Phylogeny and intercontinental distribution of the pneumocandin-producing anamorphic fungus Glarea lozoyensis

Figures & data

Table 1. Strains and sequences. GenBank entries with FJ accession codes are new sequences obtained during this work; those with DQ codes are from Baral et al. (2006).

				GenBank accession numbers
Strain code	Fungal species	Substrate/Host	Geographic origin	ITS	28S rRNA	α-actin
F-115887	Ciboria americana	Fruits of Quercus suber	Aracena, Huelva (Spain)	DQ431172	FJ005122	FJ005158
F-144619	Pezizella amenti	Female catkin of Salix atrocinerea	Montoria, Álava (Spain)	FJ005092	FJ005123	FJ005159
F-147481	Pezizella amenti	Female catkin of Salix atrocinerea	Canencia, Madrid (Spain)	FJ005093	FJ005124	FJ005160
HB 6884	Pezizella amenti	Previous year's catkins of Salix caprea	Baden-Württemberg, Tübingen (Germany)	FJ005094	FJ005125	FJ005161
F-148706	Cyathicula cacaliae	Apiaceae stems	Ontígola, Toledo (Spain)	FJ005107	FJ005126	FJ005174
F-165632	Cyathicula cacaliae	Apiaceae stems	Haro, La Rioja (Spain)	FJ005108	FJ005127	FJ005175
CBS 19762	Cyathicula coronata	Unknown	Lyon (France)	FJ005106	FJ005129	FJ005173
F-112249	Cyathicula culmicola	Unkown	Borbollón Reservoir, Cáceres (Spain)	FJ005121	FJ005130	FJ005187
F-171689	Cyathicula culmicola	Grass culm	Puente Arenas, Burgos (Spain)	FJ005117	FJ005131	-
F-171706	Cyathicula culmicola	Grass culm	Colmenarejo, Madrid (Spain)	FJ005118	FJ005132	FJ005185
F-171707	Cyathicula culmicola	Grass culm in Fraxinus forest	Colmenarejo, Madrid (Spain)	FJ005119	FJ005133	FJ005184
F-198769	Cyathicula culmicola	Herbaceous stems	Atalaya Forest Park, Ciudad Real (Spain)	FJ005120	FJ005134	FJ005186
F-169773	Cyathicula cyathoidea/cacaliae	Stems of Urtica dioica	San Millán de Juarros, Burgos (Spain)	FJ005102	FJ005128	FJ005169
F-171690	Cyathicula cyathoidea/cacaliae	Involucral scales of unidentified Compositae	Ortigosa de Cameros, La Rioja (Spain)	FJ005103	FJ005135	FJ005170
F-171698	Cyathicula cyathoidea/cacaliae	Stem of herbaceous plant	Colmenarejo, Madrid (Spain)	FJ005104	FJ005136	FJ005171
F-171699	Cyathicula cyathoidea/cacaliae	Fruits of Fraxinus angustifolia	Colmenarejo, Madrid (Spain)	FJ005105	FJ005137	FJ005172
F-155002	Cyathicula grex tomentosa	Flowering stem of Asphodelus sp.	Bachimaña, Huesca (Spain)	FJ005114	FJ005138	FJ005181
F-175201	Cyathicula pallida	Herbaceous stems	San Millán de Juarros, Burgos (Spain)	FJ005116	FJ005139	FJ005183
F-124806	Cyathicula cyathoidea	Dead stems of Urtica dioica	Piedrafita de Babia, León (Spain)	FJ005101	FJ005140	FJ005167
F-144616	Cyathicula cyathoidea	Stems of Atropa belladona	Peñacerrada, Álava (Spain)	FJ005098	FJ005141	FJ005165
F-155007	Cyathicula cyathoidea	Stems of Cirsium eryophorum	Panticosa, Huesca (Spain)	FJ005099	FJ005142	FJ005166
F-155008	Cyathicula cyathoidea	Stems of Cirsium eryophorum	Panticosa, Huesca (Spain)	FJ005100	FJ005143	FJ005168
F-155009	Cyathicula cyathoidea	Stems of Cirsium eryophorum	Panticosa, Huesca (Spain)	FJ005097	FJ005144	FJ005164
F-155011	Cyathicula cyathoidea	Stems of Urtica sp.	Panticosa, Huesca (Spain)	FJ005096	FJ005145	FJ005163
F-171704	Cyathicula cyathoidea	Dead stems of Epilobium sp.	San Millán de Juarros, Burgos (Spain)	FJ005095	FJ005146	FJ005162
F-144620	Cyathicula tomentosa	Dead culms	Izarra, Álava (Spain)	FJ005115	FJ005147	FJ005182
F-160870 (ATCC 20868)	Glarea lozoyensis	Pond water filtrates	Valle de Rio Lozoya, Madrid (Spain)	FJ005110	FJ005152	FJ005177
F-226836	Glarea lozoyensis	Leaf litter from steppe	El Calafate, Santa Cruz (Argentina)	FJ005111	FJ005149	FJ005178
F-226838	Glarea lozoyensis	Leaf litter from steppe	El Calafate, Santa Cruz (Argentina)	FJ005112	FJ005150	FJ005179
F-239379	Glarea lozoyensis	Hardwood forest soil	White Mountain National Forest, New Hampshire (USA)	FJ005113	FJ005151	FJ005180
IZ 0792	Grass endophyte	Holcus lanatus	Valvellidos, Cáceres (Spain)	FJ005109	FJ005148	FJ005176
HB 7054	Hymenoscyphus epiphyllus	Bark of Quercus sp.	Tübingen (Germany)	DQ431180	FJ005157	FJ005192
F-115882	Hymenoscyphus fructigenus	Fruits of Quercus suber	Aracena, Huelva (Spain)	DQ431171	FJ005153	FJ005190
F-137632	Hymenoscyphus immutabilis	Leaves of Ulmus sp.	Colmenarejo, Madrid (Spain)	DQ431174	FJ005154	FJ005189
F-115891	Hymenoscyphus serotinus	Branches of Fagus sylvatica	Riaza, Segovia (Spain)	DQ431173	FJ005155	FJ005191
F-110125 (BR020)	Hymenoscyphus tamaricis	Tamarix gallica debris	Alcalá de Henares, Madrid (Spain)	DQ431167	FJ005156	FJ005188

Table 2. Antimicrobial activity of four Glarea lozoyensis strains. The production media where some activity was detected is indicated in parentheses in each case. Codes for agar diffusion assays: A (clear inhibition zone >7 mm); B (clear inhibition zone < 7 mm or hazy inhibition zone >10 mm). See Vicente et al. (2009) for media formulations.

Strain code	Media used	S. aureus EPI167	H. influenzae MB5842	C. albicans MY1055	A. fumigatus MF5668
F-160870 (ATCC 20868)	CYS80, MMK2, MV8, SCAS, STP, SUPER, WHEAT, YES	A (MV8, YES)	A (MV8, YES)	A(all media)	A (MMK2, WHEAT)
F-226836	CMK, MMK2, MV8, SCAS, STP, SUPER, WHEAT, YES	_	A (MMK2)	A(all media)	_
F-226838	CMK, MMK2, MV8, SCAS, STP, SUPER, WHEAT, YES	B(CMK)	_	A(all media)	A (STP, CMK, SUPER)
F-239379	MMK2, MV8, SCAS, STP, SUPER, WHEAT, YES	A (MV8)	A (MMK2, MV8, SUPER)	A (MMK2, MV8, SCAS, SUPER, WHEAT)	B (MV8)

Figure 1. Developing conidiophores and conidia from four strains of Glarea lozoyensis. A. ATCC 20868=F-160870. B. F-226836. C. F-226838. D. F-239379. Scale bar = 20 μm.

Figure 2. Accumulation of conidial masses on aerial hyphae of Glarea lozoyensis. Strains were grown on YM medium one month at room temperature. A. ATCC 20868=F-160870. B. F-226836. C. F-226838. D. F-239379. Scale bar = 2 mm.

Figure 3. Identification of pneumocandin A₀ in fermentation extracts of strains of Glarea lozoyensis. A1. UV spectrum of pneumocandin A₀ with retention time of elution (4.78 min). A2. Positive ion mass spectrum of pneumocandin A0 (MW=1078 Da) at the same retention time. The combination of A1and A2 produce the fingerprint of pneumocandin A₀. See methods for LC–MS protocols. B1, C1, D1 and E1 are the UV spectra of extracts of the four strains at the corresponding retention time, and B2, C2, D2 and E2 are the positive ion mass spectra of the respective sample at the same retention time as for the UV spectrum. All UV spectra were similar to authentic pneumocandin A₀, except for E1 where the signal was near baseline. Similarly, all the positive ion mass spectra indicated pneumocandin A₀ was in all samples.

Figure 4. Phylogenetic tree of Glarea lozoyensis and related species generated by Bayesian analysis of combined ITS and 28S rRNA partial sequences. Ciboria americana was designated the outgroup. Clade credibility values are indicated at the branches. Colony morphology of G. lozoyensis and Cyathicula strains are mapped onto their corresponding branches.

Figure 5. Phylogenetic tree of Glarea lozoyensis and related species generated by Bayesian analysis of α-actin gene partial sequences. Clade credibility values are indicated at the branches.

Table 3. Antimicrobial activity of Cyathicula and Pezizella strains against a panel of bacteria and fungi. The media where activity was detected are indicated in parentheses in each case. Media formulations have been previously reported (Vicente et al. 2009). Codes for agar diffusion assays: A (clear inhibition zone >7 mm); B (clear inhibition zone < 7 mm or hazy inhibition zone >10 mm); C (hazy inhibition zone < 10 mm).

Strain code	Species	Media used	S. aureus EPI167	H. influenzaeMB5842	C. albicansMY1055	A. fumigatus MF5668
F-144619	P. amenti	MMK2
F-147481	P. amenti	MMK2
F-148706	Cy. cacaliae	MMK2, SUPER
F-165632	Cy. cacaliae	MMK2, SUPER, WHEAT			A (WHEAT)
CBS19762	Cy. coronata	MMK2, SUPER
F-112249	Cy. culmicola	MMK2, SUPER, MV8	B (MMK2)	A (MMK2, MV8)
F-171689	Cy. culmicola	MMK2, SUPER
F-171706	Cy. culmicola	CYS80, SUPER, YES	B (CYS80)	A (CYS80, YES)		C (CYS80)
F-171707	Cy. culmicola	SUPER
F-198769	Cy. culmicola	MMK2, SUPER
F-169773	Cy. cyathoidea/cacaliae	MMK2, SUPER
F-171690	Cy. cyathoidea/cacaliae	CYS80, SUPER		B (CYS80)
F-171698	Cy. cyathoidea/cacaliae	CYS80, MMK2, SUPER	B (CYS80)	B (CYS80)
F-171699	Cy. cyathoidea/cacaliae	SUPER
F-155002	Cy. grex tomentosa	MMK2, SUPER, SCAS		A (SCAS)
F-175201	Cy. pallida	CYS80, MMK2, SUPER
F-124806	Cy. cyathoidea	MMK2, SUPER, SCAS		A (SUPER, SCAS)
F-144616	Cy. cyathoidea	MMK2, SUPER
F-155007	Cy. cyathoidea	SUPER
F-155008	Cy. cyathoidea	CYS80, MMK2, MV8, SCAS, SUPER, WHEAT		A (WHEAT)
F-155009	Cy. cyathoidea	MMK2, SUPER
F-155011	Cy. cyathoidea	MMK2, SUPER
F-171704	Cy. cyathoidea	MMK2, SUPER
F-144620	Cy. tomentosa	MMK2, SUPER, SCY2
IZ 0792 (F-215687)	Grass endophyte	CYS80, MMK2, MV8, SCAS, SUPER, WHEAT	C (MV8)		A (all media)	B (MV8)

Figure 6. Structure of the echinocandins showing the common features and the radicals varying across the different molecular subtypes. Refer to Table 5 for R-group substitutions.

Table 4. Naturally occurring echinocandins and producing organisms.

Echinocandin type	Producer organism	Order	Reference
Echinocandins B-D	Aspergillus nidulans var. echinulatus, Aspergillus rugulosus, Emericella rugulosa, others	Eurotiales	Benz et al. (1974) Keller-Juslén et al. (1976) Traber et al. (1979)
Aculeacins A-G	Aspergillus japonicus var. aculeatus (=A. aculeatus)	Eurotiales	Mizuno et al. (1977) Satoi et al. (1977)
Mulundocandin Deoxymulundocandin	Aspergillus sydowii	Eurotiales	Roy et al. (1987) Mukhopadhyay et al. (1992)
Pneumocandin A0	Glarea lozoyensis Cryptosporiopsis spp.Pezicula spp.	Leotiales	Schwartz et al. (1989) Noble et al. (1991) Bills et al. (2009)
Other pneumocandins	Glarea lozoyensis	Leotiales	Schwartz et al. (1992) Petersen et al. (2001)
Sporiofungin	Cryptosporiopsis sp.	Leotiales	Dreyfuss et al. (1986)
Cryptocandin	Cryptosporiopsis cf. quercina	Leotiales	Strobel et al. (1999)
FR901379 (WF11899A) WF11899B-C	Coleophoma empetrii	Leotiales	Iwamoto et al. (1994) Hino et al. (2001)
FR220897 (WF14573B) FR220899 (WF14573A)	Coleophoma empetrii	Leotiales	Kanasaki et al. (2006b) Hino et al. (2001)
FR190293 (WF16616)	Tolypocladium parasiticum	Hypocreales	Kanasaki et al. (2006c) Hino et al. (2001)
FR227673 (WF22210)	Chalara sp.	Leotiales?	Kanasaki et al. (2006c) Hino et al. (2001)
FR209602-4 (WF738A-C)	Coleophoma crateriformis	Leotiales	Kanasaki et al. (2006a) Hino et al. (2001)

Table 5. Naturally occurring echincocandin subtypes and variations in lipophilic sidechains and substitutions. See Figure 5 for positions of R groups.

	Features and substitutions
Echinocandin subtype	R	R1	R2	R3	R4	R5	R6	R7	R8	R9
Echinocandin B Echinocandin C Echinocandin D	Linoleoyl Linoleoyl Linoleoyl	OH OH H	OH OH H	CH3	OH	OH H H	H	OH	CHOHCH3	CH3
Aculeacin A^+	Palmitoyl	OH	OH	CH3	OH	OH	H	OH	CHOHCH3	CH3
Mulundocandin Deoxymulundocandin	12-Methylmyristoyl	OH	OH	H	OH	OH H	H	OH	CH2OH	CH3
Pneumocandin A0* Pneumocandin A1 Pneumocandin A2 Pneumocandin A3 Pneumocandin A4 Pneumocandin B0 Pneumocandin B2	10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl 10,12-Dimethylmyristoyl	OH OH H OH H OH H	OH OH H H H OH H	CH3	OH H OH H H OH OH	OH OH OH H H OH OH	H	OH	CHOHCH2CONH2	CH3 CH3 CH3 CH3 CH3 H H
Sporiofungin	10,12-Dimethylmyristoyl	OH	OH	H	OH	OH	H	OH	CHOHCH2CONH2	CH3
Cryptocandin	Palmitoyl	OH	OH	CH3	OH	OH	H	OH	CH2CH2CONH2	CH2OH
Sulphate-containing echinocandins:
FR901379 (WF11899A) WF11899B WF11899C	Palmitoyl	OH	OH OH H	CH3	OH	OH H H	OSO3H	OH	CHOHCH2CONH2	CH3
FR209602 (WF738A) FR209603 (WF738B) FR209604 (WF738C)	Palmitoyl	OH	OH OH H	H	OH	H	OSO3H	OH	CHOHCH2CONH2	CH3 H CH3
FR220897 (WF14573B) FR220899 (WF14573A)	Palmitoyl	OH	OH	CH3	OH	H	OH	OSO3H	CHOHCH2CONH2	CH3 H
FR190293 (WF16616)	10,12-Dimethylmyristoyl	OH	OH	CH3	OH	OH	OSO3H	OSO3H	CHOHCH2CONH2	CH3
FR227673 (WF22210)	12,14-Dimethylpalmitoyl	OH	OH	CH3	OH	OH	OSO3H	OH	CHOHCH2CONH2	CH3
Notes: ^+Aculeacins B–G have been also reported in the literature (Satoi et al. 1977), but no structures are available.
* Other isomers of pneumocandins have been described where the 3-hydroxyproline (next to the hydroxyglutamine, R8) is replaced by a 4-hydroxyproline. Additional members of the pneumocandin subfamily have been reported by Petersen et al. (2001).

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