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Mycothèque de lUniversité catholique de Louvain (MBLA, MUCL2), Place Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium
| ABSTRACT |
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Morphological and molecular studies of Gliocephalotrichum collections from rain forest leaf litter in French Guyana revealed the occurrence of two undescribed species, described here as G. bacillisporum and G. longibrachium. Both species have a whorl of sterile stipe extensions directly subtending the conidiogenous penicilli. Gliocephalotrichum bulbilium also was isolated from leaf litter in French Guyana. Two sequences in GenBank, of uncultured ascomycetes from Gabon, Africa, cluster in the Gliocephalotrichum clade, suggesting the probable existence of additional undescribed taxa from the Central African rainforest.
Key words: Africa, ß-Tubulin gene, Hypocreales, internal transcribed spacer, Nectriaceae, phylogeny, South America, systematics
| INTRODUCTION |
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Four species were later added to the genus: Gliocephalotrichum cylindrosporum Wiley & Simmons, G. microchlamydosporum (J. Meyer) Wiley & Simmons, G. simplex (J. Meyer) Wiley & Simmons (Wiley and Simmons 1971
) and G. ohiense Huang & Schmitt (Huang and Schmitt 1973
).
The genus then was divided into two informal morphological groups, distinguished by the localization of the whorl of stipe extensions in relation to the conidiogenous penicillus, either directly subtending it (G. bulbilium, G. microchlamydosporum and G. ohiense) or located at some distance below it (G. cylindrosporum and G. simplex) (Wiley and Simmons 1971
). However, Ellis and Hesseltine (1962)
and Nicot (1967)
also noted that in G. bulbilium the number and location of the stipe extensions may vary according to cultural conditions. Bulbilloid aggregates are absent in those additional species, but unicellular chlamydospores are present, solitary in G. simplex and G. cylindrosporum and occasionally in short chains in G. microchlamydosporum and G. ohiense (Huang and Schmitt 1973
, Meyer 1959
, Wiley and Simmons 1971
).
So far, a connection to a sexual state has only been demonstrated for G. bulbilium, for which the teleomorph was identified as Leuconectria clusiae (Samuels & Rogerson) Rossman et al (Nectriaceae, Hypocreales) (Rossman et al 1993
).
The ecology of Gliocephalotrichum species is little known. All the known species were isolated from soil or dead plants residues in leaf litter (decaying leaves or fruits) (Ellis and Hesseltine 1962
, Huang and Schmitt 1973
, Meyer 1959
, Wiley and Simmons 1971
, Watanabe and Nakamura 2005), where they probably occur as saprophytes. Nicot (1967)
reported a culture of G. bulbilium obtained as a contaminant in an attempt to isolate a lignicolous basidiomycete. All species but G. ohiense originated from tropical or subtropical regions; Gliocephalotrichum ohiense originated from temperate area of the Northern Hemisphere (Ohio, USA, Huang and Schmitt 1973
).
During a study of leaf litter anamorphic fungi in French Guyana, several isolates of Gliocephalotrichum were examined. Two isolates were identified readily to G. bulbilium while four others, representing two distinct morphologies, could not be identified satisfactorily as any of the described species. These were determined to be new species and are described and illustrated here as Gliocephalotrichum bacillisporum sp. nov. and Gliocephalotrichum longibrachium sp. nov. In addition, ITS and partial ß-Tubulin sequences were used to infer their relationships within Gliocephalotrichum.
| MATERIAL AND METHODS |
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= arithmetic mean; R = ratio of length/width of the conidia;
R = arithmetic mean of the ratio R. Selected cultures were crossed to determine whether a teleomorph might be produced. Conditions were: V-8 juice agar (V8) and Carnation leaf agar (CLA), 25 C, with a 12 h incident light, near UV cycle.
DNA was extracted from freshly collected mycelium grown in liquid malt extract at 25 C in the dark. Extractions were carried out using the QIAGEN Dneasy plant Mini Kit (QIAGEN Inc., Hilden, Germany), and later purified with Geneclean® III kit (Q-Biogene, USA), following the manufacturers recommendations. The primer pairs NS7-ITS4 (White et al 1990
) and Bt2a-Bt2b (Glass and Donaldson 1995
) were used to amplify the ITS1-5.8S-ITS2 region of the nuclear ribosomal operon and the 5' end of the ß-Tubulin gene. Successful PCR reactions resulted in a single band observed on 0.8% agarose gel, corresponding to approximately 900 bps (ITS) and 500 bps. Polymerase chain reaction products were cleaned using the QIAquick® PCR purification kit (250) (QIAGEN Inc., Hilden, Germany), following the manufacturers protocol. Sequencing reactions were performed using CEQ DTCS Quick Start Kit® (Beckman Coulter Inc., USA), according to the manufacturers recommendations, with the primers ITS2, ITS3 and ITS4 (White et al 1990
) and Bt2a and Bt 2b. Nucleotide sequences were determined with a CEQ 2000 XL capillary automated sequencer (Beckman Coulter Inc., USA). Initially, nucleotide sequences were automatically aligned with Clustal X for MacIntosh 1.5b, then manually adjusted as necessary by the editor in PAUP* 4.0b10.
The final ITS data set comprised 32 sequences (18 taxa, TABLE I
) and 466 characters were confidently aligned and included in the analysis, including gaps (297 positions were constant, 124 were parsimony informative). The final ß-Tubulin data set comprised 25 sequences (12 taxa, TABLE I
) and 342 characters were confidently aligned and included in the analysis, including gaps (241 positions were constant, 87 were parsimony informative) (a 30 bps segments was difficult to align between the Gliocephalotrichum and other genera and was excluded from the analysis). To limit the influence of large gaps, only the first character of a multi-character gap was coded, with subsequent gap characters coded as missing. Phylogenetic analysis of the aligned sequences was performed using the maximum parsimony method of PAUP* 4.0b10 (Swofford 2002
) with gaps treated as fifth base. The most parsimonious trees were identified using heuristic searches with random addition sequence (1000), max tree set to 100, and further evaluated by bootstrap analysis, retaining clades compatible with the 50% majority-rules in the bootstrap consensus tree. Analysis conditions were: tree bisection addition branch swapping (TBR), starting tree obtained via stepwise addition, steepest descent not in effect, MulTrees effective. In the ITS analysis, Xenocalonectria serpens (Decock et al) P.W. Crous & C.L. Schoch was used as outgroup, based on the results of Schoch et al (2000)
. In the ß-Tubulin analysis, Curvicladium cygneum Decock & P.W. Crous was used as outgroup.
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| RESULTS |
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| TAXONOMY |
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Colonies on V-8 4555 mm diam in 7 d, quickly turning brown due to the production of numerous brown bulbilloid aggregates, mostly embedded in the media; aerial production of conidiophores and conidia beginning soon after mycelium produced, in whitish, poorly defined concentric rings; mycelium mostly immersed, with little or no aerial mycelium; hyphae mainly hyaline to pale yellow or pale brownish with age, 211 m wide; conidiophores mononematous, macronematous, erect, arising from wide hyphae, composed of a basal stipe, a whorl of stipe extensions (sterile setae), and an apical conidiogenous penicillus; stipe cylindrical to slightly clavate, apex slightly inflated, hyaline to faintly yellowish, especially at base, thick-walled, (145)150180(190) µm long (
= 167.4 µm long), 9.014.5 µm wide at the base (
= 11.0 µm), 11.521.0 µm wide at slightly inflated apex (
= 14.7 µm), non septate except very basal septum; stipe extensions (2)47(8), directly subtending a conidiogenous penicillus, at right angles from conidiophores but progressively bent upward, then more or less oblique, at an angle of approximately 135 outward from the stipe, hyaline, thick-walled, especially at base, septate, (200)240400 (425) µm long (
= 331 µm), 5.07.5 µm wide at base progressively narrowing to 2.83.5(5.0) µm wide at the cylindrical or slightly clavate apex; conidiogenous head complexly and densely penicillate, quadri- (quinta-) verticillate, with 3(4) successive whorls of branches and one apical whorl of phialides; whole penicilli dense, slightly divergent, 7078 µm high and 87130 µm wide; primary branches 57, cylindrical to clavate, central branch usually longer than laterals, (20)22.533.5(35) x 611(11.8) µm (
= 27.9 x 8.9 µm), thick-walled, especially near base then progressively thin-walled toward apex, hyaline, bearing 46 secondary branches; secondary branches 1015 x 2.54.0 µm, bearing 34 tertiary branches; tertiary branches 713 x 3.54.5 µm bearing 35 quaternary branches; quaternary branches bearing 58 phialides; phialides cylindrical, finger-like to slightly ventricose, straight to slightly incurved inward penicillus, what is more pronounced for phialides located outside penicillus, with a narrow apex, dome-like, and with a small apical poroid conidiogenous locus, the wall of which progressively thickening by wall accumulation, 1015 x 1.52.5 µm (
= 12.7 x 1.8 µm); conidia narrowly cylindrical to bacilliform, base slightly beveled, apex rounded, hyaline, thin-walled, without drop inclusion, (6.5) 8.010.5(12.5) x (1.5)1.52.0(2.5) µm, (
= 9.0 x 1.8 µm), R = (3.6)3.96.0(6.3), (
R = 4.9); bulbilloid aggregates of chlamydosporic cells present, abundant to very abundant (especially in MUCL 46694), commonly immersed, rarely superficial on V8, occasionally superficial on the CLA, brown to dark brown, spherical, ellipsoid or elongated, 60200 x 50110 µm, (
= 110 x 81 µm), with no internal, or superficial differentiation, made of globose, slightly thick-walled cells, yellowish to yellowish brown; solitary chlamydospores absent; teleomorph not observed in cross of FG MUCL 46693 x MUCL 46694.
Substratum. On a dead, decaying leaf of an unidentified angiosperm, in leaf litter;
Habitat. Rainforest litter.
Locality. So far known only from the type locality, French Guyana.
HOLOTYPE.. FRENCH GUYANA: Cayenne Area, Matouri, Sentier dInterprétation de la Nature "Lamirande", from a dead, decaying leaf of an unidentified angiosperm, in leaf litter, isolated by direct plating of a conidial mass, Feb 1994, C. Decock & V. Robert, # FG 1143 = MUCL 46693, as a two-week-old dried V8 culture, and slides prepared from V8 agar (living strain ex-Holotype MUCL 46693).
PARATYPE: same data, C. Decock & V. Robert, # FG 1149 = MUCL 46694, as two-week-old dried V8 culture (living strain ex-paratype MUCL 46694).
Gliocephalotrichum bacillisporum Decock et Huret sp. nov. FIGS. 2
, 1115
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Colony on V8 7080 mm diam in 5 d, first white, soon yellowish to yellow with abundant aerial production of conidiophores and conidia, homogeneously distributed over colonies; mycelium mostly immersed, superficial, with no or little aerial hyphae, submerged hyphae with a loose plumose aspect on CLA; hyphae mainly hyaline to pale yellow, 310 µm wide; conidiophores mononematous, macronematous, erect, arising from large hyphae, composed of a basal stipe, a whorl of stipe extensions (sterile hairs), and an apical conidiogenous penicillus; stipe born from superficial hyphae, either terminal or intercalary, erect (vertical), sometimes with very basal part appressed (horizontal), then abruptly bent upward, cylindrical to slightly clavate, thick-walled, hyaline to faintly yellowish at base, hyaline at apex, (75)100162(188) µm high (
= 129.5 µm), 9.015.0 µm wide at base (
= 11.5 µm), 710 µm wide at the apex (
= 8.5 µm), the latter occasionally slightly bulbous, then up to 1213 µm, mostly with a single septum, then located approximately at lower third, (10)1560(75) µm from base or rarely, in some exceptionally long stipe, two to three septa; stipe extensions (3) 57, directly subtending conidiogenous penicillus, rising at right angle from conidiophores, but progressively bent upward, then more or less oblique, at an angle of approximately approximately 135° in relation to the stipe, to upright, hyaline, septate, slightly thick-walled especially at base, progressively thin-walled toward apex, terminal cell cylindrical to slightly clavate, (87)112184(215) µm long (
= 145.9 µm), 5.06.5 µm width at base down to 2.33.0 µm wide at apex; upper part of the stipe and stipe extensions covered with yellow to pale copper-colored droplets; conidiogenous apparatus complexly and densely penicillate, (ter-) to more commonly quadri-verticillate, occasionally quinta-verticillate, with (2) 3(4) successive rows of short branches, the ultimate bearing an apical raw of phialides; whole penicilli dense, slightly divergent, 3045 µm high and 3565 µm wide; primary branches 68, cylindrical to clavate, slightly thick-walled, especially near base, apex commonly thin-walled, 9.01.0 x 4.56.0 µm (
= 12.7 x 5.1 µm), bearing apically 35 secondary branches; secondary branches 8.010.0 x 3.05.0 µm, cylindrical to slightly clavate, bearing 34 tertiary branches; tertiary branches 7.010.0 µm x 2.84.0 µm, cylindrical to slightly clavate, bearing either 23 quaternary branches or 46 phialides; quaternary branches cylindrical to slightly clavate, 57 x 23 µm; phialides cylindrical, finger-like, straight to slightly incurved inward penicillus, what is more pronounced for phialides located outside penicillus, apex narrower, dome-like, and with a small apical poroid conidiogenous locus, the wall of which progressively thickening by wall accumulation, 79 x 1.52.5 µm (
= 8.7 x 1.8 µm); conidia cylindrical to slightly ellipsoid, slightly beveled at the base, apex rounded, hyaline, thin-walled, without droplet inclusion, (5.5)6.07.0(7.5) x 1.01.5(1.8) µm, (
= 6.3 x 1.4 µm), R = (3.9)4.05.2(5.6), (
R = 4.6), accumulating in pale yellow to yellow, mucoid masses, globose at first then columnar; chlamydospores solitary, in groups of 24, or in short chains, sometimes branched or in small radiating clusters, individual cells globose to ellipsoid, thick-walled, brown, 1027 x 825 µm diam; teleomorph unknown (cross of MUCL 46554 x MUCL 46732 negative).
Substratum. On a dead, decaying leaf of an unidentified angiosperm in leaf litter;
Habitat. Rainforest litter.
Locality. So far known only from the type locality, French Guyana.
Holotype. FRENCH GUYANA: Cayenne Area, Matouri, Sentier dInterprétation de la Nature "Lamirande", from a dead, decaying leaf of an unidentified angiosperm in leaf litter, isolated by direct plating of a conidial mass, Feb 1994, C. Decock & V. Robert, # FG 1215, MUCL 46554, as two-week-old dried V8 culture and slides prepared from colonies on V8 agar (culture ex-Holotype MUCL 46554).
Paratype. same data, C. Decock & V. Robert, # FG 2157 = MUCL 46732, as two-week-old dried V8 culture (living strain exparatype MUCL 46732).
Remarks.
Gliocephalotrichum bacillisporum and G. longibrachium both have sterile stipe extensions directly subtending the conidiogenous penicillus (FIGS. 5, 6
, 11
) and produce narrowly ellipsoid to bacilliform conidia (FIGS. 1, 2
). They differ from each other in the length of the conidia and stipe extension (6.07.0 µm and 112184 µm long respectively in G. bacillisporum, and 8.010.5 µm and 240400 µm long in G. longibrachium). Gliocephalotrichum longibrachium produces large, brown, bulbilloid aggregates (FIG. 10
) whereas G. bacillisporum does not. Both species are similar to G. bulbilium, G. microchlamydosporum, and G. ohiense, in that the stipe extensions are all directly beneath the penicillus. However, the two new species differ from the three latter taxa in their bacilliform conidia (FIGS. 1, 2
) with an average L/W ratio greater than 4.5. Gliocephalotrichum bulbilium (FIG. 4
) and G. ohiense have ellipsoid conidia (Wiley and Simmons 1971
, Huang and Schmitt 1973
), with an average L/W ratio less or equal to 3 for G. bulbilium (respectively 2.6, 2.5 and 3.0 for MUCL 18575 [type strain2], MUCL 46552 and MUCL 46553). The type strain of G. ohiense at MUCL (39340) no longer sporulates, but according to published data (Huang and Schmitt 1973
), the L/W ratio does not exceed three. Gliocephalotrichum microchlamydosporum has commonly slightly sigmoid conidia (FIG. 3
), with an average L/W ratio of 3.2 (pers obs).
Gliocephalotrichum bacillisporum produces yellow conidial mucous drops, a feature only shared by G. ohiense (Huang and Schmitt 1973
), and yellow to pale copper droplets on the upper part of the stipe and sterile extensions, a feature not reported in other species. Ellis and Hesseltine (1962)
reported hyaline droplets on the stipe extensions of G. bulbilium, which were also observed in our two French Guyana isolates of that species, MUCL 46552 and MUCL 46553.
Gliocephalotrichum longibrachium is also differentiated by its long stipe extensions, the longest encountered so far in the genus, commonly reaching 400 µm in length, and the large bulbilloid aggregates (60200 x 50110 µm, FIG. 10
). Gliocephalotrichum bulbilium also produces bulbilloid structures but they are much smaller, not exceeding 110 µm in length, Wiley and Simmons 1971
).
In the present phylogenetic analyses based on ITS /5.8 S and ß-Tubulin sequence data, G. longibrachium and G. bacillisporum each form a well-defined terminal branche (FIGS. 16, 17
). Although the relationships of both taxa with other Gliocephalotrichum species are not clearly resolved, especially in the ITS-based phylogeny, both nested in a clade containing all species having the stipe extensions directly beneath the conidiogenous penicillus. This clade (FIG. 16
) also comprises two potentially undescribed, additional "taxa", so far of unknown morphology; these latter "taxa" are known from single ITS sequences (GenBank: AY 273314 and AY 273333), obtained from raw DNA extracted from leaf litter originating in Gabon, Africa (Roose-Amsaleg et al 2004
). The sequence AY 273314 proved to be very similar to that of G. ohiense (FIG. 16
) while the sequence AY 273333 in all probability represents an undescribed species.
Other Gliocephalotrichum found in French Guyana or identified in GenBank.
Gliocephalotrichum bulbilium J.J. Ellis & Hesselt., Bull. Torrey Bot Club 89:22, 1962.
Specimens examined FRENCH GUYANA: Cayenne Area, Matouri, Sentier dInterprétation de la Nature "Lamirande", from a dead, decaying leaf of an unidentified angiosperm in leaf litter, isolated by direct plating of a conidial mass, Feb 1994, C. Decock and V. Robert, FG 1159 = MUCL 46553 (as two-week-old dried cultures on V8 and CLA, living strain ex. FG1159 = MUCL 46553); same data, FG 1195 = MUCL 46552, (as two-week-old dried cultures on V8 and CLA, living strain ex FG1195 = MUCL 46552). Both isolates match the description of the species. They have ITS sequences identical to that of the type culture.
Ellis and Hesseltine (1962)
and Nicot (1967)
previously noted that the location and numbers of the stipe extensions of G. bulbilium varied according to the cultural conditions. This also was observed occasionally in our cultures of G. bulbilium on V8 media, the stipe extensions being sometimes absent or rising at variable distances below the conidiogenous penicillus. However, this unusual location of the stipe extensions occurred on abnormally long conidiophores rising mainly from aerial hyphae. It was not observed on CLA, where aerial hyphae are completely absent. This variation was not observed in G. bacillisporum or G. longibrachium.
| DISCUSSION |
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Gliocephalotrichum bacillisporum and G. longibrachium have a whorl of sterile hairs directly subtending the conidiogenous penicillus and are morphologically similar to G. bulbilium, type of the genus, G. microchlamydosporum and G. ohiense.
The position of the stipe extensions as regards the conidiogenous penicillus was used to segregate the genus into two informal groups (Wiley and Simmons 1971
). The present phylogenetic analyses, either based on ITS or ß-Tubulin sequence data, supported the present Gliocephalotrichum generic concept, that forms a well supported terminal clade in both analysis (bootstrap 99% and 94%, respectively, FIGS. 16, 17
). Furthermore, our phylogenetic inferences also tend to support the pertinence of the grouping as suggested by Wiley and Simmons (1971)
; both groups are resolved as two subclades that, although subject to the confirmation of the morphology of the unobserved African species, are homogeneous in regards to the stipe extensions position. The G. simplex and G. cylindrosporum clade is well supported by the ITS (bootstrap 91%) and moderately supported (bootstrap 59%) by ß-Tubulin based analysis. The G. bulbilium clade is not supported by our ITS based analysis (bootstrap 49%), that also did not resolve the relationships between species of this subclade. However, this second subclade (but with the unobserved African species missing) is moderately supported by the ß-Tubulin based phylogenetic inference (bootstrap 74%); in the latter, G. longibrachium appears isolated and basal to the clade, while G. bacillisporum appears related to G. bulbilium, G. microchlamydosporum and G. ohiense (bootstrap 72%).
| KEY TO THE SPECIES OF GLIOCEPHALOTRICHUM |
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| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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2 MUCL is a member of the Belgian Coordinated Collections of Microorganisms (BCCMTM). ![]()
2 The type strain of G. bulbilium at MUCL has degenerated and sporulates poorly, with aberrant conidiophores. ![]()
1 Corresponding author. E-mail: decock{at}mbla.ucl.ac.be
| LITERATURE CITED |
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Ellis JJ, Hesseltine CW. 1962. A new genus of Moniliales having penicilli subtended by sterile hairs. Bull Torr Bot Cl 89:2127.[CrossRef]
Glass NL, Donaldson G. 1995. Development of primer sets designed for use with PCR to amplify conserved genes for filamentous fungi. Appl Environm Microbiol 61:13231330.
Holmgren PK, Holmgren NL, Barnett LC. 1990. Index herbariorum. Part I: The herbaria of the world. New York Botanical Garden: 1693.
Huang LH, Schmitt JA. 1973. Gliocephalotrichum ohiense, a new species from Ohio soil. Mycologia 65:948952.[CrossRef]
Kirk PM, Cannon PF, David JC, Stalpers JA. 2001. Ainsworth and Bisbys dictionary of the fungi, 9th ed. Wallingford, UK: CABI Publishing. 624 p.
Meyer J. 1959. Moisissures du sol et des litières de la région de Yangambi (Congo belge). Pub INEAC, Ser Sc 75:1211.
Nicot J. 1967. Micromycètes de la Maboké: II.Gliocephalotrichum bulbilium Ell. Et Hess. Cahiers de la Maboké 5(2):7175.
Roose-Amsaleg C, Brygoo Y, Harry M. 2004. Ascomycetes diversity in soil-feeding termite nests and soils from a tropical rain forest. Environ Microbiol 6:462469.[CrossRef][Medline]
Rossman AY, Samuels GJ, Lowen R. 1993. Leuconectria clusiae gen. nov. and its anamorph Gliocephalotrichum bulbilium with notes on Pseudonectria. Mycologia 85:685704.[CrossRef]
Schoch CL, Crous PW, Wingfield MJ, Wingfield BD. 2000. Phylogeny of Calonectria and selected hypocrealean genera with cylindrical macroconidia. Stud Mycol 45:4562.
Swofford DL. 2002. PAUP: Phylogenetic Analysis Using Parsimony. 4.0b10. Laboratory of Molecular Systematic, Smithsonian Institute, Washington D.C.
Untereiner WA, Bonjean B, Decock C, Evrard P, de Frahan MH, Jamin N, Massart L, Nélissen L, Robert V, Bosschaerts M, Guissart F, De Brabandere J. 1998. MUCL Catalogue of Strains (Fungi-Yeast). 3rd ed.
Brussels, Belgium: Belgian Office for Scientific, Technical and Cultural Affairs, eds. Watanabe T, Nakamura K. 2005. Gliocephalotrichum microchlamydosporum and G. simplex in the Ryukyu Islands, Japan. Mycoscience 46:4648.[CrossRef]
White TJ, Bruns T, Lee S, Taylor J W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. San Diego, California: Academic Press. p 315322.
Wiley BJ, Simmons EG. 1971. Gliocephalotrichum, new combinations and a new species. Mycologia 63: 575585.[CrossRef]
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