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

: 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 Go ﬀ eauzyma 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 Go ﬀ eauzyma 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.


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
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 Isunnguata Sermia glaciers, Greenland. One to two samples were collected at each site; however, one was used for culturebased 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.
Sustainability 2020, 12, x FOR PEER REVIEW 3 of 23 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.

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 MgSO 4 , 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).

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.
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.

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.

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   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 (Tables 2 and 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 (Tables 2 and 3).

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 (Tables 2 and 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.

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 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 (Tables 1-3), 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.

Conflicts of Interest:
The authors declare no conflict of interest.