Understanding the phylogenetic diversification of the water lilies (Nymphaeales sensu lato) in deep time is currently on the rise through studies based on both molecular analyses and the fossil record. An enhanced data set of leaf architectural and vegetative characters, for example, was found to be in broad agreement with phylogenies based only on molecular characters. Yet questions remain open regarding the relationships among the genera and subgenera of Nymphaea, the minimum time of divergence of familial and subfamilial clades and the phylogenetic radiation of the nymphaeoids during the Cenozoic that led to the living genera. Here we build on the aforementioned enhanced data set by adding a suite of nine, new or previously studied, Early to Late Cretaceous and Cenozoic fossil leaves to elucidate the phylogenetic positions of the fossil taxa, to test hypotheses based on molecular studies and to resolve phylogenetic relationships within Nymphaeales. In our phylogenetic analysis, five fossil leaf species show strong relationships with Nymphaeaceae and weaker subfamilial relationships to Nymphaea subgenus Lotos and to the Victoria—Euryale clade. Three fossil leaf species have strong relationships to Cabombaceae and are stem taxa. Taken as a whole, these new phylogenetic analyses yield strong evidence for the monophyly of the living families and some subfamilial clades. They also provide support for a divergence of Cabombaceae and Nymphaeceae by the Early Cretaceous. Finally, they suggest that some subfamilial clades of Nymphaeaceae may have originated by the Albian—Cenomanian, at the time of rapid diversification of the early angiosperms.
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