mycological research 110 (2006) 941–950

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/mycres

Value of host range, morphological, and genetic

characteristics within the Entomophthora muscae
species complex

Annette Bruun JENSEN*, Lene THOMSEN, Jørgen EILENBERG

Department of Ecology, The Royal Veterinary and Agricultural University (KVL),
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

article info abstract

Article history: Entomopthora muscae sensu lato is a complex of morphologically similar fungal species path-
Received 25 November 2005 ogenic to evolutionarily advanced flies (Cyclorrhapha). To reach an operational species
Received in revised form definition and recognition of species within this complex, the values of host range,
25 April 2006 morphological and genetic characteristics are reconsidered. Within the E. muscae species
Accepted 1 June 2006 complex morphological and nuclear characteristics of the primary conidia are taxonomi-
Published online 14 August 2006 cally important. In this study we compared the dimensions and nuclear numbers of the
Corresponding Editor: primary conidia of isolates from their original (natural) hosts and after being transferred
Richard A. Humber to alternative hosts (cross-transmission) in order to check the stability of these character-
istics. The conidial characteristics change substantially when produced in alternative host
Keywords: species, but their overall range in variability still fit within the traditional morphological
Anthomyiidae species circumscriptions. The phylogenetic analyses of the ITS II and LSU rRNA gene
Diptera sequences, revealed three distinct lineages within the complex: E. schizophorae, E. muscae
Ecology and E. syrphi. Within each of these lineages sequence divergence was seen between isolates
Entomophthorales originating from different host species. Our studies on the physiological host range showed
Host range that several isolates were able to infect alternative dipteran species. Musca domestica was
Insect pathogenic fungi a particularly good receptor. The ecological host range of any individual isolate seems,
Morphology however, to be limited to one host species evidenced by the occurrence of distinct geno-
Muscidae types within each natural infected host species shown by RAPD. The high host specificity
Phylogeny of these fungi emphasizes the importance of identifying the host taxon at species level in
Taxonomy the recognition of Entomophthora species. We recommend that morphological characteris-
Zygomycota tics of fungal structures and host taxon, together with molecular data, serve as criteria for
species determination in future studies on members of the E. muscae complex.
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Fungi from the Entomophthora muscae species complex are im-
portant for the natural population regulation of many dipteran
species. Epizootics have often been reported also among pest
flies, and the fungi possess a potential to be used in biocontrol
(Klingen et al. 2000; Mullens et al. 1987; Steinkraus et al. 1993;
Watson & Petersen 1993). However, the evaluation of this
potential requires a more complete understanding of the eco-
logical interaction between host species and their pathogens.
In such ecological studies the circumscriptions of individual
fungal taxa become a central issue. Traditionally, species were

* Corresponding author. Tel.: þ45 35 28 26 82; fax: þ45 35 28 26 70

E-mail address: abj@kvl.dk.
0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2006.06.003
942 A. B. Jensen et al.

described based on recognizable morphological features and

as such E. muscae from Musca domestica was described by
Cohn in 1855. Subsequently, fungal specimens from flies of de-
rived clades (Cyclorrhapha) with E. muscae-like conidia were
designated as E. muscae (MacLeod et al. 1976). In the 1980s cyto-
logical features were considered important for the taxonomy
of the order Entomophthorales (Ben-Ze’ev & Kenneth 1982; Hum-
ber 1981, 1989; Remaudière & Keller 1980), in particular the con-
tents of heterochromatin, number and size of the nuclei.
Inspired by these new criteria, Keller (1984) suggested that
E. muscae s.l. is a complex of species separated by conidial
size, number and size of the nuclei. At present six species
are recognized and described morphologically: E. ferdinandi,
E. grandis, E. muscae s.str., E. scatophagae, E. schizophorae, and
E. syrphi (Keller 2002). Some of the species, e.g., E. schizophorae,
E. ferdinandi, and E. grandis, have distinct conidial morphology
whereas others such as E. muscae s.str. and E. scatophagae are
virtually indistinguishable using conidial characteristicistics.
The two latter species can only be differentiated morpho-
logically by the shape of their hyphal bodies, which are
reported to be consistently spherical in E. muscae s.str. (Keller
et al.1999). Whether this morphological variation is stable or
due to phenotypic modifications associated with the envi-
ronmental condition (the host species) remains unknown.
Host range has been used to elucidate entomophthoralean
species complexes. Based on transmission experiments, RFLP,
and hybridization of a DNA probe, Entomophaga grylli has been
divided into pathotypes according to host subfamily (Ramoska
et al. 1988). Several laboratory transmission experiments of
E. muscae s.l. have been performed within a range of Diptera
(Baird 1957; Eilenberg et al. 1987; Kramer & Steinkraus 1981;
Mullens 1989; Steenberg et al. 2001; Steinkraus & Kramer
1987), but no attempt to group E. muscae into pathotypes or
groups based on their host range has been made. However,
E. scatophagae is believed to have a restricted host range and
is only described from a single host species; E. grandis and
E. syrphi have only been reported from larger and smaller syr-
phid flies, respectively (Keller 2002).
A recent study of the intraspecific variation within E. mus-
cae s.str. showed that several genotypes occur and that each
type was restricted to a single host species (Jensen et al.
2001). This indicates a high degree of host specificity in the
E. muscae species complex similar to that observed in the
E. grylli complex. Phylogenetic relationships of the order En-
tomophthorales have been inferred from the SSU rRNA gene
sequences (Jensen et al. 1998; Nagahama et al. 1995); but until
now no sequence analyses have been accomplished to resolve
species complexes within Entomophthorales. Nevertheless,
PCR-RFLP of the ITS II and the 50 end of the LSU rRNA gene
support the separate origin of E. muscae, E. schizophorae, and
E. syrphi (Jensen & Eilenberg 2001).
The aim of this study was to evaluate the value of host
range, morphological, and genetic characteristics for op-
erational species recognition of species within the E. muscae
complex. Studies were made to elucidate specific topics: (1)
the plasticity of certain morphological characteristics was
explored, (2) the physiological host range among certain
host taxa was examined, and (3) the phylogeny was assessed
by sequence analyses of the ITS II and part of the LSU rRNA
gene.
Materials and methods
Field samplings
Approximately 8000 live flies (Table 1) were collected by
sweep-net in Denmark during 1999 (June–September) and
2000 (May–October) from the following habitats: cabbage and
rape fields, border vegetation, forests, stables, and houses.
Live flies were transferred individually to 30 ml cups contain-
ing 5 ml 1.5 % water-agar as described by Eilenberg & Philipsen
(1988). Condensation in the cup was avoided by cutting a hole
in the lid and covering it with gauze. Food (a paste of 9 parts
sugar, 1 part dry yeast, and little water) was applied directly
onto the gauze. Predatory flies (predominantly Coenosia tigrina
and Scatophaga stercoraria) were also supplied with live house-
flies every third day, and hover flies (Syrphidae) were provided
with flowers or pollen. The flies were incubated at room
temperature and exposed to natural day length. They were ex-
amined for death and external signs of fungal infection every
day in a 14 d period. Conidia were collected on glass slides in a
humid chamber from all the cadavers and stored dry until
morphological examination. Immediately after the conidial
collection, well-sporulating cadavers were either used in the
host range transmission experiments or were used to obtain
in vitro isolates to ensure maximum inoculum levels.
Morphological characteristics
The morphology of primary conidia discharged from the orig-
inal hosts was compared with the morphology of the same
in vivo-isolate after cross-transmission to an alternative host.
The dimensions of 20 primary conidia per isolate were mea-
sured mounted in lactophenol-cotton blue (0.01 % cotton blue)
using an Olympus Provis microscope supplemented with an
Oly-Lite computer-based system for morphometrics at
400
magnification. The number of nuclei of 20 primary conidia
was counted mounted in DAPI (4,6-diamidino-2-phenylindole,
5 mg ml1) using a Zeiss Axiophot epifluorescence microscope
(filter 0.5; violet, excitation 395–440) at
400 magnification.
Table 1 – Number of collected flies and number of flies
with external sign of Entomophthora muscae s.l.
Host taxa Approx. number Number of
of collected flies Entomophthora
infected flies
Anthomyiidae
Anthomyiidae sp. 350 25
Delia radicum 1100 250
Calliphoridae
Pollenia angustigena/rudis 900 90
Muscidae
Coenosia tigrina 300 20
Musca domestica 700 100
Scatophagidae
Scatophaga stercoraria 1650 90
Syrphidae
Syrphidae sp.1 1000 90
1 From the genera Melanostoma and Platycheirus.
Entomophthora muscae species complex 943

Standard LSD tests (PROC GLM; SAS Institute 1999) were
used to test if the interaction of host and isolate had signifi-
cant influence on the length, width, and number of nuclei in
the primary conidia of Entomophthora muscae and E. schizo-
phorae. Within each isolate comparison of mean values were
made using the Tukey test.
this study were stored in the Fungal Insect Pathogenic Culture
Collection at the Royal Veterinary and Agricultural University,
Copenhagen, by cryopreservation at 80  C according to
López Lastra et al. (2001) and are deposited in ARSEF (USDA-
ARS, Ithaca, NY). All the isolates used are listed in Tables 2
and 3.

In vitro isolation Physiological host range

In vitro isolation of the Entomophthora species was done as in
Jensen et al. (2001). Fresh, well-sporulating cadavers were
placed on a small sterile inverted Petri dish lid (3.5 cm diam)
and the base was put on top. The wings of the large flies, Musca
domestica, Myospila meditabunda, Pollenia angustigena, P. rudis,
and Scatophaga stercoraria were cut off in order to avoid con-
tamination by wings hitting the Petri dish base. Sporulation
was allowed for 30 min and the arrangement was placed in
a humid chamber in order to enhance sporulation. In a sterile
hood 1 ml GLEN (Beauvais & Latgé, 1988) including 5 % fetal
bovine serum was poured onto the discharged conidia. A
new sterile lid was used and the petri dish was sealed with
Parafilm and incubated dark at 23  C. When growth was ob-
served, the in vitro culture was transferred to 50 ml cell culture
flasks with 5 ml GLEN for further growth. The isolates used in
Laboratory transmission experiments were conducted to
obtain information on physiological host range. Two types of
transmission experiments were performed: in small cup
experiments, one to five live flies (receptors) and a well-
sporulating cadaver (donor) were placed together in a 30 ml
cup containing 5 ml 1.5 % water-agar. Cups with live flies
and sporulating cadavers were maintained at 18  C and
received a photoperiod of 16:8 (light:dark) in 24 h for fungal in-
oculation. Alternatively, in caged experiments, ten to 20 live
flies (receptors) were placed in a cylindrical cardboard
container (9 cm diam, 5 cm high). The cardboard containers
had a Petri dish base (9 cm diam) as the bottom and a 2-mm
mesh top as a cover. Two to five well-sporulating cadavers
(donors) were placed on the mesh top and a Petri dish lid
(9 cm diam) was put on top of the sporulating cadavers. The

Table 2 – Insect host and geographic origin of the in vitro isolates used for RAPD
Isolatea Fungal species Host species Collection site Year
(host family)

KVL 00-56 Entomophthora scatophagae Scatophaga stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-57b E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-58 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-59 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-60 (ARSEF 6704) E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-61 (ARSEF 6705) E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-62 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-63 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-64 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-65 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-66 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
KVL 00-67 E. scatophagae S. stercoraria (Scatophagidae) Tuse Næs, Denmark 2000
NCRI 1-99 (ARSEF 6274) Entomophthora muscae Delia radicum (Anthomyiidae) Ski, Norway 1999
ARSEF 2668 E. muscae D. radicum (Anthomyiidae) Svanholm, Denmark 1986
KVL 99-102 (ARSEF 6815)b E. muscae D. radicum (Anthomyiidae) Sengeløse, Denmark 1999
KVL 99-103 E. muscae D. radicum (Anthomyiidae) Ørslev, Denmark 1999
KVL 99-96 E. muscae D. radicum (Anthomyiidae) Sengeløse, Denmark 1999
KVL 99-85(ARSEF 6811) E. muscae D. radicum (Anthomyiidae) Hegnstrup, Denmark 1999
KVL 99-13 (ARSEF 6132)bc E. muscae s.str. Musca domestica (Muscidae) Kirke Såby, Denmark 1999
KVL 99-23 (ARSEF 6141) E. muscae s.str. M. domestica (Muscidae) Skævinge, Denmark 1999
KVL 99-110 (ARSEF 6813) E. muscae s.str. M. domestica (Muscidae) Knardrup, Denmark 1999
ARSEF 2542 E. muscae s.str. M. domestica (Muscidae) Ribe, Denmark 1986
KVL 99-63 E. muscae s.str. M. domestica (Muscidae) Sverkildstup, Denmark 1999
KVL 99-70 E. muscae s.str. M. domestica (Muscidae) Sverkildstup, Denmark 1999
KVL 99-90 E. ferdinandi Coenosia tigrina (Muscidae) Ørslev, Denmark 1999
KVL 99-92 (ARSEF 6918) E. ferdinandi C. tigrina (Muscidae) Ørslev, Denmark 1999
KVL 99-87 E. ferdinandi Pegoplata infirma (Anthomyiidae) Hegnstrup, Denmark 1999
NCRI 2-99 E. ferdinandi Botanophila fugax (Anthomyiidae) Ski, Norway 1999

a KVL, Danish abbreviation for the Royal Veterinary and Agricultural University, Copenhagen; ARSEF, ARS Collection of Entomopathogenic
Fungi, Ithaca; NCRI, Norwegian Crop Research Institute.
b Isolates also used for the sequence analyses.
c In vitro isolate made from in vivo cultures, that was also used for the transmission experiments.
944 A. B. Jensen et al.

cardboard assemblies with live flies and sporulating cadavers

Year

1999
1999
2000
1996
2000
2000
1998
were maintained 18  C and received a photoperiod of 16:8
(light:dark) in 24 h for fungal inoculation. The cage type was
only performed with donors from two in vivo cultures of Ento-

Hundested, Denmark
Hundested, Denmark
Kirke Såby, Denmark
mophthora muscae s. str (DK3) (Keller et al. 1999) and E. schizo-

Sengeløse, Denmark
Tuse Næs, Denmark
Roskilde, Denmark
Collection site

Gribskov Denmark
phorae (DPIL 150896) (Kalsbeek et al. 2001) and with Musca
domestica and Delia radicum as receptors. The two in vivo cul-
tures were maintained by continuous passages in M. domestica
as described by Kramer and Steinkraus (1981), and both had
M. domestica as original host.
After the inoculation, the flies were placed individually in
30 ml cups containing water agar and covered with gauze and
a lid with a hole. The sugar–yeast paste described above was
Accession no.d ITSII/LSU

provided as food directly on the gauze. The flies in the cups

KVL, Danish abbreviation for the Royal Veterinary and Agricultural University, Copenhagen; ARSEF, ARS Collection of Entomopathogenic Fungi, Ithaca.
DQ481217/DQ481224
DQ481218/DQ481225
DQ481219/DQ481226
DQ481220/DQ481227
DQ481221/DQ481228
DQ481222/DQ481229
DQ481223/DQ481230
Table 3 – Insect host and geographic origin of the isolates used for sequencing of the ITS II and the first part of the LSU rRNA gene

were incubated at 18  C with a photoperiod of 16:8 (light:-

dark) for three weeks, and were examined daily for death
and external signs of fungal infection. Conidia from the ca-
davers were collected and stored dry until examination. M.
domestica originated from a laboratory stock and was sup-
plied as pupae by the Danish Institute of Agricultural Sci-
ences, Department of Integrated Pest Management, Danish
Pest Infestation Laboratory Group (DPIL). The other receptor
flies originated from the field but were used only after a quar-
Scatophaga stercoraria (Scatophagidae)

antine period of at least two weeks to exclude contamination

Melanostoma mellinum (Syrphidae)

from natural infections. Thus the field collected flies were old
Delia radicum (Anthomyiidae)

and therefore those that died during the transmission exper-

Pollenia rudis (Calliphoridae)
Musca domestica (Muscidae)

M. meditabunda (Muscidae)
Host (family)

iments without external signs of Entomophthora ssp. infec-

M. domestica (Muscidae)

tions were excluded.

Logistic regression was used to analyse the effects of fungal
species and the receptor fly on the percentage of infected flies
In vitro isolate made from in vivo cultures, that was also used for the transmission experiments.

(Proc GENMOD; SAS Institute 1999) with a binomial distribu-

tion and logit as link functions. Over-dispersion was taken
into account (Collet 1991). To test the effect of receptor fly,
all receptor flies were categorized into three groups: (1) the
receptor fly was of the same species as the donor fly, (2) the
Nuclei number

receptor fly was M. domestica, and (3) the receptor fly was nei-
ther the same fly species as the donor fly or M. domistica.
16–18
14–16
16–18

20–23
17–21
5–7
5–7

Genetic characteristics

DNA extraction and amplification of ITS II and the LSU rRNA

gene of seven in vitro isolates (Table 2) were conducted as de-
Entomophthora muscae s.str.

scribed by Jensen et al. (2001). For the amplification of the ITS II

the primers nu-5.8 S-50 (Jensen & Eilenberg 2001) and ITS 4
(White et al. 1990) were used and for the amplification of the
Genbank accession numbers for the sequences.
Species

Entomophthora sp.

LSU the primers nu-LSU 0018-50 (Jensen & Eilenberg 2001)

E. schizophorae
E. schizophorae
E. scatophagae

E. syrphi s.str.

and nu-LSU 0805-30 (Kjøller & Rosendahl 2000) were used.

Isolates also used for the RAPD analysis.
E. muscae

The PCR conditions were initial denaturation for 5 min at

96  C, followed by 35 cycles with denaturation for 1 min at
96  C, annealing for 1 min at 62  C (ITS II) or 55  C (LSU), ex-
tension for 1 min at 72  C and a final extension for 10 min at
71  C. The PCR reactions were carried out in a 25 ml volume
99-13 (ARSEF 6132)b,c
99-102 (ARSEF 6815)b
00-57 (ARSEF 6704)b

with 250 mM of each dNTP, 0.8 mM of each primer, 2.5 mM

99-18 (ARSEF 6137)
00-93 (ARSEF 6817)
00-92 (ARSEF 6701)
98-19 (ARSEF 5955)

MgCl2, 1
buffer (10 mM Tris–HCl, pH 8.8 at 25  C, 50 mM KCl,

0.1 % Triton X-100), 1 unit DyNazyme II (Finnzymes, Espoo,
Finland) and 1 ml extracted DNA, diluted at 1:10 or 1:100. Before
sequencing, the PCR products were purified with the GFXtm
Isolatea

PCR DNA and Gel Purification Kit (Amersham Bioscience,

KVL
KVL
KVL
KVL
KVL
KVL
KVL

Uppsala, Sweden). Both strands of the ITS II and LSU were se-
d
b
a

quenced by GATC GmbH. To get the full sequence of the ITS II

Entomophthora muscae species complex
four internal primers ITS II 500-50 (5’-ACGTTGGCAACGAC
WAGTAA-3’), ITS II 500-3’ (5’-TTACTWGTCGTTGCCAACGT-
3’), ITS II 1000-5’ (5’-TTGTTGAAWCTTTTCTGTCGC-3’) and
ITS II 1000-30 (50 -CGACAGAAAAGWTTCAACAAT-30 ) were
used. The chromatograms were checked using Sequencer 3.1
(Gene Codes Corporations, Ann Arbor, MI). In addition the
LSU sequence of Entomophaga aulicae (Genbank accession no:
U35394) was included, but the E. aulicae ITS II sequence was
too divergent to be aligned with the sequences generated in
this study. Following initial alignment with CLUSTAL V 1.60
(Higgins et al. 1992) the alignments were adjusted manually.
MP analyses were performed with the PAUP 4.0b6 (Swof-
ford 1998) on individual data sets (ITS II and LSU) and com-
bined data set (ITS II þ LSU). The branch-and-bound search
option was used to find exact solutions. All characteristics
were equally weighted, and invariant characteristics were
ignored. In the LSU analysis E. aulicae was chosen as an out-
group. Supports for internal branches were assessed by 1000
BS replications, and the partition-homogeneity test was used
to evaluate the concordance between two individual data sets.
NJ analyses were performed with the software Treecon for
Windows (Van de Peer & De Wachter 1994) using the Jukes-
Cantor evolutionary model. Supports for internal branches
were assessed by 1000 BS replications.
RAPD was performed with the 10 commercial RAPD primers
(OPA01, OPA02, OPA05, OPA06, OPA07, OPA09, OPA10, OPA15,
OPA17,OPA20; Operon Technologies, Alameda, CA) on 11 Ento-
mophthora scatophagae, six E. muscae, six E. muscae s.str., and
four E. ferdinandi in vitro isolates originating from various
host species (Table 2) as described in Jensen et al. (2001).
Results
Morphological characteristics
The primary conidia all were campanulate and multinucleate
irrespective of host origin. The overall analysis of the primary
conidia from the cross-transmission experiments showed sig-
nificant effects on the interaction of host species and isolate on
length (F4, 266 ¼ 75.80; P < 0.0001), diameter (F4, 266 ¼ 49.16;
P < 0.0001), and number of nuclei (F4, 266 ¼ 3.83; P ¼ 0.0048) for
Entomophthora muscae and on length (F1, 133 ¼ 19.19; P < 0.0001)
and diameter (F1, 133 ¼ 89.78; P < 0.0001) for E. schizophorae.
The number of nuclei in E. schizophorae varied a little, but
was not significantly different irrespective of host species
(F1, 133 ¼ 1.29; P ¼ 0.2587).
No clear pattern in size or number of nuclei after transmis-
sion to alternative hosts was detected, except for Pollenia
angustigena/P. rudis where conidia from the original host
were slightly larger and contained more nuclei than conidia
of the same isolates produced from other dipteran hosts
(Table 4). However, the changes in morphology after transmis-
sion to alternative hosts were never so dramatic that they
influenced the classical morphological species identification.
Physiological host range
Laboratory transmission of Entomophthora muscae s.l. between
different host species was possible, but with variable success
945
(Table 5). The mortality attributable to the entomophthora-
lean fungi was significantly affected by the receptor fly
(c2 ¼ 15.89; df ¼ 2; P ¼ 0.0004). Having Musca domestica as recep-
tor fly compared with having the same receptor as donor fly
species did not show significantly increased infectivity, as
tested using Pair-wise comparisons using least squares means
to test the effects of the species of receptor fly [Pr > |t| for H0:
LSMean (category i) ¼ LSMean (category j)] (c2 ¼ 0.55; df ¼ 1;
P ¼ 0.4593), whereas all other comparisons were significantly
different (P  0.0089).
Whereas M. domestica was an excellent receptor for all iso-
lates tested (except for those from E. syrphi), Coenosia tigrina
proved difficult to infect in our study conditions, even with
E. muscae from naturally infected C. tigrina. Interestingly we
observed a decrease in the number of conidiophores, as only
one or two narrow abdominal bands of conidiophores
appeared in several of the alternative hosts, thus resulting in
moderate to restricted sporulation.
Genetic characteristics
Approximately 1440 bp of the ITS II and 900 bp of the 50 end of
the LSU rRNA gene were sequenced from the seven isolates
listed in Table 3. The ITS alignment had 306 informative sites,
whereas the LSU alignment, including Entomophthora aulicae,
had 56 informative sites. Both regions produced trees with
similar topology using maximum parsimony (Fig 1A–B). The
parsimony-based partition homogeneity test found no support
for incongruence between these two data sets (P ¼ 1.000), and
thus the two data sets were combined. The tree topology of
the combined data sets was similar to the tree topology
of the individual data sets (Fig 1C), as were also the topology
of the NJ trees (data not shown). Three major lineages of the
E. muscae species complex were resolved, each of which cen-
tres on a single species; E. muscae, E. schizophorae, and E. syrphi.
Each of the lineages was supported by BS values above 92 %.
The E. syrphi and E. muscae lineages clustered together on
a branch supported by a BS value of 74 % in the LSU analysis.
In this study E. scatophagae was isolated in vitro for the first
time. All 11 E. scatophagae isolates obtained had similar RAPD
profiles with the ten RAPD primers used, but were different
from E. muscae isolates originating from other host species
(Fig 2).
Discussion
Host range has long been regarded to be an important charac-
teristic in the circumscription of Entomophthora species. Physi-
ological host range can be reflected in laboratory transmission
experiments, and our study showed that receptor flies from
other (unnatural) host taxa generally were less susceptible
than the original (natural) host flies, which is concordant to
other transmission experiments performed (Baird 1957;
Eilenberg et al. 1987; Kramer & Steinkraus 1981; Mullens 1989;
Steenberg et al. 2001; Steinkraus & Kramer 1987). Those speci-
mens that got infected often showed a decreased sporulation,
which would consequently decrease further disease transmis-
sion. However, Musca domestica was equally susceptible to
E. muscae s.l., compared with the exposed original host species.
946 A. B. Jensen et al.

Table 4 – Dimensions and numbers of nuclei in primary conidia of Entomophthora muscae and E. schizophorae before and
after transmission to alternative host species
Transmission Fungal species Host species Mean no. Mean Mean
experiments of nuclei length (mm) width (mm)

1 Entomophthora muscae Scatophaga stercorariaa 17.4 a 27.2 a 21.8 a

1 E. muscae Musca domestica 15.6 b 28.2 b 22.4 a

2 E. muscae Delia radicuma 17.1 a 29.2 b 22.6 b

2 E. muscae M. domestica 16.1 b 25.8 a 20.1 a

3 E. muscae D. radicuma 16.6 a 24.6 a 18.5 a

3 E. muscae M. domestica 15.6 b 27.1 c 22.3 c
3 E. muscae M. domestica / D. radicumb 15.6 b 25.6 b 20.5 b

4 E. muscae D. radicuma 14.8 a 23.7 a 18.5 a

4 E. muscae M. domestica 15.5 a 25.7 b 19.4 b

5 E. muscae D. radicuma 15.3 a 24.3 a 18.5 a

5 E. muscae M. domestica 16.0 a 28.2 b 23.2 b

6 E. muscae s.str.d M. domesticaa 16.7 b 29.7 b 22.8 b

6 E. muscae s.str.d D. radicum 17.0 b 25.4 a 20.7 a
6 E. muscae s.str.d Pollenia angustigena/rudisc 19.4 a 31.4 c 25.1 c

7 E. schizophoraee M. domesticaa 5.5 a 22.6 a 17.9 a

7 E. schizophoraee D. radicum 5.5 a 23.2 a 18.0 ab
7 E. schizophoraee P. angustigena/rudisc 5.6 a 23.9 b 18.5 b

8 E. schizophorae Coenosia tigrinaa 5.6 a 20.4 a 14.5 a

8 E. schizophorae M. domestica 5.5 a 20.5 a 15.1 b

9 E. schizophorae P. angustigenaa 6.1 a 23.8 a 19.3 b

9 E. schizophorae M. domestica 5.5 a 20.7 b 15.2 a

All the cadavers used in the transmission experiments came from the field sampling, except the for the Entomophthora muscae s.str. and E. schiz-
ophorae with M. domestica as original host. Within each transmission experiment comparison of the mean values were made using a Tukey test
and mean values followed by different letters are significantly different (P < 0.05).
a The original host species.
b This isolate was transmitted from Delia radicum to Musca domestica and then back to D. radicum again.
c A mixed population of Pollenia angustigena and P. rudis.
d The cadavers came from the in vivo culture DK3.
e The cadavers came from the in vivo culture DPIL 150896.

Nevertheless, only a single genotype of E. muscae s.str. was pre-
viously documented from Danish collections of M. domestica
(Jensen et al. 2001) sampled at different locations and years,
so the high susceptibility of M. domestica to several E. muscae
genotypes seems not to be reflected in natural epizootics.
Within the E. muscae species complex morphological and
nuclear characteristics of the primary conidia are taxonomi-
cally important (Keller 1984). In this study we compared the
dimensions of the primary conidia and their number of nuclei
of isolates from their original (natural) hosts and after being
transferred to alternative hosts (cross-transmission) in order
to check the stability of these characteristics. We conclude
that even though conidial characteristics change significantly
when produced in alternative host species, the size and num-
ber of nuclei in the primary conidia are stable enough for
species circumscriptions. Interestingly, the dimensions and
number of nuclei in the primary conidia from isolates passed
through Pollenia angustigena/P. rudis increased regardless of
the original host species, but they were always within the
range of the classically morphological species descriptions
of either E. schizophorae or E. muscae s.str. (Keller et al. 1999).
Host taxa have previously been shown to have an effect on
the dimensions of the primary conidia as well as their number
of nuclei. However, the morphology of E. muscae isolates orig-
inating from different host taxa are not strongly divergent
(Jensen et al. 2001), and conidial characteristics could not be
used alone for diagnostic purposes due to the extent of their
morphological variability.
The shape of the hyphal bodies and yellowish colour of
E. scatophagae conidia are the main phenetic criteria distin-
guishing E. scatophagae from E. muscae s.str. (Keller et al. 1999;
Steinkraus & Kramer 1988). The yellowish colour of dry coni-
dia, however, results from reflection of the yellow hair of the
hirsute scatophagids (MacLeod et al.1976), and conidia of this
species are uncoloured and hyaline on a glass slide. In addi-
tion, different post-mortem characteristicistics of flies have
been suggested to have taxonomic value for separating Ento-
mophthora species, but as these characteristics are host rather
than fungus-dependent they must be considered to be invalid
for taxonomic purposes (Steenberg et al. 2001).
The phylogenetic sequence analyses of the E. muscae
species complex resolves into three major lineages, each of
which centres on a single species; E. muscae, E. schizophorae,
and E. syrphi, that had been previously recognized using tr-
aditional phenetic and pathobiological characteristics (Ba1azy
1993; Keller 2002; Keller 1987). The E. syrphi and the E. muscae
Entomophthora muscae species complex 947

Table 5 – Transmission experiments of members of the Entomophthora muscae complex to different host taxa
Fungus Original host Receptor fly No. of No. of No. of No. of
cadavers repetitions flies at risk infected (%)

Entomophthora ferdinandi Coenosia tigrina (Muscidae) C. tigrinac 2 2 9 0 (0.0)

E. ferdinandi C. tigrina (Muscidae) Delia radicum 8 8 15 2 (13.3)
E. ferdinandi C. tigrina (Muscidae) Musca domestica 12 12 53 30 (56.6)

E. muscae s.stra M. domestica (Muscidae) M. domesticac 39 8 138 133 (96.4)

E. muscae s.stra M. domestica (Muscidae) C. tigrina 5 1 19 0 (0.0)
E. muscae s.stra M. domestica (Muscidae) D. radicum 72 35 85 3 (3.5)
E. muscae s.stra M. domestica (Muscidae) Pollenia sp. 9 5 28 5 (17.9)

E. muscae D. radicum (Anthomyiidae) D. radicumc 34 18 87 16 (18.4)

E. muscae D. radicum (Anthomyiidae) M. domestica 66 50 272 222 (81.6)

E. muscae Scatophaga stercoraria (Scatophagidae) S. stercorariac 22 18 66 6 (9.1)

E. muscae S. stercoraria (Scatophagidae) D. radicum 12 8 92 0 (0.0)
E. muscae S. stercoraria (Scatophagidae) M. domestica 20 15 154 11 (7.1)

E. schizophoraeb M. domestica (Muscidae) M domestica c 33 8 151 141 (93.4)

E. schizophoraeb M. domestica (Muscidae) D. radicum 39 8 99 15 (15.2)
E. schizophoraeb M. domestica (Muscidae) Pollenia sp. 9 5 25 2 (8.0)

E. schizophorae Pollenia sp. (Calliphoridae) Pollenia sp.c 12 12 68 44 (64.7)

E. schizophorae Pollenia sp. (Calliphoridae) M. domestica 18 17 93 51 (54.8)

E. syrphi Indet. Syrphidae Indet. Syrphidae 14 6 18 0 (0.0)

E. syrphi Indet. Syrphidae D. radicum 11 11 11 0 (0.0)
E. syrphi Indet. Syrphidae M. domestica 8 8 41 0 (0.0)

All the cadavers used in the transmission experiments came from the field sampling, except for the Entomophthora muscae s.str. and E. schizo-
phorae with M. domestica as original host.
a The cadavers came from the in vivo culture DK3.
b The cadavers came from the in vivo culture DPIL 150896.
c The original host species.

s.str. lineages, which both include fungi having high numbers
of nuclei in their conidia, were more closely related to each
other than to the E. schizophorae lineage which has fewer
nuclei in the conidia. This relationship should, however, be
investigated further by including more Entomophthora species
and also species affecting hosts other than dipterans. The
molecular gave supported the existing taxonomy based on
morphological characteristics and, together with ecological
characteristics, provided evidence for a speciation that has
not yet been recognized by morphological criteria. This is
best exemplified in the E. muscae lineages, where sequence
divergence of the ITS II and to a lesser extent the first part of
the LSU rRNA gene was seen between the three isolates ori-
ginating from three different host species. The multilocus ap-
proach RAPD that was used to investigate the genetic
variation of members of the E. muscae lineages, particularly
in E. scatophagae, also supported the cryptic speciation of E.
muscae s.str. and agreed with the results from the UP-PCR,
RAPD, and PCR-RFLP analyses by Jensen et al. (2001).
Operational taxonomic concept
An operational species level concept for the taxonomic re-
solution of the Entomophthora muscae species complex that
we now propose, seeks to accommodate non-taxonomists
and researchers in applied science, as well as taxonomists.
The genus Entomophthora is monophyletic evidenced by the
ITS II and the LSU rRNA gene sequences analyses in the current
study and also by previous SSU rRNA gene sequences analyses
(Freimoser et al. 2001; Jensen et al. 1998). However, the E. muscae
species complex seems to be a paraphyletic assembly based on
PCR-RFLP analysis (Jensen & Eilenberg 2001). Even though the
host range for the multinucleate E. muscae group was expanded
to also include Hymenoptera, we suggest that the E. muscae s.l. is
restricted to species naturally found on derived clades of flies.
E. muscae s.l. is, in this definition, not to be regarded as a single
phylogenetic entity but as a workable assemblage.
E. muscae s.str. from Musca domestica is genetically very dif-
ferent from isolates originating from other naturally infected
dipteran host taxa and correspondingly significant phenetic
differences were observed (Jensen et al. 2001). Therefore we
suggest that the nomenclaturally strict sense of E. muscae
should be limited to isolates originating from M. domestica,
the host from which it was originally described (Cohn 1855)
and redescribed (Keller et al. 1999). However, it would be in-
teresting to study the biogeographic variation of E. muscae
s.str. using geographically diverse samples of the globally
distributed M. domestica.
E. muscae isolates from hosts other than M. domestica, such
as Delia radicum and Scatophaga stercoraria, clustered together
in the molecular analyses on separate branches that do not
include the fungi from M. domestica. Other studies have also
shown that each host fly species had its own distinctive fungal
pathogen genotype, but that they fall within the same mor-
phological parameter ranges as E. muscae s.str. (Jensen et al.
2001; Thomsen & Jensen 2002). This indicates that a radial
948 A. B. Jensen et al.

E. muscae s. str E. muscae s. str. E. muscae s. str.

(Md) (Md) (Md)
92 100
100
E. muscae E. muscae E. muscae
(Dr) (Dr) (Dr)
74 92 97
74 E. scatophagae E. scatophagae E. scatophagae
(Ss) (Ss) (Ss)

Entomophthora Entomophthora Entomophthora

95 sp. (Mym) sp.
98 sp. (Mym) 99
E. syrphi s. str. E. syrphi s. str.
(Mem) E. syrphi s. str.
(Mem) (Mem)
E. schizophorae
100 (Md) E. schizophorae E. schizophorae
100 (Md) 100 (Md)
E. schizophorae
(Pr) E. schizophorae E. schizophorae
50 changes 50 changes
(Pr) (Pr)
Entomophaga
50 changes
A B C
Fig 1 – Phylogenetic relationships within the Entomophthora muscae species complex inferred from parsimony analyses of
the first part of the LSU rRNA gene (A), the ITS II (B) and a combined data set (C). BS percentages over 50 % from 1000 replicates
are shown above each supported branch. A. The single most parsimonious phylogram requiring 226 steps (CI [ 0.9336
and RI [ 0.8235), Entomophaga aulicae was used as outgroup. B. The single most parsimonious phylogram requiring 500
steps (CI [ 0.9420 and RI [ 0.9254), midpoint rooting was used. C. The single most parsimonious phylogram requiring
726 steps (CI [ 0.9439 and RI [ 0.9297), midpoint rooting was used. Md [ Musca domestica; Dr [ Delia radicum;
Ss [ Scatophaga stercoraria; Mym [ Myospila meditabunda; Mem [ Melanostoma mellinum; Pr [ Pollenia rudis.

adaptation toward high host specificity may have occurred, of conidial nuclei and recognized as E. syrphi from a muscoid
and that these ecological differences can be used in the taxo- fly (Phaonia perdita) was shown to cause infection in M. domes-
nomic separation of E. muscae s.l. Based on the molecular data tica. An Entomophthora species with similar morphology but
two possible taxonomic solutions exist: either E. muscae isolated from another muscoid host species (Myospila medita-
should be maintained as one highly plastic species (which bunda) was included in the current molecular analysis, where
would require rejection of E. scatophaga as a separate species), it formed a monophyletic group together with E. syrphi. Never-
or E. muscae (as currently recognized within the E. muscae spe- theless, we do not recommend including these Entomophthora
cies complex) should be segregated into multiple new species species in E. syrphi because of their high sequence divergence
representing the distinctive genotypes affecting each of the and their different host ranges. E. syrphi, as well as E. grandis,
host fly species or genera. We recommend the latter solution. should be restricted to Entomophthora pathogenic to hoverflies.
E. schizophorae is easily recognized by both morphological Moreover the other species deserves to receive an amplified
and molecular criteria. A certain overlap between the natural and more detailed species description.
host range for E. schizophorae and E. muscae exists, e.g., in host A species can be described as a single lineage of ancestor–
taxa such as M. domestica and D. radicum, while in other host descendent populations which maintains its identity from
taxa, such as Chamaepsila rosae, only E. schizophorae infections
have so far been found (Eilenberg 2002; Eilenberg & Philipsen
1988). The sequence differences between the M. domestica and Scatophaga Delia Coenosia Musca P. infirma
P. rudis isolates used in this study suggest a similar divergence, stercoraria radicum tigrina domestica B. fugax
Neg. control
as seen within the E. muscae lineage. But until a thorough study
of the variation of E. schizophorae from different host taxa is per-
1500 bp
formed E. schizophorae should be recognized as a single species. 1200 bp
1000 bp
E. syrphi can be distinguished molecularly from the other 800 bp
members of the E. muscae species complex. Morphologically, 700 bp

however, it can be difficult to differentiate some E. syrphi 500 bp

from E. muscae s.str. (Steenberg et al. 2001), but E. syrphi gener-
ally has larger conidia containing more nuclei. E. syrphi seems
to have a restricted host range and has only been recorded from
a few hover fly species (Ba1azy 1993; Keller 1987). Variable RFLP
patterns have been observed within E. syrphi, suggesting the
existence of yet another unexplored species complex (Thom-
sen & Jensen 2002). We were not able to cross-infect E. syrphi
from hover flies to other fly species. However, in a study by
Steenberg et al. (2001) an Entomophthora with a high number
400 bp
300 bp
Fig 2 – Agarose gel electrophoresis of RAPD amplifications
obtained with the primer OPA-10. A molecular size ladder
appears in the first and last lane. The Entomophthora muscae
isolates obtained from a single host species had similar
profiles, whereas isolates from different host species had
different profiles. P. [ Pegoplata, B. [ Botanophila.
Entomophthora muscae species complex
other such lineages and which has its own evolutionary ten-
dencies and historical fate (Wiley 1978). For species within
the E. muscae complex, genetic characteristics, physiological
host range and morphology of the conidia are entities that
can be used to develop an operational taxonomic species con-
cept. This study shows that the various regions of the ribo-
somal repeat, in particular the ITS II, include variation
suitable for distinguishing species within the E. muscae species
complex. However, in future other genomic regions should be
included to perpetuate the recognition of phylogenetically
defined species (Taylor et al. 2000). Physiological host range
as evidenced by transmission experiments only provides
a hint of the ecological host range; however, the correlation
of molecular data with phenotypic evidence and host range
will continue to improve our understanding of the evolution-
ary processes and species limits within the Entomophthorales.
Acknowledgements
We wish to thank Verner Michelsen and Stig Andersen, Zoo-
logical Museum, Copenhagen for identification of fly hosts.
From the Royal Veterinary and Agricultural University, Jan
Martin is thanked for help with collection of Scatophaga ster-
coraria, Kirsten Ploug and Mette Vingaard are thanked for
technical assistance. Nicolai Vitt Meyling for comments on
the manuscript and Charlotte Nielsen for statistical support.
The Danish Pest and Infestation Laboratory is thanked for pro-
viding flies and two in vivo cultures. Richard Humber, USDA,
Ithaca and Ingeborg Klingen, Norwegian Crop Research Insti-
tute are thanked for in vitro cultures. The Danish Ministry of
Food, Agriculture and Fisheries, the Royal Veterinary and Ag-
ricultural University and the Carlsberg Foundation supported
this work financially.
references
Baird RB, 1957. Notes on a laboratory infection of Diptera caused
by the fungus Empusa muscae Cohn. Canadian Entomology 89:
432–435.
Ba1azy S, 1993. Flora of Poland, Fungi (Mycota), Vol. 24, Entomoph-
thorales. Wydawnictwa Inst. Bot. Pan., Kraków.
Beauvais A, Latgé JP, 1988. A simple medium for growing ento-
mophthoralean protoplasts. Journal of Invertebrate Pathology 51:
175–178.
Ben-Ze’ev IS, Kenneth RG, 1982. Features-criteria of taxonomic
importance in the Entomophthorales. Part I. A revision of the
Batkoan classification. Mycotaxon 14: 393–455.
Cohn F, 1855. Empusa muscae und die Krankheit der Stubenfliege.
Hedwigia 10: 57–61.
Collet D, 1991. Modeling Binary Data. Chapman & Hall, London.
Eilenberg J, 2002. Biology of fungi from the order Entomophthorales
with emphasis on the genera Entomophthora, Strongwellsea and
Eryniopsis: A contribution to insect pathology and biological control.
D.Sc. Thesis. Department of Ecology, The Royal Veterinary and
Agricultural University, Copenhagen.
Eilenberg J, Bresciani J, Martin J, 1987. Entomophthora species with
E. muscae-like primary spores on two new insect orders, Cole-
optera and Hymenoptera. Nordic Journal of Botany 7: 577–584.
949
Eilenberg J, Philipsen H, 1988. The occurrence of Entomophthorales
on the carrot flies (Psila rosae F.) in the field during two suc-
cessive seasons. Entomophaga 33: 135–144.
Freimoser FM, Jensen AB, Tuor U, Aebi M, Eilenberg J, 2001. Iso-
lation and in vitro cultivation of the aphid pathogenic fungus
Entomophthora planchoniana. Canadian Journal of Microbiology 47:
1082–1087.
Higgins DG, Bleasby AJ, Fuchs R, 1992. CLUSTAL V: improved
software for multiple sequence alignment. Cabios 8: 89–191.
Humber RA, 1981. An alternative view of certain taxonomic cri-
teria used in the Entomophthorales (Zygomycetes). Mycotaxon 13:
191–240.
Humber RA, 1989. Synopsis of a revised classification for the En-
tomophthorales (Zygomycotina). Mycotaxon 34: 441–460.
Jensen AB, Eilenberg J, 2001. Genetic variation within the insect-
pathogenic genus Entomophthora, focusing on the E. muscae
complex, using PCR-RFLP of the ITS II and LSU rDNA. Myco-
logical Research 105: 307–312.
Jensen AB, Gargas A, Eilenberg J, Rosendahl S, 1998. Relationships
of the insect-pathogenic order Entomophthorales (Zygomycota,
Fungi) based on phylogenetic analyses of nuclear small sub-
unit ribosomal DNA sequences (SSU rDNA). Fungal Genetics
and Biolology 24: 325–334.
Jensen AB, Thomsen L, Eilenberg J, 2001. Intraspecific variation
and host specificity of Entomophthora muscae s.str. isolates re-
vealed by random amplified polymorphic DNA, universal
primed PCR, PCR-restriction fragment length polymorphism,
and conidial morphology. Journal of Invertebrate Pathology 78:
251–259.
Kalsbeek V, Mullens BA, Jespersen JB, 2001. Field studies of Ento-
mophthora (Zygomycetes: Entomophthorales) d induced behav-
ioural fever in Musca domestica (Diptera: Muscidae) in Denmark.
Biological Control 21: 264–273.
Keller S, 1984. Entomophthora muscae as a species complex. Mittelung
Schweizer Entomologische Gesellschaft 57: 131–132.
Keller S, 1987. Arthropod-pathogenic Entomophthorales of Swit-
zerland. I. Conidiobolus, Entomophaga and Entomophthora. Sydo-
wia 40: 122–167.
Keller S, 2002. The genus Entomophthora (Zygomycetes, Entomoph-
thorales) with a description of five new species. Sydowia 54:
157–197.
Keller S, Kalsbeek V, Eilenberg J, 1999. Redescription of Ento-
mophthora muscae (Cohn) Fresenius. Sydowia 51: 197–209.
Kjøller R, Rosendahl S, 2000. Detection of arbuscular mycorrhizal
fungi (Glomales) in roots by nested PCR and SSCP (single
stranded conformation polymorphism). Plant and Soil 226:
189–196.
Klingen I, Meadow R, Eilenberg J, 2000. Prevalence of fungal in-
fections in adult Delia radicum and Delia floralis captured on the
edge of a cabbage field. Entomologia Experimentalis et Applicata
97: 265–274.
Kramer JP, Steinkraus DC, 1981. Culture of Entomophthora muscae
in vivo and its infectivity for six species of muscoid flies.
Mycopathology 76: 139–143.
MacLeod DM, Müller-Kögler E, Wilding N, 1976. Entomophthora
species with E. muscae-like conidia. Mycologia 68: 1–29.
López Lastra CC, Hajek AE, Humber RA, 2001. Effects of two
cryopreservation techniques on viability and pathogenicity
of entomophthoralean fungi. Canadian Journal of Botany 79:
861–864.
Mullens BA, 1989. Cross-transmission of Entomophthora muscae
(Zygomycetes: Entomophthoraceae) among naturally infected
muscoid fly (Diptera: Muscidae) hosts. Journal of Invertebrate
Pathology 53: 272–275.
Mullens BA, Rodriguez JL, Meyer JA, 1987. An epizootiological
study of Entomophthora muscae in muscoid fly populations on
Southern California poultry facilities, with emphasis on Musca
domestica. Hilgardia 55: 1–41.
950
Nagahama T, Sato H, Shimazu M, Sugiyama J, 1995. Phylogenetic
divergence of the entomophthoralean fungi: evidence from
nuclear 18 S ribosomal RNA gene sequences. Mycologia 87:
203–209.
Ramoska WA, Hajek AE, Ramos ME, Soper RS, 1988. Infection of
grasshoppers (Orthoptera, Acrididae) by members of the Ento-
mophaga grylli species complex (Zygomycetes, Entomophthor-
ales). Journal of Invertebrate Pathology 52: 309–313.
Remaudière G, Keller S, 1980. Révision systématique des genres
d’Entomophthoraceae à potentialité entomopathogène. Myco-
taxon 11: 323–338.
SAS Institute Inc, 1999. SAS/STAT User’s Guide, 1st edn., Vol. 2, SAS
Institute Inc., Cary, NC, USA, Version 8.
Steenberg T, Jespersen JB, Jensen KMV, Nielsen BO, Humber RA,
2001. Entomopathogenic fungi in flies associated with pas-
tured cattle in Denmark. Journal of Invertebrate Pathology 77:
186–197.
Steinkraus DC, Geden CJ, Rutz DA, 1993. Prevalence of Entomoph-
thora muscae (Cohn) Fresenius (Zygomycetes: Entomophthoraceae)
in houseflies (Diptera: Muscidae) on dairy farms in New York
and induction of epizootics. Biological Control 3: 93–100.
Steinkraus DC, Kramer JP, 1987. Susceptibility of sixteen species
of Diptera to the fungal pathogen Entomophthora muscae (Zygo-
mycetes: Entomophthoraceae). Mycopathology 100: 55–63.
Steinkraus DC, Kramer JP, 1988. Entomophthora scatophagae desc.
ampl. (Zygomycetes: Entomophthorales), a fungal pathogen of the
A. B. Jensen et al.
yellow dung fly, Scatophaga stercoraria (Diptera: Anthomyiidae).
Mycotaxon 32: 105–113.
Swofford DL, 1998. PAUP: Phylogenetic Analysis Using Parsimony
[Version 4]. Sinauer Assosiates, Sunderland, MA.
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,
Fisher MC, 2000. Phylogenetic species recognition and species
concepts in fungi. Fungal Genetic and Biology 31: 21–32.
Thomsen L, Jensen AB, 2002. Application of nested-PCR technique
on resting spores from the Entomophthora muscae species
complex: implications for analyses of host–pathogen popula-
tion interactions. Mycologia 94: 794–802.
Van de Peer Y, De Wachter R, 1994. TREECON for Windows:
a software package for the construction and drawing of evo-
lutionary trees for the Microsoft Windows environment.
Computer Applications in the Biosciences 10: 569–570.
Watson DW, Petersen JJ, 1993. Seasonal activity of Entomophthora
muscae (Zygomycetes: Entomophthorales) in Musca domestica L.
(Diptera: Muscidae) with reference to temperature and relative
humidity. Biological Control 3: 182–190.
Wiley EO, 1978. The evolutionary species concept reconsidered.
Systematic Zoology 27: 17–26.
White TJ, Bruns TD, Lee SB, Taylor JW, 1990. Amplification and
direct sequencing of fungal ribosomal RNA genes for phylo-
genetics. In: Innis MA, Gelfand DH, Sninsky JS, White TJ (eds),
PCR Protocols: A Guide To Methods And Applications. Academic
Press, San Diego, pp. 315–321.