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The unusual neotropical freshwater catfish Lithogenes villosusEigenmann, 1909 was described from a single specimen collected in 1908 at Aruataima Falls, a cataract of the upper Potaro River of Guyana, as a new genus and species of the armored catfish family Loricariidae. There are no subsequent published reports of additional specimens. Lithogenes villosus is unusual among the armored loricariid catfishes in being nearly naked, or unplated. The dermal plates which typically encase the head and body of other representatives of the Loricariidae are restricted in Lithogenes villosus to three paired series of small plates on the posterior portion of the trunk, plus a set of three plates on either side of the lateral cheek. Its taxonomy has been debated largely because it is intermediate in morphology between the armored loricariids and the naked astroblepids; these two families are presently regarded as sister groups among the larger assemblage of neotropical loricarioid catfishes. Nijssen and Isbrücker (1987, Revue Française d'Aquariologie et Herpétologie 13: 93–98.) transferred Lithogenes villosus, without comment or justification, from the Loricariidae to the Astroblepidae, and several recent presentations have renewed the controversy surrounding Lithogenes relationships by asserting that Lithogenes is well supported as the sister group of the Astroblepidae. Lithogenes has not been subjected to a thorough systematic investigation because it is thus far known only from the holotype, a situation that precludes rigorous comparative anatomical survey via traditional, destructive methods. Given its unusual morphology and putative basal position among the loricariids, ultimate resolution of Lithogenes relationships is crucial to resolving several issues in siluriform systematics. In this study, three-dimensional reconstructions of computed tomography (CT) scans of the holotype made at 13-μm resolution revealed details of internal anatomy informative of its relationships. Parsimony analysis of 41 characters obtained from the CT reconstructions, gross examination of external features, and previously published characters surveyed among loricarioid and outgroup catfishes indicated that Lithogenes shares a rich suite of derived characters with loricariids, most notably the presence of a mesethmoid condyle, posterior placement of the nasal capsule, closed pterotic aperture, lateral ethmoid lamina, and metapterygoid contacting the lateral ethmoid, and is the sister group of all other loricariids, exclusive of astroblepids. Evaluation of this dataset revealed that support for alternative relationships was considerably worse. A sister-group relationship between Lithogenes and astroblepids occurred in less than 1% of 10,000 bootstrap replicate trees and required 7 additional steps relative to the shortest, most parsimonious tree. A sister-group relationship between astroblepids and loricariids, exclusive of Lithogenes villosus, was not observed among the bootstrap replicates; forcing this topological constraint required nine additional steps relative to the shortest tree. Although Lithogenes villosus shares several morphological similarities with astroblepids, most of these represent symplesiomorphy and are therefore not indicative of relationships.