Understudied, underrepresented, and unknown: methodological biases that limit detection of early diverging fungi from environmental samples

Metabarcoding is an important tool for understanding fungal communities. The internal transcribed spacer (ITS) rDNA is the accepted fungal barcode but has known problems. The large subunit (LSU) rDNA has also been used to investigate fungal communities but available LSU metabarcoding primers were mostly designed to target Dikarya (Ascomycota + Basidiomycota) with little attention to early diverging fungi (EDF). However, evidence from multiple studies suggests that EDF comprise a large portion of unknown diversity in community sampling. Here we investigate how DNA marker choice and methodological biases impact recovery of EDF from environmental samples. We focused on one EDF lineage, Zoopagomycota, as an example. We evaluated three primer sets (ITS1F/ITS2, LROR/LR3, and LR3 paired with new primer LR22F) to amplify and sequence a Zoopagomycota mock community and a set of 146 environmental samples with Illumina MiSeq. We compared two taxonomy assignment methods and created an LSU reference database compatible with AMPtk software. The two taxonomy assignment methods recovered strikingly different communities of fungi and EDF. Target fragment length variation exacerbated PCR amplification biases and influenced downstream taxonomic assignments, but this effect was greater for EDF than Dikarya. To improve identification of LSU amplicons we performed phylogenetic reconstruction and illustrate the advantages of this critical tool for investigating identified and unidentified sequences. Our results suggest much of the EDF community may be missed or misidentified with “standard” metabarcoding approaches and modified techniques are needed to understand the role of these taxa in a broader ecological context.

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The Composition of the Fungal and Oomycete Microbiome of Rhododendron Roots Under Varying Growth Conditions, Nurseries, and Cultivars

Phytobiomes Journal ◽

10.1094/pbiomes-09-19-0052-r ◽

2020 ◽

Vol 4 (2) ◽

pp. 156-164 ◽

Cited By ~ 2

Author(s):

Z. S. L. Foster ◽

J. E. Weiland ◽

C. F. Scagel ◽

N. J. Grünwald

Keyword(s):

Drought Stress ◽

Community Composition ◽

Internal Transcribed Spacer ◽

Illumina Miseq ◽

Growth Conditions ◽

Fungal Communities ◽

Reference Database ◽

Agricultural Crops ◽

Microbiome Composition ◽

Root Microbiome

The microbiome of agricultural crops influences processes such as nutrient absorption, drought stress, and susceptibility to pathogens. Interactions between a plant’s genotype and its environment influence the composition of the microbiome, but these interactions are not well understood. We compared how the fungal and oomycete microbiomes of rhododendrons from Oregon nurseries differed among cultivars, growth conditions, and nurseries. Roots were sampled from randomly selected container and field-grown plants of three cultivars of rhododendron at four nurseries. The internal transcribed spacer 1 (ITS1) barcode was sequenced with the Illumina MiSeq using two sets of primers specific to fungi and oomycetes, respectively. Sequences were used to infer community composition using VSEARCH and a custom reference database combining curated fungal and oomycete sequences. Comparisons of diversity and community composition were conducted in R using the vegan and metacoder packages. Organism lifestyle was inferred using the FUNGuild database. Few oomycetes were found and fungal communities were dominated by saprobes and mutualists. Nurseries that grew plants in containers and in-field had a significantly higher diversity of fungi than those that only grew plants in containers. Microbiome composition differed significantly among growth conditions and nurseries, but not among cultivars. This suggests that, among these cultivars of rhododendron, environment is important in structuring the root microbiome, but cultivar is not.

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A new oomycete metabarcoding method using the rps10 gene

10.1101/2021.09.22.460084 ◽

2021 ◽

Author(s):

Zachary S. L. Foster ◽

Felipe E Albornoz ◽

Valerie J Fieland ◽

Meredith M Larsen ◽

Frank Andrew Jones ◽

...

Keyword(s):

Environmental Samples ◽

Illumina Miseq ◽

Taxonomic Resolution ◽

Reference Database ◽

Mock Community ◽

Operational Taxonomic Units ◽

Wide Range ◽

Dna Metabarcoding ◽

Improved Methods

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

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Cryptococcus rajasthanensis sp. nov., an anamorphic yeast species related to Cryptococcus laurentii, isolated from Rajasthan, India

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64543-0 ◽

2007 ◽

Vol 57 (2) ◽

pp. 414-418 ◽

Cited By ~ 11

Author(s):

Puja Saluja ◽

G. S. Prasad

Keyword(s):

Large Subunit ◽

Its Region ◽

Molecular Data ◽

Lsu Rdna ◽

Cryptococcus Laurentii ◽

The Novel ◽

D2 Domain ◽

Its Regions ◽

Anamorphic Yeast ◽

Physiological Tests

Two novel anamorphic yeast strains (S-15LT and 3-C1) were isolated from the inflorescences of plants collected in two different towns in Rajasthan State, India. Sequencing of the D1/D2 domains of the large-subunit (LSU) rDNA and the internal transcribed spacer (ITS) regions suggested they are strains of the same species. Phenotypic characteristics such as the absence of fermentation, the absence of sexual structures and ballistoconidia, the assimilation of myo-inositol and d-glucuronate, and positive Diazonium blue B and urease reactions indicated that these strains belong to the genus Cryptococcus. The novel strains differed from Cryptococcus laurentii in six physiological tests and differed from other related species in more than six tests. A phylogenetic analysis of the sequences of the D1/D2 domains of the LSU rDNA and the ITS regions placed these strains in the Bulleromyces clade within the order Tremellales, with C. laurentii as their closest described relative. The novel strains showed 1.6 and 7.5 % divergence in the D1/D2 domain of the LSU rDNA and ITS regions, respectively, with respect to C. laurentii. The divergence from other species was more than 3 % for the D1/D2 domain and more than 9 % for the ITS region. On the basis of the phenotypic and molecular data, strains S-15LT and 3-C1 represent a novel species within the genus Cryptococcus, for which the name Cryptococcus rajasthanensis sp. nov. is proposed. The type strain is S-15LT (=MTCC 7075T=CBS 10406T).

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Morphometrics and molecular analysis ofOzolaimus linstowin. sp. (Oxyuroidea: Pharyngodonidae) from the green lizardIguana iguana

Journal of Helminthology ◽

10.1017/s0022149x15000061 ◽

2015 ◽

Vol 90 (2) ◽

pp. 186-198 ◽

Cited By ~ 2

Author(s):

S.V. Malysheva

Keyword(s):

Body Size ◽

Large Subunit ◽

Buccal Capsule ◽

Small Subunit ◽

Present Species ◽

Lsu Rdna ◽

Adult Males ◽

Spicule Length ◽

Males And Females ◽

Characteristic Features

AbstractOzolaimus linstowin. sp. is described from the large intestine ofIguana iguanaLinnaeus, 1758 from Mexico. The present species can be easily distinguished fromO. megatyphlonandO. cirratusby the presence of a long and slender pharynx not divided into sections, more similar to the remaining two species,O. monhysteraandO. ctenosauri. Ozolaimus linstowin. sp. can be differentiated fromO. monhysteraby the shorter spicule length and smaller body size of both males and females. Males ofO. linstowin. sp. are morphologically close to those ofO. ctenosauri, but females possess a markedly smaller body size and differ in the organization of the oral cuticular armature. Adult males ofO. linstowin. sp. bear some characteristic features of the J3 juvenile morphology in terms of the cuticular organization of the oral and buccal capsule. Phylogenetic analysis ofO.linstowin. sp. using partial small subunit (SSU) and D2–D3 large subunit (LSU) rDNA shows relationships with several Oxyuridae genera.

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PrimerPooler: automated primer pooling to prepare library for targeted sequencing

Biology Methods and Protocols ◽

10.1093/biomethods/bpx006 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 7

Author(s):

Silas S. Brown ◽

Yun-Wen Chen ◽

Ming Wang ◽

Alexandra Clipson ◽

Eguzkine Ochoa ◽

...

Keyword(s):

Variant Calling ◽

Pcr Amplification ◽

Illumina Miseq ◽

Degenerate Primers ◽

Cross Hybridization ◽

Targeted Next Generation Sequencing ◽

Target Sequencing ◽

A Genome ◽

Experimental Protocols ◽

Minimal Depth

Abstract Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

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Metataxonomic comparison between internal transcribed spacer and 26S ribosomal large subunit (LSU) rDNA gene

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2018.10.010 ◽

2019 ◽

Vol 290 ◽

pp. 132-140 ◽

Cited By ~ 11

Author(s):

Jatziri Mota-Gutierrez ◽

Ilario Ferrocino ◽

Kalliopi Rantsiou ◽

Luca Cocolin

Keyword(s):

Internal Transcribed Spacer ◽

Large Subunit ◽

Lsu Rdna ◽

Rdna Gene

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Morphometrical and molecular evidence suggests cryptic diversity among hookworms (Nematoda: Uncinaria) that parasitize pinnipeds from the south-eastern Pacific coasts

Journal of Helminthology ◽

10.1017/s0022149x18000950 ◽

2018 ◽

Vol 94 ◽

Cited By ~ 1

Author(s):

M.T. González ◽

Z. López ◽

J.J. Nuñez ◽

K.I. Calderón-Mayo ◽

C. Ramírez ◽

...

Keyword(s):

Large Subunit ◽

Principal Component ◽

Eastern Pacific ◽

Lsu Rdna ◽

Molecular Evidence ◽

South American ◽

Southern Chile ◽

The South ◽

South Eastern ◽

Neophoca Cinerea

AbstractHookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

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La región de los ITS del ADN ribosomal del núcleo (nrADN), fuente de caracteres moleculares en la sistemática de las gimnospermas

Botanical Sciences ◽

10.17129/botsci.1527 ◽

2017 ◽

pp. 159 ◽

Cited By ~ 1

Author(s):

Adrián Quijada ◽

Guadalupe Méndez-Cárdenas ◽

Blanca Hernández-Baños ◽

Elena Álvarez-Buylla

Keyword(s):

Chloroplast Dna ◽

Direct Evidence ◽

Phylogenetic Reconstruction ◽

Its Region ◽

Phylogenetic Inference ◽

Reticulate Evolution ◽

Length Variation ◽

Interspecific Relationships ◽

Considerable Length

The ITS region of gymnosperm plants has shown to be longer than the typical angiosperm ITS and shows considerable length variation. The larger size relative to the ITS in angiosperms offers more characters for phylogenetic reconstruction, which make this marker suitable for inference at different taxonomic levels. Unlike chloroplast DNA (cpDNA), ITS data can provide direct evidence of interspecific hibridization. This permits the use of the ITS to obtain measurements of introgression rates among populations and to detect events of reticulate evolution. Infraspecific ITS variation in the genus Pinus has been reported, which can compromises the inference of interspecific relationships. This potential must be taken into account in phylogenetic inference of gymnosperms.

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Characterization and Phylogeny of Fungi from Industrial Wastewater Using Highly Conserved Multiple Gene Loci

10.21203/rs.3.rs-289731/v1 ◽

2021 ◽

Author(s):

Blessing Amaka Ezeonuegbu ◽

Dauda Abdullahi Machido ◽

Clement Z. Whong ◽

Wisdom S. Japhet ◽

Clement Ameh Yaro ◽

...

Keyword(s):

Phylogenetic Trees ◽

Phylogenetic Analyses ◽

Large Subunit ◽

Industrial Effluent ◽

Pcr Amplification ◽

Morphological Characterization ◽

Likelihood Method ◽

Internal Transcribed Spacer Region ◽

Fungal Isolates ◽

Fungal Dna Extraction

Abstract The aim of this study was isolation and molecular characterization of fungi from untreated industrial effluent by multigene phylogenetic analyses. The Fungi isolated were characterized based on PCR amplification and genomic sequencing of the internal transcribed spacer region (ITS), partial β-tubulin (Ben A), calmodulin (CaM), and DNA-directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization and species diversity. Fungal DNA extraction kits and primers sets for the selected genes were purchased and used following the manufacturer’s instructions. The obtained sequences were subjected to BLAST analysis and the corresponding fungal isolates were assigned species names after comparison with representative sequences available in GenBank. All the sequences from this study were deposited in GenBank and the accession number assigned. Phylogenetic trees of the fungal isolates were drawn for each gene by the Maximum Likelihood method using MEGA 7.0 software. Fifteen (15) Fungi species belonging to four genera of Aspergillus, Penicillium, Fusarium and Trichoderma with Aspergillus as the predominant genus were identified.

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Microbial Composition of Near-Boiling Silica-Depositing Thermal Springs throughout Yellowstone National Park

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5123-5135.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5123-5135 ◽

Cited By ~ 128

Author(s):

Carrine E. Blank ◽

Sherry L. Cady ◽

Norman R. Pace

Keyword(s):

Yellowstone National Park ◽

National Park ◽

Hydrogen Oxidation ◽

Large Subunit ◽

Microbial Community Composition ◽

Pcr Amplification ◽

Small Subunit ◽

Rrna Genes ◽

Small Subunit Rrna ◽

Microbial Composition

ABSTRACT The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences.

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