Inoculum 63(3) - Mycological Society of America

of the New World. Doubts about the accuracy of name applications across the
ocean have persisted, but molecular phylogenies now provide a new level of resolution.
Our research targets fleshy fungi whose names originated in Europe, but
which bear the same name and superficial form in eastern North America. Several
examples can be identified in which DNA sequences reveal differences across
the ocean, including saprophytic (Marasmius rotula; Baeospora myosura;
Sparassis crispa) and ectomycorrhizal (Strobilomyces strobilaceus; Tricholoma
populinum) fungi. In addition, it appears that genes for sexual compatibility and
recognition are more highly conserved than the internally transcribed spacer
(ITS), but morphological differences vary with the individual taxon. Implications
of this research include the necessity for new names for heretofore cryptic taxa on
both continents and resultant adjustment of biodiversity inventory reports.
Peterson, Stephen W. USDA-ARS NCAUR, 1815 North University Street, Peoria,
IL 61604. Aspergillus section Versicolores: nine new species and multilocus
DNA sequence based phylogeny
ß-tubulin, calmodulin, internal transcribed spacer and partial lsu-rDNA,
RNA polymerase, DNA replication licensing factor Mcm7, and pre-rRNA processing
protein Tsr1 were amplified and sequenced from 62 A. versicolor clade
isolates and analyzed phylogenetically using the concordance model to establish
species boundaries. We used phylogeny to define species and phenotype from 10d
CYA cultures to describe the species. Aspergillus austroafricanus, A. creber, A.
cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis
and A. venenatus are described as new species and A. amoenus, A. protuberus,
A. tabacinus and A. versicolor are accepted as distinct species on the
basis of molecular and phenotypic differences. PCR primer pairs used to detect A.
versicolor in sick building syndrome studies have a positive reaction for all of the
newly described species.
Phookamsak, Rungtiwa 1 , Amy Y Rossman 2 , Ekachai Chukeatirote 1 , and
Kevin D Hyde 1 . 1 School of science, Mae Fah Luang University, 57100, Chiang
Rai, Thailand., 2 Mycology & Microbiology Laboratory, USDA-ARS, Beltsville,
MD 20705, USA. Taxonomic and phylogenetic studies of Dothideomycetes
on bamboo in Chiang Rai Province, Northern of Thailand
Bamboos are fascinating and useful plants that have a wide range of uses.
The traditional people in Southeast Asia, China, Japan, India and South America
have used bamboos in their cultures and survival since ancient times. There are
several studies on endophytic, pathogenic and saprobic fungi on bamboo. However,
phylogenetic information based on molecular data is poorly known for many
bambusicolous fungi. We studied the taxonomy and phylogeny of Dothideomycetes
on bamboo in Chiang Rai Province, Northern Thailand. Specimens
were collected from six locations in Chiang Rai. Pure cultures were obtained
using single spore isolation. We identified bitunicate fungi on dead stems or
branches of bamboo that belong to the families of Aigialaceae, Didymosphaeriaceae,
Lophiostomataceae, Massarinaceae and Melanommataceae. Some fungi
are illustrated and a checklist of fungi examined on bamboo is presented. Among
the 33 saprobic fungi on dead materials two isolates of Aigialaceae, four of Didymosphaeriaceae,
three of Lophiostomataceae and Massarinaceae and 11 of
Melanommataceae were identified. The molecular phylogeny of Dothideomycetes
on bamboo is inferred based on combined sequences of rDNA, the
internal transcribed spacers (ITS rDNA), small subunit nuclear rDNA (18S,
SSU), large subunit nuclear rDNA (28S, LSU) and partial RNA polymerase second
largest subunit (RPB2).
Picard, Kathryn T 1 , Rowena F Stern 2 , and François Lutzoni 1 . 1 Duke University,
Durham, NC 27708, 2 Sir Alister Hardy Foundation for Ocean Science, Plymouth,
UK. Investigating early-diverging fungi from marine and estuarine
habitats in North America and Europe
Despite increasing efforts to characterize and catalog early-diverging
fungi, marine and estuarine habitats remain largely unexplored. Further, traditional
culturing methods commonly employed in taxonomic surveys provide only
limited insight into the breadth of zoosporic fungal diversity. However, with the
application of culture-independent molecular techniques, including environmental
cloning and, increasingly, high-throughput sequencing, the presence, distribution,
and variety of these previously unculturable microbes is being elucidated.
For this study, marine and estuarine habitats in North America and Europe were
surveyed for novel fungal phylotypes using next-generation sequencing methods.
North American samples originated from a yearlong sampling effort in coastal
North Carolina. Monthly sediment and plankton samples were collected from
three small estuarine islands located within the Beaufort Inlet in Pamlico Sound.
Quarter-annual collections of marine sediments from the shallow waters of Cape
Lookout Bight were also made. Using the Ion Torrent platform, the 5’ end of the
nuclear large subunit (28S) was sequenced from these samples, resulting in ~200
bp reads. European samples originated from open water collections made across
the English Channel over the course of three months. Partial nuclear small subunit
(18S) rDNA sequences (~200-400 bp) were generated from English Channel
38 Inoculum 63(3), June 2012
samples using 454 pyrosequencing. The conserved 18S and 28S markers were
chosen over more variable regions, such as ITS1 and ITS2, based on greater availability
of publicly accessible reference sequences. Preliminary analysis of both
North American and European sequence data reveals novel fungal phylotypes
from across the fungal tree.
Porras-Alfaro, Andrea 1,4 , Liu Kuan-Liang 3 , Zachary Gossage 1 , Lynnaun
Johnson 1 , Tabitha Williams 1 , Gary Xie 2 , and Cheryl R Kuske 2 . 1 Department of
Biological Sciences, Western Illinois University, Illinois, USA, 2 Los Alamos National
Laboratory, Bioscience Division, Los Alamos, New Mexico, USA, 3 Institute
of Information Management, National Cheng Kung University, Tainan City,
Taiwan, Republic of China, 4 Department of Biology, University of New Mexico,
Albuquerque, USA. ITS and LSU automated classification: the fungal RDP
naïve Bayesian classifier
The introduction of next generation sequencing (NGS) in ecological studies
has created a major revolution in fungal ecology. Analyses of large fungal
datasets are common but currently limited by the lack of databases and reliable
tools that will allow the classification of thousands of fungal sequences from environmental
samples. The main objective of this project was to create curated
databases for the Internal Transcribed Spacer (ITS) region and the large-subunit
rRNA (LSU) gene and to evaluate their performance in the Ribosomal Database
Project (RDP) naïve Bayesian classifier. We created and compared hand-curated
LSU and ITS databases (more than 8 000 fungal sequences per database). Taxonomic
gaps in fungal databases and challenges in the creation and maintenance of
these databases will be discussed. When compared with the traditional BLASTN
approach, the RDP fungal classifier was more rapid (>460-fold with our system)
with similar or superior accuracy. Performance of fungal classifier at different taxonomic
levels, sequence lengths and regions was also evaluated. Classification
was more accurate with 400-bp sequence reads than with 100-bp reads and location
of hyper variable regions played an important role in accuracy of sequence
classification. The fungal classifier shows to be a highly effective tool to analyze
large NGS datasets from environmental surveys, and to determine target regions
in LSU and ITS sequences that will improve accuracy of taxonomic classification
when short sequences are obtained. The LSU training set and tool are publicly
available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp)
Porter, Teresita M and G Brian Golding. McMaster University, Biology Department,
Life Sciences Building, 1280 Main Street West, Hamilton, ON L8S
4K1 Canada. Assigning ITS and LSU rDNA ‘barcodes’ with confidence
Whereas PCR, cloning and Sanger-based sequencing can generate libraries
comprised of thousands of sequences up to about 600-1000 bp in length;
current highly parallel sequencers are now generating amplicon libraries comprised
of millions of relatively short sequences of about 200-400 bp in length. We
are interested in how these shorter sequences affect the quality of automated taxonomic
assignments. Here we present the results from two simulation studies that
specifically address the accuracy and error rate of ITS and LSU rDNA markers
using several tools suitable for automating assignments. We compare the performance
of programs using similarity-, phylogeny-, and composition-based
methods. Generally, we find that database completeness is perhaps the biggest
factor determining assignment accuracy, followed by query sequence length, assignment
method, primer choice, and sequence error. Specifically, we find that
MEGAN lowest common ancestor (LCA) parsing produces the lowest error rate
when assigning unknown ITS and LSU rDNA sequences; and, the fungal LSU
naïve Bayesian classifier available through the Ribosomal Database Project website
performs significantly faster than BLAST-based methods.
Powell, Martha J and Peter M Letcher. Department of Biological Sciences, The
University of Alabama, Tuscaloosa, AL 35487. Using cellular structure and
biochemistry for insights into fungal cell biology and phylogeny
Chytridiomycota (chytrids) are now recognized as highly adaptable fungi,
not limited in geography or habitat by the constraints of reproduction with a flagellated,
unwalled-spore (=zoospore). As our examination of chytrid zoospore ultrastructure
has expanded, we have discovered tremendous variety in organellar
structures and organization. Molecular-based reconstructions of phylogeny have
also revealed great genetic diversity among chytrids. The intersection of molecular
phylogenies with analyses of organellar function is helping us understand how
zoospore structures have evolved within lineages. Zoospore architectural differences
are good predictors of molecular-based phylogenies. Selective pressures associated
with habitat differences may be drivers in the evolution of zoospore ultrastructure.
Evolution of zoospore architecture in the Chytridiales is discussed as
an example of how structures have been modified and lost within evolutionary lineages.
Our challenge now is discovering more about the functional roles of architectural
design differences in chytrid zoospores.
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