The North American topminnows and killifishes in the Family Fundulidae (Cyprinodontiformes) are widely distributed in freshwater, brackish, and coastal marine environments of North America, the Yucatan Peninsula, and Bermuda. Fundulid fishes are often found in habitats that undergo substantial variation in environmental conditions. Salinity tolerance of fundulids varies substantially, and some estuarine species can survive in water that is more than three times marine salinity, whereas other freshwater species can only tolerate up to one-third marine salinity. While the Family Fundulidae has been the focus of numerous phylogenetic investigations, their evolutionary relationships to other cyprinodontiforms and the evolutionary history of species within the family are in need of further investigation with robust taxonomic sampling. In this study we provide the most data-inclusive current hypothesis of evolutionary relationships for the Fundulidae, based on a combination of morphological, karyological, behavioral, and nucleotide (two mitochondrial and two nuclear genes) data. We revise the classification of the family based on this evolutionary framework. Our results indicate that the Family Fundulidae is monophyletic, and the sister group to a clade composed of the Cyprinodontidae, Profundulidae, and Goodeidae within the Cyprinodontiformes. We recognize three genera, Leptolucania, Lucania, and Fundulus, and synonymize Adinia with Fundulus. We recognize four subgenera within genus Fundulus: Plancterus, Zygonectes, Fundulus, and a newly described subgenus containing the two Pacific-coast Fundulus species. Ancestral-state reconstructions of salinity tolerance indicate that there may have been as many as five independent shifts from high to low tolerance within the family, and that salinity tolerance evolution likely was an important factor in the biogeographic history of Fundulidae, shaping current fundulid distributions.
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