1Vouchers deposited in DUKE (or otherwise noted). Collectors: AG: A. Giraldo; CC: C. Castro; MAJ: M. A. Jaramillo; RC: R. Callejas, RE: R. Elke, ROG: R. O. Gardner, DU: Collected in the greenhouses at Duke University.
LEU1 or ITS 5 and ITS 4, sequencing primers were LEU1 or ITS 5, ITS 4, ITS 3, and ITS 2. Sequencing reactions were prepared using the ABI Prism Dye terminator Cycle Sequencing Reaction Kit, according to the protocols provided by the manufacturer. Double-stranded sequences were obtained in every case, sequences were assembled using Sequencher 4.1 (GeneCodes, Ann Arbor, Michigan), and aligned by eye using previous ITS data sets for Piper as a template (Jaramillo and Manos 2001).
A Bayesian phylogenetic analysis was carried outusing MrBayes 3.0 (Ronquist and Huelsenbeck 2003). The best model of molecular evolution was selected using Modeltest (Posada and Crandall 1998). The model preferred was HKY + I + G (HKY model of substitution, Hasegawa et al. 1985; I = proportion of invariable sites; G = gamma shape parameter). Four Markov Chain Monte Carlo chains were run, 1 topology was sampled every 100 generations for 1,000,000 generations, starting from a random tree. The first 9,100 generations, before convergence, were considered the burn-in period and were not included in generating the majority-rule consensus phylogeny. The forward slash is used here as a "clade-mark" as suggested by Baum et al. (1998) to distinguish provisional clade names from ranked taxonomic classification.
Phylogenetic Analysis of the /Ottonia Using Morphological Characters
The morphological data set used by Callejas (1986) was reexamined here. Phylogenetic analyses were carried out using PAUP* (Swofford 2002) and the maximum parsimony (MP) algorithm. The parsimony analysis was performed using heuristic searches with 100 random addition replicates, TBR swapping, MULPARS, steepest descent and AMB- options as implemented in PAUP* version 4.10 b. Relative branch support was determined using 1,000 bootstrap (BS) replicates (Felsenstein 1985) and a complete heuristic search.
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