Limber Pine Research Articles

Conifers Concentrate Large Numbers of NLR Immune Receptor Genes on One Chromosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

Lethal combination for seedlings: extreme heat drives mortality of drought-exposed high-elevation pine seedlings.

Hotter drought- and biotically-driven tree mortality are expected to increase with climate change in much of the western United States, and species persistence will depend upon ongoing establishment under novel conditions or migration to track ecological niche requirements. High-elevation tree species may be particularly vulnerable to increasing water stress as snowpack declines, increasing the potential for adult mortality and simultaneous regeneration failures. Seedling survival will be determined by ecophysiological limitations in response to changing water availability and temperature. We exposed seedlings from populations of Pinus longaeva, Pinus flexilis, and Pinus albicaulis to severe drought and concurrent temperature stress in common gardens testing timing of drought onset under two different temperature regimes. We monitored seedling functional traits, physiological function, and survival. The combined stressors of water limitation and extreme heat led to conservative water use strategies and declines in physiological function, with these joint stressors ultimately exceeding species' tolerances and leading to complete episodic mortality across all species. Growing conditions were the primary determinant of seedling trait expression, with seedlings exhibiting more drought-resistant traits such as lower specific leaf area in the hottest, driest treatment conditions. Water stress-induced stomatal closure was also widely apparent. Under adequate soil moisture, seedlings endured prolonged exposure to high air and surface temperatures, suggesting broad margins for survival. The critical interaction between soil moisture and temperature suggests that rising temperatures will exacerbate growing season moisture stress. Our results highlight the importance of local conditions over population- and species-level influences in shaping strategies for stress tolerance and resistance to desiccation at this early life stage. By quantifying some of the physiological consequences of drought and heat that lead to seedling mortality, we can better understand the future effects of global change on the composition and distribution of high-elevation conifer forests.

Genome-Wide Association Study Reveals Polygenic Architecture for Limber Pine Quantitative Disease Resistance to White Pine Blister Rust.

Development of durable resistance effective against a broad range of pathotypes is crucial for restoration of pathogen-damaged ecosystems. This study dissected the complex genetic architecture for limber pine quantitative disease resistance (QDR) to Cronartium ribicola using a genome-wide association study. Eighteen-month-old seedlings were inoculated for resistance screening under controlled conditions. Disease development was quantitatively assessed for QDR-related traits over 4 years postinoculation. To reveal the genomic architecture contributing to QDR-related traits, a set of genes related to disease resistance with genome-wide distribution was selected for targeted sequencing for genotyping of single-nucleotide polymorphisms (SNPs). The genome-wide association study revealed a set of SNPs significantly associated with quantitative traits for limber pine QDR to white pine blister rust, including number of needle spots and stem cankers, as well as survival 4 years postinoculation. The peaks of marker-trait associations displayed a polygenic pattern, with genomic regions as potential resistant quantitative trait loci, distributed over 10 of the 12 linkage groups (LGs) of Pinus. None of them was linked to the Cr4-controlled major gene resistance previously mapped on LG08. Both normal canker and bole infection were mapped on LG05, and the associated SNPs explained their phenotypic variance up to 52%, tagging a major resistant quantitative trait locus. Candidate genes containing phenotypically associated SNPs encoded putative nucleotide-binding site leucine-rich repeat proteins, leucine-rich repeat-receptor-like kinase, cytochrome P450 superfamily protein, heat shock cognate protein 70, glutamate receptor, RNA-binding family protein, and unknown protein. The confirmation of resistant quantitative trait loci broadens the genetic pool of limber pine resistance germplasm for resistance breeding.

Needle bacterial community structure across the species range of limber pine.

Bacteria on and inside leaves can influence forest tree health and resilience. The distribution and limits of a tree species' range can be influenced by various factors, with biological interactions among the most significant. We investigated the processes shaping the bacterial needle community across the species distribution of limber pine, a widespread Western conifer inhabiting a range of extreme habitats. We tested four hypotheses: (i) Needle community structure varies across sites, with site-specific factors more important to microbial assembly than host species selection; (ii) dispersal limitation structures foliar communities across the range of limber pine; (iii) the relative significance of dispersal and selection differs across sites in the tree species range; and (iv) needle age structures bacterial communities. We characterized needle communities from the needle surface and tissue of limber pine and co-occurring conifers across 16 sites in the limber pine distribution. Our findings confirmed that site characteristics shape the assembly of bacterial communities across the host species range and showed that these patterns are not driven by dispersal limitation. Furthermore, the strength of selection by the host varied by site, possibly due to differences in available microbes. Our study, by focusing on trees in their natural setting, reveals real needle bacterial dynamics in forests, which is key to understanding the balance between stochastic and deterministic processes in shaping forest tree-microbe interactions. Such understanding will be necessary to predict or manipulate these interactions to support forest ecosystem productivity or assist plant migration and adaptation in the face of global change.

Drought-induced growth phenotypes are associated with genetic variation across a white pine hybrid zone

Understanding relationships among warming climate, increased drought severity, and the genetic architecture of hybrid drought resilience is necessary for forest conservation and management. We calculated three drought-related tree-ring-growth indices (dendrophenotypes) using tree-ring data from hybrid Pinus strobiformis–Pinus flexilis study trees at nine sites across Colorado, New Mexico, Arizona, and Texas. Along with hybrid index (percentage of P. strobiformis ancestry inherited by a single tree) and climate variables, we used dendrophenotypes to (Q1) examine relationships among climate and hybrid index, (Q2) examine relationships between dendrophenotypes and hybrid index, and (Q3) conduct a genotype–phenotype analysis. We observed a significant correlation between hybrid index and dendrophenotypes resulting from high-temperature drought in addition to a significant correlation between those dendrophenotypes and our dataset of single-nucleotide polymorphisms. We conclude that P. strobiformis–P. flexilis trees exhibiting higher hybrid indices are more resilient to high-temperature drought events and encourage future research that identifies a genetic linkage between relevant loci and their conferred physiological benefits.

Clark's nutcracker forest community visitation: Whitebark pine maintains a keystone seed disperser.

Clark's nutcrackers (Nucifraga columbiana) are obligate seed dispersers for whitebark pine (Pinus albicaulis), but they frequently use other conifer seed resources because of annual variability in cone production or geographic variation in whitebark pine availability. Whitebark pine is declining from several threats including white pine blister rust, leading to potential population declines in the nutcracker and the pine. We hypothesize that where there are few additional seed resources, whitebark pine becomes the key and limiting resource supporting nutcracker populations. We investigated how nutcrackers use coniferous forest community types within Yellowstone National Park to determine potential seed resources and the importance of whitebark pine. We established sites representing five forest community types, including whitebark pine, lodgepole pine (P. contorta), Engelmann spruce (Picea engelmannii), limber pine (P. flexilis), and Douglas-fir (Pseudotsuga menziesii). Each transect annually generated nutcracker point counts, conifer cone production indices, community composition data, and seed resource use observations. We compared hierarchical distance sampling models, estimating nutcracker density and its relationship to forest community type, seed harvesting time-period, year, study site, and cone seed energy. We found cone production varied across years indicating annual variability in energy availability. Nutcracker density was best predicted by forest community type and survey time-period and was highest in whitebark pine stands during the mid-harvesting season. Nutcracker density was comparatively low for all other forest community types. This finding underscores the importance of whitebark pine as a key seed resource for Clark's nutcracker in Yellowstone National Park. The decline of whitebark pine potentially leads to a downward spiral in nutcrackers and whitebark pine, arguing for continued monitoring of nutcrackers and implementation of restoration treatments for whitebark pine.

Distribution of Cronartium x flexili, an interspecific hybrid of two fungal tree rust pathogens, in subalpine forest ecosystems of western USA

Interspecific hybridization plays a key role in the evolution of novel fungal pathogens, and when it occurs between native and invasive species, can lead to potentially serious consequences. In this study, we examined the temporal and spatial distribution of a recently detected hybrid (Cronartium x flexili) of two tree pathogens, invasive to North America Cronartium ribicola and native Cronartium comandrae. In total, 726 and 1452 aecia from 178 Pinus contorta ssp. latifolia and 357 Pinus flexilis trees were collected from 26 sites in four national forests in 2019–2021. Using morphological and molecular analyses, 71 aecia collected from 25 P. flexilis trees had intermediate morphology and contained heterozygous SNPs in two genomic regions. Population analyses revealed the presence of multiple hybrid genotypes randomly distributed among sites and years. No aecia from P. contorta ssp. latifolia were identified as hybrids suggesting unidirectional gene flow from native C. comandrae to invasive C. ribicola. Aeciospores from 2 hybrid aecia produced urediniospores on Ribes nigrum. Overall, these results suggest that, even though low in frequency, C. x flexili is persistent in the region and has pathogenic potential. Hybrid expansion into the large range of susceptible pines could have cascading impacts on forest health.

Mortality thresholds of juvenile trees to drought and heatwaves: implications for forest regeneration across a landscape gradient

Tree loss is increasing rapidly due to drought- and heat-related mortality and intensifying fire activity. Consequently, the fate of many forests depends on the ability of juvenile trees to withstand heightened climate and disturbance anomalies. Extreme climatic events, such as droughts and heatwaves, are increasing in frequency and severity, and trees in mountainous regions must contend with these landscape-level climate episodes. Recent research focuses on how mortality of individual tree species may be driven by drought and heatwaves, but how juvenile mortality under these conditions would vary among species spanning an elevational gradient—given concurrent variation in climate, ecohydrology, and physiology–remains unclear. We address this knowledge gap by implementing a growth chamber study, imposing extreme drought with and without a compounding heatwave, for juveniles of five species that span a forested life zones in the Southwestern United States. Overall, the length of a progressive drought required to trigger mortality differed by up to 20 weeks among species. Inclusion of a heatwave hastened mean time to mortality for all species by about 1 week. Lower-elevation species that grow in warmer ambient conditions died earlier (Pinus ponderosa in 10 weeks, Pinus edulis in 14 weeks) than did higher-elevation species from cooler ambient conditions (Picea engelmannii and Pseudotsuga menziesii in 19 weeks, and Pinus flexilis in 30 weeks). When exposed to a heatwave in conjunction with drought, mortality advanced significantly only for species from cooler ambient conditions (Pinus flexilis: 2.7 weeks earlier; Pseudotsuga menziesii: 2.0 weeks earlier). Cooler ambient temperatures may have buffered against moisture loss during drought, resulting in longer survival of higher-elevation species despite expected drought tolerance of lower-elevation species due to tree physiology. Our study suggests that droughts will play a leading role in juvenile tree mortality and will most directly impact species at warmer climate thresholds, with heatwaves in tandem with drought potentially exacerbating mortality especially of high elevation species. These responses are relevant for assessing the potential success of both natural and managed reforestation, as differential juvenile survival following episodic extreme events will determine future landscape-scale vegetation trajectories under changing climate.

Dendroanatomy of xylem hydraulics in two pine species: Efficiency prevails on safety for basal area growth in drought-prone conditions

Xylem structure optimizes water conductivity while preventing hydraulic failure via embolism resistance, but how this process is modulated by climate variability and how it affects secondary growth in mature trees is still not fully understood, particularly in water-limited environments. Using quantitative wood anatomy techniques, we estimated xylem anatomical proxies for hydraulic efficiency (xylem specific conductivity, Ks) and safety (cell wall reinforcement, wrein) in two western US conifers, Pinus flexilis and Pinus longaeva, at a montane and subalpine location respectively. We built two large datasets (570 rings for P. flexilis, 635 rings for P. longaeva) to investigate 1) the variability of anatomical parameters (i.e lumen diameter, cell wall thickness) and hydraulic proxies along the stem in the five outermost rings (2009–2013); 2) the response of hydraulic proxies to daily climate over a period of 24 years (1990–2013); and 3) the relationship between xylem hydraulic architecture and basal area increment (BAI). Lumen diameter scaled along the stem following a power function, but the scaling patterns of cell wall thickness and hydraulic proxies differed significantly between species. From 1990–2013, Ks decreased in both species, whereas wrein increased only in P. longaeva, while no trends were observed in BAI. Climate sensitivity of Ks peaked over a longer period (84–102 days) compared to wrein (20–55 days), responding to increasing minimum temperature. In both species, Ks was a better predictor of BAI than wrein, indicating that, even under severely water-limited conditions, radial growth is linked to hydraulic efficiency rather than safety. Based on the variability of cell density along the stem, the trade-off between hydraulic efficiency and safety in P. longaeva appeared to be controlled by a strategy of space occupation. Characterizing the mechanistic relationship between xylem anatomy, plant hydraulic functioning, and stem growth is necessary to better understand climate-growth relationships in the western US and species’ growth plasticity under future climate change scenarios.

Reforestation of high elevation pines: Direct seeding success depends on seed source and sowing environment.

Forest persistence in regions impacted by increasing water and temperature stress will depend upon species' ability to either rapidly adjust to novel conditions or migrate to track ecological niches. Predicted, rapid climate change is likely to outpace the adaptive and migratory capacity of long-lived isolated tree species, and reforestation may be critical to species' persistence. Facilitating persistence both within and beyond a species' range requires identification of seed lots best adapted to the current and future conditions predicted with rapid climate change. We evaluate variation in emergent seedling performance that leads to differential survival among species and populations for three high elevation five-needle pines. We paired a fully reciprocal field common garden experiment with a greenhouse common garden study to (1) quantify variation in seedling emergence and functional traits, (2) ask how functional traits affect performance under different establishment conditions, and (3) evaluate whether trait and performance variation demonstrates local adaptation and plasticity. Among study species-limber, Great Basin bristlecone, and whitebark pines-we found divergence in emergence and functional traits, though soil moisture was the strongest driver of seedling emergence and abundance across all species. Generalist limber pine had a clear emergence advantage as well as traits associated with drought adaptation, while edaphic specialist bristlecone pine was characterized by low emergence yet high early survival once established. Despite evidence for edaphic specialization, soil characteristics alone did not explain bristlecone success. Across species, trait-environment relationships provided some evidence for local adaptation in drought-adapted traits, but we found no evidence of local adaptation in emergence or survival at this early life stage. For managers looking to promote persistence, sourcing seed from drier environments is likely to impart greater drought resistance into reforestation efforts through strategies such as greater root investment, increasing the probability of early seedling survival. This research demonstrates, through a rigorous reciprocal transplant experimental design, that it may be possible to select climate- and soil-appropriate seed sources for reforestation. However, planting success will ultimately rely on a suitable establishment environment, requiring careful consideration of interannual climate variability for management interventions in these climate and disturbance-impacted tree species.

Stomatal regulation and xylem hydraulics of limber pine and Engelmann spruce in Great Basin sky-island ecosystems

Integration of whole-plant stomatal regulation and xylem hydraulics is of critical importance for predicting species response to drought stress. Yet intraspecific variability of stomatal and hydraulic traits, and how these variabilities interact, remain largely unknown. We hypothesized that drought can drive less stomatal regulation but increase xylem hydraulic safety, resulting in stomatal-hydraulic coordination within a species. We estimated sensitivity of whole-tree canopy conductance to soil drying together with xylem hydraulic traits of two dominant conifers, i.e. limber pine (Pinus flexilis) and Engelmann spruce (Picea engelmannii). Our study was conducted using sub-hourly measurements over five consecutive years (2013–2017) at three instrumented sites with different elevations within the Nevada Eco-hydrological Assessment Network (NevCAN) in Great Basin sky-island ecosystems. Both conifers showed a reduction of stomatal sensitivity to soil drying at lower elevations, indicating an active stomatal acclimation to drought. While limber pine increased xylem embolism resistance in parallel with reduced stomatal sensitivity to soil drying, an opposite hydraulic adjustment was detected in Engelmann spruce. Our results provide evidence that mature trees can respond to climatic changes using coordinated shifts in stomatal regulation and xylem hydraulics, but such changes can differ within and between species in ways that need to be examined using in situ data. Deciphering intraspecific variability in whole-plant stomatal and hydraulic traits ultimately contributes to defining drought tolerance and vulnerability, particularly for tree species that inhabit a wide range of landscapes.

Functional traits underlie specialist-generalist strategies in whitebark pine and limber pine

Plant species life history strategies are described by functional variation spanning an acquisitive and conservative resource use continuum. Specialist species can exhibit traits promoting one end of the continuum, while generalist species can display traits promoting both acquisitive and conservative resource use. Whitebark pine (Pinus albicaulis, PIAL) and limber pine (Pinus flexilis, PIFL) are two high-elevation pines that have similar growth and morphology, yet contrasting elevational distributions with PIAL viewed as a specialist inhabiting a narrower elevation range, and PIFL as a generalist inhabiting a broader elevation range. We compared the physiological and morphological traits of greenhouse-grown 5-year-old PIAL and PIFL. Our results suggest that PIFL’s acquisitive and conservative resource use traits contribute to its generalist strategy and ability to inhabit a greater range of elevations than PIAL. PIFL had greater acquisitive resource use traits including: high-light tolerance (greater Qsat, greater fascicle density), increased biomass allocation to photosynthetic tissue (higher needle biomass, aboveground:belowground biomass, needle:branch + stem biomass), and higher C and water uptake (greater stomatal density and size, higher C assimilation rate), as well as greater conservative resource use traits including: greater physical stress resistance (shorter height, higher stem and branch diameters, greater branch and stem diameter:length), drought tolerance (higher SWC, leaf starch proportion), and drought avoidance (earlier budburst phenology, smaller hydroscape area) than PIAL. Our results suggest that PIFL may make more efficient use of high-light loads and maximize C and water uptake when moisture is abundant during spring snowmelt before the onset of dry summer conditions. Other conservative resource use traits describing cold tolerance, heat tolerance, and drought tolerance did not differ between species, suggesting that both species exhibit traits that promote similar conservative resource use enabling their overlapping persistence at higher elevations. Comparing the physiology of PIAL and PIFL within the same environment enables us to identify physiological mechanisms that underlie species establishment and survival, and how juvenile physiology contributes to their contrasting distributions and their generalist-specialist strategies.

Spatial patterns of seedlings dominated by proximity to deadwood and adult trees for Pinus flexilis and Pinus longaeva

The spatial patterns of trees are a defining feature of forests, yet because of the long lifespan of trees, the full origins of these spatial patterns remain unclear. We used 15.6 ha of a mapped forest plot to assess the local habitat characteristics and spatial structure of 1,550 naturally established seedlings and 1,955 adults of Pinus flexilis (limber pine) and Pinus longaeva (Great Basin bristlecone pine) in southern Utah, USA. For both species, proximity to deadwood (large end diameter ≥ 10 cm) and mature trees were key parameters of habitat, likely indicating abiotic or biotic facilitation. Both seedlings and mature trees were highly aggregated 0.0–2.5 m around large pieces of deadwood, which can persist in this ecosystem for centuries or millennia. Seedlings were most often located around deadwood 15–22 cm in diameter, though deadwood size had little influence on the distance at which seedlings established. Seedlings of both P. flexilis and P. longaeva were abundant near heterospecific adult Pinus. The elevational distribution of seedlings and adults of both species were not different, indicating a regeneration niche similar to the niche of mature trees and also that changing climate at this site has not yet affected species distributions. Low amounts of manganese and higher levels of pH and potassium in the mineral soil were associated with P. longaeva seedlings but were less important for P. flexilis. Edaphic patterns combined with the presence of deadwood define the overlap between seedling and mature species habitat and identify the key drivers influencing the spatial structure in this high-elevation, old-growth forest. Likely, persistence of these two Pinus species as well as regeneration or site colonization is more favorable in the presence of large deadwood.

Interactions between white pine blister rust, bark beetles, and climate over time indicate vulnerabilities to limber pine health

IntroductionLimber pine is a keystone species in the Rocky Mountains that grows on harsh, high elevation sites where few other tree species can. Recent studies suggest the species is threatened by the combined impacts of the exotic, invasive disease, white pine blister rust (WPBR), native bark beetles, and climate change. Information on changes in the health of limber pine populations and long-term impacts posed by these threats is needed to inform management efforts.MethodsWe established 106 long-term monitoring plots in 10 study areas that were surveyed three times between 2004 and 2017. We assessed site and stand factors, tree health, and regeneration over time to detect changes in limber pine abundance and health, cumulative impacts of WPBR and bark beetles, and to evaluate the drivers of WPBR occurrence and severity.ResultsLimber pine health declined significantly over the study with more than 20% of initially live limber pine trees dead by the last measurement cycle, primarily due to WPBR and bark beetles. While some recruitment occurred, mortality rates greatly outpaced recruitment of ingrowth. Disease incidence and how it changed over time was variable, but disease severity increased substantially overall and in all study areas. Limber pine regeneration was low or absent in most sites and mortality caused by WPBR increased significantly. We found strong relationships between WPBR and aridity. Trees in habitats with high vapor pressure deficit were less likely to be infected with WPBR, but trees that were already infected were more likely to develop severe symptoms and die. Longer growing seasons increased the likelihood of WPBR presence and mortality. Growing season length and vapor pressure deficit tended to increase over the study, suggesting that climate change may exacerbate disease impacts.DiscussionDeclining health of limber pine coupled with high mortality rates, increasing disease severity, and low levels of natural regeneration indicate successful recovery may not occur in some locations without management intervention. Proactive management strategies to reduce insect and disease impacts and promote stand recovery and resilience should be pursued in remaining, healthy limber pine ecosystems.

Characterization of Foliar Fungal Endophyte Communities from White Pine Blister Rust Resistant and Susceptible Pinus flexilis in Natural Stands in the Southern Rocky Mountains

Fungal endophytic communities in needles of field-grown Pinus flexilis previously inferred to carry major gene resistance (R) to white pine blister rust (WPBR) or to lack it (S) were surveyed to identify unique microbes that may be recruited by WPBR-resistant genotypes. Resistant and susceptible trees were sampled in each of 11 P. flexilis populations for a total of 50 trees sampled. Through next-generation sequencing, this study showed a diverse needle mycobiota in P. flexilis, of which many remain unknown, regardless of the presence or absence of the WPBR resistance gene, Cr4. Ascomycota dominated the mycobiota (88.9%) followed by Basidiomycota (4.4%) and Chytridiomycota (0.03%), and the remaining 6.7% were unclassified. Shared ( n = 105) and unique ( n = 48 in R and n = 49 in S) fungal taxa, including differentially abundant operational taxonomic units, were identified that could provide insights into core mycobiota and host genotype-specific fungal groups. Marginal variation of the fungal diversity and structure was observed between host genotypes, which indicates that neither Cr4 nor the physiological differences associated with the presence or absence of the gene affects mycobiota recruitment. Instead, other parameters, including host size (diameter at breast height) and site elevation, significantly influenced the variability of the composition and structure of the fungal endophytic community. Further investigations are needed to understand the relationship of unique or differentially abundant taxa with one genotype or the other, and to determine the role of the needle mycobiota in WPBR disease development in natural stands of P. flexilis.

Essential Oil Compositions of Pinus Species (P. contorta Subsp. contorta, P. ponderosa var. ponderosa, and P. flexilis); Enantiomeric Distribution of Terpenoids in Pinus Species.

Pinus species are important in traditional medicine throughout their ranges, and pine essential oils are of interest in aromatherapy and as topical treatments. In this work, the leaf (needle) essential oils of Pinus ponderosa var. ponderosa and Pinus contorta subsp. contorta from Oregon and Pinus flexilis growing in Idaho, have been obtained by hydrodistillation and analyzed by gas chromatographic techniques. The leaf essential oil of P. ponderosa was dominated by β-pinene (21.5–55.3%), methyl chavicol (8.5–41.5%), α-pinene (3.6–9.6%), δ-3-carene (3.6–6.2%), and α-terpineol (1.4–5.3%). The major components of P. contorta essential oil were β-phellandrene (23.8%), terpinen-4-ol (11.0%). The essential oil of P. flexilis was dominated by α-pinene (37.1%), β-pinene (21.9%), bornyl acetate (12.8%), and camphene (8.5%). Chiral gas chromatography revealed the enantiomeric ratios of α-pinene and limonene to be variable, but (−)-β-pinene predominated in Pinus essential oils.

Restoring a forest keystone species: A plan for the restoration of whitebark pine (Pinus albicaulis Engelm.) in the Crown of the Continent Ecosystem

The precipitous decline of the keystone species whitebark pine (Pinus albicaulis Engelm.) has resulted in dramatic changes to many high elevation ecosystems in the western U.S. and Canada. To restore these ecosystems, there is a need to establish populations of whitebark pine that will persist in the face of both natural and anthropogenic disturbances (e.g. climate change, white pine blister rust [Cronartium ribicola J.C. Fisher]). The listing of whitebark pine as endangered in Canada and its recent proposed listing as threatened in the U.S. reinforce the urgency of the need to act. This paper presents a six-step methodology for whitebark pine restoration planning in a GIS environment, that was developed as a pilot on a 5-million-acre (2-million-hectare) subset of the Crown of the Continent Ecosystem (CCE). The methodology synthesizes the best available datasets and science into a flexible, data-informed decision-making process that can be applied consistently across large geographic areas. The end-product is an action plan that guides implementation of treatments with a high likelihood of success at an appropriate scale to address the enormous need for restoration. The methodology begins by mapping where whitebark is capable of existing, then quantifies the priority conservation values it provides, the stressors affecting those conservation values, and how those stressors could affect the likelihood of restoration treatment success. Based on those results, priority core areas for restoration are identified. Finally, a restoration plan assigns and prioritizes site-specific actions that protect and restore genetically diverse, evolutionarily stable populations of whitebark pine that are well-distributed across the area of interest. This methodology can be scaled up as demonstrated by its use to identify core areas in Region 1 of the USDA Forest Service. It can also be applied to other species as demonstrated by the addition of limber pine (Pinus flexilis James) when the methodology was used to develop a restoration strategy for the full 18-million-acre (7-million-hectare) CCE (Summary Report in progress).

Comparative Association Mapping Reveals Conservation of Major Gene Resistance to White Pine Blister Rust in Southwestern White Pine (Pinus strobiformis) and Limber Pine (P. flexilis).

All native North American white pines are highly susceptible to white pine blister rust (WPBR) caused by Cronartium ribicola. Understanding genomic diversity and molecular mechanisms underlying genetic resistance to WPBR remains one of the great challenges in improvement of white pines. To compare major gene resistance (MGR) present in two species, southwestern white pine (Pinus strobiformis) Cr3 and limber pine (P. flexilis) Cr4, we performed association analyses of Cr3-controlled resistant traits using single nucleotide polymorphism (SNP) assays designed with Cr4-linked polymorphic genes. We found that ∼70% of P. flexilis SNPs were transferable to P. strobiformis. Furthermore, several Cr4-linked SNPs were significantly associated with the Cr3-controlled traits in P. strobiformis families. The most significantly associated SNP (M326511_1126R) almost colocalized with Cr4 on the Pinus consensus linkage group 8, suggesting that Cr3 and Cr4 might be the same R locus, or have localizations very close to each other in the syntenic region of the P. strobiformis and P. flexilis genomes. M326511_1126R was identified as a nonsynonymous SNP, causing amino acid change (Val376Ile) in a putative pectin acetylesterase, with coding sequences identical between the two species. Moreover, top Cr3-associated SNPs were further developed as TaqMan genotyping assays, suggesting their usefulness as marker-assisted selection (MAS) tools to distinguish genotypes between quantitative resistance and MGR. This work demonstrates the successful transferability of SNP markers between two closely related white pine species in the hybrid zone, and the possibility for deployment of MAS tools to facilitate long-term WPBR management in P. strobiformis breeding and conservation.

Co-Occurring Wyoming Pinus Species Exhibit Differing Climate–Growth Relationships

The North American Dendroecological Field week (NADEF) is an intensive dendrochronology workshop, funded in part by the National Science Foundation. The 2019 Introductory Group at NADEF developed two precisely dated tree-ring width chronologies for Pinus contorta (lodgepole pine) and Pinus flexilis (limber pine) at the Wolf Knob site ca. 5 km west of Beartooth Lake, WY, within the bounds of the Shoshone National Forest (SNF), in the Greater Yellowstone Ecosystem (GYE). Wolf Knob is a semi-arid, S- to SW-facing, mid- to high-elevation site, making it an ideal location to examine the climate sensitivity of annual tree-ring width increments. Here, we show that two co-located Pinus species exhibit differing climate–growth relationships, with P. contorta exhibiting relatively weak correlations with precipitation (r = 0.37; p < 0.01) and temperature (r = –0.23; p < 0.05) during the late summer, and P. flexilis exhibiting stronger overall correlations with both cool-season (r = 0.48; p < 0.01) and warm-season precipitation (r = 0.51; p < 0.01) as well as with snowpack records (r = 0.45; p < 0.05). Our results suggest these two Pinus species may face disparate threats in the face of regional climate change, with P. flexilis being particularly vulnerable to drought conditions and declining snowpacks in the GYE. The differing seasonal climate sensitivities of the two species is likely caused by microsite conditions (e.g. soil moisture capacity, incoming solar radiation) and distinct species-climate responses, underscoring the importance of not only site selection, but also microsite and individual selection in dendroclimatological sampling. Finally, this work contributes to identifying Snow Water Equivalent (SWE)-sensitive tree-ring proxies in the GYE, critical for understanding ongoing warming-induced snowpack declines across western North America, particularly given the projections of a largely snow-free (April 1) GYE by 2075.

Water-Use Efficiency of Co-occurring Sky-Island Pine Species in the North American Great Basin.

Water-use efficiency (WUE), weighing the balance between plant transpiration and growth, is a key characteristic of ecosystem functioning and a component of tree drought resistance. Seasonal dynamics of tree-level WUE and its connections with drought variability have not been previously explored in sky-island montane forests. We investigated whole-tree transpiration and stem growth of bristlecone (Pinus longaeva) and limber pine (Pinus flexilis) within a high-elevation stand in central-eastern Nevada, United States, using sub-hourly measurements over 5 years (2013–2017). A moderate drought was generally observed early in the growing season, whereas interannual variability of summer rains determined drought levels between years, i.e., reducing drought stress in 2013–2014 while enhancing it in 2015–2017. Transpiration and basal area increment (BAI) of both pines were coupled throughout June–July, resulting in a high but relatively constant early season WUE. In contrast, both pines showed high interannual plasticity in late-season WUE, with a predominant role of stem growth in driving WUE. Overall, bristlecone pine was characterized by a lower WUE compared to limber pine. Dry or wet episodes in the late growing season overrode species differences. Our results suggested thresholds of vapor pressure deficit and soil moisture that would lead to opposite responses of WUE to late-season dry or wet conditions. These findings provide novel insights and clarify potential mechanisms modulating tree-level WUE in sky-island ecosystems of semi-arid regions, thereby helping land managers to design appropriate science-based strategies and reduce uncertainties associated with the impact of future climatic changes.