Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-018-1066-0

REVIEW

Life at extreme elevations on Atacama volcanoes: the closest thing to Mars on Earth? S. K. Schmidt . E. M. S. Gendron . K. Vincent . A. J. Solon . P. Sommers . Z. R. Schubert . L. Vimercati . D. L. Porazinska . J. L. Darcy . P. Sowell

Received: 19 December 2017 / Accepted: 14 March 2018 Ó Springer International Publishing AG, part of Springer Nature 2018

Abstract Here we describe recent breakthroughs in our understanding of microbial life in dry volcanic tephra (‘‘soil’’) that covers much of the surface area of the highest elevation volcanoes on Earth. Dry tephra above 6000 m.a.s.l. is perhaps the best Earth analog for the surface of Mars because these ‘‘soils’’ are acidic, extremely oligotrophic, exposed to a thin atmosphere, high UV fluxes, and extreme temperature fluctuations across the freezing point. The simple microbial communities found in these extreme sites have among the lowest alpha diversity of any known earthly ecosystem and contain bacteria and eukaryotes that are uniquely adapted to these extreme conditions. The most abundant eukaryotic organism across the highest elevation sites is a Naganishia species that is metabolically versatile, can withstand high levels of UV radiation and can grow at sub-zero temperatures, and during extreme diurnal freeze–thaw cycles (e.g. - 10 to ? 30 °C). The most abundant bacterial phylotype at the highest dry sites sampled

S. K. Schmidt (&)  E. M. S. Gendron  K. Vincent  A. J. Solon  P. Sommers  Z. R. Schubert  L. Vimercati  D. L. Porazinska  J. L. Darcy  P. Sowell Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309, USA e-mail: [email protected] E. M. S. Gendron  Z. R. Schubert Molecular, Cellular, and Developmental Biology Department, University of Colorado, Boulder, CO, USA

(6330 m.a.s.l. on Volca´n Llullaillaco) belongs to the enigmatic B12-WMSP1 clade which is related to the Ktedonobacter/Thermosporothrix clade that includes versatile organisms with the largest known bacterial genomes. Close relatives of B12-WMSP1 are also found in fumarolic soils on Volca´n Socompa and in oligotrophic, fumarolic caves on Mt. Erebus in Antarctica. In contrast to the extremely low diversity of dry tephra, fumaroles found at over 6000 m.a.s.l. on Volca´n Socompa support very diverse microbial communities with alpha diversity levels rivalling those of low elevation temperate soils. Overall, the high-elevation biome of the Atacama region provides perhaps the best ‘‘natural experiment’’ in which to study microbial life in both its most extreme setting (dry tephra) and in one of its least extreme settings (fumarolic soils). Keywords Endolithic microbes  Acidic soils  Fumaroles  B12-WMSP1  Spartobacteria  Hypoliths

Introduction Here we explore the current state of knowledge concerning the identity and functioning of microbial life at extreme elevations on some of the highest volcanoes on Earth. Our initial work in these unique environments was inspired by the pioneering studies of Stephan Halloy who described oases of life

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Antonie van Leeuwenhoek

consisting of bryophyte patches in an otherwise seemingly lifeless environment at elevations up to 6050 m.a.s.l. on Volca´n Socompa on the ChileArgentina border (Halloy 1991). We were further intrigued and inspired to explore this area by the discovery of perfectly preserved mummies buried on the summit of nearby Volca´n Llullaillaco (el. 6739 m.a.s.l.) in 1999 (Reinhard and Ceruti 2010). The life-like condition of these 500-year-old mummies (Reinhard and Ceruti 2010; Wilson et al. 2013) indicated a set of environmental conditions that completely inhibited microbial activity (Vimercati et al. 2016). The juxtaposition of thriving patches of a bryophyte-dominated ecosystem with soils in which no decomposition had occurred for 500 years provided an ideal outdoor laboratory to address questions related to the cold-dry limits to life on Earth, in what is likely the best analog for Mars on Earth (Costello et al. 2009; Lynch et al. 2012; Pulschen et al. 2015). We first review what little is known about the physical environment of barren tephra (soils) at elevations above 5800 m.a.s.l. on volcanoes of the southern region of the Andean central volcanic zone— near where the borders of Argentina, Bolivia, and Chile meet (24°S, 68°W). We then discuss evidence for and against the existence of functioning microbial communities in surface soils (tephra) on Volca´n Socompa and Volca´n Llullaillaco. Lastly, we finish with a discussion of the unique microbial communities associated with fumarolic activity on Volca´n Socompa and how those communities compare to communities in other extreme fumarolic sites such as Mt. Erebus in Antarctica (e.g. Tebo et al. 2015).

The physical setting Unlike the lower elevation reaches of the Atacama Desert, high elevation environments have received very little attention from biologists. However, there is an abundance of information on the geology (e.g. Allmendinger et al. 1997; Francis et al. 1985; Richards and Villeneuve 2001), and physical geography (Richter and Schmidt 2002; Schmidt 1999; Vitry 2016) of these high elevation zones and here we draw on some of these studies to paint a picture of the physical setting, especially as it relates to understanding the potential biological functioning of the system. Volca´n Llullaillaco (24°430 S68°320 W) is the second

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highest active volcano on Earth at 6739 m.a.s.l., but currently has no known active fumaroles. Volca´n Socompa (24°230 S68°140 W) is lower in stature at 6051 m.a.s.l., but has at least 6 known areas of fumarolic activity (Halloy 1991; Costello et al. 2009). Both volcanoes are complex stratovolcanoes (Francis et al. 1985; Richards and Villeneuve 2001). Most of what we know about the microclimate of high elevation sites in the Atacama region comes from work done in the 1990s by Schmidt (1999) at elevations up to 5820 m.a.s.l. on Volca´n Saire´cabur, botanical work done across a range of lower elevations by Arroyo et al. (1988), and limited observations made by Lynch et al. (2012) and Costello et al. (2009) at elevations up to 6330 m.a.s.l. on Socompa and Llullaillaco. Figure 1 shows the vertical stratification of life zones on Volca´n Llullaillaco. As in most mountainous areas, the upslope movement of air results in cooling which increases precipitation at higher elevations and in the Atacama region this results in a zone of vegetation from about 3000 m.a.s.l. to just over 4900 m.a.s.l. (Arroyo et al. 1988; Richter and Schmidt 2002; Watson et al. 2013). Above this sparsely vegetated zone there is no plant life except for bryophyte patches associated with fumarolic activity (Costello et al. 2009; Halloy 1991; Schiavone and Sua´rez 2009). The lower elevation reaches of the vegetated zone consist of desert scrub that leads down into the Atacama Desert proper (not shown in Fig. 1). The upper un-vegetated zone is the subject of this review and receives precipitation only as snow, most of which sublimates back to the atmosphere due to intense radiation, low vapor pressure, and extremely cold temperatures (Schmidt 1999). In many respects, the environmental conditions faced by life in the upper un-vegetated zone of the Atacama are more extreme than even the hyper-arid core of the Atacama Desert. For example, levels of UV radiation at 5091 m.a.s.l. on Volca´n Saı´recabur are 25–33% higher than in lower elevation sites such as Yungay (948 m.a.s.l.) in the hyper-arid core of the Atacama (Pulschen et al. 2015). In fact, the highest ground value for UV-B irradiance ever reported was measured at 5916 m.a.s.l. on Volca´n Licancabur (Cabrol et al. 2014). In addition, the initial studies of barren tephra ‘‘soils’’ at elevations up to 5834 m.a.s.l. on Volca´n Socompa revealed extremely dry conditions with undetectable levels of water (Costello et al.

Antonie van Leeuwenhoek Fig. 1 a Photo taken in February of 2009 from about 4000 m.a.s.l. in the vegetated zone E, SE of Volca´n Llullaillaco in Argentina. b Sketch of the silhouette of Volca´n Llullaillaco viewed from the west in Chile showing that most of the prominence of the mountain lies above the sparsely vegetated zone. The dark lava flow on the S, SW side of Llullaillaco is shown for reference. Photo credit P. Sowell, drawing credit Kim Vincent

2009). Later work by Lynch et al. (2012) showed some measurable water (0.24%) to a depth of 4 cm at elevations of 6030 and 6330 m.a.s.l. on Volca´n Llullaillaco. Likewise, levels of soil carbon and nitrogen on these volcanoes are among the lowest yet measured in terrestrial soils. Table 1 compares some biogeochemical parameters measured on Llullaillaco and Socompa to the hyper-arid core of the Atacama Desert (Crits-Christoph et al. 2013), and to

the most extreme cold/dry site sampled to date in Antarctica (Goordial et al. 2016). Levels of total organic carbon are very low for all of these extreme environments, falling below about 0.02% carbon. For comparison, these values are about two orders of magnitude lower than what would be considered an oligotrophic soil, such as sparsely vegetated, highalpine soils in Colorado (cf. King et al. 2008).

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Antonie van Leeuwenhoek Table 1 Biogeochemical and microclimatic parameters measured on Llullaillaco and Socompa compared to the hyper-arid core of the Atacama Desert (Crits-Christoph et al. 2013) and to the high-elevation University Valley site in Antarctica (Goordial et al. 2016) Site

% TOC

% TON

pH

% moisture

Mean annual temp (°C)

References

Llullaillaco 6330 m

0–0.015

0.000

4.6

0–0.25

- 12.3a

Lynch et al. (2012)

Llullaillaco 6034 m

0.008–0.016

0.000

4.2

0–2.0

- 10.1a

Lynch et al. (2012)

Socompa 6049 m

ndb

nd

4.8

0–1.0

- 10.2a

Solon et al. (2018)

University Valley Antarctica

0.01–0.02

0.05–0.09

7.5

0–0.6

- 23.4

Goordial et al. (2016)

Hyper-arid core Atacama

\ 0.01

nd

7.4–7.8

nd

16.5

Crits-Christoph et al. (2013)

Erebus ice caves

0.008–0.013

nd

5.4

nd

nd

Tebo et al. (2015)

Socompa Fumaroles 5824 m

0.13–0.36

0.014–0.03

4.4–5.4

1–13.6

nd

Solon et al. (2018)

Socompa Fumaroles 6049 m

nd

nd

nd

20.5

nd

Solon et al. (2018)

a

Estimated from the data of Schmidt (1999)

b

Not determined

High elevation Atacama sites further diverge from other extreme sites in terms of parameters such as percent nitrogen (N) and pH. Total N levels were undetectable in high-elevation soils on Llullaillaco, whereas soils from the comparable sites in Table 1 have reasonably high nitrogen levels. Likewise, pH values are much lower on Llullaillaco and Socompa compared to the sites in Antarctica and the hyper-arid core of the Atacama (Table 1). The mean annual temperature in University Valley, Antarctica (- 23.4 °C) is the coldest of the three sites depicted in Table 1, but temperatures there are highly seasonal with relatively mild summers. Due to a lack of data there is much more uncertainty about temperatures above 6000 m.a.s.l. in the Atacama region. The mean annual temperatures for Llullaillaco and Socompa in Table 1 are conservative estimates based on measurements made at 5820 m.a.s.l. on Volca´n Saı´recabur and an estimated change of 0.75 °C per 100 m elevation gain (cf. Halloy 1991; Schmidt 1999). Perhaps more daunting than mean annual temperature, diurnal temperature fluctuations in high-elevation Atacama soils are among the most extreme yet measured on Earth (Schmidt 1999; Lynch et al. 2012). For example, Lynch et al. (2012) measured diurnal temperature cycles in summer (February) at the soil surface and at 4 cm depth at elevations of 5737 and 5500 m.a.s.l. on Llullaillaco and Socompa. At the soil

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surface, the lowest night-time temperature was - 14.5 °C and the highest day-time temperature was 56.2 °C or an amplitude of over 70 °C across 2 days of measurements. The amplitude was dampened (i.e. down to 47 °C) at 4 cm depth, indicating that conditions for life are likely better at deeper soil depths. Similar freeze–thaw data have been reported for 5820 m.a.s.l. on Volca´n Saı´recabur, where Schmidt (1999) reported daily freeze–thaw amplitudes of 42 °C at the soil surface and 15 °C at 5 cm depth during the mildest month of the year, December. Diurnal freeze–thaw cycles occur on a year-round basis in all of the highest-elevation mountains of the world (King et al. 2010a; Yang et al. 2002; Schmidt 1999, 2009, 2017a), and what little we know about how microorganisms cope with these daily extremes is discussed below. The study of how organisms survive freeze–thaw cycles also has relevance for the field of Astrobiology because freeze–thaw cycles likely occur in favorable sites on Mars, but they would have even larger amplitudes (e.g., as high as 165 °C, Graham 2004) than those so far recorded on Earth.

Life in ‘‘barren’’ high-elevation ‘‘soils’’ Our search for microbial life at high elevation sites in the Atacama region was inspired by the pioneering

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work of Halloy (1991) who characterized macroscopic life (mostly Bryophytes) near fumaroles at elevations up to 6050 m.a.s.l., and by the work of Archaeologists who discovered 500-year-old mummies near the summit of Volca´n Llullaillaco that showed no signs of decomposition (Reinhard and Ceruti 2010). The lack of decomposition was a strong indication that conditions in the soils near the summit of Llullaillaco were completely inhibitory to microbial life (Vimercati et al. 2016). Halloy (1991) did not specifically look for microorganisms on Socompa but reported scattered lichens at elevations up to 5620 m.a.s.l., and some ‘‘small mats of algae,’’ but these were only associated with fumaroles. On nearby Ojos del Salado (the highest active volcano on Earth), Gonzalez et al. (1987) isolated several strains of bacteria (mostly Firmicutes) from soils at elevations of over 5000 m.a.s.l. (including some from the summit) and showed that they could grow at temperatures as low as 4 °C, but did not show that these bacteria were indigenous to high elevations or active there. Otherwise, there were not any general or systematic studies of high-elevation microbes in the Atacama region until our expedition in 2005. In 2005, we accompanied Stephan Halloy and an international group of scientists to revisit some of the fumaroles described by Halloy (1991) on Socompa, and to begin studies of the apparently lifeless tephra that covers most of the mountain above 5000 m.a.s.l. (Fig. 1). Samples collected on this expedition resulted in the first molecular-phylogenetic study of microbial life on a high elevation volcano (Costello et al. 2009). Costello et al. (2009) sampled barren tephra only up to an elevation of 5285 m.a.s.l., but still showed that these ‘‘soils’’ had TOC levels of only 0.03%, and nitrogen and water levels below the limit of detection (compare to Table 1). Despite these extreme conditions, they detected (using cloning and Sanger sequencing) simple microbial communities dominated by Actinobacteria, Acidobacteria, Bacteriodetes, Verrucomicrobia, and a basidiomyceteous yeast in the genus Naganishia (formerly lumped into the genus Cryptococcus) in barren soils, and very complex communities in soils associated with fumaroles (described below). Two more recent expeditions to Socompa and Llullaillaco, in 2009 and 2016, garnered samples that have been used in a range of studies to characterize the environmental conditions (e.g. Table 1), and

microbial communities in the seemingly lifeless tephra, especially at elevations above 5800 m.a.s.l. (Lynch et al. 2012, 2014; Schmidt et al. 2017b; Solon et al. 2018; Vimercati et al. 2016). During the 2009 expedition we were able to sample soils above 6000 m.a.s.l. for the first time on both Socompa and Llullaillaco. Tephra soils collected at 6034 m.a.s.l. on Volca´n Llullaillaco were very dry (Table 1), and were intensively examined using an array of modern approaches including metagenomics, functional gene analyses, and complete analysis of 18S and 16S rRNA microbial communities using both Sanger and Illumina sequencing (King et al. 2010b; Lynch et al. 2012, 2014; Solon et al. 2018). The bacterial community at 6034 m.a.s.l. was dominated by an OTU in the genus Pseudonocardia (Actinomycetales, Actinobacteria). Metagenomics indicated that this organism is capable of using gases such as H2, CO, and CH4 as energy sources (Lynch et al. 2014), similar to organisms of other oligotrophic volcanic sediments (Weber and King 2010), perhaps indicating that Pseudonocardia may be active at 6030 m.a.s.l. on Llullaillaco. At even higher elevations (6330 m.a.s.l.) on Llullaillaco we encountered the driest and most oligotrophic soils of the expedition (Table 1). Surprisingly, the bacterial community at these sites were shifted away from an Actinobacteria-dominated community to one that was dominated by a diverse array of Chloroflexi phylotypes of which several were found to be (24% of Chloroflexi sequences) related to filamentous, non-photosynthetic members of the Ktedonobacter/Thermosporothrix clade (Fig. 2). The Ktedonobacter/Thermosporothrix clade (KT clade) also contains multiple phylotypes from fumarolic soils on Mt. Erebus in Antarctica, and phylotypes from fumarolic soil on Socompa (Fig. 2). It is also of note that, like Pseudonocardia discussed above, members of the KT clade have the ability to use CO as an energy source (King and King 2014). However, a reanalysis of the data of Lynch et al. (2012) shows that most of the Chloroflexi sequences from Llullaillaco fall into a sister clade (B12-WMSP1) to the KT clade (Fig. 2), represented by a single phylotype (OTU) that made up 70% of all Chloroflexi sequences at 6330 m.a.s.l. on Llullaillaco. Members of the B12-WMSP1 clade have also been shown to be prominent members of the fumarolic ‘‘Barry’s Dream Cave’’ on Mt. Erebus (KJ623634, Tebo et al. 2015), and the fumarole site on Socompa (FJ592907, Costello et al. 2009).

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Antonie van Leeuwenhoek Fig. 2 Phylogenetic tree made with long-read ([ 1300 bp) DNA sequences of the Chloroflexi and Verrucomicobia found on Socompa and Llullaillaco compared with sequences from other volcanoes (Mt. Erebus, Mt. Kilimanjaro). Most of the Chloroflexi at 6330 m.a.s.l. on Volca´n Llullaillaco are in the B12WMSP1 clade originally described by Costello and Schmidt (2006). The numbers in parentheses are the number of sequences in each OTU from Socompa or Llullaillaco and the NCBI accession numbers for the sequences used in the tree

The fact that some of the closest relatives of the KT clade and B12-WMSP1 clade in barren, non-fumarolic, tephra at 6330 m.a.s.l. on Llullaillaco are thermophilic and capable of oxidizing volcanic gases such as CO, raises the question as to whether these phylotypes can actually function in barren tephra or if they are just dormant propagules dispersed from fumaroles. Such a situation has been described in the Arctic where there is a constant flux of dormant, thermophilic organisms into cold regions of the deep ocean (Hubert et al. 2009). Indeed, members of the KT clade can form spores (Chang et al. 2011) which would allow for easy dispersal and perhaps long-term survival. The presence of thermophilic organisms in cold soils at 6330 m.a.s.l. may also indicate that there is current or recent geothermal activity on Llullaillaco that has gone undetected due to the extreme

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remoteness of this site. Finally, it is also possible that thermophiles can function during the brief daily intervals of high temperatures that occur due to the intense radiation received by soils at these elevations (see discussion of freeze–thaw cycles, above). This is unlikely due to the intense drying action of the same radiation that heats the soils, but growth of thermophiles may occur in hypolithic situations as has been suggested by Cockell et al. (2014). However, there has been no exploration of hypolithic environments on Llullaillaco, and it should also be noted that other members of the B12-WMSP1 clade are not necessarily thermophiles, as this clade (e.g. DQ450730) was first discovered in perennially cold alpine soils far from any volcanoes (Costello and Schmidt 2006; Freeman et al. 2009). Obviously, much more work is needed to determine if there is an active,

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bacterial microbiome at extreme elevations (away from fumaroles) on volcanoes in the Atacama region, but detailed phylogenetic analyses such as those shown in Fig. 2 offer intriguing hypotheses to be pursued in future studies. Unlike the confusing situation concerning the possible activity of prokaryotes in dry tephra at extreme elevations, the situation with eukaryotes is slightly clearer. By far, the dominant eukaryote in high-elevation tephra on Socompa and Llullaillaco is a yeast closely related to Naganishia friedmannii (formerly Cryptococcus friedmannii) from the Dry Valleys of Antarctica where it can dominate some high-elevation sites (Dreesens et al. 2014; Vishniac 1985). Lynch et al. (2012) found that N. friedmannii made up over 90% of all 18S rRNA sequences at both 6034 and 6330 m.a.s.l. on Volca´n Llullaillaco and Naganishia phylotypes also dominated dry soils at multiple elevations on Socompa (Costello et al. 2009; Solon et al. 2018). Yeasts closely related to N. friedmannii have also been isolated from a range of elevations on nearby Volca´n Saı´recabur by Pulschen et al. (2015), who demonstrated that this hardy organism can grow at - 6.5 °C and can withstand levels of UV radiation as high as the most radiation resistant bacteria. Both of these attributes bode well for this organism having the potential to be active at extreme elevations, but even more convincing evidence has come from recent work with N. friedmannii isolated from dry tephra at 6034 m.a.s.l. on Volca´n Llullaillaco (Vimercati et al. 2016; Schmidt et al. 2017b). In addition to being able to grow in the presence of high levels of UV radiation and cold temperatures as shown by Pulschen et al. (2015), N. friedmannii would also need to function during extreme freeze–thaw cycles that soils experience at high elevations. Vimercati et al. (2016) carried out a series of experiments demonstrating that N. friedmannii can grow during the most intense freeze–thaw cycles yet tested in the laboratory. They used a specially designed chamber to reproduce the daily freeze–thaw cycles (- 10 °C to ? 30 °C every 24 h) described by Schmidt (1999) during the month of December at 5820 m.a.s.l. on Volca´n Saı´recabur. During incubations of Llullaillaco tephra from 6034 m.a.s.l., Vimercati et al. (2016) showed a N. friedmannii phylotype increased in relative abundance during these diurnal freeze–thaw cycles, even when soils were incubated at

ambient water content (0.25%). After 2 months of continuous freeze–thaw cycles they then isolated N. friedmannii from these soils and showed that a pure culture of the isolate could definitively grow during freeze–thaw cycles with a doubling time of 2 days (l = 0.35 day-1), equivalent to the doubling time of the same culture kept at a constant 0 °C (Vimercati et al. 2016). This was the first demonstration that any organism can grow during realistic freeze–thaw cycles and provides the best evidence to date that N. friedmannii has the potential to grow at extreme elevations. Combined with the evidence discussed above, other traits of N. friedmanii paint a picture of an opportunistic microbe that may be able to grow in situ in high elevation tephra. Perhaps the most revealing trait is that the N. friedmannii isolate from Llullaillaco can use a wide array of organic compounds (49 out of 65 compounds tested) for growth (Vimercati et al. 2016). This may allow N. friedmannii to utilize a broad array of Aeolian deposited organic matter but likely only during the warmer months of the year and in conjunction with rare snowmelt events (Schmidt et al. 2017a, b; Vimercati et al. 2016). Despite evidence that N. friedmannii may have traits that would allow it to function in high-elevation surface soils, it must be emphasized that the microbial communities at these sites are extremely simple, and as such may contain a preponderance of dormant and or dead organisms (Lynch et al. 2012; Solon et al. 2018). More information concerning the overall structure and diversity of these communities is discussed in the following section where we compare them to communities associated with high-elevation fumaroles.

Fumaroles as oases in an extreme landscape In 2005 and 2009 we revisited some of the same fumaroles originally studied by Halloy (1991), focusing our attention on the largest area of fumarolic ground at 5824 m elevation on Socompa’s southwestern flank (called ‘‘warmspot 2’’ by Halloy 1991), and a smaller fumarole at 6049 m.a.s.l. just below the summit of Socompa. Figure 3a shows a small portion of the dense bryophyte mats associated with warmspot 2 in 2009. We did not wish to disturb these mats and therefore sampled soils from already disturbed areas

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a

b

Fig. 3 a Photo showing the downhill edge of Warm Spot 2 at 5824 m.a.s.l. on Volca´n Socompa. Note the dark color of the bryophyte-dominated mats. The green chamber used to measure gas fluxes is shown in place at one of the three sampling sites— note the buildup of water vapor inside the otherwise clear chamber after being in place for only 8 min. b CO2 flux over 8–12-min incubations of the chamber shown in (a). The three different symbol shapes represent the three replicate flux measurements spaced every 3–5 m along the lower edge of the bryophyte mat

within the mats or areas just downhill from the mats that were nevertheless still being impacted by gas and water vapor flux through the soil matrix (Fig. 3b). Table 1 shows that fumarolic soils had higher water, carbon, and nitrogen contents compared to nonfumarolic tephra. Given that the fumarolic soils were wetter and more carbon rich, it is not surprising that they contained much more complex communities than nearby dry tephra. Fumaroles at both 5825 and 6049 m.a.s.l. had significantly higher 16S and 18S

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rRNA OTU richness than dry samples from the same elevations (Solon et al. 2018). The highest species richness values were actually associated with the fumaroles at the highest elevation (6049 m.a.s.l.), where there were 803 16S rRNA OTUs compared to only 61 in nearby dry tephra. Likewise, there were 319 18S OTUs in fumarolic soils compared to only 83 in nearby dry tephra at 6049 m.a.s.l. (Solon et al. 2018). These values for OTU richness of fumarolic soils put them in the range of forest and agricultural soils examined using the same high-throughput approaches as Solon et al. (2018). Whereas the low value for 16S rRNA OTU richness in the dry tephra sites at 6049 m.a.s.l. are lower than values reported for surface soils in the University Valley of Antarctica (Goordial et al. 2016), and similar to values for extreme soils of the hyper-arid core of the Atacama (Crits-Christoph et al. 2013). The bacterial communities associated with fumaroles on Socompa were significantly different in terms of beta diversity compared to nearby dry tephra and contained most of the major phyla of bacteria normally associated with soils (Solon et al. 2018). Costello et al. (2009) also showed that the hotter fumarolic ground on Socompa had lower diversity than cooler fumarolic ground and that the hotter soils were overwhelmingly dominated (45% of sequences) by members of the Spartobacteria group of the Verrucomicrobia (Fig. 2). Closely related Spartobacteria were also common in non-fumarolic soils across all elevations sampled on both Socompa and Llullaillaco (Costello et al. 2009; Solon et al. 2018), perhaps indicating a fumarolic origin for soil bacteria in dry tephra as discussed above for some of the Chloroflexi. Further evidence for this idea is that the dominant spartobacterial phylotype from the warm fumarole site on Socompa (represented by FJ592691) is closely related to two phylotypes from fumarolic ice caves on Mt. Erebus (Acc. Numbers KJ623648 and KJ623643). In addition, a close relative was found in a microbial mat from the wall of a lava cave in the Azores (JN615785, 96% identical across 1300 BP). Our working hypothesis is that members of the Spartobacteria group are functioning in widespread fumarolic habitats across the globe and may be being dispersed both locally (i.e. to barren tephra soils on Llullaillaco and Socompa) and globally (i.e. between Socompa and Mt. Erebus in Antarctica). It is possible that the dispersal of Spartobacteria and other unusual groups of microbes that

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are abundant in both the high Atacama and the Dry Valleys of Antarctica (e.g. N. friedmannii and B12WMSP1) is via ‘‘Rossby Waves’’ that result from mixing of the subtropical and polar jet streams (Fig. 4)

that periodically join and separate (Ambrizzi et al. 1995; Madden 1979; Polvani and Saravanan 2000). More information about the biogeographic connectivity between the volcanoes of the Atacama and the Dry

Fig. 4 a Depiction of subtropical and polar jet streams that periodically mix in ‘‘Rossby Waves’’ that would facilitate dispersal of microbes and dust between high-elevation sites in the Atacama and Antarctica. Mt. Erebus and Volca´n Llullaillaco are shown in their approximate locations. b Depiction of possible subsurface connections between volcanoes on different continents. Drawing credit Kim Vincent

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Valleys of Antarctica is discussed by Schmidt et al. (2017b). The high alpha diversity of eukaryotic communities in fumarolic soils on Socompa has been viewed as indicative of active and complex food webs being present in these soils, especially at the cooler fumarolic sites (Costello et al. 2009; Solon et al. 2018). Metazoans (bdelloid rotifers and a putative micro-arthropod) made up 14% of the sequences from cooler, moister fumaroles and were essentially absent from the warm and non-fumarole soils (Costello et al. 2009). Similarly, bryozoans such as tardigrades, and a Vampyrellid amoeba (Cercozoa) were consistently detected at low percent abundance in fumarolic samples and were absent from non-fumarolic samples described by Solon et al. (2018). Figure 5 compares a fumarolic and a non-fumarolic site near the summit of Volca´n Socompa, demonstrating the potential for a complex food web with primary producers, decomposers, and predators in the fumarolic soils and only fungi (likely all decomposers) in the non-fumarolic soils. Although the presence of organisms with such high potential for dormant states does not necessarily demonstrate their activity in these environments, the presence of a complex community in fumarolic samples and not dry samples collected nearby is consistent with higher levels of trophic complexity in the fumaroles. These bryozoan communities are furthermore consistent with some of the best studied comparable environments to the Atacama fumaroles: the fumaroles on the Antarctic volcanoes Mr. Erebus, Mt. Melbourne, and Mt. Rittmann. Fumaroles in the terraced region near the summit of Mt. Erebus, Antarctica, known as Tramway Ridge, show clear zonation in the presence and type of microbial mats formed by moss, algae, and cyanobacteria, corresponding to gradients of temperature, moisture, and pH (Herbold et al. 2014a, b). Protozoa there appear to be restricted to cooler, mossier areas, just as metazoans were only detected in the cooler, moister fumaroles sampled on Socompa (Costello et al. 2009). Molecular characterization of Antarctic fumarolic microbial communities has been restricted to subsurface prokaryotes, so no directly comparable molecular datasets are available for the protozoa and meiofauna found on Socompa. However, microscopic studies have reported small colorless bdelloid rotifers and cysts of tardigrades on Mt. Erebus (Janetschek

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Fig. 5 a Rank abundance plot for the top ten eukaryotic phylotypes in soil from the fumarole at 6049 m.a.s.l. on Volca´n Socompa. Note that algae (green) dominate the site and that there is evidence of trophic complexity due to the presence of predators (red) and fungi (brown). b In contrast, a dry site at the same elevation was completely dominated by Naganishia friedmannii and other fungi, mostly in the Dothidiomycetes (‘‘Doth.’’)

1963), and Cercozoa on Mt. Melbourne (Broady et al. 1987), similar to those found using molecular methods on Socompa (Costello et al. 2009; Solon et al. 2018).

Conclusion High elevation ecosystems in the Atacama region have only recently been explored using modern molecular approaches yet we are already beginning to understand how life may be able to function in this system.

Antonie van Leeuwenhoek

Surprisingly, a eukaryotic organism, N. friedmannii, is the most promising candidate, so far, for being able to grow and function metabolically at the highest and driest sites on Volca´n Llullaillaco. This versatile organism is able to stand high levels of UV radiation, can use a wide range of organic compounds, and can grow rapidly at low temperatures and during extreme freeze–thaw cycles. These traits may explain why it, along with pigmented, drought-tolerant fungi in the Dothidiomycetes (Schmidt et al. 2012), make up the simple microbial communities found above 6000 m.a.s.l. on Llullaillaco and Socompa. It is also intriguing that the dominant (in terms of relative abundance) bacteria at 6330 m.a.s.l. on Llullaillaco are in the enigmatic B12-WMSP1 and KT clades of the Chloroflexi. It has recently been shown that a related bacterium, Ktedonobacter racemifer strain SOSP1-21, has one of the largest genomes of any bacterium (Chang et al. 2011), perhaps indicating a high degree of metabolic flexibility, much like N. friedmannii. Our working hypothesis is that organisms that function at extreme elevations must be versatile opportunists, able to take advantage of rare periods of water and nutrient availability, and remain dormant for the long intervals between such events. We also present evidence for a fumarolic origin for some of the diversity of organisms found in the most extreme sites on Llullaillaco and Socompa. Fumaroles provide warmer, more stable temperatures and more consistent water availability, even at elevations of over 6000 m.a.s.l., and host diverse and trophically complex microbial communities on a par with the most diverse soil communities found at low elevations. In addition, several of the dominant bacteria of volcanic ice caves of Mt. Erebus in Antarctica are closely related to Chloroflexi and Spartobacteria found in high relative abundances on Socompa and Llullaillaco. This intercontinental connection highlights the environmental similarities between these sites (e.g. low pH fumaroles in an extremely cold environment), but also may indicate frequent dispersal events between these environments. This intercontinental connectivity may also explain the high degree of genetic similarity between N. friedmannii strains of the high Atacama and acidic micro-environments of high-elevations sites in the Dry Valleys of Antarctica (Vimercati et al. 2016; Schmidt et al. 2017b). Finally, our work and the work of archaeologists demonstrates that there are soil environments on Earth

where conditions are too cold and too dry for microbes to function even when there is an abundant food source. The lack of any decay of mummies, food and fabrics buried for 500 years near the summit of Volca´n Llullaillaco demonstrate quite conclusively that there is a cold-dry limit to life on Earth. Future studies will hopefully address exactly what those conditions are and how life nonetheless is able to function in the extreme surface soil on these magnificent volcanoes. This review also shows that despite the exceptionally hostile conditions imposed by a dry climate and high elevations, there do exist rich patches of life above 6000 m.a.s.l. in soils receiving moisture, carbon and heat from fumaroles. Although it is not thought that fumaroles currently exist on Mars, they likely did exist in the past (Robbins et al. 2011) and such sites would be good targets to look for bio-signatures of past life on Mars (Djokic et al. 2017). Acknowledgements We thank S.R.P. Halloy, P. Ara´ns, E.K. Costello, S.C. Reed, A. Seimon, G. Jesperson, T. Harris, M.E. Farias, C. Dorador, C. Vitry, P. Maciel, M. Perez, G. Zimmerman, and T. Bowen for advice and help in the field, and D.R. Bowling for help with measuring CO2. Funding This study was funded by the National Science Foundation of the U.S.A. (Grant Numbers DEB-1258160 and PLR-1443578). Conflict of interest The authors declare no conflicts of interest.

References Allmendinger RW, Jordan TE, Kay SM, Isacks BL (1997) The evolution of the Altiplano-Puna plateau of the Central Andes. Annu Rev Earth Planet Sci 25:139–174 Ambrizzi T, Hoskins BJ, Hsu HH (1995) Rossby wave propagation and teleconnection patterns in the austral winter. J Atmos Sci 52:3661–3672 Arroyo MTK, Squeo FA, Armesto JJ, Villagran C (1988) Effects of aridity on plant diversity in the northern Chilean Andes—results of a natural experiment. Ann Mo Bot Gard 75:55–78 Broady P, Given D, Greenfield L, Thompson K (1987) The biota and environment of fumaroles on Mt Melbourne, northern Victoria Land. Polar Biol 7:97–113 Cabrol NA, Feister U, Ha¨der D, Piazena H, Grin EA, Klein A (2014) Record solar UV irradiance in the tropical Andes. Front Environ Sci. https://doi.org/10.3389/fenvs.2014. 00019 Chang YJ et al (2011) Non-contiguous finished genome sequence and contextual data of the filamentous soil

123

Antonie van Leeuwenhoek bacterium Ktedonobacter racemifer type strain (SOSP121). Stand Genom Sci 5:97–111 Cockell CS, Cousins C, Wilkinson PT, Olsson-Francis K (2014) Are thermophilic microorganisms active in cold environments? Int J Astrobiol 14:457–463 Costello EK, Schmidt SK (2006) Microbial diversity in alpine tundra wet meadow soil: novel Chloroflexi from a cold, water-saturated environment. Environ Microbiol 8:1471–1486 Costello EK, Halloy SRP, Reed SC, Sowell P, Schmidt SK (2009) Fumarole-supported islands of biodiversity within a hyperarid, high-elevation landscape on Socompa Volcano, Puna de Atacama, Andes. Appl Environ Microbiol 75:735–747 Crits-Christoph A et al (2013) Colonization patterns of soil microbial communities in the Atacama Desert. Microbiome 1:28. https://doi.org/10.1186/2049-2618-1-28 Djokic T et al (2017) Earliest signs of life on land preserved in ca. 3.5 Ga hot spring deposits. Nat Commun 8:15263 Dreesens LL, Lee CK, Cary SC (2014) The distribution and identity of edaphic fungi in the McMurdo Dry Valleys. Biology 3:466–483 Francis PW, Gardeweg M, Ramirez CF, Rothery DA (1985) Catastrophic debris avalanche deposit of Socompa volcano, northern Chile. Geology 13:600–603 Freeman KR, Pescador M, Reed SC, Costello EK, Robeson MS, Schmidt SK (2009) Soil CO2 uptake and photoautotrophic community composition in high-elevation, ‘‘barren’’ soils. Environ Microbiol 11:674–686 Gonzalez SN, Romero N, Apella MC, Pesce de Ruiz Holgado A, Oliver G (1987) Existence of lactic acid bacteria in ecological pockets in highland areas. Microbiologie Alim Nut 5:317–323 Goordial J, Davila A et al (2016) Nearing the cold-arid limits of microbial life in permafrost of an upper dry valley, Antarctica. ISME J 10:1613–1624 Graham JM (2004) The biological terraforming of Mars: planetary ecosynthesis as ecological succession on a global scale. Astrobiol 4:168–195 Halloy SRP (1991) Islands of life at 6000 m altitude: the environment of the highest autotrophic communities on Earth (Socompa Volcano, Andes). Arctic Alpine Res 23:247–262 Herbold CW, McDonald IR, Cary SC (2014a) Microbial ecology of geothermal habitats in Antarctica. In: Cowan D (ed) Antarctic terrestrial microbiology. Springer, Berlin, pp 181–215 Herbold CW, Lee CK, McDonald IR, Cary SC (2014b) Evidence of global-scale Aeolian dispersal and endemism in isolated geothermal microbial communities of Antarctica. Nat Commun 5:3875 Hubert C et al (2009) A Constant flux of diverse thermophilic bacteria into the cold Arctic seabed. Science 325:1541–1544 Janetschek H (1963) On the terrestrial fauna of the Ross-Sea area, Antarctica (preliminary report). Pac Insect 5:305–311 King CE, King GM (2014) Description of Thermogemmatispora carboxidivorans sp. nov., a novel carbon-monoxide-oxidizing member of the class Ktedonobacteria isolated from a geothermally-heated biofilm, and analysis of carbon

123

monoxide oxidation by members of the class Ktedonobacteria. Int J Syst Evol Microbiol 64:1244–1251 King AJ, Meyer AF, Schmidt SK (2008) High levels of microbial biomass and activity in unvegetated tropical and temperate alpine soils. Soil Biol Biochem 40:2605–2610 King AJ, Karki D, Nagy L, Racoviteanu A, Schmidt SK (2010a) Microbial biomass and activity in high elevation soils of the Annapurna and Sagarmatha regions of the Nepalese Himalayas. Himal J Sci. https://doi.org/10.3126/hjs.v6i8. 2303 King AJ, Freeman KR, McCormick KF, Lynch RC, Lozupone C, Knight R, Schmidt SK (2010b) Biogeography and habitat modeling of high-alpine bacteria. Nat Commun 1:53 Lynch R, King AJ, Farı´as ME, Sowell P, Vitry C, Schmidt SK (2012) The potential for microbial life in the highest-elevation ([ 6000 masl) mineral soils of the Atacama region. J Geophys Res 117:G02028 Lynch R, Darcy JL, Kane NC, Nemergut DR, Schmidt SK (2014) Metagenomic evidence for metabolism of trace atmospheric gases by high-elevation desert Actinobacteria. Front Microbiol 5:698 Madden RA (1979) Observations of large-scale traveling Rossby waves. Rev Geophys 17:1935–1949 Polvani LM, Saravanan R (2000) The three-dimensional structure of breaking Rossby waves in the polar wintertime stratosphere. J Atmos Sci 57:3663–3685 Pulschen AA, Rodrigues F, Duarte RTD, Araujo GG, Santiago IF, Paulino-Lima IG, Rosa CA, Kato MJ, Pellizari VH, Galante D (2015) UV-resistant yeasts isolated from a highaltitude volcanic area in the Atacama Desert as eukaryotic models for Astrobiology. MicrobiologyOpen 4:574–588 Reinhard J, Ceruti MC (2010) Inca rituals and sacred mountains. The Cotsen Institute of Archaeology Press, Los Angeles Richards JP, Villeneuve M (2001) The Llullaillaco volcano, northwest Argentina: construction by Pleistocene volcanism and destruction by sector collapse. J Volcanol Geotherm Res 105:77–105 Richter M, Schmidt D (2002) Cordillera de la Atacama—das trockenste Hochgebirge der Welt. Petermanns Geogr Mitt 146:48–57 Robbins SJ, Di Achille G, Hynek BM (2011) The volcanic history of Mars: high-resolution crater-based studies of the calderas of 20 volcanoes. Icarus 211:1179–1203 Schiavone MM, Sua´rez GM (2009) Globulinella halloyi (Pottiaceae), a new species from Argentina. Bryologist 112:584–588 Schmidt D (1999) Das Extremklima der nordchilenischen Hochatacama unter besonderer Beru¨cksichtigung der Ho¨hengradienten. Dresdener Geographische Beitra¨ge 4:1–122 Schmidt SK, Nemergut DR, Miller AE, Freeman KR, King AJ, Seimon A (2009) Microbial activity and diversity during extreme freeze-thaw cycles in periglacial soils, 5400 m elevation, Cordillera Vilcanota, Peru´. Extremophiles 13:807–816 Schmidt SK, Naff C, Lynch R (2012) Fungal communities at the edge: ecological lessons from high alpine fungi. Fungal Ecol 5:443–452 Schmidt SK, Darcy JL, Sommers P, Gunawan E, Knelman JE, Jager K (2017a) Freeze–thaw revival of rotifers and algae

Antonie van Leeuwenhoek in a desiccated, high elevation (5500 meters) microbial mat, high Andes, Peru´. Extremophiles 21:573–580 Schmidt SK, Vimercati L, Darcy JL, Ara´n P, Gendron EMS, Solon A, Porazinska D, Dorador C (2017b) A Naganishia in high places: functioning populations or dormant cells from the atmosphere? Mycology 8:153–163 Solon AJ, Vimercati L, Darcy JL, Ara´n P, Porazinska D, Dorador C, Farias ME, Schmidt SK (2018) Microbial communities of high-elevation fumaroles, penitentes and dry tephra ‘‘soils’’ of the Puna de Atacama Volcanic Zone. Microb Ecol. https://doi.org/10.1007/s00248-017-1129-1 Tebo BM, Davis RE, Anitori RP, Connell LB, Schiffman P, Schiffman H (2015) Microbial communities in dark oligotrophic volcanic ice cave ecosystems of Mt. Erebus, Antarctica. Front Microbiol 6:179. https://doi.org/10.3389/ fmicb.2015.00179 Vimercati L, Hamsher S, Schubert Z, Schmidt SK (2016) Growth of a high-elevation Cryptococcus sp. during extreme freeze-thaw cycles. Extremophiles 20:579–588 Vishniac HS (1985) Cryptococcus friedmannii, a new species of yeast from the Antarctic. Mycologia 77:149–153

Vitry C (2016) Contribucio´n al estudio de caminos se Sitios Arqueolo´gicos de Altura. Volca´n Llullaillaco (6739 m). Museo de Arqueologia de Alta Montana Salta, Argentina Watson JM, Cardenas MP, Flores AR, Macaya J, Jı´menez H, Barrı´a J (2013) Viola gelida, una nueva especie rosulada, rara y vulnerable del sector altoandino de la Regio´n de Atacama, Chile. Gayana Bot. 70:390–394 Weber CF, King GM (2010) Distribution and diversity of carbon monoxide-oxidizing bacteria and bulk bacterial communities across a successional gradient on a Hawaiian volcanic deposit. Environ Microbiol 12:1855–1867 Wilson AS, Brown EL, Villa C, Lynnerup N, Healey A, Ceruti MC, Reinhard J, Previgliano CH, Araoz FA, Gonzalez Diez J, Taylor T (2013) Archaeological, radiological, and biological evidence offer insight into Inca child sacrifice. Proc Natl Acad Sci USA 110:13322–13327 Yang M, Yao T, Gou X, Koike T, He Y (2002) The soil moisture distribution, thawing–freezing processes and their effects on the seasonal transition on the Qinghai-Xizang (Tibetan) plateau. J Asian Earth Sci 21:457–465

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