Over the past few decades, many natural systems have been negatively affected by anthropogenic stresses, such as overfishing and habitat degradation, and as a result, capture fisheries can no longer meet the demand for seafood (Garibaldi, 2012). Therefore, hatchery-reared organisms are often released into the wild to increase abundance and fishery yields (Leber, 2013). These restoration practices are applied for three main purposes: restocking, stock enhancement, and sea ranching (Bell et al., 2008). The mass release of juvenile hatchery-produced individuals into the wild has increased sharply in recent years worldwide, and more than 180 species were released in 20 countries from 2011 to 2016 (Kitada, 2018). In China, fisheries enhancements are practiced by both the country and the provinces along the country’s vast acreage of coastal regions, and 94.543 billion marine fish, shrimp, and scallops were released during 2004–2013 (Luo and Zhang, 2014).

Although stock enhancement programs can be demographically beneficial, they also raise numerous questions related to their eventual ecological and genetic impacts on the recipient stock (Kitada, 2018). Compared with the ecological impacts, the genetic impacts of enhanced production on wild populations are more difficult to be assessed, because such effects may not be as apparent as short-term shifts in abundance (Grant et al., 2017). Domesticated strains of a species are usually produced from a small broodstock that harbors only a small portion of the total genetic diversity within a species. Therefore, they often differ genetically from their wild counterparts by showing lower genetic diversity, particularly when they have been systematically selected for multiple generations (Champagnon et al., 2012; Lorenzen et al., 2012). As stock enhancement aims to enhance both intra- and inter-generational stocking (Kitada, 2018), there may be both short-term and long-term genetic effects. The long-term effects are mainly caused by gene flow. Hybridization between hatchery and wild individuals has the potential to reduce fitness and genetic variation and change the genetic composition and population structure of the wild population (Laikre et al., 2010). The negative genetic impacts of stock enhancement have been documented in a wide range of fish species (Laikre et al., 2010), such as red drum (Sciaenops ocellatus), red sea bream (Pagrus major), and steelhead trout (Oncorhynchus mykiss) (Araki et al., 2007; Gold et al., 2008; Hamasaki et al., 2010). Moreover, even stock enhancement that does not result in gene flow can still destroy the gene pool by decreasing the size of the wild population through competition or disease transmission in the short term. In addition, the effective population size (Ne) of the wild population may be reduced due to increasing mortality. The Ne of cultured stocks is typically much lower than that of wild stocks, potentially leading to a depletion of genetic diversity through increased random drift. Consequently, enhancing wild populations with hatchery individuals can reduce the Ne of the entire mixed population (Ryman and Laikre, 1991; Ryman et al., 1995). Thus, evaluation of the genetic variability of released stock and recipient stock in such enchainment activities can yield valuable insights and ensure a responsible stock enhancement program (Romo et al., 2006; Ward, 2006).

Black rockfish (Sebastes schlegelii) is an important commercial fish inhabiting the coasts of China, Japan, and Korea. It has strong site fidelity and limited capacity for movement (Kang and Shin, 2006; Zhang et al., 2015). Therefore, black rockfish is considered to be an ideal candidate fish species for stock enhancement. The catch per unit of effort (CPUE) and individual weight of S. schlegelii have declined since the 1980s (Xu and Jin, 2005), and stock enhancement programs were initiated in these three countries to recover overexploited stocks. In China, enhancement programs for S. schlegelii began in 1995, and this species has gradually become one of the most popular fish species for release. Millions of juveniles are released in the northern coastal region of China every year (Lü et al., 2014). However, scant information on the genetic effects of release is available, barring a few reports on enhancement technology (Nihira and Takashima, 1999), growth and movement (Nakagawa, 2008), rates of return and economic return (Nakagawa et al., 2007; Noda et al., 2012), and release habitat (Chin et al., 2013). Although a few studies have dealt with the genetic diversity and differentiation of different geographical populations (Yoshida et al., 2005; Ding et al., 2014; Zhang et al., 2016), no study has focused on the genetic variability of the released rockfish and recipient stocks. A S. schlegelii stock enhancement activity was implemented in Lidao Bay in 2013. The present study was aimed to examine the genetic variability of the hatchery-released stock, wild stock before stock enhancement and the mixed stock after enhancement of black rockfish in Lidao Bay as well as the divergence among them. The results will provide basic data to ensure a healthy and sustainable restoration grant for S. schlegelii.

The release of hatchery-reared juveniles could augment the marine fishery biomass. It could help fish stocks recover, and has been conducted in many countries. However, numerous studies have shown that the large-scale release of hatchery individuals into the wild can cause negative effects on the genetic diversity of wild populations (Laikre et al., 2010). Stock enhancement programs for black rockfish have been conducted in Asia for more than 30 years; however, scant information is available on its genetics. Therefore, more detailed genetic analyses elucidating the genetics of stock enhancement in this species were required. We conducted S. schlegelii stock enhancement analysis in 2013 at an embayment, namely Lidao Bay. Lidao bay is located in the east cost of Shandong Peninsula, China and open to the Yellow Sea. The surface seawater temperature ranged from −1 to 26°C, salinity ranged from 30.2 to 32.6‰, dissolved oxygen ranged from 7.10 to 11.36 mL/L, and pH ranged from 7.20 to 8.38 during our sampling period. This bay has some artificial reefs that may limit the dispersal of released S. schlegelii and may render small local native populations more vulnerable than those in the open sea. Thus, the possibility of genetic impact may be higher, which would offer a good opportunity for studying the genetic impacts of the black rockfish restoration. Moreover, in the present study, both mitochondrial and microsatellite makers were used to investigate the genetic effects.

Significant deviations from HWE were observed in all analyzed S. schlegelii stocks in the present study, even after sequential Bonferroni’s correction. This may be caused by heterozygote deficits, and there was heterozygote deficiency in 30 of the studied loci. Although null allele is also an important contributor for the heterozygote deviation in marine fish (Dick et al., 2014), it is unlikely that null allele is the main cause of the deviations observed in this study, because only a few loci had a high frequency of null alleles (e.g., RFs315, Kss18).

The overall genetic diversity in S. schlegelii in this study was comparable to that previously reported using mtDNA and microsatellite markers (Zhang et al., 2016; Gao et al., 2018). Zhang et al. (2016) used the mitochondrial control region marker to investigate 221 S. schlegelii individuals collected from 13 localities across its entire range in China, Japan, and Korea, and the results revealed similar genetic variability at the haplotype and nucleotide levels (overall average h = 0.91 ± 0.01 and π = 0.0063 ± 0.0092). In the present study, the genetic diversity indices of the control region in all three stocks were similar to those measured in previous studies. The 38 microsatellite loci used in this study showed slightly lower levels of polymorphism compared to other black rockfish stocks (An et al., 2012; Gao et al., 2018). Many hatchery fishes show lower numbers of alleles and less genetic variability, resulting from the limited numbers of broodstock used as founders in the hatchery stock for juvenile production (Araki and Schmid, 2010; Champagnon et al., 2012; Lorenzen et al., 2012). In this study, although some of the genetic diversity indices for both mtDNA and msDNA were lower in the hatchery-reared fish than in the wild-born fish, most of the differences were slight and not supported statistically. Therefore, no large decrease of genetic diversity was observed between the hatchery stock and the recipient wild stock, consistent with what was observed in other studies of this fish (Han et al., 2016). However, even though the hatchery stock had high gene diversity, these fish had considerably lower allelic richness relative to the wild stock. According to Araki and Schmid (2010), losses of allelic richness are more common than losses of heterozygosity in the early generations of hatchery-reared populations. As the negative genetic impacts caused by stock enhancement have been documented in several fish species (Laikre et al., 2010), the change in the genetic variability of a mixed stock of S. schlegelii after release was also examined in this study. The results of both mtDNA and msDNA analyses showed that the mixed stock exhibited nearly similar average genetic diversity, albeit slightly lower, than that of the natural stock before release. It is important to note that even if the reductions in allelic diversity and heterozygosity are limited, such reductions could gradually erode the genetic variability of the wild stock by accumulating over generations.

Three clusters were identified by DAPC in this study. Low genetic differentiation level (F_ST < 0.05) was detected between the hatchery-released stock and the wild stock. The F_ST and AMOVA results demonstrated that the wild stock before release and the mixed stock after release showed low but statistically significant levels of genetic differentiation. One possible reason for the low differentiation is that S. schlegelii in China has a relatively short domestication history, and it has not been selected in breeding programs over multiple generations. Indeed, most genitors used in artificial selective breeding were captured from natural waters and are only used once. Moreover, the black rockfish is a viviparous species, and the broodstock used to produce hatchery fish is renewed each year by sampling new genitors from the wild, such that a large number of genitors are used in a single year. These breeding practices may result in a high level of genetic diversity in the hatchery stock and low differentiation when compared with the wild stock. Another reason for the observed limited differentiation may be the absence of significant differentiation among different wild populations of this species. Black rock fish is a species that aggregates around drifting seaweed during their early development, and the both mtDNA and microsatellite marker analyses indicated no genetic divergence among different geographical populations and the existence of high gene flow (Wang et al., 2012; Zhang et al., 2016; Gao et al., 2018). The S. schlegelii adults spawn during May to July, and the larvae have a flexible feeding stage for 2–3 months (Nagasawa and Domon, 1997). Larvae of S. schlegelii might be transported by the China Coast Current. Therefore, although the swimming ability of adult S. schlegelii is limited, larval dispersal via drifting seaweed and oceanic circulation could promote genetic homogeneity (Gao et al., 2018). The lack of genetic differentiation in this species may reduce the genetic effects of stock enhancement using hatchery fish from different geographical stocks.

Estimates of Ne lack consistency and vary widely among estimation methods (Table 5). Although Ne is a key parameter for fecundity and supplementation of a population, exact estimation of the Ne based on genetic markers remains a challenge. Most methods for estimating the Ne were developed based on a hypothetical “ideal” population of constant size, with non-overlapping generations, an equal sex ratio, random mating and random variation in reproductive success (Waples and Do, 2010). These conditions may be not satisfied by black rockfish, which display complex life-history traits. Regardless, we observed that the Ne values obtained by using different estimators were systematically higher in the wild stock than in the hatchery-released stock and mixed stock after release.

There was no obvious reduction in genetic diversity in the hatchery-released stock compared with that of the wild stock, and no significant change in genetic diversity indices was detected in the local stock after enhancement in short term. Thus, our study demonstrated that black rockfish is an ideal candidate fish for releasing into the wild with an aim to increase fishery yields. However, although the observed reduction in genetic parameters, such as allelic diversity, heterozygosity and Ne, did not reach significance, a slight reduction could gradually affect the genetic diversity of the wild stock through accumulation over generations. Despite subtle genetic divergence was detected, our results also suggested that the stocks before and after release did not harbor the coincident genetic pool. Thus, the released individuals are likely to erode long-term genetic diversity of the wild stock through mating with wild individuals, and may have a lasting impact on population genetic structuring as time goes on. In this study, we only collected samples within the first year after release, and the released fishes did not reach sexual maturity. Therefore, more detailed surveys are required to elucidate the long-term genetic effects of S. schlegelii stock enhancement. Moreover, individuals sharing the same genetic architecture with the recipient population were recommended to use in subsequent enhancement activities to avoid adverse effects on the local gene pool.

The mitochondrial dataset generated for this study can be found in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/) with access numbers MN952238–MN952525. The microsatellite dataset analyzed in this study is included in the Supplementary Data Sheet S1.

FY, HL, and LW conceived and designed the experiments. LW, ZW, and FY performed the experiments, analyzed the data, prepared the figures and tables, and wrote the draft of the manuscript. LW, YW, ML, and AS carried out the sampling work in the field. All authors revised the manuscript and approved the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.