DOI: 10.5061/dryad.z8w9ghxc0
title: Lignin concentrations in phloem and outer bark are not associated with resistance to mountain pine beetle in high elevation pines

description: A key component to understanding plant-insect interactions is the nature
 of host defenses. Research on defense traits among Pinus species has
 focused on specialized metabolites and axial resin ducts, but the role of
 lignin in defense within diverse systems is unclear. We investigated
 lignin levels in the outer bark and phloem of P. longaeva, P. balfouriana,
 and P. flexilis; high elevation species in the western United States known
 to differ in susceptibility to mountain pine beetle (Dendroctonus
 ponderosae; MPB). Relative to P. flexilis, P. longaeva and P. balfouriana
 are attacked by MPB less frequently, and MPB brood production in P.
 longaeva is limited. Because greater lignification of feeding tissues has
 been shown to provide defense against bark beetles in related genera, such
 as Picea, we hypothesized that P. longaeva and P. balfouriana would have
 greater lignin concentrations than P. flexilis. Contrary to expectations,
 we found that the more MPB-susceptible P. flexilis had greater phloem
 lignin levels than the less susceptible P. longaeva and P. balfouriana. No
 differences in outer bark lignin levels among the species were found. We
 conclude that lignification in Pinus phloem and outer bark is likely not
 adaptive as a physical defense against MPB.
description: Study locations and Tree Sampling Between June and September
 2016, trees were sampled at five sites across the ranges of
 <i>P. longaeva</i> and <i>P. balfouriana</i>, four
 of them in stands with co-occurring <i>P. flexilis</i>. Four
 of the five sites were also sampled by Bentz et al. (2017), allowing a
 comparison with results from that study. Equal numbers of <i>P.
 longaeva</i> and <i>P. flexilis</i> trees were sampled
 at three geographically separated locations, and equal numbers of
 <i>P. balfouriana</i> and <i>P. flexilis</i> were
 sampled at the Sierra Nevada site. At the Klamath site <i>P.
 flexilis</i> was not present, and only <i>P.
 balfouriana</i> was sampled. At each site 15 live trees of each
 species were sampled, and diameter at breast height (DBH, ~ 1.5 m above
 ground) ranged from 30-46 cm. Study sites without signs of MPB or pathogen
 activity were chosen to avoid an influence of induced defenses. Permission
 for sampling was obtained through the Inyo, Klamath, Sierra Nevada and
 Humboldt-Toiyabe National Forests. To assess lignin
 levels (mg/g fresh weight) in outer bark and phloem, trees were sampled by
 boring into the tree at breast height with a 1” diameter circular hole saw
 (Milwaukee<sup>TM</sup>). Four samples were taken on the
 north, south, west, and east aspects of the tree trunk and pooled to
 account for potential within-tree variation. Upon tissue removal, phloem
 thickness (mm) was measured from the north and south aspect samples. Outer
 bark and phloem tissues were then separated and placed immediately in a
 sealed vial in a cooler with dry ice for transport to the Rocky Mountain
 Research Station (Logan, UT) for cold storage
 (-40<b>°</b>C).  Lignin
 extraction In the laboratory experiments, outer bark
 and phloem samples were prepared for lignin extraction using a ceramic
 mortar and pestle to grind tissue samples in liquid nitrogen. Tissues were
 ground to a fine powder and placed in vials for lignin extraction. The
 mortar and pestle were cleaned with 95% ethanol between each tissue
 sample. Lignin was extracted from the outer bark and phloem tissues using
 thioglycolic acid digestion in a modification of the method of Bruce and
 West (1989), as described by Bonello et al. (1993). Spectral absorbance of
 phloem lignin samples (n = 135) was measured at 280 nm using a NanoDrop™
 3300 Fluorospectrometer (Thermofisher Scientific) with a 1:4 dilution in
 NaOH against a standard curve of pure spruce lignin (Sigma-Aldrich) at 0,
 18, 45, 90, and 360 micrograms/mL. The spectral absorbance of outer bark
 lignin (n = 103) was measured under the same parameters using 1:64
 dilution. All phloem samples were assessed as pure and free from
 contamination, although thirty-two outer bark samples were removed from
 analysis due to residual phenolic compound contamination. In addition,
 three outliers, consisting of a single phloem sample from each species (2%
 of total samples), exhibited lignin concentration &gt; 6-fold the
 standard deviation for each species. As the outer bark contained
 remarkably higher lignin concentrations than the phloem, we removed these
 three outliers out of caution for potential tissue
 contamination.