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.

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.

Limber pine (Pinus flexilis James) genetic map constructed by exome-seq provides insight into the evolution of disease resistance and a genomic resource for genomics-based breeding.

Limber pine (Pinus flexilis) is a keystone species of high-elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non-native white pine blister rust caused byCronartium ribicola. Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome-seq was performed to construct high-density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups (LGs). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes (POGs) with localization on homologousLGs among conifer species. Gene ontology (GO) enrichment analysis showed relatively fewer constraints forPOGs with putative roles in adaptation to environments and relatively more conservation forPOGs with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide-binding site leucine-rich repeat genes (NBS-LRRs), 290 receptor-like protein kinase genes (RLKs), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co-positioned with multiple members of theR gene family, revealing the evolutionary pressure acting upon them. This high-density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genome-wide association studies (GWASs), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full-length genomes of limber pine and relatedPinus species.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence.

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

The role of hybridization during ecological divergence of southwestern white pine (Pinus strobiformis) and limber pine (P. flexilis).

Interactions between extrinsic factors, such as disruptive selection and intrinsic factors, such as genetic incompatibilities among loci, often contribute to the maintenance of species boundaries. The relative roles of these factors in the establishment of reproductive isolation can be examined using species pairs characterized by gene flow throughout their divergence history. We investigated the process of speciation and the maintenance of species boundaries between Pinus strobiformis and Pinus flexilis. Utilizing ecological niche modelling, demographic modelling and genomic cline analyses, we illustrated a divergence history with continuous gene flow. Our results supported an abundance of advanced generation hybrids and a lack of loci exhibiting steep transition in allele frequency across the hybrid zone. Additionally, we found evidence for climate-associated variation in the hybrid index and niche divergence between parental species and the hybrid zone. These results are consistent with extrinsic factors, such as climate, being an important isolating mechanism. A build-up of intrinsic incompatibilities and of coadapted gene complexes is also apparent, although these appear to be in the earliest stages of development. This supports previous work in coniferous species demonstrating the importance of extrinsic factors in facilitating speciation. Overall, our findings lend support to the hypothesis that varying strength and direction of selection pressures across the long lifespans of conifers, in combination with their other life history traits, delays the evolution of strong intrinsic incompatibilities.

Profiling methyl jasmonate-responsive transcriptome for understanding induced systemic resistance in whitebark pine (Pinus albicaulis).

KEY MESSAGE: RNA-seq analysis on whitebark pine needles demonstrated that methyl jasmonate (MeJA)-triggered transcriptome re-programming substantially overlapped with defense responses against insects and fungal pathogens in Pinus species, increasing current knowledge regarding induced systemic resistance (ISR) to pathogens and pests in whitebark pine. Many whitebark pine populations are in steep decline due to high susceptibility to mountain pine beetle and the non-native white pine blister rust (WPBR). Resistance, including induced systemic resistance (ISR), is not well characterized in whitebark pine, narrowing the current options for increasing the success of restoration and breeding programs. Exogenous jasmonates are known to trigger ISR by activating the plant's immune system through regulation of gene expression to produce chemical defense compounds. This study reports profiles of whitebark pine needle transcriptomes, following methyl jasmonate (MeJA) treatment using RNA-seq. A MeJA-responsive transcriptome was de novo assembled and transcriptome profiling identified a set of differentially expressed genes (DEGs), revealing 1422 up- and 999 down-regulated transcripts with at least twofold change (FDR corrected p < 0.05) in needle tissues in response to MeJA application. GO analysis revealed that these DEGs have putative functions in plant defense signalling, transcription regulation, biosyntheses of secondary metabolites, and other biological processes. Lineage-specific expression of defense-related genes was characterized through comparison with MeJA signalling in model plants. In particular, MeJA-triggered transcriptome re-programming substantially overlapped with defense responses against WPBR and insects in related Pinus species, suggesting that MeJA may be used to improve whitebark pine resistance to pathogens/pests. Our study provides new insights into molecular mechanisms and metabolic pathways involved in whitebark pine ISR. DEGs identified in this study can be used as candidates to facilitate identification of genomic variation contributing to host resistance and aid in breeding selection of elite genotypes with better adaptive fitness to environmental stressors in this endangered tree species.

Genetic mapping of Pinus flexilis major gene (Cr4) for resistance to white pine blister rust using transcriptome-based SNP genotyping.

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.

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.

Adaptive evolution in a conifer hybrid zone is driven by a mosaic of recently introgressed and background genetic variants.

Extant conifer species may be susceptible to rapid environmental change owing to their long generation times, but could also be resilient due to high levels of standing genetic diversity. Hybridisation between closely related species can increase genetic diversity and generate novel allelic combinations capable of fuelling adaptive evolution. Our study unravelled the genetic architecture of adaptive evolution in a conifer hybrid zone formed between Pinus strobiformis and P. flexilis. Using a multifaceted approach emphasising the spatial and environmental patterns of linkage disequilibrium and ancestry enrichment, we identified recently introgressed and background genetic variants to be driving adaptive evolution along different environmental gradients. Specifically, recently introgressed variants from P. flexilis were favoured along freeze-related environmental gradients, while background variants were favoured along water availability-related gradients. We posit that such mosaics of allelic variants within conifer hybrid zones will confer upon them greater resilience to ongoing and future environmental change and can be a key resource for conservation efforts.

Association Mapping and Development of Marker-Assisted Selection Tools for the Resistance to White Pine Blister Rust in the Alberta Limber Pine Populations.

Since its introduction to North America in the early 1900s, white pine blister rust (WPBR) caused by the fungal pathogen Cronartium ribicola 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 (Pinus flexilis), 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 C. ribicola 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 C. ribicola, MGR in these seed families appears to be controlled by Cr4 or other R genes in very close proximity to Cr4. These associated SNPs were located in genes involved in multiple molecular mechanisms potentially underlying limber pine MGR to C. ribicola, including NBS-LRR genes for recognition of C. ribicola 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.

Tracing the footprints of a moving hybrid zone under a demographic history of speciation with gene flow.

A lack of optimal gene combinations, as well as low levels of genetic diversity, is often associated with the formation of species range margins. Conservation efforts rely on predictive modelling using abiotic variables and assessments of genetic diversity to determine target species and populations for controlled breeding, germplasm conservation and assisted migration. Biotic factors such as interspecific competition and hybridization, however, are largely ignored, despite their prevalence across diverse taxa and their role as key evolutionary forces. Hybridization between species with well-developed barriers to reproductive isolation often results in the production of offspring with lower fitness. Generation of novel allelic combinations through hybridization, however, can also generate positive fitness consequences. Despite this possibility, hybridization-mediated introgression is often considered a threat to biodiversity as it can blur species boundaries. The contribution of hybridization towards increasing genetic diversity of populations at range margins has only recently gathered attention in conservation studies. We assessed the extent to which hybridization contributes towards range dynamics by tracking spatio-temporal changes in the central location of a hybrid zone between two recently diverged species of pines: Pinus strobiformis and P. flexilis. By comparing geographic cline centre estimates for global admixture coefficient with morphological traits associated with reproductive output, we demonstrate a northward shift in the hybrid zone. Using a combination of spatially explicit, individual-based simulations and linkage disequilibrium variance partitioning, we note a significant contribution of adaptive introgression towards this northward movement, despite the potential for differences in regional population size to aid hybrid zone movement. Overall, our study demonstrates that hybridization between recently diverged species can increase genetic diversity and generate novel allelic combinations. These novel combinations may allow range margin populations to track favourable climatic conditions or facilitate adaptive evolution to ongoing and future climate change.

The future of subalpine forests in the Southern Rocky Mountains: Trajectories for Pinus aristata genetic lineages.

Like many other high elevation alpine tree species, Rocky Mountain bristlecone pine (Pinus aristata Engelm.) may be particularly vulnerable to climate change. To evaluate its potential vulnerability to shifts in climate, we defined the suitable climate space for each of four genetic lineages of bristlecone pine and for other subalpine tree species in close proximity to bristlecone pine forests. Measuring changes in the suitable climate space for lineage groups is an important step beyond models that assume species are genetically homogenous. The suitable climate space for bristlecone pine in the year 2090 is projected to decline by 74% and the proportional distribution of suitable climate space for genetic lineages shifts toward those associated with warmer and wetter conditions. The 2090 climate space for bristlecone pine exhibits a bimodal distribution along an elevation gradient, presumably due to the persistence of the climate space in the Southern Rocky Mountains and exclusion at mid-elevations by conditions that favor the climate space of other species. These shifts have implications for changes in fire regimes, vulnerability to pest and pathogens, and altered carbon dynamics across the southern Rockies, which may reduce the likelihood of bristlecone pine trees achieving exceptional longevity in the future. The persistence and expansion of climate space for southern bristlecone pine genetic lineage groups in 2090 suggests that these sources may be the least vulnerable in the future. While these lineages may be more likely to persist and therefore present opportunities for proactive management (e.g., assisted migration) to maintain subalpine forest ecosystem services in a warmer world, our findings also imply heighted conservation concern for vulnerable northern lineages facing range contractions.

Carbon Costs of Constitutive and Expressed Resistance to a Non-Native Pathogen in Limber Pine.

Increasing the frequency of resistance to the non-native fungus Cronartium ribicola (causative agent of white pine blister rust, WPBR) in limber pine populations is a primary management objective to sustain high-elevation forest communities. However, it is not known to what extent genetic disease resistance is costly to plant growth or carbon economy. In this study, we measured growth and leaf-level physiology in (1) seedling families from seed trees that have previously been inferred to carry or not carry Cr4, the dominant R gene allele conferring complete, gene-for-gene resistance to WPBR in limber pine, and (2) populations that were and were not infected with C. ribicola. We found that, in the absence of C. ribicola exposure, there was no significant difference in carbon relations between families born from seed trees that harbor the resistance allele compared to those that lack it, either to plant growth and phenology or leaf-level photosynthetic traits. However, post-infection with C. ribicola, growth was significantly reduced in inoculation survivors expressing complete resistance compared to uninoculated seedlings. Furthermore, inoculation survivors exhibited significant increases in a suite of traits including photosynthetic rate, respiration rate, leaf N, and stomatal conductance and a decrease in photosynthetic water-use efficiency. The lack of constitutive carbon costs associated with Cr4 resistance in non-stressed limber pine is consistent with a previous report that the R gene allele is not under selection in the absence of C. ribicola and suggests that host resistance may not bear a constitutive cost in pathosystems that have not coevolved. However, under challenge by C. ribicola, complete resistance to WPBR in limber pine has a significant cost to plant growth, though enhanced carbon acquisition post-infection may offset this somewhat. These costs and effects on performance further complicate predictions of this species' response in warmer future climates in the presence of WPBR.

Influence of tree size on shoot structure and physiology of Pinus contorta and Pinus aristata.

This study examined the morphological and physiological characteristics of shoots of large and small mature trees of two pine species with different growth patterns and tree longevities: Rocky Mountain bristlecone pine (Pinus aristata Bailey) and lodgepole pine (Pinus contorta Dougl. ssp. latifolia Bailey). Large trees of P. aristata had 21% less annual shoot growth but retained more total foliar biomass per shoot than small trees because of a 95% longer leaf life span. Large trees of P. contorta had 69% less annual shoot growth and less foliage production per shoot, and even with a 23% longer leaf life span, they retained leass total foliar biomass per shoot than small trees. Needles of both species and sizes did not begin to abscise until they reached approximately 40-45% of their expected maximum life span. Foliar nitrogen and phosphorus concentrations, N(mass) and P(mass), of 1-year-old P. aristata leaves were significantly lower and higher, respectively, in the large than in the small trees. In P. contorta, N(mass) and P(mass) of 1-year-old leaves were similar in large and small trees. Pinus aristata also had a more consistent N(mass) with leaf age than P. contorta, and the pattern differed for small and large trees. Photosynthetic capacity and stomatal conductance of 1-year-old leaves of P. aristata were less in large trees than in small trees. The increased plasticity in leaf life span with tree size in P. aristata compared with P. contorta enabled an increase in foliar biomass per shoot in P. aristata that may offset the decrease in photosynthetic performance and contribute to the smaller reduction in shoot growth with tree size in P. aristata compared with P. contorta. Similarities in needle biomass distribution between species and tree sizes suggest that relative needle age may be of greater importance to the retention of the leaves than their chronological age. The magnitude of the changes in shoot characteristics with increasing tree size differed between the species and may reflect alternative strategies of resource allocation and use.

The interaction between leaf longevity and shoot growth and foliar biomass per shoot in Pinus contorta at two elevations.

Leaf longevity ranged from an average of 5 to 18 years in individual trees of lodgepole pine (Pinus contorta Dougl. ssp. latifolia D.K. Bailey) from six even-aged stands in central Colorado, USA, of which three were at an elevation of 2800 and three at an elevation of 3200 m. Leaf longevity was 38% greater and annual shoot growth increment was 33% less in trees growing at 3200 m than in trees growing at 2800 m elevation. There was no difference in leaf biomass per shoot between the trees at the two elevations. These results suggest that leaf longevity is greater on shoots with low annual growth potential. Thus, a slow-growing tree at high elevation (low annual shoot growth potential) can have the same amount of foliage per shoot as a fast-growing tree at lower elevation. This plasticity in leaf longevity enables a consistency in foliar biomass per shoot, which may contribute to the wide range of sites and environments that lodgepole pine occupies successfully.

Interrelation between shoot characteristics and solar irradiance in the crown of Pinus contorta ssp. latifolia.

In Pinus contorta Doug. ssp. latifolia (Engelm.), annual shoot length increment, annual foliage production per shoot, and the daily integral of photosynthetically active radiation (DPAR) at shoot tips increased with height in the crown, whereas leaf longevity decreased. There was a decrease in DPAR along the shoot from the tip to beyond the oldest leaves, and the value reached at the point just proximal to the oldest leaf cohort was the same for all shoots from all crown locations, regardless of the age of the oldest leaves. The results suggest that shoot elongation, leaf production and leaf retention are controlled by the light environment within the crown. Because high irradiance promotes leaf production and low irradiance promotes leaf shedding, foliage is not randomly distributed in the crown.