Morphological, Phylogenetic, and Ecological Analyses of the New Model Grass Species Setaria viridis (Poaceae) and its Close Relative Setaria faberi

Species limits as defined by herbarium taxonomists are nearly always based on intuitive morphological comparisons with little concrete data or statistical analysis. Setaria viridis (tribe Paniceae, subtribe Cenchrinae), an emerging model organism for the study of C4 photosynthesis, is one such inadequately defined species. In order to evaluate its relationship with the morphological intergrading Setaria faberi, a “total data” approach was taken. Statistical morphology, cytology, molecular phylogenetics, and growth experimentation were employed to examine the putative species boundary in this group. Principal components analysis of 70 morphological characters in 85 individuals revealed consistent separation between the two species in morphospace, largely driven by spikelet characters. Flow cytometry demonstrated that Setaria viridis is consistently diploid, while S. faberi is consistently tetraploid. Phylogenetic analysis of the nuclear gene knotted1 (kn1) showed that one of the two kn1 paralogues in S. faberi is identical to its orthologue in S. viridis, while the other S. faberi paralogue is only slightly differentiated. This suggests either an autopolyploid origin, or an allopolyploid origin resulting from hybridization between S. viridis and an unsampled but closely related taxon. Finally, a controlled drought stress experiment showed that drought induces morphological effects on either species, but not to the extent that the species cannot be readily differentiated