Increment core preparation for stable isotope analyses
Six sites were chosen for further analyses based on the abundance and
quality of the increment cores. The date of last year of growth was
verified for every core using visual crossdating and COFECHA software
(Holmes 1983; Yamaguchi 1991). Cross-dated cores were used to identify
the last year of growth which indicated when the tree died. The
resulting distribution of death years identified trees that died early,
when beetle population levels were building, or later during outbreak
population levels (Fig. 2; Appendix S1: Fig. S1). Specifically, early-
and late-dying trees were identified as those that died during years
occupying the respective tails of the death-year distribution within
each stand, after excluding outliers that may have died previous to
significant beetle activity (Pettit 2018; Appendix S1: Fig. S1). Cores
earmarked for stable isotope analyses were re-measured to include
earlywood- and latewood-specific growth back to 1930 using a Velmex
measuring system. Annual basal area increment (BAI) was calculated for
each tree from the outside toward the pith assuming estimates of the
diameter inside the bark from the diameter outside the bark (Myers and
Alexander 1972), and by estimating areal growth from annual rings could
be generalized as concentric circles. Trees identified for stable
isotope analyses were determined on a site-by-site basis to obtain the
greatest separation of early- and late-dying trees that included at
least five dead trees within each group, and to ensure a large enough
sample mass for stable isotope analyses (Table 1; Appendix S1: Fig. S1).
We composited the latewood of tree-rings across a site and within timing
categories (i.e., each site had two sets of samples for each year that
were distinguished by trees that died early versus late). Compositing
rings by year for stable isotope analyses assures sufficient cellulose
for stable isotope analyses when rings of trees are very small (e.g.,
Tardif et al. 2008; Voelker et al. 2014; Csank et al. 2016; Ratcliff et
al. 2018) and typically yields an expressed population signal greater
than 0.85 (Leavitt 2010). Latewood was excised from each ring to better
isolate moisture stress during the summer months (Parker and Henoch
1971; Faulstich et al. 2013). Wood was ground to a coarse powder using a
Wiley mill before extracting to holocellulose (Leavitt and Danzer 1993),
homogenized using a 500-W ultrasonic probe (Laumer et al. 2009), and
weighed into tin capsules on a microbalance. Samples were analyzed on a
Thermo Delta V Advantage isotope ratio mass spectrometer at the Newell
Stable Isotope Laboratory located at Utah State University
(http://www.usu.edu/geo/newell/Newell_Website/Stable_Isotope_Lab.html).
Carbon isotope composition, is expressed using “delta” notation as
δ13C = (Rsample /
Rstandard - 1) × 1000, where δ13C is
the molar ratio of heavy to light isotopes and Rstandardis Vienna Pee Dee Belemnite (VPDB). Precision across replicate13C analyses of cellulose was 1σ = 0.12 ‰. Stable
isotope analyses extended back to the tree-ring formed in 1960 for all
sites to include a common period of at least three decades of
inter-annual climatic variability leading up to the D. rufipennisoutbreak. Following Farquhar et al. (1989), cellulose
δ13C data were converted to Δ13C as:
Δ13C = (δ13Cair -
δ13Cplant )/(1 +
δ13Cplant / 1000), where
δ13Cair values for each year followed
those given in McCarroll and Loader (2004).