Endolithic microhabitats have been described as the last
refuge for life in arid and hyper-arid deserts where life has to deal with
harsh environmental conditions. A number of rock substrates from the
hyper-arid Atacama Desert, colonized by endolithic microbial communities
such as halite, gypsum crusts, gypcrete, calcite, granite and ignimbrite,
have been characterized and compared using different approaches. In this
work, three different endolithic microhabitats are described, each one with
a particular origin and architecture, found within a lithic substrate known
as gypcrete. Gypcrete, an evaporitic rock mainly composed of gypsum
(CaSO
The statement developed by Lourens Gerhard Marinus Baas-Becking (Baas-Becking, 1934) that “everything is everywhere but the environment selects”, which established the most referred principle for microbial biogeography, remains in discussion regarding the first half of the statement (“everything is everywhere”) (de Wit and Bouvier, 2006; O'Malley, 2008; Bass and Boenigk, 2011; Fontaneto and Hortal, 2012; van der Gast, 2015). Regarding the second half of the statement (“but the environment selects”), extreme environments present some of the most plausible scenarios since they are inhabited only by microorganisms that can survive and/or thrive in their respective physical or geochemical extremes such as temperature, solar radiation, pressure, desiccation or pH (Rothschild and Mancinelli, 2001). Hyper-arid deserts, where the aridity index is lower than 0.05 (Nienow, 2009), constitute the most extreme deserts on Earth, and they usually combine a series of simultaneous stress conditions such as water limitation, extreme high and low temperatures, scarcity of organic carbon, high solar radiation, and osmotic stress (Pointing and Belnap, 2012). While these environments are considered polyextreme, they are inhabited by microbiota able to survive under such conditions. Hence, polyextreme environments are excellent microbial ecosystem models to study adaptive mechanisms to environmental stress. The Atacama Desert (northern Chile) is perhaps the most challenging polyextreme environment on Earth and the most barren region imaginable, with scarce precipitation events (McKay et al., 2003; Wierzchos et al., 2012a) and extremely low mean annual relative humidity (RH) (Azua-Bustos et al., 2015). Further, this desert holds another world record: the highest surface ultraviolet radiation (UV), photosynthetic active radiation (PAR) and annual mean surface solar radiation (Cordero et al., 2018) in the Chilean Coastal Range and Andes sites.
In this inhospitable polyextreme desert, microbial life has been found in
different lithic habitats such as epilithic (on rocks) (Wierzchos et al. 2011), hypolithic (under rocks)
(Azua-Bustos et al., 2012) and endolithic (inside rocks) microhabitats (reviewed
by Wierzchos et al., 2018, 2012b). Three different locations
of these endolithic habitats have been described within rocks of the Atacama
Desert: cryptoendolithic (occupying pore spaces in the rock),
chasmoendolithic (living within cracks and fissures in the rock) and
hypoendolithic (living inside pores in the bottom part of the rock).
Endolithic colonization can be viewed as a stress avoidance strategy whereby
the overlying mineral substrate provides protection from incident lethal UV
and PAR levels, and it also offers enhanced moisture availability
(Walker and Pace, 2007; Wierzchos et al., 2012b). These microbial communities,
regardless of the position they occupy in the rock or the type of rock, are
supported by oxygenic phototrophic primary producers supporting a diversity
of heterotrophic microorganisms (reviewed in Wierzchos et al., 2018). Molecular
and microscopy characterization of these endolithic microbial communities
shows that, overall, these communities are dominated by Cyanobacteria,
mostly from genus
This work addresses the impact of microhabitat architecture on the diversity and composition of gypcrete endolithic microbial communities (EMCs). The concept of rock architecture was introduced by Wierzchos et al. (2015) for colonized gypcrete substrate and encompasses the internal structures of rock with all elements that are essential for microbial life. Microhabitat architecture allows for perceiving the rock interior from the existence of porous spaces of different sizes and also the solid structures that divide and support these spaces. All these components and elements are interrelated and influence one another, thus fulfilling a requisite: they might shape a suitable architecture to hold microbial life.
The study is based on the hypothesis that the different architectures of endolithic microhabitats involve small-scale differences in the micro-environmental conditions, which in turn determine the distribution of organisms in each community. The hypothesis is tested here for the first time by using a multidisciplinary approach combining microscopy and molecular tools for their characterization. The microscale dimensions and differential diversity distribution in this unique environment have led us to coin the new term “micro-biogeography”.
Colonized rocks were collected in the Atacama Desert in December 2015 from
the Monturaqui area (MTQ) (GPS coordinates: 23
Microclimate data (Meslier et al., 2018) were recorded using an Onset
HOBO® micro weather station and data logger (H21-USB), as
previously described by Wierzchos et al. (2015). Air temperature (
Colonized gypcrete samples were processed for scanning electron microscopy
in backscattered detection mode (SEM-BSE) according to methods described by
Wierzchos and Ascaso (1994) and Wierzchos et al. (2011). Light microscopy
(LM) in differential interference contrast (DIC) mode was used to examine
cell aggregates gently isolated from the cryptoendolithic, chasmoendolithic
and hypoendolithic microhabitats as well as on cyanobacterial isolates cultured
from those microhabitats. The samples were examined using an
Axio Imager M2 microscope (Carl Zeiss, Germany) in DIC mode equipped with Apochrome 63
Micro-CT scans were run on pieces of gypcrete with an X-ray computed
tomography system (CT scan) – HMXST 225 micro-CT system (Nikon Metrology,
Tring, UK) to observe volume, bulk density and variations in internal
density. For volume and bulk density measurements, a Nikon X-Tek CT-scan
device was used, with an X-ray peak voltage of 146 kV and current of 65 mA,
collecting 1583 sections at 1000
Biological material removed from endolithic colonization zones of
gypcrete was transferred to different BG11 1.5 % agar plates (purified
agar, Condalab, Spain). All samples were incubated in a growth chamber at
Three individual rocks harbouring at least two of the three endolithic
microhabitats were processed, which resulted in 11 samples, including
technical replicates: cryptoendolithic (
This DNA extraction was performed using 0.3 g of samples and the UltraClean
DNA isolation kit (Mo Bio Laboratories, Solana Beach, CA, USA) including a
three-cycle step of freezing 0.3 mL aliquots of sample suspended in buffer,
breaking them down by using an adapted drill and melting in
60
Sampling location in the Atacama Desert. Monturaqui area: MTQ (black diamond). (© Google Earth, image providers: Landsat/Copernicus).
After demultiplexing and barcode removal, sequence reads with Phred score
Sequences of 16S rRNA genes from cyanobacterial OTUs that showed significant
differences in their relative abundance between endolithic microhabitats and
16S rRNA gene sequences from cyanobacterial isolates were aligned with
sequences obtained from the US National Center for Biotechnology Information (NCBI) GenBank using the Clustal W 1.4 software
(Thompson et al., 1994). The 16S rRNA gene sequences from GenBank were selected
using the NCBI MegaBlast tool (
We combined microclimate measurements, microscopy analyses and high-throughput culture-independent molecular data to identify the effect of micro-biogeography and the factors underlying the structure and composition of microbial assemblages of gypcrete endolithic microhabitats from the hyper-arid Atacama Desert.
Gypcrete samples were collected from the Monturaqui area (MTQ), located in
the Preandean Depression of the Atacama Desert (Casero et al., 2020) (Fig. 1) in December 2015. Climate data recorded over a period of 22 months
described a mean air temperature of about 15
CT-scan images of a colonized piece of gypcrete. The 3D reconstruction of a gypcrete sample with the spatial distribution of pores (yellow colour) and complete reconstructions of the scanned volume (grey colour) on lateral, front and top views of gypcrete. Porous micromorphology is capillary-shaped in the vertical direction due to movement of the water by gravity. Arrows in top-view images point to the deepest cracks. Scale bars are 1 cm.
CT-scan images provided a 3D spatial representation of pore shapes and their distribution inside the gypcrete rock (Fig. 2). The pores revealed capillary-like micromorphology that exhibits a vertical orientation as is shown in both the top and lateral views. Detailed 3D images pointed to the apparent absence of connectivity with the surface of most of the pores (Fig. 2). However, the presence of this connectivity cannot be discarded due to the limited resolution of the CT-scan technique and the conditions of acquisition. Moreover, CT-scan images of the gypcrete surface reveal microrill weathering features (DiRuggiero et al., 2013) due to the dissolution of gypsum after scarce rains (Supplement Video S1).
Characterization of endolithic colonization zones. Series A: cryptoendolithic; series B: chasmoendolithic; series C: hypoendolithic. Series 1: macro images of gypcrete cross sections of colonized zones; series 2 and 3: SEM-BSE images of gypcrete cross sections of colonized zones; series 4: LM-DIC images of scrapped Cyanobacteria from gypcrete. Series 1: black arrows indicate green and orange coloured endolithic colonization zones of 5 mm thickness in A1 (CR), 8 mm thickness in B1 beneath the surface (CH) and 5–9 mm thickness in C1 above bottom gypsum crust (HE). Series 2: CR, CH and HE microhabitats with aggregates of endolithic microbial communities surrounded by the dotted green lines, inside the pores of gypcrete; A2 is under a white dense surface crust; B2 is inside the cracks of gypcrete, and C2 is inside the microcaves of gypcrete at the bottom of the rock. Series 3: green arrows point to aggregates of Cyanobacteria among gypcrete (Gy) crystals (A3, B3), surrounded by sepiolite (Sp), nodules (A3) and on the gypcrete (Gy) walls (C3). Series 4: aggregates of different morphotypes of cyanobacteria, shown by green, yellow and orange arrows, and gypcrete crystals (Gy).
Cross sections of the gypcrete rocks reveal the presence of three clearly differentiated microhabitats where a significant heterogeneity in micromorphology and structure was found (Fig. 3). The cryptoendolithic colonization zone is close to the compact gypcrete surface layer (up to 5 mm depth). Within cryptoendolithic microbial communities, two characteristic pigmented layers are distinguished. The observed orange colour belongs to microorganisms with a high carotenoid content closest to the gypcrete surface. The green colour layer beneath the orange layer belongs to microorganisms with chlorophyll and phycobiliprotein content. The presence of these pigments was previously reported by Wierzchos et al. (2015) and Vítek et al. (2016) (Fig. 3 A1). The chasmoendolithic colonization zone reaches a deeper (up to 8 mm depth) position in the substrate and is directly connected to the surface (Fig. 3 B1). Finally, the hypoendolithic colonization zone is located close to the compact bottom gypcrete crust, shaped like microcaves (Fig. 3 C1).
Cyanobacteria were found in the cryptoendolithic habitat among lenticular gypcrete crystals, filling up vertically elongated pores, and aggregated around sepiolite nodules (Fig. 3 A2–A3), a clay mineral with high water retention capacity, previously identified in gypcrete by Wierzchos et al. (2015). SEM-BSE images also revealed dense arrangements of cyanobacterial cells embedded in concentric sheets of exopolysacharides (EPS) (Fig. 3 A3). By contrast, the microbial assemblages inhabiting the chasmoendolithic and hypoendolithic microhabitats were coating the walls of the cracks and caves previously described (Fig. 3 B2, B3, C2, C3). Detailed SEM-BSE images (Fig. 3 A3–C3) and LM images (Fig. 3 A4–C4) of each microhabitat showed mainly Cyanobacteria with different sizes and morphology accompanied by heterotrophic bacteria.
A total of 12 cyanobacterial strains were isolated from the three different
endolithic microhabitats (Table S1): three from cryptoendolithic, three from
chasmoendolithic and six from hypoendolithic. The cyanobacterial strains were
identified, following Komárek et al. (2014), as
Diversity estimates of microbial communities in the endolithic microhabitats of gypcrete.
High-throughput sequencing of 16S rRNA gene amplicons across 11 samples and
three microhabitats resulted in a total of 385 440 V3-V4 small subunit (SSU) rDNA reads, with an
average number of paired-end reads per sample of 35 040
A total of 11 bacterial phyla with a relative abundance
Average relative abundance of sequence reads
The four main phyla constituted
Compared to other microhabitats, this phylum showed the highest relative
abundance in terms of sequences (60.4 %) but the lowest in terms of OTUs
(21.9 %), thus revealing the high abundance of a very low number of
cyanobacterial OTUs. Average Bray–Curtis distance confirmed that
dissimilarity between microhabitats (CR-CH
Maximum likelihood tree based on partial 16S rRNA sequences of Cyanobacteria OTUs above 1 % relative abundance and cyanobacterial strains isolated from the three gypcrete microhabitats. Bold indicates sequences from this study. Scale bar indicates 5 % sequence divergence.
As the major component of the endolithic communities from the three described
microhabitats, Cyanobacteria OTUs and isolates were studied in detail. A
phylogenetic analysis of the 15 major cyanobacterial OTUs (relative
abundance
Most of the OTUs (9 out of 15) and isolates (8 out of 12) were assigned to
the genus
Cluster II comprised cyanobacterial sequences belonging to the Nostocales
order from the genera
Because of the low percentage identity of OTU2 with its closest relatives in the
database (
Differentially abundant cyanobacterial OTUs across the three microhabitats
are represented by
Differential abundance analysis using the DESeq2 test revealed that 9 out of 15
of the cyanobacterial OTUs were differentially abundant in the three
microhabitats (Fig. 6). Both OTU11 (
In this study, we characterized the microbial communities inhabiting gypcrete collected from the Monturaqui area (Preandean Depression), which is of particular interest due to its location in the hyper-arid zone of the Atacama Desert. While endolithic colonization of the gypsum crust and gypcrete in this area has previously been studied (Dong et al., 2007; DiRuggiero et al., 2013; Wierzchos et al., 2015; Meslier et al., 2018), this is the first work in which cryptoendolithic, chasmoendolithic and hypoendolithic communities have been characterized separately. The novelty of this study lies in the consideration of two different EMCs inhabiting two endolithic microhabitats located in the upper part of the substrate and in the description of the structure and composition of the hypoendolithic microhabitat and its endolithic community, located at the bottom part of the substrate.
The Monturaqui region, located in the Preandean Depression of the Atacama
Desert has been found to harbour two different substrates colonized by
microbial communities, namely gypcrete (Wierzchos et al., 2015) and
ignimbrite, a volcanic rock (Wierzchos et al., 2013). Both substrates show
endolithic colonization and a lack of epilithic colonization (rock surface
colonization). The absence of this second type of colonization in any
substrates from the Monturaqui region may be explained by the extremely arid
microclimate of this area, including low relative humidity, high fluctuation
of air and surface temperature, extremely high solar irradiation, and scarce
precipitation (Wierzchos et al., 2015). Monturaqui has been described as a
hyper-arid area, showing an aridity index of 0.0075 (Wierzchos et al., 2013),
based on the ratio of mean annual precipitation (
Potential endolithic habitability is tightly linked to the porosity of a lithic substrate because the distribution and size of pores are often directly related to the substrate's water retention capacity (Cámara et al., 2014; Herrera et al., 2009; Matthes et al., 2001; Omelon 2008; Pointing et al., 2009; Meslier et al., 2018). Porosity in gypcrete allows microbial communities to survive in different microhabitats, providing sufficient space for the communities while receiving enough light and having enough water to metabolize and grow. The porous network of gypcrete restricts water loss by rapid evaporation and helps its retention by capillary forces acting in small capillary-like pores. The inner architecture of gypcrete allows for the habitability of three different locations inside the substrate. The CT-scan and SEM-BSE images from this work showed that all three types of microhabitats shared a vertical axis of morphology with vertical cracks constituting the chasmoendolithic (CH) microhabitat, and capillary-like pores constitute the cryptoendolithic (CR) and hypoendolithic (HE) microhabitats. This capillary-like pore architecture found in the CR microhabitat could be explained by the progressive substrate dissolution due to scarce rains and by the water retained and condensed within the micropores, as it occurs in halite endolithic microhabitats (Wierzchos et al., 2012a). The observed HE microhabitat architecture supports the proposal of Wierzchos et al. (2015), in which the authors described the presence of a dense crust delimiting the bottom part of the HE microhabitat. This structure reveals different dissolving and crystallization processes of the gypsum following the water displacement from the surface to the bottom of the rock (gravity flow). This water gravity flow gives rise to the cave-shaped pores, thus providing this HE microhabitat with a hard permeable bottom gypsum layer.
The larger distance between the HE microhabitat and the top surface microhabitats CR and CH might be thought of as a limiting factor for the development of HE communities, especially in terms of water availability. However, the location of the HE microhabitat at the bottom of the rock could reduce water losses due to evaporation processes. Thus, the microcave structures we observed in the HE microhabitat might retain liquid water for longer times, leading to cyanobacterial growth.
The structural characteristics of the crypto- and chasmoendolithic microhabitats, located at the top of the substrate, also allow access to water for the EMCs. Within the CR microhabitat, the labyrinth of pores directly or indirectly connected to the surface may act as cavities where water might be retained, condensed and also be present in the form of saturated water vapour (high RH) through the substrate and be available to the microbial communities. Additionally, the presence of sepiolite inclusions improves water retention in those pores, as previously described (Wierzchos et al., 2015; Meslier et al., 2018), leading to lower rates of water loses by evaporation and gravitational forces. In contrast, the CH microhabitat provides direct access to rainfall liquid and dewfall water for its community, via its fissure and cracks, while at the same time lowering water retention capacity by higher evaporation rates and losing liquid water by percolation through the rock.
Microbial communities inhabiting all three microhabitats were found in the form of large aggregates and were often embedded in an EPS matrix. These characteristics are closely linked to survival strategies under harsh environmental conditions related to low water and nutrient availability (Billi, 2009; Wright et al., 2005). Since water is the most limiting factor for the development of microbial communities inhabiting endolithic microhabitats of gypcrete, it is the component on which adaptive strategies are primarily focused. EPS is an essential adaptation strategy against hyper-aridity due to its role in hydration and dehydration processes in lithobiontic communities, as previously observed in Antarctic deserts (de los Ríos et al., 2007) and the Atacama Desert (Dong et al., 2007; Wierzchos et al., 2011, 2015; Crits-Christoph et al., 2016). The aggregate-like structure of these communities composed of Cyanobacteria and heterotrophic bacteria also helps their survival against drought, since dead cells could provide physical protection against desiccation processes (Postgate, 1967; Roszak and Colwell, 1987; Billi, 2009; de los Ríos et al., 2004). In the case of the CR community, a special strategy against dryness was observed in this work, since microorganisms were located close to the sepiolite, as previously reported concerning gypcrete endolithic communities (Wierzchos et al., 2015; Meslier et al., 2018). EPS and dead cells taking part in the aggregates can also act as a nutrient reservoir in such an oligotrophic environment, since low amounts of water-soluble ions were previously detected in the MTQ gypcrete (Meslier et al., 2018).
The absence of significant differences in diversity metrics between the three EMCs of gypcrete is in accordance with the diversity values of previously reported EMCs in the Atacama Desert (reviewed in Casero et al., 2020). At a phylum level, the community was composited of three main dominant phyla, Cyanobacteria, Proteobacteria and Actinobacteria (Fig. 4), as in other EMCs of the Atacama Desert (Wierzchos et al., 2015; Meslier et al., 2018; Dong et al., 2007). However, a switch in the Proteobacteria and Actinobacteria relative abundances was found compared to gypcrete cryptoendolithic communities previously described (Meslier et al., 2018). That difference is presumably associated with different DNA extraction methods and the inherent associated biases. While the three types of gypcrete microhabitats are exposed to the same climatic conditions, we suggest that differences in micro-architectures resulted in drastically different sets of characteristics for water retention discussed previously: CR counts on water capillary porous condensation and sepiolite water absorption properties, CH has easier access to liquid water, and HE suffers less water loss.
While the communities from the three microhabitats had similar alpha diversity
metrics, we found the composition of these communities was statistically
different, which is supported by the relative abundance of the main phyla,
Cyanobacteria, Proteobacteria and Actinobacteria, across the microhabitats
distributed differentially, exhibiting differences between the CR and CH
communities as compared to the HE community, especially regarding
cyanobacterial OTUs. This notable difference in the relative abundance of
Cyanobacteria could be related to the particular resources of the
phototrophic community. The differential access to solar irradiance could
explain the contrast between cyanobacterial proportions on both sides, at
the top (CR and CH) and bottom (HE) of the substrate. Thus, an update to the
proposal by Wierzchos et al. (2018) is here suggested, in which a causal
link is evoked to explain the higher abundances of phototrophs as opposed to
heterotrophs in EMCs, which has been observed previously (Robinson et al., 2015; DiRuggiero et al., 2013; Wierzchos et al., 2015; Meslier et al., 2018).
According to that work (Wierzchos et al., 2018), the scarcity of water was suggested to cause a lower
metabolic activity in phototrophs, thus leading to lower support of the
heterotrophic community. However, in this scenario, light intensity should
also be considered a crucial factor in understanding the differences between
the composition of top and bottom EMCs, since the HE community has notably
lower access to sun radiation. Recently, the light intensity as a driving
factor of spatial heterogeneity within halite endolithic microbial
communities was reported by Uritskiy et al. (2020). Thus, for EMCs based on phototrophic microorganisms, a limitation to one of
those resources essential for photosynthesis would further lead to low rates
of CO
In contrast with results of Wierzchos et al. (2015) in gypcrete endolithic
communities, no eukaryotic algae were found in microscopy nor
molecular analyses, with Cyanobacteria being the phototrophic phylum observed in
all gypcrete endolithic microhabitats. While we found multiple phylotypes of
Cyanobacteria among the gypcrete microhabitats, most of them belonged to the
genus
The differential distribution of key members of these EMCs among microhabitats in the same lithic substrate and the same piece of rock, as their primary producers, reveals an “environmental filtering” process (Kraft et al., 2015). This concept focuses on the relationship between an organism and the environment, recognizing that not all organisms will be able to establish themselves successfully and persist in all abiotic conditions. Thus, in this scenario, the abiotic conditions linked to the architecture and location of the endolithic microhabitat would force the development of community assemblages highly specialized to small-scale differences, thereby exhibiting a micro-biogeographical behaviour.
Our work answers the hypothesis that there are certain differences in the structure of endolithic microbial communities among crypto-, chasmo- and hypoendolithic habitats. Considering that the external climatic regime was the same for the studied pieces of rock, our results have shown that the structure of these microbial communities was different among endolithic habitats. Following the definition of microhabitat architecture by Wierzchos et al. (2015), we can distinguish different architectures of the substrate within different endolithic microhabitats. In this context, our work suggests that distinct features of microhabitat architecture that have an influence on micro-environmental variables at the microscale would shape microbial community structure.
However, we are aware that more “micro-biogeographic” studies should be done with other endolithic microhabitats from the Atacama Desert and elsewhere. These may show that gypcrete is not a peculiar case where differences in the architecture of a microhabitat play an essential role in shaping the diversity and composition of endolithic microbial communities.
This study is the first to address differences between microbial communities
inhabiting three differentiated endolithic microhabitats within the same
lithic substrate. In this study, liquid water availability was proposed to
be a driver of community composition, because the specific architectural
features of each microhabitat facilitated water input and retention in
different ways. Water, light and CO
The genus
Findings from this work reveal the importance of using an appropriate scale
for the study of microbial communities. Indeed, we found that the
microstructural and micro-architectural features of the endolithic habitats
were key factors in determining the composition of endolithic microbial
communities. Thus, this study suggests a cautious use of
“macro-environmental” parameters in characterizing differences between
endolithic communities from different deserts or substrates. Our results
point to the need for a more thorough description of the micro-environmental
conditions that directly exert an effect on microbial assemblages: light,
water and CO
All the sequencing data sets generated in this study have been submitted to the US National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and can be found under the BioProject ID PRJNA637482.
The supplement related to this article is available online at:
MCC and JW designed and performed the research. JW conceived the original project. MCC, JW and OA carried out the sampling. MCC, JW and AQ wrote the article. MCC, JW and CA performed the microscopy. TK contributed to CT-scan analysis. MCC, VMA and JDR contributed to the molecular data, analysis, and performed the sequencing. All authors contributed to editing and revising the article and approved this version for submission.
The authors declare that they have no conflict of interest.
The work of MCC was supported by grant BES 2014-069106 from the Spanish Ministry of Science and Innovation (MCINN). The MNCN-CSIC, Madrid, Spain, is acknowledged for microscopy services.
This research has been supported by the MCIU/AEI (Spain) and FEDER (UE) (grant no. PGC2018-094076-B-I00), the NSF (grant no. DEB1556574), and NASA (grant no. NNX15AP18G).We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
This paper was edited by Andreas Richter and reviewed by three anonymous referees.