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Mountain pine beetle (MPB, Dendroctonus ponderosae) is a significant mortality agent of Pinus, and climate-driven range expansion is occurring. Pinus defenses in recently invaded areas, including high elevations, are predicted to be lower than in areas with longer term MPB presence. MPB was recently observed in high-elevation forests of the Great Basin (GB) region, North America. Defense and susceptibility in two long-lived species, GB bristlecone pine (Pinus longaeva) and foxtail pine (P. balfouriana), are unclear, although they are sympatric with a common MPB host, limber pine (P. flexilis). We surveyed stands with sympatric GB bristlecone–limber pine and foxtail–limber pine to determine relative MPB attack susceptibility and constitutive defenses. MPB-caused mortality was extensive in limber, low in foxtail and absent in GB bristlecone pine. Defense traits, including constitutive monoterpenes, resin ducts and wood density, were higher in GB bristlecone and foxtail than in limber pine. GB bristlecone and foxtail pines have relatively high levels of constitutive defenses which make them less vulnerable to climate-driven MPB range expansion relative to other highelevation pines. Long-term selective herbivore pressure and exaptation of traits for tree longevity are potential explanations, highlighting the complexity of predicting plant–insect interactions under climate change.

Background Proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine ( Pinus flexilis ) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus ( Cr4 ) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown. Results To identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4 -segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4 . The tight linkage of NLRs with Cr4 -controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR. Conclusion This study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.

Since its introduction to North America in the early 1900s, white pine blister rust (WPBR) caused by the fungal pathogen has resulted in substantial economic losses and ecological damage to native North American five-needle pine species. The high susceptibility and mortality of these species, including limber pine ( ), creates an urgent need for the development and deployment of resistant germplasm to support recovery of impacted populations. Extensive screening for genetic resistance to WPBR has been underway for decades in some species but has only started recently in limber pine using seed families collected from wild parental trees in the USA and Canada. This study was conducted to characterize Alberta limber pine seed families for WPBR resistance and to develop reliable molecular tools for marker-assisted selection (MAS). Open-pollinated seed families were evaluated for host reaction following controlled infection using basidiospores. Phenotypic segregation for presence/absence of stem symptoms was observed in four seed families. The segregation ratios of these families were consistent with expression of major gene resistance (MGR) controlled by a dominant R locus. Based on linkage disequilibrium (LD)-based association mapping used to detect single nucleotide polymorphism (SNP) markers associated with MGR against , MGR in these seed families appears to be controlled by or other R genes in very close proximity to . These associated SNPs were located in genes involved in multiple molecular mechanisms potentially underlying limber pine MGR to , including NBS-LRR genes for recognition of effectors, signaling components, and a large set of defense-responsive genes with potential functions in plant effector-triggered immunity (ETI). Interactions of associated loci were identified for MGR selection in trees with complex genetic backgrounds. SNPs with tight Cr4-linkage were further converted to TaqMan assays to confirm their effectiveness as MAS tools. This work demonstrates the successful translation and deployment of molecular genetic knowledge into specific MAS tools that can be easily applied in a selection or breeding program to efficiently screen MGR against WPBR in Alberta limber pine populations.

Limber pine (Pinus flexilis James) co-evolved with the mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) and is now also challenged by the non-native pathogen Cronartium ribicola (J.C. Fisch.) that causes the lethal disease white pine blister rust (WPBR). Previous research suggests that trees infected with WPBR can be preferred hosts for MPB. Using resin duct traits associated with MPB resistance, we tested for a relationship between resistance to MPB and WPBR in limber pine, in the absence of either biological agent. These analyses will help evaluate if MPB historically may have contributed to natural selection for WPBR resistance in advance of WPBR invasion, and could help explain the unusually high frequency of the dominant Cr4 allele for complete resistance to WPBR in limber pine populations of the Southern Rocky Mountains. Resin duct production, density and relative duct area did not differ between healthy trees previously inferred to carry the dominant Cr4 allele and trees that lack it at 22 sites, though some duct traits varied with elevation. MPB resistance does not appear to have played an evolutionary role in contributing to the high frequency of Cr4 in naïve populations, however, MPB may affect the future evolution of resistance to WPBR in the pines where the two pests coincide and WPBR will affect forest recovery after MPB epidemics. MPB-WPBR interactions in a changing climate will affect the future trajectory of limber pine.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N₂‐fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis–AAB association is consistent across years, we re‐sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N₂, we incubated twigs with ¹³N₂‐enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using ¹³N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N₂ fixation in P. flexilis foliage. N₂ diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8–13.6 μg N m⁻² d⁻¹ to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low‐cost, evolutionarily stable N₂‐fixing strategy for long‐lived conifers. This novel source of biological N₂ fixation has fundamental implications for understanding forest N budgets.

Bark beetles have recently killed billions of trees, yet conifer defenses are formidable and some trees resist attack. A primary anti-insect defense of pines is oleoresin from a system of resin ducts throughout the tree. Resin defense traits are heritable, and evidence suggests that resin duct characteristics are associated with resistance to insects. However, comparisons of resin ducts in trees killed by bark beetles to trees that resisted attack are unavailable. We compared vertical resin duct characteristics (number, density, and size) and growth rates from trees that were “resistant” (survived mass attack) versus “susceptible” (killed by attack) to bark beetles in lodgepole (Pinus contorta) and limber (Pinus flexilis) pines. Resistant trees of both species had significantly more resin ducts in recent growth than susceptible trees. Discriminant analysis (DA) correctly categorized 84 % of lodgepole and 92 % of limber pines as susceptible/resistant based on combinations of resin duct and growth characteristics from recent 5- through 20-year growth intervals. DA models using measures from only the most recent 5 years of growth correctly categorized 72 and 81 % of lodgepole and limber pines, respectively. Comparing resistant to susceptible trees independent of species identity led to the correct categorization of 82 % of trees based on factors from 5- to 20-year intervals, and 73 % of trees using only resin duct counts from the most recent 5 years. We conclude that resin duct characteristics can be used to assess tree resistance to bark beetles across pine species, and offer a metric for management to enhance pest resistance.

1. Species distribution shifts in response to climate change require that recruitment increase beyond current range boundaries. For trees with long life spans, the importance of climate-sensitive seedling establishment to the pace of range shifts has not been demonstrated quantitatively. 2. Using spatially explicit, stochastic population models combined with data from long-term forest surveys, we explored whether the climate-sensitivity of recruitment observed in climate manipulation experiments was sufficient to alter populations and elevation ranges of two widely distributed, high-elevation North American conifers. 3. Empirically observed, warming-driven declines in recruitment led to rapid modelled population declines at the low-elevation, 'warm edge' of subalpine forest and slow emergence of populations beyond the high-elevation, 'cool edge'. Because population declines in the forest occurred much faster than population emergence in the alpine, we observed range contraction for both species. For Engelmann spruce, this contraction was permanent over the modelled time horizon, even in the presence of increased moisture. For limber pine, lower sensitivity to warming may facilitate persistence at low elevations — especially in the presence of increased moisture — and rapid establishment above tree line, and, ultimately, expansion into the alpine. 4. Synthesis. Assuming 21st century warming and no additional moisture, population dynamics in high-elevation forests led to transient range contractions for limber pine and potentially permanent range contractions for Engelmann spruce. Thus, limitations to seedling recruitment with warming can constrain the pace of subalpine tree range shifts.

Plants resist herbivores and pathogens by using constitutive (baseline) and inducible (change in defense after an attack) defenses. Inducibility has long been predicted to trade off with constitutive defense, reflecting the economic use of resources. However, empirical evidence for such tradeoffs is variable, and we still lack understanding about when and where defense trade-offs occur. We tested for tradeoffs between constitutive and induced defenses in natural populations of three species of long-lived pines (Pinus balfouriana, P. flexilis, P. longaeva) that differ greatly in constitutive defense and resistance to mountain pine beetle (MPB, Dendroctonus ponderosae). We also assessed how climate influenced constitutive and inducible defenses. At seven high-elevation sites in the western U.S., we simulated MPB attack to induce defenses and measured concentrations of terpene-based phloem defenses on days 0, 15, and 30. Constitutive and induced defenses did not trade off among or within species. Simulated MPB attack induced large increases in defense concentrations in all species independent of constitutive levels. MPB and its symbiotic fungi typically kill trees and thus could be selective forces maintaining strong inducibility within and among species. The contrasting constitutive concentrations in these species could be driven by the adaptation for specializing in harsh, high-elevation environments (e.g., P. balfouriana and P. longaeva) or by competition (e.g., P. flexilis), though these hypotheses have not been empirically examined. Climate influenced defenses, with the greatest concentrations of constitutive and induced defenses occurring at the coldest and driest sites. The interactions between climate and defenses have implications for these species under climate change.

1. Predictions of upslope range shifts for tree species with warming are based on assumptions of moisture stress at lower elevation limits and low-temperature stress at high-elevation limits. However, recent studies have shown that warming can reduce tree seedling establishment across the entire gradient from subalpine forest to alpine via moisture limitation. Warming effects also vary with species, potentially resulting in community shifts in high-elevation forests. 2. We examined the growth and physiology underlying effects of warming on seedling demographic patterns. We evaluated dry mass (DM), root length, allocation above- and below-ground, and relative growth rate (RGR) of whole seedlings, and their ability to avoid or endure water stress via water-use efficiency and resisting turgor loss, for Pinus flexilis, Picea engelmannii and Pinus contorta seeded below, at and above tree line in experimentally warmed, watered and control plots in the Rocky Mountains, USA. We expected that growth and allocation responses to warming would relate to moisture status and that variation in drought tolerance traits would explain species differences in survival rates. 3. Across treatments and elevations, seedlings of all species had weak turgor-loss resistance, and growth was marginal with negative RGR in the first growth phase (-0.01 to -0.04 g g⁻¹ day⁻¹). Growth was correlated with soil moisture, particularly in the relatively small-seeded P. contorta and P. engelmannii. Pinus flexilis, known to have the highest survivorship, attained the greatest DM and longest root but was also the slowest growing and most water-use efficient. This was likely due to its greater reliance on seed reserves. Seedlings developed 15% less total DM, 25% less root DM and 11% shorter roots in heated compared with unheated plots. Higher temperatures slightly increased DM, root length and RGR where soils were wettest, but more strongly decreased these variables under drier conditions. 4. Synthesis. The surprising heat inhibition of tree seedling establishment at the cold edge of forests appears to have a physiological basis: newly germinated seedlings have poor moisture stress tolerance, which appears related to marginal initial growth and heavy reliance on seed reserves. Variation in these attributes among tree species at tree line helps explain their different climate responses.

Climate change is altering plant species distributions globally, and warming is expected to promote uphill shifts in mountain trees. However, at many cold-edge range limits, such as alpine treelines in the western United States, tree establishment may be colimited by low temperature and low moisture, making recruitment patterns with warming difficult to predict. We measured response functions linking carbon (C) assimilation and temperature- and moisture-related microclimatic factors for limber pine (Pinus flexilis) seedlings growing in a heating × watering experiment within and above the alpine treeline. We then extrapolated these response functions using observed microclimate conditions to estimate the net effects of warming and associated soil drying on C assimilation across an entire growing season. Moisture and temperature limitations were each estimated to reduce potential growing season C gain from a theoretical upper limit by 15–30% (c. 50% combined). Warming above current treeline conditions provided relatively little benefit to modeled net assimilation, whereas assimilation was sensitive to either wetter or drier conditions. Summer precipitation may be at least as important as temperature in constraining C gain by establishing subalpine trees at and above current alpine treelines as seasonally dry subalpine and alpine ecosystems continue to warm.