Edited by: Kristian Forbes, University of Helsinki, Finland
Reviewed by: Stephen A. Teale, State University of New York, United States; Paul F. Gugger, University of Maryland Center for Environmental Science, United States
This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Increased mortality of forest trees, driven directly or indirectly by climate change, is occurring around the world. In western North America, whitebark pine, a high elevation keystone species, and lodgepole pine, a widespread ecologically and economically important tree, have experienced extensive mortality in recent climate-driven outbreaks of the mountain pine beetle. However, even in stands experiencing high levels of mortality, some mature trees have survived. We hypothesized that the outbreak acted as a natural selection event, removing trees most susceptible to the beetle and least adapted to warmer drier conditions. If this was the case, genetic change would be expected at loci underlying beetle resistance. Given we did not know the basis for resistance, we used inter-simple sequence repeats to compare the genetic profiles of two sets of trees, survivors (mature, living trees) and general population (trees just under the diameter preferred by the beetles and expected to approximate the genetic structure of each tree species at the site without beetle selection). This method detects high levels of polymorphism and has often been able to detect patterns associated with phenotypic traits. For both whitebark and lodgepole pine, survivors and general population trees mostly segregated independently indicating a genetic basis for survivorship. Exceptions were a few general population trees that segregated with survivors in proportions roughly reflecting the proportion of survivors versus beetle-killed trees. Our results indicate that during outbreaks, beetle choice may result in strong selection for trees with greater resistance to attack. Our findings suggest that survivorship is genetically based and, thus, heritable. Therefore, retaining survivors after outbreaks to act as primary seed sources could act to promote adaptation. Further research will be needed to characterize the actual mechanism(s) of resistance.
The capacity of forests to adapt to rapid climate change is not known. Their ability to adapt will vary greatly depending upon tree species, amount and type of genetic variation existing within and among populations, type and degree of change required, strength and type of selection pressure, heritability of desirable traits, and the timeframe over which selection is able to act. Many long-lived sessile organisms, including trees, are unlikely to be able to track shifting conditions through migration (
Bioclimatic envelope models used to predict range expansions and contractions of forest trees treat species as clones, with all individuals exhibiting identical responses (
Except for highly fragmented or relictual populations, forest trees possess moderate to high levels of standing genetic variation and often exhibit considerable local adaptation and within and among population diversity (
Adaptation in trees may be accelerated when new conditions or agents lead to high levels of mortality and directional selection in favor of heritable traits associated higher fitness and survival. For example,
The mountain pine beetle (MPB) (
In this study, we investigated whether trees that survive MPB outbreaks are genetically different than those that are selected for colonization and killed. Our overarching hypothesis was that surviving trees do not escape by chance, but rather possess genetically based characteristics that confer resistance. The basis for resistance, whether it is the ability to tolerate warmer drier conditions without a reduction in defenses, a chemical profile that negatively affects MPB host location or selection, or some other phenotypic trait, is likely to be under genetic control (
MPB outbreaks are triggered by extended periods of warm weather and drought (
We focused on two tree species that have suffered high mortality by MPB in the recent outbreak. One is a relatively naïve host, whitebark pine (
The second species studied was lodgepole pine, a co-evolved host that has experienced repeated extensive outbreaks in much of its range, likely over a long evolutionary period. Vigorous lodgepole pine typically exhibits strong constitutive and inducible defensive responses to beetle attack (
Our objective in this study was to investigate whether whitebark and lodgepole pine growing in a mixed high elevation stand that survived the outbreak are genetically distinct. If so, this may indicate an increased potential for these pines to persist in the face of the more frequent and extensive outbreaks predicted due to a changing climate. We would expect genetic change at loci underlying beetle resistance but not at a genome-wide scale. Without knowing the basis for resistance in survivors, we chose to use inter-simple sequence repeats (ISSRs) to develop genetic profiles for whitebark and lodgepole pine. ISSRs target highly variable sequences within microsatellite regions (
This study was conducted at Vipond Park, a high-elevation plateau supporting a patchwork of grassland and open forest stands located on the Beaverhead National Forest, Montana, United States (2,501 m elevation, 45.6974oN, 112.9106oW). The site is relatively xeric with an understory of sagebrush and a diverse mixture of annual and perennial forbs. Vipond Park was chosen to take advantage of the high mortality to pines that occurred there during a recent high elevation outbreak of MPB (2009–2013) when approximately 93 and 75% of mature
Transects were established in 2015 (
In initial transects, the diameter at breast height (DBH, 1.4 m above the soil line) of 100
In 2015, transects were established as previously described. Thirty survivor
The smallest diameters of
Needles (3–5) from each sample were ground to a fine powder in liquid nitrogen using a mortar and pestle. DNA was then isolated from each sample using a Qiagen DNeasy Plant Kit (Qiagen, Valencia, CA, United States) following the protocol provided by the manufacturer.
Five ISSR primers were chosen for use (
Primers used for ISSR amplification.
Primer ID | Sequence | Tree species |
---|---|---|
HB12 | CAC CAC CAC GC | |
17899A | GTG TGT GTG TGT CA | |
17901 | CAC ACA CAC ACA AG | |
UBC 807 | AGA GAG AGA GAG AGA GT | |
UBC 811 | GAG AGA GAG AGA GAG AC | |
For amplification we used a 25 μl reaction mixture consisting of 12.5 μl Promega Master Mix (Promega, Madison, WI, United States), 2.5 μl RNA-free water, 8 μl of 0.5 M primer and 2 μl of DNA template. Reactions were run individually with one of the five ISSR primers. PCR was conducted with one cycle denaturation at 95°C for 5 min, followed by 42 cycles of denaturation at 95°C for 1.3 min, annealing at 47°C for 2 min, and extension at 72°C for 1 min. A final cycle was conducted at 72°C for 1 min and final products were held at 6°C (
PCR products were visualized in a 1% agarose gel prepared using 1× tris borate buffer (TBE) to which 2 μl ethidium bromide per 100 ml gel was added. A 100 bp ladder (Promega, Valencia, CA, United States) was placed in the first lane of each gel to provide a reference for scoring bands. Amplified DNA was loaded into the remaining lanes with bromophenol blue as a running dye. Each gel was run with 1× TBE as a running buffer at 70 mA until the dye moved 3/4 of the length of the gel. Gel images were captured using a UV table. Any sample that gave ambiguous results (no, faint, or smeared bands) was repeated. Approximately 20% of samples were rerun and compared to check for consistency in results. Only samples exhibiting clear bands were included in the final analysis. Bands were scored manually.
A two-sample
Bands were scored as present (1) or absent (0) to develop a binary matrix combining data for all primers by tree species. The matrices were analyzed in Popgene v. 1.32 (
For each tree species, we examined genetic variation between groups using analysis of molecular variation (AMOVA) in GenAlEx 6.5 (
The mean, median, and range of diameters of beetle-killed and survivor
Summary statistics for diameter breast height (cm) of
Tree | Group | N | Mean ( |
Median | Minimum | Maximum |
---|---|---|---|---|---|---|
Beetle-killed | 75 | 24.5 (5.3)a | 24.2 | 12.0 | 37.3 | |
Survivor | 30 | 25.0 (5.2)a | 24.1 | 17.0 | 37.3 | |
General | 36 | 10.0 (0.6)b | 10.0 | 9.0 | 11.0 | |
Beetle-killed | 45 | 26.7 (5.0)a | 26.4 | 17.5 | 36.8 | |
Survivor | 20 | 27.5 (5.4)a | 29.9 | 18 | 37.2 | |
General | 20 | 15.3 (0.9)c | 15.2 | 13.9 | 16.8 | |
Three primers (17899A, HB12, and UBC807) resolved well for
Percent band polymorphism (BP), number of observed (Na) and effective (Ne) alleles, Shannon’s Information Index (
Tree species | Group | N | %BP | Na | Ne | HT | HS | ||
---|---|---|---|---|---|---|---|---|---|
Survivor | 30 | 96.58 | 1.97 (0.19) | 1.39 (0.25) | 0.40 (0.19 | 0.22 (0.14) | |||
General | 36 | 96.55 | 1.97 (0.19) | 1.32 (0.27) | 0.36 (0.14) | 0.25 (0.14) | |||
Combined | 66 | 96.43 | 1.96 (0.19) | 1.41 (0.19) | 0.42 (0.18) | 0.26 (0.14) | 0.26 (0.10) | 0.24 (0.01) | |
Survivor | 20 | 88.24 | 1.88 (0.32) | 1.40 (0.33) | 0.25 (0.17) | 0.39 (0.23) | |||
General | 20 | 89.41 | 1.89 (0.31) | 1.40 (0.31) | 0.26 (0.16) | 0.40 (0.22) | |||
Combined | 40 | 98.82 | 1.90 (0.11) | 1.41 (0.29) | 0.27 (0.14) | 0.42 (0.18) | 0.27 (0.02) | 0.25 (0.02) | |
HT, the total genetic diversity between the two study groups, was 0.26, and the diversity within groups, HS, was 0.24. Seven of 28 loci (25%) exhibited significant differences between observed and expected frequencies of bands between the two groups (data not shown). However, no bands were unique to either group. The Ewens–Watterson test for neutrality detected only one marginally non-neutral locus. AMOVA indicated 87% of the variation exhibited existed within groups and 13% existed between groups.
The neighbor-joining tree resolved most general population trees together in the basal clades while one major terminal clade contained all survivor trees as well as eleven general population trees that were distributed throughout the clade (
Neighbor-joining tree from ISSR data for
Results of Bayesian clustering using STRUCTURE. Individual trees are represented by vertical bars. Colored segments represent the tree’s estimated proportion similarity to each of the three clusters (red, blue, and green) optimally defined by STRUCTURE.
Principle coordinates analysis of general (blue diamonds) and survivor (orange squares) trees.
Four primers resolved well for this species (17899A, UBC807, UBC901, and UBC811). Using these primers, we were able to resolve a total of 85 bands. The mean percent BP across all primers and groups was 98.82. This was considerably higher than BP for the general population (89.4%) and survivor (88.2%) trees (
HT was 0.26 and HS was 0.25, similar to values for whitebark pine. Allele frequencies were significantly different between survivors and general population trees at 12 of 85 loci (14%) (
The neighbor-joining tree partitioned general population and survivor trees into several clades (
Neighbor-joining tree from ISSR data for
Knowledge of the effects of bark beetle outbreaks on host tree population genetic structure and resistance to attack will be increasingly valuable as climate change drives more frequent outbreaks and facilitates the movement of beetle species into naïve forests. Outbreaks of MPB seldom kill all mature trees despite high beetle numbers during population peaks. Our results suggest that surviving trees possess a wealth of information that can be used to inform our understanding of the genetic and phenotypic bases for resistance and to develop management approaches that support forest adaptation.
We found that surviving mature trees in a high elevation forest of whitebark and lodgepole pine were genetically distinct from “general population” trees that were assumed to represent the genetic structure of the population pre-outbreak and without selection by the beetle. In line with our hypothesis, a low percentage (<10%) of “survivor” genotypes were identified within the general population. The proportion of these survivors roughly mirrored the proportion of mature trees that survived the outbreak at Vipond Park. The neighbor-joining tree, the PCoA and the STRUCTURE analyses each indicated strong differentiation between survivors and “susceptible” individuals and identified the same trees as survivors within the general population. In the STRUCTURE analysis for both whitebark and lodgepole pine, susceptible trees belonged to one cluster while survivor trees belonged to two other clusters. This separation can also be seen in the PCoA. Further research will be needed to determine whether the patterns we detected are indeed indicative of resistance, and if so, whether there are multiple or overlapping factors that account for survivorship.
We found surprisingly high levels of differentiation between survivor and general population trees in both species of pine. For whitebark pine, Nei’s genetic distance between survivor and general population whitebark pines was 5%, a value that would indicate moderate differentiation if these comparisons had been made between tree populations. Likewise, AMOVA indicated 13% of the genetic variation present existed between groups. Considering that the trees in this analysis were not from different populations, but rather grew intermixed at the same site, these values seem strikingly high. Likewise, for lodgepole pine, Nei’s genetic distance was 7%, and AMOVA indicated 11% of variation occurred between the groups.
These results indicate the presence of genetically based resistance in both pine species and that trees with resistant genotypes are not selected for attack. It has been thought that once MPB achieve high population levels during outbreaks, the selection of individual trees based on tree-produced compounds and condition becomes swamped by high levels of aggregation pheromone production and competition for increasingly rare hosts (
We chose ISSR profiling as a first step to determine whether survivors were different than trees chosen by MPB for colonization. This PCR-based method detects high levels of polymorphism, is highly reproducible, and allows the screening of a large number of trees relatively rapidly and economically. Unfortunately, this method cannot tell us why survivors are different, only that they are. Further study will be needed to further investigate whether survivors are indeed highly resistant and, if so, to determine the actual basis behind resistance. Ongoing studies are investigating correlations among genetic profiles of survivor and “susceptible” trees with phenotypic traits including defensive chemistry and growth rates in relation to climate. Genomic approaches will also be extremely useful to elucidate the basis of resistance.
This study corroborated the findings of other studies that found that MPB colonizes smaller diameter whitebark pine than lodgepole pine during outbreaks (
In a previous study,
A caution is in order in interpreting our results. We were unable to amplify DNA from MPB-killed trees which forced us to use smaller diameter “general population” trees as a substitution for “susceptible” trees. These trees were mature reproductive trees and only slightly smaller than trees selected by the beetle for colonization; however, some or all may constitute a cohort that regenerated under different environmental conditions resulting in a genetic structure unrepresentative of the larger trees that were available for selection by the beetle. However, the proportional distribution of survivor and “susceptible” trees in the neighbor-joining trees, PCoAs and STRUCTURE analyses indicate that the general population samples were likely appropriate proxies.
With climate change supporting the invasion of aggressive bark beetles into naïve forests, and predictions of more frequent and severe outbreaks, it is increasingly important to understand the capacity of trees to adapt and persist (
Supporting forest adaptation is critical in this time of rapid change (
DS conceived of the project, participated in field work, conducted data analysis, and wrote the manuscript. CV conducted lab work on whitebark pine and participated in field work and data analysis. MC conducted field and lab work on lodgepole pine and contributed to data analysis.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.