Community Structures of Bacteria, Archaea, and Eukaryotic Microbes in the Freshwater Glacier Lake Yukidori-Ike in Langhovde, East Antarctica

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Article Community Structures of Bacteria, Archaea, and Eukaryotic Microbes in the Freshwater Glacier Lake Yukidori-Ike in Langhovde, East Antarctica

Aoi Chaya 1, Norio Kurosawa 1,*, Akinori Kawamata 2, Makiko Kosugi 3 and Satoshi Imura 4,5 1 Department of Science and Engineering for Sustainable Innovation, Faculty of Science and Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan 2 Ehime Prefectural Science Museum, 2133-2 Ojoin, Niihama, Ehime 792-0060, Japan 3 Astrobiology Center, National Institutes of Natural Science, 2-21-1 Mitaka, Tokyo 181-8588, Japan 4 National Institute of Polar Research, 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan 5 SOKENDAI (The Graduate University for Advanced Studies), 10–3 Midori–cho, Tachikawa, Tokyo 190-8518, Japan * Correspondence: [email protected]

Received: 3 June 2019; Accepted: 4 July 2019; Published: 6 July 2019

Abstract: Since most studies about community structures of microorganisms in Antarctic terrestrial lakes using molecular biological tools are mainly focused on bacteria, limited information is available about archaeal and eukaryotic microbial diversity. In this study, the biodiversity of microorganisms belonging to all three domains in a typical Antarctic freshwater glacier lake (Yukidori-Ike) was revealed using small subunit ribosomal RNA (SSU rRNA) clone library analysis. The bacterial clones were grouped into 102 operational taxonomic units (OTUs) and showed significant biodiversity. Betaproteobacteria were most frequently detected, followed by Cyanobacteria, Bacteroidetes, and Firmicutes as major lineages. In contrast to the bacterial diversity, much lower archaeal diversity, consisting of only two OTUs of methanogens, was observed. In the eukaryotic microbial community consisting of 20 OTUs, Tardigradal DNA was remarkably frequently detected. Genera affiliated with the phyla Ciliophora, Cryptomycota, Chlorophyta, Bacillariophyta, and Apusozoa were also detected. The biodiversity and species compositions of the whole microbial community of Lake Yukidori-Ike are similar to those of freshwater environments in temperate regions but are different from saline lakes in Antarctica, indicating that the salinity seems to affect the microbial composition more than the temperature.

Keywords: Antarctica; freshwater lake; Yukidori-Ike; bacteria; archaea; eukaryotic microorganism; SSU rRNA gene; clone library analysis

1. Introduction Approximately 98% of the Antarctic continent is permanently covered in ice, and only 0.4% of the ice-free area, the so-called “Antarctic Oases”, experiences temporary ice melts during summer [1]. The ice-free areas are colonized by various microorganisms, including bacteria, archaea, yeasts, ﬁlamentous fungi, algae, and protozoa. Previous studies on microorganisms in Antarctic lakes using molecular biological tools have revealed signiﬁcant bacterial diversity, particularly among bacteria [2–7]. For example, Bowman et al. [3] investigated diverse bacterial communities in Antarctic saline lakes located in Vestfold Hills, Eastern Antarctica, using 16S rRNA clone library analysis. They found that Proteobacteria, Cyanobacteria, and Bacteroidetes were the dominant bacterial phyla and that archaeal diversity was remarkably low. In a later study by our group [5], we also conducted 16S rRNA clone library analysis in an Antarctic meromictic lake located in Langhovde, Eastern Antarctica. That

Diversity 2019, 11, 105; doi:10.3390/d11070105 www.mdpi.com/journal/diversity Diversity 2019, 11, x FOR PEER REVIEW 2 of 14 rRNA clone library analysis in an Antarctic meromictic lake located in Langhovde, Eastern Antarctica. That study also revealed high bacterial diversity, particularly of Alpha-, Delta-, and Gammaproteobacteria, and limited archaeal diversity. However, most of these studies were conducted in marine relic saline lakes, and only limited information is available on the prokaryotic community structure in Antarctic freshwater lakes. The diversity and molecular phylogeny of eukaryotic microbes in Antarctic lakes have been much less well investigated than those of the Diversityprokaryotes.2019, 11 , 105 2 of 15 Langhovde (69°14′ S, 39°40′ E) is one of the ice-free areas located approximately 30 km south of Japanese Syowa Station, Soya Coast (the east coast of Lützow-Holm Bay) in East Antarctica. In this area,study the also Yukidori revealed Valley high bacterialis recognized diversity, as having particularly high biodiversity of Alpha-, Delta-, and is anddesignated Gammaproteobacteria, as an Antarctic Speciallyand limited Protected archaeal Area diversity. (ASPA) However, No. 141 mostto protect of these the studies fragile wereand typical conducted continental in marine Antarctic relic saline fell fieldlakes, ecosystem and only limitedand its informationcomponent species, is available some on of the which prokaryotic are endemic community to Antarctica, structure from in Antarctic human activityfreshwater in Antarctica. lakes. The diversityLong-term and monitoring molecular programs phylogeny have of eukaryoticbeen conducted microbes at this in Antarcticsite [8,9]. lakesLake Yukidori-Ike,have been much the less sampling well investigated site of this than study, those is a of freshwater the prokaryotes. glacial lake located in the middle of YukidoriLanghovde Valley (Figure (69◦140 S,1a). 39 The◦400 maximumE) is one of depth the ice-free and area areas of locatedthe lake approximately are 8.6 m and 30approximately km south of 0.041Japanese km2 Syowa, respectively. Station, The Soya bottom Coast of (the the east lake coast is cove ofred Lützow-Holm by photosynthetic Bay) in benthos East Antarctica. [10]. The InSnow this Petrelarea, the (Pagodroma Yukidori nivea Valley; “Yukidori” is recognized is the as Japanese having high name biodiversity for the Snow and Petrel), is designated a seabird, as aninhabits Antarctic the YukidoriSpecially Valley. Protected Area (ASPA) No. 141 to protect the fragile and typical continental Antarctic fell ﬁeldIn ecosystem the present and study, its component we attempted species, to analyze some the of which wholeare microbial endemic communi to Antarctica,ty of Lake from Yukidori- human Ikeactivity using in SSU Antarctica. rRNA clone Long-term library monitoring analysis, as programs a model microbiome have been conducted of Antarctic at thisfreshwater site [8,9 ].glacier Lake Yukidori-Ike,lakes. High-throughput the sampling DNA site sequencing of this study, (formerly is a freshwater called next-generation glacial lake locatedsequencing) in the has middle become of Yukidoria major tool Valley for (Figure biodiversity1a). The analyses maximum because depth of and its area high of thecomprehensiveness lake are 8.6 m and and approximately low cost in 2 processing0.041 km , respectively.a large number The of bottom samples. of the In lakethis isstudy, covered however, by photosynthetic we chose SSU benthos rRNA [10 clone]. The library Snow analysesPetrel (Pagodroma using Sanger nivea sequencing; “Yukidori” method is the Japanese to identi namefy the for microbes the Snow at the Petrel), species a seabird, level using inhabits longer the Yukidoridata sequences Valley. and to compare the results with those of previous studies.

a) b)

Figure 1. LakeLake Yukidori-Ike Yukidori-Ike ( a) and a sampling point (b).

2. MaterialsIn the present and Methods study, we attempted to analyze the whole microbial community of Lake Yukidori-Ike using SSU rRNA clone library analysis, as a model microbiome of Antarctic freshwater glacier lakes. 2.1.High-throughput Study Site and DNASampling sequencing (formerly called next-generation sequencing) has become a major tool for biodiversity analyses because of its high comprehensiveness and low cost in processing a large Lake Yukidori-Ike (Figure 1a) is located in central Langhovde on the east coast of Lutzow-Holm number of samples. In this study, however, we chose SSU rRNA clone library analyses using Sanger Bay, Antarctica (69°14′26” S, 39°45′23” E). The altitude, maximum depth, and area of Lake Yukidori- sequencing method to identify the microbes at the species level using longer data sequences and to Ike are 125, 8.6, and 0.041 km2, respectively [1]. Water samples, including surface sediment, of compare the results with those of previous studies. approximately 80 mL were collected from three points on the lakeshore both on December 26 and 31, 2012,2. Materials during andthe Methods54th Japanese Antarctic Research Expedition. The sediment mainly consisted of sandstone in diameters of approximately 0.5–5 mm. Each of the three points were 20–40 cm in depth (Figure2.1. Study 1b). Site The and water Sampling temperature and pH were 5.4 °C and 8.4 on December 26, and 10.5 °C and 8.1 on December 31. The samples were taken by using autoclaved stainless ladles and put into 100-mL Lake Yukidori-Ike (Figure1a) is located in central Langhovde on the east coast of Lutzow-Holm Bay, sterile plastic tubes. The algal mat inhabiting places on the bottom of the lake was avoided for Antarctica (69◦14026” S, 39◦45023” E). The altitude, maximum depth, and area of Lake Yukidori-Ike are 125, 8.6, and 0.041 km2, respectively [1]. Water samples, including surface sediment, of approximately 80 mL were collected from three points on the lakeshore both on December 26 and 31, 2012, during the 54th Japanese Antarctic Research Expedition. The sediment mainly consisted of sandstone in diameters of approximately 0.5–5 mm. Each of the three points were 20–40 cm in depth (Figure1b). The water temperature and pH were 5.4 ◦C and 8.4 on December 26, and 10.5 ◦C and 8.1 on December 31. The samples were taken by using autoclaved stainless ladles and put into 100-mL sterile plastic tubes. The Diversity 2019, 11, 105 3 of 15 algal mat inhabiting places on the bottom of the lake was avoided for sampling, but small amounts of algal debris might be included. The samples in the tubes were kept in freezing conditions until the DNA extraction experiments. A mixture of equal parts of the six samples was used for the extraction of environmental DNA.

2.2. Nutrient Analysis Each water sample was filtered through a membrane filter with a 0.22-µm pore size. The concentrations of nitrate (NO3), nitrite (NO2), ammonium (NH4), phosphate (PO4), and silicate (SiO2) in the filtrate were analyzed using a nutrient auto-analyzer (SWAAT, BLTEC, Osaka, Japan).

2.3. Bacterial 16S rRNA Clone Library Analysis Environmental DNA was extracted from 5.0 g of the mixed lake sample using Ultraclean Soil DNA Kit Mega Prep (Qiagen, Hilden, Germany). The extracted environmental DNA was used for the polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene using a bacterial universal primer set: forward primer 50-AGAGTTTGATCCTGGCTCAG-30 (B27F) and reverse primer 50-GGYTACCTTGTTACGACTT-30 (U1492R) [11]. Each 50 µL PCR reaction mixture consisted of ~10 ng of environmental DNA, 0.5 µmol L 1 of each primer, and 25 µL of 2 PCR Master Mix (EmeraldAmp, · − × Takara-bio, Kusatsu, Japan). PCR amplification was conducted by initial denaturation at 94 ◦C for 3 min, followed by 30 cycles of 30 s at 94 ◦C, 30 s at 58 ◦C, and 100 s at 72 ◦C, and a final extension at 72 ◦C for 5 min. The amplified PCR product was purified from agarose gel using an illustra GFX PCR Purification Kit (GE Healthcare, Chicago, IL). After clean-up, the purification product was checked by electrophoresis on 1% agarose gel with ethidium bromide staining before cloning. The PCR product was cloned into pT7 blue vector (Merck, Darmstadt, Germany), and the recombinant plasmids were transformed into Escherichia coli DH5α cells. The transformants were plated on Luria–Bertani medium (LB) plates containing 100 µg mL 1 ampicillin, 40 µg mL 1 X-gal, and 0.5 mM · − · − Isopropyl β-D-1-thiogalactopyranoside (IPTG). Randomly picked white colonies were subcultured in 100 µL of LB medium containing 100 µg mL 1 ampicillin in 96-well plates at 37 C overnight. The · − ◦ inserted 16S rRNA gene was amplified by PCR using 1 µL of the culture as a template with primers 50-TAATACGACTTCACTATAGGG-30 (T7P-F) and 50-GTTTTCCCAGTCACGACGT-30 (T7U-R). The PCR program was as follows: initial denaturation at 94 ◦C for 3 min; followed by 35 cycles of 30 s at 94 ◦C, 30 s at 51 ◦C, and 2 min at 72 ◦C; and a final extension at 72 ◦C for 5 min. Approximately 800 bp of the 50-region of each 16S rRNA clone was sequenced.

2.4. Archaeal 16S rRNA Clone Library Analysis All procedures for the archaeal 16S rRNA clone library analysis were the same as those for bacterial 16S rRNA clone library analysis except for the PCR conditions. The ﬁrst PCR for archaeal 16S rRNA genes was conducted using archaeal forward primer 5’-TCYGGTTGATCCTGCCRG-30 (Ar4f) [12] and reverse primer 5’-YCCGGCGTTGAVTCCAATT-30 (Ar958r) [11]. Then, nested PCR was further conducted using archaeal forward primer Ar4f and reverse primer 5’- CCCGCCAATTCCTTTAAGTTTC-30 (Ar9r) [13]. Both PCR stages were conducted by initial denaturation at 94 ◦C for 3 min; followed by 30 cycles of 30 s at 94 ◦C, 30 s at 58 ◦C, and 100 s at 72 ◦C; and a ﬁnal extension at 72 ◦C for 5 min.

2.5. Eukaryotic 18S rRNA Clone Library Analysis All procedures for the eukaryotic 18S rRNA clone library analysis were the same as those for bacterial and archaeal 16S rRNA clone library analysis except for the PCR conditions. The eukaryotic 18S rRNA gene was ampliﬁed with eukaryotic forward primer 50-GAAACTGCGAATGGCTC-30 (EK-82F) and reverse primer 50-CYGCAGGTTCACCTAC-30 (EK-1520R) [14]. Then, nested PCR was performed with the same forward primer and reverse primer 50-CACCTACGGAAACCTTGTTA-30 (EK-1498R) [15]. The ﬁrst PCR and nested PCR conditions comprised denaturation at 94 ◦C for 3 min, 10 cycles of touch-down PCR (denaturation at 94 ◦C for 30 s, a 30-s annealing step at decreasing Diversity 2019, 11, 105 4 of 15

temperature from 65 to 55 ◦C employing a 1 ◦C decrease with each cycle, and extension at 72 ◦C for 2 min), 20 additional cycles at 55 ◦C annealing temperature, and ﬁnal extension at 72 ◦C for 5 min.

2.6. Phylogenetic Analysis Sanger DNA sequencing of the SSU rRNA genes was conducted by Euroﬁns Genomics K.K. (Tokyo, Japan). The obtained sequences were grouped into operational taxonomic units (OTUs) using 98% sequence similarity. To identify individual clones, the obtained 16S rRNA genes were submitted to an EzTaxon search, and 18S rRNA gene sequences were submitted to the Basic Local Alignment Search Tool (BLAST). After the removal of chimeric sequences manually, the SSU rRNA gene sequences of a representative clone were aligned using Clustal W alignment program as implemented in the MEGA10 software [16], with related species published in the DDBJ/EMBL/GenBank database. The neighbor-joining tree including bootstrap probabilities (1000 samplings) was reconstructed using the MEGA10 software (gap/missing data treatment: complete deletion, Kimura 2-parameter substitution model).

2.7. Coverage and Diversity Indices The homologous coverage (C) was determined by the following equation: C = 1 (N/n), where N − is the number of OTUs and n is the total number of analyzed clones [17]. The Shannon–Wiener index and Chao1 were calculated using Estimate S software [18].

2.8. Nucleotide Sequences The nucleotide sequences of the representative clones a reported in this study are available in the DDBJ/EMBL/GenBank database under the accession numbers: Bacteria; LC489022-LC489131, Archaea; LC489132 and LC489133, and Eukarya; LC489002-LC489021.

3. Results

3.1. Nutrients

The concentrations of NO3, NO2, NH4, and SiO2 in Lake Yukidori-Ike were 5.18, 0.13, 1.27, and 25.6 µM, respectively. The PO4 concentration was below the detection limit (0.03 µM).

3.2. Bacterial Community A total of 160 bacterial clones was grouped into 101 OTUs. These OTUs were phylogenetically classified into nine phyla, 19 classes, 34 orders, 49 families, and 61 genera (Table1). The Shannon–Wiener index and Chao1 of the bacterial community were 4.5 and 302, respectively. The homologous coverage value was 0.37. The most frequently detected bacterial phylum in Lake Yukidori-Ike was Proteobacteria, which accounted for 56% of total bacterial clones and consisted of 63 OTUs (Figure2). These OTUs were classified into four classes (clonal frequencies within the Proteobacteria): Alphaproteobacteria (26%), Betaproteobacteria (58%), Deltaproteobacteria (10%), and Gammaproteobacteria (4%). The Alphaproteobacterial clones consisted of 18 OTUs. The phylogenetic analysis indicated that 12 of the 18 OTUs were classified into any of five described families: Sphingomonadaceae, Rhodobacteraceae, Micropepsaceae, Hyphomicrobiaceae, and Caulobacteraceae. The other six OTUs could not be affiliated with any known families and were classified into two unknown groups. The family Sphingomonadaceae was most frequently detected and diverse within the Alphaproteobacteria. Diversity 2019, 11, 105 5 of 15

Table 1. List of bacterial clones.

Number of Number of Phylum Class Oder Family Genus Clones OTUs Proteobacteria α-Proteobacteria Sphingomonadales Sphingomonadaceae Polymorphobacter 3 2 Novosphingobium 2 1 Sphingomonas 1 1 Sandarakinorhabdus 1 1 Sphingopyxis 1 1 Rhodobacterales Rhodobacteraceae Gemmobacter 2 1 Seohaeicola 1 1 Rhodospirillales No rank Reyranella 1 1 Micropepsales Micropepsaceae Unclassified 1 1 Rhizobiales Hyphomicrobiaceae Pedomicrobium 1 1 Caulobacterales Caulobacteraceae Phenylobacterium 1 1 Unclassified Unclassified 2 2 Unclassified Unclassified Unclassified 6 4 β-Proteobacteria Burkholderiales Comamonadaceae Polaromonas 8 3 Rhodoferax 7 3 Hydrogenophaga 1 1 Unclassified 1 1 No rank Methylibium 1 1 Burkholderiaceae Ralstonia 2 1 Oxalobacteraceae Undibacterium 6 3 Unclassified Unclassified 6 5 Rhodocyclales Rhodocyclaceae Propionivibrio 3 1 Nitrosomonadales Methylophilaceae Methylobacillus 1 1 Unclassified Unclassified 8 7 Neisseriales Neisseriaceae Neisseria 1 1 Unclassified Unclassified Unclassified 7 5 δ-Proteobacteria Myxococcales Polyangiaceae Byssovorax 1 1 Nannocystaceae Unclassified 2 1 Unclassified Unclassified 3 3 Desulfuromonadales Geobacteraceae Geobacter 1 1 Unclassified Unclassified 2 2 γ-Proteobacteria Xanthomonadales Rhodanobacteraceae Unclassified 3 1 Unclassified Unclassified 1 1 Unclassified Unclassified Unclassified Unclassified 2 2 Cyanobacteria Cyanobacteria Oscillatoriales Gomontiellaceae Crinalium 12 1 Oscillatoriaceae Phormidium 1 1 Synechococcales Leptolyngbyaceae Leptolyngbya 3 2 Phormidesmis 2 1 Unclassified 6 1 Pseudanabaenaceae Limnothrix 1 1 Chamaesiphonaceae Chamaesiphon 1 1 Nostocales Nostocaceae Nostoc 3 1 Bacteroidetes Cytophagia Cytophagales Cytophagaceae Arcicella 1 1 Unclassified Unclassified 3 3 Chitinophagia Chitinophagales Chitinophagaceae Ferruginibacter 1 1 Terrimonas 3 1 Unclassified 3 3 Unclassified Unclassified Unclassified 6 3 Firmicutes Tissierellia Tissierellales Peptoniphilaceae Anaerococcus 3 2 Unclassified Unclassified 1 1 Clostridia Clostridiales Ruminococcaceae Acetivibrio 1 1 Clostridiaceae Unclassified 1 1 Bacilli Bacillales Staphylococcaceae Staphylococcus 3 1 Negativicutes Veillonellales Veillonellaceae Veillonella 1 1 Acidobacteria Blastocatellia Blastocatellales Blastocatellaceae Aridibacter 5 2 Unclassified Unclassified Unclassified Unclassified 4 3 Ignavibacteria Unclassified Unclassified Unclassified Unclassified 2 2 Chlorobi Unclassified Unclassified Unclassified Unclassified 1 1 Actinobacteria Actinobacteria Micrococcales Microbacteriaceae Microbacterium 1 1 Armatimonadetes Chthonomonadetes Unclassified Unclassified Unclassified 1 1 Total 160 101

The Betaproteobacteria were the most diverse in Lake Yukidori-Ike and consisted of 33 OTUs. 15 of the 33 OTUs were classified into any of the five described families: Comamonadaceae, Oxalobacteraceae, Burkholderiaceae, Oxalobacteraceae, Rhodocyclaceae, and Methylophilaceae. However, the other 18 OTUs could not be affiliated with any known families and were classified into three unknown groups. The family Comamonadaceae, consisting of four genera and eight OTUs, was most frequently Diversity 2019, 11, x FOR PEER REVIEW 6 of 14

Cyanobacteria Cyanobacteria Oscillatoriales Gomontiellaceae Crinalium 12 1 Oscillatoriaceae Phormidium 1 1 Diversity 2019, 11, 105 6 of 15 Synechococcales Leptolyngbyaceae Leptolyngbya 3 2 Phormidesmis 2 1 Diversity 2019, 11, x FOR PEER REVIEW Unclassified 6 5 of1 14 detected and diverse in the Betaproteobacteria. Pseudanabaenaceae Particularly, Polaromonas Limnothrix spp. and Rhodoferax1 spp.,1 Chamaesiphonaceae Chamaesiphon 1 1 Rhodoferaxconsisting of spp., six OTUsconsisting together, of Nostocalessix accounted OTUs togeth for 29%er,Nostocaceae accounted of the Betaproteobacterial for 29%Nostoc of the Betaproteobacterial clones.3 Delta- and1 Bacteroidetesclones.Gammaproteobacteria Delta- Cytophagiaand Gammaproteobacteria were less Cytophagales frequently were detected Cytophagaceae less thanfrequently the Alpha- detectedArcicella and Betaproteobacteriathan the1 Alpha- and1 Unclassified Unclassified 3 3 Betaproteobacteriaconsisted of 10Chitinophagia OTUs. and Only consisted fourChitinopha of of these 10gales OTUs. OTUs wereOnlyChitinophagaceae identiﬁedfour of these at the OTUsFerruginibacter family were level; identified this was1 at not the possible family1 level;for the this others. was not There possible were twofor the OTUs others. (YK1B-121, There were YK1B-197) two OTUs that (YK1B-121, wereTerrimonas unable YK1B-197) to classify3 that into were1 any described classes in the Proteobacteria (Figure3). Unclassified 3 3 unable to classify into any describedUnclassified classes in the UnclassifiedProteobacteria (FigureUnclassified 3). 6 3 FirmicutesThe second secondTissierellia most most frequently frequently Tissierellales detected detected bacterial bacterial Peptoniphilaceae phylum phylum was was Cyanobacteria, Cyanobacteria,Anaerococcus which which3 accounted accounted 2 for 18%for 18%of the of total the bacterialtotal bacterial clones and clones consisted and of consistedUnclassified nine OTUs. of These nineUnclassified OTUs.OTUs were These classified1 OTUs into were1 six Clostridia Clostridiales Ruminococcaceae Acetivibrio 1 1 families—Gomontiellaceae,classiﬁed into six families—Gomontiellaceae, Oscillatoriales, Oscillatoriales, ClostridiaceaeLeptolyngbyaceae, Leptolyngbyaceae,Unclassified Pseudanabaenaceae, Pseudanabaenaceae,1 1 Chamaesiphonaceae, Bacilli and and Nostocales—showing Nostocales—showingBacillales Staphylococcaceae clonal clonal frequencies frequencies Staphylococcus of of 41, 41, 3, 3, 10, 10, 7, 7, 33, 3, 3, 3, and and 10%, 1 Negativicutes Veillonellales Veillonellaceae Veillonella 1 1 Acidobacteriarespectively, withinBlastocatellia thethe Cyanobacteria.Cyanobacteria. Blastocatellales The The third thirdBlastocatellaceae most frequently Aridibacterdetected bacterial 5 phylum was 2 Bacteroidetes, Unclassifiedwhich accounted Unclassified for 10% of bacteria bacterialUnclassifiedl clones, followed Unclassified by by the phyla Firmicutes 4 (6%), (6%), 3 IgnavibacteriaAcidobacteria (6%),Unclassified Ignavibacteria Unclassi (2%),fied ChlorobiUnclassified (1%), Actinobacteria Unclassified (1%), and Armatimonadetes2 2 AcidobacteriaChlorobi (6%),Unclassified Ignavibacteria Unclassified (2%), ChlorobiUnclassified (1%), Actinobacteria Unclassified (1%), and Armatimonadetes1 1 Actinobacteria(1%) (Figure 2 2).).Actinobacteria Micrococcales Microbacteriaceae Microbacterium 1 1 Armatimonadetes Chthonomonadetes Unclassified Unclassified Unclassified 1 1 Total 160 101

3.3. Archaeal Community A total of 72 archaeal clones were grouped into only two OTUs. These OTUs were Proteobacteria (63) Cyanobacteria (9) phylogenetically classified into one order, two families, and two genera (Table 2). The Shannon– Bacteroidetes (12) Firmicutes (7) Wiener index and Chao1 Acidobacteriawere 0.011 and (5) 2, respectively. IgnavibacteriaThe homologous (2) coverage value was 0.97. One OTU (YK1A-009), whichChlorobi accounted (1) for 97.2% of the Actinobacteriaarchaeal community, (1) was identified as the methanogenic euryarchaeonArmatimonadetes Methanosarcina (1) subterranea based on a nucleotide sequence similarity of

99.0%. The other OTU (YK1A-058) was also identified as a methanogenic euryarchaeon, Methanosaeta Figure 2. CommunityCommunity structure of bacterial clones at the phyla level. The number in parenthesis is the sp. Thenumber nucleotide of operational sequence taxonomic of OTU units YK1A-058 (OTUs) belonging showed to98.1% each phylum.similarity with that of Methanosaeta conciliinumber. No other of operational archaeal taxonomic phyla were units detected. (OTUs) belonging to each phylum.

Table 1. List of bacterial clones.

Number of Number of Phylum Class Oder Family Genus Clones OTUs

Proteobacteria α-Proteobacteria Sphingomonadales Sphingomonadaceae Polymorphobacter 3 2 Methanosarcinaceae (1) Methanosaetaceae (1) Novosphingobium 2 1 Figure 3. Community structure of archaeal clones at the family level. TheSphingomonas number in parenthesis1 is the 1 Figure 3. Community structure of archaeal clones at the family level.Sandarakinorhabdus The number in parenthesis1 is 1 number of OTUs belonging to each family. Both families are affiliated with the phylum Euryarchaeota. the number of OTUs belonging to each family. Both families areSphingopyxis affiliated with the1 phylum 1 Rhodobacterales Rhodobacteraceae Gemmobacter 2 1 3.3. Archaeal Euryarchaeota. Community Seohaeicola 1 1 Rhodospirillales No rank Reyranella 1 1 A total of 72 archaeal clonesMicropepsales wereTable grouped 2. List into ofMicropepsaceae archaeal only two clones. OTUs. TheseUnclassified OTUs were phylogenetically1 1 classified into one order, two families,Rhizobiales and two generaHyphomicrobiaceae (Table2). The Shannon–WienerPedomicrobium index1 and Chao11 Caulobacterales Caulobacteraceae Phenylobacterium Number1 of Number1 of Phylum Class Oder Family Genus were 0.011 and 2, respectively. The homologous coverageUnclassified value was 0.97.Unclassified One OTUClones (YK1A-009),2 OTUs which2 Unclassified Unclassified Unclassified 6 4 Euryarchaeotaaccounted forMethanomicrobia 97.2% of the archaeal Methanosarcinales community, Methanosarcinaceae was identified asMethanosarcina the methanogenic 70 euryarchaeon1 β-Proteobacteria Burkholderiales Comamonadaceae Polaromonas 8 3 Methanosarcina subterranea based on a nucleotideMethanosaetaceae sequence similarity MethanosaetaRhodoferax of 99.0%. 2 The 7 other OTU1 3 (YK1A-058) was also identified as a methanogenic euryarchaeon, HydrogenophagaMethanosaetaTotal sp.72 The1 nucleotide2 1 Unclassified 1 1 sequence of OTU YK1A-058 showed 98.1% similarity with that of Methanosaeta concilii. No other 3.4. Eukaryotic Community No rank Methylibium 1 1 archaeal phyla were detected. Burkholderiaceae Ralstonia 2 1 Oxalobacteraceae Undibacterium 6 3 A total of 90 eukaryotic clones were grouped Unclassifiedinto 20 OTUs. TheseUnclassified OTUs were phylogenetically6 5 classified into seven phyla, 12 Rhodocyclalesclasses,Table 13 2.orders,List ofRhodocyclaceae archaeal16 families, clones. and 18Propionivibrio genera including3 unclassified 1 Nitrosomonadales Methylophilaceae Methylobacillus 1 1 taxa (Table 3). The Shannon–Wiener index and Chao1 were 2.6 and 28, respectively.Number The homologousNumber Phylum Class OderUnclassified Family Unclassified Genus 8 7 coverage value was 0.79. Neisseriales Neisseriaceae Neisseria of Clones1 of OTUs1 Euryarchaeota The most frequently Methanomicrobia detectedUnclassified Methanosarcinales phylum was MethanosarcinaceaeTardUnclassifiedigrada, whichMethanosarcina accountedUnclassified for 53%707 of eukaryotic 1 5 δ-Proteobacteria Myxococcales Polyangiaceae Byssovorax 1 1 Methanosaetaceae Methanosaeta 2 1 clones and was classified into class EutardigradaNannocystaceae (Figure 4). MostUnclassified of the Tardigrada 2 clones were1 Total 72 2 Unclassified Unclassified 3 3 Desulfuromonadales Geobacteraceae Geobacter 1 1 Unclassified Unclassified 2 2 γ-Proteobacteria Xanthomonadales Rhodanobacteraceae Unclassified 3 1 Unclassified Unclassified 1 1 Unclassified Unclassified Unclassified Unclassified 2 2

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3.4. Eukaryotic Community A total of 90 eukaryotic clones were grouped into 20 OTUs. These OTUs were phylogenetically classiﬁed into seven phyla, 12 classes, 13 orders, 16 families, and 18 genera including unclassiﬁed taxa (Table3). The Shannon–Wiener index and Chao1 were 2.6 and 28, respectively. The homologous coverage value was 0.79.

Table 3. List of eukaryotic clones.

Number Number Phylum Class Oder Family Genus of Clones of OTUs Tardigrada Eutardigrada Parachela Hypsibiidae Diphascon 40 1 Acutuncus 8 1 Ciliophora Stichotrichia Sporadotrichida Oxytrichidae Onychodromopsis 6 1 Rigidocortex 2 1 Oligohymenophorea Peritrichia Vorticellidae Vorticellides 2 1 Philasterida Philasteridae Unclassified 1 1 Litostomatea Haptorida Spathidiidae Spathidium 1 1 Phyllopharyngia Chlamydodontida Chilodonellidae Phascolodon 1 1 Bacillariophyta Bacillariophyceae Bacillariophycidae Naviculales Humidophila 2 1 no rank no rank Bicosoecida Bicosoecidae Unclassified 1 1 Chlorophyta Chlorophyceae Chlamydomonadales Haematococcaceae Chlorogonium 3 1 Chlamydomonadaceae Oogamochlamys 1 1 Diversity 2019, 11, x FOR PEER REVIEW Actinochloridaceae Macrochloris 17 1of 14 Unclassified Unclassified 2 2 Cryptomycota identified as DiphasconUnclassified sp., which Unclassified accounted for Unclassified44% of all the Unclassified eukaryotic clones. 11 The other 1 Eutardigrada(Fungi) was identified as Acutuncus sp. Unclassified Unclassified Unclassified Unclassified 5 1 The secondUnclassified most frequently Unclassified detected eukaryot Unclassifiedic phylum was Unclassified Cryptomycota1 (fungi), which 1 accountedApusozoa for 19% no of rank all the eukaryotic no rank clones and Apusomonadidaeconsisted of three OTUs.Apusomonas The third most2 frequently 2 Total 90 20 detected phylum was Ciliophora, which accounted for 14% of eukaryotic clones and consisted of six OTUs. These OTUs were classified into five families—Oxytrichidae, Vorticellidae, Phlasteridae, Spathidiidae,The most and frequently Chilodonellidae—showing detected phylum was clonal Tardigrada, frequencies which of 62, accounted 15, 8, 8, forand 53% 8%, ofrespectively, eukaryotic withinclones the and phylum was classified Ciliophora. into The class most Eutardigrada frequently (Figuredetected4). OTU Most was of identified the Tardigrada as Onychodromopsis clones were flexilisidentified. The as Diphasconnext frequentlysp., which detected accounted phylum for 44% wa ofs all Chlorophyta the eukaryotic (8%), clones. followed The other by Eutardigrada the phyla wasBacillariophyta identified as (2%),Acutuncus Apusozoasp. (2%), and Bicosocidae (no rank in the phyla level, 1%) (Figure 4).

Tardigrada (2) Cryptomycota (3) Ciliophora (6) Chlorophyta (5) Bacillariophyta (1) Apusozoa (2) family Bicosocidae (1)

Figure 4. CommunityCommunity structure structure of eukaryal clones at the phyla level. The number in parenthesis is the number of OTUs belonging to each phylum. Th Thee family Bicosocidae has no phylum name. The second most frequently detected eukaryotic phylum was Cryptomycota (fungi), which Table 3. List of eukaryotic clones. accounted for 19% of all the eukaryotic clones and consisted of three OTUs. The third most frequently detected phylum was Ciliophora, which accounted for 14% of eukaryoticNumber of clones Number and of Phylum Class Oder Family Genus consisted of six OTUs. These OTUs were classified into five families—Oxytrichidae,Clones Vorticellidae, OTUs Phlasteridae,Tardigrada Spathidiidae,Eutardigrada and Chilodonellidae—showingParachela Hypsibiidae clonal frequenciesDiphascon of 62,40 15, 8, 8, and1 8%, respectively, within the phylum Ciliophora. The most frequently detectedAcutuncus OTU was8 identified1 as OnychodromopsisCiliophora flexilisStichotrichia. The next frequentlySporadotrichida detected Oxytrichidae phylum was ChlorophytaOnychodromopsis (8%), followed6 by1 the Rigidocortex 2 1 phyla Bacillariophyta (2%), Apusozoa (2%), and Bicosocidae (no rank in the phyla level, 1%) (Figure4). Oligohymenophorea Peritrichia Vorticellidae Vorticellides 2 1

Philasterida Philasteridae Unclassified 1 1 Litostomatea Haptorida Spathidiidae Spathidium 1 1 Phyllopharyngia Chlamydodontida Chilodonellidae Phascolodon 1 1 Bacillariophyta Bacillariophyceae Bacillariophycidae Naviculales Humidophila 2 1 no rank no rank Bicosoecida Bicosoecidae Unclassified 1 1 Chlorophyta Chlorophyceae Chlamydomonadales Haematococcaceae Chlorogonium 3 1 Chlamydomonada Oogamochlamys 1 1 ceae Actinochloridaceae Macrochloris 1 1 Unclassified Unclassified 2 2 Cryptomycota Unclassified Unclassified Unclassified Unclassified 11 1 (Fungi) Unclassified Unclassified Unclassified Unclassified 5 1 Unclassified Unclassified Unclassified Unclassified 1 1 Apusozoa no rank no rank Apusomonadidae Apusomonas 2 2 Total 90 20

4. Discussion

Diversity 2019, 11, 105 8 of 15

4. Discussion

The concentrations of NO3, NO2, NH4, and SiO2 in Lake Yukidori-Ike indicated that this lake is oligotrophic in type. In particular, the PO4 concentration was below the detection limit, suggesting that the phosphate concentration may limit the biological production in this lake. The biodiversity of bacteria was remarkably higher than those of archaea and eukarya (Figures5–7). The Shannon–Wiener index and estimated number of bacterial species (Chao1 value) were comparable to those in aquatic environments in temperate or tropical regions. The previous studies regarding bacterial diversity in Antarctic lakes also reported high bacterial diversity. These previous studies and the current study used the Sanger sequencing method to reveal the SSU rRNA clones and are not so comprehensive when compared to analysis using a high-throughput sequencing method. Therefore, most of the detected clones may actually be derived from proliferating dominant organisms in the Antarctic lakes. In Lake Yukidori-Ike, Betaproteobacteria was the most dominant and diverse class in the bacterial community (Figure5). This was also observed in some previous studies conducted in Arctic freshwater environments [19–22]. The bacterial community structures found in Antarctic saline lakes were different to those in Lake Yukidori-Ike. In the saline lakes, Alphaproteobacteria were usually dominant [2–6], and some clones were found to be common with those revealed in the sea samples. Most saline lakes located in Antarctic coastal areas were formed by separation from the ocean during the uplift of the continent after the last glacial period [23]. For example, the bacterial community structure was different from that of Lake Nurume-Ike [5], also located in the Langhovde ice-free area and known as a meromictic saline lake. In Lake Nurume-Ike, Alphaproteobacteria were dominant and Betaproteobacteria were not detected. In the community structure of Cyanobacteria, the classes Oscillatoriales and Synechococcales were dominant in Lake Yukidori-Ike, whereas the class Oscillatoriales was not detected in Lake Nurume-Ike. These contrasts are observed not only in Antarctica but also in freshwater and saline lakes in temperate areas. These results suggest that the salinity affects the structure of bacterial communities more than the temperature. The archaeal diversity was much lower than the bacterial diversity in Lake Yukidori-Ike. Only two archaeal OTUs were detected (Figure8). One, sharing 97% of archaeal clones, was identified as the methanogenic euryarchaeon Methanosarcina subterranea. The type strain of this species has been isolated from groundwater in the northernmost part of Japan. They are neither psychrophilic nor psychrotolerant archaea and may be a little different physiologically to the species detected in our study. The other OTU was also identified as a methanogenic euryarchaeon, Methanosaeta sp. The closest described species was M. concilii, but the nucleotide sequence similarity between them was 98.2%, which is below the species threshold value of 98.65% [24]. The methanogen is known to be strictly anaerobe. On the other hand, the “microenvironments” in which the micro-anaerobic zones exist, are everywhere in the aerobic samples including the aerobic sediment sample used in this study. For example, methanogens belonging to the families Methanobacteriaceae and Methanosarcinaceae were detected in the aerobic sample from Antarctic cryoconite holes [25]. Our knowledge about the diversity and community structures of eukaryotic microbes in Antarctic lakes is currently limited. Unrein et al. [26] reported the composition of planktonic eukaryotes of coastal Antarctic lakes using denaturing gradient gel electrophoresis (DGGE). They frequently detected some clones affiliated with Chrysophyceae, but this group was not detected in Lake Yukidori-Ike. This difference may be due to the different salinity levels of these lakes. The eukaryotic community structure in Lake Yukidori-Ike has common characteristics with that in the Antarctic moss pillars inhabiting an Antarctic freshwater lake, Hotoke-Ike [27]. In the Antarctic moss pillar, the first, second, and third most abundant phyla were Fungal phyla, Tardigrada, and Ciliophora, respectively. These groups were also dominant in Lake Yukidori-Ike. Diversity 2019, 11, x FOR PEER REVIEW 8 of 14 studies and the current study used the Sanger sequencing method to reveal the SSU rRNA clones and are not so comprehensive when compared to analysis using a high-throughput sequencing method. Therefore, most of the detected clones may actually be derived from proliferating dominant Diversityorganisms2019, in11 ,the 105 Antarctic lakes. 9 of 15

Proteobacteria (Beta)

Proteobacteria (Gamma)

FigureFigure 5.5. PhylogeneticPhylogenetic treetree ofof Beta-Beta- andand Gamma-proteobacteriaGamma-proteobacteriaclones. clones. TheThe DNADNA databasedatabase accessionaccession numbersnumbers forfor describeddescribed speciesspecies areare in in parentheses. parentheses. TheThe barbar indicates indicates evolutionally evolutionally distance. distance. TheThe bootstrapbootstrap valuevalue ofof eacheach nodenode is is indicated. indicated.

Diversity 2019, 11, 105 10 of 15 Diversity 2019, 11, x FOR PEER REVIEW 9 of 14

Proteobacteria (Alpha)

Planctomycetes Verrucomicrobia

Proteobacteria (Delta)

Chloroflexi

Acidobacteria

Figure 6. PhylogeneticPhylogenetic tree tree of of Alpha- Alpha- and and Delta-proteoba Delta-proteobacteria,cteria, Chloroflexi Chloroﬂexi and and Acidobacteria Acidobacteria clones. clones. The DNA database accession numbers for described species are inin parentheses.parentheses. The bar indicates evolutionally distance. The The bootstrap bootstrap value of each node is indicated.

In Lake Yukidori-Ike, Betaproteobacteria was the most dominant and diverse class in the bacterial community (Figure 5). This was also observed in some previous studies conducted in Arctic freshwater environments [19–22]. The bacterial community structures found in Antarctic saline lakes were different to those in Lake Yukidori-Ike. In the saline lakes, Alphaproteobacteria were usually dominant [2–6], and some clones were found to be common with those revealed in the sea samples. Most saline lakes located in Antarctic coastal areas were formed by separation from the ocean during the uplift of the continent after the last glacial period [23]. For example, the bacterial community structure was different from that of Lake Nurume-Ike [5], also located in the Langhovde ice-free area and known as a meromictic saline lake. In Lake Nurume-Ike, Alphaproteobacteria were dominant and Betaproteobacteria were not detected. In the community structure of Cyanobacteria, the classes Oscillatoriales and Synechococcales were dominant in Lake Yukidori-Ike, whereas the class Oscillatoriales was not detected in Lake Nurume-Ike. These contrasts are observed not only in Antarctica but also in freshwater and saline lakes in temperate areas. These results suggest that the salinity affects the structure of bacterial communities more than the temperature.

Diversity 2019, 11, 105 11 of 15 Diversity 2019, 11, x FOR PEER REVIEW 10 of 14

Firmicutes

Ignavibacteria

Bacteroidetes

Cyanobacteria

Ac nobacteria

Arma monadetes

Gemma monadetes

FigureFigure 7.7.Phylogenetic Phylogenetic tree oftree Firmicutes, of Firmicutes, Ignavibacteria, Ignaviba Bacteroidetes,cteria, Bacteroidetes, Cyanobacteria, Cyanobacteria, Actinobacteria Actinobacteriaand Armatimonadetea and Armatimonadetea clones. The DNA clones. database The DNA accession database numbers accession for describednumbers for species described are in speciesparentheses. are in Theparentheses. bar indicates The bar evolutionally indicates evolut distance.ionally The distance. bootstrap The value bootstrap of each value node of is each indicated. node is indicated. Two OTUs of Tardigrada were detected in this study. The most abundant OTU (YK1E-100) shared 84%The of Tardigradal archaeal diversity clones and was showed much 98.3%lower 18Sthan rRNA the bacterial gene sequence diversity similarity in Lake with Yukidori-Ike.Diphascon pingueOnly . twoThis archaeal OTU also OTUs showed were 99.5% detected similarity (Figure with 8). anOne, environmental sharing 97% cloneof archaeal isolated clones, from anwas Antarctic identified moss as thepillar methanogenic in Lake Hotoke-Ike, euryarchaeon located Methanosarcina in another ice-free subterranea area.in The Skarvsnes type strain [27 ].of Thesethis species results has indicate been isolatedthat OTU from YK1E-100 groundwater was derived in the fromnorthernmost the same pa speciesrt of Japan. that was They found are neither in Lake psychrophilic Hotoke-Ike but nor a psychrotolerantdiﬀerent species archaea from D. and pingue may. be The a little other different Tardigrada physiologically OTU, YK1E-099, to the showed species 96.9%detected similarity in our study.with Acutuncus The other antarcticus OTU was, whichalso identified is one of as the a mostmethanogenic widespread euryarchaeon, Antarctic tardigrade Methanosaeta species sp. The [28]. closestA. antarcticus describedis also species reported was asM. the concilii most, but common the nucleotide and dominant sequence species similarity in both between terrestrial them and was lake 98.2%,environments which is in below Antarctica the species [29–31]. threshold However, value this species of 98.65% seemed [24]. much The methanogen less dominant is than knownDiphascon to be strictlysp. in Lake anaerobe. Yukidori-Ike. On the other hand, the “microenvironments” in which the micro-anaerobic zones exist, are everywhere in the aerobic samples including the aerobic sediment sample used in this study. For example, methanogens belonging to the families Methanobacteriaceae and Methanosarcinaceae were detected in the aerobic sample from Antarctic cryoconite holes [25].

Diversity 2019, 11, 105 12 of 15

Most of the eukaryotic clones that were affiliated with the phyla Tardigrada, Ciliiophora, Bacillariophyta, Chlorophyta, and Apusozoa were identified at the genus level (Figure9). However, the fungal clones could not be classified at the class level. Because the 18S rRNA gene sequences of nearly all fungal species are available in the DNA database, the fungal clones in Lake Yukidori-Ike mayDiversity be 2019 derived, 11, x fromFOR PEER novel REVIEW uncultured species. 11 of 14

Family Methanosarcinaceae

Figure 8. Phylogenetic tree of archaeal clones. The DNA database accession numbers for described Figure 8. Phylogenetic tree of archaeal clones. The DNA database accession numbers for described species are in parentheses. The bar indicates evolutionally distance. The bootstrap value of each node species are in parentheses. The bar indicates evolutionally distance. The bootstrap value of each node is indicated. Diversityis indicated. 2019, 11, x FOR PEER REVIEW 12 of 14

Our knowledge about the diversity and community structures of eukaryotic microbes in Antarctic lakes is currently limited. Unrein et al. [26] reported the composition of planktonic eukaryotes of coastal Antarctic lakes using denaturing gradient gel electrophoresis (DGGE). They frequently detected some clones affiliated with Chrysophyceae, but this group was not detected in Lake Yukidori-Ike. This difference may be due to the different salinity levels of these lakes. The eukaryotic community structure in Lake Yukidori-Ike has common characteristics with that in the Antarctic moss pillars inhabiting an Antarctic freshwater lake, Hotoke-Ike [27]. In the Antarctic moss pillar, the first, second, and third most abundant phyla were Fungal phyla, Tardigrada, and Ciliophora, respectively. These groups were also dominant in Lake Yukidori-Ike. Two OTUs of Tardigrada were detected in this study. The most abundant OTU (YK1E-100) shared 84% of Tardigradal clones and showed 98.3% 18S rRNA gene sequence similarity with Diphascon pingue. This OTU also showed 99.5% similarity with an environmental clone isolated from an Antarctic moss pillar in Lake Hotoke-Ike, located in another ice-free area in Skarvsnes [27]. These results indicate that OTU YK1E-100 was derived from the same species that was found in Lake Hotoke-Ike but a different species from D. pingue. The other Tardigrada OTU, YK1E-099, showed 96.9% similarity with Acutuncus antarcticus, which is one of the most widespread Antarctic tardigrade species [28]. A. antarcticus is also reported as the most common and dominant species in both terrestrial and lake environments in Antarctica [29–31]. However, this species seemed much less dominant than Diphascon sp. in Lake Yukidori-Ike. Most of the eukaryotic clones that were affiliated with the phyla Tardigrada, Ciliiophora, Bacillariophyta, Chlorophyta, and Apusozoa were identified at the genus level (Figure 9). However, the fungal clones could not be classified at the class level. Because the 18S rRNA gene sequences of nearly all fungal species are available in the DNA database, the fungal clones in Lake Yukidori-Ike may FigureFigurebe derived 9.9. PhylogeneticPhylogenetic from novel treetree uncultured ofof eukaryaleukaryal species. clones.clones. TheThe DNADNA databasedatabase accessionaccession numbersnumbers forfor describeddescribed speciesspecies areare inin parentheses.parentheses. TheThe barbar indicatesindicates evolutionallyevolutionally distance.distance. TheThe bootstrapbootstrap valuevalue ofof eacheach nodenode isis indicated.indicated. In this study, many OTUs could be identiﬁed only in higher taxonomic levels such as order, class, In this study, many OTUs could be identified only in higher taxonomic levels such as order, and phylum. This is probably not caused by methods used in this study. For bacterial and archaeal 16S class, and phylum. This is probably not caused by methods used in this study. For bacterial and rRNA genes, we used 700–800 bp including highly variable regions V1, V2, V3, V4, which is more archaeal 16S rRNA genes, we used 700–800 bp including highly variable regions V1, V2, V3, V4, which is more variable than the 3′ regions [32]. Also, for eukaryotic 18S rRNA genes, data containing highly variable regions V1, V2, V3 [33] was used for BLAST. The possible reasons for the presence of many unidentified OTUs are (1) many uncultured microbes exist in this lake as well as other environments, (2) many eukaryal 18S sequences are not available in the DNA database.

5. Conclusions The bacterial, archaeal, and eukaryotic microbial community structures of the Antarctic freshwater lake Yukidori-Ike were revealed using SSU rRNA clone library analysis. The bacterial and eukaryotic microbial communities consisted of remarkably diverse species in contrast to the archaeal community. The results of the present study provide novel information about the microbial diversity of Antarctic freshwater lakes and help us to understand the living organisms in the cryosphere. This study was conducted during summer, and different microbial communities may appear in the winter season. The roles of microbial communities should also be investigated using functional genes to understand the ecology of these Antarctic lakes.

Funding: This study is a part of the Science Program of Japanese Antarctic Research Expedition (JARE). It was supported by National Institute of Polar Research (NIPR) under Ministry of Education, Culture, Sports, Science and Technology (MEXT).

Acknowledgments: We thank the expedition members of JARE 54 for support in sample collection.

Diversity 2019, 11, 105 13 of 15

variable than the 30 regions [32]. Also, for eukaryotic 18S rRNA genes, data containing highly variable regions V1, V2, V3 [33] was used for BLAST. The possible reasons for the presence of many unidentiﬁed OTUs are (1) many uncultured microbes exist in this lake as well as other environments, (2) many eukaryal 18S sequences are not available in the DNA database.

5. Conclusions The bacterial, archaeal, and eukaryotic microbial community structures of the Antarctic freshwater lake Yukidori-Ike were revealed using SSU rRNA clone library analysis. The bacterial and eukaryotic microbial communities consisted of remarkably diverse species in contrast to the archaeal community. The results of the present study provide novel information about the microbial diversity of Antarctic freshwater lakes and help us to understand the living organisms in the cryosphere. This study was conducted during summer, and diﬀerent microbial communities may appear in the winter season. The roles of microbial communities should also be investigated using functional genes to understand the ecology of these Antarctic lakes.

Author Contributions: Conceptualization, N.K.; Data curation, A.C. and N.K.; Formal analysis, A.C.; Funding acquisition, S.I.; Project administration, S.I.; Resources, N.K., A.K. and M.K.; Supervision, N.K.; Writing—original draft, A.C.; Writing—review and editing, N.K. and S.I. Funding: This study is a part of the Science Program of Japanese Antarctic Research Expedition (JARE). It was supported by National Institute of Polar Research (NIPR) under Ministry of Education, Culture, Sports, Science and Technology (MEXT). Acknowledgments: We thank the expedition members of JARE 54 for support in sample collection. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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