Mycologia, 103(1), 2011, pp. 36–44. DOI: 10.

3852/09-314
# 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

Phylogeny of Pilobolaceae

K. Michael Foos1
Nicole L. May
Dale L. Beach2
Markus Pomper
School of Natural Science and Mathematics, Indiana
University East, Richmond, Indiana 47374
Kathy B. Sheehan
Department of Molecular, Cellular and Developmental
Biology, University of Colorado, Boulder, Colorado
80309-0347
Donald G. Ruch
Department of Biology, Ball State University, Muncie,
Indiana 47306
Abstract: The three genera traditionally classified as
Pilobolaceae have been identified on the basis of
morphological characteristics. In the absence of
distinctive morphological differences phylogenetic
techniques have proven to be superior for developing
phylogenies. Molecular techniques have been used
primarily for studies of higher fungi; there are few
investigations of the Zygomycota using genetic
sequences for classification. DNA sequences coding
for three regions of rRNA were used to investigate
phylogenetic relationships of the three genera tradi-
tionally considered within the Pilobolaceae. Evidence
indicates that Pilaira should be removed from
Pilobolaceae and the family redescribed. Sporangio-
spore size is the morphological characteristic that
most closely correlates with rDNA clades of phyloge-
netic trees. This study demonstrates that traditional
morphological characteristics alone are not adequate
to differentiate species of Pilobolus.
Key words: DNA, ITS, LSU, mitochondria, Pi-
laira, Pilobolaceae, Pilobolus, ribosome, rRNA, SSU,
Utharomyces, 5.8S, 18S, 23S
INTRODUCTION
Family Pilobolaceae (Eukaryota, Fung, Fungi incertae
sedis, Basal fungal lineages, Mucoromycotina, Muco-
rales) traditionally contains three genera, Pilobolus
Tode ex Fr., Pilaira van Tiegh. and Utharomyces
Boedijn (Kirk et al. 2001). Pilaira is reported to have
five species, Utharomyces a single species and Pilobolus
Submitted 20 Dec 2009; accepted for publication 22 Jul 2010.
1
Corresponding author. E-mail: foos@iue.edu
2
Current address: Department of Biology and Environmental
Sciences, Longwood University, Farmville, VA 23909.
7–9 species (Grove 1934, Hu et al. 1989, Zycha et al.
1969). These fungi, primarily coprophilous saprobes,
are ubiquitous with the exception of Utharomyces,
which is restricted to tropical habitats. Zygospores
have been reported in Pilaira and Pilobolus but not in
Utharomyces. Because zygospores are rare in species of
this family the taxa usually are characterized by
asexual reproductive structures. All three genera have
sporangia containing large numbers of sporangio-
spores, a persistent dark pigmented sporangial wall
and papillate to broadly rounded columella. Sporan-
giophores are large and phototrophic; Pilobolus and
Utharomyces possess subsporangial swellings and
trophocysts (Kirk and Benny 1980).
Mechanisms of sporangiospore discharge also
distinguish the genera. Sporangiophores of Pilaira
lack subsporangial swellings and are the least complex
of the group. Sporangiophores of Pilaira and
Utharomyces are relatively short during early growth,
but they elongate rapidly as they mature, aiding
dissemination of sporangiospores. Pilobolus dissemi-
nates sporangiospores by ballistic discharge caused by
the elevated pressure within the subsporangial swell-
ing of the sporangiophore.
Pilaira and Utharomyces can be maintained for years
on common laboratory media, but with the exception
of a few isolates (Kubo and Mihara 1986) Pilobolus is
particularly difficult to culture on artificial media or
even on dung agar. In most instances chelated iron is
an essential ingredient, but even media enriched with
chelated iron fail to support the growth of many
isolates. Some isolates do not survive the transfer from
dung. Of the strains that can be cultivated on artificial
media most survive through only a few sequential
transfers. Furthermore only rare isolates survive long
term preservation, resulting in few isolates of Pilobolus
in culture collections worldwide, most of which are
duplicates held in multiple collections. Therefore it is
imperative to develop a rapid, reproducible and
reliable method of identification and phylogenetic
determination for these organisms that are difficult or
impossible to cultivate. White et al. (2006) noted a
need for additional data from understudied and
unculturable taxa to understand the evolutionary
history of the Zygomycota. The genera of Pilobola-
ceae are such taxa, often studied because of their
spore discharge mechanisms, but whose evolutionary
history has not been examined closely.
Historically Pilobolaceae has been categorized as
one of 12 families of Mucorales. However recent
studies indicate that this classification is highly

36
 FOOS ET AL.: PILOBOLACEAE
artificial (O’Donnell et al. 2001, Benny 2005) and
should be re-evaluated as additional molecular studies
are reported. Correct identification to species is a
prerequisite for creation of a correct phylogeny. In the
case of Pilobolus identification to species is particularly
problematic. Species descriptions traditionally have
been based on morphological characteristics, such as
length of the sporangiophore and shape, size and
pigmentation of the sporangiospores. However several
factors make many morphological characteristics used
for species identification unreliable.
Even though the size and shape of the sporangio-
spores have been found to be stable within species
(Foos and Jeffries 1988), species descriptions typically
give such a large range of sporangiospore sizes that
this character provides insufficient information to
make an identification to species. For example Grove
(1934) describes the spore lengths of P. longipes, P.
kleinii and P. sphaerosporus as 12–15 mm, 11–20 mm
and 10–20 mm respectively. In such cases where
sporangiospore dimensions overlap spore shape
(globose or elliptical) becomes the principal charac-
teristic for differentiating species. Wide variations of
the dimensions of other structures (i.e. sporangial
diameters and sporangiophore lengths) used in
species descriptions emphasize the need for the
identification of other useful taxonomic characters.
Taylor et al. (2006) observed that phylogenetic
species recognition lets biologists sort individual
organisms into species and then find which of many
variable phenotypic characters of morphological
species correlate with phylogenetic species.
Second, a typical sample of fresh dung contains
multiple species of Pilobolus (Foos 1997). Similar
structures from multiple species growing intermin-
gled on the surface of dung are nearly indistinguish-
able. Attempting to identify a species found on a
dung sample easily could lead to misidentification,
necessitating the generation of pure cultures for
accurate identification. The challenges in transferring
and culturing Pilobolus in laboratory media are
formidable and have been discussed earlier.
Finally, isolates that do survive multiple transfers
onto culture media often exhibit structures that differ
in size from those of the original isolate growing on
the original dung sample. For example we compared
the lengths of sporangiophores of P. longipes growing
on an original dung sample and the growth derived
from single sporangium transferred to dung agar.
The culture on dung agar produced sporangiophores
an order of magnitude shorter than the sporangio-
phores growing on the original dung sample.
Reliance on such malleable morphological charac-
teristics is likely to lead to incorrect identification of
isolates and has lead to the misidentification of
37
existing cultures, such as the American Type Culture
Collection isolate P. kleinii (ATCC 14499), which is
cross listed by the Centraalbureau voor Schimmelcul-
tures as P. crystallinus (CBS 272.31).
In the absence of stable morphological character-
istics for identification a more reproducible basis for
identification is required. The current study was
designed to employ genetic data to clarify relation-
ships among the three genera traditionally included
in Pilobolaceae and to develop a reliable means to
distinguish among species of Pilobolus.
O’Donnell et al. (2001) completed a comprehen-
sive study of the Mucorales with partial nucleotide
sequences of the nuclear 18S ribosomal RNA (SSU),
nuclear large subunit 28S ribosomal RNA (LSU) and
EF-1a gene exons. The phylogeny of Mucorales also
was studied by White et al. (2006), who used the
combined rRNA operon (18S + 28S + 5.8S genes) to
infer relationships. Millar et al. (2007) tested the 18S
and ITS fragments and found them to be highly
reliable in providing the correct identification of
filamentous fungi and yeasts. Seif et al. (2005)
suggested that mitochondrial DNA sequences could
be used to examine comparative relationships among
fungi. Other genes have been used to study the
genetic diversity of fungi, but few studies have
examined the individual families within Mucorales.
Based on the successful application of mitochondri-
al and nuclear genes expressing ribosomal RNAs, we
anticipated that genetic factors could be used to
identify isolates of members of Pilobolaceae. The
conservation of the rRNA coding regions implied that
the same primers used to produce amplicons in other
fungi (White et al. 1990) could be used throughout the
family, as demonstrated by O’Donnell et al. (2001) for
Mucorales. In this study we determined that nuclear
encoded ribosomal DNA (rDNA) is superior to
mitochondria-encoded rDNA for investigating the
phylogenic relationships within Pilobolaceae.
MATERIALS AND METHODS
Isolates of Pilobolus, Pilaira and Utharomyces were obtained
from the American Type Culture Collection (ATCC),
National Center for Agricultural Utilization Research
(NRRL) and University of Alberta Microfungus Collection
and Herbarium (UAMH). Other isolates were collected with
techniques described by Foos et al. (2001). All three isolates
of Pilobolus longipies were collected in Indiana, and the
isolate of Pilobolus roridus was collected in Wyoming. The
multiple isolates provide replicates that could reveal any
variation among the strains of a species (TABLE I).
All cultures were maintained at 22 6 2 C, with alternating
12 h light and dark periods of 2000 lux, cool white
fluorescent illumination. Isolates were cultured on a
synthetic hemin medium (Levetin and Caroselli 1976) in
38 MYCOLOGIA

TABLE I. Voucher number, species, source and GenBank accession numbers for DNA sequences used in this study (SSU—
fragment amplified by NS1–NS8 primers, ITS—fragment amplified by ITS5 and ITS4 primers, LSU—mitochondrial fragment
amplified by ML7 and ML8 primers). All specimens represent independent isolates of each species

Voucher number Species Source SSU GenBank ITS GenBank LSU GenBank
IUE340 Pilobolus longipes IUE 340 DQ211053 FJ160950 EF565865
IUE409 Pilobolus longipes IUE 409 DQ211054 FJ160951 EF565866
IUE563 Pilobolus longipes IUE 563 EU595654 FJ160952 EF565873
IUE410 Pilobolus umbonatus NRRL 6349 EU595657 FJ160955 EF565867
IUE519 Pilobolus umbonatus UAMH 7297 DQ211050 FJ160956 EF565870
IUE520 Pilobolus umbonatus UAMH 7298 DQ211051 DQ058412 EF565871
IUE518 Pilobolus sphaerosporus UAMH 3070 DQ211052 DQ059382 EF565869
IUE521 Pilobolus sphaerosporus UAMH 1312 EU595653 FJ160953 EF565872
IUE566 Pilobolus kleinii ATCC 14499 EU595655 FJ160954 EF565875
IUE701 Pilobolus kleinii ATCC 36185 EU595656 FJ160957 EU595646
IUE415 Pilobolus roridus IUE 415 EU595649 FJ160948 EF565868
IUE567 Pilobolus crystallinus ATCC 11505 EU595650 FJ160947 EF565876
IUE702 Pilobolus crystallinus ATCC 46942 EU595651 FJ160958 EU595647
IUE703 Pilobolus crystallinus ATCC 36186 EU595652 FJ160949 EU595648
IUE573 Pilaira anomala ATCC 36774 EU595658 FJ160942 EF565877
IUE574 Pilaira anomala ATCC 36779 EU595659 FJ160941 EF565878
IUE564 Utharomyces epallocaulus ATCC 42705 EU595660 FJ160943 EF565874
IUE605 Utharomyces epallocaulus NRRL 3168 EU595661 FJ160944 EF565879
IUE606 Utharomyces epallocaulus NRRL A-15807 EU595662 FJ160945 EF565880
IUE607 Utharomyces epallocaulus NRRL A-21843 EU595663 FJ160946 EF565881

disposable plastic Petri dishes sealed with parafilm. Mature
sporangia were collected directly from sporangiophores
with microforceps or from the lids of the Petri dishes with
inoculating needles. Sporangia were placed in 0.2 mL
microcentrifuge tubes containing 20 mL sterile collecting
water (containing 3% penicillin, 3% streptomycin and 1%
Tween 20), labeled and stored at 4 C.
DNA was extracted from sporangiospores with the MoBio
UltraClean Soil DNA Isolation Kit (Mo-Bio Laboratories
Inc., Carlsbad, California) with the following modifications.
The collecting water and sporangia from a single micro-
centrifuge tube were added to a 1.7 mL tube provided in
the kit. Spores were heat treated at 70 C for 10 min before
breaking sporangiospore walls with a FastPrep FP 120
instrument (Thermo Scientific, Waltham, Massachusetts),
set at 6.5 for 45 s for one cycle. After extraction the total
genomic DNA was stored in water at 220 C.
Total DNA was amplified with AmpliTaqH Gold DNA
polymerase (Applied Biosystems, Foster City, California)
and a dNTP mix (Promega Corp., Fitchburg, Wisconsin).
Thermal cycling was conducted in a Perkin Elmer Gene-
AmpH PCR System 2400. PCR reaction conditions for
thermal cycling were 94 C for 5 min, followed by 36 cycles
of 94 C for 1 min, 50 C for 1 min 30 s, 72 C for 2 min,
followed by an extension at 72 C for 7 min. PCR products
were purified with the QIAquickH PCR Purification Kit
(QIAGEN Inc., Valencia, California).
Primers selected to amplify and sequence specific
fragments of DNA were described originally by White et
al. (1990). Primers ML7 and ML8 were used to amplify a
fragment of mitochondrial DNA coding for a portion of the
23S fragment within the large subunit (LSU) of rRNA to
determine the extent that mitochondrial ribosomal RNA
sequences differentiate members of Pilobolaceae. Primers
NS1–NS8 were used to amplify the nuclear 18S fragment
coding for the small subunit (SSU) of rRNA to compare
with other phylogenetic studies of the family. Primers ITS5
and ITS4 were used to amplify the nuclear DNA fragment
coding for the entire ITS fragment, including the 39 end of
the SSU, the 5.8S, both of the internal transcribed spacers
and the 59 end of the LSU. PCR-amplified DNA fragments
were electrophoresed on 1% agarose gels in 13 TBE buffer
(50 mM Tris-HCl, 50 mM boric acid and 1 mM EDTA)
containing ethidium bromide and viewed with a Chemi-
ImagerTM 4400 Imaging System (Alpha Innotech, San
Leandro, California). A 100 bp DNA ladder (Takara Mirus
Bio, Madison, Wisconsin) was used as a size marker.
PCR products were sequenced with a BigDyeH Termina-
tor 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster
City, California). Each sequence reaction contained 2 mL
H2O, 3 mL 53 buffer, 1 mL BigDye, 2 mL 10 mM primer and
2 mL DNA. Thermal cycling conditions for sequencing were
25 cycles of 96 C for 10 s, 50 C for 5 s and 60 C for 4 min. An
Applied Biosystems 3700 automated fluorescence system at
the Indiana Molecular Biology Institute was used to
sequence amplicons.
Sequences were examined and compared with Codon-
Code Aligner (CodonCode Corp., Dedham, Massachusetts)
containing PHRED and PHRAP (Ewing and Green 1998,
Ewing et al. 1998) for base calling, sequence comparisons
and sequence assembly. Contigs created in CodonCode
were oriented with BLAST (Altschul et al. 1990) and aligned
with Clustal W (Thompson et al. 1994, 1997).
Where sequencing reactions produced ambiguous or
mixed results, PCR products were cloned and the resulting
plasmid inserts were sequenced. A TA-Cloning Kit (Invitro-
 FOOS ET AL.: PILOBOLACEAE 39

TABLE II. Lengths (bp) of the various rDNA fragments analyzed in this study (SSU—fragment amplified by NS1–NS8 primers,
ITS—fragment amplified by ITS5 and ITS4 primers, LSU—mitochondrial fragment amplified by ML7 and ML8 primers).
Voucher numbers refer to specimens in TABLE I

Sections of ITS sequences

Voucher number Species SSU LSU ITS 18S ITS1 5.8Sa ITS2 28S
IUE340 Pilobolus longipes 1763 370 688 39 226 153 231 39
IUE409 Pilobolus longipes 1763 370 688 39 226 153 231 39
IUE563 Pilobolus longipes 1763 370 688 39 226 153 231 39
IUE410 Pilobolus umbonatus 1764 370 706 39 180 152 296 39
IUE519 Pilobolus umbonatus 1764 370 707 39 180 152 297 39
IUE520 Pilobolus umbonatus 1764 370 707 39 180 152 297 39
IUE518 Pilobolus sphaerosporus 1764 370 694 39 234 152 230 39
IUE521 Pilobolus sphaerosporus 1764 370 694 39 234 152 230 39
IUE566 Pilobolus kleinii 1764 370 694 39 234 152 230 39
IUE701 Pilobolus kleinii 1763 370 704 39 236 152 238 39
IUE415 Pilobolus roridus 1762 370 694 39 213 152 251 39
IUE567 Pilobolus crystallinus 1761 370 695 39 215 152 250 39
IUE702 Pilobolus crystallinus 1763 370 689 39 207 152 252 39
IUE703 Pilobolus crystallinus 1763 370 657 39 213 152 214 39
IUE573 Pilaira anomala 1761 371 667 39 214 155 220 39
IUE574 Pilaira anomala 1761 371 667 39 214 155 220 39
IUE564 Utharomyces epallocaulus 1748 370 624 39 189 154 203 39
IUE605 Utharomyces epallocaulus 1748 370 624 39 189 154 203 39
IUE606 Utharomyces epallocaulus 1748 370 624 39 190 154 202 39
IUE607 Utharomyces epallocaulus 1748 370 624 39 190 154 202 39
a
Terminal bases for 5.8S fragments were AAAACAACTT and TGTTTCAGT.

gen, Carlsbad, California) was used to clone PCR fragments
into the precut cloning site of pCR2.1. Ligations were
completed with the manufacturer’s protocol and trans-
formed into chemically competent TOP10 cells (Invitrogen,
Carlsbad, California). Bacterial colonies with plasmid
pCR2.1 containing an insert were identified as white
colonies in a blue-white screen. DNA was prepared from
these colonies and submitted for sequencing with the same
primer sets as described for direct sequencing of amplicons.
Phylogenetic analyses were conducted and trees con-
structed with MEGA 4 (Tamura et al. 2007). DNA sequences
used as outgroups were obtained from GenBank. The
evolutionary history was inferred with neighbor joining
(Saitou and Nei 1987). The bootstrap consensus trees
inferred from 2000 replicates was taken to represent the
evolutionary history of the taxa analyzed (Felsenstein 1985).
Branches corresponding to partitions reproduced in less
than 50% bootstrap replicates were collapsed. The percent-
age of replicate trees in which the associated taxa clustered
together in the bootstrap test (2000 replicates) is shown
next to the branches. The trees are drawn to scale, with
branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree.
The evolutionary distances were computed with maximum
composite likelihood (Tamura et al. 2004) and are in the
units of the number of base substitutions per site. All
positions containing alignment gaps and missing data were
eliminated in pairwise sequence comparisons (pairwise
deletion option).
Sporangiospores suspended in water were examined,
measured and photographed with an Olympus BH-2 light
microscope. Kodachrome 64, 35 mm slides were digitized
with a Nikon Coolscan V. Digitized images were processed
for publication with ArcSoft Photostudio5, converted to
eight-bit grayscale, adjusted in contrast and brightness,
cropped and superimposed with a black bar representing
10 mm. All images are reproduced at the same scale.
RESULTS
DNA encoding a portion of mitochondrial LSU rRNA.—
Amplicons were obtained with ML7 and ML8 primers.
The resulting nucleotide sequences used for analysis
correlated with published fungal mitochondrial
rDNA sequences. Ribosomal DNA fragments pro-
duced from Pilobolus, Pilaira and Utharomyces dif-
fered by eight polymorphisms and one indel. Nucle-
otide sequences of the mitochondrial LSU fragments
from isolates of the same genus were identical for
Pilaira and Utharomyces, but LSU sequences from
P. umbonatus differed from other Pilobolus isolates
by a single nucleotide change. The LSU sequences of
the three isolates of P. umbonatus were identical
(TABLE II).
Sequence alignments were used to construct a
phylogram inferring the relationships of the genera.
40 MYCOLOGIA
FIG. 1. Phylogram inferred from sequences of DNA
coding for portions of the LSU (23S) mitochondrial rRNA
of Pilobolaceae with neighbor joining. A total of 371
positions were in the final dataset. Bar 5 0.002 base
substitutions per site. Voucher numbers correlating with
TABLE I are included for each isolate.
The phylogram (FIG. 1) shows the separation of
Utharomyces, Pilaira and Pilobolus. The orthologous
sequence from Rhizopus oryzae (AY863212) from
GenBank was used as outgroup. Analyses of the
mitochondrial LSU support the current circumscrip-
tion of described genera. Mitochondria-encoded LSU
is sufficient to distinguish between genera but not
species.
Nuclear DNA encoding the SSU rRNA.—Sequences of
the SSU with primers NS1–NS8 produced overlapping
segments of DNA sufficient to generate a single
contig for each species. The lengths of the fragments
of DNA coding for nuclear SSU genes for Uthar-
omyces, Pilaira and Pilobolus were characteristic for
each genus (TABLE II). Small variations were observed
within Pilaira and Utharomyces; Pilobolus demonstrat-
ed greater heterogeneity. The phylogram (FIG. 2)
inferred from the nuclear DNA coding SSU fragments
show distinct separation of the three genera with
Pilobolus and Utharomyces in one clade and Pilaira as a
separate clade. Bootstrap values suggested the ortho-
logous SSU region of Rhizopus oryzae (GU126375) as
outgroup for the phylogram.
The phylogram (FIG. 2) shows isolates of Pilobolus
separated into two main clades corresponding with
differences in spore sizes of the species in each group.
Large-spore (. 10 mm long) producing species
(FIG. 3A–D; P. longipes, P. kleinii and P. sphaeros-
FIG. 2. Phylogram inferred from sequences of DNA for
SSU (18S) rRNA of Pilobolaceae with neighbor joining. A
total of 1828 positions were in the final dataset. Bar 5 0.005
base substitutions per site. Voucher numbers correlating
with TABLE I are included for each isolate.
porus) comprise a major clade and those of small-
spore (, 10 mm long) producing species (FIG. 3E–H;
P. crystallinus, P. roridus and P. umbonatus) comprise
a second major clade. The designations of large and
small spores, consistent with the isolates under study,
were derived from spore descriptions by Grove
(1934), Zycha et al. (1969) and Hu et al. (1989).
Isolates of each species previously identified on the
basis of morphological characteristics clustered to-
gether in multiple clades strongly supported by
bootstrap values. The molecular phylogeny was
similar to the classification of morphological species.
Integration of these data with previous studies.—Be-
cause the data from this study represent a detailed
analysis of Pilobolaceae the findings were integrated
into a larger dataset containing a broad selection of
mucoralean families. The 18S rDNA sequences
determined in this study were combined with a
similar set of sequences used by O’Donnell et al.
(2001) to establish phylogenetic relationships within
Mucorales. A phylogram (not shown) constructed
from combined data of O’Donnell et al. (2001), and
this study was consistent with the previously published
study and resulted in the separation of Pilaria from
Pilobolus and Utharomyces.
Nuclear DNA encoding the ITS region of rRNA.—
Amplicons produced with ITS5 and ITS4 primers
comprise the ITS region containing the entire ITS1,
5.8S and ITS2 regions of the rDNA transcript as well
as flanking SSU and LSU regions. We provided the
composition of the ITS regions among the three
genera of Pilobolaceae (TABLE II). Isolates of Pilaira
had identical sequences. Utharomyces generated prod-
 FOOS ET AL.: PILOBOLACEAE
FIG. 3. Sporangiospores of select isolates of Pilobolus
species. A. Pilobolus longipes IUE409. B. P. kleinii IUE701.
C. P. sphaerosporus IUE521. D. P. kleinii IUE566. E. P.
crystallinus IUE703. F. P. crystallinus IUE702. G. P. roridus
IUE415. (H) P. umbonatus IUE410. Bars 5 10 mm.
ucts with the same overall length of the ITS region,
however variation in the length of the spacer regions
ITS1 and ITS2 were observed. The lengths of
amplicons from isolates of Pilobolus were not uniform
(TABLE II).
The phylogram (FIG. 4) generated from the ITS
region indicates phylogenetic relationships among
Pilobolaceae, clearly differentiating the genera. As
observed for the SSU sequence, Pilaira was distantly
separated from Utharomyces and Pilobolus. Utharo-
myces and Pilobolus occurred in separate clades
(FIG. 4). A sequence from Pilobolus crystallinus
(NBRC 8561), the only ITS sequence from Pilobolus
available from any lab other than ours, was included
in the analysis of the ITS region (NBRC 2005). The
ITS sequence of NBRC 8561 differed by several
nucleotides from IUE 703 yet formed a clade with
isolate IUE 703 separate from the other P. crystallinus
isolates. Bootstrap values suggested the orthologous
ITS region of Rhizopus oryzae (AM933544) as out-
group for the phylogram.
DISCUSSION
Molecular phylogenetics has made significant changes
to the taxonomy of microbial organisms. Identifica-
tion and description of organisms on the basis of
molecular data provide a statistically reliable and
readily accomplished methodology superior to cla-
distic analysis based on a few morphological or
biochemical characteristics for determining species.
In the fungal kingdom comparative analysis of
molecular data with phylogenies based on morpho-
logical characteristics suggests that several organisms
41
FIG. 4. Phylogram inferred from sequences of DNA
coding for ITS rRNA of Pilobolaceae with neighbor joining.
A total of 806 positions were in the final dataset. Bar 5 0.05
base substitutions per site. Voucher numbers correlating
with TABLE I are included for each isolate.
must be redescribed in light of the increased
resolution of DNA sequences (Stajich et al. 2009,
O’Donnell et al. 2001). The molecular phylogenetic
approach has been sparingly applied to the Zygomy-
cota. This report presents a detailed phylogenetic
analysis of Pilobolaceae with elements of the nuclear
encoded rRNA genes and the mitochondrial LSU
gene as described.
Phylograms constructed from the three genetic
characters (mitochondrial LSU rRNA, nuclear ITS
region and nuclear SSU rRNA) separated the genera,
placing each genus in a separate clade with strong
bootstrap support (FIGS. 1, 2, 4). The phylogram
inferred from the nuclear ITS region (FIG. 4) is
similar to the one inferred from the SSU region
(FIG. 2) and clearly segregates the three genera and
within Pilobolus separates the species with strong
bootstrap support. The inclusion of multiple genetic
loci is important for the resolution of species within
the population. Often different single traits will lead
to significantly different trees. When SSU and ITS
sequences are compared the topologies of the
resulting phylograms are not always conserved (Abe
et al. 2006). In light of the similarity of the SSU and
ITS, especially considering the variation observed
within the ITS regions, the phylograms produced by
these analyses supported the species designations
used in this paper.
The phylogram inferred from the mitochondrial
LSU delimited by the ML7 and ML8 primers (FIG. 1)
42 MYCOLOGIA
has a much lower resolution than phylograms
inferred from nuclear ribosomal DNA sequences.
The isolates used in this study segregated into three
clades corresponding to the three genera. The
sequences obtained for the mitochondrial LSU
unexpectedly contained little phylogenetic informa-
tion. The findings of the present study support
observations by Seif et al. (2005) who suggested that
mitochondrial DNA sequences could be used to
examine relationships at the upper phylogenetic
levels.
Phylograms inferred from nuclear ribosomal DNA
(FIGS. 2, 4) separated all three genera and indicated
that Pilaira is distant from the other two genera. This
conclusion agreed with O’Donnell et al. (2001),
whose comprehensive study of the Mucorales SSU
(including three isolates of species within Pilobola-
ceae) suggested that Pilaira should be removed from
Pilobolaceae. The present study strengthened that
suggestion because more isolates of each genus were
included.
The multiple isolates of Utharomyces and Pilobolus
co-segregate into distinct clades inferred from the
phylograms of both the SSU and ITS regions,
strengthening the inference that these two genera
are monophyletic. Even though it has been suggested
that all mucoralean families but one be placed in the
Mucoraceae (White et al. 2006), the results of our
examination of the ITS and SSU DNA sequences and
morphological characters such as trophocysts and
subsporangial swellings demonstrated that Pilobola-
ceae should be retained as a separate family to
include Pilobolus and Utharomyces.
The phylogram inferred from these nuclear ribo-
somal DNA sequences resolved genus Pilobolus into
two major clades, one of large spore-producing
species, the second of small spore-producing species.
The delimitation between the large and small spore-
producing species mirrors traditional morphological
descriptions (Grove 1934, Zycha et al. 1969, Hu et al.
1989). Other studies report that sporangiospore size
is one of the most stable morphological characteris-
tics (Foos and Jeffries 1988). The present data show
that this characteristic correlates with phylogenetic
relationships.
Polyphyly in Pilobolus.—In the phylograms inferred
from nuclear ribosomal DNA the clade of small spore-
producing isolates (P. crystallinus, P. roridus and P.
umbonatus) consists of two further clades, one of
which contains P. crystallinus isolates IUE703 and
NRBC 8561 and the other contains the remaining
isolates of P. crystallinus as well as P. roridus and P.
umbonatus. Separation of the isolates of P. crystallinus
into clades suggests that the morphologically de-
scribed species comprises multiple phylogenetic
species. Stajich et al. (2009) reported that strains that
are similar in multiple morphological characteristics
might be different phylogenetic species and that such
polyphyly is not uncommon. However we cannot
exclude the possibility that these isolates do in fact
express yet undetected morphological characteristics
that have not been reported in the literature. Data
indicate a polyphyletic nature of morphological
species of Pilobolus crystallinus and P. kleinii as
supported by the bootstrap values in this molecular
analysis. Wide variations in the quantitative features of
the morphological characters used to identify species
have led to misidentification or are the result of the
polyphyletic nature of isolates that have similar
morphological characteristics.
Hu et al. (1989) proposed reducing Pilobolus
umbonatus and P. roridus to a single species because
they differed only in the shape of sporangia. However
both SSU and ITS rDNA sequences of Pilobolus
umbonatus differ greatly from those of P. roridus,
placing them in different clades and thus supporting
their recognition as different species. Likewise Hu
et al. (1989) suggested reducing three species (P.
crystallinus, P. kleinii and P. hyalosporus) to one
species because they differed only in spore size and
color. However spore size is supported in the
molecular analysis as having great importance in
differentiating species of Pilobolus, and this morpho-
logical evidence should not be disregarded by
combining these three species into one. This study
reveals that P. crystallinus and P. kleinii have different
SSU and ITS rDNA sequences, in addition to different
spore sizes, and should be retained as separate
species. Because nuclear rDNA remains constant
and characteristic it provides a reproducible and
hence superior method with which to infer species of
Pilobolus.
Isolate IUE 566 was identified previously as both P.
kleinii and P. crystallinus in different culture collec-
tions. Phylograms inferred from SSU and ITS
sequences place IUE 566 in a clade with P.
sphaerosporus, however sporangiospores size and
shape are more characteristic of P. kleinii (FIG. 3B–
D). This suggests either polyphyly in P. kleinii or the
possibility of different morphological forms of P.
sphaerosporus. Additional studies will be needed to
clarify the relationship between these two species.
Further work.—More effort will be required to re-
evaluate and redescribe existing isolates of Pilobolus
available in culture collections. The traditional
methods of identification and classification of Pilo-
bolaceae based primarily on morphological charac-
ters are not sufficient to address the variability
 FOOS ET AL.: PILOBOLACEAE
associated with these organisms. Molecular phyloge-
netics can be used to determine not only the
phylogenetic relationships within Pilobolaceae but
also to identify Pilobolus isolates to species. Both
morphological and molecular information can be
used to distinguish between Utharomyces and Pilobo-
lus. The molecular information presented here, in
combination with spore size and shape, can be used
to identify species of Pilobolus. In addition the rDNA
sequences presented here will aid the characteriza-
tion of new species of this genus.
ACKNOWLEDGMENTS
This project was supported in part by an Indiana University
sabbatical grant. We thank Kerry O’Donnell (National
Center for Agricultural Utilization Research, Peoria, Illi-
nois) and Lynne Sigler (University of Alberta Microfungus
Collection and Herbarium) for providing isolates of fungal
strains used in this work and Lawrence Washington
(Indiana Molecular Biology Institute) for sequence analysis.
KMF thanks Joan Henson for her hospitality and the use of
her laboratory while developing the molecular techniques
used in this study.
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