The role of appendicularians in chordate evolution – a phylogenetic analysis of molecular and morphological characters, with remarks on ‘neoteny-scenarios’ Thomas Stach 1 and James M. Turbeville 2 1 Royal Academy of Sciences, Kristineberg’s Marine Research Station, 45034 Fiskebäckskil Sweden 2 Department of Biology, Virginia Commonwealth University, 1000 West Cary Street, P. O. Box 842012, Richmond, VA 23284-2012 USA Abstract Two major hypotheses regarding the evolution of Appendicularia exist. Garstang (1928) suggested that appendicularians evolved from a doliolid-like ancestor by way of neoteny. In this hypothesis appendicularians present an analogous model for the evolutionary steps from a sessile ascidian-like tunicate to the free-living ancestor of the notochordates. An alternative proposition, supported by sperm ultrastructural features and preliminary analyses of tunicate 18S sequences, suggests that appendicularians are primitively free-living. In the present study, we included 18S rDNA sequences from three families of Aplousobranchiata and found support for a third hypothesis. Analyses of this extensive molecular data set suggest that Appendicularia are nested within the ‘Ascidiacea’. The most probable phylogenetic position of Appendicularia is as the sister taxon to Aplousobranchiata. An additional phylogenetic analysis of traditional taxonomic characters did not contradict this interpretation and a simultaneous analysis of morphological and molecular data found weak support for this sistergroup relationship between Appendicularia and Aplousobranchiata. Potential synapomorphies of Appendicularia with aplousobranch ascidians include the horizontal orientation of the larval tail and the tendency toward accelerated ontogenetic development. Cambrian fossils can be interpreted as aplousobranch ascidians and possibly appendicularians. Thus, the fossil record agrees with the suggested phylogenetic hypothesis. Introduction More than a century after Kowalevsky (1866) demonstrated that ascidians had a larval stage that clarified their chordate affinities, tunicate phylogeny remains highly controversial. For example, Lacalli (1999; Fig. 1) suggested a radically new perspective on chordate evolution.