Susanne Erland

Diversity of Ecto-mycorrhizal Fungal Communities in Relation to the Abiotic Environment

In boreal forest ecosystems, the richness and complexity of ecto-mycorrhizal (EM) fungal communities are in striking contrast to the often species-poor stands of host trees. The factors that influence community development and maintain this high EM fungal diversity are, however, poorly understood. There are very few studies that have examined determinants of EM fungal diversity under natural undisturbed systems, with most studies examining diversity in relation to changes in abiotic factors due to pollution and/or forest management practices. In this chapter, we attempt to compile what little data are available on natural factors and suggest some areas for future studies. The great majority of the chapter is however, concerned with anthropogenic influences upon EM fungal diversity.

Ectomycorrhizal community structure in a limed spruce forest

The aim of this study was to compare the ectomycorrhizal community structure in spruce stands treated with different levels of dolomite lime. ITS-typing of randomly sampled mycorrhizas, without prior morphotyping, was used. Sixteen different ITS-RFLP patterns were found. By comparison with the available reference material, nine of these could be identified at least to genus level. Variation within treatments was large and statistically there were no significant differences between treatments with respect to specific taxa. A similarity assessment did, however, show less similarity between control and high dolomite (HD) than between low dolomite (LD) and either control or HD, suggesting a shift in the community structure as a result of the treatment. The fruitbody production at Hasslov had been recorded in a different study during 1985–92 and 28 ectomycorrhizal species had been found in the treatments examined in the present study. Except for three species, all were present in the reference material used for identification of the ITS-types. Only three species, Russula ochroleuca, Lactarius necator and Boletus chrysenteron were found as both mycorrhizas and fruitbodies. There were five taxa which occurred on over 5% of the screened roots. These were Thelephora terrestris , 21.5%; Tylopilus felleus , 13%; Tylospora fibrillosa , 13%; and two unidentified taxa, 10% and 6%. Together these five taxa colonized over 60% of the mycorrhizal roots investigated, yet none of them was found among the fruitbodies recorded in the above-ground study. Despite the differences in taxa found below and above ground, similarity tests between treatments using the fruitbody data also revealed a shift in community structure.

Insect-like olfactory adaptations in the terrestrial giant robber crab

The robber crab (Birgus latro), also known as the coconut crab, is the worlds largest land-living arthropod, with a weight reaching 4 kg and a length of over half a meter. Apart from the marine larval stage, this crab is fully terrestrial, and will actually drown if submerged in water. A transition from sea to land raises dramatically new demands on the sensory equipment of an animal. In olfaction, the stimulus changes from hydrophilic molecules in aqueous solution to mainly hydrophobic in the gaseous phase. The olfactory system of land crabs thus represents an excellent opportunity for investigating the effects of the transition from sea to land. Have land crabs come to the same solutions as other terrestrial animals, or is their olfactory sense characterized by unique innovations? Here, we show that the robber crab has evolved an olfactory sense with a high degree of resemblance to the insect system. The similarities extend to physiological, behavioral, and morphological characters. The insect nose of the robber crab is a striking example of convergent evolution and nicely illustrates how similar selection pressures result in similar adaptation.

Abundance of Tylospora fibrillosa ectomycorrhizas in a South Swedish spruce forest measured by RFLP analysis of the PCR-amplified rDNA ITS region

Polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of the ribosomal DNA, subsequent cutting with the endonuclease Hinf I and restriction fragment length polymorphism (RFLP) analysis have previously proved to be a good method for distinguishing the ectomycorrhizal Tylospora fibrillosa from a large number of other basidiomycetes. In the present study this method was used to screen single mycorrhizas sampled in a Norway spruce forest in South Sweden. In this forest 98% of randomly collected mycorrhizas had the macroscopical features of T. fibrillosa mycorrhizas. Fungal ITS was successfully amplified from 93% of the sampled mycorrhizas. Five major, distinct fungal RFLP patterns were found, clearly demonstrating the benefit of the molecular method. The T. fibrillosa RFLP pattern was present in 21%, type 1 in 27%, type 2 in 20%, type 3 in 11% and type 4 in 3% of the mycorrhizas. Fungal ITS of more than one origin was found in 22% of the amplifications. It can be concluded that T. fibrillosa is one of the main mycorrhizal fungi in the studied site and it is therefore likely to be of significant ecological importance. These results suggest that T. fibrillosa would be a suitable ‘model mycorrhiza’ for studies of anthropogenic influence, such as pollution, liming or forest fertilization, on population structure of spruce mycorrhizas in southern Sweden.

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 distinguish...

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.

Ectomycorrhizal mycelial species composition in apatite amended and non-amended mesh bags buried in a phosphorus-poor spruce forest.

We studied the effect of apatite amendment on ectomycorrhizal (EM) mycelial biomass production and species composition in a phosphorus-poor spruce forest using sand-filled mesh bags. Control and apatite-amended bags were buried in pairs in the lower part of the organic horizon for one growth season. DNA extraction, PCR of the ITS region, cloning and random sequencing were used to examine the fungal species composition in each bag. Sequences were identified by comparison with the UNITE database and GenBank. Our study confirmed previous results that the major fungal ingrowth in mesh bags was of EM origin. On average 13 species were detected in each bag. Tylospora fibrillosa, Amphinema sp., Tomentellopsis submollis, and Xerocomus badius made up almost 80% of the EM sequences. High biomass was related to increased dominance of specific species. There were no statistically significant differences in biomass production estimated from PLFA 18:2omega6, 9, or between fungal communities of apatite-amended and control bags estimated from DNA after one growth season. The potential of the mesh bag method in studies of functional diversity of EM mycelia in the field is discussed.

Molecular and anatomical evidence for a three-way association between Pinus sylvestris and the ectomycorrhizal fungi Suillus bovinus and Gomphidius roseus

Many intimate associations between different species of ectomycorrhizal fungi are inferred on the basis of the consistent cooccurrence of their fruit bodies. Suillus bovinus and Gomphidius roseus, where the latter never occurs without the former, is one example. This association was examined with PCR identification and light microscopy. S. bovinus and G. roseus were unambiguously separated on the basis of RFLPs of the PCR-amplified ITS region of ribosomal DNA. Tuberculate mycorrhizas of Pinus sylvestris sampled under fruit bodies of G. roseus and S. bovinus were investigated and the majority were identified as mixed associations involving both G. roseus and S. bovinus. Tuberculate mycorrhizas, which macroscopically resemble the ones of Suillus species, contained typical chlamydospores of G. roseus and they had haustoria where G. roseus hyphae penetrated the cortical root cells. Pine seedlings collected near the fruit bodies of the two species were mainly colonised by S. bovinus. Mycelial rhizomorphs collected under the fruit bodies of G. roseus were identified as S. bovinus, while both fungal species were present at the base of G. roseus fruit bodies. The significance of these observations and the possibility that G. roseus acts as a parasite are discussed.

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.

Effects of temperature and incubation time on the ability of three ectomycorrhizal fungi to colonize Pinus sylvestris roots

The ability of Piloderma croceum, a relatively slow growing ectomycorrhizal symbiont, to colonize Pinus sylvestris was tested in competition with two faster growing fungi, Paxillus involutus and an unidentified pink symbiont isolated from the same forest as the P. croceum isolate. The time course of infection was followed for 6 months at 12 °C and 20° P. croceum colonization of initially uninoculated plants was still increasing after 6 months growth at 20°, whereas the numbers of pink root tips remained unchanged after 2 months and the number of P. involutus mycorrhizas decreased from a maximum after 2 months. The incubation time after which maximal colonization occurred corresponded to the relative growth rates of the different fungi. The seedling root size and mycorrhizal infection after 2 months at 20° were very similar to those after 6 months at 12°.