Network construction
To detect warming effects on potential fungal interactions, we used
functional gene data to construct functional molecule ecological
networks (fMENs) for control and warmed samples separately, via the
pipeline of Molecular Ecological Network Analyses (MENA)
(http://ieg4.rccc.ou.edu/mena/) (Deng et al., 2012). Only functional
genes detected in all six biological replicates were included to ensure
network reliability.
To
compare topological characteristics between control and warmed networks,
we chose the same threshold of 0.98 during network construction, which
refers to the Spearman’s correlation coefficients between pairwise OTUs.
Network modules, which are groups of nodes that highly connected within
the group but less connected outside the group, were separated by the
fast-greedy modularity optimization. Network topological
characteristics were calculated via
MENA, including total nodes, total edges, average connectivity (node
degree), average clustering coefficient, average path distance, and
modularity. Network randomization (a total of 100 random networks) was
performed via MENA. To test whether
fMENs
of control and warmed samples have typical properties of biological
networks (e.g., small-world, hierarchy, modularity), network topological
characteristics were compared with their corresponding 100 random
networks. To test statistical differences of fMENs between control and
warmed samples, we performed two-tailed Student’s t -tests by
taking their network topological characteristics as averages and
standard deviations of their corresponding 100 random networks as
standard deviations (Wu et al., 2016). Cytoscape (version 3.5.1) was
used for network visualization. Some network scores (e.g., connectivity)
were used to identify putative key genes/species that have large
influences in maintaining community structure and function, despite that
species identities can be inaccurate owing to horizontal gene transfer.
To select network key genes, we set thresholds
based on within-module connectivity
(Zi) and among-module connectivity (Pi) of nodes: Zi ≥ 2.5 and Pi ≥ 0.62
for network hubs, Zi ≥ 2.5 and Pi < 0.62 for module hubs, Zi
< 2.5 and Pi ≥ 0.62 for connectors, Zi < 2.5 and Pi
< 0.62 for peripherals (Guimera & Amaral, 2005).
Previous studies have shown that
network key genes/species were inclined to be more responsive to
environmental changes than others (Wu et al., 2016). Therefore, we
checked whether the connectivity of network key genes was linked to
warming-induced changes in environmental factors by performing partial
Mantel tests between the matrix of connectivity of network key genes and
the matrix of environmental factors.