Full text of DOI:10.1016/S0953-7562(08)62000-6

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Mycol. Res. 105 (10): 1273–1279 (October 2001). Printed in the United Kingdom.
Merimbla humicoloides sp. nov. from conifer forest soil of
Veracruz state, Mexico
Gerald F. BILLS1*, Rosa Maria ARIAS2, Manuela REYES2 and Gabriela HEREDIA2
"
Centro de Investigacion Basica, Merck, Sharp & Dohme de Espana S.A., Josefa Valcarcel 38, E-28027, Madrid, Spain.
Instituto de Ecologıa, A.C., Apartado Postal 63, 91000, Xalapa, Veracruz, Mexico.
#
E-mail: geraldIbills!merck.com
Received 25 October 2000; accepted 3 July 2001.
Merimbla humicoloides sp. nov. was isolated after heat treatment of soil collected in a pine forest of Veracruz state, Mexico. The
fungus is characterized by pale buff to cinnamon colonies that become dark brown to black in reverse and conidiophores that vary
from irregularly asymmetrical penicilli to symmetrical penicilli with inflated metulae, and Humicola-like chlamydospores on the
submerged hyphae. Phylogenetic inferences made from the ITS1-5n8S-ITS2 rDNA sequences indicate that M. humicoloides is a
member of the Trichocomaceae and that it is related to M. ingelheimensis, Hamigera avellanea, and Penicillium species.
ITS sequence data and morphological comparisons have led us
to describe a new species, M. humicoloides.
INTRODUCTION
Heat and chemical pasteurization are used to selectively
isolate ascomycetes from soils and other organic substrata
(Warcup 1951, Warcup & Baker 1963, Samson & Luiten 1975,
Furuya & Naito 1979, 1980). Treatments of soils with heat,
ethanol, or other organic solvents reduce the numbers of
fungal colonies on isolation plates that originate from thin-
walled conidia or hyphal fragments, and increase the frequency
of isolation of fungal strains from ascospores, sclerotia, and
thick-walled conidia or hyphae. Limited evidence suggests
that pretreatment of soils with heat or small organic molecules
may stimulate ascospore germination in some fungi (Furuya &
Naito 1980). We applied pasteurization techniques during a
survey of soil fungi in Pinus hartwegii and P. patula forests of
the Volcanic Cordillera of Veracruz state, Mexico. During
study of the isolates from heat-treated soils, we encountered
a conidial fungus with aerial conidiophores that resemble the
anamorph genera Paecilomyces and Merimbla but formed dark
Humicola-like chlamydospores on the submerged hyphae.
Referral to monographs of Paecilomyces, soil fungus identi-
fication guides, and consultation with the CBS identification
service led us to believe this fungus was undescribed.
Sequence data from the ITS1-5n8S-ITS2 rDNA were obtained
to test whether the fungus was a member of the Trichocomaceae.
Comparisons of rDNA sequences of the fungus with the type
species of Paecilomyces, P. variotii, and other species of
Trichocomaceae indicated significant sequence homology with
Hamigera avellanea and Merimbla ingelheimensis. Analysis of
MATERIAL AND METHODS
Isolation and cultivation
Living cultures are maintained in the Merck Research
Laboratories Microbial Resources Culture Collection (MRL)
unless noted otherwise. For long-term storage at MRL, liquid
cultures were prepared by inoculating 250-ml Erlenmeyer
flasks containing 50 ml of Sabouraud’s maltose yeast extract
broth (neopeptone 10 g; maltose 40 g; yeast extract 4 g, agar
−
4 g l distilled H O) with agar discs from fresh cultures.
"
#
−
"
Flasks were incubated on a gyratory shaker at 220 rev min
for 5–7 d at 23 mC. Mycelial suspensions were diluted 1:1
v:v with 20% glycerol in 4-ml vials and frozen at k80 m.
For soil isolations, soil samples were collected 1–3 cm from
the surface with a soil-immersed trowel or knife and placed in
Tyvek soil collection bags. Soils were transported at ambient
temperatures to the laboratory where they were sieved and
returned to the collection bags. Soils were transported to MRL
in Rahway, NJ at ambient temperature. Soils were heat-treated
by placing a few grams of soil in an aluminum weighing pan
and incubating them at 100 m for 1 h. Soils were mixed into
molten GAN agar (Gochenaur 1978) amended with
−
10 mg g l cyclosporin A, 50 mg l each of streptomycin
−
"
"
sulfate and chlortetracycline. Plates were incubated at 23 m.
Cultural characteristics
Colony morphology was studied using standard media and
protocols for description of penicilli (Pitt 1980). G25N,
* Corresponding author.

New species of Merimbla from Mexico
1274
Table 1. Isolates analyzed and their GenBank accession codes.
purified with a QIAquick PCR Purification Kit (QIAGEN
Valencia, CA). Amplification products were checked for
correct molecular weight and purity by electrophoresis on 1%
agarose gels stained with ethidium bromide. Primers ITS5 and
ITS4 were used to sequence both strands of the ITS1, 5n8S
rDNA, and ITS2 regions. Sequence reactions were carried out
with a cycle sequencing kit (BigDye Terminator Cycle
Sequencing Ready Reaction, PE Applied Biosystems) and
cleaned with Pharmacia G-50 ultra-fine sephadex spin columns.
Eluates were dried in a vacuum centrifuge (Savant). Sequences
were obtained on an ABI Prism 377 automated sequencer
(Perkin–Elmer). Each strand of the double-stranded ampli-
fication products was sequenced at least twice to obtain a
consensus sequence. New sequences were deposited in the
GenBank database, and the accession numbers are listed in
Table 1. Additional ITS sequences used for comparative
analyses were obtained from GenBank (Table 1).
GenBank
accession no.
Species
Strain
Byssochlamys nivea
Eupenicillium pinetorum
Geosmithia argillacea
G. cylindrospora
FRR 2205
U18361
NRRL 3008
NRRL 5177
NRRL 2673
NRRL 3221
ATCC 10414, ex-holotype
GB6142
AF033411
AF033389
AF033386
AF033387
AF368295
AF368296
AF368297
AF368298
G. emersonii
Hamigera avellanea
H. avellanea
Merimbla ingelheimensis
Merimbla humicoloides
GB6187
GB6082, CBS 102854
ex-holotype
NRRL 1115
NRRL 2074
NRRL 25744
NRRL 26877
FRR 1833
Paecilomyces variotii
Penicillium sclerotiorum
P. restrictum
AF033395
AF033404
AF033459
AF038938
U18355
P. striatisporum
Neosartorya fischeri
Thermoascus crustaceus
Monascus purpureus
FRR 1328
NRRL 1596
U18353
AF033394
Sequence alignment and analyses
Sequences were aligned using Clustal X (Thompson et al.
1997) followed by visual adjustment and editing using
GeneDoc version 2n0.1 (Nicholas et al. 1997). Phylogenetic
relationships were determined from the aligned sequences
using PAUP version 4n0b3a (Swofford 2000). Nucleotide
substitutions were treated as unordered, unweighted
characters. Gaps introduced to improve alignments were
considered as missing data. Maximum parsimony trees were
inferred using the heuristic search option with stepwise
addition with the tree bisection reconnection algorithm.
Support for the internal branches of the trees was assessed by
1000 bootstrap replicates from a 50% majority-rule consensus
tree. Byssochlamys nivea was selected as an out group to root
the tree because 18S rDNA phylogenies have show it to form
a sister clade to those of Hamigera, Eupenicillium, Talaromyces,
Eurotium, and Neosartorya (Ogawa et al. 1997, Ogawa &
Sugiyama 2000). Phenetic analyses of the data was performed
using the neighbour-joining method. The aligned ITS
sequences are deposited in TreeBase in NEXUS format, with
the accession number SN782-2151.
Blakeslee’s malt, and Czapek’s yeast autolysate agars were
prepared with agar, malt extract, and yeast extract from Difco
(Detroit) according to Pitt (1980). All inorganic and defined
medium components were reagent grade from Sigma (St
Louis, MO). Strains were also studied on cornmeal (Difco),
YM agar (Difco), V8 juice agar, containing 200 ml V8 juice
(Campbell Soup, Camden, NJ), 3 g CaCO , agar 20 g, H O
$
#
0n8 l, and modified α-cellulose agar (Weitzman & Silva-Hunter
1967) containing α-cellulose 20 g, MgSO .7H O 1 g, KH PO
1 g, NaNO 1 g, tomato paste 10 g (Hunt-Wesson, Fullerton,
%
#
#
%
$
CA), baby oatmeal 10 g (Gerber Products, Fremont, MI), agar
18 g, H O 1 l, adjusted to pH 5n6 with NaOH prior to
autoclaving. Colony descriptions were made after 14 d growth
#
at 25 m in a 12 h fluorescent photoperiod, unless noted
otherwise. Colour designations in parentheses are from
Kornerup & Wanscher (1978) and in capital letters from
Ridgway (Ridgway 1912).
Extraction of genomic DNA, PCR amplification, and
sequencing
Mycelia from frozen cultures were used to inoculate 250-ml
flasks containing 50 ml of YM broth (Difco). After one week’s
RESULTS
growth at 23 m, mycelium was harvested by vacuum filtration BLAST searches using the ITS1-5n8S-ITS2 sequence of
and lyophilized. Lyophilized mycelia were pulverized in liquid GB6082 (530 bp fragment) recovered ribosomal DNA
nitrogen with a mortar and pestle. DNA was extracted from sequences of species of Penicillium, Eupenicillium, Warcupiella,
0n1 g of lyophilized cells using an Invitrogen Easy-DNA and Geosmithia. Highest scoring matches were with some
extraction kit (Invitrogen, Carlsbad, CA) following the species of Penicillium. An initial alignment of sequences from
manufacturer’s instructions. Purified DNA was stored at 4 m GB6082 with representative sequences of different genera of
pending use for PCR amplification.
the Trichocomaceae indicated the ITS region sequence had
Fragments of the ribosomal DNA repeat unit (ca 500– affinities to those of some Penicillium species. Based on BLAST
600 bp) that included the ITS1, 5n8S, and ITS2 rDNA were searches and preliminary alignments, an ITS sequences matrix
amplified using primers ITS5 and ITS4 (White et al. 1990). was compiled based on previously published data for various
PCR amplifications were carried out in 25 µl reactions using genera of the Trichocomaceae (Table 1). In addition, ITS data
Ready To Go PCR Beads (Amersham Pharmacia Biotech, were used to test whether the conidiophore morphology and
Piscataway, NJ) according to the manufacturer’s directions in colony pigments of GB6082 were more similar to those of
an Eppendorf Mastercycler Gradient thermal cycler (Eppendorf Merimbla ingelheimensis and Hamigera avellanea or to those of
Scientific, Westbury, NY). Amplification products were the type species of the genus Paecilomyces, P. variotii. Therefore,

G. F. Bills and others
1275
Byssochlamys nivea
Paecilomyces variotii
Penicillium aculeatum
Thermoascus crustaceus
Neosartorya fischeri
Hamigera avellanea GB6142
92
91
Hamigera avellanea ATCC10414
Merimbla humicoloides GB6082
99
Merimbla ingelheimensis GB6187
96
72
Penicillium striatisporum
Penicillium restrictum
Penicillium sclerotiorum
Eupenicillium pinetorum
77
76
Monascus purpureus
74
Geosmithia argillacea
93
Geosmithia cylindrospora
Geosmithia emersonii
100
10
Fig. 1. Relationships of Merimbla humicoloides, M. ingelheimensis, Hamigera avellanea, and related Trichocomaceae. Most parsimonious tree
obtained from analysis of ITS1-5n8s-ITS2 rDNA using PAUP (Length l 451 steps; CI l 0n667; RI l 0n649; HI l 0n333). Percentages
from 1000 bootstrap replications are given on tree branches.
the ITS1-5n8S-ITS2 regions of a strain of Merimbla Neosartorya fischeri, H. avellanea, M. ingelheimensis, and
ingelheimensis (GB6187) and two strains of Hamigera avellanea Eupenicillium and monoverticilliate Penicillium species (91%
(GB6142 and ATCC 10414) were sequenced and added to the bootstrap support). The unknown fungus, GB6082, and M.
ITS1-5n8S-ITS2 alignment (Table 1). Strong sequence homo- ingelheimensis occurred on the same branch (99% bootstrap
logies among M. ingelheimensis, H. avellanea, and GB6082 support). The high bootstrap support for the branch with M.
were evident in the alignment. Byssochlamys nivea was chosen ingelheimensis and the high homology between GB6082 and
as an outgroup to root the tree.
M. ingelheimensis (95% homology over a 525 bp overlap)
The ITS data matrix was 598 characters in length with 208 support the conclusion that the two strains form a mono-
variable sites, 143 of which were parsimony informative. phyletic group. Therefore, GB6082 is recognized as a new
Parsimony analysis of the set produced four trees of 451 steps species of Merimbla. The two strains of H. avellanea shared
(Fig. 1). A main clade, separate from Byssochlamys nivea and 97% sequence identity over a 490 bp region and grouped in
Paecilomyces variotti, was apparent (96% bootstrap support). a sister clade (92% bootstrap support) adjacent to that of M.
The unknown fungus, GB6082, fell within a clade containing ingelheimensis, the putative anamorph of H. avellanea. The two

New species of Merimbla from Mexico
1276
Figs 2–5. Merimbla humicoloides (GB6082) in culture after 14 d. Fig. 2. Malt yeast extract agar. Fig. 3. Cornmeal agar. Fig. 4. α-
cellulose agar. Fig. 5. V8 juice agar.
conidiogenae 1–4, enteroblasticae, phialidicae, cylindricae vel ampuli-
formes. Conidia globosa, subglobosa vel limoniformia, hyalinua,
tenuitunicata, leviter muricata, 3–4 (–6)i2n5–3 µm. Chlamydosporae
in hyphis et raro a cellulas conidiogenas formatae, Humicolae
similares, solitariae vel catenulatae, terminales vel intercalares,
hyalinae vel fusco-griseae, posterius nigrescentes, globosae vel
breviter cylindricae, 10–18 µm diam.
strains of H. avellanea exhibited 86% sequence identity with
M. ingelheimensis (452 bp overlap with each of ATCC 10414
and GB6142).
Merimbla humicoloides Bills & Heredia, sp. nov.
(Figs 2–12)
Etym.: From Humicolajoides (Latin), resembling Humicola;
referring to the morphological similarity of the chlamydosporic
synanamorph to species of soil-dwelling Humicola.
Typus: Mexico: Veracruz: Municipio de Las Vigas, Reserva
Ecologica San Juan de Montes, alt. 2300 m, heat-treated soil from
forest of Pinus hartwegii,
5 May 1999, G. Bills GB6082
(BPI – holotypus, XAL – isotypus; Merck Microbial Resource Cul-
ture Collection GB6082, CBS 102854 – cultura viva); loc. cit.
GB6083 (paratypus).
Coloniae in agaro YM lente crescentes, superficie velutina et radiatim
sulcata, partim prominentes vel umbonatae, margine submerso,
pallide luteae vel cineraceo lutae; reversum brunneum vel nigrescens.
Conidiophora ex mycelio basali oridunda, erecta, 15–75i2–5 µm;
stipites glabri, hyalini, septata; cellula pedis nulla. Rami simplices vel
irregulariter vel symmetrice verticillati. Penicilli monoverticillati vel
biverticillati, ad apicem cellulas conidiogenas vel metulas formantes.
Metulae ad apicem conidiophori in dispositione aequata vel dispari
2–5 dispositae, 8–12i2n5 µm, cylindricae vel spatulatae. Cellulae
Colonies on YM agar growing slowly, 19–23 mm diam in
14 d at 25 mC, submerged at the margins but soon raised or
umbonate, radially sulcate, dull, velvety, pale buff to grayish
buff (5D3, 5D4, Light Buff, Tilleul-Buff, Vinaceous-Buff,
Avellaneous). Reverse brown, dark grayish brown to black

G. F. Bills and others
1277
Figs 6–12. Merimbla humicoloides (GB6082). Figs 6–8. Mature symmetrical conidiophores. Fig. 9. Irregularly penicillate conidiophore
with proliferating chlamydospores. Figs 10–11. Irregularly penicillate conidiophores. Fig. 12. Chlamydospores on submerged hyphae.
Bars 6–7 l 20 µm; 8 l 25 µm; 9–11 l 20 µm; 12 l 10 µm.
(6F5, Rood’s Brown, Snuff Brown, Bister). No growth at 37 m. submerged hyphae, pale buff to light cinnamon, similar to
Colonies on CYA growing very slowly, 3–4 mm diam in 14 d colonies on cornmeal and V8 juice agars, and reverse reddish
at 25 m, partly submerged, appressed to velvety, translucent to brown (6C7, 6C6, Tawny, Vinaceous-Tawny, Pecan Brown).
pale buff (4A2, 5A2), with scant development of conidiophores Colonies on G25N agar producing neglible growth in 14 d
in the center of colonies, margin even, reverse pale to dark at 25 m.
gray, finally black as chlamydospores mature on submerged
Conidiophores arising directly from the basal mycelium, base
hyphae. No odor. No growth at 37 m. Colonies on V8 juice agar not delimited by a foot cell, erect, 15–75 µm tall, 2–5 µm
growing slowly, 14–17 mm diam in 14 d at 25 m, submerged wide, smooth- and thin-walled, septate, simple and bearing a
towards margins, becoming velvety as conidiophores develop few conidiogenous cells, to forming irregularly or sym-
at the surface, pale buff (Pale Ochraceous Buff, Light Buff, metrically branched verticils or penicilli. Penicilli simple
Light Ochraceous Buff) to cinnamon (4A2, 5A2, 5B4-5, Light monoverticillate, to symmetrical or asymmetical biverticillate,
Pinkish Cinnamon, Pinkish Buff, Pinkish Cinnamon); reverse giving rise directly to conidiogenous cells or metulae. Metulae
yellowish brown (5C7, 5CD). No odor. Colonies on cornmeal sometimes formed at apices of conidiophores, especially in
agar growing slowly to moderately, 26–30 mm diam in 14 d older colonies, arranged in symmetrical groups of 2–5 or
at 25 m, velvety with submerged hyphae, translucent, pale buff arising at different levels, 8–12i2n5–4 µm, cylindrical to
to cinnamon (5A3, 5B3, 5C4, 5D4, Light Buff, Warm Buff, spathulate, sometimes arranged in aspergilloid form, bearing
Light Ochraceous-Buff). Reverse translucent at first, grayish to 1–4 conidiogenous cells. Conidiogenous cells enteroblastic,
brown to black. Colonies on α-cellulose agar growing slowly to phialidic, cylindrical to ampulliform, tapering toward the
moderately, 26–32 mm diam in 14 d at 25 m, velvety with apical conidiogenous locus, straight, or occasionally curved or

New species of Merimbla from Mexico
1278
flexuous, 9–19i2–4 µm. In smaller Penicillium-like or Paecilo- M. ingelheimensis and H. avellanea also produce reduced
myces-like conidiophores, new conidiogenous cells may penicillate conidiophores, especially when young. Further-
proliferate from the conidiogenous locus of the previous cell. more, the light buff, cinnamon, or avellaneous colony pigments
Conidia globose, subglobose to limoniform, occasionally with of the three species, especially of the aerial mycelium and
remnants of connectors, hyaline, thin-walled, faintly conidial heads are very similar. M. ingelheimensis can develop
roughened, 3–4(–6)i2n5–3 µm, adhering in dry chains. a dark grey to nearly black colony reverse on some media,
Chlamydospores formed primarily on the surface and submerged however the colour is associated with melanized submerged
hyphae, occasionally from phialidic conidiogenous cells, filamentous hyphae, not melanized chlamydospores as in M.
Humicola-like, globose, subglobose to short cylindrical, humicoloides. Merimbla humicoloides is distinct from M.
10–18 µm diam, solitary, in pairs, or in short chains, terminal ingelheimensis and the anamorph of H. avellanea because of its
or intercalary, hyaline to dark grey or black, giving colonies black chlamydospores, smaller conidiophores, and its inability
a dark grey or black appearance in reverse view. Surface to grow at 37 m (Table 2). Possibly, the abundant chlamydo-
hyphae in some media, e.g. YM agar, with abundant large, spores permitted its survival during the heat pretreatment of
globose or vesiculate, thin-walled hyphae, up to 20 µm diam. soil from which it was isolated.
The genus Merimbla has only one formally described
Additional specimens examined. Merimbla ingelheimensis: Mexico:
species, M. ingelheimensis (syn. Penicillium ingelheimensis), but
Veracruz: Municipio de Las Vigas: Reserva Ecologica San Juan de
the unnamed anamorph of Talaromyces subinflatus was
Montes, 2300 m, heat-treated soil from forest of Pinus hartwegii, 5
described as Merimbla-like (Yaguchi et al. 1993). The literature
May 1999, GB6187, GB6197; Valle Alegre, 2500 m, heat-treated
is somewhat confused as to whether or not M. ingelheimensis
and H. avellanea are distinct species. Raper, Thom & Fennel
1949), described Penicillium avellaneum (i.e. H. avellanea) and
noted that P. ingelheimensis appeared ‘to be an unusually
coarse strain of P. avellaneum’ and with the penicilli ‘unusually
large and the number of metulae present in some structures is
soil from conifer forest, GB6487. – Hamigera avellanea: Puerto Rico:
Rincon, Highway 115, soil along road to Aguada, GB6142. USA:
Texas: San Antonio, from soil, ATCC 10414 – ex-holotype.
DISCUSSION
Irregular mono- and biverticillate conidiophores, light buff to estimated to range up to 12–15.’ When Pitt & Hocking (1979)
cinnamon colours, and dark chlamydospores are evident in described the genus Merimbla, M. ingelheimensis was desig-
young cultures of Merimbla humicoloides. The combination of nated as the type species and was equated with P. avellaneum,
characters was reminiscent of certain chlamydosporic Paecilo- the anamorph of H. avellanea. However, the distinctness of M.
myces species, e.g. P. variotii, P. clavisporus, and P. zollerniae. ingelheimensis and H. avellanea was noted by Stolk & Samson
Referral to monographs and recent descriptions of Paecilomyces, (1971). Ramırez (1982) treated P. ingelheimese and P. avellaneum
however, did not lead to a satisfactory identification to any as different species. His illustrations and comments about their
known species. Consultation with the CBS identification distinct colony morphologies agree with our observations of
service indicated that the strain may represent an undescribed these fungi in culture. Our study has only focused on the
species.
characterization of M. humicoloides and elucidation of its
The affinities of M. humicoloides to the type species of generic placement near M. ingelheimensis as indicated by both
Paecilomyces and other genera of Trichocomaceae were tested morphology and ITS data, regardless of the relationship
by comparing sequences of the ITS regions of the rDNA. The between H. avellanea and M. ingelheimensis.
strong sequence similarities among M. humicoloides, M.
Results of our ITS sequence analyses are consistent with
ingelheimensis, and H. avellanea led us to closer morphological previous analyses of the Trichocomaceae (Ogawa et al. 1997,
examination of M. humicoloides and side-by-side comparisons Ogawa & Sugiyama 2000). In previous studies employing 18S
with the latter two fungi. In mature colonies of M. humicoloides rDNA data, Eupenicillium, Eupenicillium-derived penicillia,
or in slide culture preparations, its similarities to M. Hamigera, Merimbla, Neosartorya, Monascus, Thermoascus,
ingelheimensis become evident (Table 2). Symmetrical and Talaromyces, and Talaromyces-derived penicillia (represented
asymmetrical biverticillate conidiophores with inflated metulae by P. aculeatum in this study) form a monophyletic
develop in older colonies, and a morphology-based identi- Trichocomaceae. A similar grouping of taxa was evident in our
fication as Merimbla becomes possible (Pitt & Hocking 1979). study. Comparison of the 18s rDNA sequences of H. avellanea
Table 2. Distinguishing characteristics of Merimbla species and Hamigera avellanea.
Character
Merimbla humicoloides
M. ingelheimensis
Hamigera avellanea
Ascomata
Colony reverse in
YM agar
Absent
Dark grey to black
Absent
Present
Brown to vinaceous, or purple
Buff to dark grey, black in
age on α-cellulose agar
Yes
Growth at 37 mC
Conidiophores
Metulae
No
Up to 75 µm tall
Yes
Up to 400 µm tall
Up to 1000 µm tall
Cylindrical to inflated, arising at different
levels or in true verticils of 2–7
Abundant, terminal and intercalary, black
Mostly inflated, arising at different
levels or in verticils of up to 12–15
Occasional inflated, thin walled,
intercalary swellings in old cultures
Cylindrical to inflated, mostly
arising at different levels
Absent, but inflated hyphae may be
present at the colony surface
Chlamydospores

G. F. Bills and others
1279
In Integration of Modern Taxonomic Methods for Penicillium and Aspergillus
Classification (R. A. Samson & J. I. Pitt, eds): 149–161. Harwood Academic
Publishers, Amsterdam.
and M. ingelheimensis deposited in GenBank show they differ
by a single insertion in the latter species over a 1771 bp
overlap. The two taxa were grouped together in a well-
supported clade which is consistent with their morphological
resemblance (Ogawa, Yoshimura & Sugiyama 1997, Ogawa &
Sugiyama 2000). The less conserved ITS sequence data used
in this study failed to convincingly group H. avellanea with M.
ingelheimensis, and they appear in sister clades. ITS sequence
differences contradict the suggestion of Pitt & Hocking (1979)
that M. ingelheimensis is the anamorph of H. avellanea, and
supports the contention that the P. avellaneum anamorph of H.
avellanea and M. ingelheimensis are distinct species (Stolk &
Samson 1971, Ramırez 1982).
Ogawa, H., Yoshimura, A. & Sugiyama, J. (1997) Polyphyletic origins of
species of the anamorph genus Geosmithia and the relationships of the
cleistothecial genera: Evidence from 18S, 5S and 28S rDNA sequence
analyses. Mycologia 89: 756–771.
Pitt, J. I. (1980) [‘1979’] The Genus Penicillium and its Teleomorphic States
Eupenicillium and Talaromyces. Academic Press, London.
Pitt, J. I. & Hocking, A. D. (1979) Merimbla gen. nov. for the anamorphic state
of Talaromyces avellaneus. Canadian Journal of Botany 57: 2394–2398.
Ramırez, C. (1982) Manual and Atlas of the Penicillia. Elsevier Biomedical Press,
Amsterdam.
Raper, K. B., Thom, C. & Fennel, D. I (1949) A Manual of the Penicillia.
Williams & Wilkins, Baltimore.
Ridgway, R. (1912) Color Standards and Nomenclature. Published by the author,
Washington DC.
Samson, R. A. & Luiten, B. (1975) Eleutherascus tuberculatus, a heat-resistant
ascomycete. Transactions of the British Mycological Society 64: 338–340.
Stolk, A. C. & Samson, R. A. (1971) Studies on Talaromyces and related
genera. I. Hamigera gen. nov. and Byssochlamys. Persoonia 6: 341–357.
Swofford, D. L. (2000) PAUP*. Phylogenetic Analysis Using Parsimony and
Other Methods. Version 4n0b3a. Sinauer Associates, Sunderland, MA.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.
(1997) The ClustalIX windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Research
25: 4876–4882.
ACKNOWLEDGEMENTS
We are indebted to Robert A. Samson and John I. Pitt for examining Merimbla
humicoloides and discussing its possible generic placement and to an
anonymous reviewer for correcting and improving the Latin diagnosis.
Warcup, J. H. (1951) Effect of partial sterilization by steam or formalin on the
fungus flora of an old forest nursery soil. Transactions of the British
Mycological Society 34: 520–532.
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Corresponding Editor: J. Sugiyama