Local Diversity of Heathland Cercozoa Explored by In-Depth Sequencing
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The ISME Journal (2016) 10, 2488–2497 © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 OPEN www.nature.com/ismej ORIGINAL ARTICLE Local diversity of heathland Cercozoa explored by in-depth sequencing Christoffer Bugge Harder1,2, Regin Rønn1, Asker Brejnrod3, David Bass4,5, Waleed Abu Al-Soud3 and Flemming Ekelund1 1Section of Terrestrial Ecology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; 2Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway; 3Section of Microbiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; 4Department of Life Sciences, The Natural History Museum, Cromwell Road, London, UK and 5Centre for Environment, Fisheries and Aquaculture Science (Cefas), The Nothe, Weymouth, Dorset, UK Cercozoa are abundant free-living soil protozoa and quantitatively important in soil food webs; yet, targeted high-throughput sequencing (HTS) has not yet been applied to this group. Here we describe the development of a targeted assay to explore Cercozoa using HTS, and we apply this assay to measure Cercozoan community response to drought in a Danish climate manipulation experiment (two sites exposed to artificial drought, two unexposed). Based on a comparison of the hypervariable regions of the 18S ribosomal DNA of 193 named Cercozoa, we concluded that the V4 region is the most suitable for group-specific diversity analysis. We then designed a set of highly specific primers (encompassing ~ 270 bp) for 454 sequencing. The primers captured all major cercozoan groups; and 495% of the obtained sequences were from Cercozoa. From 443 350 high-quality short reads (4300 bp), we recovered 1585 operational taxonomic units defined by 495% V4 sequence similarity. Taxonomic annotation by phylogeny enabled us to assign 495% of our reads to order level and ~ 85% to genus level despite the presence of a large, hitherto unknown diversity. Over 40% of the annotated sequences were assigned to Glissomonad genera, whereas the most common individually named genus was the euglyphid Trinema. Cercozoan diversity was largely resilient to drought, although we observed a community composition shift towards fewer testate amoebae. The ISME Journal (2016) 10, 2488–2497; doi:10.1038/ismej.2016.31; published online 8 March 2016 Introduction protozoan diversity, and the difficulty in identifying many species morphologically, it has been proble- Protozoa are essential organisms in soil ecosystems matic to evaluate such contrasting views. primarily because they have a significant role in the However, the fast development in high-throughput soil food web as bacterial grazers (Ekelund and sequencing techniques (HTS) now offers tools to Rønn, 1994). Moreover, protozoan diversity is answer such questions; still, only few such studies a good index of ecosystem function (Griffiths et al., have specifically targeted soil protozoa. One reason for 2000); in particular, it has been suggested that this is that heterotrophic soil protozoa include the high diversity allows the soil protozoan commu- members from at least four kingdoms/supergroups nity to respond to both seasonal and environ- (Baldauf et al., 2000). Therefore, they can inherently mental change (Bamforth, 1995). As fundamentally only be amplified with general eukaryotic primers, aquatic organisms, one would a priori expect which will unavoidably also amplify DNA from other protozoa to be negatively affected by drought, organisms abundant in soil. Thus, sequences from however empirical evidence is ambiguous. Whereas metazoans, fungi and plants often dominate samples, Eisenhauer et al. (2012) reported that drought and must be removed prior to further analysis. decreased protozoan abundance, Schmitt and Further, in case of micro-eukaryotes, we only have a Glaser (2011) found that drought increased the comprehensive reference database for the small sub- protozoan diversity. Because of the large unknown unit ribosomal RNA (18S) gene. The copy number of this gene may vary considerably between different eukaryotic groups (Zhu et al., 2005), which compli- Correspondence: F Ekelund, Section of Terrestrial Ecology, cates quantitative comparison of community composi- Department of Biology, University of Copenhagen, Universitet- tion based on sequence abundance. sparken 15, Copenhagen DK-2100, Denmark. E-mail: [email protected] Here, we tackle these problems by targeting a Received 14 August 2015; revised 27 November 2016; accepted specific protozoan group, the Cercozoa; for a phylo- 8 January 2016; published online 8 March 2016 genetic overview of this group see Bass et al. (2009b). Cercozoa explored by in-depth sequencing CB Harder et al 2489 Cercozoa encompass a high morphological diversity. primer to target this region. We then used this primer They include large testate amoebae such as Euglypha to target soil Cercozoa directly in an experiment and Trinema, naked filose/reticulate amoebae such where we tested the hypothesis that testate Cercozoa as vampyrellids, granofiloseans and Filoreta, and respond negatively to drought. To annotate gliding flagellates such as cercomonads, glissomo- sequences, we used a yet unpublished database nads and thaumatomonads (Bass et al., 2009a; Hess (David Bass, in preparation) that contains 966 et al., 2012; Berney et al., 2013). Owing to the high cercozoan 18S sequences of high-quality, which morphological and physiological diversity, Cercozoa cover the entire group. We hope that we provide also show high functional and ecological diversity. theoretical and practical foundations needed to Hence, changes in the relative abundance of different establish a frame for future comparative molecular cercozoan groups could potentially be a valuable analyses of cercozoan diversity. indicator of environmental change. For example, paleohydrological studies have shown that testate amoebae are sensitive indicators of water content in Materials and methods peat bogs (Charman and Warner, 1992; Booth, 2001, 2008) and there is evidence that testate amoebae also Study site and soil sampling respond negatively to drought in more arid soils Projected climate change for Denmark in the (Lousier, 1974a, b; Wilkinson and Mitchell 2010). 2100th century indicates drier summers, which Hence, it is likely that testate Cercozoa, such as are experimentally simulated on the CLIMAITE Euglypha and Trinema, would decrease in abun- study site (Larsen et al., 2011). The CLIMAITE dance in response to drought. experimental site is situated in a dry heath-/grass- ′ Morphological methods have long suggested that land 50 km NW of Copenhagen, Denmark (55° 53 ′ Cercozoa is one of the dominant groups of free-living N, 11° 58 E). The mineral fraction of the soil eukaryotic microorganisms in temperate soils consists of 92% sand, 5.8% silt and 2.2% clay (Sandon, 1927; Ekelund et al., 2001). This has been (Nielsen et al., 2009). The site is well drained with confirmed by recent HTS-based studies. Bates et al. an organic top layer (O-horizon). The pHCaCl2 in the (2013) found that Cercozoa accounted for ~ 30% of O-horizon is 3.3 increasing to 4.5 in the lower the identifiable protozoan 18S reads in arid or B-horizon. The dominant vegetation consists of the semi-arid soils and ~ 15% in more humid soils. In dwarf shrub Calluna vulgaris (c. 30% cover) and a transcriptomic analysis of soil protist activity, the perennial grass Deschampsia flexuosa (c. 70% Geisen et al. (2015) found that 40–60% of all cover). The annual mean temperature is 8 °C with identified protozoan small subunit ribosomal RNAs a mean precipitation of 613 mm (Danish Meteor- in forest and grassland soils could be assigned to ological Institute, www.dmi.dk). Since 2005, a Cercozoa. Cercozoa are also abundant in marine complete three-factorial treatment with increased benthic and interstitial communities; a recent HTS CO2, temperature and summer drought has been study using general eukaryotic primers found maintained in 12 four-chamber octagons (7 mm in Cercozoa to comprise between 9 and 24% of all diameter), where the fourth octagon is a control assigned eukaryotic operational taxonomic units plot with no treatments. Each treatment is (OTUs) on the ocean floor (Pawlowski et al., 2011). replicated seven times. These treatments are Several recent papers have named many new intended to mimic the projected climate change cercozoan taxa at species, genus and family level for the region in 2075. Drought is induced once or from temperate topsoil (Bass et al., 2009b; Howe twice a year by automatic rain shelters, which et al., 2009, 2011; Chatelain et al., 2013). Sanger exclude the precipitation continuously for sequencing of environmental DNA in Cercozoa (Bass 2–5 weeks until the water content plunges below and Cavalier-Smith, 2004) has shown a further 5% by volume in the upper 20 cm of the soil. undescribed diversity. However, the number of Larsen et al. (2011) provide a detailed description sequences obtained by this method is low compared of the experiment. – with HTS methods, and though HTS is now In November 2010, we sampled topsoil (0 8 cm, – ubiquitous in studies of microbial ecology, to our O-horizon) from two of the six drought plots 1 2 knowledge no HTS-based study has yet targeted (Dry1) and 3–1 (Dry2) and from two of the control Cercozoa directly. Reasons for this likely include plots 9–1 (Control1) and 11–4 (Control2). To mini- lack of standard protocols with consensus on using a mise the spatial variation, we pooled and mixed