Molecular Analysis Supports a Tardigrade-Arthropod Association

Abstract

The phylogenetic placement of the phylum Tardigrada among the Metazoa is somewhat uncertain. In analyses based on morphological characters, tardigrades are usually associated with arthropods but tardigrades have also been associated with a number of aschelminth phyla. We have sequenced the nearly complete 18S rRNA gene from a eutardigrade. NeighborJoining and Maximum-Parsimony analyses place tardigrades in a clade that includes arthropods and priapulids, but not other aschelminths. Additional key words: 18S rRNA, phylogeny, evolutionary relationships, metazoa The phylum Tardigrada consists of over 700 species of microscopic metazoans commonly called water bears, known for their ability to undergo cryptobiosis. The body is most often less than a millimeter in length, divided into five indistinct segments, and has four pairs of legs, each usually terminating in claws and/or suction discs. Tardigrades occupy marine or freshwater sediments and bottom debris as well as the water films of soils and cryptogams (algae, mosses, liverworts). The two major classes, Heterotardigrada and Eutardigrada, are distinguished primarily on the basis of cuticular structures. Heterotardigrades include marine and armored terrestrial species, whereas eutardigrades consist mainly of the unarmored freshwater and terrestrial tardigrades. Marine heterotardigrades are considered to have the greatest number of plesiomorphic characters and therefore form the ancestral group of all other tardigrades (Kristensen 1987). The biology of tardigrades has been reviewed by Greven (1980), Bertolani (1982), Nelson (1982, 1991), Nelson & Higgins (1990), Dewel et al. (1993), and Kinchen (1994). The phylogenetic position of tardigrades among the Metazoa has long been debated. Embryological data are not definitive and the fossil record is poor. A fossil eutardigrade and juvenile heterotardigrade were found in Cretaceous amber (Cooper 1964), and fossil eutardigrade eggs were discovered in Quaternary travertine (Durante & Maucci 1972). Cambrian fossil lobopods such as Aysheaia show similarities to both Onychophora and Tardigrada, although Aysheaia is much larger than tardigrades. The lack of sufficient homologs in a To whom correspondence should be addressed. tardigrades has hindered the determination of evolutionary relationships of tardigrades to other taxa. New methods in microscopy and molecular biology provide tools that will aid in the analysis of metazoan phylogenies (Greven 1982; Hillis & Dixon 1991; Barnes & Harrison 1993; Raff et al. 1994). The addition of DNA sequence data provides valuable new criteria for evaluating phyletic relationships, but should be analyzed in light of extensive morphological evidence already available. Although Doyere (1840) assigned the name Tardigrada to the taxon and Ramazzotti (1962) recognized the group as a phylum in his first monograph, tardigrades have been associated through the years with various taxonomic groups, including gastrotrichs, nematodes, rotifers, annelids, onychophorans, and arthropods (mites, insects, crustaceans) (reviewed in Ramazzotti & Maucci 1982, 1983). The debate between the alignment of tardigrades with aschelminths versus arthropods has been a major point of discussion in recent years ( Kristensen 1991; Nelson 1991; Dewel et al. 1993; Kinchen 1994). Morphological data obtained from classical studies of anatomy and embryology and analyzed with cladistic methods have not yet resolved the position of Tardigrada within the met-