Temporal and Spatial Variations of the Biochemical Composition of Phytoplankton and Potential Food Material (FM) in Jaran Bay, South Korea

Food material (FM) derived from biochemical components (e.g., proteins, lipids, and carbohydrates) of phytoplankton can provide important quantitative and qualitative information of the food available to filter-feeding animals. The main objective of this study was to observe the seasonal and spatial variations of the biochemical compositions of phytoplankton and to identify the major controlling factors of FM as a primary food source in Jaran Bay, a large shellfish aquaculture site in South Korea. Based on monthly sampling conducted during 2016, significant monthly variations in the depth-integrated concentrations of major inorganic nutrients and chlorophyll a within the euphotic water column and a predominance (49.9 ± 18.7%) of micro-sized phytoplankton (>20 μm) were observed in Jaran Bay. Carbohydrates were the dominant biochemical component (51.8 ± 8.7%), followed by lipids (27.3 ± 3.8%) and proteins (20.9 ± 7.4%), during the study period. The biochemical compositions and average monthly FM levels (411.7 ± 93.0 mg m−3) in Jaran Bay were not consistent among different bays in the southern coastal region of South Korea, possibly due to differences in controlling factors, such as environmental and biological factors. According to the results from multiple linear regression, the variations in FM could be explained by the relatively large phytoplankton and the P* (PO43− − 1/16 × NO3−) and NH4+ concentrations in Jaran Bay. The macromolecular compositions and FM, as alternatives food source materials, should be monitored in Jaran Bay due to recent changes in nutrient concentrations and phytoplankton communities.


Introduction
Bays are important aquatic systems that provide food resources for fisheries and aquaculture since they provide habitats and prey for various marine organisms. Rece ntly, mollusk farming, including bivalves, has contributed greatly to global farming production [1]. The present study site, Jaran Bay, is one of the largest shellfish aquaculture regions for oysters and scallops in South Korea [2], and these filter-feeding oysters and scallops feed mainly on water-dwelling phytoplankton for their growth and reproduction [3,4].
The growth and physiological conditions of phytoplankton can vary depending on environmental conditions [5][6][7]. In particular, phytoplankton synthesize biochemical components through photosynthesis

Monthly Concentrations of Nutrients and Chlorophyll a
The monthly depth-integrated nutrient concentrations within the euphotic water column from 100 to 1% light depths during the present study period are summarized in Table 1. The ranges of the NH4 + , NO2 − + NO3 − , PO4 3− and Si(OH)4 2− concentrations were 4.0−47.5, 10.9−80.0, 0.5−6.0 and 20.9−166.9 μM, respectively, in Jaran Bay from January to December 2016. The concentration ranges varied significantly during the observation period, and the highest concentrations were detected in September, except for the Si(OH)4 2− concentrations, which showed the largest peak in June and a secondary peak in September.
The total monthly chlorophyll a concentration averaged from the three light depths at seven stations ranged from 0.77 μg L −1 in September to 4.89 μg L −1 in October, with an average of 2.13 μg L −1 (S.D. = ± 1.18 μg L −1 ) ( Figure 2). Based on the different size-fractionated chlorophyll a concentrations (Figure 3), the compositions of the micro-(> 20 μm), nano-(2−20 μm) and pico-sized chlorophyll a concentrations (0.7−2 μm) varied significantly in Jaran Bay among the different months. The compositions of the micro-sized chlorophyll a concentrations ranged from the lowest value in April (23.8 ± 18.7%) to the highest value in January (77.8 ± 6.8%), whereas the nano-sized chlorophyll a compositions ranged from the lowest value in January (14.3 ± 7.0%) to the highest value in June (50.3 ± 21.3%). In comparison, the compositions of pico-sized chlorophyll a were lowest in January (7.9 ± 4.4%) and highest in April (46.0 ± 18.1%). Seasonally, the compositions of the micro-sized chlorophyll a concentrations steadily increased from spring (March−May) to winter (December−February), although significant monthly variations were present. In contrast, the compositions of the pico-sized chlorophyll a concentrations steadily decreased from spring to winter. The compositions of the nano-sized chlorophyll a concentrations were highest in summer (June−August) and lowest in winter. On average, micro-sized (>20 μm) cells contributed 49.9% (± 18.7%) of the total chlorophyll a concentration in Jaran Bay during our observation period. In comparison, the nano-and pico-sized chlorophyll a compositions contributed 28.5% (± 12.4%) and 21.6% (± 11.2%), respectively. A strong positive relationship was found between the micro-sized chlorophyll a concentrations and total chlorophyll a concentrations integrated from the euphotic water columns in this study (y = 1.31x + 5.74, r 2 = 0.82; Figure 4). The water samples that were used for determining the macromolecular compositions (e.g., carbohydrates, proteins and lipids) of particulate organic matter (POM) were filtered through 47 mm GF/F filters, and the filters were immediately preserved at −70 • C until further spectrophotometric analysis. The samples were filtered under a constant vacuum (<10 cm Hg) because live cells could be damaged during the strong vacuum filtration [23]. Carb ohydrate extraction was performed by following Dubois et al. [25]. The preground POM-retained filter paper was transferred to a polypropylene (PP) tube. Afte r the addition of 1 mL deionized water, 1 mL of a 5% phenol solution was added and allowed to rest for 40 min. Then , 5 mL of sulfuric acid (H 2 SO 4 ) was added and allowed to stand for 10 min. Next , the solutions were centrifuged at 3430 g for 10 min. The absorbance of the supernatant was measured at 490 nm. A glucose solution (1 mg mL −1 , Sigma Aldrich) was used as the standard for determining the carbohydrate concentration.
For protein extraction, each preground sample filter was transferred to a 12-mL glass tube with 1 mL deionized water (DH 2 O) and was added to 5 mL of an alkaline copper solution. Afte r the solution was well mixed, 0.5 mL of diluted Folin-Ciocalteu phenol reagent (1:1, v/v) were added and allowed to sit for 1 h 30 min at room temperature. Then , the solutions were centrifuged for 10 min at 2520 g. The absorbance of the supernatant was measured at 750 nm. Bovi ne serum albumin (2 mg mL −1 , Sigma Aldrich) was used as the standard for determining the protein concentration based on previous works in various oceans [5][6][7]16,17,21].
Last, the filters used for lipid extraction were transferred into a 16-mL glass tubes, ground with 3 mL of chloroform-methanol (1:2, v/v) and stored at 4 • C for 1 h. Afte r the solution was homogenized with 4 mL of DH 2 O, the lower (chloroform) phase of the solution was dried at 40 • C for 48 h and then heated at 200 • C for 15 min with 2 mL of H 2 SO 4 . An additional 3 mL of DH 2 O was added to the chloroform phase in the glass tubes and then they were allowed to rest for 10 min. The absorbance of the supernatant was measured at 375 nm, and a tripalmitin solution (Sigma Aldrich) was used as the standard for determining the lipid concentration. Afte r each extraction process, the concentration of each biochemical component was determined using a UV spectrophotometer (Hitachi-UH5300, Hitachi, Tokyo, Japan).

Statistical Analysis
Principal component analysis (PCA) was performed on our field-obtained data of the chemical and biological variables (i.e., nutrient concentrations and phytoplankton biomass) for their relative significance and interrelationship patterns among the various biochemical conditions measured during our sampling period. Bart lett's sphericity tests were used to determine the validity of the PCA (p < 0.01) [26,27]. Fact or analysis was conducted to obtain various factors selected by the principal component method with varimax rotation [28]. Due to the strong dependency between PO 4 3− and NO 3 − (r = 0.56, p < 0.01; Pearson's correlation coefficient), PO 4 3− was excluded but included P* (PO 4 3− − 1/16 × NO 3 − ) in the PCA. P * reflects the excess (or deficiency) of PO 4 3− versus NO 3 − [29,30].
To determine the major factors controlling the macromolecular composition and FM of POM, multiple linear regression analysis was conducted in this study based on the PCA results. The multiple linear regression equation of Pedhazur [31] is as follows: where Y denotes a dependent variable and the FM of POM is estimated from the independent variables (predictors), X 1 · · · X k . Para meter α is a constant, b 1 · · · b k are the regression coefficients for the predictors (FM in this study), and e is an error term. Insignificant variables for the controlling the FM variation were stepwise eliminated from the model by stepwise variable selection after multiple linear regression analysis. Stat istical analysis was performed with IBM SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA). t statistics were conducted for testing the regression coefficients and values of the coefficient of determination (R 2 ) were obtained for measure of goodness of fit for the FM in this study.

Monthly Concentrations of Nutrients and Chlorophyll a
The monthly depth-integrated nutrient concentrations within the euphotic water column from 100 to 1% light depths during the present study period are summarized in Table 1  The total monthly chlorophyll a concentration averaged from the three light depths at seven stations ranged from 0.77 µg L −1 in September to 4.89 µg L −1 in October, with an average of 2.13 µg L −1 (S.D. = ± 1.18 µg L −1 ) ( Figure 2). Base d on the different size-fractionated chlorophyll a concentrations (Figure 3), the compositions of the micro-(> 20 µm), nano-(2−20 µm) and pico-sized chlorophyll a concentrations (0.7−2 µm) varied significantly in Jaran Bay among the different months. The compositions of the micro-sized chlorophyll a concentrations ranged from the lowest value in April (23.8 ± 18.7%) to the highest value in January (77.8 ± 6.8%), whereas the nano-sized chlorophyll a compositions ranged from the lowest value in January (14.3 ± 7.0%) to the highest value in June (50.3 ± 21.3%). In comparison, the compositions of pico-sized chlorophyll a were lowest in January (7.9 ± 4.4%) and highest in April (46.0 ± 18.1%). Seas onally, the compositions of the micro-sized chlorophyll a concentrations steadily increased from spring (March−May) to winter (December−February), although significant monthly variations were present. In contrast, the compositions of the pico-sized chlorophyll a concentrations steadily decreased from spring to winter. The compositions of the nano-sized chlorophyll a concentrations were highest in summer (June−August) and lowest in winter. On average, micro-sized (>20 µm) cells contributed 49.9% (± 18.7%) of the total chlorophyll a concentration in Jaran Bay during our observation period. In comparison, the nano-and pico-sized chlorophyll a compositions contributed 28.5% (± 12.4%) and 21.6% (± 11.2%), respectively. A strong positive relationship was found between the micro-sized chlorophyll a concentrations and total chlorophyll a concentrations integrated from the euphotic water columns in this study (y = 1.31x + 5.74, r 2 = 0.82; Figure 4). Figure 5 shows the average of three light depth values of each macromolecular composition of POM in Jaran Bay from January to December 2016. No distinctive spatial variations were detected in the macromolecular compositions among the different stations; however, they significantly varied among the different months. Carb ohydrates were the predominant biochemical component during our observation period from January to December, with monthly proportions of carbohydrates ranging from 40.9% to 66.4%. In comparison, the protein and lipid proportions were 11.1-31.0% and 22.5-35.1%, respectively. The lipid proportion appeared to decrease steadily from January to December. Seas onally, the carbohydrate proportion were relatively variable compared to the protein and lipid proportions. The carbohydrate proportion was lowest during summer (45.6 ± 1.4%) and highest during autumn (59.1 ± 10.9%). In comparison, the protein proportion was lowest during winter (17.5 ± 5.7%) and highest during summer (28.1 ± 2.7%), while the lipid proportion was lowest in autumn (23.1 ± 0.6%) and highest in winter (31.1 ± 3.7%).

Spatial and Temporal Variations of the Macromolecular Compositions of POM
The monthly FM concentrations ranged from 297 to 630 mg m −3 , with an average of 411.7 mg m −3 (S.D. = ± 93.0 mg m −3 ), in this study (Table 2). No noticeable monthly variations were observed for the FM concentrations. Spat ial variations in the FM concentrations were not noticeable for the seven stations during the observation period except for March, April and June, which had considerably higher FM concentrations at several stations ( Figure 6). The monthly calorific values and FM contents of FM averaged from the three light depths at the seven stations did not vary significantly and ranged from 5.5-6.3 Kcal g −1 and 1.7-3.7 Kcal m −3 , respectively, in Jaran Bay (Table 2).      Figure 5 shows the average of three light depth values of each macromolecular composition of POM in Jaran Bay from January to December 2016. No distinctive spatial variations were detected in the macromolecular compositions among the different stations; however, they significantly varied among the different months. Carbohydrates were the predominant biochemical component during our observation period from January to December, with monthly proportions of carbohydrates ranging from 40.9% to 66.4%. In comparison, the protein and lipid proportions were 11.1-31.0% and 22.5-35.1%, respectively. The lipid proportion appeared to decrease steadily from January to December. Seasonally, the carbohydrate proportion were relatively variable compared to the protein and lipid proportions. The carbohydrate proportion was lowest during summer (45.6 ± 1.4%) and highest during autumn (59.1 ± 10.9%). In comparison, the protein proportion was lowest during  (Table 2). No noticeable monthly variations were observed for the FM concentrations. Spatial variations in the FM concentrations were not noticeable for the seven stations during the observation period except for March, April and June, which had considerably higher FM concentrations at several stations ( Figure 6). The monthly calorific values and FM contents of FM averaged from the three light depths at the seven stations did not vary significantly and ranged from 5.5-6.3 Kcal g −1 and 1.7-3.7 Kcal m −3 , respectively, in Jaran Bay (Table 2).   Water 2020, 12, x FOR PEER REVIEW 10 of 16 Figure 6. Water column-integrated the total FM concentrations at the sampling stations in Jaran Bay.

Principal Component Analysis (PCA)
The PCA results for our field-observed biochemical parameters are summarized in Table 3. Three PCs were selected for multiple linear regression analysis in this study. The variables shown in bold indicate the highest correlations among the 12 variables and the corresponding components. The nano-sized chlorophyll a concentrations, carbohydrates, proteins, lipids and FMs had the highest correlations with PC1, whereas the concentrations of NH4 + , NO3 − , P* and Si(OH)4 2− were highest correlated with PC2. For PC3, temperature and the micro-and pico-sized chlorophyll a concentrations showed the highest correlations. Based on the PCA results in Table 3, multiple linear regression analysis was performed to obtain the major controlling factors for the variation in the FM in Jaran Bay ( Table 4). The nano-and micro-sized chlorophyll a concentrations and P* and NH4 + concentrations were found to be the major factors for controlling the FM in Jaran Bay during our observation period ( Table 4). The concentrations of nano-and micro-sized chlorophyll a and NH4 + had positive effects whereas the P* concentration had a negative impact on the FM in Jaran Bay during the study period. In other word, a total increase in the concentrations of nano-and microsized chlorophyll a and NH4 + could bring an increase in the FM. On the other hand, an increase in P* concentration could lead to a decrease in the FM.

Principal Component Analysis (PCA)
The PCA results for our field-observed biochemical parameters are summarized in Table 3. Thre e PCs were selected for multiple linear regression analysis in this study. The variables shown in bold indicate the highest correlations among the 12 variables and the corresponding components. The nano-sized chlorophyll a concentrations, carbohydrates, proteins, lipids and FMs had the highest correlations with PC1, whereas the concentrations of NH 4 + , NO 3 − , P* and Si(OH) 4 2− were highest correlated with PC2. For PC3, temperature and the micro-and pico-sized chlorophyll a concentrations showed the highest correlations. Base d on the PCA results in Table 3, multiple linear regression analysis was performed to obtain the major controlling factors for the variation in the FM in Jaran Bay ( Table 4). The nano-and micro-sized chlorophyll a concentrations and P* and NH 4 + concentrations were found to be the major factors for controlling the FM in Jaran Bay during our observation period ( Table 4). The concentrations of nano-and micro-sized chlorophyll a and NH 4 + had positive effects whereas the P* concentration had a negative impact on the FM in Jaran Bay during the study period.
In other word, a total increase in the concentrations of nano-and micro-sized chlorophyll a and NH 4 + could bring an increase in the FM. On the other hand, an increase in P* concentration could lead to a decrease in the FM.  I = 1, 2, . . . , 12 and k = 1, 2

Discussion
The monthly-averaged concentrations of the depth-integrated nutrient concentrations measured were within the ranges previously reported from regions near Jaran Bay [32][33][34][35]. The present study indicates that each nutrient concentration showed significant seasonal variations. For example, the DIN concentrations were relatively higher during the period from September to December, whereas the silicate concentrations were higher in June−September compared to other months ( Table 1).
The monthly depth-integrated total chlorophyll a concentrations within the euphotic water column from 100% to 1% light depths ranged from 5.2 to 36.7 mg m −2 (mean ± S.D. = 17.0 ± 9.2 mg chl-a m −2 ) during the study period from January to December 2016. The largest peak was observed in October immediately, followed by the nutrient peaks observed in September (Table 1). Howe ver, the seasonal chlorophyll a concentrations did not vary greatly and ranged from 15.8 to 17.8 mg m −2 . Gene rally, the spatial variation of the total chlorophyll a concentrations appeared to be low among the seven stations in Jaran Bay during the observation period except for March (Figure 2). Over all, the phytoplankton community was dominated by micro-sized phytoplankton based on the size-fractionated chlorophyll a concentration results during our observation period. Prev ious studies have reported that the predominant species in this area consisted of diatoms [23,36]. In general, the spatial and seasonal variations of the total chlorophyll a concentrations were strongly related to the micro-sized (> 20 µm) chlorophyll a concentrations (Figure 4). This finding suggests that micro-sized cells greatly contributed to the total chlorophyll a concentration in Jaran Bay. In other words, 49.9% (± 18.7%) of total chlorophyll a was from micro-sized cells (Figure 3) during our observation period.
The overall dominant macromolecular composition of POM was carbohydrates (51.8 ± 8.7%), followed by lipids (27.3 ± 3.8%) and proteins (20.9 ± 7.4%), during our observation period ( Figure 5). The macromolecular compositions obtained from the present study fell in a similar range to those obtained from Geoje-Hansan Bay by Kim et al. [6], in which their study area was close to our research site. Howe ver, the compositions in Jaran and Geoje-Hansan bays were considerably different from those in Gwangyang Bay. The mean compositions in Gwangyang Bay were 26.4% (± 9.4%), 37.8% (± 16.1%), and 35.7% (± 13.9%) carbohydrates, proteins, and lipids, respectively [5]. Thes e differences may have been due to the influence of river-borne nutrients. The protein and lipid proportions are largely dependent on the input of dissolved inorganic nitrogen from the Seomjin River in Gwangyang Bay [5]. In comparison, there are no large river inputs in the Jaran and Geoje-Hansan bays. For coastal management plans, e.g., artificial dam construction, the potential influence of river inputs on the dominant cell size and photosynthetic end-products of phytoplankton should be considered [7].
Although the macromolecular compositions between Jaran and Geoje-Hansan Bays [6] in south Korea are similar, the monthly FM concentrations were relatively lower in Jaran Bay and ranged from 297 to 630 mg m −3 with an average of 411.7 mg m −3 (S.D. = ± 93.0 mg m −3 ), than in Geoje-Hansan Bay, which had a range of 346−1280 mg m −3 (615.5 ± 291.7 mg m −3 ; Table 5). Howe ver, the average monthly FM concentration (411.7 ± 93.0 mg m −3 ) of POM in Jaran Bay during our observation period was similar to that in Gwangyang Bay (434.5 ± 175.5 mg m −3 ) [5] despite the large difference in macromolecular compositions between the two bays. Base d on the fact that FM concentrations are derived from the total concentrations of carbohydrates, proteins and lipids [18,19] and that their relative compositions can be affected by various environmental and biological factors [5,[8][9][10][11][12][13][14], different macromolecular compositions are unlikely to be strongly related to the FM concentrations of POM. Inst ead of the compositions of the chlorophyll a concentrations, which are often used to represent phytoplankton biomass, would be more appropriate for comparisons. Howe ver, no strong relationship between the FM concentrations and total chlorophyll a concentrations was found in the present study, although a strong correlation was found in Gwangyang Bay by [7]. Simi larly, no significant linear relationship was observed between the FM and total chlorophyll a concentrations among the different bays in South Korea ( Table 5). The average chlorophyll a concentrations were 2.13 µg L −1 (S.D. = ± 1.18 µg L −1 , this study), 4.34 µg L −1 [6] and 3.45 µg L −1 [5] in the Jaran, Geoje-Hansan and Gwangyang Bays, (Table 5). Thes e bays are all in the South Sea of South Korea. In the Garolim-Asan Bay, Yellow Sea [37], the average chlorophyll a concentration (2.81 ± 2.12 µg L −1 ) was within the low range (2.13−4.34 µg L −1 ) among the three bays, but the average FM concentration (781.4 ± 228.2 mg m −3 ) was highest among the bays in this study. The chlorophyll a concentration has been used as a proxy for biomass, but may not be completely representative of phytoplankton biomass since the chlorophyll a concentration is greatly influenced by light and nutrient conditions, physiological status and species composition of phytoplankton [38][39][40][41]. Inst ead of the chlorophyll a concentration, Lee et al. [5] and Kim et al. [7] suggested that the FM concentration of POM, mainly phytoplankton, could be an alternative proxy for food sources available to higher trophic levels in bay or coastal marine ecosystems. Ther efore, the FM concentration could have a quantitatively complementary value for the amount of various food material sources available to potential consumers in estuarine or bay ecosystems [7,21]. With respect to energy aspects, the calorific content, which depends on the different macromolecular compositions of the FM concentration, should be considered as representative of the physiological or ecological conditions of higher trophic levels of consumers [5,7,21]. According to the PCA results, spatiotemporal variations in FM are primarily governed by the nano-sized chlorophyll a concentrations, carbohydrates, proteins and lipids since FM is the sum of the concentrations of the three different macromolecules. Howe ver, the positive relationship between the nano-sized chlorophyll a concentration and FM would not be predictable. In Jaran Bay, the spatiotemporal change of the total chlorophyll a concentration was primarily controlled by the micro-sized chlorophyll a concentrations because of their high contribution to the total chlorophyll a concentration. In comparison, nano-sized chlorophyll a compositions contributed 28.5% (± 12.4%) of the total chlorophyll a concentration in this study, although their monthly contributions varied somewhat broadly and ranged from 14.3% (±7.0%) in January to 50.3% (±21.3%) in June ( Figure 3). In PC2, the positive correlations among the major inorganic nutrient concentrations (e.g., NH 4 + , NO 3 − , P* and Si(OH) 4 2− ) were reasonable. Temp erature and the micro-and pico-sized chlorophyll a concentrations in PC3 indicate positive correlations among the three variables in Jaran Bay (Table 3). PCA was used in this study for ranking their relative significance (Table 3) among our field-observed biochemical parameters for multiple linear regression analysis and deriving major controlling factors (Table 4) of the FM in our study site. In this approach, we could predict the FM in our study site based on the multiple linear regression analysis. Acco rding to the multiple linear regression, approximately 60% of the variation in FM could be explained by the nano-and micro-sized chlorophyll a concentrations and P* and NH 4 + concentrations in Jaran Bay (Table 4). With this approach, the four major controlling factors were determined for the observed FM variations in Jaran Bay during our observation period from January to December 2016. Howe ver, the somewhat low prediction of up to 60% suggests that other potential factors in addition to our observed parameters should be investigated to improve the spatiotemporal variation in the FM in Jaran Bay. Sinc e this study was a pilot study, some of important parameters were not considered. For example, grazing effects from predators, such as aquaculture shellfish and zooplankton, could be highly correlated with FM, which is a main food source available to them.

Conclusions
A detailed spatiotemporal evaluation of the biochemical compositions and FM of POM of phytoplankton communities and a set of multiple linear regression analyses were conducted in Jaran Bay to understand their major controlling factors. Base d on this research, the variations in FM representing food source materials could be explained by large-cell-sized phytoplankton (>2 µm) and major inorganic nutrient concentrations. Kim et al. [42] observed progressive decreases in dissolved inorganic nutrients in the southern coastal region of South Korea in recent decades. A progressive decline of the chlorophyll a concentration has been consistently reported in several regions in the southern coastal region of South Korea [43]. At this point, we cannot assume that the changes of the species compositions or size compositions of phytoplankton are correlated with the decreases of the concentrations of nutrients and chlorophyll a. Howe ver, we may expect greater numbers of small-sized phytoplankton cells than of large cell-sized phytoplankton cells under these conditions. Thes e changes in nutrient concentrations and dominant phytoplankton communities could cause changes in FM and further alterations in potential consumers. Jara n Bay is one of the largest shellfish aquaculture sites in the South Sea of Korea. Furt her studies on the spatial and temporal variations in the macromolecular compositions and FM of POM in regard to various environmental conditions are needed to better understand the quality and quantity of the primary food source available to higher trophic animals. Funding: This research was supported by a grant (R2020048) from the National Institute of Fisheries Science (NIFS) and partly by a project titled 'Improvements of ocean prediction accuracy using numerical modeling and artificial intelligence technology' which are funded by the Ministry of Oceans and Fisheries, Republic of Korea.