Data analysis
Since data showed large year-to-year variation, we analyzed each year
separately (PerManova: F(1.313)=4.45, P=0.009). We first
tested the dispersion of chemical profiles of all trees between sampling
years (2014, 2015), and then between months within a year using NMDS
ordination with Bray-Curtis distance (metaMDS function, packagevegan v2.5-6). We used PERMANOVA to verify whether chemical
profiles changed over time with block set as strata (adonis2function, vegan package v2.5-6 in R v4.0.3). An overview of our
approach for each question is detailed below. Due to multiple
permutations within our analyses, we summarized which treatments were
compared to evaluate each research question in Table 1.
1. Do treatments influence NSCs of ponderosa pines? Since
increment cores were collected from the same trees over multiple times,
we used repeated measures analysis. We first checked the assumption of
sphericity using Mauchly’s test by testing whether starch, total sugars,
and total NSCs change with month as a function of treatment
(anova_test function, package rstatix v0.6.0). Results
showed the data met sphericity assumptions and the correlation structure
was avoided in final models. Therefore, linear mixed models were used to
test the interaction between treatments and months with blocks and trees
as nested random effects to identify whether treatments affected NSCs
(function lmer , package lme4 v1.1-23). Residuals of NSC
analyses were normally distributed and had homogeneous variances. We
reported Type III tests using Satterthwaite’s method and pairwise
comparison p-values are Tukey adjusted (package emmeans v1.5.3).
Does trenching affect NSCs?We compared starch, total sugars and total NSCs between trenched-control
(n=8) and untrenched-control (n=8) trees using the same analysis as
above.
Do bark beetle attacks affect NSCs? We compared starch, total
sugars and total NSCs between untrenched-attacked (n=8) and
untrenched-control (n=8) trees using the same analysis as above.
Do microbial inoculations affect
NSCs? We compared starch, total
sugars and total NSCs between
untrenched-inoculated (n=8) and
untrenched-control (n=8) trees using the same analysis as above.
2. Is drought-biotic stressor interaction significant for NSCs and
defense metabolites? In this and the research questions 3-5 below, we
only focused on the phloem monoterpenes as there is currently no
evidence that sesquiterpenes affect bark beetle biology.
We tested whether trenching impacted the effect of biotic stress (either
bark beetle attacks or inoculation) on NSCs and terpenes. For each
carbohydrate and defense compound, we ran separate models for bark
beetle attack vs. control and inoculation vs. control treatments. Each
repeated measures model included a full 3-way interaction with month,
trenching and either beetle attack or inoculation treatment. Defense
concentrations were log-transformed to meet assumptions of normality and
homogeneity of variance but least squared means in original scale are
presented.
3. Do changes in NSCs result in changes in defense metabolites?Using the same statistical approach as in the first question above, we
tested whether the interaction of the NSCs, month and treatments
affected concentrations of the total diterpenes or total monoterpenes
with a repeated measures ANCOVA. Both monoterpenes and diterpenes were
natural log-transformed to meet normality and homogeneity of variance
assumptions. We removed the 3-way interaction from the final analysis
because it was not significant and instead used a simpler model with
fixed effects of NSCs, treatment, month, treatment by NSCs, and
treatment by month with the same repeated measures model structure as
above. Where there were significant interactions between treatment and
NSCs, we tested whether slopes were different using Tukey’s adjusted
p-value (function lstrends in package lsmeans v2.27-2).
4. Do concentrations of NSCs and terpenes vary between dying and
live trees? We used the same type of analyses as described under the
first question above but only used the data from 2014 since six out of
eight trees attacked by bark beetles died that year. We compared six
dying trees with the two live trees in the trenched-attacked treatment
for concentrations of starch, total sugars, total NSCs, total
diterpenes, and total monoterpenes. We conducted overall pairwise
comparisons between treatments with Tukey’s adjusted p-values and then
separately for each month with Sidak adjusted p-values (emmeansv1.5.3).
5. Can interactions between NSCs and terpenes explain tree
mortality? We tested the correlation between average NSCs (starch,
total sugars, or total NSCs) and terpenes (total diterpenes or
monoterpenes) for the six dying trees in the trenched-attacked treatment
in a Pearson correlation analysis. We also conducted the same
correlation tests for live trees in the remaining six treatments
including those that survived in the trenched-attacked treatment. We
only used data from 2014 as all six dying trees in the trenched-attacked
treatment died in 2014.