Genetic divergence and migration patterns of a galatheoid squat lobster highlight the need for deep‐sea conservation

Information on genetic divergence and migration patterns of vent‐ and seep‐endemic macrobenthos can help delimit biogeographical provinces and provide scientific guidelines for deep‐sea conservation under the growing threats of anthropogenic disturbances. Nevertheless, related studies are still scarce, impeding the informed conservation of these hotspots of deep‐sea biodiversity. To bridge this knowledge gap, we conducted a population connectivity study on the galatheoid squat lobster Shinkaia crosnieri – a deep‐sea foundation species widely distributed in vent and seep ecosystems in the Northwest Pacific. With the application of an interdisciplinary methodology involving population genomics and oceanographic approaches, we unveiled two semi‐isolated lineages of S. crosnieri with limited and asymmetrical gene flow potentially shaped by the geographic settings, habitat types, and ocean currents – one comprising vent populations in the Okinawa Trough, with those inhabiting the southern trough area likely serving as the source; the other being the Jiaolong (JR) seep population in the South China Sea. The latter might have recently experienced a pronounced demographic contraction and exhibited genetic introgression from the Okinawa Trough lineage, potentially mediated by the intrusion of the North Pacific Intermediate Water. We then compared the biogeographic patterns between S. crosnieri and two other representative and co‐occurring vent‐ and seep‐endemic species using published data. Based on their biogeographical subdivisions and source‐sink dynamics, we highlighted the southern Okinawa Trough vents and the JR seep warrant imperative conservation efforts to sustain the deep‐sea biodiversity in the Northwest Pacific.


| INTRODUC TI ON
Powered by chemosynthetic primary production, hydrothermal vent and hydrocarbon seep ecosystems not only contribute to biodiversity in the deep ocean and play vital roles in global biogeochemical cycling, but also harbour a wealth of natural resources including gas hydrate, rare metals, and seafloor massive sulphides (SMSs) (Levin et al., 2016;Van Dover, 2000).With over 90 vents and 70 seeps discovered and at least 20% of macrobenthic species shared between these two habitats, the Northwest Pacific has been recognized as an evolutionary hotspot for deep-sea organisms (Beaulieu & Szafrański, 2020;Wang et al., 2022;Watanabe et al., 2010).However, owing to the growing energy demand during the past decades, vent and seep ecosystems in the Northwest Pacific have also become targets for resource exploitation.For instance, scientific drilling of gas hydrates has been initiated in the South China Sea seeps since 2007 (Zhang et al., 2021), test mining of SMSs has been attempted in the Okinawa Trough vents since 2017 (Okamoto et al., 2019), and the same for cobalt-rich crusts in the Takuyo-Daigo Seamount since 2020 (Washburn et al., 2023).These human activities can severely impact vent and seep ecosystems, including alternation of ecological functions and loss of biodiversity at the genetic, species, and ecosystem levels (Thomas et al., 2021;Van Dover, 2014;Washburn et al., 2023).
Consequently, there has been an increasing urgency in studying the population connectivity of vent-and/or seep-dwelling species, especially their genetic divergence and migration patterns.Such knowledge could cast light on the biogeographical subdivisions and source-sink dynamics of specialist species, and provide scientific guidelines for the management of deep-sea biodiversity confronted with local anthropogenic disturbances and global climate change (Baco et al., 2016;Brunner et al., 2022).Nevertheless, related studies are still scarce, which greatly hinders the data-driven planning of deep-sea conservation.
For most deep-sea macrobenthic species, population connectivity mainly occurs during their pelagic larval dispersal period (Van Dover et al., 2002).Therefore, it is crucial to investigate the long-distance migration and regional-scale differentiation of representative vent-and/or seep-endemic species from different taxonomy groups with diverse life-history traits prior to properly defining deep-sea conservation units (Baco et al., 2016;Van Dover et al., 2012).Nowadays, large-scale population connectivity studies relying on abundant single-nucleotide polymorphisms (SNPs) derived from omics-based techniques have been performed on | 3 of 16 two species of vent-and seep-dwelling macrobenthos in the Northwest Pacific (Table 1).One is the bathymodioline mussel Gigantidas platifrons (previously Bathymodiolus platifrons) which was inferred to produce planktotrophic larvae (Laming et al., 2018;Xu et al., 2018), and the other is the patellogastropod limpet Bathyacmaea nipponica which likely produce lecithotrophic larvae (Ponder et al., 2020;Xu et al., 2021).Both species have a wide distribution in the Northwest Pacific, indicating high connectivity among their geographically disjoint populations (Xu et al., 2018(Xu et al., , 2021)).Nevertheless, two cryptic semi-isolated lineages with three genetic groups were detected for G. platifrons (Xu et al., 2018): one in the Jiaolong Ridge (JR; also known as Site F or Formosa Ridge) seep in the South China Sea; the other spanning vent fields across the Okinawa Trough to the Off Hatsushima seep in the Sagami Bay, with populations in the southern Okinawa Trough exhibiting a fine-scale genetic divergence from the others of the same lineage (Xu et al., 2018).Comparatively, four habitat-linked genetic groups were identified for B. nipponica, including three seep genetic groups inhabiting the JR seep in the South China Sea, the Kuroshima Knoll seep in the Ryukyu Arc, and the Off Hatsushima seep in the Sagami Bay, along with one vent genetic group across the Okinawa Trough (Xu et al., 2021).(Watanabe et al., 2010;Zhao et al., 2020).

The galatheoid squat lobster
Shinkaia crosnieri is known to produce lecithotrophic larvae (Miyake et al., 2010), and a few studies have shed light on its population connectivity and environmental adaptation (Cheng et al., 2020;Shen et al., 2016;Xiao et al., 2020;Yang et al., 2016).However, these studies suffered from several limitations, such as including only one to two adjacent populations of S. crosnieri in the Okinawa Trough which were insufficient to illustrate their genetic diversity and source-sink dynamics in the Northwest Pacific (Cheng et al., 2020;Shen et al., 2016;Xiao et al., 2020;Yang et al., 2016), utilizing only one to a few genetic markers which failed to disentangle population admixture (Shen et al., 2016;Yang et al., 2016), and/or showing conflicted signals of migration patterns potentially due to the small sample sizes (Cheng et al., 2020;Xiao et al., 2020).
To address these issues, we collected 100 specimens of S. crosnieri from the JR seep and five representative hydrothermal vents across the Okinawa Trough, including the Waka Site of the Futagoyama Field (FF), the Tarama Knoll Field (TK), the Daisan-Kume Knoll Field (DK), the Sakai Field (SF), and the Iheya North Original Site of the Iheya North Field (IN), for a more integrative population connectivity study (Figure 1d and Table S1).With an interdisciplinary methodology involving population genomics and oceanographic approaches, we aimed to enhance our knowledge on the genetic divergence and migration patterns of S. crosnieri.Furthermore, we compared the biogeographic subdivisions and source-sink dynamics of S. crosnieri with those of G. platifrons (Xu et al., 2018) and B. nipponica (Xu et al., 2021), aiming to identify vent fields and seep areas that warrant prioritized conservation efforts in the Northwest Pacific.

| Sample collection
One hundred adults of S. crosnieri from the JR seep in the South China Sea and five representative vents (i.e., FF, TK, DK, SF, and IN) across the Okinawa Trough were collected between 2010 and 2018 using the human-occupied vehicle (HOV) SHINKAI 6500, and the remotely operated vehicles (ROVs) HYPER-DOLPHIN, KAIKO, and ROPOS (Figure 1d and Table S1).Samples were frozen at −80°C or preserved in 99.5% ethanol for later dissection and genomic DNA extraction.

| DNA extraction and genotyping-by-sequencing
Thoracic muscle of each specimen was dissected for DNA extraction using the CTAB method (Stewart Jr & Via, 1993).Purity, integrity, and quantity of DNA were examined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific), 1.0% agarose gel electrophoresis, and a Qubit® 2.0 Fluorometer (Invitrogen), respectively.

| Genome-wide SNP identification and filtration
High-quality reads of each individual were mapped to a S. crosnieri survey genome (Cheng et al., 2020; shared from the lead contact upon request) using the MEM algorithm implemented in BWA v.0.7.17 (Li & Durbin, 2009).The obtained .samfiles were transformed into .bamfiles and sorted using SAMtools v.1.9(Li et al., 2009).The depth at each base of each sorted .bamfile was calculated using the depth option of SAMtools v.1.9(Li et al., 2009).Subsequently, the depths at all bases for each individual were summarized to calculate the average value, providing a rough estimation of the mean depth.Nonunique mapped reads and duplicate reads resulting from polymerase chain reactions were removed using sambamba v.0.7.1 (Tarasov et al., 2015).

| Phylogenetic network construction and genetic divergence investigation
A phylogenetic network was constructed with the NeighborNet method implemented in SplitsTree v.4.15.1 (Huson & Bryant, 2006) based on the entire SNP dataset.The normalized distances were measured using the uncorrected P algorithm, and the Handle Ambiguous States option was set to MatchStates.
The pairwise Fixation index (F ST ) was estimated using Arlequin v.3.5.2.2 (Excoffier & Lischer, 2010) based on the entire SNP dataset to evaluate genetic differentiation between populations, running 10,000 permutations to test for statistical significance.
Population structure was investigated using three approaches relying on the single SNP dataset to avoid potential bias derived from linkage disequilibrium.Firstly, SNPRelate v.1.20.1 (Zheng et al., 2012) was utilized for principal component analysis to explore genetic discrepancies among individuals.Secondly, SpaceMix v.0.13 (Bradburd et al., 2016) was run to assess population subdivisions by estimating the geogenetic position of each individual on a geogenetic map.The allele count and the sample size of each variant nucleotide site for all individuals were used as the input, and the sampling coordinates were applied as the means of the spatial priors on the geogenetic locations.Ten fast runs with a "target" model (i.e., estimating geogenetic locations) were initially conducted, each with 1,000,000 Markov Chain Monte Carlo (MCMC) iterations.A long run with a "target" model was then performed with 10,000,000 MCMC iterations, sampling every 1000 MCMC iterations, and saving outputs every 1,000,000 MCMC iterations.
The 95% credible ellipse for the geogenetic location of each individual was generated after a 50% burn-in MCMC iterations of the long run.Finally, LEA v.3.6.0 (Frichot & François, 2015) was applied to infer ancestry coefficients of each individual and assign them to population groups.Ten runs were conducted for each K (i.e., the number of population groups) ranging from 1 to 6, with each run containing a maximum of 10,000 iterations.The cross-entropy criterion for each K was calculated using the -c option, with the smallest value indicating the optimal K (Frichot & François, 2015).
Ancestry coefficients of each individual under the optimal K were depicted using the barplot function in R (R Core Team, 2023).

| Genetic statistics estimation
Expected heterozygosity (H exp ) under Hardy-Weinberg equilibrium, H obs , and nucleotide diversity (π) were estimated for all variant nucleotide sites at both population and genetic-group levels using the POPULATIONS module incorporated in Stacks v.2.5 (Rochette et al., 2019).

| Admixture source prediction
The potential admixture source of each population was predicted using SpaceMix v.0.13 (Bradburd et al., 2016) based on the single SNP dataset by determining the geogenetic position of each population and its admixture source on a geogenetic map.The sampling coordinates served as the means of the spatial priors on the geogenetic locations.The allele count and the sample size of each variant nucleotide site from each population were used as the input.
Parameter settings for the fast and long runs were identical to the SpaceMix analysis performed at the individual level as mentioned in the section 2.4 | Phylogenetic network construction and genetic divergence investigation, except that a "source_and_target" model (i.e., estimating geogenetic locations and admixture source locations) was utilized in the long run.

| Relative migration characterization
Relative migration between populations and between genetic groups were characterized using DivMigrate-online (Sundqvist et al., 2016) relying on the single SNP dataset.The D statistic was applied to estimate the relative migration coefficients, running 100 bootstraps to test for statistical significance.

| Demographic history inferences
Demographic history of each identified genetic group was inferred using Stairway Plot v.2.1.1 (Liu & Fu, 2020) relying on the folded site frequency spectrum, which was generated based on the single SNP dataset using easySFS.py(https:// github.com/ isaac overc ast/ easySFS) with a projection size of 20 for each genetic group.
Stairway Plot analysis was executed under the default settings, except for excluding singletons and increasing the input file number to 1000 to produce the median and the 95% pseudo-confidence interval.Due to the unavailability of the mutation rate and the generation time of S. crosnieri, a mutation rate of 2.64 × 10 −9 substitutions/site/generation (Silliman et al., 2021) and a 1-year generation time (Cabezas et al., 2012) were applied tentatively to indicate the trends of recent changes for the effective population size (N e ) of each genetic group.

| Hydrodynamic modelling analyses
To gain a deeper understanding of the migration patterns and genetic divergence of S. crosnieri from an oceanographic perspective, a series of numerical particle release experiments were conducted For each experiment, approximately 3000 numerical particles within a 5 km radius were released daily for 30 days, and these particles were allowed to flow passively with ocean currents for 3 years (365 days/year × 3 years = 1095 days).Trajectories of numerical particles were calculated based on a step-adapting fourth/fifth-order Runge-Kutta method (Wang et al., 2016) and visualized using MATLAB v.R2018a (https:// www.mathw orks.com/ produ cts/ matlab.html).

| Data processing and SNP detection
Sequencing the GBS libraries yielded an average of 9.44 million raw reads per individual, and quality control resulted in an average of 9.00 million high-quality reads per individual (Table S2).Mapping the high-quality reads per individual to a survey genome of S. crosnieri (Cheng et al., 2020) resulted in a mean depth ranging from 7.02 to 15.95 (Table S2).Genotyping and filtering produced 12,977 SNPs on 6122 scaffolds (Table S3).

| Phylogenetic network and genetic divergence
SplitsTree revealed a phylogenetic network comprising two genetic clusters: one containing all individuals from the JR seep and the other including those from the five Okinawa Trough vents (Figure 2a).
Pairwise F ST values between the Okinawa Trough vent populations were all negative (i.e., −0.0187 to −0.0040) and statistically non-significant; in comparison, those between the JR seep population in the South China Sea and each Okinawa Trough vent population varied from 0.2062 to 0.2301, with statistical significance detected after Bonferroni correction (Figure 2b and Table S4).
Principal component analysis implemented in SNPRelate assigned S. crosnieri into two genetic groups along the principal component 1: all individuals from the JR seep forming a diffusely clustered genetic group, while all those from the five Okinawa Trough vents forming a densely clustered genetic group (Figure 2c).

SpaceMix revealed two clusters of geogenetic locations: one
containing those of all individuals from the JR seep and the other including those of the rest individuals inhabiting the five Okinawa Trough vents (Figure 2d).
Besides uncovering the same two genetic groups (Figure 2e,f), LEA also disclosed a minor to moderate admixture between them (Figure 2f and Table S5).Notably, individuals of the South China Sea (i.e., the JR seep) genetic group possessed an average ancestry coefficient of 7.14% ± 9.48% (maximum = 32.80%)derived from the Okinawa Trough genetic group.By contrast, individuals of the Okinawa Trough genetic group exhibited a lower average ancestry coefficient of 1.55% ± 1.91% (maximum = 8.06%) derived from the South China Sea genetic group.

| Genetic statistics
Genetic statistics for the six populations and two genetic groups of S. crosnieri are summarized in Table 2 and detailed in Tables S6 and   S7.The mean H obs and π of the JR seep population, also identified as the South China Sea genetic group, were higher than those of the five Okinawa Trough vent populations and those of the entire Okinawa Trough genetic group.

| Sources of potential admixture
SpaceMix illustrated the MAP potential source of admixture for the JR seep population was located in the 95% credible ellipses for the geogenetic locations of two Okinawa Trough vent populations (Figure 3a).Comparatively, the MAP potential source of admixture for each Okinawa Trough vent population was located in the 95% credible ellipses for the geogenetic locations of three to five Okinawa Trough vent populations (Figure 3b-f).

| Relative migration
DivMigrate-online uncovered limited and asymmetrical gene flow from the Okinawa Trough to the JR seep at both the population (Figure 4a) and the genetic-group levels (Figure 4b), while intensive gene flow was found among the Okinawa Trough vent populations (Figure 4a,c).Specifically, statistical significance was detected in the gene flow from four of the five Okinawa Trough vent populations to the JR seep population (Figure 4a), as well as in the gene flow from the Okinawa Trough genetic group to the South China Sea genetic group (Figure 4b).
The FF vent population in the southern Okinawa Trough contributed to a stronger gene flow to three other Okinawa Trough populations than reversely (Figure 4a,c).Comparatively, the TK vent population (i.e., the deepest vent examined) in the southern Okinawa Trough received a stronger gene flow from the other vent populations than in the opposite directions (Figure 4a,c).

| Demographic history
Stairway Plot unveiled an increase in the historical N e for both the South China Sea and the Okinawa Trough genetic groups (Figure 5).
Additionally, the South China Sea genetic group may have experienced a pronounced demographic contraction recently, resulting in a smaller contemporary N e compared to that of the Okinawa Trough genetic group (Figure 5).

| Regional hydrodynamics
Distribution patterns of the released numerical particles are shown in Figure 6 and Movies S1-S24.Trajectory patterns of numerical particles released from a chosen coordinate at a certain water depth varied between winter (Figure 6a-l and Movies S1-S12) and summer (Figure 6m-x and Movies S13-S24).Nevertheless, some common features of the regional hydrodynamics can still be inferred in both seasons.
For instance, with an increase in water depth, fewer numerical particles released from the JR coordinate could flow passively into the Okinawa Trough (e.g., Figure 6a,e,i in winter; Figure 6m,q,u in summer).As the water depth increases, more numerical particles released from the FF (e.g., Figure 6b   potentially undergo an early speciation process.This result is consistent with previous studies that included only one to two adjacent Okinawa Trough vent populations and/or a few gene markers (Cheng et al., 2020;Shen et al., 2016;Xiao et al., 2020;Yang et al., 2016).
However, for the first time, we uncovered the southern Okinawa Trough populations of S. crosnieri served as the potential source.
Moreover, its South China Sea lineage seems to have recently experienced a pronounced demographic contraction and exhibited genetic introgression from the Okinawa Trough lineage.
In this section, we mainly discussed the potential drivers that may have mediated the genetic differentiation and the limited and asymmetrical gene flow between the two semi-isolated lineages of S. crosnieri, and compared the biogeographic subdivisions and sourcesink dynamics of S. crosnieri with two other representative and co-occurring vent-and seep-endemic species based on published data (Xu et al., 2018(Xu et al., , 2021)).In light of these research findings, we underlined vent fields and seep areas that warrant imperative conservation efforts in the Northwest Pacific, which will be crucial for the sustainable development of deep-sea resources and the data-driven determination of marine protected areas in the global ocean.

| Genetic divergence in the Northwest Pacific
Hydrothermal vents and hydrocarbon seeps are island-like ecosystems usually distributed in tectonically active regions and along continental margins in the deep ocean, with nearby sites separated by tens to hundreds of kilometres (Van Dover et al., 2002).
Population connectivity of most vent-and/or seep-dwelling macrobenthic species is thus mainly accomplished via larval dispersal during their early life stage (Van Dover et al., 2002).For S. crosnieri, its female broods oil-rich eggs attached to the pleopods (Figure 1c) and then produce lecithotrophic larvae to achieve connectivity between their geographically disjoint populations (Miyake et al., 2010).
Therefore, one plausible biotic factor that could facilitate the formation of its two semi-isolated lineages in the Northwest Pacific is the marginal marine features of the South China Sea, which might impede the larval dispersal of S. crosnieri between its JR seep population and those inhabiting vent fields across the Okinawa Trough (Xu et al., 2018).Furthermore, habitat types may also contribute to the genetic subdivisions of S. crosnieri, enhancing the genetic divergence of its two semi-isolated lineages (i.e., one in the JR seep and the other across the Okinawa Trough) with numerous differentially expressed genes associated with stress response, immunity, and detoxification (Cheng et al., 2019;Xiao et al., 2020).

Shinkaia crosnieri was initially discovered from vent fields in the
Edison Seamount and the Okinawa Trough (Baba & Williams, 1998).
Recently, S. crosnieri has also been reported from the Haima seep in the South China Sea and the seep area in the Krishna-Godavari basin in the Indian Ocean based on morphological characterization (Gonsalves et al., 2022;He et al., 2023).Although several studies have illustrated the Indo-West Pacific colonization of marine taxa in deep-sea vent and seep ecosystems (e.g., Chen et al., 2021;Thomas et al., 2022;Tunnicliffe & Breusing, 2022), the disjunct distribution of S. crosnieri remains puzzling.Future deep-sea surveys in the Indo-West Pacific may discover more S. crosnieri populations.Population connectivity studies for them with a high-quality genome will further improve our understanding of their genetic diversity, biogeographical patterns, and adaptive evolution across different oceans and habitats.

| Genetic homogeneity and migration patterns across the Okinawa trough
Our population genomic analyses revealed that S. crosnieri inhabiting the five Okinawa Trough vents belonged to one distinct lineage, with a stronger gene flow from the shallower to the deepest vent populations examined (Figures 2 and 4 and Table S4).Between two previously studied species co-occurring with S. crosnieri across the Okniawa Trough, this pattern is more similar to that of the patellogastropod limpet B. nipponica than the bathymodioline mussel G. platifrons (Xu et al., 2018(Xu et al., , 2021)).
Both G. platifrons and B. nipponica spawn eggs into the water column where fertilization occurs (Chen et al., 2019;Laming et al., 2018;Xu et al., 2021).However, the former likely produces planktotrophic larvae that generally disperse in the upper ocean layer, while the latter was suggested to produce lecithotrophic larvae that mainly    , 2020;Xu et al., 2021) (Table 1).Shinkaia crosnieri and B. nipponica share similarities in the larval development mode (i.e., lecithotrophy), genetic divergence (i.e., a metapopulation formed across the Okinawa Trough), and gene flow patterns (i.e., a stronger gene flow from the shallower to the deepest vent population examined in the Okinawa Trough) (Xu et al., 2021).Therefore, the lecithotrophic larvae of S. crosnieri may also mainly disperse along the middle to deeper ocean currents as inferred for B. nipponica (Xu et al., 2021).
Previous biophysical modelling analyses indicated that vent fields across the Okinawa Trough are well connected, with larval transport occurring approximately every 80 years at 1000 m depth between the Hatoma Knoll vent and all other vent fields (Mitarai et al., 2016).Considering the continuous discovery of additional vent fields, the actual larval transport may occur more frequently (Mitarai et al., 2016).Moreover, our hydrodynamic modelling analyses demonstrated that an increase in water depth led to a greater retention of numerical particles within the Okinawa Trough (e.g., The lecithotrophic larvae of the deep-sea squat lobster Kiwa tyleri were suggested to undergo demersal drifting (Thatje et al., 2015).
However, the eggs and lecithotrophic larvae of S. crosnieri hatched in aquaria at atmospheric pressure were buoyant (Miyake et al., 2007).
It has been reported that the buoyancy for the lecithotrophic eggs of the vestimentiferan tubeworm Riftia pachyptila can be increased at lower pressure (Marsh et al., 2001).We thus cautiously hypothesize that the lack of in-situ pressure might explain the high buoyancy of S. crosnieri larvae observed by Miyake et al. (2007).Alternatively, the positive buoyancy displayed by the eggs and lecithotrophic larvae of S. crosnieri may allow their ascent to the mid-water zone, where the higher ambient water temperature, in comparison to the seafloor, can promote its embryonic development and larval growth (Miyake et al., 2007).Further in-situ observation and larval rearing experiments under pressurized conditions are desired to verify the larval behaviours of S. crosnieri.

| Asymmetrical gene flow from the Okinawa trough to the South China Sea
Ancestry coefficient estimation discerned some S. crosnieri belonging to the South China Sea lineage exhibited moderate ancestry coefficients derived from the Okinawa Trough lineage (Figure 2f and Table S5).This finding aligned well with the potential source of admixture (Figure 3a), the relative migration (Figure 4a

| Implications for deep-sea conservation
Large-scale population connectivity studies are promising to illuminate the genetic divergence and demographic mechanisms of deep-sea macrobenthos inhabiting vent and seep ecosystems (Baco et al., 2016).In the Northwest Pacific, such studies have been performed on three representative species co-occurring in vent and seep ecosystems, including the bathymodioline mussel G. platifrons (Xu et al., 2018), the patellogastropod limpet B. nipponica (Xu et al., 2021), and the galatheoid squat lobster S. crosnieri herein.
Based on their biogeographic subdivisions and source-sink dynamics (Table 1), we unveiled that the southern Okinawa Trough vents and the JR seep in the South China Sea warrant imperative efforts to sustain the deep-sea biodiversity in the Northwest Pacific.
Relative migration analysis performed in previous and current studies accordantly elaborated that the candidate source populations of G. platifrons (Xu et al., 2018), B. nipponica (Xu et al., 2021), and S. crosnieri (Figure 4a,c) all located in the southern Okinawa Trough (Table 1).Among them, the symbiont-bearing macrobenthos | G. platifrons and S. crosnieri are foundation species in deep-sea vent and seep ecosystems, which can create three-dimensional aggregations as substrates for microbial growth, as refuges for juveniles of other invertebrates, and as habitats for associated organisms (Van Dover, 2014;Watsuji et al., 2015).Conservation of the potential sources of these key vent-and seep-endemic macrobenthos could benefit the recruitment of their subsequent generations, support the biodiversity of regional communities, and contribute to the resilience of exploitation areas (Boschen et al., 2016).The southern Okinawa Trough also includes sites as contact zones of different deep-sea macrobenthic species.For instance, previous studies indicated that the Hatoma Knoll vent in the southern Okinawa Trough may act as a contact zone to trap G. platifrons (Xu et al., 2018) and B. nipponica (Xu et al., 2021) from two genetic groups.Besides, the Hatoma Knoll vent and all other vent fields in the Okinawa Trough could be well connected at a depth of 1000 m (Mitarai et al., 2016).
Protection of such sites will help enhance population persistence, sustain genetic diversity, and/or promote adaptive evolution (e.As one of the only three active seeps in the South China Sea found at present, the JR seep serves as a fascinating natural laboratory for us to understand the evolution and biogeography of deep-sea macrobenthos in the Northwest Pacific (Wang et al., 2022;Zhao et al., 2020).In particular, the JR seep harbours unique genetic pools of deep-sea macrobenthos endemic to vents and seeps in the Northwest Pacific, as exemplified by G. platifrons (Xu et al., 2018), B. nipponica (Xu et al., 2021), and S. crosnieri (Table 1).These three species all formed a distinct genetic group and/or semi-isolated lineage in the JR seep, which may be attributed to its partial isolation on the South China Sea continental slope and/or its local geochemical and biochemical features (Wang et al., 2022).Nonetheless, declines in local populations and a small N e of macrobenthic communities have been observed in this site.For example, abundant G.
platifrons shells were discovered in several localities of the JR seep (Zhao et al., 2020).Both B. nipponica (Xu et al., 2021) and S. crosnieri  3a, 4a, and 6), this may not be sufficient to repopulate its JR lineage if this site is completely destroyed.In addition, several seep areas in the South China Sea have become targets for gas hydrate extraction (Zhang et al., 2021).Under all these circumstances, the JR seep desires a preferential conservation effort to sustain the distinct genetic groups and/or semi-isolated lineages this site houses from ongoing and imminent anthropogenic disturbances.
Overall, the present study is one of the few large-scale empirical practices to illustrate how population genomics can be integrated with oceanographic approaches in understanding the genetic divergence and migration patterns of deep-sea macrobenthos inhabiting the island-like vent and seep ecosystems.Our research findings will contribute to future decision-making on the effective designation of deep-sea reserves and the informed establishment of marine management plans in the Anthropocene.
Shinkaia crosnieri is another foundation and dominant deep-sea macrobenthic species usually co-occurring with G. platifrons and B. nipponica in both vent and seep ecosystems in the Northwest Pacific (Figure 1a-c), including the JR seep in the South China Sea and multiple vent fields across the Okinawa Trough (a) The JR seep population, along with the bathymodioline mussel Gigantidas platifrons and the patellogastropod limpet Bathyacmaea nipponica on mussel shells (indicated by cyan arrows).(b) The dorsal view of one individual from the JR seep.(c) The ventral view of one female from the IN vent showing the yolk-rich eggs attached to its pleopods.Scale bar = 0.5 cm.(d) Sampling vent (pink dot) and seep (purple dot) sites.The map was created using Ocean Data View v.5.0 (https:// odv.awi.de).DK, Daisan-Kume Knoll Field (depth: 1340 m); FF, Futagoyama Field (depth: 1268 m); IN, Iheya North Field (depth: 1023 m); JR, Jiaolong Ridge (depth: 1122 m); SF, Sakai Field (depth: 1546 m); S min path of NPIW: The spreading path of the North Pacific Intermediate Water salinity minimum; TK, Tarama Knoll Field (depth: 1684 m).Detailed sampling information are available in Table S1.The spreading path of the NPIW salinity minimum from the northeast North Pacific southwestward toward the Luzon Strait was redraw following You et al. (2005).
here.The reproductive seasons, pelagic larval durations, and larval behaviours of S. crosnieri remain poorly understood.Nevertheless, our results of population genomic analyses indicate the lecithotrophic larvae of S. crosnieri may disperse in the intermediate to deeper water layers (details in the sections 3 Results and 4 Discussion).Therefore, two distinct release dates, one in winter (i.e., January 1, 2011) and the other in summer (i.e., July 1, 2011), four release coordinates across the distribution range of S. crosnieri in the Northwest Pacific (i.e., coordinates of the JR seep in the South China Sea and the FF, DK, and IN vents across the Okinawa Trough), and three water depths (i.e., 500, 800, and 1000 m) were selected for numerical particle release experiments.To achieve robust results, numerical particle release experiments were performed at selected coordinates and depths on designated dates relying on the daily-averaged HYCOM + NCODA Global 1/12° Reanalysis data (experiment sequence: 53.X; https:// www.hycom.org) covering 2011-2014 across a broader region (i.e., 0° N-60° N, 100° E-150° E) than that of the sampling sites.Our preliminary tests unveiled that the numerical particle trajectories were qualitatively consistent, despite variations in quantity (e.g., the exact number of numerical particles flowing from the Okinawa Trough into the South China Sea and reversely within a certain period) owing to different radii of initial numerical particle distributions, different numbers of numerical particles released, and/or different ways to release them (e.g., successively released over time or released at a specific temporal instant).Therefore, a total of 24 experiments were present as illustrative examples to indicate the prevalent hydrodynamics that may have influenced the larval dispersal and population differentiation of S. crosnieri.
,f,j in winter; Figure 6n,r,v in summer), DK (e.g., Figure 6c,g,k in winter; Figure 6o,s,w in summer), and IN (e.g., Figure 6d,h,l in winter; Figure 6p,t,x in summer) coordinates within the Okinawa Trough tended to remain within the trough region, leading to a reduced number of numerical particles flowing out of the Okinawa Trough and subsequently entering the South China Sea.Furthermore, during the same period, more numerical particles could flow passively from the South China Sea into the Okinawa Trough than in the opposite direction (Figure 6 and Movies S1-S24).

F
Genetic divergence of Shinkaia crosnieri.(a) A phylogenetic network showing genetic clusters of all individuals constructed using SplitsTree.(b) Pairwise F ST indicating genetic differentiation between populations calculated using Arlequin.Significance: *p < .00001after Bonferroni correction.(c) Principal component analysis showing genetic discrepancies among all individuals revealed using SNPRelate.(d) Geogenetic locations for all individuals inferred using SpaceMix.Each ellipse denotes the 95% confidence interval after a 50% burn-in for the geogenetic location of each individual.(e) Cross-validation errors calculated by the best run of LEA illustrated the smallest value at K = 2. (f) The optimal genetic groups (i.e., K = 2) and individual ancestries uncovered using LEA.Abbreviations for the sampling sites refer to the legend of Figure 1; OT, Okinawa Trough; SCS, South China Sea.TA B L E 2 Genetic statistics for six populations and two genetic groups of Shinkaia crosnieri.1) Region: SCS, South China Sea; OT, Okinawa Trough.(2) Pop: Refer to the legend of Figure 1.(3) Genetic group: SCS, South China Sea genetic group; OT, Okinawa Trough genetic group.(4) Genetic statistic: N, number of individuals; Variant, number of variant nucleotide sites; Polymorphic site, number of polymorphic nucleotide sites; Polymorphic (%), percentage of polymorphic sites in variant sites; Private site, number of unique SNPs; Private (%), percentage of unique SNPs in variant sites; H exp , expected heterozygosity under Hardy-Weinberg equilibrium; H obs , observed heterozygosity; π, nucleotide diversity.(5) The average value (Mean), variance (Var), and standard error (StdErr) of H exp , H obs , and π for all variant nucleotide sites of each population or each genetic group, with details available in Tables S6 and S7, respectively.F I G U R E 3 The maximum a posteriori (MAP) geogenetic locations and MAP potential sources of admixture for six Shinkaia crosnieri populations estimated using SpaceMix.(a) JR, (b) FF, (c) TK, (d) DK, (e) SF, and (f) IN.Arrows indicate the direction from the MAP source of admixture to the MAP geogenetic location of the examined populations.The MAP geogenetic location for each population is situated at the tip of the arrow and indicated by the abbreviated sampling site in dark grey, with an ellipse of its corresponding colour representing the 95% confidence interval after a 50% burn-in.The MAP potential source of admixture for each population is indicated by a black dot, with a dashed ellipse showing the 95% confidence interval after a 50% burn-in.Abbreviations for the sampling sites refer to the legend of Figure 1.F I G U R E 4 Migration patterns of Shinkaia crosnieri revealed using divMigrate-online.(a) A relative directional migration matrix between all populations.(b) Directional relative migration between the South China Sea (SCS) and the Okinawa Trough (OT) genetic groups.(c) Directional relative migration between populations across the Okinawa Trough.In (a-c), arrows indicate the direction of gene flow.Numbers within the blocks in (a) and those on the arrows in (b,c) represent the relative migration coefficients measured relying on the D statistic.Significance: *α < 0.05; **α < 0.01.Abbreviations for the sampling sites refer to the legend of Figure 1.
In the present study, we applied an interdisciplinary methodology involving population genomics and oceanographic approaches to characterize the genetic divergence and migration patterns of S. crosnieri endemic to deep-sea vent and seep ecosystems.A series of population genomic analyses concordantly elucidated that S. crosnieri dwelling in the JR seep in the South China Sea are genetically distinct from those spanning across five vent fields across the Okinawa Trough, giving rise to two semi-isolated lineages that disperse in the middle to deep water (Laming et al., 2018; Ponder F I G U R E 5 Demographic history of the South China Sea (SCS) and the Okinawa Trough (OT) genetic groups of Shinkaia crosnieri inferred using Stairway Plot.Lines depict the median estimations and shadows represent the 95% pseudo-confidence intervals of the effective population size (N e ).
Distribution patterns of the released numerical particles.Locations of numerical particles released after 3 years (i.e., 365 days/ year × 3 years = 1095 days) at the JR, FF, DK, and IN coordinates at (a-d) 500 m, (e-h) 800 m, and (i-l) 1000 m depths since January 1, 2011 (winter), as well as at (m-p) 500 m, (q-t) 800 m, and (u-x) 1000 m depths since July 1, 2011 (summer).Release sites are marked with blue pentagrams on the maps.Abbreviations for the release sites refer to the legend of Figure1.Colour coding represents the total ocean depth (unit: km).The full animations are available as Movies S1-S24.

Figure
Figure 6b-d,f-h,j-l in winter; Figure 6n-p,r-t,v-x in summer).These multiple lines of evidence collectively imply that the topography of the Okinawa Trough might have served as a physical barrier hindering the dispersal of S. crosnieri larvae beyond the trough, thereby contributing to their genetic homogeneity across this region.
,b), as well as the STRUCTURE result reported previously(Cheng et al., 2020), which all underlined a more evident southwestward gene flow of S. crosnieri from the Okinawa Trough to the South China Sea.TheNorth Pacific Intermediate Water (NPIW) is a basin-wide distribution of salinity minimum (about 34.0-34.3)located in the North Pacific subtropical gyre (depth range: 300-800 m), and the spreading path of the NPIW salinity minimum can flow from the northeast North Pacific into the South China Sea through the Luzon Strait (Figure 1d; You et al., 2005).Considering the distribution patterns of numerical particles released in the Okinawa Trough at 500 m (Figure 6b-d and Movies S2-S4 in winter; Figure 6n-p and Movies S14-S16 in summer) and 800 m depths (Figure 6f-h and Movies S6-S8 in winter; Figure 6r-t and Movies S18-S20 in summer), we speculated the southwestward gene flow of S. crosnieri from the Okinawa Trough to the South China Sea may have been mediated by the intrusion of the NPIW.Nonetheless, our hydrodynamic modelling analyses also illustrated that more released numerical particles could flow passively from the South China Sea into the Okinawa Trough than reversely during the same period (Figure6and Movies S1-S24).This result seemed contradictory to the gene flow patterns of S. crosnieri (Figures2f, 3, and 4a,b), but it provided further support to the inference that the two semi-isolated lineages of S. crosnieri potentially have different responses to environmental stresses(Cheng et al., 2019;Xiao et al., 2020).In other words, it might be easier for S. crosnieri larvae produced by the South China Sea lineage to invade the Okinawa Trough with ocean currents, as opposed to the reverse scenario.However, individuals from the South China Sea lineage may have a lower adaptability to the Okinawa Trough vents than those from the Okinawa Trough lineage to the JR seep, eventually resulting in the observed inconsonances between the regional hydrodynamics and the asymmetrical gene flow of S. crosnieri.However, this inference requires thorough verification in the future, when a high-quality S. crosnieri genome and more data on the biological characteristics of the S. crosnieri larvae (e.g., pelagic larval durations, dispersal behaviours, and environmental stress tolerances) become available.
g., hybridization between individuals from different genetic groups and/or semi-isolated lineage) of these deep-sea organisms.The first successful attempt at SMSs test mining worldwide has been performed in the Okinawa Trough (Okamoto et al., 2019), potentially enabling it to become one of the trailblazing regions in exploiting polymetallic sulphides.Owing to the aforementioned conditions, we proposed that vent fields in the southern Okinawa Trough warrant prioritized conservation efforts to protect the source populations and ensure the long-term survival of chemosynthesis-associated communities in the Northwest Pacific.

(
Figure 5) inhabiting the JR seep were estimated to process a small N e .Shinkaia crosnieri dwelling in the JR seep may also encounter a pronounced demographic contraction, likely resulting from recent bottlenecks and/or reduced connectivity (Figure 5).Although our results indicate a small amount of S. crosnieri larvae could potentially arrive from the Okinawa Trough to the South China Sea (Figures 2f,