James R. Garey, Diane R. Nelson, Laura Y. Mackey, Jia Li.
Department of Biology, University of South Florida, 4202 East Fowler Avenue SCA 110, Tampa, FL 33620 U.S.A.;  Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614 U.S.A., and  Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, U.S.A.

Abstract:   The 18S rRNA gene sequences from two tardigrades were analyzed with previously published sequences from  four other tardigrades and a variety of  metazoans.  Phylogenetic analysis confirms that tardigrades are associated with arthropods and that 18S rRNA gene phylogeny is consistent with morphology-based hypotheses of tardigrade evolutionary relationships.  Thulinia and Hypsibius group together as sister taxa to Macrobiotus, forming the order Parachela, which in turn is a sister group to Milnesium, a representative of the order Apochela.  Together, Parachela and Apochela  form a sister group to Echiniscus, a heterotardigrade that appears in the analysis as the most basal tardigrade.

Fig.  1.  Neighbor Joining tree including six tardigrade species based on an alignment of 18S rRNA gene sequences. The tardigrades appear as a monophyletic sister group to the arthropods.  Branches are drawn to scale (substitutions/site). The same topology was recovered with Maximum Parsimony and Maximum Likelihood methods.  Numbers above each fork are bootstrap values for the Neighbor Joining tree, numbers to the right of each branch are confidence probability values for the Neighbor Joining tree (see methods), and numbers below each branch are bootstrap values for the Maximum Parsimony tree.  The mollusc Placopecten magellanicus was used as an outgroup.  The question mark at Hypsibius refers to a possible species identification error (see Discussion).

Fig.  2.  Topology of a portion of the molecular tree from Fig. 1 mapped with diagnostic morphological characters.  The molecular tree is completely congruent with current morphological hypotheses of tardigrade phylogeny. The question mark at Hypsibius refers to a
possible species identification error (see Discussion).  Bootstrap and confidence probability values are the same as in Fig.1.  Branches are not drawn to scale.  Key to Characters: 1. Arthropoda + Tardigrada: supraesophageal or preoral position of the frontal appendages and their neuromeres (Dewel & Dewel 1997).  2. Arthropoda: body with articulated exoskeleton; protocerebrum with compound eyes (Nielsen 1995).  3. Tardigrada: connective between protocerebrum and ganglion of first pair of legs (Dewel & Dewel 1997).  4. Heterotardigrada: Cephalic appendages, legs with digits and/or claws (Barnes & Harrison 1993).  5. Eutardigrada: Lack of cephalic appendages; legs with claws but not digits (Barnes and Harrison 1993).  6. Apochela: With cephalic papillae and with double claws with well-separated primary and secondary branches (Schuster et al. 1980).  7. Parachela: Without cephalic papillae and with double claws in which primary and secondary branches are joined (Schuster et al. 1980).  8. Macrobiotus:  Claw branches with sequence: secondary, primary, primary, secondary; buccal tube with ventral lamina and 10 peribuccal lamellae (Schuster et al. 1980).  9. Thulinia + Hypsibius:  Claw branches with sequence: secondary, primary, secondary, primary; buccal tube without ventral lamina (Schuster et al. 1980; Bertolani 1982).  10. Thulinia: twelve peribuccal lamellae (Bertolani 1982).  11. Hypsibius:  peribuccal lamellae absent (Schuster et al. 1980).