Diverse Archaeal Community of a Bat Guano Pile in Domica Cave (Slovak Karst, Slovakia)

Folia Microbiol. 54 (5), 436–446 (2009) http://www.biomed.cas.cz/mbu/folia/

Diverse Archaeal Community of a Bat Guano Pile in Domica Cave (Slovak Karst, Slovakia) A. CHROŇÁKOVÁa,b, A. HORÁKc,d, D. ELHOTTOVÁa, V. KRIŠTŮFEKa aBiology Centre, Institute of Soil Biology, Academy of Sciences of the Czech Republic v.v.i., 370 05 České Budějovice, Czech Republic e-mail [email protected] bFaculty of Sciences, University of South Bohemia, 370 05 České Budějovice, Czech Republic cBiology Centre of the Academy of Sciences of the Czech Republic, v.v.i., Institute of Parasitology, 370 05 České Budějovice, Czech Republic dDepartment of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada

Received 25 November 2008 Revised version 19 May 2009

ABSTRACT. The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bed-rock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5–3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.

Abbreviations ANOVA analysis of variance CARD-FISH catalyzed reporter deposition-fluorescence in situ hybridization DAPI 4´,6-diamidino-2-phenylindole PCR polymerase chain reaction SCG soil crenarchaeotic group

Employment of molecular markers, especially the gene for the prokaryotic ribosomal small subunit (16S rDNA), revealed novel lineages of both Crenarchaeota and Euryarchaeota that seem to be widely distri-buted in various terrestrial and subsurface mesophilic environments (DeLong 1992, 1998; Fuhrman et al. 1992; Bintrim et al. 1997; Takai et al. 2001; Ochsenreiter et al. 2003). It belongs to the most interesting discove-ries in 16S rDNA survey and it is in misbalance with the knowledge based on unsuccessful cultivation approaches indicating the occurrence of Archaea mainly in extreme (hyperthermophilic, halophilic) or anaero-bic environments (Stetter 1994, 1995).

Recent knowledge about the ecology of non-thermophilic Crenarchaeota indicates that they are wide-spread all over the terrestrial world (soils, subsurface, river sediments, flooding area, composts), marine and freshwater habitats and even in association with animals and plants (marine sponge, termite gut, bovine rumen and plant roots) (Hershberger et al. 1996; Rudolph et al. 2001; Ochsenreiter et al. 2003; Nicol et al. 2006). The specific abiotic and biotic properties of the ecological niches of representatives of particular uncultured crenarchaeal lineages offer clues to physiology (Dawson et al. 2006). For example, Groups 1.1, 1.2 and 1.3 low-temperature Crenarchaeota are common in oxic zones such as soils and open oceans and thus likely be aerobic. On the other hand, Group 2 low-temperature Crenarchaeota occur in the anaerobic environments, so presumably conduct anaerobic metabolism. Groups 3 and 4 Crenarchaeota also are likely to be anaerobes. Still, the physiological functions and ecological roles of these microorganisms (mainly uncultured) are almost unknown (Nicol et al. 2003) or they are inferred from sporadic metagenomic projects (Treusch et al. 2005). New insights into the physiology of Crenarchaeota have been described: there is evidence that aerobic Cren-

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archaeota are likely to play a role in ammonia oxidation. The soil Crenarchaeotic metagenomic clone 54d9 carries a gene potentially encoding ammonia monooxygenase, the molecular marker of bacterial ammonia oxi-dizers (Francis et al. 2005; Schleper et al. 2005). In terrestrial ecosystems they can even predominate over bacterial ammonia oxidizers (Leininger et al. 2006).

The second phyllum Euryarchaeota has been known to be comprised of extreme halophiles, sulfur reducers, sulfate reducers, thermophilic heterotrophs, and methanogens (Bintrim et al. 1997). Novel lineages of Euryarchaeota have been detected in marine picoplankton (DeLong 1992), coastal salt marsh and conti-nental shelf sediments (Munson et al. 1997) and estuarine sediments (Abreu et al. 2001). One of these novel lineages has been shown to represent anaerobic methane-oxidizing organisms (ANME-1, ANME-2) that re-side in unique consortia with sulfate-reducing bacteria (Boetius et al. 2000; Orphan et al. 2001).

Olsen (1994) and Bintrim et al. (1997) reported that Archaea are able to occupy and survive in a broad range of biological niches, this ability being coupled with diverse metabolism of different taxonomic groups of Archaea and plays a significant role in Earth’s ecology (DeLong et al. 1994). Caves usually are nutrient-limited sites that are sequestered from the outside environment and together with bat guano samples may contain novel and diverse microbial populations (Sugita et al. 2005). Hence we focused on archaeal community composition of the important source of energy and nutrients in a cave environment.

A bat guano pile in Palmový háj Dome in Domica Cave (Slovak Karst National Park, Slovakia) was dated to the beginning of the 2nd millennium (Krištůfek et al. 2008). It was created by the activity of the Mediterranean horseshoe bat (Rhinolophus euryale), which dominates the community of bats inhabiting this cave (Uhrin et al. 1996). The Domica Cave is unique among temperate caves in terms of being an important locality for year-round colonization by almost 1600 of these bats (Uhrin et al. 1996). Their guano mainly consists of insect fragments, bat hair, pollen, and some mineral matter (Maher 2006; Krištůfek et al. 2008). Although the microbial communities in diverse cave ecosystems have being studied (Groth et al. 1999; Laiz et al. 2003; Northup et al. 2003; Sugita et al. 2005), little is known about the microbial communities of bat guano heaps, which are an important source of nutrients and energy for organisms inhabiting the cave (Gnas-pini 2005; Šustr et al. 2005; Fenolio et al. 2006). Temperate-zone caves provide a hypogean environment for its inhabitants and a bat guano pile inside the caves is a great attraction for detritovorous microbes and ani-mals. Because bat guano communities are somewhat isolated and, in general, present a simple structure of few trophic levels, they are an important field for ecological studies (Gnaspini 2005).

The aim of our work was to investigate the archaeal diversity in bat guano samples from temperate cave using rDNA sequences. The phylogenetic structure of the archaeal communities present in this habitat were compared with those obtained from other environments.

MATERIALS AND METHODS

Description of site. The Domica Cave is situated in the south of the Slovak Karst National Park, Southeast Slovakia (lat. 48°28′36″ N, 20°29′09″ E, alt. 339 m a.s.l.) in a large complex of light-colored lime-stones of the Triassic Period (http://www.ssj.sk/jaskyne/spristupnene/domica/). The summer activity of the colonies is dominated by the Mediterranean horseshoe bat (Rhinolophus euryale) (Uhrin et al. 2002). Droppings of this and Miniopterus schreibersii accumulate in the cave in heaps >1 m high and 3–4 m wide at two sites (Palmový háj Dome and Sieň indických pagôd Dome).

Collection of samples. Sampling of the Palmový háj Dome bat guano heap was carried out in the second half of September 2006. The air temperature and moisture in the cave were 10.2 °C and 97 %, res-pectively. The G2 sample was taken from the upper part of the slope of the bat guano heap (Fig. 1), while sampling site G4 originated from the foot of the guano heap (for full description of samples see Krištůfek et al. 2008). The latter site represents mixed material of bat guano, excrements of the cave invertebrates [mainly isopod Mesoniscus graniger FRIVALDSZKY 1865 (Crustaceae:Isopoda)] (Šustr et al. 2005) and bed-rock. Samples (each from 4–5 sub-sampling places) were taken using a shovel from the surface (0–30 mm).

Guano analysis. For some characteristic and site-specific chemical, physical and microbial parame-ters of the guano see Table I. Moisture of the samples was determined gravimetrically after drying at 105 °C for 5 h, pH being measured according to standard techniques (ISO 1992). Organic C (Corg) was determined using wet combustion (oxidation with chromic acid in excess of sulfuric acid) (Jackson 1958). The wet Kjel-dahl method was used to determine the total nitrogen (Ntot; Jackson 1958). Total bacterial counts were ob-tained using microscopy of DAPI-stained cells (Bloem 1995).

Scanning electron microscopy image of the bat guano was made in the Laboratory of Electron Microscopy, Institute of Parasitology (České Budějovice) using a JEOL JSM 740 1F electron microscope. The sample was prepared according to Frouz et al. (2002).

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CARD-FISH on polycarbonate filters. Guano samples (5 g) were transferred to Erlenmeyer flasks containing 45 mL of sterile 0.1 % sodium diphosphate solution, and placed in an ultrasonication bath (50 kHz, 4 min, room temperature). One mL of the dispersed sample was diluted in 9 mL 1× PBS to reach final concentration 10–4 g/mL, then the sample was fixed with formaldehyde (final concentration 2 %) and 1.6 mL of this suspension was filtered on polycarbonate filters (0.2 μm pores, 47 mm in diameter; GE Water & Process Technologies, USA) which were mounted in a plastic filtration unit (filtration area 12 cm2; Nal-gene, USA). The filters were stored at –20 °C. The CARD-FISH protocol applied here followed the pro-cedure for aquatic bacteria (Sekar et al. 2003).

Fig. 1. Overview of the bat guano pile in the Palmo-vý háj Dome (Domica Cave, Slovakia); the width of the pile is ≈4 m and the height ≈1.1 m; line – the border between sampling sites G2 and G4; photo A. Nováková.

The oligonucleotide probe ARCH915 (5´-GTG CTC CCC CGC CAA TTC CT-3´) (Stahl and Amann

1991) used for the CARD-FISH procedure was labeled with horseradish peroxidase at the 5´-end (Thermo-Fisher Scientific, Germany). Formamide (55 %, V/V) was used for the probe. The hybridization was perfor-med in a hybridization oven at 35 °C for 2 h. Hybridization signal was amplified with FITC-labeled tyr-amide for 10 min at 37 °C. Before the counting, the filter was stained with a drop of oil–DAPI mixture (800 μL Cityfluor AF1 solution (Cityfluor, UK), 200 μL Vectashield H-1000 (Vector Laboratories, Canada), and 30 μL 1 μg/mL DAPI solution). The samples were evaluated using Olympus BX60 microscope. Cell count-ing was performed on 20–25 randomly selected filter sites.

Extraction of environmental DNA. Community DNA was extracted from 0.5 g (wet mass, W/W) of bat guano sampled in triplicate using the method of Griffiths et al. (2000). Crude extracts of DNA were electrophoresed on 1 % agarose gel to check the quality and quantity of the template for downstream PCR amplification.

PCR amplification of 16S rDNA. Small subunit rRNA genes were amplified by PCR using primers specific to the domain Archaea Ar109f and Ar912r (Lueders and Friedrich 2000). The reaction mix contai-ned (in a total volume of 50 μL) 1× PCR buffer, 2.5 mmol/L MgCl2, 1× Q solution, 0.2 mmol/L dNTP (Qiagen, Germany), 10 pmol of each primer (Metabion GmbH, Germany), 1.25 U of Taq DNA polymerase (Qiagen), and 1 μL of guano DNA extract as a template. The touchdown thermal profile for the amplification of partial 16S rRNA gene sequences was as follows: initial denaturation (5 min at 95 °C); 20 cycles of 1 min at 62 °C (each cycle lowered by 0.5 K), 1 min at 72 °C; 10 cycles of 1 min at 94 °C, 1 min at 52 °C, 1 min at 72 °C; followed by a final elongation step of DNA (10 min at 72 °C). Aliquots of the 16S rRNA gene ampli-cons were visualized by gel electrophoresis on 1 % agarose gel and stained with ethidium bromide.

Cloning and sequencing of archaeal 16S rDNA. Amplified archaeal rDNA sequences from both sampling sites were pooled from 3 replicates and cloned into vector pCR2.1 by using an Original TA® Clo-ning kit (Invitrogen, USA). Clones containing inserts from the chosen colonies were identified by direct PCR analysis using M13 primers. Plasmid DNA from positive clones was extracted using NucleoSpin®-Plasmid (Macherey-Nagel, Germany). Clones were sequenced on an ABI 3130×1 Genetic Analyzer (Perkin Elmer, USA) in the Genomics Laboratory, Institute of Plant Molecular Biology (České Budějovice) using manufacturer’s chemistry.

Phylogenetic analysis and sequence accession numbers. Sequence fragments were assembled using the BioEdit 7.0.4.1 (Hall 1999) software package and consensus sequences were set up using SeqMAN (Swindell and Plasterer 1997; DNAStar, Inc.). Contigs of ≈800-bp length were compared to the DNA se-quences available in the GenBank (www.ncbi.nml.nih.gov) using the program ntBLAST. Possible chi-meric of our sequences using Bellerophon v3 through Greengenes web portal (http://greengenes.

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lbl.gov; DeSantis et al. 2006) have been tested. The 16S rDNA sequences (40) were aligned with a col-lection of archaeal sequences retrieved from the GenBank (35). Multiple alignments were created and further edited using MAFFT v6.606b (Katoh et al. 2002; Katoh and Toh 2008) and Bioedit (Hall 1999). The 16S rRNA dataset was analyzed by maximum likelihood (ML) with the use of Phyml 3.0 software (Guidon and Gascuel 2003) under the GTR+I+Γ8 model of evolution. This model was chosen according to the Akaike information criterion value computed using Modeltest 3.7 (Posada and Crandall 1998). Bootstrap support was assessed from 1000 replications by the ML algorithm, the above described model, software and by ma-ximum parsimony (MP) using PAUP 4.0 b10 (Swofford 2002). The nucleotide sequences have been deposi-ted in the GenBank under accession numbers EU098046–EU098088.

Diversity analysis. Clones with similar (>97 % sequence similarity) 16S rDNA sequences were assig-ned to the same operational taxonomic unit. Diversity coverage of 16S rDNA clone libraries was analyzed according to Hurlbert (1971), and 95 % confidence intervals were estimated (Heck et al. 1975). Rarefaction curves were produced using the Analytic Rarefaction software (ver. 1.3; S.M. Holland, University of Geor-gia, Athens; http://www.uga.edu/strata/Software.html). Richness (R) and evenness (J) were esti-mated and diversity index (H´ ) was calculated as Shannon–Weaver index (Begon et al. 1990).

Statistical analysis. Analysis of variance (ANOVA) followed by post hoc comparison between the means (Scheffé test) was used to determine the significant differences of chemical and microbiological pro-perties between the two sites. χ-Square testing was used to evaluate the significance of differences in rela-tive proportion of identified clones in constructed libraries between sampling sites. Statistical tests were per-formed in program package STATISTICA (v7.1.; StatSoft, 2005). The accepted significance level p for both statistical tests was <0.05.

RESULTS AND DISCUSSION

Characteristics of sampling sites G2 and G4 (Table I). The pH of site G4 was higher than that of G2; it seems to be the result of mixing of guano with limestone bedrock. Very low pH of site G2 is in accordance with Gnaspini (2005): he stated that guano piles are initially alkaline owing to the concentration of ammonia in the feces. They subsequently become acid as a result of the decomposition of alkaline compounds and fermentation of the piles.

Markedly higher total nitrogen content was recorded in the upper (younger) part of the slope (G2) than at the foot (G4). It is probably because relatively fresh bat guano consists of high amounts of available nitrogen in the form of uri-ne and ammonia. Therefore, it can be easily utili-zed by ammonia oxidizers community and/or might be leached from the system in the form of nitrate. In addition, fresh bat guano is generally finely granulated and consists of small pieces of insect cuticle (Fig. 2) composed of chitin rods set in a protein matrix (Ghiradella et al. 2000). Insect chitin is highly resistant to chemical attack and moist conditions are necessary to support decom-posing microorganisms capable of producing chi-tinase (McFarlane 2004). Moisture significantly decreased with ongoing decomposition, which was linked to lowering total bacterial counts, pH

increase as well as decrease of Corg and Ntot. Decu (1986) documented that piles of bat guano are qualitati-vely and microclimatically nonuniform. This is the case of our two sampling sites on the bat guano heap. Differences in moisture, pH and C:N ratio affected the number of total bacterial counts and could have mo-dified the structures of the microbial communities on both sites. Differences of all characteristics between the sites were significant (p < 0.05).

Microbial cell counts and relative abundance of Archaea. The absolute bacterial numbers determi-ned by DAPI staining varied between G2 and G4, but the difference was not significant (Table I). The pro-portions of cells hybridized by the Archaea specific probe ARCH915 ranged from 3.9 to 3.5 % of all DAPI-stained cells. We can therefore assume that archaeal abundance can differ between sites because relative pro-portion was stable but total bacterial counts decreased. These data about abundance of Bacteria and Archaea

Table I. Chemical, physical and microbial parametersa of the bat guano sampling sites G2 and G4

Parameter G2 G4

Moisture, % 77.9 ± 0.1* 60.1 ± 2.5** pH (CaCl2) 3.42 ± 0.15* 5.38 ± 0.2** Corg, % 81.7 ± 0.25* 41.0 ± 2.25** Ntot, % 8.98 ± 0.21* 3.47 ± 0.09** C:N ratio 9.09 ± 0.24* 11.8 ± 0.51* Total bacteria countsb 3.95 ± 0.78 1.64 ± 0.11 Archaeac 3.9 3.5

aMeans ± SE of triplicates. bDAPI; number × 1010/g dry matter. cPercentage of all DAPI-stained cells. *,** Significant difference (ANOVA, Scheffé test, p < 0.05).

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in bat guano deposits are unique. There have been no reports of any similar study, describing the abundance of microbial community guilds in bat guano deposits, whereas the situation in subsurface (Takai et al. 2001) or cave (Graening and Brown 2003) waters as well as cave ferromanganese deposits (Northup et al. 2003) has already been investigated in detail.

Fig. 2. Scanning electron microscopy image of bat guano sample G2; fresh bat excrements contain a large quan-tity of fragmented and nonfragmented bat hairs, butterfly or mosquito scales and insect wings; bar = 100 μm.

Archaeal community composition and phylogenetic analysis. Archaeal rDNA clones from both clone

libraries (in total 115) were characterized by sequencing (c. 800 nucleotides) and sequence similarity analy-sis using blastn search (Zhang et al. 2000). For the presence, proportion and nearest relatives from the Gen-bank of identified bat guano clones see Table II. While richness estimated as number of phylotypes present differed weakly between sites G2 and G4 (10 and 9 phylotypes, respectively), the composition of each par-ticular clone library differed significantly (χ-square test, p < 0.05). Diversity (rarefaction analysis; Fig. 3; Shannon–Weaver index H´, Table II) was markedly higher at site G2 (H´ 0.738), representing the guano ma-

Fig. 3. Rarefaction analysis of archaeal 16S rDNA clone as retrieved from bat guano samples G2 and G4. The expected number of clones [E(Sn)] was calculated from the number of clones analyzed (n) at the species level using 97 % sequence similarity. The slope of the curves indicates whether the diversity was covered (reaching a plateau) or whether further clones with <97 % sequence similarity may be expected by analyzing additional clones (steep slope).

terial of pH 3.42, in comparison with site G4 (H ´ 0.543) which represents the mixed guano with bedrocks of pH 5.38. These two investigated archaeal communities differed also in the relative proportion of SAGMCG 1 relatives, which was high in the clone library from the G4 sample (the base of the heap, 56 %), but was quite low in the clone library from the G2 sample (the upper part of the heap, 31 %). The qualitative differences

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between the composition of archaeal communities are in good correlation with chemical and microbial para-meters, e.g., total bacterial counts were also lowered in site G4, it can imply that both quantity and also the quality of microbial communities were changed during decomposition of guano deposits.

Almost all of the phylotypes were associated with nonthermophilic Crenarchaeota and only one with Euryarchaeota. The majority of clones (53.9 %) were classified as sequences related to uncultured Cren-archaeota clones associated with soil or other terrestrial habitats and belonging to SCG, which is represented here by group 1.1b Crenarchaeota. This 1.1b is assumed to be ubiquitous in soils and other terrestrial envi-ronments, while group 1.3 is found in flooded soils, sediments, freshwaters and others (Nicol and Schleper 2006). The second most abundant group 1.1a Crenarchaeota (45.2 %) was almost represented by sequences closely related to the uncultured crenarchaeotic clone SAGMA-D (43.4 %), retrieved from deep acid subsur-face waters in South Africa gold mines and representing the SAGMCG 1 cluster (Takai et al. 2001). The rest of group 1.1a Crenarchaeota clones (1.7 %) were mostly related to the uncultured crenarchaeotic clone SAGMA-Z from deep acid subsurface waters (representing the SAGMCG 2 cluster; Takai et al. 2001).

Clones representing unique phylotypes (in two repetitions per library), underwent bidirectional se-quencing analysis. Test for chimeras using Bellerophon revealed five of the sequences to be of chimeric origin and they were discarded from the dataset (three were from our clone libraries: EU098059, EU098073, EU098088). The rest (40) were used for construction of a phylogenetic tree (Fig. 4). The resulting topology divides sequences from the guano heap into three main clades (Euryarcheaota, 1.1a and 1.1b Crenarchaeota) and clearly corresponds to the results obtained by blastn. Further branching is in good congruence with cren-archaeotic groups described by Simon et al. (2000). Crenarchaeotic 1.1b sequences grouped within clades 1.1bA1, 1.1bB1, 1.1bB2 and 1.1bC but statistical support is mostly very low due to the overall sequence simi-larity and the fact that only partial sequences were available.

The majority (97 %) of the cloned sequences showed good affiliation with the above sequences (>97 % sequence similarity) and represent taxa sequences and organisms not specifically adapted to this spe-cial habitat. A few of the clones showed low affiliation with sequences currently available in the GenBank and might be thought to represent unique Crenarchaeota sequences (Table II). Cloning strategy revealed the presence of 6 crenarchaeotic and 1 euryarchaeotic clusters (Fig. 4). Two of them (RC-V and SAGMCG-2) were detected only in fresh guano samples (G2 site). Clones Gua4_45 and Gua4_53 were clustered together with group 1.1a Crenarchaeota but seem to be novel archaeal sequences sharing a common ancestor with SAGMA sequences. Northup et al. (2003) found also diverse archaeal sequences in constructed clone libra-ries from Lechugilla Cave ferromanganese deposits (New Mexico, USA). Archaeal assemblages of ferroman-ganese deposits consisted of 3 groups of Crenarchaeota (2 related to SAGMCG 1 cluster and 1 group related to SGC) and 1 group of Euryarchaeota. Our data suggest that archaeal community of bat guano deposits is more diverse. The detection of related archaeal groups in such a different environment may imply that part of the community is closely associated with the subsurface.

Crenarchaeota from group 1.1a are thought to be associated with subsurface and plant roots (Nicol and Schleper 2006). The presence of crenarchaeal sequences related to the SAGMCG 1 and 2 groups in guano samples indicates that these microorganisms are adapted to habitats with low pH and subsurface, which may explain their significant presence in the guano deposit. They were retrieved firstly from acid mine subsurface waters (pH 3.0) in South Africa gold mines (Takai et al. 2001) and now they were found to in-habit a bat guano heap in a temperate cave ecosystem. Acidophiles occur in habitats with low pH, which are based on SO42– and are generally associated with pyrite or S0 deposits and including mining regions and geothermal areas. Such acid niches are often created, at least in part, by the organisms that are present, i.e. acidophiles often catalyze acidogenic processes (Ingledew 1990). Little or nothing is known about acido-philic microbial communities of bat guano habitats. The archaeal community of acid mine waters from South Africa gold mines was restricted and dominated by crenarchaeotic clones closely related to SAGMCG 1, a major archaeal component in the dolomite aquifer and had been even assumed to play a role in sulfur oxidation (Takai et al. 2001).

The diversity of the archaeal rDNA sequences in the bat guano from the Domica Cave was not re-stricted only to this putative acidophilic Crenarchaeota cluster, as sequences related to SCG were also abun-dant (54 %). Our results indicate that a low pH habitat can be colonized also by soil microorganisms, trans-

Fig. 4. Phylogeny of archaeal samples isolated from bat guano. The maximum likelihood (ML) tree was constructed under the GTR+Γ8+I model of evolution using PhyML 3.0 (Guindon and Gascuel 2003). The bootstrap support was calculated from 1000 repli-cations using PhyML 3.0 for ML (before dash) and PAUP 4.0b10 (Swoford 2002) for maximum parsimony (after dash). The tree was rooted with 16S rDNA sequence of Methanococcus aeolicus PL-15; scale bar – 0.1 changes per site.

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ferred by running waters from soil above the cave system and/or by microorganisms associated with gut or excrements of colonizing invertebrates. The evidence of colonization of invertebrates by Archaea, for in-stance, by uncultured crenarchaeotic clone P4b-Ar-9, which was retrieved from the gut of soil-feeding ter-mite Cubitermes orthognatus, has been already documented and supports this idea (Friedrich et al. 2001). The bat guano heap in the Domica Cave is a significant food source (especially essential oligounsaturated fatty acids) for Mesoniscus graniger FRIVALDSZKY 1865 (Crustacea:Isopoda), which colonize the heap abun-dantly (Šustr et al. 2005). Thus, we can imply that the archaeal community of bat guano is enriched by archaeal community originated from the excrements of this isopod.

The occurrence of group 1.1a and 1.1b Crenarchaeota in bat guano deposit indicates that these clo-nes can be involved also in nitrogen transformation. Free ammonia is produced by decomposition of bat ex-crements and bat urine (McFarlane 2004) and can be transformed to nitrate by action of both nitrification communities of Bacteria and Crenarchaeota. Several investigators speculate that most or all of group 1.1a and 1.1b Crenarchaeota are ammonia oxidizers (Francis et al. 2005; Schleper et al. 2005; Nicol and Schleper 2006). The indication that archaeal ammonia oxidizers predominate the bacterial ones in soils (Leininger et al. 2006) signalizes that Crenarchaeota can take a significant role in nutrient cycling and their activity may influence the trophic dynamics in this particular bat guano habitat and even in the cave itself.

The low relative proportion of Euryarchaeota reported here (0.8 % of clones) could not be explai-ned easily with the specificity of the used primer pair. Galand et al. (2006) used these primers to amplify whole archaeal community and they were able to retrieve a high proportion of euryarchaeal clones in clone libraries from river sediments and coastal sea. The same primers were used by Conrad et al. (2008). They were able to gain both Crenarchaeota and Euryarchaeota, even 4 different methanogen groups from rice cul-tivars using this amplification system. We suggest that the lack of Euryarchaeota in our samples was caused by the environment explored, rather than oligonucleotide bias. Inability to produce mcrA (gene coding for methylcoenzyme-M reductase α-subunit) amplicons, used as genetic marker of methanogens (data not shown; Luton et al. 2002) proved the absence or very low abundance of this group of Archaea in the surface of bat guano deposits. Euryarchaeotic sequence (Gua2_53, EU098087) was related to clone mrR1.57 revealed from the Mackenzie River representing Rice Cluster-V (RC-V, Großkopf et al. 1998; Galand et al. 2006; 94 % sequence identity, Table II). We hypothesize that this clone is a methanogen associated with invertebrate guts or river sediments in the cave. The presence of methanogens in various cave ecosystem using culture-independent approaches was described by Mattison et al. (1998) and Northup et al. (2003).

These assumptions should be verified in the future together with a deeper characterization of micro-bial activity in the sampled material. The identification of Archaea from different habitats is important to understand their ecological significance in the biosphere and for the analysis of naturally occurring microbial communities (Bintrim et al. 1997). The composition of the archaeal community of bat guano clearly showed the presence and dominance of crenarchaeotic clones in both our sample types. It confirmed the common sug-gestion that mesophilic Crenarchaeota represent a stable and specific component in terrestrial habitats, in-cluding subsurface, and are assumed to be ubiquitous (Nicol and Schleper 2006; Weidler et al. 2007). We present here the first results that show the presence, abundance and diversity of non-thermophilic Cren-archaeota and Euryarchaeota in the surface of bat guano pile.

The authors are indebted to the Slovak Caves Administration in Liptovský Mikuláš (Slovakia) for the sampling and research permit and would like to thank to A. Lukešová and A. Nováková for help with sampling, J. Petrásek for sample preparation and V. Kasalický for assistance in applying the CARD-FISH technique, and would like to thank to Ľ. Kováč (P.J. Šafárik University, Košice, Slovakia) for his guiding and help during visits to the cave. Financial support of the Ministry of Education, Youth and Sports of the Czech Republic (LC06066) and Research Plan of the Institute of Soil Biology (AV 0Z 6066 0521) are gratefully acknowledged. The authors thank J. Hynšt and K. Edwards for critical reading of the manuscript.

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