Pilocarpus

All three methods (Parsimony, Bayesian, Maximum Likelihood) confirmed that Pilocarpus species are monophyletic, and the genus is made up of two major clades (Fig. 2). These two major clades are geographically defined: Clade 1 (P. microphyllus Stapf ex Wardlew., P. trachyllophus Holmes, P. carajaensis Skorupa, P. jaborandi Holmes, P. racemosus Vahl, P. peruvianus (J.F. Macbr.) Kaastra) present in the tropical northern region of Brazil as well as Central America, and Clade 2 (P. grandiflorus Engl., P. riedelianus Engl., P. sulcatus Skorupa, P. spicatus A. St. -Hil., P. giganteus Engl., and P. pauciflorus A. St. -Hil. ) present in mid-coastal to southern regions of Brazil. In addition, these two clades are subtended by P. pennatifolius Lem., which is determined to be the first branch of the Pilocarpus clade according to both Bayesian and Maximum Likelihood estimates. However, the Parsimony analysis resulted in the placement of both P. pennatifolius and P. peruvianus as the first branch. This uncertainty can be confirmed in both the Bayesian and Maximum Likelihood analyses, which portray less certainty (via posterior probabilities and bootstrap support) when placing P. peruvianus at the base of Clade 1 (Clade with P. microphyllus) (Fig. 2). There has only been one other study on the phylogeny of Pilocarpus, and it was estimated using Bayesian methods and with the following genes: trn G-S, 5.8S, ITS 1 and 2 (Oliveira, 2008). In this study, P. peruvianus was placed at the base, and P. pennatifolius was nested within the monophyletic clade of Pilocarpus, as a sister clade of P. spicatus (Oliveira, 2008). For our comparative analyses we chose to use the branch lengths from our Bayesian phylogenetic tree, as we calibrated the tree using Ptelea fossils (Fig. 2c).