A DNA barcoding approach in the study of tardigrades.

DNA barcoding is a technique proposed by Hebert and coworkers in 2003 and it aims to discriminate biological entities through analysis of a single gene barcode locus. The DNA barcoding system promised a better taxonomic resolution than that achieved through morphological studies, with a partial solution to the decline in taxonomic knowledge. Today DNA barcoding is a global enterprise, and the implementation of the idea has seen a rapid rise (more than 450 papers published to date on different organisms). Nonetheless, controversy still arises regarding barcoding and taxonomy. It is important to note that DNA barcoding does not focus on building a tree-of-life or on doing DNA taxonomy, even though sometimes it has been used for these purposes. DNA barcoding rather focuses on producing a universal molecular identification key based on strong taxonomic knowledge that should be included in the barcode reference library. In Phylum Tardigrada, DNA barcoding represents a recent approach to species identification and for helping to solve taxonomic problems, especially considering the diminutive size of these animals and the paucity of morphological characters useful for taxonomy. In the framework of the MoDNA Project (Morphology and DNA), carried out by our research group in collaboration with several colleagues, we are combining the study of a fragment of the mitochondrial cytochrome c oxidase subunit I gene (cox1) with morphological data, in a wide sense, to form an integrative taxonomy of tardigrades. Building of a database of reference sequences is of paramount importance for a correct application of DNA barcoding in tardigrades. Without verified reference sequences from voucher specimens that have been authenticated by qualified taxonomists, there is no reliable library for newly generated sequences with which to be compared. Methods and protocols for standardized results are focused on obtaining tight correspondence between molecular sequence and animal morphology, possibly both LM and SEM images (and egg shell morphology, when useful). This approach is particularly useful in describing new species, and important when applied on material collected in type localities. Results using this approach are presented, focusing primarily on a number of species from the so-called “Macrobiotus hufelandi group”.