Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF-ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.     

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.     

Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.-Acephala applanata species complex

Phialocephala fortinii s.1. and Acephala applanata are the dominant dark septate endophytes (DSE) in roots of many trees and shrubs. Population genetic analysis led to the discovery of morphologically indistinguishable but reproductively isolated cryptic species (CSP) within Phialocephala fortinii s.1. In the present study we show that sequence data of two coding (beta-tubulin and translation elongation factor [EF-lalpha]) and three noncoding DNA loci confirm subdivision of P. fortinii s.1. and allow to differentiate seven CSP of P. fortinii. In addition we show that strains collected throughout Europe can be classified correctly based on these sequence markers. Statistically significant differences in growth response on different media were observed among CSP of P. fortinii and A. applanata. Growth inhibition on MEA amended with 100 mgl(-1) cycloheximide had the strongest differential effect of all physiological traits examined. In contrast exoenzyme production (laccase, proteinase, pectinase, phenol-oxidase, amylase, cytochrome oxidase and tyrosinase) rarely helped to differentiate CSP of P. fortinii. However A. applanata was a strong producer of amylases, laccases and proteinases. Based on these data we propose to assign species rank to six CSP of P. fortinii: P. turiciensis, P. letzii, P. europaea, P. helvetica, P. uotolensis, P. subalpina spp. nov. and P. fortinii s.s.     

Isolation and characterization of microsatellite markers for the tree-root endophytes Phialocephala subalpina and Phialocephala fortinii s.s

Species of the Phialocephala fortinii s.l.-Acephala applanata complex are the dominant dark septate endophytes (DSE) in roots of species belonging to the Pinaceae. The two species Phialocephala subalpina and P. fortinii s.s. belong to the most widely distributed species within this complex. In the present study, 15 polymorphic microsatellite loci were developed for these two closely related species. Strains of a community which were analysed previously using single-copy restriction fragment length polymorphism were screened with the new markers. Microsatellites were suitable to classify the two species and to recognize individuals within species.     

Community structure of Phialocephala fortinii s. lat. in European tree nurseries, and assessment of the potential of the seedlings as dissemination vehicles

Patterns of colonization of conifer roots by dark septate endopyhtes of the Phialocephala fortinii s. lat. species complex in nurseries in Switzerland and Lithuania were studied. The potential for man-mediated genotype flow was estimated for two Swiss nurseries based on customers' addresses and the number of delivered plants. Two hundred and forty-nine strains from three Swiss and five Lithuanian nurseries and an afforestation site were characterized using a combination of inter-simple sequence repeat-anchored PCR (ISSR-PCR), single-copy RFLP analysis, and sequence analysis. P. fortinii s. lat. was abundant in nursery seedlings, but the frequency of seedlings colonized varied considerably among and within nurseries. Ten cryptic species (CSP) of P. fortinii s. lat. were identified, including four hitherto undiscovered CSP. P. helvetica was the dominant species in Swiss nurseries, whereas P. fortinii s. str. was the most abundant species in Lithuanian nurseries and the afforestation site. Swiss nurseries deliver plants over distances of more than 200 km indicating the high potential for man-mediated genotype flow in P. fortinii s. lat.     

Suitability of methods for species recognition in the Phialocephala fortinii-Acephala applanata species complex using DNA analysis

Sequence data of two coding and three non-coding loci were used to study the taxonomic identity within and relatedness among seven previously defined cryptic species (CSP) of Phialocephala fortinii and Acephala applanata using two approaches of species recognition. Identification of taxonomic groups corresponding to CSP was ambiguous in some cases when applying solely the genealogical concordance phylogenetic species recognition (GCPSR) concept. The definition of groups corresponding to CSP using GCPSR was complicated due to shared sequence haplotypes between CSP, unresolved CSP for several loci, and possible introgression. GCPSR in conjunction with a population genetic approach improved resolution significantly and the CSP status could be confirmed for all seven CSP of P. fortinii s.l. The most critical step in both analyses was the definition of groups. The combination of several classes of markers differing in resolution helped to define species boundaries.     

Monitoring the spatial and temporal dynamics of a community of the tree-root endophyte Phialocephala fortinii s.l

Phialocephala fortinii sensu lato was isolated from Picea abies roots that had been collected from the same 3 x 3-m forest plot in 2001 and 2004, to examine the spatial and temporal dynamics of this fungal community. RFLP analysis was used to define the multilocus haplotype (MLH) of each isolate. Pielou's measure of association and chi(2) tests of independence were employed to examine the randomness of patterns of spatial distribution of MLH observed in 2001 and 2004. Population differentiation between the two samplings was tested using the G(ST) statistic. In 2001, 144 strains of 28 MLH were isolated; in 2004, 139 strains of 29 MLH. Abundant MLH in 2001 also prevailed in 2004, and the same two cryptic species were dominant in both collections. The probability of being isolated in both years increased with increasing frequency of an MLH. The patterns of spatial distribution of most MLH did not differ between years. The G(ST) values indicated identity of the two collections. Communities of P. fortinii sensu lato remain spatially and genetically stable for at least 3 yr.     

Mycorrhiza Reduces Adverse Effects of Dark Septate Endophytes (DSE) on Growth of Conifers

Mycorrhizal roots are frequently colonized by fungi of the Phialocephala fortinii s.l. - Acephala applanata species complex (PAC). These ascomycetes are common and widespread colonizers of tree roots. Some PAC strains reduce growth increments of their hosts but are beneficial in protecting roots against pathogens. Nothing is known about the effects of PAC on mycorrhizal fungi and the PAC-mycorrhiza association on plant growth, even though these two fungal groups occur closely together in natural habitats. We expect reduced colonization rates and reduced negative effects of PAC on host plants if roots are co-colonized by an ectomycorrhizal fungus (ECM). Depending on the temperature regime interactions among the partners in this tripartite ECM-PAC-plant system might also change. To test our hypotheses, effects of four PAC genotypes (two pathogenic and two non-pathogenic on the Norway spruce), mycorrhization by Laccaria bicolor (strain S238N) and two temperature regimes (19°C and 25°C) on the biomass of the Douglas-fir (Pseudotsuga menziesii) and Norway spruce (Picea abies) seedlings were studied. Mycorrhization compensated the adverse effects of PAC on the growth of the Norway spruce at both temperatures. The growth of the Douglas-fir was not influenced either by PAC or mycorrhization at 19°C, but at 25°C mycorrhization had a similar protective effect as in the Norway spruce. The compensatory effects probably rely on the reduction of the PAC-colonization density by mycorrhizae. Temperature and the PAC strain only had a differential effect on the biomass of the Norway spruce but not on the Douglas-fir. Higher temperature reduced mycorrhization of both hosts. We conclude that ectomycorrhizae form physical and/or physiological barriers against PAC leading to reduced PAC-colonization of the roots. Additionally, our results indicate that global warming could cause a general decrease of mycorrhization making primary roots more accessible to other symbionts and pathogens.     

Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent

Root endophytes are common and genetically highly diverse suggesting important ecological roles. Yet, relative to above-ground endophytes, little is known about them. Dark septate endophytic fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are ubiquitous root colonizers of conifers and Ericaceae, but their ecological function is largely unknown. Responses of Norway spruce seedlings of two seed provenances to inoculations with isolates of four PAC species were studied in vitro. In addition, isolates of Phialocephala subalpina from two populations within and one outside the natural range of Norway spruce were also included to study the effect of the geographic origin of P. subalpina on host response. The interaction of PAC with Norway spruce ranged from neutral to highly virulent and was primarily isolate-dependent. Variation in virulence was much higher within than among species, nonetheless only isolates of P. subalpina were highly virulent. Disease caused by P. subalpina genotypes from the native range of Norway spruce was more severe than that induced by genotypes from outside the natural distribution of Norway spruce. Virulence was not correlated with the phylogenetic relatedness of the isolates but was positively correlated with the extent of fungal colonization as measured by quantitative real-time PCR.     

Microsatellite size homoplasies and null alleles do not affect species diagnosis and population genetic analysis in a fungal species complex

The suitability of 13 microsatellite loci for species diagnosis and population genetics in 11 species of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) was assessed. Two data sets were compared to test possible biases in species typing and clone detection resulting from null alleles and size homoplasies. The first data set was based on fragment lengths derived from a multiplex polymerase chain reaction (PCR) assay and the second data set was received from singleplex PCR at lower stringency and sequencing. Most null alleles observed in the multiplex PCR assay could be amplified during singleplex PCR under less stringent conditions. Size homoplasies resulting from mutations in flanking regions and differences in microsatellite structures were observed. For example, Phialocephala uotolensis possessed a (CT)(13) in addition to the (GT)(x) motif at locus mPF_0644. Despite the occurrence of null alleles and size homoplasies, species diagnosis and population genetic analysis studies were not affected. These markers will facilitate studies on population biology, ecology and biogeography of PAC species.     

Spatial distribution of discrete RAPD phenotypes of a root endophytic fungus, Phialocephala fortinii, at a primary successional site on a glacier forefront

Phialocephala fortinii is among the few identified hyphomycetes belonging to the Mycelium radicis atrovirens complex. This 'dark-septate endophyte' has a global distribution and colonizes a wide variety of host plants. In this study, the spatial distribution of discrete genets of P. fortinii on the forefront of a receding glacier was analysed using the randomly amplified polymorphic DNA (RAPD) technique to determine plants colonized and patterns of colonization. In two consecutive years of sampling, a total of 74 isolates of P. fortinii were obtained from nine plant species, typically ectomycorrhizal, ericoid mycorrhizal or non-mycorrhizal. The isolates showed substantial variation, sharing on average approx. half the RAPD markers. In the first year, three isolates belonging to a single genet were obtained from two plants separated by a distance of nearly 1.5 m. The continuity of this genet was assessed by a sampling the following year. No isolates similar to that, or any of the genets collected the year before were observed. Consequently, the identical isolates from the previous year were concluded to represent discontinuous ramets. Two additional large genets were observed during the second year of sampling, which inhabited roots of several plants representing three different species. These data suggest that the sharing of P. fortinii genets among plant species might play a fundamental role in adaptation and interaction within the whole plant community in a system undergoing primary succession.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Protein polymorphism between 2 Picea abies populations revealed by 2-dimensional gel electrophoresis and tandem mass spectrometry

In species with high gene flow and consequent low interpopulation differentiation over wide geographic ranges, differential gene expression along ecological gradients often reveals adaptive significance. We investigated potential differences in protein expression between Picea abies ecotypes adapted to contrasting altitude conditions. Protein expression patterns were compared between needles and roots of 2-month-old P. abies seedlings by means of 2-dimensional electrophoresis. Proteins exhibiting differential expression between the 2 ecotypes were analyzed by tandem mass spectrometry. A total of 19 proteins exhibited qualitative or quantitative polymorphism between the 2 populations. These proteins exhibited organ-specific expression, and the level of interpopulation protein polymorphism was organ dependent. Among differentially expressed proteins, we identified proteins involved in photosynthesis, photorespiration, root tracheary element differentiation, and transmitochondrial membrane transport. Our results show that P. abies seedlings from locally adapted ecotypes exhibit consistent differences in protein expression. The expression polymorphism of some of these proteins has potential adaptive significance.     

Quantitative analysis of ring growth in spruce roots and its application towards a more precise dating

Missing and wedging rings are common features of tree growth. They occur more frequently in roots than in stems and were reported for various species and sites. These rather frequent irregularities in roots make dendrochronological analysis and cross-dating of roots more challenging. The goal of this study was to present a compiled method for a quantitative analysis of ring-growth irregularities. The analysis was conducted on ten spruce (Picea abies L. Karst) roots taken from the Gorce Mountains (Southern Poland). A four-step cross-dating of these root samples was applied. Three to six cross-sections were analysed within each root and cross-dated with a corresponding stem and site chronology. All ring-growth analyses were conducted on micro sections. Finally, the dating method was evaluated using three control indicators. The study revealed that wedging rings occurring in both, cross-sectional and longitudinal profiles were observed in 17.3% of the rings analysed. The application of a combined zig-zag segment tracing and serial sectioning allowed to significantly reduce, compared to previous methods, the amount of undetected missing rings and revealed them as cross-sectional or longitudinal wedging rings. Thanks to the application of control indicators the irregularities occurring in rings of roots were quantified and compared with different environmental factors such as droughts, air pollution, insect outbreaks and geomorphological processes. Significant positive correlation between root age and the number of radial growth irregularities in roots was demonstrated. A detailed investigation of multiple cross-sections per root enabled to trace all types of ring irregularities in the roots and substantially reduced cross-dating subjectivity.     

Characterization of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots

Phialocephala fortinii Wang & Wilcox is a member of root-inhabiting fungi known collectively as dark septate endophytes (DSE). Although very common and distributed worldwide, few studies have documented their interaction with roots on a structural basis. The objective of this study was to determine the early colonization events and formation of microsclerotia of P. fortinii in roots of Asparagus officinalis L., a species known to have DSE. A loose network of hyphae accumulated at the root surface, and coils formed around root hairs and external to epidermal cells overlying short cells of the dimorphic, suberized exodermis. Root penetration occurred via swollen, appressorium-like structures into epidermal cells where coiling of hyphae occurred along the periphery of the cells. Hyphae penetrated from the epidermis into short exodermal cells and from these into cortical cells. Hyphae colonized the cortex up to the endodermis and sometimes entered the vascular cylinder. Some root tips were colonized as well. Microsclerotia in epidermal and exodermal short cells accumulated glycogen, protein, and polyphosphate. Energy-dispersive X-ray spectroscopy on distinct bodies visible in microsclerotial hyphae revealed high levels of phosphorus.     

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.     

Forest microsite effects on community composition of ectomycorrhizal fungi on seedlings of Picea abies and Betula pendula

Niche differentiation in soil horizons, host species and natural nutrient gradients contribute to the high diversity of ectomycorrhizal fungi in boreal forests. This study aims at documenting the diversity and community composition of ectomycorrhizal fungi of Norway spruce ( Picea abies ) and silver birch ( Betula pendula ) seedlings in five most abundant microsites in three Estonian old-growth forests. Undisturbed forest floor, windthrow mounds and pits harboured more species than brown- and white-rotted wood. Several species of ectomycorrhizal fungi were differentially represented on either hosts, microsites and sites. Generally, the most frequent species in dead wood were also common in forest floor soil. Ordination analyses suggested that decay type determined the composition of EcM fungal community in dead wood. Root connections with in-growing mature tree roots from below affected the occurrence of certain fungal species on seedling roots systems in dead wood. This study demonstrates that ectomycorrhizal fungi differentially establish in certain forest microsites that is attributable to their dispersal and competitive abilities. Elevated microsites, especially decayed wood, act as seed beds for both ectomycorrhizal forest trees and fungi, thus affecting the succession of boreal forest ecosystems.     

凉山州龙肘山锈红杜鹃与薄叶马银花根部真菌分子检测

【目的】为检验直接分子检测法用于揭示杜鹃花属(Rhododendron)植物根部真菌(Root-associated fungi,RAF)组成的有效性。【方法】采用不依赖于培养的分子检测技术直接从锈红杜鹃(R.bureavii)与薄叶马银花(R.leptothrium)的发根(Hair root)提取DNA,用真菌特异性引物扩增r DNA-ITS区、经克隆后测序,对获得的ITS序列进行分析;通过收集NCBI中与本研究的RAF相似性97%以上的所有序列对应的真菌来源(土壤或根系的身份)数据,分析真菌的生态学特性,并用FUNGuild软件提供的方法划分真菌的生态类型。【结果】从两种杜鹃花根部共检测到15种真菌,其中担子菌门(Basdiomycota)真菌3种,子囊菌门(Ascomycota)真菌12种。柔膜菌目(Helotiales)真菌在两种杜鹃花RAF群落中占据优势,并且在两种杜鹃花根系中均检测到该类真菌。此外,两种杜鹃花根部有多种生态类型的RAF共存,包括曾被频繁报道的杜鹃花类菌根菌Oidiodendron sp.和Rhizoscyphus sp.、内生真菌Phialocephala fortinii、共生一致病过渡型真菌Pezoloma ericae、外生菌根共生菌Meliniomyces sp.,以及腐生型真菌Myceana sp.、Lachnum virgineum、Herpotrichia sp.。【结论】直接分子检测法从两种杜鹃花属植物根部检测到的真菌谱系多样性较高、生态类型复杂,这一方法能较为全面地反映杜鹃花属植物RAF多样性。     

Differences in growth characteristics and dynamics of elements absorbed in seedlings of three spruce species raised on serpentine soil in northern Japan

BACKGROUND AND AIMS: Serpentine soils are characterized by the presence of heavy metals (Ni and Cr) and excess Mg; these elements often suppress plant growth. Picea glehnii is nevertheless distributed widely on serpentine soils in northern Japan. Growth characteristics were compared among P. glehnii, Picea jezoensis (distributed in the same region) and Picea abies (planted for timber production), and concentrations of elements in various tissues over time and the amount of ectomycorrhizal infection in short roots were evaluated. METHODS: Seedlings of three spruce species were planted in two types of experimental plots, comprising serpentine soil and brown forest (non-serpentine) soil, and these seedlings were grown for 3 years. Growth, ectomycorrhizal infection of short roots, and elemental composition of tissues were examined. KEY RESULTS: The total dry mass of P. glehnii planted on serpentine soil was almost the same as on brown forest soil, and a large number of needles survived to reach later age classes. By contrast, growth of P. jezoensis and P. abies in serpentine soil was significantly less than in brown forest soil, and needle shedding was accelerated. Moreover, roots of seedlings of P. glehnii on serpentine soil were highly infected with ectomycorrhiza, and the concentration of Ni in needles and roots of P. glehnii was the lowest of the three species. CONCLUSIONS: Picea glehnii has a high ability to maintain a low concentration of Ni, and the ectomycorrhizal infection may have the positive effect of excluding Ni. As a result, P. glehnii is more tolerant than the other spruce species to serpentine soil conditions.     

The effect of tree species diversity on fine-root production in a young temperate forest

The phenomenon of overyielding in species-diverse plant communities is mainly attributed to complementary resource use. Vertical niche differentiation belowground might be one potential mechanism for such complementarity. However, most studies that have analysed the diversity/productivity relationship and belowground niche differentiation have done so for fully occupied sites, not very young tree communities that are in the process of occupying belowground space. Here we used a 5–6 year old forest diversity experiment to analyse how fine-root (<2 mm) production in ingrowth cores (0–30 cm) was influenced by tree species identity, as well as the species diversity and richness of tree neighbourhoods. Fine-root production during the first growing season after the installation of ingrowth cores increased slightly with tree species diversity, and four-species combinations produced on average 94.8% more fine-root biomass than monocultures. During the second growing season, fine-root mortality increased with tree species diversity, indicating an increased fine-root turnover in species-rich communities. The initial overyielding was attributable to the response to mixing by the dominant species, Pseudotsuga menziesii and Picea abies, which produced more fine roots in mixtures than could be expected from monocultures. In species-rich neighbourhoods, P. abies allocated more fine roots to the upper soil layer (0–15 cm), whereas P. menziesii produced more fine roots in the deeper layer (15–30 cm) than in species-poor neighbourhoods. Our results indicate that, although there may be no lasting overyielding in the fine-root production of species-diverse tree communities, increasing species diversity can lead to substantial changes in the production, vertical distribution, and turnover of fine roots of individual species.