Life in extreme habitats: diversity of endolithic ...

Life in extreme habitats: diversity of endolithic microorganisms from cold desert ecosystems of eastern Pamir Nataliia KHOMUTOVSKA1, Maja JERZAK1, Iwona KOSTRZEWSKA-SZLAKOWSKA2, Jan KWIATOWSKI3, Małgorzata SUSKA-MALAWSKA1, Marcin SYCZEWSKI4 and Iwona JASSER1* Department of Plant Ecology and Environmental Conservation, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02−089 Warszawa, Poland, *e-mail: [email protected] (corresponding author) 2 Faculty of Biology, University of Warsaw, Miecznikowa 1, 02−096, Warsaw, Poland 3 Department of Molecular Phylogenetics and Evolution, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02−089 Warszawa, Poland 4 Institute of Geochemistry, Mineralogy and Petrology, Faculty of Geology, University of Warsaw, Żwirki i Wigury 93, 02−089 Warszawa, Poland 1

ARTICLE INFO

ABSTRACT

Regular research paper

The aim of this study was to identify cyanobacteria diversity in rock communities from the cold desert ecosystem in Eastern Pamir Mountains (Tajikistan) and assess if the rock type and rock`s porosity can be indicators of microbial diversity in this extreme environment. Seven samples were collected in July 2015 from hillsides (ca 4000−4500 m a.s.l.) of the Eastern Pamir Mountains. Petrographic and scanning microscopy (SEM) allowed for the characterization of the rocks inhabited by endolithic communities as granite, gneiss and limestone with variable porosity. Based on next-generation sequencing (NGS) of amplicon of V3V4 hypervariable region of 16S rRNA gene, we established that Actinobacteria, Proteobacteria and Cyanobacteria dominated the endolithic communities of microorganisms in the rocks studied, which distinguishes these communities from those described for other cold arid regions. Chroococcidiopsis and Leptolyngbya were dominant genera in the cyanobacterial communities according to culture-dependent analysis, as well as microscopic analyses of endoliths scraps from the rocks. Culture-independent metagenomic analyses revealed that Microcoleus, Acaryochloris, Chroococcidiopsis and Thermosynechococcus reads were the most abundant from all reads and dominated interchangeably in the samples. Endolithic communities of microorganisms in the rocks from the cold desert shrubland of Eastern Pamir Mts. appear to be diverse and different from communities described for other cold deserts.

Pol. J. Ecol. (2017) 65: 303-319 received after revision October 2017 doi

10.3161/15052249PJE2017.65.4.001

key words 16S rRNA Actinobacteria cold desert Cyanobacteria endolithic microorganisms NGS Proteobacteria V3-V4 region

INTRODUCTION The presence of living organisms in the harshest environments on the Earth is no longer a novelty in science. These organisms, which take on the challenge of survival at various latitudes or altitudes, have to tolerate excess solar radiation, extremely low or high temperatures,

desiccation and many other factors. Eastern Pamir Mountain region is one example of such a hostile habitat; it is a high mountain cold desert, which combines low mean annual temperatures, long winters and short summers, low precipitation, high salinity of surface and ground waters, low nutrient concentrations in waters and soils and high level of UV ra-

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Nataliia Khomutovska et al.

diation. Such cold deserts are known also from Antarctic, Greenland, Northern and Eastern China and the Nearctic areas. In Pamir variable habitats such as saline lakes and surrounding wetlands, rivers, alpine meadows and permanent glaciers represent unique alpine ecosystems, where microbial communities play an important role (Mętrak et al. 2015). Due to its high mountain topography, Pamir is isolated from the rest of the world, which creates an additionally interesting aspect regarding the diversity of microorganisms, including presence of potentially endemic taxa. Weathered rocks serve as ‘refugia’ (Friedmann 1982), which are able to protect organisms from harsh conditions and provide more stable environment for growth. In such habitat, microbial communities often develop dominant organism groups, among which cyanobacteria, that represent the autotrophs, play an important role (Wierzchos et al. 2006). Diazotrophic cyanobacteria (nitrogen-fixing) taxa, serve as pioneer organisms providing carbon and nitrogen for heterotrophic organisms forming endolithic communities with them. Such ‘consortia’ of cyanobacteria with various heterotrophic bacteria and fungi are visible as a few millimetres thick blue-green layers below the rock surface. Microorganisms colonise the matrix in three different ways: penetrate the cracks and fissures of porous and nonporous rocky substrates (chasmoendoliths); develop in pores between crystals mainly in weathered substrate cavities (cryptoendoliths); penetrates actively into the rocks matrix forming tunnels (euendolith) (Golubic et al. 1981, Wong et al. 2010). Based on his work on endoliths from the Antarctic Friedmann (1980) recognised two groups of endolithic communities’ cyanobacteria-dominated and lichen-dominated, in which the lichenised endolith communities are comprised of fungal symbionts and chlorophyte. Cyanobacteria-dominated endoliths from cold and arid regions consist mainly of Chroococcidiopsis and Leptolyngbya (Friedmann 1980, Wong et al. 2010), while hot desert communities are primarily represented by Gloeocapsa and Chroococcidiopsis (Crits-Christoph et al. 2016). Additionally, hot and cold deserts are characterised by extreme temperature changes between day and night, and as a result, endolithic communities consist mainly of microorganisms well-adapted to

these changes, such as cyanobacteria. Cyanobacteria perfectly demonstrate resistance to repeated cycles of desiccation and wetting, and apparently resume metabolic activities within minutes of rehydration (Bell 1993). Although endolithic microorganisms have been previously studied by many investigators, we report here the first study of cyanobacteria as rock-dwellers from the Eastern Pamir region, which has not been studied in this respect. Apart from classic, culturedependent studies, we also present results from metagenomic analysis, which allow for identifying these cyanobacteria, which are not easy to isolate and culture as well as other endolithic microorganisms such as heterotrophic bacteria and algae. Furthermore, we try to reveal the main factors that determine endolithic cyanobacteria diversity in this environment.

Study area habitat and sampling The investigated area is located in Eastern Tajikistan in the Central Asia mountain rage of Pamirs (Fig. 1). The majority of the Pamir Mountains is situated in Tajikistan, namely in the Gorno-Badakhshan Autonomous Oblast (GBAO), and is divided into two different geographical regions: the Western Pamir is characterized by alpine features, with high peaks, (up to 7,500 m a.s.l.), deep ravines and relatively intensive precipitation (around 1,500 mm annually, with maxima reaching 2,500 mm for the Fedchenko Meteorological Station), whereas the Eastern Pamir represents an elevated plateau with a flat relief (3,500 ± 4,000 m a.s.l.), surrounded by slightly lower (around 6,000 m a.s.l.) rounded peaks. In the Pamirs, Barrovian metamorphic rocks and associated igneous rocks also crop out in a series of domes that were exhumed by north- south extension. The Pamir domes are built of paragneiss, schist, orthogneiss, and marble and intruded by calc-alkaline igneous rock (Schwab et al. 2004). The most territory of Eastern Pamir can be characterized as a high mountainous cold desert, almost without trees, and flora with low species diversity of xerophytes and cryophiles. A dwarf shrub, teresken (Krascheninnikovia ceratoides) dominates the vegetation (Fig. 2). Exceptions are areas in the immedi-

Life in extreme habitats-endolithic microorganisms from cold desert

305

Fig. 1. Sampling stations in Eastern Pamir, Tajikistan.

ate vicinity of lakes and rivers, where variable habitats such alpine meadows, peatlands and steppes have been developed (Mętrak et al. 2015). The eastern part of the Pamir Mountains is characterized by great climatic continentality and aridity. It has low precipitation rates (between 50 and 150 mm annually) combined with high insolation, strong winds, average monthly air temperatures below zero from October to March and the aridity index between -45 and -61 (Mętrak et al. 2017). Sampling for this study was performed in July 2015. The sampling sites were situated between 37°24’‒38°26’N and 72°51’‒74°14’E, at an altitude of 4017–4485 m a.s.l., the first

two in the vicinity of Lake Kargush, third near Lake Chukurkul, fourth and fifth were near Lake Yashilkul, and sixth and seventh in the surroundings of Lake Rangkul (Fig. 1, Table 1). All rock samples were collected from the mountainsides and slopes of the mountains located near the lakes, and the sampling sites were elevated about 100−400 above the plateau. Rocks colonised by cyanobacteria (Fig. 2B) were identified based on the presence of a blue-green or green band or layer within the rock matrix after breaking the rock. Rock samples were stored in sterile plastic bags under refrigeration (3–4°C) in darkness until further analysis.

Table 1. Geographic localization of the sampling sites.

Sample

GPS Coordinates

Altitude m a.s.l.

1

37°25’28.48”N

73° 5’28.85”E

4458

2

37°24’59.34”N

73° 5’44.96”E

4485

3

37°33’29.36”N

73° 7’47.75”E

4403

4

37°42’41.66’’N

72°51’26.22’’E

4341

5

37°42’40.56’’N

72°51’26.04’’E

4341

6

38°24’39.00’’N

74°11’07.44’’E

4017

7

38°26’37.56’’N

74°14’43.32’’E

4034

306


A

B

Fig. 2. A − landscape of the Eastern Pamir with rocks colonized by cyanobacteria; B − blue-green layer in the cracks (arrows).


METHODS Isolation and cultivation of cyanobacteria The blue-green layer was separated from the rock matrix in all rock samples and isolated on WC and BG 11 media solidified with 1% agar in individual Petri dishes (Guillard and Lorenzen 1972, Rippka et al. 1979). Cyanobacterial isolates were grown at 15–16°C in a plant growth chamber with cool white light under light/dark cycles of 12/12h. Large colonies were investigated to perform isolation of individual strains by streaking. The growing strains were transferred from time to time into a fresh medium by re-streaking in order to obtain pure, but not axenic, cultures.

Microscopy analyses The organisms from the blue-green layers were prepared for microscopic analyses by scraping them off the rock samples. Organisms were analysed under 200–1000 × magnification using light and epifluorescence microscopy, which was equipped with a camera (Fi1, Nikon, Japan). The observed and identified cyanobacteria were photographed and measured using image analysis software (NIS-Element Viewer), for their determination taxonomic keys were used (Komárek and Anagnostidis 1998, 2005, Komárek 2013, Pliński and Komárek 2017). The rocks sample morphologies were examined using a FE-SIGMA VP scanning electron microscope (SEM) (Carl Zeiss Microscopy GmbH) with an energy-dispersive detector (EDS) (Quantax XFlash 3|10, Bruker Nano GmbH). Small rock fragments were placed on the aluminium mount with carbon conductive tape and coated with a 20 nm layer of gold using a Quorum 150T ES vacuum coater. Carbon tape bridges were also made for each sample in order to avoid excessive charge accumulation. Analyses were performed using a 60 μm aperture and 10 keV acceleration voltage. The beam intensity was 2.5 nA. The working distance depended on the sample height and was chosen to achieve approximately 8 mm. Thin sections were prepared from rock samples and examined using a Nikon

307

ECLIPSE LV100 POL petrographic microscope. The void fraction was calculated using optical methods of porosity measurement, based on the analysis of binary microscopic images using the Java-based image processing program, Image.

DNA extraction, sequencing and 16S rDNA metagenomic analyses Rock fragments with visible blue-green layers were powdered in sterile mortars. Liquid nitrogen was used to damage the thick cyanobacterial sheaths. Total DNA was extracted from the rock powder of colonised zones of each sample under nitrogen treatment. Extraction was carried out using a Soil DNA Purification Kit (GeneMATRIX, EURx Ltd.). The DNA was amplified using universal primers flanking the V3–V4 hyper-variable region of 16S rDNA. DNA amplification was performed by the Genomed Joint-Stock Company and the amplicon was then sequenced using the Illumina MiSeq platform. 2 × 250 bp paired reads were generated. The obtained sequences were submitted to BioProject database with ID PRJNA419148 and were analysed using BaseSpace (https://basespace.illumina.com), the Illumina cloud-based genomics computing environment for NGS (next-generation sequencing), and the GreenGene reference taxonomy database (May 2013 version) for identification of taxa.

Statistical analyses Alpha diversity indices, Shannon’s diversity index (H’) and Pielou’s evenness (E), were calculated using untransformed data. The Spearman correlation coefficient was used to study relations between alpha diversity indices, the number of operational taxonomic units (OTU) and cyanobacterial contributions compared to porosity of rock substratum. In order to compare the structure of all bacteria and cyanobacteria communities, agglomeration analyses using Ward’s method and r-Pearson’s distance measurements were applied. Results with a confidence level of P < 0.05 were identified as significant. Statistica 10 software was used for statistical analyses.


308

RESULTS Petrographic analysis and porosity Microscopic observations revealed that three samples were classified as granites (samples 1, 3, and 4), one sample as gneiss (sample 5) and the three remaining rocks were limestone (samples 2, 6, and 7). All samples were strongly weathered with exfoliation. Granites had a light grey colour in various shades. Microscopic observations showed that all granite samples had unsorted and unidirectional texture and anhedral crystals. These rocks consisted of minerals such as biotite (3–8%), quartz (20–45%), plagioclase (10–25%) and feldspar (39–50%). Brighter colours of samples 3 and 4 were due to a higher content of plagioclase and feldspar. These samples were localised in the syenogranite area in a QAPF diagram. Plagioclase and feldspar grains were strongly sericitised due to weathering processes. Minor minerals in granites included iron oxides and zircon located within biotite grains. Gneisses were fine grained with directional texture that was well highlighted by mineral separation. Brighter parts of the rocks consisted of plagioclase, feldspar and quartz. In darker parts, the rocks were comprised of amphibole, muscovite, biotite and garnet. Plagioclase and feldspar were strongly sericitised, similar to the granites. Minor minerals included iron oxides and zircons. Investigated limestones can be divided into granular limestone and microcrystalline

limestone (micrite, samples 6, and 7). The granular limestone had an unsorted texture and anhedral crystals, and was composed mainly of calcite with a very small amount of muscovite and hydromuscovite. The mica minerals sometimes turned into clay minerals. The microcrystalline limestone was composed of very fine granular calcite with a small amount of organic matter. Porosity of the substrata changed from one sample to another. The percentage of pore volume (i.e. void space) was highest in the granites where it reached a maximum of 22% and mean of 12%. In the gneiss samples, the maximum porosity was 15% with a mean of 7%, and in limestone, the maximum porosity was 11% with a mean of 3% (Table 2). In order to understand habitat selection, we further compared the rock types and porosity of the substratum with the metagenomic data.

Diversity of rock communities in culture-dependent and culture-independent analyses The analysis of microorganisms scraped off sampled rocks in fluorescent (FM) and light microscopes revealed that the Pamir communities contained unicellular forms and a small number of filamentous cyanobacteria (Fig. 3A, B, C). FM observations detected frequent Chroococcidiopsis-like taxa, mostly in cell aggregates, and rare filamentous cyanobacteria

Table 2. Sample information and species diversity based on 16S rDNA metagenomics. PF* - Number of Reads Passing Filter. Sample

Rock type

Mean No No of No of Cyano- Share of H’ for all H’ for Evenness Porosity Reads PF species bacterial Cyano- bacteria CyanoCyano(%) reads bacteria bacteria bacteria (%)

1

granite

10.1

254 617

918

99142

39.8

1.74

1.33

0.39

2

limestone

2.5

250 544

1 138

73243

30.4

2.32

1.47

0.44

3

granite

13.7

258 634

926

34411

14.2

1.91

1.76

0.51

4

granite

6.6

238 823

822

72241

31.5

1.81

1.14

0.35

5

gneiss

7.4

287 016

990

17203

6.3

1.84

1.13

0.34

6

limestone

4.3

206 505

929

6807

3.4

2.20

1.49

0.46

7

limestone

1.3

239 799

878

36523

16.2

2.16

1.98

0.62


309

A

B

C

D

E

F

Fig. 3. Culture independent analysis of cyanobacteria under fluorescence microscopy. A − Chroococidiopsis; B − Cyanobacterial sheaths and coccoid cyanobacteria; C − Leptolyngbya (red signal – cyanobacteria photosynthesic pigments, green signal – sheaths of EPS surrounding cyanobacteria aggregates, singular cells and filaments). Cultured cyanobacteria under light microscopy. D − Calothrix sp.; E − Nostoc sp. and F − Chroococcidiopsis sp.

identified as Leptolyngbya. Most of the coccoid cyanobacteria were covered by exopolymer polysaccharide sheaths (EPS), which were visible under FM as a green signal (Fig. 3). In the case of filamentous Leptolyngbya, some filaments had slightly visible EPS while others did not (Fig. 3). SEM observations of the colonised rock layers revealed that the investigated communities contained abundant unicellular bacteria, which were associated in a biofilm.

After one month of cultivation, we detected the first growth of cyanobacteria and bacteria colonies as blue-green and pink spots in the Petri dishes. During the second month, as the microorganisms were gradually growing, we examined them under light and fluorescence microscopes and performed isolation of individual taxa by re-streaking. During seven months of isolation and cultivation, we managed to isolate and identify twelve species from ten genera of cyanobacteria, the last


310

of which was Synechococcus that appeared in May 2017, seven months after the beginning of isolation and cultivation. The most frequent genera were Chroococcidiopsis (Ch. sp.) and Leptolyngbya (with L. foveolarum and L. laminosa), which were isolated from five and three of the seven samples, respectively (Table 3). Endoliths isolated from the studied rocks were generally represented by unicellular forms (Chroococcidiopsis, Synechococcus), filamentous (Leptolyngbya, Phormidium, Symploca, Microcoleus, Oscillatoria) and two filamentous genera with heterocytes (Calothrix, Nostoc) (Fig. 3D, E, F). The mean number of species isolated from the rocks was three, with the lowest number isolated from granite (one and two) and the highest from limestone (three and six). Moreover, we isolated and successfully cultured eukaryotic algae, which were identified as Trebouxia (Chlorophyta).

Diversity of endolithic communities based on 16S rDNA metagenomics In order to describe the endolithic communities at the molecular level, we generated over 649 MB of data and analysed it using BaseSpace. The total number of reads that

passed quality filtering was 1,735,938 for all samples, from which 384,490 were identified as Cyanobacteria. Actinobacteria, Proteobacteria and Cyanobacteria were detected as the most frequent phyla in all samples, with Actinobacteria being the most prominent group. The contribution of cyanobacteria varied in the studied rocks (Table 2). When rocks were grouped according to type, we found that cyanobacteria accounted for about 6% in gneiss, 17% (± 14%) in limestone and 29% (± 13%) in granite. In one of the granite samples, the contribution of cyanobacteria reached 40% of the entire bacteria community. The share of unclassified taxa at the phylum level was less than 6% (Fig. 4). Through analysing the cyanobacteria phylum for all rock samples, we found that Oscillatoriales (with Microcoleus, Phormidium and Oscillatoria) accounted for 30% of all cyanobacteria, and Synechococcales (with Acaryochloris, Synechococcus and Thermosynechococcus) and Chroococcidiopsidales, which included only one genus, Chroococcidiopsis, had similar average shares (26 and 23% respectively), while Nostocales ranked fourth in abundance with an average of 16% of reads (Fig. 5). We found from the summarised cyanobacterial reads at the order level that

Table 3. Endolithic cyanobacteria from Eastern Pamir based on isolation and microscopic analyses. + − success in isolation of a given taxon from a given sample, - − failure of isolation of a given taxon from a given sample

1

2

3

Sample 4

Chroococcidiopsis sp.

+

+

-

-

+

+

+

2

Leptolyngbya laminosa

-

+

-

-

+

-

+

3

Leptolyngbya foveolarum

-

-

+

+

-

+

-

4

Phormidium sp.

-

+

-

-

+

-

-

5

Nostoc linckia

-

+

+

-

-

-

-

7

Symploca sp.

-

+

-

-

-

-

-

8

Oscillatoria sp.

-

-

-

+

-

-

-

9

Arthrospira fusiformis

-

-

-

-

-

+

-

10

Calothrix sp.

-

-

-

-

-

+

-

11

Synechococcus elongatus

-

-

-

+

-

-

-

12

Microcoleus vaginatus

+

-

-

-

-

-

-

No

Taxa

1

5

6

7


311

Fig. 4. The structure of endolithic communities at the phylum level calculated for tree types of substrata based on 16S rDNA metagenomics.

117,238 belonged to Synechococcales, 76,025 to Oscillatoriales, 34,429 to Chroococcidiopsidales, 30,512 to Nostocales, 10,069 to Chroococcales, 6,171 to Pseudanabaenales, 723 to Spirulinales, 4 to Pleurocapsales and Gloeo-

bacterales (two for each sample). Thus, Synechococcales dominated the overall number of cyanobacterial reads. Through analysing the summarised reads of cyanobacteria for each rock sample, we found that sample 1 had the

16% 30% 3%

2%

Oscillatoriales Synechococcales Chroococcidiopsidales Pseudanabaenales Chroococcales Nostocales

23% 26% Fig. 5. Communities’ assembly of endoliths from Eastern Pamir at the order level, mean values from all seven samples.


312

Table 4. Cyanobacteria composition at genus level based on 16S rDNA metagenomics.

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Genus

Sample 4

Reads per genus

1

2

3

5

Acaryochloris Microcoleus Thermosynechococcus Chroococcidiopsis Phormidium Calothrix Halomicronema Chroococcus Nostoc Arthronema Arthrospira Limnothrix Oscillatoria Dolichospermum Scytonema Aphanizomenon Anabaena Trichodesmium Symploca Microcystis Gloeotrichia Cyanobacterium Spirulina Leptolyngbya Snowella Nodularia Gloeothece Anabaenopsis Synechococcus Lyngbya Planktothrix Rivularia Geitlerinema Xenococcus Gleobacter

62445 4479 958 2685 895 166 9836 517 525 5874 6 2422 210 261 8 259 1 143 82 268 1 119 673 245 65 26 7 2 1 1 3 0 1 0 0

4864 35561 230 5329 6833 645 3 300 621 95 1297 14 107 647 471 7 4 702 646 114 0 157 8 41 25 10 0 3 0 1 1 3 0 0 0

1905 343 604 14944 1084 513 4 95 871 153 83 15 155 1696 130 1923 1517 38 162 90 0 223 13 47 48 6 20 3 1 6 1 2 0 1 1

Sum of reads per sample Mean No of Cyanobacterial reads

93184 58739 26697 63480 12833 7550 29262

74 137 237 8 43350 107 483 8466 1581 35 1125 23 142 15 6916 149 852 1730 54 85 0 0 3119 26 3861 8 66 531 38 277 109 58 4 7 433 2 237 5 403 150 1 974 42 3 12 15 176 6 120 7 37 1 0 0 1 5 7 0 0 1 0 1 0 1 0 0 0 0 0 0

6

7

4 1317 16 1883 275 76 0 51 21 16 3526 2 9 140 15 2 0 42 69 40 1 3 1 30 5 2 1 1 0 0 0 1 1 0 0

2475 8359 57 639 1581 8603 0 146 3176 30 1258 1 60 618 1424 3 257 116 110 53 0 230 1 27 19 4 0 6 3 1 1 1 1 1 1

Mean No 10272 7186 6475 4918 1755 1593 1429 1168 1114 901 881 800 630 566 338 337 256 211 188 160 140 111 103 82 41 12 4.0 3.0 1.7 1.4 1.0 1.1 0.4 0.3 0.3

41678

Sum 71904 50304 45322 34429 12284 11151 10000 8174 7796 6307 6170 5599 4410 3959 2363 2361 1790 1476 1311 1118 977 777 723 572 289 86 28 21 12 10 7 8 3 2 2


313

100000 90000 80000

Other Nostoc Chroococcus Arthrospira Halomicronema Calothrix Phormidium Chroococcidiopsis Thermosynechococcus Acaryochloris Microcoleus

Number of reads

70000 60000 50000 40000

30000 20000 10000 0

1

2

3

4

Sample

5

6

7

Fig. 6. Structure of cyanobacterial communities at the genus level, based on reads from V3-V4 16S rDNA amplicon. Other is a sum of identified genera from Table 2.

highest number of reads (93,184), while the lowest number of reads was obtained from sample 6 (7,550) and a very low from sample 5 (12,883). The mean number of reads of cyanobacteria was 41,678 (Table 4). Analysis of the composition of cyanobacterial communities at the genus level based on 16S rDNA metagenomics shows that the majority of samples included all taxa detected in Eastern Pamir rocks (Table 4). Each sample also contained 23–46% of so-called ‘other genera’, which were taxa with the sum of less than 3.5% of all reads at this level. Additionally, 15–23% of taxa were ‘unclassified at the genus level’. The percentages of both ‘other genera’ and ‘unclassified at the genus level’ designations are given for the entire bacterial community without distinguishing the contribution of unclassified cyanobacteria. At the genus level, the largest number of reads was for Acaryochloris (71,904) and Microcoleus (50,354), followed by Thermosynechococcus (45,322) and Chroococcidopsis (34,429). Chroococcidopsis was also represented in a considerably high number of reads in all the

samples, while reads of the other taxa mentioned above varied widely. Leptolyngbya, which was isolated from almost all rock samples, was present in all samples in metagenomic analysis, though in considerably low abundance, with a total of 572 reads. Analysis of rare (but classified) taxa at the genus level in the Pamir samples shows that: Gloeothece and Geitlerinema were identified in only three samples with a low number of reads; Gloeobacter and Xenococcus were identified in two samples in only two reads and no reads were obtained of Gloeocapsa. Other taxa, such as Planktothrix, Synechococcus, Anabaenopsis, Rivularia and Lyngbya, were detected in four to seven samples but in a very low number of reads. Metagenomic analysis of the 16S rDNA amplicon detected that endolithic communities include taxa (Arthrospira, Snowella, Limnothrix, Microcystis, Planktothrix) that are typical for aquatic environments but odd for lithic habitats. Analysis of the structure of cyanobacterial communities in each sample shows that Acaryochloris, Microcoleus Chroococcidiopsis


314

Tree diagram for 830 cases; Ward's method; 1- r Pearson

A 1 4 2 6 3 7 5

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.6

1.8

Linkage distance

Tree diagram for 35 cases; Ward's method; 1- r Pearson

B 1 2 7 4 3 5 6

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Linkage distance

Fig. 7. A − agglomeration analysis of overall bacteria community and B − of cyanobacterial community.

and Thermosynechococcus were the dominant genera accounting for 60–66% of reads in various samples (Fig. 6). Arthrospira, Calothrix, Nostoc and Phormidium show a mean contribution of 5% to these communities that was occasionally much higher (up to 40%). The average contribution of other taxa was usually lower than 2% and often around 0.001%. Determination of H’ values, based on all bacterial OTUs calculated for each sample, identified samples 2, 6 and 7 as the most diverse (Table 2). Although there was no significant correlation between the number of

OTUs and H’ with the rock type, the highest and lowest OTU and H’ values were found in the limestone and granite samples, respectively. On the other hand, the percentage of cyanobacterial reads in all bacterial reads was found to be the highest in granite (99142) and the lowest in limestone (sample 3, 6807, cf. Table 2), but the correlation was not statistically significant. H’ values calculated for cyanobacterial communities varied from 1.13 in gneiss to 1.98 in one of the limestone rocks while, E values varied between 0.34 in gneiss and 0.62 limestone (Table 2).


Spearman correlation analysis did not reveal any dependence between rock porosity and diversity parameters of endolithic communities. We compared mean and maximum void fraction of the rock substrata with the total number of bacteria species, OTU number, number of cyanobacteria species in the samples and percentage of cyanobacteria in overall bacteria communities, but the obtained correlations were not statistically significant. However, the Spearman correlation coefficient calculated for all bacteria OTUs and mean and maximum porosity of rock substrata showed a negative trend between these parameters (r = -0.7, P = 0.09 and r = - 0.7, P = 0.1, respectively). A comparison of the overall bacterial community made by agglomeration analysis showed that samples 3 (granite), 7 (limestone) and 5 (gneiss) grouped together, and then grouped with the longer distance with samples 2 and 6 (both limestone). Two granite samples (1, 4) were distant from the rest of the samples and formed a separate clade with a long distance between each other (Fig. 7A). Through analysis of the community structure of cyanobacteria (Fig. 7 B), we noticed that samples 3 (granite) and 5 (gneiss) were closely grouped together and sample 6 (limestone) joined them to form one cluster, although the distance between the group of the two samples (3 and 5) and sample 6 was larger. A second cluster was formed by communities within limestone samples (2, 7), which loosely aggregated with samples 1 and 4 (granite).

DISCUSSION According to the literature, cyanobacteria play a key role as a member of endolithic communities in poor desert ecosystems because they have photosynthetic abilities and can survive desiccation and changes in temperatures (Wierzchos et al. 2006). They are also considered to dominate endoliths, especially in cold areas in high latitudes (Yung et al. 2014) and high altitudes (Sigler et al. 2003), as well as in hot deserts (DiRuggiero et al. 2013). We report here the first study of endoliths from the unexplored, in this respect, region of the cold desert of Eastern Pamir using original microscopic and culturing, as well as advanced molecular methods - next generation seguensing (NGS).

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Although endoliths are considered to be difficult to isolate, we were able to obtain ten cyanobacterial genera during our isolation. The composition of isolated cyanobacteria varied from one sample to next. Leptolyngbya and Chroococcidiopsis-like taxa were the easiest to isolate and culture, as anticipated based on previous studies. We also acquired well-growing Nostoc, Calothrix and Symploca in isolation. We also observed very high morphological differences between the cultured organisms in the genera suggesting that they contained several species (e.g. Leptolyngbya with L. foveolarum and L. laminosa). However, results concerning species affiliation should be still verified by molecular analysis in order to rule out morphological variability within the same species. In our cultures, we generally obtained the most ubiquitous and commonly described endolithic cyanobacterial genera for other cold and hot regions. Interestingly however, while other authors usually isolated only a single or a few cyanobacterial taxa from a given region (Sigler et al. 2003), we obtained all ten taxa from Eastern Pamir. Thus, the Eastern Pamir endolithic community of cyanobacteria seems to be very rich, even at the level of culture-dependent investigations. Molecular analysis based on 16S rDNA metagenomics revealed more diverse composition and structural information of Eastern Pamir endoliths. Acaryochloris, Microcoleus, Chroococcidiopsis and Thermosynechococcus were the taxa with the highest number of reads at the genus level. Acaryochloris, which was present in all samples and represented the highest number of reads in four samples, was described by Yung (2014) and Crits-Christoph with co-authors (2016) as a common genus from extreme habitats including Miers Valley and McMurdo Dry Valleys in Antarctica and the Atacama Desert in Chile. On the other hand, we did not identify sequences belonging to some taxa that are well known from microscopic and molecular studies of Antarctic endoliths, such as Gloeocapsa and Aphanocapsa (De los Rios et al. 2004, Friedmann et. al. 1988). Gloeothece, another lithobiont that is described for this region (de la Torre et al. 2003), appeared in only three of our samples with a very low number of reads.

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Based on 16S rDNA metagenomics and microscopic analyses, we can characterise Eastern Pamir endoliths as communities that are dominated by bacteria. On average, cyanobacteria account for 20% of the reads and their relative contribution varied between 3 and 40%. This result differs from previous cold desert molecular studies of lithobionts where cyanobacteria dominated communities and accounted for about 45% of the relative abundance of prokaryotes (Yung et al. 2014). Due to the similarity in locality, elevation and some similarities in climatic conditions between Eastern Pamir and Tibet, we compare the composition of cyanobacterial communities from Pamir with chasmoendolithic microorganisms studied within limestone from a high-altitude arid environment in Central Tibet as described by Wong et al. (2010). According to these authors, the Tibetan endoliths are eukaryan lichendominated with features reminiscent of both polar and alpine endoliths. Unlike Tibetan endoliths examined at the molecular level, Eastern Pamir communities did not show sequences of Chlorophycean chloroplasts. During isolation and cultivation in most of the samples, however, isolates of Trebouxia, a common lichen phycobiont that reveals their presence in endolithic communities, were obtained. However, this green alga can be easily cultivated, comparing with cyanobacteria, and thus may not reflect a real contribution of these eukaryotes in the studied communities. In this study, we did not observe evident lichenised assemblies, although some samples included isolated examples of lichenised structures, which are typical for Antarctic endoliths. Culture-dependent analyses indicate that the endolithic communities from Eastern Pamir may belong to a first class of endoliths that is cyanobacteria-dominated (Friedmann 1980), but molecular analysis demonstrates that Pamir endoliths are bacteria-dominated communities led in abundance by Actinobacteria and Proteobacteria, followed by Cyanobacteria. Thus, we assume, that the classification scheme by Friedmann (1980), which does not include a third class of endoliths, does not adequately characterise the Eastern Pamir communities.

According to the NGS sequence reads, the structure of Pamir cyanobacteria was highly variable and differed from one sample to next. The most similar structure of the community was noted for the granite sample 3 and gneiss sample 5, while other granite samples were very different. We did not detect a observable tendency of the cyanobacterial community structure towards a rock type, degree of porosity or geographical location. However, we found such a tendency for the total number of reads and Shannon diversity index, such that the highest H’ was noted in the limestone (sample 7), where it reached 1.98, and lowest in gneiss (1.13). The richness of cyanobacterial endolithic communities was quite high but the diversity index suggested communities were dominated by single taxa. In fact, E values were low, varying between 0.34 and 0.62, which confirms a very uneven distribution of taxa. However, the analysis of all cyanobacteria reads shows (Table 4) that most of the taxa were present in all samples but with varying relative abundances. This result suggests that neither a long nor short distance between sampling sites can explain the similarities or differences between the communities’ structures. Thus, similarly to Wong et al. (2010), we suggest that all the studied communities possess a similar genetic potential but that small differences between the rock substrata influence the community structures. This is in accordance with the well-known hypothesis that ‘everything is everywhere, but environment selects’ (Garcia-Pichel et al. 1996, Fenchel 2003). We also suggest that the variability of the studied endolithic communities from Eastern Pamir is much higher than usually described for cold deserts (Pointing et al. 2009). Environmental stress factors are indeed drivers of microbial communities (Billi et al. 2000) but in order to escape from harsh conditions, microorganisms might select the nearest available substrate. In this case, the architecture of microhabitats is an important element in determining the colonisation and diversity of the microbial communities, which colonise void fractions (pores) of the rock (Crits-Christoph et al. 2016). In our study, the Spearman correlation coefficient was calculated for porosity of the studied rocks and diversity parameters of


cyanobacteria for all types of substrata together and it did not reveal any significant relationship. We noticed however that granite, which was the most porous and much more inhabited by cyanobacteria than other substrata, did not host the richest community with regards to OTU. Due to a small number of analysed samples we could not verify if the remarkable differences in the community structures, which could be noted based on the metagenomic results, were statistically significant and if the rock type could explain them. Further studies of these weathered rocks might shed some light on the profound factors determining such wide variability in the endolithic communities in Eastern Pamir. For this however more rock samples and from various locations should be analysed. By examining the endolithic communities at the molecular level, we detected some taxonomical inaccuracies present in the Greengenes 13_5 database. For example, 16S rDNA metagenomics based on the Greengenes reference taxonomy database (May 2013 version) appeared outdated in that it classified many genera to orders, which are not supported by current systematics of cyanobacteria. Thus, by analysing the metagenomics results, we recalculated all data according to the more recent classification of Komárek et al. (2014), and compared our results to the current systematics of Pliński and Komárek (2017). Another interesting result from the present study is that the Pamirian endolithic communities include taxa that are mostly known from aquatic, planktonic habitats, such as Arthrospira, Trichodesmium, Snowella, Dolichospermum or Microcystis. We obtained a relatively large number of Arthrospira reads in three samples and the taxon was absent in only two samples (granite and gneiss). The presence of Arthrospira in Pamirian endolithon was also detected in the cultures. Snowella, Trichodesmium, Dolichospermum and Microcystis reads were present in all rock samples while Planktothrix, another observed planktonic taxon, did not appear in most limestones. The detection of aquatic taxa is one of the most compelling reasons to continue this study further.

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CONCLUSIONS Eastern Pamir endoliths are bacteria dominated communities with a cyanobacterial component characterised by a different structure compared to other cyanobacterial endoliths previously described from a high altitude cold desert ecosystem. Using 16S rDNA metagenomics in a culture-independent study, we observed a high diversity of microorganisms with wide variability between the investigated microenvironments. However, taxonomical inaccuracies and lack of reference sequences of endolithic cyanobacteria within the database complicates the exploration of organism diversity in such extreme environments. Analysis of endoliths from Eastern Pamir did not demonstrate a clear preference of rock type or porosity of the rock for colonisation by cyanobacteria. Additionally, although lithobionts are one of the most difficult organisms to cultivate, we isolated a substantial number of taxa, which allow in further studies for applying a polyphasic approach and will serve as reference genomes. This study is a first step in the evaluation of taxonomic composition, structure and functional diversity of microbial communities from environments as extreme as the rocks in cold desert shrubland from Eastern Pamir Mts. that may help to provide important information regarding microbial adaptation to changing conditions in one of the most challenging habitats on Earth. ACKNOWLEDGMENTS: The authors are grateful to Dovutsho Navruzshoev from the Pamir Biological Institute in Khorog (Academy of Sciences of the Republic of Tajikistan) and Mavlon Pulodovich Pulodov from National Republican Centre for Genetic Resources, Tajik Academy of Agricultural Sciences, Tajikstan. We also wish to thank the colleagues: Magdalena Malawska, Mateusz Wilk, Latif Kurbonbekov and Arsen for help in the field research and dr Andrzej Borkowski for kind assistance in geological analyses. Microscopic and molecular analyses were performed in the laboratories of Department of Microbial Ecology and Environmental Biotechnology in the Biological and Chemical Research Centre, University of Warsaw within the project under the Operational

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Programme Innovative Economy, 2007 – 2013. SEM-EDS analyses were performed in the laboratories of NanoFun, POIG.02.02.0000-025/09. This study was supported by the National Science Centre (2013/09/B/ ST10/01662 and 2015/19/B/NZ9/00473).

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