In Press at Mycologia, published on September 13, 2011 as doi:10.3852/11-056
Short title: Natural history of Amanita thiersii
Amanita thiersii is a saprotrophic fungus expanding its range in the United States
Benjamin E. Wolfe1
Harvard University, FAS Center for Systems Biology, 52 Oxford Street, Northwest Labs Room
402, Cambridge, Massachusetts 02138
Department of English, Eastern Illinois University, Charleston, Illinois 61920
Harvard University, Organismic and Evolutionary Biology, 16 Divinity Avenue, Biolabs Room
3100, Cambridge, Massachusetts 02138
Abstract: Although most species in the genus Amanita form ectomycorrhizal associations, a few
are reported to be saprotrophs living in grassland habitats. Little is known about the ecology and
distribution of these free-living Amanita species. We describe the ecology of Amanita thiersii, a
species commonly collected in lawns throughout the Mississippi River Basin. Stable isotopes of
carbon, transcriptomic sequences and patterns of growth on complex carbon sources provide
evidence for A. thiersii as a saprotrophic species. Sporocarps of A. thiersii are less depleted in
C compared to published data for ectomycorrhizal fungi, supporting a saprotrophic mode of
carbon acquisition in the field. Orthologs of cellulase genes known to play key roles in the
decomposition of cellulose in other basidiomycetes were identified in a transcriptome of A.
thiersii, establishing that this species has the genetic potential to degrade cellulose. Amanita
thiersii also can use artificial cellulose or sterile grass litter as a sole carbon source. DNA
sequences of three nuclear gene regions and banding patterns from four intersimple sequence
repeat markers were identical across 31 populations from throughout the known range of the
Copyright 2011 by The Mycological Society of America.
species, which suggests the genetic diversity of A. thiersii populations is low. Maps of A. thiersii
collections made from the 1950s until present suggest this species is experiencing a range
expansion. It was reported first in 1952 in Texas and now occurs in nine states north to Illinois.
These data provide an ecological context for interpreting the genome of A. thiersii, currently
being sequenced at the United States Department of Energy's Joint Genome Institute.
Key words: cellulase, IGS, ISSR, range expansion, stable isotope, transcriptome
Most of the 500-plus species in the genus Amanita form ectomycorrhizal associations with
woody plants (Yang et al. 1999). However sporocarps of approximately 15 species of Amanita
typically are collected at some distance from potential woody plant hosts, usually in natural or
artificial grasslands. These Amanita species have been identified tentatively as saprotrophs (Bas
1969). However the saprotrophic status of these species has never been experimentally
confirmed, and almost nothing is known about their natural histories. While most species of
Agaricales in grass-dominated ecosystems are saprotrophs that decompose grass litter, some
species are pathogens of grasses while other species form ectomycorrhizal associations with
sedges (as reviewed in Griffith and Roderick 2008). These latter cases highlight the problems
associated with making assumptions about the trophic status of fungi based simply on habitat.
Several approaches, including stable isotopes, the growth of a fungus on different carbon
sources and the identification of putative functional genes, can be used to infer the trophic status
of poorly understood fungi, including these free-living Amanita species. Stable isotopes of
carbon are widely used to distinguish between ectomycorrhizal and saprotrophic fungi (e.g. Henn
and Chapela 2001, Hobbie et al. 2001, Trudell et al. 2004) and have been used to infer the
ecology of other poorly understood species (e.g. Wilson et al. 2007). Because ectomycorrhizal
fungi use carbon taken from host plants that is more depleted in 13C, as compared to carbon from
dead organic matter, sporocarps of ectomycorrhizal fungi are generally more depleted in 13C than
saprotrophic fungi (Mayor et al. 2009). Saprotrophic fungi also generally have a much greater
capacity than ectomycorrhizal fungi for growth on complex carbon sources, for example
cellulose, and the growth of a fungus on different carbon sources can provide a useful basis for
understanding its saprotrophic potential (Maijala et al. 1991). Genome and transcriptome
sequencing can identify specific genes considered hallmarks of an ecological niche. For example
to completely decompose cellulose polymers in dead organic matter saprotrophic fungi typically
use three types of cellulases, endo-1,4--glucanases, cellobiohydrolaes, and -glucosidases
(Baldrian and Valskov 2008). Saprotrophic fungi tend to have a diversity of the genes
encoding these cellulases, while ectomycorrhizal fungi generally have fewer copies of these
genes or may lack some cellulases, such as glycoside hydrolase family 7 cellobiohydrolases
(Martin et al. 2010, Nagendran et al. 2009). The presence of these genes in a genome or
transcriptome provides strong evidence for the genetic potential of a fungus to obtain carbon
through the decomposition of organic matter.
Amanita thiersii is one apparently saprotrophic Amanita, found in lawns in the central
and southeastern United States. This species originally was described as Amanita alba, a
homonym, from a lawn in Texas (Thiers 1957). Bas (1969) renamed it Amanita thiersii. Over
the past 1015 y anecdotal reports have suggested A. thiersii as increasing in abundance in
regions of the central United States where it was not observed previously (Kuo 2007, McFarland
and Mueller 2009, Kuo and Methven 2010), suggesting that like other species of Amanita
(Pringle and Vellinga 2006, Pringle et al. 2009, Vellinga et al. 2009, Wolfe et al. 2010) the
fungus is expanding its range. However several other potentially saprotrophic Amanita species
with morphological similarities to A. thiersii, including A. manicata (Pegler 1986), A.
armillariiformis (Miller et al. 1990) and A. nauseosa (Guzmn 1981), occur in the United States.
Because specimens in historical collections of macrofungi may be misidentified (Pringle and
Vellinga 2006, Pringle et al. 2009) apparent changes in the distribution of A. thiersii in the
United States might reflect a confused species concept and not an actual range expansion.
In this paper we describe the natural history of A. thiersii in the United States, using both
field observations and laboratory experiments. We explored these questions:
(i) Does A. thiersii possess hallmarks of a saprotrophic niche, based on stable isotope
measurements of sporocarps, the presence of genes encoding cellulases in transcriptomic
sequences and patterns of growth on complex carbon sources?
(ii) What is the current distribution of A. thiersii in the United States? Does mapping the
distribution of A. thiersii records over time provide support for the perception of A.
thiersii as a species experiencing a range expansion?
(iii) How genetically diverse are populations of A. thiersii across the United States?
We also collected additional natural history data, including descriptions of pure culture
morphology, counts of nuclei and data on the effects of A. thiersii on the growth of grasses. Our
research provides the first detailed study of the ecology of a free-living Amanita species, and
these data provide an ecological context for interpreting the genome of A. thiersii, currently in
progress at the United States Department of Energy's Joint Genome Institute
MATERIALS AND METHODS
Isolation of axenic cultures of Amanita thiersii and nuclear counts of spores.A mushroom collected by Sherry
Kay from a lawn in Baldwin City, Kansas, in Jul 2009 (A thiersii 2009 Baldwin City in SUPPLEMENTARY
TABLE I) was used to generate monokaryotic (Amanita thiersii Skay4041) and dikaryotic (Amanita thiersii
Skay4041het) cultures of A. thiersii. The monokaryotic strain is being sequenced by the Department of Energy's
Joint Genome Institute and has been deposited at the Fungal Genetics Stock Center (FGSC10297).
Monokaryotic cultures were generated by pressing lamallae onto the surface of 0.2 % glucose solid MMN
medium (Marx 1969). Spores were dispersed across the surface of the medium with 20 L sterile water. Dikaryotic
cultures were generated by excising small pieces of tissue from between the pileus and lamellae of the sporocarp.
Tissues were plated onto solid, modified MMN medium (Marx 1969), with 0.2% glucose, no malt extract, and
containing 100 g/mL chloramphenicol. Plates were incubated at 27 C.
To determine the number of nuclei per spore we followed protocols outlined by Horton (2006) for staining
of nuclei with DAPI. Spores were observed with a Zeiss Axioscope fluorescence microscope (Carl Zeiss Inc., Jena,
Germany) using a DAPI filter at 400 magnification.
Stable isotopes of carbon and nitrogen. To determine whether the biomass of A. thiersii sporocarps is composed
of carbon derived from the decomposition of litter in the environment or from ectomycorrhizal associations we
measured stable isotope ratios of carbon and nitrogen in at least one sporocarp from each of 31 unique populations
collected throughout the current range of A. thiersii in the United States (SUPPLEMENTARY TABLE I).
Homogenized samples of dried gill tissue were analyzed with standard protocols (Hobbie et al. 2001).
Measurements were made with a Costech ECS4010 Elemental Analyzer configured with a DeltaPlus XP mass
spectrometer at the University of New Hampshire Stable Isotope Lab. Stable isotope data are reported as
according to this equation:
X = (Rsample/Rstandard 1) 1000 ()
where Rsample and Rstandard are the ratios of heavy to light isotopes of sampled sporocarps and standards, and X is
either carbon (C) or nitrogen (N). Vienna Pee Dee Belemnite and air were used as references for C and N
respectively. Standards for calibration included NIST 1515 (apple leaves), NIST 1575a (pine needles) and tuna
Transcriptome sequencing.To assess the genetic potential for saprotrophic decomposition by A. thiersii we
sequenced the transcriptome of Amanita thiersii Skay4041het growing on sterile grass litter. Using a QIAGEN
RNeasy Maxi Kit (QIAGEN, Valencia, California) RNA was extracted from Amanita thiersii Skay4041het mycelia
that had been grown on litter 2 wk. cDNA was generated from this RNA with the Roche rapid library preparation
method (454 Life Sciences, Branford, Connecticut) and sequenced on a Roche GS-FLX with titanium chemistry at
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