One of the biggest impediments to conducting large-scale biodiversity surveys lies in the taxonomic identification of target organisms. This is especially true when dealing with microinvertebrates, where defining morphological features are often discernible only through intensive methods such as slide preparation and microscopy. One group of organisms that exemplifies this dilemma is the small-bodied crustacean class Ostracoda. Ostracods are very common in benthic freshwater communities, but also occur in marine, intertidal, or semi-terrestrial environments. They are useful model organisms for studies on various aspects of ecology and evolution [1–5], given the high prevalence of their calcified bivalve shells in the freshwater fossil record as well as their variability in breeding systems [6, 7]. In freshwater systems alone, the class Ostracoda has been conservatively estimated to number close to 2,000 described species , with 420 freshwater species recorded for North America [9, 10]. Taxonomic keys are available to the species level for North America and Europe [10–13], and many surveys describe the regional diversity of the class (e.g. [14–20]). The projected global diversity in all habitat types is estimated to be approximately 13,000 .
An infrequently discussed challenge in conducting biodiversity surveys is how to design and implement a suitable sampling strategy. While many studies have compared the efficacy of various field collection methods for capturing accurate estimates of planktonic invertebrate community structure [21–29], there has been little discussion of the idea of sampling strategy as a whole in terms of study objectives, sampling instrumentation, time commitments, adaptation of field methods in response to environmental heterogeneity, and sorting of samples prior to identification both in the field and in the laboratory. Given that the sample size of microinvertebrate community analyses is always much greater than the resources available to identify each individual organism to the appropriate taxonomic level, this sorting of organisms representing the sample community is of utmost importance. Previous studies have demonstrated the presence of cryptic species in microinvertebrates [30–32], and highlight the potential to overlook species with cryptic morphology as well as those with low abundance .
Establishing timeframes for microinvertebrate surveys can be linked to many different factors such as limited funding associated with fieldwork, appropriate weather windows for collection, and the availability of trained personnel. These limitations are especially applicable to studies conducted in remote locations, as well as areas of intense seasonality. Conducting fieldwork in these regions should be made as efficient as possible not only to limit associated costs, but to limit human interference on the natural system. Furthermore, while there is discussion in the literature on appropriate standards for comparing sampling strategies for freshwater bodies of various size and habitat diversity [23, 29], there is less discussion on the rationale behind intensive sampling. This is a key point as more scientists participate in public research, and more research projects involve an aspect of citizen science.
Citizen science involves collaboration between scientists and volunteers to gather field and observational data , and several studies have found that these types of collaborations produce reliable data that would be difficult to gather by any individual research group or scientist [35, 36]. For biodiversity studies, citizen science projects often encompass large-scale “bioblitzes” that involve collecting a large number of organisms in a short time period, often as short as a few hours (e.g. http://www.get-to-know.org/bioblitz/). Originally coined in 1996 by Susan Rudy of the U.S. National Park Service, the term bioblitz is now widely employed, with citizen-science bioblitzes recorded in countries such as Canada, New Zealand, Portugal, and Taiwan. The results of bioblitzes are typically not published in the scientific literature, despite their widespread occurrence and potential for inclusion . This may be changing, as demonstrated by the August 2012 issue of Frontiers in Ecology and the Environment, a special issue dedicated to the publishing of citizen-science research. For these sampling campaigns to remain an effective and efficient use of citizen-scientist collaborations, sampling strategies and specific objectives that may be served by these efforts should be discussed and evaluated. Here, we quantify and compare the outcomes of different student bioblitzes within the Churchill barcoding biotas campaign to measure collection effort in relation to biodiversity yield.
For animals, DNA barcoding using a region of the mitochondrial gene cytochrome c oxidase subunit I (COI) is an increasingly common method both for identifying species and for quantifying provisional species diversity [38–41], and can be used to evaluate and compare sampling strategies for biodiversity surveys. Through separating a sample of organisms into molecular operational taxonomic units (MOTUs), it is possible to calculate provisional species richness without the need for morphology-based identifications. DNA barcoding has been previously employed to build accumulation curves for understudied taxa such as parasitoid wasps (Ichneumonidae, Braconidae, Cynipidae and Diapriidae), with barcode-based accumulation curves indicating higher diversity, but the same shape, than accumulation curves built using morphospecies . While Linnean identifications are useful for community ecology studies, due to the possibility of linking with environmental data, the rapid quantification of biodiversity lends itself nicely to answering questions of species richness, species assemblage patterns, and sampling strategy comparison. As the reference library for the Barcode of Life Data Systems (BOLD)  grows, more of these unknown MOTUs will be linked to known species and allow for more sophisticated community ecology questions to be asked.
We employed DNA barcoding to compare five sampling strategies of subarctic freshwater ostracods in Churchill, Manitoba, Canada from 2007–2011, using MOTUs as surrogates for species. The present study does not test the effectiveness of DNA barcoding in recovering species boundaries for this group; rather, we use DNA barcoding as a tool to address our main study objective. We assume here that genetic patterns in the freshwater Ostracoda of Churchill mirror those of other microcrustaceans. For example, studies of the Branchiopoda of Churchill , freshwater microcrustaceans of Mexico and Guatemala , and marine zooplanktonic ostracods  have shown strong separation of described species based upon DNA barcodes.
This study presents an a posteriori analysis evaluating the success of five sampling strategies in both capturing and estimating the regional diversity of freshwater ostracods in the Churchill region, as this site was selected for an intensive “barcoding biotas” regional biodiversity survey employing DNA barcoding methods (introduced in ). Methodological differences among the sampling strategies prevent analysis into the effect of individual variables on strategy success, but still allow for broad-scale exploration of factors influencing the success of collection events at the scale of bioblitzes. The strategies differed in their primary objectives, duration of time spent sampling, number of sites sampled, and method of sorting of samples prior to analysis and deciding which samples to submit for DNA barcoding analysis. It was predicted that our comparison of these strategies would reveal differences in their effectiveness, yielding useful information for the design of future microinvertebrate surveys, with an emphasis on student or citizen bioblitzes. Previous studies [21–29] have compared sampling methods (e.g. tow nets, D-nets, hand nets) but did not use the same methods to measure or compare sampling effectiveness. By contrast, our study provides evidence that the rationale behind a sampling strategy is as important as the equipment used during bioblitzes (especially those with non-expert volunteers). We suggest that a focus on sampling diverse microhabitats is effective and that having two rounds of specimen selection for DNA barcoding will increase efficiency of molecular resource use for quantifying species diversity.