ENVIRONMENTAL MICROBIOLOGY

Ancient Photosynthetic Eukaryote Biofilms in an AtacamaDesert Coastal Cave

A. Azúa-Bustos & C. González-Silva & R. A. Mancilla &

L. Salas & R. E. Palma & J. J. Wynne & C. P. McKay &

R. Vicuña

Received: 18 November 2008 /Accepted: 6 February 2009 /Published online: 5 March 2009# Springer Science + Business Media, LLC 2009

Abstract Caves offer a stable and protected environmentfrom harsh and changing outside prevailing conditions.Hence, they represent an interesting habitat for studying lifein extreme environments. Here, we report the presence of amember of the ancient eukaryote red algae Cyanidiumgroup in a coastal cave of the hyperarid Atacama Desert.This microorganism was found to form a seeminglymonospecific biofilm growing under extremely low photonflux levels. Our work suggests that this species, Cyanidium

sp. Atacama, is a new member of a recently proposed novelmonophyletic lineage of mesophilic “cave” Cyanidium sp.,distinct from the remaining three other lineages which areall thermo-acidophilic. The cave described in this work mayrepresent an evolutionary island for life in the midst of theAtacama Desert.

Introduction

Many cave ecosystems on Earth are known to hostheterotrophic bacteria serving as the food base [5, 6, 32,39]. Although much is known about these microorganisms[6, 34], less has been published on photosynthetic micro-organisms proliferating in caves. Recently, taxonomicsurveys of lamp-associated flora algae and cyanobacteriain electrically lit passages within show caves have beenreported [14, 44]. However, these studies were focused ontheir control rather than on their biology. Other groups havereported bioactive metabolites isolated from cyanobacteriafound in caves, with no further details on the description orthe ecology of the microorganisms [2, 3].

A similar trend is found for other phototrophs likeunicellular algae, as it is the case of the Cyanidiales, agroup of asexual, unicellular red algae usually found inacidic (pH 0.5–3.0) high-temperature (50–55°C) sitesaround hot springs [11, 36]. Most of these algae are in factblue–green due to the pigments c-phycocyanin andchlorophyll-a [45]. The Cyanidiales are classified into threegenera, Cyanidium, Cyanidioschyzon, and Galdieria [9, 40,55], of which Cyanidium caldarium is the most studiedmember. This species of alga is characterized by roundedcells with thick walls, nucleus, a single chloroplast, avacuole, and one mitochondrion [1, 16]. Cyanidium cellsreproduce by internal divisions forming daughter cells or

Microb Ecol (2009) 58:485–496DOI 10.1007/s00248-009-9500-5

A. Azúa-Bustos (*) : R. A. Mancilla : L. Salas : R. VicuñaDepartamento de Genética Molecular y Microbiología, Facultadde Ciencias Biológicas, Pontificia Universidad Católica de Chile,Alameda 340,Santiago, Chilee-mail: ajazua@uc.cl

C. González-SilvaDepartamento de Ciencias Químicas y Farmaceúticas,Universidad Arturo Prat,Iquique, Chile

J. J. WynneUSGS, Southwest Biological Science Center, and Departmentof Biological Sciences, Northern Arizona University,Flagstaff, AZ 86011, USA

C. P. McKayNASA-Ames Research Center,MS 245-3,Moffett Field, CA 94035, USA

A. Azúa-Bustos :R. VicuñaMillennium Institute for Fundamental and Applied Biology,Santiago, Chile

R. E. PalmaDepartamento de Ecología, Facultad de Ciencias Biológicas,Pontificia Universidad Católica de Chile,Santiago, Chile

autospores in the form of tetrads inside the mother cells[16]. Both the 16S ribosomal RNA (rRNA) [47, 53] and therbcL and psbA genes codified in the chloroplast [10], aswell as the 18S rRNA in the chromosomal DNA [45], havebeen previously used for identification and phylogeneticclassification of environmental samples. These phylogenet-ic studies suggest that the Cyanidiales are one of the mostancient groups of algae, having diverged about 1.3 billionyears ago at the base of the Rhodophyta [19, 42, 54].Nevertheless, it has been reported that species of theCyanidium genera have also been found growing insidecaves [9, 17]. These aerophytic epilithic “cave Cyanidium”are the mesophilic members of the clade, and currentphylogenetic analyses support the existence of fourdistinct Cyanidiales lineages: the Galdieria spp. lineage,the C. caldarium lineage, the Cyanidioschyzon merolaeplus Galdieria maxima lineage, and the novel monophy-letic lineage of mesophilic cave Cyanidium spp. [9].

Previous analyses suggest that there is a high level ofsequence divergence among Cyanidiales species based onenvironmental conditions, and, although they have a longevolutionary history, only a few recognized morphologicalspecies are known [9]. Based on morphological character-istics alone, Schwabe [41] reported in 1936 a species whichhe assigned to the Cyanidium genera in two caves on thecentral coast of Chile, which are located about 1,200 and1,500 km south of our study site. On the other hand, theAtacama Desert is located between 17° and 27° S latitudein northern Chile. It is constrained on the east by the frontranges of the Andes Mountains and on the west by theCoastal Range. The Atacama is one of the driest andprobably the oldest extant desert on Earth [25], havingexperienced hyperaridity for at least three million years andprobably 150 million years [24]. To survive in thesehyperarid conditions, life forms have adapted to very lowair humidity levels, an almost complete absence of rainevents, highly saline soils, and high solar radiation [4, 12,29]. These harsh environmental factors may explain whyparts of the Atacama Desert are almost devoid of microbiallife [12, 18, 33]. Interestingly, the Atacama Desert has beenestablished as a prime analog model for the planet Mars,and many research teams have conducted research on adiversity of astrobiological-oriented topics [43, 48].

Although much has been published about the hyperaridareas of the Atacama Desert [10, 28, 29, 50], fewer reportsexist on the microbial life present in areas of the Atacamawith a little more water availability. These areas could givevaluable information to be compared with that of other siteslocated elsewhere in the hyperarid regions of the desert.The arid Coastal Range that separates the hyperaridAtacama Desert plateau from the Pacific Ocean, acts as atopographic barrier to clouds and moisture-rich marine airmoving eastwards from the ocean. Consequently, the dry

hills of the Pacific Coastal Range have more benign sitesfor the development of microbial life, differing from thetypical desert conditions associated to the Atacama. Onlyrecently, caves in the arid areas of the Atacama have begunto be explored, and to date no microorganisms of any typehave been described living inside them [51].

Here, we describe the presence of biofilms growing atmesophilic conditions at the dimmest lighted zone of acoastal cave of the Atacama Desert. Our microscopy andmolecular biology data suggest that the photosyntheticmember of these biofilms is represented mainly, if notsolely, by a new species of the Cyanidium genus,Cyanidium sp. Atacama, closely related to the only twoother known cave Cyanidium previously described.

Methods

Biofilm Sampling

Biofilm samples were taken in situ by scraping the walls ofthe cave with a sterile scalpel and depositing the materialobtained into sterile 50-ml Falcon tubes. When working atthe dimmest lighted areas of the cave, care was taken in notdisturbing the light conditions of the site by using strongsources of artificial lights.

Temperature, RH, and pH Measurements

Temperatures of rocks inside the cave were measured witha Raytek infrared thermometer (Raytek Corporation, SantaCruz, CA, USA). Three readings per measurement weremade by placing the thermometer about 5 cm from the rockbeing analyzed. Measurements were taken every 2 m fromthe cave bottom in both the western and eastern walls. ThepH of the cave walls where biofilms develop was measuredin situ by collecting water droplets from the rocks and usingpH strips papers. Relative humidity (RH) and air temper-ature inside the cave were measured with a DO 9406thermometer–hygrometer data logger (Delta Ohm, Padua,Italy). Measurements were taken at the bottom of the cave,near the entrances, and close to the eastern and westernwalls. Measurements were taken in March and June of2008.

Transmission Electron Microscopy

Biofilm samples were centrifuged at 3,000 rpm on atabletop centrifuge. The resulting pellet was fixed with3% glutaraldehyde in sodium cacodylate buffer 0.1 MpH 7.2 during 18 h at room temperature. After threeconsecutive 20-min washes with sodium cacodylate buffer0.1 M pH 7.2, the samples were treated with 1% aqueous

486 A. Azúa-Bustos et al.

osmium tetroxide during 60 min. The samples were thendehydrated with a graded acetone series (50% to 100%),15 min for each concentration used. The samples were pre-embedded overnight with epon–acetone 1:1 and thenembedded in pure epon. The polymerization process wasdone at 60°C during 24 h. Sixty-nanometer-thin sectionswere obtained with a Sorvall MT-5000 ultramicrotome, andstained with 4% uranyl acetate in methanol and lead citrate.Observations were made with a Philips Tecnai 12 trans-mission electron microscope operated at 80 kV.

Scanning Electron Microscopy

A small rock sample covered with biofilm was fixed with2% glutaraldehyde in cacodylate buffer overnight and thenwashed with the same buffer three times, 20 min each.After critical point drying and gold coating, the sampleswere observed with a scanning electron microscope JeolJSM 25S-II at 30 kV.

Confocal Laser Scanning Microscopy

Biofilm samples of the dimmest lighted areas of the cavewere suspended in water and immediately observed usingan Olympus FV 1000 confocal laser scanning microscopeequipped with a ×100 oil immersion objective (Olympus,Hamburg, Germany). The images were analyzed with theFluoview 5.0 software.

Bright Field Microscopy

Biofilms samples were suspended in water, placed in aNeubauer cell counting chamber, and immediately observedwith a Nikon Optiphot-2 microscope equipped with aQimaging Micropublisher 3.3 RTV digital camera.

Photosynthetic Photon Flux Density Measurements

For photosynthetic photon flux density (PPFD) measure-ments, an Apogee Quantum Meter QMSW-SS calibratedfor sunlight was used as instructed by the manufacturer. Forthe wall biofilms inside the cave, the sensor was placedparallel to the biofilms and then pointed to the entrances tomeasure the amount of light reaching each sector. Eachreading was recorded when the sensor showed a stablevalue for at least 30 s.

Isolation and Sequencing of 16S rRNA, rbcL, and psbAGene Sequences

Approximately 100 mg of biofilm material were asepticallycollected and total genomic DNAwas extracted using a SoilDNA Isolation Kit (MoBio Laboratories, Solano Beach,

CA, USA) according to the manufacturer’s instructions.The 16S rRNA plastid gene present in the extracted DNAwas amplified using the Cyanidium-specific oligonucleotideforward primer Cyan100F (5′-TATAATGGAGAGTTTGATCCTGGCT-3′) and the reverse primer Cyan1450R(5′-TCCAGTACGGCTACCTTGTTACG-3′). The ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) rbcLplastid gene present in the extracted DNA was amplifiedusing the rbcL-specific oligonucleotide forward primersfwrub530 (5′-GTGACTGCTGCTACAATGGAGGA-3′)and the reverse primer rvrub1063 (5′-GCCTCTAAAGCTACCCTATTAGC-3′). These primers were designed on thebasis of sequence comparisons of known 16S rRNA andrbcL Cyanidium genes. The psbA gene was amplified aspreviously reported [9].

For amplification of the DNA templates, the Go Taqgreen and colorless Master Mix (Promega Corporation,Madison, WI, USA) were used according to the manufac-turer’s instructions, using a 1:100 dilution of the extractedDNA.

Polymerase chain reaction (PCR) conditions consistedon an initial denaturing step at 94°C for 2 min followed byfour sequential cycles of 94°C for 1 min, n°C for 1 min,and 72°C for 1 min, in which n=46–47–48–49°C. This wasthen followed by 31 cycles of 94°C for 1 min, 50°C for1 min, 72°C for 1 min, and a final extension step at 72°Cfor 15 min, thus totaling 35 cycles of amplification.

The PCR products were ligated to the pGEM-T EasyVector System (Promega Corporation, Madison, WI, USA)and cloned in Escherichia coli XL1-Blue cells. Theresulting plasmid vectors were isolated and purified usingthe Invisorb Spin Plasmid Mini Two (Invitek GmbH,Berlin, Germany) according to the manufacturer’s instruc-tions. The automated sequencing of the clones and threePCR products was done by Macrogen DNA SequencingInc. (Seoul, Korea) using the M13 forward primer site ofthe pGEM-T Easy Vector.

Phylogenetic Analysis

The nucleotide sequence of the Cyanidium sp. Atacama16S rRNA, rbcL, and psbA genes was analyzed using theMegablast option for highly similar sequences of theBLASTN algorithm against the National Center forBiotechnology Information non-rebundant database (www.ncbi.nlm.nih.gov). A multiple alignment of the orthologous16S rRNA, psbA, and rbcL genes sequences of relatedCyanidiales species was then performed using CLUS-TALW [27]. Accession numbers for the plastid genes ofpreviously sequenced rhodophytes used for phylogeneticcomparisons were: 16S rRNA: AB002583, AY882672,AY391361, AY391359, AY391360, AF170718, X52985,EU586032, and X81840, rbcL: AB002583, AY119765,

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AY391370, AY541297, AY541298, X53045, AF022186,AY391368, and AY391369, psbA: AY391365, AY391366,AB002583, AY391367, AF022186, and X14667.

Accession numbers for the 16S rRNA, rbcL, and psbAgenes of Cyanidium sp. Atacama are FJ390400, FJ402842,and FJ447339, respectively.

Phylogenetic reconstructions were performed throughmaximum parsimony using PAUP* 4.0b8. All characterswere analyzed as unordered with four possible states (A, C,G, and T), excluding phylogenetically uninformativecharacters. The most parsimonious trees were foundthrough an exhaustive search. Phylogenetic analyses werealso accomplished using the neighbor-joining andmaximum-likelihood algorithms available in PAUP. Forthe latter analysis, we first obtained the best fitting model ofsequence evolution, using the Akaike Information Criterionin Modeltest 3.06. For the 16S rRNA genes, the AICindicated that the GTR+G model (gamma: 0.2328) was thebest model to describe the evolutionary process. Valueswere: −ln L=4,847.07952 (base frequencies: A 0.2713, C0.2176, G 0.2957). In the case of the psbA genes, the AICindicated that the GTR+G+I model (alpha 3.3312) was thebest model to describe the evolutionary process. Valueswere: −ln L=3,887.80561 (base frequencies: A 0.25730, C0.18620, G 0.21670). Consistency on the nodes wasevaluated via bootstrap, performing 1,000 replicates foreach of the optimality criterion analysis. Trees were rootedwith the out-group criterion using the published sequenceof the Cyanophora paradoxa 16S rRNA gene [9].

Results

Site Description

The cave is located in the coastal range of the AtacamaDesert, about 20 km north of the city of Antofagasta, Chile.It is placed at the base of a 50-m-high cliff, facing southtowards the Pacific Ocean. The cave is approximately 35 min depth and 6 m wide, with an average height of 3 to 4 m,although in parts its height exceeds 6 m (Fig. 1). It has twoentrances; the one facing east is about 3 m wide and 3.5 mtall (Fig. 2a). The other entrance point south, confronts thePacific Ocean, and is about 4 m wide and 3 m tall. The seaenters the cave through this entrance covering no more thanone third of the ground during high tides. The walls of thecave are formed by Jurassic breccias of basaltic andesite,and the ceiling is formed by an overlaying conglomeratebed of fossilized marine sandstone and fossil shells dating35 to two million years ago (Fig. 2b) [20]. The cave has ahigh RH that varies dynamically in time and is relativelyhomogenous along its interior (Table 1). The temperature ofthe rock walls inside the cave is around 15°C during mostpart of the day and is the same at similar distances from theentrance of the cave in both the eastern and western walls(Table 2). Water droplets collected from the walls wherebiofilm is located have a pH of 4.5. These water dropletsseem to be evenly distributed along the cave and appear tobe periodic since they were observed during March of 2008but not in June, when the walls of the cave were drier.

Figure 1 Map and profile viewof the Atacama Desert cave

488 A. Azúa-Bustos et al.

Biofilm Distribution

The studied biofilms have an intense green–emerald colorand are dispersed throughout the cave, always away fromdirect light coming from the two entrances. The zoneshowing most biofilm development is located on the north–western wall, facing the east entrance but not directlyconfronting it. This zone is located about 13 m from theeast entrance, and the biofilm is located about 1.5 m fromthe cave floor, extending 1.5 m towards the cave ceiling.Part of the ceiling above the main development zone alsoshows biofilm but to a lesser extent (Fig. 2c). On this samewall, about 23 m from the east entrance and towards thebottom of the cave, there is also biofilm development,

although it is placed closer to the ground and on adjacentground rocks (Fig. 2d). The eastern wall shows nodevelopment of biofilms (Fig. 2b).

Photosynthetic Photon Flux Density Measurements

The PPFD measured outside the southeast entrance of thecave on a clear summer day was of 1,668 μmol m−2 s−1.Two different measurements were made, namely, at 11 AM

on a summer day (March) and at 14:30 PM on a winter day(June) of 2008, showing similar trends in the light profileinside the cave at the dimmest lighted zones. Five metersinside this entrance, the PPFD values on the western wallwere of 25 μmol m−2 s−1. The main biofilm development

Figure 2 Atacama cave de-scription. a East entrance. bView towards the bottom (north)of the cave. The biofilm areastudied is located on the left sidewall. c Detail of Cyanidiumbiofilm. d Biofilm on rocks atthe bottom of the cave. Note thebiofilm development only onthe rock face oriented towardsthe east entrance. In both a, b,and c, the different geologicalorigin of the walls and ceiling ofthe cave can be observed

Time Air temperature (°C) Air RH (%) IR t° (C)

Eastern wall Western wall

14:00 At main biofilm 21.7 77 – –

Cave entrance 22 80 – –

Cave bottom 22 80 – –

14:20 25.5 59 14 14

14:50 19.1 85 15 15

15:00 – 77 – –

15:40 18.8 89 15 –

Table 1 Variations in time ofair temperature and RH andinfrared temperature of walls ofthe cave interior (March 2008)

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zone showed values of 1 to 3 μmol m−2 s−1, that is, 0.06%to 0.17% of the outside incident light. The bottom of thefarthermost zone of the cave, where a thin biofilm couldstill be found, had values of 1 μmol m−2 s−1 (Table 3). Theeastern wall had much lower or received no light, asmeasured by the used PPFD sensors. Thus, the areas of thecave where the biofilm develops are subject to extremelylow light intensities, close to measuring sensitivity of thePPFD equipment.

Microscopy

Light microscopy examination of aqueous resuspendedsamples of biofilm scraped from the cave walls revealed ahomogeneous and possibly monospecific population ofphotosynthetic primary producers. The spherical cells are3–6 μm in diameter (Fig. 3a). Some autospore-containingcells are also seen (Figs. 3a and 4b). The cell’s singlechloroplasts emit an intense chlorophyll autofluorescencered signal under the confocal microscope, as observed inFig. 3b. Transmission electron microscopy (TEM) micro-graphs unveiled the typical ultrastructural elements previ-ously described for these unicellular red algae (Fig. 4a).The chloroplast, nucleus, and mitochondria are clearlydistinguished, as well as their corresponding membranes.

The thylakoid concentric membranes show the embeddedphycobillisomes that have been described for this species.Several electron-dense bodies are also observed associatedto the cell membrane (Fig. 4a), which in some cells appearto form two different chains at the opposite poles of thecell. On the other hand, scanning electron microscopy(SEM) micrographs show the Cyanidium biofilm attachedto its parent rock. Two different aggregates can beobserved: one in which the cells are close together withno supporting matrix (Fig. 5a) and another aggregate typewhere the cells are well embedded in matrix of (probably)exo-polysaccharides (EPS; Fig. 5b).

Phylogenetic Analysis of 16S rRNA, psbA, and rbcLChloroplast Genes

Using 16S rRNA, psbA, and rbcL Cyanidium-specificprimers, we amplified the 16S rRNA, psbA, and rbcLgenes consisting of 1,461-, 920-, and 548-bp products,respectively. Automated sequencing of eight of the 16SrRNA clones and three PCR products showed that all whereidentical and matched (97% identity) with a partialsequence of the 16S ribosomal RNA gene of Cyanidiumsp. Monte Rotaro, isolated from a cave in Italy. The nextmost similar sequence match (93% identity) corresponded

Table 2 Infrared temperatures of cave interior walls (June 2008)

Distance from bottom of the cave (m) IR temperature eastern wall (°C) IR temperature western wall (°C)

1 15.5 15.7

3 15.3 15.3

5 15.2 15.2

7 15.1 15

9 14.8 14.8

11 14.6 14.5

13 14.7 24.8

Table 3 Lighting profile along the length of the cave. The light available for photosynthesis was determined with a photosynthetic photon fluxdensity measuring device outside and inside the cave at different places in both the western an eastern walls of the cave

March 2008 June 2008

Distance fromcave entrance (m)

PPFD (μmol m−2 s−1) Percentage of outside light PPFD (μmol m−2 s−1) Percentage of outside light

Western wall Eastern wall Western wall Eastern wall Western wall Western wall

0 1,668 1,668 100 100 1,250 100

5 25 12 1.50 0.72 158 12.64

10 6 3 0.36 0.18 54 4.32

15 6 0 0.36 0 21 1.68

20 3 0 0.18 0 7 0.56

25 2 0 0.12 0 2 0.16

30 1 0 0.06 0 1 0.08

490 A. Azúa-Bustos et al.

to the 16S rRNA gene of Cyanidium sp. Sybil cave, alsolocated in Italy. For the 16S rRNA gene, a singlemaximum-likelihood tree was obtained through the heuris-tic search option of PAUP, and the Modeltest softwaresuggested that the best fitting model of sequence evolutionto reconstruct the likelihood tree was as observed in Fig. 6.The analysis of the 16S rRNA gene show that “caveCyanidium” species were recovered as a monophyleticgroup that included Cyanidium sp. Atacama and Cyanidiumsp. Monte Rotaro as sister species with 100% of bootstrapsupport, whereas Cyanidium sp. Sybil cave was basal tothis relationship with a bootstrap support of 91%. Similarresults were obtained when using the psbA gene, andautomated sequencing of three of the psbA clones showedthat they matched (91% identity) with a partial sequence ofthe psbA gene of Cyanidium sp. Monte Rotaro. The nextmost similar sequence match (86% identity) correspondedto the psbA gene of Cyanidium sp. Sybil cave. In the caseof the psbA gene, the cave Cyanidium species also were

recovered as a monophyletic group that included Cyanidiumsp. Atacama and Cyanidium sp. Monte Rotaro as sisterspecies with 99% of bootstrap support, whereas Cyanidiumsp. Sybil cave was basal to this relationship with abootstrap support of 92% (Fig. 7). In the case of the rbcLgene, the most similar sequence match (89% identity)corresponded to the rbcL gene of Cyanidium sp. MonteRotaro. The maximum-likelihood tree obtained for the rbcLgene did not provide sufficient resolution, as Long Branchattraction persisted for the rbcL gene of Cyanidium sp.Atacama (data not shown).

Discussion

Being in an ancient desert and a recognized Mars analog,caves of the Atacama Desert represent a prime target for thesearch of its associated microorganisms. As for photosyn-thetic microorganisms, to date, mainly cyanobacteria have

Figure 3 Micrographs of Cyanidium sp. cells found in biofilms of theAtacama cave. a Bright field micrograph of Cyanidium sp. composedof single photosynthetic cells and small fragmented colonies. Scalebar = 10 μm. b Merged CLSM micrograph of aqueous suspension ofCyanidium cells extracted from the cave biofilm. The differential

interference contrast image was merged with the red fluorescence(excitation/emission 543 nm/long pass filter <570 nm) due to theautofluorescence emitted by the cell chloroplast containing chloro-phyll. Scale bar=10 μm. The arrows in b indicate endospore-containing cells

Figure 4 TEM micrograph ofan ultrathin section ofCyanidium cells showing cellsin different developmental stateswithin the same aggregate. aNote the characteristic organ-elles and the multilayered enve-lopes surrounding the cells.Scale bar = 1.4 μm. b Cell in adividing state. Scalebar = 1.7 μm. C: chloroplast, V:vacuole, N: nucleus, M: mito-chondria, cw: cell wall, nm:nuclear membrane, eps: exo-polysaccharide layer, edb:electron-dense bodies

Atacama Cave Cyanidium 491

been described elsewhere in the Atacama [48, 50]. On theother hand, the Cyanidiales, an order of the Rhodophytaalgae, are an ancient group of microorganisms dating as faras 2,000 million years ago [52].

The finding of a member of the Cyanidiales living in acave in this area was unexpected, considering that mostknown species of this order inhabit acidic thermal springs.In our case, it was determined that the biofilms remain coolduring most part of the day (15°C measured around noon),and that water droplets associated to it have a slightly acidicpH (4.5). The internal ultrastructure of cells scraped fromthe cave biofilm and observed under TEM shows thetypical type and amount of organelles (one sphericalchloroplast and one mitochondrion, in addition to thenucleus) already described for Cyanidium species [16,30]. As for the SEM micrographs, two modes of cell

aggregations in the biofilm could be observed: one in whichthe cells appear to be loosely associated with each other,with low or no presence of extracellular materials, andanother form of aggregation in which the cells areembedded in a well-developed extracellular matrix.

Subaerial biofilm species often secrete EPS that facilitatefurther adhesion onto the substrate and, in the case of desertenvironments, the retention for longer periods of the scarcewater available [22]. In our case, the reasons for theobserved variations on biofilm conformation in an appar-ently isotropic humid microhabitat are still unclear. It hasbeen suggested that downregulating EPS production at highcell densities could allow cells to redirect energy from EPSproduction into growth and cell division prior to a dispersalevent [32]. Aside from the presence of EPS for celladhesion to the wall matrix, another interesting possibility

Figure 5 SEM micrographs of Atacama cave Cyanidium. a Biofilmwhere individual cells are forming loose well-defined aggregates canbe observed. Scale bar = 10 μm. b Biofilm conformation where the

cells are well embedded in matrix of exo-polysaccharides covering theparental rock. Scale bar = 10 μm

Figure 6 Maximum-likelihoodtree obtained from the alignedsequences of the 16S rRNAchloroplast gene for Cyanidialesspecies. Numbers above thenode represent 1,000 replicatebootstrap values

492 A. Azúa-Bustos et al.

is that EPS could be used as a medium for solublemolecules involved in intercellular communication andquorum sensing as previously suggested [26]. Being thisthe case, intercellular communication would no longer beneeded previous to a dispersal event, which could explainthe observed lack of an EPS matrix in some cases. One lastpossibility was proposed by Bellezza et al. (2006) [7] inwhich EPS, being rich in negative charges, may allow theadsorption of constituent cations from the mineral substrata.Irrespective of these possibilities, in both cases of biofilmconformation, a seemingly monospecific Cyanidium bio-film is morphologically observed. This observation issupported by the 16S rRNA analysis, which unambiguouslyshows only one type of photosynthetic species related to16S rRNA gene sequence present in the dimmest lightedareas of the cave. When using cyanobacteria 16S-rRNA-specific primers [35], no species of this phylum weredetected at this part of the cave after repeated attempts (datanot shown). This is remarkable since photosynthetic micro-organisms on rock surfaces rarely grow as coloniescomprising a single species, and epilithic biofilms arecommonly composed of several species of cyanobacteriaand algae [22]. Nevertheless, an initial molecular charac-terization of the non-photosynthetic components of thebiofilm shows that other species of heterotrophic bacteriacan be detected as forming part of the biofilm which cannotbe seen by the microscopy methods used, species ofgammaproteobacteria related to the genera Salinisphaera(98% identity by 16S rRNA sequencing) and anotherbacteria morphologically similar to Saccharomonosporaactinobacteria (data not shown). In the latter case, thisbacterium can only be detected on collected rock samples inex situ conditions, in which the water, temperature, andlight conditions have been greatly altered for extendedperiods of time.

The maximum-likelihood trees obtained for the 16SrRNA and psbA genes observed in Figs. 6 and 7 show theCyanidium sp. Atacama forming part of the proposed“Cyanidium cave” monophyletic group that includes theother two known Cyanidium cave species, Cyanidium sp.Monte Rotaro being its closest relative. Only two of theproposed 17 known species of Cyanidiales, namelyCyanidium sp. Sybil and Cyanidium sp. Monte Rotaro,appear to inhabit caves, both located near the Vesuviusvolcano [9]. These two species are considered to be the“mesophilic” members of the group. Thus, the 16S rRNAand psbA genes data support that the Atacama caveCyanidium is part of the cave Cyanidium group, which aspreviously proposed [9] may be a novel monophyleticlineage of mesophilic Cyanidium spp., distinct from theremaining three other known lineages. In the case of therbcL gene, although a much lower identity percentage isobserved, its closest reported relative still is Cyanidium sp.Monte Rotaro [9]. A maximum-likelihood tree obtained forthe rbcL gene did not provide sufficient resolution, as LongBranch attraction persisted for the rbcL gene Cyanidium sp.Atacama (data not shown). It has been previously reportedthat, in the Cyanidiales, ribosomal sequences are moreconserved than that of the rbcL gene sequences [9, 45, 46]and that rbcL gene sequences are known to be subjected tohigher rates of sequence evolution, leading to saturationevents precluding sufficient resolution in single-gene andeven combined-genes approaches [21, 22, 45, 46]. Thisdiscrepancy may be explained by the lateral transfer of therbcL gene [45]. Nevertheless, both the transmissionelectron microscopy and molecular data presented in thispaper support the contention that a species of Cyanidiuminhabits this Atacama cave.

Although Schwabe [41] reported a species which he,based on morphology, proposed as belonging to theCyanidium genera, it was found in two caves on the centralcoast of Chile located over 1,300 km south of the Atacamacave. It is known that, under the microscope, species of redalgae are difficult to identify based on morphology alone[38]. In particular, species of the genera Cyanidium andGaldieria are indistinguishable [15, 37]. On the other hand, ahigh level of sequence divergence is systematically observedamong Cyanidiales species with the partitioning of taxa,allopatric divergence, and speciation based on environmentalconditions, suggesting that new species diverge after long-term geographic isolation [9, 45, 49]. Thus, considering thedistant latitudes, it is likely that the species described in thiswork and that reported by Schwabe are not the same. This isconsistent with the discrepancies found for the rbcL genemolecular data reported in this work. Schwabe did notprovide more specific coordinates of the exact location of thecaves he studied, but efforts are now being made to locatethem, allowing future comparisons.

Figure 7 Maximum-likelihood tree obtained from the alignedsequences of the psbA chloroplast gene for Cyanidiales species.Numbers above the node represent 1,000 replicate bootstrap values

Atacama Cave Cyanidium 493

As for the general water environment internal andexternal to the cave, water relations are critical andprobably the most limiting factor for life in the hyperaridAtacama desert. The coastal area where the cave is locatedis exposed to fogs that usually arrive at late afternoon andnight. Although their influence as a source of water for thecave Cyanidium biofilms cannot be dismissed, the moredirect and constant influence of the nearby ocean spray andmist probably accounts for the long-term most stable humidenvironment inside the cave. The presence of the seawaterat the cave entrance does not seem to create a differentialbottom to entrance water gradient inside the cave since RHis homogenously high along the cave interior (Table 1).Since high humidity values (89%) were measured inside thecave after midday of clear days, it may be assumed thatthese are the minimum humidity values encountered at thislocation. Thus, the possible condensation of periodical fogsinside the cave at night should not have an appreciableimpact on biofilm development in its interior.

Interestingly, the habitat description of the caves furthersouth made by Schwabe [41] on this matter is almostidentical to the reported site in this work: “The air at theobserved locations exhibit continuously high moisture. Thesite however show only moderate damp to dry areas andapparently does not seem to be under continuing watercoverage.”

Also, the habitat description on terms of the lightconditions made by Schwabe [41] is again identical to theone described in this work: “the best development wasfound several meters from the cave entrance, at the wallsand ceilings. However, areas exposed to the light (caveentrance) show no growth”. This may be understood if therelatively low temperatures and high light conditions of theareas close to the entrances of the cave are taken intoconsideration. It is well known that the concurrence ofthese two conditions determine an important stress upon thephotosynthetic machinery [31], causing the production ofhighly deleterious reactive oxygen species and photo-inhibition. In our case, we observe a sharp transition inthe presence of Cyanidium biofilms in relation to theavailability of light. As seen in Fig. 2d, only the entrance-facing side of a rock located on the ground shows biofilmdevelopment. A similar transition is also observed from thewestern to the eastern wall. Even at places where PPFDvalues reach 1 μmol m−2 s−1, Cyanidium biofilms can stillbe observed. However, as soon as no light is measured atthe eastern wall, Cyanidium biofilms are no longerdetected. To find areas with only one photosyntheticspecies inhabiting a cave where water availability seemsnot to be the limiting factor could be explained by the verylow light levels found towards the bottom, which could bepreventing the sustained growth of other species of photo-trophs. Thus, at the bottom of the cave, only Cyanidium

biofilms can be found due to their highly efficientphotosynthetic machinery, which in turn may not be ableto cope with the higher levels of light found closer to theentrance. It would be interesting to understand themechanisms of photosynthetic quantum efficiency of thistype of cave Cyanidium. Near the cave entrances, thehigher light levels may allow the colonization of the wallsby other phototrophic microorganisms which may outcom-pete the growth of the Cyanidium biofilms. In fact, we doobserve the appearance of cyanobacteria on the wallsdirectly in front of the entrances but not at the bottom ofthe cave where the Cyanidium biofilm are located.

Finally, our findings could be placed in an astro-biological context of a Martian cave [8, 13, 23], where ahypothetical phototrophic microorganism like the reportedcave Cyanidium could be found inside a cave wellprotected from the harsh outside conditions using minimumphoton flux levels coming from a nearby entrance, but highenough for enabling the photosynthetic processes criticalfor survival.

Acknowledgments This work was supported by the MillenniumInstitute of Fundamental and Applied Biology (Chile). We also thankthe members of Rafael Vicuña’s Laboratory for critical comments andinsights which helped to improve this manuscript.

Disclosure Statement No competing financial interests exist inconnection with the submitted manuscript. This applies to all authorsof this paper.

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