Overview of microbial diversity of three habitats in the SCPE
To understand the microbial diversity in the SCPE, we sequenced 16S rRNA gene amplicons from 792 samples in 88 shrimp cultural ponds across six regional sites. A total of 38,662,478 high quality sequences (30,580 to 87,563 sequences for each sample) were obtained from all samples, and clustered into 15,197 operational taxonomic units (OTUs) with the highest number (i.e. 14,857) of OTUs in the sediment (Additional file 1: Tables S2 and S3). Such sequencing efforts were enough to capture a majority of microbial communities in all samples (Additional file 1: Table S3). The Shannon index was the highest in sediment (6.5 ± 0.3), and followed by water (4.4 ± 0.5) and shrimp intestine (3.2 ± 1.2), which significantly (P < 0.001) differed among those three habitats, and Chao1 index showed similar results (Fig. 1a; Additional file 1: Table S3). To further evaluate the overall differences among three habitats, non-metric multidimensional scaling (NMDS) analysis showed that microbial communities were clustered by water, shrimp intestine and sediment habitats, which was further corroborated by analysis of similarity (ANOSIM), revealing that the microbial community structure significantly (r = 0.8115, P < 0.001) differed between any two of compared habitats (Fig. 1b).
Core OTUs among three habitats or of each habitat in the SCPE
To examine if core microbial taxa exist among three habitats in the SCPE, we defined that core microbial taxa should occur in ≥ 90% of all 792 samples tested (see Methods for details). About 0.08% (13 out of 15,197 OTUs) constituted core taxa among three habitats that accounted for the relative abundance of 7.98% of all sequences (Fig. 2a and 2b). The core OTUs belonged to Cyanobacteria (6 OTUs and a relative abundance of 5.63%), Actinobacteria (3 OTUs and 0.96%), Proteobacteria (3 OTUs and 0.98%), and Verrucomicrobia (1 OTU and 0.41%) (Additional file 1: Table S4).
Similarly, of each habitat, we defined that core microbial taxa should occur in ≥ 90% of 264 water, 264 shrimp intestine, or 264 sediment samples, respectively. The results showed that about 0.3% (30 out of 9,874 OTUs), 0.2% (15 out of 7,466 OTUs) and 0.4% (53 out of 14,857 OTUs) constituted core microbial taxa in water (Fig. 2c and 2d), shrimp intestine (Fig. 2e and 2f) and sediment (Fig. 2g and 2h) habitats, respectively, and they accounted for the relative abundance of 33.28%, 38.76% and 9.12% of all sequences obtained. The core OTUs in water belonged to Cyanobacteria (17.87%), Actinobacteria (8.50%), Proteobacteria (2.14%), Chlorobi (4.04%) and Verrucomicrobia (0.72%); the core OTUs in shrimp intestine belonged to Proteobacteria (29.38%), Cyanobacteria (1.52%), Actinobacteria (0.43%), Tenericutes (6.97%) and Verrucomicrobi (0.46%); the core OTUs in sediment belonged to 12 phyla, including Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Ignavibacteriae accounting for 4.64%, 1.98%, 0.88%, 0.68% and 0.51%, respectively (Additional file 1: Tables S5, S6, S7 and S8). Thus, the core OTU composition of water, shrimp intestine and sediment habitats were also distinct for each habitat, suggesting that each habitat would select their core microbial taxa. Also, 15 core OTUs of shrimp intestine were also present in water and sediment habitats (table S3), suggesting possible sources (e.g., water and sediment) of shrimp intestine microbial communities. Interestingly, Vibrio OTU934, Vibrio OTU1793, Photobacterium OTU442 and Candidatus Bacilloplasma OTU384 were found to be core OTUs of shrimp intestine, and they are considered as animal opportunistic pathogens in aquatic ecosystems (Additional file 1: Table S6). Also, some functional microbes, such as Synechococcus OTU25011, Synechococcus OTU5786, Synechococcus OTU3905 and Rhodobacter OTU29858, or Synechococcus OTU25011, Synechococcus OTU5786, Rhodobacter OTU29858, Truepera OTU9796 and Desulfomicrobium OTU3310, were found to be core OTUs of water or sediment habitat, and they may enhance carbon and sulfur cycling in aquatic ecosystems (Additional file 1: Tables S5 and S7).
Comparison of the microbial community composition in three habitats
To understand the composition of microbial communities from three habitats, we compared them at the OTU level using Venn analysis. The results showed that a total of 5,997 OTUs were commonly present in three habitats, and the number of OTUs was found to be in any two habitats: 6,094 (water, 81.6%) or 7,279 (sediment, 97.5%) out of 7,466 intestine OTUs, 6 094 (intestine, 77.2%) or 9,625 (sediment, 97.5%) out of 9,874 water OTUs, and 7,279 (intestine, 49.0%) or 9,625 (water, 64.8%) out of 14,857 sediment OTUs (Fig. 3). Most OTUs were in the sediment habitat, and a high percentage (~97.5%) of OTUs in shrimp intestine and water habitats were shared with the sediment habitat. For each pond and all six regional sites, similar trends were observed (Additional file 2: Figure S1).
We further compared the composition of microbial communities among three habitats at the phylum and genus levels, showing that all detected phyla and genera were always present in any of the three habitats (Additional file 2: Figures S2 and S3), but their relative abundances significantly (P < 0.001) differed (Additional file 1: Tables S9 and S10; Additional file 2: Figure S4a and S4b). Some opportunistic pathogens, such as Vibrio, Photobacterium and Candidatus Bacilloplasma, were detected in three habitats, and their relative abundances were significantly (P < 0.001) higher in the shrimp intestine than in the water or sediment habitat (Additional file 1: Tables S10 and S11; Additional file 2: Figure S4b and S4c).
Sediment communities mainly contribute to shrimp intestine microbiota compared to water community in the SCPE metacommunity
In order to evaluate the contribution of microbial communities of three habitats to the SCPE metacommunity diversity, we used additive diversity partitioning of diversity across scales to determine whether the microbial diversity observed at the ecosystem level (ɤEcosystem) was mainly from a high microbial dissimilarity among habitats (βInterHabitats), a high dissimilarity among communities within each habitat (βIntraHabitats) or from a high microbial diversity within each local community (LocalCommunities, i.e., water, shrimp intestine or sediment sample). The results showed that the contribution of αLocalCommunities to the metacommunity diversity (ɤEcosystem) was 66.0 ± 11.2%, outweighing βInterHabitats (29.0 ± 11.0%) andIntraHabitats (5.0 ± 2.4%) in their contributions to ɤEcosystem (Fig. 4a). This high contribution of α-diversity and β-diversity to ɤEcosystem revealed thatLocalCommunities and βIntraHabitats were important for generating the microbial diversity in the SCPE.
To test if sediment communities have a decisive role to shrimp intestine microbial communities, we evaluated the contribution of different source communities to water, shrimp intestine and sediment habitats in the SCPE by SourceTracker analysis. For water microbial communities, the most dominant potential source was sediment (an average of 53.6%), followed by shrimp intestine (35.0%); for shrimp intestine communities, the most dominant potential source was also sediment (56.8%), followed by water (18.1%) (Fig. 4b). In contrast, for sediment communities, the source from water or shrimp intestine only attributed 17.9% or 15.4 %, respectively (Fig. 4b). These results indicated that each microbial community could be the source for the other two communities, and especially, sediment was found to be the most important source of water and shrimp intestine communities, and such similar trends were observed across all six regional sites (Additional file 2: Figure S5), indicating a possible general pattern in the SCPE.
The Sloan neutral community model was further applied to analyze the shared OTUs between the surrounding sediment or water and shrimp intestine. That is, neutral distribution (black points) accounted for 37.8% in sediment, and 30.8% in water microbial communities (Fig. 4c). In contrast, the proportion of under-represented (green points) and over-represented (red points) OTUs was 15.0% or 5.2%, and 44.7% or 45.7% in sediment or water, respectively (Fig. 4c). Thus, the proportion of shared and neutrally distributed OTUs between shrimp intestine and sediment was higher than these in water, suggesting that communities of shrimp intestine were tended to colonize from sediment rather than from water. More obvious similar trends were observed for the six locations across a regional scale (Additional file 2: Figure S6).
The microbial assemblies of three habitats in the SCPE were largely controlled by stochastic processes
In order to understand microbial assembly mechanisms in three habitats of the SCPE, we used the null model-based approach to calculate stochastic ratios with taxonomic (Bray-Curtis, abundance-weighted and unweighted) and phylogenetic (weighted and unweighted UniFrac) metrics. The results showed that the average stochastic ratios based on taxonomic and phylogenetic metrics were higher than 78.4% in water, 80.8% in shrimp intestine and 80.3% in sediment habitats (Fig. 5a), suggesting that stochastic factors were more important than deterministic factors in influencing microbial community composition of three habitats in the SCPE.
Variation partitioning analysis (VPA) was performed to discern the relative importance of various factors (geographic distance and environmental factors) contributing to microbial communities of water and sediment habitats in the SCPE. Water environmental factors (pH, salinity, dissolved oxygen (DO), ammonia nitrogen (NH4+-N), nitrate nitrogen (NO3--N), orthophosphate (PO43--P), concentrations of total carbon (TC), total organic carbon (TOC), total phosphorus (TP), ratio of carbon to nitrogen (C/N) and ratio of nitrogen to phosphorus (N/P)) and sediment environmental factors (pH, TC, TOC, total nitrogen (TN), TP, C/N, ratio of carbon to phosphorus (C/P) and N/P) were selected by the BioEnv procedure, which provides the highest Pearson correlation with the microbial community structure. Overall, the combination of selected water or sediment environmental factors and geographic distance showed a significant correlation (P < 0.001) with the water or sediment microbial structure. These variables explained 29.5% or 24.9% of the observed variation in water or sediment habitats, respectively, leaving 70.5% or 75.1% of the variation unexplained (Fig. 5b and 5c). The water or sediment environmental factors explained 21.7% or 14.2% (P < 0.001), and geographic distance alone explained 2.5% or 9.0% (P < 0.001) variations with 5.3% or 1.7% interaction effect detected, respectively (Fig. 5b and 5c).