Phylogenetic analyses based on nucleotide sequence data of the 12S and 16S mitochondrial ribosomal genes were performed for representatives of the Pleuronectiformes (flatfishes). Two parsimony analyses with percomorphs and basal acanthopterygians designated as outgroups were conducted; equal weighting of all nucleotides and loop regions analyzed for transversions only. A bayesian analysis was performed under the likelihood model TrN+I+Γ. A monophyletic Pleuronectiformes was resolved in all analyses. Results are consistent with the current higher level classification, recognizing the suborders Psettodoidei and Pleuronectoidei. Psettodoidei consists of a single family, Psettodidae, that is sister to all other flatfishes. Within the Pleuronectoidei three clades are identified. Bothids plus paralichthyids (in part) form a clade in all analyses. A solelike clade containing citharids, cynoglossids, samarids, soleids, Trinectes (Achiridae), and Poecilopsetta (Poecilsopsettidae), is identified in the parsimony analyses. However, a solelike clade was not recovered in the bayesian analysis. A flounderlike clade is identified containing Pleuronectidae and Paralichthyidae (in part) in all analyses. The position of Scophthalmus (Scophthalmidae) differed among all analyses. The results further support the conclusion that eye position is not a useful source of information for the classification of flatfishes. This study provides a new framework for future studies.

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