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Article Biodiversity and Abundance of Cultured Microfungi from the Permanently Ice-Covered Fryxell,

Laurie Connell 1,*, Benjamin Segee 1, Regina Redman 2, Russell J. Rodriguez 2 and Hubert Staudigel 3

1 School of Marine Sciences and Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469, USA; [email protected] 2 Adaptive Symbiotic Technologies, University of Washington, Seattle, WA 98195, USA; [email protected] (R.R.); [email protected] (R.J.R.) 3 Scripps Institute of Oceanography, La Jolla, CA 92037, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-207-581-2470

 Received: 4 June 2018; Accepted: 1 September 2018; Published: 6 September 2018 

Abstract: In this work, we explore the biodiversity of culturable microfungi from the water column of a permanently ice-covered lake in Taylor Valley, Antarctica from austral field seasons in 2003, 2008 and 2010, as well as from glacial stream input (2010). The results revealed that there was a sharp decline in total culturable fungal abundance between 9 and 11 m lake depth with a concurrent shift in diversity. A total of 29 were identified from all three water sources with near even distribution between and Basidomycota (15 and 14 respectively). The most abundant taxa isolated from Lake Fryxell in 2008 were Glaciozyma watsonii (59%) followed by Penicillium spp. (10%), both of which were restricted to 9 m and above. Although seven species were found below the chemocline of 11 m in 2008, their abundance comprised only 10% of the total culturable fungi. The taxa of isolates collected from glacial source input streams had little overlap with those found in Lake Fryxell. The results highlight the spatial discontinuities of fungal populations that can occur within connected oligotrophic aquatic habitats.

Keywords: ; fungi; Lake Fryxell; permanently ice-covered ; extreme habitats

1. Introduction Lake Fryxell in Taylor Valley, geographically part of the McMurdo Dry Valleys region of Antarctica is a permanently ice-covered lake with an average of 6 m of ice. This area is the focus of decades of research and more recently tourism, therefore may be vulnerable to human impacts. This lake is primarily fed by melt water from the Canada and Commonwealth Glaciers (Figure1—photo of area with glaciers feeding lake). A marked chemocline exists at 11 m with increases in both salts and 2− SO4 concentrations [1]. An active microbial loop exists in Lake Fryxell as described by Bowman and coworkers [2] is based on decades of microbial research on prokaryotes [3–10] and [7,11–18]. However, as with most lakes worldwide, the study of fungal biodiversity and ecological significance lags far behind [19]. One of the earliest studies of fungi associated with Lake Fryxell [20] described culturable yeasts in the algal mats that form on the lake shore but, sampling did not include either lake water or sediment. The uniqueness of the McMurdo Dry Valleys ice covered lakes and the recent human activity, both research and tourist, highlights the importance of assessing the status of and potential human impact on these microbial communities. Here we describe cultivable fungal species isolated from Lake

Life 2018, 8, 37; doi:10.3390/life8030037 www.mdpi.com/journal/life Life 2018, 8, 37 2 of 10

Life 2018, 8, x FOR PEER REVIEW 2 of 10 Fryxell water at several depths below a permanent ice cover as well as from streams at the terminus of Fryxell water at several depths below a permanent ice cover as well as from streams at the terminus of the two major glaciers feeding Lake Fryxell during austral field seasons 2003, 2008 and 2010. the two major glaciers feeding Lake Fryxell during austral field seasons 2003, 2008 and 2010.

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o 270 o 240 Figure 1. Taylor Valley Antarctica showing Lake Fryxell (LF) between Canada (CG) and FigureCommonwealth 1. Taylor Valley Glaciers Antarctica (CWG). showing The location Lake Fryxellof Taylor (LF) Valley between is shown Canada by the (CG) star and on the Commonwealth insert of GlaciersAntarctica. (CWG). The location of Taylor Valley is shown by the star on the insert of Antarctica.

2. Materials2. Materials and and Methods Methods

2.1. Field2.1. Field Location Location LakeLake Fryxell Fryxell is located is located in in Taylor Taylor Valley, Valley, McMurdoMcMurdo Dry Dry Valleys, Valleys, South South Victoria Victoria Land, Land, Antarctica Antarctica (−77.62;(−77.62; 163.15). 163.15). The The glacial glacial streams streams werewere sampledsampled within within 5 5m m of ofthe the glacial glacial front front of Canada of Canada Glacier Glacier (−77.72; 163.11), and Commonwealth Glacier (−77.69; 163.41) (Figure 1). (−77.72; 163.11), and Commonwealth Glacier (−77.69; 163.41) (Figure1). 2.2. Sample Collection and Processing 2.2. Sample Collection and Processing Water samples were collected from Lake Fryxell through a hole drilled in the lake ice during Water2003, 2008 samples and 2010 were using collected a 5-L Niskin from bottle. Lake FryxellAll sampling through was adone hole during drilled the in Austral the lake summer, ice during 2003,specifically 2008 and during 2010 using the months a 5-L Niskinof December bottle. and All January. sampling The water was donedepth duringwas determined the Austral fromsummer, the specificallylevel that during the lake the water months raised of in December the sample and hole. January. Samples The were water collected depth at was7, 8 and determined 9.5 m during from the level2003; that theat 7, lake 8, 9, water11 and raised 12 m during in the sample2008; and hole. at 7, Samples 8, 9, 10, 11, were 12, collected13 and 14 atm 7,as 8 well and as 9.5 glacier m during 2003;terminus at 7, 8, 9,streams 11 and during 12 m 2010. during Stream 2008; samples and atwere 7, 8,obtained 9, 10, 11,by collecting 12, 13 and 10 14L of m running as well stream as glacier terminuswater streams in sterile during 1 L bottles. 2010. The Stream sample samples size for all were samples, obtained regardless by collecting of source, 10 was L of considered running streamto waterbe in 5 sterileL. Each 1 5-L L bottles.sample was The filtered sample onto size Metricel for all samples,black (Gelman) regardless 47 mm of 0.45 source, sterile was membrane considered filters that were then incubated on media plates of YPD (BD, Franklin Lakes, NJ, USA) or 50% YPD to be 5 L. Each 5-L sample was filtered onto Metricel black (Gelman) 47 mm 0.45 sterile membrane each containing chloramphenicol (100 mg−1·mL) at 10 °C. The 2008 samples were filtered in 100 mL filters that were then incubated on media plates of YPD (BD, Franklin Lakes, NJ, USA) or 50% YPD aliquots with all filters plated separately and all emerging isolates were enumerated. For the other −1· ◦ eachfield containing seasonschloramphenicol some water samples (100 clogged mg themL) filters at10 withC. 100-mL The 2008 volumes samples and werethose filteredsamples inwere 100 mL aliquotsfiltered with with all smaller filters platedvolumes separately per filter and and each all complete emerging water isolates sample were was enumerated. utilized. Each filter For thewas other field seasons some water samples clogged the filters with 100-mL volumes and those samples were filtered with smaller volumes per filter and each complete water sample was utilized. Each filter was plated separately and at least five of each morphotype, if there were five on a plate, were selected from each plate for further processing. Life 2018, 8, 37 3 of 10

Culture plates were shipped refrigerated from Antarctica to University of Maine and incubated at 10 ◦C for up to one year. Each culture plate was checked for fungal growth and photographed at weekly interval for up to 18 months from time of collection. Multiple isolates of each observed morphotype were subcultured from 2003 and 2010 samples. The number of isolates investigated varied based on the number of morphotypes observed. If available at least five of each morphotype were identified. A subculture from every isolate obtained in 2008 was generated and identified using either F-ARISA (fungal-automated ribosomal intergenic spacer analysis) as previously described [21] or by DNA sequencing of the ribosomal internal transcribed spacer region (ITS) for those isolates that could not be positively identified using F-ARISA. The F-ARISA is based on the variability of ITS fragment sizes. In this method, polymerase chain reaction (PCR) is carried out and the resultant fragments are compared using high resolution capillary DNA fingerprinting. Multiple isolates from cultures acquired in 2003, 2010 and from stream samples were subcultured and identified by sequencing the ITS region. Sets of primer combinations including ITS4, ITS5, ITS1F, EF3 [22–25] were used for PCR amplification. PCR for DNA sequenced fragments were carried out as previously described [21]. Specific ITS region amplicons were produced by PCR completed with 100 ng genomic DNA in 25 µL reactions using Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Lifesciences, Pittsburgh, USA). PCR primer set ITS5-ITS4 (White et al. 1990) were used to target the ITS region for sequencing (Table1). Initial denaturation completed for 2 min at 95 ◦C and 35 cycles with a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) under the following conditions: 30 s at 95 ◦C, 30 s at 52.3 ◦C, 1 min at 72 ◦C with a final 72 ◦C 10 min extension. DNA extraction and F-ARISA from all cultured 2008 isolates, regardless of origin, was carried out as previously described [26] and at least one isolate from each taxa identified was sequenced, even for those identified using the F-ARISA method. DNA sequencing was done by the University of Maine Sequencing facility (https://umaine.edu/dnaseq/). The internal transcribed spacer (ITS) sequences, including ITS1 through ITS2, were aligned using MUSCLE [27]. The resulting alignment was used to create a phylogenetic tree through the Seaview version 4.3.1 [28]. A rooted neighbor-joining distance tree was generated using the Seaview option of Jukes–Cantor distance measure [28]. Bootstrap values were based on 100 replicates. Venn’s diagram was made using Venny 2.1 [29]. ITS sequences were submitted to GenBank [21] and are shown in Table1 for all identified taxa.

Table 1. Species identified from Lake Fryxell (LF), Canada Glacier stream (CG), and Commonwealth Glacier stream (CWG).

Species GenBank Accession # A1 B2 LF above 11 m LF below 11 m CG CWG Filobasidium magnus JX171169 X X X Debaryomyces hansenii KM816678 X X Thelebolus ellipsoideus JX171195 X X X X Thelebolus globosus JX171196 X X X X Geomyces sp. 1 KM816679 X X Acremonium sp. KM816680 X X Mrakiella aquatica JX171181 X X X Rhodotorula mucilaginosa JX171192 X X X X Vishniacozyma victoriae JX171171 X X Glaciozyma antarctica KM816681 X X Geomyces sp. 2 KM816682 X X X X Glaciozyma watsonii JX171176 X X Naganishia globosa KM819094 X X X Naganishia albidosimilis JX171168 X X X Penicillium dipodomyicola JX171186 X X Life 2018, 8, 37 4 of 10

Table 1. Cont.

Species GenBank Accession # A1 B2 LF above 11 m LF below 11 m CG CWG Holtermanniella nyarrowii KM816683 X X Aureobasidium pullulans JX171163 X X X Toxicocladosporium strelitziae KM816684 X X Cladosporium cladosporoides KM816685 X X X Penicillium commune JX171184 X X X Heydenia alpina JX171178 X X X Life 2018, 8, x FOR PEER REVIEW 4 of 10 Clavispora lusitaniae KM816686 X X

PenicilliumMrakia commune gelida JX171184KM819098 X XX X X

HeydeniaMrakia blollopsisalpina JX171178KM819093 X XX X X

Clavispora lusitaniae KM816686 X X Sporidiobolus pararoseus KM819095 X X Mrakia gelida KM819098 X X

MrakiaCladosporium blollopsis sp. KM819093KM819097 X XX X

SporidiobolusTrichoderma pararoseus atrovirde KM819095KM822752 X XX X

Cladosporium sp. KM819097 X X Trametes pubescens KM822753 X X Trichoderma atrovirde KM822752 X X

TrametesEutypa pubescens lata KM822753KM822754 X XX X

PiptoporusEutypa lata betulinus KM822754KM822755 X XX X

Piptoporus betulinus KM8227551 X 2 X A = Ascomycota, B = Basidiomycota. A1 = Ascomycota, B2 = Basidiomycota.

3.3. Results Results

3.1.3.1. Fungal Fungal Diversity Diversity ThirtyThirty species species of fungi of fungi were cultured were cultured from Lake from Fryxell Lake waters Fryxell and watersglacial streams and glacial feeding streams Lake feeding FryxellLake Fryxell (Table (Table 1, Figure1, Figure 2). There2). There was about was about equal equal representation representation between between Ascomycota Ascomycota and and BasidiomycotaBasidiomycota species species found found from from all the all sources the sources (53% to (53% 47% to respectively). 47% respectively).

N J 949 sites O bserved 100 repl. 0.05 P iptoporus_betulinus_K M 822755* 100 P iptoporus_betulinus_K C 585371 100 Agariomycetes Tram etes_pubescens_KM 822753* 100 T ram etes_pubescens_JN 164947

Filobasidium _m agnum __N R _130655 100 Filobasidium _m agnum _JX171169* B 100 N aganishia_albidosim ilis_A F145331 89 97 a N aganishia_albidosim ilis_JX 171168* s 100 4 0 N aganishia_globosa_JX 976323 i 100 N aganishia_globosa_KM 819094* d

V ishniacozym a_victoriae_JX 171171* i 100 o V ishniacozym a_victoriae_N R _073260 Tremellomycetes 2 9 m H olterm anniella_nyarrow ii__K M 816683* 100 y H olterm anniella_nyarrow ii_K Y 103594 c M rakia_blollopis_A B 916516 100 o 2 2 M rakia_blollopis_K M 819093* 100 t M rakia_gelida_A F144485 a 100 M rakia_gelida_K M 819098* 100 M rakiella_aquatica_G Q 911548 100 M rakiella_aquatica_JX 171181*

G laciozym a_antarctica__K Y 103467 99

G laciozym a_antarctica_K M 816681* 100 G laciozym a_w atsonii_JX 171176* 100 G laciozym a_w atsonii_KY 103479 97 R hodotorula_m ucilaginosa_H Q 702343 Microbotryomycetes 100 R hodotorula_m ucilaginosa_JX 171192* 5 6 S poridiobolus_pararoseus_A F417115 100 S poridiobolus_pararoseus_K M 819095* H E574403_Alexandrium _andersoni

Figure 2. Cont.

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0.1 Life 2018, NJ8, 2247 37 sites J-C 100 repl. 5 of 10 Penicillium_commune__AJ004813 Euro omycetes Life 2018, 8, x FOR PEER REVIEW Penicillium_commune_JX171184* 5 of 10 100 Penicillium_dipodomyicola_GQ161752 0.1 N J 2247 sites J-C 100 repl. Penicillium_dipodomyicola_JX171186* P enicillium _com m une__A J004813 65 Geomyces_sp._1__KM816679* Euro omycetes A P enicillium _com m une_JX 171184* 100 Geomyces_sp._20KY02_JX270556 100 P enicillium _dipodom yicola_G Q 161752 s Leo omycetes Geomyces_sp._2_KM816682*P enicillium _dipodom yicola_JX 171186* c 65 G eomGeomyces_sp._JX270614 yces_sp._1__K M 816679* A 82 100 G eom yces_sp._20KThelebolus_ellipsoideus_JX171195* Y 02_JX 270556 s o Leo omycetes S G eom yces_sp._2_KThelebolus_globosus_JX171196* M 816682* a 100 m G eom yces_sp._JX 270614 c c 82 Thelebolus_ellipsoideus_KP411553 c Thelebolus_ellipsoideus_JX 171195* o h y Thelebolus_globosus_KC485430 S Thelebolus_globosus_JX 171196* a a 100 100 Debaryomyces_hansenii_KC111444c m r c Thelebolus_ellipsoideus_K P 411553 c o 80 Debaryomyces_hansenii_KM816678*y Thelebolus_globosus_K C 485430 h m o a Clavispora_lusitaniae_KM816686* y 68 100 D ebaryom yces_hansenii_K C 111444 r c c t 93 Clavispora_lusitaniae_KY102561 o e 80 D ebaryom yces_hansenii_K M 816678* m o t 62 C lavispora_lusitaniae_KHeydenia_alpina_JX171178* M 816686* Pezizomycetesy e a 68 c t s 100 93 C lavispora_lusitaniae_KHeydenia_alpina_KF574887 Y 102561 e t 62 Aureobasidium_pullulans_AY213639H eydenia_alpina_JX 171178* Pezizomycetes e a H eydenia_alpina_KF574887 s 100 100 Aureobasidium_pullulans_JX171163* A ureobasidium _pullulans_A Y 213639 100 Cladosporium_cladosporoides__KM816685* 100 A ureobasidium _pullulans_JX 171163* 96 Cladosporium_cladosporoides_EU272532 28 Dothideomycetes 100 C ladosporium _cladosporoides__KM 816685* Cladosporium_sp._KM819097* 100 96 C ladosporium _cladosporoides_E U 272532 28 Dothideomycetes 100 Cladosporium_sp._KX757230 C ladosporium _sp._K M 819097* 100 100 C ladosporiumToxicocladosporium_strelitziae_KM816684* _sp._K X757230

100 ToxicocladosporiumToxicocladosporium_strelitziae_NR_111765 _strelitziae_K M 816684* 33 100 100 ToxicocladosporiumEutypa_lata_AY462551 _strelitziae_N R _111765 33 100 E utypa_lata_AEutypa_lata_KM822754* Y 462551 E utypa_lata_KM 822754* Sordariomycetes 100 Acremonium_sp._JX171161* 61 Sordariomycetes 100 A crem onium _sp._JX 171161* 61 Acremonium_sp._KM816680 100 Acrem onium _sp._KM 816680 Trichoderma_atrovirde_AF456912 100 Trichoderm a_atrovirde_A F456912

100 Trichoderma_atrovirde_KM822752* 100 Trichoderm a_atrovirde_K M 822752* HE574403_Alexandrium_andersoni H E 574403_A lexandrium _andersoni FigureFigure 2. 2.Neighbor Neighbor JoiningJoining treetree of of the the ITS ITS region region of rDNAof rDNA to illustrate to illustrate the relationship the relationship of sequences of sequences Figureobtainedobtained 2. Neighbor from from yeasts yeasts Joining isolated tree of fromfrom the ITSLake Lake region Fryxell Fryxell of as rDNA as well well as to Commonwealth illustrateas Commonwealth the relationship Glacier Glacier and of Canada sequencesand Canada obtainedGlacierGlacier from streams streams yeasts as as isolated aa simplesimple from cladogram.cladogra Lakem. Fryxell Isolates Isolates as from well from this as this Commonwealthwork work are identifiedare identified Glacier by asterisk andby asterisk Canada (*). Other (*). Glacier Other streamsisolatesisolates as shown a shown simple are are cladogram. from from GenBank.GenBank. Isolates TheThe from outgroups ou thistgroups work for arefor both identifiedboth Ascomycetes Ascomycetes by asterisk groups groups (*). (upper Other )( upper and isolates ) and Basidiomycetes (lower) were the alga Alexandrium andersoni. shownBasidiomycetes are from GenBank. (lower) were The outgroupsthe alga Alexandrium for both Ascomycetes andersoni. groups (upper) and Basidiomycetes (lower) were the alga Alexandrium andersoni. During 2008 we were able to determine the culture-based abundance of fungal taxa identified fromDuring Lake 2008Fryxell. we The were 2008 able Lake to Fryxelldetermine samples the showedculture-based Glaciozym abundancea species representing of fungal taxa 69% identifiedof fromtheDuring Lake total 2008Fryxell. abundance we wereThe and 2008 able Toxicocladosporidium Lake to determine Fryxell samples the, Acremonium culture-based showed, and Glaciozym abundance Geomycesa species each of fungal representing representing taxa identified 1% 69% of fromthe(Figure Laketotal Fryxell. 3).abundance The 2008and LakeToxicocladosporidium Fryxell samples, showedAcremoniumGlaciozyma, and speciesGeomyces representing each representing 69% of the 1% total(Figure abundance 3). and Toxicocladosporidium, Acremonium, and Geomyces each representing 1% (Figure3).

Toxicocladosporium, 0.69 Acremonium, 0.69 Naganishia albidosimilis, 1.39 Aureobasidium Rhodotorula mucilaginosa, 0.69 Naganishia globosa, 4.17 Toxicocladosporium, 0.69 pullulans,Acremonium, 5.56 0.69 Naganishia albidosimilis, 1.39 Aureobasidium Vishniacozyma victoriae, 1.39 Rhodotorula mucilaginosa, 0.69 Naganishia globosa, 4.17 pullulans, 5.56 Mrakiella aqua cus, 4.86 Penicillium, 10.42 Geomyces sp., 1.39 Vishniacozyma victoriae, 1.39 Holtermanniella nyarrowii, 0.69 Mrakiella aqua cus, 4.86 Penicillium, 10.42 Geomyces sp., 1.39 Holtermanniella nyarrowii, 0.69 Glaciozyma antarc ca, 9.03

Glaciozyma antarc ca, 9.03

Glaciozyma watsonii, 59.03

Glaciozyma watsonii, 59.03

Figure 3. Percent of abundance for each fungal taxa identified from Lake Fryxell during 2008.

FigureFigure 3. 3.Percent Percent of of abundance abundance for for each each fungal fungal taxa taxa identified identified from from Lake Lake Fryxell Fryxell during during 2008. 2008.

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3.2. Fungal Distribution 3.2. Fungal Distribution The distribution of fungi was explored both by depth in Lake Fryxell as well as potential seeding by The distribution of fungi was explored both by depth in Lake Fryxell as well as potential input by glacial streams. Isolates from all three field seasons were used for distribution determination. seeding by input by glacial streams. Isolates from all three field seasons were used for distribution determination.The total fungal The abundance total fungal above abundance the sharp chemocline above the ofsharp 11 m chemocline was higher thanof 11 below m was with higher little than over Glaciozyma belowall abundance with little difference over all between abundance 7 m and difference 9 m (Figures between4 and 75 ,m Table and1 ).9 Althoughm (Figures 4 and 5, Tablespp. and 1). Penicillium Although spp.Glaciozyma comprised spp. the and largest Penicillium number ofspp the. fungalcomprised abundance the largest above 11number m, they wereof the not fungal found abundancein samples belowabove 11 m, m. they There were were not several found species in samp foundles below both 11 above m. There and below were several the 11-m species chemocline found Clavispora lusitaniae, bothboundary, above howeverand below only the three 11-m species chemocline were foundboundary, exclusively however below only 11three m (species were found Holtermanniella nyarrowii Toxicocladosporium strelitziae exclusively below 11 m (Clavispora, and lusitaniae, Holtermanniella ).nyarrowii, and Toxicocladosporium strelitziae).

Figure 4. AbundanceAbundance of of cultured cultured fungi in Lake Fryxell by depth in meters from the 2008 field field season.

Potential fungalfungal seedingseeding of of Lake Lake Fryxell Fryxell by by stream stream input input was was explored explored by sampling by sampling stream stream water waternear the near fronts the of fronts the two of primarythe two glaciersprimary that glaciers feed the that lake. feed A totalthe lake. of 13 speciesA total wereof 13 identified species were from identifiedCanada Glacier from streamCanada and Glacier Commonwealth stream and GlacierCommonwealth stream (Table Glacier1). Therestream was (Table no overlap 1). There of specieswas no overlapfound between of species either found of thesebetween sources either (Figure of these5). Insour addition,ces (Figure only 5). five In of addition, these 13 only species five were of these found 13 speciesin Lake were Fryxell found samples, in Lake while Fryxell 17 species samples, found while in 17 lake species water found were notin lake found water in either were ofnot the found glacial in eitherstream of samples. the glacial stream samples.

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FigureFigure 5. 5. AA Venn’s Venn’s diagram diagram of of fungal fungal taxa taxa shown shown by by La Lakeke Fryxell Fryxell (LF) (LF) isolate isolate above above and and below below 11 11 m m depthdepth as as well well as as streams streams feeding feeding Lake Lake Fryxell Fryxell of of Canada Glacier Glacier stream stream (CG) (CG) and and Commonwealth GlacierGlacier stream stream (CWG). (CWG).

4.4. Discussion Discussion AlthoughAlthough few studiesstudies havehave examined examined fungal fungal diversity diversity in Antarcticin Antarctic lake lake water water columns, columns, there there have havebeen been studies studies that have that isolatedhave isolated fungi fromfungi soil from surrounding soil surrounding lakes, lake lakes, sediment, lake sediment, or nearby or by nearby microbial by microbialmats [30]. mats For example,[30]. For lakeexample, sediment lake samplessediment from samples the Skarvsnesfrom the Skarvsnes Oasis region Oasis of Eastregion Antarctica of East Antarcticarevealed a diversityrevealed a of diversity five Ascomycetes of five Ascomycetes and five Basidiomycetes and five Basidiomycetes genera similar genera to that similar found into Lakethat foundFryxell in [30 Lake]. Even Fryxell over such[30]. aEven large over geographic such a distancelarge geographic some of the distance same genera some (ofMrakia the same& Thelebolus genera) (wereMrakia isolated & Thelebolus from the) were Skarvsnes isolated Oasi from lake the sedimentsSkarvsnes andOasi Lake lake Fryxellsediments (the and Skarvsnes Lake Fryxell Oasi study (the Skarvsnescharacterized Oasi isolates study characterized only to genus). isolates This suggests only to genus). that these This genera suggests are coldthat adaptedthese genera and commonare cold adaptedacross Antarctica. and common More across intriguing Antarctica. are the More isolates intriguing from Skarvsnes are the isolates Oasi soils from that Skarvsnes overlap withOasi Lakesoils thatFryxell overlap isolates with (e.g., LakeGeomyces Fryxell isolates, Cryptococcus (e.g., Geomyces, Mrakia,,Theolobus Cryptococcus, Diozegia, Mrakia)., The Theolobus fact that, Diozegia glaciers). The and factstream that do glaciers not appear and tostream be a major do not source appear of fungito be to a themajor lake, source the overlap of fungi between to the the lake, studies the overlap suggest betweenthat soil maythe studies be a significant suggest that source soil of may fungi be ina significant Lake Fryxell. source Fungi of may fungi move in Lake from Fryxell. the soil Fungi to the may lake moveby wind from dispersion, the soil meltto the pond lake run-off, by wind or directdispersion, movement melt viapond snowmelt run-off, or or growth. direct movement via snowmelt or growth. Lake Fryxell has increasing H2S and decreasing dissolved O2 below the 11-m chemocline [31]. TheLake abundance Fryxell of has only increasing a few viable H2S and fungal decreasing species thatdissolved are found O2 below primarily the 11-m below chemocline the chemocline [31]. Thesuggests abundance that these of only organisms a few viable are not fungal only able spec toies tolerate that are the found local environmentprimarily below but maythe chemocline be adapted suggests that these organisms are not only able to tolerate the local environment but may be adapted to it. This is supported by the fact that, although most fungi are inhibited by H2S, a few species to it. This is supported by the fact that, although most fungi are inhibited by H2S, a few species have have been reported to metabolize and grow at high levels of H2S[32,33]. Additional research is beenneeded reported to determine to metabolize if these and fungi grow are at actively high levels metabolizing of H2S [32,33]. and/or Additional growing belowresearch the is chemocline. needed to determineThere have if beenthese severalfungi are studies actively of themetabolizing microbial and/or composition growing in Lakebelow Fryxell, the chemocline. focusing There on bacteria have beenand Archaeaseveral [studies4,5,12,14 of,34 the,35 microbial]. Methanogenic composition Archaeal in Lake diversity Fryxell, [4] focusing in Lake Fryxellon bacteria were and found Archaea to be [4,5,12,14,34,35].clustered in either Methanogenic the water column Archaeal or the diversity sediment, [4] demonstrating in Lake Fryxell functional were found separation. to be clustered In addition, in either the water column or the sediment, demonstrating functional separation. In addition, a diversity of phototropic purple bacteria was identified from the water column [5,36] and nutrient cycling both above and below the chemocline.

Life 2018, 8, 37 8 of 10 a diversity of phototropic purple bacteria was identified from the water column [5,36] and nutrient cycling both above and below the chemocline. The fact that there are at least four sources of microbial introduction into Lake Fryxell (e.g., CG, CWG, soil, and air) suggests that either: (1) The spatial distribution of source fungi is extreme and there is little or no homogenization of microbes over this area. (2) The level of detection may be so low that the error rates are high creating a detection bias from the small sample sizes that are used in these kinds of studies. (3) Soil may be a significant source of fungi as indicated above. The list of fungi found in Antarctica continues to grow with the suggestion that “fungi might be the most diverse biota in Antarctica” [37]. There remain many challenges to determine both fungal abundance and activity from Antarctic environmental samples yet is clear that they must play a role in nutrient recycling and decomposition. Based on the extreme habitat below the Lake Fryxell chemocline, it is clear that a limited number of fungal species are able to adapt yet it remains to be determined how closely the Lake Fryxell isolates are to those found in the northern hemisphere.

Author Contributions: Conceptualization, L.C., R.J.R. and R.R.; Methodology, L.C., R.J.R. and R.R.; Formal Analysis, L.C., R.J.R. and B.S.; Investigation, L.C., R.J.R., R.R., B.S. and H.S.; Resources, L.C.; Data Curation, L.C. and B.S.; Writing—Original Draft Preparation, L.C.; Writing—Review & Editing, L.C., R.J.R., R.R. and H.S.; Supervision, L.C., and R.J.R.; Project Administration, L.C.; Funding Acquisition, L.C., R.J.R., R.R. and H.S. Funding: This work was partially supported by Grant# NSF OPP-0125611 and NSF ANT-0739696 and its REU supplement. Acknowledgments: Thanks to: Scott Craig, Megan Altenritter, Leslie Astbury, Amy Chichilo, Tristy Vick-Majors, and Andrew Barber for field and laboratory help. Conflicts of Interest: The authors declare no conflicts of interest.

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