An Expanded Plastid Phylogeny of Marsilea with Emphasis on North American Species

Ferns of the genus Marsilea (water clover) are potentially invasive aquatic and wetland plants. They are difficult to identify to species because of subtle diagnostic characters, the sterile condition of many specimens, and unresolved taxonomic problems. We sequenced four plastid regions (rbcL, rps4, rps4-trnS spacer, and trnL-F spacer) from 223 accessions across ca. 38 species. Our goals were to: 1) attempt to identify problematic Marsilea specimens from the southeastern U.S., and 2) assess species delimitation using molecular data. Florida specimens previously identified as M. aff. oligospora do not match true M. oligospora (native to the western USA), and might represent an undescribed native species. The molecular data fail to resolve many species as monophyletic within the New World Marsilea section Nodorhizae. The data reveal two strongly supported clades within section Nodorhizae: 1) A western U.S./Mexican clade; and 2) A U.S. Gulf coastal plain/Florida/Caribbean clade. This DNA/morphology discordance suggests that these taxa either may have hybridized extensively or that the number of Marsilea species within these clades may be overestimated. Either case warrants the addition of nuclear data sets and reevaluation of the species boundaries within the genus.