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Article

Contrasting Patterns of Microbial Communities in Glacier Cryoconite of Nepali Himalaya and Greenland, Arctic

by
Purnima Singh
1,2,
Masaharu Tsuji
3,
Shiv Mohan Singh
4,5,* and
Nozomu Takeuchi
6
1
Parvatibai Chowgule College of Arts & Science, Goa 403602, India
2
NPDF-Science and Engineering Research Board, New Delhi 110 070, India
3
Department of Materials Chemistry, Asahikawa College, National Institute of Technology, Hokkaido 071-8142, Japan
4
Polar Biology Laboratory, National Centre for Antarctic and Ocean Research, Goa 403804, India
5
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India
6
Department of Earth Sciences, Graduate School of Science, Chiba University, Chiba 263-8522, Japan
*
Author to whom correspondence should be addressed.
Sustainability 2020, 12(16), 6477; https://doi.org/10.3390/su12166477
Submission received: 10 June 2020 / Revised: 25 July 2020 / Accepted: 30 July 2020 / Published: 11 August 2020
(This article belongs to the Special Issue Microbial Diversity in Cold Environments and Their Sustainable Use)
Browse Figures
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Abstract

:
To understand the microbial composition and diversity patterns, cryoconite granules were collected from two geographical areas, i.e., Nepali Himalaya and Greenland, Arctic. 16S rRNA, ITS and the D1/D2 domain sequencing techniques were used for characterization of microbial communities of the four glaciers. The total 13 species of bacteria such as Bacillus aryabhattai, Bacillus simplex, Brevundimonas vesicularis, Cryobacterium luteum, Cryobacterium psychrotolerans, Dermacoccus nishinomiyaensis, Glaciihabitans tibetensis, Leifsonia kafniensis, Paracoccus limosus, Polaromonas glacialis, Sporosarcina globispora, Staphylococcus saprophyticus, Variovorax ginsengisoli, and 4 species of fungi such as Goffeauzyma gilvescens, Mrakia robertii, Dothideomycetes sp., Helotiales sp. were recorded from Nepali Himalaya. Among these, 12 species of bacteria and 4 species of fungi are new contributions to Himalaya. In contrast to this, six species of bacteria such as Bacillus cereus, Cryobacterium psychrotolerans, Dermacoccus nishinomiyaensis, Enhydrobacter aerosaccus, Glaciihabitans tibetensis, Subtercola frigoramans, and nine species of fungi such as Goffeauzyma gilvescens, Mrakia robertii, Naganishia vaughanmartiniae, Piskurozyma fildesensis, Rhodotorula svalbardensis, Alatospora acuminata, Articulospora sp., Phialophora sp., Thelebolus microspores, and Dothideomycetes sp.), were recorded from Qaanaaq, Isunnguata Sermia and Thule glaciers, Greenland. Among these, five species of bacteria and seven species of fungi are new contributions to Greenland cryoconite. Microbial analyses indicate that the Nepali Himalayan cryoconite colonize higher numbers of microbial species compared to the Greenland cryoconite.

1. Introduction

Cryoconite are dark-colored, bio-inorganic dusts, transported by wind and deposited on the glaciers and Sea ice [1]. Once the dust material is settled on glacial surface, it aggregates, becomes spherical-granular structure and absorbs more solar radiation creating melt holes (cryoconite holes) [2,3]. Cryoconite is mainly composed of organic matter such as algae, cyanobacteria, bacteria, fungi, rotifers and their metabolites [4,5,6,7,8], and inorganic matter as a mixture of different elements [4,9,10,11]. The dark color of cryoconite is due to combinations of minerals, organic matters and microorganisms, which reduces the albedo of glaciers and accelerates ice melting [12,13,14,15,16]. The mineral dust composition, nutrients, climate conditions, and physical features of glaciers altogether affect colonization of the microbial community in glacier cryoconite holes [13,15,17,18,19,20,21]. Cryoconite can cover about 0.1–10% of the ice surface in the ablation zone of glaciers [6] and is distributed in many parts of the world including Arctic, Antarctic, and Himalaya. Antarctic cryoconite generally remain frozen at the glacier surface while only a few of them are exposed during summer [1,22,23,24]. Arctic, Alpine and Himalayan cryoconite holes usually remain open during summer seasons [11,25]. Cryosphere constitute a major part of the land and hydrosphere, therefore, research on the cryoconite is of great significance.
Studies on Arctic cryoconite began with the investigation of brown algae [26]. Afterward, cyanobacteria [27,28], viruses and virus-like particles [29,30], diatoms [31], Bacteria [32,33,34], yeast and filamentous fungi [35,36,37,38] were focused on in Svalbard. Cryoconite holes have also been searched from the Greenland Ice Sheet (GrIS) [39,40] and Himalayan glaciers [11].
Greenland Ice Sheet is biologically active, and covers more than 200,000 km2 [41,42]. Several studies were conducted on microbial diversity using non-culturable approach in the area of the GrIS [19,43,44,45,46,47,48,49,50,51], and reported the presence of bacteria from four different classes such as Proteobacteria, Cyanobacteria, Bacteroidetes and Actinobacteria. Besides, these studies were also conducted on ice algal blooms at GrIS supraglacial habitats [52,53,54,55,56].
The culturable approach has been used for analyses of bacterial diversity from Antarctic cryoconite [57], Svalbard, Arctic [34] and Alpine glacier [58]. Recently, Perini et al. [56] analyzed bacteria and fungi from cryoconite of Greenland Ice Sheet (GrIS), and reported nine genera of culturable bacteria (Arthrobacter sp., Bacillus sp., Cryobacterium psychrotolerans, Pseudomonas sp., Janthinobacterium sp., Rugamonas rubra, Sphingomonas sp., Rhodopseudomonas sp., Undibacterium sp., and 10 genera of culturable fungi (Aspergillus conicus, Aspergillus restrictus, Articulospora sp., Cladosporium sp., Dothiora sp., Glaciozyma sp. nov.1, Mrakia sp., Penicillium bialowiezense-like, Preussia sp., Rhodotorula svalbardensis, Vishniacozyma victoriae). The studies on culturable microbes of supraglacial ecosystems have immense importance in physiological (growth of the isolates at various pH, salt tolerance ability, optimum temperature for the growth), biochemical (carbon utilization ability, antibiotic resistance patterns, fatty acid profiles) and biotechnological (enzymes, antifreeze proteins etc.) characterizations of individual species [34].
Himalaya is a unique place in the mountain ecosystems of the world. It spreads over 2500 km, starting from west-northwest to the east. Cryoconite holes are also present over the glacier’s ablation zone of Himalaya [4,11,12,59]. The different studies were conducted on microbes belonging to Nepali Himalaya such as microbial biomass [60], bacterial diversity [61,62,63], Algal community, Cyanobacteria and Planktonic diversity [64,65,66], and microbial ecology of Himalaya [67].
Literature review suggests that a study on culturable microbial communities from cryoconite habitat of Nepali Himalaya is a gap area, and Greenland Arctic are scarce, therefore, investigation is needed. The aim of current study is to investigate microbial communities sustaining in cryoconite holes at two geographical domains such as Greenland and Himalaya. The sustainability of microbial species helps them in colonization, succession and composition of communities in an extreme glacier environment.

2. Materials and Methods

2.1. Sampling Site

Cryoconite samples used in this study were collected from three Greenland glaciers: Qaanaaq (QG), Thule (TG), Isunnguata Sermia (IS), and one Nepali Himalaya glacier, Yala (YG) (Figure 1a–d). During summer months, a number of cryoconite holes were observed on the ablation zone of all glaciers. Cryoconite were collected (Figure 1e,f) from the bottom of cryoconite holes at each study site (two sites on QG, one site on TG, two sites on IS, and two sites on YG). Cryoconite granules were loose, rounded and brownish-black in color. Cryoconite granules were collected from bottom of cryoconite holes by aspirating with a sterile disposable plastic pipette into sterile tubes, and stored at −20 °C until analyses. Sampling was carried out on 15 August 2008 from Yala glacier, Nepali Himalaya, on 15 July 2012 from Qaanaaq glacier, and on 15 August 2015 from Thule and IsunnguataSermia glaciers, Greenland. One to two samples were collected at each site; however, one was used for culture- based study at different dilutions, medias and temperature gradient. The other metadata collected was pH which ranged between 5.8 and 6.2 at Nepali Himalaya and 5.8 to 6.4 at Greenland. Water temperature of all of the cryoconite holes in both Himalaya and Greenland were zero degree. EC of the water ranged 0.6 to 0.8 µS cm−1 at Nepali Himalaya and 6.2 to 8.0 µS cm−1 at Greenland. NH4 content varied 26.70 ppb at Thule glacier, 1.2–2.4 ppb at Isunnguata Sermia glacier and 0.1 to 28.4 ppb at Qaanaaq glacier. NO3 content of 25.93 ppb at Thule, 12.5–46.8 ppb at Isunnguata Sermia, and 0.0–7.3 ppb at Qaanaaq glacier was recorded.

2.2. Isolation and Culturing of Bacteria and Fungi

One gram of cryoconite was suspended in 9 mL saline, and serial dilutions procedure (10−1, 10−2 and 103) were followed [34,35]. The spread plate method (0.1 mL) was used on different medias such as Nutrient Agar (NA), 1/10 Nutrient Agar (1/10 NA), Marine Broth (MB), 1/10 Marine Broth (1/10 MB), and R2A Medium. Similarly, 100 µL of cryoconite were plated onto different media such as Rose Bengal Agar (RBA), Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) by following pour plate and spread plate techniques. Plates were incubated at 1, 4 and 15 °C for 14–30 days. Colony forming units (CFUs) appeared after incubation was observed. Bacterial and fungal isolates showed unique morphotypes were picked from each plate. Pure cultures were obtained after 2–3 subcultures for further study.

2.3. DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing of Bacteria and Fungi

Representative isolates of bacteria were selected for molecular characterization. The pure cultures were subjected to total DNA extraction. Genomic DNA was isolated following standard protocol [68]. Universal primers 16F27 [5′-CCA GAG TTT GAT CMT GGC TCA G-3′] and 16R1492 [5′-TAC GGY TAC CTT GTT ACG ACT T-3′] were used for PCR amplification of the 16S rRNA gene. The amplified PCR product was purified by PEG-NaCl precipitation and sequenced on an ABI® 3730XL automated DNA sequencer following standard protocol. Assembly was carried out using Lasergene package, and species identification was performed by using EzBioCloud database [69]. Sequences of present study were deposited at National Center for Biotechnology Information (NCBI) GenBank database.
Representative isolates of yeasts and filamentous fungi were also selected for molecular characterization. DNA was extracted using ISOPLANT II (Wako, Japan) following manufacturer’s protocol. The extracted DNA was amplified for ITS and D1/D2 regions by PCR, using KOD-plus DNA polymerase (Toyobo, Japan). PCR reaction mixture was consisted of 10–50 ng (extracted DNA, 0.2 mM dNTPs, 2.0 mM MgSO4, 0.3 µM ITS1F (5′-GTAACAAGGTTTCCGT) and NL4 (5’-GGTCCGTGTTTCAAGACGG), and 1 U KOD-plus DNA polymerase). PCR condition (5 min at 94 °C, followed by 30 cycles for 15 s at 94 °C, 30 s at 54 °C and 90 s at 68 °C) were followed. PCR products were checked by electrophoresis using 1.5% (w/v) agarose gel. Amplicons were purified on Sephacryl S-400HR (Sigma-Aldrich, Japan), and sequenced on an ABI Prism 3130xl Sequencer (Applied Biosystems). The sequences of bacteria and fungi were deposited in the DNA data bank (NCBI).

2.4. Sequence Alignment and Phylogenetic Analyses of Bacteria and Fungi

Sequence alignments of 16S rRNA gene of different bacterial isolates with the homologous sequences (retrieved from Genbank) were performed using Clustal W Molecular Evolutionary Genetics Analysis (MEGA v4.0.) software [70]. The sequences of the isolates were subjected to a NCBI BLAST search. Sequence similarity for 16S rRNA gene was analyzed and phylogenetic trees were constructed by using Neighbor-joining method [71] and Tamura-Nei model [72]. The bootstrap consensus tree [73] represents the evolutionary history of the taxa analyzed.
ITS and D1/D2 sequences of fungi were aligned using Clustal W and MEGA 7 [74]. The pairwise alignment was performed using EMBOSS Matcher-Pairwise Sequence Alignment tool [74,75,76,77,78,79,80,81,82]. Phylogenetic trees were constructed by Neighbor-joining method [71] with the Tamura-Nei model. Tree nodes were tested by bootstrap analysis with 1000 replicates, and a bootstrap percentage ≥50%.

3. Results

3.1. Characteristics of the Microbial Strains

Several bacterial CFUs appearing on the culture media plates varied at each site. It ranged 2.09 × 103 to 3.26 × 103 at Nepali Himalaya, while at Greenland, 7.93 × 103, at Thule glacier, 7.73 × 104 to 17.02×104 at Isunnguata Sermia glacier, and 3.42 × 104 to 7.78 × 104 at Qaanaaq glacier. Cultures showed different colors (red, orange, yellow, white, cream), entire margin, convex, pulvinate, opaque, and smooth texture. Among these, 75 morphologically distinct representative isolates were purified for further analyses. Of these, 36 isolates were from YG, 10 from TG, 12 from IS, and 17 from QG glaciers.
Fungal colonies of the yeast and filamentous fungi were creamish, orange, peach, pink, and white in color, with regular and irregular margin. Yeast cells occurred singly or in groups and their shape were globose or sub-globose. Based on these morphological features, distinct strains were selected for molecular identification. Of these, 15 were from Himalaya, 8 from TG, 22 from IS, and 19 from QG glaciers.
The results of different growth media, and temperature used to obtain the different isolates information included for each isolate are shown in Supplementary Tables S1 and S3. Large number of isolates (139 strains) recorded from Himalaya and Greenland’s cryoconite granules were further analyzed.

3.2. Phylogenetic Analyses of Bacteria

The identification of bacterial taxa was based on the 16S rRNA gene domain sequencing. EMBOSS Matcher Pairwise Sequence Alignment tool was used for pairwise alignment. Total sequence lengths after alignment, positions with base changes, NCBI sequence deposition numbers, sequence similarities (%), and identification are given in Table 1.
The 16S rRNA gene sequences analyses of different strains (isolates), showed 13 genera belonging to 10 families, namely, Bacillaceae (Bacillus), Staphylococcaceae (Staphylococcus), Planococcaceae (Sporosarcina) affiliated to phylum Firmicutes; Microbacteriaceae (Cryobacterium, Glaciihabitans, Leifsonia, Subtercola); Dermacoccaceae (Dermacoccus) related to phylum Actinobacteria are Gram-positive, whereas Caulobacteraceae (Brevundimonas), Comamonadaceae (Polaromonas), Rhodobacteraceae (Paracoccus), Rhodospirillaceae (Enhydrobacter), Comamonadaceae (Variovorax) affiliated to phylum Proteobacteria are Gram-negative. The affiliations of different strains of bacteria with database have been elaborated in phylogenetic trees (Figure 2, Figure 3, Figure 4 and Figure 5).
The three Gram-positive bacteria (Bacillus, Staphylococcus and Sporosarcina) belonging to the order Bacillales formed separate clades (Figure 2a). The four other Gram-positive bacteria (Cryobacterium, Glaciihabitans, Leifsonia, Dermacoccus) were affiliated to phylum Actinobacteria of order Micrococcales (Figure 2b,c and Figure 3). One other Gram-positive group Subtercola, belonging to the phylum Actinobacteria, formed a separate clade (Figure 4 and Figure 5). The three Gram-negative bacteria (Brevundimonas, Polaromonas, Variovorax) belonging to the phylum Proteobacteria formed separate clades (Figure 2d). One other Gram-negative group Enhydrobacter was affiliated to the phylum Proteobacteria of class Alphaproteobacteria (Figure 2a,b).
Comparative analyses of four glaciers belonging to Himalaya and Greenland showed contrasting pattern in bacterial species composition. Thirteen species such as Bacillus aryabhattai, Bacillus simplex, Brevundimonas vesicularis, Cryobacterium luteum, Cryobacterium psychrotolerans, Dermacoccus nishinomiyaensis, Glaciihabitans tibetensis, Leifsonia kafniensis, Paracoccus limosus, Polaromonas glacialis, Sporosarcina globispora, Staphylococcus saprophyticus, Variovorax ginsengisoli were recorded from Yala glacier, Himalaya (Table 1). In contrast to this, at Greenland, four species such as Cryobacterium psychrotolerans, Dermacoccus nishinomiyaensis, Enhydrobacter aerosaccus, Glaciihabitans tibetensis from Thule glacier. Three species such as Cryobacterium psychrotolerans, Dermacoccus nishinomiyaensis, Subtercola frigoramans belonging to Isunnguata Sermia glacier, and four species such as Bacillus cereus, Cryobacterium psychrotolerans, Glaciihabitans tibetensis, Subtercola frigoramans were recorded from Qaanaaq glacier (Table 1).

3.3. Phylogenetic Analyses of Fungi

The results of sequence analyses such as total sequence lengths, positions with base changes, NCBI sequence deposition numbers, sequence similarities (%) and identity of yeasts and filamentous fungi are given in Table 2 and Table 3.
The sequence analyses of the D1/D2 and ITS regions of 26S rRNA gene of fungal strains (isolates) showed five species of yeasts belonging to three orders such as Filobasidiales (Goffeauzyma gilvescens, Naganishia vaughanmartiniae, Piskurozyma fildesensis), Kriegeriales (Rhodotorula svalbardensis), Cystofilobasidiales (Mrakia robertii), and four species of filamentous fungi affiliated to orders, namely, Leotiales (Articulospora sp.), Thelebolales (Thelebolus microspores) and Helotiales (Alatospora acuminata, Phialophora sp.) were recorded from Himalaya and Greenland glaciers. A few strains of filamentous fungi showed similarity only at class level (Dothideomycetes) and indicated identity 99.81–100% with Dothideomycetes sp. G2-4-2 (LC514932). The affiliations of different strains of fungi with database have been elaborated. Glaciozyma antarctica CBS5942 was used as outgroup and a phylogenetic tree of yeast isolates was constructed (Figure 6a,b). Curvularia lunata JP89B-1X was used as outgroup and a phylogenetic tree of filamentous fungi was constructed (Figure 7).
Comparative analyses of fungi from four glaciers belonging to Himalaya and Greenland showed contrasting pattern in species composition. Four species such as Goffeauzyma gilvescens, Mrakia robertii, Phialophora sp. (Helotiales) and Dothideomycetes sp. were recorded from Yala glacier, Himalaya (Table 2 and Table 3). In contrast to this, at Greenland, two species such as Goffeauzyma gilvescens and Phialophora sp. (Helotiales) were recorded from Thule glacier. Five species such as Piskurozyma fildesensis, Rhodotorula svalbardensis, Mrakia robertii, Articulospora sp. (Leotiales) and Phialophora sp. (Helotiales) were analyzed from Isunnguata Sermia glacier. Five species such as Naganishia vaughanmartiniae, Rhodotorula svalbardensis, Mrakia robertii, Thelebolus microspores, Alatospora acuminate (Helotiales) and strains resembled to class Dothideomycetes were recorded from Qaanaaq glacier (Table 2 and Table 3).

3.4. Distribution Patterns of the Bacteria and Fungi in Four Glaciers’ Cryoconite of Greenland and Himalaya

The bacterial species isolated during current study from Himalaya and Greenland cryoconite granules belonged to 13 genera such as Bacillus, Brevundimonas, Cryobacterium, Dermacoccus, Enhydrobacter, Glaciihabitans, Leifsonia, Paracoccus, Polaromonas, Sporosarcina, Staphylococcus, Subtercola and Variovorax (Table 1). Number of bacterial genera at each glacier varied: 11 from Yala glacier, 4 from Thule glacier, 3 from Isunnguata Sermia glacier and 4 from Qaanaaq glacier. Cryobacterium psychrotolerans was common to all glaciers’ cryoconite. Subtercola frigoramans, Glaciihabitans tibetensis and Dermacoccus nishinomiyaensis were the next most abundant species. Brevundimonas vesicularis, Leifsonia kafniensis, Paracoccus limosus, Polaromonas glacialis, Sporosarcina globispora, Staphylococcus saprophyticus and Variovorax ginsengisoli were only present in the Himalaya and not on the GrIS (TG, IS and QG). Enhydrobacter aerosaccus was present in TG and absent in YG, IS, QG glaciers. Genera Brevundimonas, Enhydrobacter, Leifsonia, Paracoccus, Sporosarcina, Staphylococcus and Variovorax were represented the least. Bacillus aryabhattai, Bacillus simplex and Cryobacterium luteum were present only in YG glacier; Bacillus cereus was present only in QG glacier. Glaciihabitans tibetensis was represented in YG, TG and QG glaciers and Dermacoccus nishinomiyaensis was present in YG, TG, and IS glaciers. Glacier YG indicated maximum bacterial diversity representing 13 species while IS glacier showed least species diversity, having only 3 species. The current observations reveal that distribution of bacterial species exhibit contrasting patterns along Greenland and Himalayan glaciers’ cryoconite.
The yeasts and filamentous fungi from the cryoconite granules of Greenland and Nepali Himalayan glaciers belonged to nine genera namely, Goffeauzyma, Mrakia, Naganishia, Piskurozyma, Rhodotorula, Alatospora, Articulospora, Phialophora, Thelebolus (Table 2 and Table 3). Besides these, a few isolates of many filamentous fungi also belong to class Dothideomycetes. Number of fungal genera at each glacier varied: 3 from Yala glacier, 2 from Thule glacier, 5 from Isunnguata Sermia glacier and 5 from Qaanaaq glacier. The most dominant species was Mrakia robertii followed by strains of Phialophora sp. (Helotiales). Goffeauzyma gilvescens were distributed at YG and TG glaciers. Piskurozyma fildesensis was present only in IS glacier and absent in YG, TG and QG glaciers. Naganishia vaughanmartiniae was represented only at QG and absent in YG, TG and IS glaciers. Rhodotorula svalbardensis was present in IS and QG glaciers, and absent at YG and TG. Genera Alatospora, Articulospora and Thelebolus were represented the least. Articulospora was represented in IS, and absent at QG, YG and TG glaciers. Similarly, Alatospora represented only at QG. Dothideomycetes were presented in YG and QG glaciers. Glacier IS and QG showed maximum diversity representing five species each, while TG glacier indicated least diversity having only two species.

4. Discussion

Amplicon sequence-based approach in diversity analyses documented a larger assortment of microbes [16,19,43,44,45,46,47,48,50,51,56], and reported bacterial species affiliated to four classes such as Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, and a few fungi (Microbotryomycetes and Chytridiomycota). Culturable approach, as used in current study, has importance in physiological, biochemical, and biotechnological characterizations of individual species [34]. Further, it also helps in understanding inter- and intra-species interactions in an ecosystem [75,76]. In order to maximize the recovery of culturable microbes, numerous bacteriological and fungal medias of different strengths, and low temperature gradients, were applied in the current study. It was observed that out of 139 isolates, 48 were recovered from diluted media (1/10), indicating their oligotrophic characteristic, like Svalbard [34,35].
The bacterial and fungal isolates were able to grow at low temperature between 1 and 15 °C, thus confirming their psychrophilic nature. Similar studies have also been carried out from Antarctic [57], and Arctic cryoconite [34,35] observed that the microbes grew between 4 and 22 °C. In contrast to this, bacterial isolates from the alpine glaciers’ cryoconite were psychrotolerant and grew profusely at 30 °C [58].
The bacterial species from the cryoconite of Greenland and Nepali Himalaya belonged to 13 genera namely, Bacillus, Brevundimonas, Cryobacterium, Enhydrobacter, Glaciihabitans, Dermacoccus, Leifsonia, Polaromonas, Paracoccus, Staphylococcus, Sporosarcina, Subtercola and Variovorax. Genus Cryobacterium has also been reported from the Antarctic cryoconite granules [57]. Genera such as Cryobacterium, Leifsonia, Polaromonas, Subtercola were recorded from Svalbard, Arctic [34]. Two bacterial genera (Bacillus, Cryobacterium) were analyzed from cryoconite of Greenland Ice Sheet [56]. Genus Bacillus has been previously reported from cryoconite of Alpine region [58]. Further, Polaromonas and Subtercola have also been reported from extreme habitats viz. snow, permafrost and sea ice [77,78,79,80]. The yeasts (Goffeauzyma, Mrakia, Naganishia, Piskurozyma, Rhodotorula) and filamentous fungi (Alatospora, Articulospora, Phialophora, Thelebolus) from the cryoconite of Greenland and Nepali Himalayan glaciers belonged to nine genera. Among these, genera such as Articulospora, Mrakia and Rhodotorula were recorded from Svalbard, Arctic [35]. Recently, Articulospora, Mrakia and Rhodotorula have also been reported from the cryoconite of Greenland [56]. The occurrence of similar species at geographically distant places suggests their wide distribution, and adaptation strategies at low temperatures [34,76].
The comparison of microbial communities of four glaciers indicate that YG has a higher number of species diversity (16 species: 13 species of bacteria and 3 species of fungi) followed by QG (9 species: 4 species of Bacteria and 5 species of fungi), IS (8 species: 3 species of bacteria and 5 species of fungi) and then TG (6 species: 4 species of bacteria and 2 species of fungi). The glacier surface conditions such as elevation (Table 1, Table 2 and Table 3), mineral dust [81], and microclimate [34,35] probably limit the colonization of microbial communities despite close proximity of GrIS glaciers. Further, Cameron et al. [45] stated that bacterial communities (amplicon sequence-based) across the GrIS are spatially variable due to influence of localized biological inputs and physicochemical conditions. Himalaya has more intense solar radiation due to higher sun elevation and greater impacts of anthropogenic pollutions and terrestrial dust than Greenland [81]. The results, as presented, suggest that the Himalayan site has a higher microbial diversity than the Greenland sites.

5. Conclusions

Cryoconite holes are unique habitat on the cryosphere where cold-adapted microbes sustain. The current study was focused on bacterial communities of Himalayan and Greenland cryoconite through the culture-based approach. A detailed gene sequence analysis (16S rRNA, D1/D2, ITS) contributed 12 species of bacteria (Bacillus aryabhattai, Bacillus simplex, Brevundimonas vesicularis, Cryobacterium psychrotolerans, Cryobacterium luteum, Dermacoccus nishinomiyaensis, Glaciihabitans tibetensis, Staphylococcus saprophyticus, Leifsonia kafniensis, Paracoccus limosus, Sporosarcina globispora, Variovorax ginsengisoli), and 4 species of fungi (Goffeauzyma gilvescens, Mrakia robertii, Dothideomycetes sp. Helotiales sp.) as a new record from Himalaya. Furthermore, five species of bacteria (Bacillus cereus, Enhydrobacter aerosaccus, Dermacoccus nishinomiyaensis, Glaciihabitans tibetensis, Subtercola frigoramans), and seven species of fungi (Goffeauzyma gilvescens, Mrakia robertii, Naganishia vaughanmartiniae, Piskurozyma fildesensis, Thelebolus microspores, Alatospora acuminata, Phialophora sp. and Dothideomycetes sp. are as a new contribution to Greenland. Comparison of present study with Perini et al.’s [56] GrIS results showed resemblance with five genera (Bacillus, Articulospora, Cryobacterium, Mrakia, Rhodotorula) but indicated similarity at the species level only with two taxa (Cryobacterium psychrotolerans, Rhodotorula svalbardensis). Future studies on cryoconite microbes need to be focused on ecological functioning at the molecular level. Screening and biotechnological characterization of these culturable microbes may help in health, agriculture and industry.

Supplementary Materials

The following are available online at https://www.mdpi.com/2071-1050/12/16/6477/s1, Table S1: Bacterial culture details of Nepali Himalaya and Greenland glaciers, Table S2: Yeast culture details of Nepali Himalaya and Greenland glaciers, Table S3: Filamentous fungi culture details of Nepali Himalaya and Greenland glaciers.

Author Contributions

P.S. analyzes the results and writing of the original draft preparation. M.T. constructed trees. S.M.S. involved in planning of the study. N.T. contributed in sampling. All authors have read and agreed to the published version of the manuscript.

Funding

This research received funding from SERB (PDF/2016/003707), Department of Science and Technology (SR/WOS-A/LS-419/2013(G), NCAOR, JSPS KAKENHI (19H01143 and 20K21840), and from the Arctic Challenge for Sustainability II (ArCS II), Program Grant Number JPMXD1420318865.

Acknowledgments

N.T. is thankful to Department of Hydrology and Meteorology, Govt. of Nepal, P.S. thankful to SERB and DST. S.M.S. is grateful to NCAOR and BHU for support. S.M.S. is also thankful to Ms.Simantini Naik for technical help. Authors are grateful to the reviewers for fruitful suggestions. Special thanks to Almighty for fueling us to finalize and publish this article during difficult time of pandemic Covid-19.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Landscape of glaciers and sampling locations: (a). Qaanaaq glacier, Greenland,(b) Thule glacier, Greenland, (c) Isunnguata Sermia glacier, Greenland, (d) Yala glacier, Nepali Himalaya, (e) Cryoconite holes on Isunnguata Sermia Glacier in Greenland, (f) Cryoconite granules collected from Yala Glacier, Nepali Himalaya.
Figure 1. Landscape of glaciers and sampling locations: (a). Qaanaaq glacier, Greenland,(b) Thule glacier, Greenland, (c) Isunnguata Sermia glacier, Greenland, (d) Yala glacier, Nepali Himalaya, (e) Cryoconite holes on Isunnguata Sermia Glacier in Greenland, (f) Cryoconite granules collected from Yala Glacier, Nepali Himalaya.
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Figure 2. Phylogenetic tree of Bacterial strains isolated from Yala glacier, Himalaya and their closely related reference species based on the 16S rRNA gene sequences. Accession numbers are in parentheses. Tree was constructed using neighbor-joined method. (a) Strains of genera Bacillus, Staphylococcus and Sporosarcina (b) Strains of genera Cryobacterium and Glaciihabitans, (c) Strains of genera Leifsonia and Dermacoccus, (d) Strains of genera Brevundimonas, Polaromonas and Variovorax.
Figure 2. Phylogenetic tree of Bacterial strains isolated from Yala glacier, Himalaya and their closely related reference species based on the 16S rRNA gene sequences. Accession numbers are in parentheses. Tree was constructed using neighbor-joined method. (a) Strains of genera Bacillus, Staphylococcus and Sporosarcina (b) Strains of genera Cryobacterium and Glaciihabitans, (c) Strains of genera Leifsonia and Dermacoccus, (d) Strains of genera Brevundimonas, Polaromonas and Variovorax.
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Figure 3. Phylogenetic tree of Bacterial strains of genera Cryobacterium, Dermacoccus, Enhydrobacter and Glaciihabitans analyzed from Thule glacier, Greenland, and reference species based on the 16S rRNA. The accession numbers are in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
Figure 3. Phylogenetic tree of Bacterial strains of genera Cryobacterium, Dermacoccus, Enhydrobacter and Glaciihabitans analyzed from Thule glacier, Greenland, and reference species based on the 16S rRNA. The accession numbers are in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
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Figure 4. Phylogenetic tree of Bacterial strains of genera Cryobacterium, Dermacoccus, and Subtercola analyzed from Isunnguata Sermia glacier, Greenland, and reference species based on the 16S rRNA gene sequences. The accession numbers are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
Figure 4. Phylogenetic tree of Bacterial strains of genera Cryobacterium, Dermacoccus, and Subtercola analyzed from Isunnguata Sermia glacier, Greenland, and reference species based on the 16S rRNA gene sequences. The accession numbers are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
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Figure 5. Phylogenetic tree of Bacterial strains of genera Bacillus, Cryobacterium, Glaciihabitans, and Subtercola analyzed from Qaanaaq glacier, Greenland, and reference species based on the 16S rRNA gene sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
Figure 5. Phylogenetic tree of Bacterial strains of genera Bacillus, Cryobacterium, Glaciihabitans, and Subtercola analyzed from Qaanaaq glacier, Greenland, and reference species based on the 16S rRNA gene sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
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Figure 6. Phylogenetic tree of yeast species analyzed from: (a) Yala glacier, Himalaya, (b) Greenland glaciers (Qaanaaq, Thule and Isunnguata Sermia), The closely related species are based on ITS or D1/Dr2 domain of sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
Figure 6. Phylogenetic tree of yeast species analyzed from: (a) Yala glacier, Himalaya, (b) Greenland glaciers (Qaanaaq, Thule and Isunnguata Sermia), The closely related species are based on ITS or D1/Dr2 domain of sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; each branch indicates a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
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Figure 7. Phylogenetic tree of filamentous fungi analyzed from Nepali Himalaya and Greenland glaciers, and closely related species based on ITS region and D1/Dr2 domain of sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; significance of each branch is indicated by a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
Figure 7. Phylogenetic tree of filamentous fungi analyzed from Nepali Himalaya and Greenland glaciers, and closely related species based on ITS region and D1/Dr2 domain of sequences. The accession numbers of strains are shown in parentheses. Tree was constructed using neighbor-joined method; significance of each branch is indicated by a bootstrap value, and scale bar is estimated substitutions per nucleotide position.
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Table
Table 1. Sampling points, sequence length, NCBI-GenBank accession numbers and species identity of bacterial isolates by 16S rRNA gene sequences similarity (%) from cryoconite of Nepali Himalaya (Yala glacier), Greenland (Thule glacier, Isunnguata Sermia glacier, Qaanaaq glacier).
Table 1. Sampling points, sequence length, NCBI-GenBank accession numbers and species identity of bacterial isolates by 16S rRNA gene sequences similarity (%) from cryoconite of Nepali Himalaya (Yala glacier), Greenland (Thule glacier, Isunnguata Sermia glacier, Qaanaaq glacier).
Glacier NameGPS (Latitude Longitude) and ElevationStrainSequence Deposition no.Total Sequence Length after AlignmentNo. of Base Changes16S rRNA Gene Sequences Similarity (%)
Yala glacier,
Nepal		28°24′53″ N
85°36′48″ E
Elevation: 5207 m		YNC-1
YNC-9		MK248070
MF977331		1414
1405		5
24		Bacillus aryabhattai B8W22(T)
(EF114313), 98.29–99.86%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-4MF97732614913Bacillus simplex NBRC 15720(T)
(NR_042136),99.80%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-34MK24808213033Brevundimonas vesicularis NBRC 12165(T), (NR_113586), 99.77%
28°24′53″ N
85°36′48″ E
Elevation: 5207 m		YNC-32MK24808013061Cryobacterium luteum Hh 15 (HQ845193),
99.92%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-3
YNC-5
YNC-7
YNC-11
YNC-25
YNC-26
YNC-27
YNC-28		MF977325
MF977327
MF977329
MF977333
MK248075
MK248076
MF977342
MK248077		1345
1403
1404
1411
1198
1327
1403
1402		18
27
23
35
29
16
23
22		Cryobacterium psychrotolerans 0549 (DQ515963), 97.58–98.66%
28°24′53″ N
85°36′48″ E
Elevation:5207 m		YNC-10
YNC-18
YNC-21
YNC-22
YNC-23
YNC-29
YNC-30
YNC-35
YNC-38		MF977332
MF977336
MF977338
MF977339
MF977340
MK248078
MF977343
MF977344
MF977345		1409
1409
1401
1407
1404
1401
1410
1409
1406		33
36
19
27
24
20
38
36
26		Cryobacterium psychrotolerans 0549 (DQ515963), 97.30–98.79%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-14
YNC-20		MF977335
MF977337		1457
1456		9
7		Dermacoccus nishinomiyaensis DSM 20448(T) (X87757), 99.38–99.52%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-8MF977330147860Glaciihabitans tibetensis MP203(T) (KC256953), 95.94%
28°24′53″ N
85°36′48″
E Elevation: 5207 m		YNC-15
YNC-24
YNC-3
YNC-33		MK248071
MK248074
MK248079
MK248081		843
832
832
797		11
11
11
25		Leifsonia kafniensis KFC-22(T)
(AM889135), 96.86–98.70%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-19MK24807384311
28°24′53″ N
85°36′48″ E
Elevation: 5207 m		YNC-16MK248072116913Paracoccus limosus (HQ336256), 98.89%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-2
YNC-6		MF977324
MF977328		1481
1473		16
15		Polaromonas glacialis Cr4-12(T) (HM583568), 99.45–99.98%
28°24′53″ N
85°36′48″ E
Elevation: 5207 m		YNC-36MK24808314038Sporosarcina globispora DSM 4(T), (X68415), 99.43%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-12MF97733415006Staphylococcus saprophyticus subsp.saprophyticus ATCC 15305(T) (NR_074999) 99.60%
28°24′27″ N
85°36′36″ E
Elevation: 5189 m		YNC-37MK248084139426Variovorax ginsengisoli Gsoil 3165(T), (AB245358), 98.13%
Thule glacier, Greenland76°24′17″ N
69°43′18″ W
Elevation: 536 m		TGC-2
TGC-5
TGC-7
TGC-8
TGC-9
TGC-10		MF977346
MK248088
MK248090
MK248091
MK248092
MF977347		1263
1328
1328
1326
1300
1402		10
20
20
18
22
23		Cryobacterium psychrotolerans 0549
(DQ515963), 98.36–99.21–%
76°24′17″ N
69°43′18″ W
Elevation: 536 m		TGC-4MK24808714546Dermacoccus nishinomiyaensis DSM 20448(T) (X87757), 99.59%
76°24′17″ N
69°43′18″ W
Elevation: 536 m		TGC-1MK24808513097Enhydrobacter aerosaccus LMG 21877(T) (AJ550856), 99.47%
76°24′17″ N
69°43′18″ W
Elevation: 536 m		TGC-3
TGC-6		MK248086
MK248089		1393
1386		44
42		Glaciihabitans tibetensis MP203(T), (KC256953), 96.70–96.84%
Isunnguata
Greenland 		76°11′05″ N
51°58′41″ W
Elevation: 581 m		RGC-6
RGC-7
RGC-8
RGC-11
RGC-12 RGC-13		MF977348
MK248097
MK248098
MK246105
MK246106
MK246107		1407
1402
1392
1401
1401
1401		33
22
20
20
21
21		Cryobacterium psychrotolerans 0549(DQ515963), 97.65–99.05%
76°10′35″ N
51°57′59″ W
Elevation: 575 m		RGC-10 MK246104 133814Cryobacterium psychrotolerans 0549 (DQ515963), 98.95%
76°11′05″ N
51°58′41″ W
Elevation: 581 m		RGC-5MK24809613535Dermacoccus nishinomiyaensis DSM 20448(T), (X87757), 99.63%
76°10′35″ N
51°57′59″ W
Elevation: 575 m		RGC-2
RGC-3
RGC-4
RGC-9		MK248093
MK248094
MK248095
MK246103		1006
1345
1361
1454		25
26
27
37		Subtercola frigoramans K265(T)
(AF224723), 97.46–98.07%
Qaanaaq glacier, Greenland77°30′12″ N
70°51′15″ W
Elevation: 668 m		QGC-12 MK246119 14800Bacillus cereus ATCC 14579(T), (NR_114582), 100.00%
77°30′12″ N
70°51′15″ W
Elevation: 668 m		QGC-6 QGC-7
QGC-8 QGC-9		MK246113
MK246114
MK246115
MK246116		1259
1401
1401
1402		15
20
20
23		Cryobacterium psychrotolerans 0549 (DQ515963) 98.36–98.81%
77°29′27″ N
70°44′57″ W
Elevation: 247 m		QGC-10 QGC-13MK246117
MK246120		1363
1401		15
20		Cryobacterium psychrotolerans 0549(DQ515963), 98.57–98.90%
77°30′12″ N
70°51′15″ W
Elevation: 668 m		QGC-15
QGC-16		MK246122
MK246123		1328
1488		42
49		Glaciihabitans tibetensis MP203(T), (KC256953), 96.71–96.84%
77°29′27″ N
70°44′57″ W
Elevation: 247 m		QGC-1
QGC-2
QGC-3
QGC-4
QGC-11
QGC-14		MK246108
MK246109
MK246110
MK246111
MK246118
MK246121		1393
1450
1453
1446
1454
1406		26
16
18
19
19
17		Subtercola frigoramans K265(T), (AF224723), 98.13–98.90%
77°30′12″ N
70°51′15″ W
Elevation: 668 m		QGC-5
QGC-17		MK246112
MK246124		1444
1453		17
17		Subtercola frigoramans K265(T)(AF224723), 98.82–98.83%
Table
Table 2. Species names, strain names, sampling point, and NCBI-GenBank accession number of the ITS region or D1/D2 domain sequences similarity (%) of isolated yeasts of Nepali Himalaya and Greenland, Arctic.
Table 2. Species names, strain names, sampling point, and NCBI-GenBank accession number of the ITS region or D1/D2 domain sequences similarity (%) of isolated yeasts of Nepali Himalaya and Greenland, Arctic.
Identity Based on ITS and D1/D2 GeneSampling RegionSampling Location (Latitude Longitude)StrainAccession NumberTotal Sequence Length after AlignmentNo. of Base ChangesITS region and D1/D2 Sequences Similarity (%)
Filobasidiales
Goffeauzyma gilvescensYala glacier, Nepal Himalaya28°24′27″ N,
85°36′36″ E
Elevation: 5189 m		J-20 KY782275 5310 100% with the D1/D2 region of Goffeauzyma gilvescens CBS 7525T(AF181547)
Thule glacier, Greenland76°24′17″ N 69°43′18″ W
Elevation: 536 m		J-25 KY782276 5300 100% with the D1/D2 region of Goffeauzyma gilvescens CBS 7525T (AF181547)
Naganishia vaughanmartiniae Qaanaaq glacier, Greenland77°29′27″ N
70°44′57″ W
Elevation: 247 m		J-50 KY782277 3641 99.73% with the D1/D2 region of Naganishia vaughanmartiniae CBS13,731 (KY108619)
Piskurozyma fildesensisIsunnguata Sermia glacier, Greenland76°10′35″ N
51°57′59″ W
Elevation: 575 m		J-40
J-237		 KY782278
KY782279 		579
587		0 100% with the D1/D2 region of Piskurozyma fildesensis CBS12705 (KC894160)
Kriegeriales
Rhodotorula svalbardensisIsunnguata Sermia, Greenland76°11′05″ N
51°58′41″ W
Elevation: 581 m		J-131 J-174 J-216 KY782281 KY782282 KY782284 1180
1165
1169		11
9
72		93.84–99.07% with ITS region and D1/D2 domain of Rhodotorula svalbardensis MLB-I (JF805370)
Qaanaaq glacier, Greenland77°30′12″ N
70°51′15″ W
Elevation: 668 m		J-181 J-112 KY782283 KY782280 1175
1166		10
21		99.15–98.20% with ITS Region and D1/D2 domain of Rhodotorula svalbardensis MLB-I (JF805370)
Cystofilobasidiales
Mrakia robertiiYala glacier, Himalaya28°24′27″ N
85°36′36″ E
Elevation: 5189 m		J-65
J-66
J-120
J-121
J-221		 KY782285
KY782286
KY782297
KY782298
KY782307 		596
587
587
589
588		1
1
1
1
1		 99.83% with the D1/D2 region of Mrakia robertii CBS8912T (AY038811)
Mrakia robertiiYala glacier, Himalaya28°24′53″ N
85°36′48″ E
Elevation: 5207 m		J-67
J-113
J-117
J-225		 KY782287
KY782295
KY782296
KY782308 		587
595
594
589		3
1
1
1		 99.49–99.83% with the D1/D2 region of Mrakia robertii CBS8912T (AY038811)
Mrakia robertiiIsunnguata Sermia glacier, Greenland76°10′35″ N
51°57′59″ W
Elevation: 575 m		J-82
J-86
J-92
J-127
J-229
J-31
J-34
J-36
J-39
J-89
J-93v
J-94
J-130
J-205
J-209		 KY782288
KY782289
KY782290
KY782299
KY782309
KY782310
KY782311
KY782312
KY782313
KY782314
KY782315
KY782316
KY782317
KY782318
KY782319 		589
576
595
589
587
588
595
596
588
595
593
596
596
594
592		4
4
1
1
1
2
2
2
2
3
3
3
3
2
3		 99.31–99.83% with the D1/D2 region of Mrakia robertii CBS8912T(AY038811)
Mrakia robertiiQaanaaq glacier, Greenland77°29′27″ N
70°44′57″ W
Elevation: 247 m		J-102
J-103
J-104
J-105
J-133
J-134
J-135
J-136
J-138
J-139
J-214		 KY782291
KY782292
KY782293
KY782294
KY782300
KY782301
KY782302
KY782303
KY782304
KY782305
KY782306 		590
596
594
596
596
596
596
589
596
592
589		1
2
1
1
1
2
2
1
1
1
2		 99.66–99.83% with the D1/D2 region of Mrakia robertii CBS8912T (AY038811)
Table
Table 3. Species names, strain names, sampling point, and NCBI-GenBank accession number of the ITS region and D1/Dr2 domain sequences similarity (%) of isolated filamentous fungi of Nepali Himalaya and Greenland, Arctic.
Table 3. Species names, strain names, sampling point, and NCBI-GenBank accession number of the ITS region and D1/Dr2 domain sequences similarity (%) of isolated filamentous fungi of Nepali Himalaya and Greenland, Arctic.
Identity Based on ITSand D1/D2 Gene Glacier NameGPS (Latitude Longitude) and ElevationStrainSequence Deposition No.Total Sequence Length after AlignmentNo. of Base ChangesSequences Similarity (%) with Database
DothideomycetesYala, Nepali Himalaya28°24′53″ N
85°36′48″ E
Elevation: 5207 m		J-72MF0439611067299.81% with ITS region and D1/D2 domain of Dothideomycetes sp. G2-4-2 (LC514932)
Helotiales
(Phialophora sp.)		Yala, Nepali Himalaya28°24′27″ N
85°36′36″ E
Elevation: 5189 m		J-147
J-149		MF043964
MF043965		1089
1096		18
18		98.17–98.36% with ITS region and D1/D2 domain of Phialophora sp. MLB-Phi(JN113039)
28°24′53″ N,
85°36′48″ E
Elevation: 5207 m		J-150MF043966109220
Helotiales (Phialophora sp.)Thule, Greenland76°24′17″ N
69°43′18″ W
Elevation: 536 m		J-161
J-162
J-165
J-166
J-167
J-168
J-171		MF043967
MF043968
MF043969
MF043970
MF043971
MF043972
MF043973		1095
1098
1088
1092
1097
1097
1086		15
19
18
18
18
19
19		98.25–98.63% with ITS region and D1/D2 domain of Phialophora sp. MLB-Phi(JN113039)
Leotiales (Articulospora sp.)Isunnguata Sermia, Greenland76°10′35″ N
51°57′59″ W
Elevation: 575 m		J-37
J-41		MF043957
MF043958		1066
1066		30
30		98.19% with ITS region and D1/D2 domain of Articulospora tetracladia EF18 (LC131000)
Helotiales (Phialophora sp.)Isunnguata Sermia, Greenland76°11′05″ N
51°58′41″ W
Elevation: 581 m		J-175MF04397411061998.29% with ITS region and D1/D2 domain of Phialophora sp. MLB-Phi(JN113039)
DothideomycetesQaanaaq, Greenland77°29′27″ N
70°44′57″ W
Elevation: 247 m		J-49MF0439601072099.81–100% with ITS region and D1/D2 domain of Dothideomycetes sp. G2-4-2 (LC514932)
77°30′12″ N
70°51′15″ W
Elevation: 668 m		J-140MF04396310712
Helotiales
(Alatospora acuminata)		Qaanaaq, Greenland77°30′12″ N
70°51′15″ W
Elevation: 668 m		J-182MF04397610937393.32% with ITS region and D1/D2 domain of Alatospora acuminate DSM105,546 (MK353088)
Thelebolales (Thelebolus microspores)Qaanaaq, Greenland77°29′27″ N
70°44′57″ W
Elevation: 247 m		J-48MF043959481499.17% with ITS region of Thelebolus microspores CBS137501(AY957552)
77°30′12″ N
70°51′15″ W
Elevation: 668 m		J-245MF043977481499.17% with ITS region of Thelebolus microspores CBS137501(AY957552)

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Singh, P.; Tsuji, M.; Singh, S.M.; Takeuchi, N. Contrasting Patterns of Microbial Communities in Glacier Cryoconite of Nepali Himalaya and Greenland, Arctic. Sustainability 2020, 12, 6477. https://doi.org/10.3390/su12166477

AMA Style

Singh P, Tsuji M, Singh SM, Takeuchi N. Contrasting Patterns of Microbial Communities in Glacier Cryoconite of Nepali Himalaya and Greenland, Arctic. Sustainability. 2020; 12(16):6477. https://doi.org/10.3390/su12166477

Chicago/Turabian Style

Singh, Purnima, Masaharu Tsuji, Shiv Mohan Singh, and Nozomu Takeuchi. 2020. "Contrasting Patterns of Microbial Communities in Glacier Cryoconite of Nepali Himalaya and Greenland, Arctic" Sustainability 12, no. 16: 6477. https://doi.org/10.3390/su12166477

APA Style

Singh, P., Tsuji, M., Singh, S. M., & Takeuchi, N. (2020). Contrasting Patterns of Microbial Communities in Glacier Cryoconite of Nepali Himalaya and Greenland, Arctic. Sustainability, 12(16), 6477. https://doi.org/10.3390/su12166477

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