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A molecular phylogeny of thermophilic fungi

Ingo MORGENSTERNa,b, Justin POWLOWSKIa,c, Nadeeza ISHMAELa, Corinne DARMONDa,

Sandrine MARQUETEAUa, Marie-Claude MOISANa, Genevi
eve QUENNEVILLEa,
Adrian TSANGa,b,*
a
Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, Canada
b
Department of Biology, Concordia University, Montreal, Quebec, Canada
c
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada

article info abstract

Article history: Sequences from 86 fungal genomes and from the two outgroup genomes Arabidopsis thali-
Received 21 November 2011 ana and Drosophila melanogaster were analyzed to construct a robust molecular phylogeny
Accepted 30 January 2012 of thermophilic fungi, which are potentially rich sources of industrial enzymes. To provide
Corresponding Editor: experimental reference points, growth characteristics of 22 reported thermophilic or ther-
Andrew N. Miller motolerant fungi, together with eight mesophilic species, were examined at four tempera-
tures: 22
C, 34
C, 45
C, and 55
C. Based on the relative growth performances, species
Keywords: with a faster growth rate at 45
C than at 34
C were classified as thermophilic, and species
Eurotiales with better or equally good growth at 34
C compared to 45
C as thermotolerant. We ex-
Fungal phylogeny amined the phylogenetic relationships of a diverse range of fungi, including thermophilic
Mucorales and thermotolerant species, using concatenated amino acid sequences of marker genes
Onygenales mcm7, rpb1, and rpb2 obtained from genome sequencing projects. To further elucidate
Sordariales the phylogenetic relationships in the thermophile-rich orders Sordariales and Eurotiales,
Thermophily we used nucleotide sequences from the nuclear ribosomal small subunit (SSU), the 5.8S
gene with internal transcribed spacers 1 and 2 (ITS 1 and 2), and the ribosomal large sub-
unit (LSU) to include additional species for analysis. These phylogenetic analyses clarified
the position of several thermophilic taxa. Thus, Myriococcum thermophilum and Scytalidium
thermophilum fall into the Sordariales as members of the Chaetomiaceae, Thermomyces lanugi-
nosus belongs to the Eurotiales, Malbranchea cinnamomea is a member of the Onygenales, and
Calcarisporiella thermophila is assigned to the basal fungi close to the Mucorales. The meso-
philic alkalophile Acremonium alcalophilum clusters with Verticillium albo-atrum and Verticil-
lium dahliae, placing them in the recently established order Glomerellales. Taken together,
these data indicate that the known thermophilic fungi are limited to the Sordariales, Euro-
tiales, and Onygenales in the Ascomycota and the Mucorales with possibly an additional order
harbouring C. thermophila in the basal fungi. No supporting evidence was found for thermo-
philic species belonging to the Basidiomycota.
ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Centre for Structural and Functional Genomics, 7141 Sherbrooke Street West, Montreal, Quebec, H4B 1R6
Canada. Tel.: þ1 514 848 2424x3405; fax: þ1 514 848 4504.
E-mail address: tsang@gene.concordia.ca
Abbreviations: OTU, operational taxonomic unit; SSU, rDNA (18S) from the small ribosomal subunit; LSU, rDNA (28S) from the large ribosomal
subunit; ITS, internal transcribed spacer; BSS, bootstrap support
1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.funbio.2012.01.010

Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
2 I. Morgenstern et al.
Introduction
Thermophilic organisms can be classified as those organisms
with an optimal growth temperature between 45
C and 80
C,
hyperthermophiles are those with an optimum growth tem-
perature above 80
C, and mesophiles are those that grow
optimally below 45
C (Stetter et al. 1990; Madigan & Orent
1999; Taylor & Vaisman 2010). Thermophily is common in bac-
teria and Archaea, whereas hyperthermophiles are mainly
confined to the Archaea. Only a small fraction of the estimated
600 000 fungi (Mora et al. 2011) is considered to be thermophilic
and no fungus has been described as hyperthermophilic. Most
reported thermophilic fungi have been placed into the Sordar-
iales, Eurotiales, and Mucorales (Berka et al. 2011). However,
Straatsma et al. (1994) described the existence of two thermo-
philic isolates of Basidiomycota. Furthermore, Myriococcum ther-
mophilum is listed by National Center for Biotechnology
Information (NCBI) taxonomy as a mitosporic basidiomycete
and by Index Fungorum as an agaricomycete.
The temperature preferences of thermophilic fungi have
been defined in different ways. According to Cooney &
Emerson (1964), fungi growing with a minimum temperature
of 20
C or higher and a maximum temperature of growth
above 50
C are thermophilic, whereas fungi growing below
20
C and up to about 50
C are regarded as thermotolerants.
They set the upper limit of growth for mesophiles at 40
C
(Cooney & Emerson 1964). On the other hand, Crisan (1964)
and Maheshwari et al. (2000) proposed to classify fungi as ther-
mophilic if their optimal growth temperature is above 40
C or
45
C, respectively.
Fungi are the main decomposers of lignocellulosic biomass
in terrestrial ecosystems and the enzymes they secrete
to break down lignocellulose may be useful in industrial
processes such as bleaching in the pulp and paper industry, bio-
remediation of polluted soils, clean-up of wastewater effluents,
and the production of second and third generation biofuels
(Wesenberg et al. 2003; Gianfreda & Rao 2004; Sigoillot et al.
2005; Turner et al. 2007). Thermophilic fungi are of special inter-
est for biomass conversion applications since they are potential
sources of thermostable enzymes. The advantages of biomass
conversion at high temperatures include higher reaction rates,
enhanced mass transfer, lowered substrate viscosity, and re-
duced risk of contamination (Haki & Rakshit 2003; Viikari
et al. 2007). At least some thermophilic fungi possess
cellulose-degrading capacities that are higher than those of
mesophilic reference species (Tansey 1971; Berka et al. 2011).
The screening of thermophilic fungi and other thermophilic or-
ganisms for improved enzyme varieties may contribute to low-
ering the costs of enzyme preparations (Banerjee et al. 2010).
The nomenclature and taxonomic classification of thermo-
philic fungi is in a state of disarray, often leading to misiden-
tifications and confusion (Mouchacca 1997, 2000a). New
approaches that use molecular markers combined with
efforts to establish a natural classification system that is
based solely on monophyletic groups (Doolittle 1999; Voigt &
Kirk 2011) have appreciably improved fungal taxonomy. The
most dramatic changes affect the basal fungal lineages, but
new taxonomic entities have also been introduced in the Basi-
diomycota and Ascomycota (Hibbett et al. 2007). The rapidly
Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
increasing number of available sequenced genomes has
changed the way phylogenetic analyses are conducted. Stud-
ies based solely on the analysis of a single marker locus are be-
ing replaced by multilocus and phylogenomic studies that can
produce well-resolved trees with high support values for the
majority of nodes.
The aim of this study is to produce a robust phylogenetic
framework for thermophilic fungi. A further goal is to identify
the fungal orders harbouring thermophilic species and to re-
solve the evolutionary relationships among the thermophilic
and nonthermophilic species within these orders. A detailed
knowledge of the phylogenetic distribution of thermophilic
fungi would provide insights into the evolution of thermoph-
ily in fungi and help to identify closely-related mesophiles for
comparative studies to reveal the molecular mechanisms
underlying the ability to grow at high temperature. The phylo-
genetic analyses are complemented by experimental growthe
temperature relationships for fungal species reported to be
thermophilic. Using the criterion that a thermophilic fungus
is one that grows faster at 45
C than at 34
C, our phylogenetic
analyses suggest that the known thermophilic fungi belong to
the orders Sordariales, Eurotiales, Mucorales, and Onygenales.
Moreover phylogenetic reconstructions enabled us to correct
the placement of six thermophilic species.
Materials and methods
Growth at different temperatures
Growth performance of 30 fungal strains was examined:
22 have been mentioned in the literature as thermophilic or
thermotolerant (Maheshwari et al. 2000; Mouchacca 2000a)
and the remaining species can be regarded as mesophilic.
Cultures were grown on mycobroth agar plates adjusted to
pH 5.5, a condition suitable for all but one of the tested
strains, Acremonium alcalophilum, which was grown instead
on malt extract agar plates at pH 9.0. The agar plates were in-
oculated in the centre with 2 ml of a spore solution containing
either 500 or 10 000 spores per ml, or with a 6 mm diameter
agar plug cut out from the edge of an actively growing cul-
ture. Cultures were grown at 22
C, 34
C, 45
C, and 55
C un-
til differential growth was clearly visible, which took from 1 d
(Rhizomucor miehei) to 32 d (Pleurotus ostreatus), but in most
cases took fewer than 3 d. At this time relative growth perfor-
mance was recorded for each strain by estimating the rela-
tive surface area of the agar plates covered with fungal
mycelium at the different temperatures. Thus, a simple rank-
ing from strongest to weakest (or absent) growth was
obtained for each strain.
Data mining
For the protein-coding gene analysis we selected a set of
publicly available fungal genomes representing a wide taxo-
nomic spectrum of the fungi, from basal lineages to the Asco-
mycota and Basidiomycota, to which we added ten presumed
thermophilic and three mesophilic genomes sequenced re-
cently by the Genozymes research project (http://
A molecular phylogeny of thermophilic fungi
www.fungalgenomics.ca/wiki/Fungal_Genomes). In total, 86
fungal genomes were used in this study, of which 12 belong
to known or presumed thermophilic species. Publicly avail-
able genomes were accessed via the Joint Genome Institute
portal (http://genome.jgi-psf.org/), the Broad Institute Fungal
Genome Initiative web site (http://www.broadinstitute.
org/scientific-community/science/projects/fungal-genome-
initiative/fungal-genome-initiative), and NCBI GenBank
http://www.ncbi.nlm.nih.gov/genbank/. We chose two
widely used phylogenetic markers, Rpb1 (DNA-directed
RNA polymerase II subunit RPB1) and Rpb2 (DNA-directed
RNA polymerase II subunit RPB2) (Liu & Hall 2004; Matheny
2005; James et al. 2006). Two additional markers, Tsr1
(TwentyS rRNA accumulation protein 1, Ribosome biogene-
sis protein 1) and Mcm7 (minichromosome maintenance
protein 7, DNA replication licencing factor mcm7), that had
been demonstrated by others to be high quality phylogenetic
markers (Aguileta et al. 2008; Schmitt et al. 2009) were sam-
pled initially, but only the Mcm7 sequences were kept after
topological congruence of all four markers was rejected by
a likelihood-ratio test implemented in Concaterpillar version
1.4 (Leigh et al. 2008). Thus, only the subset containing
Mcm7, Rpb1, and Rpb2 sequences with topological congru-
ence stronger than the default p cutoff value of 0.05 were
retained. The three markers generally occur as single-copy
genes in fungal genomes. However, the genome of Allomyces
macrogynus contains two nearly identical sequences each for
Rpb1 and Rpb2. In this case, we selected the copies that had
accumulated fewer changes in comparison to sequences
from other basal fungi. Blastp and tblastn (Altschul et al.
1997) searches were conducted to retrieve orthologs of
Mcm7, Rpb1, and Rpb2 protein sequences from genome re-
sources. For each search the single best hit to the query se-
quence was retrieved and identified as the ortholog. In the
few cases where multiple top hits were very similar, the
orthologs were identified by detailed manual examination.
Since most thermophilic species belong to the Sordariales and
the Eurotiales (Salar & Aneja 2007), we retrieved sequences from
additional species in these two orders to examine the relation-
ship between the thermophiles and closely-related mesophiles.
In this case, we used the rDNA sequences of the ribosomal
repeat unit, which includes the nuclear 28S and 18S genes and
the 5.8S gene with the noncoding internal transcribed spacers
(ITS) 1 and 2 for analysis. We used NCBI taxonomy (http://
www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html) to
select the species in the Sordariales and the Eurotiales. Priority
was given to taxa for which at least two of the three ribosomal
genes were available. However, to achieve a representative cov-
erage of the orders, species for which only one gene was avail-
able were also included.
PCR amplification and sequencing of rDNA
We generated sequences for the three ribosomal loci using
standard PCR protocols and primer combinations ITS1/ITS4
(White et al. 1990) or NSI1/NLB4 (Martınez et al. 2005) for the
5.8S/ITS region, NS1/NS8 (White et al. 1990), or SR4/SR8R for
the 18S rRNA gene and LR0R/LR7 or LR3R/LR3 for the 28S
rRNA genes (Vilgalys and Hester 1990, Rehner and Samuels
1995; www.biology.duke.edu/fungi/mycolab/primers.htm).
Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
3
Conditions for the PCR amplification of ITS sequences were
as follows: initial denaturation at 98
C for 2 min, followed
by 35 cycles with 98
C denaturation for 30 s, annealing at
55
C for 40 s, extension at 72
C for 1 min, followed by final
extension for 7 min at 72
C. Conditions for large subunit
(LSU) were the same, but the annealing temperature was
40
C and the extension time was 2 min. Conditions for the
PCR amplification of small subunit (SSU) used were: 35 s de-
naturation at 98
C, 55 s annealing at 53
C, and 35 s exten-
sion at 72
C for the first 14 cycles, 2 min extension in the
next 11 cycles and 3 min in the last 15 cycles, which was fol-
lowed by a final extension at 72
C for 10 min. PCR products
were purified using the Agencourt AMPure (Beckman Coulter
Genomics, Danvers, MA, U.S.A.) purification system accord-
ing to the manufacturer’s recommendations. Sequencing re-
actions were performed on an ABI 3730xl DNA Analyzer
(Applied Biosystems, Carlsbad, CA, U.S.A.). All strains and se-
quences used for this study are listed in Supplementary
Table 1.
Phylogenetic analyses
For analysis of protein-coding genes, each dataset was
aligned with the online version of MAFFT using the E-INS-
I setting (Katoh et al. 2002, 2005). Aligned sequences were
inspected manually in Mesquite (version 2.74) followed by
corrections (Maddison & Maddison 2009). Changes to the
original alignment included a trimming of the N- and C-ter-
minal ends, removal of gap-rich areas with low homology
between sequences and removal of positions occupied by
only a single sequence. The best amino acid substitution
model that did not depend on the estimation of proportion
of invariable sites was identified with ProtTest using the
Aikaike Information Criterion (AIC) (Abascal et al. 2005).
Maximum likelihood analyses were performed with RAxML
7.2.6 making use of both the rapid maximum likelihood and
rapid bootstrapping option (Stamatakis 2006). Branch sup-
port for each dataset was estimated with 100 bootstrap
replicates.
For analysis of nuclear rDNA genes, we used 129 se-
quences in the concatenated Sordariales matrix, of which 56
were ITS, 47 were LSU, and 26 were SSU sequences. Se-
quences from Diaporthe eres and Cryphonectria parasitica
were included for rooting purposes. The concatenated Euro-
tiales matrix contained 152 sequences, of which 59 were
ITS, 54 were LSU, and 39 were SSU sequences. Sequences
from the Onygenales, including the thermophile Malbranchea
cinnamomea, were included for rooting purposes. Multiple se-
quence alignments were performed in MAFFT using the auto
setting, followed by manual refinements. Each combined
dataset was analyzed in a single partition by conducting
1000 rapid bootstrap searches using the rapid bootstrap algo-
rithm followed by a rapid maximum likelihood search for
the best tree topology under the GTRGAMMA model setting
implemented in RAxML 7.2.6 (Stamatakis 2006; Stamatakis
et al. 2008). Sequences generated for this study have been de-
posited in GenBank and the assigned accession numbers are
listed in Supplementary Table 1. Alignments can be viewed
at TreeBASE (http://purl.org/phylo/treebase/phylows/study/
TB2:S12101).
4 I. Morgenstern et al.

between 45
C and 34
C (Supplementary Fig 1). The 16 species

Results
Temperature dependence of growth
All fungal species included in the temperature-dependent
growth trial grew under at least one of the selected tempera-
tures (22
C, 34
C, 45
C, 55
C) indicating viability of the inoc-
ulum sources (Supplementary Fig 1; Table 1). Optimum
growth at 55
C was recorded for only three species (Chaeto-
mium thermophilum var. coprophilum, Thermomyces ibadanensis,
and Talaromyces emersonii), whereas 16 species showed opti-
mum growth at 45
C (Supplementary Fig 1; Table 1). For two
species the optimum growth temperature was ambivalent:
Malbranchea cinnamomea performed equally well at 55
C and
45
C, and Thermomyces stellatus showed no difference
that were able to grow at 55
C but failed to grow at 22
C are
considered thermophiles according to the definition of
Cooney & Emerson (1964) (Table 1). Only two species which
were capable of growth at 55
C, Thielavia australiensis and Cor-
ynascus thermophilus, also grew at 22
C. Based on their ability
to grow above 50
C and showing better growth at 45
C than at
34
C, T. australiensis and C. thermophilus should be regarded as
thermophilic. Three species (Remersonia thermophila, Calcaris-
poriella thermophila, T. stellatus) failed to colonize the agar
plates at 55
C but grew best at 45
C. Growth at 22
C was
weak for all three species. Based on these growth characteris-
tics we suggest R. thermophila and C. thermophila could be
regarded as thermophiles, whereas T. stellatus is better de-
scribed as a thermotolerant species due to equally good
growth rates at 34
C and 45
C.

Phylogenetic analyses based on protein-coding genes

Table 1 e Temperature dependence of growth. The
results from the temperature-dependent growth trial The 88 sequenced genomes (Table 2) used for the phyloge-
presented in Supplementary Fig 1 are summarized here. nomic part of this study included 57 species from the Ascomy-
Time when growth performance was ranked is given in cota, 20 species from the Basidiomycota, and nine species from
days (d) after inoculation. The relative growth basal fungal lineages. In addition, the fruitfly Drosophila mela-
performance of each organism is indicated by numbers: nogaster and the plant Arabidopsis thaliana were included for
0, no growth; 1e4, increasing rate of growth.
rooting purposes. Orders known to harbour thermophilic spe-
Species Growth Growth temperature cies are overrepresented, whereas fungal groups with many
time genomes available but lacking known thermophilic species,
55
C 45
C 34
C 22
C
like the Saccharomycotina, were less intensely sampled.
Thermophilic
Orthologs for the phylogenetic markers Rpb1, Rpb2, and
Chaetomium thermophilum 4 d 3 2 1 0
Mcm7 were successfully identified and retrieved from every
Thermomyces ibadanensis 3 d 3 2 1 0
Talaromyces emersonii 3 d 3 2 1 0 genome studied. The markers were used to reconstruct a phy-
Malbranchea cinnamomea 11 d 3 3 1 0 logeny of the fungi (Fig 1). Of the 264 sequences retrieved, 219
Thielavia terrestris 4 d 2 3 1 0 were full-length and 45 were partial sequences (Table 2). Re-
Thermomyces lanuginosus 5 d 2 3 1 0 gions that apparently did not exhibit true homology were ex-
Myceliophthora hinnulea 3 d 2 3 1 0 cluded from analysis. Examples of these regions are areas
Paecilomyces 3 d 2 3 1 0
with low amino acid complexity, gap-rich areas or regions
byssochlamydoides
Scytalidium thermophilum 3 d 2 3 1 0
dominated by short repeat motifs. Therefore the aligned
Myriococcum thermophilum 3 d 2 3 1 0 Mcm7 sequences had a length of 1188 characters of which
Rhizomucor miehei 1 d 2 3 1 0 722 were retained for further analysis. The Rpb1 sequence
Melanocarpus albomyces 3 d 2 3 2 0 alignment had 2736 characters of which 1435 characters
Rhizomucor pusillus 2 d 1 3 2 0 were included in further analysis, and the Rpb2 dataset had
Myceliophthora thermophila 4 d 1 3 2 0
a length of 2648 characters of which 1172 characters were
Talaromyces thermophilus 6 d 1 3 2 0
retained. The total length of the concatenated alignment, in-
Thermoascus crustaceus 2 d 1 3 2 0
Corynascus thermophilus 2 d 2 4 3 1 cluding the trimmed Mcm7, Rpb1, and Rpb2 datasets was
Thielavia australiensis 5 d 2 4 3 1 thus 3329 characters and the proportion of gaps and undeter-
Calcarisporiella thermophila 6 d 0 3 2 1 mined characters was 3.3 %.
Remersonia thermophila 11 d 0 3 2 1 Maximum likelihood analysis of the combined amino acid
Thermotolerant sequence alignment resulted in a tree with high bootstrap sup-
Thermomyces stellatus 13 d 0 3 3 1 port (BSS) values even in deep internal nodes (Fig 1). Only two
Phanerochaete 3d 0 3 3 1 nodes received less than 50 % BSS, and five nodes received sup-
chrysosporium port between 50 % and 70 %. Of the remaining 78 nodes receiv-
Mesophilic ing support above 70 %, 61 nodes received 100 % BSS. More
Acremonium alcalophilum 11 d 0 0 2 1 importantly almost every major taxonomic unit from the sub-
Chaetomium globosum 5 d 0 0 2 1 kingdom level down to the class level receives 100 % BSS, in-
Amorphotheca resinae 7 d 0 0 2 1 cluding the Dikarya (subkingdom), Basidiomycota, Ascomycota
Gloeophyllum trabeum 8 d 0 0 2 1
(phyla), Saccharomycotina, Pezizomycotina, Pucciniomycotina, Usti-
Pleurotus ostreatus 32 d 0 0 1 2
Trametes versicolor 6 d 0 0 1 2
laginomycotina (subphyla), Eurotiomycetes, Leotiomycetes, Sordar-
Aureobasidium pullulans 11 d 0 0 0 1 iomycetes, Dothideomycetes, and Agaricomycetes (classes). The
Lentinula edodes 19 d 0 0 0 1 only orders in our analysis receiving less than 100 % BSS sup-
port are the Agaricales (87 % BSS with the exclusion of

Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
A molecular phylogeny of thermophilic fungi 5

Table 2 e Genomes and sequence status of marker genes used in this study. Species with genome sequence used in this
study are listed by class and within each class alphabetically. C, complete sequence used for analysis; 1, N-terminus
missing; 2, C-terminus missing; 3, internal gaps in sequence.
Species Strain Data source Marker sequences

Mcm7 Rpb1 Rpb2

Blastocladiomycetes
Allomyces macrogynus ATCC 38327 Broad C C C

Chytridiomycetes
Batrachochytrium dendrobatidis JEL 423 JGI C C 1
Spizellomyces punctatus DAOM BR117 Broad C C 2

Mucoromycotina incertae sedis

Calcarisporiella thermophila CBS 279.70 Genozymes C C C
Mucor circinelloides CBS 277.79 JGI C 1, 2 1, 2
Phycomyces blakesleeanus NRRL 1555 JGI C C C
Rhizomucor miehei CBS 182.67 Genozymes C C 2
Rhizomucor pusillus CBS 183.67 Genozymes 1 C C
Rhizopus oryzae RA99-880 Broad C 1, 2 C

Dothideomycetes
Alternaria brassicicola ATCC 96836 WUGC (via JGI) C 2, 3 C
Aureobasidium pullulans ATCC 62921 Genozymes C 1 C
Cochliobolus heterostrophus C5 JGI C C 2
Mycosphaerella fijiensis CIRAD86 JGI C C C
Mycosphaerella graminicola IPO323 JGI 1 C C
Phaeosphaeria nodorum SN15 Broad (via JGI) 1 C C
Pyrenophora tritici-repentis DW7 race 5 JGI C C 2

Eurotiomycetes
Ajellomyces capsulatus NAm1 Broad C C C
Aspergillus carbonarius ITEM 5010 JGI C 2 C
Aspergillus clavatus NRRL 1 Broad C C C
Aspergillus fumigatus Af293 JCVI (via NCBI) C C C
Aspergillus nidulans FGSC A4 Broad C C 1
Aspergillus niger ATCC 1015 JGI C C C
Aspergillus oryzae RIB 40 NITE (via NCBI) C C C
Coccidioides immitis RS Broad C C 1
Coccidioides posadasii C735 delta SOWgp JCVI (via NCBI) C C C
Malbranchea cinnamomea CBS 343.55 Genozymes C C C
Neosartorya fischeri NRRL 181 Broad C C C
Paracoccidioides brasiliensis Pb03 Broad C C C
Penicillium chrysogenum Wisc. 54-1255 DSM (via NCBI) C C C
Penicillium marneffei ATCC 18224 JCVI (via NCBI) C C C
Talaromyces stipitatus ATCC 10500 JCVI (via NCBI) C C C
Talaromyces thermophilus NRRL 2155 Genozymes C C C
Thermoascus crustaceus CBS 181.67 Genozymes C C C
Thermomyces lanuginosus ATCC 200065 Genozymes C C C
Trichophyton verrucosum HKl0517 Arthroderma Genome C C C
Project (via NCBI)
Uncinocarpus reesii 1704 Broad C 3 1, 2, 3

Leotiomycetes
Amorphotheca resinae DAOM 194228 Genozymes C C C
Botryotina fuckeliana B05.10 Broad C C 2
Pseudocercosporella herpotrichoides CBS 494.80 Genozymes C C C
Sclerotinia sclerotiorum 1980 UF-70 Broad 3 C C

Pezizomycetes
Tuber melanosporum Mel28 Genoscope (via NCBI) C 1, 3 1, 3

Saccharomycetes
Saccharomyces cerevisiae RM11-1a Broad C C C
Scheffersomyces stipites CBS 6054 JGI C C C
Lachancea thermotolerans CBS 6340 Genolevures (via NCBI) C C C

Schizosaccharomycetes
Schizosaccharomyces pombe 972h Broad C C C

(continued on next page)

Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
6 I. Morgenstern et al.

Table 2 e (continued )
Species Strain Data source Marker sequences

Mcm7 Rpb1 Rpb2

Sordariomycetes
Acremonium alcalophilum JCM 7366 JGI C C C
Chaetomium globosum CBS 148.51 Broad (via JGI) C C 2, 3
Corynascus thermophilus CBS 405.69 Genozymes 1 3 3
Cryphonectria parasitica EP 155 JGI C C C
Fusarium graminearum PH-1 Broad C C C
Magnaporthe oryzae 70-15 Broad C C C
Myceliophthora thermophila ATCC 42464 JGI C C C
Myriococcum thermophilum CBS 398.93 Genozymes C C C
Nectria haematococca mpV1, strain 77-13-4 JGI C C C
Neurospora crassa OR74A Broad C C 1, 2
Neurospora discrete FGSC 8579 matA JGI C C C
Neurospora tetrasperma FGSC 2509 matA JGI C C C
Podospora anserina S matþ Genoscope (via NCBI) C C C
Scytalidium thermophilum CBS 625.91 Genozymes C C C
Sordaria macrospora k-hell RUB (via NCBI) C C 3
Thielavia terrestris NRRL 8126 JGI C C 3
Trichoderma atroviride IMI206040 JGI C C C
Trichoderma reesei QM6a JGI 1 C C
Trichoderma virens Gv29-8 JGI C C C
Verticillium albo-atrum VaMs. 102 Broad C C C
Verticillium dahliae VdLs. 17 Broad C C C

Agaricomycetes
Agaricus bisporus H97_U1 JGI 1 C 2
Ceriporiopsis subvermispora B JGI 1 C C
Coprinopsis cinerea Okayama7#130 JGI C C 2
Gloeophyllum trabeum ATCC 11539 JGI C C C
Heterobasidion annosum TC 32-1 JGI C C C
Laccaria bicolor S238N-H82 JGI C C 1, 2
Phanerochaete chrysosporium RP-78 JGI C C C
Pleurotus ostreatus PC9 JGI C C C
Postia placenta MAD 698 JGI 1 1, 2 C
Schizophyllum commune H4-8 JGI 1 C C
Serpula lacrymans S7.3 JGI C C C
Trametes versicolor FP-101664 SS1 JGI C C C

Exobasidiomycetes
Malassezia globosa CBS 7966 P&G (via JGI) 1 1 C

Microbotryomycetes
Rhodotorula graminis WP1 JGI C C C
Sporobolomyces roseus IAM 13481 JGI 1 1 C

Pucciniomycetes
Melampsora larici-populina 98AG31_300 JGI C C C
Puccinia graminis CRL 75-36-700-3 JGI C C 1

Tremellomycetes
Cryptococcus neoformans H-99 JGI C C C
Tremella mesenterica DSM 1558 JGI 1 C C

Ustilaginomycetes
Ustilago maydis 521 Broad (via JGI) C C 1, 2

Outgroup
Arabidopsis thaliana TAIR (via NCBI) C C C
Drosophila melanogaster FlyBase (via NCBI) C C C

Abbreviations: Broad, Broad Institute of MIT and Harvard University; DSM, DSM Anti-Infectives; JCVI, J. Craig Venter Institute; JGI, DOE Joint Ge-
nome Institute; NITE, National Institute of Technology and Evaluation (Japan); P&G, Procter & Gamble; RUB, Ruhr University Bochum; TAIR, The
Arabidopsis Information Resource; WUGC, Washington University Genome Center.

Schizophyllum commune) and the Helotiales (78 % BSS). Moreover, (55 % BSS). In the Ascomycota, a clade containing Eurotiomycetes,
support for the Agaricomycotina is weak in our analysis (63 % Leotiomycetes, Sordariomycetes, to which the Dothideomycetes are
BSS), as is its sister relationship with the Ustilaginomycotina the sister clade, is supported by 88 % BSS.

Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
A molecular phylogeny of thermophilic fungi 7

Fig 1 e Maximum likelihood phylogeny inferred by amino acid sequence alignments. The amino acid sequences of Mcm7, Rpb1,
and Rpb2 were used for concatenated analysis. Bootstrap values are indicated at each node and are given in bold font for values
above 70. Thermophilic taxa are highlighted in red font. The tree is rooted with Arabidopsis thaliana and Drosophila melanogaster.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Morgenstern I, et al., A molecular phylogeny of thermophilic fungi, Fungal Biology (2012),
doi:10.1016/j.funbio.2012.01.010
8 I. Morgenstern et al.

Table 3 e Taxonomic classification of previously unresolved species.

Species GenBank taxonomy MycoBank Index Fungorum This study

Calcarisporiella Ascomycota, mitosporic Fungi, anamorphic Ascomycota Fungi, Basal fungal lineages,
thermophila Ascomycota fungi Mucoromycotina
Myriococcum Basidiomycota, mitosporic Fungi, Ascomycota Basidiomycota, Agaricomycetes Ascomycota, Sordariomycetes,
thermophilum Basidiomycota Sordariales, Chaetomiaceae
Scytalidium Ascomycota, Leotiomycetes, Anamorphic fungi Ascomycota, Leotiomycetes, Ascomycota, Sordariomycetes,
thermophilum mitosporic Leotiomycetes Helotiales Sordariales, Chaetomiaceae
Remersonia Ascomycota, Sordariomycetes, Anamorphic fungi Ascomycota, incertae sedis Ascomycota, Sordariomycetes,
thermophila Sordariales, mitosporic Sordariales Sordariales, Chaetomiaceae
Thermomyces Ascomycota; mitosporic Anamorphic fungi Ascomycota, Eurotiomycetes, Ascomycota, Eurotiomycetes,
lanuginosus Ascomycota Eurotiales Eurotiales
Malbranchea Ascomycota, Eurotiomycetes, Anamorphic fungi Ascomycota, Dothideomycetes, Ascomycota, Eurotiomycetes,
cinnamomea Onygenales, mitosporic Onygenales incertae sedis Onygenales
Acremonium Ascomycota, Sordariomycetes, Anamorphic fungi Ascomycota, Sordariomycetes, Ascomycota, Sordariomycetes,
alcalophilum Glomerellales, Plectosphaerellaceae Hypocreales, incertae sedis Glomerellales
was: Ascomycota; Sordariomycetes,
Hypocreales
Amorphotheca Ascomycota, Leotiomycetes Ascomycota, Ascomycota, Eurotiomycetes Ascomycota, Leotiomycetes
resinae Eurotiomycetes

The analysis of protein-coding genes provided clearer phylo-
genetic placement of seven species (Table 3). The thermophilic
species Calcarisporiella thermophila is supported as the sister
taxon of the Mucorales, which harbours the thermophilic spe-
cies Rhizomucor pusillus and Rhizomucor miehei along with the
mesophilic and thermotolerant taxa. Myriococcum thermophilum,
listed by NCBI taxonomy (http://www.ncbi.nlm.nih.gov/Taxon-
omy/taxonomyhome.html) as a mitosporic basidiomycete, is
now placed in the Sordariales. Scytalidium thermophilum also be-
longs to the Sordariales. Our analysis supports the inclusion of
Thermomyces lanuginosus in the Eurotiales in a sister relationship
with Talaromyces thermophilus. Thermoascus crustaceus is placed
on a different branch within the Eurotiales, basal to the asper-
gilli. The thermophile Malbranchea cinnamomea is placed in
a basal position of the Onygenales clade. The alkalophilic species
Acremonium alcalophilum are solidly supported as the sister
taxon of the two Verticillium species in the dataset and are
according to a new study placed in the Glomerellales (Reblova
et al. 2011). A recent study based on SSU and LSU sequences
also places A. alcalophilum in the Glomerellales (Summerbell
et al. 2011). Amorphotheca resinae (creosote fungus) is another
species of interest because of its unique biodegradation capac-
ity with poorly resolved taxonomic status. Our analysis sug-
gests inclusion or close relationship to the Helotiales.
Analysis of the Sordariales and Eurotiales based
on rDNA genes
Having identified the orders in which thermophilic taxa fall,
we investigated the intraorder relationship of thermophiles
and mesophiles with available nucleotide sequences from
the ribosomal repeat unit. The results from the rDNA analyses
obtained from concatenating nucleotide sequences from the
large and small ribosomal subunits and the ITS region give
a more detailed picture about the distribution of thermophilic
taxa in the Sordariales and Eurotiales. In both orders, thermo-
philes are not monophyletic but occur throughout the tree
intermixed with mesophilic taxa. Since the support values
tend to be much lower compared with the analysis based on
protein sequences, a solid interpretation of the species rela-
tionships remains difficult. This is more evident in the Sordar-
iales analysis, whereas the Eurotiales tree is comparatively
better resolved (Figs 2 and 3).
For the Sordariales we have included sequences from spe-
cies belonging to the Sordariaceae, Lasiosphaeriaceae, and Chae-
tomiaceae (Fig 2). The concatenated dataset for the Sordariales
contains 60 operational taxonomic units (OTUs), of which 21
are represented by all three genes, 27 are represented by two
genes (mostly ITS and LSU), and 12 are represented by only
one sequence. For 56 OTUs the ITS/5.8S sequence was avail-
able, for 26 OTUs the SSU was available, and 47 LSU sequences
were included. The combined dataset had a length of 3733
characters (634 ITS/5.8S, 1701 SSU, 1398 LSU).
Thermophilic species in the Sordariales belong exclusively
to the Chaetomiaceae. The tree reconstruction depicts the Chae-
tomiaceae with the exclusion of T. subthermophila as monophy-
letic and the Lasiosphaeriaceae as paraphyletic (Fig 2). However,
the low statistical support in the relevant parts of the tree does
not allow reconstruction of the relationships between the
Chaetomiaceae and Lasiosphaeriaceae with confidence. Only
the Sordariaceae is supported as a monophyletic family in the
Sordariales, but this conclusion is based on only seven species
from this family. The support values increase considerably to-
wards the terminal nodes, allowing recognition of relation-
ships for several species. Thermophilic taxa fall into three
major clades containing multiple species, and two species,
Melanocarpus thermophilus and Thielavia australiensis, which
do not cluster with other thermophiles. Thielavia australiensis
is supported as the sister taxon of Thielavia basicola (95%
BSS), the type species of the highly polyphyletic genus Thiela-
via. Myceliophthora thermophila, Myceliophthora hinnulea, Cory-
nascus thermophilus, and Myriococcum thermophilum constitute
the first major clade supported by 99 % BSS. However, the
two Myceliophthora species and the two C. thermophilus strains
do not group together. In the second major clade, Remersonia
thermophila is strongly supported as the sister taxon of the Scy-
talidium-Torula-Humicola complex (100 % BSS) (Straatsma &
Samson 1993). The third clade containing Thielavia terrestris,

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doi:10.1016/j.funbio.2012.01.010
A molecular phylogeny of thermophilic fungi 9

Fig 2 e Maximum likelihood phylogeny for the Sordariales based on concatenated SSU, ITS/5.8S, and LSU nucleotide
sequence alignments. Bootstrap values of 50 and higher are given next to each node, values above 70 are indicated in bold
font. Thermophilic taxa are highlighted in red font. Black hexagon indicates thermophilic strains included in the
temperature-dependent growth trial. Families are indicated to the right of vertical bars (monophyletic) or curly brackets
(phyletic status unresolved). The tree is rooted with Diaporthe eres and Cryphonectria parasitica belonging to the Diaporthales.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Chaetomium thermophilum, and Melanocarpus albomyces does
not receive support from the bootstrap analysis.
The concatenated dataset for the Eurotiales contains 63
OTUs, of which 28 are represented by all three genes, 33 are
represented by two genes (mostly ITS and LSU), and two
are represented by only a single gene. The combined dataset
had a length of 3565 characters (1741 SSU, 620 ITS/5.8S,
1204 LSU).
According to NCBI taxonomy (http://www.ncbi.nlm.nih.-
gov/Taxonomy/taxonomyhome.html) the Eurotiales contain
two families, the Elaphomycetaceae and the Trichocomaceae.
In our dataset, only five taxa belong to the former family,
whereas the remainder is considered to be part of the taxon-
rich Trichocomaceae. Seven species of Eurotiales included
in the analysis are regarded as thermophiles: Thermoascus aur-
antiacus, Thermoascus crustaceus, Talaromyces thermophilus,
Talaromyces leycettanus, Talaromyces byssochlamydoides, Talaro-
myces emersonii, and Thermomyces lanuginosus.
Thermophiles in the Eurotiales are not monophyletic but ap-
pear in three species pairs and as single sequences throughout
the tree (Fig 3). The two Thermoascus species receive strong
support as being monophyletic (100 % BSS). So do the species
pairs T. byssochlamydoides and T. emersonii, and T. thermophilus
and T. lanuginosus (each 100 % BSS). The two strains of T. leycet-
tanus included in this analysis occupy different positions in the
tree, indicating that they are not conspecific (Fig 3). They are

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doi:10.1016/j.funbio.2012.01.010
10 I. Morgenstern et al.

Fig 3 e Maximum likelihood phylogeny for the Eurotiales based on concatenated SSU, ITS/5.8S, and LSU nucleotide sequence
alignments. Bootstrap values of 50 and above are given next to each node, values above 70 are indicated in bold font.
Thermophilic taxa are highlighted in red font. Black hexagon indicates thermophilic strains included in the temperature-
dependent growth trial. Species belonging to the Elaphomycetaceae are indicated to the right of vertical bars. The tree is rooted
with species belonging to the Onygenales. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

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doi:10.1016/j.funbio.2012.01.010
A molecular phylogeny of thermophilic fungi
not closely related to other Talaromyces isolates or any other
thermophile. In the Onygenales outgroup, Malbranchea cinnamo-
mea comprises three strains that occupy two positions. Strains
M. cinnamomea CBS 960.72 and CBS 343.55 share the same posi-
tion based on their identical SSU sequences. Strain M. cinnamo-
mea IFM 41309 clusters with Malbranchea pulchella based on
identical LSU sequences. However, strain IFM 41309 is sup-
posed to be an alternative strain designation for CBS 343.55
(http://www.ncbi.nlm.nih.gov/nuccore/AB359414.1). Our anal-
ysis does not support this claim and suggests that IFM 41309 is
a strain of M. pulchella.
Discussion
The classification of fungi with respect to their temperature
requirements has been a topic for debate. Cooney &
Emerson (1964) based their classification solely on upper and
lower temperature boundaries, which they claimed are easier
to establish than optimum temperatures. However, deciding
where to establish boundaries is somewhat subjective and
further blurred by Cooney and Emerson’s definition of the up-
per limit of thermotolerant fungi as ‘has a thermal maximum
near 50
C.’. Setting the lower temperature limit for thermo-
phile growth at 20
C has also been controversial (Mouchacca
1999; Maheshwari et al. 2000). Species like Thielavia australien-
sis, which grow as well above 50
C as they do below 20
C, are
not accounted for by this definition. In practice, the distinction
between thermotolerant and thermophilic is not always
made and sometimes all fungi capable of growing above
45
C are considered thermophilic. This blurs the distinction
between mesophiles and thermophiles since growth up to
45
C may still be considered as within the mesophile limits
(Maheshwari et al. 2000).
Our temperature growth experiments allowed us to ad-
dress some of the controversial aspects of this issue and to
clarify the status of some species. In a modification of Emer-
son and Cooney’s definition, we regard thermophilic species
as those which grow faster at 45
C than at 34
C, even if
growth below 20
C is observed. Accordingly, T. australiensis,
Remersonia thermophila, and Calcarisporiella thermophila are
classified by us as thermophiles even though they may have
been described elsewhere as thermotolerants (De Hoog 1974;
Seifert et al. 1997; Mouchacca 2000b). We suggest the term
‘thermotolerant’ applies to fungi such as Thermomyces stellatus
and Phanerochaete chrysosporium which are capable of growth
at or above 40
C, distinguishing them from most mesophiles,
but have a temperature optimum below this threshold. Due
to high strain variability in some species, as well as differ-
ences in media and source of inoculum used to establish tem-
perature profiles, ambiguities will persist in defining the
correct temperature type for a few species (Romanelli et al.
1975; Maheshwari et al. 2000).
A number of phylogenomic and multigene studies have
led to an improved understanding of fungal phylogeny
(Fitzpatrick et al. 2006; James et al. 2006; Robbertse et al. 2006;
Wang et al. 2009) that has resulted in the adoption of a vastly
improved classification of the fungi (Hibbett et al. 2007). How-
ever, these studies are not completely congruent and the clas-
sification of certain taxonomic groups remains problematic.
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doi:10.1016/j.funbio.2012.01.010
11
The relationships depicted in our tree (Fig 1) do not deviate
widely from the composition vector tree (CVTree) (Wang
et al. 2009). In the Basidiomycota, both reconstruction methods
resolve the Ustilaginomycotina closest to the Agaricomycotina
and place the Pucciniomycotina in the basal position. However,
this architecture is supported by 55 % BSS in this study (Fig 1)
and by 35 % BSS in the CVTree (Wang et al. 2009). A conflict
between the two tree reconstructions is observed in the Asco-
mycota regarding the placement of the Dothideomycetes. Our
analysis places the Dothideomycetes with 88 % BSS basal to
the Eurotiomycetes, Leotiomycetes, and Sordariomycetes. This
topology is also supported by other studies (Fitzpatrick et al.
2006; Robbertse et al. 2006; Spatafora et al. 2006), whereas in
the CVTree the Dothideomycetes are the sister group of the Euro-
tiomycetes, a topology for which Fitzpatrick and Robbertse also
find support, depending on the tree reconstruction method
used (Fitzpatrick et al. 2006; Robbertse et al. 2006). These
results indicate that the position of the Dothideomycetes will re-
quire a more in-depth analysis to be resolved.
Various studies have investigated the phylogenetic rela-
tionship of taxa considered to constitute the Sordariales using
both morphological and molecular tools (Huhndorf et al. 2004;
Cai et al. 2006a, b; Tang et al. 2007; Greif et al. 2009). Huhndorf
et al. (2004) and Cai et al. (2006b) suggested including only the
families Chaetomiaceae, Lasiosphaeriaceae, and Sordariaceae in
the Sordariales. Statistical support for the monophyly of the
Sordariales has been reported previously (Zhang et al. 2006),
but is absent in other studies (Huhndorf et al. 2004; Cai et al.
2006b), or variable depending on data matrix used and phylo-
genetic method employed (Tang et al. 2007). Our analysis sup-
ports the Sordariales as a clade. On the family level our analysis
reveals the paraphyletic nature of the Chaetomiaceae and
Lasiosphaeriaceae. The Sordariaceae receive strong support as
a monophyletic family, but only if the genus Diplogelasinospora
is not considered belonging to this family. These findings
agree with previous studies and classification systems, associ-
ating Diplogelasinospora with the Lasiosphaeriaceae (Huhndorf
et al. 2004; Lumbsch & Huhndorf 2007).
The Eurotiales have received the attention of many phylo-
genetic studies due to the presence of the genera Aspergillus
and Penicillium and their associated teleomorphs, which are
of tremendous importance for industry and research. How-
ever, the majority of these studies centre on the genus level,
focussing on elucidating species relationships in these two
species-rich genera (Pitt & Samson 1990; Wang & Zhuang
2007). Relationships above the genus level have also been in-
vestigated and suggest the monophyly of the class Eurotiomy-
cetes (Lutzoni et al. 2004; Geiser et al. 2006; Spatafora et al. 2006),
the subclass Eurotiomycetidae (Geiser et al. 2006), and the order
Eurotiales (Geiser & LoBuglio 2001; Geiser et al. 2006). The NCBI
taxonomy lists only two families in the Eurotiales, the Trichoco-
maceae and the Elaphomycetaceae. Based on SSU sequences, the
Elaphomycetaceae may be monophyletic but support is either
weak or based on limited taxon sampling (Landvik et al.
1996; Miller et al. 2001). Our analysis, which includes se-
quences from three genera (Monascus, Pseudotulostoma, and
Xeromyces), does not support the monophyly of the Elaphomy-
cetaceae (Fig 3). Statistical support on the internal nodes in the
Eurotiales tree tends to be weak apart from nodes closer to the
tree termini, making inferences of the evolutionary history of
12
the species in this order difficult. The tree does, however, in-
dicate that we are still far away from a natural classification
system in the Eurotiales. Most genera appear as polyphyletic.
Earlier studies focussing on the anamorphs indicated that Pen-
icillium is not monophyletic, but that Aspergillus might be
(Berbee et al. 1995). Talaromyces seems to be highly polyphy-
letic. The two strains of Talaromyces leycettanus that appear
in different parts of the tree contribute to this picture of taxo-
nomic uncertainty. Talaromyces leycettanus strains NRRL 5178
and CBS 398.68 both represent the type strain (holotype CBS
398.68) (Peterson et al. 2010).
The uncertain taxonomic affiliations of many thermophilic
fungi are a challenge for applied studies investigating the enzy-
mology of these fungi. For fungi which do not belong to one of
the few orders known to harbour thermophiles, the taxonomic
lineage is often incomplete and only as specific as, e.g.
‘mitosporic Ascomycete’. Few phylogenetic studies have been
undertaken specifically focussing on thermophilic species.
Two studies use the 5.8S/ITS sequences to reconstruct the phy-
logeny of thermophilic fungi (Sharma et al. 2008; Pan et al. 2010)
and the latter study employed also LSU sequences for phyloge-
netic analysis. Both reconstructions use only a relatively small
number of taxa and do not aim to resolve the relationships to re-
lated mesophilic taxa: such relationships are important in the
context of determining the molecular basis of thermophily.
This study is thus the first to focus on the phylogeny of
thermophilic species including related mesophilic taxa. Our
results confirm that true thermophily is not widespread in
the fungal kingdom. The thermophilic taxa of uncertain taxo-
nomic status included in this study did not fall outside one of
the orders already known to harbour thermophiles, i.e. the
Sordariales, Eurotiales, Onygenales (all Ascomycota) and the
Mucorales (basal Fungi). Calcarisporiella thermophila may be
placed in an order not yet known to contain thermophiles,
but currently we cannot exclude a placement in the Mucorales.
A recent study supports our result placing C. thermophila in the
Mucoromycotina close to Endogonales and Mucorales (Hirose et al.
2011). Only a few closely-related species in the Mucorales and
Onygenales have adopted a thermophilic lifestyle. In the Euro-
tiales, thermophiles are not abundant but occur in different
positions of the phylogeny, indicating a potentially complex
evolutionary history for this trait. The Sordariales are the fun-
gal order where thermophiles have diversified the most; how-
ever, they are restricted to lineages that are recognized as
constituting the Chaetomiaceae. According to the phylogenetic
reconstructions of the Sordariales and Eurotiales (Fig. 2 and 3), it
seems plausible that thermophily in the Chaetomiaceae has
a single origin and was lost multiple times whereas multiple
independent gains seem to be more likely in the Trichocoma-
ceae (Eurotiales). However, to test this hypothesis it will be nec-
essary to obtain species phylogenies for both families that
receive solid support even for the deeper nodes.
Recently it has been demonstrated that the genera Myce-
liophthora and Corynascus are closely related and most Corynas-
cus species should be integrated into Myceliophthora (van den
Brink et al. 2011). Our analysis suggests that Myriococcum ther-
mophilum is also a member of the Myceliophthorae
Corynascus species complex.
Results from this study support the placement of thermo-
philic fungi in only three orders of Ascomycota and in the basal
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doi:10.1016/j.funbio.2012.01.010
I. Morgenstern et al.
fungi. The two isolates of Basidiomycota described by Straatsma
et al. (1994) were characterized as thermophilic based on their
ability to grow at 45
C. Without additional analyses on tem-
perature profile of growth, it is not certain whether these are
thermophilic or thermotolerant organisms. The current place-
ment of M. thermophilum in the Sordariales eliminates the ambi-
guity that this species may belong to the Basidiomycota. In
summary, there is no strong evidence to support the existence
of thermophilic species in the Basidiomycota.
Acknowledgements
This work was supported by the Bioconversion Network of the
Natural Sciences and Engineering Research Council of Can-
ada, Genome Canada, and Ge nome Que  bec. We thank Wendy
Findlay, Ian Reid, Nick O’Toole, and David Mason for bioinfor-
matics support.
Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.funbio.2012.01.010.
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