Assessing variation within and between species provides information relating to taxonomic relationships [1, 2], as well as population structure, demographics and biogeographic patterns [3, 4]. Microscopic animals that are collectively known as the meiofauna present a series of challenges to studies of variation. Despite their small size (generally captured in sieve mesh sizes between 45 μm and 1000 μm), meiofauna form an integral and vital component of the soil food web, playing a fundamental role in soil ecosystem processes, particularly in nutrient recycling and decomposition processes. Twenty of the 34 recognised animal phyla have meiofaunal representatives, 5 of which are exclusively meiofaunal , the implication being that in a handful of soil or sediment there is likely to be a high level of biodiversity. In more extreme environments, such as those of the Antarctic, meiofauna may constitute the majority of, and in some cases the only, metoazoan element present in the soil ecosystem [6–8]. Meiofauna are a focus of research from many sub-disciplines (including epidemiology, ecology, soil science, agriculture, aquaculture, and pollution monitoring), as well as providing model organisms for studies of evolutionary development (the nematode Caenorhabditis elegans) and the evolution of sex (bdelloid rotifers). These tiny animals are difficult to see, often smaller than the particulate matter that comprises the substrata they inhabit, and thus difficult to separate from sediment, detritus and non-target species. Within taxonomic groupings there may be few visible distinguishing characters, and those that are present may be subtle, requiring specialist and increasingly scarce taxonomic expertise . Nevertheless, accurate species identification is an essential first step to any scientific study.
Molecular phylogenetic techniques take advantage of developments in our ability to detect variation in DNA, effectively increasing the resolution available in comparison with morphological or phenotypic variation. This can be particularly useful when dealing with morphologically conserved groups. Each base change in a DNA sequence is analogous to a distinct morphological character in a phylogenetic analysis, which means many hundreds of characters can potentially be included in a single gene analysis. Models have been developed to take rates of evolution and homoplasy into account [10, 11] providing molecular phylogenetic inference with a robust theoretical basis . The concept that variation in a suitable and defined length of DNA sequence may be used as a bar code for species identification is becoming increasingly appreciated [13, 14].
An important advantage of molecular data is that independent replication is possible. A phylogeny based on a single gene provides information regarding the history of and relationships between the taxa sampled. However, the information is limited to the occurrence of mutations and results in what is known as the genetree/species tree problem . A significant event in the history of a lineage will only be recorded in a phylogeny if a mutation occurs at or just after the event. Conversely a high mutation rate may lead to a confused signal in cases where, for instance, an informative mutation reverts back to its ancestral state, or further changes to a state present in another lineage (homoplasy). Furthermore, there are processes, such as selection, that may affect the evolution of a gene, thereby confusing the signal of evolutionary history of the organism. These caveats may be at least partly countered by conducting independent analyses on several unlinked genes in order to generate a general consensus of phylogenies that more closely reflects the "true" evolutionary history, or by concatenating sequences to produce a super phylogeny .
It is often not appreciated that transferring molecular techniques from the few "model" organisms that are the focus of intense laboratory research to the more numerous but less studied groups is not straightforward. Conversely, molecular ecologists who work on meiofauna and other invertebrates may be surprised when they find their difficulties are not appreciated by the wider scientific community. DNA quantity is a major issue with tiny organisms. To increase DNA yield, the whole organism or, more often several pooled individuals, are used in extractions and, thus, problems with contaminants from gut contents or commensal organisms arise [17, 18]. The difficulties working with understudied species are often only evidenced by the lack of published literature available. For example, as far as we are aware, there have only been three assessments made of intraspecific variation in any species of the meiofaunal phylum Tardigrada [19–21]. This may be partially due to tardigrades and other meiofaunal groups falling outside economic and charismatic categories that influence the direction of science programs, but it is also likely to be due to the difficulties involved in obtaining genetic data from these members of the meiofaunal community. Recently a variety of techniques have been developed or demonstrated as suitable for DNA extraction from a variety of meiofauna [2, 22–25] providing a basis for basic evolutionary studies and the development of molecular tools for identification or classification [20, 22, 23, 26].
Tardigrades are found in most terrestrial, freshwater and marine habitats, including some remote Antarctic nunataks where they are found in the absence of the otherwise ubiquitous nematodes . They are one of the few phyla with representatives found from the highest and coldest terrestrial environments to the deepest oceans. They have featured in high impact publications mostly due to their uncertain phylogenetic position in the tree of life [27–30], although their ubiquitous distribution makes them ideal candidates for historical biogeographic reconstruction [31–33]. Molecular phylogenetic work to date is strongly concordant with morphological based systematics , supporting both tardigrade monophyly and monophyly in the constituent classes Eutardigrada and Heterotardigrada [17, 24, 34].
In this paper we describe a method that enables the quantitative and qualitative assessment of morphology and genetic variation among individual tardigrades. The method includes a novel technique for separating all organisms from their substrate as compared with "cherry-picking" visible organisms under a dissecting microscope, a simple DNA release technique applicable at the individual level, and a general protocol for amplifying genomic DNA from multicopy and single copy genes. Previous studies have amplified multi-copy 18S and high-copy number (CO1) genes from tardigrades [2, 20, 25, 26], but this is the first time single copy nuclear genes have been amplified from genomic DNA extracted from a single tardigrade. We demonstrate that this method allows for informed re-assessment of morphological variation, allows independent replication of phylogenetic analyses and is suitable for assessing within population variation enabling population genetic studies.