1) UVCs
A total of 69 species were observed by UVC on the coastal waters off Nodaedo Island. In another study, the composition of fish species in the bay of Tongyeong was also investigated using a method similar to the one described in the present study. An average of 44 species were identified annually, thus highlighting the substantial difference in the number of species identified by the aforementioned study and ours (Gwak et al. 2016; Lee et al. 2018). Additionally, the number of species that appeared in common with this study varied depending on the geographical characteristics of the survey area. In other words, there was a significant difference in the appearance of fish in the closed and open seas of Tongyeong. Moreover, the coast of the Nodaedo Island, located in the outer sea, had higher diversity of fish species than that observed on the bay of Tongyeong.
Among the fish species observed on the coast of the Nodaedo Island, Chromis notata was the dominant species, appearing 12 times with 7,870 individuals. Huh et al. (2013) interpreted the appearance of C. notata in the central and southeastern part of the East Sea of Korea as a result of the influx of large currents and climate change. Shin et al. (2014) reported genetic similarities between the C. notata populations of Jeju Island and the East Sea. Therefore, these observations support the hypothesis that a C. notata population that inhabited the coast of Jeju Island may have moved northwardly due to the influence of water currents. However, C. notata specimens were observed throughout the entire year. Therefore, these findings suggest that C. notata has already adapted to seasonal changes and settled on the coast of the South Sea. Lee et al. (2018) investigated the fish diversity in the bay of Tongyeong and reported different results from those of our study. The authors reported that approximately 50 individuals appeared five times a year. Although the bays of Tongyeong (Lee et al. 2018) and Nodaedo Islands are 25 km apart, they have similar water temperature and salinity. Therefore, the difference in the number of fish and their appearances is likely influenced by geographic characteristics rather than physical factors. In other words, compared to bay of Tongyeong, C. notata adapted more readily to Nodaedo Island, which is directly affected by the warm Tsushima current. These observations suggest that C. notata requires more time to settle in the closed bay of Tongyeong. Sebastes inermis, the subdominant species, is known to adapt easily to changes in water temperature and this species has already settled in the coast (Oh and Noh 2006). Many studies conducted on the south coast of South Korea have reported the occurrence of a large number of individuals belonging to these species. UVC surveys demonstrated that both S. inermis and C. notata tended to occur in groups, which is thought to be because the former is carnivorous and the latter is planktivorous, and therefore these species do not compete with each other for food (Ochi 1986; Huh and Kwak 1998).
The seasonal appearances of certain fish species are at least partially related to water temperature. However, we cannot conclusively state that species richness increases with water temperature because our findings, as well as those of previous studies (Gwak et al. 2016; Han et al. 2017; Lee et al. 2018), demonstrated the occurrence of many species between September and November, after the highest water temperature was recorded in August. This was likely because the current flows from the Jeju Island to the northeast during the summer and autumn seasons and transports fish with it (Kim and Bae 2011).
In this study, the number of species observed in August was smaller than that in July and September due to the lack of visibility during the diving survey. The UVC method is a nondestructive method that is commonly used in rocky areas. However, underwater visibility can severely affect the accuracy of this approach, which in turn depends on the weather and/or environmental conditions.
A significant difference was observed in the number of species between our study and previous studies involving UVC in the Tongyeong Island (Gwak et al. 2016; Lee et al. 2018). This is probably because water temperature fluctuates less in Nodaedo Island than at the bay of Tongyeong. A previous study reported a water temperature of <10 ℃ in January and February and a summer temperature of ≥25 ℃. In contrast, we reported a lower water temperature in the summer than that reported in the previous study. In the winter, the water temperature decreased to >10℃, confirming that water temperature fluctuates less in Nodaedo Island than at the bay of Tongyeong (Kim et al. 2016). The eastern waters of the South Sea of South Korea contain a mix of Tsushima warm current waters, cold water from the East Sea, and the coastal waters of the South Sea, resulting in complex marine characteristics. Particularly, a water temperature front is formed between the waters at Nodaedo Island and the bay of Tongyeong. Therefore, the study area provides a more stable environment than the bay of Tongyeong.
Most of the species observed only by UVC were represented by only a small number of individuals with a low frequency of occurrence. Takahara et al. (2012) reported a significant relationship between the biomass and eDNA concentration in living organisms. Therefore, the number of individuals for a given species can be so small that the eDNA concentration becomes undetectable. However, although the number of individuals of Ostorhinchus semilineatus increased considerably from September to November compared to other species, this increase was only detected through the UVC method. This was likely because the juvenile fish were schooling in a limited area at the time of underwater observation, resulting in a low concentration of eDNA.
Most of the subtropical and tropical fish species that were observed in the coast of Nodaedo Island appeared between September and December, with only 1–2 individuals per species. Lee et al. (2018) also reported mostly subtropical fish species during the same period. In general, the appearance of subtropical and tropical fish species at the temperate coast can be interpreted as the result of transport from other locations due to ocean currents (Nakamura et al. 2012). After the summer, the flow of ocean currents in the South Sea changes to the northeast from Jeju Island (Kim and Bae 2011). Considering that the period from June to September is the spawning season of Pomacentrus coelestis, Chaetodontoplus septentrionalis, and Labroides dimidiatus (Sakai and Kohda 2001; Kim et al. 2005; Chen and Tzeng 2009), it is possible that their larval and juvenile fish were transported to the coastal waters of Tongyeong. Tropical fish can survive when the water temperature is at least 16–18 ℃ (Figueira and Booth 2010). However, in the study area, the winter water temperature was <15 ℃, which is unsuitable for the survival of subtropical and tropical fish. Therefore, these fish species were not observed during the winter by UVC. Additionally, these fish likely did not reproductive in this period (Nakamura et al. 2013). In contrast, tropical species may settle in the southern coast if the annual average water temperature of the south coast of Korea continues to increase and if environmental conditions such as food and water temperature are favorable for tropical and subtropical fish species (Harriott and Banks 2002; Seong et al. 2010). Additionally, subtropical and tropical fish species observed in UVC surveys were not detected through eDNA metabarcoding, suggesting that the UVC method provides a uniquely advantageous approach for the detection of rare fish species.
2) eDNA metabarcoding
To the best of our knowledge, our study is the first to identify marine fish species using eDNA in nearly ten years, with Thomsen et al. (2012) being the last authors to conduct a similar study. Compared with traditional methods (Fyke net, underwater visual census, and trawl), the eDNA method has been proven in various studies to be both time- and cost-efficient (Shaw et al. 2016; Thomsen et al. 2016; Hinlo et al. 2017; Yamamoto et al. 2017). We used both the eDNA metabarcoding and UVC methods to overcome the limitations of existing methods for the study of fish diversity and our findings revealed the occurrence of various fish species. The fish species detected using eDNA metabarcoding were generally identified as species that use micro-habitats such as Blenniidae and Stichaeidae, which are difficult to observe by UVC, as well as fish species with migratory and schooling behaviors such as C. pallasii, Engraulis japonicus, Ammodytes personatus, and Trachurus japonicus. Additionally, a complementary relationship with the UVC method was observed. These results were similar to those of previous studies (Thomsen et al. 2012; Stat et al. 2019; Valdivia-Carrillo et al. 2019). Therefore, eDNA metabarcoding has the advantage of detecting fish that cannot be easily observed by the UVC method.
Among the fish species that appeared more than six times a year, E. japonicus and T. japonicus were well reflected in this study, as they appeared continuously in all the four seasons in the South Sea (Kim et al. 2011, 2013). In contrast, A. personatus is known to prefer relatively cold waters. Therefore, when the temperature is high during the summer, these fish become less active and tend to rest in sandy areas (Kim et al. 1994). Due to these seasonal changes in behavior, this species was detected in all seasons except in the summer. Parajulis poecilepterus inhabits places affected by warm currents. Therefore, this species did not appear in winter because of the low water temperature (Lee and Kim 1996). Clupea. pallasii migrates to the South Sea for spawning in the winter (Lee et al. 2017). However, in this study, the eDNA metabarcoding detected this species in July 2018. Further research is thus needed to clarify whether C. pallasii was included in the feed used by fish farms in the surrounding areas.
The eDNA detection results reflect fish species that appear depending on the season. However, the number of species detected per month and the water temperature did not appear to be significantly correlated. For example, although the lowest water temperature occurred in January 2019, this period exhibited the highest number of identified species in the entire year, with 18 and 8 species of fish classified at the family and genus levels. This is also different from the results of UVC, which were conducted simultaneously. Unlike in in January 2019, a small number of species were detected in December and February because there was no significant difference in the water temperature in January, December, and February. Strickler et al. (2015) reported no significant difference in DNA degradation at 5 and 20 ℃. Therefore, the effect of water temperature was likely negligible, which is also clear from the fact that a large number of species were detected in January. Stat et al. (2019) stated that eDNA detectability largely depended on DNA concentration and biological mobility. Therefore, the eDNA levels of the species detected in January were likely high due to the presence of a large number of individuals, as well as their high activity and wide DNA distribution. In fact, most of the highly active migratory fish species such as Sardinops sagax, C. pallasii, Konosirus punctatus, A. personatus, Seriola quinqueradiata, and E. japonicus appeared in January. Additionally, fish species detected more than six times during the year were generally considered active fish species such as E. japonicus, T. japonicus, A. personatus, C. pallasii, and Pagrus major, which supports our hypothesis. In other words, the eDNA is highly reflective of the fish species present rather than water temperature, whereas UVC surveys are believed to provide a snapshot of the winter species composition in limited spaces. Scomber spp., which appeared 11 times in this study, was likely misclassified as Scomber japonicus, which occurs year-round in the South Sea (Lee and Kim 2011). However, S. japonicus had never been detected through UVC. Davis and Anderson (1989) reported that the UVC survey method may underestimate fish that swim quickly or that mainly inhabit surface waters. Therefore, mackerel was not observed in the UVC survey in this study, which was likely due to the inherent limitations of the UVC method.
Sebastes spp. is presumed to be S. inermis, as it appeared 12 times in a year, as shown by UVC. Hexagrammos spp. (both H. otakii and H. agrammus) has been reported in many UVC surveys. In this study, 12S rRNA was targeted for metabarcoding analysis, following the method used by Miya et al. (2015, 2016). When the same primers as those employed in this study were used, it is possible that both Sebastes spp. and Takifugu spp. could not be classified at the species level (Miya et al. 2015; Aizu et al. 2017; Yamamoto et al. 2017). According to Aizu et al. (2017) and Kelly et al. (2014), such a result may appear depending on the primers used or may be caused by missing information related to the mitochondrial DNA data of a specific species. Therefore, the use of various primer sets and/or database verification is necessary for fish that have not been classified at the species level. Kim and Park (2002) reported that Salvelinus leucomaenis occurred in the northeastern Korean peninsula, suggesting that this species does not inhabit the South Sea. However, these are false-positive cases, which likely resulted from contamination caused by artificial inflows from ports, fishing boats, and fish markets (Ficetola et al. 2015; Yamamoto et al. 2017).
Fish detected only by eDNA are mostly bottom dwelling, such as Pholis nebulosa, as well as pelagic fish such as C. pallasii. Because the bottom is sandy and muddy at a water depth of >7 m, P. nebulosa living in the bedrock can be easily detected in the survey area using the eDNA method. However, pelagic and schooling fish are unlikely to inhabit the surveyed waters because they are highly mobile and some are migratory. Therefore, it is highly likely that the detected eDNA was transported from the outside by tides or currents. In fact, a study reported that the flow of seawater such as tides affects the detection results by transporting the eDNA outside the survey area. Therefore, in the case of the pelagic fish detected in this study, it is possible that the detected eDNA corresponded to fish that did not naturally inhabit the survey area (Valdivia-Carrillo et al. 2019). The half-life for the detection of eDNA in an artificial seawater environment has been reported to be 26 hours (Collins et al. 2018). However, the decomposition rate of DNA in the sea is expected to be faster because it depends on environmental factors such as pH, water temperature, salinity, UV radiations, and microorganisms (Strickler et al. 2015; Salter 2018). Kelly et al. (2018) compared the eDNA of fish clusters in three regions and reported that seawater flow dynamics (e.g., tidal flow) are responsible for the transport of environmental DNA from non-survey areas and affects the detection results. Andruszkiewicz et al. (2019) reported the migration of eDNA at a 10 km/day rate due to ocean currents. This suggests that the pelagic and schooling fish species detected in this study were in fact species that inhabited the southeastern sea of Korea.
In this study, the ANOSIM analysis results for presence/absence data by survey method showed significant differences between eDNA metabarcoding and the UVC. This is fish species commonly found through UVC and eDNA metabarcoding can be interpreted as the following types: 1) dive observation (number of individuals: few, number of appearances: few), eDNA (number of detections: many); 2) dive observation (number of individuals: few, number of appearances: many), eDNA (number of detections: few); 3) dive observation (number of individuals: many, number of appearances: many), and eDNA (number of detections: few) (Table 8). In the first type, Acanthopagrus schlegelii and Mugil cephalus were considered in this study. This suggests that there were more individuals in the study area, in addition to those observed in the UVC surveys. The second type includes Enneapterygius etheostoma, Rudarius ercodes, Paracentropogon rubripinnis, Semicossyphus reticulatus, Pseudolabrus sieboldi, and Pterogobius zonoleucus, which are believed to have limited detection rates because of their low DNA concentration. The third type includes C. notate, which appeared 12 times in 12 surveys. Additionally, the number of individuals was large and it occupied the dominant species position. However, eDNA metabarcoding was detected only once during the entire survey period. Miya et al. (2015) reported the detection of Chromis spp. in a tank using similar procedures as those reported herein. However, because this is a one-time detection, the DNA analysis of specific fish species is not yet fully reflected. Therefore, it is necessary to precisely analyze the presence or absence of C. notata using qPCR.
The UVC method used in this study is suitable for fish in which eDNA is not detected because of the occurrence of a small number of individuals. This method is suitable for detecting fish with pelagic and high activity. Valdivia-Carrillo et al. (2019) also showed some differences in the species composition of the two methods, and eDNA detected only fish groups with pelagic and high activity such as Istiophoridae, Scombridae, Mugilidae, and Clupeidae. In contrast, fishes observed only in UVC showed a similar pattern to the results of the present study, as fishes with strong sedentary habitat were observed. Therefore, it appears that there is a complementary relationship between the two methods. It is difficult to conclude whether the fish species that appeared and were detected in this study reflect all of the fish species that have settled on the coast of the Nodaedo Island. However, considering the appearance of fish depending on seasonal changes and the transport of eDNA by ocean currents, the results of this study likely do not exclusively reflect the biodiversity of Nodaedo Island but also that of nearby areas such as the southeastern coast of Korea.