Mycologia, 104(6), 2012, pp. 1456–1465. DOI: 10.

3852/11-402
# 2012 by The Mycological Society of America, Lawrence, KS 66044-8897

Two new species of Acremonium from Spanish soils

Alejandra Giraldo conidia are usually small, unicellular and aggregate in

Josepa Gené1 slimy heads or chains; sometimes heads and chains
Josep Cano are present in a single culture. Characteristic chla-
Unitat de Micologia, Facultat de Medicina i Ciències de mydospores and sclerotia are produced in some
la Salut, Universitat Rovira i Virgili, IISPV, C/Sant species (Gams 1971, 1975; Domsch et al. 2007;
Llorenç 21, 43201 Reus, Tarragona, Spain
Perdomo et al. 2011).
Sybren de Hoog Recent molecular studies have demonstrated that
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan Acremonium is polyphyletic, with species belonging to
8, 3584 CT Utrecht, the Netherlands different orders of Sordariomycetes. Most species,
Josep Guarro including the type species A. alternatum Link: Fr.,
Unitat de Microbiologia, Facultat de Medicina i
belong to the Hypocreales, some to the Sordariales
Ciéncies de la Salut, Universitat Rovira i Virgili, IISPV, and a small group to the family Plectosphaerellaceae
C/Sant Llorenç 21, 43201 Reus, Tarragona, Spain in the Glomerellales (Glenn et al. 1996, Zare et al.
2007, Schoch et al. 2009, Perdomo et al. 2011,
Summerbell et al. 2011). In a recent phylogenetic
Abstract: In a survey on the diversity of microfungi study, which included many species of Acremonium
in Spanish soils, two new species of Acremonium were and related genera, Summerbell et al. (2011) re-
found. Both species were characterized as having accommodated some hypocrealean species of Acre-
more or less erect, mostly branched conidiophores monium in Sarocladium W. Gams & D. Hawksw. and in
bearing whorls of acicular phialides. In addition, one the reinstated genus Gliomastix Guég.
of these species, Acremonium asperulatum, produced In a survey on the diversity of microfungi from
abundant chlamydospores and globose rough-walled Spanish soils, several strains morphologically as
conidia. The other species, Acremonium variecolor, attributed to the genus Acremonium, denoted as
produced a brownish diffusible pigment and smooth- species I and species II, were isolated. Because the
walled, subglobose conidia with apiculate base; sessile relevant features of these isolates did not match any
conidia inserted directly on vegetative hyphae also described species, we carried out a detailed study to
were present. The analysis of the sequences of the ITS characterize them, with sequences of several genes
region, the D1/D2 domains of the 28S rRNA gene combined with phenotypic characteristics.
and a fragment of the actin gene revealed relation-
ships of both species with members of the Bionec-
MATERIALS AND METHODS
triaceae (Hypocreales). Genetic differences were
observed with morphologically similar species. Site, sampling and fungal isolation.—Soil samples were
Key words: Hypocreales, phylogeny, soil fungi, collected in two areas in northern Spain, in the provinces of
taxonomy Huesca (Aragon) and Lugo (Galicia). Aragonese samples
were collected in the Ordesa valley, the Añisclo Canyon and
in the valleys of Escuaı́n and Bujaruelo. The first three sites
INTRODUCTION belong to the Ordesa y Monte Perdido National Park. The
Acremonium Link is an anamorph genus that current- abundant forest is 750–2100 m, and its vegetation comprises
ly contains more than 100 species. Most of these are mainly oaks, pines, hazel and beech trees. Average tempera-
saprobic and are isolated mainly from dead plant ture is 0.4–0.7 C in the coldest months (January, February) to
13 C in the warmest months (July, August), with an average
material and soil (Gams 1971, Domsch et al. 2007).
annual rainfall of approximately 1735 mm. Galician samples
Some taxa can cause opportunistic infections in
were from the Sierra de Os Ancares Natural Reserve, an area
humans and animals (de Hoog et al. 2000, Guarro formed by high mountains, valleys, and influenced by some
et al. 2009, Das et al. 2010). Cultures of Acremonium rivers. The area covers 53 664 ha and its altitude is 300 m in
species generally grow slowly and form narrow, the valley and 1925 m at the highest peak. Its average
tapered phialides on creeping hyphae, although temperature is 7–8 C in the coldest months to 18 C in the
differentiated conidiophores with or without verticil- warmest, and the average annual rainfall is approximately
late branching may be observed in some species. The 2042 mm. The tree vegetation comprises mainly poplar,
birch, chestnut, walnut, oak, ash and alder.
Submitted 2 Dec 2011; accepted for publication 26 Apr 2012. Samples were taken from the superficial layer of soil and
1
Corresponding author. E-mail: josepa.gene@urv.cat from river sediments with sterilized polyethylene bags

1456
 GIRALDO ET AL.: NEW SPECIES OF ACREMONIUM 1457

closed with rubber bands. In the laboratory samples were NITE Biological Resource Center (NBRC) and GenBank
stored at 4–7 C until they were processed. One gram of each databases. Sequences were aligned with ClustalX 1.8
soil sample was washed repeatedly with 10 mL sterilized water (Thompson et al. 1997) with default parameters, followed
to reduce excessive microbial growth. After the final wash, by manual adjustments with a text editor. The phylogenetic
excess water was decanted and the remaining soil was relationships were predetermined with ITS sequences. A
distributed among three Petri dishes. Potato dextrose agar multilocus sequence analysis of a selected group subsequently
(PDA, Difco Laboratories, Detroit, Michigan), supplemented was carried out to confirm the results obtained from ITS data.
with chloramphenicol (200 mg/L) and cycloheximide at a For the first analysis we used Gblocks 0.91b software with
final concentration of 2 g/L at 45 C, was mixed with the soil, relaxing selection parameters (Castresana 2000, Talavera and
and once solidified the cultures were incubated at 25 C in the Castresana 2007) to remove ambiguous (unalignable) parts.
dark. All cultures were examined weekly with a stereomicro- The phylogenetic analysis was carried out with MEGA 4.0
scope up to 1 mo. To purify isolates conidia were transferred (Tamura et al. 2007), with neighbor joining (NJ) (Saitou and
with a sterile dissection needle from isolation plate cultures Nei 1987) and the algorithm Kimura 2-parameter to obtain
to Petri dishes containing potato carrot agar (PCA; 20 g the distance tree. Gaps were treated as pairwise deletion.
potatoes, 20 g carrot; 20 g agar, 1000 mL distilled water) Support for internal branches was assessed by a search of 1000
prepared by ourselves following the procedure in Onions bootstrapped sets of data. The multilocus sequence analysis
and Pitt (1988) and incubated at 25 C in the dark. comprised 19 of the 51 isolates, including the soil isolates and
phylogenetically related type and reference strains of
Fungal isolates.—In addition to the isolates obtained with
Acremonium and other genera. Other randomly selected type
the above procedure, numerous reference strains of
or reference strains were included in the analysis. The most
morphologically similar species were included in the study
parsimonious trees of the combined dataset, including ITS,
(TABLE I). They were provided mainly by the CBS-KNAW
D1/D2 and actin gene, were performed with PAUP* 4.0b10
Fungal Biodiversity Centre (CBS) (Utrecht, the Nether-
(Swofford 2002). One hundred heuristic searches were
lands) and the Mycothèque de l’Université Catholique de
conducted with random sequence addition and tree bisec-
Louvain (MUCL) (Louvain-la-Neuve, Belgium).
tion-reconnection branch-swapping algorithms, collapsing
DNA extraction, amplification and sequencing.—Isolates zero-length branches and saving all minimal-length trees
were grown on yeast extract sucrose agar (YES; yeast extract (MULTREES). Gaps were treated as missing data. The internal
2%, sucrose 15%, agar 2%, 1000 mL water) 5 d at 25 C, and branch support was assessed with a heuristic parsimony search
DNA was extracted with a PrepMan Ultra sample prepara- of 1000 bootstrapped datasets. Tree length, consistency,
tion reagent (Applied Biosystems, Foster City, California), homoplasy and retention indexes (CI, HI, RI respectively)
according to the manufacturer’s protocol. DNA was were recorded. The combined dataset was tested for
quantified with GeneQuant pro (Amersham Pharmacia incongruence with the partition homogeneity test (PHT), as
Biotech, Cambridge, UK). The internal transcribed spacer implemented in PAUP*.
(ITS) region and D1/D2 domains of the 28S of the nuclear
Phenotypic studies.—Macro- and microscopic features of the
rRNA gene were amplified respectively with the primer pairs
soil isolates were studied on oatmeal agar (OA; 30 g filtered
ITS5/ITS4 and NL1/NL4, following the protocols of Cano
oat flakes after 1 h simmering, 20 g agar, 1000 mL distilled
et al. (2004) and Gilgado et al. (2005). A fragment of the
water) prepared by ourselves following the procedure of
actin gene was amplified with the primer pairs Act1/Act4
Onions and Pitt (1988), malt extract agar 2% (MEA, Difco
(Voigt and Wöstermeyer 2000). PCR products were purified
Laboratories) and PDA, incubated at 25 C in the dark 7–14 d
with a GFXTM PCR DNA kit (Pharmacia Biotech, Cerda-
up to 1 mo. The isolates’ ability to grow at 5, 15, 20, 25, 30,
nyola, Spain) and were stored at 220 C until sequencing.
32, 35 and 37 C was tested on PDA. Color notations in
PCR products were sequenced with the same primers used
parentheses were taken from Kornerup and Wanscher
for amplification and following the Taq DyeDeoxy Termi-
(1978). Microscopic features were examined by making
nator cycle sequencing kit protocol (Applied Biosystems,
direct wet mounts with 85% lactic acid or lactophenol
Gouda, the Netherlands). DNA sequencing reaction mix-
cotton blue or by slide cultures on OA, with an Olympus
tures were analyzed on a 310 DNA sequencer (Applied
CH-2 light microscope. Photomicrographs were obtained
Biosystems). In addition, some amplified fragments were
with a Zeiss Axio-Imager M1 light microscope with phase
purified and sequenced at Macrogen Inc. (Seoul, South
contrast and Nomarski differential interference.
Korea) with a 3730XL DNA analyzer (Applied Biosystems).
The program SeqMan (7.0.0 DNASTAR, Madison, Wiscon- Nucleotide sequence accession numbers.— The new DNA
sin) was used to obtain consensus sequences of each isolate. sequences generated in this study were deposited in
Some ITS sequences, corresponding to several species of GenBank (TABLE I). The alignment used in the phyloge-
Acremonium or other genera phylogenetically related in netic analysis was deposited in TreeBASE (www.treebase.
Summerbell et al. (2011) or that were morphologically org, submission number 12146).
similar to our isolates, were retrieved from GenBank and
included in the phylogenetic analysis (TABLE I).
RESULTS
Aligment and phylogenetic analysis.— BLAST sequence
identity queries (Altschul et al. 1990) were carried out to Phylogenetic analysis.—The BLAST query revealed
compare the soil isolates with other fungi deposited in the that ITS sequences of our unidentified Acremonium
1458 MYCOLOGIA

TABLE I. Species and strains included in the study, their origin and GenBank accession numbers

GenBank accession number

D1/D2 domains of
Species Strains Source ITS region 28S rRNA gene Actin
T
Acremonium alternatum CBS 407.66 Hypoxylon deustum, Austria HE798150
Acremonium antarcticum CBS 987.87 Hypogymnia physodes, DQ825970
Luxembourg
Acremonium blochii CBS 993.69 Skin, the Netherlands HE608636 HE608654 HE608628
Acremonium borodinense CBS 101148T Soil in sugarcane field, Japan HE608635 HE608653 HE608624
Acremonium sp. I (5 A. CBS 130362T Forest soil, refuge San Nicolas, HE608641 HE608649 HE608620
asperulatum sp. nov.) Bujaruelo Valley, Huesca, Spain
FMR 11135 Forest soil, Añisclo canyon, HE608642
Huesca, Spain
FMR 11136 Forest soil, Broto, Ordesa HE608643
Valley, Huesca, Spain
FMR 11137 Forest soil, Añisclo canyon, HE608644
Huesca, Spain
FMR 11138 Forest soil, Ordesa Valley, HE608645
Huesca, Spain
FMR 11139 Forest soil, Torla, Ordesa HE608646
Valley, Huesca, Spain
CBS 130363 Sediments, Ara River, Bujaruelo HE608650 HE608621
Valley, Huesca, Spain
Acremonium sp. II (5 A. CBS 130360T Forest soil, Garganta de Escuaı́n, HE608647 HE608651 HE608622
variecolor sp. nov.) Huesca, Spain
CBS 130361 Forest soil, Lugo, Spain HE608648 HE608652 HE608623
Acremonium curvulum CBS 430.66T Wheat field soil, Germany HE608638 HE608656 HE608630
Acremonium domschii CBS 764.69T Inonotus obliquus, Germany
Acremonium egyptiacum CBS 114785T Ground, Egypt FN706550
Acremonium exuviarum CBS 113360T Corucia zebrata, California AY882946
Acremonium fuci CBS 112868T Blade of Fucus serratus, AY632653
Germany
Acremonium furcatum MUCL 9745T Sand, France
Acremonium fusidioides CBS 840.68T Dung of antelope, Central FN706542
African Republic
Acremonium hansfordii CBS 390.73 Periconia cookei on AB540578
Dendrocalamus sp., India
Acremonium implicatum MUCL 4112 Soil, Georgia, USA FN706553 HE608659 HE608632
Acremonium incoloratum CBS 146.62T Soil, Poona, Maharashtra
Acremonium inflatum CBS 439.70 Sandy soil under permanent
wheat, the Netherlands
Acremonium longisporum CBS 993.87 Ulcer on arm and back, the
Netherlands
Acremonium persicinum CBS 310.59T Coastal sand under Ammophila FN706554
arenaria, France
Acremonium pinkertoniae CBS 157.70T Soil from tropical greenhouse, HE608660 HE608625
the Netherlands
Acremonium potronii CBS 379.70F Skin lesion in dolphin, Belgium
Acremonium recifei MUCL 9696T Mycetoma, Brazil
Acremonium roseolum CBS 289.62T Dead stems, England
Acremonium sclerotigenum CBS 124.42T Dune sand under Ammophila FN706552
and Convolvulus, France
Acremonium spinosum CBS 136.33T Toe nail, Argentina HE608637 HE608655 HE608629
Bulbithecium hyalosporum CBS 318.91T Dung of horse, Cuzco, Perú HE608634 HE608661 HE608626
Cosmospora butyri CBS 301.38T Butter, Denmark DQ286652
 GIRALDO ET AL.: NEW SPECIES OF ACREMONIUM 1459

TABLE I. Continued

GenBank accession number

D1/D2 domains of
Species Strains Source ITS region 28S rRNA gene Actin
Cosmospora coccinea CBS 114050 Dead crust, on fallen branch of FJ474072
Fagus sylvatica, Bavaria,
Germany
Geosmithia morbida CBS 124663 Pityophthorus juglandis, FN434082
Colorado, USA
Gibellulopsis nigrescens CBS 120949 Soil under lawn, Baarn, the EF543857
Netherlands
Gibellulopsis piscis CBS 892.70T Granuloma in gold- fish DQ825985
(Carassius auratus), Brazil
Gliomastix polychroma MUCL 9834T Bark of Hevea brasiliensis, FN706547
Sumatra, Indonesia
Haematonectria — Grevillea robusta, Kenya HQ651171
haematococca
Leucosphaerina arxii CBS 737.84T Dung of horse, North HE608640 HE608662 HE608627
Carolina, USA
Nalanthamala diospyri CBS 430.89 Diospyros virginiana, AY554209
Mississippi, USA
Neonectria fuckeliana — Picea abies, Austria AJ557573
Pochonia suchlasporia var. CBS 248.83T Egg of Heterodera avenae, AJ292406
catenata Sweden
Pochonia suchlasporia var. CBS 251.83T Egg of Heterodera avenae, AJ292402
suchlasporia Sweden
Sarocladium bacillisporum CBS 425.67T Soil, Ontario, Canada HE608639 HE608658 HE608633
Sarocladium bactrocephalum CBS 749.69T Ustilago sp., Manitoba, Canada
Sarocladium glaucum CBS 796.69T Woollen overcoat, Solomon Islands FN691454 HE608657 HE608631
Sarocladium kiliense MUCL 9724T Skin, Germany FN691446
Sarocladium strictum CBS 346.70T Old leaf on Triticum aestivum,
Germany
Sarocladium zeae CBS 800.69T Stalk of Zea mays, Nebraska, USA FN691451

FMR Faculty of Medicine Reus, Spain; CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; MUCL,
Mycothèque de l’Université Catholique de Louvain, Louvain-la Neuve, Belgium; T type strain; Accession numbers of sequences
newly determined in this study are indicated in boldface.

isolates did not match significantly any sequence other the ex-type strains of Acremonium borodinense
deposited in GenBank or NBRC. The phylogenetic Tad. Ito, Okane, Nagak. & W. Gams and A.
tree inferred from neighbor joining analysis of the pinkertoniae W. Gams and a reference strain of A.
ITS sequences revealed that the seven isolates of blochii W. Gams. In the phylogenetic study, numerous
Acremonium sp. I and the two isolates of Acremonium species were included that showed morphological
sp. II were clustered with different members of the similarities with the two possible new species but all
Bionectriaceae, where the ex-epitype strain of A. were distributed distantly from Acremonium species I
alternatum (CBS 407.66) was included. Acremonium sp and II in the ITS tree.
I and II grouped into a highly supported terminal For the multilocus sequence analysis, we selected
clade (100% bootstrap support [bs]) located far from strains that were closely related to the soil isolates in
remaining species included in this study (FIG.1). The the first analysis, supplemented with reference strains
clade was split into two well supported sister clades of Acremonium and related taxa (TABLE I). With our
(92% and 100% bs, respectively), each of them primer set we were able to amplify and sequence 434–
including one of the two mentioned species. A highly 550 bp, 435–584 bp and 712–835 bp of the ITS
supported (94% bs) sister clade of I and II, although regions, D1/D2 of 28S rRNA and the partial actin
phylogenetically distant, also included two subclades. gene respectively. Of the 1668 characters from the
One of them comprised the ex-type strains of the three loci used in this analysis, 303 were parsimony
ascomycetes Bulbithecium hyalosporum Udagawa & T. informative. The lowest number was 47 for D1/D2
Muroi and Leucosphaerina arxii Malloch and the and the highest was 133 for ITS. The result of the
1460 MYCOLOGIA

FIG. 1. Neighbor joining tree constructed with sequences of the ribosomal internal transcribed spacer (ITS) regions and
5.8S rRNA gene. Branch lengths are proportional to distance. Bootstrap support values above 60% are indicated at the nodes.
T
5 type strain.

partition homogeneity test showed that the datasets 0.3411 and a RI of 0.8046, were produced from
for the three loci were congruent (P 5 0.16) and heuristic searches with the combined dataset from the
could be combined. A total of 1142 most parsimoni- three loci (FIG. 2). The isolates identified as Acremo-
ous trees, 465 steps long, with a CI of 0.6589, an HI of nium sp. I and II clustered in a well supported clade
 GIRALDO ET AL.: NEW SPECIES OF ACREMONIUM 1461

FIG. 2. One of 1142 most parsimonious trees obtained from heuristic searches based on analysis produced from the
combined ITS, D1/D2 and actin datasets. Bootstrap support values are indicated at the nodes. CI 5 consistency index; HI 5
homoplasy index; RI 5 retention index. Bootstrap support values above 70% are indicated at the nodes. T 5 type strain.

divided in two subclades representing each of the two between the sequences of the two species were 3–
species. The combined tree topology was similar to 4.6% for the three loci.
the one observed in the trees of individual genes
analyzed with NJ (data not shown). The sequences of Phenotypic studies.—Cardinal temperatures after 14 d
the seven strains of Acremonium sp. I had 100% in the dark were similar for Acremonium sp. I and
homology in each of the three loci studied. Those of Acremonium sp. II, that is the minimum, optimum
the two strains of Acremonium sp. II showed 99.8% and maximum growth temperatures were respectively
homology for the ITS region and 100% for the D1/ 5 C (2–7 mm diam), 20–25 C (26–44 mm diam) and
D2 domains and the actin fragment. The differences 32 C (2–4 mm diam). No isolate of either species grew
1462 MYCOLOGIA

FIG. 3. Acremonium asperulatum FMR 11136 (A, B), CBS 130362 (C–F), FMR 11137 (G) and A. variecolor CBS 130360 (H–
N). A, B. Colonies on PDA and OA respectively at 25 C after 22 d. C. Branched conidiophores. D. Phialide with a basal septum
and conidia arranged in slimy heads. E. Phialides with collarettes. F, G. Globose rough- and thick-walled conidia and
chlamydospores. H–J. Colonies on PDA (obverse and reverse) and OA respectively at 25 C after 22 d. K, L. Branched
conidiophores and conidia arranged in slimy heads. M. Phialide with conidia arranged in slimy heads. N. Subglobose conidia
with apiculate base and a sessile conidium on vegetative hyphae (arrow). Bars: C–G, K–N 5 10 mm.
 GIRALDO ET AL.: NEW SPECIES OF ACREMONIUM 1463

at 35 C. Colonies at 20–30 C were flat, slightly cottony de Bujaruelo, isolated from forest soil, Jun 2009, M.
and whitish. They were characterized microscopically Hernández, J. Mena & J. Cano (HOLOTYPE, IMI 500816;
by more or less erect conidiophores with whorls of culture ex-type, CBS 130362 5 MUCL 53781 5 FMR
acicular phialides. Acremonium sp. I produced abun- 11065); isolated from sediments of Ara River, Mar 2011,
M. Hernández, A. Giraldo & X. Capilla (CBS 130363 5
dant chlamydospores, and its conidia were rough-
MUCL 53782 5 FMR 11783). Ordesa y Monte Perdido
walled and globose. Acremonium sp. II produced a
National Park: Añisclo canyon, isolated from forest soil, Jun
brownish diffusible pigment on PDA, and its conidia 2009, M. Hernández, J. Mena & J. Cano (FMR 11135, FMR
were smooth-walled, subglobose with an apiculate 11137); Ordesa Valley, Broto, isolated from forest soil, Jun
base. In addition, sessile conidia growing directly 2009, M. Hernández, J. Mena & J. Cano (FMR 11136 and
from vegetative hyphae were observed in the latter FMR 11138); Ordesa Valley, Torla, isolated from forest soil,
species on all media tested. Jun 2009, M. Hernández, J. Mena & J. Cano (FMR 11139).

TAXONOMY Acremonium variecolor A. Giraldo, Guarro, Gené &

On the basis of the phylogenetic analysis and Cano, sp. nov. FIG. 3H–N
phenotypic features, we conclude that the species MycoBank MB563320
Acremonium sp. I and Acremonium sp. II are different Etymology. Refers to the variable colony color, ranging
from any previously described species in this genus from olive to brown.
and therefore are proposed as new. Colonies on OA and MEA at 25 C, attaining 43–
45 mm diam after 14 d in the dark, white (1A1) to
yellowish white (4A2), flat, glabrous to slightly
Acremonium asperulatum A. Giraldo, Guarro, Gené & cottony; reverse on OA brownish orange (5C4), on
Cano, sp. nov. FIG. 3A–G MEA pinkish white (7A2–3) or olive gray (3E2) at the
MycoBank MB563319 center, yellowish white (4A2) toward the periphery.
Etymology. Refers to the wall ornamentation of the On PDA reaching 41–44 mm diam after 14 d in the
conidia. dark at 25 C, yellowish white (3A2) to grayish yellow
Colonies on OA and MEA at 25 C, attaining 44– (4B4), often zonate, radially folded, velvety at the
60 mm and 31–63 mm diam respectively after 14 d in center, slightly cottony toward the periphery; diffusi-
the dark, white (1A1) to yellowish white (3–4A2), ble pigment brownish gray (5–8F1) or brown (6E5) in
often zonate, flat, velvety to slightly cottony; reverse the center, olive (1F4–5) toward the periphery was
brownish orange (5C4–6, 6C5) or yellowish white produced on PDA at 20, 25 and 30 C but not at #15 C.
(3A2). On PDA at 25 C, attaining 31–39 mm diam Vegetative hyphae septate, hyaline, smooth- and thin-
after 14 d in the dark, white (1A1) or yellowish white walled, 2 mm wide. Sporulation abundant, phalacro-
(4A2), radially folded or rugose, at first glabrous genous to nematogenous. Conidiophores erect, most-
becoming velvety to slightly cottony; reverse yellowish ly branched, bearing whorls of 2–5 phialides, septate,
(4A2–5) or amber yellow (4B6). Vegetative hyphae up to 290 mm long, hyaline, smooth, with walls usually
septate, hyaline, smooth- and thin-walled, 1.5–3 mm thicker than those of the vegetative hyphae. Phialides
wide. Sporulation abundant, phalacrogenous to ne- terminal or lateral, straight, acicular, 18–95 mm long,
matogenous. Conidiophores erect, simple or mostly 1–2 mm wide at the base, with periclinal thickening at
branched, bearing whorls of 2–4 phialides, septate, up the apex, collarette inconspicuous, thin- and smooth-
to 105 mm long, hyaline, smooth, with cell walls walled, hyaline; some phialidic conidiogenous cells
usually thicker than those of the vegetative hyphae. without a basal septum (adelophialides) were ob-
Phialides terminal or lateral, straight or slightly bent, served on OA. Conidia unicellular, subglobose or
acicular, 28–68 mm long, 1–2 mm wide at the base, with ovoid, 3–4(–5) 3 2–4 mm, slightly apiculate base,
minute collarette and distinct periclinal thickening at hyaline to subhyaline, thick- and smooth-walled,
the apex, thin- and smooth-walled, hyaline. Conidia chromophilic, arranged in slimy heads. Sessile conid-
unicellular, globose, 3–4(–5) mm diam, hyaline to ia growing directly on vegetative hyphae, solitary,
subhyaline, rough- and thick-walled, chromophilic, blastic, unicellular, cylindrical or ellipsoidal, 5–7(–9)
arranged in slimy heads. Chlamydospores abundant 3 2–3(–4) mm, hyaline and smooth-walled. Chlamydo-
on PDA and OA, few on MEA, terminal or intercalary, spores and teleomorph not observed.
single or in chains, unicellular, subglobose or oval, 5– Specimens examined: SPAIN. ARAGÓN REGION,
10 3 5–9 mm, hyaline to subhyaline, smooth- and HUESCA PROVINCE: Ordesa y Monte Perdido National
thick-walled, strongly chromophilic. Teleomorph not Park, Garganta de Escuaı́n, isolated from forest soil, Jun
observed. 2009, M. Hernández, J. Mena & J. Cano (HOLOTYPE, IMI
Specimens examined: SPAIN. ARAGÓN REGION, 500815; ex-type culture, CBS 130360 5 MUCL 53779 5
HUESCA PROVINCE: Bujaruelo Valley: refuge San Nicolas FMR 11140). GALICIA REGION, LUGO PROVINCE:
1464 MYCOLOGIA

Nature Reserve Sierra de Os Ancares, isolated from forest logically by having smaller and simple conidiophores
soil, May 2010, M. Hernández, J. Mena & J. Guarro (CBS (25–30 and 17–27 mm long respectively), by absence
130361 5 MUCL 53780 5 FMR 11141). of chlamydospores, and by colonies with a colorless
reverse (Gams 1971, Ito et al. 2000). In addition, A.
DISCUSSION borodinense produces two types of phialidic conidia,
both with morphological features different from the
Based on the combination of a multilocus analysis species described here (Gams 1971, Ito et al. 2000).
and phenotypic features, we propose and describe The two types of conidia observed in A. variecolor
two new species of Acremonium, A. asperulatum and A. differ in their ontogeny: One is phialidic and the
variecolor. Apart from morphological features, the other is blastic. The latter kind of conidia also was
reason for including these fungi in Acremonium was described in Pochonia suchlasporia W. Gams & Dack-
based mainly on the recent phylogenetic study carried man (Gams 1988, Zare et al. 2001), but this species is
out by Summerbell et al. (2011). After discussing phylogenetically distant from A. variecolor (FIG. 1).
several alternatives concerning the epitypification of Acremonium blochii, which was nested in a clade close
Acremonium, Summerbell et al. (2011) designated to that of A. variecolor, has morphologically similar
CBS 407.66 as epitype of A. alternatum, the type phialidic conidia, but these are arranged in long
species of the genus. The phylogentic D1/D2 tree chains or slimy heads and it has only single phialides.
given in that study shows that the strain belonged to Other Acremonium species included in Bionectria-
the family Bionectriaceae, where a large group of ceae, with some morphological features similar to our
species currently accepted in Acremonium also were new taxa, are Acremonium fusidioides (Nicot) W. Gams
placed. Therefore, all those Acremonium-like fungi and A. hansfordii (Deighton) W. Gams. Both produce
that are phylogenetically close to CBS 407.66 should a characteristic brownish diffusible pigment (Gams
be recognized as Acremonium sensu stricto. Our ITS 1971, 1975), but they usually have simple conidio-
sequences analyses showed that the two new species phores; A. hansfordii has fusiform and more or less
make up a well supported clade, with some members brown-pigmented conidia with truncate ends; A.
of the Bionectriaceae being related at the same time fusidioides produces two types of phialidic conidia,
to the epitype of A. alternatum. the most abundant ones being slightly pigmented and
These novel Acremonium species are characterized fusiform and the less common ones globose and
mainly by the production of verticillately branched hyaline (Gams 1971, 1975). In our ITS analysis both
conidiophores, long phialides and by the production species formed a well supported lineage distant from
of globose or ovoid, thick-walled conidia. Key our new species.
morphological features that differentiate the two
fungi are that A. asperulatum produces chlamydo-
spores and has globose conidia with rough walls ACKNOWLEDGMENTS
arranged in slimy heads, while A. variecolor produces We are indebted to the curators of the CBS-KNAW Fungal
a characteristic diffusible brownish pigment on PDA Biodiversity Centre (Utrecht, the Netherlands) and the
and has two types of conidia, subglobose with an Mycothèque de l’Université Catholique de Louvain (Lou-
apiculate base grouped in slimy heads on the vain-la-Neuve, Belgium) for supplying many of the strains
phialides and solitary and sessile elongated conidia, used in the study. This study was supported by the Spanish
arising directly on vegetative hyphae. Chlamydospores Ministerio de Educación y Ciencia, grants CGL 2009-08698/
were not observed in the latter species. The conjunc- BOS and CGL 2011-27185/BOS.
tion of such characteristics with their phylogenetic
distances from the other species of Acremonium and
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