerous wild boar remains have been excavated on the Japanese mainland (Honshu, Shikoku, and Kyushu) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, the earliest known occurrences of wild boars on Okinawa and Ishigaki Islands date back to the Late Pleistocene [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Archeological remains of wild boars from the Late Pleistocene have been found in the Shiraho Saonetabaru Cave on Okinawa Island, dating from around 24,000 to 16,000 years ago [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, WBs remains have been reported from the Sakitari Cave on Ishigaki Island, dating to approximately 20,000 years ago [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These archaeological evidences indicate that wild boars likely arrived in the Japanese archipelago between 10,000 and 30,000 years ago, which is concordant with this study.\u003c/p\u003e \u003cp\u003eIn the JWB clade, the Saga (Kyushu) individual is positioned as basal, followed by the Shikoku individual, and then the Honshu individuals. This tree topology corresponds with the geographical distribution of these individuals and supports previous research by microsatellite markers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the Last Glacial Maximum, Honshu, Shikoku, and Kyushu were connected by land due to lower sea levels. Sea level rose during the postglacial period, resulting in the separation of Honshu and Shikoku around 7,000 years ago and of Honshu and Kyushu around 5,000 years ago [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These geological events are likely responsible for the population divergence observed among the JWB. Similar patterns of population structure have been reported in other mammals, such as the red fox (\u003cem\u003eVulpes vulpes\u003c/em\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the case of RWB, individuals from Okinawa and Ishigaki formed distinct clusters. Notably, these two islands have never been connected by land, even during periods of low sea levels. It is likely that the ancestral RWB first colonized one island before being dispersed to others, where the isolated populations subsequently differentiated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIntrogression event of the JWB\u003c/h2\u003e \u003cp\u003eOur analysis indicates that there is gene flow between JWB and WB populations in Eastern Russia and Northern China. However, it remains unclear in which direction this gene flow occurred: whether individuals from eastern Russia and northern China introgressed into JWB, or vice versa. Furthermore, the \u003cem\u003ef\u003c/em\u003e4 values between JWB and individuals from Eastern Russia and Northern China did not show significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), leaving us uncertain about which specific WB populations on the continent have experienced gene flow with JWB. Collecting more modern and ancient genomic data will help clarify the details of gene flow.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we newly determined the whole genome sequences of five Japanese wild boars and two Ryukyu wild boars and investigated their genetic relationships with other \u003cem\u003eSus\u003c/em\u003e populations. Our results showed that JWB and RWB form a monophyletic clade diverging from the common ancestor with the Eastern Russian/Northern Chinese wild boar lineage, and that JWB and RWB each form distinct clades. Furthermore, we detected gene flow between JWB and wild boars from Eastern Russia/Northern China, suggesting WB migration in Northern East Asia. This initial NGS-based genomic analysis of JWB and RWB establishes a fundamental baseline for addressing the still-uncertain origins of their ancestors, and dispersion across the islands. Further genomic data from modern and ancient WB populations will elucidate the unresolved aspects of the historical and geographical distribution of \u003cem\u003eSus Scrofa\u003c/em\u003e in Asia.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eJWB: Japanese wild boar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDP: domestic pig\u003c/p\u003e\n\u003cp\u003ePCA: principal component analysis\u003c/p\u003e\n\u003cp\u003eRWB: Ryukyu wild boar\u003c/p\u003e\n\u003cp\u003eSNP: single nucleotide polymorphism\u003c/p\u003e\n\u003cp\u003eWB: wild boar\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild boar samples were purchased from a commercial source, with their origin information. No sampling of live animals was performed in this study, and therefore no ethical approval was required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences in this study have been deposited in the DDBJ Sequenced Read Archive under accession number PRJDB40278.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number 24H01588 under the Grant-in-Aid for Transformative Research Areas (A) Integrative bioarchaeological studies on human prehistory in the Japanese archipelago to MT, and Japan Society for the Promotion of Science (JSPS) International Fellowships for Research in Japan Short-term (PE21054) to DG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Takehisa Yamamoto from National Institute of Animal Health, National Agriculture and Food Research Organization, for the provision of wild boar samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRI performed population genetic analyses using the whole genome sequence data and wrote and edited manuscript. DG selected and purified genomic DNA samples based on the basic genetic analysis of Japanese and Ryukyu wild boar, obtained WGS data, and edited manuscript. YT edited manuscript and supported genomic data and population genetic analyses. JG performed and supervised population genetics analyses, and edited manuscript. MT analyzed and supervised the basic genetic analysis on Japanese and Ryukyu wild boar population, sample selection, WGS data acquisition, and edited manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarrios-Garcia MN, Ballari SA: \u003cstrong\u003eImpact of wild boar (Sus scrofa) in its introduced and native range: a review\u003c/strong\u003e. \u003cem\u003eBiological invasions \u003c/em\u003e2012, \u003cstrong\u003e14\u003c/strong\u003e(11):2283-2300.\u003c/li\u003e\n\u003cli\u003eZhang M, Yang Q, Ai H, Huang L: \u003cstrong\u003eRevisiting the evolutionary history of pigs via de novo mutation rate estimation in a three-generation pedigree\u003c/strong\u003e. \u003cem\u003eGenomics, Proteomics \u0026amp; Bioinformatics \u003c/em\u003e2022, \u003cstrong\u003e20\u003c/strong\u003e(6):1040-1052.\u003c/li\u003e\n\u003cli\u003eLarson G, Cucchi T, Fujita M, Matisoo-Smith E, Robins J, Anderson A, Rolett B, Spriggs M, Dolman G, Kim T-H: 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Within this archipelago, there are two subspecies: the Japanese wild boar (JWB, \u003cem\u003eSus scrofa leucomystax\u003c/em\u003e) and the Ryukyu wild boar (RWB, \u003cem\u003eSus scrofa riukiuanus\u003c/em\u003e), which exhibit differences in morphology and habitat. Until now, the whole genomes of JWB and RWB individuals with known origins had not been sequenced, leaving their genetic relationships with other populations unclear. In this study, we resequenced the whole genomes of five JWB individuals from Honshu (specifically Tochigi, Aichi, and Kyoto), one from Shikoku (Tokushima), and one from Kyushu (Saga). Additionally, we resequenced two RWB individuals from Okinawa and Ishigaki Islands.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe analyzed the genomes to clarify the genetic relationships between JWB, RWB, and other lineages of the genus \u003cem\u003eSus\u003c/em\u003e. Phylogenetic trees based on genomic data indicate that the JWB and RWB populations form distinct clades, and these two clades form a monophyletic clade. The JWB/RWB clade diverged from a common ancestor with the Eastern Russian and Northern Chinese wild boars. Principal component analysis confirmed distinct genetic clusters for JWB and RWB. These results suggest that JWB and RWB represent independent boar lineages that evolved following isolation within the Japanese archipelago. However, the \u003cem\u003ef4\u003c/em\u003e statistic indicates gene flow between JWB and Eastern Russian/Northern Chinese boars, suggesting migration across Northern East Asia.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverall, our results show that JWB and RWB diverged from East Asian boars through dispersal and isolation within the Japanese archipelago.\u003c/p\u003e","manuscriptTitle":"Whole-genome analyses reveal the phylogeny and gene flow of Japanese and Ryukyu wild boars","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-17 18:04:50","doi":"10.21203/rs.3.rs-9264188/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-18T06:24:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T16:16:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T04:52:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111867886604686836400123531661988112531","date":"2026-04-11T12:34:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"227988306772820318576662807988431292719","date":"2026-04-11T05:37:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-09T12:30:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T04:48:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-06T21:53:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-06T10:22:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ecology and Evolution","date":"2026-04-06T09:53:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ecology-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evob","sideBox":"Learn more about [BMC Ecology and Evolution](http://bmcevolbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/evob/default.aspx","title":"BMC Ecology and Evolution","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60a5d2d7-1152-4765-ad2a-2992ce297020","owner":[],"postedDate":"April 17th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T06:24:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T16:16:05+00:00","index":29,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T06:39:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-17 18:04:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9264188","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9264188","identity":"rs-9264188","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}