GrayiaGünther 1858 is a genus of relatively large (1.2–2.5 m) aquatic Afrotropical snakes. Recent molecular phylogenies recovered Grayia in its own distinct subfamily (Grayiinae), which was supported as the sister group to Colubrinae. Tropical African snakes are generally understudied, so the relationships within Grayia are poorly known. High degrees of intra- and interspecies variation can make identification difficult, and previous studies involving Grayia included misidentified specimens in other genera. The goal of this study is to create a phylogenetic tree that can be used to understand the relationships and taxonomy of Grayia via an integrative taxonomic approach that combines molecular data for 60 specimens and morphological data for 719 specimens. Two nuclear (BDNF, NT3) and four mitochondrial genes (COI, cyt b, 16S, and ND4) were used to construct phylogenetic trees with Maximum Likelihood and Bayesian Inference algorithms. The phylogenetic trees recovered two clades, Grayia caesar + G. tholloni and G. ornata + G. smythii, which the time-calibrated Bayesian Evolutionary Analysis Sampling Tree (BEAST) analysis estimated to have diverged from each other in the mid-Oligocene. This deep divergence, combined with distinct morphological differences, led us to resurrect the name XenurophisGünther 1863 as a subgenus [G. (Xenurophis) caesar, G. (Xenurophis) tholloni]. Molecular and morphological evidence further supports a new cryptic species of Grayia from the Upper and Middle Congo River and its tributaries. This new species is estimated to have diverged from its nearest sister species, G. ornata, in the Late Miocene—which coincides with the divergence dates of sister taxa within other Central African snake genera. Grayia ornata sensu stricto was found to consist of several evolutionary lineages, which mirror the patterns recovered in other Central African vertebrates.

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