Short title: Natural history of Amanita thiersii

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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

Michael Kuo

Department of English, Eastern Illinois University, Charleston, Illinois 61920

Anne Pringle

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
13
     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

INTRODUCTION

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

(http://www.jgi.doe.gov/sequencing/why/athiersii.html).

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

muscle.

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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