Abstract Geological evidence supports the occurrence of an epicontinental Trans-Saharan Seaway bisecting the African continent during the Late Cretaceous to early Paleogene. The seaway formed a wide saltwater channel connecting the Neotethys with the South Atlantic, yet no previous study has investigated its impact on freshwater fish diversification. Phylogenomic data and time-calibrated trees indicate a Late Cretaceous signature for the appearance of three modern lineages of characiform fishes. Phylogenetic analyses using ultraconserved elements of 83 characiforms reveals that Alestidae, Hepsetus, and Lepidarchidae fam. nov. originated during the Santonian-Campanian of the Late Cretaceous (84–77.5 million years ago; Ma). Lepidarchidae consists of two monotypic taxa not previously recognized as sister species: the Niger tetra Arnoldichthys endemic to the lower Niger and Ogun rivers of Nigeria, and the dwarf jellybean tetra Lepidarchus from coastal rivers of Ghana, Côte d'Ivoire, Liberia, Sierra Leone, and Guinea. Microcomputed tomography scans (µCT) of 117 characiforms provide three novel morphological characters supporting Hepsetus and Lepidarchidae, four characters for monophyly of Lepidarchidae and five for a restricted Alestidae. The Santonian-Campanian divergence indicates allopatric speciation processes influenced by the Trans-Saharan Seaway, partitioning the African ichthyofauna in a west-east orientation. The timing for African characiform cladogenesis aligns with the Cenomanian fossil record and is circa 16–23 Ma younger than the earliest characiform-like fossils from Late Cretaceous outcrops of Morocco and Sudan. This study highlights the magnitude of Cretaceous transgression events shaping the freshwater biota and gaps in our understanding of the evolutionary history and paleobiogeography of ray-finned fishes across the African continent.
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