Diversity, Distribution, and Ecology of Fungi in the Seasonal Snow of Antarctica.

We characterized the fungal community found in the winter seasonal snow of the Antarctic Peninsula. From the samples of snow, 234 fungal isolates were obtained and could be assigned to 51 taxa of 26 genera. Eleven yeast species displayed the highest densities; among them, Phenoliferia glacialis showed a broad distribution and was detected at all sites that were sampled. Fungi known to be opportunistic in humans were subjected to antifungal minimal inhibition concentration. Debaryomyces hansenii, Rhodotorula mucilaginosa, Penicillium chrysogenum, Penicillium sp. 3, and Penicillium sp. 4 displayed resistance against the antifungals benomyl and fluconazole. Among them, R. mucilaginosa isolates were able to grow at 37 °C. Our results show that the winter seasonal snow of the Antarctic Peninsula contains a diverse fungal community dominated by cosmopolitan ubiquitous fungal species previously found in tropical, temperate, and polar ecosystems. The high densities of these cosmopolitan fungi suggest that they could be present in the air that arrives at the Antarctic Peninsula by air masses from outside Antarctica. Additionally, we detected environmental fungal isolates that were resistant to agricultural and clinical antifungals and able to grow at 37 °C. Further studies will be needed to characterize the virulence potential of these fungi in humans and animals.

1. Introduction

Antarctica is composed of special ecosystems that allow for the study of the taxonomy and ecology of resident life forms under extreme conditions [1]. Among the eukaryote organisms living in Antarctica, fungi have received special attention due their ability to colonize and survive in different Antarctic habitats/substrates. Most studies on fungi in Antarctica have focused on fungi living in terrestrial and marine environments, such as lakes [2,3], soils [4,5], historic woods [6], plants [7,8], and macroalgae [9,10]. In these Antarctic habitats, fungi are represented mostly by the taxa of the phyla Ascomycota and its anamorphs, followed by Basidiomycota, Mortierellomycota, and Chytridiomycota [11,12].

Snow habitats are ecosystems characterized by permanently low temperatures, low nutrient composition, and high levels of ultraviolet radiation at high altitudes [13]. Snow represents an important component of the cryosphere and occupies about 35% of the Earth’s surface during the Northern Hemisphere winter [14], covering over 99% of the 13.8 million km2 of the Antarctic continent. Antarctic seasonal snow, that is the snow deposited in the last winter season, can be considered a cryptic microhabitat for microorganisms directly related to the atmosphere, the precipitation of dust, and microbial cells, as well as other organic materials suspended in the air [14].

According to Kang et al. [15], falling snow greatly contributes to the deposition of airborne contaminants via the removal of atmospheric aerosols and the absorption of gas phase vapors. Industrial and agricultural activities have a great impact on these natural habitats due the dispersion of greenhouse gases, nitrogen compounds, and other synthetic chemicals, which, when released in high amounts, are dispersed in the atmosphere and transported by winds to remote areas of the planet, including the polar regions [16]. Previous studies have already demonstrated that several pesticides and semi-volatile organic compounds circulate and accumulate in remote ecosystems across the world, including high-elevation and high-latitude habitats [17,18,19]. Kang et al. [15] detected 22 persistent organic pollutants (POPs), identified as organochlorine pesticides (OCPs), in snow in Antarctica; among them, α-hexachlorocyclohexane (HCH), γ-HCH, and hexachlorobenzene (HCB) were frequently found in snow at different concentration ranges. The Antarctic Peninsula is close to South America, which is known for its intense agricultural and industrial activities and which employs the use of chemicals to combat plant and animal pests and diseases. These chemicals are then transported by winds to the Antarctic atmosphere to be then deposited in the snow by precipitation [16].

Though studies on the fungi found in snow in Antarctica are scarce, the presence of cold-tolerant cosmopolitan and psychrophilic endemic fungi living at the edge of life has been demonstrated [20,21,22,23,24], which are in contact with different compounds transported by the air and deposited in the snow, including pollutants with chemical structures similar to agricultural and clinical antifungal drugs. These pollutants may work as selective antimicrobial agents on Antarctic fungi living in snow. In this context, the present study aimed to characterize the fungal diversity present in the seasonal snow of the Antarctic Peninsula islands and investigate the possible resistance of species and strains against agricultural and clinical antifungal drugs.

2. Methods

2.1. Snow Sampling, Concentration, and Fungal Isolation

Seasonal snow samples (snow deposited during the last winter season) were collected in six different regions of the Antarctic Peninsula and in the South Shetland Islands between November and December 2015 (Table S1; Figure S1). Approximately 10 kg of snow from the top, middle, and base layers (10 cm above soil) were collected in triplicate using a sterile plastic scoop, and these were stored in sterilized 20 L plastic bags (Figure S2). The samples were thawed in a microbiology laboratory on board the Brazilian polar ship Admiral Maximiano over a period of 12 h. After snow thaw, a mix of 10 L of water were obtained, and the physicochemical properties of the melted snow sample were measured using the Hanna multi-parameter probe HI 9828 (manufacture Hanna instruments, Woonsocket, RI, USA). A total of 1.5 L of melted snow was filtered through a membrane with a diameter of 47 mm (Millipore, Billerica, MA, USA) in triplicate experiments. The membranes were inoculated on a Sabouraud agar (Acumedia Lab, Lansing, MI, USA) and on a minimal Medium (6.98 g K2HPO4; 5.44 g KH2PO4; 1.0 g (NH4)2SO4; 1.1 g MgSO4 7H2O, 0.25 g peptone, 20 g agar per L, pH 6.8 ± 0.2), containing 200 µg mL−1 of chloramphenicol (Sigma-Aldrich, Saint Louis, MO, USA), and these membranes were incubated at 10 °C for 30 days. The fungi were purified in fresh Petri dishes containing the Sabouraud agar and deposited in the Collection of Microorganisms and Cells of the Universidade Federal de Minas Gerais, Brazil, under the code UFMGCB in cryotubes at −80 °C and in distilled sterilized water [25] at room temperature.

2.2. Fungal Identification

The protocol for the DNA extraction of the cultivable fungi was conducted according to that procedure of Rosa et al. [1]. For the filamentous fungi, the internal transcribed spacer (ITS) region was amplified using ITS1 and ITS4 primers [26], which were amplified as described by Rosa et al. [1]. The amplification of the β-tubulin [27] and ribosomal polymerase II genes (RPB2) [28] were utilized for fungal taxa with low intraspecific variation according to protocols established by Gonçalves et al. [29]. The yeasts were identified according to methods proposed by Kurtzman et al. [30]. Yeast molecular identities were confirmed as described by Lachance et al. [31]. The fungal sequences were analyzed with the program MEGA7 [32]. All sequences were compared with type and/or annotated fungal sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (BLAST at http://www.ncbi.nlm.nih.gov) [33]. Representative consensus sequences of the fungal taxa were deposited into the GenBank database (Table 1). To achieve species-rank identification based on ITS, β-tubulin data, and ribosomal polymerase B2, the consensus sequence was aligned with all sequences from related species retrieved from the NCBI GenBank database using BLAST [34]. The information about fungal classification generally followed the databases of dictionary Kirk et al. [35], the website MycoBank (http://www.mycobank.org) [36], and the Index Fungorum (http://www.indexfungorum.org) [37].

Table 1

Fungi obtained from Antarctic snow segments and identified by sequence comparison with the BLASTn match with the NCBI GenBank database.

Region	Snow Segment	Density (CFU L−1)	UFMGCB a	Top BLAST Search Results (GenBank Accession Number)	Query Cover (%)	Identity (%)	N° of bp Analyzed	Proposed Taxa (GenBank acc. n°)
Deception Island (62° 58′ 55.6″ S; 60° 33′ 11.5″ W)	1	>300	18371	Leucosporidium fragarium (NG058330) e	100	99	831	Leucosporidium fragarium (MN0655470) j
		36.66	12598	Penicillium cavernicola (MH862709) b	100	100	466	Penicillium sp. 1 (MK981319) g
		10	12377	Mortierella elongatula (MH859811) b	100	96	483	Mortierella sp. (MK889363) g
		3.3	18377	Rhodotorula mucilaginosa (MH636067) e	100	99	495	Rhodotorula mucilaginosa (MN0655160) j
	2	>300	18378	Phenoliferia glacialis (LC203727) e	100	100	815	Phenoliferia glacialis (MN065548) j
		9.9	12592	Cladosporium phyllactiniicola (MH863929) b	100	100	432	Cladosporium sp. 1 (MK981325) g
		6	12376	Antarctomycespsychrotrophicus (MH874317) b	100	100	432	Antarctomycespsychrotrophicus (MK889356) g
		4	12597	Thelebolus balaustiformis (NR159056) b	99	100	443	Thelebolus balaustiformis (MK889369) g
		2	12375	Penicillium cavernicola (MH862709) bPenicillium albocoremium (KU896812) cPenicillium solitum (KU904363) d	10099100	10097100	430405689	Penicillium solitum (MK981306) g (MN017378) h (MN056972) i
	3	>300	18368	Leucosporidium golubevii (KY108283) e	99	98	670	Leucosporidium sp. (MN065549) j
		41.3	12371	Leohumicola minima (NR121307) b	100	91	474	Helotiales sp. 1 (MK889360) g
		4.6	12428	Thelebolus balaustiformis (NR159056) b	100	100	487	Thelebolus balaustiformis (MK889370) g
		5.3	12741	Penicillium cavernicola (MH862709) b	100	99	468	Penicillium sp. 1 (MK981334) g
		4	12372	Penicillium rubens (NR111815) bPenicillium glycyrrhizacola (KF021538) c	10099	10098	464398	Penicillium sp. 2 (MK981307) g (MN0173379) h
		2	12380	Penicillium tardochrysogenum (MH865983) bPenicillium chrysogenum (AY495981) c	100100	9997	470377	Penicilliumchrysogenum (MK981309) g (MN045349) h
		1	12373	Penicillium roqueforti (MH855127) bPenicillium carneum (AY674386) cPenicillium psychrosexualis (KU904362) d	10096100	1009799	463321554	Penicillium cf. psychrosexualis (MK981308) g (MN045348) h (MN379931) i
King George Island (62° 06′ 211″ S; 058° 27’ 627″ W)	1	>300	18388	Phenoliferia psychrophenolica (KY108774) e	100	100	551	Phenoliferia psychrophenolica (MN065550) j
		>300	18404	Phenoliferia glacialis (NG058369) e	94	99	555	Phenoliferia cf. glacialis (MN065514) j
		>300	18386	Phaeococcomyces mexicanus (NG058078) e	100	98	490	Phaeococcomyces sp. (MN065551) j
		>300	18400	Rhodotorula mucilaginosa (KY218728) e	100	99	622	Rhodotorula mucilaginosa (MN065517) j
		2.66	12430	Penicillium tardochrysogenum (MH865983) b	100	100	476	Penicillium sp. 3 (MK981320) g
		2.66	12388	Thelebolus globosus (MH862951) b	100	99	499	Thelebolus globosus (MK889371) g
		13.3	12391	Alternaria multiformis (MH862776) b	100	100	447	Alternaria multiformis (MK889355) g
		4.6	12395	Penicillium rubens (NR111815) bPenicillium chrysogenum (JF909937) d	9898	9999	469936	Penicillium chrysogenum (MK981310) g (MN379932) i
		3.5	12390	Neomicrosphaeropsis italica (NR158247) b	100	98	503	Neomicrosphaeropsis cf. italica (MK889364) g
		2.6	12397	Penicilliumtardochrysogenum (MH865983) bPenicillium chrysogenum (JF909937) d	100100	99100	467920	Penicillium chrysogenum (MK981311) g(MN379933) i
		3.3	12389	Ijuhya vitelina (NR154100) b	100	88	511	Bionectriaceae sp. 1 (MK889358) g
		1.3	12393	Thelebolus balaustiformis (NR159056) b	100	100	474	Thelebolus balaustiformis (MK889374) g
		1.3	18399	Mrakia gelida (KY108585) e	98	99	554	Mrakia sp. (MN065528) j
	2	>300	18387	Hamamotoa singularis (KY107777) e	100	99	435	Hamamotoa singularis (MN065527) j
		>300	18483	Rhodotorula mucilaginosa (KY218730) e	100	100	673	Rhodotorula mucilaginosa (MN065518) j
		>300	18391	Hamamotoa singularis (KY107777) e	100	97	551	Hamamotoa sp. (MN065526) j
		>300	18478	Vishniacozyma victoriae (LC203739) e	100	99	642	Vishniacozyma victoriae (MN0655520) j
		88	18381	Glaciozyma antarctica (NG057664) e	100	100	553	Glaciozyma antarctica (MN065525) j
		7.3	12734	Ijuhya vitelina (NR154100) b	100	87	453	Bionectriaceae sp. 1 (MK889359) g
		6	12385	Penicillium cavernicola (MH862709) bPenicillium glandicola (KU896814) c	10093	10082	465407	Penicillium sp. 1 (MK981312) g (MN045350) h
		1.3	12594	Cladosporium phyllactiniicola (MH863929) b	100	100	465	Cladosporium sp. 1 (MK981333) g
		1	12384	Neomicrosphaeropsis italica (NR158247) b	100	98	482	Neomicrosphaeropsis cf. italica (MK889365) g
	3	>300	18395	Phenoliferia psychrophenolica (KY108774) e	100	100	565	Phenoliferia psychrophenolica (MN065553) j
		>300	18393	Rhodotorula mucilaginosa (MF927654) e	100	99	546	Rhodotorula mucilaginosa (MN065519) j
		>300	18403	Glaciozyma antarctica (LC202043) e	100	100	501	Glaciozyma antarctica (MN065524) j
		>300	18493	Phenoliferia glacialis (LC203727) e	100	100	590	Phenoliferia glacialis (MN0655540) j
		8	12386	Penicillium cavernicola (MH862709) bPenicillium albocoremium (KU896812) c	10095	10097	425370	Penicillium sp. 1 (MK981313) g (MN045351) h
		2.6	12735	Penicillium caseifulvum (MH862722) b	100	100	373	Penicillium sp. 4 (MK981321) g
		4	12387	Penicillium commune (NR111143) bPenicillium palitans (KU904360) d	100100	100100	466577	Penicillium palitans (MK981314) g (MN379934) i
		4	12582	Cladosporiumasperulatum (MH863916) b	100	100	475	Cladosporium sp. 2 (MK981326) g
		4	12740	Leohumicola minima (NR121307) b	100	91	482	Helotiales sp. 1 (MK889361) g
Trinity Peninsula (63° 24’ 45.6″ S; 57° 00’ 164″ W)	1	>300	18481	Phenoliferia glacialis (LC203723) e	100	99	678	Phenoliferia glacialis (MN065531) j
		>300	18417	Bannozymayamatoana (AF189896) e	100	99	563	Bannozymayamatoana (MN065523) j
		>300	18489	Vishniacozyma victoriae (LC203739) e	100	100	559	Vishniacozyma victoriae (MN065555) j
		>300	18480	Cystobasidium pallidum (NG059006) e	100	93	529	Cystobasidium sp. (MN065556) j
		110	18410	Phenoliferia psychrophenolica (KY108774) e	100	99	543	Phenoliferia psychrophenolica (MN065530) j
		46	12405	Penicillium caseifulvum (MH862722) bPenicillium longisporum (KJ834467) c	10088	10098	495336	Penicillium sp. 4 (MK981316) g (MN075199) h
		31.3	18408	Dioszegia hungarica (KY107643) e	100	99	488	Dioszegia hungarica (MN065557) j
		34	18423	Dioszegia crocea (KX096670) e	100	100	448	Dioszegia crocea (MN065520) j
		12.6	18411	Genolevuria bromeliarum (NG058291) eGenolevuria bromeliarum (NG058291) b	9861	9495	542860	Genolevuria sp. (MN065564) j (MN065510) g
		6	12429	Penicillium oregonense (MH865652) bPenicillium glandicola (KU896814) c	10093	9682	419407	Penicillium sp. 5 (MK981315) g (MN045352) h
		2	12404	Thelebolus balaustiformis (NR159056) b	99	100	488	Thelebolus balaustiformis (MK889373) g
	2	>300	18413	Rhodotorula mucilaginosa (KP223715) e	100	100	596	Rhodotorula mucilaginosa (MN065512) j
		>300	18425	Phenoliferia psychrophenolica (KY108774) e	100	100	508	Phenoliferia psychrophenolica (MN065535) j
		106	12427	Penicillium tardochrysogenum (NR1383081) bPenicillium chrysogenum (JF909937) cPenicillium chrysogenum (AY495981) d	100100100	99100100	504651342	Penicillium chrysogenum (MK981317) g (MN075200) h (MN379935) i
		8.6	18426	Debaryomyces hansenii (KX981201) e	100	100	712	Debaryomyces hansenii (MN065522) j
		2.6	18416	Leucosporidium muscorum (KY108280) eLeucosporodium creatinivorum (KC455908) b	10099	99100	599654	Leucosporidium sp. (MN121386) e (MN121387) g
		1.3	18422	Candida sake (KY102375) e	100	100	656	Candida sake (MN065558) j
	3	>300	18414	Phenoliferia glacialis (LC203723) e	100	99	653	Phenoliferia cf glacialis (MN0655320) j
		>300	18415	Phenoliferia psychrophenolica (KY108774) e	100	99	499	Phenoliferia psychrophenolica (MN065533) j
		5.3	12402	Penicillium tardochrysogenum (MH865983) b	100	99	419	Penicillium sp. 3 (MK981335) g
		2.6	12732	Cladosporium phyllactiniicola (MH863929) b	100	100	428	Cladosporium sp. 1 (MK981327) g
Arctowski Peninsula (64 45 ’0″S; 62 25’ 0″ W)	1	>300	18430	Phenoliferia psychrophenolica (KY108774) e	100	100	533	Phenoliferia psychrophenolica (MN065534) j
		120	18428	Genolevuria amylolytica (NG057728) e	100	99	455	Genolevuria amylolytica (MN065559) j
		3.65	12432	Antarctomycespsychrotrophicus (MH874317) b	100	100	435	Antarctomycespsychrotrophicus (MK889357) g
		2	12433	Cladosporiumphaenocomae (MH865096) bCladosporium longisporum (KJ834467) c	10094	10097	435355	Cladosporium sp. 3 (MK981324) g (MN075202) h
		2	18436	Debaryomyces hansenii (LC219506) e	100	100	546	Debaryomyces hansenii (MN065521) j
	2	>300	18431	Phenoliferia psychrophenolica (KY108774) e	100	100	571	Phenoliferia psychrophenolica (MN065536) j
		>300	18477	Phenoliferia glacialis (LC203727) e	100	100	510	Phenoliferia glacialis (MN065537) j
		15.33	12431	Cladosporium phyllactiniicola (MH863929) b	100	100	462	Cladosporium sp. 1 (MK981330) g
		7.3	12408	Cladosporiumvariabile (MH863132) b	100	100	481	Cladosporium sp. 3 (MK981331) g
		1.3	12587	Penicillium caseifulvum (MH862722) b	100	100	488	Penicillium sp. 4 (MK981323) g
		1	12407	Thelebolusbalaustiformis (NR159056) b	98	100	439	Thelebolus cf. balaustiformis (MK889366) g
Snow Island (62° 46’ 300″ S; 06° 15’ 240″ W)	1	>300	18455	Phenoliferia psychrophenolica (KY108774) e	100	100	527	Phenoliferia psychrophenolica (MN065538) j
		157.3	18439	Mrakia gelida (LC203698) e	100	100	618	Mrakia gelida (MN065529) j
		19.95	18441	Phenoliferia glacialis (LC203727) e	100	100	631	Phenoliferia glacialis (MN065539) j
		28	18475	Holtermanniella wattica (LC203693) e	100	100	458	Holtermanniella wattica (MN065560) j
		6.3	18443	Rhodotorula mucilaginosa (KY218730) e	100	100	543	Rhodotorula mucilaginosa (MN065513) j
		3.3	18491	Genolevuria bromeliarum (NG058291) e	96	98	542	Genolevuria sp. (MN065563) j
		1.3	18440	Kabatiella bupleuri (JN886792) eKabatiella bupleuri (JN886792) b	98100	9898	420577	Kabatiella cf. bupleuri (MN065565) j (MN655511) g
		2	12590	Clathrosphaerina zalewskii (MH856474) b	100	91	423	Hyaloscyphaceae sp. (MK889362) g
	2	>300	18445	Phenoliferia psychrophenolica (KY108774) e	100	100	553	Phenoliferia psychrophenolica (MN065540) j
		>300	18484	Phenoliferia glacialis (LC203727) e	100	100	571	Phenoliferia glacialis (MN065561) j
		0.6	18448	Vishniacozyma victoriae (LC203739) e	100	99	599	Vishniacozyma victoriae (MN065562) j
		2	12434	Cladosporiumtenuissimum (MH864840) b	100	99	471	Cladosporium sp. 4 (MK981332) g
		2	12411	Penicillium tardochrysogenum (NR1383081) bPenicillium chrysogenum (AY4959811) c	100100	10099	523416	Penicillium chrysogenum (MK981318) g (MN075201) h
	3	>300	18437	Phenoliferia glacialis (LC203727) e	100	100	571	Phenoliferia glacialis (MN065541) j
		>300	18456	Phenoliferia psychrophenolica (FN550378) e	100	99	514	Phenoliferia psychrophenolica (MN065542) j
		6.65	12739	Pseudogymnoascus destructans (EU88492) b	100	99	453	Pseudogymnoascus destructans (MK889375) g
		2.66	12412	Thelebolus globosus (KX576510) b	100	99	410	Thelebolus globosus (MK889367) g
		2.6	12589	Pseudogymnoascus verrucosus (KJ755525) b	100	100	400	Pseudogymnoascus verrucosus (MK889376) g
		4	12588	Pseudogymnoascus pannorum (MH861038) b	99	98	414	Pseudogymnoascus sp. (MK889378) g
Robert Island (62 °37’ 941″ S; 059° 70’ 400″ W)	1	>300	18470	Phenoliferia psychrophenolica (FN550378) e	100	100	525	Phenoliferia psychrophenolica (MN065543) j
		>300	18461	Phenoliferia glacialis (KX773532) e	100	100	506	Phenoliferia glacialis (MN065544) j
		3.3	12420	Cladosporium phyllactiniicola (MH863929)b	98	100	430	Cladosporium sp. 1 (MK981328) g
		4	12419	Thelebolusbalaustiformis (NR159056) b	100	99	511	Thelebolus cf. balaustiformis (MK889368) g
		1.3	12423	Pseudocamarosporium africanum (NR154294) bPseudocamarosporium africanum (JX496368) c	9990	10099	528422	Pseudocamarosporium africanum (MK889379) g (MN075203) h
	2	>300	18464	Phenoliferia psychrophenolica (KY108774) e	100	99	538	Phenoliferia psychrophenolica (MN065545) j
		2.66	12414	Thelebolus balaustiformis (NR159056) b	100	100	425	Thelebolus balaustiformis (MK889372) g
		1.33	12413	Cladosporium phyllactiniicola (MH863929) b	100	100	469	Cladosporium sp. 1 (MK981329) g
		1.3	12733	Penicillium rubens (NR111815) b	98	100	434	Penicillium sp. 2 (MK981322) g
	3	>300	18472	Phenoliferia psychrophenolica (KY108774) e	100	100	528	Phenoliferia psychrophenolica (MN065546) j
		>300	18490	Rhodotorula mucilaginosa (KY218730) e	100	100	605	Rhodotorula mucilaginosa (MN065515) j
		14	12416	Antarctomyces pellizariae (KX576510) b(KX790790) c (KY100007) d	100	100	545	Antarctomyces pellizariae (KX576510) g (KX790790) h (KY100007) i
		1	12438	Pseudogymnoascus verrucosus (KJ755525) b	98	99	488	Pseudogymnoascus cf. verrucosus (MK889377) g

a UFMGCB, Culture of Microorganisms and Cells from the Federal University of Minas Gerais. Taxa subjected to BLAST analysis based on the b ITS, c β-tubulin, d Polymerase II, and e D1/D2 regions for the elucidation of taxonomic positions. f Taxonomic position suggested. Sequences of g ITS, h β-tubulin and/or i Polymerase II and j D1/D2 sequences deposited in GenBank database.

2.3. Morphological Characterization

After sequence analysis, Penicillium species with unknown taxonomic positions were characterized according to their macroscopic properties (colony color, texture, reverse color, border type, sporulation, and the production of yellow soluble pigments). The diameters of the colonies were observed on yeast extract agar (YES, yeast extract 3 g, peptone 5 g, agar 20 g per L) and Czapek yeast extract agar (CYA, Himedia, Mumbai, India) media at 25 and 30 °C, respectively, for 14 days [38]. The resulting colors were graded according to the specifications proposed by Kornerup and Wanscher [39].

2.4. Diversity, Richness, Dominance, and Distribution

The diversity, richness, and evenness indices of (i) Fisher’s α, (ii) Margalef’s, and (iii) Simpson’s, respectively, were calculated to estimate the fungal assembles diversity in Antarctic snow. In addition, the distribution among the fungal assemblages were determinates by Sorensen (QS) and Bray-Curtis (B) indices. All indices were calculated with 95% confidence and a bootstrap of 1.000 iterations using the program PAST, version 1.90 [40]. Additionally, a Venn diagram was produced according to the work of Bardou et al. [41] to illustrate the distribution of the fungal assemblages of the seasonal snow Antarctic.

2.5. Fungal Growth Responses to Temperature

Fungi known to be opportunistic in humans were grown on a Sabouraud agar at 10, 15, 20, 25, 30, 35, and 37 °C on Petri dishes to determine their growth profile at different temperatures. The filamentous fungi were inoculated by transferring blocks (4 mm2) from 10-day-old pre-cultures grown at 10 °C. Microtiter plates were incubated in triplicate for 7 days. Yeast was cultured on YM for 72 h at 10 °C. Then, 10 µL of cells with a turbidity of 0.5 on the MacFarland scale (1.5 × 106 cells mL−1 at 70% transmittance) were inoculated at the center of the Petri dishes containing Sabouraud media. All plates were incubated in triplicate for 14 days to detect the presence or absence of growth.

2.6. Minimum Inhibitory Concentration (MIC) of Antifungal Drugs Benomyl, Fluconazole, and Amphotericin B

Fungi known to be opportunistic in humans were subjected to minimum inhibitory concentration (MIC) determination for the antifungal drugs benomyl (Sigma, Steinheim, North Rhine-Westphalia, Germany) and fluconazole (Sigma, Saint Louis, MO, USA). Isolates with an MIC ≥ 64 µg/mL for fluconazole that also exhibited growth at 37 °C were subjected to MIC determination for the antifungal drug amphotericin B (Sigma, Saint Louis, MO, USA). The MIC protocol was performed using a modified version of the method used for yeast [42] and filamentous fungi [43] in a Roswell Park Memorial Institute (RPMI-1640) medium (INLAB, Diadema, SP, Brazil) using 96-well microtiter plates. The yeast cells and fungal spores were treated with benomyl concentrations ranging from 0.078125 to 40 µg mL−1 [44], fluconazole from 0.062 to 64 µg mL−1, and amphotericin B from 0.007 to 8 µg mL−1. Microtiter plates containing spores and yeast cells were incubated at 25 °C for 48 h for benomyl and fluconazole and at 37 °C for 48 h for amphotericin B. All MIC assays were performed in duplicate.

3. Results

3.1. Snow Fungal Community

From the different snow samples, 234 fungal isolates were obtained and identified as 51 taxa of 26 genera (Table 1). Among the yeast species identified, Bannozyma yamatoana, Cystobasidium pallidum, Glaciozyma antarctica, Leucosporidium fragarium, Leucosporidium golubevii, Phenoliferia glacialis, Phenoliferia psychrophenolica, Phaeococcomyces sp., Rhodotorula mucilaginosa, Hamamotoa singularis, and Vishniacozyma victoriae displayed the highest densities (≤300 CFU L−1). In contrast, filamentous fungi taxa showed the lowest density values. Twenty-nine fungi (56.8%) occurred in low densities (≤50 CFU L−1); these represented singletons and were considered rare taxa within the seasonal snow fungal community. Twenty-two taxa showed low molecular similarities or inconclusive taxonomic definitions when compared with the sequences of known fungi deposited in the GenBank database. Among them, Bionectriaceae sp., Cystobasidium sp., Genolevuria sp., Helotiales sp., Galactomyces sp., and Hyaloscyphaceae sp. showed the lowest query coverage and identity percentages, and these may represent new species.

3.2. Diversity and Distribution

The seasonal snow fungal community showed high values of diversity (Fisher α), richness (Margalef’s), and dominance (Simpson’s) (Table 2). The highest ecological indices were found in the snow of King George Island, followed by Snow Island, Trinity Island, Deception Island, Robert Island, and the Arctowski Peninsula. In addition, the similarities between the fungal assemblages of the different snow segments of each site showed that, except for those of King George Island, Segments 2 and 3 were more similar than Segment 1 (Figure S3). Eleven yeast species displayed the highest densities; among them, Phenoliferia glacialis showed a broad distribution and was detected at all sites that were sampled (Figure S4).

Table 2

Physicochemical parameters of melted snow sampled in different sites of Antarctic Peninsula and diversity indices fungal.

Parameters/Diversity Indices/Density	Sites Sampled
	Deception Island	King George Island	Trinity Peninsula	Arctowski Peninsula	Snow Island	Robert Island
Conductivity (µS cm−1)	50	173	20	28	52	53
Resistivity (MΩ cm−1)	0.024	0.006	0.047	0.023	0,026	0.039
Total dissolved solids (ppm)	24	86	11	14	14.6	27
Oxidation Reduction Potential (mV)	190.9	310	256.8	255.9	361.2	230.2
pH	6.76	6.3	6.74	6.24	6.1	6.5
Salinity (ppt)	0.02	0.08	0.01	0.01	0.02	0.02
Number of taxa	16	31	21	11	20	14
Fisher α	2.69	4.63	3.09	1.71	3.17	2.06
Margalef’s	2.16	3.65	2.52	1.44	2.55	1.73
Simpson’s	0.74	0.92	0.90	0.74	0.84	0.84

3.3. Opportunistic Virulence Potential

Five isolates of R. mucilaginosa, one of P. chrysogenum, Penicillium sp. 3, and Penicillium sp. 4 showed capacity for resistance to the clinical antifungal drug fluconazole at 25 °C. Two isolates of Debaryomyces hansenii were resistant to benomyl but susceptible to fluconazole (Table 3). In addition, isolates of R. mucilagina displayed MIC values of 0.03 and 0.125 µg mL−1 against amphotericin B at 37 °C (Table 3). However, we did not detect a clear correlation between the antifungal benomyl, used in agriculture, with the cross-resistance of fungi against fluconazole and amphotericin B, used in clinical therapy.

Table 3

The minimal inhibitory concentration of benomyl, fluconazole, and amphotericin B against fungi isolated from seasonal snow of Antarctica.

Fungi	UFMGCB a	MIC b at 25 °C		MIC b at 37 °C d
		Benomyl	Fluconazole	Amphotericin B
Debaryomyces hansenii	18426	40	8	−
D. hansenii	18436	40	4	−
Rhodotorula mucilaginosa	18377	0.3125	64	0.03
R. mucilaginosa	18400	0.3125	64	0.03
R. mucilaginosa	18393	0.3125	64	0.03
R. mucilaginosa	18413	0.3125	64	0.03
R. mucilaginosa	18443	0.3125	64	0.125
Penicillium chrysogenum	12380	0	32	−
P. chrysogenum	12427	0	64	−
Penicillium sp. 3	12430	2.5	64	−
Penicillium sp. 2	12733	2.5	1	−
Penicillium sp. 4	12405	2.5	64	−

a UFMGCB, Culture of Microorganisms and Cells from the Federal University of Minas Gerais. b MIC, Minimal inhibitory concentration at µg mL−1. d for selected fungi with MIC ≥ 64 µg mL−1 that were able to grow at 37 °C. 0, Absence of growth. −, Fungi incapable to grow at 37 °C.

4. Discussion

4.1. Taxonomy, Diversity, and Distribution

In the winter seasonal snow of the Antarctic Peninsula, we detected a rich fungal community. Within this community, the yeasts B. yamatoana, C. pallidum, G. antarctica, L. fragarium, L. golubevii, P. glacialis, P. psychrophenolica, R. mucilaginosa, H. singularis, and V. victoriae, all of which are known to occur in tropical, temperate, and polar environments, were found at high densities. Generally, in extreme environments, fungal assemblages are dominated by Ascomycota.

Rhodotorula includes pigmented yeast species that are found worldwide and can abundantly grow in extreme environments [45,46]. Rhodotorula mucilaginosa is ubiquitous and is present in different habitats and substrates, including cold and extreme environments [47]. In Antarctica, R. mucilaginosa is commonly isolated from terrestrial and marine substrates [48,49]. Despite the extensive snow cover of the Antarctic continent, our study represents the first report on the high density of R. muscilagionsa in snow samples of different sites in Antarctica.

Of all 51 taxa found in the Antarctic snow, only Phenoliferia glacialis (a synonym of Rhodotorula glacialis) was isolated from all sites sampled in this work and with high densities. Phenoliferia glacialis is a psychrophilic yeast that exhibits high metabolic versatility, and together with P. psychrophenolica (Rhodotorula psychrophenolica), was originally obtained from the Etendard Glacier in France and the Stubaier Glacier and cryoconite in Austria [46]. Recently, Ferreira et al. [50] isolated P. glacialis and P. psychrophenolica from the leaves of Deschampsia antarctica, mosses, and different biofilms present in the Antarctic Peninsula. Cystobasidium pallidum (Rhodotorula pallida) was obtained from different cold environments, such as the rhizosphere in Russia [51] and the seawater of the Indian Ocean [52]. In Antarctica, C. pallidum was detected in snow, rocks, and the Ross Dependency in the Antarctic Peninsula [53,54].

Vishniacozyma victoriae (a synonym of Cryptococcus victoriae) was first described in Antarctica [55] but was subsequently detected in penguin guano, soil, sediment, freshwater samples, the rhizosphere of D. antarctica [48], macroalgae [10], lichens [56], and as endophytes of D. antarctica and Colobanthus quitensis [57]. In addition, V. victoriae was detected in environments outside of Antarctica, such as soil and rhizosphere soil in Korea [58], seawater in Portugal [59], the gut of the insect Chrysoperla rufilabris in the USA [60], an industrial malting area and indoor air in Finland [61,62], in glacial ice from the Arctic [63], in a dry meat processing factory in Norway [64], and in the Italian Alps [65,66]. Garcia et al. [67] proposed that V. victoriae represents a generalist species with the ability to tolerate different stressful environments. Antony et al. [22] previously reported V. victoriae in the snow of Victoria Land, Antarctica.

The Glaciozyma antarctica species represents a re-classification of Leucosporidium antarcticum, originally described by Turchetti et al. [68]. In Antarctica, G. antarctica was previously isolated from Antarctic marine waters [69], soils around to Lake Fletcher, Lichen and Taylor Valleys, and on dead sponge [68], seawater of the Weddell Sea near Joinville Island, in the soils of South Victoria Land and Lake Fletcher [66], and in the seawater of the Northern Antarctic Peninsula [70].

Leucosporidium fragarium was obtained from the melt water river of the Frias Glacier of Mount Tronador [71,72]. Leucosporidium golubevii was isolated from the river water in the north of Portugal [73] and as endophytic fungus present in leaves of C. quitensis [57]. Bannozyma yamatoana (Bensingtonia yamatoana) was reported in Antarctica, present in soil and ornithogenic soil [48], and associated with thalli of lichen Usnea antarctica [56].

Generally, in extreme environments fungal communities are dominated by Ascomycota filamentous fungi [74]. However, our results demonstrate that the basidiomycetous yeast assemblage was the dominant form in the winter seasonal snow of Antarctica. Similar results were obtained by de García et al. [71] from yeasts isolated from glacial melt water rivers originating from glaciers in the Argentinean Patagonia. Butinar et al. [62] detected viable yeasts in the ice layers of Arctic glaciers in the Svalbard Islands (Norway), and Turchetti et al. [65] detected viable yeasts in Alpine glaciers; however, in both studies, the yeasts occurred at low densities.

4.2. Resistance against Antifungal Drugs at 37 °C

We screened fungi previously reported as potential pathogenic taxa against the antifungal compounds benomyl (used as a pesticide in agriculture), fluconazole, and amphotericin B (used in medicine), which have structures composed of aromatic rings and which are similar to some POPs and OCPs previously found in snow [15]. Debaromyces hansenii, R. mucilaginosa, Penicillium chrysogenum, and Penicillium sp. 3 and sp. 4 showed resistance against the antifungal drugs tested.

Debaryomyces hansenii is considered a cosmopolitan yeast species that is able to occupy and colonize different ecological niches and ecosystems. In Antarctica, D. hansenni was previously recovered from the rhizosphere of D. antarctica and soil [48], as well as the soil and thalli of lichens [75], macroalgae [76], freshwater, and marine water [77]. However, D. hansenii (also reported as Candida famata, its anamorph) has been previously found to cause opportunistic human infections [78,79,80]. Moreover, there have been reports of higher MIC values of fluconazole for D. hansenii than for pathogenic yeasts species [81]. Though several isolates found in the snow show susceptibility to fluconazole and are unable to grow at 37 °C, D. hansenii is able to synthesize or tolerate several toxins and has been reported to show resistance against several environmental pesticides, among them benomyl [81]. Similarly, we found isolates of D. hansenii that were resistant to benomyl.

Rhodotorula mucilaginosa has recently emerged as an opportunistic fungal pathogen. Rhodotorula mucilaginosa is able to cause outbreaks of skin infections in chickens and lung infections in sheep [82,83], and it is able to worsen the condition of immunosuppressed patients [84]. Here, Rhodotorula mucilaginosa present in snow in Antarctica was found to be resistant to fluconazole and susceptible to amphotericin B. Similar results were reported by Spiliopoulou et al. [85], who found that opportunistic Rhodotorula species was resistant to fluconazole but susceptible to echinocandins and amphotericin B. Generally, opportunistic R. mucilaginosa has been reported to be able grow at 30 °C but not at 37 °C. However, R. mucilaginosa isolates found in the snow in Antarctica were resistant to fluconazole and able to grow at 37 °C.

Penicillium chrysogenum was previously reported as an opportunistic fungus in immunocompromised patients, resulting in otomycoses, endophthalmitis, keratitis, endocarditis, cutaneous infections, and systemic infection [86]; it was also able to cause bloodstream infection in immunocompromised patients [87] and cerebral disease in healthy individuals [88]. Penicillium taxa recovered from the ornithogenic soil samples and rocks of from continental and Antarctic Peninsula [89,90,91] displayed different pathogenic potential to human in vitro.

Kang et al. [15] detected organochlorine pesticides (OCPs) at different concentrations in the snow of East Antarctica and suggested that the air masses that arrive at the sampling sites mainly originate from the Indian and Atlantic Oceans which travel over the Antarctic continent, suggesting that the OCPs were subjected to long-range atmospheric transport and were deposited in surface snow. In recent years, several studies have focused on fungal cross-resistance between agricultural pesticides and clinical antifungal drugs [92,93,94]. Opportunistic fungi are also ubiquitous in the environment and can come into contact with pesticides and antifungal agents used in crops [94]. Since OCPs have chemical structures that are similar to those of antifungal azoles, their presence in the snow in Antarctica may explain the detection of D. hansenii, R. mucilacinosa, and P. chrysogenum resistant to agricultural and clinical antifungal drugs.

5. Conclusions

Our results show that the winter seasonal snow in the Antarctic Peninsula contains a diverse fungal community dominated by cosmopolitan ubiquitous basidiomycetous yeasts species previously found in tropical, temperate, and polar ecosystems. These species were found to display specific adaptations for their successful survival and colonization in the Antarctic snow microhabitat. The high densities of these cosmopolitan fungi suggest that they may be present in the air masses arriving at the Antarctic Peninsula from outside Antarctica, and these air masses are then pushed down by snow precipitation and compacted at high concentrations in the snow each year. Additionally, we detected environmental fungal isolates resistant to agricultural and clinical antifungals, some of which were able to grow at 37 °C. Further studies will be needed to characterize the innate virulence potential of these fungi against humans and animals.