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.

Study locations and Tree Sampling

Between June and September 2016, trees were sampled at five sites across the ranges of P. longaeva and P. balfouriana, four of them in stands with co-occurring P. flexilis. Four of the five sites were also sampled by Bentz et al. (2017), allowing a comparison with results from that study. Equal numbers of P. longaeva and P. flexilis trees were sampled at three geographically separated locations, and equal numbers of P. balfouriana and P. flexilis were sampled at the Sierra Nevada site. At the Klamath site P. flexilis was not present, and only P. balfouriana 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^TM). 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°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 > 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.