Population genetic dynamics of the Ivory Snail (Babylonia areolata): Insights from coastal waters of Vietnam for conservation and aquaculture

Population genetic dynamics of the Ivory Snail (Babylonia areolata): Insights from coastal waters of Vietnam for conservation and aquaculture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Population genetic dynamics of the Ivory Snail (Babylonia areolata): Insights from coastal waters of Vietnam for conservation and aquaculture Sang Vu, Sarifah Aini, Angkasa Putra, Soo Rin Lee, Nguyen Duc Long, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6937476/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The ivory snail Babylonia areolata has experienced a significant population decline in marine ecosystem due to the overharvesting, habitat loss, and climate change. Despite its ecological significance and commercial value, the population genetic studies on this gastropod remain limited. This study provides the first genetic insight into B. areolata based on 105 newly generated mitochondrial cytochrome c oxidase subunit I ( COI ) gene sequences collected from Vietnam. The analysis revealed the lowest interspecific genetic distance (11.2%) between B. areolata and Babylonia borneensis , and the highest (18.5%) with Babylonia zeylanica . The Bayesian and Maximum-likelihood phylogenetic analyses showed B. areolata as a distinct monophyletic lineage, while species delimitation methods recovered multiple operational taxonomic units, suggesting the potential presence of cryptic diversity. The low inter-population divergence (0.2–0.3%) further indicated a high level of genetic connectivity among B. areolata populations across coastal waters of Thailand, Vietnam, and China. The haplotype network analysis revealed 26 haplotypes, including a dominant central haplotype, supporting the hypothesis that larval dispersal and regional ocean currents have shaped gene flow of B. areolata . Additionally, the presence of several unique haplotypes in the study regions may reflect historical geological events and demographic isolation, which likely shaped the distinct populations of B. areolata , especially in enclosed coastal areas like Cam Ranh Bay in central Vietnam. These findings underscore the importance of molecular tools in elucidating population structure and offer critical insights for the conservation and sustainable aquaculture of B. areolata in Southeast and East Asia. Marine gastropod Mitochondrial DNA Genetic diversity Phylogeny Population structure Fisheries management Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The marine gastropods under the genus Babylonia are regarded as an economically and ecologically important group, particularly within the tropical and subtropical coastal ecosystems of Asia (Di et al. 2021 ; Zhao et al. 2022 ). Taxonomically, Babylonia is classified within the family Babyloniidae under the order Neogastropoda, a relatively small group of marine gastropods distinguished by their large, clearly patterned shells and high morphological variability (Fraussen and Stratmann 2013 ). Presently, the family Babyloniidae is divided into two genera viz., Babylonia , comprising 17 recognized species, and Zemiropsis , with five species (MolluscaBase 2025 ). While Babylonia species are broadly distributed from the northern Indian Ocean to the Indo-West Pacific, Zemiropsis species have a more restricted range, primarily inhabiting the southwestern Indian Ocean, including coastal waters of South Africa and southern Mozambique (Altena and Gittenberger 1981 ; Fraussen and Rosado 2013 ). Typically, Babylonia species occupy continental shelf habitats at depths ranging from 4 to 80 meters, exhibiting diverse morphological traits such as variations in umbilicus structure, convex sutures, and distinctive apical shell shapes (Zou et al. 2024 ; Yen et al. 2025 ). Although Babylonia was initially placed in the family Buccinidae based on shell morphology, further molecular phylogenetic analyses using mitochondrial genes ( COI and 16S rRNA ) have confirmed its current classification under the family Babyloniidae (Harasewych and Kantor 2001 ; Hayashi 2005 ). Among the extant species diversity, Babylonia areolata , commonly known as the ivory snail, is widely recognized for its substantial commercial value due to its rapid growth, short reproductive cycle, excellent taste, rich nutritional composition, and potential pharmacological properties (Periyasamy et al. 2011 ; Wang et al. 2011 ; Dobson et al. 2021 ; Sutthinon et al. 2023 ; Zou et al. 2025 ). Geographically, B. areolata is distributed across South, Southeast, and East Asia, including regions viz., Sri Lanka, the Nicobar Islands (India), the Gulf of Thailand, coastal Vietnam, southeastern China, and Taiwan (Lü et al. 2016 ; Zou et al. 2024 ). In Southeast Asia, particularly Vietnam, B. areolata has emerged as a priority species for aquaculture development, with production reaching 11,195 tons in 2024 and market prices ranging from 10 to 15 USD/kg (Vietnam Ministry of Agriculture and Environment 2024 ). Although aquaculture practice of B. areolata has emerged as a sustainable approach to alleviate pressure on wild stocks, market demand continues to contribute to significant declines in natural populations due to overharvesting and habitat degradation (Lü et al. 2020 ; Di et al. 2021 ). Thus, the scientific investigation into the taxonomy, ecology, and reproductive biology of Babylonia species has long been a focal point of research (Harasewych and Kantor 2001 ; Yen et al. 2025 ). Advances in molecular systematics utilizing microsatellite, mitochondrial, and nuclear markers have been largely employed in resolving morphological variation (Zou et al. 2011a , 2011b ), species delimitation (Yen et al. 2025 ), phylogenetic relationships (Hou et al. 2013 ; Wang 2017 ; Ran et al. 2020 ), and population genetic structure (Hualkasin et al. 2008 ; Wang et al. 2011 ). More recently, the transcriptomic approaches have been also employed to elucidate immune responses to bacterial infections by integrating molecular, biochemical, and histological data (Yu et al. 2024 ; Zhang et al. 2024 ). In addition, the proteomic analyses have also enhanced understanding of larval physiological responses to environmental stressors such as hypoxia and ocean acidification (Mukherjee et al. 2013 ; Di et al. 2019 ). Despite these comprehensive investigations into the genetic diversity and population genetic structure of B. areolata across its geographic range remain limited, particularly in populations from Vietnam. Therefore, this study aims to (i) analyze genetic diversity and phylogenetic clustering pattern, (ii) estimate the Operational Taxonomic Units (OTUs), and (iii) evaluate haplotype diversity and population dynamics of B. areolata populations across the coastal regions of Thailand, Vietnam, and China. This population level genetic information is expected to provide a deeper understanding of intra-species genetic variation among distant populations of B. areolata . The findings offer valuable insights for fisheries resource management, facilitate genetically informed aquaculture programs, and contribute to enhancing the economic value of this marine gastropods through sustainable aquaculture practices in Southeast Asia. Materials and methods Sample collection and genomic DNA extraction A total of 105 individuals of B. areolata were collected from four distinct coastal provinces in Vietnam, Ninh Thuận (11.582222°N, 109.055833°E), Khánh Hòa (11.897778°N, 109.156111°E), Thua Thien Hue (16.241111°N, 108.082500°E), and Bình Thuận (10.573889°N, 107.582500°E) (Fig. 1 ; Online Resource Table S1 ). All collected specimens were transported to the Institute of Biology, Vietnam Academy of Science and Technology, where approximately 100 mg of tissue was carefully dissected from the foot muscle of each specimen and preserved in 80% ethanol for subsequent molecular analyses. The tissue samples were finely sectioned into small fragments and homogenized in DNA extraction buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM EDTA, 1% SDS, and 1 mg/mL proteinase K. The genomic DNA was isolated using the AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) in accordance with the manufacturer’s instructions. The concentration and purity of the extracted DNA were evaluated with a NanoDrop spectrophotometer (Thermo Fisher Scientific D1000, Waltham, MA, USA). PCR amplification and sequencing To amplify a ~ 650 bp fragment of the partial COI gene, a published primer pair (LCO and HCO) was used in PCR (Folmer et al. 1994 ). The PCR amplification was carried out in a 50 µL reaction mixture containing 1 µL each of forward and reverse primers, 0.9 µL of 3% dimethyl sulfoxide (DMSO), 19.9 µL of sterile deionized water, 3 µL of 10× ExTaq buffer, 0.2 µL of ExTaq HS DNA polymerase, 3 µL of dNTP mix, and 1 µL of genomic DNA as the template. The experiment was carried out using a Takara PCR Thermal Cycler Dice® Gradient (Takara Korea Biomedical Inc., Seoul, Republic of Korea). The PCR program included an initial denaturation at 94°C for 3 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 40°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The PCR products were separated on a 1.5% agarose gel, and the desired DNA bands were excised and purified using the AccuPrep® PCR/Gel Purification Kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer’s guidelines. The purified fragments were then submitted to Macrogen (Daejeon, Republic of Korea; https://dna.macrogen.com/ ) for Sanger sequencing in both directions. Dataset construction, genetic distance, and phylogenetic inference The partial COI gene sequences generated in this study were initially validated through nucleotide BLAST searches on the NCBI platform ( https://blast.ncbi.nlm.nih.gov ) and the consensus sequences were subsequently submitted to the GenBank to acquire unique accession numbers. For downstream analyses, a comprehensive dataset was assembled, comprising 105 newly generated COI sequences and 34 additional sequences of B. areolata and closely related congeners retrieved from GenBank (Online Resource Tables S1–S2). The sequences alignment was performed using CLUSTAL X software, resulting in a consensus alignment of 620 bp (Thompson et al. 1997 ). Both the inter- and intra-species genetic distance was estimated using the Kimura 2-Parameter (K2P) model implemented in MEGA version 12 (Kumar et al. 2024 ). To infer phylogenetic relationships, a total of 11 Babylonia species were analyzed, with Zemiropsis papillaris (Accession no. OR677031) designated as the outgroup. The phylogenetic trees were constructed using both Bayesian (BA) and Maximum Likelihood (ML) approaches. The optimal nucleotide substitution model General Time Reversible model (GTR) + gamma-distributed rate heterogeneity (G) and a proportion of invariant sites (I) was selected based on the lowest BIC (Bayesian Information Criterion) score recognized through PartitionFinder 2 and jModelTest v2 (Darriba et al. 2012 ; Miller et al. 2015 ; Lanfear et al. 2017 ). The BA analysis was carried out by using MrBayes version 3.1.2, applying the General Time Reversible model with gamma-distributed rate heterogeneity and a proportion of invariant sites (nst = 6). The Markov Chain Monte Carlo (MCMC) procedure was run for 1 million generations, employing four chains (one cold and three heated), with sampling every 100 generations and discarding the first 25% as burn-in (Ronquist et al. 2012 ). For ML approach, phylogenetic inference was carried out using the PhyML 3.0 web server ( http://www.atgc-montpellier.fr/phyml/ ), incorporating 1000 bootstrap replicates to assess node support (Guindon et al. 2010 ; Trifinopoulos et al. 2016 ). Both BA and ML phylogenetic trees were visualized using the Interactive Tree of Life (iTOL) platform, version 4 ( https://itol.embl.de/ ) (Letunic and Bork 2024 ). OTUs estimation, haplotype diversity, and population genetic differentiation To accurately delineate OTUs, a combination of species delimitation methods was employed, encompassing Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), Poisson Tree Processes (PTP), and Generalized Mixed Yule Coalescent (GMYC) framework for the B. areolata dataset (105 generated + 14 database). The alignment file (.fasta) was utilized as input for both ABGD and ASAP analyses with Jukes-Cantor (JC69) model (Puillandre et al. 2012 , 2021 ). In PTP and GMYC analyses, an unrooted ultrametric ML phylogeny in Newick format (.nwk) was used to estimate the putative number of OTUs (Fujisawa and Barraclough 2013 ; Zhang et al. 2013 ). The species delimitation using ABGD and ASAP was conducted via the SPART web server (Miralles et al. 2022 ), while PTP and GMYC analyses were performed using iTaxoTools v0.1 (Vences et al. 2021 ). To further investigate the population genetic structure, a total of 119 COI sequences were analyzed to assess the number of haplotypes, haplotype diversity (Hd), and nucleotide diversity (π) using DnaSP version 6.0 (Rozas et al. 2017 ). Subsequently, the haplotype relationships were visualized using the Templeton, Crandall, and Sing (TCS) network method implemented in POPART version 1.7 (Clement et al. 2000 ; Leigh and Bryant 2015 ). The tests of selective neutrality were also conducted for each sampling locality using Tajima’s D and Fu and Li’s F and D statistics, implemented through the DnaSP version 6.0 (Tajima 1989 ; Fu and Li 1993 ). Results Genetic distance and phylogeny The genetic distance analysis based on the partial COI gene within the genus Babylonia revealed notable variation at both inter- and intra- species level. The mean inter-species genetic distances within the genus ranged from 3.0–20.5%, with the lowest recorded between B. semipicta and B. spirata (3.0%) and the highest between B. zeylanica and B. formosae (20.5%). Notably, B. areolata exhibited the lowest inter-species genetic distance with B. borneensis at 11.2%, while the highest distance was observed with B. zeylanica at 18.5% (Table 1 ). Furthermore, the genetic distance analysis indicated low mean intra-species genetic variation (0.2%) in B. areolata . The BA and ML phylogenetic analyses showed monophyletic clustering of B. areolata , which was distinctly separated from other congeners. Nevertheless, the resulting tree topology indicated a close evolutionary relationship between B. areolata and B. formosae , B. japonica , B. lutosa , and B. borneensis in the present dataset. Conversely, B. spirata , a species native to the Indo-West Pacific, occupied a basal position in the current phylogenetic analyses and was inferred to represent a potential ancestral lineage within the genus Babylonia (Fig. 2 ; Online Resource Fig. S1 ). Table 1 The mean inter-species genetic divergences among Babylonia species, based on K2P distances estimated from partial mitochondrial COI gene sequences. Species Inter- Babylonia areolata Babylonia lutosa 12.2 Babylonia spirata 15.9 15.0 Babylonia semipicta 15.4 14.5 3.0 Babylonia formosae 13.8 12.8 17.6 16.9 Babylonia zeylanica 18.5 16.7 17.6 16.9 20.5 Babylonia japonica 14.6 15.6 18.2 18.3 5.6 18.7 Babylonia borneensis 11.2 12.2 15.2 14.5 14.7 15.9 16.1 Babylonia feicheni 13.7 14.8 18.0 17.8 15.7 19.3 17.3 13.1 Babylonia valentiana 15.5 16.4 18.1 16.3 19.1 15.4 18.7 14.4 14.2 Babylonia pieroangelai 13.3 14.3 16.3 14.8 18.4 16.9 18.3 15.7 15.3 15.3 Species delimitation and population structure Species delimitation analyses of B. areolata revealed varying numbers of OTUs depending on the different methods applied. Both ABGD and ASAP methods each identified 26 OTUs, while the PTP method detected a higher count of 42 OTUs, and the GMYC method delineated only two OTUs (Online Resource Tables S3–S5). The haplotype network analysis conducted on B. areolata sequences from three countries (Vietnam, China, and Thailand) identified Hd = 0.6948, π = 0.0024, and 26 distinct haplotypes (Fig. 3 A; Online Resource Table S6). Among the detected haplotypes, Hap_1 was the most dominant, occurring in numerous individuals across all sampling sites, including four populations in Vietnam, as well as in three sequences from China and four sequences from Thailand. This haplotype occupied a central position within the network and exhibited direct connections to many other haplotypes, indicating its shared genetic characteristics. In addition to Hap_1, Hap_6 also showed a relatively high frequency and comprised sequences from several populations in Vietnam and China. Conversely, the majority of other haplotypes were unique, each found in only one sequence from a single sampling location (Fig. 4 ). Overall, the two species delimitation methods (ABGD and ASAP), along with population structure analyses, revealed a consistent pattern in the detection of OTUs and haplotypes within the present B. areolata dataset. The inter-population genetic distances were 0.2% between both the Vietnam–China and China–Thailand populations, whereas the distance between Vietnam and Thailand populations was 0.3% (Fig. 3 B). Genetic diversity indices and neutrality test parameters based on the partial COI sequences of B. areolata from six locations (four in Vietnam, as well as China and Thailand) revealed distinct genetic variation among populations. Among the Vietnamese populations, Khánh Hòa exhibited the highest Hd = 0.83669 and π = 0.00299, with 17 polymorphic sites and 15 haplotypes identified from 32 COI sequences. Conversely, the most southern Bình Thuận population showed the lowest genetic diversity, with six haplotypes, eight polymorphic sites, Hd = 0.51077, and π = 0.00149 (Table 2 ). The Chinese and Thai populations, although represented by smaller sample sizes ( n = 7), displayed moderate levels of genetic diversity. The Chinese population showed Hd = 0.80952 and π = 0.00193, while the Thai population had Hd = 0.71429 and π = 0.00246, with three and four polymorphic sites, respectively. The neutrality tests revealed negative Tajima’s D values across all populations, ranging from − 0.65405 (China) to − 2.12186 (Khánh Hòa). The Fu and Li’s F values were also negative in all populations, with the most extreme values observed in Khánh Hòa (–3.40989) and Bình Thuận (–3.36766) in Vietnam (Table 2 ). Table 2 Genetic diversity indices and neutrality test results based on partial mitochondrial COI sequences of B. areolata populations from Vietnam, China, and Thailand. N: number of sequences, P: polymorphic sites, H: number of haplotypes, K: average number of nucleotide differences, Hd: haplotype diversity, π: nucleotide diversity. Locations Divesity indices Netrality test N P H K Hd π Tajima’s D Fu and Li’s F Fu and Li’s D Vietnam Ninh Thuận 29 10 8 1.20690 0.62808 0.00223 -1.67864 -2.30591 2.32583 Khánh Hòa 32 17 15 1.61895 0.83669 0.00299 -2.12186 -3.40989 -3.26510 Thua Thien Hue 18 11 8 1.58824 0.69935 0.00293 -1.83747 -2.35660 -2.38333 Bình Thuận 26 8 6 0.80923 0.51077 0.00149 -2.06988 -3.36766 -3.23909 China 7 3 4 1.04762 0.80952 0.00193 -0.65405 -0.59207 -0.51900 Thailand 7 4 4 1.33333 0.71429 0.00246 -0.87642 -0.87602 -0.78927 Discussion The present phylogenetic analyses confirm the monophyly of B. areolata , consistent with earlier multi-locus studies employing partial mitochondrial genes ( COI and 16S rRNA ) and the nuclear H3 gene (Yen et al. 2025 ). Genetic distance analysis based on COI sequences across various Babylonia species revealed substantial interspecific divergence (3.0–20.5%), indicating marked evolutionary differentiation and supporting the distinct taxonomic status of B. areolata . The species delimitation analyses identified variation in the number of OTUs, suggesting potential cryptic diversity within B. areolata populations inhabiting the coastal waters of Thailand, Vietnam, and China. This cryptic diversity may be driven by local adaptation and oceanographic heterogeneity, as similarly reported in other gastropod taxa (e.g., Conus , Littoraria , and Nerita ) (Postaire et al. 2014 ; Ameri et al. 2023 ; Shin and Allmon 2023 ; Xu et al. 2024 ). Beyond cryptic diversity, the genetic patterns in marine taxa often reflect the complex interplay between environmental factors and biological traits (Faria et al. 2021 ). Ocean currents, sea surface temperature (SST), and bathymetry can either enhance population connectivity or act as barriers to gene flow, depending on localized hydrodynamic regimes (Fontana et al. 2024 ; Li et al. 2025 ). In this study, population genetic analyses of B. areolata from Thailand, Vietnam, and China revealed remarkably low intra-specific (0.2%) and inter-population (0.2–0.3%) genetic distances. This high connectivity is consistent with findings in other marine gastropods, where extensive larval dispersal and seasonal ocean currents facilitate widespread gene flow (Cho et al. 2025 ; Haslam et al. 2025 ). Such gene flow among geographically distant populations may pose risks of inbreeding depression and the erosion of unique wild genetic resources, concerns critical to sustainable aquaculture and broodstock management. The haplotype network of B. areolata revealed the presence of unique haplotypes restricted to Fujian, China, implying partial genetic isolation likely caused by geographic or oceanographic barriers, parallel to patterns seen in Nerita yoldii in Chinese coastal waters (Tang et al. 2024 ). Furthermore, in the coastal waters of Vietnam, B. areolata populations exhibited both shared and unique haplotypes. The sequences generated from Khánh Hòa in central Vietnam displayed shared haplotypes with Chinese and Thai populations, possibly due to seasonal current-driven larval dispersal in deeper waters (Bashevkin et al. 2020 ). Simultaneously, a few sequences from the same locality exhibited unique haplotypes, potentially confined to the shallow waters in close proximity to Cam Ranh Bay (Fig. 4 ). These localized haplotypes may reflect adaptation to spatially and temporally variable environments and interactions with coexisting biotic components (Chen et al. 2016 ; Quang et al. 2017 ; Nguyen and Jutta 2019 ). Conversely, the B. areolata populations from Bình Thuận, Ninh Thuận, and Thừa Thiên Huế demonstrated high degree of gene flow with populations from China and Thailand. This genetic mixing may be influenced by the Philippine Sea and northern ocean currents, which converge during the northeast monsoon and enter the Gulf of Thailand via the Vietnamese coast (Pongparadon et al. 2015 ). Furthermore, the SSTs of Khánh Hòa, Ninh Thuận, and Thừa Thiên Huế are comparable to those in the Taiwan Strait, potentially supporting the persistence of shared haplotypes between distant populations. Notably, the shared thermal conditions between Bình Thuận and the Gulf of Thailand may have facilitated gene flow, a pattern commonly observed in other marine taxa (Masanja et al. 2023 ) (Fig. 4 ). It is evidenced that the life history traits of B. areolata , particularly its planktonic larval phase, contribute to long-distance dispersal via oceanic currents (Shen et al. 2018 ). Our findings underscore the significant influence of oceanographic variability, hydrodynamic regimes, and demographic processes may shape the genetic structure of B. areolata across coastal Thailand, Vietnam, and China. Nevertheless, the potential impact of human-mediated translocations, particularly through aquaculture activities, must not be overlooked. Such anthropogenic pressure may have led to population admixture, thereby lost their genetic distinctiveness in marine environment. Despite this, the presence of a few unique haplotypes in Thailand, Vietnam, and China suggests historical and biogeographic influences requiring further investigation. The geological history and paleoclimatic changes, particularly the sea-level oscillations during the Pleistocene, likely played a crucial role in shaping present-day genetic patterns of marine organisms in Southeast and East Asia by fragmenting habitats and isolating populations within shallow bays and gulfs separated by sills and ridges, thereby limiting gene flow (He et al. 2014 ; Niu et al. 2019 ). In summary, this study offers essential insights into the population genetics of B. areolata , with direct implications for broodstock selection in aquaculture, especially from Vietnam. The comprehensive understanding of genetic differentiation can guide selective breeding programs targeting desirable traits such as rapid growth, reproductive efficiency, disease resistance, and environmental resilience (Robledo et al. 2018 ). Moreover, the observed population structure emphasizes the importance of strict quarantine measures and management strategies to conserve genetically distinct populations across Thailand, Vietnam, and China. Conclusion This study presents a molecular assessment of B. areolata populations from the coastal waters of Thailand, Vietnam, and southern China, revealing low intra- and inter-population genetic divergences based on COI gene sequences (0.2–0.3%). The phylogenetic analysis identified B. areolata as a distinct monophyletic lineage, whereas species delimitation analyses revealed multiple OTUs, suggesting the existence of potential cryptic diversity within this marine gastropod. The presence of a single dominant haplotype broadly distributed across the study regions indicates strong gene flow, likely mediated by larval dispersal and prevailing oceanographic currents. In contrast, the detection of unique haplotypes in specific localities across Thailand, Vietnam, and China suggests regional genetic structuring, potentially shaped by historical demographic events and subsequent colonization processes, particularly in semi-enclosed systems such as bays and gulfs. These findings underscore the utility of mitochondrial DNA markers in elucidating population structure and uncovering hidden genetic diversity, offering crucial insights for conservation genetics and informing the development of sustainable aquaculture practices to preserve the genetic integrity of wild B. areolata populations in Southeast and East Asia. Declarations Acknowledgements The authors also wish to express their gratitude to Ms. Nguyen Thi Ngoc and Ms. Do Thi Thanh Trung of the Institute of Biology, Vietnam Academy of Science and Technology, Vietnam; Professor Le Duc Minh, Dr. To Thanh Thuy, and Mr. Le Toan Thang of University of Science, Vietnam National University, Hanoi, Vietnam; Dr. Nguyen Thi Anh Thu of Nha Trang University, undergraduate student (Nguyen Thi Huong Tra) and Ms. Kieu Thi Hong of Hanoi University of Pharmacy, Vietnam; Dr. Vu Van In, Vietnam Japan University, Vietnam National University, Hanoi, Vietnam for their valuable assistance during the experimental procedures. Jingda Kang is supported by the master program of University of Stirling, UK and Nguyen Duc Long is supported by the master program of University of Science, Vietnam National University, Hanoi, Vietnam. Funding This study is supported by the Chey Institute for Advanced Studies’ International Scholar Exchange Fellowship for the academic year of 2024–2025 in South Korea. Sang Van Vu is also supported by the Postdoctoral Scholarship Program of Vingroup Innovation Foundation (VINIF), under grant number VINIF.2024.STS.52. Authorship Contributions Conceptualization, S.V.V., S.K., H.-W.K.; software, S.K., S.A., A.P.; validation, N.D.L., J.K.; formal analysis, S.V.V., S.A., A.P., S.R.L.; data curation, N.D.L., T.T.L., S.V.V.; writing—original draft preparation, S.V.V., S.A., A.P.; writing—review and editing, H.-W.K., S.K.; visualization, E.S., A.G., A.S.; supervision, S.K., H.-W.K., H.H.N.; project administration, S.V.V., S.K., H.-W.K.; funding acquisition, S.V.V., H.-W.K. All authors have read and agreed to the published version of the manuscript. Data availability The DNA sequences analyzed in this study were deposited in GenBank under the accession numbers PV478301–PV478405. Conflict of interest The authors declare that there are no conflicts of interest related to this work. Consent to participate All authors provided their consent to participate in the project and to contribute to the preparation of this manuscript. Supplementary information The online version contains supplementary material available at ORCID Ids Sang Van Vu: https://orcid.org/0000-0003-1929-4419 Sarifah Aini: https://orcid.org/0009-0001-1065-4724 Angkasa Putra: https://orcid.org/0000-0002-5533-9437 Soo Rin Lee: https://orcid.org/0000-0002-6443-855X Nguyen Duc Long: https://orcid.org/0009-0008-4285-4115 Thanh Tat Le: https://orcid.org/0009-0004-2702-1482 Hoang Huy Nguyen: https://orcid.org/0000-0002-6284-5813 Eric Saillant: https://orcid.org/0000-0002-1480-4515 Almas Gheyas: https://orcid.org/0000-0002-7682-4394 Armin Sturm: https://orcid.org/0000-0003-2632-1999 Jingda Kang: https://orcid.org/0000-0002-6018-5532 Hyun-Woo Kim: https://orcid.org/0000-0003-1357-5893 Shantanu Kundu: https://orcid.org/0000-0002-5488-4433 References Altena COR, Gittenberger E (1981) The genus Babylonia (Prosobranchia: Buccinidae). 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Zool Scr 54:17–32. https://doi.org/10.1111/zsc.12694 Yu J, Lü W, Zhang L et al (2024) Effects of Vibrio harveyi infection on the biochemistry, histology, and transcriptome in the hepatopancreas of ivory shell ( Babylonia areolata ). Fish Shellfish Immunol 153:109856. https://doi.org/10.1016/j.fsi.2024.109856 Zhang J, Kapli P, Pavlidis P et al (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29:2869–2876. https://doi.org/10.1093/bioinformatics/btt499 Zhang J, Wang J, Gu Z et al (2024) Transcriptome analysis of different aquaculture substrates on the immune response of Babylonia areolata . Mar Biotechnol 26:609–622. https://doi.org/10.1007/s10126-024-10324-w Zhao Q, Gu H, Wang W et al (2022) Gastrointestinal tract microbial community of Babylonia areolata and its diversity are closely correlated with the outbreak of disease. Aquacult Res 53:1636–1648. https://doi.org/10.1111/are.15694 Zou S, Li Q, Kong L (2011b) Additional gene data and increased sampling give new insights into the phylogenetic relationships of Neogastropoda, within the caenogastropod phylogenetic framework. Mol Phylogenet Evol 61:425–435. https://doi.org/10.1016/j.ympev.2011.07.014 Zou S, Li Q, Kong L et al (2011a) Comparing the usefulness of distance, monophyly, and character-based DNA barcoding methods in species identification: A case study of Neogastropoda. PLoS One 6:e26619. https://doi.org/10.1371/journal.pone.0026619 Zou W, Gan Y, Hong J et al (2025) A study on the chemosensory organs, feeding behavior, and attractant substances of Babylonia areolata . Aquac Rep 43:102906. https://doi.org/10.1016/j.aqrep.2025.102906 Zou Y, Fu J, Liang Y et al (2024) Chromosome-level genome assembly of the ivory shell Babylonia areolata . Sci Data 11:1201. https://doi.org/10.1038/s41597-024-03085-y Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6937476","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":474019032,"identity":"8a820f29-2622-486a-b7a2-ab3393532bb8","order_by":0,"name":"Sang Vu","email":"","orcid":"","institution":"University of Science, Vietnam National University","correspondingAuthor":false,"prefix":"","firstName":"Sang","middleName":"","lastName":"Vu","suffix":""},{"id":474019033,"identity":"90ed7efb-dc9b-4a21-9930-14459cbd1532","order_by":1,"name":"Sarifah Aini","email":"","orcid":"","institution":"Pukyong National 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09:23:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6937476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6937476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85170222,"identity":"bf76db1e-d03c-4c91-9aeb-10a2bb68578e","added_by":"auto","created_at":"2025-06-23 05:31:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6501270,"visible":true,"origin":"","legend":"\u003cp\u003eThe map shows the sampling locations of \u003cem\u003eB. areolata\u003c/em\u003e at four coastal sites in Vietnam, marked with red stars, alongside an image of \u003cem\u003eB. areolata\u003c/em\u003e sourced from the public domain free repository Wikimedia Commons. The map was created using ArcGIS version 10.6, with global administrative boundary shapefiles obtained from the DIVA-GIS platform.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/1da92f9b1ca0cbfd7f24c970.jpg"},{"id":85170218,"identity":"63a81df6-dc27-49b2-9bb0-773e4407e4e1","added_by":"auto","created_at":"2025-06-23 05:31:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6758515,"visible":true,"origin":"","legend":"\u003cp\u003eThe Bayesian phylogenetic tree reconstructed using partial mitochondrial \u003cem\u003eCOI\u003c/em\u003e gene sequences, showing a distinct clade for \u003cem\u003eB. areolata\u003c/em\u003e in comparison to other \u003cem\u003eBabylonia\u003c/em\u003e species. Posterior probability supports are shown at each node as gray dots, indicating the confidence in the inferred evolutionary relationships.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/82cc5ddf1730a952a47ffcdc.jpg"},{"id":85170221,"identity":"da3836b3-90a0-407f-a042-e4e0ea17f031","added_by":"auto","created_at":"2025-06-23 05:31:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2430546,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The TCS haplotype network of \u003cem\u003eB. areolata\u003c/em\u003e, constructed from mitochondrial COI gene sequences collected along the coasts of Thailand, Vietnam, and China, shows circle sizes proportional to haplotype frequencies, with colors representing sampling locations as indicated in the legend. Mutational steps are shown in parentheses, black circles represent median vectors (hypothetical haplotypes), and the values inside the first brackets indicate inferred mutational connections. (B) The intra-population genetic distances of \u003cem\u003eB. areolata \u003c/em\u003efrom Thailand, Vietnam, and China, calculated using the K2P model.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/5074761fb13a275fc8e478c7.jpg"},{"id":85171150,"identity":"a94c232a-7b56-41b2-8912-05ff585897b0","added_by":"auto","created_at":"2025-06-23 05:39:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5139003,"visible":true,"origin":"","legend":"\u003cp\u003eThe left map illustrates the haplotype distribution across \u003cem\u003eB. areolata\u003c/em\u003e populations, with pie charts showing the proportional composition of haplotypes as defined by the TCS network at each sampling location. The right map displays mean sea surface temperatures along with the Philippine Sea and northern ocean currents (labeled 1 and 2), which meet during the northeast monsoon and flow into the Gulf of Thailand via the Vietnamese coast. An inset map of Cam Ranh Bay highlights a distinct ecosystem in central Vietnam.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/c9a81ae987fadc00e41c5aef.jpg"},{"id":85615588,"identity":"a906cc6b-eab0-4110-9e89-8c57fa210147","added_by":"auto","created_at":"2025-06-29 14:32:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21731838,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/f4340ead-820c-44ea-b080-fe71c369bee7.pdf"},{"id":85170215,"identity":"2e7d5349-63c8-439c-bf0c-327af8112741","added_by":"auto","created_at":"2025-06-23 05:31:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":388833,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6937476/v1/5a401236a671468b8316f621.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Population genetic dynamics of the Ivory Snail (Babylonia areolata): Insights from coastal waters of Vietnam for conservation and aquaculture","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe marine gastropods under the genus \u003cem\u003eBabylonia\u003c/em\u003e are regarded as an economically and ecologically important group, particularly within the tropical and subtropical coastal ecosystems of Asia (Di et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Taxonomically, \u003cem\u003eBabylonia\u003c/em\u003e is classified within the family Babyloniidae under the order Neogastropoda, a relatively small group of marine gastropods distinguished by their large, clearly patterned shells and high morphological variability (Fraussen and Stratmann \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Presently, the family Babyloniidae is divided into two genera viz., \u003cem\u003eBabylonia\u003c/em\u003e, comprising 17 recognized species, and \u003cem\u003eZemiropsis\u003c/em\u003e, with five species (MolluscaBase \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While \u003cem\u003eBabylonia\u003c/em\u003e species are broadly distributed from the northern Indian Ocean to the Indo-West Pacific, \u003cem\u003eZemiropsis\u003c/em\u003e species have a more restricted range, primarily inhabiting the southwestern Indian Ocean, including coastal waters of South Africa and southern Mozambique (Altena and Gittenberger \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Fraussen and Rosado \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Typically, \u003cem\u003eBabylonia\u003c/em\u003e species occupy continental shelf habitats at depths ranging from 4 to 80 meters, exhibiting diverse morphological traits such as variations in umbilicus structure, convex sutures, and distinctive apical shell shapes (Zou et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yen et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although \u003cem\u003eBabylonia\u003c/em\u003e was initially placed in the family Buccinidae based on shell morphology, further molecular phylogenetic analyses using mitochondrial genes (\u003cem\u003eCOI\u003c/em\u003e and \u003cem\u003e16S rRNA\u003c/em\u003e) have confirmed its current classification under the family Babyloniidae (Harasewych and Kantor \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hayashi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the extant species diversity, \u003cem\u003eBabylonia areolata\u003c/em\u003e, commonly known as the ivory snail, is widely recognized for its substantial commercial value due to its rapid growth, short reproductive cycle, excellent taste, rich nutritional composition, and potential pharmacological properties (Periyasamy et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Dobson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sutthinon et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Geographically, \u003cem\u003eB. areolata\u003c/em\u003e is distributed across South, Southeast, and East Asia, including regions viz., Sri Lanka, the Nicobar Islands (India), the Gulf of Thailand, coastal Vietnam, southeastern China, and Taiwan (L\u0026uuml; et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zou et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In Southeast Asia, particularly Vietnam, \u003cem\u003eB. areolata\u003c/em\u003e has emerged as a priority species for aquaculture development, with production reaching 11,195 tons in 2024 and market prices ranging from 10 to 15 USD/kg (Vietnam Ministry of Agriculture and Environment \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although aquaculture practice of \u003cem\u003eB. areolata\u003c/em\u003e has emerged as a sustainable approach to alleviate pressure on wild stocks, market demand continues to contribute to significant declines in natural populations due to overharvesting and habitat degradation (L\u0026uuml; et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Di et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, the scientific investigation into the taxonomy, ecology, and reproductive biology of \u003cem\u003eBabylonia\u003c/em\u003e species has long been a focal point of research (Harasewych and Kantor \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Yen et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Advances in molecular systematics utilizing microsatellite, mitochondrial, and nuclear markers have been largely employed in resolving morphological variation (Zou et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e), species delimitation (Yen et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), phylogenetic relationships (Hou et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ran et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and population genetic structure (Hualkasin et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). More recently, the transcriptomic approaches have been also employed to elucidate immune responses to bacterial infections by integrating molecular, biochemical, and histological data (Yu et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, the proteomic analyses have also enhanced understanding of larval physiological responses to environmental stressors such as hypoxia and ocean acidification (Mukherjee et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Di et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite these comprehensive investigations into the genetic diversity and population genetic structure of \u003cem\u003eB. areolata\u003c/em\u003e across its geographic range remain limited, particularly in populations from Vietnam. Therefore, this study aims to (i) analyze genetic diversity and phylogenetic clustering pattern, (ii) estimate the Operational Taxonomic Units (OTUs), and (iii) evaluate haplotype diversity and population dynamics of \u003cem\u003eB. areolata\u003c/em\u003e populations across the coastal regions of Thailand, Vietnam, and China. This population level genetic information is expected to provide a deeper understanding of intra-species genetic variation among distant populations of \u003cem\u003eB. areolata\u003c/em\u003e. The findings offer valuable insights for fisheries resource management, facilitate genetically informed aquaculture programs, and contribute to enhancing the economic value of this marine gastropods through sustainable aquaculture practices in Southeast Asia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and genomic DNA extraction\u003c/h2\u003e \u003cp\u003eA total of 105 individuals of \u003cem\u003eB. areolata\u003c/em\u003e were collected from four distinct coastal provinces in Vietnam, Ninh Thuận (11.582222\u0026deg;N, 109.055833\u0026deg;E), Kh\u0026aacute;nh H\u0026ograve;a (11.897778\u0026deg;N, 109.156111\u0026deg;E), Thua Thien Hue (16.241111\u0026deg;N, 108.082500\u0026deg;E), and B\u0026igrave;nh Thuận (10.573889\u0026deg;N, 107.582500\u0026deg;E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Online Resource Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All collected specimens were transported to the Institute of Biology, Vietnam Academy of Science and Technology, where approximately 100 mg of tissue was carefully dissected from the foot muscle of each specimen and preserved in 80% ethanol for subsequent molecular analyses. The tissue samples were finely sectioned into small fragments and homogenized in DNA extraction buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM EDTA, 1% SDS, and 1 mg/mL proteinase K. The genomic DNA was isolated using the AccuPrep\u0026reg; Genomic DNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) in accordance with the manufacturer\u0026rsquo;s instructions. The concentration and purity of the extracted DNA were evaluated with a NanoDrop spectrophotometer (Thermo Fisher Scientific D1000, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePCR amplification and sequencing\u003c/h3\u003e\n\u003cp\u003eTo amplify a\u0026thinsp;~\u0026thinsp;650 bp fragment of the partial \u003cem\u003eCOI\u003c/em\u003e gene, a published primer pair (LCO and HCO) was used in PCR (Folmer et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The PCR amplification was carried out in a 50 \u0026micro;L reaction mixture containing 1 \u0026micro;L each of forward and reverse primers, 0.9 \u0026micro;L of 3% dimethyl sulfoxide (DMSO), 19.9 \u0026micro;L of sterile deionized water, 3 \u0026micro;L of 10\u0026times; ExTaq buffer, 0.2 \u0026micro;L of ExTaq HS DNA polymerase, 3 \u0026micro;L of dNTP mix, and 1 \u0026micro;L of genomic DNA as the template. The experiment was carried out using a Takara PCR Thermal Cycler Dice\u0026reg; Gradient (Takara Korea Biomedical Inc., Seoul, Republic of Korea). The PCR program included an initial denaturation at 94\u0026deg;C for 3 minutes, followed by 40 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 40\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 1 minute, with a final extension at 72\u0026deg;C for 5 minutes. The PCR products were separated on a 1.5% agarose gel, and the desired DNA bands were excised and purified using the AccuPrep\u0026reg; PCR/Gel Purification Kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer\u0026rsquo;s guidelines. The purified fragments were then submitted to Macrogen (Daejeon, Republic of Korea; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dna.macrogen.com/\u003c/span\u003e\u003cspan address=\"https://dna.macrogen.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for Sanger sequencing in both directions.\u003c/p\u003e\n\u003ch3\u003eDataset construction, genetic distance, and phylogenetic inference\u003c/h3\u003e\n\u003cp\u003eThe partial \u003cem\u003eCOI\u003c/em\u003e gene sequences generated in this study were initially validated through nucleotide BLAST searches on the NCBI platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the consensus sequences were subsequently submitted to the GenBank to acquire unique accession numbers. For downstream analyses, a comprehensive dataset was assembled, comprising 105 newly generated \u003cem\u003eCOI\u003c/em\u003e sequences and 34 additional sequences of \u003cem\u003eB. areolata\u003c/em\u003e and closely related congeners retrieved from GenBank (Online Resource Tables S1\u0026ndash;S2). The sequences alignment was performed using CLUSTAL X software, resulting in a consensus alignment of 620 bp (Thompson et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Both the inter- and intra-species genetic distance was estimated using the Kimura 2-Parameter (K2P) model implemented in MEGA version 12 (Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To infer phylogenetic relationships, a total of 11 \u003cem\u003eBabylonia\u003c/em\u003e species were analyzed, with \u003cem\u003eZemiropsis papillaris\u003c/em\u003e (Accession no. OR677031) designated as the outgroup. The phylogenetic trees were constructed using both Bayesian (BA) and Maximum Likelihood (ML) approaches. The optimal nucleotide substitution model General Time Reversible model (GTR)\u0026thinsp;+\u0026thinsp;gamma-distributed rate heterogeneity (G) and a proportion of invariant sites (I) was selected based on the lowest BIC (Bayesian Information Criterion) score recognized through PartitionFinder 2 and jModelTest v2 (Darriba et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Miller et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lanfear et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The BA analysis was carried out by using MrBayes version 3.1.2, applying the General Time Reversible model with gamma-distributed rate heterogeneity and a proportion of invariant sites (nst\u0026thinsp;=\u0026thinsp;6). The Markov Chain Monte Carlo (MCMC) procedure was run for 1\u0026nbsp;million generations, employing four chains (one cold and three heated), with sampling every 100 generations and discarding the first 25% as burn-in (Ronquist et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For ML approach, phylogenetic inference was carried out using the PhyML 3.0 web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.atgc-montpellier.fr/phyml/\u003c/span\u003e\u003cspan address=\"http://www.atgc-montpellier.fr/phyml/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), incorporating 1000 bootstrap replicates to assess node support (Guindon et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Trifinopoulos et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Both BA and ML phylogenetic trees were visualized using the Interactive Tree of Life (iTOL) platform, version 4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Letunic and Bork \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eOTUs estimation, haplotype diversity, and population genetic differentiation\u003c/h3\u003e\n\u003cp\u003eTo accurately delineate OTUs, a combination of species delimitation methods was employed, encompassing Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), Poisson Tree Processes (PTP), and Generalized Mixed Yule Coalescent (GMYC) framework for the \u003cem\u003eB. areolata\u003c/em\u003e dataset (105 generated\u0026thinsp;+\u0026thinsp;14 database). The alignment file (.fasta) was utilized as input for both ABGD and ASAP analyses with Jukes-Cantor (JC69) model (Puillandre et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In PTP and GMYC analyses, an unrooted ultrametric ML phylogeny in Newick format (.nwk) was used to estimate the putative number of OTUs (Fujisawa and Barraclough \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The species delimitation using ABGD and ASAP was conducted via the SPART web server (Miralles et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), while PTP and GMYC analyses were performed using iTaxoTools v0.1 (Vences et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To further investigate the population genetic structure, a total of 119 \u003cem\u003eCOI\u003c/em\u003e sequences were analyzed to assess the number of haplotypes, haplotype diversity (Hd), and nucleotide diversity (π) using DnaSP version 6.0 (Rozas et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Subsequently, the haplotype relationships were visualized using the Templeton, Crandall, and Sing (TCS) network method implemented in POPART version 1.7 (Clement et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Leigh and Bryant \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The tests of selective neutrality were also conducted for each sampling locality using Tajima\u0026rsquo;s D and Fu and Li\u0026rsquo;s F and D statistics, implemented through the DnaSP version 6.0 (Tajima \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Fu and Li \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic distance and phylogeny\u003c/h2\u003e \u003cp\u003eThe genetic distance analysis based on the partial \u003cem\u003eCOI\u003c/em\u003e gene within the genus \u003cem\u003eBabylonia\u003c/em\u003e revealed notable variation at both inter- and intra- species level. The mean inter-species genetic distances within the genus ranged from 3.0\u0026ndash;20.5%, with the lowest recorded between \u003cem\u003eB. semipicta\u003c/em\u003e and \u003cem\u003eB. spirata\u003c/em\u003e (3.0%) and the highest between \u003cem\u003eB. zeylanica\u003c/em\u003e and \u003cem\u003eB. formosae\u003c/em\u003e (20.5%). Notably, \u003cem\u003eB. areolata\u003c/em\u003e exhibited the lowest inter-species genetic distance with \u003cem\u003eB. borneensis\u003c/em\u003e at 11.2%, while the highest distance was observed with \u003cem\u003eB. zeylanica\u003c/em\u003e at 18.5% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, the genetic distance analysis indicated low mean intra-species genetic variation (0.2%) in \u003cem\u003eB. areolata\u003c/em\u003e. The BA and ML phylogenetic analyses showed monophyletic clustering of \u003cem\u003eB. areolata\u003c/em\u003e, which was distinctly separated from other congeners. Nevertheless, the resulting tree topology indicated a close evolutionary relationship between \u003cem\u003eB. areolata\u003c/em\u003e and \u003cem\u003eB. formosae\u003c/em\u003e, \u003cem\u003eB. japonica\u003c/em\u003e, \u003cem\u003eB. lutosa\u003c/em\u003e, and \u003cem\u003eB. borneensis\u003c/em\u003e in the present dataset. Conversely, \u003cem\u003eB. spirata\u003c/em\u003e, a species native to the Indo-West Pacific, occupied a basal position in the current phylogenetic analyses and was inferred to represent a potential ancestral lineage within the genus \u003cem\u003eBabylonia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Online Resource Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe mean inter-species genetic divergences among \u003cem\u003eBabylonia\u003c/em\u003e species, based on K2P distances estimated from partial mitochondrial \u003cem\u003eCOI\u003c/em\u003e gene sequences.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eInter-\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia areolata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia lutosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia spirata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia semipicta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia formosae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia zeylanica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia japonica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia borneensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia feicheni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia valentiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e14.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBabylonia pieroangelai\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e18.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e15.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e15.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpecies delimitation and population structure\u003c/h3\u003e\n\u003cp\u003eSpecies delimitation analyses of \u003cem\u003eB. areolata\u003c/em\u003e revealed varying numbers of OTUs depending on the different methods applied. Both ABGD and ASAP methods each identified 26 OTUs, while the PTP method detected a higher count of 42 OTUs, and the GMYC method delineated only two OTUs (Online Resource Tables S3\u0026ndash;S5). The haplotype network analysis conducted on \u003cem\u003eB. areolata\u003c/em\u003e sequences from three countries (Vietnam, China, and Thailand) identified Hd\u0026thinsp;=\u0026thinsp;0.6948, π\u0026thinsp;=\u0026thinsp;0.0024, and 26 distinct haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Online Resource Table S6). Among the detected haplotypes, Hap_1 was the most dominant, occurring in numerous individuals across all sampling sites, including four populations in Vietnam, as well as in three sequences from China and four sequences from Thailand. This haplotype occupied a central position within the network and exhibited direct connections to many other haplotypes, indicating its shared genetic characteristics. In addition to Hap_1, Hap_6 also showed a relatively high frequency and comprised sequences from several populations in Vietnam and China. Conversely, the majority of other haplotypes were unique, each found in only one sequence from a single sampling location (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, the two species delimitation methods (ABGD and ASAP), along with population structure analyses, revealed a consistent pattern in the detection of OTUs and haplotypes within the present \u003cem\u003eB. areolata\u003c/em\u003e dataset.\u003c/p\u003e\u003cp\u003eThe inter-population genetic distances were 0.2% between both the Vietnam\u0026ndash;China and China\u0026ndash;Thailand populations, whereas the distance between Vietnam and Thailand populations was 0.3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Genetic diversity indices and neutrality test parameters based on the partial \u003cem\u003eCOI\u003c/em\u003e sequences of \u003cem\u003eB. areolata\u003c/em\u003e from six locations (four in Vietnam, as well as China and Thailand) revealed distinct genetic variation among populations. Among the Vietnamese populations, Kh\u0026aacute;nh H\u0026ograve;a exhibited the highest Hd\u0026thinsp;=\u0026thinsp;0.83669 and π\u0026thinsp;=\u0026thinsp;0.00299, with 17 polymorphic sites and 15 haplotypes identified from 32 \u003cem\u003eCOI\u003c/em\u003e sequences. Conversely, the most southern B\u0026igrave;nh Thuận population showed the lowest genetic diversity, with six haplotypes, eight polymorphic sites, Hd\u0026thinsp;=\u0026thinsp;0.51077, and π\u0026thinsp;=\u0026thinsp;0.00149 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Chinese and Thai populations, although represented by smaller sample sizes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7), displayed moderate levels of genetic diversity. The Chinese population showed Hd\u0026thinsp;=\u0026thinsp;0.80952 and π\u0026thinsp;=\u0026thinsp;0.00193, while the Thai population had Hd\u0026thinsp;=\u0026thinsp;0.71429 and π\u0026thinsp;=\u0026thinsp;0.00246, with three and four polymorphic sites, respectively. The neutrality tests revealed negative Tajima\u0026rsquo;s D values across all populations, ranging from \u0026minus;\u0026thinsp;0.65405 (China) to \u0026minus;\u0026thinsp;2.12186 (Kh\u0026aacute;nh H\u0026ograve;a). The Fu and Li\u0026rsquo;s F values were also negative in all populations, with the most extreme values observed in Kh\u0026aacute;nh H\u0026ograve;a (\u0026ndash;3.40989) and B\u0026igrave;nh Thuận (\u0026ndash;3.36766) in Vietnam (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic diversity indices and neutrality test results based on partial mitochondrial \u003cem\u003eCOI\u003c/em\u003e sequences of \u003cem\u003eB. areolata\u003c/em\u003e populations from Vietnam, China, and Thailand. N: number of sequences, P: polymorphic sites, H: number of haplotypes, K: average number of nucleotide differences, Hd: haplotype diversity, π: nucleotide diversity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eLocations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eDivesity indices\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003eNetrality test\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eπ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTajima\u0026rsquo;s D\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFu and Li\u0026rsquo;s F\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eFu and Li\u0026rsquo;s D\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eVietnam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNinh Thuận\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.20690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.62808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-1.67864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-2.30591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.32583\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKh\u0026aacute;nh H\u0026ograve;a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.61895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.83669\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-2.12186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-3.40989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-3.26510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThua Thien Hue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.58824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.69935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-1.83747\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-2.35660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-2.38333\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u0026igrave;nh Thuận\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.80923\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.51077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-2.06988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-3.36766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-3.23909\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eChina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.04762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.80952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.65405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.59207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-0.51900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eThailand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.33333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.71429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.00246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.87642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.87602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-0.78927\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present phylogenetic analyses confirm the monophyly of \u003cem\u003eB. areolata\u003c/em\u003e, consistent with earlier multi-locus studies employing partial mitochondrial genes (\u003cem\u003eCOI\u003c/em\u003e and \u003cem\u003e16S rRNA\u003c/em\u003e) and the nuclear H3 gene (Yen et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Genetic distance analysis based on \u003cem\u003eCOI\u003c/em\u003e sequences across various \u003cem\u003eBabylonia\u003c/em\u003e species revealed substantial interspecific divergence (3.0\u0026ndash;20.5%), indicating marked evolutionary differentiation and supporting the distinct taxonomic status of \u003cem\u003eB. areolata\u003c/em\u003e. The species delimitation analyses identified variation in the number of OTUs, suggesting potential cryptic diversity within \u003cem\u003eB. areolata\u003c/em\u003e populations inhabiting the coastal waters of Thailand, Vietnam, and China. This cryptic diversity may be driven by local adaptation and oceanographic heterogeneity, as similarly reported in other gastropod taxa (e.g., \u003cem\u003eConus\u003c/em\u003e, \u003cem\u003eLittoraria\u003c/em\u003e, and \u003cem\u003eNerita\u003c/em\u003e) (Postaire et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ameri et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shin and Allmon \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond cryptic diversity, the genetic patterns in marine taxa often reflect the complex interplay between environmental factors and biological traits (Faria et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ocean currents, sea surface temperature (SST), and bathymetry can either enhance population connectivity or act as barriers to gene flow, depending on localized hydrodynamic regimes (Fontana et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, population genetic analyses of \u003cem\u003eB. areolata\u003c/em\u003e from Thailand, Vietnam, and China revealed remarkably low intra-specific (0.2%) and inter-population (0.2\u0026ndash;0.3%) genetic distances. This high connectivity is consistent with findings in other marine gastropods, where extensive larval dispersal and seasonal ocean currents facilitate widespread gene flow (Cho et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Haslam et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Such gene flow among geographically distant populations may pose risks of inbreeding depression and the erosion of unique wild genetic resources, concerns critical to sustainable aquaculture and broodstock management. The haplotype network of \u003cem\u003eB. areolata\u003c/em\u003e revealed the presence of unique haplotypes restricted to Fujian, China, implying partial genetic isolation likely caused by geographic or oceanographic barriers, parallel to patterns seen in \u003cem\u003eNerita yoldii\u003c/em\u003e in Chinese coastal waters (Tang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, in the coastal waters of Vietnam, \u003cem\u003eB. areolata\u003c/em\u003e populations exhibited both shared and unique haplotypes. The sequences generated from Kh\u0026aacute;nh H\u0026ograve;a in central Vietnam displayed shared haplotypes with Chinese and Thai populations, possibly due to seasonal current-driven larval dispersal in deeper waters (Bashevkin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Simultaneously, a few sequences from the same locality exhibited unique haplotypes, potentially confined to the shallow waters in close proximity to Cam Ranh Bay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These localized haplotypes may reflect adaptation to spatially and temporally variable environments and interactions with coexisting biotic components (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Quang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nguyen and Jutta \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conversely, the \u003cem\u003eB. areolata\u003c/em\u003e populations from B\u0026igrave;nh Thuận, Ninh Thuận, and Thừa Thi\u0026ecirc;n Huế demonstrated high degree of gene flow with populations from China and Thailand. This genetic mixing may be influenced by the Philippine Sea and northern ocean currents, which converge during the northeast monsoon and enter the Gulf of Thailand via the Vietnamese coast (Pongparadon et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, the SSTs of Kh\u0026aacute;nh H\u0026ograve;a, Ninh Thuận, and Thừa Thi\u0026ecirc;n Huế are comparable to those in the Taiwan Strait, potentially supporting the persistence of shared haplotypes between distant populations. Notably, the shared thermal conditions between B\u0026igrave;nh Thuận and the Gulf of Thailand may have facilitated gene flow, a pattern commonly observed in other marine taxa (Masanja et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is evidenced that the life history traits of \u003cem\u003eB. areolata\u003c/em\u003e, particularly its planktonic larval phase, contribute to long-distance dispersal via oceanic currents (Shen et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our findings underscore the significant influence of oceanographic variability, hydrodynamic regimes, and demographic processes may shape the genetic structure of \u003cem\u003eB. areolata\u003c/em\u003e across coastal Thailand, Vietnam, and China. Nevertheless, the potential impact of human-mediated translocations, particularly through aquaculture activities, must not be overlooked. Such anthropogenic pressure may have led to population admixture, thereby lost their genetic distinctiveness in marine environment. Despite this, the presence of a few unique haplotypes in Thailand, Vietnam, and China suggests historical and biogeographic influences requiring further investigation. The geological history and paleoclimatic changes, particularly the sea-level oscillations during the Pleistocene, likely played a crucial role in shaping present-day genetic patterns of marine organisms in Southeast and East Asia by fragmenting habitats and isolating populations within shallow bays and gulfs separated by sills and ridges, thereby limiting gene flow (He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Niu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In summary, this study offers essential insights into the population genetics of \u003cem\u003eB. areolata\u003c/em\u003e, with direct implications for broodstock selection in aquaculture, especially from Vietnam. The comprehensive understanding of genetic differentiation can guide selective breeding programs targeting desirable traits such as rapid growth, reproductive efficiency, disease resistance, and environmental resilience (Robledo et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the observed population structure emphasizes the importance of strict quarantine measures and management strategies to conserve genetically distinct populations across Thailand, Vietnam, and China.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a molecular assessment of \u003cem\u003eB. areolata\u003c/em\u003e populations from the coastal waters of Thailand, Vietnam, and southern China, revealing low intra- and inter-population genetic divergences based on \u003cem\u003eCOI\u003c/em\u003e gene sequences (0.2\u0026ndash;0.3%). The phylogenetic analysis identified \u003cem\u003eB. areolata\u003c/em\u003e as a distinct monophyletic lineage, whereas species delimitation analyses revealed multiple OTUs, suggesting the existence of potential cryptic diversity within this marine gastropod. The presence of a single dominant haplotype broadly distributed across the study regions indicates strong gene flow, likely mediated by larval dispersal and prevailing oceanographic currents. In contrast, the detection of unique haplotypes in specific localities across Thailand, Vietnam, and China suggests regional genetic structuring, potentially shaped by historical demographic events and subsequent colonization processes, particularly in semi-enclosed systems such as bays and gulfs. These findings underscore the utility of mitochondrial DNA markers in elucidating population structure and uncovering hidden genetic diversity, offering crucial insights for conservation genetics and informing the development of sustainable aquaculture practices to preserve the genetic integrity of wild \u003cem\u003eB. areolata\u003c/em\u003e populations in Southeast and East Asia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also wish to express their gratitude to Ms. Nguyen Thi Ngoc and Ms. Do Thi Thanh Trung of the Institute of Biology, Vietnam Academy of Science and Technology, Vietnam; Professor Le Duc Minh, Dr. To Thanh Thuy, and Mr. Le Toan Thang of University of Science, Vietnam National University, Hanoi, Vietnam; Dr. Nguyen Thi Anh Thu of Nha Trang University, undergraduate student (Nguyen Thi Huong Tra) and Ms. Kieu Thi Hong of Hanoi University of Pharmacy, Vietnam; Dr. Vu Van In, Vietnam Japan University, Vietnam National University, Hanoi, Vietnam for their valuable assistance during the experimental procedures. Jingda Kang is supported by the master program of University of Stirling, UK and Nguyen Duc Long is supported by the master program of University of Science, Vietnam National University, Hanoi, Vietnam.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the Chey Institute for Advanced Studies\u0026rsquo; International Scholar Exchange Fellowship for the academic year of 2024\u0026ndash;2025 in South Korea. Sang Van Vu is also supported by the Postdoctoral Scholarship Program of Vingroup Innovation Foundation (VINIF), under grant number VINIF.2024.STS.52.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S.V.V., S.K., H.-W.K.; software, S.K., S.A., A.P.; validation, N.D.L., J.K.; formal analysis, S.V.V., S.A., A.P., S.R.L.; data curation, N.D.L., T.T.L., S.V.V.; writing\u0026mdash;original draft preparation, S.V.V., S.A., A.P.; writing\u0026mdash;review and editing, H.-W.K., S.K.; visualization, E.S., A.G., A.S.; supervision, S.K., H.-W.K., H.H.N.; project administration, S.V.V., S.K., H.-W.K.; funding acquisition, S.V.V., H.-W.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA sequences analyzed in this study were deposited in GenBank under the accession numbers PV478301\u0026ndash;PV478405.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest related to this work.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors provided their consent to participate in the project and to contribute to the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSang Van Vu: https://orcid.org/0000-0003-1929-4419\u003c/p\u003e\n\u003cp\u003eSarifah Aini: https://orcid.org/0009-0001-1065-4724\u003c/p\u003e\n\u003cp\u003eAngkasa Putra: https://orcid.org/0000-0002-5533-9437\u003c/p\u003e\n\u003cp\u003eSoo Rin Lee: https://orcid.org/0000-0002-6443-855X\u003c/p\u003e\n\u003cp\u003eNguyen Duc Long: https://orcid.org/0009-0008-4285-4115\u003c/p\u003e\n\u003cp\u003eThanh Tat Le: https://orcid.org/0009-0004-2702-1482\u003c/p\u003e\n\u003cp\u003eHoang Huy Nguyen: https://orcid.org/0000-0002-6284-5813\u003c/p\u003e\n\u003cp\u003eEric Saillant: https://orcid.org/0000-0002-1480-4515\u003c/p\u003e\n\u003cp\u003eAlmas Gheyas: https://orcid.org/0000-0002-7682-4394\u003c/p\u003e\n\u003cp\u003eArmin Sturm:\u0026nbsp;https://orcid.org/0000-0003-2632-1999\u003c/p\u003e\n\u003cp\u003eJingda Kang: https://orcid.org/0000-0002-6018-5532\u003c/p\u003e\n\u003cp\u003eHyun-Woo Kim: https://orcid.org/0000-0003-1357-5893\u003c/p\u003e\n\u003cp\u003eShantanu Kundu: https://orcid.org/0000-0002-5488-4433\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAltena COR, Gittenberger E (1981) The genus \u003cem\u003eBabylonia\u003c/em\u003e (Prosobranchia: Buccinidae). 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Genet Mol Res 10:3230\u0026ndash;3235. https://doi.org/10.4238/2011.December.21.5\u003c/li\u003e\n \u003cli\u003eXu JW, Wang J, Dong YW (2024) Genetic determination of a cryptic species in the \u003cem\u003eLittoraria\u003c/em\u003e genus with whole-genome molecular resolution. Ecol Evol 14:e70715. https://doi.org/10.1002/ece3.70715\u003c/li\u003e\n \u003cli\u003eYen Y-H, Joseph J, Liu S-YV (2025) Delimiting species boundaries within the Babyloniidae (Mollusca: Gastropoda: Neogastropoda) using multi-locus phylogenetic analysis. Zool Scr 54:17\u0026ndash;32. https://doi.org/10.1111/zsc.12694\u003c/li\u003e\n \u003cli\u003eYu J, L\u0026uuml; W, Zhang L et al (2024) Effects of \u003cem\u003eVibrio harveyi\u003c/em\u003e infection on the biochemistry, histology, and transcriptome in the hepatopancreas of ivory shell (\u003cem\u003eBabylonia areolata\u003c/em\u003e). 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Aquacult Res 53:1636\u0026ndash;1648. https://doi.org/10.1111/are.15694\u003c/li\u003e\n \u003cli\u003eZou S, Li Q, Kong L (2011b) Additional gene data and increased sampling give new insights into the phylogenetic relationships of Neogastropoda, within the caenogastropod phylogenetic framework. \u003cem\u003eMol Phylogenet Evol\u0026nbsp;\u003c/em\u003e61:425\u0026ndash;435. https://doi.org/10.1016/j.ympev.2011.07.014\u003c/li\u003e\n \u003cli\u003eZou S, Li Q, Kong L et al (2011a) Comparing the usefulness of distance, monophyly, and character-based DNA barcoding methods in species identification: A case study of Neogastropoda. \u003cem\u003ePLoS One\u003c/em\u003e 6:e26619. https://doi.org/10.1371/journal.pone.0026619\u003c/li\u003e\n \u003cli\u003eZou W, Gan Y, Hong J et al (2025) A study on the chemosensory organs, feeding behavior, and attractant substances of \u003cem\u003eBabylonia areolata\u003c/em\u003e. Aquac Rep 43:102906. https://doi.org/10.1016/j.aqrep.2025.102906\u003c/li\u003e\n \u003cli\u003eZou Y, Fu J, Liang Y et al (2024) Chromosome-level genome assembly of the ivory shell \u003cem\u003eBabylonia areolata\u003c/em\u003e. Sci Data 11:1201. https://doi.org/10.1038/s41597-024-03085-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Marine gastropod, Mitochondrial DNA, Genetic diversity, Phylogeny, Population structure, Fisheries management","lastPublishedDoi":"10.21203/rs.3.rs-6937476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6937476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ivory snail \u003cem\u003eBabylonia areolata\u003c/em\u003e has experienced a significant population decline in marine ecosystem due to the overharvesting, habitat loss, and climate change. Despite its ecological significance and commercial value, the population genetic studies on this gastropod remain limited. This study provides the first genetic insight into \u003cem\u003eB. areolata\u003c/em\u003e based on 105 newly generated mitochondrial cytochrome c oxidase subunit I (\u003cem\u003eCOI\u003c/em\u003e) gene sequences collected from Vietnam. The analysis revealed the lowest interspecific genetic distance (11.2%) between \u003cem\u003eB. areolata\u003c/em\u003e and \u003cem\u003eBabylonia borneensis\u003c/em\u003e, and the highest (18.5%) with \u003cem\u003eBabylonia zeylanica\u003c/em\u003e. The Bayesian and Maximum-likelihood phylogenetic analyses showed \u003cem\u003eB. areolata\u003c/em\u003e as a distinct monophyletic lineage, while species delimitation methods recovered multiple operational taxonomic units, suggesting the potential presence of cryptic diversity. The low inter-population divergence (0.2\u0026ndash;0.3%) further indicated a high level of genetic connectivity among \u003cem\u003eB. areolata\u003c/em\u003e populations across coastal waters of Thailand, Vietnam, and China. The haplotype network analysis revealed 26 haplotypes, including a dominant central haplotype, supporting the hypothesis that larval dispersal and regional ocean currents have shaped gene flow of \u003cem\u003eB. areolata\u003c/em\u003e. Additionally, the presence of several unique haplotypes in the study regions may reflect historical geological events and demographic isolation, which likely shaped the distinct populations of \u003cem\u003eB. areolata\u003c/em\u003e, especially in enclosed coastal areas like Cam Ranh Bay in central Vietnam. These findings underscore the importance of molecular tools in elucidating population structure and offer critical insights for the conservation and sustainable aquaculture of \u003cem\u003eB. areolata\u003c/em\u003e in Southeast and East Asia.\u003c/p\u003e","manuscriptTitle":"Population genetic dynamics of the Ivory Snail (Babylonia areolata): Insights from coastal waters of Vietnam for conservation and aquaculture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 05:31:47","doi":"10.21203/rs.3.rs-6937476/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31194f64-5eaf-4c1a-bfc1-4b47737b70f9","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-29T14:23:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-23 05:31:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6937476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6937476","identity":"rs-6937476","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}