Soil Biology & Biochemistry 41 (2009) 849–857 Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio Molecular profiling of soil animal diversity in natural ecosystems: Incongruence of molecular and morphological results Tiehang Wu a, Edward Ayres b, Grace Li b, Richard D. Bardgett c, Diana H. Wall b, James R. Garey a,* a Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA b Department of Biology and Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523, USA c Soil and Ecosystem Ecology, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK article info abstract Article history: A major problem facing ecologists is obtaining a complete picture of the highly complex soil community. Received 2 October 2008 While DNA-based methods are routinely used to assess prokaryote community structure and diversity in Received in revised form soil, approaches for measuring the total faunal community are not yet available. This is due to difficulties 24 January 2009 such as designing primers specific to a range of soil animals while excluding other eukaryotes. Instead, Accepted 2 February 2009 scientists use laborious and specialized taxonomic methods for extracting and identifying soil fauna. We Available online 25 February 2009 examined this problem using DNA sequencing to profile soil animal diversity across two Alaskan ecosystems and compare the results with morphological analyses. Of 5267 sequences, representing 549 Keywords: Biodiversity operational taxonomic units (OTU), only 18 OTUs were common to both sites. Representatives included 8 Soil phyla, dominated by arthropods and nematodes. This is the most comprehensive molecular analysis of DNA sequence soil fauna to date, and provides a tool to rapidly assess a missing component of soil biodiversity. Community structure Ó 2009 Elsevier Ltd. All rights reserved. Phylogenetic analysis Morphological analysis 1. Introduction Where soil animal biodiversity has been studied at high resolution (i.e. species level) it has been restricted to a limited number of There is growing interest in assessing soil animal biodiversity ecosystems and/or taxonomic groups (Bardgett, 2005). This dearth (Wall et al., 2001; Fitter et al., 2005), largely because soils are of information results partly from a lack of methods to rapidly and recognized as one of the most species rich animal habitats on Earth easily measure soil animal biodiversity at high resolution (Behan- (Behan-Pelletier and Newton, 1999; Wardle, 2002). For example, up Pelletier and Newton, 1999). to 89 nematode species were found in <90 cm3 soil from a tropical Current methods for characterizing animal biodiversity in soils forest in Cameroon (Bloemers et al., 1997) and 159 mite species are based on traditional morphological identification. Thus, they were found in 579 cm2 soil in a grassland in Kansas (St John et al., require specialist knowledge for each taxonomic group (e.g. 2006a). These soils have many additional invertebrate species that termites, mites, nematodes, rotifers, tardigrades etc.), making were not identified. Moreover, since soil animals and their inter- identification extremely labor intensive. For example, six person- actions with micro-organisms mediate many ecosystem properties years were required to identify individuals of one soil animal group and processes, including decomposition, nutrient cycling, carbon (mites) to the species level in a single experiment conducted at one sequestration, plant community dynamics, and maintenance of soil grassland site in Kansas, USA (St John et al., 2006b). In addition, due structure, an understanding of their diversity is required to main- to the large range in body size and natural history of animals in tain ecosystem services from which humans benefit (Wall, 2004). soils, different extraction methods are needed for different animal Despite the vast diversity of animals in soil, most biodiversity groups, precluding the identification of all animal species from research has focused on aquatic and aboveground organisms individual soil samples. Finally, the challenge of identifying soil (Behan-Pelletier and Newton, 1999; Wardle, 2002) and little is animal species morphologically is compounded by the fact that known about factors that influence patterns of biodiversity in soils most species are undescribed (Behan-Pelletier and Newton, 1999). at local, regional, or global scales (Ettema and Wardle, 2002). Thus, molecular methods that rapidly assess ‘‘species’’-level diversity of a range of animal taxa from a single soil sample could potentially be an important tool in the study of soil biodiversity. * Corresponding author. Tel.: þ1 813 974 7103; fax: þ18139741614. Molecular techniques for the study of soil biodiversity (typi- E-mail address: [email protected] (J.R. Garey). cally microbial), such as denaturing gradient gel electrophoresis, 0038-0717/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2009.02.003 850 T. Wu et al. / Soil Biology & Biochemistry 41 (2009) 849–857 terminal restriction fragment length polymorphism and phos- From each plot, we collected 20 soil cores (3.4 cm diameter; 10 cm pholipid fatty acid analysis, provide only a broad overview of deep), which were bulked for a single soil sample for each plot. diversity due to their relatively low level of resolution; in contrast, sequencing provides a detailed measure of biodiversity 2.2. Molecular methods at a resolution analogous to species level (O’Brien et al., 2005). Today, microbial ecologists often study diversity by extracting The bulked samples were processed to produce the sieve frac- bulk DNA from soil, sediment or water, amplifying the bacterial tions. Within 24 h of collection, 200 g of fresh soil sample from each 16S rRNA gene and constructing a sequence-based clone library of the four plots at each site was hand-mixed and added to 500 ml (Chen and Pachter, 2005; Tringe et al., 2005). However, due 95% ethanol which acted as a suspension medium and a preserva- primarily to a lack of primers specific to a range of animals, this tive. The soil–ethanol suspension for molecular analysis was mixed, approach has been limited to relatively small datasets (100– left to stand for 2 h, and remixed prior to washing through a series 1000 sequences) utilizing fungi (O’Brien et al., 2005), algae and of progressively finer sieves (mesh sizes: 2 mm, 1 mm, 0.5 mm, and protists (Lawley et al., 2004; Lara et al., 2007), nematodes (Floyd 0.1 mm) to separate animals of different body size. The material on et al., 2002; Griffiths et al., 2006) or mixed microeukaryotes (Fell the 1 mm, 0.5 mm and 0.1 mm was collected and stored in 95% et al., 2006). Our study reports a dataset of more than 5000 ethanol, resulting in 12 samples from each of the two sites. metazoan sequences from two sites, each represented by four Metazoan specific primers for the 18S rDNA gene were designed replicates. from an alignment of 80 species of eukaryote rDNA sequences from We developed a primer to amplify the 18S rRNA gene (rDNA) GenBank including 69 sequences representative of metazoan phyla from a broad array of animals and to exclude other eukaryotes and 11 sequences representing non-metazoan phyla. The forward (plants, fungi, and protozoa) and bacteria. We used the eukary- primer 18S11b (50-GTC AGA GGT TCG AAG GCG-30) corresponds to otic 18S rDNA gene as a target because it is a homologue of the positions 1037–1054 of the human sequence (NR_003286 in Gen- bacterial 16S rDNA gene, is widely used for phylogenetic study Bank) and to a region that is relatively constant among metazoans, (Blaxter et al., 1998; Griffiths et al., 2006), and there are existing but has positions that vary considerably in other eukaryotes. The databases (e.g. GenBank) related to this gene (Cole et al., 2003). reverse primer 18S2a (50-GAT CCT TCC GCA GGT TCA CC-30) corre- The 18S rDNA gene has some highly conserved regions used to sponds to positions 1848–1867 of the human sequence. The discern deep phylogenetic relationships at the phylum level, primers amplify approximately a 830 bp segment. while other regions vary at the species level. Although full- DNA was extracted from each sample using a modified CTAB length 18S rDNA sequences have been used extensively in extraction procedure (Gawel and Jarret, 1991). Metazoan-specific metazoan phylogenetics, the short sequences used in this paper primers amplified the 18S rDNA (an initial 2 min denaturing step at are not expected to reconstruct metazoan phylogeny. Partial 18S 95 C, followed by 40 cycles of denaturation at 95 C for 15 s, rDNA sequences have been used in barcoding (Floyd et al., 2002) annealing at 55 C for 30 s, extension at 72 C for 1 min). PCR and thus can be used to identify operational taxonomic units products were cloned into libraries using Topo TA cloning kits (OTUs) and can discriminate among even closely related species. (Invitrogen, Carlsbad, CA) and approximately 200 single pass These partial 18S rDNA sequences can also be used to identify sequences (519 base pairs after trimming) were obtained from each the closest match in GenBank that can provide some degree of library using a commercial sequencing facility. The animal-specific taxonomic information. Some studies have shown a congruence primers minimized amplification of plant, protozoa, fungi or between morphological and molecular analysis in simple bacteria DNA. systems (Eyualem and Blaxter, 2003). We utilized a molecular Raw sequences were processed with custom software devel- approach to assess soil animal diversity in two contrasting arctic oped in our lab that finds a highly conserved region of the 18S ecosystems in Alaska: a boreal forest and arctic tundra. We also rDNA sequence as an anchor point and then trims upstream and identified soil animals morphologically and compared the downstream from that point.