Bull. Natl. Mus. Nat. Sci., Ser. B, 34(4), pp. 161–173, December 22, 2008

Molecular Phylogenetics of Geastrales with Special Emphasis on the
Position of Sclerogaster
Kentaro Hosaka1 and Michael A. Castellano2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba 305–0005, Japan
2
USDA, Forest Service, Northern Research Station, Forestry Sciences Laboratory,
3200 Jefferson Way, Corvallis, OR 97331, USA

Abstract Molecular phylogenetic analyses for the order Geastrales (Agaricomycetes, Basidiomycota, Fungi) were conducted based on a two gene dataset with increased taxon sampling of thegenus Sclerogaster. The placement of Sclerogaster in Geastrales, and the monophyly of the genus
were strongly supported in all analyses. Sclerogaster forms one of the distinct lineages within
Geastrales, but its exact position within Geastrales remains unresolved. Bayesian analyses support
the sister relationship between Geastraceae and Sclerogaster without significant posterior probabilities. Most analyses under parsimony criterion supported the sister relationship between Sclerogaster and Schenella, but lack significant bootstrap values. The topology of Geastrales indicatedthat the truffle-like form may be an ancestral morphology of the earthstar fruit body-type. Biogeographical patterns of Sclerogaster indicate that long distance dispersal may be the most importantfactor, and this may be associated with its saprotrophic habit.
Key words : atp6, Basidiomycota, fungi, LSU, Phallomycetidae, Phylogeny, Sclerogaster, Systematics.

Introduction
The subclass Phallomycetidae (Agaricomycetes, Basidiomycota) is commonly known as
the gomphoid-phalloid fungi (Hibbett and Thorn,2001). Because of its morphological diversity,
traditional taxonomy failed to recognize the gomphoid-phalloid fungi as a single entity. Hibbett etal. (1997) first demonstrated the monophyly of
the gomphoid-phalloid fungi, which were subsequently supported by additional studies (Binderand Hibbett, 2002; Hibbett and Binder, 2002; Hibbett and Thorn, 2001; Hosaka et al., 2006;
Humpert et al., 2001; James et al., 2006; Lutzoni
et al., 2004; Matheny et al., 2007; Moncalvo etal., 2002; Pine et al., 1999). The first attempt toincorporate these results into a formal classification scheme by treating the gomphoid-phalloid
fungi as a single order Phallales was made by
Kirk et al. (2001). Later Hosaka et al. (2006) analyzed a 5-gene dataset of the gomphoid-phalloid

fungi. Their results clearly showed that there are
four major clades within the larger gomphoidphalloid clade, and each of the smaller clades
were well-supported and in two cases, i.e., Phallales and Gomphales, with a long history of ordinal status. Hosaka et al. (2006) elevated Phallales
sensu Kirk et al. to subclass status, and proposed
the new subclass Phallomycetidae. Four major
clades within the Phallomycetidae each received
ordinal status; Phallales, Gomphales, Hysterangiales, and Geastrales.
These phylogenetic analyses revealed many
previously unexpected lineages, and taxonomic
revisions for several taxa were made accordingly.
However, the taxonomic revisions for many taxa
have not been conducted yet. The order Geastrales is especially problematic because several
genera, such as Phialastrum and Trichaster, were
not included in the phylogenetic analyses although they traditionally have been placed inGeastraceae (Sunhede, 1989). In addition, Zeller

162

Kentaro Hosaka and Michael A. Castellano(1948) described Broomeiaceae in Lycoperdales,
but it is unclear whether this family is moreclosely related to Geastrales or to other homobasidiomycetes.
One of the unexpected lineages within Geastrales includes the genus Sclerogaster Hesse,
which possesses hypogeous (truffle-like) basidiomata with a yellow or green tinted gleba (Fig.
3) and yellow to brown, warty basidiospores (Fig.
4) (Castellano et al., 1989). When the genus was
described by Hesse (1891), it was placed in the“Hymenogastreen”, which was considered an artificial assemblage of truffle-like basidiomycetes(Clémençon et al., 2007). Fischer (1900) later
placed the genus in the family Hymenogastraceae, but its phylogenetic affinity remained unresolved. Since then, several attempts were madeto phylogenetically place the genus. Some authors (Jülich, 1981; Castellano et al., 1989) considered Sclerogaster to be closely related to Octaviania (Boletales), but others (Dodge andZeller, 1936; Zeller and Dodge, 1935; Kreisel,1969) implied close affinity of Sclerogaster withHydnangium(Agaricales).
Furthermore,
Malençon (1931) and Heim (1971) implied aclose affinity of the genus with Russulales.
Therefore, Sclerogaster have been placed in atleast three distinct orders of Basidiomycota.
Molecular phylogenetic analyses strongly suggested placement of the genus in Geastrales withclose affinity to the Geastraceae and Schenellaceae (Pyrenogastraceae) (Hosaka et al., 2006).
This was surprising because a close relationshipbetween Sclerogaster and Geastraceae had neverbeen postulated previously. Morphologically,
Sclerogaster, Geastraceae and Schenellaceae allpossess globose spores with a surface ornamented with spines or warts (Domínguez de Toledo
and Castellano, 1996; Sunhede, 1989; Castellano
et al., 1989). Sclerogaster differs from Geastraceae and Schenellaceae in having a green toyellow or orange gleba (Fig. 3) versus brown to
black gleba that turns powdery at maturity. However, only one species (Sclerogaster xerophilus)
was sampled from approximately 15 described
species, and more critically, no type species were

included. Additional taxon sampling is necessary
for a more definitive placement of the genus.
This study attempts to answer the questions of:(1) monophyly of the genus Sclerogaster, and (2)phylogenetic placement of the genus.
Materials and Methods
Taxon sampling, PCR, and DNA sequencing
Taxa sampled, along with GenBank accession
numbers, are listed in Table 1. A total of 51 taxa
(four outgroup and 47 ingroup taxa) were sampled for this study. The selection of ingroup andoutgroup taxa was based on the phylogeny ofprevious studies (Hosaka et al., 2006) to cover
the diversity of Geastrales.
DNA was extracted from glebal tissue of freshor dried fruiting bodies. The protocol generally
follows that of Doyle and Doyle (1987) but withthe following modifications. For fresh materials,
immature glebal tissue was soaked in DMSO
buffer (Seutin et al., 1991) with an addition of
100 mM Tris-HCl (pH 8.0) and 0.1 M sodium
sulfite (Na2SO3) under 4°C until extraction was
conducted. For dried materials, immature glebal
tissue was soaked overnight in modified DMSO
buffer under room temperature.
Soaked tissue samples were then ground in liquid nitrogen using mortar and pestle. After grinding, samples were immediately transferred to 1.5
mL tubes with 1,000 mL of 2X CTAB buffer(Doyle and Doyle, 1987) with an addition of
0.1M Na2SO3. Some taxa of Geastrales have apowdery spore mass at maturity, and the powdery
spore mass cannot easily be ground in liquid nitrogen. If the spore mass was the only tissue
available for DNA extraction, spores wereground using the bead beating protocol modified
from Munkacsi et al. (2006). Approximately 30
milligrams of dry basidiospores were mixed with
0.2 mg of 0.1 mm glass beads (BioSpec), 20 g of
1 mm chrome steel beads (BioSpec) and two 3
mm chrome steel beads (BioSpec), and frozen inliquid nitrogen for 5 minutes. The mixture wasbeaten at 30 Hz for 1 minute using the TissueLyser (Qiagen), frozen in liquid nitrogen for 3

Sclerogaster phylogeny

minutes, and then beaten again at 30 Hz for 1
minute. After grinding, 1,000 mL of 2X CTAB
buffer with an addition of 0.1 M Na2SO3 wasadded directly to the sample tubes.
Samples were incubated at 55°C overnight,
and centrifuged at 12,000 rpm for 5 minutes.
Only the aqueous phase was transferred to a new
tube, and precipitated tissue debris were discarded. The equal volume of the mixture of chloroform: isoamylalcohol (24 : 1) was added to thebuffer, mixed vigorously for two minutes, andcentrifuged at 12,000 rpm for 15 minutes. The
aqueous phase was pipetted out and transferred
to a new tube. This step of using chloroform wasconducted only once.
After transferring ca. 300 mL of the aqueous
phase, 1,000 mL of 6M sodium idodine buffer (6M NaI, 50 mM Tris-HCl (pH 7.4), 10 mM EDTA,
0.1 M Na2SO3) was added and mixed gently for 1
minute. Silica mixture was prepared following
the protocol of Rogstad (2003), and 25 mL of the
mixture was added to the samples. Samples wereincubated at 55°C for 1 hour, and centrifuged atfull speed for ca. 10 seconds. The supernatant
was discarded and 750 mL of washing buffer (10mL Tris-HCl (pH 7.4), 1mM EDTA, 100 mM
NaCl, 50% EtOH) was added, mixed briefly, andcentrifuged at full speed for ca. 5 seconds. Thiswashing step was repeated twice. After finishing
the washing step, samples were centrifuged one
more time at full speed for 10 seconds, the remaining washing buffer was pipetted out, andprecipitated silica was dried at room temperaturefor 30 minutes to 1 hour. Final elution was performed by adding 100 mL of ultra pure water,
mixed briefly, and incubated at 65°C for 15 minutes. Samples were centrifuged at 12,000 rpm for
1 minute, and supernatant layer was transferred
to a new tube and stored at 20°C until PCR
was performed.
DNA sequence data were obtained from two
independent loci: LR0R–LR3 region for nuclear
large subunit of rDNA (LSU) and ATPase subunit 6 (atp6). The primers and PCR protocols
were described previously (Kretzer and Bruns
1999; Vilgalys and Hester 1990). Cycle sequenc-

163

ing was performed following the manufacturer’s
instructions (Big Dye ver. 3.1, Applied Biosystems) using the same primers described above.
Phylogenetic analyses
DNA sequences were initially aligned using
Muscle v.3.6 (Edgar, 2004a, b), followed by manual alignment in the data editor of BioEdit ver.
7.0.1 (Hall 1999). Ambiguously aligned regions
and introns were excluded from the analyses. To
test for incongruence between the two individual
datasets, parsimony analyses of individual lociwere compared. First, 70 % bootstrap (BS) trees
were calculated (100 BS replicates with five random addition sequences, TBR and Multrees options off) including only the taxa with sequencesfrom both loci (36 taxa). These trees were used
as constraints in a different dataset (for example,
parsimony analysis of the atp6 dataset with the
LSU tree as a constraint), using the ‘Load Constraints’ option in PAUP version 4.0b10 (Swofford 2002). Parsimony analyses were conductedunder these constraints, keeping only the treesthat are compatible with these constraints. Comparisons of constraint and unconstraint trees weremade using the ‘Tree Scores’ option in PAUP
version 4.0b10 (Swofford 2002). Parsimony
based comparisons were performed by the Templeton test (Templeton 1983), using nonparametric pairwise tests option. Likelihood-based comparisons were performed by the Shimodaira–
Hasegawa test (Shimodaira and Hasegawa 1999),
using RELL optimization with 1,000 BS replicates. Significance of results was determined bya P-value of less than 0.05. After testing for incongruence, the individual gene datasets werecombined and phylogenetic (both parsimony andBayesian) analyses were conducted with a combined dataset of two loci as described above.
Parsimony analyses (Analyses 1–6, Table 2)
were conducted under the equally weighted parsimony criterion using PAUP version 4.0b10(Swofford 2002). Phylogenetic analyses wereconducted with a combined dataset of LSU andatp6 either with (51 taxaAnalysis 1, Table 1) orwithout (36 taxaAnalysis 2, Table 1) missing

164

Kentaro Hosaka and Michael A. Castellano

Fig. 1. A 50% majority rule consensus of Geastrales phylogeny derived from Bayesian analysis using combined
atp6 and LSU dataset for a supermatrix of 51 taxa (Analysis 1). Taxon names followed by voucher numbers,
and by area of distribution in bold face (only for the Sclerogastraceae clade). Nodes supported by 0.95
Bayesian posterior probability (PP) and by 70% parsimony bootstrap (BS) values are indicated by black
circles. Numbers on branches are nodal supports (Bayesian PP/parsimony BS values).

Sclerogaster phylogeny
Table 1.

165

Sequence data newly generated for this study and associated GenBank accession numbers.
Taxon

Herbarium

Specimen No.

LSU

atp6

Geastrum hariottii
Geastrum saccatum
Geastrum subiculosum
Geastrum subiculosum
Geastrum sp.
Geastrum sp.
Geastrum sp.
Geastrum sp.
Geastrum sp.
Geastrum sp.
Geastrum sp.
Geastrum sp.
Sclerogaster columellatus
Sclerogaster compactus
Sclerogaster compactus
Sclerogaster lanatus
Sclerogaster minor
Sclerogaster pacificus
Sclerogaster xerophilus
Sclerogaster sp.
Sclerogaster sp.
Sclerogaster sp.
Sclerogaster sp.
Sclerogaster sp.
Sclerogaster sp.

BR
OSC
BR
BR
BR
CUW
OSC
TNS
TNS
TNS
TNS
TNS
OSC
TNS
OSC
OSC
OSC
OSC
NY
OSC
OSC
NY
NY
TNS
TNS

33661/85
Trappe 23765
33313/34
B517
339380/95
MCA 1723
Trappe 26298
KH-6182003-7
KH-NC04-007
KH-NZ06-006
KH-NZ06-052
KH-NZ06-135
Trappe 8098
WSL-KH01
Trappe 6136
Hintz 783
Trappe 8720
Trappe 9011
Wright 1956
H4595
Trappe 15701
Zeller 7425
Zeller 8462
KH-NZ06-209
KH-NZ06-210——
FJ435966
FJ435967
FJ435968
FJ435969——
FJ435970————
FJ435971————
FJ435972
FJ435973
FJ435974
FJ435975
FJ435976
FJ435977
FJ435978
FJ435979
FJ435980
FJ435981
FJ435982
FJ435983
FJ435984

FJ435985
FJ435986
FJ435987
FJ435988
FJ435989
FJ435990
FJ435991
FJ435992
FJ435993
FJ435994
FJ435995
FJ435996——
FJ435997————
FJ435998——————
FJ435999————
FJ436000
FJ436001

data. The analyses 3–6 were conducted with asingle locus dataset with a minimum number oftaxa (36 taxaAnalyses 4 & 6) or more taxa
(Analyses 3 & 5). The analyses were performed
under the heuristic search option with tree bisection–reconnection (TBR) and Multrees option
on, and 1,000 replicates of random addition sequence were conducted. Support for the individual nodes was tested with BS analysis under theequally-weighted parsimony criterion. BS analysis was based on 500 BS replicates using theheuristic search option (TBR and Multrees options on), with five random addition sequences.
Bayesian analyses (Analyses 7–12, Table 3)
were conducted using MrBayes version 3.0b4(Huelsenbeck and Ronquist 2001). Phylogenetic
analyses were conducted with a combined
datasets of LSU and atp6 either with (51taxaAnalysis 7, Table 1) or without (36taxaAnalysis 8, Table 1) missing data. The
analyses 9?12 were conducted with a single locus
dataset with a minimum number of taxa (36taxaAnalyses 10 & 12) or more taxa (Analyses

9 & 11). Four data partitions, including LSU and
one for each codon position for the protein-coding locus (atp6), were delimited for Bayesiananalyses. The GTRGI model was employed
separately for each of the four data partitions.
Bayesian analyses were run with 2 million
MCMCMC generations with four chains, sampling trees every 100th generation. The log-likelihood scores of sample points against generation
time were plotted using TRACER version 1.3(http://evolve.zoo.ox.ac.uk/software.html) to determine if the run reached stationarity. We also
observed the average standard deviation of split
frequencies (ASDSF) and verified that the values
dropped below 0.01. The support of nodes wastested by posterior probabilities (PP), obtained
from a 50 % majority rule consensus after deleting the trees in the burn-in period.
Results and Discussion
PCR amplification and sequencing
Most DNA samples produced a clear, single

Fig. 2. Phylogeny of Geastrales. Each tree is a 50% majority consensus based on analysis of: A. Analyses 1 & 7, B. Analyses 2 & 8, C. Analyses 3 & 9, D. Analyses 4& 10, E. Analyses 5 & 11, and F. Analyses 6 & 12. Nodes supported by 0.95 Bayesian PP and by 70% parsimony BS values are indicated by black circles. Numbers on branches are nodal supports (Bayesian PP/parsimony BS values).

166
Kentaro Hosaka and Michael A. Castellano

Sclerogaster phylogeny

band of LSU (ca. 650 bp). However, direct sequencing of some PCR products resulted in ambiguous peaks, suggesting the presence of heterogeneous copies of LSU. This needs to be resolved by cloning the PCR products. On theother hand, PCR of the atp6 failed to amplify
eight DNA samples of Sclerogaster spp. Because
the primers for the atp6 successfully amplified
most species of Phallomycetidae (Hosaka et al.,2006), this suggests substitutions of nucleotide
sequences at the primer regions for Sclerogaster.
Taxon specific primers for Sclerogaster need to
be designed to obtain sequences of atp6. In addition, some Sclerogaster species (not included inthis study) resulted in PCR products longer than
1000 bp. Because most PCR products of atp6
were ca. 700 bp long for Geastrales, this suggests
the presence of introns in the atp6. Further research is necessary to confirm these findings.
Test of dataset incongruence
The Templeton test and Shimodaira-Hasegawa
test did not reveal any major conflicts between
datasets. This suggests the two datasets of LSU
and atp6 are combinable. However, not all
species in this study have sequences for bothgenes, and the presence of missing data may
cause conflicts between datasets. To test this, theindividual dataset of LSU and atp6, as well as the
combined dataset, were analyzed using different
numbers of taxa (Table 2, 3). Phylogenetic analyses under different taxon and character sampling
schemes (analyses 1–12) produced almost identical topologies (Fig. 2), suggesting the presenceof missing data did not cause any major conflicts
Table 2.

167

between datasets. All analyses showed the monophyly of Geastrales, and four clades within theorder (Geastraceae, Sclerogaster, Schenella, andSphaerobolus) were also strongly supported as
monophyletic (Figs. 1, 2).
Parsimony analyses
Although the number of included characters
are almost the same between the LSU (572 bp)
and atp6 (648 bp) datasets, the number of parsimony informative characters were almost threetimes more for the atp6 dataset than LSU dataset(Table 2). This implies that the atp6 dataset has
more resolution power for reconstructing thephylogeny of Geastrales. This is consistent withthe fact that the LSU dataset produced much
more equally parsimonious trees than the atp6
dataset (Table 2).
However, both datasets strongly supported(BS70%) the monophyly of Geastrales, as wellas the monophyly of four major clades (Geastraceae, Sclerogaster, Schenella, and Sphaerobolus) within it. This suggests that the lack of resolution power of the LSU dataset is problematic
only for the lower level phylogeny (e.g., within
the Geastraceae clade). Parsimony analyses, except the analysis 4, supported a sister relationshipof Sclerogaster and Schenella, but no significant
bootstrap values were observed. More data andtaxa need to be sampled to resolve this potential
conflict between parsimony and Bayesian analyses.
Bayesian analyses
After 2 million generations of MCMCMC

Parsimony analyses using various combinations of datasets and taxon numbers (Analyses 1 to 6).

Analysis

Datasetnumber of
taxa

1
2
3
4
5
6

atp6LSU
atp6LSU
LSU
LSU
atp6
atp6

51
36
45
36
42
36

number of
number of
number ofparsimony
included
trees
informative
characters
generatedcharacters
1,220
1,220
572
572
648
648

434
420
127
119
307
301

4
4
12
450
2
1

Tree
Length

CI

RI

RC

1,831
1,550
388
331
1,414
1,190

0.37900.42260.44590.48640.36850.41510.67830.69010.78060.74470.65040.68680.25710.2916
0.3481
0.36220.23960.2851

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Kentaro Hosaka and Michael A. Castellano

Fig. 3. Basidiomata of Sclerogaster spp. A. S. sp. from New Zealand (KH-NZ06-209). Note yellowish gleba. B.
Immature basidiomata of S. sp. (KH-NZ06-209). C. S. sp. (KH-NZ06-209). Note thick, white rhizomorphs
attaching to woody debris. D. S. xerophilus from USA (Trappe 7346). Bars0.5 cm.

runs, all analyses reached stationarity. The average standard deviation of split frequencies(ASDSF) dropped below 0.01 after ca. 1 million
generations for the analyses 7 and 9, ca. 800,000
generations for the analysis 8, ca. 580,000 generations for the analysis 11, and ca. 350,000 generations for the analyses 10 and 12. Accordingly,
the number of trees discarded as the burn-inphase are 10,000 for the analyses 7 and 9, 8,000
for the analysis 8, 6,000 for the analysis 11, and
4,000 for the analyses 10 and 12 (Table 3). Afterdiscarding the trees in the burn-in phase, the potential scale reduction factor was 1.000–1.002 for
all parameters, indicating that the analyses wererun for a sufficient number of generations.
All analyses strongly supported (PP0.95) the
monophyly of Geastrales, as well as the mono-

phyly of four major clades (Geastraceae, Sclerogaster, Schenella, and Sphaerobolus) within it(Figs. 1, 2). In contrast to the parsimony analyses, however, all Bayesian analyses (analyses
7–12) supported a monophyly of Geastraceae
and Sclerogaster (Fig. 2). The result is inconsistent with the previous study by Hosaka et al.
(2006), which showed Sclerogaster was sister toGeastraceae and Schenella (as Pyrenogaster)
combined. However, both studies failed to obtainsignificant (PP0.95) support for these relationships, and therefore more data are necessary to
make a final determination.
Taxonomic implication
Although significant supports are lacking,Bayesian analyses consistently showed that Scle-

Sclerogaster phylogeny
Table 3.

169

Bayesian analyses using various combinations of datasets and taxon numbers (Analyses 7 to 12).

Analysis

Datasetnumber of
taxa

number ofincludedcharactersnumber of trees
retained after
the burn-in

Arithmetic
mean(log-likelihood)

Harmonicmean(log-likelihood)

7
8
9
10
11
12

atp6LSU
atp6LSU
LSU
LSU
atp6
atp6

51
36
45
36
42
36

1,220
1,220
572
572
648
648

10,00012,00010,00016,00014,00016,00010646.84
9358.01
3092.83
2750.82
7236.25
6294.62

10730.00
9436.54
3168.34
2811.69
7280.97
6330.75

Fig. 4. Basidiospores of Sclerogaster spp. A. S. columellatus from USA (Trappe 8098). Note warty ornaments.
B. S. minor from USA (Trappe 8720). C. S. pacificus from USA (Trappe 9011). D. S. xerophilus from USA(Wright 1956). Bars10 m m.

rogaster is a distinct lineage within Geastrales.
This supports the recognition of family status forthis group. In fact, Sclerogastraceae was published by Locquin (1974), but it was without aLatin diagnosis and therefore regarded as invalid
in accordance with Article 36.1 of the ICBN.

More recently, Kirk et al. (2008) validated thefamily Sclerogastraceae. The family is monotypic, containing the genus Sclerogaster Hesse.
Traditionally, Sclerogaster has been considered
to be closely related to Octaviania (Jülich, 1981;
Castellano et al., 1989; Watling, 2006), Hyd-

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Kentaro Hosaka and Michael A. Castellano

nangium (Dodge and Zeller, 1936; Zeller andDodge, 1935), or Russulales (Malençon, 1931;
Heim, 1971) but this study strongly suggests
placement in the Geastrales with close affinity to
the Geastraceae and Schenellaceae (Figs. 1, 2).
This is consistent with the fact that Sclerogastraceae, Geastraceae and Schenellaceae all possess globose spores with a surface ornamented
with spines or warts (Fig. 4; Domínguez de Toledo and Castellano, 1996; Sunhede, 1989; Castellano et al., 1989). In addition, Agerer and Iosifidou (2004) and Clémençon et al. (2007) demonstrated the similarity of rhizomorph structure between Sclerogaster and Geastraceae.
This study strongly showed the monophyly of
the genus, including the type species, S. lanatus.
The exact placement of Sclerogaster in Geastrales, however, remains unresolved. According
to the parsimony analyses, Sclerogaster is potentially sister to Schenellaceae, which was known
as Pyrenogastraceae until recently, but the former
name has a nomenclatural priority (Estrada–Torres et al., 2005). If Sclerogaster and Schenellaceae are demonstrated as the sister groups, it
may be preferable to recognize only one familyname because both Schenellaceae and Sclerogastraceae are monotypic. More taxon sampling isnecessary for a definitive placement of thesetaxa.
Trends in morphological evolution of fruiting
bodies
It is generally believed that sequestrate (trufflelike) fruiting bodies were derived from nonsequestrate ancestors (Thiers, 1984). It is usually
attributed to the loss of forcible spore discharge(ballistospory), and clearly demonstrated to betrue for Russulales (Miller et al., 2001) andCortinariaceae (Peintner et al., 2001). For Phallomycetidae, however, this general theory does
not hold because all representative taxa in Phallales, Hysterangiales and Geastrales are gastroid(including sequestrate) and lack a forcible spore
discharge mechanism. Hosaka et al. (2006) reconstructed the ancestral character state of fruiting body morphology, and demonstrated that se-questrate fruiting bodies are ancestral to aboveground, stinkhorn morphology. They also tried toreconstruct the ancestral character state for Geastrales, but failed to reconstruct the basal nodes
unambiguously. To obtain unambiguous reconstruction, more robust phylogeny is necessary.
However, presence of two major sequestrate lineages (Sclerogaster and Schenellaceae) at a basal
position within Geastrales (Figs. 1, 2) suggeststhat sequestrate fruiting bodies may be ancestral
to above-ground, earthstar morphology.
Biogeographical implication
Although only 14 taxa were included in theanalyses, biogeographical patterns of Sclerogaster (Fig. 1) clearly show some differencesfrom those of Hysterangiales (Hosaka et al.,2008). Phylogeny of Hysterangiales strongly supported basal paraphyletic assemblage of theSouthern Hemisphere taxa, suggesting the Southern Hemisphere (Gondwanan) origin of thegroup, with subsequent range expansion to theNorthern Hemisphere (Hosaka et al., 2008). On
the other hand, Sclerogaster did not show such aclear pattern (Fig. 1). However, extensive taxon
sampling has not been conducted from theSouthern Hemisphere for this study, and it may
therefore be premature to draw any biogeographical conclusions at this time.
As expected, taxa from New Zealand (KHNZ06-209, 210) and Australia (Trappe 15701)
were shown to be sister taxa (Fig. 1). It is againpremature to draw any conclusions at this time,
but this relationship may be due to long distancedispersal between Australia and New Zealand,
which was demonstrated for both some ectomycorrhizal fungi (Hosaka et al., 2008; Moyersoen etal., 2003) and saprotrophic fungi (Hibbett, 2001).
One of the intriguing relationships was the sister
relationship of taxa from Australia (H4595) andUSA (Zeller 8462) (Fig. 1). Although this relationship was not strongly supported, this may be
due to long distance dispersal between the Northern and Southern Hemispheres. Although it
seems unlikely for truffle-like fungi to have theability to disperse such a long distance, the im-Sclerogaster phylogeny

portance of oceanic dispersal for many organisms has recently been revisited (de Queiroz,
2005).
It is noteworthy to mention that Sclerogaster ispresumably saprotrophic. Although direct evidence is lacking, their habitat on rotten wood andbranches (Fig. 3), or in thick humus layers indicates that they are not ectomycorrhizal fungi. In
addition, nearly all taxa within Geastrales are reported as saprotrophic (Sunhede, 1989;
Domínguez de Toledo and Castellano, 1996; Flegler, 1984; but see Agerer and Beenken, 1998).
Saprotrophic fungi are generally more widespread in distribution than ectomycorrhizal fungi
because they do not require the presence of compatible host plants. The study of Sphaerobolus,
which belongs to Geastrales and therefore isclosely related to Sclerogaster, showed little phylogeographic pattern within the Northern Hemisphere, suggesting on-going long distance dispersal (Geml et al., 2005). Sphaerobolus is undoubtedly saprotrophic because they easily produce
fruiting bodies on artificial media (Flegler,1984). A similar nutritional mode (saprotrophic)
shared by Sphaerobolus and Sclerogaster may
also indicate that they might have a similar phylogeographical pattern. More extensive taxon
sampling for Sclerogaster is required to understand the biogeography of the genus.

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