University of Missouri, St. LouisIRL @ UMSLDissertations UMSL Graduate Works12-11-2018Systematics of Malesian-Pacific Piper (Piperaceae)Rani AsmarayaniUniversity of Missouri - St. Louis, rasmarayani@yahoo.comFollow this and additional works at: https://irl.umsl.edu/dissertationPart of the Botany CommonsThis Dissertation is brought to you for free and open access by the UMSL Graduate Works at IRL @ UMSL. It has been accepted for inclusion inDissertations by an authorized administrator of IRL @ UMSL. For more information, please contact marvinh@umsl.edu.Recommended CitationAsmarayani, Rani, "Systematics of Malesian-Pacific Piper (Piperaceae)" (2018). Dissertations. 812.https://irl.umsl.edu/dissertation/812 Systematics of Malesian-Pacific Piper (Piperaceae) Rani Asmarayani M.S. in Biology, 2006, Institut Teknologi Bandung, Indonesia A Dissertation Submitted to The Graduate School at the University of Missouri-St. Louis in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biology with an emphasis in Ecology, Evolution, and Systematics May 2019 Advisory Committee Peter F. Stevens, Ph.D. Chairperson Elizabeth A. Kellogg, Ph.D. Robert J. Marquis, Ph.D. Nathan C. Muchhala, Ph.D. Richard C. Keating, Ph.D. Copyright, Rani Asmarayani, 2019 To my grandmas, Kwik Rien Nio & Tjan Hoa Loan iv ACKNOWLEDGEMENTS I am deeply indebted to my dissertation advisor, Peter F. Stevens, for his expertise, guidance, patient, and tremendous support. I also thank my committee, Elizabeth A. Kellogg, Robert J. Marquis, Richard C. Keating, and Nathan C. Muchhala for their expertise, guidance, and valuable discussion and feedback for the development of the manuscript. I thank Iván Jiménez for his expertise and valuable discussion for species delimitation. Fieldwork, herbarium study, and laboratory work were made possible with the help of the keepers, staff, and collaborators of herbaria ANDA, BO, DUKE, K, KEP, L, MO, PTBG, SAN, SING, and SRP, as well as Bantimurung-Bulusaraung National Park (Indonesia), Bogor Botanical Garden (Indonesia), Economic Planning Unit (Malaysia), Lore Lindu National Park (Indonesia), National Parks Board (Singapore), PT. Freeport Indonesia, Sabah Biodiversity Council (Malaysia), especially Eko B. Walujo, Joeni S. Rahajoe, James C. Solomon, P. Mick Richardson, Peter C. Hoch, Kate Armstrong, Charlie D. Heatubun, Peter B. Phillipson (Weda Bay Botanical Inventory Project), Khoon-Meng Wong, David Middleton, Hock-Keong Lua, Serena Lee, Leng-Guan Saw, Rafidah A. Rahman, John B. Sugau, Joan T. Pereira, Pratita Puradyatmika, Marie Briggs, Roxali Bijmoer, Nurainas, Mohammad Amir, Agus Sujadi, Imin Kamin, Jeisin Jumian, Yunita N. Esthi, and Susila. Halim Susanto and the family of Aris B. Prasetyo are also thanked for their help with fieldwork in Central Java. I thank various parties for providing me funding, both for doctoral fellowships and research scholarships, without which this dissertation research would never happen. I would like to express my gratitude to Fulbright Foreign Student Program, Whitney R. Harris World Ecology Center (UMSL), Des Lee Plant Systematic Laboratories (UMSL) through Elizabeth A. Kellogg and Patricia G. Parker, Missouri Botanical Garden, Herbarium Bogoriense, Singapore Botanic Garden, PT. Freeport Indonesia, Systematics Research Fund – The Systematics Association and The Linnean Society, American Society of Plant Taxonomy, and International Association for Plant Taxonomy. My family and friends have been very supportive and helpful during this long ordeal, to whom I owe my gratitude. v DISSERTATION ABSTRACT Comprised of ~2400 spp., Piper is a major clade in the magnoliid angiosperms. Three major groups are recognized in Piper, i.e., the Neotropical, Asian and South Pacific. Unlike Neotropical Piper, relationships in the Paleotropical Piper remain enigmatic. This study focused on the Paleotropical Piper with emphasize on the Malesian-Pacific Piper, Malesia being the center of diversity of the genus in the Paleotropics. The goals are to evaluate relationships within Paleotropical Piper (chapter 1), to evaluate characters, morphological (chapter 1) and stem anatomical (chapter 2), that may distinguish clades, and to investigate the species boundaries focusing on the well-supported Muldera clade emphasizing the Malay Peninsula species (chapter 3). I used eleven out of 14 infrageneric groups in Paleotropical Piper and 14% of the species and inferred their phylogenetic relationships based on nuclear (ITS, g3pdh) and chloroplast (trnL-F) regions. Within the Asian clade, species predominantly distributed west of Wallace’s Line (WWL) form a moderately/strongly supported clade in the ITS phylogeny, embedded within a grade of species from east of Wallace’s Line (EWL). The ITS phylogeny also resolves 12 and 5 clades in the WWL clade and the EWL grade, respectively. G3pdh and trnL-F phylogenies resolve some of the same clades, but some relationships among and within clades are incongruent. Almost no currently recognized infrageneric groups in Asian Piper are monophyletic. Most of the morphological characters previously used to delimit infrageneric groups are homoplasious, and I also found that there is much parallelism in the stem anatomical characters. Characters that may distinguish clade include cupular bracts and a ring of sclereids surrounding sclerenchymatous cap of peripheral vascular bundles of the stems for Muldera clade, angular stems for Sarcostemon s.l. clade, very vi broad primary medullary rays in the stems for Pseudochavica sensu Ridley s.str. clade, spherical stigmas for core E2 clade, and peripheral mucilage canals interior to the large vascular bundles in the stems of E1 clade. The species limits within Muldera clade remains unclear, there are some shared characters between morphospecies and common occurrence of paraphyly, suggesting hybridization or lateral transfer among species of Muldera clade. Keywords: Paleotropical Piper; classification; phylogeny; morphology; stem anatomy; Piper section Muldera; species limits vii TABLE OF CONTENTS Acknowledgements …………………………..…………………………......................... iv Dissertation abstract ……………………………………………………………………... v Table of contents ……………………...………………………………………..………. vii Chapter 1: Phylogenetic relationships in Malesian-Pacific Piper (Piperaceae).………… 1 Chapter 2: On the anatomy of Malesian-Pacific Piper and its implications for systematics 1: Stems ………………………………………………………………………....…….. 123 Chapter 3: Piper section Muldera (Piperaceae) of the Malay Peninsula: variation and species limits .................................................................................................................. 174 1 Chapter 1: Phylogenetic relationships in Malesian-Pacific Piper (Piperaceae) Rani Asmarayani Department of Biology, University of Missouri – St. Louis, 1 University Boulevard, St. Louis, Missouri 63121, U.S.A.; Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, U.S.A.; Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor KM 46, Cibinong 16911, West Java, Indonesia; rasmarayani@yahoo.com NOTE: Manuscript received 13 January 2018, accepted 18 June 2018, and published in print and online issues 29 August 2018 in Taxon. Reference: Asmarayani, R., 2018. Phylogenetic relationships in Malesian-Pacific Piper (Piperaceae) and their implications for systematics. Taxon 67: 693-724. https://doi.org/10.12705/674. 2 Abstract. Comprised of ~2000 spp., Piper is a major clade in the magnoliid angiosperms. Three major groups are recognized in Piper, i.e., the Neotropical, Asian and South Pacific. Unlike Neotropical Piper, relationships in the Paleotropical Piper remain enigmatic. This study evaluates the relationships within Paleotropical Piper by focusing sampling efforts in the Malesian region. This is the center of diversity of the genus in the Paleotropics, yet has been barely represented in previous phylogenetic studies. Eleven out of 14 infrageneric groups in Paleotropical Piper and 14% of the species were included in this study and their phylogenetic relationships were inferred based on nuclear (ITS, g3pdh) and chloroplast (trnL-F) regions. The goals were to test the monophyly of infrageneric groups, to examine how the main characters used to delimit these infrageneric groups have shaped classifications past and present, and to understand the origin of bisexual flowers and the shrubby habit in the predominantly dioecious Asian clade of climbers. The results show that the Asian clade is monophyletic in all phylogenies and the South Pacific clade is monophyletic in nuclear phylogenies. Within the Asian clade, species predominantly distributed west of Wallace’s Line (WWL) form a moderately/strongly supported clade in the ITS phylogeny, embedded within a grade of species from east of Wallace’s Line (EWL). The ITS phylogeny also resolves 12 and 5 clades in the WWL clade and the EWL grade, respectively. G3pdh and trnL-F phylogenies resolve some of the same clades, but some relationships among and within clades are incongruent. Almost no currently recognized infrageneric groups in Asian Piper are monophyletic. Most of the characters previously used to delimit infrageneric groups are homoplasious, including fruit type, bract type, direction of anther dehiscence, and number of anther valves. However, bract and fruit types are still of some value in that many clades in the Asian Piper are characterized by single bract and fruit types. In 3 Paleotropical Piper, bisexual flowers have evolved numerous times in almost every clade, and shrubby habit has evolved at least six times, but is restricted to four clades in the WWL clade. The 17 clades resolved in this study are described along with their potential diagnostic characters. Keywords: classification; climber; dioecy; Paleotropical Piper; phylogeny § INTRODUCTION With ~2000 spp. (Quijano-Abril & al., 2006), Piper L. (Piperaceae) is a major clade in the magnoliid angiosperms. It includes the “king of spices”, black pepper (P. nigrum L.), and an emerging herbal medicine, kava (P. methysticum G.Forst.). The leaves or fruits of the betel leaf plant (P. betle L.) are chewed with betel nut (Areca catechu L.) and lime in South and Southeast Asia. This potentially addictive betel-chewing habit (Burton-Bradley, 1979; Chu, 2001, 2002) is important in the cultural life of many indigenous peoples. Moreover, many other Piperspecies have long been used in traditional medicine and as condiments (e.g., Heyne, 1927; Burkill, 1935). Piper is pantropical, but only three species occur in Africa and on the island of Réunion, while a few species reach subtropical areas (e.g., Hao & al., 2012). The plants grow in lowland to montane, sometimes almost subalpine, forests up to 4000 m altitude (P. subcanirameum C.DC.; Chew, 1992). Many Piper species, typically those growing in primary forests, are endemic to individual islands or mountains (e.g., Quisumbing, 1930; Marquis, 2004), but some of them grow in secondary open forests and sometimes become invasive, e.g., the Neotropical P. aduncum L. which is now invasive in Malesia and the South Pacific (Lepš & al., 2002). In Neotropical forests, Piper is often abundant and 4 speciose (Gentry & Emmons, 1987), and is ecologically important. They have well known associations with frugivore/disperser specialists (Carollia bats; e.g., Fleming, 2004), ants (Pheidole spp.; e.g., Tepe & al., 2007), and herbivore specialists (Eois moths; e.g., Wilson & al., 2012). In contrast, the ecology of Paleotropical Piper is barely known, albeit ant-plant associations occur in P. myrmecophilum C.DC. (Quisumbing, 1930; Gardner, 2006). With such species diversity and properties, Piper has been proposed as a model genus to study phytochemistry, ecology, and evolution (see Dyer & Palmer, 2004). Phylogenetic analyses confirm that the genus is monophyletic and it is divided into Neotropical (ca. 1300 spp.) and Paleotropical (ca. 700 spp.) clades (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Asmarayani & Pancoro, 2005; Wanke & al., 2007a; Jaramillo & al., 2008; Smith & al., 2008). The Paleotropical clade is further divided into the Asian (ca. 600 spp.) and South Pacific (ca. 100 spp.) clades. These divisions are supported by morphological characters. The Neotropical species are shrubs/treelets with bisexual flowers, the South Pacific species are dioecious shrubs/treelets, and the Asian species are dioecious root climbers. Members of the genus Piper are easily recognized morphologically by their swollen nodes, entire distichous leaves, and terminal spikes that become leaf-opposed. The spikes vary in how they are held (i.e., erect vs. pendulous) and their length, thickness, and color, but all consist of minute perianthless flowers subtended by bracts. Sexuality, stamen number, anther morphology, style length, bract type, fruit type, inflorescence position, and leaf venation have been used to differentiate infrageneric groups within Piper. The small size of the flowers, however, may have caused instability in the classification, and the difficulty in observing the number and position of the stamens—the main characters of Candolle’s system (1869, 1923), along with bract type—in living, and even more so in herbarium 5 specimens, has often been mentioned (Bentham & Hooker, 1880; Trelease & Yuncker, 1950; Yuncker, 1958). As a result, Candolle’s system was abandoned by some botanists (e.g., Trelease & Yuncker, 1950). Indeed, the number of stamens is largely uninformative in Paleotropical Piper, with most having 2 or 3 stamens (Fig. 1I), and the number is often not constant in flowers even from a single spike. Despite these problems, current phylogenetic studies (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Jaramillo & al., 2008) confirm the monophyly of seven infrageneric groups sensu Miquel (1843–1844) within Neotropical Piper, including the most species-rich one, section Radula Miq. In addition, several phylogenetic studies focusing on specific infrageneric groups within the Neotropical clade have also been carried out (Jaramillo & Callejas, 2004b; Tepe & al., 2004; Molina-Henao & al., 2016). Relationships in the Paleotropical clade, however, remain largely unresolved. Taxonomic history. Paleotropical Piper has been classified in up to 14 infrageneric groups, one (Macropiper Miq.) includes the species in the South Pacific clade, while the other 13 make up the Asian clade (Table 1). Paleotropical Piper was classified by both Miquel (1843–1844) and Candolle (1923) (Table 1), while Engler (1889) combined the two systems. Miquel (1843–1844) relied on variation in fruit and bract types to separate most infrageneric groups, while Candolle (1923) mostly used stamen number and morphology. The only two Asian infrageneric groups admitted by both systems, Coccobryon Klotzsch ex Miq. and Muldera Miq., were based on having bisexual flowers and distinctive cupular bracts, respectively. Particular classification problems in Paleotropical Piper come from the disagreements about the importance of specific characters in defining infrageneric groups, and often the same species can be placed in different infrageneric groups, depending on the author and the characters used. Two 6 examples of these classification problems are described below (see Electr. Suppl.: Appendix S1 for expanded version of taxonomic history of Paleotropical Piper). First, Candolle’s (1923) subgenus “Eupiper” C.DC. (not validly published), characterized by having 2 or 3 stamens, included about 80% of the Asian species, but in Miquel’s system (1843–1844) those species were placed in one of four genera, Chavica Miq., Cubeba Raf., Piper L. and Rhyncholepis Miq., based mostly on differences in bract and fruit types. According to Miquel, Chavica had round, peltate, pedicellate bracts and small crowded sessile fruits, Cubeba had oblong or round, peltate, sessile bracts and stalked fruits (Fig. 1F), Piper (sensu Miquel, 1844; “Piper Miq.”, hereafter) had oblong bracts adnate to the rachis and loose infructescences with large sessile fruits (Fig. 1D), and Rhyncholepis had subulate, peltate, pedicellate bracts and elongated styles. Chavica was further divided into sections “Euchavica” Miq. (not validly published) with cylindrical spikes (Fig. 1A, B), Sphaerostachyon Miq. with globular pistillate spikes, and Peltandron Miq. with enlarged anther connectives (rather like Fig. 1L). Miquel included the majority of Chavica, 41 species, in section “Euchavica”, while he placed only 4 and 1 species in sections Sphaerostachyon and Peltandron, respectively (Miquel, 1843). In 1869, Candolle had recognized Chavica as a separate genus, but he circumscribed it differently: Piper species had quadrivalved anthers that dehisce laterally, and Chavica species had bivalved anthers that dehisce apically (“extrorsely” in the original description). Only six Chavica species were listed in Candolle’s treatments in 1869 and 1898, and the majority of Miquel’s Chavica he included in his “Eupiper” (“Eupiper**”, see Table 1). Later in 1923, however, he abandoned Chavica entirely and placed its two species in “Eupiper”. Other classification problems stem from species delimitation issues, not surprising 7 in such a species-rich group, and changing emphases on the characters chosen to place a species in a particular infrageneric group. Perhaps one of the most egregious cases is P. rostratum Roxb. (Fig. 1H). Roxburgh (1814, 1820) described this as a plant from, perhaps erroneously, the Moluccas based on a plant cultivated in the Calcutta Botanical Garden (no collector listed). It was placed by Miquel (1843) in Rhyncholepis as R. roxburghii Miq. Hooker (1886) placed specimens collected by Griffith and Maingay from the Malay Peninsula in P. rostratum in his section Chavica and synonymized P. stylosum Miq. from Sumatra with it. Nevertheless, Candolle (1914) rejected this synonymy despite the similarities in the descriptions of P. rostratum and P. stylosum, both being shrubs or creepers with beaked fruits and elongated styles. Indeed, P. stylosum had been placed in many infrageneric groups. Starting off in Piper Miq. (Miquel, 1863), it was placed in both “Eupiper” and Coccobryon in the same treatment (Candolle, 1923), and in Pseudochavica (Ridley, 1924). Piper rostratum does have elongated styles (which would place it in Rhyncholepis), round and peltate bracts (Chavica), partially concrescent fruits (Pseudochavica sensu Ridley, 1924; “Pseudochavica Ridl.”, hereafter) which look like free fruits when young (Piper Miq.) and tightly packed fruits when fully mature (Chavica), and sometimes it has bisexual flowers (Coccobryon). Hardly surprisingly, most recent local treatments use no infrageneric classifications of Piper (e.g., Backer & Bakhuizen van den Brink, 1963 [Java]; Chew, 1972 [New Guinea, Solomon Islands, Australia]; Van Royen, 1982 [New Guinea highlands]; Cheng & al., 1999 [China]; Gardner, 2003 [New Guinea], 2006 [the Philippines], 2010 [Solomon Islands], 2013 [New Guinea]). Molecular phylogenetic studies do not support the monophyly of Chavica, Piper, and Cubeba sensu Miquel (1843–1844), implying bract and fruit types are homoplasious 8 characters (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Asmarayani & Pancoro, 2006; Jaramillo & al., 2008; Smith & al., 2008). The only Paleotropical group supported in the phylogenies as being monophyletic is Quisumbing’s expanded Peltandron (Jaramillo & Manos, 2001). Piper capense of Africa, previously thought to belong to Coccobryon (Miquel, 1844; Candolle, 1869, 1923), has been found to be part of the Macropiper clade (Jaramillo & al., 2008; Smith & al., 2008). The phylogenies also place P. methysticum, part of Candolle’s (1869, 1923) “ Eupiper”, in Macropiper (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Jaramillo & al., 2008; Smith & al., 2008), confirming Miquel’s (1843) classification. Piper capense and P. methysticum are indeed shrubby like other species of Macropiper. Malesia. The Malesian floristic region sensu Van Steenis (1950) extends from the Malay Peninsula to the Solomon Islands, and is considered to be the most geographically complex tropical region on earth (Lohman & al., 2011). The phytogeographical patterns of this area have been recognized in broad-scale floristic analyses (Van Welzen & al., 2005, 2011; Van Welzen & Slik, 2009), confirming that Merrill-Dickerson/Huxley’s Line (a variant of Wallace’s Line) and Lydekker’s Line separate the West Malesian, Wallacean, and East Malesian areas. About two-thirds of Paleotropical Piper (ca. 450 spp.) occur in Malesia and include both the Asian and South Pacific clades, the latter being distributed from New Guinea eastward (e.g., P. methysticum; Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Jaramillo & al., 2008; Smith & al., 2008). Indeed, of the 14 infrageneric groups of Paleotropical Piper, 12 occur in Malesia. This study evaluates relationships within Paleotropical Piper based on three unlinked molecular markers, and examines the distribution of morphological characters 9 on the resulting phylogenies to understand their importance in classification. Sampling efforts focused in the Malesian region, the center of diversity of Paleotropical Piper (Yuncker, 1958), yet barely—except for some Philippine species—represented in previous phylogenetic studies. In this study, I included species from 11 of the 12 Malesian infrageneric groups—section Sphaerostachyon is not included—and infer their phylogenetic relationships using the internal transcribed spacer of nuclear ribosomal DNA (ITS), the low-copy nuclear gene encoding glyceraldehyde 3-phosphate dehydrogenase (g3pdh), and the chloroplast trnL-F regions. Using these phylogenies, I examined the monophyly of infrageneric groups. In addition, by plotting morphological characters and their states as they have been used in the past (see above, Taxonomic history) on to the phylogenetic trees, I examined whether the characters of bracts, fruits, and stamens demarcate infrageneric groups. I also considered the origin of bisexual flowers and the shrubby habit in the predominantly dioecious and climbing Asian clade. Altogether, this phylogenetic study provides a foundation towards revising the classification and understanding the evolution of the Paleotropical members of this fascinating genus. § MATERIAL AND METHODS Material and molecular dataset. One hundred and forty-eight individuals of 78 species were newly sampled for this study. Plant material was collected from across the Malesian region and obtained from Bogor Botanic Garden (Indonesia) and the Missouri Botanical Garden. DNA material of four species from China, Ghana, Japan, and New Caledonia were obtained from Xishuangbanna Tropical Botanical Garden (China), Missouri Botanical Garden, and Makino Botanical Garden (Japan). To complement the data, 10 sequences of additional species from other parts of Asia, Africa, and the South Pacific were obtained from GenBank (http://www.ncbi.nlm. nih.gov). Species identifications or group affiliations of the voucher specimens of these sequences were checked and only sequences with vouchers whose identity could be confirmed were used in the analyses. Combining the newly collected material and the GenBank sequences, 211 individuals of 104 species (14% of Paleotropical species) were included in this study (see Appendix 1 for complete list). Widespread species were represented by 2 to 7 individuals from different localities/islands, and by 11 individuals for P. caninum Blume and 19 for P. betle. The sampled species represent members of all major infrageneric groups in Malesia (Piper Miq., Cubeba, and “Euchavica”, including Chavica sensu Candolle, 1869), seven of the other ten infrageneric groups in Asia (Muldera, Peltandron, Rhyncholepis, Coccobryon, Pseudochavica Ridl., Sarcostemon, and Oligandropiper), and the South Pacific Macropiper (Electr. Suppl.: Table S1). Shrubby species from highland New Guinea (P. gibbilimbum C.DC., P. montivagum Ridl.) and species with disputable infrageneric placements, like P. borneense, P. fragile, and P. rostratum (see Introduction and Electr. Suppl.: Appendix S1), were also included. I was unable to obtain species placed in section Pseudochavica Hook.f., but species with similar characters (= Chavica sensu Koorders, 1908 in part; “Chavica Koord.”, hereafter; see Electr. Suppl.: Appendix S1) are included. I also was unable to obtain section Sphaerostachyon (but see Discussion: Systematic implications), and no voucher was made for P. mullesua Buch.-Ham. ex D.Don. (GenBank accession: AF203634), a species of Sphaerostachyon, so this was also excluded. Subgenus Piperoides (Candolle, 1866) and section Apopiper (Candolle, 1869) were not included in this study; the three species 11 in these groups are from India, Australia, and Mexico. Houttuynia cordata Thunb. And Saururus cernuus L. of Saururaceae were used as outgroups. Plant material of Saururaceae and other Piperaceae was collected in the field or obtained from Herbarium Bogoriense (Indonesia). Sequences of additional species of other Piperaceae and Saururaceae, as well as all Neotropical Piper, were acquired from GenBank (Appendix 2); Neotropical species were chosen to represent all the major clades recovered by Jaramillo & al. (2008) DNA was obtained from (young) leaves preserved in silica gel. DNA extraction used a modified CTAB protocol (Porebski & al., 1997) or Qiagen DNeasy Plant Mini Kit following the manufacturer’s protocol. PCR for DNA amplification used Promega GoTaq Flexi DNA Polymerase or AmpliTaq 360 DNA Polymerase kits. The PCR mixture comprised 2 (to 12) μl DNA template, 5 μl of 5X Green GoTaq Flexi Buffer or 2.5 μl of 10X AmpliTaq 360 PCR Buffer, 2 (to 2.5) μl 25 mM MgCl2, 0.5 (to 1) μl 10 mM dNTPs, 1 μl of each 10 mM primer, 0.125 μl Taq Polymerase, and deionized water for a final volume of 25 μl. For difficult samples (i.e., having high GC content), 2.5 μl of 360 GC Enhancer (AmpliTaq 360) or 1.25 μl dimethyl sulfoxide (DMSO) and 2.5 μl betaine was added to the PCR mixture. The thermal cycling conditions for each marker followed previous suggestions (see below). Purification of PCR products was carried out using Qiagen QIAquick Gel Extraction Kit in 1% agarose gel or was carried out by sequencing companies (see below) in cases of direct sequencing. PCR products of nuclear markers were cloned using Promega pGEM-T Easy Vector System I and Promega JM109 Competent Cells following the manufacturer’s protocol. Samples were then Sanger sequenced in both forward and reverse directions at Macrogen (Korea), Beckman Coulter Genomics (U.S.A.), or Genewiz (U.S.A.). For nuclear markers, 2–14 clones were 12 sequenced (average of six sequences). In addition, the internal transcribed spacer of nuclear ribosomal DNA (ITS, hereafter) was sequenced directly for 66 individuals. Sequences were assembled and checked manually for quality using Geneious v.7.1.9 (Kearse & al., 2012). Sequences were checked against GenBank with BLAST to confirm that they belonged to Piper. Alignment used MUSCLE (Edgar, 2004) implemented in MEGA v.7.0.18 (Kumar & al., 2015) and was manually refined by eye. Initial marker screening of 8 nuclear and 13 chloroplast regions was performed on a set of seven species representing different infrageneric groups, biogeographic regions, fruit types, bract types, and growth forms; the set included P. celebicum Blume RA557, P. gibbilimbum AK1350, P. macropiper Pennant RA383, P. pentandrum C.DC. RA593, P. porphyrophyllum N.E.Br. RA66, P. quinqueangulatum RA482, and P. sulcatum Blume RA123. Regions and primers tested included ITS (Kita & Ito, 2000), six low-copy nuclear genes (Strand & al., 1997), the ribosomal protein S8e (Wagner & al., 2012), petA-psbJ (L. Prince in Jaramillo & al., 2008), matK (S. Wanke, pers. comm.; pair of MG15 [ATC TGG GTT GCT AAC TCA ATG] and Pe_matK_2340R [TTT TCT TTT GAC ATT GAA CCA A], pair of Pe_matK_2140F [ACT TTA TCT ATT TAT GGC AAT G] and PSBAR [CGC GTC TCT CTA AAA TTG CAG TCA T], using PCR conditions in Wanke & al., 2007a), trnL intron and trnL-trnF intergenic spacer (trnL-F , hereafter; Taberlet & al., 1991), and ten other chloroplast markers (Wagner & al., 2012). Of those 21 markers, only 6 could be amplified readily and had consistent band lengths despite modification of PCR mixture and thermal cycling condition. Ultimately I chose two nuclear markers, ITS and low-copy g3pdh, and a chloroplast marker, trnL-F, based on the ease with which they amplified and their level of variability (Kimura 2-parameter distances for nuclear ITS, g3pdh, S8e were 9.3%, 6.9%, and 5.8%, respectively; those of 13 chloroplast trnL-F, rpl16 intron, and psbT gene + psbT-psbN spacer + psbN gene + psbN-psbH spacer were 1.4%, 1.2%, and 1.0%, respectively). The three markers chosen have been previously used in phylogenetic studies of Piper (e.g., Jaramillo & Manos, 2001; Smith & al., 2008). Identical sequence(s) from an individual when using nuclear markers, both from cloning and direct sequencing (ITS only), were removed. In ITS, putative pseudogenes were defined as having one or more of the following conditions: (1) loss of one or more conserved motifs in ITS1 and 5.8S (Liu & Schardl, 1994; Jobes & Thien, 1997; Harpke & Petersen, 2008), (2) one or more mismatch(es) in the 5.8S conserved motifs, (3) two or more mismatch base pairings in the hairpin structure of the ITS1 conserved motif, or (4) the presence of a ≥ 20 bp deletion (when compared with other intragenomic clones/intraspecies sequences) since this may cause instability in the secondary structure. The putative pseudogenes and their recombinants were removed from subsequent analyses; recombinants lack the conditions mentioned above, but have partial sequences identical to those of the putative pseudogenes in other regions not scrutinized, i.e., ITS2 and other parts of ITS1 and 5.8S. Two copies of g3pdh were retrieved from Asian Piper and each copy was treated as a separate marker, A and B. Only a single copy was detected in the South Pacific and Neotropical Piper (this study; J.F. Smith, pers. comm.; Smith & al., 2008). In addition to complete datasets that include all clones and direct sequences, simplified datasets containing a single sequence per individual were assembled to make nuclear datasets comparable with each other and to the chloroplast dataset. Preliminary phylogenetic analyses of ITS and g3pdh showed that most clones clustered together with other intragenomic clones or their conspecific clones. The single sequences chosen were 14 the ones showing the fewest mutations, and/or representing the majority sequences if the copy was represented by more than one clone sequence. Phylogenetic analyses. The data partitions for ITS and trnL-F were defined a priori, that is ITS1, 5.8S, and ITS2 for ITS, and trnL intron, trnL, and trnL-trnF intergenic spacer for trnL-F. The boundaries of the regions in ITS were inferred based on the comparison with published sequences (Yokota & al., 1989; Baldwin & al., 1992), while those of trnL-F were determined based on comparison with the published chloroplast sequence of P. cenocladum (Cai & al., 2006) annotated by DOGMA (Wyman & al., 2004). G3pdh was treated as a single partition and analyzed as nucleotide sequences. Phylogenetic relationships among species were estimated using Bayesian and maximum likelihood approaches. Bayesian analyses were carried out using MrBayes v.3.2.6 (Ronquist & al., 2012) on the CIPRES cluster (https://www.phylo.org/). The appropriate data partitioning scheme and the best model of sequence evolution for each partition and marker were assessed using the corrected Akaike information criterion in PartitionFinder v.2.1.1 (Lanfear & al., 2016). The best partitioning scheme and best-fitting model of sequence evolution is GTR + I + Γ for ITS1 + ITS2 and SYM + I + Γ for 5.8S, GTR + I + Γ for g3pdh, and GTR + Γ for trnL intron + trnL + trnL-F spacer. Analyses were performed in four separate runs, each with four Markov chains, for as many as 25 million generations, sampled every 1000 generations and terminated after the four tree samples converged indicated by an average standard deviation of split frequencies < 0.01. The majority-rule consensus tree was obtained after excluding the first 25% of the trees. Maximum likelihood analyses were carried out using RAxML v.8.2.9 on the CIPRES cluster, using the GTR + Γ model of evolution (Stamatakis, 2014). 15 The partitioning schemes in these analyses followed those produced by PartitionFinder. Support for the optimum maximum likelihood tree was assessed using a rapid bootstrap analysis of 1000 replicates. Phylogenetic node support values of ≥ 85 maximum likelihood bootstrap percentage (BP) and ≥ 0.98 Bayesian posterior probability (PP) were considered strong, 70–84 BP and 0.95–0.97 PP were considered moderate, and 50–69 BP and 0.90–0.94 PP were considered weak. These values were based on simulation studies by Hillis & Bull (1993), Erixon & al. (2003), and Huelsenbeck & Rannala (2004). Phylogenetic trees were visualized using Dendroscope v.3.5.9 (Huson & Scornavacca, 2012) and FigTree v.1.4.3 (Rambaut, 2016). Shimodaira-Hasegawa (SH) and Shimodaira approximately unbiased (AU) tests, implemented in PAUP* v.4.0a152 (Swofford, 2002), were performed to test the topological congruence among genetic markers. Similar tests were also performed to test the monophyly of conflicting clades among genetic markers by examining alternative suboptimal topologies from constrained trees. Constrained phylogenetic analyses were carried out in RAxML using the settings described above. Character distribution and evolution. The distribution of six characters were examined for Paleotropical Piper. Prior to the analyses, the tree was pruned so that each species was represented by only a single individual. The character states were obtained from the literature and from herbarium studies (Electr. Suppl.: Table S2). Bract, fruit, and stamen morphology have been used to delimit infrageneric groups within Paleotropical Piper, and the character states were plotted on the phylogenetic trees to understand their utility as diagnostic characters. Bracts were scored as round to elliptic and peltate (0), oblong and adnate to the rhachis, with free margins (1), or completely adnate to the 16 rhachis (2, including cupular bracts). Ripe fruits were scored as sessile and free (0), partially concrescent (1), fully concrescent (2, the fruits are concrescent more than half way), or free with constricted base (3, stalked fruits). Stamen characters explored in this study included the number of valves by which the sporangia opened, whether two (0) or four (1), and the direction of the dehiscence, lateral (0) or apical (1, = extrorse, see Candolle, 1869). The anthers of Piper are very small and how they dehisce may be difficult to determine. When there is no distinct direction and the anther thecae are longer than wide and/or at an angle of < 90° to each other, dehiscence was coded as “lateral”; when the anther thecae are about the same length vertically and horizontally, dehiscence was interpreted as “apical”. Stamen number, two or three stamens, is mostly invariable within Paleotropical Piper, exceptions being in monotypic Sarcostemon (P. quinqueangulatum, one stamen) and Muldera (two to ten stamens), so its use was not explored further. Plotting of these four characters on the phylogenetic trees was carried out using ape, geiger, and phytools packages in R version v.3.0.2 (R Development Core Team, 2014; script written by Isabel Loza, University of Missouri St. Louis). Although most species of Asian Piper are dioecious climbers, some have bisexual flowers and/or a shrubby habit. The species were assigned as being strictly bisexual (0), polygamous (1, having populations with bisexual and unisexual flowers), or strictly dioecious (2). Habit types were assigned based on the presence of roots on the nodes along the shoots, either shrubs/ treelets (0, without roots) or climbers (1, with roots). Sexuality and habit type were mapped on the phylogenetic trees and the evolution of bisexual flowers and shrubby habit was examined using Saururaceae as the outgroup. Character state distribution and evolution were explored using the likelihood approach in Mesquite v.3.3 (Maddison & Maddison, 2017); the Markov k-state 1 parameter model 17 was chosen to perform ancestral character reconstruction. § RESULTS Molecular dataset. Sequences of all three markers were obtained for 118 individuals of 75 Paleotropical species. Within that number, however, only 38 individuals of 28 species from the Asian clade have both copies of g3pdh (see section “g3pdh data”). The characteristics of all molecular datasets used in this study are given in Table 2. ITS data. Eight hundred and ninety-eight clones and 65 PCR products of Paleotropical Piper were sequenced from 145 individuals in this study. Forty-six individuals have both clones and direct sequences. All 56 sequences obtained from GenBank were products of direct sequencing. Putative pseudogenes and recombinants were detected in 147 sequences. Two putative pseudogenes were detected in direct sequences from P. betle JFS5808 (GenBank accession: EU581126) and P. ramipilum C.DC. WSW3 (GenBank accession: EU581346). In four individuals, P. caninum RA512, P. majusculum Blume RA197, P. mollissimum Blume RA458 and P. sp. RA809, putative pseudogenes alone were recovered from cloning, but a putative functional paralog was recovered from direct sequencing of P. mollissimum RA458. In preliminary phylogenetic analyses, the divergent paralogs were either clustered with conspecific putative functional paralogs, or clustered with closely related species, albeit separated from their conspecific putative functional paralogs, or forming distinct clades with other putative pseudogenes from different species. Sequences which have mismatch(es) in the 5.8S conserved motifs were usually considered to be putative pseudogenes and removed; however, some mismatches, presented hereafter, in these 18 conserved motifs seem to characterize clades and were kept in the analyses. The last base of a 14 bp-M2 motif (Harpke & Petersen, 2008) in a subclade within E2 (P. chlorostachyum C.DC. RA202, P. novoguineense Warb. CH1875m, and P. subcanirameum RA425) and in clade Peltandron (based on Peltandron Miq.; P. celtidiforme Opiz RA538, also including sequences from GenBank not included in this study [see Results: Phylogenetic analyses]) has changed from C to A. In clade E1, the first base of the 10 bp-M3 motif (Harpke & Petersen, 2008) has changed from T to C. Additionally, Peperomia spp. and Zippelia begoniifolia have substitutions in the 16 bp M1 motif at base 13 (from C to T) and in the last base (from C to T), respectively. After removing identical sequences, putative pseudogenes, and recombinants, the complete ITS dataset (Paleotropical species) produced in this study comprises 641 sequences from 200 individuals of 103 species (Table 2). The number of putative functional paralogs within cloned individuals are 2–10 (the latter in P. austrocaledonicum C.DC. JM677 and P. bojonyum C.DC. RA541), and the uncorrected distances (p-distance) among those paralogs vary from 0.2% to 3.8% (the latter in P. puberulirameum C.DC RA616), but are mostly below 1%, a level that could be attributed to polymerase errors (Speksnijder & al., 2001). Multiple distinct sets of paralogs (i.e., a group of sequences with similar mutations) were observed in some individuals, like P. puberulirameum RA616 and P. fragile RA438 (p-distance = 1.8%). Putative functional paralogs from the same individual or species cluster together in phylogenies, except in P. bojonyum RA537, P. flavimarginatum C.DC. SING2015-105, P. pentandrum RA593, P. puberulirameum RA616, P. rubro-venosum hort. ex Rodigas RA764, P. sarcopodum C.DC. RA862, and P. sp. RA547 (Electr. Suppl.: Fig. S4). The reciprocal monophyly of the paralogs of these individuals is not supported; furthermore, there is weakly supported 19 separation of a paralog in P. rubro-venosum RA764 (51 BP/– PP and 63 BP/– PP). In the simplified dataset, variation among Asian and South Pacific species (Kimura 2-parameter distance) is 7.6% and 6.2%, respectively (Table 2), slightly lower than that of Neotropical species used in this study (8%, from 125 individuals of 74 species). g3pdh data. Sequences of g3pdh were cloned from 114 individuals of 75 species of Paleotropical Piper resulting in 638 sequences, while 15 sequences obtained from GenBank were all products of direct sequencing. Upon removal of identical sequences, the g3pdh complete dataset comprises 550 sequences from 129 individuals of 79 species. Copy A and B were retrieved as sister clades, each with strong support (96 BP/1.0 PP and 97 BP/ 1.0 PP, respectively; Fig. 4; Electr. Suppl.: Fig. S2). Direct sequences clustered either with copy A or B clades. Both copies differ by point mutations and indels, but species in clades Muldera (based on Muldera Miq.), Cubeba s.str. (based on Cubeba Raf.), W2, W6, and W5, and P. sp. RA863 appear to have lost about 52 bp (38–64 bp) in the 3′ region of their copy A. Copy B was most commonly recovered, being found in 109 individuals compared to only 54 individuals with copy A, and some clades, clades W1, E2, E3, and Peltandron, appeared to have only copy B. In P. bantamense Blume, copy A alone was recovered from one individual, the other three individuals had copy B only. In P. austrocaledonicum, all 9 clones in this study (JM677) were copy A, while the direct sequence from GenBank (GM19190) was copy B. Only 41 individuals of 28 species produced both copies, and there the Kimura 2-parameter distance between the copies ranged from 2.2% (P. oreophilum Ridl. RA584) to 16% (P. pedicellosum Wall. RA644, P. ribesioides Wall. RA635). Recombination between copies A and B was found in P. fragrans Trel. RA409. 20 One to seven paralogs were retrieved in copy A per individual, and the uncorrected distances among those paralogs vary from 0.1% to 3.3% (the latter in P. celebicum RA448). Phylogenetic analysis with this complete dataset shows that most paralogs from the same individual or species cluster together, except P. baccatum Blume RA116, P. bantamense RA464, P. caninum (6 individuals), P. cilibracteum C.DC. (3 individuals), P. cubeba L.f. RA161, P. flavimarginatum SING2015-105, and P. oreophilum RA584 (Electr. Suppl.: Fig. S5). While the reciprocal monophyly of P. baccatum RA116 and P. oreophilum RA584 has no support, the paralogs of the other species show strong paraphyly (Electr. Suppl.: Fig. S2). In copy B, 1–8 paralogs (the latter in P. abbreviatum Opiz RA359) are retrieved per individual, and the uncorrected distances among those paralogs are 0.1%–3.9% (in P. febrifugum C.DC. RA578). Again, most of the paralogs from the same individual or species cluster together. However, the paralogs of P. betle (8 individuals), P. celebicum RA448, P. rostratum RA479, P. sp. RA873, and P. sp. MC1080 are not reciprocally monophyletic or show weak paraphyly, while those of P. caninum (5 individuals), P. oreophilum RA584, P. quinqueangulatum RA482, P. ramipilum RA473, P. rubro-venosum RA764, and P. sp. RA547 show weak to strong paraphyly (Electr. Suppl.: Fig. S5). In the simplified dataset, variation among the South Pacific species and Asian copy A (Kimura 2-parameter distance) is 8% and 7.1%, respectively (Table 2), slightly higher than that of Neotropical species used in this study (6.5%, from 34 individuals of 24 species), but that of Asian copy B is as low as 3.7%. trnL-F data. The region was sequenced in 142 individuals of 78 species. GenBank added 15 individuals of 12 species, of which 4 were species I had not sequenced, making the total 157 individuals from 82 species. Variation among Asian and 21 South Pacific species (Kimura 2-parameter distance) are 1% and 2.2%, respectively (Table 2), lower than that of Neotropical species used in this study (2.7%, from 34 individuals of 24 species). This chloroplast marker has the lowest variation among the markers used in this study. Phylogenetic analyses. Both Bayesian and maximum likelihood analyses produced trees with similar topologies. In general, ITS (Fig. 2; Electr. Suppl.: Figs. S1, S4) produced the most resolved phylogenies with strongly supported branches, followed by g3pdh (Fig. 3; Electr. Suppl.: Figs. S2, S5). The backbones of the trees, particularly the ITS trees, also received moderate to strong support. The chloroplast tree, on the other hand, lacked backbone support, although a few clades within Asian Piper were well supported (Fig. 4; Electr. Suppl.: Fig. S3). Using pairwise comparisons, all four markers showed significantly different topologies from each other (P < 0.01), and upon visual inspection the discrepancies included moderately to strongly supported branches at a variety of levels. Because hard incongruences were detected, the trees were not concatenated but were analyzed separately (Seelanan & al., 1997). The following discussion of the ITS and g3pdh phylogenies is based on analyses of simplified datasets (Figs. 2, 3; Electr. Suppl.: Figs. S1, S2), and results from analyses of the complete datasets (Electr. Suppl.: Figs. S4, S5) will be mentioned only if they differ substantially. The phylogenies corroborate previous phylogenetic studies on the relationships among genera in Piperaceae, supporting the monophyly of Piper, the resolution of three major biogeographic groups within Piper, and the non-monophyly of the largest Asian infrageneric groups, namely Chavica sect. “Euchavica”, Piper Miq., and Cubeba (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Asmarayani & Pancoro, 2006; 22 Wanke & al., 2007a; Jaramillo & al., 2008; Smith & al., 2008) (Figs. 2–4; Electr. Suppl.: Figs. S1–S3). Within Piperaceae, Manekia is resolved as sister to Zippelia, but with only weak to moderate support (ITS: 50 BP/– PP; trnL-F: 79 BP/ 0.92 PP), and this clade is sister to the Peperomia + Piper clade. In all nuclear phylogenies, Piper is resolved as a monophyletic group (ITS: 89 BP/ 1.0 PP; g3pdh: 100 BP/ 1.0 PP) and sister to Peperomia. Although the trnL-F phylogeny does not support the monophyly of Piper, Piper + Peperomia formed a monophyletic group (100 BP/ 1.0 PP). Within Piper, only the Asian clade is strongly supported as a monophyletic group in all phylogenies (ITS: 99 BP/1.0 PP, g3pdh: 99 BP/ 1.0 PP, trnL-F: 85 BP/ 1.0 PP; Figs. 2–4; Electr. Suppl.: Figs. S1–S3). Piper borneense and P. fragile (Macropiper sensu Candolle, 1923) are part of the Asian clade. All phylogenies also show that shrubby species from highland New Guinea are more closely related to P. methysticum, part of the South Pacific clade (Jaramillo & Manos, 2001), than to other species in the Asian clade. The monophyly of the whole South Pacific clade is supported in ITS and g3pdh phylogenies (94 BP/1.0 PP and 83 BP/ 1.0 PP, respectively), but not in the trnL-F phylogeny. In the trnL-F phylogeny, P. methysticum and shrubby species from highland New Guinea are weakly supported as a clade sister to the Asian clade (60 BP/– PP), and this combined clade forms a strongly supported clade with other Piper from the South Pacific and Africa (i.e., the Paleotropical clade, 95 BP/ 1.0 PP). Constraining all species from New Guinea, South Pacific, and Africa to be a monophyletic group in the trnL-F phylogeny cannot be rejected with SH and AU tests. There is also support for the monophyly of Paleotropical Piper in the ITS phylogeny (72 BP/ 0.99 PP), but not in the g3pdh phylogeny. Constraining both Asian and South Pacific clades to be monophyletic in the g3pdh phylogeny, however, cannot be rejected by the SH and AU tests. The 23 Neotropical species are not recovered as monophyletic in any phylogeny, Paleotropical Piper being embedded within a Neotropical grade. Nevertheless, constraining Neotropical species to be monophyletic cannot be rejected by the SH and AU tests. The South Pacific clade is biogeographically structured in all phylogenies, being made up of the South Pacific (= Macropiper s.str., based on Macropiper Miq.), New Guinean, and Coccobryon s.str. (based on Coccobryon Klotzsch) clades (Figs. 2–4; Electr. Suppl.: Figs. S1–S3). Piper capense, the only member of Coccobryon s.str., is resolved as sister to the rest of the South Pacific clade in the g3pdh phylogeny (support for the rest is 100 BP/ 1.0 PP), but only very weakly supported as sister to the Macropiper s.str. clade in the ITS phylogeny (55 BP/– PP). Within the Asian clade, species predominantly distributed west of Wallace’s line (WWL) form a moderately/strongly supported clade in the ITS phylogeny (77 BP/ 1.0 PP), embedded within a grade of species from east of Wallace’s line (EWL). However, monophyly of the WWL species is only weakly supported in the g3pdh A phylogeny (– BP/ 0.94 PP) and is not supported in the g3pdh B and trnL-F phylogenies. EWL species never form a monophyletic group, but monophyly of EWL species in the ITS analysis cannot be rejected by the SH and AU tests. The ITS phylogeny resolves 12 and 5 clades, each with at least two species, in the WWL clade and the EWL grade, respectively (Table 3, Fig. 2; Electr. Suppl.: Fig. S1). However, of these 17 Asian clades, only 5, 12, and 5 clades are supported in the g3pdh A, g3pdh B, and trnL-F phylogenies, respectively (Figs. 3, 4; Electr. Suppl.: Figs. S2, S3). The W5 clade (of the WWL clade), as well as the E1 and E4 clades (of the EWL grade), receive moderate to strong support in all trees. In the EWL grade, Peltandron (based on Peltandron Miq.) is represented by only P. celtidiforme from Sulawesi. However, in 24 preliminary results, it formed a clade with other representatives of Peltandron with sequences from GenBank, the Philippines P. celtidiforme and P. penninerve C.DC. (GenBank accessions: EU581148, AF275205, AF275206; ITS: 100 BP/ 1. 0 PP). Nevertheless, since the voucher specimens of those species were not available for this study, they were excluded from subsequent analyses. Besides P. celtidiforme, six species are not consistently resolved as part of any clade (unaffiliated species, hereafter), namely P. buruanum Miq. RA196 (EWL), P. uncinulatum Ridl. RA599 (WWL), P. sp. RA568 (WWL), P. sp. RA809 (WWL), P. sp. RA838 (WWL), and P. sp. RA863 (WWL). Among the three major non-monophyletic infrageneric groups in Asian Piper, Cubeba is found exclusively in the WWL clade, where it forms two closely related clades, Cubeba s.str. and W6, while P. griffithii C.DC. is part of the W2 clade otherwise made up predominantly by species of Piper Miq. (Figs. 2, 3; Electr. Suppl.: Figs. S1, S2, Table S1). Most Piper Miq. species are found in four clades within the WWL clade, the W1, W2, Piper sensu Miquel (1844) s.str., and W5 clades, while P. buruanum is the only member found in the EWL grade although it is unplaced there (note that although P. cilibracteum of W5 clade was described by Candolle, it was published by Koorders (1908) who placed it in Chavica, but his concept differed from that of Miquel—see Electr. Suppl.: Appendix S1). Chavica sect. “Euchavica” (including Hooker’s, 1886, Koorders’s, 1908 & 1912, and Ridley’s, 1924, sect. Chavica in Figs. 2–4; Electr. Suppl.: Figs. S1–S3) is the most scattered in that its species are found in 4 clades—W1, W3, W4, and Euchavica s.str. (based on “Euchavica ” Miq.)—in the WWL clade and in all 5 clades—E1, E2, E3, E4, and E5—in the EWL grade. The EWL grade consists mostly of species from Chavica (“Euchavica”) in addition to Peltandron and one species of Piper Miq., while the WWL clade includes 25 species from all infrageneric groups used in this study (Peltandron from WWL clade is represented by P. sp. RA863 [Fig. 1L, unaffiliated species], placed there because of its enlarged anther connective). Within the WWL clade, Muldera (Muldera clade, hereafter) is a strongly supported monophyletic group in the ITS phylogeny (100 BP/ 1.0 PP; Fig. 2; Electr. Suppl.: Fig. S1, Table S1), albeit with the inclusion of the Philippines P. medinillifolium Quisumb. MAJ196 (which is morphologically similar to “Euchavica”), and weakly to strongly supported monophyletic group in the g3pdh B complete dataset phylogeny (57 BP/ 1.0 PP; Electr. Suppl.: Fig. S5). Pseudochavica Ridl. is non-monophyletic in that P. boehmeriifolium (Miq.) Wall. ex C.DC. (in the W1 clade) and P. uncinulatum (unplaced) do not cluster with the other species; on the other hand, P. rostratum, previously placed in various infrageneric groups including Pseudochavica Ridl. and Rhyncholepis, forms a strongly supported clade with P. cyrtostachys Ridl. and P. oreophilum in the remaining Pseudochavica Ridl. (Pseudochavica sensu Ridley [1924] s.str. clade, hereafter; ITS: 98 BP/ 1.0 PP, g3pdh B: 93 BP/ 1.0 PP, trnL-F: 86 BP/ 1.0 PP; Figs. 2–4; Electr. Suppl.: Figs. S1–S3, Table S1). Beside P. rostratum, P. sp. RA838 has elongated styles so I placed it in Rhyncholepis. However, the two species are not monophyletic. Species with the characteristics of Pseudochavica Hook.f. (= Chavica sensu Koorders, 1908 in part; e.g., P. arcuatum Blume, P. cilibracteum) are not monophyletic. Piper quinqueangulatum, the monotypic Sarcostemon, is in the WWL clade and forms a strongly supported clade with P. brevifolium C.DC. (Sarcostemon s.l. clade, hereafter) in all nuclear phylogenies (99–100 BP/ 1.0 PP; Figs. 2, 3; Electr. Suppl.: Figs. S1, S2, Table S1). Representatives of Coccobryon included in this study are scattered in distantly related clades in Paleotropical Piper: P. capense and P. methysticum are part of the South Pacific clade, and P. rostratum is part of the WWL clade (Figs. 2–4; 26 Electr. Suppl.: Figs. S1–S3, Table S1). When considering only subgenera within Candolle’s system (1923), the presence of Muldera, Sarcostemon, and Coccobryon among “Eupiper” sensu Candolle (1923—it included Miquel’s [1843–1844] Piper Miq., Cubeba, Chavica, and Rhyncholepis) made “Eupiper” paraphyletic (Figs. 2–4; Electr. Suppl.: Figs. S1–S3, Table S1). Chavica sensu Candolle (1869) also is not monophyletic: P. blumei (Miq.) Backer is part of the W4 clade, while P. sarmentosum and P. thomsonii (C.DC.) Hook.f. are part of the W1 clade (Fig. 2; Electr. Suppl.: Fig. S1, Table S1). Finally, Engler’s (1889) Oligandropiper is not monophyletic: P. methysticum is part of the South Pacific clade, P. retrofractum Vahl and P. macropiper are part of the EWL grade, and P. betle, P. nigrum, and P. sarmentosum are part of the WWL clade (Figs. 2–4; Electr. Suppl.: Figs. S1–S3, Table S1). Relationships among the clades in the EWL grade are unclear. The E1 clade is resolved as sister to the rest of the Asian clade in the g3pdh B and trnL-F phylogenies (support for the rest of the Asian clade is 77 BP/ 0.99 PP and –/0.96 PP, respectively; Figs. 3, 4; Electr. Suppl.: Figs. S2, S3). The unplaced P. buruanum is sister to the E4 clade in the ITS and g3pdh B phylogenies (72 BP/ 0.99 PP and 88 BP/ 1.0 PP, respectively; Figs. 2, 3; Electr. Suppl.: Figs. S1, S2). The E2, E3, and Peltandron clades form a weakly supported clade in the ITS complete dataset phylogeny (51 BP/– PP; Electr. Suppl.: Fig. S4), but not in the simplified dataset phylogeny. In contrast to relationships within the EWL grade, clade relationships within the WWL clade show some phylogenetic structure, with most support being in the ITS phylogeny (Fig. 2; Electr. Suppl.: Fig. S1). The W1 clade is resolved as the sister to the rest of the WWL clade in the ITS phylogeny (support for the rest of the WWL clade is 86 BP/ 1.0 PP). Sarcostemon s.l., the W2 clade, and P. uncinulatum form a weakly to 27 strongly supported clade (ITS: 78 BP/ 1.0 PP, g3pdh B: 67 BP/ 0.98 PP). Relationships among them, however, are incongruent: in the ITS phylogeny, P. uncinulatum is sister to the W2 clade (80 BP/0.91 PP), but it forms a clade with Sarcostemon s.l. in the g3pdh B and trnL-F phylogenies (99 BP/ 1.0 PP and 59 BP/– PP, respectively; Fig. 3, 4; Electr. Suppl.: Figs. S2, S3). In the ITS phylogeny, eight clades—W3, W4, Euchavica s.str., Piper sensu Miquel (1844) s.str., W5, W6, Cubeba s.str., Muldera—and three unaffiliated species—P. sp. RA568, P. sp. RA838, P. sp. RA863—form a strongly supported clade (84 BP/ 1.0 PP) within which P. sp. RA568 is resolved as sister to the rest (74 BP/0.98 PP). Furthermore, Piper sensu Miquel (1844) s.str., W5, W6, Cubeba s.str., Muldera, P. sp. RA838, and P. sp. RA863, form a moderately to strongly supported clade (74 BP/ 1.0 PP). Within that clade, P. sp. RA838 is resolved as sister to the W6 clade (72 BP/ 1.0 PP), and this relationship has some support in the trnL-F phylogeny, in that P. sp. RA838 forms a clade with some species of the W6 clade (61 BP/ 0.99 PP) although the W6 clade as a whole is not supported as monophyletic. In the g3pdh B phylogeny, however, P. sp. RA838 is sister to the W5 clade, albeit very weakly supported (57 BP/– PP). Slightly different clade relationships are recovered in the g3pdh A phylogeny, where P. sp. RA863 and five clades—Piper sensu Miquel (1844) s.str., W2, W5, W6, Cubeba s.str., Muldera—form a moderately to strongly supported clade (82 BP/ 1.0 PP). Within that clade, Piper sensu Miquel (1844) s.str. is resolved as sister to the rest (69 BP/ 0.98 PP), and the [Muldera + P. sp. RA863], [Cubeba s.str. + W2], and [[Cubeba s.str. + W2] + W5] clades are all strongly supported (100 BP/ 1.0 PP, 100 BP/ 1.0 PP, and 88 BP/1.0 PP, respectively). Here, incongruity occurs regarding the position of Cubeba s.str. and P. sp. RA863. The g3pdh B phylogeny weakly to strongly supports [[Cubeba s.str. + Muldera] + W6] (57 BP/ 1.0 PP), while the trnL-F phylogeny weakly 28 supports [Cubeba s.str. + P. sp. RA863 + Muldera] (58 BP/ 0.94 PP). In trnL-F phylogeny, P. sp. RA863 is sister to P. mollissimum RA458, the clade being part of a tetratomy that is Cubeba s.str. (61 BP/ 0.99 PP). Comparing the four phylogenies, relationships among species within each clade show some strongly supported incongruences. Hence, P. celebicum, P. fragrans, P. majusculum, P. febrifugum, and P. abbreviatum are always included in the E1 clade, but in the g3pdh A phylogeny, P. febrifugum is resolved as sister to the rest (support for the rest is 76 BP/ 0.99 PP; Fig. 3; Electr. Suppl.: Fig. S2), while in the ITS phylogeny, [P. abbreviatum + P. sp. MAJ194] is resolved as sister to the rest (support for the rest is 98 BP/ 1.0 PP; Fig. 2; Electr. Suppl.: Fig. S1). In the g3pdh B and trnL-F phylogenies, the E1 clade shows no to little backbone support, but P. febrifugum forms a clade with P. majusculum RA551 and RA362 (the latter in the trnL-F phylogeny only; 100 BP/ 1.0 PP and 62 BP/ 0.99 PP, respectively; Figs. 3, 4; Electr. Suppl.: Figs. S2, S3), also in the ITS phylogeny (100 BP/ 1.0 PP). In the E4 clade of the ITS phylogeny, P. macropiper, P. aff. macropiper, and P. cf. dumiformans C.DC. form a clade with P. truncatibaccum C.DC. (91 BP/ 1.0 PP), but the three species form a clade with P. arfakianum C.DC. in the g3pdh B phylogeny (74 BP/ 1.0 PP). While most species with multiple individuals—sometimes from different localities—show reciprocal monophyly, there are a few exceptions with strong to moderate support: P. muricatum Blume (see ITS and g3pdh B phylogenies; Figs. 2, 3; Electr. Suppl.: Figs. S1, S2), and P. cilibracteum and P. caninum (see g3pdh A phylogeny; Fig. 3; Electr. Suppl.: Fig. S2). In addition, in the trnL-F phylogeny, P. muricatum WSW2 (GenBank accessions EU519796 and EU519616) is more closely related to the W4 clade (85 BP/ 1.0 PP) than to other P. muricatum individuals or the 29 other species of the W6 clade (dashed arrows in Fig. 4 and Electr. Suppl. Fig. S3). A similar pattern, but with weaker support, is also shown by P. nigrum WSW1 (GenBank accessions EU519773 and EU519593), which is more closely related to the W6 clade (– BP/ 1.0 PP) than to other P. nigrum individuals or the other species of the Piper sensu Miquel (1844) s.str. clade (solid arrows in Fig. 4 and Electr. Suppl. Fig. S3). The chloroplast phylogeny also shows some distinctive geographical patterns in that P. cilibracteum from Java is more closely related to P. acre Blume from Java (83 BP/ 1.0 PP) than to other P. cilibracteum from the Malay Peninsula, and P. quinqueangulatum from Java and Sumatra are more closely related to P. brevifolium from Java (86 BP/ 1.0 PP) than to other P. quinqueangulatum from Malay Peninsula. Character distribution and evolution. Character states (Electr. Suppl.: Table S2) were plotted or mapped on the ITS phylogeny, the most resolved tree, of 96 Asian and 8 South Pacific species. Most species in this phylogeny are reciprocally monophyletic (see section “ITS data” for species with unsupported reciprocal monophyly and Appendix 1 for specimens chosen). Habit, sexuality, bract type, and fruit type have only 1.9%, 5.8%, 5.8%, and 11.5% missing data, respectively, the missing data being mostly because specimens are juvenile or sterile or the species has never been found in flower (i.e., P. rubro-venosum). There are more missing data for direction of anther dehiscence and number of anther valves, 42.3% and 41.3%, respectively, because many species of Piper have been described from a single sex alone, mostly from female plants, or male specimens were not available in this study. In some cases, only by synonymizing species based on material from separate sexes could the character states from both staminate and pistillate flowers be scored. This taxonomic work will be published separately 30 (Asmarayani, in prep.). Most species of Paleotropical Piper have peltate and round to elliptic bracts (Fig. 5; Electr. Suppl.: Fig. S6). Other bract morphologies are found only in subsets of WWL species. Oblong bracts adnate to the rhachis but with free margins occur in all species in Sarcostemon s.l. and some species in Cubeba s.str., W5, Piper sensu Miquel (1844) s.str., and W2 clades, while completely adnate bracts occur in all species of Muldera (but not P. medinillifolium, see Discussion) and P. ribesioides of Cubeba s.str. Most South Pacific species have sessile and free fruits, except P. excelsum G.Forst. of Macropiper s.str. which has fully concrescent fruits (Fig. 5; Electr. Suppl.: Fig. S6). In the Asian species, fruit variation is more extensive. In general, most clades are characterized by a single fruit morphology: Muldera, W5, Piper sensu Miquel (1844) s.str., Sarcostemon s.l., and E4 clades have sessile and free fruits, Pseudochavica sensu Ridley (1924) s.str. clade has partially concrescent fruits, Euchavica s.str., W4, E3, E2, and E1 clades have fully concrescent fruits, while the W6 and Cubeba s.str. clades have free fruits with constricted bases. Nevertheless, some clades comprise species with two fruit types. Both partially and fully concrescent fruits are found in the W3 and E5 clades, free fruits both with and without constricted bases in the W2 clade, and sessile fruits both free and partially concrescent in the W1 clade. Apical anther dehiscence is found in many clades across the Paleotropical Piper (Fig. 5; Electr. Suppl.: Fig. S6). Some clades seem to have exclusively lateral or apical dehiscence, thus the W3 clade has lateral dehiscence and Euchavica s.str., W2, E5, and New Guinean clades have apical dehiscence (note, however, there are missing data, and the conclusions may break down). The most common number of anther valves is two, and four-valved anthers occur in at least six clades of Paleotropical Piper. No clade has 31 exclusively four-valved anthers. Polygamy has evolved at least once in the EWL grade, in P. breviamentum C.DC. of the E1 clade, and nine times in all clades in the WWL clade except the Muldera , W5, and W2 clades (Electr. Suppl.: Fig. S7). In the South Pacific clade, polygamy has evolved at least twice, in both the Macropiper s.str. and New Guinean clades, and P. capense of Coccobryon s.str. has bisexual flowers. The climbing habit is the ancestral state in the Asian Piper and all species in the EWL grade are climbers (Electr. Suppl.: Fig. S8). The shrubby habit has evolved at least six times in four clades in the WWL clade: the W1, W2, and W6 clades have some shrubby species, while all Pseudochavica sensu Ridley (1924) s.str. species sampled are shrubs. § DISCUSSION The results, mainly based on the ITS phylogenies, support the monophyly of two Paleotropical clades, the Asian and South Pacific clades, but not the monophyly of almost any infrageneric groups in Asian Piper, implying that most of the characters previously used to delimit infrageneric groups are homoplasious. Furthermore, the phylogenies provide support for biogeographic division within the Asian clade, the west of Wallace’s line (WWL) clade and east of Wallace’s line (EWL) grade, including their 12 and 5 clades, respectively, and place the shrubby New Guinean species in the South Pacific clade. Some of these results are also supported by the g3pdh and trnL-F phylogenies, but some of the relationships among and within clades are incongruent. Incongruence between relationships suggested by different phylogenetic markers implies a complicated evolutionary history for Piper, including polyploidization and potential hybridization. 32 ITS, g3pdh, trnL-F, and Piper. Despite several drawbacks, ITS has been widely used to infer phylogenies of angiosperms (e.g., Alvarez & Wendel, 2003; Nieto Feliner & Rosselló, 2007), including Piper (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Asmarayani & Pancoro, 2006; Jaramillo & al., 2008). Precautions have been taken when using ITS in this study by cloning, eliminating pseudogenes and their recombinants, and utilizing additional unlinked molecular markers, low-copy nuclear g3pdh and chloroplast trnL-F. In this study, 2–11 paralogs were retrieved from cloned individuals. Of these paralogs, in addition to direct sequences, putative ITS pseudogenes and their recombinants were detected in Asian Piper, mostly through cloning; 16% of all clone sequences produced were pseudogenes and their recombinants, compared to 2.5% of direct sequences, which may indicate that the consensus ITS sequences in most individuals are putative functional paralogs. The pseudogenes and their recombinants were not detected in the South Pacific Piper, perhaps because only a few samples of this clade were included here. Although the majority of the putative pseudogenes and their recombinants do not suggest evolutionary relationships that differ from those suggested by their putative functional paralogs, some of them do form distinct clades such that if they had been used alone they would support different phylogenetic inferences (e.g., Mayol & Rosselló, 2001). In addition, the multiple copies of putative functional paralogs in a single individual indicate that concerted evolution in nuclear ribosomal DNA is incomplete in Piper. Nevertheless, most of the paralogs of an individual or species are reciprocally monophyletic, and no case of non-reciprocal monophyly is strongly supported. The g3pdh gene used in this study, gapC, is part of the g3pdh gene family which occurs in nearly all organisms and is best known for its role as a key enzyme in the 33 glycolytic pathway, among other crucial cellular processes (Wall, 2002; Nicholls & al., 2012). Within Asian Piper, two copies of gapC, A and B (different from gapA and gapB, the other genes in the g3pdh gene family), were recovered, and each has its own evolutionary trajectory. The duplication is likely to have occurred immediately before the diversification of Asian Piper, given that (a) only one copy is found in the South Pacific and Neotropical Piper, (b) Asian Piper is monophyletic, and (c) the two copies coalesce at the ancestral node of Asian Piper. However, the two copies are not recovered in all individuals of Asian Piper, which may be because cloning efforts were insufficient or because of PCR bias (e.g., Polz & Cavanaugh, 1998), although the consistent lack of copy A in all species of the Euchavica s.str., W1, E2, E3, and Peltandron clades might indicate that the loss of this copy is potentially an apomorphy of those clades. Indeed, close relationships among E2, E3, and Peltandron clades, albeit weakly supported, were suggested by the ITS phylogeny of the complete dataset (Electr. Suppl.: Fig. S4). Furthermore, within each copy of gapC , up to eight paralogs were recovered from an individual, and not all of them form reciprocally monophyletic groups with the other paralogs from that individual or species. These paraphyletic groups might be explained by incomplete lineage sorting. Hard incongruences in the relationships among clades in g3pdh A and B phylogenies (see Results; Fig. 3; Electr. Suppl.: Figs. S2, S5) may be accounted for by the problem of paralogy, involving duplication and random loss of paralogs in different clades (Sang, 2002), which has occurred after the duplication of the gapC gene, although other scenarios like hybridization cannot be ruled out. Cytological studies suggest that Paleotropical Piper are a mixture of diploids and polyploids. Indeed, ploidy levels range from 2x to 12x in Asian Piper, although most are polyploids (reviewed by Samuel, 1987; 34 Samuel & Morawetz, 1989; Mathew & al., 1999; Menachery, 1999; T.N. Praptosuwiryo., pers. comm.), while South Pacific Piper are either diploids (e.g., Macropiper s.str.: P. melchior and P. excelsum [Gardner, 1997]) or decaploids (e.g., the New Guinean P. methysticum and P. gibbilimbum [Lebot & al., 1991]). Polyploidization might explain the different copies of gapC gene in Asian Piper, but since I detected only a single copy of the gene in decaploid South Pacific Piper (P. methysticum and P. gibbilimbum, both cloned in this study), this may suggest different polyploidization processes, allopolyploidization in the Asian Piper vs. autopolyploidization in the South Pacific Piper, respectively. However, evidence of natural hybridization in Piper is not particularly convincing. Samuel (1987) found that the chromosomes of Asian tetraploid species tended to form bivalents, suggesting that they were allotetraploids; but she also suggested the possibility of autotetraploidy, the small size of the chromosomes in Piper making it easy to form bivalents (also see Lebot & al., 1991 and Tepe & al., 2004 for possible hybridization in the South Pacific P. methysticum and the Neotropical P. imperiale C.DC., respectively). Furthermore, artificial hybridization experiments carried out between commercial tetraploid P. nigrum and other tetraploid Asian species (Sasikumar & al., 1999) or diploid Neotropical species (Vanaja & al., 2008) had low success and were only partly fertile (Vanaja & al., 2008). Hard incongruence between the phylogenies of ITS and both copies of g3pdh (see Results; Figs. 2, 3; Electr. Suppl.: Figs. S1, S2, S4, S5) might also be explained by both paralogy and hybridization, although the various ways in which ITS evolves complicate the matter further. Given that putative pseudogenes and their recombinant sequences were removed and based on what we know about the occurrence of polyploids in Paleotropical Piper, there are still two concerns to be addressed regarding the evolution 35 of ITS in the group (see Álvarez & Wendel, 2003). First, ITS is located in multiple DNA arrays, and the number of arrays might be correlated with ploidy level and each array might undergo further duplication. Second, upon hybridization or lateral transfer (for possible mechanisms, see Gao & al., 2014), concerted evolution might make the ITS sequences resemble that of one of the parents only, or there might be recombination between the sequences. Chloroplast DNA has evolved slowly compared with nuclear DNA (e.g., Wolfe & al., 1987), and although it has been widely used in plant phylogenetic studies (e.g., Shaw & al., 2005), its use in phylogenetic studies of Piper has not been extensive. In their study of the phylogeny of Piperales, Wanke & al. (2007a) found that the trnK/matK region of chloroplast DNA in Piper has the lowest interspecific variation within Piperales; Piper has only one-third to half the parsimony-informative characters of Peperomia (Piperaceae, sister genus of Piper) and Aristolochia (Aristolochiaceae). Indeed, the trnL-F marker used in this study does not vary much, with only 5 out of the 16 clades recognized in the ITS phylogenies being resolved (the W3 clade is not included as it is represented only by one species in the phylogeny) and there is not much support along the backbone. TrnL-F was chosen in this study because of the ease of its amplification in Paleotropical Piper and it has slightly more variation than the other chloroplast markers tried (see Methods). Combining several chloroplast markers or using whole chloroplast genomes in the future will likely result in a better-resolved phylogeny. The hard incongruence between nuclear and chloroplast trnL-F phylogenies is mostly correlated with geography (see Results; Figs. 2–4; Electr. Suppl.: Figs. S1–S5). In the trnL-F phylogeny, individuals from adjacent areas are more closely related regardless of the species, as shown by species pairs of P. cilibracteum – P. acre and P. 36 quinqueangulatum – P. brevifolium (Fig. 4). This may suggest chloroplast capture, either through hybridization or lateral transfer. Similar causes might lead to the difference between P. muricatum WSW2 and P. nigrum WSW1 and their other conspecifics/clade members in the trnL-F phylogeny (dashed and solid arrows, respectively, in Fig. 4; Electr. Suppl.: Fig. S3), since the same individuals cluster together with their respective species/clade in the g3pdh A phylogeny. Possible chloroplast capture in Piper is also suggested by the results of Chaveerach & al. (2016) in their search for potential barcode markers for Piper of Thailand. Piper kraense Ridl., morphologically close to P. majusculum of the E1 clade, often was identical with other species in the WWL clade in the chloroplast markers examined. Systematic implications. The monophyly of Piper has been evident since the first molecular phylogenetic study on this group (Jaramillo & Manos, 2001), and it is confirmed here by the ITS and g3pdh phylogenies. Monophyly was also confirmed by the chloroplast marker trnK/matK (Wanke & al., 2007a), whereas the chloroplast marker used here, trnL-F, can only confirm a close relationship between Piper and Peperomia. Furthermore, the division of Piper into three major monophyletic clades, Neotropical, South Pacific, and Asian, has been widely accepted (Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004; Asmarayani & Pancoro, 2006; Jaramillo & al., 2008). As in the analyses of Wanke & al. (2007a) and Smith & al. (2008), this study, however, does not recover a monophyletic Neotropical clade, although monophyly cannot be rejected. In addition, the relationships among these three major groups have rarely received consistent support in previous phylogenetic studies. In their study of the diversification of Piper, Martínez & al. (2015) found that the Asian clade was sister to the rest of the genus. 37 Here, however, there is a basal Neotropical grade and also support for a sister relationship between the Asian and South Pacific clades, in agreement with Smith & al. (2008). These results, if confirmed, will have important implications for understanding the biogeography and diversification of the genus. This study presents the first insight into the phylogeny of Paleotropical Piper, particularly Asian Piper (otherwise called Piper s.str. in earlier studies of Jaramillo & al., 2008 and Wanke & al., 2007a). The results emphasize the non-monophyly of almost all infrageneric groups in Asian Piper, at the same time underlining that most of the characters previously used to delimit infrageneric groups, namely fruit and bract type, direction of anther dehiscence, and the number of anther valves, are homoplasious. However, bract and fruit types are still of some value in that many clades in Asian Piper recovered here are characterized by particular bract and fruit types (Fig. 5; Electr. Suppl.: Fig. S6). On the other hand, no clade has exclusively two or four anther valves, and whether there is any clade that has exclusively lateral or apical anther dehiscence awaits additional data, although the number of parallelisms for this character make this unlikely. The two infrageneric groups that had been explicitly delimited by these anther characters, Piper and Chavica sensu Candolle (1869), are not monophyletic, and literature and herbarium studies show that species in Piper sensu Candolle (1869) have all four combinations of the number of valves and the direction of dehiscence. Hence, P. caninum, P. celtidiforme, P. retrofractum, and P. betle, all in Piper sensu Candolle (1869), all have different combinations of these anther characters. In Neotropical Piper, seven of Miquel’s (1843–1844) infrageneric groups (two also recognized by Candolle, 1923) are supported by phylogeny (Jaramillo & al., 2008; also Jaramillo & Manos, 2001 and Jaramillo & Callejas, 2004a), and they are mostly 38 distinguished by their leaf morphology and venation, floral arrangement (i.e., loosely vs. tightly packed flowers), number of stamens, and some distinctive characters like cucullate bracts with pedicellate flowers. In Paleotropical Piper, however, not all of these characters are useful in diagnosing clades. Leaf morphology varies in these species, and while leaf shape is usually diagnostic only at the species level, leaf texture is useful in that coriaceous leaves characterize both Muldera and Cubeba s.str. Penninerved leaves are a diagnostic character for the Peltandron and E1 clades. Neotropical Piper have both loosely and tightly packed flowers, and the latter are very distinctive, forming horizontal bands along the inflorescence. In Paleotropical Piper, which predominantly have loosely packed flowers, tightly packed flowers (which result in small and tightly packed fruits in infructescences, although not forming horizontal bands) may have evolved in the E1 (e.g., P. majusculum), E2 (e.g., P. versteegii C.DC.), E4 (e.g., P. lessertianum C.DC.), and Peltandron (e.g., P. celtidiforme) clades of the EWL grade. Furthermore, stamen number in Paleotropical Piper is not as informative as in Neotropical Piper, where it can be used to distinguish Enckea Kunth. (4–6 stamens) and Pothomorphe C.DC. (2 stamens). On the other hand, bract and fruit types are not particularly diverse in Neotropical Piper (Callejas, 1986; Jaramillo & Manos, 2001). Subpeltate bracts, where the stipe is attached near the margin of the bract, are the most common bract type in Neotropical Piper (Callejas, 1986), and only Enckea and Ottonia Spreng. are characterized by having distinctive cucullate bracts (Callejas, 1986; Jaramillo & Manos, 2001; Jaramillo & Callejas, 2004a; Jaramillo & al., 2008). Similarly, most infrageneric groups in Neotropical Piper have free fruits, and concrescent fruits occur only in Isophyllon Miq. (included in Radula Miq.) and some species of Enckea (Callejas, 1986; Jaramillo & al., 2008). Another character that is often mentioned as being valuable 39 in distinguishing clades and species in Neotropical Piper is prophyll morphology (Burger, 1972; Callejas, 1986; Tebbs, 1989). In Paleotropical Piper, prophyll morphology has rarely been explored, although prophyll size may be included in species description (e.g., Gardner, 2013 [New Guinean climbers]; Cheng & al., 1999 [China]). Gardner (2003) found that there are no clear size differences supporting species in the New Guinean shrubby group (= the New Guinean clade in the South Pacific clade). Gajurel & al. (2008) in their account of Indian species found that the shape and the texture of the prophyll could be used to differentiate species. Previously, Paleotropical species with bisexual flowers had been placed in Coccobryon, but this study found that it is not monophyletic. Bisexual flowers have been observed in species in all three groups within the South Pacific clade, the E1 clade of the EWL grade, and scattered in almost all clades in the WWL clade (Electr. Suppl.: Fig. S7). Furthermore, with the exception of P. capense from the South Pacific clade which has bisexual flowers only, all Paleotropical Piper species that have individuals with bisexual flowers also have other individuals with male and/or female flowers, i.e., they are polygamous. Bisexual flowers in the Asian Piper occur when the staminodial primordia are not suppressed during the development of pistillate flowers or vice versa (Lei & Liang, 1998). In a few cases, however, these apparently bisexual flowers are functionally unisexual because pollen is not developed (e.g., in some of the bisexual flowers of P. sulcatum; Backer & Bakhuizen van den Brink, 1963) or the ovary remains sterile (e.g., in all bisexual flowers of P. philippinum Miq.; Quisumbing, 1930). The shrubby habit is rather uncommon in Asian Piper. It has evolved at least six times in four clades of the WWL clade only (Electr. Suppl.: Fig. S8). Shrubs in Asian Piper are generally much smaller and more slender than their counterparts in the 40 Neotropics (except species traditionally belonging to Neotropical Trianaeopiper Trel.) and the South Pacific, being less than 1 m tall. In addition, despite the strong correlation of dwarfism in Trianaeopiper with low light habitats (Jaramillo & Callejas, 2004b), Asian shrubs are generally found in the areas where light can penetrate, such as forest edges or by paths in the forest—conditions similar to those needed to those by most, if not all, Asian Piper. Despite the phylogenetic incongruence between different molecular markers, there is very little conflict in species membership in the 17 resolved clades. Below are the descriptions of these clades recovered in the phylogenies, and also the unaffiliated species, with potential distinctive characters for the clades. They will form the basis of future sections/subgenera in subsequent work. Note that in many cases estimates of total species numbers of these clades cannot be given, largely owing to uncertainties in species limits (see Introduction). Species limits within Paleotropical Piper have been subject to debate, originating from lumping (see Chew, 1972, 1992, 2003; Gardner 2006, 2013) and also disagreement about species limits between botanists working on the same species complex (see Gardner, 2013 vs. Chew, 1972, 1992, 2003). Muldera clade. Muldera is the only infrageneric group that has been consistently recognized by all botanists working on Asian Piper(Table 1). Its species are distinctive in having bracts that appear to be fused to the flower and form a cup-like structure (Miquel, 1843; Candolle, 1869; Quisumbing, 1930) (Fig. 1G, H). There are about 20 species in this clade distributed mainly in West Malesia and India. All are climbers, usually with stout stems, and may reach 25 m height. Their leaves are coriaceous with multiplinerve venation (i.e., plinerve venation with two or more lateral veins arising from above the 41 base of the leaf). The juveniles, often abundant on the forest floor, are easily differentiated from other Piper by their cordate leaves colored dark green above and pale green beneath and by their red-spotted stems. Muldera species are usually dioecious, but the Indian P. galeatum (Miq.) C.DC. (not sampled here) also has bisexual flowers (C.A. Barber 5485 in K). The stamens vary from two, mostly in Indian species, to ten in P. recurvum Blume (Blume, 1826), the most stamens reported from Piper. Some species of Muldera, such as P. polygynum C.DC., develop what appear to be multiple fruits from a single flower, while all other Piper have a single-seeded fruit. The inclusion of the Philippines P. medinillifolium (a sequence from GenBank) in the Muldera clade warrants further investigation. The voucher specimen examined, albeit sterile, has oblong leaves with prominent abaxial venation resembling those of P. macropiper in the E4 clade of the EWL grade. Piper medinillifolium has small free fruits and round peltate bracts with a long stipe (Quisumbing, 1930), which are also signatures of the E4 clade of the EWL grade. Cubeba s.str. clade. Cubeba s.str. forms a weakly supported clade with Muldera in g3pdh copy B and trnL-F phylogenies (54 BP/ 0.92 PP and 58 BP/ 0.94 PP, respectively; it also includes P. sp. RA863 in the trnL-F phylogeny). Vegetatively, species in this clade resemble Muldera in having coriaceous leaves with multiplinerve venation, although leaf anatomy differs between the two (Asmarayani, in prep.). They are also all climbers, often with stout stems like Muldera. Their juveniles resemble those of Muldera but the leaves are usually more elongated, and sometimes oblong. Cubeba s.str. species are mostly dioecious, but P. mollissimum rarely has bisexual flowers (Backer & Bakhuizen van der Brink, 1963). The flowers differ from Muldera in having round to 42 elongate bracts that are subsessile, but in P. ribesioides they are completely adnate to the rhachis like Muldera although they do not form a cup. The base of the fruit is constricted when mature, and this character, along with bract morphology, is shared with the W6 clade. Cubeba s.str. is mainly West Malesian, extending to Myanmar, and includes the medicinal P. cubeba. W6 clade. This clade includes the rest of genus/section Cubeba s.l., except P. griffithii (part of the W2 clade). Despite similarities in bract and fruit type with Cubeba s.str., this clade is more diverse in habit and leaf morphology. Members of this clade are either climbers or shrubs and with quite different morphologies. In one case sister species have different habits, the shrubby P. philodendron Ridl. and climbing P. curtisii C.DC. being moderately to strongly supported as sister in the ITS and g3pdh B phylogenies at least (Figs. 2, 3; Electr. Suppl.: Figs. S1, S2). Climbers in this clade are slender and generally dioecious. The shrubs can be very small, about 30 cm tall, or up to 2 m tall and stout (P. philodendron), often hairy, and some species have bisexual flowers. The leaves are ovate or oblong-obovate, membranaceous, chartaceous or subcoriaceous, with plinerve, multiplinerve, or penninerve venation and prominent venation underneath (less obvious in P. caninum and P. curtisii). In shrubby species, the peduncle and rachis are distinctively thick and robust, and the infructescences are usually erect. The bracts vary from orbicular to oblong. Climbers in this clade include P. guineense from Africa and P. caninum (Fig. 1F), widely distributed from the Solomon Islands and Australia, through Malesia, to Myanmar and India. Piper pullibaccum Trel. from New Guinea and P. tenuipedunculum C.DC. from the Philippines are also part of this clade. The shrubby species are mostly West Malesian, with many species in Borneo and some in Sumatra 43 and the Malay Peninsula. Piper sensu Miquel (1844) s.str., W5, and W2 clades. These three clades were part of Piper Miq. s.l., and have free, sessile, and more less loosely arranged fruits. An exception is P. griffithii , the inclusion of which in the W2 clade is surprising considering that its free fruit has a constricted base forming a stalk, otherwise the hallmark of Cubeba s.l. In all three clades, only about half of the species analyzed have oblong bracts which are adnate to the rhachis, a diagnostic character of Piper Miq. s.l. (Fig. 1D), while the other half have round to elliptic bracts which are peltate and sessile or with a short stipe (Fig. 1E). Species with the latter bract type would be placed in Pseudochavica Hook.f. and Chavica, instead of Piper Miq. s.l., by Hooker (1886) and Koorders (1908, 1912), respectively. The spikes are generally pendulous (more or less erect in P. kurzii Ridl. of the W2 clade) and may greatly elongate in some species, being more than 20 cm long (e.g., in P. porphyrophyllum of the W5 clade, P. flaviflorum C.DC. of Piper sensu Miquel (1844) s.str., and P. bantamense of the W2 clade). The species are mostly dioecious, except P. nigrum of Piper sensu Miquel (1844) s.str., and are mostly climbers, except P. kurzii (Fig. 1D). The leaves are generally broadly or narrowly ovate with multiplinerve venation. Piper sensu Miquel (1844) s.str. and the W5 clade are part of a polytomy, while the W2 clade is separate in ITS and g3pdh A phylogenies (Fig. 3; Electr. Suppl.: Figs. S2, S5). Further research is needed to differentiate these three clades. Species of the W5 clade are West Malesian, while those of Piper sensu Miquel (1844) s.str. and the W2 clade are distributed from West Malesia northward to subtropical areas (P. kadsura (Choisy) Ohwi from Japan) and possibly India (P. nigrum). Many species, mostly from China and India, have similar fruits and bracts. 44 Euchavica s.str., W4, and W3 clades. These three clades were part of “Euchavica ” in the WWL clade. All species are climbers, and there are polygamous species in all three clades. Juvenile leaves of all three clades, where known, are ovate and deeply cordate. The leaf venation is multiplinerved and prominent underneath. The spikes are pendulous. The bracts are always round to elliptic and subsessile to sessile, and the fruits are fully or partially concrescent. Included in Euchavica s.str. is P. betle, which has a pungent aromatic smell caused by chavibetol (Das & al., 2016), and P. bojonyum (Euchavica s.str.) and P. argyrites (W3 clade) with a similar smell. Further research is needed to differentiate these three clades. The W4 clade is West Malesian, Euchavica s.str. grows in West Malesia, Philippines, and Sulawesi, and the W3 clade grows from the Malay Peninsula to Indochina and China. Sarcostemon s.l. clade. Included in this clade are P. quinqueangulatum and P. brevifolium. They are dioecious climbers, but in P. quinqueangulatum some populations have bisexual flowers (pers. obs.). The leaves are membranaceous to subcoriaceous with multiplinerve venation which is prominent underneath. The spikes are elongated and pendulous. Both species have elliptic/oblong bracts adnate to the rachis and loosely arranged sessile free fruits, typical of Piper Miq. Piper quinqueangulatum has a single stamen with a swollen filament (Fig. 1K), a diagnostic character of Sarcostemon s.str.; the staminate spike of P. brevifolium is unknown. Both species have angular stems, uncommon in Piper. Piper quinqueangulatum is a widespread species in West Malesia and Thailand, while P. brevifolium is endemic to Java. 45 Pseudochavica sensu Ridley (1924) s.str. clade. Three species included in the analyses are small and slender shrubs: P. oreophilum and P. rostratum are generally less than 1 m tall, but P. cyrtostachys may reach 1.8 m height. Piper cyrtostachys rarely branches and has subpeltate leaves, both very unusual characters in Piper. The species are generally dioecious, but P. rostratum also has bisexual flowers (Fig. 1F). The bracts in all species are round and peltate with a short stipe, and the fruits are partially concrescent (Figs. 1C1, C2). Pseudochavica sensu Ridley (1924) s.str. is perhaps a small group (see W1 clade below), distributed in Sumatra and the Malay Peninsula up to Thailand. Piper cyrtostachys is endemic to the Cameron Highlands of the Malay Peninsula. Likely included in this clade is P. penangense (Miq.) C.DC. (not included in this study), a member of Chavica sect. Sphaerostachyon and morphologically similar to P. rostratum; Suwanphakdee & Chantaranothai (2008) distinguished the two species by the amount of pubescence. W1 clade. The species in this clade include both climbers and shrubs, both dioecious and polygamous. The infructescences vary in size from ca. 1.5 cm long in P. thomsonii (C.DC.) Hook.f. (Cheng & al., 1999) to 30 cm long in P. boehmeriifolium (Miq.) Wall. ex C.DC. (Cheng & al., 1999). The bracts are round, peltate, subsessile to sessile, and the fruits are generally free but crowded, so appearing to be concrescent. Included in this clade are the widespread Asian P. sarmentosum and P. boehmeriifolium. Piper sarmentosum is widely used in traditional cuisine and medicine. Species in this clade have many similarities with those in Pseudochavica sensu Ridley (1924) s.str. and one might think they would fall in the same clade (also see Suwanphakdee & Chantaranothai, 2008 for P. penangense and P. sarmentosum). Nevertheless, 46 phylogenetic analyses, mainly ITS (Fig. 2, Table 3; Electr. Suppl.: Fig. S1), do not recover them as sister taxa. Pseudochavica sensu Ridley (1924) s.str. seems to have diversified in the Malay Peninsula and places nearby while the W1 clade seems to have diversified to the north and west, in Indochina, China, and India. Unaffiliated species of the WWL clade: P. uncinulatum Ridl., P. sp. RA568, P. sp. RA838, P. sp. RA863, and P. sp. RA809. These five species are all dioecious climbers with round and peltate subsessile to sessile bracts. The fruits are sessile and free in P. sp. RA809, and possibly also in P. sp. RA568 and P. sp. RA838 (the specimens have immature fruits), but likely concrescent in P. uncinulatum, while the pistillate spike of P. sp. RA863 is unknown. The anthers in P. sp. RA809 and P. sp. RA863 are distinctive in that the anther connectives are enlarged (Fig. 1L), a character of Peltandron of the EWL grade. The staminate spike of P. uncinulatum and P. sp. RA838 is unknown. Piper uncinulatum and P. sp. RA568 are known from the Malay Peninsula, while the other three species are from Borneo. E5 clade. Species in this clade are all dioecious climbers, and the bisexual flowers of P. philippinum are functionally unisexual (Quisumbing, 1930; not included in this study). The leaves have multiplinerve to penninerve venation. The bracts are round, peltate, subsessile to sessile, and the fruits are partially or fully concrescent (Figs. 1B1, B2). This clade is distributed in the Philippines, New Guinea, the Bismarck Archipelago, Solomon Islands, and New Caledonia, and possibly extending to Sulawesi (P. elbertii C.DC. [Chew, 2003], not included in this study). 47 E4 clade. The E4 clade includes species previously placed in “Euchavica”, and some in Piper Miq. All species in this clade are dioecious climbers. The leaves vary in size from 5 cm long in P. subcanirameum and P. arfakianum to 50 cm in P. decumanum, but they are all chartaceous with plinerve to multiplinerve venation which is prominent underneath. The spikes are usually pendulous and can be greatly elongated, up to 64 cm long in P. decumanum, but they are erect and short, only 1.5 cm long, in P. arfakianum. The bracts are generally round and peltate with a long stipe in pistillate flowers, but the stipe is shorter in staminate flowers (see Quisumbing, 1930). The species in this clade have small, oblong, and free fruits, but tightly packed so appearing to be concrescent (Figs. 1A1, A2). This is a large clade with many undescribed species, distributed mostly in the Philippines, Moluccas, and New Guinea. Included in this clade is P. macropiper Pennant, which is more widely distributed, also being known from West Malesia and the South Pacific; Gardner (2013) synonymized several other species from New Guinea under P. macropiper s.l. E3 clade. The two species in this clade are both climbers. The juveniles are distinct in that they have subpeltate, glossy leaves that usually have white and red spots when they are in shady areas, and this makes P. rubro-venosum and P. fragile (the juvenile is mostly known as P. ornatum N.E.Br.) popular as ornamental plants. Flowers of P. rubro-venosum are unknown, but P. fragile has round, peltate bracts with short stipes, and its fruits are fully concrescent. While P. rubro-venosum has rarely, if ever, been recorded in the wild, P. fragile is mostly found in coastal areas of Sulawesi and New Guinea, to the South Pacific, with one record from Borneo (Quisumbing, 1930; pers. obs. in SAN). 48 E2 clade. All species in this clade are dioecious climbers. The leaves have multiplinerve venation. Piper versteegii C. DC. has multiple inflorescences arranged in fascicles, a reduced fertile shoot leaving the sheathing petiole alone (Chew, 1972; Gardner, 2013). The bracts are round and peltate with a short stipe, and the fruits are completely concrescent. The stigmas in this clade are atypical in that they usually are spherical, except in P. versteegii which has long filiform stigmas. Piper mestonii F.M.Bailey has bilobed stigmas, also unusual in Piper, while P. sp. RA758 has 4- to 5-lobed stigmas. This clade seems to have diversified in New Guinea and their distribution extends to Australia. E1 clade. All members of this clade are climbers, but P. abbreviatum can also be small creeping shrubs on the forest floor. Most are dioecious, except P. breviamentum which has bisexual flowers. The leaves are penninerved (less obvious in P. abbreviatum) with prominent venation underneath, and sometimes resemble those of Peltandron. Infructescences varying from short and rounded (in P. abbreviatum) to up to 50 cm long (in P. majusculum), and from erect (in P. celebicum) to pendulous (in P. majusculum). The bracts are round and peltate, with a short stipe or sessile. The fruits are fully concrescent when ripe. This clade is distributed throughout Malesia, also occurs in Thailand. Included in this clade are P. celebicum, endemic to Sulawesi, the widespread P. abbreviatum and P. majusculum, and at least two species morphologically close to P. majusculum from West Malesia/Indochina, P. febrifugum and P. kraense (the latter not included in this study). 49 Unaffiliated species of the EWL grade: P. buruanum Miq. This species is consistently supported as sister to the E4 clade in the ITS and g3pdh B phylogenies (Figs. 2, 3; Electr. Suppl.: Figs. S1, S2, S4, S5). Piper buruanum, however, differs from the E4 clade in that it has free large fruits in short abbreviated spikes. It is distributed in the Moluccas to New Guinea, and is closely related, if not similar, to P. bosnicanum C.DC. from New Guinea and the Solomon Islands. South Pacific clade. This clade comprises shrubs and treelets up to 8 m tall (Gardner, 2003) from the South Pacific (= Macropiper s.str.), Africa (= Coccobryon s.str.), and the New Guinea highlands where they grow up to 4000 m in altitude (= New Guinean clade). These form three groups in all phylogenies (Figs. 2–4; Electr. Suppl.: Figs. S1–S5), but distinguishing characters for them are hard to find. The leaves have plinerve venation in the Macropipers.str. and monotypic Coccobryon s.str. clades, and multiplinerve or plinerve in the New Guinean clade. Most species are dioecious, but monoecious individuals of P. guahamense and P. latifolium of Macropiper s.str. have been observed (Smith, 1975). While some populations of Macropiper s.str. (e.g., in P. puberulum [not included in this study] and P. guahamense; Smith, 1975) and New Guinean clades (e.g., in P. methysticum, P. recessum R.O.Gardner [not included in this study], and P. gibbilimbum; Gardner, 2003) have individuals with bisexual flowers, the African P. capense (Coccobryon s.str.) is perhaps the only species characterized by perfect flowers, thus it has traditionally been placed in Coccobryon (Candolle, 1923; Miquel, 1843). The spikes are solitary in Coccobryon s.str. and New Guinean clades (Gardner, 2003), but most often come in pairs or more and appear to be axillary in Macropiper s.str., a feature that caused it to be segregated as a separate genus in the past. 50 However, the spikes in Macropiper s.str. are actually terminal with reduced axillary branches (Gardner, 1997). The bracts are all peltate and the fruits are mostly free and tightly packed (fully concrescent in P. excelsum of Macropiper s.str.). There are 11 species recognized within Macropiper s.str. (Smith, 1975; Sykes, 1992; Green, 1993), only 1 in Coccobryon s.str., and 7 from the New Guinea highlands (Gardner, 2003; Asmarayani, in prep). § ACKNOWLEDGEMENT I especially thank Peter F. Stevens for valuable discussion, feedback, and continuous support. I also thank Elizabeth A. Kellogg for valuable discussion and feedback. Nathan C. Muchhala, Robert J.Marquis, and Richard C. Keating are thanked for providing helpful comments in the development of the manuscript. Fieldwork, herbarium study, and laboratory work were made possible with the help of the keepers, staff, and collaborators of herbaria ANDA, BO, DUKE, K, KEP, L, MO, PTBG, SAN, SING, and SRP, as well as Bantimurung-Bulusaraung National Park (Indonesia), Bogor Botanical Garden (Indonesia), Economic Planning Unit (Malaysia), Lore Lindu National Park (Indonesia), National Parks Board (Singapore), PT. Freeport Indonesia, Sabah Biodiversity Council (Malaysia), especially Eko B. Walujo, Joeni S. Rahajoe, James C. Solomon, P. Mick Richardson, Peter C. Hoch, Kate Armstrong, Charlie D. Heatubun, Khoon-Meng Wong, David Middleton, Hock-Keong Lua, Serena Lee, Leng-Guan Saw, Rafidah A. Rahman, John B. Sugau, Joan T. Pereira, Pratita Puradyatmika, Marie Briggs, Roxali Bijmoer, Nurainas, Mohammad Amir, Agus Sujadi, Imin Kamin, Jeisin Jumian, Yunita N. Esthi, and Susila. Halim Susanto and the family of Aris B. Prasetyo are thanked for help with fieldwork. Samples from Moluccas were obtained from Weda Bay Botanical Inventory Project through Peter B. Phillipson. This paper is part of my dissertation at the University of Missouri St. Louis (UMSL), which was supported by Fulbright Foreign Student Program and Whitney R. Harris World Ecology Center – UMSL. This work was funded by Des Lee Plant Systematic Laboratory – UMSL, Missouri Botanical Garden, Whitney R. Harris World Ecology Center – UMSL, Herbarium Bogoriense, Singapore Botanic Garden, PT. Freeport Indonesia, American Society of Plant Taxonomy, and International Association for Plant Taxonomy. The laboratory work was carried out at the Desmond E. Lee and Family Laboratories at the Department of Biology, UMSL, and the Plant Molecular Systematics Laboratory at Herbarium Bogoriense (Indonesia). § LITERATURE CITED Álvarez, I. & Wendel, J.F. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molec. Phylogen. Evol. 29: 417–434. Asmarayani, R. & Pancoro, A. 2005. Phylogenetic study of Piper based on ITS regions 51 of nuclear ribosomal DNA. Floribunda 2: 202–208. Backer, C.A. & Bakhuizen van den Brink, R.C., Jr. 1963. Piper. Pp. 168–173 in: Flora of Java, vol. 1. Groningen: N.V.P. Nordhoff. Baldwin, B.G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae. Molec. Phylogen. Evol. 1: 3–16. 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Within Miquel’s and Candolle’s treatments, arrows denote the movement of some species affiliations in the subsequent treatments. “ * ”, “ ** ”, and “ *** ” mark different concepts although the same name is used.Two major classification systems were recognized, that of Miquel (1843–1844; followed by Hooker, Koorders, and Ridley) and of Candolle (1923; followed by Quisumbing), while Engler (1889) combined the two systems (see Electr. Suppl.: Appendix S1 for his classification system). Engler’s Oligandropiperincluded Miquel’s Piper, “Euchavica”, and Coccobryon. Hooker, Koorders, Ridley, and Quisumbingworked on local floras so their classification systems may not include all infrageneric groups in the Paleotropical clade. “Eupiper” (Candolle, 1866, 1869, 1923), Coccobryon(Candolle, 1869, 1923) and Apopiper(Candolle, 1869) included Neotropical and Paleotropical species. All groups have representatives in Malesia bar Piperoidesand Apopiper, which grow in India, and in Australia and Mexico, respectively.57Table 2. Characteristics of molecular datasets used in this study. Calculated with MEGA 7.0.18 (Kumar & al., 2015). For ITS and g3pdh, calculations are based on simplified datasets.* based on complete sequences only.58Table 3. Support of clades (BP/PP) resolved in this study. “- -” indicates support < 0.90 PP. “ *” in Muldera in ITS phylogenies mark the inclusion of P. mediniifolium Quis., thought to be member of E4 clade (see Discussion). N/A indicates that no species in that clade is included in the phylogeny. Only a single copy of g3pdh was detected in the South Pacific clade.59HA1A2 C2C1B2B1DEFGI J K L60Fig. 1. Spike morphology in Asian Piper. A1 & A2, Free and small crowded fruits with round peltate bracts (red arrowhead) in P. truncatibaccum C.DC. (“Euchavica”), A2 is transverse section of the spike; B1 & B2, Concrescent fruits with round peltate bracts (green arrowhead) in P. retrofractum Vahl (“Euchavica”), B2 is transverse section of the spike; C1 & C2, Partially concrescent fruits with round peltate bracts (red arrowhead) in P. cyrtostachys Ridl. (Pseudochavica Ridl.), C2 is transverse section of the spike; D, Free fruits with oblong bracts adnate to the rachis but with free margins (yellow arrowhead) in P. kurzii Ridl. (Piper Miq.); E, Free large fruits with peltate bracts (yellow arrowhead) in P. puberulirameum C.DC. (Piper Miq., Pseudochavica Hook.f., Chavica Koord. in part); F, Stalked fruits with peltate bracts (red arrowhead) in P. caninum Blume (Cubeba); G, Free fruits with cupulate bracts (red arrowhead) in P. sp. RA837 (Muldera); H, Elongated styles (which become part of the fruits) and bisexual flowers in P. rostratum Roxb. (Rhyncholepis, Chavica, Piper Miq., Pseudochavica Ridl., Coccobryon), yellow arrowhead points to a stamen; I, Staminate spike of P. cyrtostachys, with the predominant stamen number and morphology of Asian Piper, 2 or 3 stamens (red arrowhead), oblong filaments with no obvious enlarged anther connective, and bivalved anthers; J, Seven stamens (red arrowhead) borne within cupular bracts in staminate spike of P. flavimarginatum C.DC. (Muldera); K, Single stamen with greatly swollen filament (red arrowhead) in P. quinqueangulatum Miq. (Sarcostemon); L, Stamens with obvious enlarged anther connectives (red arrowhead) in P. sp. RA863, similar character found in Peltandron. — All photos: R. Asmarayani.61Fig. 2. ITS phylogeny of simplified dataset. Numbers above branches are maximum-likelihood bootstrap support percentages (BP)/Bayesian posterior probabilities (PP). Only values ≥ 70 BP and ≥ 0.95 PP are shown, 50–69 BP or 0.90–0.94 PP are indicated by “-”, < 50 BP or < 0.90 PP are indicated by “- -”. Species in grey highlight are species without any consistent affiliation. Multiple individuals within the one species are collapsed when they are reciprocally monophyletic, the number within bracket indicating the number of individuals; “ * ” in the end of the tip labels or next to the clade names indicates that not all individuals of the species are included (i.e., all specimens of a species are not reciprocally monophyletic) or the clade is not monophyletic, respectively. To be continued in next page. 62Fig 2. Continued. The two letters after species name and collection number are collection locality: Br = Borneo (Indonesia and Malaysia), Jv = Java (Indonesia), Ml = Moluccas (Indonesia), Mp = Malay Peninsula (Malaysia and Singapore), Ng = New Guinea (Indonesia and Papua New Guinea), Sl = Sulawesi (Indonesia), Su = Sumatra (Indonesia), AU = Australia, GH = Ghana, KE = Kenya, MY = Malaysia, NC = New Caledonia, NZ = New Zealand, PH = Philippines, UG = Uganda, TH = Thailand, KRB = Bogor Botanic Garden, MBGK = Makino Botanical Garden, MO = Missouri Botanical Garden, XTBG = Xishuangbanna Tropical Botanical Garden, Cu = Cultivated. The last two letters are traditional infrageneric affiliations based on Miquel’s (1843–1844) and Candolle’s (1923) systems (separated by “ / ”), and a third affiliation is added if they have been placed in Engler’s Oligandropiper (1889): Ch = Chavica, Co = Coccobryon, Cu = Cubeba, Ec = Chavica sect. “Euchavica”, Ep = “Eupiper” sensuCandolle (1923), Ma = Macropiper, Mu = Muldera (including Schizonephros), Ol = Oligandropiper, Pc = Pseudochavica Ridl., Pe = Chavica sect. Peltandron (including Penninervia), Pi = Piper Miq. (including “Eupiper” sensu Hooker, 1886; Koorders, 1908, 1912; Ridley, 1924, 1925), Rh = Rhyncholepis, Sa = Sarcostemon, ? = unknown. When multiple infrageneric groups have been assigned to a species, one infrageneric group was chosen to best represent the affiliation of the species and “+” is added (see Electr. Suppl.: Table S1 for references). An expanded version of this tree is in Electr. Suppl.: Fig. S1.63Fig. 3. G3pdhphylogeny of simplified dataset. Annotation as in Fig. 2. Expanded version of this tree is in Electr. Suppl. Fig. S2.64Fig 3. Continued.65Fig. 4. TrnL-F phylogeny. Annotation as in Fig. 2. Solid and dashed arrows indicate specimens which are not clustered with their conspecifics/clades. An expanded version of this tree is in Electr. Suppl.: Fig. S3.66Fig 4. Continued.67Fig. 5. Character plots of bract types (), fruit types (), direction of anther dehiscence (), and the number of anther valves () on the Paleotropical Piper clades resolved in the ITS phylogeny. Species in grey highlight are species without any consistent affiliation. “ * ” indicates that the character state was obtained from only a single species. The character states of P. medinillifolium Quisumb. is omitted in the Muldera clade (see Discussion: Systematic implications). Only branches ≥ 50 BP or ≥ 0.9 PP are shown in the tree. A detailed version of this tree in species is in Electr. Suppl.: Fig. S6.68Table S1. Species of PaleotropicPiperincluded in this study and their traditional and current infrageneric classifications. Infrageneric affiliations listed pertain only to the accepted names and/or, if necessary, well-accepted synonyms.69Table S1. Continued.70Table S1. Continued.71Table S1. Continued.72Table S1. Continued.73Table S1. Continued.74Table S1. Continued.75Table S1. Continued.76Table S1. Continued.77Table S1. Continued.78Table S1. Continued.79Table S1. Continued.80Table S1. Continued.81Table S1. Continued.82Table S1. Continued.83Table S1. Continued.Traditional infrageneric affiliation assignments are based on Miquel’s (1843–1844) and Candolle’s (1923) systems (separated by “/ ”), and if the species has never been assigned to a group, predicted affiliations are based on Miquel’s (1843–1844) and Candolle’s (1923) concepts and there are no superscripts. Species traditionally placed in Engler’s Oligandropiper(1889) are separated by “/ ” after Candolle’s system assignments. In both systems, when multiple infrageneric groups had been assigned to a species, an infrageneric group (in bold) was chosen to best represent the affiliation of the species in Results and Discussion. Collector abbreviations as in Appendix 1. Referencesfor the infrageneric group: 1Miquel, 1843–1844; 2Hooker, 1886; 3Koorders, 1908, 1912; 4Ridley, 1924a, 1925; ACandolle, 1923; BCandolle, 1869; CQuisumbing, 1930.84§ Literature cited in Table S1Bailey, F.M. 1889. Botany of the Bellenden-Ker expedition. Pp. 29–80 in: [Meston, A.], Report of the Government Scientific Expedition to Bellenden-Ker Range upon the flora and fauna of that part of the colony. Brisbane: … Government Printer. Candolle, C. de 1866. Piperaceae novae. J. Bot. 4: 161–167, 210–219.Candolle, C. de 1869. Ordo 186: Piperaceae, Tribus Pipereae. Pp. 240–391 in: Candolle, A., Prodromus systematis naturalis regni vegetabilis, vol. 16(1). Parisiis [Paris]: sumptibusVictoris Masson et Filii.Candolle, C. de 1898. Piperaceae novae. Annuaire Conserv. Jard. Bot. Genève 2: 1–37.Candolle, C. de 1910. Philippine Piperaceae. Leafl. Philipp. Bot. 8: 759–789.Candolle, C. de 1923. Piperacearum clavis analytica. Candollea 1: 65–415. Engler, A. 1889. Piperaceae. Pp. 3–11 in: Engler, A. & Prantl, K., Die natürlichen Pflanzenfamilien nebstihren Gattungen wichtigeren Arten, insbesondere den Nutzpflanzen, vol. 3(1). Leipzig: Engelmann. Green, P.S. 1993. Notes relating to the floras of Norfolk and Lord Howe Islands, IV. Kew Bull. 48: 316–320. Hooker, J.D. 1886 (“1890”). Piper. Pp. 78–96 in: Flora of British India, vol. 5, Chenopodiaceae to Orchideae. London: L. Reeve & Co. Koorders, S.H. 1908. Die Piperaceae von Java. Amsterdam: Johannes Muller.Koorders, S.H. 1912. Piper. Pp. 14–33 in: Exkursionflora von Java, umfassend die Blütenpflanzen, Dikotyledonen (Archichlamydeae). Jena: Fischer.Miquel, F.A.G. 1854. Piperaceae. Pp. 83–86 in: Zollinger, H. (ed.), SystematischesVerzeichniss der im Indischen Archipel in den Jahren 1842–1848 gesammelten sowie der aus Japan empfangenen Pflanzen, vol. 2. Zürich: Druck und Verlag von E. Kiesling. Miquel, F.A.W. 1859. Subordo Pipereae Miq. Pp. 436–456 in: Flora van Nederlandsch Indie, vol. 1(2). Amsterdam: C. G. van der Post; etc. Miquel, F.A.G. 1863. Piperaceae. Ann. Mus. Bot. Lugduno-Batavi 1: 134–141.Quisumbing, E. 1930. Philippine Piperaceae. Philipp. J. Sci. 43: 1–246.Ridley, H.N. 1916. Report of the botany of the Wollaston Expedition to Dutch New Guinea 1912–13. Trans. Linn. Soc. London, Bot. 9: 1–269.Ridley, H.N. 1923. A botanical excursion to Northern Sumatra, Piperaceae. J. Malayan Branch Roy. Asiat. Soc. 1: 87–89.Ridley, H.N. 1924a. Piper. Pp. 27–51 in: The Flora of the Malay Peninsula, vol. 3. London: Reeve.Ridley, H.N. 1925. Piper. Pp. 327–329 in: The Flora of the Malay Peninsula, vol. 5. London: Reeve.Sykes, W.R. 1992. Two new names in Macropiper Miq. (Piperaceae) from New Zealand. New Zealand J. Bot. 30: 231–236. Trelease, W. 1928. Some nondescript Pipers from New Guinea. J. Arnold Arbor. 9: 146–150.85Table S2. Character states of each species examined for ancestral character reconstruction (sexuality and habit) and character plotting (bract, fruit, and stamen): Sexuality: 0, strictly bisexual; 1, polygamous; 2, strictly dioecious. Habit: 0, shrubs/treelets; 1, climbers. Bract: 0, round to elliptic and peltate; 1, oblong and adnate to the rachis with free margin; 2, completely adnate to the rachis (including cupular). Fruit: 0, sessile and free; 1, partially concrescent; 2, fully concrescent (more than 1/2); 3, free with constricted base. Direction of anther dehiscence: 0, lateral; 1, apical. Number of anther valves: 0, two; 1, four.86Table S2. Continued.87Table S2. Continued.88Fig. S1. For caption, see p. 67. 89Fig. S1. Continued.90Fig. S1. Continued.91Fig. S1. ITS phylogeny of simplified dataset, expanded version. Numbers above branches are maximum-likelihood bootstrap support percentage (BP)/Bayesian posterior probabilities (PP). Only ≥ 70 BP and ≥ 0.95 PP values are shown, 50–69 BP or 0.90–0.94 PP are indicated by “-”, < 50 BP or < 0.90 PP are indicated by “- -”. Species in grey highlight are species without any consistent affiliation. “ * ” in the end of the tip labels or next to the clade names indicates that all specimens of a species are not reciprocally monophyletic or the clade is not monophyletic, respectively. The two letters after species name and collection number are collection locality: Br = Borneo (Indonesia and Malaysia), Jv = Java (Indonesia), Ml = Moluccas (Indonesia), Mp= Malay Peninsula (Malaysia and Singapore), Ng = New Guinea (Indonesia and Papua New Guinea), Sl = Sulawesi (Indonesia), Su = Sumatra (Indonesia), AU = Australia, GH = Ghana, KE = Kenya, MY = Malaysia, NC = New Caledonia, NZ = New Zealand, PH = Philippines, UG = Uganda, TH = Thailand, KRB = Bogor Botanic Garden, MBGK = Makino Botanical Garden, MO = Missouri Botanical Garden, XTBG = Xishuangbanna Tropical Botanical Garden, Cu = Cultivated. The last two letters are traditional infrageneric affiliations based on Miquel’s (1843–1844) and Candolle’s (1923) systems (separated by “ / ”), and a third affiliation is added if they have been placed in Engler’s Oligandropiper (1889): Ch = Chavica, Co = Coccobryon, Cu = Cubeba, Ec = Chavica sect. “Euchavica”, Ep = “Eupiper” sensuCandolle (1923), Ma = Macropiper, Mu = Muldera (including Schizonephros), Ol = Oligandropiper, Pc = Pseudochavica Ridl., Pe = Chavica sect. Peltandron (including Penninervia), Pi = Piper Miq. (including “Eupiper” sensu Hooker, 1886; Koorders, 1908, 1912; Ridley, 1924a, 1925), Rh = Rhyncholepis, Sa = Sarcostemon, ? = unknown. When multiple infrageneric groups have been assigned to a species, one infrageneric group was chosen to best represent the affiliation of the species and “+” is added (see Electr. Suppl.: Table S1 for references).92Fig. S2. G3pdhphylogeny of simplified dataset, expanded version. Annotation as in Fig. S1.93Fig. S2. Continued.94Fig. S2. Continued.95Fig. S3. TrnL-F phylogeny, expanded version. Annotation as in Fig. S1. Solid and dashed arrows indicate specimens which are not clustered with their conspecifics/clades.96Fig. S3. Continued.97Fig. S3. Continued.98Fig. S4. ITS phylogeny of complete dataset. Numbers above branches are maximum-likelihood bootstrap support percentage (BP)/Bayesian posterior probabilities (PP). Only ≥ 50 BP and ≥ 0.90 PP values are shown, < 50 BP or < 0.90 PP are indicated by “- -”. Species in grey highlight are species without any consistent affiliation. “ * ” next to the clade names indicates that the clade is not monophyletic. The two letters after species name and collection number are collection locality: Br = Borneo (Indonesia and Malaysia), Jv = Java (Indonesia), Ml = Moluccas (Indonesia), Mp = Malay Peninsula (Malaysia and Singapore), Ng = New Guinea (Indonesia and Papua New Guinea), Sl = Sulawesi (Indonesia), Su = Sumatra (Indonesia), AU = Australia, GH = Ghana, KE = Kenya, MY = Malaysia, NC = New Caledonia, NZ = New Zealand, PH = Philippines, UG = Uganda, TH = Thailand, KRB = Bogor Botanic Garden, MBGK = Makino Botanical Garden, MO = Missouri Botanical Garden, XTBG = Xishuangbanna Tropical Botanical Garden, Cu = Cultivated. Numbers and letters after the locality are clone identities.99Fig. S4. Continued.100Fig. S4. Continued.101Fig. S4. Continued.102Fig. S4. Continued.103Fig. S4. Continued.104Fig. S4. Continued.105Fig. S4. Continued.106Fig. S5. G3pdhphylogeny of complete dataset. Annotation as in Fig. S4.107Fig. S5. Continued.108Fig. S5. Continued.109Fig. S5. Continued.110Fig. S5. Continued.111Fig. S5. Continued.112Fig. S6. Character plots of bract types (), fruit types (), direction of anther dehiscence (), and the number of anther valves () on ITS phylogeny of Paleotropical Piper. Only branches ≥ 50 BP are shown in the tree. Species in grey highlight are species without any consistent affiliation.113Fig. S7. Likelihood ancestral state reconstruction of sexuality types using the ITS phylogeny. Only branches ≥ 50 BP are shown in the tree. Estimated ancestral states at the nodes are depicted as pie diagrams. Species in grey highlight are species without any consistent affiliation.114Fig. S8. Likelihood ancestral state reconstruction of habit types using the ITS phylogeny. Annotation as in Fig. S7.115 116 Appendix S1. TAXONOMIC HISTORY OF INFRAGENERIC GROUPS IN PALEOTROPICAL PIPER Paleotropical Piper has been classified into up to 14 infrageneric groups, one (Macropiper Miq.) includes the species in the South Pacific clade, while the other 13 make up the Asian clade (Table 1). Paleotropic Piper was classified by both Miquel (1843–1844) and Candolle (1923) (Table 1), while Engler (1889) combined the two systems. Miquel (1843–1844) relied on variation in fruit and bract types to separate most infrageneric groups, while Candolle (1923) mostly used stamen number and morphology. The only two Asian infrageneric groups admitted by both systems, Coccobryon Klotzsch ex Miq. and Muldera Miq., were based on having bisexual flowers and distinctive cupular bracts, respectively. Particular classification problems in Paleotropical Piper come from the disagreements about the importance of specific characters in defining infrageneric groups, and often the same species can be placed in different infrageneric groups, depending on the author and the characters used. Macropiper was based on M. latifolium (G.Forst.) Miq. (P. latifolium G.Forst.) and M. methysticum (G.Forst.) Miq. (P. methysticum G.Forst.; Miquel, 1840). The group was characterized by being dioecious and having persistent petiolar stipules and axillary spikes. Axillary spikes also define Pothomorphe Miq. of the Neotropics and Candolle initially placed Macropiper in Pothomophe (Candolle, 1869). This character was deemed to be very important within Piper (Yuncker, 1958) and many botanists maintained Macropiper, as well as the Neotropical Pothomorphe, as separate genera (e.g., Smith, 1975; Sykes, 1992; Green, 1993; Gardner, 1997). Smith (1975) restricted Macropiper to nine species from the South Pacific in his revision of the genus. However, other authors have also included a shrubby species from Borneo (P. borneense N.E.Br.; Candolle, 1869, 1923), and two climbers from the Moluccas (M. reinwardtianum Miq. [P. reinwardtianum (Miq.) C.DC.]; Miquel, 1848) 117 and New Guinea (P. barclayanum C.DC. [P. fragile Benth.]; Candolle, 1869, 1923), all of which were removed from Macropiper by Smith (1975) along with P. methysticum which he thought did not have axillary spikes. Within Asian Piper, groups such as genus/subgenus/section Muldera are widely recognized by all botanists because of its distinctive cupulate bracts (Fig. 1G, J), while subgenus Piperoides C.DC. and section Apopiper C.DC. are barely known. Piperoides, characterized by being monoecious and having bivalved anthers, appeared in only one of Candolle’s treatments (Candolle, 1866); its only species, the Indian P. petiolatum C.DC., was placed in the genus Chavica in his next treatment (Candolle, 1869) (Table 1), but was only mentioned in the index in his last treatment (Candolle, 1923). Apopiper was characterized by having two stamens with nonarticulated anthers, and included both the Paleotropical P. hederaceum C.DC., from Australia, and the Neotropical P. membranaceum C.DC., from Mexico (Candolle, 1869). Later, however, Candolle (1923) placed these species in sections “Eupiper” and Coccobryon, respectively. Genus/subgenus/section Coccobryon was characterized by its perfect flowers (Fig. 1H), unusual for Paleotropical Piper. Whereas Miquel (1844) included only a single African species, P. capense L.f., in Coccobryon, Candolle (1869, 1923) included both Neotropical and Paleotropical species in it. The latter made up of the Neotropical genera Artanthe, Sphaerostachys and Peltobryon of Miquel (1844). First, Candolle’s (1923) subgenus “Eupiper” C.DC. (not validly published), characterized by having 2 or 3 stamens, included about 80% of the Asian species, but in Miquel’s system (1843–1844) those species were placed in one of four genera, Chavica Miq., Cubeba Raf., Piper L. and Rhyncholepis Miq., based mostly on differences in bract and fruit types. According to Miquel, Chavica had round, peltate, pedicellate bracts and 118 small crowded sessile fruits, Cubeba had oblong or round, peltate, sessile bracts and stalked fruits (Fig. 1F), Piper (sensu Miquel, 1844; “Piper Miq.”, hereafter) had oblong bracts adnate to the rachis and loose infructescences with large sessile fruits (Fig. 1D), and Rhyncholepis had subulate, peltate, pedicellate bracts and elongated styles. Chavica was further divided into sections “Euchavica” Miq. (not validly published) with cylindrical spikes (Fig. 1A, B), Sphaerostachyon Miq. with globular pistillate spikes, and Peltandron Miq. with enlarged anther connectives (rather like Fig. 1L). Miquel included the majority of Chavica, 41 species, in section “Euchavica”, while he placed only 4 and 1 species in sections Sphaerostachyon and Peltandron, respectively (Miquel, 1843). In 1869, Candolle had recognized Chavica as a separate genus, but he circumscribed it differently: Piper species had quadrivalved anthers that dehisce laterally, and Chavica species had bivalved anthers that dehisce apically (“extrorsely” in the original description). Only six Chavica species were listed in Candolle’s treatments in 1869 and 1898, and the majority of Miquel’s Chavica he included in his “Eupiper” (“Eupiper**”, see Table 1). Later in 1923, however, he abandoned Chavica entirely and placed its two species in “Eupiper”. Candolle’s system (1923) was later adopted by Quisumbing (1930) for his revision of Philippine Piper, although he recognized Miquel’s Chavica sect. Peltandron (= sect. Penninervia Quis.). Miquel’s system (1843–1844) was applied with slight modifications to revisions of Piper of India, Java, and the Malay Peninsula by Hooker (1886), Koorders (1908, 1912), and Ridley (1924a, 1925), respectively. The most recent classification of Piper by Callejas (1986), without listing any species, recognized seven subgenera of Paleotropical Piper, and it is also based on slight modifications of Miquel’s system (1843–1844). None of these workers recognized sections in Chavica, and Hooker and Callejas further synonymized 119 Rhyncholepis under Chavica. Callejas (1986) also listed Miquel’s Chavica sect. Peltandron as a separate subgenus. Engler (1889) combined Candolle’s (1869) and Miquel’s (1843–1844) systems and rearranged the classification. In his system, Paleotropical Piper were placed in three genera: Macropiper, Piper L., and Chavica sensu Candolle (1869). He further broke down Piper into four subgenera, the Neotropical Peltobryon and Steffensia, and the Paleotropical Rhyncholepis and “Eupiper” (““Eupiper”***”, see Table 1). “Eupiper” contained three sections: Cubeba, Oligandropiper Engl., and Muldera. Engler listed few species in previously known infrageneric groups, but he listed some species in his new section, Oligandropiper, which comprised species previously belonging to Euchavica, Piper Miq. (including the type of Piper L., P. nigrum), and Coccobryon; he also placed P. methysticum in his Oligandropiper. Pseudochavica Hook.f. was established by Hooker (1886) and included species from “Eupiper” of Candolle (1869), which in turn were part of Piper Miq. and Chavica sect. Euchavica of Miquel (1843–1844). Hooker distinguished Pseudochavica from Chavica by its larger fruits, the former accommodating species with fruits like Piper Miq. but with round peltate bracts like Euchavica (Fig. 1E); species with similar characters were placed in Chavica by Koorders (1908, 1912). Later, Ridley (1924a) moved the only species of Hooker’s Pseudochavica in the Malay Peninsula to “Eupiper” (= Piper Miq.), and added the character of having fruits partially immersed in the rachis to the circumscription of Pseudochavica. Except for three species described by Ridley (1920, 1924b), all species in Pseudochavica sensu Ridley (1924a, 1925; “Pseudochavica Ridl.”, hereafter) (Fig. 1C) had been part of “Eupiper” in Candolle (1869, 1923), and some were part of Piper Miq. and Chavica sect. Euchavica and sect. Sphaerostachyon in Miquel (1843–1844). The fruit 120 character added by Ridley (1924a) to Pseudochavica made his and Hooker’s (1886) concepts of Pseudochavica fundamentally different. Even some infrageneric concepts used by a single botanist changed across time, causing shifts of species’ memberships as their concepts developed (Table 1). Miquel’s Cubeba (1840) included species that were later placed in Chavica, Piper, and Cubeba in his Systema piperacearum (1843–1844). “Eupiper” of Candolle (1866) was characterized by having quadrivalved anthers and included both Neotropical and Paleotropical species, but some of the latter were later placed in his Coccobryon (Candolle, 1869, 1923). Candolle’s (1923) exclusively Neotropical Nematanthera previously included both Paleotropical and Neotropical species (Candolle, 1869), similar to his Pothomorphe (see above), with the erstwhile Paleotropical members being either placed in “Eupiper” or not treated at all. Other classification problems stem from species delimitation issues, not surprising in such a species-rich group, and changing emphases on the characters chosen to place a species in a particular infrageneric group. Monotypic Sarcostemon C.DC. (Candolle, 1910, 1923), characterized by having a single stamen with a swollen filament (Fig. 1K), was based on P. korthalsii Miq., which had been placed in “Eupiper” in Candolle (1869). However, P. quinqueangulatum Miq., later synonymized under P. korthalsii by Backer & Bakhuizen van den Brink (1963), remained part of “Eupiper” in Candolle’s system (1923). Perhaps one of the most egregious cases is P. rostratum Roxb. (Fig. 1H). Roxburgh (1813, 1820) described this as a plant from, perhaps erroneously, the Moluccas based on a plant cultivated in the Calcutta Botanical Garden (no collector listed). It was placed by Miquel (1843) in Rhyncholepis as R. roxburghii Miq. Hooker (1886) placed specimens collected by Griffith and Maingay from the Malay Peninsula in P. rostratum in his section Chavica and synonymized P. stylosum Miq. from Sumatra with it. Nevertheless, Candolle (1914) rejected 121 this synonymy despite the similarities in the descriptions of P. rostratum and P. stylosum, both being shrubs or creepers with beaked fruits and elongated styles. Piper stylosum had been placed in many infrageneric groups. Starting off in Piper Miq. (Miquel, 1863), it was placed in both “Eupiper” and Coccobryon in the same treatment (Candolle, 1923), and in Pseudochavica (Ridley, 1924a). Piper rostratum does have elongated styles (which would place it in Rhyncholepis), round and peltate bracts (Chavica), partially concrescent fruits (Pseudochavica Ridl.) which look like free fruits when young (Piper Miq.) and tightly packed fruits when fully mature (Chavica), and sometimes it has bisexual flowers (Coccobryon). Hardly surprisingly, most recent local treatments use no infrageneric classifications of Piper (e.g., Backer & Bakhuizen van den Brink, 1963 [Java]; Chew, 1972 [New Guinea, Solomon Islands, Australia]; Van Royen, 1982 [New Guinea highlands]; Cheng & al., 1999 [China]; Gardner, 2003 [New Guinea], 2006 [the Philippines], 2010 [Solomon Islands], 2013 [New Guinea]). Literature cited in Appendix S1 Backer, C.A. & Bakhuizen van den Brink, R.C., Jr. 1963. Piper. Pp. 168–173 in: Flora of Java, vol. 1. Groningen: N.V.P. Nordhoff. Callejas, R. 1986. Taxonomic revision of Piper subgenus Ottonia (Piperaceae). Dissertation, City University of New York, New York City, New York, U.S.A. Candolle, C. de 1869. Ordo 186: Piperaceae, Tribus Pipereae. Pp. 240–391 in: Candolle, A., Prodromus systematis naturalis regni vegetabilis, vol. 16(1). Parisiis [Paris]: sumptibus Victoris Masson et Filii. Candolle, C. de 1910. Philippine Piperaceae. Leafl. Philipp. Bot. 8: 759–789. Candolle, C. de 1914. Materials for a Flora of the Malayan Peninsula No. 24, Piperaceae. J. Asiat. Soc. Bengal 75: 288–339. Candolle, C. de 1923. Piperacearum clavis analytica. Candollea 1: 65–415. Cheng, C., Nianhe, X. & Gilbert, M.G. 1999. Piperaceae. Pp. 110–131 in: Wu, Z.Y. & Raven, P.H. (ed.), Flora of China, vol. 4. Beijing: Science Press; St. Louis: Missouri Botanical Garden Press. Chew, W.L. 1972. Studies in West Malesian Piperaceae I. Blumea 20: 145–149. Gardner, R.O. 1997. Macropiper (Piperaceae) in the south-west Pacific. New Zealand J. Bot. 35: 293–307. Gardner, R.O. 2006. Piper (Piperaceae) in the Philippine Islands: The climbing species. Blumea 51: 569–586. 122 Gardner, R.O. 2010. Piper (Piperaceae) in the Solomon Islands: The climbing species. Blumea 55: 4–13. Gardner, R.O. 2013. Piper (Piperaceae) in New Guinea: The climbing species. Blumea 57: 275–294. Green, P.S. 1993. Notes relating to the floras of Norfolk and Lord Howe Islands, IV. Kew Bull. 48: 316–320. Engler, A. 1889. Piperaceae. Pp. 3–11 in: Engler, A. & Prantl, K., Die natürlichen Pflanzenfamilien nebst ihren Gattungen wichtigeren Arten, insbesondere den Nutzpflanzen, vol. 3(1). Leipzig: Engelmann. Hooker, J.D. 1886 (“1890”). Piper. Pp. 78–96 in: Flora of British India, vol. 5, Chenopodiaceae to Orchideae. London: L. Reeve & Co. Koorders, S.H. 1908. Die Piperaceae von Java. Amsterdam: Johannes Muller. Koorders, S.H. 1912. Piper. Pp. 14–33 in: Exkursionflora von Java, umfassend die Blütenpflanzen, Dikotyledonen (Archichlamydeae). Jena: Fischer. Miquel, F.A.G. 1840 (“1838–1840”). Observationes de Piperaceis et Melastomaceis. Pp. 31–65 in: Commentarii phytographici. Lugduni Batavorum [Leiden]: apud S. et J. Luchtmans. Miquel, F.A.G. 1843–1844. Systema Piperacearum. Rotterdam: apud H. A. Kramers. Miquel, F.A.G. 1848. Piperaceae Reinwardtianae. Linnaea 5: 480–486. Miquel, F.A.G. 1863. Piperaceae. Ann. Mus. Bot. Lugduno-Batavi 1: 134–141. Ridley, H.N. 1920. New and rare species of Malayan Plants, Piperaceae. J. Straits Branch Roy. Asiat. Soc. 82: 188–190. Ridley, H.N. 1924a. Piper. Pp. 27–51 in: The flora of the Malay Peninsula, vol. 3. London: Reeve. Ridley, H.N. 1924b. New Malayan plants, Piper uncinulatum Ridl. J. Bot. 62: 300. Ridley, H.N. 1925. Piper. Pp. 327–329 in: The flora of the Malay Peninsula, vol. 5. London: Reeve. Roxburgh, W. 1814 (“1813”). Piper. P. 80 in: A catalogue of plants described by Dr. Roxburgh, in his mss. Flora Indica. Pp. 77–105 in: Hortus Bengalensis. Serampore: printed at the Mission Press. Roxburgh, W. 1820. Piper. Pp. 153–163 in: Flora Indica, vol. 1. Serampore: printed at the Mission Press. Smith, A.C. 1975. The genus Macropiper (Piperaceae). Bot. J. Linn. Soc. 71: 1–38. Sykes, W.R. 1992. Two new names in Macropiper Miq. (Piperaceae) from New Zealand. New Zealand J. Bot. 30: 231–236. Van Royen, P. 1982. Piperaceae. Pp. 1267–1289 in: The alpine flora of New Guinea, vol. 3, Taxonomic part Winteraceae to Polygonaceae. Germany: J. Cramer. Yuncker, T.G. 1958. The Piperaceae – A family profile. Brittonia 10: 1–7. 123 Chapter 2: On the anatomy of Malesian-Pacific Piper and its implications for systematics 1: Stems Rani Asmarayani Department of Biology, University of Missouri – St. Louis, 1 University Boulevard, St. Louis, Missouri 63121, USA; Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, USA; Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor KM 46, Cibinong 16911, West Java, Indonesia; rasmarayani@yahoo.com Abstract. Stem anatomy of 48 species of Malesian-Pacific Piper is investigated in a phylogenetic context to identify potential anatomical stem characters that may distinguish clades. Similar variation occurs in all three major groups of Paleotropical Piper, the West of Wallace’s Line, East of Wallace’s Line, and South Pacific groups, and there is much parallel evolution. Potential synapomorphies of clades include a ring of sclereids surrounding the lignified pericyclic caps of peripheral vascular bundles (Muldera), the angular stem (Sarcostemon s.l.), very broad primary medullary rays – the rays are 2-3 times wider than the vascular bundles – (Pseudochavica sensu Ridley s.str.), and the arrangement of the peripheral mucilage canals (E1 and core E2). Introduction Piper L. (Piperaceae) is a major clade in the magnoliids, comprising ~2400 species. It includes several economically valuable species, such as the king of spices, black pepper (P. nigrum L.), the Pacific herbal, kava (P. methysticum G. Forster), and betel leaf (P. betle L.), the main ingredient of betel-chewing in Asia. In addition, many of the species are important in traditional medicines and in condiments (e.g., Heyne 1927; Burkill 1966). Piper is pantropical, although with very few species in Africa, and species can be 124 found from lowland coastal to montane forests. In Neotropical forests, Piper is ecologically important through its association with specialized bat frugivores/dispersers (Carollia spp., Phyllostomidae; e.g., Fleming 2004), moth caterpillar herbivores (Eois spp., Geometridae; e.g., Marquis 1991; Wilson et al. 2012), beetle herbivores (Curculionidae; Marquis 1991), and ants (Pheidole bicornis Forel, 1899, Formicidae, and perhaps other Pheidole spp.; e.g., Tepe et al. 2004; Tepe et al. 2009). Much less is known about Paleotropical Piper. There is an ant-plant association in P. myrmecophilum C. DC. (Quisumbing 1930; Gardner 2006), although it has been little studied. Piper is divided into three major biogeographical and morphological groups, and each of them has been supported by recent molecular phylogenetic studies: the Neotropical group (1804 spp.; Ulloa Ulloa et al. 2017) includes shrubs/treelets with bisexual flowers, the South Pacific group (ca. 20 spp.; Asmarayani 2018) includes dioecious shrubs/treelets, and the Asian group (ca. 600 spp.; Quijano-Abril et al. 2006) includes dioecious root climbers (Jaramillo and Manos 2001; Jaramillo and Callejas 2004a; Asmarayani and Pancoro 2006; Wanke et al. 2007; Jaramillo et al. 2008; Smith et al. 2008; Asmarayani 2018). Within each of these groups, past infrageneric groups may be supported (in the Neotropical group; Jaramillo et al. 2008) or novel groupings can be recognized (in the South Pacific and Asian groups; Asmarayani 2018). The Asian group is divided into an East of Wallace’s Line grade and a West of Wallace’s Line clade with 12 and 5 clades, respectively, while the South Pacific group is divided into three clades (Asmarayani 2018). Piper species are easily recognized by their swollen nodes, distichous entire leaves, and leaf-opposed spikes. The flowers are minute, perianthless, and subtended by bracts. Most species of Piper grow in shady areas although they usually flower only in 125 sunlight (e.g., Callejas 1986; pers. obs.). They have diverse growth forms, being scandent or erect plants, stem twiners or root climbers, and small perennial herbs to shrubs to treelets (e.g., Jaramillo and Callejas 2004; Isnard et al. 2012). Nonetheless, they all have the same basic construction, Piper having the architectural model of Petit (Hallé et al. 1978; Blanc and Andraos 1983), where the monopodial orthotropic main axis bears sympodial plagiotropic lateral shoots. These plagiotropic shoots are made up of a series of modules, each comprised of a short internode with an adaxial prophyll, a long internode with an expanded leaf, and an internode with a terminal inflorescence; each module develops in the axil of the expanded leaf of the previous module (Blanc and Andraos 1983). In many species, particularly the Asian root climbers (Blanc and Andraos 1983), as well as some erect Neotropical species (e.g., Callejas 1986; Jaramillo and Callejas 2004), there is strong leaf dimorphism, the leaves of the orthotropic shoots being more symmetrical than those of the plagiotropic shoots. In many cases, the leaves of orthotropic and plagiotropic shoots have different shapes, e.g., ovate and cordate versus obovate in P. sarmentosum Roxb., and size (Callejas 1986; pers. obs.). Stem anatomy and its relation to morphological features of Piper Distinguishing Piper species is not easy, especially when the material is sterile, as it all so often is, and many botanists have used anatomy to help in identifying species (e.g., Konoshima and Miyagawa 1977; Albiero et al. 2005; Duarte and Siebenrock 2005; Raman et al. 2012; dos Santos et al. 2015). The stem of Piper consists of (from the outside in) an epidermis, cortex – consisting of parenchyma externally, then collenchyma, and then more parenchyma –, an endodermis, a ring of peripheral vascular bundles, an undulating sclerenchyma band, and medullary/pith parenchyma with one or more ring(s) 126 of medullary vascular bundles and sometimes one or several mucilage canal(s) (e.g., Solereder 1908; Metcalfe and Chalk 1972). These rings of vascular bundles, as well as the undulating sclerenchyma band (= medullary fibrous ring, Bond 1931), distinguish most Piperaceae from other angiosperms (e.g., Metcalfe and Chalk 1972; Isnard et al. 2012; Trueba et al. 2015). However, Peperomia and the basal lineage of Piperaceae, Verhuellia, do not have undulating sclerenchyma bands. The latter lacks peripheral bundles and has only an amphicribral vascular bundle in the center of the stem (Isnard et al. 2012; Trueba et al. 2015). In addition, all Piperaceae have secretory cells with translucent oily contents in the stem and other parts of the plants (e.g., Solereder 1908; Metcalfe & Chalk 1972). Sclereids (e.g., Metcalfe and Chalk 1972; Tepe et al. 2007), calcium oxalate crystals (e.g., Metcalfe and Chalk 1972; Tepe et al. 2007), and starch grains (e.g., Ironside 1911; Bond 1931; Tepe et al. 2007; Gajurel et al. 2008) are commonly found in the stem. The epidermal layer of Piper is protected by a cuticle (e.g., Gajurel et al 2008; Weiss 1876), and is sometimes covered by unicellular or multicellular, glandular or eglandular hairs (e.g., Weiss 1876; Mohandas 1982; Gajurel et al 2008). Cork formation is superficial (Weiss 1876), being derived from the epidermis (e.g., Neotropical [NT, hereafter] P. carpunya Ruiz & Pav., Asian [AS, hereafter] P. longum) or the outermost cortical cells (e.g., NT P. arboreum Aubl. [P. geniculatum Sw.], NT P. dilatatum Rich. [Artanthe corylifolia Miq.], AS P. betle L. [Chibber 1913], and South Pacific [SP, hereafter] P. excelsum G. Forst. [Ironside 1911]). Lenticels are also observed on stems that have undergone secondary thickening (e.g. AS P. betle [Chibber 1913]). Beneath the epidermis are layers of parenchyma cells, the outer and inner cortex, between which is band of collenchyma cells (e.g., Chibber, 1912; Mohandas, 1982). The 127 parenchyma cells in the inner cortex are generally smaller and more tightly arranged than those in the outer cortex (Mohandas, 1982). The collenchyma, the main mechanical tissue in the young stem of Piper, is made up of layers of elongated cells that are prismatic in transverse section (Weiss 1876). The collenchyma may be in either a continuous ring or isolated bundles (e.g., AS P. longum [Weiss 1876]); the continuous ring will eventually become interrupted during stem expansion (e.g., Weiss 1876; Solereder 1908; Bond 1931; Metcalfe and Chalk 1972). As the stem ages, these collenchyma cells may retain their characteristics (e.g., NT P. arboreum [Weiss 1876] and P. peltatum L. [Solereder 1908]), or the 2-3 innermost layers (e.g., NT P. dilatatum and P. bredemeyeri J. Jaq., AS P. betle L. and P. longum) or all collenchymatous cells (e.g., NT P. carpunya and P. rivinoides Kunth) may become lignified (= sclerenchymatous fibers, Weiss 1876). In Piper, there is a cauline endodermis that occurs only in association with the peripheral vascular bundles (Weiss 1876; Bond 1931). Its distribution, however, varies. Weiss (1876, eight species) and Bond (1931, eight species) divided the endodermis into three types: (1) a complete endodermal ring surrounding the peripheral vascular bundle ring (e.g., NT P. arboreum and P. bredemeyeri [Weiss 1876], AS P. nigrum and P. porphyrophyllum N. E. Br. [Bond 1931]) , (2) an incomplete endodermal ring, mostly adjacent to the peripheral vascular bundles (e.g., NT P. tiliaefolium Schlecht., AS P. retrofractum, and SP P. excelsum [Bond 1931], and (3) no endodermal cells s.str., instead, a layer of large cells lacking Casparian strips surrounding the peripheral vascular bundle ring (e.g., NT P. rivinoides [Weiss 1876] and P. celtidifolium Kunth [Bond 1931]). However, in some species the endodermis may be present or absent in different shoots from the same individual (e.g., SP P. excelsum; Bond 1931; Balfour 1958). 128 The peripheral vascular bundles are all collateral and are often alternately larger and smaller (e.g., Debray 1885; Hoffstadt 1916). There are zero to five smaller bundles between two larger bundles, and they are branches of the larger bundles (Hoffstadt 1916), their exact number depending on the age and the size of the stem (Debray 1885; Chibber 1913; Hoffstadt 1916). Not surprisingly, the number of these vascular bundles can vary within a single plant (Weiss 1876). An undulating sclerenchyma band at the margin of the pith is adjacent to the xylem of the peripheral bundles (Van Tieghem 1908); the amplitude of the sinuations depends on the sizes of the bundles, the larger the bundle the greater the amplitude (Chibber 1913). The phloem of the peripheral bundles is capped by pericyclic fibers (Weiss, 1876; Van Tieghem 1908; Bond 1931). This cap may differentiate during primary (e.g., NT P. celtidifolium, AS P. retrofractum Vahl [=P. chaba Hunter]) or secondary growth (e.g. NT P. aduncum L. [=P. angustifolium Ruiz & Pav.], SP P. excelsum) (Bond 1931). It is usually lignified, forming semilunar groups of sclerenchymatous fibers outside the phloem (=bast fibers) as in NT P. dilatatum and P. arboreum, AS P. betle and P. longum, although in NT P. bullatum Vahl, for example, no lignin is laid down (Weiss 1876). In some species, there are only a few sclerenchymatous fibers and they are found mainly in association with the large bundles (e.g., NT P. carpunya and P. bredemeyeri; Weiss 1876). In other species, e.g., AS P. retrofractum, these fibers merge with the undulating sclerenchyma band so that the vascular bundles are completely surrounded by fibers (Bond 1931). The medullary vascular bundles are generally about equal in size (e.g., Debray 1885; Hoffstadt 1916). Although they are mostly collateral and normally oriented (i.e., 0°), occasionally bundles furthest away from the axis of the stem may twist up to 180° (Weiss 1876; Debray 1885; Chibber 1913), as in NT P. arboretum and P. carpunya 129 (Weiss 1876), and AS P. betle (Chibber 1913). The bundles often have sclerenchymatous fibers at both the xylem and phloem poles, although usually fewer fibers are developed at the latter (Weiss 1876; Solereder 1908). The vascular bundles from both systems may branch at the nodes and there are anastomoses between neighboring bundles in the same or different systems (Weiss 1876; Debray 1885). Rarely, branching and anastomosis of the bundles occur in/along the internodes (Debray 1885; Ironside 1911; Hoffstadt 1916; Murty 1958), and so on occasion two medullary vascular bundles in the internode region may be particularly close together (Weiss 1876). Some medullary bundles move to the peripheral ring and remain there for at least an internode before leaving as leaf traces (Weiss 1876; Debray 1885; Hoffstadt 1916). There are five to twenty, usually nine, leaf traces in Piper (e.g., Rousseau 1927; Hoffstadt 1916; Murty 1958), which all come from the peripheral bundle system (Weiss 1876; Debray 1885; Hoffstadt 1916). Secondary thickening of the stem in Piper may occur extensively in the peripheral vascular bundles, but usually not or only to a very limited extent and then much slower in the medullary bundles (e.g., Weiss 1876; Debray 1885; Solereder 1908; Van Tieghem 1908; Hoffstadt 1916; Bond 1931; Ravindran & Remashree 1998; Isnard et al. 2012). However, Solereder (1908) reported that NT P. geniculatum Sw. and AS P. blumei (Miq.) Baker (Chavica frustrata Miq.) had considerable secondary thickening in the medullary bundles. The fascicular and interfascicular cambium in the peripheral vascular bundles is most active adaxially producing xylem and primary medullary rays, but it produces only a little phloem and a few short rays abaxially (Weiss, 1876; Bond 1931). The primary medullary rays are very broad, up to 40 cells wide (e.g., Weiss 1876; Solereder 1908; Chibber 1913; Ironside 1911; Balfour 1958), and become lignified early 130 (Bond 1931). The pericyclic sclerenchymatous semilunar fiber groups may form a complete ring by merging with the lignified parenchyma produced by the interfascicular cambium (e.g., AS P. nigrum [Solereder 1908; Van Tieghem 1908] and P. betle [Chibber 1913]; note that this pericyclic sclerenchymatous semilunar fiber can merge with the undulating sclerenchyma band during primary growth [see above]). When medullary vascular bundles have secondary thickening, the parenchymatous cells between the bundles stretch radially and are compressed tangentially by the growth of the bundles (Weiss, 1876). Enlargement of the stem is also caused by the division of parenchyma cells in the pith (Hoffstadt, 1916). During stem expansion, the collenchyma, the pericyclic fiber ring, and the undulating sclerenchyma band may become interrupted (e.g., Solereder 1908; Van Tieghem 1908; Ironside 1911; Bond 1931). Mucilage canals in Piper are lysigenous (Solereder 1908; Van Tieghem 1908; Chibber 1913; Gardner 1997). There is often a single central mucilage canal which occupies the center of the medullary parenchyma, and there are several peripheral canals which are adaxial to the small peripheral vascular bundles (vanTieghem 1908; Chibber 1913, Mohandas 1982). The canals are bordered by flattened cells that are smaller than the pith parenchyma cells (Van Tieghem 1908). The canals are uninterrupted along the internode, but they end abruptly at the nodes and then reappear in the next internode in the same position as the previous node (Van Tieghem 1908). However, Murty (1958) found in AS P. betle that the canals may merge, branch, as well as change size at the nodes. Stem anatomical characters and the taxonomy of Piper 131 Weiss (1876) investigated the stem of eight species from the Neotropical and Asian clades, and he noticed differences in the collenchyma and in the number and arrangement of the vascular bundles. Since then, the stem anatomy of Piper, and its vegetative anatomy in general, has been investigated widely with the aim to aid in species identification. Vegetative anatomical studies have been carried out, most notably, to differentiate economically valuable species (e.g., Konoshima and Miyagawa 1977; Raman et al., 2012; Silva et al 2017, dos Santos et al. 2018), and to help identify species growing in particular regions (e.g., Mohandas 1982; Gajurel et al. 2008; Tihurua et al. 2012). De Candolle (1869 onwards) had used characters of primary growth in his species descriptions; these included the nature of the collenchyma (continuity of the band, shape of the collenchyma cells), the number of medullary vascular bundle rings, and the presence and the number of mucilage canals. Few studies have tried to use such anatomical features to characterize infrageneric groups within Piper. However, Debray (1885) noticed that the three sampled species of Artanthe (a Neotropical infrageneric group, but now thought to be non-monophyletic [Jaramillo et al. 2008]) differed from other Piper (Neotropical, Asian, and South Pacific species studied) in having less secondary growth in the stems of the same size and with the same number of medullary vascular bundle rings. A few studies focusing on specific Neotropical clades have also been carried out (i.e., Callejas 1986 [Ottonia Spreng.], Tepe et al. 2007 [Macrostachys Miq.], Quijano-Abril et al. 2013 [Enckea Kunth]). Within Paleotropical Piper, however, almost none of the previously recognized infrageneric groups are monophyletic (Asmarayani 2018), thus any attempt to use anatomical features as diagnostic characters for infrageneric groups previously recognized is largely irrelevant. Here, I examine the diversity of stem anatomy of Malesian-Pacific Piper in the 132 light of the results of the current phylogenetic study of the Paleotropical Piper (Asmarayani 2018). By studying representatives of clades resolved in the phylogeny, I aim to identify potential anatomical characters that may distinguish clades. Material and Methods Material. Material was collected from across the Malesian region and obtained from Bogor Botanic Garden (Indonesia) and the Missouri Botanical Garden by Asmarayani and colleagues (Appendix 1). In addition, preserved vegetative material of P. kadsura (Choisy) Ohwi was obtained from Makino Botanical Garden (by N. Tanaka). Anatomical observations on stems were carried out on sixty-four individuals of 48 species, representing 12 clades and 2 unaffiliated species of West of Wallace’s Line clade (WWL, hereafter), 5 clades of East of Wallace’s Line grade (EWL, hereafter), and the New Guinea clade of the South Pacific clade (SP, hereafter) in Asmarayani (2018). Methods. Material was fixed in 70% ethanol or formaldehyde-acetic acid alcohol (FAA; 50 parts of ethanol 95%, 5 parts of glacial acetic acid, 10 parts of formaldehyde 37%, and 35 parts of deionized water; Ruzin 1999) in the field. Stem sections were prepared by hand and stained following Keating (2014, pp. 1-12). For comparative purposes, all stem samples were taken from the middle of the internode that is immediately before the expanded leaf. When possible stems were sampled from several internodes, both plagiotropic and orthotropic (Table S1). They were stained with cresyl violet acetate (CVA), and mounted in CaCl2 or glycerin (Keating, 2014). CVA will stain lignin bright blue and parenchyma tan (Keating, 2014) and a combination of CVA and CaCl2 gives better coloration than that of CVA and glycerin. The anatomical slides were observed using an Olympus CX31 microscope. 133 Photos were taken from each slide using a Leica ICC50 HD camera, and assembled using Stitching (Preibisch et al. 2009) or MosaicJ (Thévenaz & Unser 2007) implemented in Fiji (Schindelin et al., 2012). Measurements were carried out using ImageJ 1.52e. Results and Discussion General anatomy of Malesian-Pacific Piper. The stem characters observed are presented in Table S1. I found no differences between internodes of orthotropic and plagiotropic shoots. All Paleotropical Piper observed have two anatomical characteristics of Piper: two systems of vascular bundles and an undulating sclerenchyma band immediately adaxial to the outermost ring of bundles. Most of the variation observed here is within the scope of that already known in Piper spp., although several new features were observed. Below is the description of variation found in the stem tissue of clades in Paleotropical Piper; the names of the clades follow those used by Asmarayani (2018; fig. 2A). Stem outline. When young, Piper spp. have a more or less terete stem. While some species retain this shape during their development, P. quinqueangulatum Miq. (WWL, Sarcostemon s.l. clade) becomes distinctly angular (figs. 1E, 2E), and P. brevifolium C. DC. (not sampled), another member of the same clade, also has an angular stem. The young stem of species like P. puberulirameum C. DC. (WWL, W2 clade) may be flattened in the plane of distichy of the leaves; this character may not be retained, as older stems can be more or less terete. Cortex. There are two layers of cortex, the outer and inner, separated by the collenchyma. Here I treat the two cortical layers together. The number of layers of the outer cortex varies among species, from zero (e.g., P. decumanum L. [EWL, E4 clade]) to 134 many (e.g., 6-8 layers in P. philodendron Ridl. [WWL, W6 clade] and P. methysticum [SP]), and the number is generally higher in older stems, the cortical cells remaining meristematic (e.g., P. cilibracteum C. DC. [WWL, W5 clade], P. macropiper Pennant [WWL, E4 clade], P. methysticum [SP]). In general, the parenchyma cells in the outer cortex are smaller and more tightly packed than those in the inner cortex, however, in P. albamentum C. DC. (EWL, E1 clade; fig. 1H) the parenchyma cells of the outer cortex are larger and are elongated radially, although this character is not obvious in older stems. In many species like P. cubeba L. f. (WWL, Cubeba s.str. clade), P. nigrum (WWL, Piper sensu Miquel clade) and P. aff. macropiper Pennant (EWL, E4 clade), the walls of the parenchyma cells of the outer cortex are thicker than those of the inner cortex. In P. decumanum L. and P. cf. dumiformans C. DC. (both EWL, E4 clade), cells of the outer cortex have collenchymatous thickening although they are larger than the collenchyma proper. All Muldera (WWL) examined, and also some other species in other clades (e.g., P. porphyrophyllum N. E. Br. [WWL, W5 clade], P. majusculum Blume [EWL, E1 clade]), have brown cortex cells, most prominently in the outer cortex and collenchyma (see Collenchyma). Collenchyma (fig. 4B). The collenchyma may be in distinct bundles (e.g., P. ramipilum C. DC. [WWL, W4 clade], P. retrofractum Vahl [EWL, E1 clade], P. methysticum [SP, fig. 1L, 3C]; figs. 1E, 1H, 2E) or in a continuous ring which will become interrupted by parenchyma produced by the division of cortical cells as the stem expands (e.g. P. pedicellosum Wall. [WWL, Cubeba s.str. clade], P. celtidiforme Opiz [EWL, Peltandron clade]). The parenchyma between interrupted collenchyma bands sometimes becomes lignified (i.e. sclereids) during stem development (see Pericyclic fiber cap and sclereids in the cortex). The shape of the stem sometimes reflects the 135 location of collenchyma bundles, thus in P. quinqueangulatum (see above) they are located at the ridges of the stem, as for species with less prominent ridges like P. albamentum C. DC. (EWL, E1 clade). However, in P. versteegii C. DC. (EWL, E2 clade), the collenchyma ring is interrupted at the angles of the stem. The collenchyma cells in distinct bundles sometimes have massive wall thickenings, especially at the angles (i.e. angular thickening) and usually the majority of the cells maintain their collenchymatous characteristics (i.e. not lignified). On the other hand, species with, initially, continuous ring of collenchyma generally have less thickened walls and most of the innermost layers or all the collenchyma becomes lignified (= sclerenchymatous fibers, Weiss 1876; e.g., P. pentandrum C. DC. [WWL, Muldera clade], P. versteegii [EWL, E2 clade, fig. 1J]). The number of layers of collenchyma cells varies slightly both among species and among stems of different sizes and ages from the same plant. In general, there are 2-6 layers, but some species have >6 layers. Species that have >6 layers of collenchyma (e.g., P. philodendron [WWL, W6 clade], P. decumanum [EWL, E4 clade], P. methysticum [SP]) usually have the collenchyma in distinct bundles with massive wall thickenings and the cells maintain their collenchyma characteristics or only rarely become lignified. The collenchymal cells in many species are generally whitish in color, but they are brown in some species (see Cortex). Endodermis (Casparian strips). Some species in which multiple shoots were sampled always have a complete endodermal ring (e.g., P. betle L. [WWL, Euchavica s.str. clade], P. albamentum C. DC. [EWL, E1 clade]), or always lack endodermal cells s.str. (e.g., P. cubeba L.f. [WWL, Cubeba s.str. clade], P. methysticum [SP]). Sometimes there is an incomplete endodermal ring, but other shoots of the same species sampled 136 may have a complete ring (e.g., P. puberulirameum C. DC. [WWL, W2 clade], P. macropiper Pennant [EWL, E4 clade]) or no endodermal cells s.str. at all (e.g., P. argyrites Ridl. [WWL, W3 clade]). The inconsistency of endodermal cells distribution among species and between different shoots of the same individual creates question of their function. Sometimes what appear to be a flow of cellular substances is observed between Casparian strips in stem with complete endodermal ring. Cauline endodermis is an apomorphy for the genus Piper (Metcalfe and Chalk 1972; Stevens 2001 onwards; note that Bond [1931] concluded that the stem endodermis is a vestigial structure in Piper). Pericyclic fiber cap (fig. 4C) and sclereids in the cortex. Pericyclic fibers become lignified during stem development and often form a semilunar cap, or arrow-like cap in P. decumanum, on top of the phloem of peripheral vascular bundles, first on the larger bundles, then on the smaller bundles. In many species, these lignified pericyclic fibers have thick walls. In most species, the caps eventually unite with each other. They are linked by an undulating sclerenchyma band, in a few species during their primary growth, e.g., all three species in E2 clade (fig. 1J, 1K) and the only species sampled in Peltandron clade, P. celtidiforme Opiz (fig. 1F). More commonly, they are united by lignified parenchyma produced by the interfascicular cambium so forming a continuous lignified ring (this may happen during secondary growth, as in P. celtidiforme). In stems with massive secondary thickening (see below), the lignification of this parenchyma sometimes extends deeply inwards into the primary medullary rays between the phloem (figs. 2A-D, 2F, 2H). In many species of Muldera (e.g., P. flavimarginatum C. DC. [fig. 2A], P. pentandrum C. DC.), a species of Cubeba s.str. (P. ribesioides Wall., fig. 2C), and Peltandron (P celtidiforme), this lignified ring is associated with a variable number of 137 very thick-walled sclereids externally; these sclereids seem to have originated from the endodermis layer, which often to have periclinal division prior to lignification. However, in all shrubby species including P. philodendron (WWL, W6 clade), P. oreophilum Ridl. and P. cyrtostachys Ridl. (WWL, Pseudochavica sensu Ridley s.str. clade), P. sarmentosum (WWL, W1 clade) and P. methysticum (SP), the pericyclic fiber caps are poorly lignified and never merge. Many species, particularly in Muldera but also in many other species in other clades, e.g., P. ribesioides Wall. (WWL, Cubeba s.str. clade), P. puberulirameum C. DC. (WWL, W2 clade), P. celebicum Blume (EWL, E1 clade, fig. 1G), have abundant sclereids in the cortex. They are particularly prominent in the outer and inner cortexes, and also may fill gaps between interrupted collenchyma bands. Secondary thickening in peripheral vascular bundles and the primary medullary rays (fig. 4D). Generally, Piper spp. have secondary thickening in the peripheral vascular bundles (fig. 2). This secondary thickening can be very extensive, especially in species that are climbers (figs. 2C, D, F), and the undulating sclerenchyma band may become interrupted because of the increase in stem width that results (figs. 2C, F). Although shrubby species can also have secondary thickening in the peripheral vascular bundles (e.g., P. oreophilum and P. cyrtostachys [WWL, Pseudochavica sensu Ridley s.str. clade, fig. 2G], P. methysticum [SP, fig. 3C]), this secondary thickening is less common in shrubs than in the climbers. Shrubby species like P. sarmentosum (WWL, W1 clade, fig. 3A) and P. philodendron (WWL, W6 clade, fig. 3B) seem to have no secondary thickening at all, or have only very limited secondary thickening, despite the fact that samples were taken near the base of the plants: P. sarmentosum and P. philodendron may reach 1 and 2 m tall, respectively. 138 When secondary thickening has begun, the primary medullary rays soon become lignified (also see Introduction), and the later formed rays are also usually lignified (figs. 2, D, G, H, I, J). However, in species like P. ribesioides Wall. (WWL, Cubeba s.str. clade, fig. 2C), P. cilibracteum (WWL, W5 clade, fig. 2B), P. puberulirameum C. DC. (WWL, W2 clade, fig. 2F), and P. quinqueangulatum (WWL, Sarcostemon s.l. clade, fig. 2E), the later formed rays are not lignified at all or a bit so, although the secondary thickening is very extensive. The pattern of lignification in the primary medullary rays is correlated with the vessel arrangement in the vascular bundles: the species with lignified later-formed rays have the vessels in the xylem arranged in 1-2 files (e.g., figs. 2A, D, I) and the trans-sectional area of the vessels is less than that of the rays within the xylem (i.e., secondary medullary rays), while those with unlignified later-formed rays have dense vessels arranged in multiple files and the trans-sectional area of the vessels is larger than that of the rays within the xylem (e.g., figs. 2B, C, E, F). The primary medullary rays of Piper spp. are very broad (see also Introduction), being about the width of the vascular bundles in which the secondary thickening has become massive (e.g., figs. 2C, D) or somewhat narrower (e.g., figs. 2B, F, I). However, in the two species of the shrubby clade of Pseudochavica sensu Ridley s.str. examined, the rays are much wider than the vascular bundles and consists of as many as 40 cells across (fig. 2G). Medullary vascular bundles (fig. 4E). Most Piper spp. have a single ring of medullary vascular bundles (e.g., figs. 2A-E, 2G-K). However, some species like P. pentandrum (WWL, Muldera clade), P. celtidiforme Opiz (EWL, Peltandron clade, figs. 1F, 2I), and P. methysticum (SP, figs. 1L, 3C) have multiple rings of medullary vascular bundles. In these species, the vascular bundles in the outer ring, rarely in the inner ring, 139 are often twisted up to 180°. The medullary vascular bundles may branch or anastomose along the internodes, hence the bundles that are in pairs (“paired” in Table S1) or very close to each other or even partly fused (“joined” in Table S1); in species with two or more rings, such bundles are in the outer ring. Piper ribesioides (WWL, Cubeba s.str. clade, fig. 2C) has massive secondary thickening in its medullary vascular bundles, and the bundles, both peripheral and medullary, bifurcate in the outer part forming a Y-shape, the arms of the Y being separated by unlignified secondary rays. In P. flavimarginatum C. DC. (WWL, Muldera clade, fig. 2A) and P. versteegii (EWL, E2 clade, fig. 1J), secondary thickening in the medullary vascular bundles is much more obvious than in the peripheral bundles, at least as the stem begins to enlarge. However, secondary growth in the former later ceases while in the latter it continues (see also Yang and Chen 2017). Arrangement of mucilage canals (fig. 4F). In general, Piper spp. have mucilage canals. However, the presence or absence of mucilage canals may vary in different shoots of the same individual (e.g., P. pedicellosum [WWL, Cubeba s.str. clade], P. rubro-venosum Rodigas [EWL, E3 clade], P. albamentum [EWL, E1 clade]), so the presence or absence of the mucilage canals is not a reliable diagnostic character. When present, there is often a central mucilage canal and/or peripheral mucilage canals that are rather regularly arranged in the tissue surrounding the medullary vascular bundles. These peripheral canals are usually located interior to the small vascular bundles of the peripheral ring (e.g., figs. 1A, B, D, K, L). However, in P. albamentum (fig. 1H), P. abbreviatum, P. celebicum (fig. 1G), and P. sp. RA 559 – all members of E1 clade of EWL grade – they are interior to the large vascular bundles of the peripheral ring. Piper argyrytes Ridl. (WWL, E3 clade) has mucilage canals located both interior to the small and large vascular bundles of the peripheral ring. Piper versteegii of the EWL grade (E2 140 clade) is distinctive in that the canals occur next to the inpushings of the undulating sclerenchyma band adjacent to the large peripheral bundles (fig. 1J), while the core E2 clade, P. mestonii and P. sp. RA758, are also distinctive in having two canals interior to the small vascular bundles of the peripheral ring (fig. 1K). Pattern of lignification in pith parenchyma. In almost all stems investigated, at least 1-2 cell layers of the outermost part (peripheral) of the outer pith (i.e. the area between the undulating sclerenchyma band and the medullary vascular bundle ring) become lignified during development/enlargement, intergrading with the undulating sclerenchyma band. In most species, the lignification then progresses centripetally towards the center of the pith (central pith). The pith of P. cilibracteum (WWL, W5 clade, fig. 2B), P. sp. RA811 (WWL, unaffiliated), and P. celtidiforme (EWL, Peltandron clade, fig. 2I) becomes almost completely lignified during its development/enlargement. However, the pattern of lignification varies between shoots in P. pentandrum C. DC. (WWL, Muldera clade) and P. puberulirameum C. DC. (WWL, W2 clade, fig. 2F), that of the younger stems perhaps paradoxically showing more extensive lignification across the pith – the outer pith, the area between the undulating sclerenchyma band and the medullary vascular bundle ring, is lignified – than in the older/bigger stems. The lignification of the cells is generally very modest, but in some members of Cubeba s.str. clade (P. cubeba and P. ribesioides) and W2 clade (P. puberulirameum), the lignified walls are very thick (i.e., slereids). Piper puberulirameum is rather distinctive in that groups of sclereids are arranged in a discontinuous ring in the outer pith and near mucilage canals. The outer pith of older stems of P. puberulirameum is barely lignified, but it has many scattered groups of sclereids. In P. decumanum L. (EWL, E4 clade), the lignification in the pith occurs just inside the inner vascular bundle ring. The pith of most 141 shrubby species investigated, e.g., P. philodendron (WWL, W6 clade), P. sarmentosum (WWL, W1 clade, fig. 3A), and P. methysticum [SP, fig. 3C]), is not lignified at all. Shrubby species of Pseudochavica sensu Ridley s.str. clade (WWL), P. oreophilum and P. cyrtostachys (fig. 2G), have limited lignification in the 1-4 peripheral layers of the outer pith only. Systematic implications. Most of the variation in the stem anatomy, such as the nature of collenchyma, the pattern of lignification of the primary medullary rays and the vessel arrangement and trans-sectional area in the secondary thickening, the number of rings of medullary vascular bundles, the extent of secondary thickening in the medullary vascular bundles, and the pattern of lignification in the pith parenchyma, occurs in all two major groups of Asian Piper, the WWL and EWL (fig. 4B, D-G). There is a high level of parallel evolution in stem anatomy and it may not be very useful as a diagnostic character for major groups. Nevertheless, combination of characters from stem anatomy can be useful in species identification. Just as the shrubby habit evolved several times in Paleotropical Piper (Asmarayani 2018), the stem anatomy of shrubs from four different clades, W6, W1, Pseudochavica sensu Ridley s.str., and SP clades, show parallelisms: they all have limited or no secondary thickening in the peripheral vascular bundles, minimal lignification of the pericyclic cap and pith, and a lot of parenchyma cells in pith and cortex (figs. 2G, 3A-C). These characters seem to be correlated with the shrubby herbaceous habit. A similar pattern is also observed in the Neotropical clade Enckea, the erect herbaceous species have limited secondary growth compared to species that are treelets (Quijano-Abril et al. 2013). Characters that may be synapomorphies of smaller clades include a ring of 142 sclereids surrounding lignified pericyclic cap of peripheral vascular bundles for Muldera clade, the angular stem for Sarcostemon s.l. clade, very broad primary medullary rays – the rays are 2-3 times wider than the vascular bundles – for Pseudochavica sensu Ridley s.str. clade, and the arrangement of the peripheral mucilage canals for E1 and core E2 clades. Acknowledgements I especially thank Peter F. Stevens for valuable discussion, feedback, and continuous support. I thank Richard C. Keating for discussion and valuable insights in the field of anatomy. I also thank my committee – Elizabeth A. Kellogg, Nathan C. Muchhala, and Robert J. Marquis – for providing helpful comments and direction in the development of the manuscript. Fieldwork and laboratory work were made possible with the help of the keepers, staff, and collaborators of herbaria ANDA, BO, KEP, MO, SAN, and SING, as well as Bantimurung-Bulusaraung National Park (Indonesia), Economic Planning Unit (Malaysia), National Parks Board (Singapore), PT. Freeport Indonesia, Sabah Biodiversity Council (Malaysia), especially Eko B. Walujo, Joeni S. Rahajoe, James C. Solomon, P. Mick Richardson, Peter C. Hoch, Charlie D. Heatubun, Khoon-Meng Wong, David Middleton, Hock-Keong Lua, Serena Lee, Leng-Guan Saw, Rafidah A. Rahman, John B. Sugau, Joan T. Pereira, Pratita Puradyatmika, Nurainas, Mohammad Amir, Imin Kamin, and Jeisin Jumian. This paper is part of my dissertation at the University of Missouri St. Louis (UMSL), which was supported by Fulbright Foreign Student Program and Whitney R. Harris World Ecology Center – UMSL. This work was funded by Missouri Botanical Garden, Whitney R. 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Wilson, JS, ML Forister, LA Dyer, JM O’Connor, K Burls, CR Feldman, MA Jaramillo, JS Miller, G Rodriguez-Castaneda, EJ Tepe, JB Whitfield, B Young 2012 Host conservatism, host shifts and diversification across three trophic levels in two Neotropical forests. J Evol Biol 25: 532-546. Yang S-Z, P-H Chen 2017 Cambial variations of Piper (Piperaceae) in Taiwan. Bot Stud 58:17. A B CD E FJ K L146G H ID E FG H IFig. 2. Secondary growth in stems of Piper spp. A, P. flavimarginatum RA662 (WWL, Muldera clade), insert is peripheral vascular bundle with lignified pericyclic fiber cap (white arrowhead) covered by sclereids (yellow arrowhead). B, P. cilibracteumRA679 (WWL, W5 clade). C, P. ribesioides RA635 (WWL, Cubeba s. str. clade). D, P. pedicellosum RA675 (WWL, Cubeba s. str. clade). E, P. quinqueangulatum RA573 (WWL, Sarcostemon s. l. clade). F, P. puberulirameum RA616 (WWL, W2 clade), insert is younger stem with lignified primary medullary rays. G, P. cyrtostachys Ridl. RA579 (WWL, Pseudochavica sensu Ridley (1924) s. str. clade ). H, P. macropiper RA707 (EWL, E4 clade). I, P. celtidiforme RA533 (EWL, Peltandron clade). Scale bars = 0.5 mm. ß Fig. 1. Primary growth in stems of Piper spp.. A, P. sp. RA636 (WWL, Muldera clade). B, P. caninum RA881 (WWL, W6 clade). C, P. kadsura NTsn (WWL, W2 clade). D, P. mollissimum RA629 (WWL, Cubeba s.str. clade). E, P. quinqueangulatumRA812 (WWL, Sarcostemon s. l. clade). F, P. celtidiforme RA533 (EWL, Peltandron clade). G, P. celebicum RA557 (EWL, E1 clade), insert is the cortex of the species showing groups of sclereids in the inner cortex (yellow arrowhead). H, P. albamentumRA736 (EWL, E1 clade), insert is cortex of the species showing the radially elongated cells of outer cortex (yellow arrowhead). I, P. cf. dumiformans C. DC. RA735 (EWL, E4 clade). J, P. versteegii RA731 (EWL, E2 clade). K, P. mestonii F.M. Bailey (EWL, E2 clade). L, P. methysticum RA711 (SP, New Guinea). Scale bars = 0.5 mm.147A B CFig. 3. Stems of shrubby Piper spp. A, P. sarmentosum RA765 (WWL, W1 clade). B, P. philodendron RA685 (WWL, W6 clade). C, P. methysticum RA711 (SP, New Guinea). Scale bars = 0.5 mm. ABC148Fig. 4. ITS phylogeny of Paleotropical Piper, adopted from Asmarayani (2018), and anatomical character plots: A, ITS phylogeny with clade names and the number of species examined in this anatomical study (in brackets); B, Plot of the arrangement of collenchyma; C, Plot of pericyclic sclerenchyma cap exterior to the peripheral vascular bundles; D, Plot of pattern of primary medullary ray lignification in secondary thickening; E, Plot of number of rings of medullary vascular bundles; F, Plot of the position of peripheral mucilage canals relative to the peripheral vascular bundles (note that this includes only those species which have peripheral mucilage canals). “ * ” in B-F indicates that the character state is known from only a single species. Vb(s) = vascular bundle(s).= clades in WWL clade= clades in EWL grade= clades in South Pacific cladeA**********************= distinct bundles= continuous ring= retain collenchymalcharacteristics= become lignifiedB*********** = discrete= mergingC***********= interior to large vbs= interior to small vbs= next to large vb= interior to small and large vbsF*********** = >1= 1E***********= unlignified(mostly)= lignifiedD149CLADE SPECIES SPECIMENStem typeStem outlineStem diameter (long X short, mm)Outer cortex layersOuter cortex cells Collenchyma bandCollenchyma layersCollenchyma cell thickeningLignified collenchyma layersSclereidsEndodermis (Casparian strips)MulderaP. schizonephros C. DC.R. Asmarayani 674Plag. Terete 3.159 X 2.732 2-3Brown, equal size to/larger than collenchymaDiscontinuous 4-5 Angular, brown 1-4, in discontinuous bandsOuter and inner cortex, between collenchymaNoneOrth. Terete 5.903 X 5.604 3-4Brown, equal size to collenchymaDiscontinuous 5-6Angular, brown, little thickening walls2-5, in discontinuous bandsOuter (abundant) and inner cortex, between collenchyma (abundant)NoneMulderaP. flavimarginatum C. DC.R. Asmarayani 662Plag. Terete 2.292 X 1.719 3-4Brown, larger than collenchymaDiscontinuous 1-3Not seen (all lignified)1-3, in discontinuous bandsSome: outer and inner cortex, between collenchymaNoneOrth. Terete 6.192 X 5.912 5-6Brown, larger than collenchymaDiscontinuous 1-7Not seen (all lignified)1-7, in discontinuous bandsAbundant: outer cortex, between collenchyma (groups of cells filling NoneMuldera P. pentandrum C. DC.R. Asmarayani 593Plag. Terete 3.286 X 2.913 3-5Brown, larger than collenchymaDiscontinuous 1-5 Angular, brown1-5, in discontinuous bandsOuter (abundant) and inner cortex (some), between collenchyma (some)NonePlag. Terete 5.112 X 4.481 3-5Brown, larger than collenchymaDiscontinuous 2-5 Angular, brown2-5, in discontinuous bandsSome groups of cells: inner cortexNonePlag. Terete 5.921 X 5.406 4-5Brown, larger than collenchymaDiscontinuous 2-6Not seen (all lignified)2-6, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between NonePlag. Terete 7.093 X NA 4-5Brown, larger than collenchymaDiscontinuous 1-5Not seen (all lignified)1-5, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between collenchymaNoneMuldera P. sp.R. Asmarayani 547Plag. Terete 2.748 X 2.725 2-3Brown, larger than collenchymaContinuous 2-4Angular, dark brown2-4, in discontinuous bandsSome: outer collenchymaNoneOrth. Terete 4.827 X 4.646 2-3Brown, larger than collenchymaDiscontinuous 4-5Angular, dark brown3-5, in discontinuous bandsFew: inner cortex ContinuousPlag. Terete 7.257 X 7.246 3-4Brown, larger than collenchymaDiscontinuous 4-5 Angular3-5, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between collenchymaNoneMuldera P. sp.R. Asmarayani 634Orth. Terete 3.256 X 2.952 3-5Brown, larger than collenchymaDiscontinuous 1-4Angular, little thickening walls1-4, in discontinuous bandsFew: outer cortex, between collenchymaContinuousMuldera P. sp.R. Asmarayani 636Orth. Terete 4.549 X 4.346 2-3Brown, larger than collenchymaDiscontinuous 4-6Angular, little thickening walls1-5, in discontinuous bandsFew: outer cortex, between collenchymaNoneCubeba s.str. P. cubeba L. f.R. Asmarayani 768Plag. Terete 1.795 X 1.766 1-2Dark brown, equal size to/larger than collenchymaDiscontinuous 3-5 Angular1-4, in discontinuous bandsOuter (some) and inner cortex (few), outermost layer of collenchyma (some)NoneCortexTable S1. Stem anatomical characters observed. NA=not applicable (in some cases the specimens are too degraded to observe the character); orth.=orthotropic shoot; plag.=plagiotropic shoot; usb=undulating sclerenchyma band; vb(s)=vascular bundle(s); "+" in the number of medullary vb ring indicates that the additional ring does not form a complete ring.150Orth. Terete 3.404 X 3.285 2-4Dark brown, equal size to/larger than collenchymaDiscontinuous 3-5Angular, little thickening walls1-4, in discontinuous bandsOuter cortex (some), between or outermost layer of collenchyma (some)NoneCubeba s.str. P.mollissimum BlumeR. Asmarayani 629Orth.Terete, ± furrowed4.228 X 3.606 2-3Innermost cells have thick walls, larger than collenchymaDiscontinuous 4-6 Angular2-5, in discontinuous bandsNone ContinouosCubeba s.str. P. pedicellosum Wall.R. Asmarayani 644-LHK12-47Plag. Terete 1.721 X 1.44 1-2Rather thick walls, larger than collenchymaDiscontinuous 1-3Angular, little thickening walls1-3, in discontinuous bandsNone NoneOrth.Terete, ovale3.494 X 2.585 2-4Rather thick walls, larger than collenchymaDiscontinuous 1-4Angular, little thickening walls1-4, in discontinuous bandsOuter cortex (few) NoneR. Asmarayani 675Plag. Terete 1.259 X 1.162 1Brown, thick walls, larger than collenchymaDiscontinuous 2-4 Angular1-3, in disontinuous bandsNone NonePlag. Terete 1.451 X 1.399 1-2Brown, thick walls, larger than collenchymaDiscontinuous 1-3 Angular1-4, in discontinuous bandsNone NonePlag. Terete 3.521 X 2.92 1Brown, thick walls, larger than collenchymaDiscontinuous 3-4 Angular1-3, in discontinuous bandsGroups of cells: outer (few) and inner (some) cortex, within collenchyma (some)NoneCubeba s.str. P. ribesioides Wall.R. Asmarayani 635Plag. Terete 2.671 X 2.432 2-3Dark brown, equal size to/larger than collenchymaDiscontinuous 2-4Not seen (all lignified)2-4, in discontinuous bandsAbundant: outer cortex NoneOrth. Terete 10.45 X 8.894 2-10Dark brown, equal size to/larger than collenchymaDiscontinuous 2-5Not seen (all lignified)2-5, in discontinuous bandsAbundant big groups of cells: outer and inner cortex, between collenchymaNoneW6 P. caninum BlumeR. Asmarayani 597Plag. Terete 1.576 X 1.491 1-2Rather thick walls, larger than collenchymaDiscontinuous 2-3 Angular1-2, in scattered groups of cellsNone ContinuousOrth. Terete 2.825 X 2.612 1-2Rather thick walls, larger than collenchymaDiscontinuous 3-5Angular, little thickening walls1-3, in discontinuous bandsNone ContinuousW6 P. caninum BlumeR. Asmarayani 881Plag. Terete 2.319 X 2.285 1-2Rather thick walls, larger than collenchymaDiscontinuous 2-3 Angular1-3, in discontinuous bandsNone NoneW6 P. curtisii C. DC.R. Asmarayani 651Plag. Terete 1.192 X 1.09 1-2Rather thick walls, larger than collenchymaDiscontinuous 1-3Angular, little thickening walls1-2, in scattered groups of cellsFew (seen 1): between collenchymaNoneW6 P. muricatum BlumeR. Asmarayani 630Plag. Terete 2.319 X 2.237 2-3Larger than collenchymaDistinct bundles 3-5Angular, rather massive1-4, some scattered cellsNoneMostly vb-associatedW6 P. philodendron Ridl.R. Asmarayani 685Orth. Terete 11.554 X 11.229 6-8Equal size to/larger than collenchymaDistinct bundles 8-12 Angular None None ContinuousW6 P. pullibaccum Trel.R. Asmarayani 742Plag. Terete 2.125 X 2.085 1-2Larger than collenchymaDistinct bundles 3-5Angular, some cells have massive thickening walls1-4, in groups of cells or discontinuous bandsNoneDiscontinuous, not always vb-associatedW5 P. cilibracteum C. DC.R. Asmarayani 624Plag. Terete 1.773 X 1.746 2-3Thick walls, larger than collenchymaDiscontinuous 1-3Angular, little thickening walls1-2, in discontinuous bandsSome: outer and inner cortexNone151R. Asmarayani 679Plag. Terete 2.585 X 2.445 2-3Rather thick walls, larger than collenchymaDiscontinuous 1-4 Angular1-3, in discontinuous bandsNoneVb-associated, uncommon (seen in 1 vb)Orth. Terete 7.647 X 6.861 4-6Rather thick walls, larger than collenchymaDiscontinuous 2-6 Angular2-6, in discontinuous bandsNone ContinuousW5P. porphyrophyllum N. E. Br.R. Asmarayani 797Plag. Terete 3.003 X 2.864 2-3Brown, rather thick walls, larger than collenchymaDiscontinuous 1-3 Angular, brown1-3, in discontinuous bandsFew: inner cortex NoneEupiper s.str. P. nigrum L.R. Asmarayani 510Plag. Terete 2.363 X 1.92 2-3Rather thick walls, larger than collenchymaDiscontinuous 2-4 Angular1-4, in discontinuous bandsSomer: outer cortex NoneW4 P. ramipilum C. DC.R. Asmarayani 590Plag. Terete 3.037 X 2.667 2-3Thick walls, larger than collenchymaDistinct bundles 3-5 Angular1-3, in scattered solitary or groups of cellsNone NoneOrth. Terete 5.98 X 5.57 4-5Thick walls, larger than collenchymaDiscontinuous (not clear in distinct bundles or not)3-6 Angular1-3, in scattered solitary or groups of cellsNoneVb-associated, uncommon (seen in 2 vbs)Euchavica s.str.P. betle L. MO 2002-1050 Plag. Terete 3.024 X 3.017 2-3Innermost cells have thick walls, larger than collenchymaDistinct bundles 3-5Angular, massive thickening walls1-4, in big groups of cells in some collenchyma bundles (none in some bundles)None ContinuousPlag.Terete, ± reniform3.763 X 3.329 2-3Innermost cells have thick walls, larger than collenchymaDistinct bundles 4-6Angular, massive thickening walls1-4, in big groups of cellsNone ContinuousOrth.Terete, ± ovale4.258 X 3.112 2-3Innermost cells have thick walls, larger than collenchymaDistinct bundles 4-5 Angular1-4, in big groups of cellsNone ContinuousPlag. Terete 4.705 X 4.305 2-3Innermost cells have thick walls, larger than collenchymaDistinct bundles 4-5Angular, massive thickening walls1-5, in big groups of cellsNone ContinuousW3 P.argyrites Ridl.R. Asmarayani 637Plag. Terete 3.79 X 3.349 2-4Thick walls, larger than collenchymaDistinct bundles 4-6 Angular1-3, some scattered cellsNone NoneOrth. Terete 9.234 X 9.234 2-3Thick walls, larger than collenchymaDistinct bundles 4-7 Angular1-4, some scattered cellsNoneMostly vb-associatedW2P. puberulirameum C. DC.R. Asmarayani 616Plag. Terete 2.942 X 2.716 1-2Thick walls, equal size to/larger than collenchymaDistinct bundles 1-4Angular, little thickening walls1-4, in discontinuous bandsSome groups of cells: within collenchyma, inner cortexVb-associated, uncommonOrth.Terete, biconcave6.305 X 3.817 1Rather thick walls, equal size to/larger than collenchymaDiscontinuous 1-5Angular, little thickening walls1-5, in scattered groups of cellsAbundant groups of cells: within collenchyma, inner cortexContinuousOrth. Terete 12.394 X 10.039 1-2Larger than collenchymaDiscontinuous 1-5Not seen (all lignified)1-5, in scattered groups of cellsAbundant groups of cells: within collenchyma, inner cortexVb-associated, uncommon152W2P. kadsura (Choisy) OhwiN. Tanaka s. n. Plag. Terete 2.149 X 2.098 1Equal size to/larger than collenchymaDistinct bundles 3-4 Angular 1, few scattered cells None ContinuousOrth. Terete 3.105 X 2.82 1-2Rather thick walls, equal size to/larger than collenchymaDistinct bundles 1-4Angular, little thickening walls1-3, in discontinuous bandsNone ContinuousSarcostemon s.l.P. quinqueangulatum Miq.R. Asmarayani 573Plag.Terete, ± pentagonal5.134 X 5.125 2-4Larger than collenchymaDistinct bundles, located in the ridges of the stem5-7 AngularVery few scattered cellsNone NoneOrth.Terete, 10-ridged10.016 X 8.8235 (furrow) - 20 (riLarger than collenchymaDistinct bundles, located in the ridges of the stem10-11 Angular Scattered cells Outer and inner cortex ContinuousOrth.Terete, ± 7-ridged10.414 X 10.0626 (furrow) - 18 (riLarger than collenchymaDistinct bundles, located in the ridges of the stem9-10 Angular Scattered cells Outer and inner cortex ContinuousR. Asmarayani 812Plag.Terete, 8-ridged3.21 X 3.3363-4 (furrow) - 17 (riLarger than collenchymaDistinct bundles, located in the ridges of the stem2-6 Angular None NoneFew, not always vb-associatedPseudochavica sensu Ridley s.str.P. oreophilum Ridl.R. Asmarayani 567Plag. Terete 1.914 X 1.851 3Rather thick walls, equal size to/larger than collenchymaDistinct bundles 3-4 Angular, massive None None ContinuousPlag. Terete 3.417 X 3.322 3-4Rather thick walls, equal size to/larger than collenchymaDistinct bundles 5-6 Angular, massive Scattered cells None ContinuousOrth. Terete 8.122 X 8.108 3Brown, rather thick walls, equal size to/larger than collenchymaDiscontinuous 4-7 Angular, massive Scattered cellsSome: outer cortex, between collenchymaContinuousPseudochavica sensu Ridley s.str.P. cyrtostachys Ridl.R. Asmarayani 579Plag. Terete 1.538 X 1.512 1Rather thick walls, equal size to/larger than collenchymaDistinct bundles 3-4 Angular 1-4, few scattered cells None ContinuousPlag. Terete 2.612 X 2.463 1Rather thick walls, equal size to/larger than collenchymaDistinct bundles 4 Angular, massive 1-4, few scattered cellsFew cells/groups of cells: inner cortexContinuousOrth. Terete 4.728 X 4.601 1-2Larger than collenchymaDiscontinuous 4 Angular1-4, in some groups of cellsSome: outer and inner cortex, between collenchymaContinuousW1 P. sarmentosum Roxb.R. Asmarayani 765Orth. Terete 2.536 X 2.527 1Rather thick walls, equal size to/larger than collenchymaDistinct bundles 3-6 Angular None None ContinuousW1 P. sp.R. Asmarayani 602Orth. Terete 1.34 X 1.2 0 NA Discontinuous 2-3 Angular1-3, in scattered solitary or groups of cellsFew: between collenchyma, inner cortexContinuousUnafiliated WWLP. sp.R. Asmarayani 809Plag. Terete 4.142 X 3.919 2-4Larger than collenchymaDiscontinuous 1-5Angular, little thickening walls1-5, in discontinuous bandsSome scattered cells: outer cortexNoneUnafiliated WWLP. sp.R. Asmarayani 568Plag. Terete 1.688 X 1.498 ≥1Brown, NA for other charactersNot seen 1-4 Angular1-3, in some groups of cells or discontinuous bandsGroups of cells: inner cortexNAPlag. Terete 1.81 X 1.583 2-3Larger than collenchymaDiscontinuous 1-4 Angular1-4, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between collenchymaContinuous153Orth. Terete 5.939 X 4.881 2-3Larger than collenchymaDiscontinuous 1-4 AngularGroups of cells or discontinuous bandsGroups cells: inner cortexContinuousUnafiliated WWLP. sp.R. Asmarayani 811Plag. Terete 1.424 X 1.275 2-3Brown, larger than collenchymaDiscontinuous 1-4Angular, little thickening walls1-4, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between collenchymaNoneOrth.± Terete, flattened in one side4.773 X 3.647 2-4Brown, larger than collenchymaDiscontinuous 1-6Angular, little thickening walls1-6, in discontinuous bandsAbundant groups of cells: outer and inner cortex, between collenchymaContinuousE5 P. retrofractum VahlR. Asmarayani 767Orth. Terete 1.44 X 1.431 0-1Thick walls, equal size to/larger than collenchymaDistinct bundles 2-8 Angular1-6, some scattered cellsSome scattered cells: 1-3 outermost layers of collenchymaNonePlag. Terete 2.398 X 2.373 1Rather thick walls, equal size to collenchymaDistinct bundles 2-6 Angular 1-2, few scattered cellsFew scattered cells: 1-2 outermost layers of collenchymaNoneR. Asmarayani 884Plag. Terete 1.956 X 1.888 0-1Thick walls, equal size to/larger than collenchymaDistinct bundles 5-7Angular, rather massive thickening wallsNone None NoneE4 P. macropiper PennantR. Asmarayani 607Plag. Terete 1.681 X 1.583 2-3Larger than collenchymaDiscontinuous 1-4Angular, little thickening walls1-3, in discontinuous bandsSome groups of cells: inner cortexVb-associated, uncommonOrth. Terete 3.336 X 3.302 2-3Larger than collenchymaDiscontinuous 1-4Angular, little thickening walls1-4, in discontinuous bandsGroups of cells: outer (abundant) and inner (few) cortex, between collenchymaContinuousR. Asmarayani 707Plag. Terete 3.046 X 2.594 3-4Brown, larger than collenchyma, slightly elongated radiallyDiscontinuous 1-4Angular, brown, massive thickening walls1-4, most cells, in discontinuous bandsAbundant: outer and inner cortex, between collenchymaNoneOrth.± Terete, flattened in one side5.505 X 5.234 5-7Brown, larger than collenchyma, slightly elongated radiallyDiscontinuous 1-6Angular, brown, massive thickening walls1-6, in many groups of cellsNone ContinuousE4P. aff. macropiper PennantR. Asmarayani 548Plag. Terete 1.824 X 1.759 1-2Rather thick walls, larger than collenchymaDiscontinuous 2-3Angular, little thickening walls1-3, in discontinuous bandsNone ContinuousE4 P. decumanum L.R. Asmarayani 756Plag. Terete 6.64 X 6.457 2-4Collenchymal thickened walls (similar thickness with collenchyma), larger than collenchymaDistinct bundles 6-8Angular, rather massive thickening wallsNone Few: inner cortex ContinuousE4P. cf. dumiformans C. DC.R. Asmarayani 735Plag.Terete, ± furrowed2.095 X 2.02 1-2Collenchymal thickened walls (similar thickness with collenchyma), larger than collenchymaDistinct bundles 3-5 Angular 1-2, few scattered cells None ContinuousE3P. rubro-venosum Hort. ex RodigasR. Asmarayani 764Orth. Terete 1.878 X 1.702 0-1Rather thick walls, equal size to/larger than collenchymaDistinct bundles 2-5 Angular None NoneMostly vb-associatedOrth. Terete 3.505 X 3.275 0-1Rather thick walls, equal size to/larger than collenchymaDistinct bundles 2-5 Angular None NoneVb-associated, uncommon154R. Asmarayani 774Orth. Terete 4.054 X 4.02 1-2Rather thick walls, larger than collenchymaDiscontinuous 3-5 Angular NoneScattered cells: outer cortex (some), inner cortex (few)Vb-associatedE2 P. mestonii BaileyR. Asmarayani 749Plag. Terete 3.58 X 3.505 2-4Rather thick walls, larger than collenchymaDiscontinuous 2-4 Angular1-3, in discontinuous bandsSome scattered cells: outer cortexNoneE2 P. versteegii C. DC.R. Asmarayani 731Orth.Terete, ± hexagonal4.061 X 4.027 3-4Larger than collenchymaDiscontinuous, breaking in the angles of the stem4-6Angular, little thickening walls1-4, all cells, in discontinuous bandsNone ContinuousE2 P. sp.R. Asmarayani 758Plag. Terete 5.198 X 4.881 3-4Rather thick walls, larger than collenchymaDiscontinuous 2-7Angular, little thickening walls1-5, in discontinuous bandsNone NonePeltandron P. celtidiforme OpizR. Asmarayani 533Plag. Terete 2.129 X 1.946 2Brown, rather thick walls, larger than collenchymaDiscontinuous 2-3 Angular None None NonePlag. Terete 3.168 X 2.852 2Brown, rather thick walls, larger than collenchymaDiscontinuous 4 Angular None None NoneOrth. Terete 7.035 X 6.606 NA (≥1)Brown, NA for other charactersDiscontinuous 4 Angular None None NoneE1 P. abbreviatum OpizR. Asmarayani 511Plag. Terete 1.834 X 1.722 2-3Larger than collenchymaDistinct bundles 1-4 Angular 1-2, few scattered cells None ContinuousE1R. Asmarayani 519Plag. Terete 1.711 X 1.662 1-2Larger than collenchymaDiscontinuous 3-4 Angular None None ContinuousE1 P. albamentum C. DC.R. Asmarayani 736Plag.Terete, ± ridged1.53 X 1.412 3-4Elongated radially, larger than collenchymaDistinct bundles, located in the ridges of the stem1-5 Angular 1- 4, few scattered cellsFew: outer and inner cortexContinuousPlag.Terete, ± flattened in one side2.546 X 2.241 3-5Some cells (mainly the ones in the ridges of the stem) are elongated radially, larger than collenchymaDistinct bundles, located in the ridges of the stem3-5Angular, some cells have massive thickening walls1- 3, few scattered cellsSome: outer and inner cortexContinuousE1 P. celebicum BlumeR. Asmarayani 557Plag. Terete 2.558 X 2.472 2-3Larger than collenchymaDistinct bundles 2-5Angular, massive thickening walls1-2, some scattered cellsSome cells/groups of cells: inner cortexContinuousE1 P. majusculum BlumeR. Asmarayani 737Plag. Terete 5.614 X 5.162 4-5Dark brown, rather thick walls, larger than collenchymaDiscontinuous 5-7Angular, dark brown3-7, in discontinuous bandsBetween collenchyma (filling the gaps), inner cortex (few)NoneE1 P. sp.R. Asmarayani 559Plag. Terete 2.78 X 2.741 2-3Larger than collenchymaDiscontinuous 2-3 Angular2-3, in scattered groups of cellsAbundant big groups of cells: inner cortexNot necessarily vb-associatedNew GuineaP. methysticum G. Foster var. wichmannii (C. DC.) LebotR. Asmarayani 711Plag. Terete 4.38 X 3.939 5-6Larger than collenchymaDistinct bundles 5-8Angular, massive thickening walls1-3, few scattered groups of cellsNone NoneOrth. Terete 11.788 X 11.336 6-8Larger than collenchymaDistinct bundles 5-9Angular, massive thickening walls1, few scattered cells None None155Table S1. ContinuedCLADE SPECIES SPECIMENStem typeShape of lignified pericyclic capContinuity of lignified pericyclic capHow lignified pericyclic caps around the vbs are unitedNumber of vbsNumber of smaller vbs between large vbsSecondary thickeningPrimary medullary rays Pattern of lignification in primary medullary raysMulderaP. schizonephros C. DC.R. Asmarayani 674Plag.Semilunar, thick walls, few areas are covered by sclereidsDiscontinuousLignified parenchyma, some cells have thick walls, some caps are not united31 1-4 Yes, early stageSome interfascicular areasMost rays between the xylemOrth.Semilunar, thick walls, most areas are covered by sclereidsContinuousLignified parenchyma, thick walls, some areas are covered by sclereids42 2-3 Yes Yes Most rays between the xylemMulderaP. flavimarginatum C. DC.R. Asmarayani 662Plag.Semilunar, few caps are not lignified, few areas are covered by sclereidsDiscontinuous Lignified parenchyma, most caps are not united22 2-3 Some vbs, early stageSome interfascicular areasMostly not lignifiedOrth.Semilunar, thick walls, all are covered by sclereidsContinuousLignified parenchyma, thick walls, some areas are covered by sclereids32 2-3 Yes Yes Rays between the xylemMuldera P. pentandrum C. DC.R. Asmarayani 593Plag.Semilunar, thick walls, few areas are covered by sclereidsDiscontinuousLignified parenchyma, some cells have thick walls31 2-4 Some vbs, early stageSome interfascicular areasMostly rays between the xylemPlag.Semilunar, thick walls, few areas are covered by DiscontinuousLignified parenchyma, some cells have thick walls, few areas are 40 1-4 Some vbs, early stageSome interfascicular areasSome, mostly rays between the xylemPlag.Semilunar, thick walls, some areas are covered by sclereidsContinuousLignified parenchyma, very thick walls, some areas are covered by slcereids35 2-5 Yes YesRays between the xylem, also some outermost rays between the phloemPlag.Semilunar, thick walls, some areas are covered by sclereidsContinuousLignified parenchyma, very thick walls, some areas are covered by sclereids52 1-3 Yes YesRays between the xylem, also some outermost rays between the phloemMuldera P. sp.R. Asmarayani 547Plag. Semilunar Discontinuous Lignified parenchyma 27 2-4 Some vbs, early stageSome interfascicular areasRays between the xylemOrth.Semilunar, some caps are not lignifiedDiscontinuous Lignified parenchyma 30 1-3 Some vbs, early stageSome interfascicular areasSome rays between the xylemPlag.Semilunar, thick walls, all are covered by sclereidsContinuous Lignified parenchyma, very thick wall 29 2-4 Yes YesRays between the xylem, also some outermost rays between the phloemMuldera P. sp.R. Asmarayani 634Orth.Semilunar, some caps are not lignifiedDiscontinuous None 24 1-4 No No NAMuldera P. sp.R. Asmarayani 636Orth.Semilunar, some caps have thick wall, some caps are not lignifiedDiscontinuousLignified parenchyma, thick-very thick walls31 2-4 Some vbs, early stageSome interfascicular areasSome rays between the xylemCubeba s.str. P. cubeba L. f.R. Asmarayani 768Plag. Semilunar, thick walls Discontinuous Lignified parenchyma 21 1-5 Yes Yes Rays between the xylemOrth. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 22 1-4 Yes YesMost, especially innermost, rays between the xylem, also most outermost rays between the phloemPeripheral vascular bundles (vbs)156Cubeba s.str. P.mollissimum BlumeR. Asmarayani 629Orth.NA (only few pericyclic cells in few vbs are lignified)Discontinuous None 23 2-3 Few vbs, early stage No NACubeba s.str. P. pedicellosum Wall.R. Asmarayani 644-LHK12-47Plag.Semilunar, few caps are not lignifiedDiscontinuousLignified parenchyma, all caps are not united18 1-3, 5 Some vbs, early stage No NAOrth.Semilunar, few caps are not lignifiedDiscontinuousLignified parenchyma, all caps are not united30 2-4 Some vbs, early stage No NAR. Asmarayani 675Plag. Semilunar Discontinuous None 16 2-3 No No NAPlag.Semilunar, few caps have thick wallsDiscontinuous None 20 1-3 Yes, early stage Yes Rays between the xylemPlag. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 23 1-3 Yes Yes Rays between the xylemCubeba s.str. P. ribesioides Wall.R. Asmarayani 635Plag.Semilunar, most caps are not lignifiedDiscontinuous Usb, most caps are not united 28 1-4 Few vbs, early stage No NAOrth.Semilunar, very thick wallsContinuousLignified parenchyma, very thick walls26 2-3Yes; some vbs bifurcate halfwayYesOutermost rays and few scattered groups between the xylem, most rays between the phloemW6 P. caninum BlumeR. Asmarayani 597Plag.Semilunar, some caps have thick walls, some caps are not lignifiedDiscontinuous None 16 1-4 Few vbs, early stage No NAOrth.Semilunar, some caps have thick wallsDiscontinuous Lignified parenchyma 23 1-3 Most vbs, early stageSome interfascicular areasInnermost rays between the xylemW6 P. caninum BlumeR. Asmarayani 881Plag.Semilunar, some caps have thick walls, few areas are covered by sclereidsDiscontinuousLignified parenchyma, most caps are not united, few areas are covered by sclereids24 1-2, 4 Most vbs, early stage Yes Rays between the xylemW6 P. curtisii C. DC.R. Asmarayani 651Plag.Semilunar, most caps are not lignifiedDiscontinuousLignified parenchyma, all caps are not united15 1-2, 4 Few vbs, early stage No NAW6 P. muricatum BlumeR. Asmarayani 630Plag.NA (all caps are not lignified)NA None 21 1-3 Few vbs, early stageFew interfascicular areas, very early stageNoneW6 P. philodendron Ridl.R. Asmarayani 685Orth.NA (all caps are not lignified)NA None 45 1-5 No (very limited) No (very limited) None (very limited)W6 P. pullibaccum Trel.R. Asmarayani 742Plag.Semilunar, some caps have thick walls, some ccaps are not lignifiedDiscontinuousLignified parenchyma, some cells have thick walls22 1-2, 4 Most vbs, early stageSome interfascicular areasRays between the xylemW5 P. cilibracteum C. DC.R. Asmarayani 624Plag.Semilunar, some caps have thick wallsContinuous Lignified parenchyma 23 1-4 Yes Yes Rays between the xylemR. Asmarayani 679Plag. Semilunar Discontinuous None 22 1-3 Yes, early stageFew interfascicular areasYes157Orth. Semilunar Discontinuous Lignified parenchyma 33 1-2 Yes YesFew innermost rays between the xylem, also few outermost rays between the phloemW5P. porphyrophyllum N. E. Br.R. Asmarayani 797Plag.Semillunar, some caps have thick wallsDiscontinuousLignified parenchyma, some caps are fully united34 2-4 Yes, early stage YesMost innermost rays between the xylemEupiper s.str. P. nigrum L.R. Asmarayani 510Plag. Semillunar, thick wall DiscontinuousLignified parenchyma, some cells have thick walls19 1-4 Yes YesMost innermost rays between the xylemW4 P. ramipilum C. DC.R. Asmarayani 590Plag.NA (only few pericyclic cells in few vbs are lignified)Discontinuous NA 37 0-3 Few vbs, early stage No NAOrth. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 27 1Yes; some vbs bifurcate halfwayYes Rays between the phloemEuchavica s.str.P. betle L. MO 2002-1050 Plag.NA (all caps are not lignified)NA None 32 2-5 Few vbs, early stageFew interfascicular areas, very early stageNonePlag.Semilunar, most caps are not lignifiedDiscontinuous None 34 2-4(-7) Some vbs, early stageFew interfascicular areas, very early stageNoneOrth.Spot, most caps are not lignifiedDiscontinuous None 37 2-4 No No NAPlag.Semilunar, some caps are not lignifiedDiscontinuous Lignified parenchyma 41 3-5 Some vbs, early stageSome interfascicular areas, very early stageNoneW3 P.argyrites Ridl.R. Asmarayani 637Plag.Semilunar, some caps are not lignifiedDiscontinuous Lignified parenchyma 27 0-3 Some vbs, early stageFew interfascicular areas, very early stageNoneOrth. Semilunar Discontinuous Lignified parenchyma 48 1-3 Yes YesSome innnermost rays between the xylem, also some outermost rays between the phloemW2P. puberulirameum C. DC.R. Asmarayani 616Plag. Semilunar, thick walls DiscontinuousLignified parenchyma, thick walls, few caps are not united23 1-3 Yes Yes Rays between the xylemOrth. Semilunar, thick walls ContinuousLignified parenchyma, thick walls, few caps are not united30 1-2 Yes YesMost outermosts rays between the phloemOrth. Semilunar, thick walls ContinuousLignified parenchyma, thick walls, few caps are not united17 1-2 Yes YesSome innermost rays between the xylem, also some outermost rays between the phloemW2P. kadsura (Choisy) OhwiN. Tanaka s. n. Plag. None NA None 15 1-4 Few vbs, early stage No NA158Orth. None NA None 15 1-2, 4 Yes, early stageFew interfascicular areas, very early stageNoneSarcostemon s.l.P. quinqueangulatum Miq.R. Asmarayani 573Plag.NA (only few pericyclic cells in some vbs are lignified)DiscontinuousLignified parenchyma, only few parenchyma cells are lignified262, 4; large vbs are opposite the ridgesSome vbs, early stageSome interfascicular areasMost rays between the xylemOrth. Semilunar DiscontinuousLignified parenchyma, some caps are not united452-4; large vbs are opposite the ridgesFew vbs, early stageFew interfascicular areasSome innermost rays between the xylemOrth. Semilunar Continuous Lignified parenchyma 361-4; large vbs are opposite the ridgesYes YesSome innermost layers of interxylem, but mostly not lignifiedR. Asmarayani 812Plag.NA (only few pericyclic cells in most vbs are lignified)DiscontinuousLignified parenchyma, all caps are not united201-2; large vbs are opposite the ridgesYes, early stageFew interfascicular areas, very early stageSome innermost layersPseudochavica sensu Ridley s.str.P. oreophilum Ridl.R. Asmarayani 567Plag.NA (all caps are not lignified)NA None 21 1-3 No No NAPlag.NA (only few pericyclic cells in few vbs are lignified)Discontinuous None 24 1-3 Some vbs, early stageFew interfascicular areasRays between the xylemOrth. Semilunar DiscontinuousLignified parenchyma, all caps are not united34 1-3 Yes Yes Rays between the xylemPseudochavica sensu Ridley s.str.P. cyrtostachys Ridl.R. Asmarayani 579Plag.NA (all caps are not lignified)NA None 13 0-1 No No NAPlag.NA (all caps are not lignified)NA None 15 0-1 Yes Yes Rays between the xylemOrth.NA (only few pericyclic cells in some vbs are lignified)Discontinuous None 18 1-2 Yes Yes Rays between the xylemW1 P. sarmentosum Roxb.R. Asmarayani 765Orth.Semilunar, most caps are not lignifiedDiscontinuous None 24 1-4 Some vbs, early stageFew interfascicular areas, very early stageNoneW1 P. sp.R. Asmarayani 602Orth.NA (only few pericyclic cells in few vbs are lignified)Discontinuous None 15 0-2 Few vbs, early stageFew interfascicular areasSome innermost rays between the xylemUnafiliated WWLP. sp.R. Asmarayani 809Plag.Semilunar, most caps are not lignifiedDiscontinuousLignified parenchyma, most caps are not united29 0-4 Few vbs, early stage Yes, very early stageSome innermost Rays between the xylemUnafiliated WWLP. sp.R. Asmarayani 568Plag. Semilunar Discontinuous NA 22 2-4 No No NAPlag. Semilunar Discontinuous Lignified parenchyma 24 2-3 Yes, early stage Yes Rays between the xylemOrth. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 30 2-3 Yes Yes Few scattered cells159Unafiliated WWLP. sp.R. Asmarayani 811Plag.Semilunar, some caps are not lignifiedDiscontinuousLignified parenchyma, most caps are not united26 1-2, 4 Few vbs, early stage Few interfascicular areas, very early stageFew innermost rays between the xylemOrth. Semilunar, thick walls Continuous Lignified parenchyma 35 1-4 Yes YesMost rays between the xylem, some outermost rays between the phloemE5 P. retrofractum VahlR. Asmarayani 767Orth. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 26 1-2 Yes Yes NoPlag. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 24 1-3 Yes, early stage Yes Rays between the xylemR. Asmarayani 884Plag. Semilunar Discontinuous None 17 1-2 Some vbs, early stageSome interfascicular areasInnermost rays between the xylemE4 P. macropiper PennantR. Asmarayani 607Plag. Semilunar DiscontinuousLignified parenchyma, all caps are not united20 1-4 Few vbs, early stage Few interfascicular areas, very early stageRays between the xylemOrth.Semilunar, some caps have thick wallsContinuous Lignified parenchyma 26 1-4 Yes YesRays between the phloem, rays between the xylem scarcely lignifiedR. Asmarayani 707Plag. Semilunar DiscontinuousUsb or lignified parenchyma when rays have been formed; some caps are not united25 1, 3, 8 Few vbs, early stage Few interfascicular areas, very early stageNoOrth. Semilunar Continuous Lignified parenchyma 61 1-4 Yes YesRays between the xylem some outermost rays between the phloemE4P. aff. macropiper PennantR. Asmarayani 548Plag.Semilunar, most caps have thick wallsDiscontinuousLignified parenchyma, some caps are not united27 1-2 Most vbs, early stageMost interfascicular areasRays between the xylemE4 P. decumanum L.R. Asmarayani 756Plag. Semilunar/arrow Discontinuous None 69 2-5 Some vbs, early stageSome interfascicular areasRays between the xylemE4P. cf. dumiformans C. DC.R. Asmarayani 735Plag.NA (only 1 pericyclic cell in 1 vb is lignified)NA None 18 1-3, 6 Some vbs, early stageFew interfascicular areas, very early stageNoE3P. rubro-venosum Hort. ex RodigasR. Asmarayani 764Orth.NA (only few pericyclic cells in few vbs are lignified)Discontinuous None 11 2, 4 Few vbs, early stage Few interfascicular areas, very early stageNoOrth. Semilunar Discontinuous None 20 0-1 Some vbs, early stageFew interfascicular areasNoR. Asmarayani 774Orth. Semilunar DiscontinuousLignified parenchyma, some caps are not united21 1 Yes YesInnermost rays (starting to lignify)160E2 P. mestonii BaileyR. Asmarayani 749Plag.Semilunar, few caps are not lignifiedDiscontinuous Usb, some caps are not united 30 1-4 Some vbs, early stageSome interfascicular areas, very early stageSome innermost rays between the xylemE2 P. versteegii C. DC.R. Asmarayani 731Orth. Semilunar Discontinuous Usb, some caps are not united 39 1-3 Some vbs, early stage No NAE2 P. sp.R. Asmarayani 758Plag. Semilunar Continuous Usb 45 1-3 Some vbs, early stage No NAPeltandron P. celtidiforme OpizR. Asmarayani 533Plag.Semilunar, thick walls, some caps are covered by sclereidsContinuousUsb, some areas are covered by sclereids24 1-2 Some vbs, early stage No NAPlag.Semilunar, thick walls, some caps are covered by sclereidsContinuousUsb, some areas are covered by sclereids39 1-5 Some vbs, early stage No NAOrth. Semilunar Continuous Lignified parenchyma 68 2-3Yes; a vb bifurcates halfwayYesMost half innermost rays between the xylemE1 P. abbreviatum OpizR. Asmarayani 511Plag.Semilunar, most caps are not lignifiedDiscontinuous None 14 1 No No NAE1R. Asmarayani 519Plag.Semilunar, most caps are not lignifiedDiscontinuous None 16 0-1 No No NAE1 P. albamentum C. DC.R. Asmarayani 736Plag. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 26 1-3 Some vbs, early stageSome interfascicular areasRays between the xylemPlag. Semilunar, thick walls Continuous Lignified parenchyma, thick walls 34 1-3 Yes YesRays between the xylem, some outermost rays between the phloemE1 P. celebicum BlumeR. Asmarayani 557Plag.Semilunar; thick-very thick wallsContinuousLignified parenchyma, thick-very thick walls30 1-4 Yes Yes Rays between the xylemE1 P. majusculum BlumeR. Asmarayani 737Plag. Semilunar Continuous Lignified parenchyma 40 1-4 Yes Yes Rays between the xylemE1 P. sp.R. Asmarayani 559Plag. Semilunar Continuous Lignified parenchyma 28 1-3 Few vbs, early stageFew interfascicular areas,very early stageNoNew GuineaP. methysticum G. Foster var. wichmannii (C. DC.) LebotR. Asmarayani 711Plag.Semilunar/spot, some caps are not lignifiedDiscontinuous None 37 1-3 Yes, early stage Yes, very early stage Rays between the xylemOrth.Semilunar/spot, some caps are not lignifiedDiscontinuous None 96 1-5 Yes Yes Rays between the xylem161Table S1. ContinuedCLADE SPECIES SPECIMENStem typeVessel number across top of vbs (Vessel arrangement)Vessels vs. secondary medullary ray trans-sectional area (qualitative)Radial width of primary medullary rays (number of cells) Undulating sclerenchyma band (usb) layersSclerenchyma cap on phloemSclerenchyma cap on xylemNumber of ringsNumber of vbs (outermost; innermost, if applicable)Vbs with altered orientation/close to each otherSecondary thickening Ray-like cells between vascular bundlesMulderaP. schizonephros C. DC.R. Asmarayani 674Plag. NA NA NA 3-5 Yes Yes 2 5; 10All outer vbs have 180° orientation, 1 outer vb pairedInner vbs only, equally massive as peripheral vbsSome interfascicular areas in inner ringOrth. 1(-2)Vessels < Rays7-16 5-7 Yes Yes 2 12; 16Some outer vbs have 90° orientationYes, inner vbs are equally massive as the peripheral vbsInterfascicular areas in inner ringMulderaP. flavimarginatum C. DC.R. Asmarayani 662Plag. NA NA NA 1-3 Yes Yes 1 8 None Some vbs, early stageSome interfascicular areasOrth. 1(-2)Vessels < Rays8-15 3-5 Yes Yes 1 1; 18Outer vb has 160° orientation, 2 inner vbs are in a pairYes, rather massive YesMuldera P. pentandrum C. DC.R. Asmarayani 593Plag. NA NA NA 3-5Some vbs, others have only few lignified cellsYes 1 14 1 vb paired Some vbs, early stageSome interfascicular areasPlag. NA NA NA 4-7 Yes Yes 1 + 6; 151 outer vb has 45° orientationMost inner vbs, early stageSome interfascicular areas in inner ringPlag. 1(-2)Vessels < Rays6-18 4-7Some vbs in inner ring, others have only few lignified Yes 2 6; 122 outer vbs have 45° and 90° orientationSome inner vbs, early stageSome interfascicular areas in inner ring and between inner and outer Plag. 1Vessels < Rays8-16 4-6 Yes Yes 2+ 3; 12; 17Most vbs in middle and outer rings are in altered orientation (up to 100°)Yes, early to advanced stages, inner vbs are more developed than middle vbsSome interfascicular areas in inner ring and between inner and middle vbMuldera P. sp.R. Asmarayani 547Plag. NA NA NA 4-5Some vbs, others have only few lignified cellsMost vbs 1 10 None Few vbs, early stageSome interfascicular areasOrth. NA NA NA 5-8 Yes Yes 1 11 None Yes, early stageSome interfascicular areasPlag. 1(-2)Vessels < Rays12-17 6Some vbs, others have only few lignified cellsYes 1 10 None Some vbs, early stageSome interfascicular areasMuldera P. sp.R. Asmarayani 634Orth. NA NA NA 4-5 Yes Yes 1+ 2; 10 None Most vbs, early stageSome interfascicular areasMuldera P. sp.R. Asmarayani 636Orth. NA NA NA 4-5 Yes Yes 1 16A small vb, slightly outside the ring, has 30° orientationYes, early stagesSome interfascicular areasCubeba s.str. P. cubeba L. f.R. Asmarayani 768Plag. NA NA NA 6-8Most vbs have small group of lignified cellsYes 1 8 None Yes, early stage Few interfascicular areasOrth. 1-2Vessels < Rays7-18 5-6Most vbs have small group of lignified cellsYes 1 8 None Yes, early stageSome interfascicular areasMassive secondary thickening in Medullary vascular bundles162Cubeba s.str. P.mollissimum BlumeR. Asmarayani 629Orth. NA NA NA 3-5 No Yes 1 6 None Some vbs, early stage NoCubeba s.str. P. pedicellosum Wall.R. Asmarayani 644-LHK12-47Plag. NA NA NA 4-6Some vbs have only few lignified cellsYes 1 61 vb has 60° orientationYes, early stage Few interfascicular areasOrth. NA NA NA 5-7 Most vbs Yes 1 12 None Yes, early stageMost interfascicular areasR. Asmarayani 675Plag. NA NA NA 3-5 No Yes 1 6 No Few vbs, early stageSome interfascicular areasPlag. NA NA NA 4-6 No Yes 1 8 No Most vbs, early stageSome interfascicular areasPlag. 1(-2)Rays > Vessels7-15 4-7Few vbs, few others have only few lignified cellsYes 1 6 No Yes, early stage Few interfascicular areasCubeba s.str. P. ribesioides Wall.R. Asmarayani 635Plag. NA NA NA 4-7 No Yes 1 13 None Yes, early stage NoOrth. 1-2(-3)Vessels = Rays13-294-6, discontinuous, sometimes inserted by clustered cells of thickly lignified cellsYes Yes 1 10 NoneYes; some vbs bifurcate halfwayYesW6 P. caninum BlumeR. Asmarayani 597Plag. NA NA NA 5-9Some vbs have only few lignified cellsYes 1 6 None Yes, early stageSome interfascicular areasOrth. NA NA NA 9-11 Yes Yes 1 7 None Yes, early stageSome interfascicular areasW6 P. caninum BlumeR. Asmarayani 881Plag. NA NA NA 5-71 vb has only few lignified cellsYes 1 6 None Yes, early stage Few interfascicular areasW6 P. curtisii C. DC.R. Asmarayani 651Plag. NA NA NA 4-6Some vbs have only few lignified cellsYes 1 7 None Some vbs, early stage NoW6 P. muricatum BlumeR. Asmarayani 630Plag. NA NA NA 3-4Few vbs have only have 1 lignified cellYes 1 9 None Yes, early stage Few interfascicular areasW6 P. philodendron Ridl.R. Asmarayani 685Orth. NA NA NA 3-6 No Some vbs 1+ 3; 132 outer vbs have 45 and 180° orientationNo (very limited) No (very limited)W6 P. pullibaccum Trel.R. Asmarayani 742Plag. NA NA NA 4-7 No Some vbs 1 8 None Yes, early stage An interfascicular areaW5 P. cilibracteum C. DC.R. Asmarayani 624Plag. NA NA NA 3-5Few vbs have only few lignified cellsYes 1 1, 9 None Yes, early stage Few interfascicular areasR. Asmarayani 679Plag. NA NA NA 5-8 No Yes 1+ 4; 93 outer vbs has 30-90° orientationSome vbs, early stageSome interfascicular areas163Orth. 1-4 Vessels > Ray 7-13 8-10All vbs have small group of lignified cellsYes 1 171 vb has 90° orientationYes, early stage YesW5P. porphyrophyllum N. E. Br.R. Asmarayani 797Plag. NA NA NA 4-6, discontinuousFew vbs, others have only few lignified cellsMost vbs 1+ 3; 102 outer vbs has 45-90° orientationSome vbs, early stageSome interfascicular areasEupiper s.str. P. nigrum L.R. Asmarayani 510Plag. NA NA NA 3-5 Yes Yes 1 2 NA Yes, early stage YesW4 P. ramipilum C. DC.R. Asmarayani 590Plag. NA NA NA 5-6Few vbs have only few lignified cellsSome vbs 1 221 vb has 45° orientationYes, early stageSome interfascicular areasOrth. 1-2Vessels > Rays12-15 5-8 Yes Yes 1+ 2; 129 vbs are in groups of 3Some vbs, early stageSome interfascicular areasEuchavica s.str.P. betle L. MO 2002-1050 Plag. NA NA NA 2-4 No Few vbs 1 12 None No NoPlag. NA NA NA 3-6 No Some vbs 1+ 1; 11Outer vb has 180° orientationSome vbs, early stage Few interfascicular areasOrth. NA NA NA 4-5 No Yes 1 12 None Most vbs, early stage Few interfascicular areasPlag. NA NA NA 5-7 No Yes 1+ 3; 111 outer vb has 90° orientationYes, early stage NoW3 P.argyrites Ridl.R. Asmarayani 637Plag. NA NA NA 5-6Some vbs have small group of lignified cellsYes 1+ 1; 102 inner vbs are in a pairYes, early stage Few interfascicular areasOrth. 1-2(-4)Vessels > Rays14-28 10-15All vbs have small group of lignified cellsYes 1 22 None Yes, early stages YesW2P. puberulirameum C. DC.R. Asmarayani 616Plag. NA NA NA 3-6All vbs have only few lignified cellsYes 1+ 1; 8 2 vbs are in a pair Yes, early stage Few interfascicular areasOrth. NA NA NA 5-7All vbs have only some lignified cellsYes 1 9 None Yes, early stageSome interfascicular areasOrth. (6-)8-9(-13)Vessels > Rays10-24 3-4, discontinuous Yes Yes 1 6 None Yes, early stage NoW2P. kadsura (Choisy) OhwiN. Tanaka s. n. Plag. NA NA NA 2-4 NoFew vbs have only few lignified cells1 8 None No NoOrth. NA NA NA 4-7 No Yes 1 7 None Yes, early stage No164Sarcostemon s.l.P. quinqueangulatum Miq.R. Asmarayani 573Plag. NA NA NA 6-7 No 1 vb only 1+ 19; 4Outer ring has most vbs (1 ring); 1 outer vb has 90° orientationMost vbs, early stageSome interfascicular areasOrth. NA NA NA 6-9Some vbs have small group of lignified cellsSome vbs 1+ 3; 17Outer vbs have 70-90° orientationYes, early stageSome interfascicular areasOrth. (1-)2(-3)Vessels > Rays8-40, uneven width across s 8-13 Yes Yes 1+ 1 (seen); 16 None Yes, early stageSome interfascicular areasR. Asmarayani 812Plag. NA NA NA 5-72 vbs have only few lignified cellsSome vbs 1 13 1 vb paired Yes, early stageSome interfascicular areasPseudochavica sensu Ridley s.str.P. oreophilum Ridl.R. Asmarayani 567Plag. NA NA NA 3-6 No Few vbs 1 8 None No NoPlag. NA NA NA 7-8Few vbs have only few lignified cellsMost vbs 1 9 None Some vbs, early stage NoOrth. 1-2Vessels < Rays15-36 6-7 Yes Yes 1 6 None Yes NoPseudochavica sensu Ridley s.str.P. cyrtostachys Ridl.R. Asmarayani 579Plag. NA NA NA (2-)4-5 NoFew vbs, others have only few lignified cells1 9 None Few vbs, early stage NoPlag. NA NA NA 7-8 NoSome vbs, others have only few lignified cells1 8 None Few vbs, early stage NoOrth. 3-4Vessels = Rays21-40 6-8 1 vb Yes 1 7 None Yes, early stage NoW1 P. sarmentosum Roxb.R. Asmarayani 765Orth. NA NA NA 2-4 No No 1 7 None No NoW1 P. sp.R. Asmarayani 602Orth. NA NA NA 3-4 No Yes 1 4 None No NoUnafiliated WWLP. sp.R. Asmarayani 809Plag. NA NA NA 3-7 Yes Yes 1 8 None Some vbs, early stage NoUnafiliated WWLP. sp.R. Asmarayani 568Plag. NA NA NA 5 Yes Yes 1 8 NA No NoPlag. NA NA NA 4-6 Yes Yes 1 8 2 vbs are in a pair Some vbs, early stageSome interfascicular areasOrth. (2-)4Vessels > Rays3-12 7-10 Yes Yes 1 9 None Yes, early stageSome interfascicular areasUnafiliated WWLP. sp.R. Asmarayani 811Plag. NA NA NA 3-51 vb, others have only few lignified cellsYes 1 8 None Some vbs, early stage Few interfascicular areas165Orth. 1(-2)Vessels = Rays7-15 5-7 Yes Yes 1 15 None Yes, early stage YesE5 P. retrofractum VahlR. Asmarayani 767Orth. 2-3(-4)Vessels > Rays3-14 5-7All vbs have small group of lignified cellsYes 1 9 None Yes, early stageMost interfascicular areasPlag. NA NA NA 4-6Few vbs have only few lignified cellsYes 1 11 None Yes, early stageSome interfascicular areasR. Asmarayani 884Plag. NA NA NA 5-6 No Most vbs 1 7 None Some vbs, early stageSome interfascicular areasE4 P. macropiper PennantR. Asmarayani 607Plag. NA NA NA 2-4Few vbs have only few lignified cellsYes 1 9 None No Few interfascicular areasOrth. NA NA NA 4-5 Yes Yes 1+ 1, 11 None Yes, early stage YesR. Asmarayani 707Plag. NA NA NA 4-6 Most vbs Yes 1+ 5, 161 outer vb have 45° orientationMost vbs, early stageSome interfascicular areasOrth. NA NA NA 5-8Few vbs have only few lignified cellsYes 1+ 13, 221 outer vb have 90° orientationYes, early stageMost interfascicular areas in inner ringE4P. aff. macropiper PennantR. Asmarayani 548Plag. NA NA NA 5-6 No Yes 1 121 vb has 90° orientation, 2 vbs pairedYes, early stage YesE4 P. decumanum L.R. Asmarayani 756Plag. NA NA NA 6-7Few vbs have only few lignified cellsYes 3 21, 21, 171 outer vb and 3 middle vbs have 45-90° orientation, 1 inner vb has 180° orientation; 1 outer vb pairedSome vbs (mostly inner vbs), early stageFew interfascicular areas in inner and middle ringsE4P. cf. dumiformans C. DC.R. Asmarayani 735Plag. NA NA NA 4-5 NoFew vbs have only few lignified cells1 8 None Yes, early stage Few interfascicular areasE3P. rubro-venosum Hort. ex RodigasR. Asmarayani 764Orth. NA NA NA 4-6 No Yes 1 5 None Yes, early stageSome interfascicular areasOrth. NA NA NA 4-8Few vbs have only few lignified cellsYes 1 8 2 vbs are in a pair Some vbs, early stageSome interfascicular areasR. Asmarayani 774Orth. NA NA NA 5-8Some vbs have small group of lignified cellsYes 1 11 None Yes YesE2 P. mestonii BaileyR. Asmarayani 749Plag. NA NA NA 4-7Few vbs have only few lignified cellsYes 1 141 vb paired, attached in the phloem side (xylem-phloem-xylem)Some vbs, early stageSome interfascicular areasE2 P. versteegii C. DC.R. Asmarayani 731Orth. NA NA NA 4-7 Yes Yes ttened ring arrange 16 NoneYes, early stage and more massive than peripheral vbsYes (interfascicular areas are barely exist)166E2 P. sp.R. Asmarayani 758Plag. NA NA NA 6-8Most vbs, few vbs have only few lignified cellsYes 2 13; 143 outer vbs have 45-90° orientationMost vbs, early stage Few interfascicular areasPeltandron P. celtidiforme OpizR. Asmarayani 533Plag. NA NA NA 2-4 NoFew vbs have only few lignified cells2 4; 93 outer vbs have 30-90° orientation, 1 inner vb has 30° orientationYes, early stage Few interfascicular areasPlag. NA NA NA 4-7 Yes Yes 2 8; 132 outer vbs have 30-45° orientationYes, early stage Few interfascicular areasOrth. 1(-2)Vessels < Rays5-9 7-11 Most vbs Yes 2 18; 14 None Yes, early stageSome interfascicular areasE1 P. abbreviatum OpizR. Asmarayani 511Plag. NA NA NA 5-6 No No 1 7 1 vb paired Some vbs, early stage An interfascicular areaE1R. Asmarayani 519Plag. NA NA NA 3-4 No 2 vbs 1 91 vb has 90° orientation, 1 vb paired, 2 vbs are in a pairFew vbs, early stage NoE1 P. albamentum C. DC.R. Asmarayani 736Plag. NA NA NA 3-4Few vbs have only few lignified cellsYes 1 7 3 vbs paired Few vbs, early stage NoPlag. 1(-2)Vessels < Rays4-11 5-6 Yes Yes 1+ 12; 11 outer vb has 90° orientation, 4 inner vbs have 30-90° orientationNo Few interfascicular areasE1 P. celebicum BlumeR. Asmarayani 557Plag. NA NA NA 7-8 No Yes 1 14 None Yes, early stage Few interfascicular areasE1 P. majusculum BlumeR. Asmarayani 737Plag. NA NA NA 4-6 Yes Yes 1 17 None Most vbs, early stageMost interfascicular areasE1 P. sp.R. Asmarayani 559Plag. NA NA NA 7-8 Yes Yes 1 12 None Yes, early stage Few interfascicular areasNew GuineaP. methysticum G. Foster var. wichmannii (C. DC.) LebotR. Asmarayani 711Plag. NA NA NA 3-7Some vbs have only few lignified cellsYes 1+ 3; 121 outer vb has 45° orientationYes, early stage Few interfascicular areasOrth. 1(-2)Vessels < Rays5-11 5-6, discontinuous2 vbs have only a lignified cellSome vbs 3 24; 28; 241 outer vb has 45° orientationYes, early stageSome interfascicular areas167Table S1. ContinuedCLADE SPECIES SPECIMENStem typePattern of lignification in pith parenchymaNumber of mucilage canals (central; peripheral)Position of peripheral mucilage canalsSecretory (ethereal oil) cellsMulderaP. schizonephros C. DC.R. Asmarayani 674Plag.Most cells except peripheral areas and surrounding mucilage canals0; 8 (1 ring)Interior to small vbsNot seenOrth. Peripheral areas; middle pithNA; 12 (1 ring)Interior to small vbsFew: inner cortex, phloem, pithMulderaP. flavimarginatum C. DC.R. Asmarayani 662Plag. None None NAOuter (abundant) and inner (some) cortex, phloem (some)Orth.Peripheral to vb ring except surrounding mucilage canals0; 11 (1 ring)Interior to small vbsOuter cortex (abundant), inner cortex (some), primary medullary rays Muldera P. pentandrum C. DC.R. Asmarayani 593Plag. Peripheral to vb ring 1; 9 (1 ring)Interior to small vbsOuter and inner cortex (abundant), phloem (few), pith (some)Plag.Some cells 1-5 peripheral layers; some cells in the vb 1; 14 (1 ring, 4 canals look Interior to small vbsSome: inner cortex, peripheral phloem, Plag.Most cells in the peripheral to inner vb ring1; 9 (1 ring)Interior to small vbsFew: inner cortex, peripheral phloem, pithPlag.Most cells except surrounding mucilage canals1; 14 (1 ring)Interior to small vbsInner cortex (few), pith (some)Muldera P. sp.R. Asmarayani 547Plag.Peripheral areas; scattered group of cells in pith1; 8 (1 ring)Interior to small vbsNot seenOrth.Some cells in peripheral areas; scattered group of cells in outer pith1; 10 (1 ring)Interior to small vbsNot seenPlag. Most cells 0; 7 (1 ring)Interior to small vbsNot seenMuldera P. sp.R. Asmarayani 634Orth. None 0; 3 (1 ring)Interior to small vbsAbundant: cortex, pithMuldera P. sp.R. Asmarayani 636Orth. Some cells peripheral areas 1; 10 (1 ring)Interior to small vbsFew: inner cortex, pithCubeba s.str. P. cubeba L. f.R. Asmarayani 768Plag.Peripheral to vb ring, intergrading with usb1; 0 NA Not seenOrth.Most cells except 1-4 layers surrounding mucilage canals and 2 areas elsewhere1; 2 (partial ring)Interior to small vbsNot seen168Cubeba s.str. P.mollissimum BlumeR. Asmarayani 629Orth.Few cells in peripheral areas, intergrading with usb1;7 (1 ring)Interior to small vbsOuter (some) and inner (few) cortex, pith (few)Cubeba s.str. P. pedicellosum Wall.R. Asmarayani 644-LHK12-47Plag.Peripheral areas, intergrading with usb; a large clustered of cells in outer pith1; 2 (partial ring)Interior to small vbsFew: cortexOrth.Peripheral areas, intergrading with usb, few small clustered of cells in outer pith1; 0 NAFew: medullary phloem, pithR. Asmarayani 675Plag. Peripheral areas None NA Not seenPlag. Peripheral areas 1; 0 NA Not seenPlag. Peripheral areas None NA Not seenCubeba s.str. P. ribesioides Wall.R. Asmarayani 635Plag. None 1; 8 (1 ring)Interior to small vbsFew: inner cortex, pith (outer cortex too dark to be observed)Orth.Few scattered groups of cells in the outer pith and between vbs1; 8 (1 ring)Interior to small vbsFew: inner cortex, primary medullary rays, pith (outer cortex too dark to be observed)W6 P. caninum BlumeR. Asmarayani 597Plag.1-2 peripheral layers, intergrading with usb1; 2 (1 ring) or 3 (half ring)Interior to small vbsFew: cortex (dark)Orth.1-2 peripheral layers, intergrading with usb1; 0 NA Few: cortex (dark)W6 P. caninum BlumeR. Asmarayani 881Plag.Peripheral to outer pith, intergrading with usb1; 7 (1 ring)Interior to small (mostly) and large vbsNot seenW6 P. curtisii C. DC.R. Asmarayani 651Plag. None 1; 0 NASome: cortex, peripheral phloem, pithW6 P. muricatum BlumeR. Asmarayani 630Plag.Some cells in peripheral areas, intergrading with usb1; 8 (1 ring, 2 canals are a pair)Interior to small vbsFew: cortexW6 P. philodendron Ridl.R. Asmarayani 685Orth. None 2; 0 NAOuter and inner cortex (abundant), pith (abundant), vbs (few)W6 P. pullibaccum Trel.R. Asmarayani 742Plag.2-8 peripheral layers, intergrading with usb1; 1Interior to small vbNot seenW5 P. cilibracteum C. DC.R. Asmarayani 624Plag. All cells 0; 0 NASome: cortex, phloem, pithR. Asmarayani 679Plag. Peripheral areas 1; 0 NAAbundant: cortex, pith, medullary phloem169Orth.Most cells except some scattered groups of cells1; 0 NAAbundant: cortex and pith, medullary phloemW5P. porphyrophyllum N. E. Br.R. Asmarayani 797Plag. All cells 1; 10 (1 ring)Interior to small vbsFew: phloemEupiper s.str. P. nigrum L.R. Asmarayani 510Plag.1-2 peripheral layers, intergrading with usb0; 2 (partial ring)Interior to small vbsNot seenW4 P. ramipilum C. DC.R. Asmarayani 590Plag. Few scattered cells 1; 0 NA Abundant: all partsOrth.Scattered cells in the central pithNone NASome; inner cortex, phloemEuchavica s.str.P. betle L. MO 2002-1050 Plag. None 1; 5 (1 ring)Interior to small vbsAbundant: cortex, phloem, pithPlag. None 1; 6 (1 ring)Interior to small vbsAbundant: cortex, vbs, pithOrth. None 1; 8 (1 ring)Interior to small vbsAbundant: cortex, vbs, pithPlag.Peripheral areas, intergrading with usb1; 8 (1 ring)Interior to small vbsAbundant: cortex, phloem, pithW3 P.argyrites Ridl.R. Asmarayani 637Plag.Some 1-3 layers in peripheral, intergrading with usb1; 1Interior to large vbAbundant: cortex, phloem, pithOrth. 2-10 peripheral layers2; NA (seen 4)Interior to small and large vbsPhloem (some), pith (few)W2P. puberulirameum C. DC.R. Asmarayani 616Plag.Outer pith; some clustered cells surrounding vbs and mucilage canal (not 1-2 layers next to the canal)1; 1Interior to small vbSome: cortexOrth.1 peripheral layer; scattered groups of sclereids arranged in a ring in outer pith and surrounding mucilage canals (not 1-2 layers next to the canals)1; 5 (half ring)Interior to small vbsFew: inner cortex, peripheral phloemOrth.1 peripheral layer; scattered groups of sclereids arranged in a ring in outer pith and surrounding mucilage canal (not 1-2 layers next to the canal)1; 0 NAMany: cortex (some), peripheral phloem and rays (abundant), pith (few)W2P. kadsura (Choisy) OhwiN. Tanaka s. n. Plag. None 1; 0 NA Not seen170Orth.1-2 peripheral layers, intergrading with usb1; 0 NA Not seenSarcostemon s.l.P. quinqueangulatum Miq.R. Asmarayani 573Plag. Peripheral areas1; 6 (partial ring, 2 canals are a pair)Interior to small vbsAbundant: all part including lignified onesOrth. Peripheral areas1; 14 (1 ring, 6 canals are 3 pairs)Interior to small vbsAbundant: cortex, pith, medullary phloemOrth.Peripheral areas; many scattered cells/small group of cells elsewhere1; 10 (1 ring)Interior to small vbsAbundant: all part including lignified onesR. Asmarayani 812Plag.Peripheral areas, intergrading with usb1; 0 NAAbundant: cortex, phloem, pithPseudochavica sensu Ridley s.str.P. oreophilum Ridl.R. Asmarayani 567Plag. None 1; 0 NA Not seenPlag.1-4 peripheral layers, intergrading with usb1; 0 NA Some: inner cortexOrth.1-4 peripheral layers, intergrading with usb1; 0 NA Some: cortexPseudochavica sensu Ridley s.str.P. cyrtostachys Ridl.R. Asmarayani 579Plag.1-2 peripheral layers, intergrading with usbNA; 0 NA Not seenPlag.2-3 peripheral layers, intergrading with usbNA; 0 NA Not seenOrth.1-3 peripheral layers, intergrading with usb; few cells surrounding a vbNA; 0 NA Not seenW1 P. sarmentosum Roxb.R. Asmarayani 765Orth. None 1; 0 NA Not seenW1 P. sp.R. Asmarayani 602Orth. None 1; 0 NA Not seenUnafiliated WWLP. sp.R. Asmarayani 809Plag.2-4 peripheral layers, intergrading with usbNone NAMany: cortex, pith, phloemUnafiliated WWLP. sp.R. Asmarayani 568Plag.Most cells except some 1-2 layer of cells surrounding mucilage canals1; 6 (1 ring)Interior to small vbsNot seenPlag.Most cells except some 1-2 layer of cells surrounding mucilage canals1; 3 or 4 (partial ring)Interior to small vbsNot seenOrth.Most cells except 1-2 layer of cells surrounding mucilage canals1; 8 (1 ring)Interior to small vbsNot seen171Unafiliated WWLP. sp.R. Asmarayani 811Plag.Some 1-6 peripheral layers, intergrading with usb; some scattered groups of cells elsewhere0; 2 (partial ring)Interior to small vbsFew: inner cortexOrth.Most cells except 1-2 layers surrounding mucilage canals1; 2 (partial ring)Interior to small vbsFew: inner cortex, pithE5 P. retrofractum VahlR. Asmarayani 767Orth.Few scattered small groups of cells in the outer pith1; 0 NA Not seenPlag.1-3 peripheral layers, intergrading with usb1; 0 NA Not seenR. Asmarayani 884Plag.1-8 peripheral layers, intergrading with usbNone NA Not seenE4 P. macropiper PennantR. Asmarayani 607Plag.Peripheral areas; scattered/clustered sclereids in the middle pithNone NASome: cortex, phloem, pithOrth. Most cells None NASome; cortex, phloem, pithR. Asmarayani 707Plag.Most cells except surrounding mucilage canal1; 0 NA Abundant: cortex, pithOrth.Most cells in peripheral to inner vb ring1; 0 NA Abundant: cortex, pithE4P. aff. macropiper PennantR. Asmarayani 548Plag.Most cells except 1-2 layers surrounding central mucilage canal1; 0 NASome: outer and inner cortex, pithE4 P. decumanum L.R. Asmarayani 756Plag.1-2 peripheral layers, intergrading with usb; some cells in the middle pith; some between pith vbs1 or 2; 22 (1 ring)Interior to small vbsNot seenE4P. cf. dumiformans C. DC.R. Asmarayani 735Plag.2-4 peripheral layers, intergrading with usbNone NA Not seenE3P. rubro-venosum Hort. ex RodigasR. Asmarayani 764Orth. None None NA Not seenOrth. None 1; 0 NA Few: pithR. Asmarayani 774Orth.1-6 peripheral layers, intergrading with usb1; 0 NACortex (few), phloem (few), pith (abundant)172E2 P. mestonii BaileyR. Asmarayani 749Plag.1-2 peripheral layers, intergrading with usb1; 12 (1 ring)Interior to small vbsCortex (some), pith (few)E2 P. versteegii C. DC.R. Asmarayani 731Orth.Peripheral areas, intergrading with usb; few scattered cells elsewhere1; 6 (1 ring)Interior and adjacent to large vbsFew: cortexE2 P. sp.R. Asmarayani 758Plag.Some 1-3 layers in peripheral intergrading with usb; scattered groups of cells in vb rings1; 15 (1 ring)Interior to small vbsNot seenPeltandron P. celtidiforme OpizR. Asmarayani 533Plag. Peripheral areas1; 2 (partial ring)Interior to small vbsAbundant: all parts including sclerenchymal tissue surrounding peripheral vbsPlag. Peripheral areas 1; 9 (1 ring)Interior to small vbsAbundant: all parts including sclerenchymal tissue surrounding peripheral vbsOrth.Most cells except surrounding central mucilage canal1; 15 (1 ring)Interior to small vbsAbundant: innermost part of peripheral vbs, pithE1 P. abbreviatum OpizR. Asmarayani 511Plag.1-2 layer in peripheral, intergrading with usb1; 0 NAOuter cortex (some), medullary phloem (few)E1R. Asmarayani 519Plag.Peripheral areas, intergrading with usb1; 0 NA Cortex, phloemE1 P. albamentum C. DC.R. Asmarayani 736Plag. Few clustered of cells 1; 1Interior to large vbCortex (some), pith (few)Plag.1-2 layer in peripheral, intergrading with usb1; 8 (1 ring)Interior to large vbsCortex (abundant), peripheral phloem (some), pith (some)E1 P. celebicum BlumeR. Asmarayani 557Plag.Most cells in peripheral to outer pith1; 5 (partial ring)Interior to large vbsNot seenE1 P. majusculum BlumeR. Asmarayani 737Plag.1-2 peripheral layers, intergrading with usb1; 0 NA Few: inner cortex, pithE1 P. sp.R. Asmarayani 559Plag.Peripheral areas, intergrading with uusb few cells elsewhere1; 7 (1 ring)Interior to large vbsNot seenNew GuineaP. methysticum G. Foster var. wichmannii (C. DC.) LebotR. Asmarayani 711Plag. None 1; 1Interior to small vbFew: outer cortexOrth. None 1; 16 (1 ring)Interior to large and small vbsOuter cortex (abundant), peripheral phloem (few), pith (some)173 174 Chapter 3: Piper section Muldera (Piperaceae) of the Malay Peninsula: variation and species limits Rani Asmarayani Department of Biology, University of Missouri – St. Louis, 1 University Boulevard, St. Louis, Missouri 63121, USA; Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, USA; Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor KM 46, Cibinong 16911, West Java, Indonesia; rasmarayani@yahoo.com Abstract. Species limits in Piper spp. have been unstable, with specimens often being assigned to different species. Using a strongly supported clade in Asian Piper, section Muldera (Miq.) Benth. & Hook.f., I investigated species boundaries in the eight Malay Peninsula species using morphological, anatomical, and molecular approaches. I assessed morphological variation using principal component analysis (PCA) and a model-based clustering method. Herbarium specimens and material collected in the field were examined, and the latter were also used to examine variation in vegetative anatomy. Evidence of reciprocal monophyly of the species came from the molecular phylogenetic analyses of nuclear ITS and g3pdh and chloroplast trnL-F. Morphological analyses do not lend support for hypothesized species from the Malay Peninsula. Nuclear phylogenetic trees support a combination of P. schizonephros, P. polygynum, and P. maingayi as a morphospecies that may also have a distinct distribution of calcium oxalate crystals in the mesophyll of the leaf. Nevertheless, shared characters between this morphospecies and some, but not all, of the other morphogroups, and paraphyly of other morphogroups in the phylogenetic analyses, may suggest hybridization or lateral transfer among species of Muldera. In-depth population studies coupled with examination of morphological variation for all Muldera species, including those from outside the Malay 175 Peninsula, are necessary to disentangle the species limits and ultimately the evolutionary history in this group. Introduction Piper L. is a major clade of angiosperms. It includes ~2400 spp., nearly all in the tropics, a figure much larger than the recent estimate of ~1000 spp. (e.g., Callejas 1986, Tebbs, 1993), due to an increase in the estimated numbers of Neotropical species (Ulloa Ulloa et al., 2017). The number of Paleotropical species is uncertain, with many species newly described, and many more synonymized (e.g., 61 synonyms in Chew 1972a, 1972b, 1992, 2003; reduction of 83 Philippine species to 15 by Gardner 2006), even including recently described species (e.g., P. maculaphyllum Chaveer. & Sudmoon [2008] = P. argyrites Ridl. in Suwanphakdee & Chantaranotai [2011]). Even when botanists recognize the same species, they may differ in the specimens they assign to those species (see Gardner, 2013 vs. Chew, 1972, 1992, 2003). In much recent literature, whether a group of organisms is called a distinct species or not is a matter of personal opinion, and any value a general consensus might have is not obvious (Peter Stevens, pers. comm.). Naming species has always been an integral part in communicating knowledge about plants. However, species concepts, and hence the criteria used to delimit species, remain the center of debate, “the species concept problem” (Queiroz, 2007). Queiroz (1998, 2007) suggested that all species concepts share an underlying common element, that they all consider species to be separately evolving segments of a metapopulation lineage. This view, the unified concept of species, uses a variety of criteria, some emphasized by particular species concepts (e.g., reproductive isolation in the biological 176 species concept), to provide evidence for delimiting species (Queiroz, 1998, 2007). Agreement using independent lines of evidence provides better corroboration of species boundaries (e.g., Queiroz, 2007, Schlick-Steiner et al., 2010). Here, I investigate the species boundaries of a group of Paleotropical Piper, section Muldera (Miq.) Benth. & Hook.f. in the Malay Peninsula, in the context of the unified species concept. Piper sect. Muldera Section Muldera (synonym: section Schizonephos [Griff.] C. DC.) is part of the Asian clade of Piper (Muldera clade; Asmarayani, 2018). It is the only infrageneric group consistently recognized by all botanists working on Asian Piper and its monophyly is well supported (Asmarayani, 2018). Currently, there are 26 species (33 names including synonyms) described as Muldera, however, at least four species of those 26 do not belong in this section (Gamble, 1925; Hooker, 1886; pers. obs.). Twenty-two species have been described from West Malesia (Sumatra, Malay Peninsula, Java, Borneo, and the Philippines), Thailand (the Isthmus of Kra), India, and Sri Lanka, but from herbarium material examined the distribution of this section extends northwards to Cambodia and eastwards to the Moluccas. Muldera species grow well in primary forest and are rarely found in disturbed forest. All species are root climbers, usually with stout stems (diameter at breast height may reach 12 cm – pers. obs.), and sometimes reaching 25 m tall; in fact, Muldera species are the largest and tallest climbers in Asian Piper. Their juveniles, often abundant on the forest floor, are easily distinguished from other Piper by their cordate leaves colored dark green above and pale green beneath and their red spotted stems. The plants are usually dioecious, although some individuals of P. galeatum C. DC., an Indian 177 species, also produce bisexual flowers (C.A. Barber 5485 in K). The stamens vary from two, mostly in Indian species, to ten, reported in P. recurvum Blume (Blume, 1826), the most stamens reported from Piper (Piper spp. usually have 2-3, 4, or 6 stamens). The carpels in Piper, often 3-4, are usually fused and have a single ovule per flower, yet P. polygynum C. DC. develops multiple fruits from a single flower, also observed in a few flowers of P. sp. R. Asmarayani 836. The most striking aspect of Muldera is the cup-shaped structure surrounding the flower, quite unlike anything in other Piperaceae, that has been interpreted – but with no real evidence – as being formed from a bract that is somehow fused to the flower (Miquel, 1843-44; Candolle, 1869; Quisumbing, 1930). Although Muldera is so distinctive, assigning an individual to a species is almost impossible without flowers and fruits. The vegetative parts show much variability even within individuals (pers. obs.), and there are no obvious differences among the species. The dioecy of Piper raises another problem. Since what appear to be two species of Muldera, i.e. individuals of the same sex but with quite different floral morphologies, grow in the Nee Soon Swamp Forest, Singapore (Malay Peninsula; pers. obs.), geography cannot be used as a proxy to deduce the identity of plants of the opposite sex. In addition, although some species have distinctive flowers (e. g. P. schizonephros C. DC. and P. sarcopodum C. DC.), most of them have such subtle differences that Hooker (1886) in his account of 5 species of Muldera in the Flora of British India suggested that male and female plants might be incorrectly associated. Fifteen of the 22 species of Muldera have been described from staminate or carpellate individuals only. To add insult to injury, recently six species of Muldera (P. recurvum Blume of Java, P. firmum [Miq.] C. DC. of Sumatra, P. pachyphyllum Hook. f. and P. flavimarginatum C. DC. of the Malay Peninsula, P. protrusum of Thailand, and P. ceylanicum C. DC. of Sri Lanka) have been 178 lumped into P. baccatum Blume (Suwanphakdee et al., 2016), although without detailed analyses. Here I aim to decipher species limits in Piper section Muldera, focusing on the Malay Peninsula, where eight species have been recognized in past treatments: three are known from staminate individuals (P. flavimarginatum, P. rarispicum C. DC., P. schizonephros C. DC.), three from carpellate individuals (P. firmum, P. pachyphyllum, and P. polygynum) and two from both staminate and carpellate individuals (P. maingayi Hook. f. and P. pentandrum C. DC.) (Hooker, 1886; Candolle, 1914; Ridley, 1924). The variation I grouped the specimens from the Malay Peninsula, both those I collected and herbarium specimens, into eleven morphogroupsa, using overall similarity and mostly based on known species in Muldera. There are five staminate morphogroupsa: S1 (P. schizonephros and P. rarispicum group), S2 (P. maingayi group), S3 (P. flavimarginatum group), S4 (P. pentandrum group), and S5 (P. sp. J. Sinclair s.n. group); and six carpellate morphogroupsa: C1 (P. polygynum group), C2 (P. maingayi group), C3 (P. flavimarginatum group), C4 (P. pachyphyllum-pentandrum group), C5 (P. firmum group), and C6 (P. sp. E. Soepadmo 763 group) (see Table S1 for the list of the specimens and their morphogroups). Furthermore, I hypothesize that four morphogroupa pairs, S1-C1, S2-C2, S3-C3, and S4-C4, are distinct morphospecies. Here, I examined various aspects of species variation – morphology, anatomy, and genomes – and used each separately and together to provide evidence for species boundaries. Anatomical and molecular data were taken from specimens collected in the field and morphological data were collected from both the field and herbarium specimens. Morphological variation was assessed using principal component analysis (PCA) and a model-based clustering method using mclust in R (Scrucca et al., 2016) 179 following Cadena et al. (2018). Variation in vegetative anatomy and evidence of reciprocal monophyly from the molecular phylogenetic analyses were also used to evaluate the species limits in this group. Material and Methods Morphology dataset and analyses. I investigated 80 sheets of 41 herbarium specimens and high quality images of herbarium specimens from G, K, L, BO, MO, CAL, SAN, SING, and 11 specimens collected in the field (Appendix 1). The fertile specimens represent all morphogroupsa from the Malay Peninsula, and include ten type specimens of the names described from there. I also investigated an additional ten sheets of three specimens from Sumatra (the carpellate type specimen of P. firmum and a staminate specimen identified as P. firmum) and Java (the carpellate type specimen of P. recurvum Blume) to assess their morphological similarity with the material from the Malay Peninsula. Because some herbaria do not allow destructive sampling of specimens (i.e., cutting specimens for resuscitation), measurements were taken from dry material. Seven vegetative characters were measured from the leaves of plagiotropic shoots only, since the shape and size of the leaves on the orthotropic shoots both vary greatly and are often similar in closely related species of Piper. The length of the internodes varies within a single shoot, and there is no obvious variation in the height and habit of Muldera species, thus these features were not considered. Seventeen quantitative characters of the flowers and fruits (Fig. 1.1) were measured at comparable stages, i.e., at anthesis for staminate specimens and at flower/small fruit stage for carpellate specimens. Because of the small size of the flowers, photos were taken of the flowers and measurements were carried out 180 using ImageJ 1.52e (Schneider et al., 2012). When possible, three measurements of each character were taken from each herbarium sheet and averaged for each specimen. A qualitative character, surface texture of various parts of the plant, was observed on both vegetative and floral parts, but was used for descriptive purposes only. In the end, six datasets were generated and analyzed separately (Suppl. Table S1): (1) vegetative characters of both staminate and carpellate specimens (VEG; 7 characters), (2) floral characters of both staminate and carpellate specimens (FLSC; 14 characters), (3) vegetative and floral characters of staminate specimens (VEGFLS; 24 characters), (4) staminate floral characters (FLS; 17 characters), (5) vegetative and floral characters of carpellate specimens (VEGFLC; 24 characters), and carpellate floral characters (FLC; 17 characters; Suppl. Table 1). To analyze the data, I used the pipeline implemented in Cadena et al. (2018). To assess morphological space, principal component analysis (PCA) was carried out on the covariance matrix of log-transformed data. The PC axes were then analyzed using the R package clustvarsel version 2.3.2 (Scrucca & Raftery, 2018), in backward/forward fashion to select a set of principal components which can discriminate groups without a priori information about their existence. The multivariate analyses were then used to define morphogroupsb using the normal mixture model (NMM) that carried out in R package mclust version 5.4.1 (Scrucca et al., 2016) using PC axes selected by clustvarsel. NMM is a probabilistic model which assumes that phenotypic variation is a mixture of distinct normal distributions, where each distinct normal distribution represents a distinct morphogroupb (i.e., species) (McLachlan and Peel, 2000; Cadena et al., 2018). NMM was set to allow 1-11 morphogroupsb in VEG and VEGFLS dataset, 1-5 in VEGFLS and FLS, and 1-6 in VEGFLC and FLC; the number of morphogroupsb allowed is the number of 181 morphogroupsa hypothesized for the aforementioned datasets. Empirical support for different NMMs was assessed using the Bayesian Information Criterion (BIC; Schwarz 1978). Anatomical dataset and observations. Twelve specimens representing three staminate (S1, S3, and S5) and four carpellate (C1, C2, C3, and C4) morphogroupsa were investigated for their stem and leaf anatomy (Appendix 1). The fresh materials were fixed in ethanol 70% or formaldehyde-acetic acid alcohol (FAA; 50 parts of ethanol 95%, 5 parts of glacial acetic acid, 10 parts of formaldehyde 37%, and 35 parts of deionized water; Ruzin 1999) in the field. In some cases, stem material from herbarium specimens was resuscitated using the ammonium hydroxide method (Keating 2014), soaking in detergent for two days and then in ammonium hydroxide for four days. For comparative purposes, all sections of the stem and leaf were taken from the middle of the internodes and middle of the lamina, respectively. When possible stems were sampled from several internodes, both plagiotropic and orthotropic (Table S2.1 and S2.2). The slides were prepared using hand sections stained following Keating (2014) using cresyl violet acetate (CVA), which stains lignin bright blue and parenchyma tan, and mounted in glycerin. General anatomical features were observed using an Olympus CX31 microscope, while the leaf calcium oxalate crystals were observed using a polarizing lens filter. Photos were taken from each slide using a Leica ICC50 HD camera, and assembled using Stitching (Preibisch et al. 2009) or MosaicJ (Thévenaz & Unser 2007) implemented in Fiji (Schindelin et al., 2012). Measurements were carried out using ImageJ 1.52e. 182 Molecular dataset and phylogenetic analyses. Fourteen fertile specimens were collected from Singapore for this study. To complement the data, I added 14 specimens of Muldera I collected from Borneo, and an additional seven sets of sequences (including clones) of Muldera from GenBank (http://www.ncbi.nlm.nih.gov/; from Asmarayani [2018]; Appendix 2) that I collected from the Malay Peninsula (three sets), Borneo (two sets), Java (one set), and Sulawesi (one set). Altogether, this study included 38 individuals representing three staminate (S1, S3, and S5) and four carpellate (C1, C2, C3, and C4) morphogroupsa from the Malay Peninsula and four other staminate (S1, S6, S7, and S8) and two carpellate (C7 and C8) morphogroupsa from elsewhere in Malesia. The Asian P. caninum Blume and P. fragrans Trel. and the South Pacific P. methysticum G. Forst. were used as outgroups; their sequences were obtained from GenBank (Appendix 1). DNA was obtained from (young) leaves preserved in silica gel. DNA extraction used Qiagen DNeasy Plant Mini Kit following the manufacturer’s protocol. Three unlinked molecular markers were used based on Asmarayani (2018), the internal transcribed spacer of nuclear ribosomal DNA (ITS), a low copy nuclear gene encoding glyceraldehyde 3-phosphate dehydrogenase (g3pdh), and the chloroplast trnL intron and trnL-trnF intergenic spacer (trnL-F). Preparation of the PCR mixture for DNA amplification, the thermal cycling conditions for each marker, and the purification of PCR products followed Asmarayani (2018). PCR products of nuclear markers were cloned using Promega pGEMâ-T Easy Vector System I and Promega JM109 Competent Cells following the manufacturer’s protocol. Up to 16 clones per specimen (average of eight and six clones for ITS and g3pdh, respectively) were then Sanger sequenced in both forward and reverse directions at Beckman Coulter Genomics (USA) or Genewiz (USA). 183 Sequences were assembled and checked manually for quality using Geneious v.7.1.9 (Kearse & al., 2012). Sequences were checked against GenBank with BLAST to confirm that they belonged to Piper. Identical sequence(s) from individuals obtained from cloning were removed. In ITS, putative pseudogenes and their recombinants were removed based on criteria used by Asmarayani (2018). Two copies of g3pdh were retrieved from Muldera, A and B, and they were retrieved as sister clades, thus each copy was treated separately (Asmarayani, 2018). For alignments I used MUSCLE (Edgar, 2004) implemented in MEGA (Kumar & al., 2015), and they were manually refined by eye. In addition to complete datasets that included all the clones, simplified datasets containing a single sequence per individual were assembled to make nuclear datasets comparable to each other and to the chloroplast dataset. Phylogenetic analyses of ITS and g3pdh using complete datasets showed that most clones did not cluster with other intragenomic clones or their conspecific clones. The single sequences chosen for the simplified datasets were those showing the fewest mutations, and/or representing the majority sequence if the copy was represented by more than one clone sequence (see Figs. S1-S2 for the positions of the selected clones in the phylogenetic trees of the complete dataset). The data partitions for ITS and trnL-F were defined a priori, that is ITS1, 5.8S, and ITS2 for ITS, and the trnL intron, trnL, and trnL-trnF intergenic spacer for trnL-F. The boundaries of the regions in ITS were inferred based on the comparison with published sequences (Yokota & al., 1989; Baldwin & al., 1992), while those of trnL-F were determined based on comparison with the published chloroplast sequence of Piper 184 cenocladum (Cai & al., 2006) annotated by DOGMA (Wyman & al., 2004). G3pdh was treated as a single partition and analyzed as nucleotide sequences. Phylogenetic relationships among species were estimated using Bayesian and maximum likelihood approaches. Bayesian analyses were carried out using MrBayes 3.2.6 (Ronquist & al., 2012) on the CIPRES cluster (https://www.phylo.org/). The appropriate data partitioning scheme and the best model of sequence evolution for each partition and marker were assessed using the corrected Akaike information criterion in PartitionFinder 2.1.1 (Lanfear & al., 2016). The best partitioning scheme and best-fitting model of sequence evolution is GTR+I+Γ for g3pdh and ITS1 + ITS2, SYM+I+Γ for 5.8S, and GTR+Γ for trnL intron + trnL + trnL-F spacer. Analyses were performed in six separate runs, each with four Markov chains, for as many as 25 million generations, sampled every 1000 generations and terminated after the six runs converged indicated by an average standard deviation of split frequencies <0.01. The majority-rule consensus tree was obtained after excluding the first 25% of the trees. Maximum likelihood analyses were carried out using RaxML 8.2.9 on the CIPRES cluster, using the GTR+Γ model of evolution (Stamatakis, 2014). The partitioning schemes in these analyses followed those produced by PartitionFinder 2.1.1. Support for the optimum maximum likelihood tree was assessed using a rapid bootstrap analysis of 1000 replicates. Phylogenetic node support values of ≥85 maximum likelihood bootstrap percentage (BP) and ≥0.98 Bayesian posterior probability (PP) were considered strong, 70--84 BP and 0.95--0.97 PP were considered moderate, and 50--69 BP and 0.90-0.94 PP were considered weak. These values were based on simulation studies by Hillis & Bull (1993), Erixon & al. (2003), and Huelsenbeck & Rannala (2004). Phylogenetic trees were visualized using Dendroscope 185 v3.5.9 (Huson & Scornavacca, 2012) and FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Shimodaira-Hasegawa (SH) and Shimodaira Approximately Unbiased (AU) tests, implemented in PAUP* 4.0a152 (Swofford, 2002), were performed to test the topological congruence among genetic markers. Similar tests were also performed to test the monophyly of conflicting clades among genetic markers by examining alternative suboptimal topologies from constrained trees. Constrained phylogenetic analyses were carried out in RAxML using the settings described above. Results Morphology. In the VEG dataset, clustvarsel found five PC axes to be valuable for separating morphogroupsb (suppl. table S1). Using these PC axes, NMM resolved two morphogroupsb; support values for the other models were lower (∆BIC ≥ 6; suppl. fig. 1.1A) and the second best model supported only a single morphogroupb. The two morphogroupsb were not congruent with the hypothesized morphospecies (fig. 1.2A); most of the specimens fell into morphogroupb 1, but several members of S3-C3 and C5 morphospecies fell into morphogroupb 2. The uncertainty values of specimen assignments in morphogroupb 2 are considerably higher than those of morphogroupb 1 (8.13X10-3-1.44X10-1 vs. 0-1.28X10-5; suppl. table S1). The characters that contributed most to morphogroupb separation in this dataset are leaf apex and petiole length, morphogroupb 1 having a shortly acuminate leaf and morphogroupb 2 having a long petiole (suppl. fig. 1.2). In each of the FLSC and VEGFLS datasets, clustvarsel found 10 PC axes to be valuable for separating morphogroupsb (suppl. table S1). However, using these PC axes, 186 NMM resolved only a single morphogroupb for each dataset (suppl. fig. 1.1B-C). Supports for the other models with different numbers of morphogroupsb were much lower (∆BIC ≥ 23 and ∆BIC ≥ 8 for FLSC and VEGFLS, respectively; suppl. fig. 1.1B-C). In the FLS dataset, clustvarsel found eight PC axes to be valuable for separating morphogroupsb (suppl. table S1). Using these PC axes, NMM resolved three morphogroupsb; support for the other models with different numbers of morphogroupsb was lower (∆BIC ≥ 46; suppl. fig. 1.1D). The three morphogroupsb were not congruent with the hypothesized morphogroupsa (fig. 1.2B), all morphogroupsb being mixtures of members of at least two morphogroupsa. Morphogroupb 3 in particular included all members of the S2 and S5 morphogroupsa (1 and 2 specimens, respectively) and also comprised some members of S1, S3, and S4 morphogroupsa. There is no uncertainty in the specimen assignment to any morphogroupb (suppl. table S1). Among the characters which contributed to morphogroupb separation, some characterize groups: morphogroupb 1 and 2 have a long peduncle, a long and thick rachis, and numerous stamens, and these two morphogroupsb are distinguishable by floral cup characters: morphogroupb 1 has shorter upper cup and lip to base distances than morphogroupb 2, while morphogroupb 3 has a short peduncle, a short and thin rachis, and few stamens (suppl. fig. 1.3). In the VEGFLC dataset, clustvarsel found eight PC axes to be valuable for separating morphogroupsb (suppl. table S1). Using these PC axes, NMM resolved two morphogroupsb; supports for the other models with different numbers of morphogroupsb were lower (∆BIC ≥ 57; suppl. fig. 1.1E). The two morphogroupsb were not congruent with the hypothesized morphogroupsa (fig. 1.2C); most of the specimens fell into morphogroupb 1, the C1 and C2 morphogroupsa belonging exclusively here, while the C6 morphogroupa (a single specimen only) was in morphogroupb 2. The uncertainty values 187 of specimen assignment in all morphogroupsb were low (0-2.86X10-3; suppl. table S1). Characters which contributed significantly to morphogroupb separation included both vegetative and floral characters: morphogroupb 1 has a shortly acuminate leaf and morphogroupb 2 has a long peduncle (suppl. fig. 1.4). In the FLC dataset, clustvarsel found seven PC axes to be valuable for separating morphogroupsb (suppl. table S1). Using these PC axes, NMM resolved six morphogroupsb; support for the second best model with two morphogroupsb was lower (∆BIC ≥ 57; suppl. fig. 1.1F). The six morphogroupsb were not congruent with the hypothesized morphogroupsa (fig. 1.2D); morphogroupsb 1, 2, and 6 were mixtures of some members of at least two morphogroupsa, morphogroupb 3 included some members of the C3 morphogroupa, morphogroupb 4 included the C2 and C6 morphogroupsa and some members of the C4 and C5 morphogroupsa, and morphogroupb 5 included but a single specimen from Java of the C4 morphogroupa, the type specimen of P. recurvum. There were low uncertainty values over specimen assignments to morphogroupsb 1, 2, and 4 (0-3.27X10-3; suppl. table S1). Among the characters which contributed to morphogroupb separation, a short upper cup characterizes morphogroupb 4 (suppl. fig. 1.5). Anatomy of leaf and stem. Anatomical data of leaves and stems are represented in suppl. table S2.1 and S2.2, respectively. There is no difference in the anatomy of orthotropic and plagiotropic shoots. The leaves of Muldera are dorsiventral, and are made up of a single layer of epidermis, 3-5 layers of hypodermis and then mesophyll in the middle (fig. 2.1A-D). The adaxial hypodermis generally is made up of fewer layers than the abaxial hypodermis, but its cells are larger and in P. sp. RA674 (S1 morphogroupa; 188 fig. 2.1A) and P. sp. RA662 (C3 morphogroupa; fig. 2.1C) there are a few exceptionally large hypodermal cells next to the palisade tissue of the mesophyll. However, these large hypodermal cells are not always present. Piper sp. RA593 (C4 morphogroupa; fig. 2.1B) has the most hypodermal layers, 4 and 6 layers on the adaxial and abaxial sides, respectively, compared to the other morphogroupsa. The mesophyll includes 1-2 layers of adaxial palisade tissue and spongy parenchyma in the abaxial part. The difference between these two tissues sometimes is not obvious, however, they are easily differentiated by the size and abundance of calcium oxalate crystals. The mesophyll of Muldera mostly has calcium oxalate crystals of the styloid type. Generally, the palisade cells have larger styloid crystals than do the spongy cells, and the styloid crystals in the spongy cells are sometimes shorter (<0.01 mm) when mixed with crystal sand (CSSTYL sensu Horner et al. 2012); in the C4 morphogroupa (P. sp. RA593), however, the size difference is not obvious. The abundance of calcium oxalate crystals in the mesophyll seems to be correlated with the age of the leaf, the older leaves having more abundant crystals (e.g., C4 [P. sp. RA593] and S3 [P. sp. RA661] morphogroupsa). However, in the S1 (P. sp. RA674), C1 (P. sp. RA670 and P. sp. SING 2014-300) and C2 (P. sp. RA664) morphogroupsa and a member of the C3 (P. sp. SING 2015-073) morphogroupa the spongy cells have only a very few crystals while the palisade cells have abundant crystals; all leaves examined from the same individual show the same differences. Calcium oxalate crystals are also found in the epidermal and hypodermal cells where they are invariably crystal sand. The stems of Muldera consist (from the outside in) of an epidermis, cortical parenchyma with several layers of collenchyma, a ring of peripheral vascular bundles, an undulating sclerenchyma band, and medullary/pith parenchyma with one or more ring(s) 189 of medullary vascular bundles and one or several mucilage canal(s). The cortical parenchyma is generally brown, perhaps tanniniferous. The collenchyma of Muldera comprises 1-7 layers and appears to form a continuous ring which later becomes interrupted as the stem enlarges. The walls of the collenchyma cells are little thickened and the innermost or all layers become lignified with age. The cells of the pericyclic fiber caps adjacent to the phloem of the peripheral vascular bundles are lignified forming semilunar caps with very much thickened walls. The parenchyma cells produced by the interfascicular cambium are eventually lignified, linking the lignified pericyclic fiber caps into a ring. During secondary growth, very thick-walled sclereids develop outside the pericyclic fiber caps. There are mucilage canals in the central and peripheral parts of the medullary parenchyma, although the central mucilage canal is not always present; the peripheral canals are located interior to the smaller vascular bundles. There is generally one ring of medullary vascular bundles, but the S1 (P. sp. RA674, fig. 2.2A) and C4 (P. sp. RA593, fig. 2.2F) morphogroupsa have 2 rings. Muldera varies in the extent of secondary thickening in both peripheral and medullary vascular bundles: some samples have more extensive secondary thickening in the peripheral bundles and very limited thickening in the medullary bundles (i.e., C3 [P. sp. RA673, fig. 2.2E] and C4 [P. sp. RA593, fig.2.2F] morphogroupsa), while in other samples secondary thickening in the medullary bundles is rather extensive, sometimes exceeding that in the peripheral bundles, at least initially (i.e., S1 [P. sp. RA674, fig. 2.2A], C1 [P. sp. RA670, fig. 2.2B], C2 [P. sp. RA664, fig. 2.2C], C3 [P. sp. RA662, chapter 2 fig. 2A], and S3 [P. sp. RA661, fig. 2.2D] morphogroupsa). The extent of lignification in the primary medullary rays also varies: they are usually lignified, but in C1 (P. sp. RA670, fig. 2.2B) and C2 (P. sp. RA664, fig. 2.2C) morphogroupsa they are unlignified. 190 Molecular dataset. Of 38 individuals examined, sequences of ITS, g3pdh, and trnL-F were obtained from 34 individuals of all seven staminate and six carpellate morphogroupsa. However, only 19 individuals of five staminate (two from the Malay Peninsula) and five carpellate (three from the Malay Peninsula) morphogroupsa have both copies of the g3pdh gene. The characteristics of all molecular datasets used in this study are given in suppl. table 3.1. In ITS, putative pseudogenes and recombinants were detected in 32 out of 155 sequences newly produced in this study. Upon removal of identical sequences along with putative pseudogenes and their recombinants and inclusion of GenBank sequences, the complete Muldera ITS dataset comprises 111 sequences from 38 individuals of all morphospecies (Table 1). The number of putative functional paralogs within cloned individuals is 1-7, and the uncorrected distances (p-distance) among those paralogs vary from 0.2-2.4%, but are mostly below 1%, a level that could be attributed to polymerase errors (Speksnijder & al., 2001). Most putative functional paralogs from the same individual or morphospecies do not receive support for reciprocal monophyly, but few of them are strongly paraphyletic (see P. flavimarginatum RA 666, P. sp. SING 2014-104, and P. pachyphyllum SING 2016-114). 110 sequences of g3pdh were newly produced for this study. Upon removal of identical sequences and inclusion of GenBank sequences, the complete Muldera g3pdh dataset comprises 120 sequences. Copy B was more commonly recovered, being found in 28 individuals from all morphogroupsa, compared to 26 individuals from five staminate and five carpellate morphogroupsa for copy A. Only 19 individuals of five staminate and 191 five carpellate morphogroupsa produced both copies, and there the Kimura 2-parameter distance between the copies ranged from 5.6-9.2%. One to five paralogs of copy A per individual were retrieved, and the uncorrected distances among those paralogs vary from 0-3.2% (the latter in P. polygynum RA670). Phylogenetic analyses of the complete dataset show that few paralogs from the same individual or morphogroupsa are supported as being reciprocally monophyletic, and all members of the C3 morphogroupa (P. sp. RA662, P. sp. RA673, and P. sp. SING2015-105) are weakly to strongly paraphyletic. In copy B, one to five paralogs are retrieved per individual, and the uncorrected distances among those paralogs are 0-3.9% (the latter in P. sp. RA815). Again, most paralogs from the same individual or morphogroupsa are not reciprocally monophyletic, and a few are weakly to strongly paraphyletic (P. schizonephros RA650 and P. sp. RA547). Twenty eight sequences of trnL-F were newly produced in this study. This chloroplast marker has very low variation within Muldera (the Kimura 2-parameter distance is 0.1%), the least variation among the markers used in this study. Molecular phylogenetic analyses. Both Bayesian and maximum likelihood analyses produced trees with similar topologies. In general, nuclear markers (Fig. 3.1; Suppl. Figs. S3.1-S3.4) produced more resolved phylogenies compared to the chloroplast tree (Fig. 3.2). Using pairwise comparisons, the trees produced by nuclear markers, i.e., ITS, g3pdh copy A, and g3pdh copy B, are similar but they differed significantly in topology from the chloroplast trnL-F tree (P<0.01). Thus, the nuclear markers were concatenated and the nuclear and chloroplast trees were analyzed separately (Seelanan & al., 1997). The following discussion of the nuclear phylogeny is based on the 192 concatenated tree (Fig. 3.1) or simplified datasets (Suppl. Figs. S3.1, S3.2), results from analyses of the complete datasets (Suppl. Figs. S3.3, S3.4) being mentioned when relevant. Muldera species are resolved as a strongly supported monophyletic group (Muldera clade) in most phylogenies, except in the g3pdh copy A complete dataset where they form a strongly supported clade that also included P. caninum (95 BP/ 1.0 PP; Suppl. Fig. S3.4). The trnL-F phylogeny resolved two clades within the Muldera clade which coincide with geography: a weakly supported Malay Peninsula-Sumatra-Java clade (65 BP/- PP) and a moderately to strongly supported Bornean-Sulawesi clade (84 BP/1.0 PP). However, there is no biogeographical signal in any of the nuclear phylogenies (Fig. 3.1; Suppl. Figs. S3.1-S3.4). Considering only the Malay Peninsula morphogroupsa, the concatenated nuclear phylogeny (Fig. 3.1) resolved four weakly-moderately supported clades. The first clade (S1-C1-C2 clade, - BP/0.97 PP) also included a sterile specimen from Sumatra, U. This clade is particularly strongly supported by the ITS phylogeny (Suppl. Fig. S3.1; 97 BP/1.0 PP), but not by the g3pdh and trnL-F phylogenies. The other three clades in the concatenated nuclear phylogeny each includes members of carpellate C3 morphogroupa with (i) one of two members of the S3 morphogroupa (77 BP/0.94 PP), (ii) all members of the S5 morphogroupa and one of two members of the C4 morphogroupa (51 BP/0.91 PP), and (iii) an undetermined young staminate specimen (Sy RA659, likely part of the S3 morphogroupa; - BP/0.97 PP), respectively. The other members of S3 and C4 morphogroupsa are unresolved in the concatenated nuclear phylogeny. In the ITS phylogeny two clades are resolved (- BP/0.98 PP and 84 BP/1.0 PP), each comprising members of C3, C4, and S3 morphogroupsa, and S5 members are also 193 clustered in one of them (Suppl. Fig. S3.1 and S3.3). In the g3pdh copy A phylogeny there were two clades, one, very poorly supported (54 BP/- PP), comprises members of the C3 and S3 morphogroupsa and the undetermined young staminate specimen Sy RA659, the other, strongly supported (100 BP/1.0 PP), comprises members of the C3 and C4 morphogroupsa, with each clade being sister to specimens from Borneo (Suppl. Fig. S3.2 and S3.4). The g3pdh copy B phylogeny resolved two clades, one comprises C3 and S5 morphogroupsa (61 BP/0.96 PP), and the other comprises C4 and S3 morphogroupsa (67 BP/0.99 PP), while the Malay Peninsula undetermined young staminate specimen Sy RA659 formed a clade with Bornean undetermined young staminate specimen Sy RA796 (Suppl. Fig. S3.2 and S3.4). Finally, in the trnL-F phylogeny C3, S3 (including Sy RA659), and S5 morphogroupsa formed a moderately-strongly supported clade (84 BP/1.0 PP) within a Malay Peninsula-Sumatra-Java clade, the C4 morphogroupa being unresolved in the latter (Fig. 3.2). Discussion No results from the multivariate analyses of morphological data were congruent with my hypotheses of 11 morphogroupsa or 7 morphospecies from the Malay Peninsula. Except for the type specimen of the Javanese P. recurvum, which was in a separate morphogroupb in the FLC dataset, all morphogroupsb in the analyses were mixtures of some members of different morphogroupsa/morphospecies (fig. 1.2; suppl. table S1). More morphogroupsb were recovered when considering only the staminate or carpellate character datasets individually (FLS or FLC, respectively) than in combination with vegetative character datasets (VEGFLS or VEGFLC, respectively; 3 versus 1 and 6 versus 2 morphogroupsb, respectively). However, vegetative characters in combination 194 datasets (i.e., VEGFLC) did sometimes contribute substantially to morphogroupb separation (e.g., in plots of PC2-PC3, PC2-PC16; suppl. fig. S1.4). Paradoxically, in the joint staminate and floral datasets combined (FLSC), only a single morphogroupb was recovered. There are only slight differences between morphogroupsa/morphospecies, not enough to separate either morphogroupsa or the morphospecies. On the other hand, molecular data, particularly the nuclear markers (fig. 3.1l suppl. figs. S3.1 and S3.2), suggest that the S1-C1 morphospecies and C2 morphogroupa could be combined. The C2 morphogroupa is similar to the C1 morphogroupa, except that it has only single fruit per flower as opposed to up to four fruits in the C1 morphogroupa. Anatomically, they both have more calcium oxalate crystals in the palisade and fewer in the spongy cells of the leaf (usually they are equally abundant in both tissues) as well as early-developing extensive secondary thickening in the medullary vascular bundles in the stem. Nevertheless, these anatomical characters are not exclusive to the S1-C1-C2 clade, as some, but not all, members of the S3 and C3 morphogroupsa (S3-C3 morphospecies) also share them. The C1 and C2 morphogroupsa of the S1-C1-C2 clade have unlignified primary medullary rays, but S1 morphogroupa has lignified rays. Neither the molecular analyses nor the anatomical studies provide any evidence for the monophyly of the S3-C3 morphospecies. Phylogenetic studies in Paleotropical Piper suggest a complicated evolutionary history with polyploidization and potential hybridization or lateral gene transfer (Asmarayani 2018). Cytological studies of two Muldera species from India, P. galeatum (Miq.) C. DC. and P. trichostachyon (Miq.) C. DC. suggested that they are tetraploid, 2n = 52 respectively (Mathew et al. 1999). Most Paleotropical species of Piper were found to be reciprocally monophyletic (Asmarayani 2018), while most Muldera 195 morphogroupsa/morphospecies are not reciprocally monophyletic, suggesting that an additional recent evolutionary event, likely hybridization or lateral gene transfer, had occurred in this group. Potential hybridization or lateral gene transfer is strongly indicated by the chloroplast phylogeny, which, despite low variation, resolved two clades that were also geographically circumscribed. It is worth mentioning that the specimens from Borneo, which were placed in S1 morphogroupa, do not cluster with any member of the S1-C1-C2 clade in any phylogeny. With hybridization, or lateral gene transfer, intermediate phenotypes may result. Anatomical characters shared between some members of the S3-C3 morphospecies and members of other morphogroupsa maybe the outcome of such an event. Intermediate characters, both shape and size, may obscure variation in multidimensional space so that NMM is unable to separate morphogroupsb (e.g., Kleindorfer et al. 2014). Hooker (1896) himself had doubted the existence of species in Muldera by doubting the pairing of staminate and carpellate specimens. Botanists have been inclined to place all Muldera specimens from the same region into the same species (e.g. P. flavimarginatum for all Muldera from Singapore, P. baccatum for all Muldera from Borneo; pers. obs.), an approach that was eventually formalized into the lumping of many species of Muldera, including the two Malay Peninsula species, P. flavimarginatum and P. pentandrum, into P. baccatum (Suwanphakdee et al., 2016) Species limits within Muldera remain unclear. Even using the unified species concept in which a single line of evidence can be used to delimit species (“any evidence of lineage separation is sufficient to infer the existence of separate species” in Queiroz 2007), the species properties I examined do not give clear-cut limits for lineage separation. Furthermore, “a highly corroborated hypothesis … requires multiple lines of 196 evidence” (Queiroz 2007). Clearly, to understand this complicated situation, in-depth population studies – i.e., much more extensive sampling – coupled with examination of the morphological variation for all Muldera species from outside the Malay Peninsula are necessary. In addition there are some other lines of evidence which have not been explored, including cytology and fruit and seed anatomy, although preliminary analysis of the latter suggests that it will help little in delimiting species. Acknowledgements I especially thank Peter F. Stevens for valuable discussion, feedback, and continuous support. I thank Iván Jiménez for discussion and valuable insights in species delimitation. I also thank my committee – Elizabeth A. Kellogg, Nathan C. Muchhala, Robert J. Marquis, and Richard C. Keating –for providing helpful comments and direction in the development of the manuscript. Fieldwork, herbarium study, and laboratory work were made possible with the help of the keepers, staff, and collaborators of herbaria BO, K, KEP, L, MO, SAN, and SING, as well as Bantimurung-Bulusaraung National Park (Indonesia), Economic Planning Unit (Malaysia), National Parks Board (Singapore), Sabah Biodiversity Council (Malaysia), especially Eko B. Walujo, Joeni S. Rahajoe, James C. Solomon, P. Mick Richardson, Peter C. Hoch, Charlie D. Heatubun, Khoon-Meng Wong, David Middleton, Hock-Keong Lua, Serena Lee, Leng-Guan Saw, Rafidah A. Rahman, John B. Sugau, Joan T. Pereira, Marie Briggs, Roxali Bijmoer, Mohammad Amir, Imin Kamin, and Jeisin Jumian. 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Microscopy Research and Technique 70: 135-146. Ulloa Ulloa C., Acevedo-Rodríguez P., Beck S., Belgrano M.J., Bernal R., Berry P.E., Brako L., Celis M., Davidse G., Forzza R.C., Gradstein S.R., Hokche O., León B., León-Yánez S., Magill R.E., Neill D.A., Nee M., Raven P.H., Stimmel H., Strong M.T., Villaseñor J.L., Zarucchi J.L., Zuloaga F.O., Jørgensen P.M. 2017. An integrated assessment of the vascular plant species of the Americas. Science 358: 1614-1617. Fig. 1.1. Floral measurements taken from herbarium specimen as seen in staminate flower: a, lower cup length; b, upper cup length; c, cup base length; d, cup length; e, lip to base distance; f, lip to base length; g, lip span; h, lip width; i, cup width; j, cup angle. Cup length (d) measurements were only taken from the staminate flowers since this character in carpellate flowers is higly affected by fruit development. Note that in this specimen, lip and cup width are the same.Fig. 1.2. The numbers of specimen from each hypothetized morphogroupa/morphospecies in the morphogroupsb resolved by NMMs. A, VEG dataset; B, FLS dataset; C, VEGFLC dataset; D, FLC dataset. FLSC and VEGFLS datasets generated only a single morphogroupb, thus are not shown.200A BDCMorphogroupbMorphogroupb MorphogroupbMorphogroupbFig. 2.1. Leaves of Muldera. A, S1 morphogroupa, RA674. B, C4 morphogroupa, RA593. C, C3 morphogroupa, RA662. D, S3 morphogroupa, RA666. Scale bars = 0.5 mm. A BC D201202Fig. 2.2. Secondary growth in stems of Muldera. A, S1 morphogroupa, RA674. B, C1 morphogroupa, RA670. C, C2 morphogroupa, RA664. D, S3 morphogroupa, RA661. E, C3 morphogroupa, RA673. F, C4 morphogroupa, RA593. Scale bars = 0.5 mm. ABCDEFFig. 3.1. Concatenation of ITS and g3pdh copy A and B phylogeny. Numbers above branches are maximum-likelihood bootstrap support percentage (BP)/Bayesian posterior probabilities (PP). Only ≥ 50 BP and ≥ 0.90 PP values are shown, < 50 BP or < 0.90 PP are indicated by “-”. The first letter and number combined refer to morphogroupsa (see Introduction), Sy and Cy indicate undetermined young staminate and carpelate specimen, respectively, U indicates undetermined sterile specimen. The two letters after the collection number refer to the collection locality: Br = Northeast Borneo (Malaysia), Jv= Java (Indonesia), Mp = the Malay Peninsula (Malaysia and Singapore), Sl = Sulawesi (Indonesia), Sm = Sumatra (Indonesia).203Fig. 3.2. TrnL-F phylogeny. Annotation as in Fig. 3.1.204A BC DE FFig. S1.1. BIC values of the number of morphogroupsb (“components”) resolved by NMMs. Legends are the models in mclust. A, VEG dataset; B, FLSC dataset;, C, VEGFLS dataset; D, FLS dataset; E, VEGFLC dataset; F, FLC dataset. 205Fig. S1.2. Pairwise two-dimensional plots on PC axes of two morphogroupsb identified by the best normal mixture model in VEG dataset. In the left panels of a pair, blue and yellow small open circles represent specimens assigned to morphogroupb 1 and 2, respectively, and ellipses delineate 95% high-density regions for normal distribution for each morphogroupb. In the right panels of a pair, arrows represent the contribution of each character to each principal component; circles delineate the length of arrows expected if all characters contributed equally to two-dimensional principal component spaces, and only arrows exceeding this value are labeled.206Fig. S1.3. Pairwise two-dimensional plots on PC axes of three morphogroupsb identified by the best normal mixture model in FLS dataset. In the left panels of a pair, blue, yellow, and pink small open circles represent specimens assigned to morphogroupsb 1, 2, and 3, respectively. Annotation as in Fig. S1.2. Continued on next page.207Fig. S1.3. Continued.208Fig. S1.4. Pairwise two-dimensional plots on PC axes of two morphogroupsb identified by the best normal mixture model in VEGFLC dataset. Annotation as in Fig. S1.2. Continued on next page.209Fig. S1.4. Continued.210Fig. S1.5. Pairwise two-dimensional plots on PC axes of six morphogroupsb identified by the best normal mixture model in FLC dataset. Annotation as in Fig. S1.2. Continued on next page.211Fig. S1.5. Continued.212Fig. S3.1. ITS phylogeny of simplified dataset. Numbers above branches are maximum-likelihood bootstrap support percentage (BP)/Bayesian posterior probabilities (PP). Only ≥ 50 BP and ≥ 0.90 PP values are shown, < 50 BP or < 0.90 PP are indicated by “-”. The first letter and number combined refer to morphogroupsa (see Introduction), Sy and Cy indicate undetermined young staminate and carpelate specimen, respectively, U indicates undetermined sterile specimen. The two letters after the collection number refer to the collection locality: Br = Northeast Borneo (Malaysia), Jv = Java (Indonesia), Mp = the Malay Peninsula (Malaysia and Singapore), Sl = Sulawesi (Indonesia), Sm = Sumatra (Indonesia).213Fig. S3.2. G3pdh phylogeny of simplified dataset. Annotation as in Fig. S3.1.214ABFig. S3.3. ITS phylogeny of complete dataset. Annotation as in Fig. S3.1. Numbers and letters after the locality are clone identities. Sequences in red letters were used for simplified dataset. 215Fig. S3.4. G3pdhphylogeny of complete dataset. Annotation as in Fig. S3.1. Numbers and letters after the locality are clone identities. Sequences in red letters were used for simplified dataset. 216ABSPECIMENMORPHOGROUPaMORPHOSPECIESASSIGNMENTUNCERTAINTY VALUEASSIGNMENTUNCERTAINTY VALUEASSIGNMENTUNCERTAINTY VALUEASSIGNMENTUNCERTAINTY VALUEASSIGNMENTUNCERTAINTY VALUEASSIGNMENTUNCERTAINTY VALUEW. Griffith 4400 S1 S1_C1 1 0 1 0 1 0 1 0 NA NA NA NAC. Curtis 1771, Sungei Penang S1 S1_C1 1 0 1 0 1 0 2 0 NA NA NA NAC. Curtis 1771, Government Hill S1 S1_C1 1 0 1 0 1 0 2 0 NA NA NA NAR. Asmarayani 650 S1 S1_C1 NA NA 1 0 NA NA 2 0 NA NA NA NAR. Asmarayani 674 S1 S1_C1 1 0 1 0 1 0 2 0 NA NA NA NAE. Gardette 2042 S1 S1_C1 1 1.19E-08 1 0 1 0 2 0 NA NA NA NAI. Hassan SING-2011-222 S1 S1_C1 NA NA 1 0 NA NA 2 0 NA NA NA NAH.-K. Lua & I. Ali SING-2012-354 S1 S1_C1 1 0 1 0 1 0 3 0 NA NA NA NAA.C. Maingay 1336 S2 S2_C2 1 1.81E-12 1 0 1 0 3 0 NA NA NA NAH.N. Ridley 3772 S3 S3_C3 1 6.59E-14 1 0 1 0 3 0 NA NA NA NAR. Asmarayani 659 S3 S3_C3 1 8.1486E-07 NA NA NA NA NA NA NA NA NA NAR. Asmarayani 661 S3 S3_C3 1 1.28E-05 1 0 1 0 3 0 NA NA NA NAR. Asmarayani 666 S3 S3_C3 1 2.22E-16 1 0 1 0 1 0 NA NA NA NAJ.F. Maxwell 82-260 S3 S3_C3 1 0 1 0 1 0 1 0 NA NA NA NAH.N.Ridley s.n. (1894) S3 S3_C3 2 4.32E-03 NA NA NA NA NA NA NA NA NA NASidek & Ali 618 S3 S3_C3 1 6.66E-16 1 0 1 0 1 0 NA NA NA NAA. Yee & al. SING-2012-312 S3 S3_C3 1 1.18E-14 1 0 1 0 1 0 NA NA NA NAB. Scortechini 114a S4 S4_C4 1 1 0 1 0 3 0 NA NA NA NAL. Wray, Jr. 3655 S4 S4_C4 NA NA 1 0 NA NA 2 0 NA NA NA NAC. Boden-Kloss SFN-10595 S4 S4_C4 1 3.12E-13 1 0 1 0 2 0 NA NA NA NAM. Nur SFN-18801 S4 S4_C4 1 2.20749E-07 1 0 1 0 2 0 NA NA NA NAH.N. Ridley s.n. (1904) S4 S4_C4 1 0 1 0 1 0 3 0 NA NA NA NAC.L.C. Lee & H.-K. Lua SING-2015-104 S5 S5 1 3.75E-14 1 0 1 0 3 0 NA NA NA NAJ. Sinclair s.n. S5 S5 1 1.02069E-08 1 0 1 0 3 0 NA NA NA NAL. Wray, Jr. 4068 C1 S1_C1 1 8.70E-10 1 0 NA NA NA NA 1 0 1 7.18E-13R. Asmarayani 664a C1 S1_C1 NA NA 1 0 NA NA NA NA NA NA 2 9.42E-07R. Asmarayani 670 C1 S1_C1 1 0 1 0 NA NA NA NA 1 0 2 0I.H. Burkill & M. Haniff SFN-15659 C1 S1_C1 1 0 1 0 NA NA NA NA 1 0 1 2.00E-08E.J.H. Corner SFN-30593 C1 S1_C1 1 0 1 0 NA NA NA NA 1 0 1 0H.-K. Lua SING-2012-353 C1 S1_C1 NA NA 1 0 NA NA NA NA NA NA 1 2.47E-10H.-K. Lua SING-2014-300 C1 S1_C1 1 2.72E-12 1 0 NA NA NA NA 1 0 2 1.15E-03A.C. Maingay 1334 C2 S2_C2 1 0 1 0 NA NA NA NA 1 0 4 0J. Wyatt-Smith KEP-71898 C2 S2_C2 1 3.94E-11 1 0 NA NA NA NA 1 0 4 0R. Asmarayani 649 C3 S3_C3 2 1.22E-02 1 0 NA NA NA NA 2 2.86E-03 3 0R. Asmarayani 662 C3 S3_C3 2 8.13E-03 1 0 NA NA NA NA 1 0 3 0R. Asmarayani 673 C3 S3_C3 2 1.22E-02 1 0 NA NA NA NA 1 3.79E-05 3 0E.J.H. Corner SFN-34996 C3 S3_C3 1 1.12E-10 1 0 NA NA NA NA 1 0 1 0E.J.H. Corner s.n. C3 S3_C3 1 2.59E-07 1 0 NA NA NA NA 1 1.93E-14 4 0C.L.C. Lee & H.-K. Lua SING-2015-105 C3 S3_C3 2 1.44E-01 1 0 NA NA NA NA 1 1.06E-11 3 0H.-K. Lua & I. Hassan SING-2012-060 C3 S3_C3 2 8.64E-02 1 0 NA NA NA NA 1 5.97E-08 1 0H.-K. Lua SING-2015-073 C3 S3_C3 2 5.15E-02 1 0 NA NA NA NA 1 0 3 0A. Samsuri & al. NES 398 C3 S3_C3 1 0 1 0 NA NA NA NA 1 0 3 0W. Griffith 4427 C4 S4_C4 1 3.33E-15 1 0 NA NA NA NA 1 0 2 0B. Scortechini 779 C4 S4_C4 1 2.18E-07 1 0 NA NA NA NA 1 6.77E-14 4 0C.L. Blume s.n. C4 S4_C4 1 0 1 0 NA NA NA NA 2 0 5 0R. Asmarayani 593 C4 S4_C4 1 0 1 0 NA NA NA NA 1 0 2 9.67E-13I.H. Burkill & M. Haniff SFN-17065 C4 S4_C4 1 0 1 0 NA NA NA NA 1 5.22E-05 4 1.37E-11E.J.H. Corner SFN-30323 C4 S4_C4 1 9.42E-05 1 0 NA NA NA NA 1 0 6 0R. Ismail KEP-98543 C4 S4_C4 1 0 1 0 NA NA NA NA 1 6.40E-13 2 3.27E-03H.-K. Lua & B.-C. Ho SING-2016-114 C4 S4_C4 1 4.70E-09 1 0 NA NA NA NA 1 0 2 0H.N. Ridley s.n. (1915) C4 S4_C4 1 2.22E-16 1 0 NA NA NA NA 1 0 6 0P.W. Korthals s.n. C5 C5 2 2.72E-01 1 0 NA NA NA NA 2 2.96E-06 6 0Kiah SFN-32023 C5 C5 1 2.93E-09 1 0 NA NA NA NA 2 9.63E-09 4 0T.C. Whitmore FRI-0720 C5 C5 1 4.67E-10 1 0 NA NA NA NA 1 2.22E-16 4 0E. Soepadmo 763 C6 C6 1 0 1 0 NA NA NA NA 2 2.74E-05 4 0Table S1.1. Univariate analyses for each character observed. Specimen assignments from hypothesized morphogroupa and morphospecies into morphogroupsb resolved in NMMs. "S" and "C" in morphogroupa/morphospecies name indicates staminate and carpellate specimens, respectively.SPECIMEN INFORMATION VEG (2 morphogroupsb) FLSC (1 morphogroupb) VEGFLS (1 morphogroupb) FLS (3 morphogroupsb) VEGFLC (2 morphogroupsb) FLC (6 morphogroupsb)PC2, PC3, PC4, PC7, PC8, PC17, PC5PRINCIPAL COMPONENT AXES CHOSEN BY clustvarsel PC2, PC3, PC4, PC5, PC6PC2, PC4, PC5, PC6, PC7, PC8, PC9, PC12, PC14, PC1PC2, PC3, PC5, PC6, PC9, PC10, PC13, PC14, PC15, PC17PC3, PC4, PC6, PC8, PC11, PC12, PC14, PC15PC2, PC3, PC7, PC9, PC14, PC16, PC19, PC24217MORPHOSPECIESMORPHOGROUPSPECIMEN Leaf typeHeight of midrib (mm)Thickness of leaf, 2 mm from edge of midrib (measured from edge of abaxial bump)Hypodermal Layers, Adaxial; AbaxialHeight of Mesophyll, 2 mm from the middle of midrib (mm)Calcium oxalate crystals in palisade (type; size [mm]; abundance)Calcium oxalate crystals in spongy cells (type; size [mm]; abundance)S1-C1 S1 R. Asmarayani 674 Plag. 0.60 0.405 3; 3 0.150ST; 0.05; abundantST; 0.028; fewPlag. 0.64 0.470 3; 3-4 0.192ST; 0.06; abundantNoneOrth. 0.75 0.530 3; 3-4 0.230ST; 0.072; abundantST; 0.035; fewS1-C1 C1 R. Asmarayani 670 Plag. 0.64 0.400 3; 3-4 0.138ST; 0.077; abundantST; 0.03; very fewOrth. 0.62 0.430 3; 3-4 0.152ST; 0.09; abundantST; 0.05; very fewS1-C1 C1H.-K. Lua SING-2014-300Plag. 0.58 0.395 3-4; 3-4 0.168ST; 0.058; abundantST; 0.025; fewS2-C2 C2 R. Asmarayani 664 Orth. 0.78 0.500 2-3; 3-4 0.135ST; 0.082; abundantCSST; <0.01; very fewS3-C3 S3 R. Asmarayani 661 Plag. 0.69 0.450 3; 4-5 0.165 ST; 0.04; fewST; 0.01; quite abundantOrth. 0.85 0.675 3; 4-5 0.280ST; 0.048; abundantST; 0.037; abundantS3-C3 S3 R. Asmarayani 666 Plag. 0.62 0.525 2-3; 4 0.260ST; 0.043; abundantST; 0.038; abundantPlag. 0.79 0.645 3; 4-5 0.295ST; 0.067; abundantST; 0.052; abundantS3-C3 C3 R. Asmarayani 662 Plag. 0.54 0.410 3; 4 0.188ST; 0.055; abundantST; 0.02; abundantS3-C3 C3 R. Asmarayani 673 Plag. 0.98 0.650 2-3; 4-5 0.208ST; 0.048; abundantST; 0.01; abundantS3-C3 C3C.L.C. Lee & H.-K. Lua SING-2015-105Plag. 0.76 0.540 2; 3-4 0.170ST; 0.093; abundantST; 0.04; abundantPlag. 0.88 0.590 2; 3-4 0.210ST; 0.075; abundantST; 0.04; abundantPlag. 1.10 0.715 3-4; 4-5 0.225ST; 0.072; abundantST; 0.038; abundantS3-C3 C3H.-K. Lua SING-2015-073Plag. 0.75 0.520 3; 3-4 0.165ST; 0.062; abundantST; 0.03; fewC4 C4 R. Asmarayani 593 Plag. 1.05 0.750 3; 5 0.275ST; 0.02; very fewST; 0.017; very few? 1.15 0.780 3-4; 5-6 0.270ST; 0.028; quite abundantST; 0.02; quite abundantS5 S5C.L.C. Lee & H.-K. Lua SING-2015-104Plag. 0.78 0.560 3; 5 0.200ST; 0.095; abundantST; 0.032; abundantPlag. 0.91 0.600 3-4; 4-5 0.275ST; 0.052; abundantST; 0.043; abundantTable S2.1. Leaf anatomical characters observed. Calcium oxalate crystal type following Horner et al. (2012): ST=styloid, CSST=short styloid. Orth.=orthotropic shoot; plag.=plagiotropic shoot.218MORPHOSPECIESMORPHOGROUPSPECIMENStem typePosition in plagiotropic shootStem outlineStem diameter (long X short, mm)Outer cortex layersOuter cortex cells 1 Collenchyma bandCollenchyma cell layersCollenchyma cell thickening 1, 2Lignified collenchyma cell layersSclereidsEndodermis (Casparian strips)S1-C1 S1R. Asmarayani 674*Plag. No information Terete 3.159 X 2.732 2-3Brown, equal to/larger than collenchymaDiscontinuous 4-5 Angular, brown 1-4, discontinuous bandsOuter (few) and inner (some) cortex, between collenchyma (few)NoneOrth. NA Terete 5.903 X 5.604 3-4Brown, equal to collenchymaDiscontinuous 5-6Angular, brown, thickening slight2-5, discontinuous bandsOuter (abundant) and inner (some) cortex, between collenchyma (abundant)NoneS1-C1 S1E. Gardette 2042Plag. No information NA >1.452 X >1.079 2-3Brown, larger than collenchymaDiscontinuous 2-3 NA1-3, discontinuous bandsOuter and inner cortex, between collenchymaNAS1-C1 C1R. Asmarayani 670Plag.Next younger module than belowTerete 1.766 X 1.566 2-3Brown, larger than collenchymaDiscontinuous 2-3 Angular, brown1-3, discontinuous bandsFew: outer and inner cortex, between collenchymaNonePlag.Fruiting moduleTerete 2.147 X 1.971 2-3Brown, larger than collenchymaDiscontinuous 2-4 Angular, brown1-4, discontinuous bandsFew: outer and inner cortex, between collenchymaNoneOrth. NA Terete 3.525 X 3.322 3-4Brown, equal to/larger than collenchymaDiscontinuous 4-6 Angular, brown2-5, discontinuous bandsSome: outer and inner cortex, between collenchymaNoneOrth. NA Terete 4.89 X 4.511 4-6Brown, larger than collenchymaDiscontinuous 5-6 Angular, brown3-5, discontinuous bandsAbundant groups: outer and inner cortex, between collenchymaNoneS1-C1 C1H.-K. Lua SING-2014-300Plag. No information Terete >1.781 X >1.609 2-3Brown, larger than collenchymaDiscontinuous 2-3 NA1-3, discontinuous bandsOuter and inner cortex, between collenchymaNAS2-C2 C2R. Asmarayani 664Orth. NA NA 2.661 X 2.227 2-4Brown, larger than collenchymaDiscontinuous 3-6 Angular, brown2-5, discontinuous bandsSome: outer and inner cortex, between collenchymaNoneOrth. NA Terete 4.287 X 4.172 2-5Brown, larger than collenchymaDiscontinuous 3-6 Angular?, brown3-6, discontinuous bandsOuter (abundant) and inner (few) cortex, between collenchyma (abundant)NoneS2-C2 C2J. Wyatt-Smith KEP-71898Plag. No information Terete 1.936 X 1.898 2-4Larger than collenchymaDiscontinuous 2-5 Angular, brown1-4, discontinuous bandsOuter and inner cortex NoneS3-C3 S3R. Asmarayani 661Plag.Young flowering moduleTerete 2.814 X 2.43 2-3Brown, larger than collenchymaDiscontinuous 1-5Angular, thickening slight1-5, discontinuous bandsFew: outer cortex NoneCortexTable S2.2. Stem anatomical characters observed. NA=not applicable (in some cases the specimens are incomplete or too degraded to observe the character); orth.=orthotropic shoot; plag.=plagiotropic shoot; usb=undulating sclerenchyma band; vb(s)=vascular bundle(s). 1 Where no color is mentioned, the cell wall is normal/whitish in color. 2 No mention of thickening means that thickening is "normal". 3 There were only a single or a very few cells lignified. 4 "+" in the number of medullary vb rings indicates that there is an additional incomplete ring. "*" behind specimen number means that the data were taken from Chapter 2.219Orth. NA Terete 5.867 X 5.189 4Brown, larger than collenchymaDiscontinuous 2-5 Angular2-5, discontinuous bandsAbundant: outer (in discontinuous bands) and inner cortex (in groups), between collenchyma (in groups)NoneS3-C3 S3R. Asmarayani 666Plag. No information Terete 2.373 X 2.19 2-4Brown, equal to/larger than collenchymaDiscontinuous 3-4 Angular, brown1-4, discontinuous bandsFew: between collenchymaNonePlag. No information Terete 3.417 X 3.193 2-4Brown, equal to/larger than collenchymaDiscontinuous 2-5 Angular, brown1-5, discontinuous bandsOuter (some) and inner (few) cortex, between collenchyma (some)NoneS3-C3 S3J.F. Maxwell 82-260Plag.S3-C3 C3R. Asmarayani 662*Plag. No information Terete 2.292 X 1.719 3-4Brown, larger than collenchymaDiscontinuous 1-3Obscured by lignification1-3, discontinuous bandsSome: outer and inner cortex, between collenchymaNoneOrth. NA Terete 6.192 X 5.912 5-6Brown, larger than collenchymaDiscontinuous 1-7Obscured by lignification1-7, discontinuous bandsAbundant: outer cortex, between collenchymaNoneS3-C3 C3R. Asmarayani 673Plag. No information Terete 2.061 X 1.986 2-4Brown, equal to/larger than collenchymaDiscontinuous 3-4 Angular, brown1-4, discontinuous bandsOuter (few) and inner cortex (abundant), between collenchyma (few)NonePlag. No information Terete 3.654 X 3.363 3-4Brown, equal to/larger than collenchymaDiscontinuous 3-5 Angular, brown1-5, discontinuous bandsOuter (some) and inner cortex (few), between collenchyma (few)NonePlag. No information Terete 4.956 X 0.4794 3-5Brown, larger than collenchymaDiscontinuous 3-6Obscured by lignification3-6, discontinuous bandsOuter (abundant) and inner (some) cortex, between collenchyma (some)NoneS3-C3 C3H.-K. Lua SING-2015-073Plag. No information Terete >2.748 X >2.386 3-4Brown, larger than collenchymaDiscontinuous 3-4Obscured by lignification3-4, discontinuous bandsInner cortex NonePlag. No information Terete >3.641 X 3.234 3-4Brown, larger than collenchymaDiscontinuous 3-4Obscured by lignification3-4, discontinuous bandsInner cortex NoneS3-C3 C3C.L.C. Lee & H.-K. Lua SING-2015-105Plag. No information Terete >1.995 X >1.514 3-4Brown, larger than collenchymaDiscontinuous 2-3 Angular1-3, discontinuous bandsOuter and inner cortex NonePlag. No information Terete 3.205 X 2.716 3-4Larger than collenchymaDiscontinuous 4-5Angular, thickening slight2-5, discontinuous bandsOuter and inner cortex, between collenchymaNoneS4 S4M. Nur SFN-18801Plag. NA >1.803 X > 1.239 1-3Brown, equal to/larger than collenchymaContinuous NA NA1-3, discontinuous bandsNone NA220C4 C4R. Asmarayani 593*Plag.Immediately younger module is fruitingTerete 3.286 X 2.913 3-5Brown, larger than collenchymaDiscontinuous 1-5 Angular, brown1-5, discontinuous bandsOuter (abundant) and inner cortex (some), between collenchyma (some)NonePlag.Next older module than aboveTerete 5.112 X 4.481 3-5Brown, larger than collenchymaDiscontinuous 2-5 Angular, brown2-5, discontinuous bandsSome groups: inner cortexNonePlag. No information Terete 5.921 X 5.406 4-5Brown, larger than collenchymaDiscontinuous 2-6Obscured by lignification2-6, discontinuous bandsAbundant groups: outer and inner cortex, between collenchymaNonePlag. No information Terete 7.093 X NA 4-5Brown, larger than collenchymaDiscontinuous 1-5Obscured by lignification1-5, discontinuous bandsAbundant groups: outer and inner cortex, between collenchymaNoneS5 S5C.L.C. Lee & H.-K. Lua SING-2015-104Plag. No information NA >1.873 X >1.207 2-3Brown, larger than collenchymaDiscontinuous 2-3 Angular1-3, discontinuous bandsOuter and inner cortex, between collenchymaNonePlag. No information Terete >3.092 X >2.599 3-4Brown, larger than collenchymaDiscontinuous 3-4 Angular2-4, discontinuous bandsInner cortex NoneC5 C5Kiah SFN-32023Plag. NA >1.975 X >1.46 2-3Brown, equal to/larger than collenchymaDiscontinuous 1-3 NA1-3, discontinuous bandsOuter and inner cortex, between collenchymaNAC6 C6E. Soepadmo 763? ? Terete 1.514 X 1.491 3-4Brown, larger than collenchymaDiscontinuous NA NA1-2, discontinuous bandsOuter and inner cortex, between collenchymaNAU UR. Asmarayani 634*Orth. NA Terete 3.256 X 2.952 3-5Brown, larger than collenchymaDiscontinuous 1-4Angular, thickening slight1-4, discontinuous bandsFew: outer cortex, between collenchymaContinuousU UR. Asmarayani 636*Orth. NA Terete 4.549 X 4.346 2-3Brown, larger than collenchymaDiscontinuous 4-6Angular, thickening slight1-5, discontinuous bandsFew: outer cortex, between collenchymaNone221Table S2.2. Continued.MORPHOSPECIESMORPHOGROUPSPECIMENStem typeLignified pericyclic cap2; associated sclereids if presentContinuity of lignified pericyclic capHow lignified pericyclic caps are united2; associated sclereids if presentNumber of vbsNumber of smaller vbs between large vbsSecondary thickeningPrimary medullary rays in interfascicular areasPattern of lignification in primary medullary raysS1-C1 S1R. Asmarayani 674*Plag.Semilunar, thick walled; few sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma, some cells are thick walled31 1-4 All vbs, early stage Some areas Most between the xylemOrth.Semilunar, thick walled; almost continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids42 2-3 All vbs All areas Most between the xylemS1-C1 S1E. Gardette 2042Plag.Semilunar, sometimes unlignified; some sclereidsDiscontinuous NA 26 2-3 Some vbs, early stage NA NAS1-C1 C1R. Asmarayani 670Plag.Semilunar, thick walled; few sclereidsPartly discontinuous, most caps not (fully) unitedLignified parenchyma, thick walled; few sclereids23 1-2 Most vbs, early stage Some areas NonePlag.Semilunar, thick walled; few sclereidsPartly discontinuous, most caps not (fully) unitedLignified parenchyma, thick walled; few sclereids26 1-3 Most vbs, early stage Some areasFew in adaxial area between the xylemOrth.Semilunar, thick walled; few sclereidsContinuousLignified parenchyma, thick walled; few sclereids33 2-3 All vbs All areasMost between the xylem, some in abaxial area between the phloemOrth.Semilunar, thick walled; some sclereidsContinuousLignified parenchyma, thick walled; some sclereids34 1-3 All vbs All areas Most between the phloemS1-C1 C1H.-K. Lua SING-2014-300Plag.Semilunar, sometimes thick walled; few sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma 22 1-3 All vbs, early stage Some areasFew in adaxial area between the xylemS2-C2 C2R. Asmarayani 664Orth.Semilunar, thick walled; almost continuous sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma, thick walled; some sclereids24 2-3 Some vbs, early stage Some areas Some between the xylemOrth.Semilunar, thick walled; almost continuous sclereidsContinuousLignified parenchyma, thick walled; almost continuous sclereids30 2-4 All vbs All areasSome in abaxial area between the phloemS2-C2 C2J. Wyatt-Smith KEP-71898Plag.Semilunar, thick walled; some sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma 28 1-4 Some vbs, early stage NA NAPeripheral vascular bundles (vbs)222S3-C3 S3R. Asmarayani 661Plag.Semilunar, sometimes unlignifiedPartly discontinuous, all caps not fully unitedLignified parenchyma 28 1-5 Some vbs, early stage Some areas NoneOrth.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids39 1-5 All vbs All areas All between the xylemS3-C3 S3R. Asmarayani 666Plag.Semilunar, sometimes thick walled; few sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma; few sclereids 27 1-4 Some vbs, early stage All areasFew in adaxial area between the xylemPlag.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids32 1-4 All vbs All areas Some between the xylemS3-C3 S3J.F. Maxwell 82-260Plag. 23S3-C3 C3R. Asmarayani 662*Plag.Semilunar, sometimes unlignified; few sclereidsPartly discontinuous; most caps not (fully) unitedLignified parenchyma 22 2-3 Some vbs, early stage Some areas Few between the xylemOrth.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids32 2-3 All vbs All areas All between the xylemS3-C3 C3R. Asmarayani 673Plag.Semilunar, usually unlignified; some sclereidsPartly discontinuous; most caps not (fully) unitedLignified parenchyma; some sclereids 24 1-4 Few vbs, early stage None NonePlag.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids29 1-4 Most vbs, early stage All areas All between the xylemPlag.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids30 1-3 All vbs All areasAll between the xylem, some in abaxial area between the phloemS3-C3 C3H.-K. Lua SING-2015-073Plag.Semilunar, thick walled; continuous sclereidsPartly discontinuous, few caps not (fully) unitedLignified parenchyma, thick walled; some sclereids22 1-2, 4 Some vbs Some areas All between the xylemPlag.Semilunar, thick walled; continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids29 1-4 All vbs All areas All between the xylemS3-C3 C3C.L.C. Lee & H.-K. Lua SING-2015-105Plag.Semilunar, thick walled; almost continuous sclereidsPartly discontinuous, most caps not (fully united)Lignified parenchyma 18 1-3 Some vbs, early stage Few areasSome between the xylem/phloem223Plag.Semilunar, thick walled; almost continuous sclereidsContinuousLignified parenchyma, thick walled; some sclereids24 1-4 Most vbs, early stage Some areas All between the xylemS4 S4M. Nur SFN-18801Plag. None NA NA 24 1-3 Some vbs, early stage NA NAC4 C4R. Asmarayani 593*Plag.Semilunar, thick walled; few sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma, some cells are thick walled31 2-4 Some vbs, early stage Some areas Most between the xylemPlag.Semilunar, thick walled; few sclereidsPartly discontinuous, few caps not (fully) unitedLignified parenchyma, some cells are thick walled; few sclereids40 1-4 Some vbs, early stage Some areasSome between the xylem, few between the phloemPlag.Semilunar, thick walled; some sclereidsContinuousLignified parenchyma, very thick walled; some slcereids35 2-5 All vbs All areasAll between the xylem, some in abaxial area between the phloemPlag.Semilunar, thick walled; some sclereidsContinuousLignified parenchyma, very thick walled; some sclereids52 1-3 All vbs All areasAll between the xylem, some in abaxial area between the phloemS5 S5C.L.C. Lee & H.-K. Lua SING-2015-104Plag.Semilunar, thick walled; continuous sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma, thick walled; some sclereids17 1-4 Few vbs, early stage None NAPlag.Semilunar, thick walled; continuous sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma, thick walled; some sclereids24 1-4 Some vbs, early stage Few areas Most between the xylemC5 C5Kiah SFN-32023Plag. Semilunar; few sclereidsPartly discontinuous, some caps not (fully) unitedLignified parenchyma 19 1-4 Some vbs, early stage NA NAC6 C6E. Soepadmo 763?Semilunar, sometimes thick walled; some sclereidsPartly discontinuous, few caps not (fully) unitedLignified parenchyma 24 1-2, 4-5 Some vbs Some areasAll between the xylem, NA between the phloemU UR. Asmarayani 634*Orth.Semilunar, sometimes unlignifiedDiscontinuous NA 24 1-4 No None NAU UR. Asmarayani 636*Orth.Semilunar, sometimes thick walled, sometimes unlignifiedPartly discontinuous, some caps not (fully) unitedLignified parenchyma, thick-very thick walled31 2-4 Some vbs, early stage Some areas Some between the xylem224Table S2.2. Continued.MORPHOSPECIESMORPHOGROUPSPECIMENStem typeVessel number across top of vbsVessels vs. secondary medullary ray trans-sectional area (qualitative)Width of primary medullary rays (number of cells) Undulating sclerenchyma band (usb) layersNumber of rings 4Number of vbs (outermost; innermost, if applicable)Sclerenchyma cap on phloemSclerenchyma cap on xylemVbs with altered orientation/close to each otherSecondary thickening Ray-like cells in areas between vascular bundlesS1-C1 S1R. Asmarayani 674*Plag. NA NA NA 3-5 2 5; 10 All vbs All vbsAll outer vbs have 180° orientation, 1 outer vb joinedInner vbs only, massive like peripheral vbsSome areas in inner ringOrth. 1(-2)Vessels < Rays7-16 5-7 2 12; 16 All vbs All vbsSome outer vbs have 90° orientationAll vbs, inner vbs are massive like peripheral vbsIn inner ringS1-C1 S1E. Gardette 2042Plag. NA NA NA 3-5 1 8 All vbs All vbs 4 vbs are paired Some vbs, early stage NAS1-C1 C1R. Asmarayani 670Plag. NA NA NA 3-5 1 9 All vbs All vbs None All vbs, early stage Some areasPlag. NA NA NA 2-5 1 8 All vbs All vbs 1 vb joined All vbs, early stage Some areasOrth. NA NA NA 4-7 1 16 All vbs All vbs None All vbs Most areasOrth. 1-2(-3)Vessels > Rays5-14 4-7 1 16 All vbs All vbs 1 vb joinedAll vbs, some are massive like peripheral vbsAll areasS1-C1 C1H.-K. Lua SING-2014-300Plag. NA NA NA 3-5 1 6 All vbs All vbs None All vbs, early stage NAS2-C2 C2R. Asmarayani 664Orth. NA NA NA 3-6 1 8 All vbs All vbs None No Few areasOrth. NA NA NA 3-7 1 15 All vbs All vbs NoneAll vbs, more massive than peripheral vbsAll areasS2-C2 C2J. Wyatt-Smith KEP-71898Plag. NA NA NA 4-7 1 NA (>7) All vbs All vbs 1 vb joined All vbs, early stage Some areasS3-C3 S3R. Asmarayani 661Plag. NA NA NA 4-5 1 7 All vbs All vbs None All vbs, early stage Few areasMassive secondary thickening in peripheral vascular bundles (vbs)Medullary vascular bundles225Orth. NA NA NA 4-7 1 16 All vbs All vbs None All vbs, rather massive All areasS3-C3 S3R. Asmarayani 666Plag. NA NA NA 3-5 1+ 1; 10 All vbs All vbs1 outer vb has 90° orientation, 2 inner vbs are pairedAll vbs, early stage and more massive than peripheral vbsSome areasPlag. NA NA NA 3-6 1 11 All vbs All vbs 2 vbs joinedAll vbs, some are more massive than peripheral vbsSome areasS3-C3 S3J.F. Maxwell 82-260Plag.S3-C3 C3R. Asmarayani 662*Plag. NA NA NA 1-3 1 8 All vbs All vbs None Some vbs, early stage Some areasOrth. 1(-2)Vessels < Rays8-15 3-5 1 1; 18 All vbs All vbsOuter vb has 160° orientation, 2 inner vbs are pairedAll vbs, rather massive All areasS3-C3 C3R. Asmarayani 673Plag. NA NA NA 2-4 1 7 All vbs All vbs None No NonePlag. NA NA NA 2-5 1 11 All vbs All vbs NoneAll vbs, some are more massive than peripheral vbsSome areasPlag. 1(-2)Vessels < Rays6-16 4-6 1 11 All vbs All vbs NoneAll vbs, some are rather massive (but less massive than peripheral vbs)Some areasS3-C3 C3H.-K. Lua SING-2015-073Plag. NA NA NA 4-5 NA NA (>9) All vbs All vbs NA NA NAPlag. NA NA NA 4-6 NA NA (>10) All vbs All vbs NAAll vbs, rather massive like peripheral vbsNAS3-C3 C3C.L.C. Lee & H.-K. Lua SING-2015-105Plag. NA NA NA 2-4 1 6 All vbs All vbs None NA NAPlag. NA NA NA 2-6 1 9 All vbs All vbs None Few vbs, early stage Some areasS4 S4M. Nur SFN-18801Plag. NA NA NA 3-5 1 10 All vbs All vbs None NA NA226C4 C4R. Asmarayani 593*Plag. NA NA NA 3-5 1 14Some vbs, others have only few lignified cellsAll vbs 1 vb joined Some vbs, early stage Some areasPlag. NA NA NA 4-7 1 + 6; 15 All vbs All vbs1 outer vb has 45° orientationMost inner vbs, early stageSome areas in inner ringPlag. 1(-2)Vessels < Rays6-18 4-7 2 6; 12Some vbs in inner ring, others have only few lignified cellsAll vbs2 outer vbs have 45° and 90° orientationSome inner vbs, early stageSome areas in inner ring and between inner and outer vbsPlag. 1Vessels < Rays8-16 4-6 2+ 3; 12; 17 All vbs All vbsMost vbs in middle and outer rings have up to 100° orientationAll vbs, early to advanced stages, inner vbs more developed than middle vbsSome areas in inner ring and between inner and middle vbsS5 S5C.L.C. Lee & H.-K. Lua SING-2015-104Plag. NA NA NA 2-4 NA NA (>6) All vbs All vbs NA Some vbs, early stage NAPlag. NA NA NA 4-6 1 NA (>9) All vbs All vbs NAAll vbs, rather massive like peripheral vbsSome areasC5 C5Kiah SFN-32023Plag. NA NA NA 3-4 NA NA (>6) All vbs All vbs NA Some vbs, early stage NAC6 C6E. Soepadmo 763? NA NA NA 2-4 1+ 1; 7 All vbs All vbs NoneSome vbs, rather massive (but less than peripheral vbs)Some areasU UR. Asmarayani 634*Orth. NA NA NA 4-5 1+ 2; 10 All vbs All vbs None Most vbs, early stage Some areasU UR. Asmarayani 636*Orth. NA NA NA 4-5 1 16 All vbs All vbsA small vb, slightly outside the ring, has 30° orientationAll vbs, early stages Some areas227Table S2.2. Continued.MORPHOSPECIESMORPHOGROUPSPECIMENStem typePattern of lignification in pith parenchymaNumber of mucilage canals (peripheral; central)Position of peripheral mucilage canalsSecretory (ethereal oil) cellsS1-C1 S1R. Asmarayani 674*Plag.Some cells in 1-3 peripheral layers; some groups of cells elsewhere8 (1 ring); 0Interior to small vbsNot seenOrth.Most cells in outer pith except surrounding mucilage canals; NA for other areas12 (1 ring); NAInterior to small vbsFew: inner cortex, phloem, pithS1-C1 S1E. Gardette 2042Plag. NA 7 (1 ring); 0Interior to small vbsNot seenS1-C1 C1R. Asmarayani 670Plag.Most cells except some cells in outer pith and surrounding mucilage canals3 (partial ring); 0Interior to small vbsNot seenPlag.Some cells in 1-6 peripheral layers; some groups of cells elsewhere2 (partial ring); 0Interior to small vbsFew: inner cortexOrth.Most cells in outer pith except surrounding mucilage canals; few groups of cells in central pith10 (1 ring); 1Interior to small vbsNot seenOrth.Some cells in 1-2 peripheral layers10 (1 ring); 0Interior to small vbsFew: outer cortex, peripheral phloemS1-C1 C1H.-K. Lua SING-2014-300Plag.Some groups of cells in outer and central pith4 (partial ring); 0Interior to small vbsNot seenS2-C2 C2R. Asmarayani 664Orth.Some cells in 1-6 peripheral layers6 (1 ring); 0Interior to small vbsFew: inner cortex, peripheral phloemOrth.Most cells in 1 peripheral layer9 (1 ring); 1Interior to small vbsFew: outer cortexS2-C2 C2J. Wyatt-Smith KEP-71898Plag. NANA (seen 5); NAInterior to small vbsOuter cortex (abundant), NA for other228S3-C3 S3R. Asmarayani 661Plag.Some cells in 1 peripheral layer3 (half ring); 0Interior to small vbsOuter cortex (abundant), elsewhere (some)Orth.Most cells in outer pith except surrounding mucilage canals; some cells in vb ring 10 (1 ring); 1Interior to small vbsOuter cortex (abundant), inner cortex (some), primary medullary rays (some)S3-C3 S3R. Asmarayani 666Plag.Some groups of cells in outer and central pith3 (partial ring); 0Interior to small vbsFew: cortex, phloem, pithPlag.Some cells in 1-4 peripheral layers; some groups of cells in outer and central pith9 (1 ring); 0Interior to small vbsSome: outer and inner cortex, phloemS3-C3 S3J.F. Maxwell 82-260Plag. NA; NAS3-C3 C3R. Asmarayani 662*Plag.Some cells in 1 peripheral layer0; 0 NAOuter (abundant) and inner (some) cortex, phloem (some)Orth.Most cells in outer pith and vb ring except surrounding mucilage canals11 (1 ring); 0Interior to small vbsOuter cortex (abundant), inner cortex (some), primary medullary rays (some)S3-C3 C3R. Asmarayani 673Plag.Some cells in 1-6 peripheral layers; some groups of cells surrounding few vbs1; 0Interior to small vbOuter (some) and inner (few) cortex, phloem (few), pith (few)Plag.Most cells from outer pith to vb peripheral area of central pith6 (partial ring); 0Interior to small vbsOuter cortex (some), rays (few)Plag.Most cells from outer pith to vb peripheral area of central pith except surrounding mucilage canals8 (1 ring); NAInterior to small vbsOuter (some) and inner (few) cortex, pith (few)S3-C3 C3H.-K. Lua SING-2015-073Plag.Some cells in 1-3 peripheral layers; NA for other areaNA; NA NAOuter (abundant) and inner (few) cortex, phloem (at least some in few phloem, NA for other phloem), NA for pithPlag.Some cells in 1-7 peripheral layers; NA for other areaNA (seen 4); NAInterior to small vbsOuter (abundant) and inner (some) cortex, vbs (at least some in some vbs, NA for other vbs), pith (at least few in intact pith)229S3-C3 C3C.L.C. Lee & H.-K. Lua SING-2015-105Plag.Some cells from outer pith to vb ring; NA for other area0; 0 NAOuter cortex, inner cortex, phloem, NA for pithPlag.Some cells in 1-3 peripheral layers; some groups of cells elsewhere5 (partial ring); NAInterior to small vbsOuter cortex, phloemS4 S4M. Nur SFN-18801Plag. NANA (seen 2); 0Interior to small vbsCortex, phloem, pithC4 C4R. Asmarayani 593*Plag. Outer pith to vb ring 9 (1 ring); 1Interior to small vbsOuter and inner cortex (abundant), phloem (few), pith (some)Plag.Some cells in 1-5 peripheral layers and vb rings; few groups of cells elsewhere14 (1 ring, 4 canals are paired); 1Interior to small vbsSome: inner cortex, peripheral phloem, primary medullary rays; few: pith, medullary phloemPlag.Most cells from outer pith to inner vb ring9 (1 ring); 1Interior to small vbsFew: inner cortex, peripheral phloem, pithPlag.Most cells except surrounding mucilage canals14 (1 ring); 1Interior to small vbsInner cortex (few), pith (some)S5 S5C.L.C. Lee & H.-K. Lua SING-2015-104Plag.Some cells in 1-2 peripheral layers; NA for other areaNA; NA NA Cortex, pithPlag.Some cells in 1 peripheral layer; NA for other areaNA (seen 2); NAInterior to small vbsCortex, phloem, pithC5 C5Kiah SFN-32023Plag.Some cells in 1-2 peripheral layers; NA for other areaNA (seen 1); NAInterior to small vbsCortex, phloem, pithC6 C6E. Soepadmo 763? Most cells NA; 0 NA Cortex, phloem, pithU UR. Asmarayani 634*Orth.Some cells in 1 peripheral layer3 (1 ring); 0Interior to small vbsAbundant: cortex, pithU UR. Asmarayani 636*Orth.Some cells in 1-7 peripheral layers10 (1 ring); 1Interior to small vbsFew: inner cortex, pith230Table S3.1. Characteristic of molecular datasets used in this study, calculated with MEGA 7.0.18 (Kumar & al., 2015). For ITS and g3pdh, calculations are based on simplified datasets. ITS g3pdh Copy A g3pdh Copy B trnL-F Number of morphospecies (male + female) 13 10 13 13 Number of individuals 38 26 28 34 Sequence length* 652-657 bp 940-990 bp 1015-1036 bp 903-906 bp Alignment length 665 bp 992 bp 1046 bp 909 bp Variable characters 45 bp (6.8%) 103 bp (10.4%) 113 bp (10.8%) 4 bp (0.4%) Average Kimura 2-parameter distance 1.1% 1.5% 1.7% 0.1% * based on complete sequences only 231

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