**3. Results**

#### *3.1. Microbial Total Biomass and Community Composition*

Total PLFA concentrations, a proxy for microbial biomass, increased significantly (*p* < 0.05) in the nature forest floor, the intermittent stream and the headwater stream from the 28th day of decomposition to the 119th day. The total PLFA concentrations in the headwater stream were significantly higher than those in the intermittent stream and the natural forest floor during the 62nd day of decomposition. However, the total PLFA

concentrations decreased greatly on the 95th day of decomposition in both the intermittent stream and the headwater stream, which made habitat type don't significantly affect the concentrations of total PLFA at the end of litter decomposition (Figure 1a).

**Figure 1.** The concentrations of microbial total PLFA (**a**) and PLFA of microbial groups (**b**–**e**,**g**,**h**) in the natural forest floor, intermittent stream, and headwater stream leaf litter and the ratios of biomarkers (**f**,**i**). The blue font indicates the number of days the intermittent stream was dry. The *p* values showed the results from repeated measures ANOVA testing for the effect of habitat type over time. The expression data were shown as mean ± SE. *NS*: not significant. Asterisks denote significant differences among habitat types: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

Some microbial community compositions varied over time, and this variation showed different patterns among habitat types. In three habitats, the G+ bacterial and actinomycetes PLFA and the ratios of G+:G− bacteria PLFA (G+/G−) peaked on the 119th day and its concentrations and ratios in the stream and the intermittent stream were significantly higher than that in the nature forest floor. Meanwhile the G+ bacterial PLFA concentrations and the ratios of G+/G− peaked on the 62nd day in the stream (Figure 1d,g,i)). At this point, the G+ bacterial PLFA concentrations in the headwater stream were significantly higher than those in the other two habitats. The ratios of G+/G− in the headwater stream and the intermittent stream were significantly higher than that in the nature forest floor. In the nature forest floor, the fungal PLFA concentrations were significantly higher than those in the other two habitats during the early and late stages of decomposition (Figure 1c). The ratios of fungal: bacterial PLFA (F/B) had a similar trend as the fungal PLFA (Figure 1f). In the headwater stream, the eukaryotic PLFA concentrations were significantly higher than those in the other two habitats during the late stages of decomposition (Figure 1h).

#### *3.2. Distribution Patterns of PLFA*

We used the PCA to illustrate the differences among the three habitats for selected PLFAs related to litter decomposition (Figure 2 and Table 1). The PCA performed on the composition of PLFAs showed that 61.0% and 13.2% of variations were explained by the first and second axes respectively. The total biomass, bacteria, G+ bacteria, G− bacteria, and actinomycete had positive loadings on the PCA-1 in the three habitats. However, fungal and eukaryotic PLFA was unspecific in three habitats. The fungal PLFA had positive loadings on PCA-1 in the intermittent stream and headwater stream and had positive loadings on PCA-2 in the nature forest floor. The eukaryotic PLFA had positive loadings on PCA-1 in the headwater stream and had positive loadings on PCA-2 in both intermittent stream and headwater streams.

**Figure 2.** Principal components analyses of PLFA profiles (log10 transformed mol) on litter at three habitats. PLFAs in green, orange and blue font represent the natural forest floor, the intermittent stream and stream samples, respectively. TB: Total Biomass, B: Bacteria, F: Fungi, G+: G+ bacteria, G−: G− bacteria, A: Actinomycete.

**Table 1.** Linear regression for three habitats between the first and second principal components (PCA-1, PCA-2) after the PCA and the concentrations of microbial PLFA. Asterisks denote significant influences of the concentrations of microbial PLFA on the data variation represented by PCA-1 and PCA-2: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.


#### *3.3. Individual PLFA Analysis*

The comparison of individual PLFAs in terms of the whole decomposition period considered revealed a major change in the composition of the microbial communities in the three habitats. We identified more individual PLFAs in the natural forest floor and the intermittent stream over the decomposition of the litter (Figure 3). Among them, the concentrations of individual PLFA indicative of G+ bacteria (i15:0, a15:0) increased steadily with decomposition time in the forest floor and the intermittent stream. The concentrations of PLFA indicative of G− bacteria (18:1 w5c) fluctuated greatly with decomposition time (Figure 3a,b). In contrast, there were only two PLFAs (n19:0, 18:2 w6c) in the headwater stream (Figure 3c).

Under the same decomposition period, we identified more individual PLFAs in the later stage of decomposition (Figure 4). The individual PLFA differed more markedly among the three habitats with the increase of decomposition time. In the early stage of decomposition, the difference of individual PLFA was mainly reflected between the forest floor and the aquatic system (Figure 4a,b). There was insignificant difference between

the headwater stream and the intermittent stream. On the contrary, in the later stage of decomposition, the difference of individual PLFA was mainly reflected in the headwater stream and the intermittent stream (Figure 4c,d).

**Figure 3.** Concentrations of individual PLFA in (**a**) the natural forest floor and (**b**) the intermittent stream and (**c**) the headwater stream leaf litter. Asterisks indicate significant differences among decomposition time: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. Dots indicate the mean and whiskers the standard error.

**Figure 4.** Concentrations of individual PLFA on (**a**) the 28th day of decomposition and (**b**) the 62nd day of decomposition and (**c**) the 95th day of decomposition and (**d**) the 119th day of decomposition leaf litter. Letters indicate significant differences among transect regions (*p* < 0.05). Dots indicate the mean and whiskers the standard error.

#### *3.4. PLFA Biomass and Litter Decomposition*

Total PLFA did not correlate with mass loss in three habitats (*p* > 0.05) (Figure 5a). However, the communities of microbial decomposers showed a strong correlation with litter mass loss in three habitats. Among them, the PLFA concentrations of microbial groups and the ratios of biomarkers showed the highest correlation with mass loss in the headwater stream (Figure 5). Specifically, the concentrations of bacterial PLFA, fungal PLFA, G+ bacterial PLFA, G− bacterial PLFA, eukaryotic PLFA, and the G+/G− ratios were related to the loss of litter quality (*p* < 0.05) (Figure 5b,c,e–g,i)). However, only the concentrations of G+ bacterial PLFA, actinomycetes PLFA, and the G+/G− ratios correlated with mass loss in the natural forest floor (*p* < 0.001) (Figure 5d,e,i)). Only the G+/G− ratios correlated with mass loss in the intermittent stream (*p* < 0.05) (Figure 5i).

**Figure 5.** Linear regression for three habitats between litter mass loss and the concentrations of microbial total PLFA (**a**) and PLFA of microbial groups (**b**–**g**), and the ratios of biomarkers (**h**,**i**).
