Piperaceae) Revealed by Molecules

Annals of Botany 99: 1231–1238, 2007 doi:10.1093/aob/mcm063, available online at www.aob.oxfordjournals.org From Forgotten Taxon to a Missing Link? The Position of the Genus Verhuellia (Piperaceae) Revealed by Molecules S. WANKE1 , L. VANDERSCHAEVE2 ,G.MATHIEU2 ,C.NEINHUIS1 , P. GOETGHEBEUR2 and M. S. SAMAIN2,* 1Technische Universita¨t Dresden, Institut fu¨r Botanik, D-01062 Dresden, Germany and 2Ghent University, Department of Biology, Research Group Spermatophytes, B-9000 Ghent, Belgium Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021 Received: 6 December 2006 Returned for revision: 22 January 2007 Accepted: 12 February 2007 † Background and Aims The species-poor and little-studied genus Verhuellia has often been treated as a synonym of the genus Peperomia, downplaying its significance in the relationships and evolutionary aspects in Piperaceae and Piperales. The lack of knowledge concerning Verhuellia is largely due to its restricted distribution, poorly known collection localities, limited availability in herbaria and absence in botanical gardens and lack of material suitable for molecular phylogenetic studies until recently. Because Verhuellia has some of the most reduced flowers in Piperales, the reconstruction of floral evolution which shows strong trends towards reduction in all lineages needs to be revised. † Methods Verhuellia is included in a molecular phylogenetic analysis of Piperales (trnT-trnL-trnF and trnK/matK), based on nearly 6000 aligned characters and more than 1400 potentially parsimony-informative sites which were partly generated for the present study. Character states for stamen and carpel number are mapped on the combined molecular tree to reconstruct the ancestral states. † Key Results The genus Peperomia is generally considered to have the most reduced flowers in Piperales but this study shows that this is only partially true. Verhuellia, with almost equally reduced flowers, is not part of or sister to Peperomia as expected, but is revealed as sister to all other Piperaceae in all analyses, putting character evolution in this family and in the perianthless Piperales in a different light. A robust phylogenetic analysis including all relevant taxa is presented as a framework for inferring patterns and processes of evolution in Piperales and Piperaceae. † Conclusions Verhuellia is a further example of how a molecular phylogenetic study can elucidate the relationships of an unplaced taxon. When more material becomes available, it will be possible to investigate character evolution in Piperales more thoroughly and to answer some evolutionary questions concerning Piperaceae. Key words: Verhuellia, Peperomia, Piper, Piperales, Piperaceae, character evolution, morphology, phylogeny, ancestral state reconstruction, stochastic character mapping. INTRODUCTION synonyms of the remaining species of Verhuellia. A detailed taxonomic discussion will be published elsewhere. Verhuellia, first described by Miquel (1843) in tribe Verhuellia has only been superficially studied. Only one Peperomieae, is a species-poor genus in Piperaceae, historical literature source describes some morphological known from very few collections and localities on Cuba characters in more detail (Schmitz, 1872a,b) and thorough and Hispaniola (Haiti and Dominican Republic). Besides anatomical studies are lacking. Tebbs (1993) synonymized Peperomia and Verhuellia, three other genera have been Verhuellia with Peperomia without much argumentation. included in this tribe (Acrocarpidium, Erasmia and In contrast, Saralegui Boza (2004) considered Verhuellia Phyllobryon; Miquel, 1852, 1868; de Candolle, 1869). as a separate genus, although discussion is lacking. Based on synapomorphies such as the unicarpellate ovary Phylogenetic analyses using molecular data have proved and the two disporangiate anthers, Acrocarpidium, useful in elucidating relationships of unplaced and mis- Erasmia and Phyllobryon have now been included in placed taxa, putting evolutionary trends in many plant Peperomia (Samain et al., 2007). This is also substantiated groups in a different light and often leading to new insights by molecular results (Wanke et al., 2006a). in generally accepted concepts (e.g. APG II, 2003). We use Miquel (1843) included three species in Verhuellia, this approach in Piperales to clarify the relationships of V. brasiliensis, V. elegans and V. serpens. As a consequence Verhuellia. During the last 5 years, several studies dedi- of the transfer of V. brasiliensis and V. serpens to Peperomia, cated to the phylogeny and evolution of Piperales or V. elegans has to be considered as the type of the genus. Piperaceae have been published (Jaramillo and Manos, Saralegui Boza (2004) correctly, but without argumentation, 2001; Jaramillo et al., 2004; Neinhuis et al., 2005; Wanke designated this as the type species. In total, nine Verhuellia et al., 2006a, 2007), but the relationships of Verhuellia species names have been published of which only two are were not discussed because it was not included. Hence, still accepted, V. elegans and V. hydrocotylifolia. All other its position remains unclear and needs reinvestigation species are now included in Peperomia or treated as based on molecular inference and a re-evaluation of * For correspondence. E-mail [email protected] morphological characters. # The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 1232 Wanke et al. — Affinities of Verhuellia Revealed by Molecules As reported by Jaramillo et al. (2004), the perianthless in SeqState (Mu¨ller, 2005). The alignment and the indel species of Piperales are being used as a model for examin- matrix are available from TreeBASE (www.treebase.org). ing floral development and evolution, because the simple Phylogenetic hypotheses were generated using maximum flowers facilitate ontogenetic studies and inferences parsimony and Bayesian inferences (as a basis for the about their evolution. With the exception of Manekia and ancestral state reconstruction). Phylogenetic reconstructions Verhuellia, the floral morphology and ontogeny of all using heuristic searches under maximum parsimony (MP) genera in this group have been studied comprehensively were performed using PAUP* 4.0b10 (Swofford, 2002) (Tucker, 1975, 1976, 1979, 1980, 1981, 1982a, 1982b, via a ratchet approach (Nixon, 1999) implemented in 1985; Liang and Tucker, 1989, 1990, 1995; Tucker et al., PRAP (Mu¨ller, 2004) for easy handling. The following 1993; Lei and Liang, 1998, 1999). ratchet settings were employed: ten random addition Piperales are one of the most species-rich and hetero- cycles of 500 iterations each with a 25 % of upweighting geneous clades in the magnoliids and include much varia- of the characters in the iterations. Evaluation of support Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021 bility in growth form and life history (Wanke et al., of the MP tree was performed using the bootstrap approach 2007) and morphological characters (inflorescence position, (Felsenstein, 1985), conducting 1000 replicates and random presence or absence of perianth, number of stigmas; e.g. addition searches with ten iterations per cycle, and with Igersheim and Endress, 1998; Doyle and Endress, 2000). decay values using PRAP in combination with PAUP* For interpretation of character evolution in Piperales, a and the same options in effect as for the ratchet. All par- robust phylogenetic analysis including all relevant taxa is titions of the datasets were analysed separately and in needed. The flowers of Verhuellia are among the most combination. reduced in the perianthless Piperales, although those of For Bayesian inference, the program MrBayes v3.1 Peperomia have been assumed to be the most reduced (Ronquist and Huelsenbeck, 2003) was used, assuming a (Jaramillo et al., 2004). As a consequence, the reconstruc- general time reversible model (GTR) and rate variation tion of floral evolution, showing strong trends towards among sites following a gamma distribution. The model reduction in all lineages of Piperales, needs to be revised. GTR þ G þ I was chosen as the one that best fits the data as determined using Modeltest v3.6 (Posada and Crandall, 1998) employing the interface MTgui (Nuin, 2005). MATERIALS AND METHODS Chains were sampled every ten generations and the result- Fewer than 30 herbarium collections of Verhuellia are ing trees were written to a tree file. Calculation of the available and most of these have been investigated for the consensus tree and the posterior probability (PP) of clades present study (B, BM, C, G, GH, K, NY, MA, S, U, US). was based upon the trees sampled after the chains Moreover, apart from one recently collected specimen of converged (25 %). Only PPs of 0.95 and higher were con- V. elegans, all of these specimens are at least 60 years sidered significant (alpha ¼ 0.05). Trees were compiled old. Due to their considerable age and their preservation, and drawn using TreeGraph (Mu¨ller and Mu¨ller, 2004). the DNA is likely to be severely fragmented and detailed Character states for stamen and carpel number were com- morphological and anatomical studies are seriously piled in a dataset using Mesquite v.1.11 (Maddison and hindered. Maddison, 2006) and mapped on the combined molecular DNA isolation followed methods described in Borsch Bayesian phylogeny with SIMMAP

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