Distinctive molecular ecological network structure and connectivity across four mangrove root-associated compartments
To explore potential microbe-microbe interactions in mangrove roots, we analyzed intra-domain and inter-domain microbial interactions based on sequencing datasets of bacterial and fungal communities (Fig. 1a; Additional file 2: Table S2), which were represented as single bacterial and fungal network, and bacterial-fungal interaction (BFI) networks. Across four mangrove root-associated compartments, we obtained a total of 12 microbial co-occurrence networks and their R2 of power-law ranged from 0.860 to 0.929 (Additional file 3: Table S3), suggesting that those network connectivity distributions were fitted well with the scale-free property .
Multiple network topological metrics consistently revealed that microbial co-occurrence patterns differed profoundly across four root-associated compartments (Fig. 1). The assemblages of three peripheral compartments (non-rhizosphere, rhizosphere and episphere) formed larger and more complex networks with more nodes and edges than that of the endosphere, especially for BFI (Fig. 1b, c). More interestingly, network complexity also differed between intra-domain and inter-domain interaction networks. The BFI networks exhibited a lower average connectivity than their corresponding bacterial and fungal networks in three peripheral compartments (Fig. 1d). However, in the endosphere, a significantly (P < 0.05, two-tailed Fisher’s exact test) higher average connectivity was detected in the BFI network (2.986) than in the fungal (2.419) network (Fig. 1d; Additional file 3: Table S3), and more nodes and edges were also observed in the BFI network (426 and 636) than in the single bacterial or fungal networks (210 and 276 for bacteria; 186 and 225 for fungi) (Fig. 1b, c). The results revealed that the endosphere was the only compartment in which the BFI network harbored more intensive interactions than the single bacterial or fungal networks, creating a distinctive niche inside root with the enhanced inter-domain interactions.
We next identified inter-domain microbial assemblages that potentially interacted or shared niches across four root-associated compartments. Four representative BFI networks contained modules with modularity > 0.5 (Additional file 3: Table S3), and we concentrated on modules with more than 15 nodes. Similar to the overall BFI network structure (Fig. 1a), the number of modules became smaller in transition from the non-rhizosphere, rhizosphere to episphere, but became larger again in the endosphere (Additional file 4: Fig. S1). Notably, we found that module composition did not show clear differences across four root-associated compartments, in which the proportions of bacteria and fungi were nearly identical (Additional file 4: Fig. S1). The results indicated that the bacteria and fungi contributed similar proportion to the inter-domain microbial interactions in the soil-mangrove root continuum.
Putative keystone taxa across four mangrove root-associated compartments
On the basis of within-module connectivity (Zi) and among-module connectivity (Pi) values, we classified all network nodes into four parts: module hubs, network hubs, peripherals and connectors . Due to the roles in network topology, the taxa detected as module hubs, network hubs and connectors were proposed to be keystone taxa in the complex microbial communities. The results showed that most nodes from each network were peripherals, and no network hubs were identified (Fig. 2). Intriguingly, in three peripheral compartments, more module hubs and connectors were detected in the single bacterial and fungal networks than in the BFI networks (Additional file 5: Fig. S2); however, in the endosphere compartment, the corresponding BFI network harbored more keystone taxa (Additional file 5: Fig. S2), which is consistent with its more complicated networks (Fig. 1a).
To investigate the role of keystone taxa in microbial inter-domain interactions, we focused on BFI networks and their associated keystone taxa across four root-associated compartments. We found that 15, 23, and 13 keystone taxa were observed in the BFI networks of the non-rhizosphere, rhizosphere and endosphere, respectively, but no keystone taxa were detected in the episphere (Fig. 2). Consistently, most of these putative keystone taxa had low relative abundances (Additional file 2: Table. S2; Additional file 5: Fig. S2), suggesting that low-abundant taxa may significantly contribute to mangrove root functions. Further analysis of keystone taxa indicated that fungi accounted for a larger proportion (60.5%) in module hubs and connectors of BFI networks than bacteria (39.5%). Nonetheless, they displayed distinct importance across those four compartments. In the non-rhizosphere and rhizosphere, keystone taxa with top three highest degrees (the number of links for a particular node) were all monopolized by fungi (Additional file 6: Fig. S3). However, in the endosphere, keystone taxa with top three highest degrees belonged to bacteria, and they were affiliated with Vibrio (OTUB_1491), Anaerolineae (OTUB_472) and Desulfarculaceae (OTUB_260) (Fig 2; Additional file 2: Table S2).
Possible microbial interaction mechanisms across four mangrove root-associated compartments
In order to understand possible mechanisms of microbial interactions across compartments, we investigated the functional profile of root-associated microbial communities by shotgun metagenome sequencing. We focused on functional genes and pathways involved in quorum sensing and cobamide biosynthesis as they have been considered as typical strategies of microbial interactions possessed by various communities [22, 51].
Quorum sensing served as a cell-cell communication device, which was exploited by many microflora . Here, we detected quorum sensing circuits with two modules represented by Gram-negative bacteria, and found that genes involved in quorum sensing were unevenly distributed across four continuous compartments (Fig. 3). In module 1, the genes individually related to the production of autoinducer (S)-3-hydroxytridecan-4-one (CAI-1) and the mediation of group behaviors, cqsA and luxR, were highly abundant in the episphere and endosphere, respectively. As the most abundant gene in module 1, tdh relates to the production of autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) and its abundance continuously decreased from the non-rhizosphere to the endosphere. In module 2, lasI and pqsH, related to production of autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and 2-heptyl-3-hydroxy-4-quinolone (PQS), were detected across four compartments, but showed a relatively higher abundance in the endosphere. More strikingly, the endosphere was the only compartment that simultaneously contained rhlI and lasR, which were involved into the syntheses of autoinducers N-butyryl-L-homoserine lactone (C4-HSL) and the mediation of group behaviors, respectively. The divergent pattern of genes involved in quorum sensing indicated that three peripheral compartments held a greater potential in conducting module 1, whereas module 2 was more likely to be conducted in the endosphere (Fig. 3). Combined with the intensive microbial interactions in the endosphere (Fig. 1a), we infer that module 2 of quorum sensing was closely associated with the enhanceding inter-domain microbial interactions in the endosphere of mangrove roots.
Cobamide, as one of the sharing valuable nutrients, was widely used by microbes to interact with each other . Our metagenome sequencing analysis revealed that among 14 cobamide biosynthesis genes, six genes (cysG, cobF, cbiB, cobC, cobA, and cobD) were significantly (P < 0.05, two-tailed Fisher’s exact test) higher in the endosphere than in other three compartments, and they were involved in each of key steps for cobamide biosynthesis, including the tetrapyrrole precursor biosynthesis, corrin ring adenosylation, nucleotide loop assembly and aminopropanol phosphate production (Fig. 4a). Intriguingly, a compartment specificity was detected for anaerobic or aerobic corrin ring adenosylation. Compared to three peripheral compartments, the endosphere harbored a higher abundance of genes associated with aerobic adenosylation (cobG, cobF and cobA) and a lower abundance of genes associated with anaerobic adenosylation (cbiT and cbiE) (Fig. 4a). Furthermore, the taxonomic composition of all cobamide-related genes across four compartments (Fig. 4b) revealed a high detection frequency of Rhodobacteraceae and Pseudomonadaceae, highlighting their key roles in mediating microbial interactions by producing cobamides. It is important to note that these two families were characterized by a nearly equal detection frequency across four root-associated compartments (Fig. 4b), with a low abundance (2.98%-6.81%) in the whole bacterial community (Additional file 7: Fig. S4). Therefore, these results indicated that the intensified microbial interactions in the endosphere were linked to aerobic cobamide biosynthesis performed by specialists (Rhodobacteraceae and Pseudomonadaceae).