Genetic architecture of disease resistance and tolerance in Douglas-fir trees.

Understanding the genetic basis of how plants defend against pathogens is important to monitor and maintain resilient tree populations. Swiss needle cast (SNC) and Rhabdocline needle cast (RNC) epidemics are responsible for major damage of forest ecosystems in North America. Here we investigate the genetic architecture of tolerance and resistance to needle cast diseases in Douglas-fir (Pseudotsuga menziesii) caused by two fungal pathogens: SNC caused by Nothophaeocryptopus gaeumannii, and RNC caused by Rhabdocline pseudotsugae. We performed case-control genome-wide association analyses and found disease resistance and tolerance in Douglas-fir to be polygenic and under strong selection. We show that stomatal regulation as well as ethylene and jasmonic acid pathways are important for resisting SNC infection, and secondary metabolite pathways play a role in tolerating SNC once the plant is infected. We identify a major transcriptional regulator of plant defense, ERF1, as the top candidate for RNC resistance. Our findings shed light on the highly polygenic architectures underlying fungal disease resistance and tolerance and have important implications for forestry and conservation as the climate changes.

Transcription through the eye of a needle: daily and annual cyclic gene expression variation in Douglas-fir needles.

BACKGROUND: Perennial growth in plants is the product of interdependent cycles of daily and annual stimuli that induce cycles of growth and dormancy. In conifers, needles are the key perennial organ that integrates daily and seasonal signals from light, temperature, and water availability. To understand the relationship between seasonal cycles and seasonal gene expression responses in conifers, we examined diurnal and circannual needle mRNA accumulation in Douglas-fir (Pseudotsuga menziesii) needles at diurnal and circannual scales. Using mRNA sequencing, we sampled 6.1 × 109 reads from 19 trees and constructed a de novo pan-transcriptome reference that includes 173,882 tree-derived transcripts. Using this reference, we mapped RNA-Seq reads from 179 samples that capture daily and annual variation. RESULTS: We identified 12,042 diurnally-cyclic transcripts, 9299 of which showed homology to annotated genes from other plant genomes, including angiosperm core clock genes. Annual analysis revealed 21,225 circannual transcripts, 17,335 of which showed homology to annotated genes from other plant genomes. The timing of maximum gene expression is associated with light intensity at diurnal scales and photoperiod at annual scales, with approximately half of transcripts reaching maximum expression +/- 2 h from sunrise and sunset, and +/- 20 days from winter and summer solstices. Comparisons with published studies from other conifers shows congruent behavior in clock genes with Japanese cedar (Cryptomeria), and a significant preservation of gene expression patterns for 2278 putative orthologs from Douglas-fir during the summer growing season, and 760 putative orthologs from spruce (Picea) during the transition from fall to winter. CONCLUSIONS: Our study highlight the extensive diurnal and circannual transcriptome variability demonstrated in conifer needles. At these temporal scales, 29% of expressed transcripts show a significant diurnal cycle, and 58.7% show a significant circannual cycle. Remarkably, thousands of genes reach their annual peak activity during winter dormancy. Our study establishes the fine-scale timing of daily and annual maximum gene expression for diverse needle genes in Douglas-fir, and it highlights the potential for using this information for evaluating hypotheses concerning the daily or seasonal timing of gene activity in temperate-zone conifers, and for identifying cyclic transcriptome components in other conifer species.

Photoperiod cues and patterns of genetic variation limit phenological responses to climate change in warm parts of species' range: Modeling diameter-growth cessation in coast Douglas-fir.

The phenology of diameter-growth cessation in trees will likely play a key role in mediating species and ecosystem responses to climate change. A common expectation is that warming will delay cessation, but the environmental and genetic influences on this process are poorly understood. We modeled the effects of temperature, photoperiod, and seed-source climate on diameter-growth-cessation timing in coast Douglas-fir (an ecologically and economically vital tree) using high-frequency growth measurements across broad environmental gradients for a range of genotypes from different seed sources. Our model suggests that cool temperatures or short photoperiods can induce cessation in autumn. At cool locations (high latitude and elevation), cessation seems to be induced primarily by low temperatures in early autumn (under relatively long photoperiods), so warming will likely delay cessation and extend the growing season. But at warm locations (low latitude or elevation), cessation seems to be induced primarily by short photoperiods later in autumn, so warming will likely lead to only slight extensions of the growing season, reflecting photoperiod limitations on phenological shifts. Trees from seed sources experiencing frequent frosts in autumn or early winter tended to cease growth earlier in the autumn, potentially as an adaptation to avoid frost. Thus, gene flow into populations in warm locations with little frost will likely have limited potential to delay mean cessation dates because these populations already cease growth relatively late. In addition, data from an abnormal heat wave suggested that very high temperatures during long photoperiods in early summer might also induce cessation. Climate change could make these conditions more common in warm locations, leading to much earlier cessation. Thus, photoperiod cues, patterns of genetic variation, and summer heat waves could limit the capacity of coast Douglas-fir to extend its growing season in response to climate change in the warm parts of its range.

Will changes in phenology track climate change? A study of growth initiation timing in coast Douglas-fir.

Under climate change, the reduction of frost risk, onset of warm temperatures and depletion of soil moisture are all likely to occur earlier in the year in many temperate regions. The resilience of tree species will depend on their ability to track these changes in climate with shifts in phenology that lead to earlier growth initiation in the spring. Exposure to warm temperatures ('forcing') typically triggers growth initiation, but many trees also require exposure to cool temperatures ('chilling') while dormant to readily initiate growth in the spring. If warming increases forcing and decreases chilling, climate change could maintain, advance or delay growth initiation phenology relative to the onset of favorable conditions. We modeled the timing of height- and diameter-growth initiation in coast Douglas-fir (an ecologically and economically vital tree in western North America) to determine whether changes in phenology are likely to track changes in climate using data from field-based and controlled-environment studies, which included conditions warmer than those currently experienced in the tree's range. For high latitude and elevation portions of the tree's range, our models predicted that warming will lead to earlier growth initiation and allow trees to track changes in the onset of the warm but still moist conditions that favor growth, generally without substantially greater exposure to frost. In contrast, toward lower latitude and elevation range limits, the models predicted that warming will lead to delayed growth initiation relative to changes in climate due to reduced chilling, with trees failing to capture favorable conditions in the earlier parts of the spring. This maladaptive response to climate change was more prevalent for diameter-growth initiation than height-growth initiation. The decoupling of growth initiation with the onset of favorable climatic conditions could reduce the resilience of coast Douglas-fir to climate change at the warm edges of its distribution.

Climate-related genetic variation in drought-resistance of Douglas-fir (Pseudotsuga menziesii).

There is a general assumption that intraspecific populations originating from relatively arid climates will be better adapted to cope with the expected increase in drought from climate change. For ecologically and economically important species, more comprehensive, genecological studies that utilize large distributions of populations and direct measures of traits associated with drought-resistance are needed to empirically support this assumption because of the implications for the natural or assisted regeneration of species. We conducted a space-for-time substitution, common garden experiment with 35 populations of coast Douglas-fir (Pseudotsuga menziesii var. menziesii) growing at three test sites with distinct summer temperature and precipitation (referred to as 'cool/moist', 'moderate', or 'warm/dry') to test the hypotheses that (i) there is large genetic variation among populations and regions in traits associated with drought-resistance, (ii) the patterns of genetic variation are related to the native source-climate of each population, in particular with summer temperature and precipitation, (iii) the differences among populations and relationships with climate are stronger at the warm/dry test site owing to greater expression of drought-resistance traits (i.e., a genotype × environment interaction). During midsummer 2012, we measured the rate of water loss after stomatal closure (transpiration(min)), water deficit (% below turgid saturation), and specific leaf area (SLA, cm(2) g(-1)) on new growth of sapling branches. There was significant genetic variation in all plant traits, with populations originating from warmer and drier climates having greater drought-resistance (i.e., lower transpiration(min), water deficit and SLA), but these trends were most clearly expressed only at the warm/dry test site. Contrary to expectations, populations from cooler climates also had greater drought-resistance across all test sites. Multiple regression analysis indicated that Douglas-fir populations from regions with relatively cool winters and arid summers may be most adapted to cope with drought conditions that are expected in the future.

Impact of climate change on cold hardiness of Douglas-fir (Pseudotsuga menziesii): environmental and genetic considerations.

The success of conifers over much of the world's terrestrial surface is largely attributable to their tolerance to cold stress (i.e., cold hardiness). Due to an increase in climate variability, climate change may reduce conifer cold hardiness, which in turn could impact ecosystem functioning and productivity in conifer-dominated forests. The expression of cold hardiness is a product of environmental cues (E), genetic differentiation (G), and their interaction (G × E), although few studies have considered all components together. To better understand and manage for the impacts of climate change on conifer cold hardiness, we conducted a common garden experiment replicated in three test environments (cool, moderate, and warm) using 35 populations of coast Douglas-fir (Pseudotsuga menziesii var. menziesii) to test the hypotheses: (i) cool-temperature cues in fall are necessary to trigger cold hardening, (ii) there is large genetic variation among populations in cold hardiness that can be predicted from seed-source climate variables, (iii) observed differences among populations in cold hardiness in situ are dependent on effective environmental cues, and (iv) movement of seed sources from warmer to cooler climates will increase risk to cold injury. During fall 2012, we visually assessed cold damage of bud, needle, and stem tissues following artificial freeze tests. Cool-temperature cues (e.g., degree hours below 2 °C) at the test sites were associated with cold hardening, which were minimal at the moderate test site owing to mild fall temperatures. Populations differed 3-fold in cold hardiness, with winter minimum temperatures and fall frost dates as strong seed-source climate predictors of cold hardiness, and with summer temperatures and aridity as secondary predictors. Seed-source movement resulted in only modest increases in cold damage. Our findings indicate that increased fall temperatures delay cold hardening, warmer/drier summers confer a degree of cold hardiness, and seed-source movement from warmer to cooler climates may be a viable option for adapting coniferous forest to future climate.

Generalized provisional seed zones for native plants.

Deploying well-adapted and ecologically appropriate plant materials is a core component of successful restoration projects. We have developed generalized provisional seed zones that can be applied to any plant species in the United States to help guide seed movement. These seed zones are based on the intersection of high-resolution climatic data for winter minimum temperature and aridity (as measured by annual heat : moisture index), each classified into discrete bands. This results in the delineation of 64 provisional seed zones for the continental United States. These zones represent areas of relative climatic similarity, and movement of seed within these zones should help to minimize maladaptation. Superimposing Omernik's level III ecoregions over these seed zones distinguishes areas that are similar climatically yet different ecologically. A quantitative comparison of provisional seed zones with level III ecoregions and provisional seed zones within ecoregions for three species showed that provisional seed zone within ecoregion often explained the greatest proportion of variation in a suite of traits potentially related to plant fitness. These provisional seed zones can be considered a starting point for guidelines for seed transfer, and should be utilized in conjunction with appropriate species-specific information as well as local knowledge of microsite differences.

Differences in branch hydraulic architecture related to the aridity of growing sites and seed sources of coastal Douglas-fir saplings.

To better understand hydraulic adaptations of coastal Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco) to local climate, we examined genetic (G) and environmental (E) responses of branch hydraulic architecture of 7-year-old saplings from dry and wet climates of origin grown at a relatively dry and a relatively wet common garden site in western Oregon. We sampled 2 years of branch growth from three dry-source and three wet-source families grown at both sites (72 branches, total). Overall, only 4 of the 11 traits had significant genetic (G) effects, whereas 9 traits had significant environmental (E) effects (P < 0.05). Both dry and wet sources had higher leaf-specific conductance (kl) at the dry than the wet site, but the values were achieved by different mechanisms and driven by G × E effects for leaf area/sapwood area (Al/As), shoot length (L), specific conductivity (Ks) and leaf-specific conductivity (Kl). Dry sources achieved higher kl in the dry site through higher Kl (via a lower Al/As and no change in Ks) with no difference in L. Wet sources achieved higher kl at the dry site through no difference in Kl (via no effect on Al/As, despite decreases in Al and As, and lower Ks) with lower L. Vulnerability to embolism (measured as percentage loss of conductivity at 4 MPa) had no G effect but an E effect, with slightly lower values at the dry site. Specific leaf area had G and E effects, with lower values for the dry sources and site. There were no G or E effects on wood density. The different responses of dry and wet sources to site aridity suggest that populations are differentially adapted to the aridity of growing sites. Population variation in response to aridity should be considered when selecting seed sources for establishing forests for future climates.

Genetic variation of piperidine alkaloids in Pinus ponderosa: a common garden study.

BACKGROUND AND AIMS: Previous measurements of conifer alkaloids have revealed significant variation attributable to many sources, environmental and genetic. The present study takes a complementary and intensive, common garden approach to examine genetic variation in Pinus ponderosa var. ponderosa alkaloid production. Additionally, this study investigates the potential trade-off between seedling growth and alkaloid production, and associations between topographic/climatic variables and alkaloid production. METHODS: Piperidine alkaloids were quantified in foliage of 501 nursery seedlings grown from seed sources in west-central Washington, Oregon and California, roughly covering the western half of the native range of ponderosa pine. A nested mixed model was used to test differences among broad-scale regions and among families within regions. Alkaloid concentrations were regressed on seedling growth measurements to test metabolite allocation theory. Likewise, climate characteristics at the seed sources were also considered as explanatory variables. KEY RESULTS: Quantitative variation from seedling to seedling was high, and regional variation exceeded variation among families. Regions along the western margin of the species range exhibited the highest alkaloid concentrations, while those further east had relatively low alkaloid levels. Qualitative variation in alkaloid profiles was low. All measures of seedling growth related negatively to alkaloid concentrations on a natural log scale; however, coefficients of determination were low. At best, annual height increment explained 19.4 % of the variation in ln(total alkaloids). Among the climate variables, temperature range showed a negative, linear association that explained 41.8 % of the variation. CONCLUSIONS: Given the wide geographic scope of the seed sources and the uniformity of resources in the seedlings' environment, observed differences in alkaloid concentrations are evidence for genetic regulation of alkaloid secondary metabolism in ponderosa pine. The theoretical trade-off with seedling growth appeared to be real, however slight. The climate variables provided little evidence for adaptive alkaloid variation, especially within regions.

Growth phenology of coast Douglas-fir seed sources planted in diverse environments.

The timing of periodic life cycle events in plants (phenology) is an important factor determining how species and populations will react to climate change. We evaluated annual patterns of basal-area and height growth of coast Douglas-fir (Pseudotusga menziesii var. menziesii (Mirb.) Franco) seedlings from four seed sources that were planted in four diverse environments as part of the Douglas-fir Seed-Source Movement Trial. Stem diameters and heights were measured periodically during the 2010 growing season on 16 open-pollinated families at each study installation. Stem diameters were measured on a subset of trees with electronic dendrometers during the 2010 and 2011 growing seasons. Trees from the four seed sources differed in phenology metrics that described the timing of basal-area and height-growth initiation, growth cessation and growth rates. Differences in the height-growth metrics were generally larger than differences in the basal-area growth metrics and differences among installations were larger than differences among seed sources, highlighting the importance of environmental signals on growth phenology. Variations in the height- and basal-area growth metrics were correlated with different aspects of the seed-source environments: precipitation in the case of height growth and minimum temperature in the case of basal-area growth. The detailed dendrometer measurements revealed differences in growth patterns between seed sources during distinct periods in the growing season. Our results indicate that multiple aspects of growth phenology should be considered along with other traits when evaluating adaptation of populations to future climates.

Association genetics of coastal Douglas fir (Pseudotsuga menziesii var. menziesii, Pinaceae). I. Cold-hardiness related traits.

Adaptation to cold is one of the greatest challenges to forest trees. This process is highly synchronized with environmental cues relating to photoperiod and temperature. Here, we use a candidate gene-based approach to search for genetic associations between 384 single-nucleotide polymorphism (SNP) markers from 117 candidate genes and 21 cold-hardiness related traits. A general linear model approach, including population structure estimates as covariates, was implemented for each marker-trait pair. We discovered 30 highly significant genetic associations [false discovery rate (FDR) Q < 0.10] across 12 candidate genes and 10 of the 21 traits. We also detected a set of 7 markers that had elevated levels of differentiation between sampling sites situated across the Cascade crest in northeastern Washington. Marker effects were small (r(2) < 0.05) and within the range of those published previously for forest trees. The derived SNP allele, as measured by a comparison to a recently diverged sister species, typically affected the phenotype in a way consistent with cold hardiness. The majority of markers were characterized as having largely nonadditive modes of gene action, especially underdominance in the case of cold-tolerance related phenotypes. We place these results in the context of trade-offs between the abilities to grow longer and to avoid fall cold damage, as well as putative epigenetic effects. These associations provide insight into the genetic components of complex traits in coastal Douglas fir, as well as highlight the need for landscape genetic approaches to the detection of adaptive genetic diversity.

Genecology of Douglas fir in western Oregon and Washington.

BACKGROUND AND AIMS: Genecological knowledge is important for understanding evolutionary processes and for managing genetic resources. Previous studies of coastal Douglas fir (Pseudotsuga menziesii var. menziesii) have been inconclusive with respect to geographical patterns of variation, due in part to limited sample intensity and geographical and climatic representation. This study describes and maps patterns of genetic variation in adaptive traits in coastal Douglas fir in western Oregon and Washington, USA. METHODS: Traits of growth, phenology and partitioning were measured in seedlings of 1338 parents from 1048 locations grown in common gardens. Relations between traits and environments of seed sources were explored using regressions and canonical correlation analysis. Maps of genetic variation as related to the environment were developed using a geographical information system (GIS). KEY RESULTS: Populations differed considerably for adaptive traits, in particular for bud phenology and emergence. Variation in bud-set, emergence and growth was strongly related to elevation and cool-season temperatures. Variation in bud-burst and partitioning to stem diameter versus height was related to latitude and summer drought. Seedlings from the east side of the Washington Cascades were considerably smaller, set bud later and burst bud earlier than populations from the west side. CONCLUSIONS: Winter temperatures and frost dates are of overriding importance to the adaptation of Douglas fir to Pacific Northwest environments. Summer drought is of less importance. Maps generated using canonical correlation analysis and GIS allow easy visualization of a complex array of traits as related to a complex array of environments. The composite traits derived from canonical correlation analysis show two different patterns of variation associated with different gradients of cool-season temperatures and summer drought. The difference in growth and phenology between the westside and eastside Washington Cascades is hypothesized to be a consequence of the presence of interior variety (P. menziessii var. glauca) on the eastside.