FIGURE 5 Average MGDG unsaturation (number of double bonds (DB)) in the Krka River Estuary (triangles) and the Wenchang River Estuary (squares).
Our aim was to investigate the influential parameters responsible for the sMGDG accumulation, which MGDG species increase under salinity stress and which phytoplankton group is possibly responsible for their synthesis. To this end, we first performed PCA to determine the environmental variables (salinity (S), temperature (T), DIN and PO43¯) responsible for sMGDG accumulation (Figure 6a and b, Tables S4 and S5). For this analysis, we have taken data from the estuaries and not from the rivers. The SiO44- concentrations were not considered in this analysis as diatoms did not appear to be important for sMGDG accumulation. First two principal components in the principal component analysis between five variables for the Krka River Estuary and the Wenchang River Estuary explained 90.95% and 92.37% of the total variability, respectively. The position of the salinity and PO43¯ variables indicates that salinity changes and the availability of DIN contribute the most to the observed results. Temperature shows opposite trends in the PCA with salinity for both estuaries. The reason for this temperature distribution in the Krka River Estuary is the mixing of cold river water with warmer seawater in September, while the opposite occurred in the Wenchang River Estuary in May, when warmer river water mixed with colder seawater.
Second, we performed a PCA considering sMGDG (%), salinity, DIN, pigments and 28 variables of MGDG with fatty acid double bond combinations for the Krka River Estuary and Wenchang River Estuary, respectively. After a preliminary PCA, the MGDG species that correlated significantly with other variables were selected for further PCA (Figures. 6c and d, Table S6 and S7). First two principal components in the PCA for the Krka River Estuary explained 58.80% of the total variability among 34 variables, while for the Wenchang River Estuary they explained 53.81% of the total variability between 28 variables.
In the Krka River Estuary, in addition to sMGDG and DIN,peridinin and violaxanthin together with MGDG species with the fatty acid double bond combinations 6+6, 5+6, 1+4 and 0+5 have the largest negative PC1 loadings. These results indicate that dinoflagellates (pigment peridinin ) and chlorophytes and prasinophytes (pigment viola ) were probably the main contributors for the accumulation of sMGDG at low salinity.
PCA for the Wenchang River Estuary variables shows that the greatest positive PC1 loadings next to sMGDG (%) and DIN, have lutein(chlorophytes and prasinophytes), and 4+5, 3+4 and 1+3 MGDG species (Figure 6d). This indicates that chlorophytes and prasinophytes with the MGDG double bond combinations 4+5, 3+4 and 1+3 probably contributed to the accumulation of sMGDG at low salinity.
The main MGDG fatty acids that contributed to increased MGDG unsaturation (MGDG (%)) in the Krka River Estuary were fatty acids with the combination of double bonds 6+6, 5+6, 1+4 and 0+5. In the Wenchang River Estuary fatty acids with a combination of double bonds 4+5, 3+4 and 1+3.