Accepted Manuscript Molecular phylogeny and morphology of Elatostema s.l. (Urticaceae): implications for inter- and infrageneric classifications Yu-Hsin Tseng, Alex K. Monro, Yi-Gang Wei, Jer-Ming Hu PII: DOI: Reference: S1055-7903(18)30010-1 https://doi.org/10.1016/j.ympev.2018.11.016 YMPEV 6350 To appear in: Molecular Phylogenetics and Evolution Received Date: Revised Date: Accepted Date: 8 January 2018 22 October 2018 20 November 2018 Please cite this article as: Tseng, Y-H., Monro, A.K., Wei, Y-G., Hu, J-M., Molecular phylogeny and morphology of Elatostema s.l. (Urticaceae): implications for inter- and infrageneric classifications, Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev.2018.11.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Molecular phylogeny and morphology of Elatostema s.l. (Urticaceae): implications for inter- and infrageneric classifications Yu-Hsin Tsenga, Alex K. Monro b,c, Yi-Gang Weid, Jer-Ming Hua, * a Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10617, Taiwan b Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK c Herbarium, Royal Botanic Gardens, Kew TW9 3AE, UK d Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China Corresponding author at: Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10617, Taiwan. Email addresses: jmhu@ntu.edu.tw (J. M. Hu) 1 Abstract Elatostema s.s. (Urticaceae) comprises approximately 500 species of herbs and subshrubs distributed in tropical and subtropical Asia, Australasia, and Africa. The delimitation of Elatostema s.s. and the closely related genera Elatostematoides, Pellionia, and Procris has long been problematic because of the large number of taxa and presumed homoplasy among diagnostic morphological characters. In the present study, we refer to these four genera together as Elatostema s.l. To evaluate the circumscription of Elatostema s.l. and its generic and subgeneric classification, we conducted phylogenetic analyses of DNA sequence data from the internal transcribed spacer of the nuclear genome (nrITS) and two markers from the plastid genome (psbA-trnH and psbM-trnD) for 126 taxa, representing 88 species of Elatostema s.s., four of Elatostematoides, nine of Pellionia, and five of Procris. Ten selected morphological characters were investigated using ancestral state reconstructions. Our results show that Elatostema s.l. can be divided into three well-supported and morphologically distinct genera: Procris, Elatostematoides, and Elatostema sensu auct. The results of our molecular phylogeny suggest four strongly supported clades within this newly defined Elatostema s.a.: core Elatostema, Pellionia, Weddellia, and an as yet undescribed clade African Elatostema. Homoplasy among the morphological characters used in this study makes it impossible to circumscribe genera using synapomorphies, but combined suites of characters do enable the morphological diagnosis of Elatostema s.a., Elatostematoides, and Procris. Keywords: Elatostema, Elatostematoides, molecular phylogeny, morphology, Pellionia, Procris 2 Introduction Elatostema J.R. Forst. & G. Forst. (Urticaceae), here referred to as Elatostema s.s., is a group of approximately 500 species (Wang, 2012; Wei et al., 2011) of understory herbs, subshrubs, and shrubs. Elatostema s.s. is most common in areas of deep shade, particularly in dense forests, along streams, at/near waterfalls, and in caves, where they can become a dominant element of the forest understory. They are distributed in tropical and subtropical Africa, East and Southeast Asia, and Australasia, with centers of species richness in Southeast Asia and Southwest China (Wang, 2012; Wei et al., 2011). The delimitation of Elatostema s.s. (acronyms for Elatostema here is E.) and the closely related genera Elatostematoides C.B. Rob. (acronyms for Elatostematoides here is Eld.), Pellionia Gaudich. and Procris Comm. ex Juss. in Urticaceae has long been problematic, probably due to the high number of taxa and the use of homoplastic morphological characters in their taxonomy (Hadiah and Conn, 2009; Hadiah et al., 2003, 2008; Wang, 1980a, b, 2012; Wu et al., 2013, 2015). In this paper, we refer to these four taxa together as Elatostema s.l., and the definition of “Elatostema” without s.s. or s.l. is based on previous taxonomic treatments, including either two or three related taxa. Elatostema s.l. is characterized by alternate leaves (Fig. 1C, D, F) and minute green to white unisexual flowers that form in dense clusters (Fig. 1A, B, D, E, G-I). However, some features of the flowers and inflorescence, especially tepal presence and length, inflorescence architecture, receptacle shape, and involucre presence can vary between taxa. In a pistillate inflorescence, flowers have receptacles distinct from each other (Fig. 3C, D) or fused into a fleshy, shared receptacle that is lobed to discoid or subglobose (Fig. 1I), which may or may not be surrounded by an involucre (Fig. 4A). When present, the involucre is formed by fused or closely associated bracts 3 and resembles a collar-like structure under the inflorescence (Fig. 1A). Tepals in pistillate flowers can be absent (Fig. 4C) or present (Figs. 1D, 4C, 4D). Staminate flowers form in cymes (Fig. 4B) or heads (Fig. 1B, E, G, H) with obvious tepals (Fig. 1E), and the presence and length of the receptacle can vary by taxon. These characters together with habit and the absence/presence of nanophylls (Fig. 1C, F) have been the main focus for generic and subgeneric classification in previous studies (Schröter and Winkler, 1935, 1936; Wang, 1980b). Elatostema s.l. was first revised by Weddell (1869). Since then, most taxonomic work on Elatostema s.l. has been published in regional revisions and flora accounts, with the inter- and infrageneric delimitation remaining controversial (Table 1). Hallier (1896) reduced Pellionia and Procris to subgenera of Elatostema when dealing with Malesian Urticaceae. Schumann and Lauterbach (1900) maintained Elatostema s.s, Pellionia, and Procris as distinct genera in the treatment of Urticaceae from former German protectorates in the South Pacific. Based on the investigation of Philippine species, Robinson (1910) provided a detailed discussion of generic delimitation between Elatostema s.s., Elatostematoides, Pellionia, and Procris and suggested that Procris and Pellonia should be retained at a generic level based on the lack of a true involucre in the staminate inflorescence. He also distinguished Elatostematoides from Elatostema s.s. by the lack of true involucres in the staminate and pistillate inflorescences and the presence of very unequal opposite leaves. Winkler’s revision of New Guinean Urticaceae (Winkler, 1922) concluded that Pellionia and Procris should be treated as subgenera of Elatostema. However, in the first monograph of Elatostema (Schröter and Winkler, 1935, 1936), Procris was recognized as a distinct genus on the basis of the fleshy pistillate receptacle not being enclosed in an involucre of bracts (Schröter, 1938a). Wang (1980a, b) proposed that 4 Pellionia and Procris should be maintained as distinct genera. Pellionia was treated as distinct on the basis of the pistillate flowers with obvious perianths and the usually tuberculate achene. Wang’s classification has been followed in the Flora of Japan (Tateishi, 1993) as well as by some Taiwanese researchers (Shih et al., 1995; Yang et al., 1995). Attempts to address inter- and infrageneric delimitations of Elatostema s.l. have used phylogenetic analyses based on morphological characters (Beaman, 2001; Hadiah and Conn, 2009; Wu et al., 2015) and DNA sequence data (Hadiah et al., 2003 and 2008; Wu et al., 2013), but relationships between Elatostema s.s., Elatostematoides, Pellionia, and Procris remain uncertain. In summary, the major taxonomic treatments of Elatostema s.l. are the following: (1) Elatostema s.s., Elatostematoides, Pellionia, and Procris as distinct genera (Robinson, 1910; Schumann and Lauterbach, 1900; Wang, 1980a, 1980b; Weddell, 1869; Yang et al., 1995); (2) Pellionia and Procris as subgenera of Elatostema (Hallier, 1896; Winkler, 1922); and (3) Procris as a distinct genus and Pellionia and Elatostematoides as subgenera of Elatostema (Schröter, 1938; Schröter and Winkler, 1935, 1936). The relationships between taxa within Elatostema s.s. are controversial. This controversy has resulted in two systems being proposed for infrageneric relationships based on different morphological characters. Schröter and Winkler (1935, 1936) recognized Procris and four subgenera within Elatostema: Elatostematoides, Elatostema, Pellionia, and Weddellia H. Schröter. Of these four subgenera, Wang (1980a, b) recognized two as genera (Elatostematoides and Pellionia) and two as sections of Elatostema s.s. (Elatostema and Weddellia). Wang (1980a, b) also proposed three additional sections in Elatostema s.s.: Androsyce Wedd., Laevisperma (Hatus.) T. Yamaz. and Pellionioides W.T. Wang. Wang (2012) later subsumed Laevisperma into the section Weddellia. Furthermore, Wang (1980b) suggested that 5 the section Pellionioides is the most “primitive” group because its staminate inflorescence morphology was intermediate between Pellionia and Elatostema s.s. and proposed Androsyce as the most derived section based on its pyriform staminate receptacles. Researchers have performed phylogenetic analyses of Schröter and Winkler’s system using morphological characters (Hadiah and Conn, 2009) and DNA sequences (Hadiah et al., 2003, 2008), but Wang’s taxonomic proposals have not yet been evaluated. In summary, studies at the intergeneric level within Elatostema s.l. and infrageneric level within Elatostema s.s. utilizing morphological characters or molecular data have included proposals of relationships, but most of these studies have had a restricted geographic focus and sampling of taxa. The phylogenies recovered in these investigations did little to resolve generic or infrageneric groupings. The main goal of the present study was to construct a robust phylogeny for Elatostema s.l. based on DNA sequence data, with sampling across a broad range of taxa and geographical localities. Furthermore, we performed ancestral state reconstructions of morphological characters traditionally considered to be key in the taxonomic analysis of these genera. 2. Materials and Methods 2.1. Taxon sampling Representatives of Schröter and Winkler’s (1935, 1936) four subgenera and Wang’s (1980b, 2012) five sections were selected from Elatostema s.s., Elatostematoides, Pellionia, and Procris. Sequence data were obtained for 92 Elatostema s.s. taxa (88 species and four varieties, representing approximately 18.4% of the species), as well as four Elatostematoides (approximately 10%), nine Pellionia 6 (approximately 13%), and five Procris (approximately 25%). To maximize the geographical coverage, our samples were obtained from both fresh and herbarium materials. Fresh leaves were collected and preserved in silica gel, and vouchers of freshly collected material were deposited at BM, HAST, IBK, TAI, and TAIF. Herbarium specimens were sampled from A, BM, IBK, K, KYO, MO, and TAIF (Supplementary Text 1). Boehmeria macrophylla Hornem., Debregeasia orientalis C.J. Chen, Lecanthus peduncularis (Royle) Wedd., Leucosyke quadrinervia C.B. Rob., Nanocnide japonica Blume, and Poikilospermum acuminatum (Trécul) Merr. were chosen as outgroups to represent each of the major Urticaceae clades (Wu et al., 2013). Species names, the accession numbers of sequences downloaded from GenBank, and newly generated sequences used in this study are shown in Supplementary Text 1. 2.2. Genomic DNA (gDNA) extraction and PCR Total gDNA was extracted from fresh or dried materials following Li et al. (2005). Two plastid regions (psbA-trnH intergenic spacer and psbM-trnD intergenic spacer) and the nuclear ribosomal internal transcribed spacer (nrITS) were used for phylogenetic analysis. PCR reactions were performed in 50 μL of final volumes with 5 μL of 10 PCR buffer, 1 μL of 10 mM dNTP (2.5 mM each), 1 μL of each specific primer (10 μM each), 35–50 ng of template, and 0.2 μL of MDBio Taq DNA Polymerase (MDBio, Taipei, Taiwan). The psbA-trnH spacer was amplified using primers psbA3’f” and trnH (Kress et al., 2005). The psbM-trnD spacer was amplified using primers psbMF and trnDGUCR (Shaw et al., 2005). Primers for the nrITS region amplification were 18S 1830 (Nickrent et al., 1994) and 26S 307R (Soltis and Kuzoff, 1995) for most accessions. For problematic samples, we used primers MKT56 and 7 MKT57 instead (Thomson et al., 1995). PCR amplifications were set at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. PCR products were purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and were sequenced using an ABI PRISM 337 automated sequencer (Applied Biosystems). The nrITS dataset comprised 130 accessions: 104 Elatostema s.s., four Elatostematoides, ten Pellionia, six Procris, and six outgroup species. For psbA-trnH and psbM-trnD, two additional accessions, E. ellipticum Wedd. and E. rugosum A. Cunn., were included. Elatostema variolaminosum var. latum H. Schröter. was not sampled for psbM-trnD because the PCR amplification was not successful. Five outgroup species were sampled for psbAtrnH and four species for psbM-trnD. The combined dataset for the three markers comprised 132 sequences representing 88 species: four varieties of Elatostema s.s., four species of Elatostematoides, nine of Pellionia, and five of Procris; these were defined as ingroup taxa and other six species from Urticaceae were used as outgroup taxa. Some herbarium samples did not yield DNA of sufficient quality or suffered from contamination of fungal DNA in PCR amplification (approximately 80% nrITS sequences tested). In these cases, PCR products were cloned using pGEM-easy Easy Vector System (Promega, Madison, WI, USA) and the ligation products were transformed into Escherichia coli DH5- or ECOS-competent cells (Yeastern Biotech, Taipei, Taiwan) following the manufacturer’s instructions. The plasmids with corrected sizes were extracted using a Mini-MTM Plasmid DNA Extraction system (Viogene, Taipei, Taiwan), and the inserted sequences were sequenced as mentioned. 2.3. Phylogenetic analysis 8 Raw sequences were edited and assembled using Sequencher 5.2.4 (Gene Codes Corp., Ann Arbor, MI, USA) with subsequent manual adjustments. The output DNA sequences were then aligned using MUSCLE v3.6 (Edgar, 2004) with a first round of multiple alignments and posterior rounds of refinement under default settings. The three datasets (nrITS, psbA-trnH, and psbM-trnD) were aligned independently. Alignments were adjusted manually in MacClade 4.06 (Maddison and Maddison, 2000). Phylogenies were reconstructed based on the nrITS dataset by itself, the combined plastid datasets (psbA-trnH and psbM-trnD), and all three datasets combined (nrITS, psbA-trnH, and psbM-trnD). All of these reconstructions were analyzed using maximum parsimony (MP), Bayesian inference (BI), and maximum likelihood (ML) methods to evaluate the phylogenetic congruence among markers. The two best tree topologies from ML analyses of cpDNA and nrITS were compared for topological incongruence using the compat.py script (Kauff and Lutzoni, 2002, 2003). A conflict in tree topologies of each tree was considered significant when incongruent topologies both received maximum likelihood bootstrap (MLBS) values of 80% or greater. MP analyses were performed using PAUP* v4.0b10 (Swofford, 2002), in which all characters were unordered and equally weighted, and gaps were treated as missing data. Heuristic searches of MP were conducted with 1,000 random addition replicates followed by tree bisection–reconnection branch swapping with the steepestdescent option. Branch supports were assessed using 1,000 bootstrap replicates (maximum parsimony bootstrap; MPBS) with the sample settings the same as those for heuristic searches. Best-fit DNA substitution models were selected using the Akaike Information Criterion (AIC) in Modeltest v 2.7 (Posada and Crandall, 1998) for each data partition. 9 The substitution model of the sequences was set to GTR+G+I for the combined dataset, determined by Modeltest. BI analyses were based on a Markov chain algorithm implemented in MRBAYES 3.2.6 (Huelsenbeck and Ronquist, 2001). Four chains of the Markov chain Monte Carlo (MCMC) simulation were performed for 5,000,000 generations each with trees sampled every 200 generations. Before the node probability was calculated (posterior probability), the first 6,250 sampled trees were discarded. These parameters were also visually inspected with an R version of AWTY (https://github.com/danlwarren/RWTY) (Nylander et al., 2008). Effective sample size values for all parameters were examined using Tracer v1.6 (Rambaut et al., 2014) to ensure that they are >200. To avoid being frozen on local optima, two independent runs were performed. For the combined dataset, sequences of nrITS and combined plastids were treated as two partitions. ML analyses with 1,000 bootstrap resampling (MLBS) were conducted using the online version of RAxML-HPC2 v8.2.9 (Stamatakis et al., 2008) available at the CIPRES Science Gateway version 3.3 (http://www.phylo.org/index.php/portal/) (Miller et al., 2010) with the gamma model of rate heterogeneity. For MP and ML analyses, 70%–79%, 80%–90%, and 90%–100% bootstrap supports were considered as weak, moderate, and strongly supported, respectively, and values lower than 70% were considered as providing insufficient support. For BI analyses, the posterior probabilities of <0.9, 0.9–0.94, 0.95–0.99, and 1.0 were considered as providing no, weak, moderate, and strong support, respectively. To examine the topological conflicts between MP, ML, and BI trees, both SH tests (Shimodaira and Hasegawa, 1999) and KH tests (Kishino and Hasegawa, 1989) were performed using PAUP* v4.0b10. 10 2.4. Ancestral character state reconstruction of morphological characters Ten morphological characters that represent the principal character states used by Schröter and Winkler (1935, 1936) and Wang (1980b) were selected for studying character evolution. Characters were scored based on examination of herbarium specimens, flora accounts, taxonomic revisions, and fieldwork (Fig. S3). To logically code the morphological characters and avoid confusing characters and character states, we followed the conventional coding described by Hawkin et al. (1997), which codes absence of a structure as an independent state (e.g., receptacle absent or present) and additional characters in the same structure as another independent state (e.g., receptacle shape). Following this rule, ten binary characters and their states were defined as follows: (1) habit: herb or shrub (including subshrub); (2) nanophyll: absent or present; (3) receptacle in pistillate inflorescence: absent or present; (4) receptacle shape in pistillate inflorescence: lobed to discoid, or subglobose; (5) involucre in pistillate inflorescence: absent or present; (6) staminate inflorescence architecture: unbranched or branched; (7) tepal in pistillate flower: absent or present; (8) the ratio of tepal length to ovary length in pistillate flower: <1/3 or ≥1; (9) receptacle in staminate inflorescence: absent or present; (10) the ratio of receptacle length to a staminate flower length (including pedicel, filament, and stamen): <1 or ≥1. Ancestral states of these characters were reconstructed using ML methods in Mesquite v.3.0.3 (Maddison and Maddison, 2015) and also applying a Bayesian method (stochastic character mapping) in the R package Phytools v. 6-44 (Revell, 2012). Any state change of character was assumed equally probable and unordered. There may be biological doubts about this assumption, but no sufficient information is currently available on any specific character change to suggest alternative hypotheses. 11 In the ML construction, we randomly sampled 1,000 trees from the post burnin set of the Bayesian analysis to conduct asymmetry likelihood ratio tests in Mesquite to select a model of character evolution for each character. A two-rate model was selected for Character 10 (receptacle/staminate flower ratio) (AsymmMk; P < 0.05 in 1,000 trees examined). An equal rate model (Mk1) was selected for all other characters, even though some references have shown the potential problems of using Mk models for ancestral state reconstructions. For example, equally rate model for test evolutionary hypotheses may not be appropriate when character gain or loss is directional. It may not only decrease the accuracy of reconstructions with misleading certainty, but also often be undetectable (e.g., Cunningham et al., 1998; Oakley et al., 2000). To account for phylogenetic uncertainty, we used “Trace character over trees.” All reconstructions were integrated over the 1,000 trees from the post burn-in set and summarized on the Bayesian consensus tree made from the combined data of all three datasets. The results were summarized as a percentage of changes of character states using the option of “Average frequencies across trees.” For the BI reconstruction, we chose the Phytools’ make.simmap function (Revell, 2012) to allow for missing character states. We used a subset of 100 trees from the post burn-in set; each tree included 80 stochastic character maps (charactermapping simulations), resulting in 8,000 simulations. The posterior distribution of character state histories was summarized using the describe.simmap function of Phytools. 12 3. Results 3.1. Phylogenetic reconstruction The characteristics and statistics of the datasets used in this study are summarized in Table 2. The comparison of trees for nrITS and cpDNA datasets revealed only one incongruence. In the nrITS tree, E. banahaense was sister to a clade of E. lutescens and E. calcareum (MLBS = 100) (Fig. S1); but in the cpDNA tree, it was sister to E. edule (MLBS = 82) (Fig. S2). Because no major incongruence was detected and the phylogeny of the combined dataset showed more resolved tree topologies with higher support values in most clades than either of the contributing datasets, we used the all combined datasets for subsequent analyses. MP, ML, and BI analyses of the combined dataset all recovered three identical major ingroup clades—Clades A, B, C—together with four largely congruent subclades within Clade C: C1–4 (Fig. 2). (Detailed descriptions are shown in the next section.) The SH and KH tests indicate that the MP (data not shown) and ML (Fig. 6) trees are consistent with each other (P > 0.05 in all pairwise tests) but have a significantly different topology from the BI tree (P < 0.05 in all pairwise tests). The conflicts between the BI tree and MP and ML trees are in Clade C4, and these conflicted branches are with weak or no supports. 3.2. Relationships within Elatostema s.l. Elatostema s.s. and allied taxa (Elatostematoides, Pellionia, and Procris) formed a strongly supported monophyletic clade in the combined analyses (MLBS/MPBS/PP: 94/96/1) (Fig. 2). The ingroups were divided into three strongly supported major clades (Clade A: 100/100/1, Clade B: 100/99/1, and Clade C: 100/100/1). There were five species in Clade A (denoted as Procris in Fig. 2), six 13 species in Clade B (denoted as Elatostematoides in Fig. 2), and 100 taxa in Clade C (Fig. 2) including 96 species and four varieties of Elatostema. Clade A/Procris were strongly supported, and Pellionia repens (Lour.) Merr. was recovered as sister to it. Clade B included E. variolaminosum var. latum and E. filicoides (Seems.) Schröt., both of which were treated under subgenus Pellionia by Schröter and Winkler (1935, 1936), together with one species assigned to Elatostematoides by Robinson (1910) (Eld. vittatum) and three to the subgenus Elatostematoides by Schröter and Winkler (1935, 1936) (E. lonchophyllum, E. fruticulosum and E. australe). Clade C comprised four strongly supported subclades (C1: 100/100/1; C2: 96/98/1; C3: 91/100/1; and C4: 100/100/1 in Fig. 2). Clade C1 corresponded to Weddellia sensu Schröter and Winkler (1935, 1936) (denoted as Weddellia), C2 to Pellionia (nine accessions, eight species, denoted as Pellionia), and C3 to African species of Elatostema s.s. (denoted as African Elatostema). Clade C4 corresponded to the majority of Elatostema s.s. taxa included in this study (93 accessions, 80 species, and two varieties) and was denoted as core Elatostema. 3.3. Morphological characters The pattern and distribution of the ten selected morphological characters across the Elatostema s.l. phylogeny are shown to the right of the phylogenetic tree in Fig. 2. The reconstructions of the ten characters are summarized in Figs. 3–5 and as supplementary data (Figs. S3–S22). The results were synthesized according to the six clades recovered for Elatostema s.l. (Clade A, B, C1, C2, C3, C4) and Pellionia repens (Figs. 3–5). 14 The ML construction by Mesquite and the BI construction by Phytools showed the similar results. Shrubby habitat was mostly found in the early diverged clades A and B and in 13 species in Clade C4. Although most of the species in C4 were herbaceous, our results indicated at least nine reversals to woodiness during the evolution of Elatostema species (Figs. 3A, S3 and S13). The frequent presence of nanophylls was commonly found in the early diverged lineages of Elatostema s.l. (i.e., Clades A, B, and C1 in Figs 3B and S4). Note that nanophylls were also present in the derived clade (i.e. E. monandrum (Buch.-Ham. ex D. Don) H. Hara in Clade C4, Figs. 2, S4 and S14), which suggests a reversal for this character state. Clades B and C2 had no receptacle in pistillate inflorescences, but this was found in other clades (Figs. 3C, S5, and S15). Species in Clade A had subglobose receptacles, whereas, with the exception of C2, the remainders of Clade C had lobed to discoid receptacles (Figs. 3D, S6, and S16). The presence of an involucre in pistillate inflorescences appeared to be a synapomorphy for Elatostema s.l. (Figs. 4A, S7, and S17), and a reversal to an absence of involucre was in the species of Clade C2. Clades B and C2 were characterized by branched inflorescence compared with most species in Elatostema s.l. in which the staminate inflorescences were unbranched. The branched staminate inflorescences were also found in some species of Clades A and C4 (i.e. E. oblongifolium Fu and E. huanjiangense W.T.Wang & Y.G.Wei, Figs. 4B, S8, and S18). Most species in the early diverged clades had obvious tepals in pistillate flowers, but this structure in Clades C1, C3, and C4 was either absent or much shorter than the ovary (Figs. 4C, S9, S19, and 4D, S10, S20). 15 Clades C1 and C3 had staminate inflorescence with receptacles; Clades B and C2, however, had staminate inflorescence without receptacle (Figs. 5A, S11, and S21). Because there were many missing data in Character 10 (receptacle/staminate flower ratio), we only focused on Clade C4 for this character in Fig. 5B (detailed showed in S12 and S22). Within Clade C4, significant variations in staminate inflorescence morphology were found. A summary of diagnosed morphological characters combined with clades is shown in Table 3. 16 4. Discussion Previous studies (Beaman, 2001; Hadiah and Conn, 2009; Hadiah et al., 2003, 2008; Wu et al., 2013, 2015) did not recover well-supported phylogenies for Elatostema s.l. that could underpin a robust classification, likely due to poor taxon and character sampling. The present study represents the first molecular phylogeny based on a broad representation of geographical, taxonomic, and morphological sampling of Elatostema s.l. Our results confirm the monophyly of Elatostema s.l. and recover strongly supported morphologically diagnosable clades, which we use to recognize Procris, Elatostematoides, and Elatostema sensu auct. at the generic rank. Key morphological characters based on our phylogenetic framework and their taxonomic implications are discussed in the following sections. 4.1. Morphological characters Consistent with previous studies (Beaman, 2001; Hadiah and Conn, 2009; Wu et al., 2015), our results confirmed several homoplasies for the morphological characters previously used to define infrageneric and intergeneric groupings in Elatostema s.l. For example, according to the evolutionary scenario proposed by Wang (2010), species with bracts that fused into pear-shaped receptacles in the staminate inflorescences belong to the section Androsyce, which is the most “advanced” section in Elatostema. Our phylogeny showed that the species with pearshaped receptacles (E. ficoides Wedd. and E. brachyodontum (Hand.-Mazz.) W.T. Wang) were not monophyletic (see Clade C4). Similarly, a plesiomorphy for early diverged lineages of Elatostema s.l., the presence of nanophylls (Clades Procris, Elatostematoides, Weddellia) was found in the core Elatostema clade (E. monandrum), suggesting a reversal of this character state during the course of 17 evolution. Homoplasy among the principle morphological characters used to classify Elatostema s.l. probably explains the century of disagreement over the classification of these taxa. Identifying morphological synapomorphy is obviously challenging where there is a high degree of homoplasy. However, our resolved phylogeny provided a framework with which to evaluate the character evolution in Elatostema s.l. and enabled us to recover the following putative plesiomorphies for Elatostema s.l.: (1) the presence of opposite nanophylls, (2) the absence of an involucre in the pistillate inflorescence, and (3) the presence of obvious tepals in the pistillate flowers. The frequent presence of nanophylls in the early diverged lineages of Elatostema s.l. (i.e., Clades Procris, Elatostematoides, and Weddellia) suggested that opposite leaves are also a plesiomorphy for Elatostema s.l. By contrast, caducous nanophyll provided a weak support for the origin of alternate leaves in Elatostema s.a., being the product of the anisophyllous reduction of an opposite-leaved ancestor as suggested by Schröter and Winkler (1935, 1936) and Beaman (Beaman, 2001). However, the presence of nanophylls in the core Elatostema Clade (E. monandrum) suggested that the presence or absence of nanophylls is a reversible character state; therefore, this alone is not a stable character for classification. We also found that the persistence of nanophylls is difficult to score consistently as caducous nanophylls are easily damaged during the collection and herbarium mounting process. It may, therefore, be that this character has been overlooked in previous reports. Two types of pistillate inflorescences in Elatostema s.l. were found through character reconstruction using tepal and involucre characters (characters 5, 7, and 8): (1) inflorescence lacking an involucre and composed of pistillate flowers bearing 18 usually conspicuous tepals (Clades Procris, Elatostematoides, and Pellionia); (2) inflorescence subtended by an involucre and composed of pistillate flowers lacking tepal or bearing highly reduced tepals (Clade Weddellia, African Elatostema, and core Elatostema). The perianth (tepals) usually serves to protect the immature reproductive organs or to attract pollinators (Culley et al., 2002). Elatostema s.l. is wind pollinated, so the role of tepals is likely to be protective rather than as an attractant. This is supported by the mutual exclusion of fully developed tepals and involucres in the inflorescence of Elatostema s.l. Homoplasy among the morphological characters recovered in this study makes it impossible to circumscribe genera using ordinary synapomorphies. Combined suites of characters, however, did support the morphological diagnosis of Elatostema and allied taxa (Table 3). Elatostema s.l. can be divided into five morphologically circumscribed groups sharing several morphological synapomorphies (Clades Procris, Elatostematoides, Weddellia, Pellionia, African Elatostema + core Elatostema) and consistent with our results of molecular phylogenetic relationships. Among these groupings, Clades C2 (Pellionia) and B (Elatostematoides) share similar character states, suggesting parallel character state reversals in these two clades. 4.2. Phylogenetic relationships within Elatostema s.l. Taking into account the distribution of morphological characters in our molecular phylogenies, we recognized three genera within Elatostema s.l.: Clade A as Procris, Clade B as Elatostematoides, and the remainder as Elatostema (sensu auct., Clade C). Based on molecular phylogeny, the following subgroupings were recognized in Elatostema s.a.: Weddellia (Clade C1), Pellionia (Clade C2), African Elatostema (Clade C3), and core Elatostema (Clade C4). However, clades African 19 Elatostema and core Elatostema were morphologically indistinguishable. Within the core Elatostema clade, there was a high degree of homoplasy among all ten of the morphological characters reviewed. Since no key morphological character can distinguish these clades, we maintained them as four clades within Elatostema s.a. rather than generating a formal taxonomic rank (e.g., subgenus) to accommodate them. Our classification is mostly compatible with that of Schröter and Winkler (1935, 1936) and Schröter (1938a, b), which also recognized Elatostematoides, Weddellia, Pellionia, and Procris as distinct taxa. The rank may differ, but it is associated with the same complement of species as those found in this study. 4.2.1. Generic delimitation within Elatostema s.l. The major differences between our classification of Elatostema s.l. and previous attempts (Table 1) are the inclusion of Pellionia within Elatostema s.a. and the recognition of Elatostematoides and Procris as distinct genera. Procris is characterized by the presence of distinct nanophylls and a subglobose fleshy pistillate receptacle (Weddell, 1856, 1869; Robinson, 1910; Schröter, 1938a, b; Friis, 1993). Procris has been treated as a distinct genus by many taxonomists, such as Weddell (1856, 1869), Robinson (1910), Schröter and Winkler (1935, 1936), Schröter (1938a, b), Friis (1993), Shih et al. (1995), and Chen et al. (2003), and it has been treated as a subgenus of Elatostema by Hallier (1896) and Winkler (1922). We found Procris to be a strongly supported monophyletic group sister to all of Elatostema s.l. (Elatostema s.a. + Elatostematoides) and the most early diverged lineage of Elatostema s.l., thus supporting Robinson’s (1910) view that Procris is the most basal genus based on the presence of tepals and the absence of an involucre in the pistillate inflorescence. 20 We recognized the genus Elatostematoides, first proposed by Robinson (1910) based on his interpretation of inflorescence and floral morphology. Schröter and Winkler (1935, 1936), however, considered it as a subgenus of Elatostema. Wang (1980b) recognized Elatostematoides at generic rank based on pistillate inflorescence, tepal, and achene morphology. We recovered a monophyletic Elatostematoides that could be diagnosed morphologically as follows: shrub, bearing nanophylls, with conspicuous tepals in pistillate flowers and branched staminate inflorescences. Elatostematoides can therefore be distinguished from Procris on the basis of having pistillate inflorescences lacking receptacles. 4.2.2. Infrageneric classification of Elatostema s.a. The four subgroupings of Elatostema s.a., Clades C1–4, were largely congruent with the classification of Schröter and Winkler (1935, 1936) (Fig. 6), with the exception of the recognition of Elatostematoides at generic rank in our study. It is, however, largely incongruent with the infrageneric classification of Wang (1980b, 2014). In particular, our results do not support Wang’s hypothesis (1980b, 2012, 2014) that Pellionioides is the most primitive group within Elatostema s.s. and that Androsyce is the most derived (Fig. 6). Although Wang’s (1980b, 2012, 2014) groupings are not suitable for an infrageneric classification, they are of great use for field identification. We recovered a monophyletic Weddellia clade, albeit based on a sample of only two species. This supports Schröter and Winkler’s (1935, 1936) circumscription of subgenus Weddellia. In their classification, the subgenus Weddellia was morphologically most similar to the subgenus Elatostema, from which it could be distinguished by the presence or absence of nanophylls and the merosity of the 21 staminate flowers. Within Weddellia, nanophylls are always present and the staminate flowers are five-parted; within Elatostema, nanophylls are absent and the staminate flowers are usually four- or rarely five-parted (Schröter and Winkler, 1935, 1936). In our sampling, species in the Weddellia clade also had short-petiolate nanophylls that were typically lacking in most species of the core Elatostema clade. However, 5parted staminate flowers were found both within Weddellia and the core Elatostema clade (e.g. E. albopilosoides Q. Lin & L.D. Duan, E. acuteserratum B.L. Shih & Yuen P. Yang, E. hirtellipedumculatum B.L. Shih & Yuen P. Yang, E. hypoglaucum B.L. Shih & Yuen P. Yang). Therefore, the merosity of the staminate flowers was not useful for delimitating subgroupings. The recognition of Pellionia at the generic rank has long been controversial. Our data strongly support the monophyly of Pellionia within Elatostema s.a., with three exceptions: E. variolaminosum var. latum, E. filicoides, and Pellionia repens (discussed later). Morphologically, Pellionia is congruent with Elatostema s.a. with its herbaceous habit and the absence of nanophyll. It differs from other Elatostema species in bearing pistillate flowers with prominent tepals and the absence of receptaculate staminate inflorescences, although these characters in Pellionia are similar to those in Elatostematoides. Wang (2016) treated Elatostematoides as a section under Pellionia. Based on our results, the key morphological difference between Pellionia and Elatostematoides was that Elatostematoides are shrubs with nanophylls, whereas Pellionia are mostly herbaceous and lack nanophylls, based on current sampling. However, although Pellionia includes five sections according to Wang (2016) (i.e., Pileoides, Elatostematoides, Pellionia, Leiolaena, and Elatostematopsis), we only sampled two (Pellionia and Elatostematoides). More samplings from other sections are still required to evaluate our classification and the 22 relationship within Pellionia. We recovered two species assigned to Pellionia, namely Elatostema variolaminosum var. latum and Elatostema filicoides, which were nested within Elatostematoides. These taxa have been treated as subgenera of Pellionia by Schröter and Winkler (1935, 1936) and Smith (1981). After examining specimens and reviewing their original descriptions, we found that both species share the same morphological character states that we used to delimit Elatostematoides: shrubs with opposite nanophylls (the latter uncertain in E. filicoides). This observation is congruent with our interpretation of the morphological characters used to distinguish Elatostematoides and Pellionia. Notably, nanophylls have also been recorded for some other Pellionia species (e.g., E. filicinum Ridl. and E. sinuatum Hassk.) not sampled in this study. Therefore, including them in future studies should further confirm or refute our application of these characters to circumscribe these two genera. Our study recovered a clade, African Elatostema, which contains all but one Elatostema of the species sampled from Africa and Madagascar. This clade shares morphological character states with the species in the core Elatostema clade, and we could not find any diagnosable characters or suites of characters to separate these two taxa. In all African Elatostema sampling, only one species, E. welwitschii, was not included in the African Elatostema clade, but in the core Elatostema clade. Elatostema welwitschii is commonly found in tropical Africa from Uganda and Tanzania to Sao Tome and Nigeria (Friis, 1989), and we speculate that it may be the product of a unique colonization event from Asia to Africa. The core Elatostema clade comprises the majority of Elatostema s.a. species (84%, 82/98 species in this study). The phylogeny of this clade suggests five bursts of diversification in Asia and Oceania with the greatest accumulation of species in China 23 and Southeast Asia. This is in contrast to the sister genera, Procris and Elatostematoides, which has relatively few species (20 in Procris and 20–40 in Elatostematoides). All three genera are associated with similar habitats, so it is unclear how the differential diversification happened; thus, the drivers of speciation require further investigation. 4.3. The position of Pellionia repens Pellionia repens is a perennial herb found in Southeast Asia and South China (Chen et al., 2003). Its position is problematic because all previous molecular (Hadiah et al., 2003, 2008; Wu et al., 2013) and morphological phylogenies (Wu et al., 2015) have recovered it as more closely related to Procris than Elatostema. Conn and Hadiah (2011) proposed a new combination for the name, Procris repens (Lour.) B.J. Conn & Hadiah, based on the conclusion of Hadiah et al. (2008) that Pellionia is a synonym of Procris. Our morphological analysis, however, did not support this. Consistent with Wu et al. (2013, 2015), we found that Pellionia repens is a sister to Procris. Morphologically, Pellionia repens should belong to Pellionia sensu (Schröter and Winkler, 1935, 1936; Chen et al., 2003). On the basis that the unreduced embryo sac is derived from somatic cells, Fagerlind (1944) reported that this species (synonym: E. repens (Lour.) Hallier f. var. viride (N.E. Br.) H.Schröet.) is a tetraploid (2n = 52) with an apomictic hybrid origin. The hybrid nature of this species could explain the conflict between the molecular and morphological characters and highlights the need for further research to better understand the origins of this species before taking nomenclatural action. Although hybridization is rarely found in Urticaceae species, we recently documented an example, E. ×hybrida Y. H. Tseng & J. M. Hu, using morphological, phylogenetic, and cytological observations (Tseng and 24 Hu 2014). We suggest that such an approach should be applied to a comprehensive sample of Pellionia repens and allied species to clarify the hybrid history. 5. Conclusion Our robust phylogeny based on nrITS and plastid DNA sequence data combined with morphological characters greatly improved our understanding of the evolution of Elatostema s.l. and supported a stable classification for a group of plants whose circumscription has remained controversial for over a century. Our phylogeny of Elatostema s.l. provides a framework for further taxonomic, evolutionary, and biogeographical research. Nevertheless, further taxon and character sampling are required to improve resolution and support values within Elatostema s.a., especially with respect to species in the African Elatostema clade. Synopsis of genera The following generic synopsis of Elatostema s.l. is based on the present results. Elatostema J.R. Forster & J.G.A. Forster, Charact. Gen. [105]. 1 Mar 1776. Type: E. sessile J.R. & J.G.A. Forster. Syn.: Pellionia Gaudichaud-Beaupré. Note: This genus comprises approximately 570 species, including four major clades: core Elatostema, Pellionia, Weddellia, and African Elatostema. Elatostema is mainly distributed in tropical and subtropical Asia, Australasia, and Africa. Procris Commerson ex A.L. Jussieu, Gen. 403. 4 Aug 1789. Type: P. axillaris J.F. Gmelin (Syst. Nat. 2: 267. Sep (sero)-Nov 1791). 25 Note: This genus comprises approximately 20 species, which are distributed throughout the Old World tropics. Elatostematoides Robinson, Philipp. J. Sci., C 5: 497. 20 Jan 1911. Type: E. manillense (Weddell) Robinson. Note: The number of species in this genus is approximately 20–40. Elatostematoides is restricted to Southeast Asia and Pacific Islands. Taxonomic Treatment Elatostematoides australe (Wedd.) Y. H. Tseng, A. K. Monro, Y. G. Wei, & J. M. Hu, comb. nov. Pellionia australis Wedd. DC. Prodr. 16(1): 169. 1869. Elatostema australe (Wedd.) Hallier f. Ann. Jard. Bot. Buitenzorg 13: 316 1896. TYPE: Fiji. Ovalaho. 1851, Vieillard, E. 50 (Holotype: P photo!). Elatostematoides filicoides (Seem.) Y. H. Tseng, A. K. Monro, Y. G. Wei, & J. M. Hu, comb. nov. Pellionia filicoides Seem. Fl. Vit. 239. 1868. Elatostema filicoides var. eufilicoides (Seems.) Schröter. Repert. Spec. Nov. Regni Veg. Beih. 83(2): 69. 1936. TYPE: Fiji. 1980. Seemann, B. 421 (Holotype: K!). Elatostematoides fruticulosum (K. Schumann) Y. H. Tseng, A. K. Monro, Y. G. Wei, & J. M. Hu, comb. nov. Elatostema fruticulosum K. Schumann. Fl. Schutzgeb. Südsee 254. 1905. TYPE: Papua New Guinea. Morobe Privience, Satelberg. July 24 1890, Lauterbach, C.A.G. 532. 26 Elatostematoides lonchophyllum (H. Schröter) Y. H. Tseng, A. K. Monro, Y. G. Wei, & J. M. Hu, comb. nov. Elatostema variolaminosum var. latum H. Schröter, Repert. Spec. Nov. Regni Veg. Beih. 83(2): 147. 1936. TYPE: Malaysia. Borneo, Sarawak, Kuching. Aug. 29 1929, Clemens, J. & Clemens, M.S., 20816 (Holotype: K!). Elatostematoides variolaminosum var. latum (H. Schröter) Y.H. Tseng, A. K. Monro, Y. G. Wei, & J. M. Hu, comb. nov. Elatostema variolaminosum var. latum H. Schröter, Repert. Spec. Nov. Regni Veg. Beih. 83(2): 68. 1936. TYPE: Malaysia. Borneo, Mount Kinabalu. Jan. 04 1933, Clemens, J. & Clemens, M.S. 30722 (Isolectotype: BM!) Acknowledgments We gratefully thank A, BO, BM, K, KYO, IBK, MO, PE, TAI, TAIF, and HAST herbaria for providing plant materials. This work was supported by the Natural Science Foundation of China (grant number 31160039) and Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain. We are grateful to David Taylor for suggestions and English editing. This manuscript was edited by Wallace Academic Editing. 27 References Beaman, R.S., 2001. Phylogeny and biogeography of Elatostema (Urticaceae) from Mount Kinabalu. Sabah Parks Nat. J. 4, 71-93. Chen, J., Lin, Q., Friis, I., Wilmot-Dear, C.M., Monro, A.K., 2003. Urticaceae. In: Wu, Z.Y., Raven, P.H. (Eds.), Flora of China. 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Ancestral state reconstruction reveals rampant homoplasy of diagnostic morphological characters in Urticaceae, conflicting with current classification schemes. PloS One 10, e0141821. http:/doi.org/10.1371/journal.pone.0141821. Wu, Z.Y., Monro, A.K., Milne, R.I., Wang, H., Yi, T.S., Liu, J., Li, D.Z., 2013. Molecular phylogeny of the nettle family (Urticaceae) inferred from multiple loci of three genomes and extensive generic sampling. Mol. Phylogenet. Evol. 69, 814-827. http:/doi.org/10.1016/j.ympev.2013.06.022. Yang, Y.P., Shih, B.L., Liu, H.Y., 1995. A revision of Elatostema (Urticaceae) of Taiwan. Bot. Bull. Acad. Sinica 36, 259-279. 34 Figure legends Fig. 1. Morphological diversity of Elatostema s.l. A, E. yaoshanense (pistillate inflorescence with involucre); B, E. lineolatum var. majus (staminate inflorescence and flower); C, E. parvum (with opposite nanophyll); D, Pellionia scabra (tepals longer than ovary); E. Pellionia radicans (branched staminate inflorescence); F, Procris frutescens (staminate inflorescence and opposite nanophyll); G, Procris crenata (staminate inflorescence); H, E. cyrtandrifolium (staminate flowers on a receptacle); I, E. cyrtandrifolium (pistillate flowers on receptacle). a: anther; i: involucre; n: nanophyll; o: ovary; r: receptacle; t: tepals. D, E, and G were photographed by Li-Hsien Yang; F by Jer-Ming Hu; and others by Yu-Hsin Tseng. Fig. 2. Phylogenetic tree of Elatostema s.l. generated from Bayesian analysis of combined nrITS and plastid psbA-trnH and psbM-trnD sequence data. Numbers on the branches indicate bootstrap values (≥60%) of the maximum likelihood and the maximum parsimony analyses and the posterior probability (≥0.8) of Bayesian inference analysis. Morphological characters are indicated for the ten selected traits: (1) Habit; (2) nanophyll; (3) receptacle in pistillate inflorescence; (4) receptacle shape in pistillate inflorescence; (5) involucre in pistillate inflorescence; (6) staminate inflorescence architecture; (7) tepal in pistillate flower; (8) the ratio of tepal length to ovary length in pistillate flower; (9) receptacle in staminate inflorescence; (10) the ratio of receptacle length to a staminate flower length (including pedicel, filament, and stamen). Polymorphic characters are coded by more than one color in the bracket. Fig. 3. Ancestral state reconstruction for Elatostema s.l. performed using Mesquite using likelihood methods (larger circle on the tree) and Phytools using Bayesian 35 method (stochastic character mapping) (smaller circle). The backbone of the tree is based on the BI consensus tree from the combined molecular analysis. A, habit; B, nanophyll; C, receptacle of pistillate inflorescence; D, receptacle shape in pistillate inflorescence. The illustrations are representative types of the major groups of species within Elatostema s.l. r: receptacle. Fig. 4. Ancestral state reconstruction for Elatostema s.l. performed using Mesquite (larger circle on the tree) and Phytools (smaller circle). A, involucre in pistillate inflorescence; B, staminate inflorescence architecture; C, tepal in pistillate flower; D, the ratio of tepal length to ovary length in pistillate flower. The illustrations are representative types of the major groups of species within Elatostema s.l. i: involucre; t: tepal; o: ovary. Fig. 5. Ancestral state reconstruction for Elatostema s.l. performed using Mesquite (larger circle on the tree) and Phytools (smaller circle). A, receptacle in staminate inflorescence; B, the ratio of receptacle length to a staminate flower length (including pedicel, filament, and stamen). Only showed the Mesquite result in Clade C4. The illustrations are representative types of the major groups of species within Elatostema s.l. r: receptacle. Fig. 6. Comparison of the current infrageneric classification within Elatostema of Schröter and Winkler (1935, 1936) and Wang (1980b) with the phylogenetic tree of Elatostema generated from maximum likelihood analysis of combined nrITS, plastid psbA-trnH, and psbM-trnD sequence data. Color of branches indicates the genus and section assignation of species in each infrageneric classification. The black dashed 36 line indicates that the assigned section of a species or the relationship between taxa remains uncertain. The inset is the same tree depicted as a phylogram to show the branch lengths. Figure S1. Phylogenetic tree of Elatostema s.l. generated from Bayesian analysis of nrITS. Numbers on the branches indicate the bootstrap values (≥60%) of maximum likelihood and maximum parsimony and the posterior probability (≥0.7) of Bayesian inference analyses. Figure S2. Phylogenetic tree of Elatostema s.l. generated from Bayesian analysis of combined plastid sequences (psbA-trnH and psbM-trnD). Numbers on the branches indicate the bootstrap values (≥60%) of maximum likelihood and maximum parsimony and the posterior probability (≥0.7) of Bayesian inference analyses, respectively. Figure S3. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of habit. Morphological characters that were scored based on field observations (F), examination of herbarium specimens (H), or data in the literature (L) were marked following each species name. Polymorphic characters are coded by more than one color in the bracket. Figure S4. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the nanophyll by Mesquite. Figure S5. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the receptacle in pistillate inflorescence by Mesquite. Figure S6. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the receptacle shape in pistillate inflorescence by Mesquite. Figure S7. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the involucre in pistillate inflorescence by Mesquite. 37 Figure S8. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the staminate inflorescence architecture by Mesquite. Figure S9. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the tepal in pistillate flower by Mesquite. Figure S10. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the ratio of tepal length to ovary length in pistillate flower by Mesquite. Figure S11. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the receptacle in staminate inflorescence by Mesquite. Figure S12. Ancestral state reconstruction for Elatostema s.l. based on maximum likelihood optimization of the ratio of receptacle length to a staminate flower length (including pedicel, filament, and stamen). Figure S13. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of habit by Phytools. Figure S14. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the nanophyll by Phytools. Figure S15. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the receptacle in pistillate inflorescence by Phytools. Figure S16. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the receptacle shape in pistillate inflorescence by Phytools. Figure S17. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the involucre in pistillate inflorescence by Phytools. Figure S18. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the staminate inflorescence architecture by Phytools. 38 Figure S19. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the tepal in pistillate flower by Phytools. Figure S20. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the ratio of tepal length to ovary length in pistillate flower by Phytools. Figure S21. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the receptacle in staminate inflorescence by Phytools. Figure S22. Ancestral state reconstruction for Elatostema s.l. based on Bayesian inference of the ratio of receptacle length to a staminate flower length (including pedicel, filament, and stamen) by Phytools. 39 1 2 3 E. albopilosoides 1. Habit E. binatum Herb E. tenuinerve -/-/.87 E. oblongifolium Shrub -/-/.74 E. oblongifolium 63/77/.98 E. hechiense 2. Nanophyl E. huanjiangense Absent E. gyrocephalum Present 100/100/1 E. gyrocephalum var. pubicaule E. androstachyum 3. Receptacle in pistillate inflorescence E. sinopurpureum 100/100/1 E. microcephalanthum Absent E. microcephalanthum Present E. ficoides 97/-/E. xanthophyllum 4. Receptacle shape in pistillate inflorescence E. hypoglaucum Lobed or discoid E. yakushimense 99/98/1 67/-/.92 100/100/1 E. laetevirens subglobose E. lungzhouense E. brachyodontum 5. Involucre in pistillate inflorescence 100/100/1 E. brachyodontum Absent E. malacotrichum Present E. myrtillus 87/68/1 E. oligophlebium 6. Staminate inflorescence architecture E. asterocephalum Unbranched E. tianeense E. hezhouense Branched E. sublineare -/-/.9 E. yachense -/-/.97 7. Tepal in pistillate flower 100/100/1 E. multicaule Absent E. lithoneurum 100/100/1 Present Elatostema sp. 4 Elatostema sp. 1 Elatostema sp. 3 97/90/1 8. Tepal length / ovary length in pistillate flower 100/99/1 100/100/1 Elatostem sp.2 < 1/3 E. morobense ≥1 E. grande 100/100/1 E. grande 96/86/1 9. Receptacle in staminate inflorescence E. grandifolium 100/100/1 E. kraemeri Absent 97/99/1 E. strictum Present E. samoense 94/89/1 E. banahaense 10. Receptacle length / staminate flower length 100/100/1 E. lutescens 100/100/1 in staminate inflorescence 100/100/1 E. calcareum 97/99/1 100/100/1 E. edule <1 E. edule ≥1 100/100/1 100/100/1 E. villosum E. strigillosum 100/100/1 E. strigillosum Character uncertain/inapplicable 100/100/1 Elatostema sp. 5 E. acuteserratum E. hirtellipedunculatum 100/100/1 E. suzukii 98/87/1 100/89/1 100/100/1 E. suzukii E. involucratum E. japonicum 100/100/1 E. subcoriaceum E. serra 64/-/.84 71/78/.97 E. thalictroides 68/-/.99 E. insulare 99/72/1 E. rugosum E. reticulatum 98/75/1 E. lineare -/-/.75 E. pinnatum 99/98/1 E. welwitschii E. welwitschii E. penibukanense E. lineolatum var. majus 93/-/1 99/62/.9 100/100/1 E. cyrtandrifolium E. cyrtandrifolium E. balansae 98/97/1 E. fengshanense 100/95/1 E. garrettii E. macintyrei Elatostema sp. 6 96/90/1 E. integrifolium E. integrifolium 100/100/1 E. longistipulum 100/100/1 C4 E. platyphyllum 94/86/1 100/100/1 E. platyphyllum E. laevissimum 100/100/1 E. tenuicaudatum E. dissectum 70/62/.98 E. grandidentatum E. hookerianum -/68/.91 100/98/1 E. nasutum 79/76/1 99/100/1 E. yaoshanense 89/98/1 E. ellipticum 100/99/1 E. monandrum 100/100/1 E. pusillum 100/100/1 E. obtusum var. trilobulatum 0.06 E. obtusum Core Elatostema 4 5 6 7 8 9 10 100/100/1 C4 Core Elatostema 96/95/1 1 2 3 4 5 6 7 8 9 10 E. goudotianum E. incisum E. madagascariense /1 98/98/1 E. paivaeanum 91/100/1 E. orientale C3 E. monticola 82/87/.85 5 E. paivaeanum -/62/.98 100/100/1 Pellionia viridis C 2 -/-/.92 Pellionia grijsii -/86/.91 Pellionia scabra Pellionia acutidentata -/-/.91 .91 Pellionia minima C2 Pellionia retrohispida Pellionia heteroloba 96/98/1 Pellionia radicans 79/-/.99 -/-/.8 /-/.8 Pellionia radicans E. sinense var. xinningense 7/.92 92/97/.92 E. sinense var. longicornutum 100/100/1 C1 E. sinense E. parvum E. variolaminosum var. latum 73/64/.99 100/100/1 Eld. vittatum E. lonchophyllum p y 100/99/1 B E. fruticulosum 100/100/1 E. filicoides 100/100/1 E. australe E 100/100/1 Procris crenata sp. Procris 72/-/.98 100/100/1 Procris crenata 100/100/1 Procris frutescens A 100/98/1 Procris archboldiana 99/97/1 Procris montana Pellionia repens Lecanthus peduncularis Poikilospermum acuminata Nanocnide japonica Boehmeria macrophylla Debregeasia orientalis Leucosyke quadrinervia 0.06 1 99/97/1 AfricanElatostema Pellionia Weddellia 94/96/1 94/-/.98 Elatostematoides Procris -/-/1 94/-/1 100/97/1 (A) Habit (B) Nanophyll Core Elatostema (C4) African Elatostema (C3) Pellionia (C2) Weddellia (C1) Elatostematoides (B) Herb Shrub Absent Present Procris (A) Pelllionia repens (C) Receptacle in pistillate inflorescence Core Elatostema (C4) (D) Receptacle shape in pistillate inflorescence African Elatostema (C3) Pellionia (C2) Weddellia (C1) Lobed to discoid Absent Elatostematoides (B) Subglobose Procris (A) Present Ambiguous r r Pelllionia repens Character uncertain/inapplicable (A) Involucre in pistillate inflorescence Core Elatostema (C4) (B) Staminate inflorescence architecture African Elatostema (C3) Pellionia (C2) Weddellia (C1) Unbranched Absent Elatostematoides (B) Present Procris (A) Branched Pelllionia repens i (C) Tepal in pistillate flower Core Elatostema (C4) (D) Tepal / ovary length in pistillate flower African Elatostema (C3) Pellionia (C2) Weddellia (C1) Absent Elatostematoides (B) < 1/3 o Present t Procris (A) t Pelllionia repens ≥1 Ambiguous (A) Receptacle in staminate inflorescence (B) Receptacle / staminate flower length in staminate inflorescence E. lungzhouense E. sinopurpureum E. microcephalanthum E. microcephalanthum E. ficoides E. xanthophyllum E. hypoglaucum E. yakushimense E. laetevirens E. androstachyum E. huanjiangense E. gyrocephalum E. gyrocephalum var. pubicaule E. malacotrichum E. myrtillus E. oligophlebium E. asterocephalum E. tianeense E. hezhouense E. sublineare E. yachense E. multicaule E. hechiense E. brachyodontum E. brachyodontum E. binatum E. tenuinerve E. oblongifolium E. oblongifolium E. albopilosoides E. lithoneurum Elatostema sp. 4 Elatostema sp. 1 Elatostema sp. 3 Elatostem sp.2 E. morobense E. grande E. grande E. grandifolium E. kraemeri E. strictum E. samoense E. banahaense E. lutescens E. calcareum E. edule E. edule E. villosum E. strigillosum E. strigillosum Elatostema sp. 5 E. acuteserratum E. hirtellipedunculatum E. suzukii E. suzukii E. involucratum E. japonicum E. subcoriaceum E. reticulatum E. serra E. thalictroides E. insulare E. rugosum E. lineare E. pinnatum E. welwitschii E. welwitschii E. penibukanense E. lineolatum var. majus E. cyrtandrifolium E. cyrtandrifolium E. balansae E. fengshanense E. garrettii E. macintyrei Elatostema sp. 6 E. integrifolium E. integrifolium E. longistipulum E. platyphyllum E. platyphyllum E. laevissimum E. tenuicaudatum E. dissectum E. grandidentatum E. hookerianum E. nasutum E. yaoshanense E. ellipticum E. monandrum E. pusillum E. obtusum var. trilobulatum E. obtusum Core Elatostema (C4) African Elatostema (C3) Pellionia (C2) Weddellia (C1) Elatostematoides (B) Procris (A) Pelllionia repens Present r <1 ≥1 Ambiguous Character uncertain Schröter and Winkler (1935, 1936) subg. Elatostema subg. Elatostematoides subg. Pellionia subg. Weddellia Wang (1980) E. binatum E. tenuinerve E. oblongifolium E. oblongifolium E. huanjiangense E. albopilosoides E. hechiense E. androstachyum E. ficoides E. microcephalanthum E. microcephalanthum E. sinopurpureum E. gyrocephalum E. gyrocephalum var. pubicaule E. xanthophyllum E. hypoglaucum E. yakushimense E. laetevirens E. multicaule E. yachense E. hezhouense E. sublineare E. malacotrichum E. asterocephalum E. oligophlebium E. tianeense E. myrtillus E. lungzhouense E. brachyodontum E. brachyodontum E. lithoneurum Elatostema sp. 4 Elatostema sp. 1 Elatostema sp. 3 Elatostema sp. 2 E. grande E. grande E. morobense E. samoense E. grandifolium E. kraemeri E. strictum E. edule E. edule E. banahaense E. calcareum E. lutescens E. strigillosum E. strigillosum E. villosum E. acuteserratum Elatostema sp. 5 E. involucratum E. hirtellipedunculatum E. suzukii E. suzukii E. japonicum E. subcoriaceum E. rugosum E. thalictroides E. insulare E. serra E. reticulatum E. lineare E. pinnatum E. garrettii E. fengshanense E. balansae E. cyrtandrifolium E. cyrtandrifolium E. macintyrei E. welwitschii E. welwitschii E. lineolatum var. majus E. penibukanense Elatostema sp. 6 E. integrifolium E. integrifolium E. platyphyllum E. platyphyllum E. longistipulum E. tenuicaudatum E. laevissimum E. pusillum E. monandrum E. ellipticum E. nasutum E. yaoshanense E. hookerianum E. grandidentatum E. dissectum E. obtusum var. trilobulatum E. obtusum E. monticola E. madagascariense E. incisum E. paivaeanum E. goudotianum E. orientale E. paivaeanum Pellionia minima Pellionia scabra Pellionia acutidentata Pellionia viridis Pellionia grijsii Pellionia retrohispida Pellionia radicans Pellionia radicans Pellionia heteroloba E. sinense var. longicornutum E. sinense var. xinningense E. sinense E. parvum E. fruticulosum E. filicoides E. australe E. lonchophyllum Eld. vittatum E. variolaminosum var. latum Procris archboldiana Procris montana Procris frutescens Procris crenata Procris sp. Procris crenata Pellionia repens sect. Androsyce sect. Elatostema sect. Laevisperma sect. Pellionioides sect. Weddellia Table 1 History of generic classification of Elatostema s.l., species diversity and sampling for this study. Taxon Generic classification based on: Weddell Hallier Robinson (1910) Distribution Winkler Schröter and Wang (1980a, b) Shih et al. Hadiah et al. Wu et al. No. of No. of species in This study species (1869) (1896) (1922) Winkler (1935, (1995) (2008) our sampling (2013) in taxon 1936) Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Elatostema Warm parts 500 of old world Elatostematoides - - Elatostematoides - Elatostema Elatostematoides - - - Elatostematoides 88 species, 4 varieties Southeast 20– Asia and 40 4 Pacifica Islands Pellionia Pellionia Elatostema Pellionia Elatostema Elatostema Pellionia Pellionia Procris Pellionia Elatostema Tropical and 60 9 subtropical 40 Asia and Pacific Islands Procris Procris Elatostema Procris Elatostema Procris Procris Procris Procris Pellionia Procris Warm and 20 5 tropical parts of old world Table 2 Statistics for the molecular datasets used in this study. Number of sequences Aligned length (bp) Length variation (bp) Variable characters Parsimony- Model selected (ingroup/outgroup) informative (AIC) (bp) characters (bp) ITS 124/6 1249 640–964 703 (56.3%) 587 (47.0%) GTR+I+G psbA-trnH 126/5 610 234–347 295 (48.4%) 175 (28.7%) TVM+G psbM-trnD 125/4 802 368–579 325 (40.5%) 141 (17.6%) TVM+G 41 Combined 126/6 1412 324–860 620 (43.9%) 316 (25.6%) TVM+I+G 126/6 2661 760–1832 1326 (49.8%) 903 (33.9%) GTR+I+G plastid Combined Table 3 Diagnosed morphological characters combined with clades in this study Clade Habit Procris (Clade A) herb/sh Nanophyll present Receptacle in Involucre in Tepal length in Staminate Receptacle in pistillate pistillate pistillate flower inflorescence staminate inflorescence inflorescence subglobose absent rub inflorescence much shorter/as unbranched/br long as or longer anched absent/fused than ovary Elatostematoides shrub present absent absent as long as or longer branched absent 42 (Clade B) Weddellia (Clade than ovary herb present lobed to discoid present C1) absent/much unbranched fused branched absent unbranched fused absent/much unbranched, fused shorter than ovary rarely shorter than ovary Pellionia (Clade herb, C2) rarely absent absent absent as long as or longer than ovary shrub African- herb absent lobed to discoid present much shorter than ovary Elatostema (Clade C3) Core Elatostema herb/sh absent, (Clade C4) rub rarely present lobed to discoid present branched 43 44 Highlights  We represent the most robust molecular phylogeny for Elatostema to date.  Elatostema s.a., Elatostematoides and Procris are distinct genera in Elatostema s.l.  Four strongly supported clades are within this newly defined Elatostema s.a. 45