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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 () 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 , 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 -poor and little-studied genus Verhuellia has often been treated as a synonym of the genus , downplaying its significance in the relationships and evolutionary aspects in Piperaceae and . 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 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, , 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 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 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 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 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 , 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 v.1.0 Beta 2.1 et al. (2003). Specimens used in the phylogenetic study (Bollback, 2006), a program implementing stochastic char- are listed in Table 1. The trnT-trnL sequences were acter mapping (Nielsen, 2002; Huelsenbeck et al., 2003). largely generated for the present study, whereas those for The morphological characters were set with equal priors the trnL-F and trnK/matK were mostly taken from on the bias parameter [p(0) ¼ 0.5 and p(1) ¼ 0.5] and Neinhuis et al. (2005) and Wanke et al. (2007). The trnK/ without priors on the rate parameter, i.e. using branch matK region was generally amplified in two parts with an lengths as a rate for the occurrence of morphological change overlap of 250–400 bp, using the primers listed in among taxa (Schultz and Churchill, 1999; Huelsenbeck Table 2 as described by Wanke et al. (2007). Similarly, et al., 2003). the trnT-F region was also amplified in two parts, with a minimal overlap in the 50 trnL gene as described by Neinhuis et al. (2005) for the trnL-F region. In some species, the trnK/matK and the trnT-F regions were ampli- RESULTS fied in three parts due to long insertions of AT-rich micro- Data sets and tree statistics satellites. After gel electrophoresis, the PCR products were purified using a gel extraction kit (Macherey-Nagel). Direct Because of uncertain homology, eight mutational hotspots sequencing used the CEQ DTCS Quick Start Kit (Beckman were excluded from the trnK/matK matrix and nine from Coulter) with a CEQ 8000 sequencer, following standard the trnT-L-F matrix (Table 3), mostly mononucleotide protocols for each kit. repeats (plastid microsatellites). Within the trnK/matK Analyses were based on manually aligned sequence data, dataset, seven hotspots were observed within the 50 trnK guided by analysis of microstructural changes, as performed intron and one in the 30 trnK intron. For the trnT-L-F by Borsch et al. (2003) or Wanke et al. (2007). An indel region, four hotspots were excluded from the trnT-L matrix was prepared using the ‘simple indel coding’ (SIC) spacer, three from the trnL intron and two from the approach (Simmons and Ochoterena, 2000) as implemented trnL-F spacer. TABLE 1. Taxa used in the present study, including the origin of the material studied (field or collection), voucher information and the herbarium where the voucher is deposited, as well as GenBank accession numbers (including source), are given

GenBank accession number Family Species Origin Voucher (herbarium) trnK/matK trnT-L trnL-F Downloaded fromhttps://academic.oup.com/aob/article/99/6/1231/2769300bygueston28September2021

Aristolochiaceae albida Duch. BG Bonn, 17419 Neinhuis 92 (DR) DQ5320644 EF422813 AY6891536 Linden BG Bonn, 02560 Neinhuis 93 (DR) DQ5320444 EF422810 AY6891756 Aristolochia eriantha Mart. & Zucc. BG Bonn, 12952 Neinhuis 99 (DR) DQ5320544 EF422812 AY6891636 L. France, Cassis, Calenque d’en Veau leg. Kreft, Wanke 037 (DR) DQ5320625 EF422814 DQ5320245 Mart. & Zucc. BG Bonn, 02099 Neinhuis 101 (DR) DQ8821874 EF422811 AY6891656 yakusimense Masam. BG Bonn, 14276 Neinhuis 91 (DR) DQ8821971 EF422808 AY6891506 Wanke henryi Oliver BG Bonn, 02618 Neinhuis 120 (DR) DQ5320334 AY1453407 AY1453407 siliquosa (Lamk.) Ding Hou , Kerala (BG Bonn, 09037) Neinhuis 121 (DR) DQ5320354 EF422809 AY6891516

Lactoridaceae fernandeziana Phil. Chile, Masatierra Island (Juan Fernandez) Crawford & Stuessy 11950 DQ8821954 AY1453247 AY1453426 — al. et Piperaceae Manekia naranjoana (C.DC.) Costa Rica O. Vargas s.n. (DUKE) DQ8822394 EF422821 EF422821 Callejas Manekia sydowii (Trel.) Arias, Columbia, Antioquia MAJ038 (DUKE) DQ8822384 EF422820 EF422820 Callejas & Bornstein of Affinities Peperomia gracillima S. Watson BG Bonn, 06005 Wanke 060 (DR) DQ2127163 EF422825 EF422825 Peperomia graveolens Rauh & Ecuador, El Oro Rauh & Barthlott 35122 (HEID) DQ2127223 EF422826 EF422826 Barthlott Peperomia maypurensis Kunth BG Bonn, 11132 Wanke 006 (DR) DQ2127353 EF422827 AY6891466 Peperomia pitcairnensis C.DC. BG Bonn, 17744 Wanke 007 (DR) DQ2127623 EF422828 AY6891456 Verhuellia Peperomia marmorata Hook. f. BG Bonn, 17527 Wanke 064 (DR) DQ2127253 EF422829 EF422829 Piper angustum#1 Rudge Miss. Bot. Gard. Acc. 910150 – EF422824 EF422824 Piper betle#2 L. BG Cologne Neinhuis s.n. (DR) – EF422823 EF422823 Piper cf. magnificum#2 Hort. ex BG Bonn, 05020 Wanke 069 (DR) DQ8822094 Gentil #1 N.E.Br. BG Bonn, 18144 Wanke 005 (DR) DQ8822114 – Molecules by Revealed Piper sp. BG Bonn, 00854 Borsch 3475 (BONN) DQ8822254 AY1453467 AY1453467 Verhuellia elegans Miq. Dominican Rep., Sierra de Bahoruco Jime´nez & Garcı´a 3560 (GENT) coll. 24.12.2003 EF422831 EF422819 EF422819 Verhuellia sp. Haiti, Massife du Nord Ekman 8928 (US, GH), coll. 29.05.1927 EF422830 EF422818 EF422818 Zippelia begoniifolia Blume BG Kunming, s.n. Wanke & Neinhuis s.n. (DR) DQ8822304 EF422822 EF422822 californica (Nutt.) Hook. BG Bonn, 06422 Wanke 002 (DR) DQ8821984 EF422817 AY6891426 &Arn. Gymnotheca chinensis Decne. BG Bonn, 17072 Wanke 004 (DR) DQ8821994 EF422816 AY6891416 cordata Thunb. BG Bonn, 08120 Borsch 3481 (BONN) DQ2127123 AY1453447 AY1453447 cernuus L. USA, Borsch & Wilde 3108 (VPI, FR) DQ8822002 AY1453437 AY1453437 (Lour.) Baill. BG Bonn, 00312 Wanke 001 (DR) DQ2127133 EF422815 AY6891406 Outgroup Canella winterana Gaertn. BG Bonn, 15293 Borsch 3466 (BONN) DQ8822402 AY1453487 AY1453487 Winteraceae Drimys winteri#3 J.R. Forst. & BG Bonn Borsch 3479 (BONN) – EF422807 EF422807 G. Forst. Tasmannia lanceolata#3 (Poir.) BG Bonn, 00769 Borsch 3484 (BONN) DQ8822412 – A.C.Sm.

1 Hilu et al., 2003; 2 Mu¨ller et al., 2006; 3 Wanke et al.,2006a; 4 Wanke et al. 2007; 5 Wanke et al.,2006b; 6 Neinhuis et al., 2005; 7 Borsch et al., 2003. # Certain species have been replaced with closely related species within the two datasets (trnK/matK versus trnT-F), indicated by numbers following the names. (#1: Piper ornatum has been replaced with P. angustum; #2: Piper cf. magnificum has been replaced with P. betle and #3: Tasmannia lanceolata has been replaced with Drimys winteri) 1233 1234 Wanke et al. — Affinities of Verhuellia Revealed by Molecules

TABLE 2. Primers used in this study for the amplification and sequencing of the newly generated data (from previous publications) and newly designed for this study

Primer Direction Region Sequence (50 –30) Design

Ver-matK-3000R Reverse trnK/matK CTC TAA AAA CCC CGA ACC TAA T This study Ver-matK-1800F Forward trnK/matK TTC AGT CAT TGT AGA AAT TCC This study MG1 Reverse trnK/matK AAC TAG TCG GAT GGA GTA GAT Liang and Hilu (1996) MG15 Forward trnK/matK ATC TGG GTT GCT AAC TCA ATG Liang and Hilu (1996) Pi-matK-730R Reverse trnK/matK ATA GAA ATG GA(CT) TCG TTC AAG Wanke et al. (2006a) Pi-matK-1060F Forward trnK/matK ACT T(AG)T GGT CTC AAC (CT)G Wanke et al. (2006a) Pi-matK-1480F Forward trnK/matK TCG TAA ACA (CT)AA AAG TAC Wanke et al. (2006a) AR-matK-1850R Reverse trnK/matK CCA GGC AAG ATA CTA AT Wanke et al. (2007) Pi-matK-1820R Reverse trnK/matK ACA CTA ATT GGA AGG AGA ATG G Wanke et al. (2007) Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021 AR-matK-1200F Forward trnK/matK TTC CAA AGT CAA AAG AGC G Wanke et al. (2007) Pi-matK-2800F Forward trnK/matK AAT CTT TCT CAT TAT TAC AGT GG Wanke et al. (2007) AR-matK-1510R Reverse trnK/matK TAG ACT CCT GAA A(AG)A GAA GTG G Wanke et al. (2007) Pe-matK-2500R Reverse trnK/matK TTC GCA ATA AAT GCA AAG AGG Wanke et al. (2007) trnTF-50F Forward trnT-L-F TAC AAA TGC GAT GCT CTA ACC This study trnL110R Reverse trnT-L-F GAT TTG GCT CAG GAT TGC CC Borsch et al. (2003) trnTc Forward trnT-L-F CGA AAT CGG TAG ACG CTA CG Taberlet et al. (1991) trnTf Reverse trnT-L-F ATT TGA ACT GGT GAC ACG AG Taberlet et al. (1991) Ver-trnTL-500R Reverse trnT-L-F CGA ATG AAA CCA TAG GTA T This study Ver-trnTL-480F Forward trnT-L-F GGT TGC AAT TCA AAT AAT AAT This study

TABLE 3. Location of mutational hotspots and characterization of trees

trnT-L-F trnK/matK

Position in alignment Uncertain homology due to Position in alignment Uncertain homology due to Total evidence

H1 300–345 Poly T 369–544 Poly AT – H2 587–632 Poly A 652–660 Poly C þ poly T – H3 689–1116 Poly T 815–828 Poly A – H4 1301–1315 Poly A 961–968 Poly A – H5 1993–2055 Poly A þ poly T 977–994 Poly A – H6 2085–2095 Poly A 1071–1087 Poly T – H7 2390–2401 AT repeats 1102–1164 Variability – H8 2816–2827 Variability 3115–3170 Variability – H9 2877–2993 AT repeats – – – No of trees (MP) 4 (1) 3 (6) 1 (1) Length 1582 (2377) 2626 (2965) 4170 (5307) Total characters 2892 (3372) 3057 (3270) 5949 (6640) Variable characters 932 (1411) 1252 (1463) 2176 (2865) MP info. characters 587 (853) 895 (1011) 1470 (1850) No. of indels 480 213 693 CI 0.782 (0.722) 0.666 (0.661) 0.712 (0.689) RI 0.868 (0.816) 0.831 (0.822) 0.839 (0.814) RC 0.678 (0.589) 0.553 (0.544) 0.597 (0.561)

CI ¼ consistency index; RI ¼ retention index; RC ¼ rescaled consistency index. Values between brackets are calculated based on substitutions and coded length mutations (indels).

For the combined datasets (excluding the hotspots), parsimonious trees were found (Table 3). Incongruence the calculations were performed on 1470 potentially among trees was generally only found within Piper parsimony-informative characters, based on substitutions (a polytomy, not shown). Verhuellia is not part of or only or on 1850 potentially parsimony-informative charac- sister to Peperomia, as expected, but appears as sister to ters including coded length mutations as additional all other Piperaceae (Zippelia þ Manekia and Piper þ characters. Peperomia) (Fig. 1). This has consequences for the interpretation of character evolution in Piperaceae and Piperales, which can be seen from character mapping Phylogenetic relationships of the genus Verhuellia based on the ancestral state reconstruction approach All analyses revealed nearly identical topologies in (Fig. 2). The node of perianthless Piperales is charac- the strict consensus trees and only a few equally most terized by a hexamerous androecium and a trimerous Wanke et al. — Affinities of Verhuellia Revealed by Molecules 1235 Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021

F IG. 1. Phylogram of the single most-parsimonious tree obtained with the combined dataset. Independent analyses of the different regions revealed virtually identical topologies to the one obtained from the combined analyses. Support values differ only marginally and are given based on the total evidence above the branches. Below the branches, posterior probabilities (Bayesian analysis) (first), and decay values from the parsimony analysis are given, both calculated from the combined data set. Asar. ¼ Asaroideae; Lac. ¼ Lactoridaceae. Certain species have been replaced by closely related species within the two datasets (trnK/matK versus trnT-F) – see Table 1. , whereas the root node of Piperaceae is superficial similarities to Peperomia. Both the placement characterized by a dimerous androecium and a trimerous near or within Peperomia and the synonymy of Verhuellia gynoecium. with Peperomia were based on characters apparently repre- senting parallel evolution, e.g. the occurrence of only two . However, there are no detailed observations, as DISCUSSION thorough studies have not been performed. Authors who Position of Verhuellia in Piperales have regarded Verhuellia as a separate genus considered it to be closely related to Peperomia as both were placed in Despite the clarification of the phylogenetic relationships of the tribe Peperomieae rather than Pipereae (Piper, Zippelia Verhuellia, living material required for detailed morphologi- and several taxa now synonymized with Piper; e.g. Miquel, cal and ontogenetic studies to elucidate character evolution is 1843, 1868; de Candolle, 1869). This historical placement still not available. The sister group position of Verhuellia to of Verhuellia has not previously been questioned. the rest of Piperaceae is unexpected because it shows some 1236 Wanke et al. — Affinities of Verhuellia Revealed by Molecules Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021

F IG. 2. Ancestral state reconstruction, based on a Bayesian approach using the combined trnK/matK region and the trnT-L-F region in Piperales showing (A) stamen number and (B) carpel number. Colour changes between nodes indicate transitional states of the given characters (branch lengths are used asa rate for the occurrence of morphological change among taxa). Certain species have been replaced by closely related species within the two datasets (trnK/ matK versus trnT-F) – see Table 1.

Morphological affinities to Peperomia and other Piperaceae show extrorse dehiscence. However, the most conspicuous difference is the number of stigmas (three or four in In contrast to the species-rich, pantropical genus Verhuellia vs. one in Peperomia, sometimes two-lobed; Peperomia, the species-poor genus Verhuellia has only Dahlstedt, 1900; Yuncker, 1933; Skottsberg, 1947; been the subject of limited studies due to its restricted dis- Sastrapradja, 1968; Remizowa et al., 2005). All these chara- tribution, limited availability in herbaria, absence in botanic cters provide support for Verhuellia as a distinct genus in gardens and inaccessibility of material. These genera have Piperaceae, as reported by Saralegui Boza (2004) in the been considered as closely related because they are both most recent publication in which the genus is mentioned, characterized by some of the most reduced flowers in although the discrepancy with Tebbs (1993) is not discussed. Piperaceae, superficially similar to each other. Specifically, the similarity in inflorescence morphology and the flowers with a dimerous androecium appear to be strong characters uniting Verhuellia with Peperomia. However, in a clade Reappraising evolution of floral characters where all representatives are characterized by marked All lineages of Piperales show strong trends towards reduction of floral organs, convergent evolution could dis- reduction of floral organs, complicating the use of morpho- guise true relationships and this could be confounded by logical data in reconstruction of their evolution. This has the limited observation that has been possible. also been the main reason for different relationships of Although Tebbs (1993) synonymized Verhuellia with members of Piperales in classification systems of angios- Peperomia on the basis of ‘similar habit, and perms prior to molecular approaches. Clades with similar fruits’, the two genera show a strikingly different habit patterns of reduction of flower organs have also been and differences in floral morphology. The stamens in placed near to or within Piperales, thus providing evidence Verhuellia are tetrasporangiate and show latrorse dehis- for parallel evolution of certain traits. A prominent example cence whereas those in Peperomia are disporangiate and is the placement of within Piperales, mainly Wanke et al. — Affinities of Verhuellia Revealed by Molecules 1237 based on the reduced flowers (Cronquist, 1988). Similarly, Bollback JP. 2006. SIMMAP: stochastic character mapping of discrete floral details in Saururaceae superficially resemble those traits on phylogenies. BMC Bioinformatics 7: 88. Borsch T, Hilu KW, Quandt D, Wilde V, Neinhuis C, Barthlott W. of Acoraceae (Igersheim et al., 2001). A trimerous perianth 2003. Noncoding plastid trnT-trnF sequences reveal a well resolved and adaxial prophylls occur in Piperales, monocots and phylogeny of . Journal of Evolutionary Biology , suggesting a close relationship and giving 16: 558–576. rise to the so-called paleoherb hypothesis (Taylor and de Candolle C. 1869. Prodromus Systematis Naturalis Regni Vegetabilis Hickey, 1990, 1992). 16: Piperaceae. Paris: Masson, 235–471. Cronquist A. 1988. The evolution and classification of flowering . Generally, Peperomia is considered to have the most , NY: New York Botanic Garden. reduced flowers in Piperales (Jaramillo et al., 2004), but Dahlstedt H. 1900. Studien u¨ber su¨d- und central-amerikanische this study shows that this is only partially true. As a conse- Peperomien mit besonderer Beru¨cksichtigung der brasilianischen quence of the position of the almost equally reduced genus Sippen. Kongligen Svenska Vetenskaps-Akademiens Handlingar 33: 1–218. Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021 Verhuellia as sister to all other Piperaceae, character evol- Doyle JA, Endress PK. 2000. Morphological phylogenetic analysis of ution in this family and in the perianthless Piperales basal angiosperms: comparison and combination with molecular needs new attention. data. International Journal of Plant Sciences 161: 121–151. The androecium at the basal node of the perianthless Felsenstein J. 1985. Confidence limits on phylogenies: an approach using Piperales is hexamerous both with (this study) and the bootstrap. Evolution 39: 783–791. Hilu KW, Borsch T, Mu¨ller K, Soltis DE, Soltis PS, Savolainen V, et al. without (Jaramillo et al., 2004) inclusion of Verhuellia. 2003. Angiosperm phylogeny based on matK sequence information. However, stamen number at the basal node of Piperaceae American Journal of Botany 90: 1758–1776. differs between the two studies: two with (this study) and Huelsenbeck JP, Nielsen R, Bollback JP. 2003. Stochastic mapping of four without the inclusion of Verhuellia (Jaramillo et al., morphological characters. Systematic Biology 52: 131–158. 2004). Igersheim A, Endress PK. 1998. Gynoecium diversity and systematics of paleoherbs. Botanical Journal of the Linnean Society 127: 289–370. In contrast to Jaramillo et al. (2004), gynoecial evolution Igersheim A, Buzgo M, Endress PK. 2001. Gynoecium diversity and sys- in Piperaceae is not characterized by the reduction from four tematics in basal monocots. Botanical Journal of the Linnean Society carpels, as in Zippelia and Manekia ( ¼ Sarcorhachis), to 136: 1–65. three in Piper, and one in Peperomia. As depicted in Jaramillo MA, Manos PS. 2001. Phylogeny and patterns of floral diver- sity in the genus Piper (Piperaceae). American Journal of Botany Fig. 2B, the gynoecium at the root node of Piperaceae is 88: 706–716. trimerous and changes to four carpels in the Manekia– Jaramillo MA, Manos PS, Zimmer EA. 2004. Phylogenetic relationships Zippelia clade. The same trend is observed in the of the perianthless Piperales: reconstructing the evolution of floral Gymnotheca–Saururus clade in Saururaceae and could be development. International Journal of Plant Sciences 165: 403–416. interpreted as parallel evolution. Lei LG, Liang HX. 1998. Floral development of dioecious species and trends of floral evolution in Piper sensu lato. Botanical Journal of Once living material of Verhuellia becomes available, it the Linnean Society 127: 225–237. will be possible to investigate character evolution in the Lei LG, Liang HX. 1999. Variations in floral development in Peperomia order Piperales more thoroughly. This presents the possi- (Piperaceae) and their taxonomic implications. Botanical Journal of bility of answering some evolutionary questions relating the Linnean Society 131: 423–431. Liang H, Hilu KW. 1996. Application of the matK gene sequences to to the family Piperaceae. grass systematics. Canadian Journal of Botany 74: 125–134. Liang HX, Tucker SC. 1989. Floral development in Gymnotheca chinen- sis (Saururaceae). American Journal of Botany 76: 806–819. ACKNOWLEDGEMENTS Liang HX, Tucker SC. 1990. Comparative study of floral vasculature in Saururaceae. American Journal of Botany 77: 607–623. Financial support for this study came from the German Liang HX, Tucker SC. 1995. Floral ontogeny of Zippelia begoniaefolia Science Foundation (DFG NE 681/5-1), the Research and its familial affinities: Saururaceae or Piperaceae? American Foundation-Flanders (FWO G.0172.07), the Special Journal of Botany 82: 681–689. Research Fund (BOF, Ghent University) the Department Maddison WP, Maddison DR. 2006. Mesquite version: a modular system for evolutionary analysis. Version 1.11. http://mesquiteproject.org. of Biology, Ghent University, and the Friends of the Miquel FAW. 1843. Systema Piperacearum. Rotterdam: H.A. Kramers. Botanic Garden, Ghent. We thank F. Jime´nez and Miquel FAW. 1852. Piperaceae. 7–26. In: von Martius CFP ed. Flora R. Garcı´a (Dominican Republic), Daniel Crawford, Tod Brasiliensis, Vol. 4. Munich & Leipzig. Stuessy, Thomas Borsch, O. Vargas, Wilhelm Barthlott, Miquel FAW. 1868. De Piperaceis Novae Hollandiae. Verslagen en the curator of the herbarium S and the Botanic Gardens Mededeelingen der Koninklijke Akademie van Wetenschappen. Afdeeling Natuurkunde ser. 2, 2: 53–64. of Ghent and Dresden for providing material for Mu¨ller J, Mu¨ller K. 2004. TreeGraph: automated drawing of complex tree DNA extraction.The curators of the herbaria B, BM, C, figures using an extensible tree description format. Molecular Ecology G, GH, K, NY, MA, S, U and US made their Verhuellia Notes 4: 786–788. specimens available for study. Finally, we thank Mike Mu¨ller K. 2004. PRAP – computation of Bremer support for large data- sets. Molecular Phylogenetics and Evolution 31: 780–782. Fay and two anonymous reviewers for their extensive sug- Mu¨ller K. 2005. SeqState – primer design and sequence statistics for phy- gestions to improve this manuscript and A. M. Muasya logenetic DNA data sets. Applied Bioinformatics 4: 65–69. and Alexander Vrijdaghs for their support. Mu¨ller KF, Borsch T, Hilu KW. 2006. Phylogenetic utility of rapidly evolving DNA at high taxonomical levels: contrasting matK, trnT-F, and rbcL in basal angiosperms. Molecular Phylogenetics and LITERATURE CITED Evolution 41: 99–117. Neinhuis C, Wanke S, Hilu KW, Mu¨ller K, Borsch T. 2005. Phylogeny APG II. 2003. An update of the Angiosperm Phylogeny Group classifi- of based on parsimony, likelihood, and Bayesian cation for the orders and families of flowering plants: APG II. analyses of trnL-trnF sequences. Plant Systematics and Evolution Botanical Journal of the Linnean Society 141: 399–436. 250: 7–26. 1238 Wanke et al. — Affinities of Verhuellia Revealed by Molecules

Nielsen R. 2002. Mapping mutations on phylogenies. Systematic Biology Tebbs MC. 1993. Piperaceae. In: Kubitzki K, Rohwer JG, Bittrich V eds. 51: 729–739. The families and genera of vascular plants, Vol. 2. Berlin: Nixon KC. 1999. The parsimony ratchet, a new method for rapid parsi- Springer-Verlag, 516–520. mony analysis. Cladistics 15: 407–414. Tucker SC. 1975. Floral development in Nuin PAS. 2005. MTgui – a simple interface to ModelTest. Program dis- (Saururaceae). 1. Floral initiation and stamen development. tributed by the author. University of Toronto. http://www.genedrift. American Journal of Botany 62: 993–1007. org/mtgui.php. Tucker SC. 1976. Ontogeny of the inflorescence of Saururus cernuus Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA sub- (Saururaceae). 2. Carpel initiation and floral vasculature. American stitution. Bioinformatics 14: 817–818. Journal of Botany 63: 289–301. Remizowa M, Rudall PJ, Sokoloff D. 2005. Evolutionary transitions Tucker SC. 1979. Ontogeny of the inflorescence of Saururus cernuus among flowers of perianthless Piperales: inferences from inflores- (Saururaceae). American Journal of Botany 66: 227–236. cence and flower development in the anomalous species Peperomia Tucker SC. 1980. Inflorescence and flower development in the Piperaceae. fraseri (Piperaceae). International Journal of Plant Sciences 166: I. Peperomia. American Journal of Botany 67: 686–702. 925–943. Tucker SC. 1981. Inflorescence and floral development in Houttuynia Downloaded from https://academic.oup.com/aob/article/99/6/1231/2769300 by guest on 28 September 2021 Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic cordata (Saururaceae). American Journal of Botany 68: 1017–1032. inference under mixed models. Bioinformatics 19: 1572–1574. Samain MS, Mathieu G, Vanderschaeve L, Wanke S, Neinhuis C, Tucker SC. 1982a. Inflorescence and flower development in the Goetghebeur P. 2007. Nomenclature and typification of subdivisional Piperaceae. II. Inflorescence development of Piper. American names in the genus Peperomia (Piperaceae). Taxon 56: 229–236. Journal of Botany 69: 743–752. Saralegui Boza H. 2004. Piperaceae. Flora de la Repu´blica de Cuba 9: Tucker SC. 1982b. Inflorescence and flower development in the 57–60. Piperaceae. III. Floral ontogeny of Piper. American Journal of Sastrapradja SN. 1968. On the morphology of the flower in Peperomia Botany 69: 1389–1401. (Piperaceae) species. Annales Bogoriensis 4: 235–244. Tucker SC. 1985. Initiation and development of inflorescence and flower Schmitz F. 1872a. Der morphologische Aufbau von Verhuellia Miq. Flora in Anemopsis californica (Saururaceae). American Journal of Botany 26: 402–415. 72: 20–31. Schmitz F. 1872b. Der morphologische Aufbau von Verhuellia Miq. Flora Tucker SC, Douglas AW, Liang HX. 1993. Utility of ontogenetic and 27: 417–424. conventional characters in determining phylogenetic relationships of Schultz TR, Churchill GA. 1999. The role of subjectivity in reconstruct- Saururaceae and Piperaceae (Piperales). Systematic Botany 18: ing ancestral character states: a Bayesian approach to unknown rates, 614–641. states, and transformation asymmetries. Systematic Biology 48: Wanke S, Samain MS, Vanderschaeve L, Mathieu G, Goetghebeur P, 651–664. Neinhuis C. 2006a. Phylogeny of the genus Peperomia (Piperaceae) Skottsberg C. 1947 Peperomia berteroana Miq. and P. tristanensis inferred from the trnK/matK region (cpDNA). Plant Biology 8: Christoph., an interesting case of disjunction. Acta Horti 93–102. Gothoburgensis 16: 251–288. Wanke S, Gonzalez F, Neinhuis C. 2006b. Systematics of pipevines – Simmons MP, Ochoterena H. 2000. Gaps as characters in sequence based combining morphological and fast-evolving molecular characters to phylogenetic analyses. Systematic Biology 49: 369–381. investigate the relationships within subfamily Aristolochioideae Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (Aristolochiaceae). International Journal of Plant Sciences 167: . (*and other methods), version 4 0b10. Sunderland, MA: Sinauer. 1215–1227. Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universal primers for Wanke S, Jaramillo MA, Borsch T, Samain MS, Quandt D, Neinhuis amplification of three non-coding regions. Plant C. 2007. Evolution of the Piperales – matK and trnK intron sequence Molecular Biology 17: 1105–1109. data reveal lineage specific resolution contrast. Molecular Taylor DW, Hickey LJ. 1990. An Aptian plant with attached and Phylogenetics and Evolution 42: 477–497. flowers: implications for angiosperm origin. Science 247: 702–704. Yuncker TG. 1933. Revision of the Hawaiian species of Peperomia. Taylor DW, Hickey LJ. 1992. Phylogenetic evidence for the herbaceous Bernice P. Bishop Museum Bulletin 112: 3–131. origin of angiosperms. Plant Systemetics and Evolution 180: 137–156.