The Marsileaceae is a small family of semi-aquatic ferns displaying numerous traits commonly observed in angiosperms, including heterospory, sophisticated hydraulic architecture, and high rates of atmospheric gas exchange. Despite these similar traits, Marsileaceae is comparatively ecologically limited. Most species are found in Marsilea which is sister to Regnellidium and Pilularia, together these two genera include only seven species. Here we studied the anatomy and physiology of Marsileaceae to better understand the potential constraints on ecological and species diversity in this family. We focused on epidermal anatomy and stomatal responses to changes in light and water availability, which are unique amongst ferns. We found two evolutionary strategies in Marsileaceae, one of morphological simplification, physiological inflexibility, and aquatic specialization in Pilularia; which contrasts with a strategy of maximizing photosynthetic carbon gain at the expense of high rates of water loss in Marsilea and Regnellidium. We conclude that aquatic environments provide evolutionary opportunities for physiological innovation with regard to stomatal function, as well as selective pressures that drive the canalized evolution of highly specialized aquatic forms.