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The phylogenetic relationships within the Order Aplousobranchiata (Ascidiacea) are largely unexplored. In this work, we study the phylogenetic status of the genera Clavelina and Pycnoclavella. Traditionally, both genera had been included in the family Clavelinidae, until the new family Pycnoclavellidae was defined, removing the genus Pycnoclavella from Clavelinidae. Not all authors accept the validity of Clavelina and Pycnoclavella as distinct genera, let alone their belonging to different families. In addition, the assignment of species to these genera, as well as to the genus Archidistoma, has been controversial. We analyzed sequences of the mitochondrial gene cytochrome c oxidase subunit I belonging to ten species of Pycnoclavella (including several formerly assigned to Archidistoma and Clavelina), 11 species of Clavelinidae, and ten species of other aplousobranch genera belonging to seven families, plus two outgroups. Two different tree construction methods (maximum likelihood and Bayesian inference) showed similar results. Pycnoclavella and Clavelina appeared in distinct clades but formed a monophyletic group relative to representatives of the main families of the order Aplousobranchiata. Our phylogenetic results indicate that both genera are valid but should be included within a single family, with the name Clavelinidae having precedence. The monotypic clavelinid genus Nephtheis branches in our trees within the clade of the genus Clavelina. Our results also confirm that some forms assigned to Archidistoma and Clavelina have been misplaced and belong to the genus Pycnoclavella. Pycnoclavella martae n.sp. is described. [ABSTRACT FROM AUTHOR]