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.     

Purification and properties of glutamine synthetase from Douglas fir roots

Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40-fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non-denaturing gradient-PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 m M . respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K_m values which were 11 and 75 μ M in the presence of ammonium concentrations lower and higher than 1.3 m M , respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio-synthetic activities, respectively.     

Comparative anatomy of ectomycorrhizas synthesized on Douglas fir by Rhizopogon spp. and the hypogeous relative Truncocolumella citrina

The morphology and anatomy of ectomycorrhizas of Rhizopogon parksii , Rhizopogon vinicolor and Rhizopogon subcaerulescens , and a hypogeous relative, Truncocolumella citrina , synthesized on Douglas fir in glasshouse conditions using spore slurries as inoculum, are described and compared. Mycorrhizas formed with R. parksii and R. vinicolor did not exhibit their characteristic subtuberculate morphology in these tests, but rather had a pinnate form. All species had diagnostic features of ectomycorrhizas: a well-developed Hartig net and a fungal mantle. In addition, several species exhibited crystal inclusions in the outer mantle, usually at the interface between the mantle and soil. Truncocolumella citrina had crystal-like inclusions within the mantle but external to fungal hyphae, a feature rarely described in ectomycorrhizas.     

A neural network based pattern recognition system for somatic embryos of Douglas fir

A pattern recognition system was developed to classify Douglas fir somatic embryos by employing an image analysis system and two neural network based classifiers. The contour of embryo images was segmented, digitalized and converted to numerical values after the discrete and fast Fourier transformation. These values, or Fourier features, along with some other shape factors, were used for embryo classification. The pattern recognition system used a hierarchical decision tree to classify Douglas fir embryos into three normal and one abnormal embryo classes. An accuracy of greater than 80% was achieved for normal embryos. This system provides an objective and efficient method of classifying embryos of Douglas fir. It will be a useful tool for kinetic studies and process optimization of conifer somatic embryogenesis.     

In vitro effect of formaldehyde on Douglas fir pollen

Formaldehyde (HCHO) concentrations of up to 479 mmol m^–3 have been reported in urban fogwater, yet little is known about the phytotoxicity of HCHO at such concentrations. We report that formaldehyde at target concentrations of 300, 600, 900 and 1200mmol m^–3 reduced germination of Douglas fir [ Pseudotsuga menziesii (Mirbel) Franco.] pollen in vitro . Formaldehyde concentrations and pH in media containing pollen decreased over 25 h. These effects were not seen with heat-killed pollen nor with media lacking pollen, which may suggest uptake or detoxification by live pollen. Stress was indicated by ion leakage from pollen, which increased within 20 h in all HCHO treatments. Reduced pollen viability was indicated by TTC staining of pollen, which decreased after HCHO treatment.     

Molecular analysis of bacterial communities associated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycorrhizal fungi

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.     

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

Uptake and translocation of manganese in seedlings of two varieties of Douglas fir (Pseudotsuga menziesii var. viridis and glauca)

Douglas fir (Pseudotsuga menziesii) variety glauca (DFG) but not the variety viridis (DFV) showed symptoms of manganese (Mn) toxicity in some field sites. We hypothesized that these two varieties differed in Mn metabolism. To test this hypothesis, biomass partitioning, Mn concentrations, subcellular localization and 54Mn-transport were investigated. Total Mn uptake was three-times higher in DFG than in DFV. DFV retained > 90% of 54Mn in roots, whereas > 60% was transported to the shoot in DFG. The epidermis was probably the most efficient Mn barrier since DFV contained lower Mn concentrations in cortical cells and vacuoles of roots than DFG. In both varieties, xylem loading was restricted and phloem transport was low. However, sieve cells still contained high Mn concentrations. DFV displayed higher biomass production and higher shoot : root ratios than DFG. Our results clearly show that both varieties of Douglas fir differ significantly in Mn-uptake and allocation patterns rendering DFG more vulnerable to Mn toxicity.     

Fire, fuels and restoration of ponderosa pine–Douglas fir forests in the Rocky Mountains, USA

Aim Forest restoration in ponderosa pine and mixed ponderosa pine–Douglas fir forests in the US Rocky Mountains has been highly influenced by a historical model of frequent, low‐severity surface fires developed for the ponderosa pine forests of the Southwestern USA. A restoration model, based on this low‐severity fire model, focuses on thinning and prescribed burning to restore historical forest structure. However, in the US Rocky Mountains, research on fire history and forest structure, and early historical reports, suggest the low‐severity model may only apply in limited geographical areas. The aim of this article is to elaborate a new variable‐severity fire model and evaluate the applicability of this model, along with the low‐severity model, for the ponderosa pine–Douglas fir forests of the Rocky Mountains. Location Rocky Mountains, USA. Methods The geographical applicability of the two fire models is evaluated using historical records, fire histories and forest age‐structure analyses. Results Historical sources and tree‐ring reconstructions document that, near or before ad 1900, the low‐severity model may apply in dry, low‐elevation settings, but that fires naturally varied in severity in most of these forests. Low‐severity fires were common, but high‐severity fires also burned thousands of hectares. Tree regeneration increased after these high‐severity fires, and often attained densities much greater than those reconstructed for Southwestern ponderosa pine forests. Main conclusions Exclusion of fire has not clearly and uniformly increased fuels or shifted the fire type from low‐ to high‐severity fires. However, logging and livestock grazing have increased tree densities and risk of high‐severity fires in some areas. Restoration is likely to be most effective which seeks to (1) restore variability of fire, (2) reverse changes brought about by livestock grazing and logging, and (3) modify these land uses so that degradation is not repeated.     

Phytoremediation of Cadmium-Contaminated Soils by Young Douglas Fir Trees: Effects of Cadmium Exposure on Cell Wall Composition

Douglas fir trees grown on an artificially Cd-contaminated soil, can tolerate this trace element (up to 68 mg/kg in soil) during several months. Most of the absorbed Cd is retained in roots (25 mg/kg DM), but transfer to aerial part is also effective. Showing the highest content, up to 6 mg/kg DM, among all the aboveground parts, barks seem to be a preferred storage compartment. However, the transfer factor is quite low, about 0.3. Another objective of this study was to compare the cell wall components of trees exposed to increasing Cd amounts in soil. A decrease in lignin and an increase in pectin contents were observed in response to increasing soil cadmium concentration. A concurrent reduction in methyl-esterification of pectin suggests than the structure of this major binding site could therefore be modified as a reaction to cadmium contamination. Future prospects will focus on the modulation of pectin composition in response to Cd exposure.     

Detection and identification of gibberellins in Douglas fir (Pseudotsuga menziesii) shoots

Extracts of Douglas fir ( Pseudotsuga menziesii [Mirb.] Franco) shoots were purified by reversed and normal phase HPLC; gibberellin (GA)-like compounds detected by radioimmunoassay with antibodies against GA₄ and the Tan-ginbozu dwarf rice micro-drop biossay were analyzed by GC-MS. Three major components were identified as GA₄, GA₇, and GA₉ while smaller amounts of GA¹, GA₃ and putative GA⁹-glucosyl ester were also present.     

Differential male reproductive success in Douglas fir

Summary Differential male reproductive success was studied in clones at two seed orchards of Douglas fir, Pseudotsuga menziesii (Mirb.) Franco. The performance of tester pollen parents was compared in controlled pollinations with two-parent pollen mixes. Marker pollen homozygous for a rare IDH allele was the genetic marker in each pollen mix. The resulting seeds were analyzed electrophoretically. At both seed orchards, the proportion of seeds sired by tester pollen significantly varied among the tester pollen parents. Tester pollen parents did not perform the same across all seed parents. The significant interaction effect was evidence of male-female complementarity. These results suggest a genetic basis to differential male reproductive success in Douglas fir.     

Pseudotsuga menziesii chemical races of California and Oregon

Cortical oleoresin from more than 300 Douglas-fir trees sampled in over 30 localities throughout California and southern Oregon was analyzed for its monoterpene hydrocarbon composition. The results confirmed the existence of a distinct chemical race found in its pure unhybridized form throughout the Sierra Nevada as well as near Lompoc in the Purissima Hills of coastal California. Chemical features characterizing this race included near-absence of sabinene and presence of larger amounts of - and β-pinenes in the cortical essential oil. Elsewhere in California and southernmost Oregon, the Sierra Nevada race has extensively intergraded with the coastal Douglas-fir race from western Oregon, Washington, and British Columbia (var. menziesii), a race producing large amounts of sabinene and lesser amounts of - and β-pinenes. On the basis of chemical composition, the Sierra Nevada race was judged to be closer to the inland Douglas-fir races (var. glauca) than to the coastal race. Evolution of the Sierra Nevada race is explained by the emergence of the Great Basin arid regions during the PliocenePleistocene epochs which disjoined Western Douglas-fir populations from their eastern inland counterparts.     

Silver fir and Douglas fir are more tolerant to extreme droughts than Norway spruce in south‐western Germany

Improving our understanding of the potential of forest adaptation is an urgent task in the light of predicted climate change. Long‐term alternatives for susceptible yet economically important tree species such as Norway spruce (Picea abies) are required, if the frequency and intensity of summer droughts will continue to increase. Although Silver fir (Abies alba) and Douglas fir (Pseudotsuga menziesii) have both been described as drought‐tolerant species, our understanding of their growth responses to drought extremes is still limited. Here, we use a dendroecological approach to assess the resistance, resilience, and recovery of these important central Europe to conifer species the exceptional droughts in 1976 and 2003. A total of 270 trees per species were sampled in 18 managed mixed‐species stands along an altitudinal gradient (400–1200 m a.s.l.) at the western slopes of the southern and central Black Forest in southwest Germany. While radial growth in all species responded similarly to the 1976 drought, Norway spruce was least resistant and resilient to the 2003 summer drought. Silver fir showed the overall highest resistance to drought, similarly to Douglas fir, which exhibited the widest growth rings. Silver fir trees from lower elevations were more drought prone than trees at higher elevations. Douglas fir and Norway spruce, however, revealed lower drought resilience at higher altitudes. Although the 1976 and 2003 drought extremes were quite different, Douglas fir maintained consistently the highest radial growth. Although our study did not examine population‐level responses, it clearly indicates that Silver fir and Douglas fir are generally more resistant and resilient to previous drought extremes and are therefore suitable alternatives to Norway spruce; Silver fir more so at higher altitudes. Cultivating these species instead of Norway spruce will contribute to maintaining a high level of productivity across many Central European mountain forests under future climate change.     

Hydraulic control of stomatal conductance in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and alder [Alnus rubra (Bong)] seedlings

Experiments were conducted on 1-year-old Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and 2- to 3-month-old alder [Alnus rubra (Bong)] seedlings growing in drying soils to determine the relative influence of root and leaf water status on stomatal conductance (g_c). The water status of shoots was manipulated independently of that of the roots using a pressure chamber that enclosed the root system. Pressurizing the chamber increases the turgor of cells in the shoot but not in the roots. Seedling shoots were enclosed in a whole-plant cuvette and transpiration and net photosynthesis rates measured continuously. In both species, stomatal closure in response to soil drying was progressively reversed with increasing pressurization. Responses occurred within minutes of pressurization and measurements almost immediately returned to pre-pressurization levels when the pressure was released. Even in wet soils there was a significant increase in g_c with pressurization. In Douglas fir, the stomatal response to pressurization was the same for seedlings grown in dry soils for up to 120 d as for those subjected to drought stress over 40 to 60 d. The stomatal conductance of both Douglas fir and alder seedlings was less sensitive to root chamber pressure at higher vapour pressure deficits (D), and stomatal closure in response to increasing D from 1.04 to 2.06 kPa was only partially reversed by pressurization. Our results are in contrast to those of other studies on herbaceous species, even though we followed the same experimental approach. They suggest that it is not always appropriate to invoke a ‘feedforward’ model of short-term stomatal response to soil drying, whereby chemical messengers from the roots bring about stomatal closure.     

Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata)

The ectomycorrhizal fungal associations of Douglas fir ( Pseudotsuga menziesii D. Don) and bishop pine ( Pinus muricata D. Don) were investigated in a mixed forest stand. We identified fungi directly from field-collected ectomycorrhizal (ECM) root tips using PCR-based methods. Sixteen species of fungi were found, of which twelve associated with both hosts. Rhizopogon parksii Smith was specific to Douglas fir. Three other species colonized only one of the hosts, but were too infrequent to draw conclusions about specificity. Seventy-four percent of the biomass of ECM root tips sampled in the stand were colonized by members of the Thelephoraceae and Russulaceae. All 12 species of fungi that associated with both tree species did so within a 10×40 cm soil volume, suggesting that individual fungal genotypes linked the putatively competing tree hosts.