Microscopic eukaryotes are abundant, diverse, and fill critical ecological roles across every ecosystem on earth, yet there is a well-recognized gap in our understanding of their global biodiversity. Fundamental advances in DNA sequencing and bioinformatics now allow accurate en masse biodiversity assessments of microscopic eukaryotes from environmental samples.Despite a promising outlook, the field of eukaryotic marker gene surveys faces significant challenges: how to generate data that is most useful to the community, especially in the face of evolving sequencing technology and bioinformatics pipelines, and how to incorporate an expanding number of target genes. Microscopic Eukaryotes: Global dominance, scant knowledgeMicroscopic eukaryotic taxa are abundant and diverse, playing a globally important role in the functioning of ecosystems [1,2] and host-associated habitats [3]. Here, we consider taxa generally represented by individuals <1mm in size; the term "microscopic eukaryotes" thus encompasses meiofaunal metazoa (e.g. Nematoda, Platyhelminthes, Gastrotricha and Kinorhyncha), fungi, microbial eukaryotes including deep protist lineages (Alveolata, Rhizaria, Amoebozoa, Algal taxa in the Chlorophyta and Rhodophyta, etc.), and eggs and juvenile stages of some larger metazoan species. These ubiquitous eukaryote groups play key roles as decomposers, predators, producers and parasites, yet we know little about their biology, ecology, and diversity. Analyses of eukaryotic community structure often reveal divergent lineages [4][5][6] and long lists of previously undiscovered sequences [7,8].© 2011 Elsevier Ltd. All rights reserved. * Corresponding Author: holly.bik@gmail.com.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptNematodes, for instance, account for 80-90% of all metazoa on Earth, yet less than 4% of the estimated >1 million species are formally known and described [9]. This discrepancy between known and estimated diversity is common for all microscopic eukaryote groups, and generally stems from the difficulty of applying traditional approaches in species identification to high-throughput sequence data. Traditional approaches, though wellvalidated, do not scale to the large numbers of sequences now being collected [6,[9][10][11][12].In many ways, the problems faced in the study of microscopic eukaryotes mirror those facing studies of archaea and bacteria. The exploration of archaeal and bacterial diversity long ago adopted a molecular taxonomy [13] -early uses of high...

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.