BACKGROUND: Myxozoans are metazoan parasites whose traditional spore morphology-based taxonomy conflicts DNA based phylogenies. Freshwater species of the genus Hoferellus are parasites of the excretory system, with several members infecting food and ornamental fish species, as well as amphibians. This study aims to increase our understanding of their molecular diversity and development, aspects about which little is known, and to generate a molecular diagnostic tool to discriminate between different pathogenic and non-pathogenic Hoferellus spp. METHODS: SSU and ITS rDNA phylogeny, along with morphological descriptions using light and electron microscopy were used to identify and characterize Hoferellus species collected from the urinary system of fishes and frogs. A PCR-based diagnostic assay was designed to differentiate between cryptic Hoferellus spp in cyprinid fishes commonly cultured in Central Europe. RESULTS: Our phylogenetic results separate the species of Hoferellus into two phylogenetic sublineages which are indistinguishable on the basis of generic morphological traits: 1) The Hoferellus sensu stricto sublineage, which is composed of the type species Hoferellus cyprini, Hoferellus carassii and a cryptic species, Hoferellus sp. detected only molecularly in common carp. 2) The Hoferellus sensu lato sublineage into which the new species we described in this study, Hoferellus gnathonemi sp. n. from the kidney of the elephantnose fish and Hoferellus anurae from reed frogs, are placed together with Hoferellus gilsoni previously sequenced from European eel. Apart from phylogenetic analyses, we also provide novel ultrastructural data on the phagocytotic nature of some Hoferellus plasmodia and on the elusive intracellular stages ascribed to the presporogonic development of this genus. CONCLUSIONS: We provide molecular evidence of the polyphyly of the genus Hoferellus and provide novel morphological details of its members. Based on the presented data, we revise and propose emendation of the genus Hoferellus.

Institute of Parasitology Biology Centre of the Czech Academy of Sciences České Budějovice 37005 Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences České Budějovice 37005 Czech Republic Marine Zoology Unit Cavanilles Institute of Biodiversity and Evolutionary Biology Science Park University of Valencia Paterna 46980 Spain Department of Microbiology Oregon State University Corvallis OR 97331 USA

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