Lignin concentrations in phloem and outer bark are not associated with resistance to mountain pine beetle among high elevation pines

A key component in 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; tree species growing at high elevations in the western United States known to differ in susceptibility to mountain pine beetle (Dendroctonus ponderosae; MPB). Pinus longaeva and P. balfouriana are attacked by MPB less frequently than P. flexilis, 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.


Introduction
Bark beetles (Coleoptera: Curculionidae, Scolytinae) are forest disturbance agents globally and include many tree-killing species [1]. Overcoming tree defenses is a central challenge for bark beetles which feed on living phloem and requires the destruction of tree vascular tissue for offspring survival. Tree defenses provide protection against insect attack, thereby maintaining the functional integrity of two subcortical high-fitness-value tissue types: phloem, which is responsible for transport and distribution of photosynthate produced in leaves and needles; risk due to climate change. We attempted to fill this gap by quantifying lignin in the outer bark (i.e., rhytidome) and phloem of co-occurring P. longaeva, P. balfouriana and P. flexilis from multiple sites and compared lignin concentrations within and among species and between the two tissue types. We hypothesized that the more MPB-resistant P. longaeva and P. balfouriana would have greater lignin concentrations than co-occurring P. flexilis.

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 (Fig 1; Table 1). Four of the five sites were also sampled by Bentz et al. (2017) [10], 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).  Table 1). Pine distributions are based on Little (1971) [69]. https://doi.org/10.1371/journal.pone.0250395.g001

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) [70], as described by Bonello et al. (1993) [71]. 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 (S1 Fig). 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. Adjusted sample sizes for outer bark and phloem samples are shown in Table 1.

Statistical analysis
Differences among tree species in phloem and outer bark lignin concentrations, phloem thickness, and DBH were assessed with a hierarchical mixed effect analysis of variance (ANOVA), that accounts for variation among sites, using the package "lme4" [72] in R version 4.0.0 [73]. Multiple comparisons among sites were assessed using the package "multcomp" [74]. Linear regression (package "lme4") was used to assess the relationships between phloem and outer bark lignin concentrations, phloem lignin concentration and phloem thickness, DBH and phloem thickness, DBH and phloem lignin concentration, and DBH and outer bark lignin concentration.

Results
Phloem lignin concentrations did not differ between P. longaeva and P. balfouriana, but, contrary to our hypotheses, P. flexilis had significantly higher (~2-fold) phloem lignin concentrations than the other two species (Fig 2; Table 2). We found no differences among the species in Table 1. Site locations (see Fig 1) and stand metrics including species sampled, number of phloem and bark samples analyzed, and mean ± standard error of DBH (diameter breast height).

Site
Pinus outer bark lignin concentrations (Fig 2; Table 2). P. flexilis had thinner phloem than both P. longaeva and P. balfouriana, but there were no differences in phloem thickness between P. longaeva and P. balfouriana (Fig 3; Table 2). P. flexilis trees with thicker phloem tended to have lower phloem lignin levels, but we found no relationship between phloem thickness and phloem lignin levels in P. longaeva or P. balfouriana (Table 3). We also found no relationship between phloem thickness and outer bark lignin levels in any species (Table 3). P. flexilis and P. balfouriana were generally smaller than P. longaeva (Table 2), although DBH had no effect on phloem or lignin concentrations in any of the species (Table 3). There was also no significant relationship between phloem and outer bark lignin concentrations among trees, although P. balfouriana with more phloem lignin tended to have less outer bark lignin (Table 3). There were no significant differences among the sites in phloem lignin concentration for any species (P. flexilis: p > 0.238; P. longaeva: p > 0.095; P. balfouriana: p = 0.101), although P. flexilis outer bark lignin concentration differed at two sites (S1 Table).

Discussion
Contrary to our expectations, P. flexilis exhibited the highest levels of constitutive phloem lignin relative to co-occurring P. longaeva and P. balfouriana¸although there were no differences  among the species in outer bark lignin. We also found no consistent relationship between phloem and outer bark lignin concentrations at the tree species level. Because P. flexilis is considered more susceptible to MPB and produces greater numbers of offspring than P. longaeva and P. balfouriana, our results suggest that in these species constitutive lignin may not function as a direct defense against MPB attack or brood production. Our findings are similar to previous studies that showed phloem lignification did not differ among ash species (Fraxinus spp.) with varying resistance to the phloephagous emerald ash borer (Agrilus planipennis Fair.) [75,76]. Although constitutive phloem lignin, as measured in our study, may not provide a significant defense, methyl jasmonate-induced lignification of F. americana and F. pennsylvanica phloem/outer bark was associated with resistance to the emerald ash borer [77]. The potential  Table 1). Different letters (i.e., a,b) denote statistically significant differences among species means (p < 0.05). See Table 2   for induced lignification to act as an active defense in the Pinus species we sampled has not been investigated and should be part of future studies. Pinus flexilis has consistently been found to have less constitutive and induced LMW specialized metabolites (i.e., terpenes and their derivations) than other species, including P. longaeva and P. balfouriana at the sites sampled for this study [10], P. contorta and P. ponderosa [78], and the closely related bristlecone species P. aristata (Soderberg et al. in review). Although interspecific differences in selective pressure may have led to differences in investment in phloem specialized metabolite defenses [10,79,80], our findings suggest an inverse relationship between lignification and phloem chemical defenses that are known to provide defense against bark beetles [26,31]. In our study, P. flexilis had thinner phloem, but greater lignin concentrations and absolute abundance than P. longaeva and P. balfouriana, the latter two having thicker phloem. Moreover, P. flexilis with the thickest phloem had the lowest lignin concentrations, further suggesting a negative relationship between phloem thickness and lignification. That outer bark lignin concentrations did not differ among the tree species but phloem concentrations did, suggest that lignification within the phloem may be under different selective pressures relative to outer bark. Trait associations and underlying mechanisms facilitating phloem lignification may be unique to the functions of nutrient transport or defense against invading bacteria or pathogens [81].
In summary, if defense against bark beetle attack were a strong selective driver for higher lignification in Pinus, higher lignin levels would be expected within both outer bark and phloem tissues of species considered less susceptible to MPB. This expectation is supported by prior research in Picea that was focused on species that are generally not considered primary mortality agents of mature trees, including Pissodes larva that feed in terminal buds [53][54][55][56]69], H. abietis that girdle seedlings [58], and the base-feeding D. micans [32,57]. MPB is a bole feeder that often kills mature trees. Our results showing that the more frequently MPB-attacked P. flexilis had greater phloem lignin concentrations relative to the less MPB-susceptible P. longaeva and P. balfouriana suggest that the defensive function of lignin may be dependent on the plant tissue consumed and aggressiveness of the insect. We also found that the species with the greatest constitutive phloem lignin concentrations, P. flexilis, was previously found to produce lower levels of constitutive LMW specialized metabolites than the other two species. While increased tissue lignification may have an additive effect with specialized metabolites on host defenses against MPB, there may be metabolic tradeoffs that are not accounted for between LMW specialized metabolites and lignin. Therefore, greater lignification within feeding tissues does not appear to be generally adaptive as a defense against MPB. Moreover, interspecific differences in phloem but not outer bark lignin concentrations highlight that the benefits and costs of lignification in Pinus are likely specific to phloem tissue. High elevation Pinus species are increasingly threatened by MPB as a result of warming temperatures. Our results enhance the important knowledge base of defense strategies employed by MPB-susceptible high elevation Pinus.
Supporting information S1 Fig. Lignin extracts of phloem and outer bark samples. All phloem samples were clear and colorless and therefore assumed pure (left vial). Outer bark samples were assumed to be pure when clear and colorless to light pink (right vial), but incompletely digested and/or contaminated when dark red (middle vial). (JPG) S1 Table. Model estimates testing for differences in phloem and bark lignin concentrations (mg/g FW) among sample sites of P. flexilis, P. longaeva, and P. balfouriana (see Table 1, Fig 1). Effect size (Est.) and 95% confidence interval (95% CI) estimates between comparison samples are shown. P-values (p) presented describe the likelihood of statistical difference with values < 0.05 presented in bold. (DOCX)