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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.

Members of the Pinus genus are well known for their medicinal properties, which can be attributed to their essential oils. In this work, we have examined the leaf essential oils of five understudied Pinus species collected from various locations in western North America. The essential oils were obtained by hydrodistillation and analyzed by gas chromatographic methods, including enantioselective gas chromatography. Pinus albicaulis was dominated by (+)-δ-3-carene; Pinus flexilis was dominated by α-pinene (mostly (+)-α-pinene) and (−)-β-pinene; Pinus lambertiana was dominated by (−)-β-pinene; Pinus monticola was dominated by (−)-β-pinene, (+)-δ-3-carene, and (−)-α-pinene; and Pinus sabiniana was rich in (−)-α-pinene and limonene. While this work adds to our knowledge of Pinus essential oils, additional research is needed to more fully appreciate the geographic and altitudinal variations in the volatile compositions of these 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.

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.

Annually dated tree-rings of 509 live and deadwood limber pine (Pinus flexilis) samples from the semi-arid Wassuk Range, Nevada, yielded a 3996-yr record extending from 1983 BC to AD 2013. Correlations of radial growth with climate were positive for water relations and negative for summer temperatures. Long-term trends of ring-width corresponded to climate variability documented from other proxies, including low growth during the Late Holocene Dry Period and Medieval Climate Anomaly (MCA) and elevated growth during cool, wet periods of the Neoglacial and Little Ice Age. Spline fit of the data indicated that growth decrease in the last 20 years was second lowest on record, surpassed by lowest growth at 20 BC—AD 150. Demographics of limber pine by aspect and elevation were not strongly related to long-term climate dynamics, except in the case of extirpations on all but north aspects at the end of the MCA. Pines occurred persistently on north aspects, where a continuous record existed to present. Elevation shifts were not obvious on any aspect, and no evidence existed for migration above current treeline. Non-climatic factors appear to interact with climate to make north slopes refugial for upland pines in semi-arid regions across four millennia.

Tree-rings representing annual dates from live and deadwood Pinus flexilis at ten sites across the central Great Basin (~38°N) yielded a cumulative record across 4002 years (1983 BC–AD 2019). Individual site chronologies ranged in length from 861–4002 years; all were continuous over their sample depths. Correlations of growth with climate were positive for water relations and mostly negative for summer temperatures. Growth was generally correlated across sites, with the central Nevada stands most distinct. Although growth was low during the Late Holocene Dry Period, variability marked this interval, suggesting that it was not pervasively dry. All sites had low growth during the first half of the Medieval Climate Anomaly, high growth during the mid-interval pluvial, and low growth subsequently. Little synchrony occurred across sites for the early Little Ice Age. After AD 1650, growth was depressed until the early twentieth century. Growth at all sites declined markedly ca. AD 1985, was similar to the lowest growth period of the full records, and indicative of recent severe droughts. A small rebound in growth occurred after ca. AD 2010. A strong signal for Atlantic Multidecadal Oscillation (AMO) occurred in growth response at most sites. The persistence of all stands despite climate variability indicates high resilience of this species.

Background Linkage of DNA markers with phenotypic traits provides essential information to dissect clustered genes with potential phenotypic contributions in a target genome region. Pinus flexilis E. James (limber pine) is a keystone five-needle pine species in mountain-top ecosystems of North America. White pine blister rust (WPBR), caused by a non-native fungal pathogen Cronartium ribicola (J.C. Fisch.), has resulted in mortality in this conifer species and is still spreading through the distribution. The objective of this research was to develop P. flexilis transcriptome-wide single nucleotide polymorphism (SNP) markers using RNA-seq analysis for genetic mapping of the major gene ( Cr4 ) that confers complete resistance to C. ribicola . Results Needle tissues of one resistant and two susceptible seedling families were subjected to RNA-seq analysis. In silico SNP markers were uncovered by mapping the RNA-seq reads back to the de novo assembled transcriptomes. A total of 110,573 in silico SNPs and 2,870 indels were identified with an average of 3.7 SNPs per Kb. These SNPs were distributed in 17,041 unigenes. Of these polymorphic P. flexilis unigenes, 6,584 were highly conserved as compared to the genome sequence of P. taeda L (loblolly pine). High-throughput genotyping arrays were designed and were used to search for Cr4 -linked genic SNPs in megagametophyte populations of four maternal trees by haploid-segregation analysis. A total of 32 SNP markers in 25 genes were localized on the Cr4 linkage group (LG). Syntenic relationships of this Cr4 -LG map with the model conifer species P. taeda anchored Cr4 on Pinus consensus LG8, indicating that R genes against C. ribicola have evolved independently in different five-needle pines. Functional genes close to Cr4 were annotated and their potential roles in Cr4 -mediated resistance were further discussed. Conclusions We demonstrated a very effective, low-cost strategy for developing a SNP genetic map of a phenotypic trait of interest. SNP discovery through transcriptome comparison was integrated with high-throughput genotyping of a small set of in silico SNPs. This strategy may be applied to mapping any trait in non-model plant species that have complex genomes. Whole transcriptome sequencing provides a powerful tool for SNP discovery in conifers and other species with complex genomes, for which sequencing and annotation of complex genomes is still challenging. The genic SNP map for the consensus Cr4 -LG may help future molecular breeding efforts by enabling both Cr4 positional characterization and selection of this gene against WPBR.

Smooth bark on trees and shrubs was historically hypothesized to be an anatomical defence against epiphytic vegetation and phytophagous insects. This hypothesis has fallen from favour, yet no clear tests of bark texture as a defence against insects have been published. We tested the smooth bark defence hypothesis using bark beetles specialized in attacking pine trees as model insects, and Pinus flexilis (limber pine) – a widespread tree that can have both smooth and rough bark surfaces on the same stem – as the model tree. We investigated the effects of bark texture on the locations of bark beetle attacks on trees with a combination of field surveys and experiments in the Colorado Rocky Mountains, USA. Bark beetle attacks were overwhelmingly located on rough bark surfaces and virtually absent from smooth bark. Increasing proportional coverage by smooth bark was negatively related to bark beetle attacks per square metre of bark surface. Experimental tests of bark beetles' ability to grip smooth versus rough bark revealed that bark beetles have difficulty gripping and quickly fell from smooth bark but not from rough bark. Smooth bark was negatively related to increasing tree size, but our models indicated that even partial coverage by smooth bark on a tree's trunk can significantly reduce total bark beetle attacks – this reduction likely improves tree fitness as bark beetles must aggregate to overcome tree defences. Synthesis. Our results indicate that smooth bark on trees can act as an anatomical defence against insects by reducing their ability to grip a tree's surface – even for insects specialized in attacking tree stems. Similar to other forms of anti‐insect defence (i.e. secondary chemistry, leaf toughness), smooth bark appears to be influenced by plant ontogeny whereby younger trees have greater defences than older trees. Understanding the adaptive significance of bark texture will require continued field and genetic study. Nevertheless, our results revealed that smooth bark texture increases tree resistance to phytophagous insects calling for the resurrection and vetting of the smooth bark defence hypothesis.

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.