Characterization of Bacterial Communities in Lithobionts and Soil Niches from Victoria Valley, Antarctica Marc W
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FEMS Microbiology Ecology, 92, 2016, fiw051 doi: 10.1093/femsec/fiw051 Advance Access Publication Date: 4 March 2016 Research Article RESEARCH ARTICLE Characterization of bacterial communities in lithobionts and soil niches from Victoria Valley, Antarctica Marc W. Van Goethem1, Thulani P. Makhalanyane1,†, Angel Valverde1, Stephen C. Cary2 and Don A. Cowan1,∗ 1Department of Genetics, Centre for Microbial Ecology and Genomics, University of Pretoria, Lynwood Road, Pretoria 0028, South Africa and 2Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton 3120, New Zealand ∗Corresponding author: Department of Genetics, Centre for Microbial Ecology and Genomics, University of Pretoria, Pretoria, 0028, South Africa. Tel: +27-12-420-5873; E-mail: [email protected] One sentence summary: Characterization of general bacterial and cyanobacterial populations from soil and lithibiontic microbial communities (hypoliths and endoliths) of Victoria Valley, Antarctica. Editor: Dirk Wagner †Thulani P. Makhalanyane, http://orcid.org/0000-0002-8173-1678 ABSTRACT Here we provide the first exploration of microbial diversity from three distinct Victoria Valley edaphic habitats, namely lithobionts (hypoliths, endoliths) and surface soils. Using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene amplicon pyrosequencing we assess community structure and diversity patterns, respectively. Our analysis revealed that habitat type (endolithic versus hypolithic versus surface soils) significantly influenced bacterial community composition, even though dominant phyla such as Actinobacteria (41%of total reads) were common to all samples. Consistent with previous surveys in other Dry Valley ecosystems, we found that lithobionts were colonized by a few highly dominant phylotypes (such as Gemmatimonas and Leptolyngbya). Our analyses also show that soil bacteria were more diverse and evenly distributed than initially expected based on previous evidence. In contrast to total bacteria, the distribution of Cyanobacteria was not strongly influenced by habitat type, although soil- and endolith-specific cyanobacterial lineages were found. The detection of cyanobacterial lineages in these habitats appears to be influenced by the dispersal of aquatic inocula from lacustrine communities or benthic mats which are abundantin Victoria Valley. Together, our results provide insights into the phylogenetic variation and community structure across niche habitats in Victoria Valley. Keywords: Antarctica; bacteria; Cyanobacteria; endolith; hypolith; soil INTRODUCTION Valleys of Antarctica are an ideal model system for assess- ing how adverse conditions may shape microbial diversity and Despite the recent advances in molecular analysis, we know functional processes (Cowan et al. 2014). Extremely low mois- comparatively little regarding the effect of environmental stres- ture availability, below-freezing temperatures, katabatic wind sors on microbial diversity and function. The McMurdo Dry Received: 18 December 2015; Accepted: 25 February 2016 C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected] 1 Downloaded from https://academic.oup.com/femsec/article-abstract/92/4/fiw051/2198003 by University of California, Berkeley/LBL user on 03 July 2018 2 FEMS Microbiology Ecology, 2016, Vol. 92, No. 4 episodes, frequent freeze-thaw cycles and very low levels of pre- cyanobacterial populations may be influenced by the transport cipitation all combine to render the Dry Valleys inhospitable to of lineages from surrounding aquatic systems on the basis of most higher organisms (Cowan and Ah Tow 2004), resulting in previous findings (Wood et al. 2008). To enhance our understand- a simple ecosystem dominated by microbial communities (Cary ing of community structure and diversity we performed micro- et al. 2010). There is evidence suggesting that abiotic factors are bial fingerprinting analysis, using terminal restriction fragment strong drivers of microbial community composition, which in- length polymorphisms (T-RFLP), and 16S rRNA gene amplicon clude the scarcity of water (Fell et al. 2006), local soil geochem- sequencing of bacterial communities. istry (Lee et al. 2012) and temperature (Hopkins et al. 2006). We now know that the controls on microbial community structure MATERIALS AND METHODS extend from local (Pointing et al. 2009) to regional scale differ- ences (Van Horn et al. 2013; Bottos et al. 2014). Whilst the growing Study site and sampling body of knowledge has suggested significant differences exist between lithobiontic and soil communities, very little is known All samples were retrieved from the Victoria Valley region of ◦ ◦ regarding community dynamics across various habitats espe- Eastern Antarctica (77 20 S, 161 39 E) during the austral sum- cially in understudied Dry Valleys such as the Victoria Valley. mer season of January 2013 (Fig. 1). A total of 15 samples, The Antarctic Dry Valleys are largely colonized by a variety five each of surface soils, endoliths and hypoliths were col- of cold-adapted microorganisms (Cowan et al. 2014; Williams et lected. Ventrally-colonized quartz rocks (hypoliths) and inter- al. 2014; Makhalanyane et al. 2015b) that survive in an array of nally colonized sandstone rocks (endoliths) were randomly re- habitats such as hypolithic and endolithic environments (com- trieved and stored in sterile Whirl-Pak sample bags (Nasco, WI, munities that inhabit undersides and interstices of rocks, re- USA). The endolithic samples included both fungal-dominated spectively), moranic soils, permafrost and ice-covered lakes. Re- and Cyanobacteria-dominated communities. Soil samples were cent phylogenetic surveys have shown significant differences in transferred into sterile 50 ml tubes. Samples were maintained < ◦ hypolithic and surface soil community compositions (Pointing at 0 C in the field and during transit to the laboratory. Sample − ◦ et al. 2009; Makhalanyane et al. 2013a), which are broadly con- material was stored at 80 C upon arrival until required. sistent with the differences in community structure found be- tween these habitats in hot hyperarid deserts (Azua-Bustos et Environmental DNA isolation and T-RFLP analysis al. 2011; Makhalanyane et al. 2013b). In addition, it is known that Dry Valleys are highly heterogenous in terms of physicochemi- DNA was isolated from duplicate 1 g samples using a cal conditions, although the effect of these differences in shap- modified phenol/chloroform extraction method (Miller et al. ing microbial diversity is not yet fully understood (Lee et al. 2012; 1999). Hypolithic and endolithic sample material was re- Van Horn et al. 2013). moved from rock substrates with sterile razor blades. DNA was Lithic-associated communities are often dominated by eluted in 20 μl nuclease-free water and quantified using the Cyanobacteria, reviewed by Chan et al.(2012), which are al- NanoDrop 2000 spectrophotometer (NanoDrop Products, Wilm- most certainly the major drivers of photosynthetic carbon fixa- ington, DE, USA). For T-RFLP analysis, 6 carboxyfluorescein- tion and nitrogen cycling in desert ecosystems (Warren-Rhodes labelled forward (341F) and unlabelled reverse (908R) primers et al. 2006). Moreover, lithons comprise simple trophic systems amplified universal bacterial 16S rRNA genes (Lane et al. 1985) of primary producers involved in ecosystem processes (almost under cycling conditions described previously (Makhalanyane exclusively Cyanobacteria) and heterotrophic consumers and et al. 2013a). Following purification and digestion with MspIen- degraders (Pointing et al. 2009; Robinson et al. 2015). Further- donuclease, electrophoresis on an ABI3500xl genetic analyser more, lithons harbour variable microbial compositions (Makha- (Applied BiosystemsR , CA, USA) was performed to separate lanyane et al. 2013a, 2015b), with some endoliths dominated by restriction fragments. Fragments were analysed at SeqServe lichens rather than Cyanobacteria (De Los R´ıos, Wierzchos and (Bioinformatics and Computational Biology, University of Pre- Ascaso 2014), and experience unique levels of environmental toria, South Africa; http://seqserve.bi.up.ac.za/). R scripts were stress resulting from lithic buffering (Warren-Rhodes et al. 2006). used to bin T-RF profiles into operational taxonomic units (OTUs) Cyanobacteria are also generally ubiquitous and widespread using increments previously prescribed (Abdo et al. 2006). A in Antarctic aquatic environments including lakes and ponds nested PCR protocol was used to evaluate cyanobacterial com- (Cowan and Ah Tow 2004). Lacustrine and benthic habitats are munities with the Cyanobacteria-specific primers 6 FAM-359F reported to serve as important sources of novel cyanobacterial and 781R (Nubel,¨ Garcia-Pichel and Muyzer 1997). The ampli- (and to a lesser extent heterotrophic bacterial) diversity, which fication of 442 bp products was achieved using the following ◦ may alter terrestrial community structures after introduction thermal cycling conditions: 5 min denaturation at 95 C, followed ◦ and colonization events occur (Wood et al. 2008). by 30 cycles of the following three steps; 95 C denaturation for ◦ ◦ Exposed Dry Valley soils generally support fewer cyanobac- 30 s, 64 C annealing for 30 s and 72 C elongation for 90 s; with a ◦ terial lineages than lithobiontic consortia and are dominated by final elongation step at 72 C for 10 min. All downstream analy- heterotrophic species (Lee