Contrasting Diversity and Host Association of Ectomycorrhizal Basidiomycetes versus Root-Associated Ascomycetes in a Dipterocarp Rainforest

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity.     

Intragenomic variation in nuclear ribosomal markers and its implication in species delimitation,identification and barcoding in fungi

Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.     

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a <Emphasis Type="Italic">Quercus</Emphasis> woodland

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessment of soil fungal communities using pyrosequencing

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.     

Sample richness and genetic diversity as drivers of chimera formation in nSSU metagenetic analyses

Eukaryotic diversity in environmental samples is often assessed via PCR-based amplification of nSSU genes. However, estimates of diversity derived from pyrosequencing environmental data sets are often inflated, mainly because of the formation of chimeric sequences during PCR amplification. Chimeras are hybrid products composed of distinct parental sequences that can lead to the misinterpretation of diversity estimates. We have analyzed the effect of sample richness, evenness and phylogenetic diversity on the formation of chimeras using a nSSU data set derived from 454 Roche pyrosequencing of replicated, large control pools of closely and distantly related nematode mock communities, of known intragenomic identity and richness. To further investigate how chimeric molecules are formed, the nSSU gene secondary structure was analyzed in several individuals. For the first time in eukaryotes, chimera formation proved to be higher in both richer and more genetically diverse samples, thus providing a novel perspective of chimera formation in pyrosequenced environmental data sets. Findings contribute to a better understanding of the nature and mechanisms involved in chimera formation during PCR amplification of environmentally derived DNA. Moreover, given the similarities between biodiversity analyses using amplicon sequencing and those used to assess genomic variation, our findings have potential broad application for identifying genetic variation in homologous loci or multigene families in general.     

Influence of DNA extraction and PCR amplification on studies of soil fungal communities based on amplicon sequencing

Most studies involving next-generation amplicon sequencing of microbial communities from environmental studies lack replicates. DNA extraction and PCR effects on the variation of read abundances of operational taxonomic units generated from deep amplicon 454 pyrosequencing was investigated using soil samples from an agricultural field with diseased pea. One sample was extracted four times, and one of these samples was PCR amplified four times to obtain eight replicates in total. Results showed that species richness was consistent among replicates. Variation among dominant taxa was low across replicates, whereas rare operational taxonomic units showed higher variation among replicates. The results indicate that pooling of several extractions and PCR amplicons will decrease variation among samples.     

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.     

Compensatory Base Changes in ITS2 Secondary Structures Correlate with the Biological Species Concept Despite Intragenomic Variability in ITS2 Sequences – A Proof of Concept

Compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) rDNA secondary structures correlate with Ernst Mayr’s biological species concept. This hypothesis also referred to as the CBC species concept recently was subjected to large-scale testing, indicating two distinct probabilities. (1) If there is a CBC then there are two different species with a probability of ∼0.93. (2) If there is no CBC then there is the same species with a probability of ∼0.76. In ITS2 research, however, the main problem is the multicopy nature of ITS2 sequences. Most recently, 454 pyrosequencing data have been used to characterize more than 5000 intragenomic variations of ITS2 regions from 178 plant species, demonstrating that mutation of ITS2 is frequent, with a mean of 35 variants per species, respectively per individual organism. In this study, using those 454 data, the CBC criterion is reconsidered in the light of intragenomic variability, a proof of concept, a necessary criterion, expecting no intragenomic CBCs in variant ITS2 copies. In accordance with the CBC species concept, we could demonstrate that the probability that there is no intragenomic CBC is ∼0.99.     

An investigation of the biodiversity of thermophilic and thermotolerant fungal species in composts using culture-based and molecular techniques

In this study, the biodiversity of thermophilous fungi in two different commercial composts was investigated using culture-based methods, denaturing gradient gel electrophoresis (DGGE) and tag-encoded pyrosequencing. 454 pyrosequencing of the internal transcribed spacer (ITS) region recovered a total of 175 OTUs between the two composts. The Ascomycota was the dominant phylum in both composts (90 % of all sequences recovered) with the thermophilic-rich orders Sordariales and Eurotiales being the most numerous. Molecular studies demonstrated the frequent presence of several thermophilic (Scytalidium thermophilum, Myriococcum thermophilum) and thermotolerant (Pseudallescheria boydii, Corynascus verrucosus and Coprinopsis sp.) fungi in the composts, despite the absence of these species from the culture-based analysis. Conversely, Aspergillus fumigatus and Mycocladus corymbifer, which were the dominant species in cultivation analyses, had very low representation in molecular studies. The results show that the previous picture of the dominant thermophilous fungi in compost communities derived from culture-based analysis has been biased, and that composting environments represent a potentially rich resource of novel fungi.     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

Endophytic Fungal Communities Associated with Vascular Plants in the High Arctic Zone Are Highly Diverse and Host-Plant Specific

This study assessed the diversity and distribution of endophytic fungal communities associated with the leaves and stems of four vascular plant species in the High Arctic using 454 pyrosequencing with fungal-specific primers targeting the ITS region. Endophytic fungal communities showed high diversity. The 76,691 sequences obtained belonged to 250 operational taxonomic units (OTUs). Of these OTUs, 190 belonged to Ascomycota, 50 to Basidiomycota, 1 to Chytridiomycota, and 9 to unknown fungi. The dominant orders were Helotiales, Pleosporales, Capnodiales, and Tremellales, whereas the common known fungal genera were Cryptococcus, Rhizosphaera, Mycopappus, Melampsora, Tetracladium, Phaeosphaeria, Mrakia, Venturia, and Leptosphaeria. Both the climate and host-related factors might shape the fungal communities associated with the four Arctic plant species in this region. These results suggested the presence of an interesting endophytic fungal community and could improve our understanding of fungal evolution and ecology in the Arctic terrestrial ecosystems.     

Ascomycota has a faster evolutionary rate and higher species diversity than Basidiomycota

Differences in rates of nucleotide or amino acid substitutions among major groups of organisms are repeatedly found and well documented. A growing body of evidence suggests a link between the rate of neutral molecular change within populations and the evolution of species diversity. More than 98% of terrestrial fungi belong to the phyla Ascomycota or Basidiomycota. The former is considerably richer in number of species than the latter. We obtained DNA sequences of 21 protein-coding genes from the lichenized fungus Rhizoplaca chrysoleuca and used them together with sequences from GenBank for subsequent analyses. Three datasets were used to test rate discrepancies between Ascomycota and Basidiomycota and that within Ascomycota: (i) 13 taxa including 105 protein-coding genes, (ii) nine taxa including 21 protein-coding genes, and (iii) nuclear LSU rDNA of 299 fungal species. Based on analyses of the 105 protein-coding genes and nuclear LSU rDNA datasets, we found that the evolutionary rate was higher in Ascomycota than in Basidiomycota. The differences in substitution rates between Ascomycota and Basidiomycota were significant. Within Ascomycota, the species-rich Sordariomycetes has the fastest evolutionary rate, while Leotiomycetes has the slowest. Our results indicate that the main contribution to the higher substitution rates in Ascomycota does not come from mutualism, ecological conditions, sterility, metabolic rate or shorter generation time, but is possibly caused by the founder effect. This is another example of the correlation between species number and evolutionary rates, which is consistent with the hypothesis that the founder effect is responsible for accelerated substitution rates in diverse clades.     

Geographic and Tissue Influences on Endophytic Fungal Communities of Taxus chinensis var. mairei in China

Endophytc fungi were collected from the barks, branches and leaves of Taxus chinensis var. mairei from the Jiangxi, Zhejiang and Chongqing regions of China and their influences on geographic and tissue investigated. A total of 145 fungal taxa were identified based on molecular techniques, of these 125 taxa (86.2 %) belonging to Ascomycota, 14 (9.7 %) to Basidiomycota, 5 (3.4 %) to Zygomycota, and 1 (0.7 %) to undefined fungi. The species richness and diversity of endophytic fungi were significantly affected by tissue, and were 1.2–2.5-fold higher in the branches and barks when compared to the leaves. The locality affected the species richness per tree and the shannon diversity index per tree by longitude. The endophyte assemblages were strongly shaped by locality and tissue according to partial least squares discriminant analysis. In addition, the distributions of dominant fungi at orders and genera levels differed as a function of locality and tissue. Most of the dominant taxa showed spatial heterogeneity and tissue specificity or preference and many fungal taxa with low frequency were special to one locality or one tissue.     

DNA分子标记技术在真菌系统学研究中的应用及影响

DNA分子标记技术为真菌系统进化研究提供了许多新的方法,真菌分子系统学已成为一门成熟的学科。简述了真菌分子系统学的发展简史和代表性的研究方法以及对真菌系统学的主要贡献,包括将广义的真菌划分为3个类群,粘菌和卵菌不再属于真菌界成员。真菌生命之树项目的研究结果对真菌界高阶分类系统作出重大调整,将先前的4个门(壶菌门、接合菌门、子囊菌门和担子菌门)变为7个门(微孢子虫门、壶菌门、新丽鞭毛菌门、芽枝霉门、球囊菌门、子囊菌门和担子菌门)和4个亚门,并对真菌各类群概念作出修订。此外,DNA分子标记技术对真菌种概念的认识、有性型-无性型关联及分子生态学等研究领域产生了重要影响。     

Arctic root‐associated fungal community composition reflects environmental filtering

There is growing evidence that root‐associated fungi have important roles in Arctic ecosystems. Here, we assess the diversity of fungal communities associated with roots of the ectomycorrhizal perennial herb Bistorta vivipara on the Arctic archipelago of Svalbard and investigate whether spatial separation and bioclimatic variation are important structuring factors of fungal community composition. We sampled 160 plants of B. vivipara from 32 localities across Svalbard. DNA was extracted from entire root systems, and 454 pyrosequencing of ITS1 amplicons was used to profile the fungal communities. The fungal communities were predominantly composed of Basidiomycota (55% of reads) and Ascomycota (35%), with the orders Thelephorales (24%), Agaricales (13.8%), Pezizales (12.6%) and Sebacinales (11.3%) accounting for most of the reads. Plants from the same site or region had more similar fungal communities to one another than plants from other sites or regions, and sites clustered together along a weak latitudinal gradient. Furthermore, a decrease in per‐plant OTU richness with increasing latitude was observed. However, no statistically significant spatial autocorrelation between sites was detected, suggesting that environmental filtering, not dispersal limitation, causes the observed patterns. Our analyses suggest that while latitudinal patterns in community composition and richness might reflect bioclimatic influences at global spatial scales, at the smaller spatial scale of the Svalbard archipelago, these changes more likely reflect varied bedrock composition and associated edaphic factors. The need for further studies focusing on identifying those specific bioclimatic and edaphic factors structuring root‐associated fungal community composition at both global and local scales is emphasized.     

Host Identity Impacts Rhizosphere Fungal Communities Associated with Three Alpine Plant Species

Fungal diversity and composition are still relatively unknown in many ecosystems; however, host identity and environmental conditions are hypothesized to influence fungal community assembly. To test these hypotheses, we characterized the richness, diversity, and composition of rhizosphere fungi colonizing three alpine plant species, Taraxacum ceratophorum, Taraxacum officinale, and Polemonium viscosum. Roots were collected from open meadow and willow understory habitats at treeline on Pennsylvania Mountain, Colorado, USA. Fungal small subunit ribosomal DNA was sequenced using fungal-specific primers, sample-specific DNA tags, and 454 pyrosequencing. We classified operational taxonomic units (OTUs) as arbuscular mycorrhizal (AMF) or non-arbuscular mycorrhizal (non-AMF) fungi and then tested whether habitat or host identity influenced these fungal communities. Approximately 14% of the sequences represented AMF taxa (44 OTUs) with the majority belonging to Glomus groups A and B. Non-AMF sequences represented 186 OTUs belonging to Ascomycota (58%), Basidiomycota (26%), Zygomycota (14%), and Chytridiomycota (2%) phyla. Total AMF and non-AMF richness were similar between habitats but varied among host species. AMF richness and diversity per root sample also varied among host species and were highest in T. ceratophorum compared with T. officinale and P. viscosum. In contrast, non-AMF richness and diversity per root sample were similar among host species except in the willow understory where diversity was reduced in T. officinale. Fungal community composition was influenced by host identity but not habitat. Specifically, T. officinale hosted a different AMF community than T. ceratophorum and P. viscosum while P. viscosum hosted a different non-AMF community than T. ceratophorum and T. officinale. Our results suggest that host identity has a stronger effect on rhizosphere fungi than habitat. Furthermore, although host identity influenced both AMF and non-AMF, this effect was stronger for the mutualistic AMF community.     

Comparisons of the Fungal and Protistan Communities among Different Marine Sponge Holobionts by Pyrosequencing

To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.     

Molecular diversity of fungal phylotypes co-amplified alongside nematodes from coastal and deep-sea marine environments

Nematodes and fungi are both ubiquitous in marine environments, yet few studies have investigated relationships between these two groups. Microbial species share many well-documented interactions with both free-living and parasitic nematode species, and limited data from previous studies have suggested ecological associations between fungi and nematodes in benthic marine habitats. This study aimed to further document the taxonomy and distribution of fungal taxa often co-amplified from nematode specimens. A total of 15 fungal 18S rRNA phylotypes were isolated from nematode specimens representing both deep-sea and shallow water habitats; all fungal isolates displayed high pairwise sequence identities with published data in Genbank (99-100%) and unpublished high-throughput 454 environmental datasets (>95%). BLAST matches indicate marine fungal sequences amplified in this study broadly represent taxa within the phyla Ascomycota and Basidiomycota, and several phylotypes showed robust groupings with known taxa in phylogenetic topologies. In addition, some fungal phylotypes appeared to be present in disparate geographic habitats, suggesting cosmopolitan distributions or closely related species complexes in at least some marine fungi. The present study was only able to isolate fungal DNA from a restricted set of nematode taxa; further work is needed to fully investigate the taxonomic scope and function of nematode-fungal interactions.