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Genetic analysis of the frozen microbiome at 7900 m a.s.l., on the South Col of Sagarmatha (Mount Everest)

Genetic analysis of the frozen microbiome at 7900 m a.s.l., on the South Col of Sagarmatha (Mount... ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 2023, VOL. 55, NO. 1, 2164999 https://doi.org/10.1080/15230430.2023.2164999 Genetic analysis of the frozen microbiome at 7900 m a.s.l., on the South Col of Sagarmatha (Mount Everest) a,b c a c d Nicholas B. Dragone , L. Baker Perry , Adam J. Solon , Anton Seimon , Tracie A. Seimon , and Steven K. Schmidt a b Ecology and Evolutionary Biology, University of Colorado Boulder, Boulder, Colorado, USA; Cooperative Institute for Research in Environmental Science, University of Colorado Boulder, Boulder, Colorado, USA; Department of Geography and Planning, Appalachian State University, Boone, North Carolina, USA; Wildlife Conservation Society, Zoological Health Program, Bronx, New York, USA ABSTRACT ARTICLE HISTORY Received 1 July 2022 Microbial communities in alpine environments >7,500 m.a.s.l. have not been well studied using Revised 8 December 2022 modern cultivation-independent sequencing approaches due to the challenges and danger asso- Accepted 1 January 2023 ciated with reaching such high elevations. For this reason, we know little about the microorganisms found in sediments on Earth’s tallest mountains, how they reach these surfaces, and how they KEYWORDS survive and remain active at such extreme elevations. Here, we explore the microbial diversity Mount Everest; alpine; recovered from three sediment samples collected from the South Col (~7,900 m.a.s.l.) of microbial ecology; Sagarmatha (Mount Everest) using both culturing and next generation sequencing approaches microbiology (16S rRNA gene, internal transcribed spacer [ITS] region, and 18S rRNA gene sequencing). Both approaches detected very low diversity of bacteria, protists, and fungi that included a combination of cosmopolitan taxa and specialized microorganisms often found at high elevations like those of the genera Modestobacter and Naganishia. Though we managed to grow viable cultures of many of these taxa, it remains likely that few, if any, can be active in situ at the South Col. Instead, these high- elevation surfaces may act as deep-freeze collection zones of organisms deposited from the atmo- sphere or left by climbers scaling the Earth’s highest mountain. Introduction elevation increases. In the Himalayas, fungi dominate sediment communities at elevations from 3,000 to High-elevation microbial communities must survive a variety of challenging conditions (D. Singh et al. 3,500 m (Petrovič, Gunde-Cimerman, and Zalar 2000; 2012; Merino et al. 2019). With an increase in elevation Margesin and Miteva 2011), archaeal ammonia oxidizers between 4,000 and 5,400 m, and bacterial ammonia comes a corresponding shift in altitude-dependent environmental conditions, including colder tempera- oxidizers up to 6,500 m (L. Zhang et al. 2009). In addi- tures, lower atmospheric pressures, lower oxygen avail- tion, phototrophic microbes are thought to be missing in dry high-elevation soils, but active photosynthetic com- ability, and lower water activity (Merino et al. 2019). Sustaining the levels of metabolic activity required to be munities have been detected in periglacial soils above active becomes more challenging as elevation increases 5,500 m in the Himalayas (S. K. Schmidt et al. 2011) and (Margesin et al. 2009; Margesin and Miteva 2011) and, above 6,000 m in soils receiving water vapor from fumaroles (Solon et al. 2018). as a result, microbial diversity tends to decrease corre- spondingly (Bryant et al. 2008; L. Zhang et al. 2009; Analysis of samples that have been collected from the Margesin and Miteva 2011). For example, in the highest alpine environments around the world (>7,000 m) suggests that these environments are typi- Transantarctic Mountains, microbial communities in higher elevation soils are significantly less diverse, have cally dominated by a small number of polyextremophilic lower biomass, and even grow more slowly than those in taxa (Lynch et al. 2012; Merino et al. 2019). Psychrophiles, like the fungus Naganishia friedmannii, lower elevation soils (Dragone et al. 2022). The identity and function of the microorganisms can also change as which has been shown to remain active in alpine CONTACT Nicholas B. Dragone nidr7164@colorado.edu; Steven K. Schmidt steve.schmidt@colorado.edu Ecology and Evolutionary Biology, University of Colorado Boulder, 1900 Pleasant Street, 334 UCB, Boulder, CO 80309. Supplemental data for this article can be accessed online at https://doi.org/10.1080/15230430.2023.2164999. © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 N. B. DRAGONE ET AL. environments above 6,000 m (S. K. Schmidt et al. 2017), of marker gene sequencing and more traditional culture- or the bacterial genus Geodermatophilus, whose type based methods, we add to previous microbiology strain was isolated in 1970 from sediment collected at research on Mt. Everest by asking two questions: Can 8,400 m.a.s.l. on Everest (Ishiguro and Fletcher 1975), we find evidence of microorganisms on terrestrial sur- are common. However, not all of the microorganisms faces >7,000 m and, if so, are these organisms viable and identified above 7,000 m are highly adapted to such an could any be active and able to survive at such extreme extreme environment. Surface snow samples collected at elevations? ~8,000 m on the north side of Everest were found to contain high biomass communities of Proteobacteria Materials and methods and Actinobacteria, including the genera Actobacterium, Acinetobacter, and Kocuria (Liu et al. Sample collection 2007), which can be found in temperate and tropical Field sampling was performed as part of the National soils around the world (Delgado-Baquerizo et al. 2018). Geographic and Rolex Perpetual Planet Everest Though Liu et al. (2007) suggested that these detected Expedition in April to May 2019. A total of three surface organisms were deposited by snowfall or eolian trans- sediment samples (0–5 cm depth) were collected in port and are probably not active or even viable in situ, sterile polyethylene bags using aseptic techniques. their findings highlight that many questions still remain Sediments were collected about 170 m SE of Camp IV about the microbial communities of the world’s highest (27.9728°N, 86.9315°E, 7,944 m.a.s.l.; Figure 1). alpine environments. The three samples were sent to the University of Mt. Everest (Sagarmatha, Chomolungma), located on Colorado Boulder, Colorado. Though the samples the China–Nepal border within the Mahalangur Himal remained sealed after collection and throughout the subrange of the Himalayas, is the world’s highest moun- entire shipping process, they did not remain frozen tain. With an elevation of 8,849 m.a.s.l., Everest’s high- throughout the shipment. After arriving at the elevation environments likely represent some of the University of Colorado, Boulder the samples were refro- most “extreme” terrestrial surfaces on Earth. Though zen and remained in storage at −70°C until they could be we know that diverse and active microbial communities processed. have been described in many lower elevation environ- ments on the mountain (L. Zhang et al. 2009; Margesin and Miteva 2011) and bacteria have been isolated from Cultivation-independent analysis samples collected at 8,400 m (Ishiguro 1969; Ishiguro Samples were removed from the freezer and particles and Fletcher 1975), questions still remain about the <1 mm in size were manually separated from the larger habitability of the mountain’s highest elevations, in par- fragments using a sterilized sediment sieve. DNA was ticular of exposed sediment surfaces >7,500 m on the then extracted from 0.5 g of these finest particles in mountain where microbial communities have never a laminar flow hood using the Qiagen DNeasy been studied using next-generation sequencing Powersoil Kit following the manufacturer’s recommen- approaches. We expect that many microorganisms may dations. An extraction blank was included to test for any never be active in such surface environments, restricted possible contamination introduced during the DNA by the extremely challenging conditions experienced at extraction. such high elevations. The DNA aliquots extracted from each of the three To better understand the impact that elevation has on sediment samples and the associated extraction blank microbial habitability and community structure, we ana- were used for a variety of polymerase chain reaction lyzed three samples collected from the South Col (PCR) amplifications to assess the archaeal/bacterial, (~7,900 m.a.s.l) of Everest. At the South Col, a ridge fungal, and microeukaryotic communities. The primer between Everest’s summit and the neighboring Lhotse, sets that were used for PCR amplification included temperatures regularly reach −33°C with an air pressure a primer pair that targets the hypervariable V4 region one-third that at sea level (~380 hPA at time of collec- of the archaeal and bacterial 16S rRNA gene (515 F: 5′- tion; Moore and Semple 2004; Matthews et al. 2020). GTGCCAGCMGCCGCGGTAA-3′ and 806-R: 5′- Additionally, measurements taken by a weather station GGACTACHVGGGTWTCTAAT-3′; Walters et al. installed at the South Col in 2019 have recorded 2016), a primer pair that targets the internal transcribed a maximum wind speed of 66.5 m/s and a maximum spacer of the fungal rRNA operon (ITS1-F: 5′- insolation of 1,500 W/m , which is greater than the CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2-R: 5′- values expected from the top of the atmosphere due to GCTGCGTTCTTCATCGATGC-3′; Bellemain et al. reflectance (Matthews et al. 2020). Using a combination 2010), and a primer pair that targets the hypervariable ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 3 Figure 1. The South Col of Mount Everest (27.9728°N, 86.9315°E). (A) Map displaying the sampling location along the Everest climbing route. Samples were collected ~170 m southeast of Camp IV from an exposed surface environment at 7,944 m.a.s.l. (B) View from sampling location back toward Camp IV. (C) The surface at the collection site was made up of fragments of variable grain size, with the smallest fragments <0.5 mm in diameter and the largest fragments ~2-5 cm in diameter. Photo credit: L. Baker Perry/National Geographic. V9 region of the 18S rRNA gene (1391-F: 5′- amplifications. All three primer sets included the appro- GTACACACCGCCCGTC-3′ and EukBR: 5′- priate Illumina adapters and unique 12 bp barcode TGATCCTTCTGCAGGTTCACCTAC-3′). One no- sequences to permit multiplexed sequencing (Caporaso template PCR control was run with each set of et al. 2012). PCRs were performed in 25 µL reaction 4 N. B. DRAGONE ET AL. volumes. PCR amplification using the ITS1-F/ITS2-R used for analysis of the 16S rRNA sequences and 18S and 1391-F/EukBR primer sets was performed using rRNA sequences and a minimum bootstrapping thresh- GoTaq Hot Start PCR Master Mix and PCR amplifica - old of 85 percent was used for ITS region sequences. tion using the 515 F/806-R pair used the Platinum II Downstream analysis of the DADA2 processed data Hot-Start PCR Master Mix (2X). Cycling parameters for was performed in R v4.0.5 (R Core Team 2017) using the the ITS1F/ITS2R primer sets consisted of an initial package “mctoolsr” (https://github.com/leffj/mctoolsr/ ) denaturation step at 94°C for 3 minutes, followed by (Leff 2016). For the 16S rRNA sequencing, ASVs asso- thirty-five cycles of denaturation at 94°C (45 seconds), ciated with chloroplasts, mitochondria, and eukaryotes annealing at 50°C (60 seconds), extension at 70°C (90 (sixty-two ASVs) were removed. ASVs that made up at seconds), and a final extension step at 72°C for 10 min- least 1 percent of the extraction blank or no-template utes. Cycling parameters for the 515 F/806 R primer set PCR control and were at least 1 percent of the samples consisted of an initial denaturation step at 94°C for on average were discarded as likely lab contaminants 2 minutes, followed by thirty-five cycles of denaturation (ASV-17). After this filtering, a total of fifty-seven bac- at 94°C (15 seconds), annealing at 60°C (15 seconds), terial ASVs remained. No archaeal sequences were iden- extension at 68°C (60 seconds), and a final extension tified from the next-generation sequencing. step at 72°C for 10 minutes. Cycling parameters for the For the fungal ITS region sequencing data, ASVs that 1391-F/EukBR primer set consisted of an initial dena- could not be classified to the phylum level (only classi- turation step at 94°C for 3 minutes, followed by thirty- fied as “fungi”) were removed prior to downstream five cycles of denaturation at 94°C (45 seconds), anneal- analysis (three ASVs). No reads were identified in the ing at 57°C (60 seconds), extension at 72°C (90 seconds), extraction blank. The thirteen reads identified in the no- and a final extension step at 72°C for 10 minutes. template PCR control were associated with the second Amplified product was visualized on a gel and then most abundant ASV in the samples (ASV-2, Naganishia) cleaned and normalized to equimolar concentrations that are often dominant members of high-altitude, low- using SequalPrep Normalization Plates. Sequencing temperature environments (S. K. Schmidt et al. 2017). was performed by the University of Colorado Boulder’s Therefore, these reads were likely not a result of labora- next-generation sequencing facility using the Illumina tory contamination but are instead most likely derived MiSeq platform. The V2 2 × 150 bp Illumina sequencing from the soil samples and represent “tag switching” kit was used for the 16S rRNA gene and the 18S rRNA events (Schnell, Bohmann, and Gilbert 2015). After gene sequencing and the 2 × 250 bp paired-end Illumina this filtering, sixteen fungal ASVs remained. sequencing kits were used for the internal transcribed For downstream analysis of the 18S rRNA gene spacer (ITS) region amplicons. Raw sequencing data can sequences, we note that two ASVs were associated with be accessed in the National Center for Biotechnology human DNA. Only one of these ASVs was identified in Information Sequence Read Archive, project accession the blanks, with 90 percent of the human reads coming number PRJNA882470. from the samples themselves. Based on this, we do not believe that these were contaminated during processing and were likely in the samples at collection. For down- Bioinformatics stream analysis, these human ASVs were removed. Additionally, two ASVs in the blank samples were not 16S rRNA gene sequences, ITS region sequences, and found in any of the three South Col samples and were 18S rRNA gene sequences from the soil extractions were removed from the data set. After this filtering, fourteen processed using DADA2 pipeline v3.8 (Callahan et al. eukaryotic ASVs remained. 2016). Sequences were quality filtered and clustered into amplicon sequence variants (ASVs). Taxonomic infor- mation was assigned to ASVs using a naive Bayesian Culturing study/culture identification classifier method (Wang et al. 2007) that takes the set of ASVs generated and compares them to a training set of To determine whether any microorganisms in these reference sequences: the 16S rRNA bacterial and samples were viable, the three sediment samples were archaeal SILVA database v132 (Quast et al. 2013; plated on seven different types of solid culture media in Yilmaz et al. 2014), the UNITE database v8.3 for fungi conditions designed to promote growth. Antarctic bac- (Nilsson et al. 2019), and SILVA 18S SSU rRNA v132 terial medium (P. Singh, Singh, and Roy 2016), modified Ref NR 99 database for eukaryotes, which includes fungi nutrient broth agar (Pulschen et al. 2017), variable nine- (Quast et al. 2013; Yilmaz et al. 2014). A minimum salt solution (Egan, Kjelleberg, and Holmström 2015), bootstrapping threshold required to return and a modified variable nine-salt solution without the a taxonomic classification of 50 percent similarity was nine-salt solution (Egan, Kjelleberg, and Holmström ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 5 2015) were chosen because they were designed for the 25 µL reaction volumes. PCR amplification of the ITS culturing bacteria from oligotrophic environments. region of the rRNA operon was performed using GoTaq Antartic bacterial medium and modified nutrient broth Hot Start PCR Master Mix in 25, while PCR amplifica - agar were designed to target organisms found in nutri- tion of the 16S rRNA gene was performed using ent-poor, cold, and dry Antarctic sediments (P. Singh, Platinum II Hot-Start PCR Master Mix (2X). Cycling Singh, and Roy 2016; Pulschen et al. 2017) and have parameters for the ITS1F/ITS4R primer sets consisted of been used successfully to grow Proteobacteria, an initial denaturation step at 94°C for 3 minutes, fol- Actinobacteria, Firmicutes, and the fungus Naganishia lowed by thirty-five cycles of denaturation at 94°C (45 (Pulschen et al. 2015; Dragone et al. 2021), which were seconds), annealing at 50°C (60 seconds), extension at the most abundant taxa identified through our 16S 70°C (90 seconds), and a final extension step at 72°C for rRNA gene and ITS region sequencing effort 10 minutes. Cycling parameters for the S_27 F/ (Figure 2). Samples were also plated on three nutrient- S_1429_R primer set consisted of an initial denaturation rich media variants, Luria-Bertani agar (Atlas 2004) and step at 94°C for 2 minutes, followed by thirty-five cycles R2A agar at pH 7 and pH 9 (Reasoner and Geldreich of denaturation at 94°C (15 seconds), annealing at 60°C 1985), which were chosen to target the copiotroph taxa (15 seconds), extension at 68°C (60 seconds), and a final identified in our next-generation sequencing approach extension step at 72°C for 10 minutes. Amplified PCR that we felt may be missed if we just used oligotrophic product from all of the cultured isolates, the six extrac- media (Figure 1, Data S1). All media were created fol- tion blanks, and the three no-template PCR controls lowing the methods described in the cited literature, but were sequenced by GENEWIZ Sanger sequencing ser- more information about the specific pH and conditions vice. Fungal DNA was sequenced using the ITS1_F can be found in Dataset S3. primer and the bacterial/archaeal DNA was sequenced One gram of each sample was vortexed and then using the S_27 F primer to capture the full-length shaken for 5 minutes in 1 mL of a 7.2 pH phosphate- sequence of the ITS rRNA operon and 16S rRNA gene, buffered solution and 60 µL of the resulting soil slurry respectively. was pipetted onto each plate (surface area of each plate: Sanger sequence data from the isolates were pro- 58 cm ) and spread across the plates using flame- cessed following the “swabs to genomes” workflow sterilized cell spreaders. Two blank plates inoculated described in Dunitz et al. (2015). No sequences from with 60 µL PBS were prepared for each media type and any of the six extraction blanks or three no-template handled in an identical manner to the twenty-eight controls were recovered. Briefly, sequences were first plates inoculated with the soil slurries. Plates of each filtered based on their length, with only sequences different media type were incubated at 4°C with regular >700 bp kept for identifications based on the 16S nocturnal/diurnal light cycles. Plated samples and the rRNA gene and only those >200 bp kept for identifica - uninoculated control plates remained under these con- tions based on the ITS region. These remaining ditions for four weeks. All unique colonies that grew sequences were cleaned with SeqTrace v0.9.1 (Stucky were isolated using a streak plate isolation technique. To 2012), after which thirty-eight 16S rRNA sequences ensure purity, cultures from the first isolation plate were and six ITS sequences remained. Taxonomy was streaked for isolation two subsequent times. assigned to all sequences using the RDP classifier DNA from each of the isolated colonies was extracted v2.10.2 (Wang et al. 2007) and the basic local alignment using the Qiagen DNeasy Ultraclean 96 Microbial Kit search tool (Altschul et al. 1990). Recovered sequences following the manufacturer’s recommendations. A total for all fungal and bacterial isolates, as well as their likely of six extraction blanks were included to control for taxonomic classification, are reported in Dataset S3. contamination introduced during the DNA extraction. The DNA aliquots extracted from each of the isolated Geodermatophilaceae phylogenetic tree creation cultures and associated extraction blanks were PCR amplified in duplicate using a primer pair that targets The phylogenetic relationship of our two Modestobacter the archaeal and bacterial 16S rRNA gene (S_27 F: 5′- isolates to previously described Modestobacter taxa was GTGCCAGCMGCCGCGGTAA-3′ and S_1429 r: 5′- determined from 850 bp sequences of the 16S rRNA GGACTACHVGGGTWTCTAAT-3′) and using the pri- gene via maximum likelihood with RaxML (Stamatakis mer pair (ITS1-F: 5′-CTTGGTCATTTAGAGG 2014). Sequences of 850 bp in length were extracted AAGTAA-3′ and ITS4-R: 5′-GCTGCGTTCTTCATC from partial or complete sequences accessed through GATGC-3′) that targets the ITS of the fungal rRNA the National Center for Biotechnology Information operon. Three no-template PCR controls were run for Nucleotide Database, including eight species of each set of amplifications. PCRs were performed in Modestobacter and ten species of Geodermatophilus 6 N. B. DRAGONE ET AL. Figure 2. The microganisms identified in the three South Col samples. (A) The bacterial genera identified through the 16S rRNA gene sequencing and the culturing study. (B) The fungal families identified through the ITS sequencing and the culturing study. (C) The identity of the microeukaryotic ASVs identified through the 18S rRNA gene sequencing. For all heat maps, the number of ASVs (or cultures) assigned with each taxonomic identification is displayed in the corresponding box and colored based on the magnitude. “NA” indicates that a taxonomic classification at that level is not avaliable. ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 7 that have been isolated from soils and sediments in cold, Results dry environments around the world, including the Environmental properties of South Col sediment G. obscurus that was previously isolated from a high- elevation surfaces on Mount Everest by Ishiguro and The properties of these sediments create a unique lithic Fletcher (1975). Other sequences included M. lapidis environment for microbial communities, most notably (Trujillo et al. 2015), M. multiseptatus (Mevs et al. defined by its extremely high elevation (>7,900 m). 2000), M. muralis (Trujillo et al. 2015), M. marinus Unsurprising, given the high elevation, the sediments (Xiao et al. 2011), M. roseus (Qin et al. 2013), were dry (water content <1.0 percent for all samples, M. italicus (Montero-Calasanz et al. 2019), mean = 0.31 percent water content) and contained low M. altitudinis (Golinska et al. 2020), M. excelsi measured concentrations of organic carbon (average = (Golinska et al. 2020), G. poikilotrophi (Montero- 0.06 percent g/g) and no detectable nitrogen. Sediment Calasanz et al. 2014), G. africanus (Montero-Calasanz, pH was an average of 9.4 across the three samples, suggest- Göker, Pötter et al. 2013a), G. siccatus (Montero- ing a basic environment. Though DNA extraction and Calasanz, Göker, Pötter et al. 2013b), G. ruber (Y. culturing were only performed on the smallest sediment Zhang et al. 2011), G. pulveris (Hezbri et al. 2016), fragments (<1 mm diameter), all three samples were made G. arenaris (Montero-Calasanz et al. 2012), up of fragments of variable grain side, with the largest G. nigrescens (Nie et al. 2012), and G. dictyosporus sediment fragments ~2-5 cm in diameter (Figure 1) show- (Montero-Calasanz et al. 2015), G. saharensis (Montero- ing the rocky nature of the substrate. Calasanz, Göker, Rohde et al. 2013). First, sequences Additional analysis of the three samples that were col- were aligned using MUSCLE v3.8.31 (Edgar 2004). lected from the South Col show that the mineralogical Aligned reads were used to construct a tree with composition (weight percentage) was on average 59.1 per- RaxML v8.0.0 (raxmlHPC -f a -m GTRGAMMA -p cent SiO , 15.7 percent Al O , 6.4 percent Fe O , 5.2 percent 2 2 3 2 3 12345 -x 12345 -number 100), including Blastococcus CaO, 2.6 percent MgO, 3.5 percent K O, and 2.1 percent deserti (NR_169414.1) as an outgroup for tree rooting Na O, with P O , TiO , and MnO each making up <1 per- 2 2 5 2 (Stamatakis 2014). The tree was visualized and anno- cent (Table 1). These “Everest series greenschists,” which tated using iTOL v6.3.2 (Letunic and Bork 2016). were described by Searle (1999) in more detail, are a type of metamorphic rock formed under the lowest temperatures and pressures experienced by the Himalayan region. These Geochemical analysis rocks also contained several trace elements, the most abun- dant of which included barium (average = 596 ppm), Sediment pH was determined according to the method strontium (average = 393 ppm), zirconium (average = 221 described in King et al. (2010). Specifically, 5 g of soil and ppm), rubidium (average = 181 ppm), zinc (average = 100 5 g of deionized water were placed in a 15-mL conical tube ppm), cerium (average = 98 ppm), chromium (average = 87 and shaken for 2 hours at 200 rpm. pH was then measured ppm), vanadium (average = 80 ppm), and lanthanum with an Orion Star A211 Benchtop pH meter. Total water (average = 51 ppm). For more details on mineralogical content of samples was measured following a method and elemental composition of samples see Dataset S1. described in Vimercati, Darcy, and Schmidt (2019). In summary, 5 g of sediment of each sample were placed in sterile glass tubes. The tubes were left open to dry at 60°C Cultivation-independent marker gene sequencing in an oven for 48 hours. Water content was measured as the percentage difference between the wet and dry sample. All three samples yielded enough PCR-amplifiable DNA Other geochemical measurements were performed on to characterize the bacterial and eukaryotic taxa using freeze-dried and crushed aliquots of the three samples. marker gene sequencing (see Dataset S2). 16S rRNA Total nitrogen (TN) content measurements were measured by the Arikaree Laboratory at the University of Colorado Table 1. Minerological composition of the three South Col sediments. Boulder using a Shimadzu TOC-L/TNM-L TOC/TN ana- Mineral Composition (weight percentage ± 1 SD) lyzer. Total organic carbon (TOC) content was measured SiO 59.15 ± 0.78 Al O 15.72 ± 0.27 by Activation Laboratories Ltd.. Lithogeochemistry analysis 2 3 Fe O 6.35 ± 0.18 2 3 was also performed by Activation Laboratories Ltd. Mineral CaO 5.17 ± 0.92 composition of the samples was performed via lithium K O 3.48 ± 0.07 MgO 2.56 ± 0.09 borate fusion/inductively couple plasma-optical emission Na O 2.10 ± 0.07 spectroscopy and the composition of trace elements was TiO 0.76 ± 0.01 P O 0.13 ± 0.01 2 5 measured via lithium borate fusion/inductively coupled MnO 0.08 ± 0.003 plasma-mass spectrometry. 8 N. B. DRAGONE ET AL. gene reads averaged 4,699 reads/sample (3,101–6,708), were streaked for isolation after the four-week incuba- ITS region reads averaged 9,556 reads/sample (661– tion. Sanger sequencing revealed that a total of thirty- 25,284 reads), and 18S rRNA gene reads averaged eight unique bacterial isolates and six fungal isolates 14,450 reads/sample (480–28,173 reads). As expected, were grown from the three sediment samples the number of taxa identified per sample (microbial (Dataset S3). richness) was low, with average richness of twenty- All bacterial cultures grown were identified as being three 16S ASVs (fifteen to thirty-three), six ITS ASVs of the phyla Actinobacteria (twenty-seven isolates), (two to fourteen), and five 18S ASVs (one to twelve). No Firmicutes (six isolates), or Proteobacteria (five isolates). archaeal sequences were identified using these methods. Eleven different families were identified, including The thirty-six bacterial ASVs identified by cultiva- Micrococcaceae (eight isolates), Propionibacteriaceae tion-independent marker gene sequencing included (seven), Microbacteriaceae (five), Bacillaceae (four), taxa of the phyla Firmicutes (sixteen ASVs), Oxalobacteraceae (three), Nocardioidaceae (three), Proteobacteria (sixteen ASVs), Actinobacteria (ten Planococcaceae (two), Intrasporangiaceae (two), ASVs), Bacteroidetes (four ASV), Chloroflexi (two Geodermatophilaceae (two), Azospirillaceae (one), and ASVs), Acidobacteria (two ASVs), Verrucomicrobia Comamonadaceae (one; Figure 2A). (two ASVs), Planctomycetes (one ASV), All of the fungal isolates were identified as being of Abditibacteriota (one ASV), and Gemmatimonadota the cold-adapted yeast genus Naganishia (Figure 2B), (one ASV). In total, thirty-one families were repre- including three isolates most closely related to sented, the most common of which were Bacillaceae N. vishniacii and two other isolates most closely related (six ASVs), Burkholderiaceae (six ASVs), to N. albidosimilis and N. adeliensis. Carnobacteriaceae (three ASVs), Corynebacteriaceae (three ASVs), Chitinophagaceae (one ASV), and Paenibacillaceae (three ASVs). All other families were Discussion represented by only one ASV (Figure 2A). All sixteen of the fungal ASVs identified through the The environmental properties of the South Col sedi- next-generation marker gene sequencing effort were of ments we analyzed in this study are not unique to either the phyla Ascomycota (nine ASVs) or Everest. Many high alpine environments experience lim- Basidiomycota (seven ASVs). The most common fungal ited water and nutrients and high pH, among other family was Filobasidiaceae (six ASVs), all of which were elevation-influenced properties (Merino et al. 2019), identified to the genus Naganishia. The seven other and still have diverse microbial communities (D. Singh fungal families included Nectriaceae (three ASVs), et al. 2012; Yuan et al. 2014; Vimercati, Darcy, and Pleosporaceae (two ASVs), Teratosphaeriaceae (one Schmidt 2019). Our three samples, however, are notable ASV), Sporormiaceae (one ASV), Cladosporiaceae (one for the extremely high elevation from which they were ASV), Trichomeriaceae (one ASV), and one collected (>7,900 m). There are only fifteen mountains Basidiomycota ASV not identified to a fungal family in the world, including Mt. Everest, with peaks higher in (Figure 2B). elevation than the South Col. To our knowledge, these The 18S rRNA gene sequencing effort identified four- samples represent the highest elevation sediment envir- teen ASVs. Most of these sequences (ten ASVs) were onment to be explored for microorganisms using culti- fungal (Figure 2C) and 77 percent of all reads recovered vation-independent next-generation sequencing from the three samples were one ASV that had 100 per- methods. Organisms found at such high elevations, if cent identity over the very short read of 129 bp with any, must be able to cope with extremely high insolation members of the genera Piskurozyma and Naganishia. (Matthews et al. 2020), cold temperatures, low atmo- Nonfungal ASVs were identified as being of the genera spheric pressures, and low oxygen availability, among Rosulabryum (one ASV) and Crassostrea (one ASV), the other corresponding environmental conditions (Merino order Ostreoida (one ASV), and an unidentified ASV et al. 2019). from the phylum Cercozoa (one ASV; Figure 2C). Though the three samples South Col sediment sam- ples were collected using aseptic techniques and sterile sampling material and remained sealed from the Culture dependent methods moment of collection until they were processed, the Growth of at least one colony occurred on all plates that samples did not remain frozen through the entire ship- were inoculated with samples. No growth occurred on ping process. For this reason, our analyses focused on any of the fourteen control plates. One hundred eighty- the diversity of organisms identified in the samples and six bacterial colonies and twenty-nine fungal colonies not on their abundance. ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 9 Taxonomic diversity of bacteria identified as being most closely related to M. roseus (Qin et al. 2013). Organisms of the genus Modestobacter are Both the Illumina marker gene sequencing and culture- often identified in other cold, dry soils and sediments based identifications yielded similar microbial diversity in (Mevs et al. 2000; Busarakam et al. 2016; Golinska et al. the South Col sediments, though the next-generation 2020) and, like the closely related genus Geodermatophilus sequencing approach identified a greater diversity of (Montero-Calasanz et al. 2017), have recently been found to microorganisms than the culturing study (ten phyla ver- have many psychrophilic and oligotrophic adaptations sus three phyla; Figure 2). Both techniques revealed that (Golinska et al. 2020; Tarlachkov et al. 2020). Genomic the most common bacterial phyla were Firmicutes, analysis of Modestobacter taxa (see Golinska et al. [2020] Proteobacteria, and Actinobacteria. At finer taxonomic for detailed summary) has found genes related to cold- levels, Illumina sequencing identified a greater diversity shock regulation (e.g., Csp protein family; Essoussi et al. of Firmicutes, whereas the culturing methods identified 2010), pathways related to desiccation tolerance via the a greater diversity of Actinobacteria, though both meth- uptake of trehalose (Reina-Bueno et al. 2012), and multiple ods identified many of the same taxa (e.g., copies of the uvrD genes that have previously been linked to Geodermatophilaceae, Nocardioidaceae). These differ - increased ultraviolet (UV) tolerance (Normand et al. 2012). ences are not surprising, because culturing methods are Of additional note is the presence of genes that allow for the inherently selective. Many of the media we used for our use of carbon monoxide and other trace gases as a source of study were designed for culturing of organisms in cold carbon and energy (e.g., coxD, coxE, coxG; Lorite et al. soil environments, where Actinobacteria often are the 2000). These potential functional attributes suggest adapta- dominant taxa (Babalola et al. 2009; D. J. Smith et al. tions that might allow for survival in environments like the 2012; Dragone et al. 2021). South Col. Both methods captured a multitude of taxa with closely Along with Geodermatophilaceae and the other cold- related sequences that have been retrieved from other cold, associated organisms described previously, the diversity dry, lithic environments. The dominant phyla identified in of bacteria we identified in the South Col samples our South Col sediments were Actinobacteria, Firmicutes, included more cosmopolitan species that are not char- and Proteobacteria, taxonomic groups that often dominate acteristic of high alpine or cold soil/sediment environ- microbial communities in Antarctic soils (Cowan et al. ments. Many taxa, including those of the families 2014). We see few Acidobacteria and Bacteroidetes, phyla Paenibacillaceae and Bacillaceae, are endospore- that are common to Arctic tundra (Nemergut et al. 2005). forming species (Montes et al. 2004; Mandic-Mulec, At a finer taxonomic resolution, the families Stefanic, and van Elsas 2015). Bradyrhizobium is Nocardioidaceae, Planococcaceae, Oxalobacteraceae, a nitrogen-fixing genus that is most often found in Geodermatophilaceae, and Chitinophagaceae have closely symbiotic association with legumes, though it is also related sequences that have previously been retrieved from a common soil taxon (Klepa et al. 2021). Alloiococcus, the Transantarctic Mountains and the McMurdo Dry Staphylococcus, and Streptococcus are genera that Valleys (J. J. Smith et al. 2006; Dragone et al. 2021, 2022), include human pathogens (Aguirre and Collins 1992; as well as from cryoconite holes on Antarctic glaciers Becher et al. 2009; Palmieri, Varaldo, and Facinelli (Sommers et al. 2018). We also see taxa that have been 2011), though the latter two often are often found in identified in other high alpine sediment environments certain soil environments (Delgado-Baquerizo et al. around the world, including near the top of Kilimanjaro 2018). (Vimercati, Darcy, and Schmidt 2019) and the Puna de Atacama Volcanic Zone (Solon et al. 2018). Other genera, Taxonomic diversity of eukaryotes like Auraticoccus and Kribbella, have been found on rocks (Cheema et al. 2020), in deserts (Saygin et al. 2019), and on Both the Illumina marker gene sequencing of the ITS the walls of stone monuments and catacombs (Alonso- region and culture-based identifications yielded similar Vega et al. 2011). microbial diversity in the South Col and showed that Of special note is our identification of members of the most of the diversity of these samples was made up of family Geodermatophilaceae, including Geodermatophilus the fungal family Filobasidiaceae. More specifically, the obscurus, which was originally isolated from a sediment majority of the fungal ASVs identified in the marker sample taken at 8,400 m.a.s.l. by the American Everest gene sequencing and all of the fungi that grew in culture expedition of 1963 and later described by Ishiguro and were of the genus Naganishia. This is not surprising, Fletcher (1975). Though G. obscurus was not identified in because Naganishia is a genus of cold-adapted poly- our samples, we did identify taxa of the related genus extremophile yeasts that are commonly found in cold Modestobacter (Figure 3), with one cultured isolate and dry ecosystems, including in Antarctica (Duarte 10 N. B. DRAGONE ET AL. Figure 3. Modestobacter and Geodermatophilus phylogenetic tree. Phylogenetic relationships displayed are based on partial 16S rRNA gene sequences of at least 800 bp in length. The twenty sequences represented in this tree include two isolates recovered during the culturing study, eight representatives of the genus Modestobacter, and ten representatives of the genus Geodermatophilus, including G. obscurus, the organism isolated from a sediment sample taken at 8,400 m.a.s.l. by the American Everest expedition of 1963. The two cultured Geodermatophilaceae isolates were most closely related to M. excelsi and M. altitudinus, taxa most often associated with cold, dry, high alpine environments like soils of the Atacama Desert and Antarctica. The tree is rooted by Blastococcus deserti, a representative of another Geodermatophilaceae family. et al. 2018; Nizovoy et al. 2021), on Kilimanjaro notable, 77 percent of all 18S rRNA gene reads were one (Vimercati, Darcy, and Schmidt 2019), and in high- ASV that was a 100 percent match to several different elevation sediments of the hyperarid Puna de Atacama yeasts in the Filoblasidiales, including numerous (Pulschen et al. 2015; S. K. Schmidt et al. 2018). In these Naganishia like N. vishniacii and the genus other environments, Naganishia is thought to survive by Piskurozyma, a fungus closely related to Naganishia. periodically coming out of dormancy during brief peri- Given the very short reads from the Illumina sequencing ods of favorable conditions (S. K. Schmidt et al. 2017). run (129 bp), we cannot definitively assign a specific This genus of cold-adapted poly-extremophilic yeasts is taxonomy to this ASV at this time, but given results commonly found in a variety of extreme cold, dry envir- from the ITS and long-read 18S data from cultures, it onments and is adapted to be resistant to high doses of is very likely that this ASV is a member of the genus UV radiation, can grow at temperatures below 0°C, and Naganishia. can survive and even grow during extreme daily freeze– thaw cycles (Vimercati et al. 2016; S. K. Schmidt et al. Implications for microbial survival in extremely 2017). high-altitude environments The non-Naganishia fungal taxa identified in the samples are mostly cosmopolitan, many of which are Our results add to previous work describing the micro- commonly found in association with plants (Gibberella, bial communities on Mount Everest and in the Alternaria, Cladosporium; Zanne et al. 2020). One taxon, Himalayas (Freeman et al. 2009; L. Zhang et al. 2009; Knufia , belongs to a genus that includes extremotolerant S. K. Schmidt et al. 2011). Previous studies have found fungi often found on rocks (Breitenbach et al. 2018). that Himalayan microbial communities are dominated The 18S rRNA gene sequencing results generally by psychrophilic fungi at elevations from 3,000 to supported the patterns described from the ITS data. 5,300 m (Petrovič, Gunde-Cimerman, and Zalar 2000; Though many of the other ASVs were not classified Margesin and Miteva 2011). At higher elevations past the phylum level, this sequencing effort revealed between 4,000 m and 6,500 m, communities of ammonia a similar mix of cosmopolitan taxa and extremotolerant oxidizers were dominated by archaeal taxa below taxa like that revealed by the ITS region sequencing. The 5,400 m and bacteria taxa above (L. Zhang et al. 2009), genus Sporisorium, for example, includes plant patho- whereas even algae and chytrids were found in soils wet gens and mutualists (Ghareeb et al. 2011) and the genus by snowmelt above 5,500 m (Freeman et al. 2009; Mrakia includes cryotolerant and xerophilic species S. K. Schmidt et al. 2011). Across all studies, microbial (Thomas-Hall et al. 2010; Turchetti et al. 2011). Most diversity decreased with increasing elevation. Our three ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 11 sediments from the South Col contained extremely low 2022). However, previous work at high elevations in the microbial diversity, much more like the microbial diver- Atacama has shown that even when air temperatures are sity found in the dry tephra “soils” on the volcanic peaks below −10°C (at 2 m height), surface soil temperatures can of the inner Atacama Desert (Costello et al. 2009; reach +5°C due to the intense insolation (D. Schmidt 1999; S. K. Schmidt et al. 2018; Solon et al. 2018) than the Schubert 2014). Organisms like Naganishia sp. may be able diversity found at lower elevations on the mountain. to take advantage of even these short periods of water This is not surprising given that microorganisms at availability beause they can grow during repeated freeze– such an extreme elevation would have to be able to thaw cycles and at constant temperatures as low as −6°C cope with correspondingly high UV radiation, extreme (Vimercati et al. 2016). Obviously, it would be hard to test cold temperatures, low atmospheric pressures, and this idea in situ, but more work on the unique microbes lower oxygen availability, among other corresponding isolated in the present study could illuminate the potential environmental conditions (Merino et al. 2019). for organisms to function during brief thaw periods at We expect that most of the organisms we identified extreme elevations. It also very likely that the South Col may have been seeded by eolian transport from other and other extremely high-elevation environments may just less extreme terrestrial surfaces. Wind-driven transport be deep-freeze collection points for deposited organisms of microorganisms and DNA is well described in (either from the atmosphere or by transiting humans). a variety of environments (Aalismail et al. 2019; Souza Given the challenge, and danger, of performing in situ et al. 2019) and has been shown to lead to the detection tests at such extreme high-elevation sites, we cannot say of organisms in environments where they cannot grow whether the brief periods when free water would be avail- (Archer et al. 2019; Maki et al. 2019). This includes on able are enough to support microbial growth. Perhaps Mount Everest, where Liu et al. (2007) found many extended monitoring by the weather station installed at cosmopolitan bacterial taxa in snow at 8,000 m. Our this site where these samples were collected will shed identification of cosmopolitan taxa and foreign plant some light on this mystery in the future (Matthews et al. and animal DNA (e.g., Rosulabryum and Crassostrea 2020; Potocki et al. 2022). DNA; Figure 2C) in these samples supports this theory. Though our culturing study could not re-create the In fact, the most dominant prokaryotic phyla extreme conditions at the South Col, our results show that (Proteobacteria, Firmicutes, and Actinobacteria) and many of the organisms, including the most ubiquitous fungal phyla (Ascomycota) identified in our samples fungal and bacterial taxa identified, are in fact viable and are the same phyla that typically dominate microbial could be grown at 4°C. Though the average air temperature sequences recovered by bioaerosol studies (Aalismail from 1991 to 2022 at the South Col was −22.6°C, well below et al. 2019; Archer et al. 2019; Souza et al. 2019; Ruiz- our culturing conditions, air temperatures in the Mount Gil et al. 2020) and include some of the taxa Liu et al. Everest region have been increasing at a rate of ~0.33°C per (2007) suggested could not be active at such high eleva- decade (Kang et al. 2022), and a record high of −1.4°C was tions on Everest. Even the most highly cold-adapted measured at the South Col in July 2022 (National genera (e.g., Modestobacter, Naganishia, or Ishiguro’s Geographic Society 2022). Organisms that are not active Geodermatophilus as described in Ishiguro and in situ now may become active in the future if the current Fletcher [1975] and Ishiguro and Wolfe [1970]) are rate of warming continues (described in Kang et al. 2022). likely to have been seeded from lower elevation source populations. Representatives of these genera have been Evidence of human-associated contamination of detected, and even isolated, from air samples collected Mount Everest around the world (J. J. Smith et al. 2006; Amato et al. 2007; S. K. Schmidt et al. 2017). The microorganisms identified in these South Col sedi- The prospect should also be raised that all, or most, ments included taxa that are often associated with organisms found at the South Col in this study rarely, if human contamination. Many of the sequences recov- ever, grow there. The organisms we isolated and grew likely ered could be deposited from mountaineers through only came out of dormancy due to the less extreme, con- coughing, nasal discharge, and associated sputum sistent conditions of the laboratory setting. Though the (Grice and Segre 2011; Wade 2013; Ibironke et al. intense insolation does indicate that melt occurs at the 2020). Staphylococcus, for example, is one of the most South Col, consistent with reports from climbers, it common skin and nose bacteria, and Streptococcus is the remains unclear whether this free water would be enough dominant genus in the human mouth (Otto 2010; to support brief periods of microbial growth, given that air Gevers et al. 2012; Ursell et al. 2013; Escapa et al. temperatures rarely rise above −10°C at such high eleva- 2018). Though these genera are widespread in nature tions on Mount Everest (Matthews et al. 2020; Potocki et al. and are not always associated with humans (e.g., Ko 12 N. B. DRAGONE ET AL. et al. 2011; Zhao et al. 2015), the sequences that were was conducted in partnership with Tribhuvan University, with approval from all relevant agencies of the Government of identified as being most closely related to our taxa based Nepal (Letter numbers 075/076, 576, and 2351, issued by the on analysis using blastn are human associated. For Department of National Parks and Wildlife Conservation of example, the Staphylococcus sequence identified in our the Government of Nepal). We also thank the communities of samples shares a 100 percent identity with the skin the Khumbu region, our field support team, and Shangri-La colonizers S. hominis and S. epidermis (Nagase et al. Nepal Trek. We also thank Jessica Henley, Matthew Gebert, Caihong Vanderburgh, and Claire Mastrangelo for help with 2002; Otto 2009), and the Streptococcus sequence shares the laboratory work; the University of Colorado Boulder Next a 100 percent identity with oral cavity colonizer S. oralis Generation Sequencing Facility and GENEWIZ for help with (Kennedy et al. 2000). Because none of these human- the sequencing effort; and The Arikaree Laboratory at CU associated taxa were identified in our extraction blanks Boulder and Activation Laboratories for assistance with the and no-template PCR controls and none grew on our geochemical analyses. Finally, we thank Sam Guilford for his control plates in our culturing study, it remains likely help creating the map in Figure 1. that these organisms were from the collected sediments. Mount Everest is heavily trafficked by climbers and Disclosure statement guides each year, prompting concerns that humans have irrevocably changed the high alpine environment. Our No potential conflict of interest was reported by the authors. study provides evidence that climbing activity may increase the transport of human-associated microorganisms onto Funding the mountain’s highest surfaces. However, we expect that at such high elevations these microorganisms are not active. This work was supported by funds provided to NBD from the We predict that if we sampled in the more human-utilized University of Colorado Boulder’s Department of Ecology and areas on the mountain we may find even more microbial Evolutionary Biology and the University of Colorado Boulder Graduate School. Publication of this article was funded by the evidence of human impact on the environment, as has been University of Colorado Boulder Libraries Open Access Fund. well described at lower elevations in the Himalayas (Rashid and Romshoo 2013; Ahmad et al. 2021; Rather et al. 2022) and around the world (Sinton, Finlay, and Hannah 1998; ORCID Joergensen and Emmerling 2006; Harwood et al. 2014). Nicholas B. Dragone http://orcid.org/0000-0002-8983- Conclusions The South Col of Everest represents a unique surface Data and materials availability environment, most notably for being >7,900 m. Despite All data used in this study are available in the main text or the the extreme elevation and corresponding high UV radia- supplementary material with the raw sequence data available tion, cold temperatures, low atmospheric pressures, lower in the National Center for Biotechnology Information (NCBI) oxygen availability, we have identified bacterial and fungal Sequence Read Database, project accession number DNA sequences and have grown bacterial and fungal PRJNA882470, BioSample accessions SAMN30937384, cultures from sediments collected from the South Col. SAMN30937385, SAMN30937386. The mix of cold-adapted and cosmopolitan species iden- tified using our methods suggests that most of these References organisms are inactive in situ and may have been seeded by eolian transport from other less extreme terrestrial Aalismail, N. A., D. K. Ngugi, R. Díaz-Rúa, I. Alam, M. Cusack, and C. M. Duarte. 2019. Functional metage- surfaces. 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Kilimanjaro physico-chemical properties. Chemosphere 119:52–58. supports both cosmopolitan and endemic microbial doi:10.1016/j.chemosphere.2014.05.060. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arctic Antarctic and Alpine Research Taylor & Francis

Genetic analysis of the frozen microbiome at 7900 m a.s.l., on the South Col of Sagarmatha (Mount Everest)

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Abstract

ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 2023, VOL. 55, NO. 1, 2164999 https://doi.org/10.1080/15230430.2023.2164999 Genetic analysis of the frozen microbiome at 7900 m a.s.l., on the South Col of Sagarmatha (Mount Everest) a,b c a c d Nicholas B. Dragone , L. Baker Perry , Adam J. Solon , Anton Seimon , Tracie A. Seimon , and Steven K. Schmidt a b Ecology and Evolutionary Biology, University of Colorado Boulder, Boulder, Colorado, USA; Cooperative Institute for Research in Environmental Science, University of Colorado Boulder, Boulder, Colorado, USA; Department of Geography and Planning, Appalachian State University, Boone, North Carolina, USA; Wildlife Conservation Society, Zoological Health Program, Bronx, New York, USA ABSTRACT ARTICLE HISTORY Received 1 July 2022 Microbial communities in alpine environments >7,500 m.a.s.l. have not been well studied using Revised 8 December 2022 modern cultivation-independent sequencing approaches due to the challenges and danger asso- Accepted 1 January 2023 ciated with reaching such high elevations. For this reason, we know little about the microorganisms found in sediments on Earth’s tallest mountains, how they reach these surfaces, and how they KEYWORDS survive and remain active at such extreme elevations. Here, we explore the microbial diversity Mount Everest; alpine; recovered from three sediment samples collected from the South Col (~7,900 m.a.s.l.) of microbial ecology; Sagarmatha (Mount Everest) using both culturing and next generation sequencing approaches microbiology (16S rRNA gene, internal transcribed spacer [ITS] region, and 18S rRNA gene sequencing). Both approaches detected very low diversity of bacteria, protists, and fungi that included a combination of cosmopolitan taxa and specialized microorganisms often found at high elevations like those of the genera Modestobacter and Naganishia. Though we managed to grow viable cultures of many of these taxa, it remains likely that few, if any, can be active in situ at the South Col. Instead, these high- elevation surfaces may act as deep-freeze collection zones of organisms deposited from the atmo- sphere or left by climbers scaling the Earth’s highest mountain. Introduction elevation increases. In the Himalayas, fungi dominate sediment communities at elevations from 3,000 to High-elevation microbial communities must survive a variety of challenging conditions (D. Singh et al. 3,500 m (Petrovič, Gunde-Cimerman, and Zalar 2000; 2012; Merino et al. 2019). With an increase in elevation Margesin and Miteva 2011), archaeal ammonia oxidizers between 4,000 and 5,400 m, and bacterial ammonia comes a corresponding shift in altitude-dependent environmental conditions, including colder tempera- oxidizers up to 6,500 m (L. Zhang et al. 2009). In addi- tures, lower atmospheric pressures, lower oxygen avail- tion, phototrophic microbes are thought to be missing in dry high-elevation soils, but active photosynthetic com- ability, and lower water activity (Merino et al. 2019). Sustaining the levels of metabolic activity required to be munities have been detected in periglacial soils above active becomes more challenging as elevation increases 5,500 m in the Himalayas (S. K. Schmidt et al. 2011) and (Margesin et al. 2009; Margesin and Miteva 2011) and, above 6,000 m in soils receiving water vapor from fumaroles (Solon et al. 2018). as a result, microbial diversity tends to decrease corre- spondingly (Bryant et al. 2008; L. Zhang et al. 2009; Analysis of samples that have been collected from the Margesin and Miteva 2011). For example, in the highest alpine environments around the world (>7,000 m) suggests that these environments are typi- Transantarctic Mountains, microbial communities in higher elevation soils are significantly less diverse, have cally dominated by a small number of polyextremophilic lower biomass, and even grow more slowly than those in taxa (Lynch et al. 2012; Merino et al. 2019). Psychrophiles, like the fungus Naganishia friedmannii, lower elevation soils (Dragone et al. 2022). The identity and function of the microorganisms can also change as which has been shown to remain active in alpine CONTACT Nicholas B. Dragone nidr7164@colorado.edu; Steven K. Schmidt steve.schmidt@colorado.edu Ecology and Evolutionary Biology, University of Colorado Boulder, 1900 Pleasant Street, 334 UCB, Boulder, CO 80309. Supplemental data for this article can be accessed online at https://doi.org/10.1080/15230430.2023.2164999. © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 N. B. DRAGONE ET AL. environments above 6,000 m (S. K. Schmidt et al. 2017), of marker gene sequencing and more traditional culture- or the bacterial genus Geodermatophilus, whose type based methods, we add to previous microbiology strain was isolated in 1970 from sediment collected at research on Mt. Everest by asking two questions: Can 8,400 m.a.s.l. on Everest (Ishiguro and Fletcher 1975), we find evidence of microorganisms on terrestrial sur- are common. However, not all of the microorganisms faces >7,000 m and, if so, are these organisms viable and identified above 7,000 m are highly adapted to such an could any be active and able to survive at such extreme extreme environment. Surface snow samples collected at elevations? ~8,000 m on the north side of Everest were found to contain high biomass communities of Proteobacteria Materials and methods and Actinobacteria, including the genera Actobacterium, Acinetobacter, and Kocuria (Liu et al. Sample collection 2007), which can be found in temperate and tropical Field sampling was performed as part of the National soils around the world (Delgado-Baquerizo et al. 2018). Geographic and Rolex Perpetual Planet Everest Though Liu et al. (2007) suggested that these detected Expedition in April to May 2019. A total of three surface organisms were deposited by snowfall or eolian trans- sediment samples (0–5 cm depth) were collected in port and are probably not active or even viable in situ, sterile polyethylene bags using aseptic techniques. their findings highlight that many questions still remain Sediments were collected about 170 m SE of Camp IV about the microbial communities of the world’s highest (27.9728°N, 86.9315°E, 7,944 m.a.s.l.; Figure 1). alpine environments. The three samples were sent to the University of Mt. Everest (Sagarmatha, Chomolungma), located on Colorado Boulder, Colorado. Though the samples the China–Nepal border within the Mahalangur Himal remained sealed after collection and throughout the subrange of the Himalayas, is the world’s highest moun- entire shipping process, they did not remain frozen tain. With an elevation of 8,849 m.a.s.l., Everest’s high- throughout the shipment. After arriving at the elevation environments likely represent some of the University of Colorado, Boulder the samples were refro- most “extreme” terrestrial surfaces on Earth. Though zen and remained in storage at −70°C until they could be we know that diverse and active microbial communities processed. have been described in many lower elevation environ- ments on the mountain (L. Zhang et al. 2009; Margesin and Miteva 2011) and bacteria have been isolated from Cultivation-independent analysis samples collected at 8,400 m (Ishiguro 1969; Ishiguro Samples were removed from the freezer and particles and Fletcher 1975), questions still remain about the <1 mm in size were manually separated from the larger habitability of the mountain’s highest elevations, in par- fragments using a sterilized sediment sieve. DNA was ticular of exposed sediment surfaces >7,500 m on the then extracted from 0.5 g of these finest particles in mountain where microbial communities have never a laminar flow hood using the Qiagen DNeasy been studied using next-generation sequencing Powersoil Kit following the manufacturer’s recommen- approaches. We expect that many microorganisms may dations. An extraction blank was included to test for any never be active in such surface environments, restricted possible contamination introduced during the DNA by the extremely challenging conditions experienced at extraction. such high elevations. The DNA aliquots extracted from each of the three To better understand the impact that elevation has on sediment samples and the associated extraction blank microbial habitability and community structure, we ana- were used for a variety of polymerase chain reaction lyzed three samples collected from the South Col (PCR) amplifications to assess the archaeal/bacterial, (~7,900 m.a.s.l) of Everest. At the South Col, a ridge fungal, and microeukaryotic communities. The primer between Everest’s summit and the neighboring Lhotse, sets that were used for PCR amplification included temperatures regularly reach −33°C with an air pressure a primer pair that targets the hypervariable V4 region one-third that at sea level (~380 hPA at time of collec- of the archaeal and bacterial 16S rRNA gene (515 F: 5′- tion; Moore and Semple 2004; Matthews et al. 2020). GTGCCAGCMGCCGCGGTAA-3′ and 806-R: 5′- Additionally, measurements taken by a weather station GGACTACHVGGGTWTCTAAT-3′; Walters et al. installed at the South Col in 2019 have recorded 2016), a primer pair that targets the internal transcribed a maximum wind speed of 66.5 m/s and a maximum spacer of the fungal rRNA operon (ITS1-F: 5′- insolation of 1,500 W/m , which is greater than the CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2-R: 5′- values expected from the top of the atmosphere due to GCTGCGTTCTTCATCGATGC-3′; Bellemain et al. reflectance (Matthews et al. 2020). Using a combination 2010), and a primer pair that targets the hypervariable ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 3 Figure 1. The South Col of Mount Everest (27.9728°N, 86.9315°E). (A) Map displaying the sampling location along the Everest climbing route. Samples were collected ~170 m southeast of Camp IV from an exposed surface environment at 7,944 m.a.s.l. (B) View from sampling location back toward Camp IV. (C) The surface at the collection site was made up of fragments of variable grain size, with the smallest fragments <0.5 mm in diameter and the largest fragments ~2-5 cm in diameter. Photo credit: L. Baker Perry/National Geographic. V9 region of the 18S rRNA gene (1391-F: 5′- amplifications. All three primer sets included the appro- GTACACACCGCCCGTC-3′ and EukBR: 5′- priate Illumina adapters and unique 12 bp barcode TGATCCTTCTGCAGGTTCACCTAC-3′). One no- sequences to permit multiplexed sequencing (Caporaso template PCR control was run with each set of et al. 2012). PCRs were performed in 25 µL reaction 4 N. B. DRAGONE ET AL. volumes. PCR amplification using the ITS1-F/ITS2-R used for analysis of the 16S rRNA sequences and 18S and 1391-F/EukBR primer sets was performed using rRNA sequences and a minimum bootstrapping thresh- GoTaq Hot Start PCR Master Mix and PCR amplifica - old of 85 percent was used for ITS region sequences. tion using the 515 F/806-R pair used the Platinum II Downstream analysis of the DADA2 processed data Hot-Start PCR Master Mix (2X). Cycling parameters for was performed in R v4.0.5 (R Core Team 2017) using the the ITS1F/ITS2R primer sets consisted of an initial package “mctoolsr” (https://github.com/leffj/mctoolsr/ ) denaturation step at 94°C for 3 minutes, followed by (Leff 2016). For the 16S rRNA sequencing, ASVs asso- thirty-five cycles of denaturation at 94°C (45 seconds), ciated with chloroplasts, mitochondria, and eukaryotes annealing at 50°C (60 seconds), extension at 70°C (90 (sixty-two ASVs) were removed. ASVs that made up at seconds), and a final extension step at 72°C for 10 min- least 1 percent of the extraction blank or no-template utes. Cycling parameters for the 515 F/806 R primer set PCR control and were at least 1 percent of the samples consisted of an initial denaturation step at 94°C for on average were discarded as likely lab contaminants 2 minutes, followed by thirty-five cycles of denaturation (ASV-17). After this filtering, a total of fifty-seven bac- at 94°C (15 seconds), annealing at 60°C (15 seconds), terial ASVs remained. No archaeal sequences were iden- extension at 68°C (60 seconds), and a final extension tified from the next-generation sequencing. step at 72°C for 10 minutes. Cycling parameters for the For the fungal ITS region sequencing data, ASVs that 1391-F/EukBR primer set consisted of an initial dena- could not be classified to the phylum level (only classi- turation step at 94°C for 3 minutes, followed by thirty- fied as “fungi”) were removed prior to downstream five cycles of denaturation at 94°C (45 seconds), anneal- analysis (three ASVs). No reads were identified in the ing at 57°C (60 seconds), extension at 72°C (90 seconds), extraction blank. The thirteen reads identified in the no- and a final extension step at 72°C for 10 minutes. template PCR control were associated with the second Amplified product was visualized on a gel and then most abundant ASV in the samples (ASV-2, Naganishia) cleaned and normalized to equimolar concentrations that are often dominant members of high-altitude, low- using SequalPrep Normalization Plates. Sequencing temperature environments (S. K. Schmidt et al. 2017). was performed by the University of Colorado Boulder’s Therefore, these reads were likely not a result of labora- next-generation sequencing facility using the Illumina tory contamination but are instead most likely derived MiSeq platform. The V2 2 × 150 bp Illumina sequencing from the soil samples and represent “tag switching” kit was used for the 16S rRNA gene and the 18S rRNA events (Schnell, Bohmann, and Gilbert 2015). After gene sequencing and the 2 × 250 bp paired-end Illumina this filtering, sixteen fungal ASVs remained. sequencing kits were used for the internal transcribed For downstream analysis of the 18S rRNA gene spacer (ITS) region amplicons. Raw sequencing data can sequences, we note that two ASVs were associated with be accessed in the National Center for Biotechnology human DNA. Only one of these ASVs was identified in Information Sequence Read Archive, project accession the blanks, with 90 percent of the human reads coming number PRJNA882470. from the samples themselves. Based on this, we do not believe that these were contaminated during processing and were likely in the samples at collection. For down- Bioinformatics stream analysis, these human ASVs were removed. Additionally, two ASVs in the blank samples were not 16S rRNA gene sequences, ITS region sequences, and found in any of the three South Col samples and were 18S rRNA gene sequences from the soil extractions were removed from the data set. After this filtering, fourteen processed using DADA2 pipeline v3.8 (Callahan et al. eukaryotic ASVs remained. 2016). Sequences were quality filtered and clustered into amplicon sequence variants (ASVs). Taxonomic infor- mation was assigned to ASVs using a naive Bayesian Culturing study/culture identification classifier method (Wang et al. 2007) that takes the set of ASVs generated and compares them to a training set of To determine whether any microorganisms in these reference sequences: the 16S rRNA bacterial and samples were viable, the three sediment samples were archaeal SILVA database v132 (Quast et al. 2013; plated on seven different types of solid culture media in Yilmaz et al. 2014), the UNITE database v8.3 for fungi conditions designed to promote growth. Antarctic bac- (Nilsson et al. 2019), and SILVA 18S SSU rRNA v132 terial medium (P. Singh, Singh, and Roy 2016), modified Ref NR 99 database for eukaryotes, which includes fungi nutrient broth agar (Pulschen et al. 2017), variable nine- (Quast et al. 2013; Yilmaz et al. 2014). A minimum salt solution (Egan, Kjelleberg, and Holmström 2015), bootstrapping threshold required to return and a modified variable nine-salt solution without the a taxonomic classification of 50 percent similarity was nine-salt solution (Egan, Kjelleberg, and Holmström ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 5 2015) were chosen because they were designed for the 25 µL reaction volumes. PCR amplification of the ITS culturing bacteria from oligotrophic environments. region of the rRNA operon was performed using GoTaq Antartic bacterial medium and modified nutrient broth Hot Start PCR Master Mix in 25, while PCR amplifica - agar were designed to target organisms found in nutri- tion of the 16S rRNA gene was performed using ent-poor, cold, and dry Antarctic sediments (P. Singh, Platinum II Hot-Start PCR Master Mix (2X). Cycling Singh, and Roy 2016; Pulschen et al. 2017) and have parameters for the ITS1F/ITS4R primer sets consisted of been used successfully to grow Proteobacteria, an initial denaturation step at 94°C for 3 minutes, fol- Actinobacteria, Firmicutes, and the fungus Naganishia lowed by thirty-five cycles of denaturation at 94°C (45 (Pulschen et al. 2015; Dragone et al. 2021), which were seconds), annealing at 50°C (60 seconds), extension at the most abundant taxa identified through our 16S 70°C (90 seconds), and a final extension step at 72°C for rRNA gene and ITS region sequencing effort 10 minutes. Cycling parameters for the S_27 F/ (Figure 2). Samples were also plated on three nutrient- S_1429_R primer set consisted of an initial denaturation rich media variants, Luria-Bertani agar (Atlas 2004) and step at 94°C for 2 minutes, followed by thirty-five cycles R2A agar at pH 7 and pH 9 (Reasoner and Geldreich of denaturation at 94°C (15 seconds), annealing at 60°C 1985), which were chosen to target the copiotroph taxa (15 seconds), extension at 68°C (60 seconds), and a final identified in our next-generation sequencing approach extension step at 72°C for 10 minutes. Amplified PCR that we felt may be missed if we just used oligotrophic product from all of the cultured isolates, the six extrac- media (Figure 1, Data S1). All media were created fol- tion blanks, and the three no-template PCR controls lowing the methods described in the cited literature, but were sequenced by GENEWIZ Sanger sequencing ser- more information about the specific pH and conditions vice. Fungal DNA was sequenced using the ITS1_F can be found in Dataset S3. primer and the bacterial/archaeal DNA was sequenced One gram of each sample was vortexed and then using the S_27 F primer to capture the full-length shaken for 5 minutes in 1 mL of a 7.2 pH phosphate- sequence of the ITS rRNA operon and 16S rRNA gene, buffered solution and 60 µL of the resulting soil slurry respectively. was pipetted onto each plate (surface area of each plate: Sanger sequence data from the isolates were pro- 58 cm ) and spread across the plates using flame- cessed following the “swabs to genomes” workflow sterilized cell spreaders. Two blank plates inoculated described in Dunitz et al. (2015). No sequences from with 60 µL PBS were prepared for each media type and any of the six extraction blanks or three no-template handled in an identical manner to the twenty-eight controls were recovered. Briefly, sequences were first plates inoculated with the soil slurries. Plates of each filtered based on their length, with only sequences different media type were incubated at 4°C with regular >700 bp kept for identifications based on the 16S nocturnal/diurnal light cycles. Plated samples and the rRNA gene and only those >200 bp kept for identifica - uninoculated control plates remained under these con- tions based on the ITS region. These remaining ditions for four weeks. All unique colonies that grew sequences were cleaned with SeqTrace v0.9.1 (Stucky were isolated using a streak plate isolation technique. To 2012), after which thirty-eight 16S rRNA sequences ensure purity, cultures from the first isolation plate were and six ITS sequences remained. Taxonomy was streaked for isolation two subsequent times. assigned to all sequences using the RDP classifier DNA from each of the isolated colonies was extracted v2.10.2 (Wang et al. 2007) and the basic local alignment using the Qiagen DNeasy Ultraclean 96 Microbial Kit search tool (Altschul et al. 1990). Recovered sequences following the manufacturer’s recommendations. A total for all fungal and bacterial isolates, as well as their likely of six extraction blanks were included to control for taxonomic classification, are reported in Dataset S3. contamination introduced during the DNA extraction. The DNA aliquots extracted from each of the isolated Geodermatophilaceae phylogenetic tree creation cultures and associated extraction blanks were PCR amplified in duplicate using a primer pair that targets The phylogenetic relationship of our two Modestobacter the archaeal and bacterial 16S rRNA gene (S_27 F: 5′- isolates to previously described Modestobacter taxa was GTGCCAGCMGCCGCGGTAA-3′ and S_1429 r: 5′- determined from 850 bp sequences of the 16S rRNA GGACTACHVGGGTWTCTAAT-3′) and using the pri- gene via maximum likelihood with RaxML (Stamatakis mer pair (ITS1-F: 5′-CTTGGTCATTTAGAGG 2014). Sequences of 850 bp in length were extracted AAGTAA-3′ and ITS4-R: 5′-GCTGCGTTCTTCATC from partial or complete sequences accessed through GATGC-3′) that targets the ITS of the fungal rRNA the National Center for Biotechnology Information operon. Three no-template PCR controls were run for Nucleotide Database, including eight species of each set of amplifications. PCRs were performed in Modestobacter and ten species of Geodermatophilus 6 N. B. DRAGONE ET AL. Figure 2. The microganisms identified in the three South Col samples. (A) The bacterial genera identified through the 16S rRNA gene sequencing and the culturing study. (B) The fungal families identified through the ITS sequencing and the culturing study. (C) The identity of the microeukaryotic ASVs identified through the 18S rRNA gene sequencing. For all heat maps, the number of ASVs (or cultures) assigned with each taxonomic identification is displayed in the corresponding box and colored based on the magnitude. “NA” indicates that a taxonomic classification at that level is not avaliable. ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 7 that have been isolated from soils and sediments in cold, Results dry environments around the world, including the Environmental properties of South Col sediment G. obscurus that was previously isolated from a high- elevation surfaces on Mount Everest by Ishiguro and The properties of these sediments create a unique lithic Fletcher (1975). Other sequences included M. lapidis environment for microbial communities, most notably (Trujillo et al. 2015), M. multiseptatus (Mevs et al. defined by its extremely high elevation (>7,900 m). 2000), M. muralis (Trujillo et al. 2015), M. marinus Unsurprising, given the high elevation, the sediments (Xiao et al. 2011), M. roseus (Qin et al. 2013), were dry (water content <1.0 percent for all samples, M. italicus (Montero-Calasanz et al. 2019), mean = 0.31 percent water content) and contained low M. altitudinis (Golinska et al. 2020), M. excelsi measured concentrations of organic carbon (average = (Golinska et al. 2020), G. poikilotrophi (Montero- 0.06 percent g/g) and no detectable nitrogen. Sediment Calasanz et al. 2014), G. africanus (Montero-Calasanz, pH was an average of 9.4 across the three samples, suggest- Göker, Pötter et al. 2013a), G. siccatus (Montero- ing a basic environment. Though DNA extraction and Calasanz, Göker, Pötter et al. 2013b), G. ruber (Y. culturing were only performed on the smallest sediment Zhang et al. 2011), G. pulveris (Hezbri et al. 2016), fragments (<1 mm diameter), all three samples were made G. arenaris (Montero-Calasanz et al. 2012), up of fragments of variable grain side, with the largest G. nigrescens (Nie et al. 2012), and G. dictyosporus sediment fragments ~2-5 cm in diameter (Figure 1) show- (Montero-Calasanz et al. 2015), G. saharensis (Montero- ing the rocky nature of the substrate. Calasanz, Göker, Rohde et al. 2013). First, sequences Additional analysis of the three samples that were col- were aligned using MUSCLE v3.8.31 (Edgar 2004). lected from the South Col show that the mineralogical Aligned reads were used to construct a tree with composition (weight percentage) was on average 59.1 per- RaxML v8.0.0 (raxmlHPC -f a -m GTRGAMMA -p cent SiO , 15.7 percent Al O , 6.4 percent Fe O , 5.2 percent 2 2 3 2 3 12345 -x 12345 -number 100), including Blastococcus CaO, 2.6 percent MgO, 3.5 percent K O, and 2.1 percent deserti (NR_169414.1) as an outgroup for tree rooting Na O, with P O , TiO , and MnO each making up <1 per- 2 2 5 2 (Stamatakis 2014). The tree was visualized and anno- cent (Table 1). These “Everest series greenschists,” which tated using iTOL v6.3.2 (Letunic and Bork 2016). were described by Searle (1999) in more detail, are a type of metamorphic rock formed under the lowest temperatures and pressures experienced by the Himalayan region. These Geochemical analysis rocks also contained several trace elements, the most abun- dant of which included barium (average = 596 ppm), Sediment pH was determined according to the method strontium (average = 393 ppm), zirconium (average = 221 described in King et al. (2010). Specifically, 5 g of soil and ppm), rubidium (average = 181 ppm), zinc (average = 100 5 g of deionized water were placed in a 15-mL conical tube ppm), cerium (average = 98 ppm), chromium (average = 87 and shaken for 2 hours at 200 rpm. pH was then measured ppm), vanadium (average = 80 ppm), and lanthanum with an Orion Star A211 Benchtop pH meter. Total water (average = 51 ppm). For more details on mineralogical content of samples was measured following a method and elemental composition of samples see Dataset S1. described in Vimercati, Darcy, and Schmidt (2019). In summary, 5 g of sediment of each sample were placed in sterile glass tubes. The tubes were left open to dry at 60°C Cultivation-independent marker gene sequencing in an oven for 48 hours. Water content was measured as the percentage difference between the wet and dry sample. All three samples yielded enough PCR-amplifiable DNA Other geochemical measurements were performed on to characterize the bacterial and eukaryotic taxa using freeze-dried and crushed aliquots of the three samples. marker gene sequencing (see Dataset S2). 16S rRNA Total nitrogen (TN) content measurements were measured by the Arikaree Laboratory at the University of Colorado Table 1. Minerological composition of the three South Col sediments. Boulder using a Shimadzu TOC-L/TNM-L TOC/TN ana- Mineral Composition (weight percentage ± 1 SD) lyzer. Total organic carbon (TOC) content was measured SiO 59.15 ± 0.78 Al O 15.72 ± 0.27 by Activation Laboratories Ltd.. Lithogeochemistry analysis 2 3 Fe O 6.35 ± 0.18 2 3 was also performed by Activation Laboratories Ltd. Mineral CaO 5.17 ± 0.92 composition of the samples was performed via lithium K O 3.48 ± 0.07 MgO 2.56 ± 0.09 borate fusion/inductively couple plasma-optical emission Na O 2.10 ± 0.07 spectroscopy and the composition of trace elements was TiO 0.76 ± 0.01 P O 0.13 ± 0.01 2 5 measured via lithium borate fusion/inductively coupled MnO 0.08 ± 0.003 plasma-mass spectrometry. 8 N. B. DRAGONE ET AL. gene reads averaged 4,699 reads/sample (3,101–6,708), were streaked for isolation after the four-week incuba- ITS region reads averaged 9,556 reads/sample (661– tion. Sanger sequencing revealed that a total of thirty- 25,284 reads), and 18S rRNA gene reads averaged eight unique bacterial isolates and six fungal isolates 14,450 reads/sample (480–28,173 reads). As expected, were grown from the three sediment samples the number of taxa identified per sample (microbial (Dataset S3). richness) was low, with average richness of twenty- All bacterial cultures grown were identified as being three 16S ASVs (fifteen to thirty-three), six ITS ASVs of the phyla Actinobacteria (twenty-seven isolates), (two to fourteen), and five 18S ASVs (one to twelve). No Firmicutes (six isolates), or Proteobacteria (five isolates). archaeal sequences were identified using these methods. Eleven different families were identified, including The thirty-six bacterial ASVs identified by cultiva- Micrococcaceae (eight isolates), Propionibacteriaceae tion-independent marker gene sequencing included (seven), Microbacteriaceae (five), Bacillaceae (four), taxa of the phyla Firmicutes (sixteen ASVs), Oxalobacteraceae (three), Nocardioidaceae (three), Proteobacteria (sixteen ASVs), Actinobacteria (ten Planococcaceae (two), Intrasporangiaceae (two), ASVs), Bacteroidetes (four ASV), Chloroflexi (two Geodermatophilaceae (two), Azospirillaceae (one), and ASVs), Acidobacteria (two ASVs), Verrucomicrobia Comamonadaceae (one; Figure 2A). (two ASVs), Planctomycetes (one ASV), All of the fungal isolates were identified as being of Abditibacteriota (one ASV), and Gemmatimonadota the cold-adapted yeast genus Naganishia (Figure 2B), (one ASV). In total, thirty-one families were repre- including three isolates most closely related to sented, the most common of which were Bacillaceae N. vishniacii and two other isolates most closely related (six ASVs), Burkholderiaceae (six ASVs), to N. albidosimilis and N. adeliensis. Carnobacteriaceae (three ASVs), Corynebacteriaceae (three ASVs), Chitinophagaceae (one ASV), and Paenibacillaceae (three ASVs). All other families were Discussion represented by only one ASV (Figure 2A). All sixteen of the fungal ASVs identified through the The environmental properties of the South Col sedi- next-generation marker gene sequencing effort were of ments we analyzed in this study are not unique to either the phyla Ascomycota (nine ASVs) or Everest. Many high alpine environments experience lim- Basidiomycota (seven ASVs). The most common fungal ited water and nutrients and high pH, among other family was Filobasidiaceae (six ASVs), all of which were elevation-influenced properties (Merino et al. 2019), identified to the genus Naganishia. The seven other and still have diverse microbial communities (D. Singh fungal families included Nectriaceae (three ASVs), et al. 2012; Yuan et al. 2014; Vimercati, Darcy, and Pleosporaceae (two ASVs), Teratosphaeriaceae (one Schmidt 2019). Our three samples, however, are notable ASV), Sporormiaceae (one ASV), Cladosporiaceae (one for the extremely high elevation from which they were ASV), Trichomeriaceae (one ASV), and one collected (>7,900 m). There are only fifteen mountains Basidiomycota ASV not identified to a fungal family in the world, including Mt. Everest, with peaks higher in (Figure 2B). elevation than the South Col. To our knowledge, these The 18S rRNA gene sequencing effort identified four- samples represent the highest elevation sediment envir- teen ASVs. Most of these sequences (ten ASVs) were onment to be explored for microorganisms using culti- fungal (Figure 2C) and 77 percent of all reads recovered vation-independent next-generation sequencing from the three samples were one ASV that had 100 per- methods. Organisms found at such high elevations, if cent identity over the very short read of 129 bp with any, must be able to cope with extremely high insolation members of the genera Piskurozyma and Naganishia. (Matthews et al. 2020), cold temperatures, low atmo- Nonfungal ASVs were identified as being of the genera spheric pressures, and low oxygen availability, among Rosulabryum (one ASV) and Crassostrea (one ASV), the other corresponding environmental conditions (Merino order Ostreoida (one ASV), and an unidentified ASV et al. 2019). from the phylum Cercozoa (one ASV; Figure 2C). Though the three samples South Col sediment sam- ples were collected using aseptic techniques and sterile sampling material and remained sealed from the Culture dependent methods moment of collection until they were processed, the Growth of at least one colony occurred on all plates that samples did not remain frozen through the entire ship- were inoculated with samples. No growth occurred on ping process. For this reason, our analyses focused on any of the fourteen control plates. One hundred eighty- the diversity of organisms identified in the samples and six bacterial colonies and twenty-nine fungal colonies not on their abundance. ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 9 Taxonomic diversity of bacteria identified as being most closely related to M. roseus (Qin et al. 2013). Organisms of the genus Modestobacter are Both the Illumina marker gene sequencing and culture- often identified in other cold, dry soils and sediments based identifications yielded similar microbial diversity in (Mevs et al. 2000; Busarakam et al. 2016; Golinska et al. the South Col sediments, though the next-generation 2020) and, like the closely related genus Geodermatophilus sequencing approach identified a greater diversity of (Montero-Calasanz et al. 2017), have recently been found to microorganisms than the culturing study (ten phyla ver- have many psychrophilic and oligotrophic adaptations sus three phyla; Figure 2). Both techniques revealed that (Golinska et al. 2020; Tarlachkov et al. 2020). Genomic the most common bacterial phyla were Firmicutes, analysis of Modestobacter taxa (see Golinska et al. [2020] Proteobacteria, and Actinobacteria. At finer taxonomic for detailed summary) has found genes related to cold- levels, Illumina sequencing identified a greater diversity shock regulation (e.g., Csp protein family; Essoussi et al. of Firmicutes, whereas the culturing methods identified 2010), pathways related to desiccation tolerance via the a greater diversity of Actinobacteria, though both meth- uptake of trehalose (Reina-Bueno et al. 2012), and multiple ods identified many of the same taxa (e.g., copies of the uvrD genes that have previously been linked to Geodermatophilaceae, Nocardioidaceae). These differ - increased ultraviolet (UV) tolerance (Normand et al. 2012). ences are not surprising, because culturing methods are Of additional note is the presence of genes that allow for the inherently selective. Many of the media we used for our use of carbon monoxide and other trace gases as a source of study were designed for culturing of organisms in cold carbon and energy (e.g., coxD, coxE, coxG; Lorite et al. soil environments, where Actinobacteria often are the 2000). These potential functional attributes suggest adapta- dominant taxa (Babalola et al. 2009; D. J. Smith et al. tions that might allow for survival in environments like the 2012; Dragone et al. 2021). South Col. Both methods captured a multitude of taxa with closely Along with Geodermatophilaceae and the other cold- related sequences that have been retrieved from other cold, associated organisms described previously, the diversity dry, lithic environments. The dominant phyla identified in of bacteria we identified in the South Col samples our South Col sediments were Actinobacteria, Firmicutes, included more cosmopolitan species that are not char- and Proteobacteria, taxonomic groups that often dominate acteristic of high alpine or cold soil/sediment environ- microbial communities in Antarctic soils (Cowan et al. ments. Many taxa, including those of the families 2014). We see few Acidobacteria and Bacteroidetes, phyla Paenibacillaceae and Bacillaceae, are endospore- that are common to Arctic tundra (Nemergut et al. 2005). forming species (Montes et al. 2004; Mandic-Mulec, At a finer taxonomic resolution, the families Stefanic, and van Elsas 2015). Bradyrhizobium is Nocardioidaceae, Planococcaceae, Oxalobacteraceae, a nitrogen-fixing genus that is most often found in Geodermatophilaceae, and Chitinophagaceae have closely symbiotic association with legumes, though it is also related sequences that have previously been retrieved from a common soil taxon (Klepa et al. 2021). Alloiococcus, the Transantarctic Mountains and the McMurdo Dry Staphylococcus, and Streptococcus are genera that Valleys (J. J. Smith et al. 2006; Dragone et al. 2021, 2022), include human pathogens (Aguirre and Collins 1992; as well as from cryoconite holes on Antarctic glaciers Becher et al. 2009; Palmieri, Varaldo, and Facinelli (Sommers et al. 2018). We also see taxa that have been 2011), though the latter two often are often found in identified in other high alpine sediment environments certain soil environments (Delgado-Baquerizo et al. around the world, including near the top of Kilimanjaro 2018). (Vimercati, Darcy, and Schmidt 2019) and the Puna de Atacama Volcanic Zone (Solon et al. 2018). Other genera, Taxonomic diversity of eukaryotes like Auraticoccus and Kribbella, have been found on rocks (Cheema et al. 2020), in deserts (Saygin et al. 2019), and on Both the Illumina marker gene sequencing of the ITS the walls of stone monuments and catacombs (Alonso- region and culture-based identifications yielded similar Vega et al. 2011). microbial diversity in the South Col and showed that Of special note is our identification of members of the most of the diversity of these samples was made up of family Geodermatophilaceae, including Geodermatophilus the fungal family Filobasidiaceae. More specifically, the obscurus, which was originally isolated from a sediment majority of the fungal ASVs identified in the marker sample taken at 8,400 m.a.s.l. by the American Everest gene sequencing and all of the fungi that grew in culture expedition of 1963 and later described by Ishiguro and were of the genus Naganishia. This is not surprising, Fletcher (1975). Though G. obscurus was not identified in because Naganishia is a genus of cold-adapted poly- our samples, we did identify taxa of the related genus extremophile yeasts that are commonly found in cold Modestobacter (Figure 3), with one cultured isolate and dry ecosystems, including in Antarctica (Duarte 10 N. B. DRAGONE ET AL. Figure 3. Modestobacter and Geodermatophilus phylogenetic tree. Phylogenetic relationships displayed are based on partial 16S rRNA gene sequences of at least 800 bp in length. The twenty sequences represented in this tree include two isolates recovered during the culturing study, eight representatives of the genus Modestobacter, and ten representatives of the genus Geodermatophilus, including G. obscurus, the organism isolated from a sediment sample taken at 8,400 m.a.s.l. by the American Everest expedition of 1963. The two cultured Geodermatophilaceae isolates were most closely related to M. excelsi and M. altitudinus, taxa most often associated with cold, dry, high alpine environments like soils of the Atacama Desert and Antarctica. The tree is rooted by Blastococcus deserti, a representative of another Geodermatophilaceae family. et al. 2018; Nizovoy et al. 2021), on Kilimanjaro notable, 77 percent of all 18S rRNA gene reads were one (Vimercati, Darcy, and Schmidt 2019), and in high- ASV that was a 100 percent match to several different elevation sediments of the hyperarid Puna de Atacama yeasts in the Filoblasidiales, including numerous (Pulschen et al. 2015; S. K. Schmidt et al. 2018). In these Naganishia like N. vishniacii and the genus other environments, Naganishia is thought to survive by Piskurozyma, a fungus closely related to Naganishia. periodically coming out of dormancy during brief peri- Given the very short reads from the Illumina sequencing ods of favorable conditions (S. K. Schmidt et al. 2017). run (129 bp), we cannot definitively assign a specific This genus of cold-adapted poly-extremophilic yeasts is taxonomy to this ASV at this time, but given results commonly found in a variety of extreme cold, dry envir- from the ITS and long-read 18S data from cultures, it onments and is adapted to be resistant to high doses of is very likely that this ASV is a member of the genus UV radiation, can grow at temperatures below 0°C, and Naganishia. can survive and even grow during extreme daily freeze– thaw cycles (Vimercati et al. 2016; S. K. Schmidt et al. Implications for microbial survival in extremely 2017). high-altitude environments The non-Naganishia fungal taxa identified in the samples are mostly cosmopolitan, many of which are Our results add to previous work describing the micro- commonly found in association with plants (Gibberella, bial communities on Mount Everest and in the Alternaria, Cladosporium; Zanne et al. 2020). One taxon, Himalayas (Freeman et al. 2009; L. Zhang et al. 2009; Knufia , belongs to a genus that includes extremotolerant S. K. Schmidt et al. 2011). Previous studies have found fungi often found on rocks (Breitenbach et al. 2018). that Himalayan microbial communities are dominated The 18S rRNA gene sequencing results generally by psychrophilic fungi at elevations from 3,000 to supported the patterns described from the ITS data. 5,300 m (Petrovič, Gunde-Cimerman, and Zalar 2000; Though many of the other ASVs were not classified Margesin and Miteva 2011). At higher elevations past the phylum level, this sequencing effort revealed between 4,000 m and 6,500 m, communities of ammonia a similar mix of cosmopolitan taxa and extremotolerant oxidizers were dominated by archaeal taxa below taxa like that revealed by the ITS region sequencing. The 5,400 m and bacteria taxa above (L. Zhang et al. 2009), genus Sporisorium, for example, includes plant patho- whereas even algae and chytrids were found in soils wet gens and mutualists (Ghareeb et al. 2011) and the genus by snowmelt above 5,500 m (Freeman et al. 2009; Mrakia includes cryotolerant and xerophilic species S. K. Schmidt et al. 2011). Across all studies, microbial (Thomas-Hall et al. 2010; Turchetti et al. 2011). Most diversity decreased with increasing elevation. Our three ARCTIC, ANTARCTIC, AND ALPINE RESEARCH 11 sediments from the South Col contained extremely low 2022). However, previous work at high elevations in the microbial diversity, much more like the microbial diver- Atacama has shown that even when air temperatures are sity found in the dry tephra “soils” on the volcanic peaks below −10°C (at 2 m height), surface soil temperatures can of the inner Atacama Desert (Costello et al. 2009; reach +5°C due to the intense insolation (D. Schmidt 1999; S. K. Schmidt et al. 2018; Solon et al. 2018) than the Schubert 2014). Organisms like Naganishia sp. may be able diversity found at lower elevations on the mountain. to take advantage of even these short periods of water This is not surprising given that microorganisms at availability beause they can grow during repeated freeze– such an extreme elevation would have to be able to thaw cycles and at constant temperatures as low as −6°C cope with correspondingly high UV radiation, extreme (Vimercati et al. 2016). Obviously, it would be hard to test cold temperatures, low atmospheric pressures, and this idea in situ, but more work on the unique microbes lower oxygen availability, among other corresponding isolated in the present study could illuminate the potential environmental conditions (Merino et al. 2019). for organisms to function during brief thaw periods at We expect that most of the organisms we identified extreme elevations. It also very likely that the South Col may have been seeded by eolian transport from other and other extremely high-elevation environments may just less extreme terrestrial surfaces. Wind-driven transport be deep-freeze collection points for deposited organisms of microorganisms and DNA is well described in (either from the atmosphere or by transiting humans). a variety of environments (Aalismail et al. 2019; Souza Given the challenge, and danger, of performing in situ et al. 2019) and has been shown to lead to the detection tests at such extreme high-elevation sites, we cannot say of organisms in environments where they cannot grow whether the brief periods when free water would be avail- (Archer et al. 2019; Maki et al. 2019). This includes on able are enough to support microbial growth. Perhaps Mount Everest, where Liu et al. (2007) found many extended monitoring by the weather station installed at cosmopolitan bacterial taxa in snow at 8,000 m. Our this site where these samples were collected will shed identification of cosmopolitan taxa and foreign plant some light on this mystery in the future (Matthews et al. and animal DNA (e.g., Rosulabryum and Crassostrea 2020; Potocki et al. 2022). DNA; Figure 2C) in these samples supports this theory. Though our culturing study could not re-create the In fact, the most dominant prokaryotic phyla extreme conditions at the South Col, our results show that (Proteobacteria, Firmicutes, and Actinobacteria) and many of the organisms, including the most ubiquitous fungal phyla (Ascomycota) identified in our samples fungal and bacterial taxa identified, are in fact viable and are the same phyla that typically dominate microbial could be grown at 4°C. Though the average air temperature sequences recovered by bioaerosol studies (Aalismail from 1991 to 2022 at the South Col was −22.6°C, well below et al. 2019; Archer et al. 2019; Souza et al. 2019; Ruiz- our culturing conditions, air temperatures in the Mount Gil et al. 2020) and include some of the taxa Liu et al. Everest region have been increasing at a rate of ~0.33°C per (2007) suggested could not be active at such high eleva- decade (Kang et al. 2022), and a record high of −1.4°C was tions on Everest. Even the most highly cold-adapted measured at the South Col in July 2022 (National genera (e.g., Modestobacter, Naganishia, or Ishiguro’s Geographic Society 2022). Organisms that are not active Geodermatophilus as described in Ishiguro and in situ now may become active in the future if the current Fletcher [1975] and Ishiguro and Wolfe [1970]) are rate of warming continues (described in Kang et al. 2022). likely to have been seeded from lower elevation source populations. Representatives of these genera have been Evidence of human-associated contamination of detected, and even isolated, from air samples collected Mount Everest around the world (J. J. Smith et al. 2006; Amato et al. 2007; S. K. Schmidt et al. 2017). The microorganisms identified in these South Col sedi- The prospect should also be raised that all, or most, ments included taxa that are often associated with organisms found at the South Col in this study rarely, if human contamination. Many of the sequences recov- ever, grow there. The organisms we isolated and grew likely ered could be deposited from mountaineers through only came out of dormancy due to the less extreme, con- coughing, nasal discharge, and associated sputum sistent conditions of the laboratory setting. Though the (Grice and Segre 2011; Wade 2013; Ibironke et al. intense insolation does indicate that melt occurs at the 2020). Staphylococcus, for example, is one of the most South Col, consistent with reports from climbers, it common skin and nose bacteria, and Streptococcus is the remains unclear whether this free water would be enough dominant genus in the human mouth (Otto 2010; to support brief periods of microbial growth, given that air Gevers et al. 2012; Ursell et al. 2013; Escapa et al. temperatures rarely rise above −10°C at such high eleva- 2018). Though these genera are widespread in nature tions on Mount Everest (Matthews et al. 2020; Potocki et al. and are not always associated with humans (e.g., Ko 12 N. B. DRAGONE ET AL. et al. 2011; Zhao et al. 2015), the sequences that were was conducted in partnership with Tribhuvan University, with approval from all relevant agencies of the Government of identified as being most closely related to our taxa based Nepal (Letter numbers 075/076, 576, and 2351, issued by the on analysis using blastn are human associated. For Department of National Parks and Wildlife Conservation of example, the Staphylococcus sequence identified in our the Government of Nepal). We also thank the communities of samples shares a 100 percent identity with the skin the Khumbu region, our field support team, and Shangri-La colonizers S. hominis and S. epidermis (Nagase et al. Nepal Trek. We also thank Jessica Henley, Matthew Gebert, Caihong Vanderburgh, and Claire Mastrangelo for help with 2002; Otto 2009), and the Streptococcus sequence shares the laboratory work; the University of Colorado Boulder Next a 100 percent identity with oral cavity colonizer S. oralis Generation Sequencing Facility and GENEWIZ for help with (Kennedy et al. 2000). Because none of these human- the sequencing effort; and The Arikaree Laboratory at CU associated taxa were identified in our extraction blanks Boulder and Activation Laboratories for assistance with the and no-template PCR controls and none grew on our geochemical analyses. Finally, we thank Sam Guilford for his control plates in our culturing study, it remains likely help creating the map in Figure 1. that these organisms were from the collected sediments. Mount Everest is heavily trafficked by climbers and Disclosure statement guides each year, prompting concerns that humans have irrevocably changed the high alpine environment. Our No potential conflict of interest was reported by the authors. study provides evidence that climbing activity may increase the transport of human-associated microorganisms onto Funding the mountain’s highest surfaces. However, we expect that at such high elevations these microorganisms are not active. This work was supported by funds provided to NBD from the We predict that if we sampled in the more human-utilized University of Colorado Boulder’s Department of Ecology and areas on the mountain we may find even more microbial Evolutionary Biology and the University of Colorado Boulder Graduate School. Publication of this article was funded by the evidence of human impact on the environment, as has been University of Colorado Boulder Libraries Open Access Fund. well described at lower elevations in the Himalayas (Rashid and Romshoo 2013; Ahmad et al. 2021; Rather et al. 2022) and around the world (Sinton, Finlay, and Hannah 1998; ORCID Joergensen and Emmerling 2006; Harwood et al. 2014). Nicholas B. Dragone http://orcid.org/0000-0002-8983- Conclusions The South Col of Everest represents a unique surface Data and materials availability environment, most notably for being >7,900 m. 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Journal

Arctic Antarctic and Alpine ResearchTaylor & Francis

Published: Dec 31, 2023

Keywords: Mount Everest; alpine; microbial ecology; microbiology

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