Results
Photosynthetic active radiation (PAR) in the water column was lower in the sections with larger shaded areas throughout the experiment (Fig. 2a). Water temperature varied from 18 to 25ºC during the experiment but showed no notable differences in mean values of the water columns or the vertical profiles among the four treatments of the two ponds regardless of degree of the shading (Fig. S1(a), S2). In all treatments, pH values gradually decreased towards the end of experiment and were higher in pond 218 (Fig. S1(b)). Dissolved oxygen (DO) concentration varied among the treatments and between the ponds, but were within the range of 5 to 12 mg L-1 (Fig. S1(c)).
Phytoplankton biomass (mg C L-1) correlated significantly with chlorophyll a (µg L-1) (r =0.702, p <0.001), varied temporally (Fig. S3), and was generally higher in the no shade treatments, followed by the low shade treatments in both Pond 217 and 218 (Fig. 2b). Zooplankton biomass also varied temporally (Fig. S4) and was generally lower in low shade treatments compared with other treatments (Fig. 2b). To remove effects of the initial conditions, we calculated mean phytoplankton (P ) and zooplankton biomasses (H ) in samples collected during the period from June 10 to August 28. Phytoplankton biomass was lower in Pond 218 regardless of the treatments, but such a notable difference between the ponds was not found in zooplankton biomass. Accordingly, no significant relationship was found between the mean values of phytoplankton and zooplankton biomass (Fig. 2a).
Both for zooplankton and phytoplankton, the community compositions were similar among the four treatments within the same pond (PERMANOVA,F =0.993, p =0.42 for algae; F =1.23, p =0.34 for zooplankton) but significantly differed between the two ponds (F =1.59, p =0.017 for algae; F =3.82, p =0.047 for zooplankton). In zooplankton communities, copepods predominated in pond 217, while large cladocerans including Daphnia occurred abundantly in pond 218 (Fig. S5). In phytoplankton communities, Euglenophyceae and Chrysophyceae occurred abundantly in pond 217, and Euglenophyceae and Dinoflagellata dominated in pond 218 (Fig. S6). In all treatments, cyanobacteria biomass was less than 10%. According to previous knowledge (Lampert and Sommer 2007), we defined that phytoplankton smaller than 30 µm for the major axis of the cell or colony were edible. Then, we estimated the ratio of edible phytoplankton biomass to total phytoplankton biomass as a fraction of the edible phytoplankton (αedi ). It varied from near zero to almost one in all the treatments of both ponds (Fig. S3). Seston carbon to phosphorus ratio varied from 90 to 310 (Fig. S7) and was higher for treatments with less shade in pond 217, while in pond 218 seston carbon to phosphorus ratio did not vary among the treatments (Fig. 3b).
Chlorophyll a specific daily production rate estimated from the P-I curve (Fig. S8) varied temporally depending on weather conditions but was, in general, higher in treatments with less shade (Fig. 3c). Daily primary production rates also varied and were higher in treatments with less shade in pond 217, although among the treatments in pond 218 the levels were similar (Fig. S4).
Observations of fish abundance in each treatment section, determined using minnow traps, showed that banded killifish (Fundulus diaphanus ) and fathead minnow (Pimephales promelas ) were present (Fig. S9). Both fish species were collected on all sampling dates in pond 217 but were not caught after June 21 in pond 218 (Fig. S4). Thus, mean abundance of these fish species (θ ) was higher in pond 217 than in pond 218 (Fig. 3d). In the former pond, fish abundance also varied among the treatments, and was greater in no shade treatments than in any of other treatments. Neither mean of zooplankton biomass (r = 0.310, p = 0.45) nor mean of specific production rate (μ ) (r = 0.247, p = 0.56) were significantly related to mean fish abundance (θ ).
Throughout the study period, the mass ratio of zooplankton and phytoplankton varied temporally (Fig. S4). Among the treatments, the temporal mean of this ratio (H/P ratio) was highest in the mid shade treatment and lowest in the low shade treatment in both ponds (Fig. 3). However, the H/P mass ratio was higher in pond 218 than in pond 217. A significant relationship was not detected between theH/P mass ratio and mean PAR in the water column (Fig. 2a;r = 0.155, p = 0.714), mean frequency of edible phytoplankton (αedi ) (Fig. 3a; r = 0.241,p = 0.565), mean seston carbon to phosphorus ratio (αnut ) (Fig. 3b; r = −0.265, p = 0.523), and mean specific production rate (μ ) (Fig. 3c; r= 0.081, p = 0.849), whilst a significantly negative relationship was detected between the H/P mass ratio and mean fish abundance (θ ) (Fig. 3d; r = −0.818, p = 0.013).
We fitted the H/P mass ratio by αedi ,αnut , μ , and θ among treatments in the two ponds using a multiple regression linear model. The variance inflation factors (VIFs) for these explanatory variables ranged from 1.05 to 2.38, indicating a low probability of multicollinearity among explanatory variables. Moreover, an analysis with the generalized linear model showed that the model including all of these parameters had the lowest value of Akaike’s Information criterion (Table S2), indicating that it was the best model. The multiple regression analysis revealed that all of these four variables were indeed significant, as evidenced by the 95% confidence intervals (CIs) that were smaller or larger than zero, and explained 94% of variance in the H/P ratio (Table 1). In addition, partial regression analysis showed that all the partial correlation coefficients of these factors were significant (Fig. 4), indicating that effects of these explanatory variables on the H/Pmass ratio were independent of each other. More importantly, the regression coefficient was significantly smaller than zero for seston carbon to phosphorus ratio (αnut ) while it did not significantly differ from one for edible phytoplankton frequency (αedi ) and specific production rate (μ ), and was smaller than one but larger than zero for fish abundance (θ ). To examine the effect sizes of these explanatory variables on H/P mass ratio, we estimated standardized regression coefficients. The absolute value of the coefficients showed that the effect size on the H/P mass ratio was highest for θ , followed by αnut (Table 1).