Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus

Paxillus involutus (basidiomycetes, Boletales) is a common ectomycorrhizal fungus in the Northern Hemisphere. The fungus displays significant variation in phenotypic characters related to morphology, physiology, and ecology. Previous studies have shown that P. involutus contains several intersterility groups and morphological species. In this study, we have used concordance of multiple gene genealogies to identify genetically isolated species of P. involutus. Fragments from five protein coding genes in 50 isolates of P. involutus collected from different hosts and environments in Europe and one location in Canada were analysed using phylogenetic methods. Concordance of the five gene genealogies showed that P. involutus comprises at least four distinct phylogenetic lineages: phylogenetic species I (with nine isolates), II (33 isolates), III (three isolates), and IV (five isolates). The branches separating the four species were long and well supported compared with the species internodes. A low level of shared polymorphisms was observed among the four lineages indicating a long time since the genetic isolation began. Three of the phylospecies corresponded to earlier identified morphological species: I to P. obscurosporus, II to P. involutus s. str., and III to P. validus. The phylogenetic species had an overlapping geographical distribution. Species I and II differed partly in habitat and host preferences.     

Speciation and Phylogenetic Distances within Paxillus s. str. (Basidiomycetes, Boletales)

Abstract: This study aimed to elucidate the phylogenetic relationships within Paxillus 5. str. Collections of the Paxillus involu-tus complex from different habitats have been compared. DNA sequence data were generated from the nuclear encoded 28S gene and the ITS region. Phylogenetic analyses of the two data sets showed three clearly differentiated groups within Paxillus s. str. Collections from gardens and park areas under Betula, Cory-lus, Populus, Quercus and Tub showed to have a very close relationship to the North American species Paxillus vernalis , but are separated from P. involutus collections found in forests as well as from P. filamentosus . Possible taxonomic conclusions are discussed.     

Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots

Roots of clonal birches ( Betula pendula ) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro . Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum , whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.     

Screening for rapidly evolving genes in the ectomycorrhizal fungus Paxillus involutus using cDNA microarrays

We have examined the variations in gene content and sequence divergence that could be associated with symbiotic adaptations in the ectomycorrhizal fungus Paxillus involutus and the closely related species Paxillus filamentosus. Strains with various abilities to form mycorrhizae were analysed by comparative genomic hybridizations using a cDNA microarray containing 1076 putative unique genes of P. involutus. To screen for genes diverging at an enhanced and presumably non-neutral rate, we implemented a simple rate test using information from both the variations in hybridizations signal and data on sequence divergence of the arrayed genes relative to the genome of Coprinus cinereus. C. cinereus is a free-living saprophyte and is the closest evolutionary relative to P. involutus that has been fully sequenced. Approximately 17% of the genes investigated were detected as rapidly diverging within Paxillus. Furthermore, 6% of the genes varied in copy numbers between the analysed strains. Genome rearrangements associated with this variation including duplications and deletions may also play a role in adaptive evolution. The cohort of divergent and duplicated genes showed an over-representation of either orphans, genes whose products are located at membranes, or genes encoding for components of stress/defence reactions. Some of the identified genomic changes may be associated with the variation in host specificity of ectomycorrhizal fungi. The proposed procedure could be generally applicable to screen for rapidly evolving genes in closely related strains or species where at least one has been sequenced or characterized by expressed sequence tag analysis.     

Cation exchange capacity and lead sorption in ectomycorrhizal fungi

Two ectomycorrhizal fungi, Paxillus involutus 533 and Laccaria bicolor S238, differing greatly in their mycelial characteristics, were investigated with regard to their cation exchange capacity and Pb-binding capacity in vitro after growth with either NO₃ ^- or NH₄ ⁺ as N source. The CECs of 800–1200 mol g-1 dry weight for Paxillus involutus 533 and 2000–3000 mol g-1 dry weight for Laccaria bicolor S238, were high compared to plant roots. The fungal mycelium also had a high Pb sorption capacity. It was higher in Laccaria bicolor S238 than in Paxillus involutus 533 and higher after pregrowth in NO₃ ^- compared to NH₄ ⁺. Both the higher CEC and the higher Pb sorption capacity of Laccaria bicolor S238 compared to Paxillus involutus 533 might have been the result of the hydrophilic nature of the of Laccaria bicolor S238 mycelium. It would have absorbed the solutions better than the hydrophobic mycelium of Paxillus involutus 533. X-ray microanalysis of the cell walls revealed that the Pb content of the cell walls was higher in Paxillus involutus 533 than in Laccaria bicolor S238. Nevertheless, electron dense deposits in the cell walls of Laccaria bicolor S238 contained large amounts of Pb, P and S. Thus, while Pb was evenly distributed in the cell walls of Paxillus involutus 533, Pb was accumulated in electron dense deposits in Laccaria bicolor S238. The results are discussed in view of their significance for the mycorrhizal symbiosis.     

Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains.     

Boron uptake by ectomycorrhizas of silver birch

Boron (B) is an essential micronutrient for plants but it is thought not to be essential for fungi. We studied whether the extraradical mycelia of Paxillus involutus in symbiosis with silver birch (Betula pendula) take up B and transport it to the host plant. We grew mycorrhizal plants in flat microcosms with a partitioning wall, below which there was only extraradical mycelium. A boric acid solution enriched in ¹⁰B was applied to these mycelia. Increased ¹⁰B/¹¹B isotope ratios were subsequently measured in birch leaves, stems, and roots plus mycorrhizas in the upper compartment. Boron was therefore taken up by the mycorrhizal mycelia and transported to the host plant in this species combination.     

ALLELOPATHIC POTENTIAL OF RASPBERRY FOLIAR LEACHATES ON GROWTH OF ECTOMYCORRHIZAL FUNGI ASSOCIATED WITH BLACK SPRUCE

In vitro bioassays on seven species of ectomycorrhizal fungi associated with black spruce (Picea mariana (Mill.) BSP) were tested under allelopathic action of raspberry (Rubus idaeus L.). Radial growth inhibitions were observed when fungi were confronted with increasing concentrations (0%–0.1%–0.5%–2.5%) of foliar leachates made from May, July, and September material (1986). For the 2.5% treatment, species Paxillus involutus, Laccaria proximo, Laccaria bicolor, Thelephora terrestris, and Cortinarius pseudonapus grew only 6, 8, 32, 42, and 46% of their control, whereas Hebeloma cylindrosporum and Cenococcum geophilum were stimulated. Tree seedlings from raspberry invaded plantation showed mycorrhizal infection rate over 75%, with fine roots mainly colonized by C. geophilum. In combination with efforts of tree improvement and based on the results of this study, it seems important to select a well-adapted my-corrhizae, when interference by raspberry is involved.     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

Paxillus involutus/Pinus sylvestris Mycorrhizae from Heavily Polluted Forest II. Ultrastructural and Cytochemical Observations

Paxillus involutus/Pinus sylvestris mycorrhizae, collected from experimental plots (situated in a Pino-Quercetum forest) treated with cadmium dust, were previously reported to accumulate metals like Cd, P, S, N, Al and Si in vacuolar bodies. The Gomori-Swift test was applied to the same material to reveal if cysteine-rich proteins might be involved in element detoxification. Vacuolar deposits of high and low phosphate level were both stained. Higher intensity of the Swift reaction was found in the fungal mycelium from mycorrhizae sampled from polluted plots than in mycorrhizae from unpolluted sites. In both cases a higher intensity of the test was revealed in the host cytoplasm close to the Hartig net than in internal parts of roots. Different intensities of the Gomori-Swift reaction were also observed when the Hartig net was compared to the fungal mantle. A PATAg test was additionally processed and revealed an accumulation of glycogen in rosettes and net-like structures when samples from cadmium dust treated plots were observed.     

Testing an Adaptive Hypothesis Through Context-Dependence: Effects of Water Depth on Foraging Behaviour in Three Species of Garter Snakes

Adaptive hypotheses based on interspecific comparisons can be tested by evaluating the context‐dependence of the behaviour of individual organisms. Drummond (Behaviour, 86, 1983, 1) categorized garter snake species (Thamnophis) as terrestrial–aquatic generalists or aquatic specialists based on diet and aquatic foraging behaviour. He hypothesized that the characteristic foraging behaviours of aquatic specialists – including frequent crawling on the underwater substrate and a high rate of underwater predatory strikes – are adaptations for feeding on relatively widely dispersed aquatic prey. Drummond's hypothesis based on interspecific comparisons suggests that individual snakes might change their foraging in the direction of aquatic specialist behaviour with an increase in water depth (which increases prey dispersion). I tested this prediction through laboratory observations of Mexican Pacific lowlands garter snakes (T. validus) feeding on minnows in shallow (2 cm) and deep (3–7 cm) water. Members of this species are appropriate subjects because they are ecologically intermediate between the generalists and aquatic specialists studied by Drummond, and thus might be expected to show more variation in aquatic foraging behaviour than those species. T. validus showed significantly higher frequencies of crawling on the underwater substrate and of underwater strikes in the deep water than in the shallow water; i.e. increased water depth shifted the behaviour of these snakes toward that of aquatic specialists, thus supporting Drummond's hypothesis. Individuals of an aquatic specialist species, the narrow‐headed garter snake (T. rufipunctatus), showed less pronounced changes in behaviour with increased water depth. Western ribbon snakes (T. proximus), which feed primarily at the land–water interface (and are expected to act like terrestrial–aquatic generalists), typically refused to feed in deep water. Interspecific differences in underwater visual acuity may underly the behavioural differences among the three species by determining whether changes in foraging behaviour with water depth are advantageous. Information on phylogenetic relationships suggests that the facultative behaviour of T. validus may represent an intermediate stage in the evolution of aquatic specialization.     

Boron and other elements in sporophores of ectomycorrhizal and saprotrophic fungi

Fungi are usually thought not to have a boron (B) requirement. It is not known if mycorrhizas take up B from low concentrations that are common in forest soils, as fungi might also immobilise B. Here, we studied the B concentrations in sporophores of 49 ectomycorrhizal and 10 saprotrophic fungi to assess whether B is translocated in mycelium or not. Additionally, P and metal concentrations were measured for comparison. Variability both within species and between species was very large, as the lowest measured B concentration was 0.01 mg kg⁻¹ in Amanita muscaria, and the highest was 280 mg kg⁻¹ in Paxillus involutus. There was no clear difference between saprotrophic and mycorrhizal fungi. The majority of species did not accumulate B at more than 0.01–3 mg kg⁻¹, but there were some species that consistently had median concentration values higher than 5–6 mg kg⁻¹ and much higher maximum values, particularly Paxillus involutus, Lactarius necator and several Russula species. Most species increased their B concentration in B fertilised plots, but there were exceptions, particularly Rozites caperatus and Lactarius camphoratus. Boron concentrations did not correlate with those of other elements. In conclusion, B is translocated in the mycelia of most of the studied species. The differences between species may be due to differences in their water use, or carbohydrates used in translocation. It remains to be studied, if B concentrations in mycorrhizas or mycelia in soil are in the same order of magnitude as the larger ones found here, and if this has any effects on the host plants.     

Host species and strain combination determine growth reduction of spruce and birch seedlings colonized by root-associated dark septate endophytes

Interactions of Betula pendula and Picea abies with dark septate endophytes of the Phialocephala fortinii-Acephala applanata species complex (PAC) were studied. PAC are ubiquitous fungal root symbionts of many woody plant species but their ecological role is largely unknown. Sterile birch and spruce seedlings in monoculture and mixed culture were exposed to four PAC strains, added either singularly or paired in all possible combinations at 18°C and 23°C. Plant and fungal biomass was determined after 4 months. The most significant factors were strain and host combination. One of the strains significantly reduced biomass gain of spruce but not of birch. Plant biomass was negatively correlated with total endophytic fungal biomass in half of the strain - plant combinations. Endophytic PAC biomass was four times higher in spruce (≈ 40 mg g(-1) drw) than in birch (≈ 10 mg g(-1) drw). Competition between strains was strain-dependent with some strains significantly reducing colonization density of other strains, and, thus, attenuating adverse effects of 'pathogenic' strains on plant growth in some strain - plant combinations. Biomass gain of spruce but not of birch was significantly reduced at higher temperature. In conclusion, host, fungal genotype, colonization density and presence of a competing PAC strain were the main determining factors for plant growth.     

Ocean acidification has little effect on developmental thermal windows of echinoderms from Antarctica to the tropics

As the ocean warms, thermal tolerance of developmental stages may be a key driver of changes in the geographical distributions and abundance of marine invertebrates. Additional stressors such as ocean acidification may influence developmental thermal windows and are therefore important considerations for predicting distributions of species under climate change scenarios. The effects of reduced seawater pH on the thermal windows of fertilization, embryology and larval morphology were examined using five echinoderm species: two polar (Sterechinus neumayeri and Odontaster validus), two temperate (Fellaster zelandiae and Patiriella regularis) and one tropical (Arachnoides placenta). Responses were examined across 12–13 temperatures ranging from ?1.1 °C to 5.7 °C (S. neumayeri), ?0.5 °C to 10.7 °C (O. validus), 5.8 °C to 27 °C (F. zelandiae), 6.0 °C to 27.1 °C (P. regularis) and 13.9 °C to 34.8 °C (A. placenta) under present‐day and near‐future (2100+) ocean acidification conditions (‐0.3 pH units) and for three important early developmental stages 1) fertilization, 2) embryo (prehatching) and 3) larval development. Thermal windows for fertilization were broad and were not influenced by a pH decrease. Embryological development was less thermotolerant. For O. validus, P. regularis and A. placenta, low pH reduced normal development, albeit with no effect on thermal windows. Larval development in all five species was affected by both temperature and pH; however, thermal tolerance was not reduced by pH. Results of this study suggest that in terms of fertilization and development, temperature will remain as the most important factor influencing species' latitudinal distributions as the ocean continues to warm and decrease in pH, and that there is little evidence of a synergistic effect of temperature and ocean acidification on the thermal control of species ranges.     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Effects of cadmium on growth and glucose utilisation of ectomycorrhizal fungi in vitro

Effects of Cd on growth and glucose utilisation of Paxillus involutus, Rhizopogon subcaerulescens and Suillus bovinus were investigated in vitro in liquid culture. S. bovinus was the species most sensitive to Cd in terms of dry matter production and P. involutus was less sensitive than R. subcaerulescens. Greater production of hyphae of P. involutus than the other fungi appeared to confer some degree of Cd resistance, possibly by binding Cd onto cell walls. Growth of the three fungi was increased by glucose addition. While Cd significantly reduced dry matter production of the fungi, there were no significant differences in glucose consumption caused by Cd treatment. This suggests that the use of glucose might have been diverted to detoxification and/or repair mechanisms. Further studies on respiration rates and energy metabolites of these fungi under Cd exposure are needed in order to clarify the results of the present study.     

Micronutrient levels in some temperate European tree species: a comparative field study

Micronutrient concentrations in foliage and stemwood (including bark) of six European tree species (Betula pendula Roth., Quercus robur L., Fraxinus excelsior L., Fagus sylvatica L., Tilia cordata Mill. and Picea abies (L.) Karst.) planted on the same type of soil at six sites in three different countries were studied. Micronutrient concentrations in foliage were considerably higher than in stemwood for all elements and species studied, except for Fe in spruce. Interspecies comparisons revealed significant differences in concentrations both in foliar and stemwood biomass, as well as in stemwood:foliage nutrient ratios. Lime foliage showed a considerably higher concentration of B than all other species, while the stemwood concentration of this element was highest in ash. Mn concentration in both foliar and stemwood biomass of ash was extremely low compared with concentrations in other species. Birch stemwood showed nearly double the level of Zn in other species at all sites. Zn concentrations in the birch foliage were also higher than in other species, with the exception of the Lithuanian sites, which showed lower EDTA-extractable Zn concentrations in the soils. The concentration of Cu was lowest in spruce foliage, while Cu concentrations in stemwood were similar in all species. It was concluded that species-related differences in microelement nutrition must be taken into account when evaluating the nutrient status of common European forest tree species, and when using them as bio-indicators of the effects of environmental pollution.     

Habitat associations of <Emphasis Type="Italic">Agathidium pulchellum</Emphasis>, an endangered old-growth forest beetle species living on slime moulds

The beetle genus Agathidium is the largest insect group documented that principally feeds on slime moulds. Agathidium pulchellum, one of the rarest Agathidium species in Europe, is listed in the EU’s Habitats Directive. We studied the habitat associations of A. pulchellum in 44 sites located in old-growth and managed forests in eastern Finland. Agathidium pulchellum occurred exclusively on the slime-mould species Trichia decipiens. The host was associated with mid-decayed aspen, spruce and birch logs, and its incidence grew with both increasing log diameter and stand-level log density of spruce and aspen. We also observed that even if its host was present, the beetle was absent from sites with less than 80 aspen and spruce logs per hectare. All sites with A. pulchellum were natural forests of high conservation value. Our results show that it is possible to systematically survey the occurrence of A. pulchellum.     

Classifying seedlots of Picea sitchensis and P. glauca in zones of introgression using restriction analysis of chloroplast DNA

Summary Chloroplast DNA (cpDNA) restriction analysis was used to classify five reforestation seedlots as to species. The material included two Sitka spruce (Picea sitchensis (Bong.) Carr.), one white spruce (P. glauca (Moench) Voss) from interior British Columbia, and two putative hybrid seedlots from the coast-interior introgression zone in British Columbia. The cpDNA patterns generated by Bam-HI and Bc1-I from individual trees of Sitka spruce, white spruce, western white spruce (P. glauca var. albertiana (S. Brown)), and Engelmann spruce (P. engelmanni (Parry)) were species-specific. They were used as reference patterns for comparisons. In addition, two controlled crosses between white and Sitka spruce were analyzed to demonstrate the paternal inheritance of cpDNA in spruces. The cpDNA restriction patterns for the five seedlots were obtained from composite samples of seedlings from each lot and compared to the typical cpDNA patterns of each species. Restriction patterns for the two Sitka spruce seedlots agreed with those from the Sitka spruce tree, while patterns for the white spruce seedlots from British Columbia agreed with those from the white spruce tree, lacking evidence of any Engelmann spruce component in the sample. On the other hand, one putative hybrid seedlot showed cpDNA patterns similar to white spruce while the other showed fragments unique to both Sitka and white spruce, indicating that this was a hybrid seedlot. The analysis of cpDNA restriction polymorphism has proven to be an effective tool for classifying seedlots in regions of introgression. To our knowledge, these results provide the first demonstration of the use of cpDNA analysis for solving practical forestry problems.