Dokdo sea lion Zalophus japonicus genome reveals its evolutionary trajectory before extinction

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Little is known about its origins and speciation compared to other Otariidae species or how the rapid decline affected the species’ genetic diversity. Results To raise the Dokdo sea lion from this relative obscurity, we sequenced DNA from 16 Z. japonicus ’ bone fragments, obtained from Dokdo and Ulleungdo islands in Korea. Our genome-wide SNP-based analyses establish Z. japonicus as the earliest diverged species within its genus, significantly redefining its evolutionary relationship with the California ( Z. californianus ) and Galapagos ( Z. wollebaeki ) sea lions. Our research further elucidates the phylogeny of Z. japonicus , shedding light on the complexity of the genetic isolation process within its genus that was prompted by the geographic isolation of the three populations of Zalophus ancestral stock. Conversely, the genetic signature of Dokdo sea lion genome can be modeled as an evolutionary pathway involving gene flow from Otariidae species with shared range. In addition, we discovered, population decline of the Z. japonicus started already over 1,000 years ago, however, Z. japonicus genome maintained a relatively high heterozygosity despite nearing extinction. Conclusions Our genome-scale analysis has eliminated ambiguity in the phylogeny of Z. japonicus and shed light on the evolutionary pathways underlying its speciation and the genetic diversity before its extinction. Broadly, this study highlights the importance of genome-scale analysis for the extinct marine megafauna to elucidate the complexity of their gene flow and subsequent genetic diversities among extant species. Furthermore, this study offers retrospective genomic insights into the extinction process of a carnivorous marine mammal, information that could aid conservation efforts towards extant Otariidae species. Zalophus japonicus Dokdo sea lion Japanese sea lion Otariidae speciation whole genome sequencing extinction marine mammal introgression paleogenomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The Dokdo sea lion – Zalophus japonicus (Otariidae: Carnivora), also known as the Japanese sea lion, was a significant species native to East Asia, inhabiting the Dokdo and Ulleungdo islands within Korean territorial waters (Fig. 1 A). Known locally as Gangchi, this species, along with other Otariidae such as the Northern fur seal ( Callorhinus ursinus ) and Steller sea lion ( Eumetopias jubatus ), thrived in the coastal habitats of the northwest Pacific Ocean, spanning from Russia to the coastal waters of Korea and Japan [ 1 ]. Despite the absence of dated fossil records for Z. japonicus , the discovery of other extant pinniped fossils, such as E. jubatus , suggests a long-standing habitation in these regions, potentially since the Pliocene [ 2 ]. Historical sources indicate that Z. japonicus was hunted for human consumption in Hokkaido since the Jomon period, but it's widely believed that this subsistence hunting didn't significantly impact their population dynamics [ 3 ]. Over the past two centuries, the Z. japonicus population experienced a drastic decline. In the mid-19th century, estimates suggested a healthy population of 30,000 to 50,000 animals, comparable to later counts of Galapagos and California sea lions [ 3 , 4 ]. However, by the 1950s, their numbers plummeted to just 50–60, leading to their classification as extinct by the International Union for Conservation of Nature (IUCN) in 1990 [ 3 ]. This sharp decrease was largely due to extensive hunting for meat, skin, and oil between 1904 and 1925, particularly targeting females and pups [ 5 ]. Z. japonicus is recognized as a member of the genus Zalophus . This genus comprises three distinct species: the western Dokdo sea lion ( Z. japonicus ), the eastern California sea lion ( Z. californianus ), and the Galapagos sea lion ( Z. wollebaeki ) found in the North Pacific Galapagos archipelago (Fig. 1 B) [ 4 ]. Z. japonicus was initially classified as a subspecies of the California sea lion, however, it was reclassified based on unique cranial features [ 6 ] and mitochondrial DNA (mtDNA) differences [ 7 ]. Evolutionary studies suggest a divergence of merely 2.2 million years between Z. japonicus and Z. californianus , indicating a close genetic relationship [ 7 ]. The specific evolutionary pathways leading to the isolation of these sea lion species remain unclear. Complicating their classification further, pinnipeds exhibit a wide range of individual cranial variations [ 8 ] and documented interspecific sexual behaviors [ 9 – 12 ], potentially leading to introgression and morphological diversity. While such behaviors haven't been directly observed in Z. japonicus , the possibility is supported by observed introgression events in other pinniped species [ 12 , 13 ]. To date, genomic research on Z. japonicus has predominantly relied on mtDNA, which captures only a limited scope of the species' genomic diversity. Consequently, comprehensive whole-genome sequencing has become essential to fully understand the Dokdo sea lion's genetic makeup and its phylogenetic relationships within the Otariidae [ 14 ]. Dokdo island, a vital habitat for Z. japonicus in the 1900s and one of their last sighting locations, preserved biological materials that may offer novel insights. In this study, 16 bone fragments of Dokdo sea lions were unearthed from Dokdo and the nearby Ulleungdo islands, situated 87.4 km apart in the East Sea of Korea. We compared the genomes of extinct Z. japonicus with those of other sequenced Otariidae species. Our analyses not only reveal the genetic diversity and detailed phylogeny of Z. japonicus but also for the first-time shed light on the evolutionary processes of speciation and introgression in this species and its genus. Results Dokdo sea lion samples and genomic data Our study focused on the genomic analysis of Dokdo sea lion using 16 bone samples (Z1, Z3-Z9, Z11-Z18) excavated from Dokdo and Ulleungdo islands (Figure 1a, Additional file 1: Fig. S1, Additional file 1: Fig. S2). These skeletal remains, mainly limb and rib bones, underwent DNA extraction and deep DNA sequencing using next-generation sequencing (NGS) methods, despite challenges posed by the small size of the fragments (Additional file 2: Data S1). Detailed information about laboratory techniques used is presented in Supplementary Material. Initial tests on sample Z3 using single-end (SE) and paired-end (PE) NGS revealed higher mapping rates with PE against a Z. californianus reference genome, leading us to construct PE NGS libraries for all samples. We generated 8.4 Tb of data with individual mapping rates ranging from 0.1% to 1.3%. Mapping Z. japonicus reads to E. jubatus genome covered 83.35% of its genome and 90.83% of protein-coding genes (Figure 1c, Additional file 2: Data S2). This coverage was compared to other Otariidae species, including Z. californianus, Z. wollebaeki, E. jubatus , and C. ursinus . For the bioinformatics analysis, we utilized the PALEOMIX pipeline [15], aligning 43G reads to the Z. californianus genome (Additional file 1: Table S1), with an average read length of 140 bp (Additional file 1: Fig. S3). DNA misincorporation levels were low (Additional file 1: Fig. S4), consistent with the species' fairly recent extinction. Additionally, we sequenced modern Z. californianus and obtained other pinniped species’ DNA for comprehensive comparative analysis (Additional file 2: Data S2) of Z. japonicus and broader knowledge on Otariidae phylogenetics and evolutionary history. Congeneric Zalophus speciation involved introgression Dokdo sea lion genetic ancestry and relationship with other Otariidae species, is not yet well understood and has not yet been studied outside the morphological classification and mtDNA analysis [7, 16, 17]. To elucidate the genomic composition of Zalophus sea lions beyond mtDNA [7, 17], we estimated the gene flow and possible admixture scenarios among Zalophus and with other pinnipeds. Firstly, the f4 -statistics unsurprisingly show that congeneric Zalophus species share the highest genetic affinities among each other compared to the E. jubatus (Additional file 1: Table S2). Secondly, Z. japonicus exhibits a relatively more distinct genetic makeup compared to its closest species, Z. californianus and Z. wollebaeki (Fig. 2a-c). In Z. japonicus , we could not identify gene flow or introgression from E. jubatus , which was observed in both Z. californianus and Z. wollebaeki (Fig. 2a, b, Additional file 1: Table S3, and Additional file 1: Table S4). Consistently, genetic modeling using qpGraph suggests that Z. japonicus diverged early in the evolution of the Zalophus genus, with 98% of its genetic components derived from the common ancestor of Zalophus and 2% derived from the lineage of E. jubatus and C. ursinus (Fig. 2c). After the divergence, we observed no additional genetic admixture between Z. japonicus and other Zalophus species. Third, we suggest that gene flow came from E. jubatus and the common ancestor of Z. californianus and Z. wollebaeki as well as after Z. japonicus divergence (Figure 2a, b). E. jubatus appeared to share significantly less genetic affinity and gene flow with Z. japonicus compared to Z. californianus and Z. wollebaeki (Figure 2a, b). Interestingly, the latter two species not only received more gene flow and derived alleles from E. jubatus , but also their levels of genetic affinity to E. jubatus were nearly identical (Figure 2a). This provides further support to a scenario wherein E. jubatus introgressed into the common ancestor of Z. californianus and Z. wollebaeki prior to the divergence and isolation of Z. californianus and Z. wollebaeki as it is quite unlikely that E. jubatus introgressed the two modern geographically isolated Zalophus species at the same rate (while not sharing any known habitats with Z. wollebaeki ). Furthermore, following the divergence of Z. japonicus , we suggest that more than one major introgression event had occurred between Z. californianus and Z. wollebaeki (Fig. 2a, b). Lastly, we observed an inconsistent and complex genetic relationship between Z. japonicus and other Zalophus species, likely due to repeated interspecific and intergeneric introgression events. f4 -statistics revealed a higher genetic affinity between Z. japonicus with Z. californianus compared to Z. wollebaeki (Fig. 2a). However, admixture f3 -statistics showed no genetic admixture between Z. japonicus and either C. ursinus or E. jubatus in the Z. californianus genome (Fig. 2b). These contradictory finding suggest Z. californianus played a significant role in the evolution of the Zalophus genus, potentially neutralizing previous admixture effects with Z. japonicus through recent genetic interactions with Z. wollebaeki (Fig. 2c). In contrast, Z. wollebaeki still retains genetic traces of ancestral genetic admixture with Z. japonicus . It includes a 17% genetic component shared with all Zalophus species (Fig. 2c), indicating a lesser divergence. Additionally, Z. wollebaeki did not experience significant genetic exchange with other species (which include introgression from either C. ursinus or E. jubatus ) until its admixture with the common ancestor of Z. californianus . These lines of evidence suggest that Z. japonicus not only is a unique species with a distinct genetic makeup, that for major part evolved directly from the common ancestor of the Z. californianus and Z. wollebaeki , but also that Z. japonicus may be the earliest diverged species in this genus. Complex introgressive speciation of Zalophus species explains their phylogenetic ambiguities Our study for the first time validated Z. japonicus phylogeny using 1,581,963 autosomal SNVs (Additional file 2: Data S5) and compared it with whole-mtDNA-based classification (Fig. 3). The mtDNA phylogeny of Z. japonicus specimens revealed two genetically uniform and nearly indistinguishable mtDNA haplotypes (Additional file 1: Fig. S6). The genetic distances between them were sufficient to identify different mtDNA haplotypes but subtle enough to be possibly derived from the same maternal Z. japonicus ancestor (0.0007 vs 0.0005). Both autosomal SNV and mtDNA-based phylogenetic trees presented the same topology with two distinct Otariidae clades and Northern fur seal ( C. ursinus ) as an outgroup: one of Northern pinnipeds composed of Zalophus and Eumetopias sea lions, and one of Southern pinnipeds composed of Phocarctos and Neophoca sea lions with Arctocephalus fur seals (Fig. 3a, b). In this context, Z. japonicus showed almost equal phylogenetic distance to Z. wollebaeki and Z. californianus (mtDNA: 0.014 and 0.014; WGS: 0.015 and 0.015, respectively) (Additional file 2: Data S6). Even though Z. japonicus was similarly related to its congenerics, the genetic distance between the Z. wollebaeki and Z. japonicus was about 40% greater compared to the distance between the Z. wollebaeki and Z. californianus . This observation held true regardless of whether the genetic distances were estimated using autosomal SNVs or mtDNA (Additional file 2: Data S6). Our and previously reported [18] intrageneric Zalophus phylogeny aligns with f4 -statistic that showed higher genetic affinity between Z. wollebaeki and Z. californianus compared to the affinity each of them had with Z. japonicus . The genetic distances between Z. japonicus and its extant genetic donors, C. ursinus and E. jubatus , showed significant disparities between estimates based on mtDNA and autosomal SNVs (Fig. 3C). The genetic distances based on mtDNA were approximately two times greater than those based on the autosomal SNVs, and reflected in the phylogenetic tree branching (Fig. 3a-c, Additional file 2: Data S5). However, this disparity in genetic distance from Z. japonicus did not apply to the already mentioned Z. japonicus’ congenerics and other phylogenetically distant species, such as those under the genera Phoca and Pusa (Fig. 3c, Additional file 2: Data S6). These findings imply either a significant divergence of maternal lineages between Zalophus and the pair of C. ursinus and E. jubatus or a genetic legacy of Z. japonicus introgression from C. ursinus and E. jubatus . The introgression scenario obscures phylogenetic relationships, as the relative reduction in autosome-based genetic distance may falsely suggest a more recent common ancestry than it actually is. Nevertheless, both scenarios may be true, and it underscores the multifaceted nature of evolutionary dynamics within the Otariidae, which is also largely consistent with our previously shown genetic ancestry modeling (Fig. 2). Heterozygosity of extinct Dokdo sea lion Our subsequent objective was to elucidate the genetic diversity of the Z. japonicus samples within the context of population analyses. To accurately estimate heterozygosity (theta) from low depth Z. japonicus data, we pooled reads from multiple individuals, enabling genome-wide calculation of heterozygosity across more than 200M loci with a depth of coverage exceeding ten (Additional file 2: Data S6). We obtained moderate heterozygosity value of Z. japonicus (0.00101) which was greater than any other Zalophus species and several other marine mammal species that are recognized as “Least concern” regarding their vulnerability to extinction according to the International Union for Conservation of Nature and Natural Resources (IUCN) (Fig. 4). Interestingly, the heterozygosity levels of C. ursinus and E. jubatus (“Vulnerable” and “Near threatened”, respectively) differed drastically from each other (Fig. 4). Despite C. ursinus having been extirpated from most of its range over the past 200–800 years due to hunting and environmental factors, its heterozygosity remains at a relatively high level, which corresponds with the historical DNA analysis previously published [19]. In contrast, the heterozygosity estimate for E. jubatus was almost as low as that of Z. wollebaeki (“Endangered”) (Fig. 4). We suggest that this fact is related to the nowadays population decline of E. jubatus , which began in the 1980s and continues to this day across its distribution range [20, 21]. Moreover, one of Z. japonicus’ closest living relatives, the non-endangered Z. californianus , exhibited about two times lower heterozygosity (0.000491 and 0.000595), suggesting that even a halved estimate for Z. japonicus (to compensate for sample merging effect) would not indicate low genetic diversity. Among Z. californianus , relatively low heterozygosity value was observed in a potentially inbred individual from a Korean zoo exhibit (0.000491, SRR11434789). This analysis suggests Z. japonicus’ heterozygosity (0.00101) is not so low as to raise concerns about extinction, and therefore does not indicate severe inbreeding. Figure 4. Heterozygosity of Dokdo sea lion in the context of other marine mammals. The average heterozygosity values in log scale (from the left to right) for: Steller’s sea cow – H. gigas , Dokdo sea lion – Z. japonicus , Galapagos sea lion – Z. wollebaeki , Northern fur seal – C. ursinus , dugong – D. dugon , West Indian manatee – T. manatus , walrus – O. rosmarus , Steller sea lion – E. jubatus , beluga whale – D. leucas , grey seal – H. grypus , narwhal – M. monoceros , California sea lion – Z. californianus , and California sea lion – Z. californianus (from zoo). Samples are colored according to IUCN conservation status (2024). Population decline of Dokdo sea lion started over 1,000 years ago One crucial determinant of a species' vulnerability to extinction is its population size, therefore, we inferred the retrospective history of effective population size ( Ne ) changes in Z. japonicus and other Otariidae species ( Z. californianus , Z. wollebaeki , E. jubatus and C. ursinus ) for comparison, using the pairwise sequentially Markovian coalescent (PSMC) algorithm [22] (Fig. 5). For reliable Ne inference over time, we used Z. japonicus SNVs identified from genomic loci with a minimum depth of more than ten reads. The population dynamics of Z. japonicus present a unique pattern compared to its Otariidae counterparts. While other Otariidae mammals showed a population decline around 10,000 years ago, possibly due to the climatic shifts of the Holocene including warming temperatures and sea level rises, Z. japonicus experienced this decline later, around 4,500 years ago. This suggests a different adaptative response to environmental changes. Notably, the distinct population trajectory of Z. japonicus could be linked to genetic introgression with E. jubatus and C. ursinus . We also observed a unique small rebound in population numbers about 1,500 years ago, which has not been observed in the other species. This resurgence was short-lived as a continued decrease in Z. japonicus population numbers is apparent ever since. In addition to the PSMC analysis, demographic dynamics inferred using PopSizeABC further ascertained Z. japonicus population decline since one thousand years ago with 95% confidence interval. While a more robust dataset of Z. japonicus and other Otariidae species is essential to fully understand the interplay between climatic factors and population trends, our analysis provides the first whole-genome-based insights into the downward trend in of Z. japonicus ’ demographic history. Discussion The deep DNA sequencing and advances in bioinformatics allowed us to understand the basics of evolution better, and to describe traces of genetic introgression and the events that accompanied them, e.g., rapid speciation, multiple ecological radiations, and rapid adaptation to the changing environment [ 23 – 25 ]. Intergeneric fertile hybridization in pinnipeds is well-known fact, which adds an additional layer of complexity analyzing their speciation, phylogeny, and ancestry [ 12 , 26 ]. Our study sheds light on the evolution of extant Otariidae species inhabiting the Northern Pacific Ocean, with a special focus on the genus Zalophus , especially the Dokdo sea lion, an extinct member of this genus. Through f3 and f4 admixture tests, we describe an introgression from C. ursinus and/or E. jubatus to Z. californianus and Z. wollebaeki (Fig. 2 a-c). Moreover, we find ancient introgression events between the extinct Dokdo sea lion and E. jubatus/C. ursinus . While there are no remaining historical or scientific records on Z. japonicus hybridization, Z. californianus as a species has a rich hybridization history in zoo enclosures with mixed-species pinniped exhibits. On a larger scale, recently, compelling evidence emerged suggesting that smaller wild E. jubatus body size found specifically in Oregon population in United States could be attributed to a paternal genetic input from male Z. californianus that opportunistically mate with E. jubatus females during their seasonal migrations [ 27 ]. It is also known that there was a significant overlap not only in the species ranges (Fig. 1 b) but also an ecological niche between extinct Z. japonicus and extant C. ursinus , and E. jubatus [ 3 , 4 , 28 ]. We suppose that such gene flow between Otariidae species in the northern part of the Pacific Ocean increased their genetic diversity and could have had an adaptive effect in the changing environment for tens of thousands of years; for example, it could have helped the Zalophus species survive the Pleistocene-Holocene extinction of megafauna event. Unfortunately, increasing human activities over the last few hundred years have led to ecosystem degradation, the destruction of native habitats, and the direct extinction of many animal species. The Dokdo sea lion is one of the species that is historically considered a victim of theriocide [ 3 ]. Our study not only confirms that Z. japonicus , a recently extinct iconic species in Korea and Japan, is genetically and evolutionarily very distinct from Z. californianus and Z. wollebaeki , but also supports the written history, insisting that Dokdo sea lion population does not appear to have had a natural evolutionary dead end. Among evidence we report relatively high heterozygosity suggesting that the theriocide inflicted on the Z. japonicus population was faster than the inbreeding rate. Moreover, the demographic history of the Dokdo sea lion has a relatively different trajectory compared with extant Otariidae species. This extinct marine mammal was influenced by a radical decrease in effective population size around 4,500 years ago, while the Northern fur seal, Steller sea lion, California sea lion, and Galapagos sea lion went through genetic bottleneck around 10,000 years ago. Dokdo sea lion is an illustrative example of how human activity can lead a seemingly genetically stable populations to the verge of extinction. We acknowledge that our study has many limitations such as small sample number, and low sequencing depth, which precluded us from performing certain analyses, but hopefully will give an introduction for future in-depth studies. Notably, all the Z. japonicus bones were fragmented and affected by sea water, factors that may negatively contribute to the preservation of DNA and consequently to NGS sequencing yields (51, 52). Secondly, we could not comprehensively validate relative sample dating and DNA misincorporation estimates with other methods due to expected sample age estimates falling within modern-historical range. Moreover, we acknowledge that there was no overall correlation between the species conservation status and the heterozygosity in our study. This goes on to show that estimating species genetic diversity and conservation-needs requires a holistic approach that considers many intricate factors such as population numbers, habitat and food supply availability, pollution, hunting and poaching rates, reproductive behavior and fertility rates, accumulation of pathogenic alleles; heterozygosity is just one of them. Conclusions This study provides novel genomic, evolutionary, and demographic insights based on the whole genome sequencing and analysis of an extinct sea lion from East Asia, Z. japonicus. Z. japonicus genome fills a significant gap in the collective knowledge on the sea lion genus Zalophus and, more broadly, the eared seal family Otariidae. These data significantly elucidate the speciation process within genus Zalophus , suggesting that: 1) Z. japonicus was the earliest diverged species in its genus; 2) Z. californianus and Z. wollebaeki populations had genetic exchanges with each other upon their initial separation; 3) the genomes of all the three Zalophus species show signs of introgression from the Otariidae lineage of E. jubatus / C. ursinus , with a particularly strong signal present in Z. japonicus. Moreover, the demographic estimates of Z. japonicus , such as an effective population size and the heterozygosity, did not suggest an elevated risk of extinction. These estimates provide an important complementary line of evidence to the sparsely documented knowledge on the Z. japonicus extinction. Materials and methods Experimental Design For our genomic comparison study of the extinct Z. japonicus , we used 16 Z. japonicus bones (Additional file 1: Fig. S1 , Additional file 1: Fig. S2 ) - three from Gajaegul in Ulleungdo (Gaze Cave, latitude 37.51° and longitude 130.79°) and 13 from Seodo Gajaegul in Dokdo islands (Gaze Cave, latitude 37.24° and longitude 131.86°) (Additional file 1: Table S1 ). Both sites are named “Gajaegul”, meaning “sea lion cave” in Ulleungdo county dialect. The Z. japonicus bones were provided by Cetacean Research Institute of National Institute of Fisheries Science in Republic of Korea. The collection of Dokdo sea lion bones was conducted under the permission granted by the Gyeoungbuk province local government for the collection of protected marine organisms (Permit No. 2019-2). We also collected a Z. californianus muscle sample from the Seoul Grand Park, Republic of Korea, obtained during the necropsy process (Permit No. Seoul Grand Park Scientific Research 2020-009). As our study involved extinct animals and cadavers, ethical approval was not required. In addition, we downloaded 12 pinniped genomes (Additional file 2: Data S5), which were used to construct phylogenetic tree, ancestry analysis, and in the genetic diversity studies. DNA extraction and next generation sequencing To avoid contamination, only endogeneous bone tissue was collected after UV radiation and ethanol treatment. The genomic DNA from the extinct species was extracted using DNeasy Tissue & Blood Kit (Qiagen, Valencia, CA) and the Cetrimonium bromide (CTAB) manual. To generate Illumina NGS data, we constructed PE and SE libraries using the KAPA Hyper Library Preparation Kit (Kapa Biosystems, Woburn, MA, USA) and the Accel NGS 1S plus DNA kit (Swift BioSciences, Washtenaw County, Michigan, USA), respectively, according to manufacturer’s instructions. The Illumina-based NGS sequencing was performed with Illumina NovaSeq 6000 (Illumina, CA, USA) and NextSeq 500 (Illumina, CA, USA). For MGI-Seq, we constructed paired-end libraries with MGIEasy DNA Library Prep Kit (MGI, Shenzhen, China) and sequenced them on the DNBSEQ-T7 sequencing platform. Dokdo sea lion mitochondrial genome assembly Upon assessing the read quality with Trimmomatic (ver. 0.39) [ 29 ], we extracted mtDNA reads by mapping all the short DNA reads to the mito-genome of Z. californianus (Acc. NC_006416). We assembled the mitochondrial genome (mito-genome) of the Z. japonicus (Z6) sample using NOVOPlasty (ver. 4.2) [ 30 ]. Minor gaps of the mito-genome assembly were filled in by conducting Sanger sequencing (Additional file 1: Table S5, Additional file 1: Table S6) followed by assembly using Cap3 program (updated on December 21, 2007) [ 31 ]. We predicted and annotated Z. japonicus’ mito-genome using the MITOS program ( http://mitos.bioinf.uni-leipzig.de/index.py ) (Additional file 1: Fig S5, Additional file 2: Data S3) [ 32 ]. Complete mtDNA genomes were aligned with Mummer (ver. 4.0.0rc1) (Additional file 1: Fig. S5) [ 33 ] and Dendroscope (ver. 3.5.10) [ 34 ]. We additionally obtained ten mtDNA genome consensus sequences by aligning our samples’s NGS reads to Z6 deep-sequenced mtDNA genome (Additional file 2: Data S4). The consensus sequences were obtained from high quality SNV data (mapping quality > 30, genotype quality > 20, and coverage > 10) by implementing samtools consensus utility (ver. 1.9) [ 35 ]. Five samples (Z1, Z5, Z7, Z8, and Z12) were excluded in the process due to significantly lower (insufficient) amount of NGS reads (Additional file 2: Data S4). We then constructed the phylogenetic tree of the ten Z. japonicus mtDNA genomes along with other closely related species (Fig. 3 a, Additional file 1: Fig. S6 and Additional file 2: Data S6). We aligned CDS sequences using muscle program (ver. 3.8.31) [ 36 ] and constructed the phylogenetic tree using phyML (ver. 3.1) with default parameters (ver. 3.1) [ 37 ]. Phylogeny, admixture and genomic composition analyses of Dokdo sea lion and other pinnipeds To construct phylogenetic tree and analyze ancestry, we aligned reads to an outgroup species that is the most distantly related to Z. japonicus , namely, the walrus, Odobenus rosmarus (acc. ANOP00000000) [ 38 ]. Specifically for Z. japonicus DNA samples, we applied PALEOMIX pipeline [ 15 ] by mapping all Z. japonicus reads to the O. rosmarus genome [ 38 ]. We estimated deamination patterns using mapDamage (ver. 2.0) [ 39 ]. For the modern mammal genomes, their NGS reads were aligned to the same reference genome using bwa mem (ver. 0.7.17) [ 40 ] after filtering out low quality reads using Trimmomatic with Quality < 30 and read length < 70 (ver. 0.39) [ 29 ]. We then utilized Picard [ 41 ] (ver. 2.27.5) to eliminate PCR duplicates and employed the GATK (ver. 4.1.3.0) for variant calling [ 42 ]. We constructed consensus sequence using the vcf2phylip [ 43 ] utilizing only genomic loci in the Z. japonicus bam files with read depth larger than five. We then constructed phylogenetic tree using the PhyML (ver. 3.1) [ 37 ] as in mitochondrial genome. The f3- and f4- statistics (table S2 -4) were conducted using a ADMIXTOOLS (ver. 2.0) algorithm [ 44 ]. An admixture graph was constructed with qpGraph model [ 44 ] with admix = 2. The qpGraph [ 44 ] was automatically optimized for genetic admixture of our admixture model. The maps used for the Otariinae species’ distribution were obtained from https://mapstyle.withgoogle.com . The graphic representations of each species in Fig. 1 and Fig. 2 as well as the those of Ulleungdo and Dokdo islands were created based on royalty-free images under the Creative Commons (CC) license. Estimation of the effective population size of Dokdo sea lion We used PSMC algorithm [ 22 ] to estimate the effective population size of Dokdo sea lion in the last 20,000 years. For higher mapping rates, for this analysis, we aligned reads from all ancient and extant Zalophus and E. jubatus species to the E. jubatus reference genome (acc. GCA_004028035.1, ver. ASM402803v1), which shares more recent common ancestry with these species compared to the previously used walrus. Using the PALEOMIX pipeline [ 15 ] we aligned all Z. japonicus reads to the E. jubatus reference and selected only high confidence SNVs, with more than 10x coverage for the PSMC analysis. The stringent data pre-filtering aimed to reduce biases stemming from possible over-representation of heterozygous sites in the ancient DNA. This was conducted as a necessary step, because PSMC [ 22 ] infers the Ne changes over time using the density of heterozygous sites throughout the diploid genome of a single individual. For the PSMC analysis of modern genomes, we aligned reads with bwa mem (ver. 0.7.17) [ 40 ] after trimming low quality reads using Trimmomatic (ver. 0.39) [ 29 ]. We proceeded by utilized Picard (ver. 2.27.5) to eliminate PCR duplicates ( https://broadinstitute.github.io/picard/ ) and employed the GATK (ver. 4.1.3.0) for variant calling [ 42 ]. We applied a generation time of 10 [ 45 ] and a mutation rate of 0.27 * 10 − 8 [ 46 ]. Calculation of the heterozygosity of Dokdo sea lion genomes To accurately identify the heterozygous regions in the Z. japonicus genome, we calculated the distribution of heterozygous positions across the genomic loci from a bam file, wherein Z. japonicus reads were mapped to the most closely related reference genome, Z. californianus (Acc. GCF_00976235.2) using the PALEOMIX pipeline [ 15 ]. We called SNVs utilizing samtools mpileup [ 35 ] with a minimum base quality of 20 (-Q 20) and mapping quality 20 (-q 20). We included genomic loci with a DP of 10 or greater. We cleaned all reads from the modern mammal genomes using the Trimmomatic (ver. 0.39) [ 29 ] and mapped them using the bwa mem (ver. 0.7.17) [ 40 ] to the most closely related reference genomes (Additional file 2: Data S7). After removing the PCR duplicates using Picard (ver. 2.27.5), we applied the same mpileup criteria with Z. japonicus . This way, we implemented mlRho (ver. 2.9) [ 47 ] to estimate the heterozygosity only on the high-quality loci with high quality variants. f -statistics analyses for Dokdo sea lion and other Otariidae species For ancestry analysis, we used f4 -statistics and admixture f3 -statistics. The admixture f3 - and f4 -statistics were conducted using a ADMIXTOOLS (ver. 2.0) algorithm [ 44 ]. All formulars employed are detailed in Additional file 2: Data S5-S7. Our criterion for significance was set at an absolute Z-score less than three. Abbreviations IUCN: International Union for Conservation of Nature mtDNA: mitochondrial DNA NGS: next-generation sequencing SE: single-end PE: paired-end Ne : effective population size Declarations Ethics approval and consent to participate The collection of Dokdo sea lion bones was conducted under the permission granted by the Gyeoungbuk province local government for the collection of protected marine organisms (Permit No. 2019-2). We also collected a Z. californianus muscle sample from the Seoul Grand Park, Republic of Korea, obtained during the necropsy process (Permit No. Seoul Grand Park Scientific Research 2020-009). As our study involved extinct animals and cadavers, ethical approval was not required. Consent for publication Not applicable Availability of data and materials All data are publicly available for scientific research. Sequencing data have been deposited in the NCBI SRA with accession number PRJNA982545. All the other data are available either in the main text or in the supplementary materials. Competing interests C.K. and S.J. are employees and J.B. is the CEO of Clinomics Inc. Other authors declare that they have no competing interests. Funding National Institute of Fisheries Science, Ministry of Ocean and Fisheries, Korea (R2020024, R2021030, R2022033, R2024004). Promotion of Innovative Business for Regulation-Free Special Zones funded by the Ministry of SMEs and Startups (MSS, Korea) (grant number [P0016195, P0016193] (1425156792, 1425157301) (2.220035.01, 2.220036.01)). Ulsan City Research Fund (1.200047.01). Fedor Sharko and Artem Nedoluzhko were supported by ARCTIC SIRENIA RESEARCH FOUNDATION. Author’s contributions Conceptualization: J. K., J. C., A. B, S. J and F. S., A. N.; Methodology: C. K., J. K, F. S; Investigation: E. K, H.-W. K., M. Y., J.-H. L, K. L., and H. S.; Visualization: A. B, J. C., J. K.; Supervision: J. K, A. B; Writing—original draft: J. K., J. C., A. B; Writing—review & editing: A. N, J. B Acknowledgements Not applicable References Rice DW: Marine mammals of the world: Systematics and distribution (Special publication / the Society for Marine Mammalogy) vol. 1: Society for Marine Mammalogy; 1998. Valenzuela-Toro A, Pyenson ND: What do we know about the fossil record of pinnipeds? A historiographical investigation . 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Other authors declare that they have no competing interests. Supplementary Files Additionalfile1BMCBiology.docx Additional file 1: Fig. S1. Excavation of Dokdo sea lion bones Fig. S2. Dokdo sea lion bones used in this study Fig. S3. Read length distribution mapped to California sea lion ( Z. californianus ) genome Fig. S4. Postmortem DNA damage pattern in the DNA-libraries of Dokdo sea lion generated by PALEOMIX pipeline Fig S5. Assembly and annotation of the complete mitochondrial genome of Dokdo sea lion using the Z6 individual specimen Fig. S6. Phylogenetic tree of the mitogenomes of Dokdo sea lion and related species Fig. S7. Comparison of mitochondrial genomes between Z. japonicus and Z. californianus using mummer (ver. 4.0.0rc1) Table S1. Statistics of the PALEOMIX pipeline mapping to California sea lion ( Z. californianus ) reference genome Table S2. Gene flow between Steller sea lion ( E. jubatus ) and Zalophus species with a form of f4 (A,B;C,D). Table S3. f4 -statistics of Otariidae species with a form of f4(A,B;C,D). Table S4. Genetic admixture of Zalophus species compared to their related species. Table S5. Primers to filling the mito-genome gap of Dokdo sea lion (Z6 individual) and melting temperature (Tm) used Table S6. Sanger sequencing reads to fill mito-genome gap of Z6 individual Additionalfile2.xlsx Additional file 2: Data S1. Statistics of DNA sequencing and mapping statistics to California sea lion reference genome. Data S2. Number (No) of mapped read to Steller sea lion reference genome and coverage statistics. Data S3. Annotation of the mitochondrial genome of Dokdo sea lion. Data S4.Coverage of the mitochondrial genomes based on reads with mapping quality > 30, genotype quality > 20, and coverage >10. Data S5. Mammal genome datasets used for the ancestry analysis. Data S6. Genetic distance matrix for mtDNA (A) and nuclear genome (B). Data S7. Average genome-wide autosomal heterozygosity values. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviews received at journal 03 Sep, 2024 Reviewers agreed at journal 25 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers invited by journal 19 Jul, 2024 Editor invited by journal 18 Jul, 2024 Editor assigned by journal 11 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 10 Jul, 2024 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-4721400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335346454,"identity":"101ee559-1f44-4b5e-b2d0-4d139f5c9bee","order_by":0,"name":"Jungeun Kim","email":"","orcid":"","institution":"Genome Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Jungeun","middleName":"","lastName":"Kim","suffix":""},{"id":335346455,"identity":"94362d49-5cc4-4691-9c64-d7b04c6ee06d","order_by":1,"name":"Asta Blazyte","email":"","orcid":"","institution":"Korean Genomics Center (KOGIC), Ulsan National Institute of Science 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03:10:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4721400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4721400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61770301,"identity":"87e1e595-63af-44d7-8339-cf74ce4edf87","added_by":"auto","created_at":"2024-08-05 11:17:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":384661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDokdo sea lion habitat with sampling locations and sequencing (mapping) overview. \u003c/strong\u003e(a) The map shows the Dokdo sea lion distribution range (striped area), Ulleungdo and Dokdo islands (red dots), and sample sites in Ulleungdo (latitude 37.51° and longitude 130.79°) and Dokdo (latitude 37.24° and longitude 131.86°) islands (red markers). Blue markers mark the two locations where Dokdo sea lions were last seen before being declared extinct and denote the year of last sighting in the location. (b) The species ranges and geographic distribution of \u003cem\u003eZalophus\u003c/em\u003e and other Otariidae species relevant in this study. Dark red denotes the species range of the Dokdo sea lion (\u003cem\u003eZ. japonicus\u003c/em\u003e), pink - species range of California sea lion (\u003cem\u003eZ. californianus\u003c/em\u003e), and yellow – Galapagos sea lion (\u003cem\u003eZ. wollebaeki\u003c/em\u003e). Purple semi-transparent area denotes species range of the Steller sea lion (\u003cem\u003eE. jubatus\u003c/em\u003e), which partially overlaps ranges of Dokdo sea lion and California sea lion. The species distribution range of Northern fur seal (\u003cem\u003eC. ursinus\u003c/em\u003e) is not shown because it overlaps the range of Steller sea lion almost entirely. (c) Dokdo sea lion (\u003cem\u003eZ. japonicus\u003c/em\u003e) genome sequencing coverage (breadth of coverage; of protein coding regions – CDS, green, and whole genome – WGS, blue, respectively) comparison with California sea lion (\u003cem\u003eZ. californianus\u003c/em\u003e) and Northern fur seal (\u003cem\u003eC. ursinus\u003c/em\u003e), utilizing Steller sea lion (\u003cem\u003eE. jubatus\u003c/em\u003e) genome as a reference.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/df7301c52573275852e29272.png"},{"id":61770822,"identity":"535ea86a-b64f-4b41-b526-550c62e125da","added_by":"auto","created_at":"2024-08-05 11:25:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":366635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic ancestry of Dokdo sea lion\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e(a) Genetic ancestry of Otariidae species based on \u003cem\u003ef4\u003c/em\u003e(A,B;C,D) statistics. The B genome has a higher genetic affinity with the D genome when the Z-score is \u0026gt; 3. On the other hand, the B genome has higher genetic affinity with the C genome when Z-score is \u0026lt; -3. The red text in the table indicates statistically identical genetic ancestry. The raw data for \u003cem\u003ef4\u003c/em\u003e-statistics is presented in Additional file 1: Table S3. (b) Admixture \u003cem\u003ef3\u003c/em\u003e statistics using notation \u003cem\u003ef3\u003c/em\u003e(T;A,B). In this statistic, negative Z-scores \u0026lt; -3 indicate A and B genomes were admixed in the target genome (T). Instances with negative \u003cem\u003ef3\u003c/em\u003e-score are presented in red color. The raw data for \u003cem\u003ef3\u003c/em\u003e-statistics is presented in Table S4. (c) Genetic ancestry of \u003cem\u003eZalophus\u003c/em\u003e species based on a qpGraph algorithm [48].\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/169618215cfd3d6fa98a110a.png"},{"id":61770823,"identity":"8e2e1123-eeec-419e-90ea-787d7c9552a1","added_by":"auto","created_at":"2024-08-05 11:25:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183825,"visible":true,"origin":"","legend":"\u003cp\u003eDokdo sea lion\u003cem\u003e \u003c/em\u003ephylogeny. (a) mtDNA-based phylogenetic tree of Otariidae in the context of sea lions and other pinnipeds. The subfamily Otariinae, that consists of \u003cem\u003eZ. japonicus, Z. wollebaeki\u003c/em\u003e, \u003cem\u003eZ. californianus\u003c/em\u003e, and\u003cem\u003e E. jubatus\u003c/em\u003e, is denoted by blue colored branches in the phylogenetic tree. (b) Autosomal SNV-based (WGS) phylogenetic tree of Otariidae in the context of sea lions and other pinnipeds (Additional file 2: Data S5). The subfamily Otariinae, is denoted by blue colored branches in the phylogenetic tree. (c) Phylogenetic distance comparison between mtDNA and autosomal SNV-based (WGS) phylogeny (Additional file 2: Data S6). Colors mark cases of the genetic distances differing more than 40% between mtDNA (red) and autosomal SNV-based (green) distance matrices.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/efdb2b493246165683637bfb.png"},{"id":61770302,"identity":"267b7b57-01b5-4636-8a3f-6877928f86ba","added_by":"auto","created_at":"2024-08-05 11:17:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterozygosity of Dokdo sea lion in the context of other marine mammals. \u003c/strong\u003eThe average heterozygosity values in log scale (from the left to right) for: Steller’s sea cow – \u003cem\u003eH. gigas\u003c/em\u003e, Dokdo sea lion – \u003cem\u003eZ. japonicus\u003c/em\u003e, Galapagos sea lion – \u003cem\u003eZ. wollebaeki\u003c/em\u003e, Northern fur seal – \u003cem\u003eC. ursinus\u003c/em\u003e, dugong – \u003cem\u003eD. dugon\u003c/em\u003e, West Indian manatee – \u003cem\u003eT. manatus\u003c/em\u003e, walrus – \u003cem\u003eO. rosmarus\u003c/em\u003e, Steller sea lion – \u003cem\u003eE. jubatus\u003c/em\u003e, beluga whale – \u003cem\u003eD. leucas\u003c/em\u003e, grey seal – \u003cem\u003eH. grypus\u003c/em\u003e, narwhal – \u003cem\u003eM. monoceros\u003c/em\u003e, California sea lion – \u003cem\u003eZ. californianus\u003c/em\u003e, and California sea lion – \u003cem\u003eZ. californianus\u003c/em\u003e (from zoo). Samples are colored according to IUCN conservation status (2024).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/2c115725516a2f898fc87985.png"},{"id":61770306,"identity":"57460b2b-9f49-48f5-8e1b-cd09d1ef6ddf","added_by":"auto","created_at":"2024-08-05 11:17:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":331348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDemographic history of Dokdo sea lion and extant Otariidae species. \u003c/strong\u003eThe extant Otariidae species analyzed: California sea lion – \u003cem\u003eZ. californianus\u003c/em\u003e, Galapagos sea lion – \u003cem\u003eZ. wollebaeki\u003c/em\u003e,Northern fur seal – \u003cem\u003eC. ursinus\u003c/em\u003e, Steller sea lion – \u003cem\u003eE. jubatus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/b3301d90b0093cbf7d90f991.png"},{"id":61771359,"identity":"397c9a29-7f16-45ed-89d2-e2f5000af234","added_by":"auto","created_at":"2024-08-05 11:33:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3265812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/fcc6c822-45b4-45e1-9142-42ef2b3516bc.pdf"},{"id":61770307,"identity":"8f524ff0-7eda-4fa5-9305-a9ae7d83ece7","added_by":"auto","created_at":"2024-08-05 11:17:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2504414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1. \u003c/strong\u003eExcavation of Dokdo sea lion bones\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2. \u003c/strong\u003eDokdo sea lion\u003cem\u003e \u003c/em\u003ebones used in this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3. \u003c/strong\u003eRead length distribution mapped to California sea lion (\u003cem\u003eZ. californianus\u003c/em\u003e) genome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S4. \u003c/strong\u003ePostmortem DNA damage pattern in the DNA-libraries of Dokdo sea lion generated by PALEOMIX pipeline\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig S5. \u003c/strong\u003eAssembly and annotation of the complete mitochondrial genome of Dokdo sea lion using the Z6 individual specimen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S6. \u003c/strong\u003ePhylogenetic tree of the mitogenomes of Dokdo sea lion\u003cem\u003e \u003c/em\u003eand related species\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S7. \u003c/strong\u003eComparison of mitochondrial genomes between \u003cem\u003eZ. japonicus\u003c/em\u003e and \u003cem\u003eZ. californianus\u003c/em\u003e using mummer (ver. 4.0.0rc1)\u003c/p\u003e\n\u003cp\u003eTable S1. Statistics of the PALEOMIX pipeline mapping to California sea lion (\u003cem\u003eZ. californianus\u003c/em\u003e) reference genome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e Gene flow between Steller sea lion (\u003cem\u003eE. jubatus\u003c/em\u003e) and \u003cem\u003eZalophus\u003c/em\u003especies with a form of \u003cem\u003ef4\u003c/em\u003e(A,B;C,D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003e \u003cem\u003ef4\u003c/em\u003e-statistics of Otariidae species with a form of f4(A,B;C,D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003e Genetic admixture of \u003cem\u003eZalophus\u003c/em\u003especies compared to their related species.\u003c/p\u003e\n\u003cp\u003eTable S5. Primers to filling the mito-genome gap of Dokdo sea lion (Z6 individual) and melting temperature (Tm) used\u003c/p\u003e\n\u003cp\u003eTable S6. Sanger sequencing reads to fill mito-genome gap of Z6 individual\u003c/p\u003e","description":"","filename":"Additionalfile1BMCBiology.docx","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/c8514ebfe7abf7f2fdcb56b6.docx"},{"id":61770305,"identity":"1b737186-a1b5-43d4-9e67-5b23cd3a17b5","added_by":"auto","created_at":"2024-08-05 11:17:24","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":76791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S1\u003c/strong\u003e. Statistics of DNA sequencing and mapping statistics to California sea lion reference genome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S2\u003c/strong\u003e. Number (No) of mapped read to Steller sea lion reference genome and coverage statistics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S3. \u003c/strong\u003eAnnotation of the mitochondrial genome of Dokdo sea lion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S4.\u003c/strong\u003eCoverage of the mitochondrial genomes based on reads with mapping quality \u0026gt; 30, genotype quality \u0026gt; 20, and coverage \u0026gt;10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S5.\u003c/strong\u003e Mammal genome datasets used for the ancestry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S6.\u003c/strong\u003e Genetic distance matrix for mtDNA (A) and nuclear genome (B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData S7.\u003c/strong\u003e Average genome-wide autosomal heterozygosity values.\u003c/p\u003e","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4721400/v1/63ea08d54698d5ac0c000c8a.xlsx"}],"financialInterests":"Competing interest reported. C.K. and S.J. are employees and J.B. is the CEO of Clinomics Inc. Other authors declare that they have no competing interests.","formattedTitle":"Dokdo sea lion Zalophus japonicus genome reveals its evolutionary trajectory before extinction","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Dokdo sea lion \u0026ndash; \u003cem\u003eZalophus japonicus\u003c/em\u003e (Otariidae: Carnivora), also known as the Japanese sea lion, was a significant species native to East Asia, inhabiting the Dokdo and Ulleungdo islands within Korean territorial waters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Known locally as Gangchi, this species, along with other Otariidae such as the Northern fur seal (\u003cem\u003eCallorhinus ursinus\u003c/em\u003e) and Steller sea lion (\u003cem\u003eEumetopias jubatus\u003c/em\u003e), thrived in the coastal habitats of the northwest Pacific Ocean, spanning from Russia to the coastal waters of Korea and Japan [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite the absence of dated fossil records for \u003cem\u003eZ. japonicus\u003c/em\u003e, the discovery of other extant pinniped fossils, such as \u003cem\u003eE. jubatus\u003c/em\u003e, suggests a long-standing habitation in these regions, potentially since the Pliocene [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Historical sources indicate that \u003cem\u003eZ. japonicus\u003c/em\u003e was hunted for human consumption in Hokkaido since the Jomon period, but it's widely believed that this subsistence hunting didn't significantly impact their population dynamics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Over the past two centuries, the \u003cem\u003eZ. japonicus\u003c/em\u003e population experienced a drastic decline. In the mid-19th century, estimates suggested a healthy population of 30,000 to 50,000 animals, comparable to later counts of Galapagos and California sea lions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, by the 1950s, their numbers plummeted to just 50\u0026ndash;60, leading to their classification as extinct by the International Union for Conservation of Nature (IUCN) in 1990 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This sharp decrease was largely due to extensive hunting for meat, skin, and oil between 1904 and 1925, particularly targeting females and pups [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eZ. japonicus\u003c/em\u003e is recognized as a member of the genus \u003cem\u003eZalophus\u003c/em\u003e. This genus comprises three distinct species: the western Dokdo sea lion (\u003cem\u003eZ. japonicus\u003c/em\u003e), the eastern California sea lion (\u003cem\u003eZ. californianus\u003c/em\u003e), and the Galapagos sea lion (\u003cem\u003eZ. wollebaeki\u003c/em\u003e) found in the North Pacific Galapagos archipelago (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eZ. japonicus\u003c/em\u003e was initially classified as a subspecies of the California sea lion, however, it was reclassified based on unique cranial features [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and mitochondrial DNA (mtDNA) differences [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Evolutionary studies suggest a divergence of merely 2.2\u0026nbsp;million years between \u003cem\u003eZ. japonicus\u003c/em\u003e and \u003cem\u003eZ. californianus\u003c/em\u003e, indicating a close genetic relationship [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The specific evolutionary pathways leading to the isolation of these sea lion species remain unclear. Complicating their classification further, pinnipeds exhibit a wide range of individual cranial variations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and documented interspecific sexual behaviors [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], potentially leading to introgression and morphological diversity. While such behaviors haven't been directly observed in \u003cem\u003eZ. japonicus\u003c/em\u003e, the possibility is supported by observed introgression events in other pinniped species [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To date, genomic research on \u003cem\u003eZ. japonicus\u003c/em\u003e has predominantly relied on mtDNA, which captures only a limited scope of the species' genomic diversity. Consequently, comprehensive whole-genome sequencing has become essential to fully understand the Dokdo sea lion's genetic makeup and its phylogenetic relationships within the Otariidae [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDokdo island, a vital habitat for \u003cem\u003eZ. japonicus\u003c/em\u003e in the 1900s and one of their last sighting locations, preserved biological materials that may offer novel insights. In this study, 16 bone fragments of Dokdo sea lions were unearthed from Dokdo and the nearby Ulleungdo islands, situated 87.4 km apart in the East Sea of Korea. We compared the genomes of extinct \u003cem\u003eZ. japonicus\u003c/em\u003e with those of other sequenced Otariidae species. Our analyses not only reveal the genetic diversity and detailed phylogeny of \u003cem\u003eZ. japonicus\u003c/em\u003e but also for the first-time shed light on the evolutionary processes of speciation and introgression in this species and its genus.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDokdo sea lion\u003cem\u003e\u0026nbsp;\u003c/em\u003esamples and genomic data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study focused on the genomic analysis of Dokdo sea lion using 16 bone samples (Z1, Z3-Z9, Z11-Z18) excavated from Dokdo and Ulleungdo islands (Figure 1a, Additional file 1: Fig. S1, Additional file 1: Fig. S2). These skeletal remains, mainly limb and rib bones, underwent DNA extraction and deep DNA sequencing using next-generation sequencing (NGS) methods, despite challenges posed by the small size of the fragments (Additional file 2: Data S1). Detailed information about laboratory techniques used is presented in Supplementary Material. Initial tests on sample Z3 using single-end (SE) and paired-end (PE) NGS revealed higher mapping rates with PE against a \u003cem\u003eZ. californianus\u003c/em\u003e reference genome, leading us to construct PE NGS libraries for all samples. We generated 8.4 Tb of data with individual mapping rates ranging from 0.1% to 1.3%. Mapping \u003cem\u003eZ. japonicus\u003c/em\u003e reads to \u003cem\u003eE. jubatus\u0026nbsp;\u003c/em\u003egenome covered 83.35% of its genome and 90.83% of protein-coding genes (Figure 1c, Additional file 2: Data S2). This coverage was compared to other Otariidae species, including \u003cem\u003eZ. californianus, Z. wollebaeki, E. jubatus\u003c/em\u003e, and \u003cem\u003eC. ursinus\u003c/em\u003e. For the bioinformatics analysis, we utilized the PALEOMIX pipeline [15], aligning 43G reads to the \u003cem\u003eZ. californianus\u003c/em\u003e genome (Additional file 1: Table S1), with an average read length of 140 bp (Additional file 1: Fig. S3). DNA misincorporation levels were low (Additional file 1: Fig. S4), consistent with the species\u0026apos; fairly recent extinction. Additionally, we sequenced modern \u003cem\u003eZ. californianus\u003c/em\u003e and obtained other pinniped species\u0026rsquo; DNA for comprehensive comparative analysis (Additional file 2: Data S2) of \u003cem\u003eZ. japonicus\u003c/em\u003e and broader knowledge on Otariidae phylogenetics and evolutionary history.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCongeneric \u003cem\u003eZalophus\u003c/em\u003e speciation involved introgression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDokdo sea lion genetic ancestry and relationship with other Otariidae species, is not yet well understood and has not yet been studied outside the morphological classification and mtDNA analysis [7, 16, 17]. To elucidate the genomic composition of \u003cem\u003eZalophus\u003c/em\u003e sea lions beyond mtDNA [7, 17], we estimated the gene flow and possible admixture scenarios among \u003cem\u003eZalophus\u003c/em\u003e and with other pinnipeds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirstly, the \u003cem\u003ef4\u003c/em\u003e-statistics unsurprisingly show that congeneric \u003cem\u003eZalophus\u003c/em\u003e species share the highest genetic affinities among each other compared to the \u003cem\u003eE. jubatus\u003c/em\u003e (Additional file 1: Table S2). Secondly, \u003cem\u003eZ. japonicus\u003c/em\u003e exhibits a relatively more distinct genetic makeup compared to its closest species, \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u0026nbsp;\u003c/em\u003e(Fig. 2a-c). In \u003cem\u003eZ. japonicus\u003c/em\u003e, we could not identify gene flow or introgression from \u003cem\u003eE. jubatus\u003c/em\u003e, which was observed in both \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e (Fig. 2a, b, Additional file 1: Table S3, and Additional file 1: Table S4). Consistently, genetic modeling using qpGraph suggests that \u003cem\u003eZ. japonicus\u003c/em\u003e diverged early in the evolution of the \u003cem\u003eZalophus\u003c/em\u003e genus, with 98% of its genetic components derived from the common ancestor of \u003cem\u003eZalophus\u003c/em\u003e and 2% derived from the lineage of \u003cem\u003eE. jubatus\u003c/em\u003e and \u003cem\u003eC. ursinus\u003c/em\u003e (Fig. 2c). After the divergence, we observed no additional genetic admixture between \u003cem\u003eZ. japonicus\u003c/em\u003e and other \u003cem\u003eZalophus\u003c/em\u003e species. Third, we suggest that gene flow came from \u003cem\u003eE. jubatus\u003c/em\u003e and the common ancestor of \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e as well as after \u003cem\u003eZ. japonicus\u003c/em\u003e divergence (Figure 2a, b). \u003cem\u003eE. jubatus\u003c/em\u003e appeared to share significantly less genetic affinity and gene flow with \u003cem\u003eZ. japonicus\u003c/em\u003e compared to \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e (Figure 2a, b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, the latter two species not only received more gene flow and derived alleles from \u003cem\u003eE. jubatus\u003c/em\u003e, but also their levels of genetic affinity to \u003cem\u003eE. jubatus\u003c/em\u003e were nearly identical (Figure 2a). This provides further support to a scenario wherein \u003cem\u003eE. jubatus\u003c/em\u003e introgressed into the common ancestor of \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e prior to the divergence and isolation of \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e as it is quite unlikely that \u003cem\u003eE. jubatus\u003c/em\u003e introgressed the two modern geographically isolated \u003cem\u003eZalophus\u003c/em\u003e species at the same rate (while not sharing any known habitats with \u003cem\u003eZ. wollebaeki\u003c/em\u003e). Furthermore, following the divergence of \u003cem\u003eZ. japonicus\u003c/em\u003e, we suggest that more than one major introgression event had occurred between \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e (Fig. 2a, b). Lastly, we observed an inconsistent and complex genetic relationship between \u003cem\u003eZ. japonicus\u003c/em\u003e and other \u003cem\u003eZalophus\u003c/em\u003e species, likely due to repeated interspecific and intergeneric introgression events. \u003cem\u003ef4\u003c/em\u003e-statistics revealed a higher genetic affinity between \u003cem\u003eZ. japonicus\u003c/em\u003e with \u003cem\u003eZ. californianus\u003c/em\u003e compared to \u003cem\u003eZ. wollebaeki\u003c/em\u003e (Fig. 2a). However, admixture \u003cem\u003ef3\u003c/em\u003e-statistics showed no genetic admixture between \u003cem\u003eZ. japonicus\u003c/em\u003e and either \u003cem\u003eC. ursinus\u003c/em\u003e or \u003cem\u003eE. jubatus\u003c/em\u003e in the \u003cem\u003eZ. californianus\u003c/em\u003e genome (Fig. 2b). These contradictory finding suggest \u003cem\u003eZ. californianus\u003c/em\u003e played a significant role in the evolution of the \u003cem\u003eZalophus\u003c/em\u003e genus, potentially neutralizing previous admixture effects with \u003cem\u003eZ. japonicus\u003c/em\u003e through recent genetic interactions with \u003cem\u003eZ. wollebaeki\u0026nbsp;\u003c/em\u003e(Fig. 2c). In contrast, \u003cem\u003eZ. wollebaeki\u003c/em\u003e still retains genetic traces of ancestral genetic admixture with \u003cem\u003eZ. japonicus\u003c/em\u003e. It includes a 17% genetic component shared with all \u003cem\u003eZalophus\u003c/em\u003e species (Fig. 2c), indicating a lesser divergence. Additionally, \u003cem\u003eZ. wollebaeki\u003c/em\u003e did not experience significant genetic exchange with other species (which include introgression from either \u003cem\u003eC. ursinus\u003c/em\u003e or \u003cem\u003eE. jubatus\u003c/em\u003e) until its admixture with the common ancestor of \u003cem\u003eZ. californianus\u003c/em\u003e. These lines of evidence suggest that \u003cem\u003eZ. japonicus\u003c/em\u003e not only is a unique species with a distinct genetic makeup, that for major part evolved directly from the common ancestor of the \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e, but also that \u003cem\u003eZ. japonicus\u003c/em\u003e may be the earliest diverged species in this genus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplex introgressive speciation of \u003cem\u003eZalophus\u003c/em\u003e species explains their phylogenetic ambiguities\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study for the first time validated \u003cem\u003eZ. japonicus\u003c/em\u003e phylogeny using 1,581,963 autosomal SNVs (Additional file 2: Data S5) and compared it with whole-mtDNA-based classification (Fig. 3). The mtDNA phylogeny of \u003cem\u003eZ. japonicus\u003c/em\u003e specimens revealed two genetically uniform and nearly indistinguishable mtDNA haplotypes (Additional file 1: Fig. S6). The genetic distances between them were sufficient to identify different mtDNA haplotypes but subtle enough to be possibly derived from the same maternal \u003cem\u003eZ. japonicus\u003c/em\u003e ancestor (0.0007 vs 0.0005). Both autosomal SNV and mtDNA-based phylogenetic trees presented the same topology with two distinct Otariidae clades and Northern fur seal (\u003cem\u003eC. ursinus\u003c/em\u003e) as an outgroup: one of Northern pinnipeds composed of \u003cem\u003eZalophus\u003c/em\u003e and \u003cem\u003eEumetopias\u003c/em\u003e sea lions, and one of Southern pinnipeds composed of \u003cem\u003ePhocarctos\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNeophoca\u003c/em\u003e sea lions with \u003cem\u003eArctocephalus\u003c/em\u003e fur seals (Fig. 3a, b). In this context, \u003cem\u003eZ. japonicus\u003c/em\u003e showed almost equal phylogenetic distance to \u003cem\u003eZ. wollebaeki\u003c/em\u003e and \u003cem\u003eZ. californianus\u003c/em\u003e (mtDNA: 0.014 and 0.014; WGS: 0.015 and 0.015, respectively) (Additional file 2: Data S6). Even though \u003cem\u003eZ. japonicus\u003c/em\u003e was similarly related to its congenerics, the genetic distance between the \u003cem\u003eZ. wollebaeki\u003c/em\u003e and \u003cem\u003eZ. japonicus\u003c/em\u003e was about 40% greater compared to the distance between the \u003cem\u003eZ. wollebaeki\u003c/em\u003e and \u003cem\u003eZ. californianus\u003c/em\u003e. This observation held true regardless of whether the genetic distances were estimated using autosomal SNVs or mtDNA (Additional file 2: Data S6). Our and previously reported [18] intrageneric \u003cem\u003eZalophus\u003c/em\u003e phylogeny aligns with \u003cem\u003ef4\u003c/em\u003e-statistic that showed higher genetic affinity between \u003cem\u003eZ. wollebaeki\u003c/em\u003e and \u003cem\u003eZ. californianus\u003c/em\u003e compared to the affinity each of them had with \u003cem\u003eZ. japonicus\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe genetic distances between \u003cem\u003eZ. japonicus\u003c/em\u003e and its extant genetic donors, \u003cem\u003eC. ursinus\u003c/em\u003e and \u003cem\u003eE. jubatus\u003c/em\u003e, showed significant disparities between estimates based on mtDNA and autosomal SNVs (Fig. 3C). The genetic distances based on mtDNA were approximately two times greater than those based on the autosomal SNVs, and reflected in the phylogenetic tree branching (Fig. 3a-c, Additional file 2: Data S5). However, this disparity in genetic distance from \u003cem\u003eZ. japonicus\u003c/em\u003e did not apply to the already mentioned \u003cem\u003eZ. japonicus\u0026rsquo;\u003c/em\u003e congenerics and other phylogenetically distant species, such as those under the genera \u003cem\u003ePhoca\u003c/em\u003e and \u003cem\u003ePusa\u003c/em\u003e (Fig. 3c, Additional file 2: Data S6). These findings imply either a significant divergence of maternal lineages between \u003cem\u003eZalophus\u003c/em\u003e and the pair of \u003cem\u003eC. ursinus\u003c/em\u003e and \u003cem\u003eE. jubatus\u0026nbsp;\u003c/em\u003eor a genetic legacy of \u003cem\u003eZ. japonicus\u003c/em\u003e introgression from \u003cem\u003eC. ursinus\u003c/em\u003e and \u003cem\u003eE. jubatus\u003c/em\u003e. The introgression scenario obscures phylogenetic relationships, as the relative reduction in autosome-based genetic distance may falsely suggest a more recent common ancestry than it actually is. Nevertheless, both scenarios may be true, and it underscores the multifaceted nature of evolutionary dynamics within the Otariidae, which is also largely consistent with our previously shown genetic ancestry modeling (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHeterozygosity of extinct Dokdo sea lion\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur subsequent objective was to elucidate the genetic diversity of the \u003cem\u003eZ. japonicus\u003c/em\u003e samples within the context of population analyses. To accurately estimate heterozygosity (theta) from low depth \u003cem\u003eZ. japonicus\u003c/em\u003e data, we pooled reads from multiple individuals, enabling genome-wide calculation of heterozygosity across more than 200M loci with a depth of coverage exceeding ten (Additional file 2: Data S6). We obtained moderate heterozygosity value of \u003cem\u003eZ. japonicus\u003c/em\u003e (0.00101) which was greater than any other \u003cem\u003eZalophus\u003c/em\u003e species and several other marine mammal species that are recognized as \u0026ldquo;Least concern\u0026rdquo; regarding their vulnerability to extinction according to the International Union for Conservation of Nature and Natural Resources (IUCN) (Fig. 4). Interestingly, the heterozygosity levels of \u003cem\u003eC. ursinus\u003c/em\u003e and \u003cem\u003eE. jubatus\u003c/em\u003e (\u0026ldquo;Vulnerable\u0026rdquo; and \u0026ldquo;Near threatened\u0026rdquo;, respectively) differed drastically from each other (Fig. 4). Despite \u003cem\u003eC. ursinus\u003c/em\u003e having been extirpated from most of its range over the past 200\u0026ndash;800 years due to hunting and environmental factors, its heterozygosity remains at a relatively high level, which corresponds with the historical DNA analysis previously published [19]. In contrast, the heterozygosity estimate for \u003cem\u003eE. jubatus\u003c/em\u003e was almost as low as that of \u003cem\u003eZ. wollebaeki\u0026nbsp;\u003c/em\u003e(\u0026ldquo;Endangered\u0026rdquo;) (Fig. 4). We suggest that this fact is related to the nowadays population decline of \u003cem\u003eE. jubatus\u003c/em\u003e, which began in the 1980s and continues to this day across its distribution range\u0026nbsp;[20, 21].\u003c/p\u003e\n\u003cp\u003eMoreover, one of \u003cem\u003eZ. japonicus\u0026rsquo;\u003c/em\u003e closest living relatives, the non-endangered \u003cem\u003eZ. californianus\u003c/em\u003e, exhibited about two times lower heterozygosity (0.000491 and 0.000595), suggesting that even a halved estimate for \u003cem\u003eZ. japonicus\u003c/em\u003e (to compensate for sample merging effect) would not indicate low genetic diversity. Among \u003cem\u003eZ. californianus\u003c/em\u003e, relatively low heterozygosity value was observed in a potentially inbred individual from a Korean zoo exhibit (0.000491, SRR11434789). This analysis suggests \u003cem\u003eZ. japonicus\u0026rsquo;\u003c/em\u003e heterozygosity (0.00101) is not so low as to raise concerns about extinction, and therefore does not indicate severe inbreeding. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4. Heterozygosity of Dokdo sea lion in the context of other marine mammals.\u0026nbsp;\u003c/strong\u003eThe average heterozygosity values in log scale (from the left to right) for: Steller\u0026rsquo;s sea cow \u0026ndash; \u003cem\u003eH. gigas\u003c/em\u003e, Dokdo sea lion \u0026ndash; \u003cem\u003eZ. japonicus\u003c/em\u003e, Galapagos sea lion \u0026ndash; \u003cem\u003eZ. wollebaeki\u003c/em\u003e, Northern fur seal \u0026ndash; \u003cem\u003eC. ursinus\u003c/em\u003e, dugong \u0026ndash; \u003cem\u003eD. dugon\u003c/em\u003e, West Indian manatee \u0026ndash; \u003cem\u003eT. manatus\u003c/em\u003e, walrus \u0026ndash; \u003cem\u003eO. rosmarus\u003c/em\u003e, Steller sea lion \u0026ndash; \u003cem\u003eE. jubatus\u003c/em\u003e, beluga whale \u0026ndash; \u003cem\u003eD. leucas\u003c/em\u003e, grey seal \u0026ndash; \u003cem\u003eH. grypus\u003c/em\u003e, narwhal \u0026ndash; \u003cem\u003eM. monoceros\u003c/em\u003e, California sea lion \u0026ndash; \u003cem\u003eZ. californianus\u003c/em\u003e, and California sea lion \u0026ndash; \u003cem\u003eZ. californianus\u003c/em\u003e (from zoo). Samples are colored according to IUCN conservation status (2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePopulation decline of Dokdo sea lion started over 1,000 years ago\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne crucial determinant of a species\u0026apos; vulnerability to extinction is its population size, therefore, we inferred the retrospective history of effective population size (\u003cem\u003eNe\u003c/em\u003e) changes in \u003cem\u003eZ. japonicus\u003c/em\u003e and other Otariidae species (\u003cem\u003eZ. californianus\u003c/em\u003e, \u003cem\u003eZ. wollebaeki\u003c/em\u003e, \u003cem\u003eE. jubatus\u003c/em\u003e and \u003cem\u003eC. ursinus\u003c/em\u003e) for comparison, using the pairwise sequentially Markovian coalescent (PSMC) algorithm [22] (Fig. 5). For reliable \u003cem\u003eNe\u003c/em\u003e inference over time, we used \u003cem\u003eZ. japonicus\u003c/em\u003e SNVs identified from genomic loci with a minimum depth of more than ten reads. The population dynamics of \u003cem\u003eZ. japonicus\u003c/em\u003e present a unique pattern compared to its Otariidae counterparts.\u003c/p\u003e\n\u003cp\u003eWhile other Otariidae mammals showed a population decline around 10,000 years ago, possibly due to the climatic shifts of the Holocene including warming temperatures and sea level rises, \u003cem\u003eZ. japonicus\u003c/em\u003e experienced this decline later, around 4,500 years ago. This suggests a different adaptative response to environmental changes. Notably, the distinct population trajectory of \u003cem\u003eZ. japonicus\u003c/em\u003e could be linked to genetic introgression with \u003cem\u003eE. jubatus\u003c/em\u003e and \u003cem\u003eC. ursinus\u003c/em\u003e. We also observed a unique small rebound in population numbers about 1,500 years ago, which has not been observed in the other species. This resurgence was short-lived as a continued decrease in \u003cem\u003eZ. japonicus\u003c/em\u003e population numbers is apparent ever since. In addition to the PSMC analysis, demographic dynamics inferred using PopSizeABC further ascertained \u003cem\u003eZ. japonicus\u003c/em\u003e population decline since one thousand years ago with 95% confidence interval. While a more robust dataset of \u003cem\u003eZ. japonicus\u003c/em\u003e and other Otariidae species is essential to fully understand the interplay between climatic factors and population trends, our analysis provides the first whole-genome-based insights into the downward trend in of \u003cem\u003eZ. japonicus\u003c/em\u003e\u0026rsquo; demographic history.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe deep DNA sequencing and advances in bioinformatics allowed us to understand the basics of evolution better, and to describe traces of genetic introgression and the events that accompanied them, e.g., rapid speciation, multiple ecological radiations, and rapid adaptation to the changing environment [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Intergeneric fertile hybridization in pinnipeds is well-known fact, which adds an additional layer of complexity analyzing their speciation, phylogeny, and ancestry [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our study sheds light on the evolution of extant Otariidae species inhabiting the Northern Pacific Ocean, with a special focus on the genus \u003cem\u003eZalophus\u003c/em\u003e, especially the Dokdo sea lion, an extinct member of this genus. Through \u003cem\u003ef3\u003c/em\u003e and \u003cem\u003ef4\u003c/em\u003e admixture tests, we describe an introgression from \u003cem\u003eC. ursinus\u003c/em\u003e and/or \u003cem\u003eE. jubatus\u003c/em\u003e to \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Moreover, we find ancient introgression events between the extinct Dokdo sea lion and \u003cem\u003eE. jubatus/C. ursinus\u003c/em\u003e. While there are no remaining historical or scientific records on \u003cem\u003eZ. japonicus\u003c/em\u003e hybridization, \u003cem\u003eZ. californianus\u003c/em\u003e as a species has a rich hybridization history in zoo enclosures with mixed-species pinniped exhibits. On a larger scale, recently, compelling evidence emerged suggesting that smaller wild \u003cem\u003eE. jubatus\u003c/em\u003e body size found specifically in Oregon population in United States could be attributed to a paternal genetic input from male \u003cem\u003eZ. californianus\u003c/em\u003e that opportunistically mate with \u003cem\u003eE. jubatus\u003c/em\u003e females during their seasonal migrations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is also known that there was a significant overlap not only in the species ranges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) but also an ecological niche between extinct \u003cem\u003eZ. japonicus\u003c/em\u003e and extant \u003cem\u003eC. ursinus\u003c/em\u003e, and \u003cem\u003eE. jubatus\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We suppose that such gene flow between Otariidae species in the northern part of the Pacific Ocean increased their genetic diversity and could have had an adaptive effect in the changing environment for tens of thousands of years; for example, it could have helped the \u003cem\u003eZalophus\u003c/em\u003e species survive the Pleistocene-Holocene extinction of megafauna event.\u003c/p\u003e \u003cp\u003eUnfortunately, increasing human activities over the last few hundred years have led to ecosystem degradation, the destruction of native habitats, and the direct extinction of many animal species. The Dokdo sea lion is one of the species that is historically considered a victim of theriocide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Our study not only confirms that \u003cem\u003eZ. japonicus\u003c/em\u003e, a recently extinct iconic species in Korea and Japan, is genetically and evolutionarily very distinct from \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e, but also supports the written history, insisting that Dokdo sea lion population does not appear to have had a natural evolutionary dead end. Among evidence we report relatively high heterozygosity suggesting that the theriocide inflicted on the \u003cem\u003eZ. japonicus\u003c/em\u003e population was faster than the inbreeding rate. Moreover, the demographic history of the Dokdo sea lion has a relatively different trajectory compared with extant Otariidae species. This extinct marine mammal was influenced by a radical decrease in effective population size around 4,500 years ago, while the Northern fur seal, Steller sea lion, California sea lion, and Galapagos sea lion went through genetic bottleneck around 10,000 years ago. Dokdo sea lion is an illustrative example of how human activity can lead a seemingly genetically stable populations to the verge of extinction.\u003c/p\u003e \u003cp\u003eWe acknowledge that our study has many limitations such as small sample number, and low sequencing depth, which precluded us from performing certain analyses, but hopefully will give an introduction for future in-depth studies. Notably, all the \u003cem\u003eZ. japonicus\u003c/em\u003e bones were fragmented and affected by sea water, factors that may negatively contribute to the preservation of DNA and consequently to NGS sequencing yields (51, 52). Secondly, we could not comprehensively validate relative sample dating and DNA misincorporation estimates with other methods due to expected sample age estimates falling within modern-historical range. Moreover, we acknowledge that there was no overall correlation between the species conservation status and the heterozygosity in our study. This goes on to show that estimating species genetic diversity and conservation-needs requires a holistic approach that considers many intricate factors such as population numbers, habitat and food supply availability, pollution, hunting and poaching rates, reproductive behavior and fertility rates, accumulation of pathogenic alleles; heterozygosity is just one of them.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides novel genomic, evolutionary, and demographic insights based on the whole genome sequencing and analysis of an extinct sea lion from East Asia, \u003cem\u003eZ. japonicus. Z. japonicus\u003c/em\u003e genome fills a significant gap in the collective knowledge on the sea lion genus \u003cem\u003eZalophus\u003c/em\u003e and, more broadly, the eared seal family Otariidae. These data significantly elucidate the speciation process within genus \u003cem\u003eZalophus\u003c/em\u003e, suggesting that: 1) \u003cem\u003eZ. japonicus\u003c/em\u003e was the earliest diverged species in its genus; 2) \u003cem\u003eZ. californianus\u003c/em\u003e and \u003cem\u003eZ. wollebaeki\u003c/em\u003e populations had genetic exchanges with each other upon their initial separation; 3) the genomes of all the three \u003cem\u003eZalophus\u003c/em\u003e species show signs of introgression from the Otariidae lineage of \u003cem\u003eE. jubatus\u003c/em\u003e/\u003cem\u003eC. ursinus\u003c/em\u003e, with a particularly strong signal present in \u003cem\u003eZ. japonicus.\u003c/em\u003e Moreover, the demographic estimates of \u003cem\u003eZ. japonicus\u003c/em\u003e, such as an effective population size and the heterozygosity, did not suggest an elevated risk of extinction. These estimates provide an important complementary line of evidence to the sparsely documented knowledge on the \u003cem\u003eZ. japonicus\u003c/em\u003e extinction.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eFor our genomic comparison study of the extinct \u003cem\u003eZ. japonicus\u003c/em\u003e, we used 16 \u003cem\u003eZ. japonicus\u003c/em\u003e bones (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional file 1: Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) - three from Gajaegul in Ulleungdo (Gaze Cave, latitude 37.51\u0026deg; and longitude 130.79\u0026deg;) and 13 from Seodo Gajaegul in Dokdo islands (Gaze Cave, latitude 37.24\u0026deg; and longitude 131.86\u0026deg;) (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Both sites are named \u0026ldquo;Gajaegul\u0026rdquo;, meaning \u0026ldquo;sea lion cave\u0026rdquo; in Ulleungdo county dialect. The \u003cem\u003eZ. japonicus\u003c/em\u003e bones were provided by Cetacean Research Institute of National Institute of Fisheries Science in Republic of Korea. The collection of Dokdo sea lion bones was conducted under the permission granted by the Gyeoungbuk province local government for the collection of protected marine organisms (Permit No. 2019-2). We also collected a \u003cem\u003eZ. californianus\u003c/em\u003e muscle sample from the Seoul Grand Park, Republic of Korea, obtained during the necropsy process (Permit No. Seoul Grand Park Scientific Research 2020-009). As our study involved extinct animals and cadavers, ethical approval was not required. In addition, we downloaded 12 pinniped genomes (Additional file 2: Data S5), which were used to construct phylogenetic tree, ancestry analysis, and in the genetic diversity studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and next generation sequencing\u003c/h2\u003e \u003cp\u003eTo avoid contamination, only endogeneous bone tissue was collected after UV radiation and ethanol treatment. The genomic DNA from the extinct species was extracted using DNeasy Tissue \u0026amp; Blood Kit (Qiagen, Valencia, CA) and the Cetrimonium bromide (CTAB) manual. To generate Illumina NGS data, we constructed PE and SE libraries using the KAPA Hyper Library Preparation Kit (Kapa Biosystems, Woburn, MA, USA) and the Accel NGS 1S plus DNA kit (Swift BioSciences, Washtenaw County, Michigan, USA), respectively, according to manufacturer\u0026rsquo;s instructions. The Illumina-based NGS sequencing was performed with Illumina NovaSeq 6000 (Illumina, CA, USA) and NextSeq 500 (Illumina, CA, USA). For MGI-Seq, we constructed paired-end libraries with MGIEasy DNA Library Prep Kit (MGI, Shenzhen, China) and sequenced them on the DNBSEQ-T7 sequencing platform.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDokdo sea lion mitochondrial genome assembly\u003c/h2\u003e \u003cp\u003eUpon assessing the read quality with Trimmomatic (ver. 0.39) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we extracted mtDNA reads by mapping all the short DNA reads to the mito-genome of \u003cem\u003eZ. californianus\u003c/em\u003e (Acc. NC_006416). We assembled the mitochondrial genome (mito-genome) of the \u003cem\u003eZ. japonicus\u003c/em\u003e (Z6) sample using NOVOPlasty (ver. 4.2) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Minor gaps of the mito-genome assembly were filled in by conducting Sanger sequencing (Additional file 1: Table S5, Additional file 1: Table S6) followed by assembly using Cap3 program (updated on December 21, 2007) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We predicted and annotated \u003cem\u003eZ. japonicus\u0026rsquo;\u003c/em\u003e mito-genome using the MITOS program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mitos.bioinf.uni-leipzig.de/index.py\u003c/span\u003e\u003cspan address=\"http://mitos.bioinf.uni-leipzig.de/index.py\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Additional file 1: Fig S5, Additional file 2: Data S3) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Complete mtDNA genomes were aligned with Mummer (ver. 4.0.0rc1) (Additional file 1: Fig. S5) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Dendroscope (ver. 3.5.10) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We additionally obtained ten mtDNA genome consensus sequences by aligning our samples\u0026rsquo;s NGS reads to Z6 deep-sequenced mtDNA genome (Additional file 2: Data S4). The consensus sequences were obtained from high quality SNV data (mapping quality\u0026thinsp;\u0026gt;\u0026thinsp;30, genotype quality\u0026thinsp;\u0026gt;\u0026thinsp;20, and coverage\u0026thinsp;\u0026gt;\u0026thinsp;10) by implementing samtools consensus utility (ver. 1.9) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Five samples (Z1, Z5, Z7, Z8, and Z12) were excluded in the process due to significantly lower (insufficient) amount of NGS reads (Additional file 2: Data S4). We then constructed the phylogenetic tree of the ten \u003cem\u003eZ. japonicus\u003c/em\u003e mtDNA genomes along with other closely related species (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Additional file 1: Fig. S6 and Additional file 2: Data S6). We aligned CDS sequences using muscle program (ver. 3.8.31) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and constructed the phylogenetic tree using phyML (ver. 3.1) with default parameters (ver. 3.1) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogeny, admixture and genomic composition analyses of Dokdo sea lion and other pinnipeds\u003c/h2\u003e \u003cp\u003eTo construct phylogenetic tree and analyze ancestry, we aligned reads to an outgroup species that is the most distantly related to \u003cem\u003eZ. japonicus\u003c/em\u003e, namely, the walrus, \u003cem\u003eOdobenus rosmarus\u003c/em\u003e (acc. ANOP00000000) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Specifically for \u003cem\u003eZ. japonicus\u003c/em\u003e DNA samples, we applied PALEOMIX pipeline [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] by mapping all \u003cem\u003eZ. japonicus\u003c/em\u003e reads to the \u003cem\u003eO. rosmarus\u003c/em\u003e genome [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We estimated deamination patterns using mapDamage (ver. 2.0) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For the modern mammal genomes, their NGS reads were aligned to the same reference genome using bwa mem (ver. 0.7.17) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] after filtering out low quality reads using Trimmomatic with Quality\u0026thinsp;\u0026lt;\u0026thinsp;30 and read length\u0026thinsp;\u0026lt;\u0026thinsp;70 (ver. 0.39) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We then utilized Picard [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (ver. 2.27.5) to eliminate PCR duplicates and employed the GATK (ver. 4.1.3.0) for variant calling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We constructed consensus sequence using the vcf2phylip [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] utilizing only genomic loci in the \u003cem\u003eZ. japonicus\u003c/em\u003e bam files with read depth larger than five. We then constructed phylogenetic tree using the PhyML (ver. 3.1) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] as in mitochondrial genome.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ef3-\u003c/em\u003e and \u003cem\u003ef4-\u003c/em\u003estatistics (table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e-4) were conducted using a ADMIXTOOLS (ver. 2.0) algorithm [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. An admixture graph was constructed with qpGraph model [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] with admix\u0026thinsp;=\u0026thinsp;2. The qpGraph [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] was automatically optimized for genetic admixture of our admixture model.\u003c/p\u003e \u003cp\u003eThe maps used for the Otariinae species\u0026rsquo; distribution were obtained from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mapstyle.withgoogle.com\u003c/span\u003e\u003cspan address=\"https://mapstyle.withgoogle.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The graphic representations of each species in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e as well as the those of Ulleungdo and Dokdo islands were created based on royalty-free images under the Creative Commons (CC) license.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of the effective population size of Dokdo sea lion\u003c/h2\u003e \u003cp\u003eWe used PSMC algorithm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] to estimate the effective population size of Dokdo sea lion in the last 20,000 years. For higher mapping rates, for this analysis, we aligned reads from all ancient and extant \u003cem\u003eZalophus\u003c/em\u003e and \u003cem\u003eE. jubatus\u003c/em\u003e species to the \u003cem\u003eE. jubatus\u003c/em\u003e reference genome (acc. GCA_004028035.1, ver. ASM402803v1), which shares more recent common ancestry with these species compared to the previously used walrus. Using the PALEOMIX pipeline [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] we aligned all \u003cem\u003eZ. japonicus\u003c/em\u003e reads to the \u003cem\u003eE. jubatus\u003c/em\u003e reference and selected only high confidence SNVs, with more than 10x coverage for the PSMC analysis. The stringent data pre-filtering aimed to reduce biases stemming from possible over-representation of heterozygous sites in the ancient DNA. This was conducted as a necessary step, because PSMC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] infers the \u003cem\u003eNe\u003c/em\u003e changes over time using the density of heterozygous sites throughout the diploid genome of a single individual. For the PSMC analysis of modern genomes, we aligned reads with bwa mem (ver. 0.7.17) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] after trimming low quality reads using Trimmomatic (ver. 0.39) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We proceeded by utilized Picard (ver. 2.27.5) to eliminate PCR duplicates (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://broadinstitute.github.io/picard/\u003c/span\u003e\u003cspan address=\"https://broadinstitute.github.io/picard/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and employed the GATK (ver. 4.1.3.0) for variant calling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We applied a generation time of 10 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and a mutation rate of 0.27 * 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of the heterozygosity of Dokdo sea lion genomes\u003c/h2\u003e \u003cp\u003eTo accurately identify the heterozygous regions in the \u003cem\u003eZ. japonicus\u003c/em\u003e genome, we calculated the distribution of heterozygous positions across the genomic loci from a bam file, wherein \u003cem\u003eZ. japonicus\u003c/em\u003e reads were mapped to the most closely related reference genome, \u003cem\u003eZ. californianus\u003c/em\u003e (Acc. GCF_00976235.2) using the PALEOMIX pipeline [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We called SNVs utilizing samtools mpileup [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] with a minimum base quality of 20 (-Q 20) and mapping quality 20 (-q 20). We included genomic loci with a DP of 10 or greater. We cleaned all reads from the modern mammal genomes using the Trimmomatic (ver. 0.39) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and mapped them using the bwa mem (ver. 0.7.17) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] to the most closely related reference genomes (Additional file 2: Data S7). After removing the PCR duplicates using Picard (ver. 2.27.5), we applied the same mpileup criteria with \u003cem\u003eZ. japonicus\u003c/em\u003e. This way, we implemented mlRho (ver. 2.9) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] to estimate the heterozygosity only on the high-quality loci with high quality variants.\u003c/p\u003e \u003cp\u003e \u003cb\u003ef\u003c/b\u003e \u003cb\u003e-statistics analyses for Dokdo sea lion and other Otariidae species\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor ancestry analysis, we used \u003cem\u003ef4\u003c/em\u003e-statistics and admixture \u003cem\u003ef3\u003c/em\u003e-statistics. The admixture \u003cem\u003ef3\u003c/em\u003e- and \u003cem\u003ef4\u003c/em\u003e-statistics were conducted using a ADMIXTOOLS (ver. 2.0) algorithm [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. All formulars employed are detailed in Additional file 2: Data S5-S7. Our criterion for significance was set at an absolute Z-score less than three.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIUCN: International Union for Conservation of Nature\u003c/p\u003e\n\u003cp\u003emtDNA: mitochondrial DNA\u003c/p\u003e\n\u003cp\u003eNGS: next-generation sequencing\u003c/p\u003e\n\u003cp\u003eSE: single-end\u003c/p\u003e\n\u003cp\u003ePE: paired-end\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNe\u003c/em\u003e: effective population size\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collection of Dokdo sea lion bones was conducted under the permission granted by the Gyeoungbuk province local government for the collection of protected marine organisms (Permit No. 2019-2). We also collected a \u003cem\u003eZ. californianus\u003c/em\u003e muscle sample from the Seoul Grand Park, Republic of Korea, obtained during the necropsy process (Permit No. Seoul Grand Park Scientific Research 2020-009). As our study involved extinct animals and cadavers, ethical approval was not required.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are publicly available for scientific research. Sequencing data have been deposited in the NCBI SRA with accession number PRJNA982545. All the other data are available either in the main text or in the supplementary materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.K. and S.J. are employees and J.B. is the CEO of Clinomics Inc. Other authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Institute of Fisheries Science, Ministry of Ocean and Fisheries, Korea (R2020024, R2021030, R2022033, R2024004).\u0026nbsp;Promotion of Innovative Business for Regulation-Free Special Zones funded by the Ministry of SMEs and Startups (MSS, Korea) (grant number [P0016195, P0016193] (1425156792, 1425157301) (2.220035.01, 2.220036.01)).\u0026nbsp;Ulsan City Research Fund (1.200047.01). Fedor Sharko and Artem Nedoluzhko were supported by ARCTIC SIRENIA RESEARCH FOUNDATION.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: J. K., J. C., A. B, S. J and F. S., A. N.; Methodology: C. K., J. K, F. S; Investigation: E. K, H.-W. K., M. Y., J.-H. \u0026nbsp;L, K. L., and H. S.; Visualization: A. B, J. C., J. K.; Supervision: J. K, A. B; Writing\u0026mdash;original draft: J. K., J. C., A. B; Writing\u0026mdash;review \u0026amp; editing: A. N, J. B\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRice DW: \u003cstrong\u003eMarine mammals of the world: Systematics and distribution (Special publication / the Society for Marine Mammalogy) \u003c/strong\u003evol. 1: Society for Marine Mammalogy; 1998.\u003c/li\u003e\n\u003cli\u003eValenzuela-Toro A, Pyenson ND: \u003cstrong\u003eWhat do we know about the fossil record of pinnipeds? 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Genschoreck T, Webster T, Reich D: \u003cstrong\u003eAncient Admixture in Human History\u003c/strong\u003e. \u003cem\u003eGenetics \u003c/em\u003e2012, \u003cstrong\u003e192\u003c/strong\u003e(3):1065-1093.\u003c/li\u003e\n\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":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Zalophus japonicus, Dokdo sea lion, Japanese sea lion, Otariidae, speciation, whole genome sequencing, extinction, marine mammal, introgression, paleogenomics","lastPublishedDoi":"10.21203/rs.3.rs-4721400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4721400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe Dokdo sea lion (\u003cem\u003eZalophus japonicus\u003c/em\u003e), commonly referred to as Gangchi in Korea also known as the Japanese sea lion, was endemic to the Northwest Pacific coast before becoming extinct in the 1950s. Little is known about its origins and speciation compared to other Otariidae species or how the rapid decline affected the species\u0026rsquo; genetic diversity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTo raise the Dokdo sea lion from this relative obscurity, we sequenced DNA from 16 \u003cem\u003eZ. japonicus\u003c/em\u003e\u0026rsquo; bone fragments, obtained from Dokdo and Ulleungdo islands in Korea. Our genome-wide SNP-based analyses establish \u003cem\u003eZ. japonicus\u003c/em\u003e as the earliest diverged species within its genus, significantly redefining its evolutionary relationship with the California (\u003cem\u003eZ. californianus\u003c/em\u003e) and Galapagos (\u003cem\u003eZ. wollebaeki\u003c/em\u003e) sea lions. Our research further elucidates the phylogeny of \u003cem\u003eZ. japonicus\u003c/em\u003e, shedding light on the complexity of the genetic isolation process within its genus that was prompted by the geographic isolation of the three populations of \u003cem\u003eZalophus\u003c/em\u003e ancestral stock. Conversely, the genetic signature of Dokdo sea lion genome can be modeled as an evolutionary pathway involving gene flow from Otariidae species with shared range. In addition, we discovered, population decline of the \u003cem\u003eZ. japonicus\u003c/em\u003e started already over 1,000 years ago, however, \u003cem\u003eZ. japonicus\u003c/em\u003e genome maintained a relatively high heterozygosity despite nearing extinction.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur genome-scale analysis has eliminated ambiguity in the phylogeny of \u003cem\u003eZ. japonicus\u003c/em\u003e and shed light on the evolutionary pathways underlying its speciation and the genetic diversity before its extinction. Broadly, this study highlights the importance of genome-scale analysis for the extinct marine megafauna to elucidate the complexity of their gene flow and subsequent genetic diversities among extant species. Furthermore, this study offers retrospective genomic insights into the extinction process of a carnivorous marine mammal, information that could aid conservation efforts towards extant Otariidae species.\u003c/p\u003e","manuscriptTitle":"Dokdo sea lion Zalophus japonicus genome reveals its evolutionary trajectory before extinction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 11:17:19","doi":"10.21203/rs.3.rs-4721400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-11T16:51:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T12:19:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-03T21:16:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142108324988431276999940794995379492182","date":"2024-07-25T17:37:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152444605290519680942860007362925513704","date":"2024-07-19T08:02:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-19T07:02:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-18T14:58:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T14:33:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T07:32:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2024-07-11T03:09:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6b1286f-ee07-4fe2-a3f6-b22c4cbcc881","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T16:12:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 11:17:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4721400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4721400","identity":"rs-4721400","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}