Genetic structure of swimming boars (Sus scrofa): genome-wide single nucleotide polymorphisms revealed invasion of two lineages into Shodoshima Island

Genetic structure of swimming boars (Sus scrofa): genome-wide single nucleotide polymorphisms revealed invasion of two lineages into Shodoshima Island | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic structure of swimming boars (Sus scrofa): genome-wide single nucleotide polymorphisms revealed invasion of two lineages into Shodoshima Island Shintaro Ishizuka, Eiji Inoue, Takeo Kuriyama, Jun J Sato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6850685/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2025 Read the published version in European Journal of Wildlife Research → Version 1 posted 8 You are reading this latest preprint version Abstract Wild boars ( Sus scrofa ) have substantial impacts on island ecosystems and economies worldwide because of their rapid population growth and ability to expand their distribution. Although many studies have focused on the invasion of artificially introduced boars into islands, the natural processes of boar invasion into islands have rarely been studied. This study investigated the natural process of the recent swimming invasion of boars into Shodoshima Island in the Seto Inland Sea, Japan. We investigated whether wild boars on this island invaded the Honshu (northern side) or Shikoku (southern side) islands. We performed mitochondrial and genome-wide single-nucleotide polymorphism (SNP) analyses of boars living on these islands. Two mtDNA haplotypes were found on Shodoshima Island, and were shared on Shikoku Island, but not on Honshu Island. SNP analysis showed that boars on Shodoshima and Shikoku Islands possessed similar genomic compositions, and two ancestral populations were present within the boars of Shodoshima Island. These findings suggest that the two genetic lineages of boars invaded Shodoshima Island from Shikoku, and the population primarily expanded through the reproduction of the two ancestry lineages. This study contributes to a better understanding of the patterns of wild boar invasion into islands and the management of boars on small isolated islands. Sus scrofa Shodoshima Island Seto Island Sea single-nucleotide polymorphism analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Biological invasions into islands cause various issues. This can occur for several reasons, such as artificial introduction (e.g. common wall lizard, Podarcis muralis : Michaelides et al. 2013 ; small Indian mongoose, Herpestes auropunctatus : Hays and Conant 2007 ) or range expansion (e.g. black-tailed deer, Odocoileus hemionus : Martin et al. 2011 ). Biological invasions, irrespective of whether they result from artificial or natural processes, can damage island ecosystems (Reaser et al. 2007 ), such as a reduction in native flora (Relva et al. 2010 ) or predation of endangered species (Maeda et al. 2019 ), thereby leading to species extinction and biodiversity loss on islands (Savidge 1987 ; Johnson and Stattersfield 1990 ; Sax and Gaines 2008 ). It is also expected to trigger economic damage, such as crop damage (Naylor 1996 ) and zoonotic disease transmission (Popiolek et al. 2011; Zhang et al. 2022 ). For the conservation of native ecosystems on islands that are vulnerable to biological invasion and to mitigate economic damage, a better understanding and efficient management of biological invasion is necessary. Biological invasions comprise four phases: the appearance of invaders, population growth, range expansion, and invader impacts (Crooks 2005 ). The appearance of invaders is thought to occur through several potential processes. If the initial invasion occurs at a single time and a small number of initial invaders share the same origin, the original populations would experience founder effects and show low genetic diversity (Nei et al. 1975 ; Dlugosch and Parker 2008 ). In contrast, if the appearance of invaders can occur multiple times and these invaders have different origins, population growth can be achieved by the reproduction of multiple lineages, and a relatively high genetic diversity is expected (Dlugosch and Parker 2008 ). Furthermore, if the invasion of islands from other areas occurs continuously, even after the appearance of an initial invader, it can also contribute to population growth and the maintenance of genetic diversity. Thus, genetic analyses are useful for clarifying the processes of invader appearance and population growth. The wild boar ( Sus scrofa ) is an invasive game species that is the most widely distributed mammal worldwide (Massei and Genov 2004 ; Barrios-Garcia and Ballari 2012 ). As they are fecund and reproduce vigorously, their population size often increases rapidly (Coblentz and Baber 1987 ; Taylor et al. 1998 ; Náhlik and Sándor 2003 ). Their significant population growth can result in range expansion and negative effects on local ecosystems and economies (Lewis et al. 2019 ; Risch et al. 2021 ; Fulgione and Buglione 2022 ). For example, their rooting behavior can reduce plant species diversity (Tierney and Cushman 2006 ; Siemann et al. 2009 ) and regeneration (Sweitzer and Van Vuren 2002 ; Bongi et al. 2017 ). Their predation can be critical for endangered reptile populations (Fordham et al. 2006 ) and birds (Carpio et al. 2016; Senserini and Santilli 2016 ; Mori et al. 2021 ). They also have negative impacts on local economies, causing issues such as crop damage (Herrero et al. 2006 : Schley et al. 2008 ; Amici et al. 2012 ), hybridization with domestic pigs (Šprem et al. 2014 ; Iacolina et al. 2018 ), or spreading boar-borne diseases (Gortázar et al. 2007 ; Bosch et al. 2017). It has been reported that they have already invaded several islands (Barrios-Garcia and Ballari 2012 ) and have caused several harmful impacts, such as hybridization with native subspecies (Long 2003 ) or reducing the number of native species (Roemer et al. 2002 ). To effectively manage wildlife and conserve local ecosystems and economies on islands, it is important to understand the processes of boar invasion into islands and their existing population structures. The invasion of boars on Pacific islands has been intensively studied (Webr et al. 2018) and Europe (Canu et al. 2018 ; Ballouard et al. 2021 ). Boars have been artificially introduced onto Pacific Islands as a source of bushmeat or commercial hunting (Courchamp et al. 2003 ). However, they sometimes invade islands by swimming (e.g. Ballouard et al. 2021 ) (Takahashi and Tisdell 1992 ; Fujita et al. 2014 ). The process of boar invasion into islands via swimming and their population structures is poorly understood because such an invasion is artificially uncontrolled. If the islands are surrounded by multiple neighboring islands, it is possible to assume that boars invaded either from multiple islands or a single island. Therefore, boars on these islands may be genetically similar to those on multiple or single islands. Furthermore, boars are a male-biased dispersing species (Truvé and Lemel 2003 ; Podgórski et al. 2014 ), suggesting that only males may continuously invade islands after population foundation. In this case, male gene flow between islands could occur frequently and the genetic diversity of males may be higher than that of females on the island. Previous studies have assessed invasion of boars using genetic analyses with focuses on population structures on continuous landmasses (Sagua et al. 2018 ; Herna ́ndez et al. 2018 ; Mangan et al. 2021 ; Delgado-Acevedo et al. 2021 ; Saito et al. 2022 ; Nomura et al. 2023 ; Sawai et al. 2023 ), adaptation to urban areas (Hagemann et al. 2022 ; Zsolnai et al. 2022 ), or hybridization (Smyser et al. 2024 ; Acosta et al. 2024). However, the invasion of boars into islands has been infrequently investigated using genetic analyses, and the effect of invasion via swimming on genetic structure has rarely been examined, except for a few studies (e.g. Canu et al. 2018 ). Information on the swimming invasion of boars into islands contributes to a broader understanding of the natural mechanisms underlying the biological invasion of islands by terrestrial mammals. Japan is an ideal location for studying swimming boar invasion. Two subspecies of wild boar, Japanese wild boar ( S. s. leucomystax ) and Ryukyu wild boar ( S. s. riukiuanus ), are distributed throughout Japan. The Japanese Archipelago comprises 14,125 islands (Geospatial Information Authority of Japan, https://www.gsi.go.jp/top.html ). Recently, the population of boars in Japan has rapidly increased, resulting in their invasion into various novel areas, including islands (Ministry of the Environment 2021 ). Especially in the Seto Inland Sea, recent boar invasions into islands are considered to occur via swimming as “swimming boars” have been sporadically observed (Takahashi 2014 , Fig. 1). Thus, this study focused on Shodoshima Island, a part of Kagawa Prefecture located in the Seto Inland Sea, to investigate the pattern of boar invasion into islands via swimming. The island is surrounded by neighboring landmasses, including Honshu Island on the northern side and Shikoku Island on the southern side (Fig. 1). On Shodoshima Island, wild boars were considered extinct by approximately 1875 (Takahashi 2001 ). It was confirmed that boars were absent until 1990 (Kagawa Prefecture 2012 ), and the current IUCN Red List shows that wild boars endemic to this island have been designated as extinct. However, wild boars have been observed on the island since around 2010, and the harvest size increased from 42 to 2,154 between 2010 and 2020 (Kagawa Prefecture 2023 ), suggesting that the population of boars on the island had rapidly grown. Therefore, the island provides a suitable model to assess the effect of swimming boars on their genetic structure and understand their invasion routes into islands. In this study, nuclear and mitochondrial genetic analyses of wild boars on Shodoshima Island and its surrounding areas were performed to investigate the genetic structure of wild boars inhabiting a wide area, including Shodoshima Island and its surrounding islands, and to infer the route of invasion by swimming. Second, we investigated the genetic structure of boars on the island to determine the number of genetic lineages of wild boars that had invaded the island. Third, we analyzed sex differences in genetic diversity within the island to test whether males are more likely to be invaders owing to the male-biased dispersal seen in this species. Materials and methods Study site and sample collection The study sites were Shodoshima Island, Odeshima Island (a small island near Shodoshima Island), and the areas surrounding these two islands, including Honshu and Shikoku Islands (Fig. 2). A total of 48 boar tissue samples were collected from December 2020 and February 2023. All the samples were preserved in 70% ethanol at room temperature or frozen. Approximate sampling localities within the islands (30: Shodoshima Island, 1: Odeshima Island, 5: Shikoku Island, and 12: Honshu Island) and sexes (32 males and 16 females) were known for all samples. The location and sex of each sample are listed in Table S1 . Mitochondrial genetic analysis DNA was extracted from the tissue samples using a DNeasy Blood & Tissue Kit (Qiagen, CA, USA). To analyze mtDNA nucleotide sequences, we amplified an approximately 550-bp portion of the mtDNA D-loop region with the forward primer mitL76 (5’-AATATGCGACCCCAAAAATTTAACCATT-3’) and the reverse primer mitH62 (5’-CCTGCCAAGCGGGTTGCTGG-3’), as reported in a previous study (Watanobe et al. 2003). PCR was performed in 10-µl reaction volumes comprising 2 µl of genomic DNA and 5 µl of QIAGEN Multiplex PCR Master Mix, with 200 nM each of the forward and reverse primers. The amplification parameters were as follows: 94°C for 5 min; 45 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s; 60°C for 30 min. PCR products were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA). We outsourced the FASMAC sequencing service (Kanagawa, Japan) and sequenced the products using the dye-termination method on an ABI 3100xl sequencer (Applied Biosystems, Foster City, CA, USA). For sequencing, we used the same two primers (mitL76 and mitH62) used in PCR. Finally, a 548-bp nucleotide sequence encompassing part of the mtDNA control region with a second hypervariable domain was obtained. We aligned the nucleotide sequences of each species using the “MUSCLE” program implemented in MEGA11 (Tamura et al. 2021 ). Based on this alignment, we determined mtDNA haplotypes. To assess genetic dissimilarities among the mtDNA haplotypes, a median-joining network was constructed using NETWORK ver 10.2 ( https://www.fluxus-engineering.com/sharenet.htm ). The obtained DNA sequence data have been deposited to DDBJ/ENA/Genbank international DNA database with accession numbers, LC876760–LC876765. Next generation sequencing We outsourced the Bioengineering Lab (Kanagawa, Japan) to perform the GRAS-Di analysis (Enoki and Takeuchi 2018 ) and analyzed genome-wide single nucleotide polymorphisms (SNP). This method has recently been used to analyze population genetic structures in several vertebrates (e.g. Hosoya et al. 2019 ; Sato and Yasuda 2022 ). DNA concentration was calculated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The library used for next-generation sequencing (NGS) was prepared using the 2-step PCR method. The first PCR was performed in 25-µl reaction volumes comprising 5 µl 5× PrimeSTAR Buffer (Mg 2+ ), 2 µl dNTPs (each 2.5 mM), 10 µl primer mix (100 pmol/µl), 1 µl genomic DNA (15 ng/µl), 0.25 µl PrimeSTAR HS DNA Polymerase (2.5U/µl), and 6.75 µl nuclease-free water. The sequences of each primer in the mixture are listed in Table S2. The amplification parameters were as follows: 98°C for 2 min; 30 cycles of 98°C for 10 s, 50°C for 15 s, and 72°C for 20 s. The second PCR was performed in 50-µl reaction volumes comprising 10 µl 5× PrimeSTAR Buffer (Mg 2+ ), 4 µl dNTPs (each 2.5 mM), 1.5 µl template DNA (1st PCR product), 0.5 µl PrimeSTAR HS DNA Polymerase (2.5U/µl), and 31.5 µl nuclease-free water, with 0.25 pM of forward (5’-GAACGACATGGCTACGATCCGACTT-NN-TAAGAGACAG-3’) and reverse (5’-TGTGAGCCAAGGAGTTG-Index-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-NN-AAGAGACAG-3’) primers (where “NN” represents 0–2 random nucleotides). The index sequences of the reverse primers are listed in Table S2. The amplification parameters were as follows: 98°C for 2 min; 25 cycles of 98°C for 15 s, 53°C for 15 s, and 72°C for 20 s; 72°C for 1 min. The amplification products were mixed in equal volumes and purified using the Min Elute PCR Purification Kit (Qiagen, CA, USA). The concentration of the library was calculated using a Qubit 3.0 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher Scientific). Library quality was confirmed using an Agilent 2100 Bioanalyzer and a High-Sensitivity DNA Kit (Agilent Technologies). The library was transformed into a single-standard circular DNA library using the MGIEasy Circulation Kit (MGI Tech Co., Ltd.). A Dynamical Network Biomarker (DNB) was prepared using a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech Co., Ltd.). The prepared DNB was sequenced using a DNBSEQ-G400 (MGI Tech Co., Ltd.) under 2 × 200 bp conditions. The obtained DNA sequence data have been deposited with links to BioProject accession number PRJDB20530 in the DDBJ BioProject database. SNP calling To exclude primer sequences, we trimmed the first 15 bp of each read using the “fastx_trimmer” function in the FASTX-toolkit ver 0.0.14 ( http://hannonlab.cshl.edu/fastx_toolkit/ index.html). Adaptor sequences were removed using cutadapt ver 4.1 (Martin 2011 ). For quality control, reads with quality < Q30 and length < 50 bp were trimmed using sickle ver 1.33 (Joshi and Fass 2011 ). To unify the length of sequences for data analysis, sequences beyond 50 bp were trimmed using the “fastx_trimmer” function in the FASTX-toolkit ver 0.0.14. We then adopted the “denovo_map.pl” program in the Stacks ver 2.59 (Catchen et al. 2011 , 2013 ). The program included six stages: building loci (ustacks), creating a catalog of all loci across the population (cstacks), matching each sample against the catalog (sstacks), transposing the data oriented by locus (tsv2bam), assembling a contig and calling single nucleotide polymorphisms (SNPs) (gstacks), and population genomics analysis (populations). Genetic diversity within islands and the genetic distance between islands were calculated using Genodive ver 3.06 (Meirmans et al. 2020). Population genetic structure analysis ADMIXTURE analysis was carried out to investigate the population genetic structures (Alexander et al. 2009 ). The genotype file was exported and processed using PLINK ver 1.9 (Chang et al. 2015 ) for ADMIXTURE analysis. We used Admixture ver 1.3.0, and investigated population genetic structures with the number of coalescent ancestry populations (K = 2 to 5). ADMIXTURE analyses were performed using two different scales. First, the genetic structure of the study area, including Shodoshima Island and its surrounding islands, was investigated using SNP data from all 48 individuals sampled in this study. Secondly, the genetic structure of Shodoshima Island was investigated using SNP data from 30 individuals collected on the island. The ADMIXTURE results were visualized using the “pophelper” package in R ver 4.4.4 (Francis 2017 ). Results Mitochondrial DNA analysis Nucleotide sequences of the mtDNA control region were successfully determined for all 48 samples. Six haplotypes (A–F) were identified (Fig. 3 and Table S1 ). The number of nucleotide substitutions among the haplotypes and the proportion of haplotypes in each population are shown in Fig. 3. Two haplotypes (A and B), which differed by nine nucleotides from each other, were found on Shodoshima Island. Haplotype A was widely distributed across the island, whereas haplotype B was observed only in the central and northern parts of the island (Fig. 4). These two haplotypes were also found on Shikoku Island, but not on Honshu Island. One haplotype (D) was shared between the Shikoku and Honshu Islands. Haplotype A was detected on Odeshima Island. Genome-wide SNP analysis A total of 8,410 SNPs were obtained from samples collected from the entire study area, including Shodoshima Island and its surrounding islands. A summary of the genetic diversity, including the number of alleles, effective number of alleles, observed heterozygosity, heterozygosity within the population, and inbreeding coefficients, is shown in Table S3. According to the ADMIXTURE analysis of the entire study area, including Shodoshima Island and its surrounding islands, the cross-validation error of K = 2 was the lowest, indicating that the assumption of two ancestral populations is reasonable (Table S4). Samples from Shodoshima and Shikoku Islands were mostly assigned to the same cluster (colored black, Fig. 5), whereas those from Honshu Island were largely assigned to another cluster (colored gray, Fig. 5). The genetic distance (Fst) between Shodoshima Island and Shikoku Island was 0.031, which was lower than that between Shodoshima Island and Honshu Island (0.201), and between Shikoku Island and Honshu Island (0.176). A total of 8,276 SNPs were obtained from samples from Shodoshima Island. According to the ADMIXTURE analysis of Shodoshima Island, the cross-validation error of K = 2 was the lowest, indicating that assuming two ancestral populations was reasonable (Fig. 6, Table S4). The ADMIXTURE plot revealed that samples from “01” to “18,” which showed mtDNA haplotype A, contained a relatively large proportion of black color, while those from “19” to “30,” which showed mtDNA haplotype B, contained a relatively large proportion of gray color, suggesting that they were genetically roughly divided from each other (Fig. 6). In contrast, 20 of the 30 samples shared the genetic components of both clusters (Fig. 6). The number of alleles, effective number of alleles, observed heterozygosity, heterozygosity within the population, and inbreeding coefficient, which are measures of genetic diversity, did not differ notably between the sexes (Table 1 ). Table 1 Sex differences in genetic diversity of boars on Shodoshima Island. Male Female Sample size 11 19 Number of alleles 1.86 1.93 Effective number of alleles 1.46 1.49 Observed heterozygosity 0.27 0.29 Heterozygosity within population 0.29 0.30 Inbreeding coefficient 0.09 0.06 Discussion This study found six mtDNA haplotypes in the study area, including Shodoshima Island and its neighboring islands. Two mtDNA haplotypes (A and B) were shared in Shodoshima and Shikoku Islands, but not in Honshu Island. ADMIXTURE analysis based on genome-wide SNPs also showed that boars in the entire study area could be genetically divided into two ancestral populations, and those in Shodoshima and Shikoku were mostly assigned to the same population. The genetic distance between Shodoshima and Shikoku was smaller than that between Shodoshima and Honshu. The overall results suggest that boars in Shodoshima and Shikoku Islands are genetically close to each other and that they invaded from Shikoku to Shodoshima Island. One possible reason for this direction of invasion may be the excessive boar population size on Shikoku Island. Since the estimated number of boars in the Kagawa Prefecture excluding islands according to surveys in 2020 was 39,996 (Kagawa Prefecture 2023 ) and the area of the island is 1,692.72 km 2 (Geospatial Information Authority of Japan), the density was calculated to be 23.63 individuals per km 2 . The population size of boars in the area is considered to have increased rapidly in recent years, as the total number of captures in Kagawa Prefecture, excluding islands, drastically increased from 5,451 to 10,494 between 2010 and 2020 (Kagawa Prefecture 2023 ). In contrast, the population density in Okayama Prefecture was estimated to be 6.25 individuals per km 2 , as the estimated number of boars was 44,452 (Okayama Prefecture 2022 ) and the area of this prefecture was 7,114.44 km 2 (Geospatial Information Authority of Japan). The large population size of boars on Shikoku Island, especially around Kagawa Prefecture, might have caused the invasion of boars into Shodoshima Island. Furthermore, given that one mtDNA haplotype was shared between the Shikoku and Honshu Islands, the invasion of boars from Shikoku Island might reach Honshu Island. The genetic structure of boars around Shodoshima Island in this study was consistent with previous studies showing that boars were genetically differentiated between the Shikoku and Honshu Islands (Sawai et al. 2022; Nomura et al. 2023 ). However, the trend observed in wild boar was different from that in most other terrestrial mammals on Shodoshima Island. Studies on mtDNA in Japanese wood mouse species ( Apodemus speciosus and A. argenteus ), Japanese macaques ( Macaca fuscata ), and sika deer ( Cervus nippon ) have suggested that Shodoshima populations are similar to those of Honshu Island (Suzuki et al. 2004 ; Kawamoto et al. 2007 ; Ishizuka et al. 2024 ). A similar trend was observed in A . speciosus on the islands of the western Seto Inland Sea (Sato et al. 2017 ). This difference may be due to the method of invasion of the area. One study suggested that terrestrial mammals, such as Japanese macaques and sika deer, invaded the island, as the Seto Inland Sea did not appear or was shallow until the Holocene glacial retreat (Ishizuka et al. 2024 ). In contrast to these species, boars can swim (Takahashi and Tisdell 1992 ; Fujita et al. 2014 ) and the surrounding water in the sea is not likely to be a barrier against their movements. This is likely why they have successfully invaded the island recently, even though the Seto Inland Sea was present. However, it should be noted that a terrestrial mammal, the lesser Japanese mole ( Mogera imaizumii ) on Shodoshima Island was genetically close to those on Shikoku Island (Mitsuhashi et al. 2024 ). Ecological or behavioral differences among mammals might be a factor in the differences in phylogenetic history. This study found two mtDNA haplotypes from Shodoshima Island that were genetically distant from each other, with nine substitutions present between them. ADMIXTURE analysis based on genome-wide SNPs also showed that boars on Shodoshima Island could be divided into two ancestral populations. This suggests that the boars that invaded Shodoshima Island belonged to two distinct lineages. Given that the boar invasion occurred from the southern side (Shikoku Island) and that haplotype B was distributed in the northern part of the island compared to haplotype A, a lineage with haplotype B might have invaded the island prior to the invasion of another lineage with haplotype A. Moreover, 20 of the 30 individuals shared genetic components of the two ancestry populations, suggesting that the two lineages had already hybridized with each other. Sexual maturity in female boars is achieved before one year of age (Gethöffer et al. 2007 ; Fonseca et al. 2011 ), indicating a short generation time. After invasion, the two lineages may hybridize with each other for a short period. The invasion of multiple lineages enhances the success of a newly settled population by increasing genetic diversity (Barret and Husband 1990). Similarly, the establishment of a boar population on Shodoshima Island may have been due to the presence of multiple ancestral lineages. Invasion by multiple lineages has also been reported in various animals (e.g. brushtail possum Trichosurus vulpecula : Triggs and Green 1989 ; brown rat Rattus norvegicus : Calmet et al. 2001 ; brown anole Anolis sagrei : Kolbe et al. 2004; oriental fruit fly Bactrocera dorsalis : Barr et al. 2014 ). Therefore, we concluded that founders with different origins might explain the recent population growth of wild boar on Shodoshima Island. The extent of genetic diversity was similar between sexes. As boars show male-biased dispersals (Truvé and Lemel 2003 ; Podgórski et al. 2014 ), it is possible to assume that males invading the island is more likely, and that the genetic diversity of males was higher than that of females. However, the results of the present study do not support this hypothesis. The frequency of boar invasion on the island might not differ between sexes. On the other hand, another possibility is that sex differences in genetic diversity might be masked by their vigorous reproduction (Coblentz and Baber 1987 ; Taylor et al. 1998 ; Náhlik and Sándor 2003 ). The reproduction of boars within the island is expected to increase the population size of both sexes equally. Population growth of males by reproduction on the island might equalize the genetic diversity between sexes, even though the frequency of invasion into the island is higher in males than in females. To clarify these possibilities, future genetic and observational studies are required to investigate sex differences in the patterns of invasion into islands by swimming boars on other islands. The present study clarified the route and genetic diversity of boars that invaded Shodoshima Island, Japan. Because understanding the population structure based on genetic diversity and genetic boundaries is important for wildlife management (DeYoung and Honeycutt 2005 ), our results are expected to be utilized for the management of boars and to reduce damage to various crops, such as rice, fruits, and vegetables, on the island (Kagawa Prefecture 2023 ). Given that their invasion could occur from Shikoku Island to Shodoshima Island, the southern parts of the island should be key areas in preventing their invasion. Furthermore, this study can serve as an important case in which terrestrial mammal species naturally invade an island in addition to artificial introduction. Our results provide insights into the invasion patterns of terrestrial mammals into islands. Similar to boars, several ungulate species are also good at swimming (e.g. chamois Rupicapra spp .: Kavčić et al. 2020 ). Future studies are required to investigate the natural process of invasion into islands by other terrestrial mammals and to develop comprehensive management strategies to combat mammalian invasion into islands. Declarations Acknowledgements We express our gratitude to the staff of Tonosho Town, especially Mr. M. Nakaue, Mr. M. Inaba, and the staff of Shodoshima Town, especially Mr. Y. Kubota, for their cooperation in collecting samples from Shodoshima Island. We also thank Mr. S. Nieda and Mr. Y. Furuichi for their cooperation in collecting samples from Honshu and Shikoku Islands, respectively. We also thank Mrs. A. Nishio, C. Saeki, M. Ishii, Y. Kaji, K. Miyashita, K. Hida, M. Nakatsuka, and T. Horinouchi for their help with fieldwork. This study was financially supported by a Japan Society for the Promotion of Science Grant-in-Aid for JSPS fellows (21J00922 to SI). English language editing was performed using Editage (https://www.editage.jp). Author contributions SI designed the study, collected the genetic data, conducted data analysis, and wrote the manuscript. EI, TK, and JJS supported the design of this study and analysis of genetic data. All the authors have approved the final manuscript for publication. Funding This study was financially supported by the Japan Society for the Promotion of Science (21J00922 to SI). Data availability Data supporting the findings of this study are available in the supplementary file of this article. Conflict of interest We have no competing interests. Ethics All methods used in this study were noninvasive to animals. Tissue samples were collected from the carcasses that were legally hunted by local residents or licensed hunters. 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Supplementary Files Supplementaryfile.xlsx Cite Share Download PDF Status: Published Journal Publication published 12 Nov, 2025 Read the published version in European Journal of Wildlife Research → Version 1 posted Editorial decision: Revision requested 02 Oct, 2025 Reviews received at journal 28 Jul, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor assigned by journal 12 Jun, 2025 Submission checks completed at journal 12 Jun, 2025 First submitted to journal 09 Jun, 2025 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. We do this by developing innovative software and high quality services for the global research community. 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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-6850685","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491064292,"identity":"b9bd6d5d-dd51-4704-8455-516697bfb416","order_by":0,"name":"Shintaro Ishizuka","email":"data:image/png;base64,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","orcid":"","institution":"Fukuyama University","correspondingAuthor":true,"prefix":"","firstName":"Shintaro","middleName":"","lastName":"Ishizuka","suffix":""},{"id":491064296,"identity":"7f70f88c-0a4d-4edd-bc3a-4a4041d95094","order_by":1,"name":"Eiji Inoue","email":"","orcid":"","institution":"Toho University","correspondingAuthor":false,"prefix":"","firstName":"Eiji","middleName":"","lastName":"Inoue","suffix":""},{"id":491064299,"identity":"ab0a55b3-d811-45a5-84e2-cdfa26c8ee8f","order_by":2,"name":"Takeo Kuriyama","email":"","orcid":"","institution":"University of Hyogo","correspondingAuthor":false,"prefix":"","firstName":"Takeo","middleName":"","lastName":"Kuriyama","suffix":""},{"id":491064302,"identity":"1a0a19fe-84db-4edb-ba5a-33202ff1b36c","order_by":3,"name":"Jun J Sato","email":"","orcid":"","institution":"Fukuyama University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"J","lastName":"Sato","suffix":""}],"badges":[],"createdAt":"2025-06-09 04:53:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6850685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6850685/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10344-025-02024-0","type":"published","date":"2025-11-12T15:58:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87744541,"identity":"152bac1f-62e6-4783-ae46-eaabbc942420","added_by":"auto","created_at":"2025-07-28 14:02:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27875,"visible":true,"origin":"","legend":"\u003cp\u003eA photograph of a boar swimming in the Seto Inner Sea.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/435a4a7535792d01574d385e.jpg"},{"id":87744549,"identity":"77d3416f-783e-4a07-93ef-939ab93a5bc9","added_by":"auto","created_at":"2025-07-28 14:02:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":435635,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the study area. Arabic numbers indicate cities in which samples were collected. White parts represent the Seto Inland Sea. Key: “1: Tonosho”, “2: Shodoshima”, “3: Takamatsu”, “4: Tamano”, “5: Okayamaminami”, “6: Okayamanaka”, “7: Okayamakita”, “8: Kichibichuo”, “9: Misaki”, and “10: Ihara”.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/69fa019b6747070e296d07d6.jpg"},{"id":87744550,"identity":"638f3b4c-2e0c-4f17-b7f0-bafaac421013","added_by":"auto","created_at":"2025-07-28 14:02:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96828,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial DNA haplotype network based on the neighbor-joining method. Circles indicate each haplotype. Circle sizes represent sample sizes. Black, dark gray, bright gray, and white colors in the circles represent haplotypes found in Shodoshima Island, Odeshima Island, Shikoku Island, and Honshu Island, respectively.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/77306d67de7c0c01455d72a8.jpg"},{"id":87745666,"identity":"a626e265-4232-4b7b-a303-b57bdc055710","added_by":"auto","created_at":"2025-07-28 14:10:33","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48951,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of mitochondrial DNA haplotypes on Shodoshima Island. Black and white shapes represent haplotypes A and B, respectively. Circles and triangles represent females and males, respectively. Note that sampling localities in this figure are approximate.\u003c/p\u003e","description":"","filename":"Fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/27b2c1671e4e843bb370bf5b.jpeg"},{"id":87745664,"identity":"acca7d9f-a1b7-43f0-869b-4adaf54ec253","added_by":"auto","created_at":"2025-07-28 14:10:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163864,"visible":true,"origin":"","legend":"\u003cp\u003eADMIXTURE analysis of boars in the comprehensive area including Shodoshima Island and other surrounding lands. Samples from “01” to “30”, “31”, “32” to “36”, and “37” to “48” were obtained in Shodoshima Island, Odeshima Island, Shikoku Island, and Honshu Island, respectively.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/03fdc69ac162d0746f4e8d57.jpg"},{"id":87746263,"identity":"13f9e31b-f0d8-41a2-89c4-4e6da6cfa3ca","added_by":"auto","created_at":"2025-07-28 14:18:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":150751,"visible":true,"origin":"","legend":"\u003cp\u003eADMIXTURE analysis of boars on Shodoshima Island. Samples from “01” to “18” and “19” to “30” showed mtDNA haplotypes A and B, respectively.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/b721854b57910dd7727d864b.jpg"},{"id":96105119,"identity":"bcc3bb04-8a1a-4292-bef3-350113f9e79c","added_by":"auto","created_at":"2025-11-17 16:08:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1459701,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/32f80dbc-d302-4498-b0f6-0f753e55d2ec.pdf"},{"id":87744542,"identity":"9c90583b-bea6-4dd5-aa35-99a3637ba1e8","added_by":"auto","created_at":"2025-07-28 14:02:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17269,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6850685/v1/cfc9bfc538527110fc6629dc.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenetic structure of swimming boars (Sus scrofa): genome-wide single nucleotide polymorphisms revealed invasion of two lineages into Shodoshima Island\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiological invasions into islands cause various issues. This can occur for several reasons, such as artificial introduction (e.g. common wall lizard, \u003cem\u003ePodarcis muralis\u003c/em\u003e: Michaelides et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; small Indian mongoose, \u003cem\u003eHerpestes auropunctatus\u003c/em\u003e: Hays and Conant \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) or range expansion (e.g. black-tailed deer, \u003cem\u003eOdocoileus hemionus\u003c/em\u003e: Martin et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Biological invasions, irrespective of whether they result from artificial or natural processes, can damage island ecosystems (Reaser et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), such as a reduction in native flora (Relva et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) or predation of endangered species (Maeda et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), thereby leading to species extinction and biodiversity loss on islands (Savidge \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Johnson and Stattersfield \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Sax and Gaines \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is also expected to trigger economic damage, such as crop damage (Naylor \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and zoonotic disease transmission (Popiolek et al. 2011; Zhang et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For the conservation of native ecosystems on islands that are vulnerable to biological invasion and to mitigate economic damage, a better understanding and efficient management of biological invasion is necessary.\u003c/p\u003e\u003cp\u003eBiological invasions comprise four phases: the appearance of invaders, population growth, range expansion, and invader impacts (Crooks \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The appearance of invaders is thought to occur through several potential processes. If the initial invasion occurs at a single time and a small number of initial invaders share the same origin, the original populations would experience founder effects and show low genetic diversity (Nei et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Dlugosch and Parker \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In contrast, if the appearance of invaders can occur multiple times and these invaders have different origins, population growth can be achieved by the reproduction of multiple lineages, and a relatively high genetic diversity is expected (Dlugosch and Parker \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, if the invasion of islands from other areas occurs continuously, even after the appearance of an initial invader, it can also contribute to population growth and the maintenance of genetic diversity. Thus, genetic analyses are useful for clarifying the processes of invader appearance and population growth.\u003c/p\u003e\u003cp\u003eThe wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) is an invasive game species that is the most widely distributed mammal worldwide (Massei and Genov \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Barrios-Garcia and Ballari \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As they are fecund and reproduce vigorously, their population size often increases rapidly (Coblentz and Baber \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Taylor et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; N\u0026aacute;hlik and S\u0026aacute;ndor \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Their significant population growth can result in range expansion and negative effects on local ecosystems and economies (Lewis et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Risch et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fulgione and Buglione \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, their rooting behavior can reduce plant species diversity (Tierney and Cushman \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Siemann et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and regeneration (Sweitzer and Van Vuren \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bongi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Their predation can be critical for endangered reptile populations (Fordham et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and birds (Carpio et al. 2016; Senserini and Santilli \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). They also have negative impacts on local economies, causing issues such as crop damage (Herrero et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e: Schley et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Amici et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), hybridization with domestic pigs (Šprem et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Iacolina et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), or spreading boar-borne diseases (Gort\u0026aacute;zar et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Bosch et al. 2017). It has been reported that they have already invaded several islands (Barrios-Garcia and Ballari \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and have caused several harmful impacts, such as hybridization with native subspecies (Long \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) or reducing the number of native species (Roemer et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). To effectively manage wildlife and conserve local ecosystems and economies on islands, it is important to understand the processes of boar invasion into islands and their existing population structures.\u003c/p\u003e\u003cp\u003eThe invasion of boars on Pacific islands has been intensively studied (Webr et al. 2018) and Europe (Canu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ballouard et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Boars have been artificially introduced onto Pacific Islands as a source of bushmeat or commercial hunting (Courchamp et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, they sometimes invade islands by swimming (e.g. Ballouard et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Takahashi and Tisdell \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The process of boar invasion into islands via swimming and their population structures is poorly understood because such an invasion is artificially uncontrolled. If the islands are surrounded by multiple neighboring islands, it is possible to assume that boars invaded either from multiple islands or a single island. Therefore, boars on these islands may be genetically similar to those on multiple or single islands. Furthermore, boars are a male-biased dispersing species (Truv\u0026eacute; and Lemel \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Podg\u0026oacute;rski et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting that only males may continuously invade islands after population foundation. In this case, male gene flow between islands could occur frequently and the genetic diversity of males may be higher than that of females on the island. Previous studies have assessed invasion of boars using genetic analyses with focuses on population structures on continuous landmasses (Sagua et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Herna ́ndez et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mangan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Delgado-Acevedo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saito et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nomura et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sawai et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), adaptation to urban areas (Hagemann et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zsolnai et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), or hybridization (Smyser et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Acosta et al. 2024). However, the invasion of boars into islands has been infrequently investigated using genetic analyses, and the effect of invasion via swimming on genetic structure has rarely been examined, except for a few studies (e.g. Canu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Information on the swimming invasion of boars into islands contributes to a broader understanding of the natural mechanisms underlying the biological invasion of islands by terrestrial mammals.\u003c/p\u003e\u003cp\u003eJapan is an ideal location for studying swimming boar invasion. Two subspecies of wild boar, Japanese wild boar (\u003cem\u003eS. s. leucomystax\u003c/em\u003e) and Ryukyu wild boar (\u003cem\u003eS. s. riukiuanus\u003c/em\u003e), are distributed throughout Japan. The Japanese Archipelago comprises 14,125 islands (Geospatial Information Authority of Japan, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsi.go.jp/top.html\u003c/span\u003e\u003cspan address=\"https://www.gsi.go.jp/top.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Recently, the population of boars in Japan has rapidly increased, resulting in their invasion into various novel areas, including islands (Ministry of the Environment \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Especially in the Seto Inland Sea, recent boar invasions into islands are considered to occur via swimming as \u0026ldquo;swimming boars\u0026rdquo; have been sporadically observed (Takahashi \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Fig.\u0026nbsp;1). Thus, this study focused on Shodoshima Island, a part of Kagawa Prefecture located in the Seto Inland Sea, to investigate the pattern of boar invasion into islands via swimming. The island is surrounded by neighboring landmasses, including Honshu Island on the northern side and Shikoku Island on the southern side (Fig.\u0026nbsp;1). On Shodoshima Island, wild boars were considered extinct by approximately 1875 (Takahashi \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It was confirmed that boars were absent until 1990 (Kagawa Prefecture \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and the current IUCN Red List shows that wild boars endemic to this island have been designated as extinct. However, wild boars have been observed on the island since around 2010, and the harvest size increased from 42 to 2,154 between 2010 and 2020 (Kagawa Prefecture \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), suggesting that the population of boars on the island had rapidly grown. Therefore, the island provides a suitable model to assess the effect of swimming boars on their genetic structure and understand their invasion routes into islands.\u003c/p\u003e\u003cp\u003eIn this study, nuclear and mitochondrial genetic analyses of wild boars on Shodoshima Island and its surrounding areas were performed to investigate the genetic structure of wild boars inhabiting a wide area, including Shodoshima Island and its surrounding islands, and to infer the route of invasion by swimming. Second, we investigated the genetic structure of boars on the island to determine the number of genetic lineages of wild boars that had invaded the island. Third, we analyzed sex differences in genetic diversity within the island to test whether males are more likely to be invaders owing to the male-biased dispersal seen in this species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eStudy site and sample collection\u003c/p\u003e\u003cp\u003eThe study sites were Shodoshima Island, Odeshima Island (a small island near Shodoshima Island), and the areas surrounding these two islands, including Honshu and Shikoku Islands (Fig.\u0026nbsp;2). A total of 48 boar tissue samples were collected from December 2020 and February 2023. All the samples were preserved in 70% ethanol at room temperature or frozen. Approximate sampling localities within the islands (30: Shodoshima Island, 1: Odeshima Island, 5: Shikoku Island, and 12: Honshu Island) and sexes (32 males and 16 females) were known for all samples. The location and sex of each sample are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eMitochondrial genetic analysis\u003c/p\u003e\u003cp\u003eDNA was extracted from the tissue samples using a DNeasy Blood \u0026amp; Tissue Kit (Qiagen, CA, USA). To analyze mtDNA nucleotide sequences, we amplified an approximately 550-bp portion of the mtDNA D-loop region with the forward primer mitL76 (5\u0026rsquo;-AATATGCGACCCCAAAAATTTAACCATT-3\u0026rsquo;) and the reverse primer mitH62 (5\u0026rsquo;-CCTGCCAAGCGGGTTGCTGG-3\u0026rsquo;), as reported in a previous study (Watanobe et al. 2003). PCR was performed in 10-\u0026micro;l reaction volumes comprising 2 \u0026micro;l of genomic DNA and 5 \u0026micro;l of QIAGEN Multiplex PCR Master Mix, with 200 nM each of the forward and reverse primers. The amplification parameters were as follows: 94\u0026deg;C for 5 min; 45 cycles of 94\u0026deg;C for 30 s, 57\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s; 60\u0026deg;C for 30 min. PCR products were purified using ExoSAP-IT (Affymetrix, Cleveland, OH, USA). We outsourced the FASMAC sequencing service (Kanagawa, Japan) and sequenced the products using the dye-termination method on an ABI 3100xl sequencer (Applied Biosystems, Foster City, CA, USA). For sequencing, we used the same two primers (mitL76 and mitH62) used in PCR. Finally, a 548-bp nucleotide sequence encompassing part of the mtDNA control region with a second hypervariable domain was obtained. We aligned the nucleotide sequences of each species using the \u0026ldquo;MUSCLE\u0026rdquo; program implemented in MEGA11 (Tamura et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Based on this alignment, we determined mtDNA haplotypes. To assess genetic dissimilarities among the mtDNA haplotypes, a median-joining network was constructed using NETWORK ver 10.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fluxus-engineering.com/sharenet.htm\u003c/span\u003e\u003cspan address=\"https://www.fluxus-engineering.com/sharenet.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The obtained DNA sequence data have been deposited to DDBJ/ENA/Genbank international DNA database with accession numbers, LC876760\u0026ndash;LC876765.\u003c/p\u003e\u003cp\u003eNext generation sequencing\u003c/p\u003e\u003cp\u003eWe outsourced the Bioengineering Lab (Kanagawa, Japan) to perform the GRAS-Di analysis (Enoki and Takeuchi \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and analyzed genome-wide single nucleotide polymorphisms (SNP). This method has recently been used to analyze population genetic structures in several vertebrates (e.g. Hosoya et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sato and Yasuda \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). DNA concentration was calculated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The library used for next-generation sequencing (NGS) was prepared using the 2-step PCR method. The first PCR was performed in 25-\u0026micro;l reaction volumes comprising 5 \u0026micro;l 5\u0026times; PrimeSTAR Buffer (Mg\u003csup\u003e2+\u003c/sup\u003e), 2 \u0026micro;l dNTPs (each 2.5 mM), 10 \u0026micro;l primer mix (100 pmol/\u0026micro;l), 1 \u0026micro;l genomic DNA (15 ng/\u0026micro;l), 0.25 \u0026micro;l PrimeSTAR HS DNA Polymerase (2.5U/\u0026micro;l), and 6.75 \u0026micro;l nuclease-free water. The sequences of each primer in the mixture are listed in Table S2. The amplification parameters were as follows: 98\u0026deg;C for 2 min; 30 cycles of 98\u0026deg;C for 10 s, 50\u0026deg;C for 15 s, and 72\u0026deg;C for 20 s. The second PCR was performed in 50-\u0026micro;l reaction volumes comprising 10 \u0026micro;l 5\u0026times; PrimeSTAR Buffer (Mg\u003csup\u003e2+\u003c/sup\u003e), 4 \u0026micro;l dNTPs (each 2.5 mM), 1.5 \u0026micro;l template DNA (1st PCR product), 0.5 \u0026micro;l PrimeSTAR HS DNA Polymerase (2.5U/\u0026micro;l), and 31.5 \u0026micro;l nuclease-free water, with 0.25 pM of forward (5\u0026rsquo;-GAACGACATGGCTACGATCCGACTT-NN-TAAGAGACAG-3\u0026rsquo;) and reverse (5\u0026rsquo;-TGTGAGCCAAGGAGTTG-Index-TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-NN-AAGAGACAG-3\u0026rsquo;) primers (where \u0026ldquo;NN\u0026rdquo; represents 0\u0026ndash;2 random nucleotides). The index sequences of the reverse primers are listed in Table S2. The amplification parameters were as follows: 98\u0026deg;C for 2 min; 25 cycles of 98\u0026deg;C for 15 s, 53\u0026deg;C for 15 s, and 72\u0026deg;C for 20 s; 72\u0026deg;C for 1 min. The amplification products were mixed in equal volumes and purified using the Min Elute PCR Purification Kit (Qiagen, CA, USA). The concentration of the library was calculated using a Qubit 3.0 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher Scientific). Library quality was confirmed using an Agilent 2100 Bioanalyzer and a High-Sensitivity DNA Kit (Agilent Technologies). The library was transformed into a single-standard circular DNA library using the MGIEasy Circulation Kit (MGI Tech Co., Ltd.). A Dynamical Network Biomarker (DNB) was prepared using a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech Co., Ltd.). The prepared DNB was sequenced using a DNBSEQ-G400 (MGI Tech Co., Ltd.) under 2 \u0026times; 200 bp conditions. The obtained DNA sequence data have been deposited with links to BioProject accession number PRJDB20530 in the DDBJ BioProject database.\u003c/p\u003e\u003cp\u003eSNP calling\u003c/p\u003e\u003cp\u003eTo exclude primer sequences, we trimmed the first 15 bp of each read using the \u0026ldquo;fastx_trimmer\u0026rdquo; function in the FASTX-toolkit ver 0.0.14 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hannonlab.cshl.edu/fastx_toolkit/\u003c/span\u003e\u003cspan address=\"http://hannonlab.cshl.edu/fastx_toolkit/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e index.html). Adaptor sequences were removed using cutadapt ver 4.1 (Martin \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For quality control, reads with quality\u0026thinsp;\u0026lt;\u0026thinsp;Q30 and length\u0026thinsp;\u0026lt;\u0026thinsp;50 bp were trimmed using sickle ver 1.33 (Joshi and Fass \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To unify the length of sequences for data analysis, sequences beyond 50 bp were trimmed using the \u0026ldquo;fastx_trimmer\u0026rdquo; function in the FASTX-toolkit ver 0.0.14. We then adopted the \u0026ldquo;denovo_map.pl\u0026rdquo; program in the Stacks ver 2.59 (Catchen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The program included six stages: building loci (ustacks), creating a catalog of all loci across the population (cstacks), matching each sample against the catalog (sstacks), transposing the data oriented by locus (tsv2bam), assembling a contig and calling single nucleotide polymorphisms (SNPs) (gstacks), and population genomics analysis (populations). Genetic diversity within islands and the genetic distance between islands were calculated using Genodive ver 3.06 (Meirmans et al. 2020).\u003c/p\u003e\u003cp\u003ePopulation genetic structure analysis\u003c/p\u003e\u003cp\u003eADMIXTURE analysis was carried out to investigate the population genetic structures (Alexander et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The genotype file was exported and processed using PLINK ver 1.9 (Chang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) for ADMIXTURE analysis. We used Admixture ver 1.3.0, and investigated population genetic structures with the number of coalescent ancestry populations (K\u0026thinsp;=\u0026thinsp;2 to 5). ADMIXTURE analyses were performed using two different scales. First, the genetic structure of the study area, including Shodoshima Island and its surrounding islands, was investigated using SNP data from all 48 individuals sampled in this study. Secondly, the genetic structure of Shodoshima Island was investigated using SNP data from 30 individuals collected on the island. The ADMIXTURE results were visualized using the \u0026ldquo;pophelper\u0026rdquo; package in R ver 4.4.4 (Francis \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eMitochondrial DNA analysis\u003c/p\u003e\u003cp\u003eNucleotide sequences of the mtDNA control region were successfully determined for all 48 samples. Six haplotypes (A\u0026ndash;F) were identified (Fig.\u0026nbsp;3 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The number of nucleotide substitutions among the haplotypes and the proportion of haplotypes in each population are shown in Fig.\u0026nbsp;3. Two haplotypes (A and B), which differed by nine nucleotides from each other, were found on Shodoshima Island. Haplotype A was widely distributed across the island, whereas haplotype B was observed only in the central and northern parts of the island (Fig.\u0026nbsp;4). These two haplotypes were also found on Shikoku Island, but not on Honshu Island. One haplotype (D) was shared between the Shikoku and Honshu Islands. Haplotype A was detected on Odeshima Island.\u003c/p\u003e\u003cp\u003eGenome-wide SNP analysis\u003c/p\u003e\u003cp\u003eA total of 8,410 SNPs were obtained from samples collected from the entire study area, including Shodoshima Island and its surrounding islands. A summary of the genetic diversity, including the number of alleles, effective number of alleles, observed heterozygosity, heterozygosity within the population, and inbreeding coefficients, is shown in Table S3. According to the ADMIXTURE analysis of the entire study area, including Shodoshima Island and its surrounding islands, the cross-validation error of K\u0026thinsp;=\u0026thinsp;2 was the lowest, indicating that the assumption of two ancestral populations is reasonable (Table S4). Samples from Shodoshima and Shikoku Islands were mostly assigned to the same cluster (colored black, Fig.\u0026nbsp;5), whereas those from Honshu Island were largely assigned to another cluster (colored gray, Fig.\u0026nbsp;5). The genetic distance (Fst) between Shodoshima Island and Shikoku Island was 0.031, which was lower than that between Shodoshima Island and Honshu Island (0.201), and between Shikoku Island and Honshu Island (0.176).\u003c/p\u003e\u003cp\u003eA total of 8,276 SNPs were obtained from samples from Shodoshima Island. According to the ADMIXTURE analysis of Shodoshima Island, the cross-validation error of K\u0026thinsp;=\u0026thinsp;2 was the lowest, indicating that assuming two ancestral populations was reasonable (Fig.\u0026nbsp;6, Table S4). The ADMIXTURE plot revealed that samples from \u0026ldquo;01\u0026rdquo; to \u0026ldquo;18,\u0026rdquo; which showed mtDNA haplotype A, contained a relatively large proportion of black color, while those from \u0026ldquo;19\u0026rdquo; to \u0026ldquo;30,\u0026rdquo; which showed mtDNA haplotype B, contained a relatively large proportion of gray color, suggesting that they were genetically roughly divided from each other (Fig.\u0026nbsp;6). In contrast, 20 of the 30 samples shared the genetic components of both clusters (Fig.\u0026nbsp;6). The number of alleles, effective number of alleles, observed heterozygosity, heterozygosity within the population, and inbreeding coefficient, which are measures of genetic diversity, did not differ notably between the sexes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSex differences in genetic diversity of boars on Shodoshima Island.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of alleles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEffective number of alleles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObserved heterozygosity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeterozygosity within population\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInbreeding coefficient\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study found six mtDNA haplotypes in the study area, including Shodoshima Island and its neighboring islands. Two mtDNA haplotypes (A and B) were shared in Shodoshima and Shikoku Islands, but not in Honshu Island. ADMIXTURE analysis based on genome-wide SNPs also showed that boars in the entire study area could be genetically divided into two ancestral populations, and those in Shodoshima and Shikoku were mostly assigned to the same population. The genetic distance between Shodoshima and Shikoku was smaller than that between Shodoshima and Honshu. The overall results suggest that boars in Shodoshima and Shikoku Islands are genetically close to each other and that they invaded from Shikoku to Shodoshima Island. One possible reason for this direction of invasion may be the excessive boar population size on Shikoku Island. Since the estimated number of boars in the Kagawa Prefecture excluding islands according to surveys in 2020 was 39,996 (Kagawa Prefecture \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the area of the island is 1,692.72 km\u003csup\u003e2\u003c/sup\u003e (Geospatial Information Authority of Japan), the density was calculated to be 23.63 individuals per km\u003csup\u003e2\u003c/sup\u003e. The population size of boars in the area is considered to have increased rapidly in recent years, as the total number of captures in Kagawa Prefecture, excluding islands, drastically increased from 5,451 to 10,494 between 2010 and 2020 (Kagawa Prefecture \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, the population density in Okayama Prefecture was estimated to be 6.25 individuals per km\u003csup\u003e2\u003c/sup\u003e, as the estimated number of boars was 44,452 (Okayama Prefecture \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the area of this prefecture was 7,114.44 km\u003csup\u003e2\u003c/sup\u003e (Geospatial Information Authority of Japan). The large population size of boars on Shikoku Island, especially around Kagawa Prefecture, might have caused the invasion of boars into Shodoshima Island. Furthermore, given that one mtDNA haplotype was shared between the Shikoku and Honshu Islands, the invasion of boars from Shikoku Island might reach Honshu Island.\u003c/p\u003e\u003cp\u003eThe genetic structure of boars around Shodoshima Island in this study was consistent with previous studies showing that boars were genetically differentiated between the Shikoku and Honshu Islands (Sawai et al. 2022; Nomura et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the trend observed in wild boar was different from that in most other terrestrial mammals on Shodoshima Island. Studies on mtDNA in Japanese wood mouse species (\u003cem\u003eApodemus speciosus\u003c/em\u003e and \u003cem\u003eA. argenteus\u003c/em\u003e), Japanese macaques (\u003cem\u003eMacaca fuscata\u003c/em\u003e), and sika deer (\u003cem\u003eCervus nippon\u003c/em\u003e) have suggested that Shodoshima populations are similar to those of Honshu Island (Suzuki et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kawamoto et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ishizuka et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A similar trend was observed in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003especiosus\u003c/em\u003e on the islands of the western Seto Inland Sea (Sato et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This difference may be due to the method of invasion of the area. One study suggested that terrestrial mammals, such as Japanese macaques and sika deer, invaded the island, as the Seto Inland Sea did not appear or was shallow until the Holocene glacial retreat (Ishizuka et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast to these species, boars can swim (Takahashi and Tisdell \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Fujita et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and the surrounding water in the sea is not likely to be a barrier against their movements. This is likely why they have successfully invaded the island recently, even though the Seto Inland Sea was present. However, it should be noted that a terrestrial mammal, the lesser Japanese mole (\u003cem\u003eMogera imaizumii\u003c/em\u003e) on Shodoshima Island was genetically close to those on Shikoku Island (Mitsuhashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Ecological or behavioral differences among mammals might be a factor in the differences in phylogenetic history.\u003c/p\u003e\u003cp\u003eThis study found two mtDNA haplotypes from Shodoshima Island that were genetically distant from each other, with nine substitutions present between them. ADMIXTURE analysis based on genome-wide SNPs also showed that boars on Shodoshima Island could be divided into two ancestral populations. This suggests that the boars that invaded Shodoshima Island belonged to two distinct lineages. Given that the boar invasion occurred from the southern side (Shikoku Island) and that haplotype B was distributed in the northern part of the island compared to haplotype A, a lineage with haplotype B might have invaded the island prior to the invasion of another lineage with haplotype A. Moreover, 20 of the 30 individuals shared genetic components of the two ancestry populations, suggesting that the two lineages had already hybridized with each other. Sexual maturity in female boars is achieved before one year of age (Geth\u0026ouml;ffer et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Fonseca et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), indicating a short generation time. After invasion, the two lineages may hybridize with each other for a short period. The invasion of multiple lineages enhances the success of a newly settled population by increasing genetic diversity (Barret and Husband 1990). Similarly, the establishment of a boar population on Shodoshima Island may have been due to the presence of multiple ancestral lineages. Invasion by multiple lineages has also been reported in various animals (e.g. brushtail possum \u003cem\u003eTrichosurus vulpecula\u003c/em\u003e: Triggs and Green \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; brown rat \u003cem\u003eRattus norvegicus\u003c/em\u003e: Calmet et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; brown anole \u003cem\u003eAnolis sagrei\u003c/em\u003e: Kolbe et al. 2004; oriental fruit fly \u003cem\u003eBactrocera dorsalis\u003c/em\u003e: Barr et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, we concluded that founders with different origins might explain the recent population growth of wild boar on Shodoshima Island.\u003c/p\u003e\u003cp\u003eThe extent of genetic diversity was similar between sexes. As boars show male-biased dispersals (Truv\u0026eacute; and Lemel \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Podg\u0026oacute;rski et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), it is possible to assume that males invading the island is more likely, and that the genetic diversity of males was higher than that of females. However, the results of the present study do not support this hypothesis. The frequency of boar invasion on the island might not differ between sexes. On the other hand, another possibility is that sex differences in genetic diversity might be masked by their vigorous reproduction (Coblentz and Baber \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Taylor et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; N\u0026aacute;hlik and S\u0026aacute;ndor \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The reproduction of boars within the island is expected to increase the population size of both sexes equally. Population growth of males by reproduction on the island might equalize the genetic diversity between sexes, even though the frequency of invasion into the island is higher in males than in females. To clarify these possibilities, future genetic and observational studies are required to investigate sex differences in the patterns of invasion into islands by swimming boars on other islands.\u003c/p\u003e\u003cp\u003eThe present study clarified the route and genetic diversity of boars that invaded Shodoshima Island, Japan. Because understanding the population structure based on genetic diversity and genetic boundaries is important for wildlife management (DeYoung and Honeycutt \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), our results are expected to be utilized for the management of boars and to reduce damage to various crops, such as rice, fruits, and vegetables, on the island (Kagawa Prefecture \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Given that their invasion could occur from Shikoku Island to Shodoshima Island, the southern parts of the island should be key areas in preventing their invasion. Furthermore, this study can serve as an important case in which terrestrial mammal species naturally invade an island in addition to artificial introduction. Our results provide insights into the invasion patterns of terrestrial mammals into islands. Similar to boars, several ungulate species are also good at swimming (e.g. chamois \u003cem\u003eRupicapra spp\u003c/em\u003e.: Kavčić et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Future studies are required to investigate the natural process of invasion into islands by other terrestrial mammals and to develop comprehensive management strategies to combat mammalian invasion into islands.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our gratitude to the staff of Tonosho Town, especially Mr. M. Nakaue, Mr. M. Inaba, and the staff of Shodoshima Town, especially Mr. Y. Kubota, for their cooperation in collecting samples from Shodoshima Island. We also thank Mr. S. Nieda and Mr. Y. Furuichi for their cooperation in collecting samples from Honshu and Shikoku Islands, respectively. We also thank Mrs. A. Nishio, C. Saeki, M. Ishii, Y. Kaji, K. Miyashita, K. Hida, M. Nakatsuka, and T. Horinouchi for their help with fieldwork. This study was financially supported by a Japan Society for the Promotion of Science Grant-in-Aid for JSPS fellows (21J00922 to SI). English language editing was performed using Editage (https://www.editage.jp).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSI designed the study, collected the genetic data, conducted data analysis, and wrote the manuscript. EI, TK, and JJS supported the design of this study and analysis of genetic data. All the authors have approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Japan Society for the Promotion of Science (21J00922 to SI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available in the supplementary file of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods used in this study were noninvasive to animals. Tissue samples were collected from the carcasses that were legally hunted by local residents or licensed hunters. 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Mamm Biol 102:221\u0026ndash;234\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-wildlife-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejwr","sideBox":"Learn more about [European Journal of Wildlife Research](http://link.springer.com/journal/10344)","snPcode":"10344","submissionUrl":"https://submission.nature.com/new-submission/10344/3","title":"European Journal of Wildlife Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sus scrofa, Shodoshima Island, Seto Island Sea, single-nucleotide polymorphism analysis","lastPublishedDoi":"10.21203/rs.3.rs-6850685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6850685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWild boars (\u003cem\u003eSus scrofa\u003c/em\u003e) have substantial impacts on island ecosystems and economies worldwide because of their rapid population growth and ability to expand their distribution. Although many studies have focused on the invasion of artificially introduced boars into islands, the natural processes of boar invasion into islands have rarely been studied. This study investigated the natural process of the recent swimming invasion of boars into Shodoshima Island in the Seto Inland Sea, Japan. We investigated whether wild boars on this island invaded the Honshu (northern side) or Shikoku (southern side) islands. We performed mitochondrial and genome-wide single-nucleotide polymorphism (SNP) analyses of boars living on these islands. Two mtDNA haplotypes were found on Shodoshima Island, and were shared on Shikoku Island, but not on Honshu Island. SNP analysis showed that boars on Shodoshima and Shikoku Islands possessed similar genomic compositions, and two ancestral populations were present within the boars of Shodoshima Island. These findings suggest that the two genetic lineages of boars invaded Shodoshima Island from Shikoku, and the population primarily expanded through the reproduction of the two ancestry lineages. This study contributes to a better understanding of the patterns of wild boar invasion into islands and the management of boars on small isolated islands.\u003c/p\u003e","manuscriptTitle":"Genetic structure of swimming boars (Sus scrofa): genome-wide single nucleotide polymorphisms revealed invasion of two lineages into Shodoshima Island","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 14:02:28","doi":"10.21203/rs.3.rs-6850685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-02T15:16:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T06:53:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168568491758836157003279517353748052677","date":"2025-07-25T22:10:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303422943802159979368037113772117984196","date":"2025-07-25T02:29:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T20:17:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-13T01:20:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-13T01:18:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Wildlife Research","date":"2025-06-09T04:39:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-wildlife-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejwr","sideBox":"Learn more about [European Journal of Wildlife Research](http://link.springer.com/journal/10344)","snPcode":"10344","submissionUrl":"https://submission.nature.com/new-submission/10344/3","title":"European Journal of Wildlife Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"91e9221a-d682-4a47-aec7-865049d3889f","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:03:30+00:00","versionOfRecord":{"articleIdentity":"rs-6850685","link":"https://doi.org/10.1007/s10344-025-02024-0","journal":{"identity":"european-journal-of-wildlife-research","isVorOnly":false,"title":"European Journal of Wildlife Research"},"publishedOn":"2025-11-12 15:58:26","publishedOnDateReadable":"November 12th, 2025"},"versionCreatedAt":"2025-07-28 14:02:28","video":"","vorDoi":"10.1007/s10344-025-02024-0","vorDoiUrl":"https://doi.org/10.1007/s10344-025-02024-0","workflowStages":[]},"version":"v1","identity":"rs-6850685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6850685","identity":"rs-6850685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}