910 Int. J. Plant Sci. 174(6):910–936. 2013.  2013 by The University of Chicago. All rights reserved. 1058-5893/2013/17406-0006$15.00 DOI: 10.1086/670668 COMBINED MORPHOLOGICAL AND MOLECULAR PHYLOGENY OF THE CLUSIOID CLADE (MALPIGHIALES) AND THE PLACEMENT OF THE ANCIENT ROSID MACROFOSSIL PALEOCLUSIA Brad R. Ruhfel, 1, * Peter F. Stevens,† and Charles C. Davis* *Department of Organismic and Evolutionary Biology, Harvard University Herbaria, Cambridge, Massachusetts 02138, USA; and †Department of Biology, University of Missouri, and Missouri Botanical Garden, St. Louis, Missouri 63166-0299, USA Premise of research. The clusioid clade is a member of the large rosid order Malpighiales and contains ∼1900 species in five families: Bonnetiaceae, Calophyllaceae, Clusiaceae sensu stricto (s.s.), Hypericaceae, and Podostemaceae. Despite recent efforts to clarify their phylogenetic relationships using molecular data, no such data are available for several critical taxa, including especially Hypericum ellipticifolium (previously recognized in Lianthus), Lebrunia, Neotatea, Thysanostemon, and the second-oldest rosid fossil (∼90 Ma), Paleoclusia chevalieri. Here, we (i) assess congruence between phylogenies inferred from morphological and molecular data, (ii) analyze morphological and molecular data simultaneously to place taxa lacking molecular data, and (iii) use ancestral state reconstructions (ASRs) to examine the evolution of traits that have been important for circumscribing clusioid taxa and to explore the placement of Paleoclusia. Methodology. We constructed a morphological data set including 69 characters and 81 clusioid species (or species groups). These data were analyzed individually and in combination with a previously published molecular data set of four genes (plastid matK, ndhF, and rbcL and mitochondrial matR) using parsimony, maximum likelihood (ML), and Bayesian inference. We used ML ASRs to infer the evolution of morphological characters. Pivotal results. Our phylogeny inferred from morphology alone was poorly supported but largely in agreement with molecular data. Moreover, our combined analyses were much better supported and largely confirm taxonomic hypotheses regarding relationships of extant taxa newly included here. The extinct Paleo- clusia was placed as a member of stem group Clusiaceae s.s. or within crown group Clusiaceae s.s. as sister to one of its two major subclades. Conclusions. Despite poor overall bootstrap support for the placement of Paleoclusia, ancestral character state reconstructions are generally in agreement with our placements. Our recommendation is that Paleoclusia be treated as either a minimum stem group or a crown group age constraint of Clusiaceae s.s. Keywords: Clusiaceae, combined analysis, Guttiferae, morphology, Paleoclusia, rosids. Online enhancements: appendixes, figures, supplementary table. Introduction The clusioid clade belongs to the large angiosperm order Malpighiales (Ruhfel et al. 2011). It includes five families (Bon- netiaceae, Calophyllaceae, Clusiaceae sensu stricto [s.s.], Hy- pericaceae, and Podostemaceae; APG III 2009; Wurdack and Davis 2009; Xi et al. 2012) representing 89 genera (Ruhfel et al. 2011) and ∼1900 species (Stevens 2001–). Habitats and growth forms in the clusioid clade show extreme variation, from large tropical rainforest trees to diminutive aquatic plants of swift-flowing waterways. Their distribution is nearly cos- mopolitan, but species diversity is greatest in the tropics. The 1 Author for correspondence; current address: Department of Bio- logical Sciences, Eastern Kentucky University, Richmond, Kentucky 40457, USA; e-mail: brad.ruhfel@eku.edu. Manuscript received May 2012; revised manuscript received February 2013; electronically published June 7, 2013. clade is important ecologically and economically. Terrestrial members of the clade (i.e., all but Podostemaceae) are an im- portant component of tropical rainforests worldwide (Davis et al. 2005; CTFS 2009). Podostemaceae, on the other hand, are the largest strictly aquatic plant family (Philbrick and Nov- elo 1995; Cook 1996) and play a key ecological role in river systems via their interactions with fish and invertebrates (Allan 1995; Machado-Allison et al. 2003). Species from Calophyl- laceae, Clusiaceae s.s., and Hypericaceae are variously used in horticulture, tropical fruit, and timber production and in the pharmaceutical industry (Ernst 2003; Stevens 2007a, 2007b; Ruhfel et al. 2011). Recent molecular studies have sought to clarify relationships within the clusioid clade (Gustafsson et al. 2002; Wurdack and Davis 2009; Ruhfel et al. 2011; Xi et al. 2012). Ruhfel et al. (2011) produced the first well-resolved, taxon-rich phylogeny of the group. This study greatly improved our understanding of intrafamilial relationships within the clusioid families and in- This content downloaded from 140.247.0.42 on Thu, 20 Jun 2013 13:54:02 PM All use subject to JSTOR Terms and Conditions