Evolution of the ascidian anural larva: Evidence from embryos and molecules

Most ascidians pass through a tadpole (urodele) larval stage, although some species have derived a tailless (anural) larva. New insights into the evol ution of anural larvae in the Roscovita clade of molgulid ascidians were ob tained from studing embryonic development of the transitional anural specie s Molgula bleizi and from phylogenetic analysis based on muscle and cytoske letal actin gene sequences. By observing in vitro fertilized eggs, we found that M. bleizi, previously described as a typical anural developer, actual ly forms a short immotile tail during embryogenesis. The short tail contain s notochord lineage cells, which undergo abbreviated morphogenetic movement s but eventually arrest in development. Molgula bleizi tail muscle lineage cells produce the muscle enzyme acetylcholinesterase (AChE) but do not expr ess muscle actin genes. The presence of a short tail, a vestigial notochord , and AChE-positive muscle cells suggest that M. bleizi is a recently deriv ed anural species. An M, bleizi larval muscle actin gene (MbMA1) was isolat ed, sequenced, and shown to be a pseudogene based on critical deletions in its coding region that would result in a nonfunctional actin protein. The m utations in MbMA1 are distinct from and have evolved independent of the lar val muscle actin pseudogenes MoccMA1a and MoccMA1b in Molgula occulta, anot her anural developer in the Roscovita clade. Pseudogene formation explains the absence of muscle actin mRNA in M. bleizi embryos. The 3' untranslated region of an M. bleizi cytoskeletal actin gene was also isolated and sequen ced, Phylogenetic trees reconstructed using muscle and cytoskeletal actin s equences suggest that the anural developer M. bleizi evolved prior to the d ivergence of the urodele developer Molgula oculata and the anural developer M. occulta in the Roscovita clade. Since M. bleizi lives attached to hard substrata in the tidal zone, whereas M. oculata and M. occulta live buried in subtidal sand flats, our results suggest that the anural larva evolved a t least twice in the Roscovita clade of molgulid ascidians as an adaptation to different habitats.