Belowground Ectomycorrhizal Community Research Articles

Temporal variation in below-ground ectomycorrhizal communities in dry dipterocarp forests, Thailand

Temporal variation in below-ground ectomycorrhizal communities in dry dipterocarp forests, Thailand

Community structure and dynamics of ectomycorrhizal fungi in a dipterocarp forest fragment and plantation in Thailand

Background: Ectomycorrhizal (EcM) associations have been reported with trees of the plant family Dipterocarpaceae. We hypothesised that a dipterocarp forest fragment, with more host species, would yield a higher diversity of associated EcM fungal species than that of a Dipterocarpus alatus monoculture plantation.Aims: To examine the above- and below-ground EcM community structure of a dry dipterocarp forest fragment and a D. alatus plantation in Thailand.Methods: Sporocarps were collected and identified, and EcM root tips were collected in the dry and wet seasons and identified by terminal restriction fragment length polymorphism and internal transcribed spacer sequencing.Results: Higher EcM fungal diversity was observed in the dry dipterocarp forest fragment than in the D. alatus plantation, with a larger number of species found in the wet seasons than the dry seasons. Sporocarp species richness was greater than root-tip EcM richness. Different dominant EcM taxa were observed in the above- and below-ground communities.Conclusions: Our results suggest that host tree diversity may explain the greater EcM fungal diversity in the dry dipterocarp forest fragment than the D. alatus monoculture plantation. Seasonal dynamics also influences the composition of the EcM communities, with different dominant taxa and relative abundance in the dry and wet seasons. Further research would be of great help in providing useful information for management of dipterocarp forests as well as restoration of disturbed landscapes.

Below-Ground Ectomycorrhizal Community Structure in the Postfire Successional Pinus koraiensis Forests in the Central Sikhote-Alin (the Russian Far East)

Below-Ground Ectomycorrhizal Community Structure in the Postfire Successional Pinus koraiensis Forests in the Central Sikhote-Alin (the Russian Far East)

Below-ground ectomycorrhizal communities: the effect of small scale spatial and short term temporal variation

Ectomycorrhizal (ECM) associations are an important form of symbiosis for the majority of tree species. In forest ecosystems where nutrients are often limited, ECM associations are vital for seedling establishment and tree survival. ECM communities are often very species rich and frequently follow the log normal distribution—with a few species being abundant and many species being rare. Much of the current knowledge of ECM communities has been revealed through a combination of above-ground sporocarp and below-ground root tip analyses. However, above-ground surveys of ECM communities reveal largely different findings regarding ECM diversity and community structure to below-ground surveys. Therefore below-ground surveys are vital to our understanding of ECM biology, ecology and community dynamics. In this article I review the recent findings regarding how ECM communities vary both spatially and temporally in forests. Spatial variation occurs at the centimetre scale both horizontally and vertically in forests, and can be explained by the separation of the ECM community into distinct niches. Temporal variation occurs over relatively short time scales, with ECM communities showing large changes even on a monthly basis. I then apply the niche concept to ECM fungi, and review a recent theory, ECM functional morphology, and examine how this may be used to explain a significant amount of spatial and temporal variation in forest ECM communities. The functional morphology theory is particularly useful in explaining patterns of ECM community variation across the distinct successional stages of the forest cycle. However, the effect of other abiotic and biological variables on ECM communities should not be ignored. Finally, as ECM communities are non-randomly distributed and vary widely in species richness over time, I lay out a sampling strategy to provide representative samples of the actual ECM community in the study area. Using (i) an extensive sampling methodology, (ii) separation of samples at distances greater than levels of spatial autocorrelation, and (iii) samples collected throughout the year, over a number of years, an ample picture of the ECM community in temperate forests can be collected.

Below-ground ectomycorrhizal community in natural Tuber melanosporum truffle grounds and dynamics after canopy opening

The ectomycorrhizal fungus Tuber melanosporum fruits in association with Quercus in natural forests of Spain. Some of these stands are managed to keep an open canopy and meet the habitat requirements of the fungus. However, there are few quantitative studies analysing in these forests the relationship between soil environment and T. melanosporum. Eight forest stands which produce T. melanosporum have been monitored for 6 years in order to characterise the below-ground ectomycorrhizal community and to assess its temporal dynamics after experimental canopy opening. The brûlé, the ground where T. melanosporum fruits, shows a distinct ectomycorrhizal community, characterised by lower density of active ectomycorrhizal tips, lower morphotype richness per soil volume, higher abundance of T. melanosporum and lower abundance of Cenococcum geophilum than soil closest to the trunk of the host Quercus ilex. Opening the canopy has not stimulated an increase in T. melanosporum, suggesting that a shift in the soil environment alone will not trigger the formation of new truffières in the short term. The dry climate of these truffières may be a factor as T. melanosporum abundance appears to be sensitive to annual weather conditions.

Species and functional diversity of ectomycorrhizal fungal communities on Scots pine (Pinus sylvestris L.) trees on three different sites

In the present study, we describe the aboveground (epigeous sporocarps) and belowground (ectomycorrhizal root tips and their exploration types) composition of ectomycorrhizal (ECM) fungal species on three 20-year-old Scots pine sites. The aim of this study was to relate the composition of ECM species to history of the site, stand density and soil chemistry (pH, toxic metal content, etc.) and to show how variable site condition contribute to above- and belowground ECM community changes. ECM fungi were identified by combination of morphotyping and direct sequencing of the PCR-amplified internal-transcribed spacer of n-rDNA. By identifying sporocarps and mycorrhizas, we detected a total of 54 taxa of ECM fungi: 28, 30, and 23 species at the Kórnik (control), Luboń (near the chemical plant) and Głogów (near the Copper Smelter, heavy metal influenced) sites, respectively. The sporocarp survey weakly reflected the analysis of ECM tips in terms of species composition, and largely supplemented the belowground view. Wilcoxina rehmii was the belowground-dominating species at all three sites and was found at the highest abundance at the site established after the clear cut of poplars and with the highest tree density (Kórnik). At the Luboń site, subdominant mycorrhizas were formed by Phialophora finlandia. On the site near the Copper Smelter in Głogów, a significant shift in species composition was observed compared with the two other sites and a very large proportion of medium-fringe exploration type belonging to Atheliaceae was found. Despite the partial overlap in the nonmetric multi-dimensional scaling ordination, analysis of similarity revealed significant differences between tested sites. Because of the number of covarying site properties which may influence ECM communities, it is difficult, if not unfeasible, to separate the precise reasons of the distribution of ECM fungi. Continued research on the biodiversity of ECM fungi in field sites along an environmental gradient would greatly improve our understanding of the effects of ecological conditions on the functional diversity of mycorrhizal fungi.

The AD-type ectomycorrhizas, one of the most common morphotypes present in truffle fields, result from fungi belonging to the Trichophaea woolhopeia species complex

Belowground ectomycorrhizal communities are often species rich. Characterization of the ectomycorrhizas (ECMs) underneath native truffle areas and/or cultivation sites is particularly relevant to identifying fungal species that might interfere with or promote truffle propagation and fruiting. Fungal identification at the genus/species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. In a survey of the mycorrhizal biodiversity of Tuber melanosporum orchards and inoculated host plants in nurseries, we repeatedly sampled ECMs with morphological features resembling those of the ECMs widely known as the AD type. Despite the fact that the AD type is regarded as one of the most competitive fungal species towards Tuber spp., its taxonomical rank has yet to be resolved. By analyzing the 28S and internal transcribed spacer (ITS) rDNA regions, here, we show that AD-type ECMs result from host plant colonization by the pyronemataceous species Trichophaea woolhopeia. Further to this, the 28S and ITS phylogenetic trees built from the AD-type ECMs analyzed sustain the hypothesis that T. woolhopeia is a species complex.

Belowground ectomycorrhizal fungal communities respond to liming in three southern Swedish coniferous forest stands

In this study we report on changes in the belowground ectomycorrhizal fungal communities in southern Swedish coniferous forests as a consequence of liming with 3–7 ton limestone per hectare 16 years prior to the study. A total of 107 ectomycorrhizal fungi were identified from 969 independently sampled root tips by sequencing the internal transcribed spacer region of the ribosomal DNA. Forty, 59 and 51 species were identified in three pine and spruce forests. Within all sites only about 25% of the species overlapped between the limed and the reference areas. However, the most abundant species were often found in both limed and reference plots and 60–70% of the root tips at each site were colonised by species occurring in both limed and reference plots. Across all three sites, fungal species belonging to the genus Tylospora and the order Pezizales became significantly more frequent in limed plots, while species belonging to the genera Russula and Lactarius decreased in frequency. At individual sites, several species within the genera Amphinema, Piloderma, Inocybe and Hygrophorus were also significantly influenced by liming. We estimate that, at the root tip level, about 30% of the roots were colonised by a different fungal species as a consequence of liming. The observed shift in ectomycorrhizal fungal communities was related to an increased pH and base saturation with liming. We conclude that even a moderate level of liming have long lasting effects on belowground ectomycorrhizal community composition. We discuss the observed community changes in relation to ectomycorrhizal functionality and biodiversity in limed forests.

Belowground ectomycorrhizal communities in three Norway spruce stands with different degrees of decline in the Czech Republic

We investigated the ectomycorrhizal communities on the roots of adult trees and seedlings associated with three Norway spruce stands in the Czech Republic using morphological and molecular tools. The stands had different degrees of forest decline due to air pollution. The aims of the study were to obtain information about the belowground ectomycorrhizal community in a heavily damaged spruce forest and to assess whether missing ectomycorrhizal fungal partners could be one of the reasons for the observed lack of regeneration. The ectomycorrhizal species richness on the roots of adult trees was significantly lower in the heavily damaged site Mumlavska hora than in the other two, but less drastically so than that found in a fruitbody survey. The roots of adult trees and seedlings were fully mycorrhizal at this site although they were less species-rich. The most abundant ectomycorrhizal species on the root system of adult trees in all three forest stands was Tylospora fibrillosa, a member of the athelioid clade. It made up over 60% of root tips in Mumlavska hora and its proportion was at least twice that in the other two sites. This species was also an efficient colonizer of roots from seedlings, in particular, in the most damaged site. The different soil properties in this site may have caused the observed differences in the ectomycorrhizal species richness and composition. For example, cation exchange capacity and soil base saturation were lower and the soil more often saturated. However, the number of living trees and their defoliation status may well directly impact the ectomycorrhizal species composition by presumably affecting the amount of carbon delivered to the symbiotic fungal partners. Athelioids and thelephoroids are an important component of the belowground ectomycorrhizal community in most temperate and boreal forests, but the role they play might even be more crucial in stressed forest ecosystems. Based on our results, we suggest that factors other than missing ectomycorrhizal inoculum constrain natural regeneration in the heavily damaged site Mumlavska hora.

Types of Ectomycorrhiza of Mature Beech and Spruce at Ozone-Fumigated and Control Forest Plots

In the Kranzberg forest near Freising (Germany) a novel "Free-Air Canopy O3 Exposure" system has been employed for analysing O3-induced responses from sub-cellular to ecosystem levels that are relevant for carbon balance and CO2 demand of 60-year-old beech trees. The below-ground ectomycorrhizal community was studied in two-fold ambient O3 concentrations (five cores per sampling) and in a control plot with an ambient O3 concentration (four cores per sampling). Five samplings were taken throughout two vegetation seasons (2003 and 2004). Types of ectomycorrhiza were determined by their morphological, anatomical and molecular characteristics and quantified by counting. The total number of mycorrhizal fine roots was higher at the fumigated plot as compared with the control site. The numbers of ectomycorrhizal types at the fumigated and control plots were 28 and 26, respectively. Cenococcum geophilum was present in all soil cores at all sampling times with a significant increase in abundance under ozone-fumigated trees. Other mycorrhizal types present at higher abundance at the fumigated than at the control plot were identified as Russula densiflora, R. fellea, R. illota, Tuber puberulum, Lactarius sp. 2 and Russula sp. 2. Some mycorrhizal types were present exclusively at the fumigated plot (Fagirhiza fusca, F. setifera, Lactarius acris, Piceirhiza nigra and Russula sp. 1). A possible ecological role for the abundant types of ectomycorrhiza and their putative application in bio-indication is discussed.

Morphological and molecular analyses of ectomycorrhizal diversity in a man-made T. melanosporum plantation: description of novel truffle-like morphotypes

Below-ground ectomycorrhizal communities are often species-rich, and monitoring their dynamics is important to understand the conditions that promote truffle fructification. Characterization of the different ectomycorrhizas (ECM) at the species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. Following this strategy, we have characterized ectomycorrhizal biodiversity in an artificial Tuber melanosporum plantation. Although the plantation was unproductive, T. melanosporum mycorrhizas were the most present and two Tuber-like mycorrhizal morphotypes, named ECMm1 and ECMm3, showing distinctive features were found. Internal transcribed spacer (ITS) phylogenetic analysis demonstrated that ECMm3 is related to the Tuber rufum/Tuber ferrugineum species complex, whereas ECMm1 shows the highest ITS similarity with Tuber scruposum and fungi-colonizing Epipactis roots. The results presented here provide more insights into genetic variability, mycorrhizal morphology, and below-ground distribution of fungi associated with artificial truffle plantations.

The mycorrhizal community in a forest chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northern England

Demography and fungal diversity of the belowground ectomycorrhizal community in a chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northumberland, Northern England, were analysed; mycorrhizal root samples were taken from 6-, 12-, 30- and 40-year-old stands, and fungal fruiting bodies were collected in autumn to complement the survey. Naturally germinated seedlings less than 1 year of age (taken from the 30-year-old stand) were also examined. A total of 118,000 mycorrhizal root tips were extracted from 40 soil cores (ten per age class) and from the complete root systems of 25 seedlings and separated into active and senescent root tips according to their morphology and anatomy. Active tips were distinguished according to their mycobionts which were characterised and identified microscopically. Although almost 100% of all fine roots were mycorrhizal, EM fungal diversity throughout the chronosequence was low, consisting of a total of 16 species of which three were only found as fruiting bodies. Of the six mycobionts found most regularly below ground, Tylospora fibrillosa was the most common, colonising about 70% of all root tips and more than 90% of those of seedlings and young trees. Root density and mycorrhizal diversity increased, but percentage of vital root tips decreased with increasing tree age, levelling off in the 30- and 40-year-old stand. Among the five subdominant fungal species, Dermocybe crocea was found to have its peak of distribution in the 12-year-old stand and Russula emetica, Lactarius rufus, Hymenoscyphus ericae agg. and the unidentified Piceirhiza sulfo-incrustata in the 30- and 40-year-old stands. The possible correlations between the mycorrhizal community structure and biotic and abiotic factors are discussed.

Ectomycorrhizas associated with a relict population of Dryas octopetala in the Burren, western Ireland II. Composition, structure and temporal variation in the ectomycorrhizal community

The composition, structure and temporal variation of ectomycorrhizal (EM) communities associated with mountain avens (Dryas octopetala) in grass heaths of the Burren, western Ireland were assessed by using soil core sampling in two permanent plots and 30 other sites (196 cores in total). Of the 34 different EM types observed, 11 were common and constituted over 80% of the EM biomass. Four EM types, Craterellus lutescens, Tomentella sp., Dryadirhiza fulgens and Cenococcum geophilum were the most abundant as measured by EM length and frequency of occurrence in cores. The species profile and relative abundances were very similar in cores from the permanent plots and different sites in the Burren, indicating that they were all representative of the same EM community. The below-ground EM community in both plots was compared with production of basidiomes, and the latter was found to be an unreliable indicator of EM community structure. Temporal variation in the EM community was assessed by repeated core sampling of the two permanent plots over a 14-month period (between March 1998 and May 1999). No statistically significant shifts in EM abundance were found between sampling dates, probably as a consequence of the large variation in EM abundance between core samples over the sampling period. No significant relationship was found between rainfall, soil moisture or soil temperature and fluctuations in EM abundance. Patterns of total EM abundance and fluctuations in EM diversity were strongly correlated between the two permanent plots over the sampling period. Temporal fluctuations in the dominant EM type, Craterellus lutescens, were similar in both plots with respect to mycorrhizal length, biomass and relative abundance, and the patterns between both plots were positively correlated. EM diversity was negatively correlated with biomass of ectomycorrhizas of Craterellus lutescens in both plots, but it was significant only in plot 1.

Belowground ectomycorrhizal community structure of mature lodgepole pine and mixed conifer stands in Yellowstone National Park

Forest development patterns following wildfire are known to influence the physical and chemical attributes of soils at different points in time, and are further thought to influence ectomycorrhizal (ECM) community structure. We used molecular methods to compare belowground ECM species richness, composition, and abundance between adjacent stands of homogenous lodgepole pine (established after a fire around 1867) and old growth mixed conifer (around 300 years old) in Yellowstone National Park (YNP). In each stand type, we collected soil cores to both identify mycorrhizae and assess soil chemistry. Although no statistical difference was observed in the mean number of ECM root tips per core between stand types, the total number of species identified (81 versus 35) and the mean number of species per core (8.7 ± 0.5 versus 2.5 ± 0.3) were significantly higher in lodgepole pine. Species compositions were widely disparate between stands where only four of 112 species were shared. Soil analysis revealed that mixed conifer was significantly lower in mean pH, but higher in mean organic matter, potassium, phosphorus, and ammonium when compared to lodgepole pine. Although analysis of covariance did not statistically demonstrate that soil chemistry is driving ECM community structure with certainty, our data are, nonetheless, consistent with this hypothesis. Our data further suggest that fungal richness declines and composition shifts some time after Engelmann spruce and subalpine fir colonize mature lodgepole pine stands, and that time since last wildfire influences soil chemistry in this system. Moreover, because of data limitations, future field experiments will be necessary to determine if soil chemistry, as well as other biotic and abiotic factors not examined in this study, is a primary influence on ECM community structure at our study site in YNP.

Short-term effects of seasonal prescribed burning on the ectomycorrhizal fungal community and fine root biomass in ponderosa pine stands in the Blue Mountains of Oregon

The effects of seasonal prescribed fire on the belowground ectomycorrhizal community and live fine root biomass were investigated before, 1 year after, and 2 years after prescribed underburning. Ectomycorrhizas were sampled from four replications of three treatments (fall underburning, spring underburning, and a nonburned control) in a randomized complete block design. Samples were separated in two subsamples representing the upper 5 cm and lower 5 cm of a soil core. Molecular tools were used to distinguish 140 restriction fragment length polymorphism (RFLP) species of fungi directly from the ectomycorrhizas. Prior to underburning, the number of RFLP species and amount of live root biomass were similar among treatment units and between upper and lower core samples. Fall underburning largely removed live root biomass to a depth of 10 cm and significantly reduced ectomycorrhizal species richness compared with spring underburning and the nonburned control for at least 2 years. RFLP species richness and live root biomass following spring underburning were generally similar to the nonburned treatment. The successful reintroduction of fire to the ecosystem to retain high species diversity of ectomycorrhizal fungi and achieve the desired future condition of large-tree ponderosa pine retention with low fuel loads may require more than underburning in a single season.

Elevated atmospheric CO2 alters root symbiont community structure in forest trees.

• Changes in below-ground ectomycorrhizal (ECM) community structure in response to elevated CO2 and balanced nutrient addition were investigated in a 37-yr-old Picea abies forest. • Trees in whole-tree chambers were exposed to factorial combinations of ambient/elevated CO2 (700ppm) and fertilization (+/-). ECM fungal community structure was determined in 1997 and 2000 using a combination of morphotyping and molecular analyses. Samples were taken both from chambers and from reference trees receiving the same fertilization treatments but without chambers. • Significant effects on ECM community structure were found in response to elevated CO2 . Neither elevated CO2 nor fertilization altered species richness; however, there was considerable variation among samples, which may have masked treatment effects on individual species. After 3yr, the effects of elevated CO2 on community composition were of the same magnitude as those seen after 15yr of fertilization treatment. • Our results show that increasing atmospheric CO2 concentrations affect the community structure of root symbionts colonizing forest trees. The potential effects of altered ECM community structure on allocation and turnover of carbon and nutrients within forest ecosystems are discussed.

Effects of continuous optimal fertilization on belowground ectomycorrhizal community structure in a Norway spruce forest.

Studies of effects of fertilizer treatment on ectomycorrhizal fungal community structure have predominantly been based on large, single additions of nitrogen. Studies involving chronic additions of nutrients in combination with irrigation are much less common. We used morphotyping to study effects of balanced additions of a nutrient solution on ectomycorrhizal fungal community structure in a 36-year-old stand of Picea abies (L.) Karst. Despite high variability among individual samples, principal components analysis revealed a clear shift in community structure in response to fertilization. Irrigated plots receiving only water did not differ significantly from untreated control plots. Mycorrhizal root tips colonized by Cenococcum geophilum Fr. were significantly more common in fertilized plots than in control plots. Possible responses by other ectomycorrhizal species were masked by high variability. Over sixty morphotypes were distinguished, but there was no measurable effect of either fertilizer or irrigation treatment on morphotype richness or total number of root tips.

Below-ground Ectomycorrhizal Community Structure in Two Picea abies Forests in Southern Sweden

Ectomycorrhizal community structure is likely to be influenced by factors such as anthropogenic stress, site history and degree of isolation. Information about the mycorrhizal community at the level of individual mycorrhizal roots is needed, since these are the organs for nutrient exchange. To identify mycorrhizal species we used morphotyping based on colour and external anatomy of the mycorrhizal roots. The morphotypes were further divided using restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA extracted and amplified from the mycobionts colonizing single mycorrhizal roots. The communities studied were in two similar 60 - year - old Picea abies (L.) Karst. forests located in southern Sweden approximately 50 km apart at sites with different N deposition. The level of mycorrhizal colonization was almost 100% at both sites, but the total number of mycorrhizas was 30 - 42% higher at the northern site. Six morphotypes were distinguished at the northern site with the lower N deposition (Vedby), and four at the southern site (Skrylle). Some morphotypes consisted of several ITS types and identical ITS types were also found in roots assigned to different morphotypes. The RFLP data revealed a total of 16 ITS types. Eleven of these were identified at least to genus by comparison with local or regional reference material. Five ectomycorrhizal taxa, Cenococcum geophilum, Thelephora terrestris, Tylosporafibrillosa, Tylopilus felleus and Ve-95-3 were common to the two sites but the total number of taxa recorded at Vedby was twice as high as at Skrylle. Possible reasons for the differences between sites are discussed.

Species diversity and distribution of biomass above and below ground among ectomycorrhizal fungi in an old-growth Norway spruce forest in south Sweden

The structure of an ectomycorrhizal community was assessed on a 100-m2 plot in a 100-year-old, oligotrophic Norway spruce, Picea abies (L.) Karst., forest in southern Sweden. During the 6-year study (1986–1992) sporocarps were identified and their biomass determined. Late in the fall of 1993, we identified mycorrhizas and estimated their abundance. Forty-eight epigeous, ectomycorrhizal taxa were identified based on the examination of sporocarps. Hygrophorus olivaceoalbus (Fr.:Fr.) Fr. and six species of Cortinarius, i.e., C. acutus (Pers.:Fr.) Fr., C. brunneus (Pers.:Fr.) Fr., C. evernius (Fr.:Fr.) Fr., C. obtusus (Fr.) Fr., C. paleaceus Fr., and C. strobilaceus Moser, were found every year. For the period as a whole, they accounted for 32% of the annual sporocarp biomass. Twenty-one species were observed during 1 year only. Cenococcum geophilum Fr. and Piloderma croceum Erikss. & Hjortst. accounted for 18 and 19%, respectively, of the mycorrhizal abundance of the mycorrhizal root tips examined. Using polymerase chain reaction (PCR) based molecular methods, we were able to distinguish 25 taxa forming mycorrhiza from soil cores covering a total of 22.5 cm2 of the forest floor. Twelve of these taxa were identified using a sporocarp or mycelial culture based reference data base containing 25 of the sporocarp-producing species. These 12 species accounted for an average of 74% of the sporocarp biomass. In contrast, their share of the estimated mycorrhizal abundance and biomass was about 30%. At least half of the abundance of the belowground ectomycorrhizal community was accounted for by species that did not produce conspicuous epigeous sporocarps. Ascomycetes accounted for about 20% of the mycorrhizal abundance. Calculations showed that on a per hectare basis there was 8.8 kg of fungal biomass in the form of sporocarps (average annual cumulative production), an estimated 250–400 kg as mycorrhiza (standing crop) and 440 kg in the form of sclerotia of Cenococcum geophilum (standing crop). Key words: ectomycorrhizal community structure, ITS–RFLP, Picea abies.