Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada

To isolate individual tardigrades from their habitat substrate we used a non-toxic density gradient media that did not interfere with downstream biochemical processes. Using a simple DNA release technique and nested polymerase chain reaction with universal primers we were able amplify multi-copy and, to some extent, single copy genes from individual tardigrades. Maximum likelihood trees from ribosomal 18S, mitochondrial cytochrome oxidase subunit 1, and the single copy nuclear gene Wingless support a recent study indicating that the family Hypsibiidae is a non-monophyletic group. From these sequences we were able to detect variation between individuals at each locus that allowed us to identify the presence of cryptic taxa that would otherwise have been overlooked.Molecular results obtained from individuals, rather than pooled samples, are a prerequisite to enable levels of variation to be placed into context. In this study we have provided a proof of principle of this approach for meiofaunal tardigrades, an important group of soil biota previously not considered amenable to such studies, thereby paving the way for more comprehensive phylogenetic studies using multiple nuclear markers, and population genetic studies.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 [5], the implication being that in a handful of soil or sed