Two new species of Cladorrhinum

Abstract

Two new species of Cladorrhinum, C. flexuosum and C. microsclerotigenum, are described and their sequences of the 5.8S and 28S ribosomal DNA and internal transcribed spacers 1 and 2 of the ribosomal RNA gene cluster analyzed. Cladorrhinum flexuosum, isolated from soil in Spain, forms fast-growing, dull yellow colonies, flexuous conidiophores and globose to dacryoid conidia. Cladorrhinum microsclerotigenum, isolated from Musa sp. in Turkey, strongly resembles Cladorrhinum phialophoroides in the production of abundant terminal phialides, ellipsoid conidia and microsclerotia in culture. It differs from C. phialophoroides in its ability to grow at 36 C and high number of intercalary phialides, which only infrequently occur in C. phialophoroides.

Full text

Two new species of
Cladorrhinum
Hugo Madrid
Josep Cano
Josepa Gene´
1
Josep Guarro
Unitat de Micologia, Facultat de Medicina i Cie`ncies de
la Salut, IISPV, Universitat Rovira i Virgili, C/ Sant
Llorenc¸ 21, 43201 Reus, Tarragona, Spain
Abstract
:Two new species of
Cladorrhinum
,
C.
flexuosum
and
C. microsclerotigenum
, are described
and their sequences of the 5.8S and 28S ribosomal
DNA and internal transcribed spacers 1 and 2 of the
ribosomal RNA gene cluster analyzed.
Cladorrhinum
flexuosum
, isolated from soil in Spain, forms fast-
growing, dull yellow colonies, flexuous conidiophores
and globose to dacryoid conidia.
Cladorrhinum
microsclerotigenum
, isolated from
Musa
sp. in Turkey,
strongly resembles
Cladorrhinum phialophoroides
in
the production of abundant terminal phialides,
ellipsoid conidia and microsclerotia in culture. It
differs from
C. phialophoroides
in its ability to grow at
36 C and high number of intercalary phialides, which
only infrequently occur in
C. phialophoroides
.
Key words:
Ascomycota, Lasiosphaeriaceae, mi-
tosporic fungi, Sordariales, taxonomy
INTRODUCTION
The species of the anamorphic genus
Cladorrhinum
Sacc. & Marchal form slimy masses of one-celled
conidia on intercalary conidiogenous cells with lateral
phialidic openings. Fertile hyphae can accumulate
into more or less branched conidiophores. Some-
times terminal and lateral phialides and microscler-
otia also can be observed in culture (de Hoog et al.
2000, Domsch et al. 2007). In a revision of the genus
by Mouchacca and Gams (1993) five named species
were treated, that is
C. foecundissimum
Sacc. &
Marchal (type species),
C. brunnescens
W. Gams,
C.
bulbillosum
W. Gams & Mouch.,
C. phialophoroides
Mouch. & W. Gams and
C. samala
(Subram. &
Lodha) W. Gams & Mouch. The unnamed ana-
morphs of
Cercophora samala
Udagawa & T. Muroi
and certain species of
Apiosordaria
Arx & W. Gams
also were ascribed to
Cladorrhinum
by those authors.
Cladorrhinum
species were distinguished mainly on
the basis of the ability to produce microsclerotia, the
relative abundance of intercalary versus terminal
phialides, growth rates at different temperatures, the
pigmentation of the mycelium and conidial shape. In
the concept of
Cladorrhinum
Mouchacca and Gams
(1993) gave importance to the tufted aggregation of
the conidiophores. Therefore the anamorph of
Apodospora viridis
Cain & J.H. Mirza and those of
several species of
Podospora
Ces., which produce only
scattered intercalary phialides in culture, were not
included. Later
Cladorrhinum
anamorphs matching
Mouchacca’s and Gams’ circumscription were de-
scribed for
Podospora fimiseda
(Ces. & de Not) Niessl
(Bell and Mahoney 1997) and
Cercophora striata
(Ellis
& Everh.) N. Lundq. (Miller and Huhndorf 2001).
Cladorrhinum
species are found as saprobes on
dung and plant material and in soil, but at least one
species of the genus,
C. bulbillosum
, occasionally is
involved in opportunistic infections of mammals
(Zapater and Scattini 1979, Chopin et al. 1997).
Cladorrhinum foecundissimum
has been proposed
recently as a potential biocontrol agent due to its
strong antagonistic activity against
Rhizoctonia solani
J.G. Ku¨hn and
Pythium ultimum
Trow (Lewis and
Larkin 1998).
Little is known about the evolutionary relationships
of
Cladorrhinum
species to other ascomycetes. The
known associated teleomorphs belong to genera in
the Lasiosphaeriaceae, Sordariales (Mouchacca and
Gams 1993, Bell and Mahoney 1997, Miller and
Huhndorf 2001). However the family appeared as an
artificial group in several phylogenetic studies (Huhn-
dorf et al. 2004; Miller and Huhndorf 2004, 2005).
Cai et al. (2006) demonstrated that four species with
Cladorrhinum
anamorphs, that is
Apiosordaria verru-
culosa
(C.N. Jensen) Arx & W. Gams,
Cercophora
samala
,
C. striata
and
Podospora fimiseda
, belonged to
a well supported lineage named clade A. This group
also included other species that produce scattered
intercalary phialides in culture, such as
Podospora
austroamericana
(Speg.) J.H. Mirza & Cain and
Podospora pauciseta
(Ces.) Traverso, as well as other
members of the Lasiosphaeriaceae without known
anamorphs. Nevertheless the phylogenetic placement
of the anamorphic species of
Cladorrhinum
is
unknown.
In the present study we addressed the taxonomic
position of two
Cladorrhinum
isolates whose morpho-
logical features did not seem to match any of the
currently described species of this genus. One of
them, CBS 126090, recently was obtained from a soil
sample collected in Spain, and the other, CBS 290.75,
Submitted 5 Nov 2010; accepted for publication 19 Jan 2011.
1
Corresponding author. E-mail: josepa.gene@urv.cat
Mycologia,
103(4), 2011, pp. 795–805. DOI: 10.3852/10-150
#2011 by The Mycological Society of America, Lawrence, KS 66044-8897
795

was isolated from
Musa
sp. in Turkey. The latter had
been studied by Mouchacca and Gams (1993) but was
considered an atypical isolate of
C. phialophoroides
.
MATERIALS AND METHODS
Sampling and fungal isolation.—
The
Cladorrhinum
isolate
CBS 126090 was obtained from a forest soil sample collected
in Pola de Somiedo, Asturias Province, Spain, in 2006. The
fungus was isolated with a wood bait technique described by
Calduch et al. (2004). The isolate CBS 290.75 was obtained
from
Musa
sp. in Adana, Adana Province, Turkey, in 1975
(Mouchacca and Gams 1993). No information is available
on the isolation technique used to obtain this isolate.
Phenotypic characterization.—
Colony morphology and
growth rates were studied on 85 mm wide Petri dishes with
2%malt extract agar (MEA, Oxoid) in duplicate. The plates
were centrally inoculated with 2 mm wide plugs of mycelium
and incubated in the dark 5 d at 6–36 C at intervals of 6 C.
We recorded colony diameters as minimum and maximum
values obtained for isolates belonging to each taxon. Color
notations in parentheses are from Kornerup and Wanscher
(1978). Microscopic morphology was studied in lactic acid
from cultures on oatmeal agar (OA; 30 g filtered oat flakes,
20 g agar, 1 L distilled water) and sterilized wood after 14 d
at 24 C with an Olympus CH-2 light microscope. We shot
photomicrographs with a Zeiss Axio Imager M1 light
microscope.
DNA sequence analysis.—
DNA extraction was performed
directly from fungal colonies by following the Fast DNA Kit
protocol (Bio 101 Inc., Vista, California) with the homog-
enization step repeated five times. Amplification of the 5.8S
ribosomal DNA and the internal transcribed spacers 1 and
2 of the ribosomal RNA gene cluster (hereafter referred to
as ITS region) was performed with the primer pair ITS5/
ITS4 as described by Gilgado et al. (2005). The D1/D2
domains of the 28S rDNA were amplified and sequenced
with primers NL1/NL4 as described by Cano et al. (2004).
PCR products were purified with a GFXTM PCR DNA kit
(Pharmacia Biotech, Cerdanyola, Spain) and were stored at
220 C until sequencing. PCR products were sequenced
with the same primers employed for amplification and
following the
Taq
DyeDeoxy Terminator cycle sequencing
kit protocol (Applied Biosystems, Gouda, the Netherlands).
We analyzed DNA sequencing reaction mixtures on a 310
DNA sequencer (Applied Biosystems). We used the
program SeqMan (Lasergene, Madison, Wisconsin) to
obtain consensus sequences from the complementary
sequences of each isolate. BLAST sequence homology
queries (Altschul et al. 1990) were performed to compare
data of the isolates studied with those of other fungi
deposited in the GenBank database. Nucleotide sequence
alignments were performed with Clustal X 1.81 (Thompson
et al. 1997), followed by manual adjustments with a text
editor. Distance trees were constructed with neighbor
joining (Saitou and Nei 1987), excluding ambiguously
aligned regions and using the Kimura-2-parameter substi-
tution model with pairwise deletion of gaps, as implement-
ed in MEGA 4.0 (Tamura et al. 2007). We assessed the
robustness of branches by bootstrap analysis of 1000
replicates. The sequences generated in this study and the
alignments used in the cladistic analysis were deposited
respectively in GenBank and TreeBASE (www.treebase.org,
submission number SN4946).
RESULTS
DNA sequence analyses.—
BLAST queries revealed that
the ITS sequence of isolate CBS 126090 (FN662474)
showed a relatively high percent identity to that of
Cladorrhinum samala
(FM955447, 93%) while the
sequence of the D1/D2 domains was closest to
Podospora fimiseda
and
Cercophora costaricensis
(G.C.
Carroll & Munk) O. Hilber & R. Hilber (AY346296
and AY780059, both 98%identical). The ITS
sequence of isolate CBS 290.75 (FN662475) had a
relatively high percent identity to those of
Cercophora
samala
and
Zopfiella tetraspora
(J.N. Rai, Mukerji &
J.P. Tewari) S. Ahmad (AY999134 and AY999130, both
93%), while the closest matches for its D1/D2
sequence were
Cercophora samala
(AY999111, 98%
identical) and
C. striata
(AY780065, 97%identical).
The ITS sequences of
Cladorrhinum
spp. and
associated teleomorphs were too divergent to be
aligned with confidence. Therefore we performed the
cladistic analysis with the D1/D2 dataset. Isolates in
the analysis, their origins and EMBL accession
numbers are included (TABLE I). The manually
edited alignment of the D1/D2 locus included 496
positions. The neighbor joining tree (FIG. 1) showed
five main groups, which corresponded to the clades
named A, B, C, Chaetomiaceae and Sordariaceae by
Cai et al. (2006). In this study we used the same
nomenclature for these groups. Clade A had 71%
bootstrap support and included all the anamorphic
species of
Cladorrhinum
, except
C. brunnescens
, which
grouped with members of the Chaetomiaceae in a
clade with low bootstrap support. In our opinion it is
not necessary to reallocate
C. brunnescens
to a
different genus because it belongs to the same order
as the type species of
Cladorrhinum
. Similar taxonom-
ic decisions have been made with other anamorphic
genera, such as
Cladophialophora
Borelli (Crous et al.
2007, Badali et al. 2008) and
Ramichloridium
Stahel
ex de Hoog (Arzanlou et al. 2007), to avoid the
proliferation of genus names. Clade A also included
all the teleomorph species with
Cladorrhinum
ana-
morphs studied and some taxa without known
anamorphs. Nevertheless it also included
Cercophora
terricola
S. Ueda, which produces a
Chrysosporium
-like
anamorph with lateral blastoconidia (Ueda 1994).
This kind of anamorph, also produced by other
members of the Lasiosphaeriaceae, was interpreted by
796 MYCOLOGIA

Mouchacca and Gams (1993) as some kind of
reduction of the intercalary phialides of
Cladorrhi-
num
, in which the conidiogenous opening becomes
blocked after producing a single conidium.
Cladorrhinum samala
proved to be distantly related
to
Cercophora samala
, which was proposed by Uda-
gawa and Muroi (1979) as its possible teleomorph. In
addition
C. foecundissimum
was at considerable
TABLE I. Strains, sources, and sequences used in the phylogenetic analysis
Species Collection number Origin
EMBL accession
#(28S rDNA)
Apiosordaria backusii
CBS 106.77 Sandy soil, Japan AY780051
Apiosordaria longicaudata
CBS 390.84 (T) Soil, Japan FR692340
Apiosordaria tetraspora
CBS 363.84 (T) Soil, Thailand FR692341
Apiosordaria verruculosa
F-152365 Ethanol-pasteurized soil, Spain AY346258
Apiosordaria verruculosa
var.
maritima
CBS 550.66 Salt-marsh soil, England FR692345
Cercophora coprophila
S.M.H. 3794 Puerto Rico AY780058
Cercophora costaricensis
S.M.H. 4021 Costa Rica AY780059
Cercophora newfieldiana
S.M.H. 2622 USA AF064642
Cercophora samala
CBS 109.93 (T) Dung of cow, Japan AY999111
Cercophora striata
S.M.H. 3431 Unidentified branch, Panama AY780065
Cercophora sulphurella
S.M.H. 2531 Illinois, USA AY587938
Cercophora terricola
ATCC 200395 (T) River sediment, Japan AY780067
Chaetomidium cephalothecoides
MUCL 40270 (T) Mouse dung, USA AF286413
Chaetomidium leptoderma
CBS 538.74 (T) Soil, England AF096186
Chaetomium globosum
CBSC 15-5973 NA AY545729
Chaetomium strumarium
CBS 333.67 (T) Soil, India AY681170
Cladorrhinum brunnescens
CBS 643.75A (T) Fibres of
Cocos nucifera
, the Netherlands FR692346
Cladorrhinum bulbillosum
CBS 304.90 (T) Desert sand, Egypt FR692339
Cladorrhinum flexuosum
sp. nov. CBS 126090 (T)
(5FMR 10415)
Soil, Spain FN662477
Cladorrhinum foecundissimum
CBS 180.66 (NT) Agricultural soil, the Netherlands FR692343
Cladorrhinum
microsclerotigenum
sp. nov.
CBS 290.75 (T)
(5FMR 10045)
Musa
sp., Turkey FN662476
Cladorrhinum phialophoroides
CBS 301.90 (T) Desert soil, Egypt FR692344
Cladorrhinum samala
CBS 303.90 Desert soil, Egypt FR692338
Immersiella caudata
CBS 606.72 Soil, the Netherlands AY999113
Podospora austroamericana
CBS 724.68 Flower of
Carica papaya
, India AY999101
Podospora comata
ATCC 36713 Cow dung, Venezuela AY780072
Podospora didyma
CBS 232.78 Rabbit dung, Canada AY999100
Podospora fimiseda
CBS 990.96 Horse dung, New Zealand AY346296
Podospora pauciseta
CBS 124.78 Dung of goat, India EU999216
Ramophialophora humicola
CBS 124563 (T) Forest soil, Spain FR692337
Sordaria fimicola
CBS 508.50 Dung, Canada AY681160
Sordaria lappae
CBS 154.97 Soil, Hungary AY681137
Sordaria macrospora
Buck s.n. Caribou dung, Canada AY346301
Zopfiella erostrata
CBS 255.71 Dung, Central African Republic AY999110
Zopfiella longicaudata
NBRC 30296 Soil, Japan AY999109
Zopfiella tabulata
CBS 230.78 Porcupine dung, Canada AY999105
Zopfiella tetraspora
CBS 158.97 Garden soil, India AY999108
Valsella salicis
BPI 748461
Salix fragilis
, Italy AF408389
Sequences generated in this study appear in boldface. T, ex-type strain; NT, ex-neotype strain (Mouchacca and Gams 1993);
NA, not available; ATCC, American Type Culture Collection, Bethesda, Maryland, USA; BPI, Systematic Botany and Mycology
Laboratory, USDA, Beltsville, Maryland, USA; Buck, William Buck, Jacksonville, Florida, USA; CBS, Centraalbureau voor
Schimmelcultures, Utrecht, the Netherlands; CBSC, Carolina Biological Supply Company, Burlington, North Carolina, USA;
F, Field Museum of Natural History, Chicago, Illinois, USA; FMR, Faculty of Medicine collection, Reus, Spain; MUCL,
Mycothe`que de L’Universite´ Catholique de Louvain, Faculte´ des Sciences Agronomiques, Louvain-la-Neuve, Belgium; NBRC,
Biological Resource Center, Department of Biotechnology, National Institute of Technology and Evaluation, Chiba, Japan;
S.M.H., Sabine M. Huhndorf, Field Museum, Chicago, USA.
MADRID ET AL.: NEW SPECIES OF
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LADORRHINUM
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FIG. 1. Neighbor joining tree constructed with sequences of the partial 28S rRNA gene. Branch lengths are proportional to
distance. Bootstrap values above 70%are indicated near the internodes.
Valsella salicis
was used as outgroup. Teleomorph
species known to produce
Cladorrhinum
anamorphs are indicated by an asterisk. Clades were labeled according to Cai et
al. (2006).
798 MYCOLOGIA

genetic distance from
Apiosordaria verruculosa
, which
was indicated by Cannon et al. (1985) as its
teleomorph. These molecular results support the
conclusions of Mouchacca and Gams (1993) who
suspected on the basis of morphology and mating
studies that these anamorph-teleomorph connections
had been suggested in error.
The D1/D2 neighbor joining tree clearly distin-
guished isolates CBS 290.75 and CBS 126090 from the
accepted species of
Cladorrhinum
and related tele-
omorphs. Isolate CBS 290.75 clustered with
Cerco-
phora samala
and
C. striata
in a subclade of clade A
with low bootstrap support. On the other hand isolate
CBS 126090 was placed in a well supported subclade
that also included
Podospora fimiseda
,
Cercophora
costaricensis
and
Cladorrhinum samala
. Based on the
results of the cladistic analysis and phenotypic
differences, we propose two new species of
Clador-
FIG.2.
Cladorrhinum flexuosum
(CBS 126090). A. Densely branched, flexuous conidiophore. B. Intercalary and terminal
phialides. C. Lateral phialide. D. Conidia. Bars 55mm.
MADRID ET AL.: NEW SPECIES OF
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LADORRHINUM
799

FIG.3.
Cladorrhinum flexuosum
(CBS 126090). A. Conidiophore. B–D. Conidiogenous cells. E. Conidia. Bars 55mm.
800 MYCOLOGIA

rhinum
to accommodate isolates CBS 290.75 and CBS
126090.
TAXONOMY
Cladorrhinum flexuosum H. Madrid, Cano, Gene´&
Guarro, sp. nov. FIGS.2,3
MycoBank MB516710
Etymology.
In reference to the flexuose conidio-
phores produced by the fungus.
Coloniae in agaro maltoso ad 24 C post 5 dies 63–65 mm
diam, griseo-luteae, velutinae vel floccosae. Hyphae septa-
tae, ramosae, hyalinae vel pallide olivaceae, laeves, 1.5–5 mm
latae. Microsclerotia absentia. Conidiophora semimacrone-
mata, septata, dense ramosa, plerumque flexuosa, pallide
olivacea vel pallide brunnea, laevia, saepe crassitunicata,
longitudine indeterminata, 2.5–5 mm lata, in floccis aggre-
gata. Phialides intercalares, laterales vel terminales; inter-
calares cylindricae vel subcylindricae, 5–21 32–4 mm,
collulo laterali plerumque 2–5 mm longo; laterales et
terminales subcylindricae vel lageniformes, laterales 4–11
32–3.5 mm, terminales 9–15 33–4 mm. Conidia unicellu-
laria, subhyalina vel pallide olivaceo-brunnea, laevia, tenui-
tunicata, plerumque globosa vel dacryoidea, 2–4 32–3 mm,
guttulata, in capitulis mucidis aggregata. Teleomorphosis
ignota.
Colonies on MEA 63–65 mm diam in 5 d at 24 C,
6 mm at 36 C, dull yellow (3B4), radiating, flat, with
scarce aerial mycelium, producing floccose conidial
tufts at the center, velvety toward the periphery;
reverse yellowish white (3A2) at the center, yellowish
gray (3B2) toward the periphery. Vegetative hyphae
septate, branched, hyaline to pale olive, smooth- and
thin-walled, 1.5–5 mm wide. Microsclerotia not ob-
served. Conidiophores semimacronematous, septate,
profusely branched, branches usually flexuous and
densely entangled, often ending in sterile cells with
obtuse apices, pale olivaceous to pale brown, smooth,
with cell walls usually thicker than those of the
vegetative hyphae, length indeterminate, 2.5–5 mm
wide, aggregated in tufts. Phialides mostly intercalary
on the conidiophore axis and branches but also
terminal or lateral, commonly flexuous, light olive to
light brown, with one or rarely two conidiogenous
openings, bearing conspicuous collarettes; intercalary
phialides cylindrical to subcylindrical, 5–21 32–4 mm,
with a subcylindrical to lageniform lateral neck mostly
2–5 mm long, rarely longer, up to 9 mm; lateral and
terminal phialides subcylindrical to lageniform, later-
al phialides 4–11 32–3.5 mm, terminal phialides 9–15
33–4 mm. Conidia one-celled, subhyaline to pale
olive-brown, smooth- and thin-walled, mostly globose
to dacryoid, 2–4 32–3 mm, guttulate, often truncate
at the base or bearing a subcylindrical hilum,
aggregated in slimy masses. Teleomorph not ob-
served.
Holotype.
SPAIN. ASTURIAS PROVINCE: Pola de
Somiedo, from soil, Oct 2006,
C. Silvera &A. Mercado
(IMI 397930, ex-type cultures CBS 126090 and FMR
10415).
This fungus is similar to other members of
Cladorrhinum
producing globose to dacryoid conidia,
such as
Cladorrhinum samala
,
C. bulbillosum
,
C.
brunnescens
,
C. foecundissimum
, and the anamorphs
of
Cercophora striata
and
Podospora fimiseda
.In
contrast to
C. flexuosum C. bulbillosum
and the
anamorph of
Cercophora striata
produce blackish
microsclerotia in culture (Mouchacca and Gams
1993, Miller and Huhndorf 2001). The dark, thick-
walled setiform hyphae that characterize
Cladorrhi-
num samala
(Subramanian and Lodha 1964, Ellis
1971) were not observed in cultures of
C. flexuosum
;
C. samala
grew faster than
C. flexuosum
at all
temperatures (TABLE II). While
C. flexuosum
produc-
es fast-growing, flat colonies on MEA,
C. brunnescens
grows slowly (TABLE II) and its colonies show abun-
dant aerial mycelium (Mouchacca and Gams 1993).
Unlike
C. flexuosum
,
C. foecundissimum
has regular,
not markedly flexuose conidiophores (Marchal 1885,
Mouchacca and Gams 1993, Domsch et al. 2007).
Podospora fimiseda
is a homothallic fungus that
TABLE II. Colony diameter of
Cladorrhinum
spp. at different temperatures
Species
Colony diameter (mm) on MEA after 5 d
6 C 12 C 18 C 24 C 30 C 36 C
Cladorrhinum brunnescens
CBS 643.75A 3 5–6 7–9 9–11 6–7 0
Cladorrhinum bulbillosum
CBS 304.90 4 34 71–78 .85 .85 .85
Cladorrhinum flexuosum
CBS 126090 0 11–14 33–36 63–65 .85 6
Cladorrhinum foecundissimum
CBS 180.66 5 13–14 24–27 34–36 24 0
Cladorrhinum microsclerotigenum
CBS 290.75 0 9–10 19–21 26–29 54–55 21–23
Cladorrhinum phialophoroides
CBS 301.90 3 6–7 13–16 21–23 17–21 0
Cladorrhinum samala
CBS 303.90 4 26–27 63–66 .85 .85 15–18
MEA, malt extract agar. Colony diam .85 indicate full plates after 5 d incubation.
MADRID ET AL.: NEW SPECIES OF
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801

produces perithecia on agar media. Its anamorph
seems to require media enriched with dung to
achieve abundant sporulation (Bell and Mahoney
1997). In addition the anamorphs of other species of
Podospora
and that of
Apodospora viridis
produce
conidiogenous cells similar to those of
C. flexuosum
,
but they appear scattered on the mycelium (Mou-
chacca and Gams 1993), not in dense clusters as in
our isolate. Two species with this type of anamorph,
P.
pauciseta
and
P. austroamericana
(Mirza and Cain
1969), were included in our cladistic analysis. These
taxa clustered within clade A in concordance with Cai
et al. (2006). The anamorph of
Arnium calymmatos-
porum
Jeng & J.C. Krug is also similar to
C. flexuosum
,
but the former species produces light green colonies
and the conidiophores illustrated in its protolog are
FIG.4.
Cladorrhinum microsclerotigenum
(CBS290.75).A.Microsclerotium.B–C. Conidiophores and phialidic
conidiogenous cells. D. Phialide with two conidiogenous openings (arrows). E. Conidia. Bars 55mm.
802 MYCOLOGIA

not flexuous (Jeng and Krug 1976). We were not able
to locate strains or DNA sequences of
Arnium
calymmatosporum
and
Apodospora viridis
for compar-
ison.
Ramophialophora humicola
H. Madrid, Stchigel,
Gene´ & Guarro is a morphologically similar anamor-
phic fungus that also belongs to the Sordariales and
shows terminal, lateral and intercalary phialides that
produce dacryoid conidia. Nevertheless, unlike
C.
flexuosum
,
R. humicola
is a slow-growing taxon that
produces conidiophores that often end in sterile,
setiform, sometimes swollen apices (Madrid et al.
2010).
Ramophialophora humicola
belongs to a differ-
ent lineage of Sordariales (FIG. 1), which corresponds
to clade B in Cai et al. (2006).
FIG.5.
Cladorrhinum microsclerotigenum
(CBS 290.75). A. Microsclerotium. B, C. Conidiophores and conidiogenous cells.
D. Conidia. Bars 55mm.
MADRID ET AL.: NEW SPECIES OF
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LADORRHINUM
803

Cladorrhinum microsclerotigenum H. Madrid, Gene´,
Cano & Guarro, sp. nov. FIGS.4,5
MycoBank MB516711
Etymology.
In reference to the microsclerotia
formed by the fungus.
Coloniae in agaro maltoso ad 24 C post 5 dies 26–29 mm
diam, brunneae vel luteo-griseae, pro partim albae, planae,
velutinae. Hyphae septatae, ramosae, hyalinae vel olivaceo
brunneae, laeves, 1–9 mm latae. Microsclerotia numerosa,
immersa vel paulo erumpentia, discreta vel confluentia,
atrobrunnea, subglobosa vel forma irregularia, usque ad
55 mm diam. Conidiophora semimacronemata, septata,
simplicia vel ramosa, pallide olivacea vel pallide olivaceo-
brunnea, laevia, saepe crassitunicata, longitudine indetermi-
nata, 2–4 mm lata, cum phialides intercalares, laterales vel
terminales. Phialides intercalares cylindricae vel subcylindri-
cae, 4–8 33–4 mm, collulo laterali usque ad 6 mmlongae;
laterales et terminales plerumque lageniformes, 4–11 32.5–
4mm. Conidia unicellularia, hyalina vel pallide olivacea,
laevia, plerumque ellipsoidea, 2–4 31.5–2.5 mm, guttulata, in
capitulis mucidis aggregata. Teleomorphosis ignota.
Colonies on MEA 26–29 mm diam in 5 d at 24 C,
21–23 mm at 36 C, brown (5F4) at the center,
yellowish gray (3B2) toward the periphery, with
patches of short, white mycelium, flat, velvety;
reverse brownish gray (4F2) at the center, yellowish
gray (3B2) toward the periphery. Vegetative hyphae
septate, branched, hyaline to olive-brown, smooth-
and mostly thin-walled, 1–9 mm wide, giving rise to
terminal, lateral or intercalary clumps of inflated,
irregularly shaped cells that differentiate into
microsclerotia. Mature microsclerotia immersed to
erumpent, discrete to confluent, subglobose or
irregularly shaped, up to 55 mm wide, composed of
dark brown, rounded to polygonal, thick-walled cells
up to 17 mm diam. Conidiophores semimacronema-
tous, septate, simple or branched, pale olive to pale
olivaceous brown, smooth, with cell walls usually
thicker than those of the vegetative hyphae, length
indeterminate, 2–4 mm wide. Phialides intercalary,
lateral and terminal, pale olive to pale olivaceous
brown, with one or rarely two conidiogenous
openings, bearing conspicuous collarettes; when
intercalary, cylindrical to subcylindrical, 4–8 33–
4mm, with a subcylindrical to lageniform lateral
neck up to 6 mm long; lateral and terminal phialides
mostly lageniform, but also subcylindrical or cen-
trally swollen, 4–11 32.5–4 mm. Conidia one-celled,
hyaline to pale olive, smooth- and thin-walled,
mostly ellipsoid, but also obovoid, clavate or
cylindrical, 2–4 31.5–2.5 mm, guttulate, often
truncate at the base, aggregated in slimy masses.
Teleomorph not observed.
Holotype.
TURKEY. ADANA PROVINCE: Adana,
from
Musa
sp., May 1975,
A. Cengiz
(IMI 397929, ex-
type cultures CBS 290.75 and FMR 10045).
Isolate CBS 290.75 previously was considered a
deviating strain of
C. phialophoroides
, which differed
from typical isolates by having conidiophores similar
to those of
C. bulbillosum
, that is composed mainly of
‘‘short phialidic cells’’ (the size was not indicated,
Mouchacca and Gams 1993). We compared
C.
microsclerotigenum
with the type strain of
C. phialo-
phoroides
and found evident length differences only in
the terminal phialides, which were up to 19 mm in the
latter species but did not exceed 11 mmin
C.
microsclerotigenum
. However the phialides of
C.
phialophoroides
were mostly terminal and lateral and
only a few of them were intercalary while in
C.
microsclerotigenum
intercalary phialides were abun-
dant. Furthermore while
C. microsclerotigenum
grew at
36 C,
C. phialophoroides
did not grow at that
temperature (TABLE II). We also examined the type
strain of
C. bulbillosum
. This species could be
distinguished easily from
C. microsclerotigenum
on
the basis of conidial morphology, subglobose to
dacryoid in
C. bulbillosum
, and mostly ellipsoidal in
C. microsclerotigenum
. The latter species also is similar
to other
Cladorrhinum
anamorphs with ellipsoid
conidia, such as those of
Cercophora samala
(Mou-
chacca and Gams 1993) and certain
Apiosordaria
species (Krug et al. 1983). Nevertheless, in contrast to
C. microsclerotigenum
, these species do not produce
microsclerotia in culture (Mouchacca and Gams
1993).
ACKNOWLEDGMENTS
We are indebted to the curators of the Centraalbureau voor
Schimmelcultures (CBS, Utrecht, the Netherlands) for
providing some of the isolates studied. The first author
thanks Dr Sybren de Hoog and Bert Gerrits van den Ende
for guidance during a stay at CBS. This work was supported
by the Spanish Ministerio de Ciencia e Innovacio´ n, grants
CGL 2008-04226 and CGL 2009-08698/BOS.
LITERATURE CITED
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990.
Basic local alignment search tool. J Mol Biol 215:403–
410.
Arzanlou M, Groenewald JZ, Gams W, Braun U, Shin H-D,
Crous PW. 2007. Phylogenetic and morphotaxonomic
revision of
Ramichloridium
and allied genera. Stud
Mycol 58:57–93, doi:10.3114/sim.2007.58.03
Badali H, Gueidan C, Najafzadeh MJ, Bonifaz A, Gerrits van
den Ende AHG, de Hoog GS. 2008. Biodiversity of the
genus
Cladophialophora
. Stud Mycol 61:175–191,
doi:10.3114/sim.2008.61.18
Bell A, Mahoney DP. 1997. Coprophilous fungi in New
Zealand II.
Podospora
species with coriaceous perithe-
cia. Mycologia 89:908–915, doi:10.2307/3761111
804 MYCOLOGIA

Cai L, Jeewon R, Hyde K. 2006. Molecular systematics of
Zopfiella
and allied genera: evidence from multigene
sequence analyses. Mycol Res 110:359– 368,
doi:10.1016/j.mycres.2006.01.007
Calduch M, Gene´ J, Stchigel A, Cano J, Guarro J. 2004.
Ramophialophora
, a new anamorphic genus of Sordar-
iales. Stud Mycol 50:83–88.
Cannon PF, Hawksworth DL, Sherwood-Pike MA. 1985. The
British Ascomycotina, an annotated checklist. Slough:
Commonwealth Agricultural Bureaux. 302 p.
Cano J, Guarro J, Gene´ J. 2004. Molecular and morpholog-
ical identification of
Colletotrichum
species of clinical
interest. J Clin Microbiol 42:2450–2454, doi:10.1128/
JCM.42.6.2450-2454.2004
Chopin B, Sigler L, Connole MD, O’Boyle DA, Mackay B,
Goldstein L. 1997. Keratomycosis in a percheron cross
horse caused by
Cladorrhinum bulbillosum
. J Med Vet
Mycol 35:53–55, doi:10.1080/02681219780000871
Crous PW, Schubert K, Braun U, de Hoog GS, Hocking AD,
Shin D, Groenewald JZ. 2007. Opportunistic, human-
pathogenic species in the Herpotrichiellaceae are
phenotypically similar to saprobic or phytopathogenic
species in the Venturiaceae. Stud Mycol 58:185–217,
doi:10.3114/sim.2007.58.07
de Hoog GS, Guarro J, Gene´ J, Figueras MJ. 2000. Atlas of
clinical fungi. 2nd ed. Utrecht/Reus: Centraalbureau
voor Schimmelcultures/Universitat Rovira i Virgili.
1126 p.
Domsch KH, Gams W, Anderson TH. 2007. Compendium
of soil fungi. 2nd ed. Eching: IHW Verlag. 672 p.
Ellis MB. 1971. Dematiaceous hyphomycetes. Kew: Com-
monwealth Mycological Institute. 608 p.
Gilgado F, Cano J, Gene´ J, Guarro J. 2005. Molecular
phylogeny of the
Pseudallescheria boydii
species com-
plex: proposal of two new species. J Clin Microbiol 43:
4930–4942, doi:10.1128/JCM.43.10.4930-4942.2005
Huhndorf SM, Miller AN, Ferna´ ndez FA. 2004. Molecular
systematic of the Sordariales: the order and the family
Lasiosphaeriaceae redefined. Mycologia 96:368–387,
doi:10.2307/3762068
Jeng RS, Krug JC. 1976. New records and new species of
Arnium
. Can J Bot 55:83–95, doi:10.1139/b77-015
Kornerup A, Wanscher JH. 1978. Methuen handbook of
color. 3rd ed. London: Eyre Methuen. 252 p.
Krug JC, Udagawa S, Jeng RS. 1983. The genus
Apiosordaria
.
Mycotaxon 17:533–549.
Lewis JA, Larkin RP. 1998. Formulation of the biocontrol
fungus
Cladorrhinum foecundissimum
to reduce the
damping-off disease caused by
Rhizoctonia solani
and
Pythium ultimum
. Biol Control 12:182–190,
doi:10.1006/bcon.1998.0639
Madrid H, Cano J, Stchigel A, Gene´ J, Guarro J. 2010.
Ramophialophora humicola
and
Fibulochlamys chilensis
,
two new microfungi from soil. Mycologia 102:605–612,
doi:10.3852/09-128
Marchal E. 1885. Champignons coprophiles de la Belgique.
Bull Soc R Bot Belg 24:57–77.
Miller AN, Huhndorf SM. 2001. Neotropical ascomycetes
10. New and interesting
Cercophora
species. Sydowia 53:
211–226.
———, ———. 2004. A natural classification of
Lasio-
sphaeria
based on nuclear LSU rDNA sequences. Mycol
Res 108:26–34, doi:10.1017/S0953756203008864
———, ———. 2005. Multigene phylogenies indicate
ascomal wall morphology is a better predictor of
phylogenetic relationships than ascospore morpho-
logy in the Sordariales (Ascomycota, Fungi). Mol
Phylogenet Evol 35:60–75, doi:10.1016/j.ympev.2005.
01.007
Mirza JH, Cain RF. 1969. Revision of the genus
Podospora
.
Can J Bot 47:1999–2048, doi:10.1139/b69-293
Mouchacca J, Gams W. 1993. The hyphomycete genus
Cladorrhinum
and its teleomorph connections. Myco-
taxon 48:415–440.
Saitou N, Nei M. 1987. The neighbor joining method: a new
method for reconstructing phylogenetic trees. Mol Biol
Evol 4:406–425.
Subramanian CV, Lodha BC. 1964. Four new coprophilous
hyphomycetes. Antonie van Leeuwenhoek 30:317–330,
doi:10.1007/BF02046737
Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA 4:
molecular evolutionary genetics analysis (MEGA).
Version 4.0. Mol Biol Evol 24:1596–1599.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,
Higgins DG. 1997. The Clustal X windows interface:
flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Res 25:
4876–4882, doi:10.1093/nar/25.24.4876
Udagawa S, Muroi T. 1979. Coprophilous Pyrenomycetes
from Japan V. Trans Mycol Soc Japan 20:453–468.
Ueda S. 1994. A new
Cercophora
with a
Chrysosporium
-like
anamorph. Mycoscience 35:287–290, doi:10.1007/
BF02268451
Zapater RC, Scattini F. 1979. Mycotic keratitis by
Cladorrhinum
. Sabouraudia 17:56–69, doi:10.1080/
00362177985380081
MADRID ET AL.: NEW SPECIES OF
C
LADORRHINUM
805