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.

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 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 co-localized with Cr4 on the Pinus consensus linkage group 8 (LG-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 non-synonymous SNP, causing amino acid change (Val376Ile) in a putative pectin acetylesterase (PAE), 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 (QR) 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.

Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family

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.

Community Structure and Functional Role of Limber Pine (Pinus flexilis) in Treeline Communities in Rocky Mountain National Park

Research Highlights: Limber pine (Pinus flexilis) is abundant in some alpine treeline ecotone (ATE) communities east of the Continental Divide in Rocky Mountain National Park (RMNP) and the Colorado Front Range. Limber pine may be able to colonize the ATE under changing climate aided by directed seed dispersal by Clark’s nutcrackers (Nucifraga columbiana). Cronartium ribicola, white pine blister rust, is a growing threat to limber pine and may affect its functional role within the ATE. Background and Objectives: The ATE is sensitive, worldwide, to increasing temperature. However, the predicted advance of treeline under a changing climate may be modified by tree species composition and interactions. We aimed to (1) examine the conifer species composition and relative abundances in treeline communities with limber pine; (2) assess which functional roles limber pine assumes in these communities—tree island initiator, tree island component, and/or solitary tree; and (3) determine whether limber pine’s occurrence as a tree island initiator can be predicted by its relative abundance as a solitary tree. Materials and Methods: We selected four study sites in RMNP above subalpine forest limber pine stands. We sampled the nearest tree island to each of forty random points in each study site as well as solitary tree plots. Results: Across study sites, limber pine comprised, on average, 76% of solitary trees and was significantly more abundant as a solitary tree than Engelmann spruce (Picea engelmannii) or subalpine fir (Abies lasiocarpa). Limber pine was a frequent component of multi-tree islands in three study sites, the major component in one study site, and dominated single-tree islands at two study sites. At three of four study sites, no species had significantly greater odds of being a tree island initiator. Limber pine was found less often as a tree island initiator than predicted from its relative abundance as a solitary tree, given the likely role of solitary trees in tree island formation.

An Economical Approach to Distinguish Genetically Needles of Limber from Whitebark Pine

Whitebark pine is difficult to distinguish from limber pine when seed cones are not present. This is often the case because of young stand age, growth at environmental extremes, or harvesting by vertebrate species. Developing an economical genetic identification tool that distinguishes non-cone-bearing limber from whitebark pine, therefore, could aid many kinds of research on these species. Phylogenetic studies involving limber and whitebark pine suggest that chloroplast DNA sequences differ between these species. We therefore wanted to identify chloroplast loci that could differentiate limber from whitebark pine trees by taking an economical approach involving restriction-site analysis. We generated chloroplast DNA barcode sequences sampled from limber and whitebark pine trees that we identified using attached seed cones. We searched for nucleotide differences associated with restriction endonuclease recognition sites. Our analyses revealed that matK and the psbA-trnH spacer each readily amplified and harbored multiple DNA-sequence differences between limber and whitebark pine. The matK coding sequence of whitebark pine has a BsmAI restriction site not found in limber pine. The psbA-trnH spacer of limber pine has two PsiI restriction sites, neither of which is found in whitebark pine. DNA-sequence and restriction-site analysis of the psbA-trnH spacer from 111 trees showed complete congruence between visually and genetically identified limber (n = 68) and whitebark (n = 43) pine trees. We conclude that restriction site analysis of the chloroplast psbA-trnH spacer and matK involves both minimal technical expertize and research funds. These findings should be of value to foresters interested in species identification and distribution modeling, as well as the analysis of fossil pine pollen, given that gymnosperms transmit chloroplast DNA paternally.