ORIGINAL ARTICLE Environmental microarray analyses of Antarctic soil microbial communities Etienne Yergeau1,6, Sung A Schoondermark-Stolk1,7, Eoin L Brodie2, Sebastien �� Dejean3,�� Todd Z DeSantis2, Olivier Gonc ��alves4, Yvette M Piceno2, Gary L Andersen2 and George A Kowalchuk1,5 1Netherlands Institute of Ecology (NIOO-KNAW), Centre for Terrestrial Ecology, Heteren, The Netherlands 2Ecology Department, Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA 3Institut de mathematiques, �� Universite �� Paul Sabatier, Toulouse, France 4Laboratoire ���Microorganismes: Genome �� et Environnement���, UMR CNRS 6023, Universite �� Blaise Pascal, Clermond-Ferrand II, France and 5Institute of Ecological Science, Free University of Amsterdam, Amsterdam, The Netherlands Antarctic ecosystems are fascinating in their limited trophic complexity, with decomposition and nutrient cycling functions being dominated by microbial activities. Not only are Antarctic habitats exposed to extreme environmental conditions, the Antarctic Peninsula is also experiencing unequalled effects of global warming. Owing to their uniqueness and the potential impact of global warming on these pristine systems, there is considerable interest in determining the structure and function of microbial communities in the Antarctic. We therefore utilized a recently designed 16S rRNA gene microarray, the PhyloChip, which targets 8741 bacterial and archaeal taxa, to interrogate microbial communities inhabiting densely vegetated and bare fell-field soils along a latitudinal gradient ranging from 51 1S (Falkland Islands) to 72 1S (Coal Nunatak). Results indicated a clear decrease in diversity with increasing latitude, with the two southernmost sites harboring the most distinct Bacterial and Archaeal communities. The microarray approach proved more sensitive in detecting the breadth of microbial diversity than polymerase chain reaction-based bacterial 16S rRNA gene libraries of modest size (B190 clones per library). Furthermore, the relative signal intensities summed for phyla and families on the PhyloChip were significantly correlated with the relative occurrence of these taxa in clone libraries. PhyloChip data were also compared with functional gene microarray data obtained earlier, highlighting numerous significant relationships and providing evidence for a strong link between community composition and functional gene distribution in Antarctic soils. Integration of these PhyloChip data with other complementary methods provides an unprecedented understanding of the microbial diversity and community structure of terrestrial Antarctic habitats. The ISME Journal (2009) 3, 340���351 doi:10.1038/ismej.2008.111 published online 20 November 2008 Subject Category: integrated genomics and post-genomics approaches in microbial ecology Keywords: Antarctic soil ecosystems GeoChip microarray microbial community structure microbial diversity PhyloChip microarray Introduction Antarctic environments are extraordinary in the harshness of their climates, far more severe than northern climates at similar latitudes (Convey, 2001). Antarctic food webs are consequently rela- tively simple, with a general absence of insect and mammalian herbivores (Davis, 1981 Heal and Block, 1987). Cold temperatures and low moisture availability are probably the main limiting factors responsible for the depauperate status of Antarctic habitats (Kennedy, 1996). The relatively simplified food-web structure of Antarctic terrestrial habitats provides a reasonably tractable system to disentan- gle the drivers of soil microbial activities and the consequences of system perturbation. Recent studies on the soils of this area have aimed to establish baseline knowledge of microbial community struc- ture and function across a range of environments, and to assess the impacts of global warming (Lawley et al., 2004 Brinkmann et al., 2007 Bokhorst et al., 2007a, 2008 Yergeau et al., 2007a, b, c Yergeau and Kowalchuk, 2008). Environmental conditions, such Received 4 August 2008 revised 8 October 2008 accepted 9 October 2008 published online 20 November 2008 Correspondence: GA Kowalchuk, Netherlands Institute of Ecology (NIOO-KNAW), Centre for Terrestrial Ecology, PO BOX 40, Heteren 6666 ZG, The Netherlands. E-mail: g.kowalchuk@nioo.knaw.nl 6Present address: Biotechnology Research Institute, National Research Council of Canada, Montreal, �� Canada. 7Present address: Diakonessenhuis, Medical Microbiology and Immunology, Unit Molecular Diagnostics, Utrecht, The Netherlands. The ISME Journal (2009) 3, 340���351 & 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00 www.nature.com/ismej

as temperature and freeze���thaw cycles, appear to have profound effects on soil microbial commu- nities (Bokhorst et al., 2007a Yergeau and Kowal- chuk, 2008). Bacterial diversity, community structure, abundance and functional gene density were all reported to be affected to different degrees by environmental conditions, most of the time in interaction with the type of aboveground cover (Yergeau et al., 2007a, b, c). The application of microarrays to study complex microbial communities is a relatively new practice, but the rapid increase in genetic databases (Cole et al., 2005 DeSantis et al., 2006) has facilitated the development of comprehensive platforms encom- passing the known range of bacterial and archaeal diversity based on 16S rRNA gene sequences (for example, the PhyloChip, Brodie et al., 2006 DeSan- tis et al., 2007). The PhyloChip platform allows for the simultaneous detection of 8741 bacterial and archaeal taxa and has been shown to reveal a broader range of diversity than modestly sized 16S rRNA gene libraries for soil, water and aerosol samples (Brodie et al., 2006, 2007 DeSantis et al., 2007). However, it is not yet clear how such PhyloChip results might be compared with more traditional molecular methods like PCR-DGGE, T-RFLP, cloning���sequencing and quantitative PCR, or how such data can be integrated into studies of microbial community ecology. Furthermore, micro- array platforms are highly dependent on the amount of information already known and cannot detect taxa that have not been described earlier in databases. Thus, it is imperative that such methods be tested across novel environments, such as the Antarctic soils examined in this study. Most earlier reports about microbial communities in Antarctic soils have relied on relatively labor- intensive methods with low levels of taxonomic resolution. With the increasing interest in linking microbial identity and function, PhyloChip analyses also offer the opportunity to link microbial commu- nity composition with analyses of enzyme activity, density of functional gene families and the distribu- tion of nutrient-cycle-related functional gene sequences. Thus, the aims of this study were: (1) to determine the suitability of 16S rRNA gene microarrays to monitor Antarctic soil bacteria and archaea, (2) to describe Antarctic soil-borne bacterial and archaeal communities using microarrays, there- by providing a more complete description of bacterial and archaeal diversity than possible ear- lier, (3) to relate bacterial and archaeal community composition and diversity to important environ- mental parameters and (4) to assess the feasibility of linking functional gene and 16S rRNA gene micro- array data. To achieve these ends, the recently expanded PhyloChip of DeSantis et al. (2007) was used on PCR-amplified DNA directly extracted from soils sampled at five different sites ranging from the Falkland Islands (51 1S) to Coal Nunatak (72 1S), with a comparison of extensively vegetated patches vs bare, fell-field environments. The resulting patterns of bacterial and archaeal community com- position and diversity were compared with similar data recovered from clone libraries and real-time PCR assays and integrated into studies of function, including functional gene microarray analyses. Materials and methods Sampling sites During the austral summer of 2003���2004, 2 2 m plots were established at the following sites (see Supplementary Figure S1 for a map): the Falklands Islands (cool temperate zone 511760S 591030W), Signy Islands (South Orkney Islands, maritime Antarctic 601430S, 451380W) and Anchorage Islands (near Rothera Research Station, western Antarctic Peninsula 671340S, 681080W). At each location, two habitat types were selected for soil sampling: (1) ���vegetated���, where dense vegetation cover was present with retention of underlying soil, and (2) ���fell-field���, with rocky or gravel terrain and scarce vegetation or cryptogam coverage. Data with respect to vegetation cover within these environments were reported earlier (Bokhorst et al., 2007b). Twelve plots were delineated per location, with half of the plots positioned over each soil type. The Falkland Islands fell-field habitat was not sufficiently exten- sive to allow for such a design, and nine of the twelve plots were therefore placed in the vegetated environment. Two additional sites were chosen for sampling, but without delineation of permanent plots. Six frost-sorted soil polygons at two different sites were sampled near Fossil Bluff (711190S, 681180W) and five adjacent polygons were sampled at Coal Nunatak (721030S, 681310W). Soil samples For molecular analyses, five 1-cm-diameter (from 2���3 to 15 cm deep, depending on the depth of soil per habitat) cores were sampled from each plot or polygon. They were frozen to 20 1C as soon as possible (within 24 h) and maintained at that temperature until further analysis. Material for soil analyses was collected from a 10-cm-diameter core taken directly adjacent to the established plots in order to minimize destructive sampling in the long- term plots. Sampling was conducted during 26���28 October 2004 for the Falkland Islands, during 2���3 January 2005 for Signy Islands, during 18���19 January 2005 for Anchorage Islands and during 22���23 February 2005 for Coal Nunatak and Fossil Bluff. Nucleic acid extractions DNA was extracted from 500 mg soil samples after bead-beating for 30 s at 50 m s 1 in a hexadecyl- trimethyl-ammonium bromide (CTAB) buffer using a phenol���chloroform purification protocol as PhyloChip analysis of Antarctic soil microbes E Yergeau et al 341 The ISME Journal