2.5 Statistical analysis
We analyzed and plotted our data in the statistical software R v4.0.3 (R
Core Team, 2020)
(http://www.r-project.org) using
the packages ‘DESeq2’ (Love et al., 2014), ‘vegan’ (Oksanen et al.,
2020), ‘mixOmics’ (Rohart et al., 2017), and ‘ggplot2’ (Wickham, 2016).
We tested for the overall differences in foliar, root, and induced
metabolome composition among the plant diversity or soil legacy levels
by calculating permutational multivariate analyses of variance using
distance matrices. We log + 1 transformed the metabolite intensity data
to achieve multivariate normality, and used Bray-Curtis dissimilarity to
calculate the distance matrices. All analyses were permuted 9999 times.
We used the same approach to test for the differences in the foliar
metabolome composition between control and induced plants. We calculated
each analysis separately for each species and experiment.
To test for the regulation of metabolites, we calculated differential
expression analyses between the monoculture treatment level and each
plant diversity or soil legacy mixture level. We used the ‘DESeq’
function provided by the ‘DESeq2’ package with default argument
structure and values. We defined a metabolite to be significantly
up-regulated when the log2 fold change was above 0.6 (1.5 x higher than
in control) and the p-value below 0.05. Conversely, we defined a
metabolite to be significantly down-regulated when the log2 fold change
was below -0.6 (less than 0.66 x control) and the p-value below 0.05. We
used the same approach to test for the regulation of metabolites between
control and induced plants. We calculated each analysis separately for
each species and experiment.
Subsequently, we assigned the putative molecular formula
(https://www.chemcalc.org/mf-finder) and compound name
(https://pubchem.ncbi.nlm.nih.gov) based on the high-resolution
mass-to-charge values generated by liquid chromatography quadrupole
time-of-flight mass spectrometry for 95 out of 362 up-or down regulated
metabolites. In cases where our search query returned multiple candidate
compounds, we limited the selection to compounds with a mass difference
of less than 2 ppm and a verified description in at least one plant
species.