**4. Discussion**

A large number of molecular markers were obtained by the 2b-RAD genome sequencing of yellowtail kingfish samples from five collections. A comparative analysis of the genetic characters of the wild and farmed Chinese collections, the wild Japanese collection, and the wild and farmed Australian collections was performed using these markers. The results of the population genetic structure analysis, PCA, and a neighbor-joining phylogenetic tree all supported the same conclusions. Genetic differentiation of the wild and farmed Chinese populations, wild Japanese populations, and the wild and farmed Australian populations was high. The calculated gene flow values for the wild and farmed Australian populations compared with the wild and farmed Chinese populations and wild Japanese population were much less than 1, which indicates almost no gene exchange. These results are consistent with the findings of Ai et al. [11], who showed, by comparing nuclear loci and morphological characters in samples from the Bohai Sea, that the Chinese and Australian yellowtail kingfish formed two completely distinct clusters and that there was no genetic introgression from the Australian yellowtail kingfish into the Chinese yellowtail kingfish population based on nuclear gene markers and morphological characteristics.

Although yellowtail kingfish are an oceanic species that can migrate more than 2000 km, their migration routes are confined to continental shelf waters within the same hemisphere, and adult fish mostly migrate at regional scales; thus, the likelihood of migration across the open ocean is low [31,32]. In addition, the equator acts as a natural geographic barrier to yellowtail kingfish migration in the Pacific Ocean because the equatorial water temperatures (18–24 ◦C in continental shelf areas) exceed those tolerated by yellowtail kingfish [33]. Therefore, the gene flow between Northern Hemisphere and

Southern Hemisphere yellowtail kingfish is blocked [10]. The equator has also been shown to act as a natural barrier to gene flow in other wide-ranging or migratory teleosts. For example, mtDNA analyses indicated a lack of genetic communication between Pacific jack mackerel (*Trachurus symmetricus*) populations from the North and South Pacific Ocean [34], and genetic differentiation between the North Pacific and South Pacific populations of yellowfin tuna (*Thunnas albacares*) in the two oceans was attributed to equatorial isolation [35]. Similarly, our data showed high genetic differentiation between the Chinese, Japanese and Australian populations, indicating that these populations are geographically (and therefore reproductively) isolated. Sepúlveda et al. (2017) pointed out that the temporal movements of yellowtail kingfish in the southeastern Pacific revealed a particular life strategy (i.e., reproductive or habitat segregation) for this species [36]. This was consistent with previous studies of genetic differentiation among Australian, Japanese, and/or Chinese yellowtail kingfish populations analyzed by mtDNA or nuclear genes [9,31]. Using mitochondrial and nuclear genetic markers and morphometric analysis, Martinez-Takeshita et al. (2015) revealed significant genetic and morphological divergence between yellowtail kingfish (*Seriola lalandi*) specimens collected from the Northeast Pacific, Northwest Pacific, and Southern Hemisphere [10]. Even if it were possible for yellowtail kingfish to migrate across the equator, gene exchange remains unlikely. Yellowtail kingfish in the Northern Pacific spawn between March and October (e.g., the Chinese wild populations spawn from April to June) [37,38], but in the Southern Hemisphere they typically spawn between October and January [39].

Although previous studies speculated that the Chinese and Japanese yellowtail kingfish populations are indistinct based on their geographic distributions [1], genetic and quantitative morphological evidence for this was lacking. Previously, we found that the mitochondrial genome structure of Chinese yellowtail kingfish was highly similar to that of Japanese yellowtail kingfish, except that the mitochondrial genome of the Chinese population was slightly longer, and that variation in the COX1 and NAD5 genes was observed between Chinese and Japanese populations [14,40]. This awakened our interest in exploring the population genetic structure of Chinese and Japanese yellowtail kingfish. Here, our population genetic structure analysis showed no genetic differentiation between the wild and farmed Chinese populations and the wild Japanese population, suggesting that they are derived from the same population. The phylogenetic analysis showed that the first branch of the Chinese wild population was closely related to the wild Japanese population, while the wild Japanese population was linked to the farmed Chinese population. Interestingly, gene flow between the Chinese and Japanese wild populations (*Nm* 256.4499) was much higher than that between the Chinese wild and farmed populations. The *Nm* value between the farmed Chinese and wild Japanese populations was slightly higher than that between the wild Chinese and wild Japanese populations. The calculated *Fst* and *DR* values also suggested that the wild populations in China and Japan are most closely related to each other. This may be because China and Japan are located in Current and Kuroshio Current, of which are relatively high. However, the Northern Pacific Ocean currents are colder and thus more suitable for the migration of yellowtail kingfish adults, juveniles, and larvae. Indeed, two of the main factors affecting the migratory routes of yellowtail kingfish are ocean currents and temperature [41–43]. Our preliminary surveys indicated that the Dasha fishing ground in the Yellow Sea (~32◦00 –34◦00 N, ~122◦30 –125◦00 E) is the spawning grounds of the wild Chinese yellowtail kingfish population. Furthermore, fishermen in various coastal Chinese cities report catching yellowtail kingfish adults, juveniles, and larvae at staggered periods throughout the year: April–June at Qingdao, May–August at Dalian, July–August at Weihai, and August–October at Zhoushan. From this we inferred that yellowtail kingfish migrate south to north along the Chinese coast, following food resources and the Kuroshio Current, and then swim either northwestward into the Yellow Sea with the Yellow Sea Warm Current or northwestward around the Korean peninsula with the Tsushima Current. However, further genetic data from yellowtail kingfish populations in the East China Sea and the Yellow Sea are required to verify this speculation. Therefore, in our next study, we plan to establish 8–10 large-scale sampling stations in various locations along the hypothesized yellowtail kingfish migration route (e.g., the East China Sea, Yellow Sea, Bohai Sea, Japan Sea, and Korean Peninsula coastal waters) to capture individuals at various spatial and temporal points during their migration. These data will also help to further clarify the phylogenetic relationship between the Chinese and Japanese populations.

When a fish population is influenced by factors such as mutation, selection pressure, migration, random genetic drift or non-random mating, the genotype frequencies within the population can be affected. All populations conform to the Hardy-Weinberg equilibrium, but the Hardy–Weinberg equilibrium (*HW-P*) value for the total sample was lower than the *HW-P* values for each cluster. This is consistent with the result of *He* (0.2013) being higher than *Ho* (0.0824). All clusters were divided into two populations, with gene flow occurring within each. Gene flow between the Chinese and Japanese populations displayed an especially high level at *Nm* 256.449. The Northwest Pacific yellowtail kingfish has a common spawning ground and follows similar migration routes. We calculated the *F*-statistics, *Fit* (0.3298) is greater than *Fst* (0.2898), which is greater than *Fis* (0.0627). There was no inbreeding between individuals and there was genetic differentiation between each population. Fernández et al. (2015) studied the genetic structure of the Chilean population of *Seriola lalandi* to detect moderate inbreeding (*Fis* = 0.12). Previous studies [9,44] consistently yielded higher *Ho* and *He* values for yellowtail kingfish populations in comparison to the present findings. Significantly higher values than obtained in this study might be an outcome of different sequencing methods. The size of the sample may also be one of the factors that influence the results. It has been suggested that sample sizes of more than 50 are best for population analysis.

In the Chinese populations, the genetic diversity values were only slightly higher for the farmed versus wild fish. This could reflect the influence of aquaculture domestication on Chinese yellowtail kingfish, even though the culture of this species in China is still in an initial stage and the culture effort has not yet undertaken direct breeding. As a result, there is no significant genetic differentiation attributed to kinship within the population. In general, cultured fish populations frequently originate from a limited number of individuals, leading to a constrained genetic background. This species tends to show a small degree of schooling behavior [36]. In contrast, aquaculture efforts in Australia have focused on the impact of escaped farmed fish on the wild population [8], although no significant genetic differentiation has yet been detected between wild and farmed populations there. Although the phylogenetic results show distinct distributions between the wild and farmed fish in Australia. In the PCA, individual outliers could be observed, but their impact seems minimal. Structure to *K* = 3 or 4, there are no clear clusters within the two populations. Genetic diversity is a valuable piece of information for fish farmers to use to improve and manage their fish stocks [36]. Based on the results of our low genetic diversity, the underlying reasons for this outcome could be complex and may also be influenced by the experimental methods used. Nevertheless, this serves as a reminder for us to pay attention to improving the genetic diversity of farmed populations and preserving the integrity of the habitats and wild populations. This highlights supplementing new individuals to farmed populations to increase gene flow between individuals, and should also remind us of the benefit of implementing targeted breeding strategies. Additionally, comprehensive genetic management plans should be developed and implemented, including monitoring and maintaining genotype frequencies, genetic diversity, and genetic health, along with appropriate intervention measures.

#### **5. Conclusions**

We isolated and identified SNP markers in yellowtail kingfish populations from China (wild and farmed), Japan (wild), and Australia (wild and farmed). The genetic structure of each population was analyzed using these SNP markers. Our analyses show that: (1) no significant genetic differentiation was detected between the wild and farmed Chinese and wild Japanese populations, and both of those populations showed significant genetic differentiation from the wild and farmed Australian populations; (2) no obvious genetic differentiation was observed between the wild and farmed Chinese populations, whereas the wild and farmed Australian populations are likely becoming genetically differentiated because of longer farming generations or genetic bottleneck. Moreover, the genetic relationship between the Chinese and Japanese wild populations was closer than that between the wild and farmed Chinese populations. Our data will provide theoretical and technical support for genetic breeding, ensuring the sustainable production of wild and farmed yellowtail kingfish in China.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jmse11081583/s1, Figure S1: Boxplot showing cross-validation error levels for various numbers of subpopulations (*K* = 1–10).

**Author Contributions:** Conceptualization, Y.X. and A.C.; methodology, Y.X. and A.C.; validation, Y.X., K.K. and Y.J.; formal analysis, A.C. and Y.X.; investigation, A.C. and Y.X.; data curation, K.K., Y.J., B.W., T.K. and X.L.; writing—original draft preparation, A.C.; writing—review and editing, A.C. and Y.X.; supervision, Y.X.; funding acquisition, Y.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from National Key R&D Program of China (2022YFD2401102), Marine Science and Technology Fund of Shandong Province for Qingdao Marine Science and Technology Center (2022QNLM030001), Taishan Industrial Experts Program, Central Public-interest Scientific Institution Basal Research Fund, CAFS (2020TD47), and Earmarked Fund for China Agriculture Research System (CARS-47).

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Animal Care and Use Committee of Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (Approval code: YSFRI-2022025, and date: 3 July 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data in this study are available from the authors upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
