Nitrate input simplified interactive network
Ten networks based on Spearman’s correlation coefficient were
constructed to identify possible interactions. Key network topologies
were listed in Table S1, from which we selected network size, modularity
and connectivity to correlate three community metrics (Figure 2A, 2B,
2C). Sizes of control networks remained time-steady (428.8 ± 23.7 nodes,P >> 0.05) while nitrate networks kept
shrinking, from 308 (Day 0) to 94 nodes (Day 32), meaning that fewer OTU
pairs were generating significant abundance covariations. Such changing
patterns applied to all metrics in Figure 2 that elevated nitrate was
simplifying community compositions (with respect to richness and
α-diversity) and microbial interactions. To visualize this
simplification, all networks were plotted (Figure 3). The Fruchterman
Reingold layout (Figure 3A) well exhibited the shrinking sizes of
nitrate networks, while the Yifan Hu layout (Figure 3B) emphasized the
decreasing modularity and node-node interactions.
Putative keystone nodes referred to those of great network connectivity,
either within a module (i.e. module hub) or among modules (i.e.
connector). Their taxa were therefore putative keystone taxa. We plotted
the distribution of keystone nodes in Figure S2A. Of all the 3352 nodes,
most were insignificant peripherals (97.67%), 44 were module hubs and
34 were connectors (Table S2). No network hub was detected. When
correlating node significance to OTU relative abundances (Figure S2B),
we found that abundance did not necessarily determine its network
significance, although keystone nodes turned to be sparse OTUs. Of the
78 keystone nodes, 31 were rare (< 0.1% relative abundance),
27 were moderately rare (0.1 - 0.5%), 13 were moderately abundant (0.5
- 1%), and only 7 were abundant (>1%). In addition, the
relative abundances of module hubs and connectors indicated no
significant difference (P > 0.05).
To investigate the putative keystone taxa, we prepared a phylogenic tree
that indicated (i) all the putative keystone taxa at the phylum or genus
level; (ii) their phylogenic distance based on neighbor-joining method;
and (iii) their keystone type (Figure S3). Most keystone nodes (85.5%)
could be assigned to a phylum but only 28.9% could be assigned to a
genus. Members of phylum Proteobacteria (e.g. genusSmithella and Syntrophorhabdus ) and Chloroflexi(genus Bellilinea , Leptolinea and Pelolinea )
constituted the majority of keystone taxa. Genus Smithella in
particular, was found highly interactive in networks since 5 OTUs
belonging to Smithella were keystone nodes. In addition, four
keystone taxa were methanogenic archaea (Lee et al. 2015) and
were members of genus Methanolinea , Methanothrix , and Methanobacterium.
Generally, the number of keystone nodes decreased as the nitrate
amendment proceeded longer (Figure S4). That is, the network
simplification occurred alongside the loss of keystone taxa. Recall thatThiobacillus was barely detectable in the control communities
(< 0.02% relative abundance) but dominated the late nitrate
communities (~60.88%). However, it was never a keystone
genus, showing no significant covariations in any network (Table S3).