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Botanical Journal of the Linnean Society, 2013, 173, 535–548. With 3 figures

Phylogenetics, character evolution, and distribution patterns of the greenbriers, Smilacaceae (Liliales), a near-cosmopolitan family of monocots ZHECHEN QI1,2,†, KENNETH M. CAMERON3*, PAN LI4, YUNPENG ZHAO1,2, SHICHAO CHEN5, GUANGCUN CHEN1,2 and CHENGXIN FU1,2* 1

The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education, College of Life Sciences, Zhejiang University, Hangzhou 310058, China 2 Laboratory of Systematic and Evolutionary Botany, Institute of Plant Sciences and Conservation Center for Gene Resources of Endangered Wildlife, Zhejiang University, Hangzhou 310058, China 3 Wisconsin State Herbarium, Department of Botany, University of Wisconsin, Madison, WI 53706, USA 4 Research and Development Centre, Firmenich Aromatics (China), Shanghai 201108, China 5 College of Life Sciences and Technology, Tongji University, Shanghai 200092, China Received 28 February 2013; revised 18 May 2013; accepted for publication 2 August 2013

Smilacaceae, composed of Smilax and Heterosmilax, are a cosmopolitan family of > 200 species of mostly climbing monocots with alternate leaves characterized by reticulate venation, a pair of petiolar tendrils and usually prickly stems. Although there has been a long history of studying Smilax since Linnaeus named the genus in 1753, the phylogenetic history of this dioecious family remains unclear. Here we present results based on nuclear ribosomal internal transcribed spacer (nrITS) and plastid matK and rpl16 intron DNA sequence data from 125 taxa of Smilacaceae. Our taxon sampling covers all sections of Smilax and Heterosmilax and major distribution zones of the family; species from Ripogonaceae and Philesiaceae are used as outgroups. Our molecular analysis indicates that phylogenetic relationships largely contradict the traditional morphological classification of the family, instead showing a conspicuous geographical pattern among the species clades. The previously recognized genus Heterosmilax was found to be embedded in Smilax. Species in the family are separated into primarily New World and Old World clades, except for a single species lineage, Smilax aspera, that is sister to the remaining species of the family, but with poor statistical support. Ancestral character state reconstructions and examination of distribution patterns among the clades provide important information for future taxonomic revisions and historical biogeography of the group. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548.

ADDITIONAL KEYWORDS: Heterosmilax – molecular phylogeny – Philesiaceae – Ripogonaceae – Smilax.

INTRODUCTION Smilacaceae are a monocot family of lianas, shrubs and herbs widely distributed across mostly tropical and subtropical, but also temperate, regions of the

*Corresponding author. E-mail: [email protected]; [email protected] †Present address: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China.

world in the Southern and Northern Hemispheres (see Fig. 1). Typically, they are characterized by tuberous or stoloniferous rhizomes, leaves alternate with reticulate venation, paired petiolar tendrils, stems often armed with prickles, unisexual flowers with six tepals and either six fertile stamens or staminodes in the case of pistillate flowers, umbellate infloresences, fruits fleshy berries and a woody, climbing or erect habit. The family was proposed first by Ventenat in 1799. Early taxonomic studies placed them in various

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Figure 1. Geographic distribution of Smilacaceae illustrated by colours corresponding to the four primary lineages (A, B, C, D) recognized in the molecular phylogenetic reconstruction presented in Figure 2. Red-lined region: clade A, represents tri-continental disjunction of Smilax aspera; Brown-lined regions: clade B, most species of this clade are distributed in the New World, with two notable exceptions; Green-lined regions: clade C, prickleless herbs and semi-herb species of Smilax mostly from the Old World, with one disjunction in North America. Blue-lined regions: clade D, species from this clade occur only in the Old World tropics and subtropics. The dash-lined areas indicate three perceived distribution realms of Smilacaceae, as discussed.

positions: in Liliales sensu lato (s.l.) (Hutchinson, 1979; Thome, 1983; Goldberg, 1989; Cronquist, 1991); in Dioscoreales (Dahlgren & Clifford, 1982; Thorne, 1992); or in Asparagales (Huber, 1969). In the narrowest sense, Takhtajan (1987) treated Smilacaceae as an element of Smilacales, which included three additional families: Philesiaceae (Philesia Comm. ex Juss., Lapageria Ruiz & Pav.), Ripogonaceae (Ripogonum J.R.Forst. & G.Forst) and Luzuriagaceae (Luzuriaga Ruiz & Pav.). As the publication of molecular systematic studies focused on monocot phylogeny during the past two decades, Smilacaceae have been confirmed to be a member of a monophyletic Liliales, closely related to Philesiaceae, Ripogonaceae and Liliaceae sensu stricto (s.s.) (Chase et al., 1995; Patterson & Givnish, 2002; Fay et al., 2006; Kim et al., 2013; Petersen, Seberg & Davis, 2013). However, the exact position of Smilacaceae in Liliales has been somewhat controversial. In some studies (Vinnersten & Bremer, 2001; Chen & Hong, 2007; Kim et al., 2013), they form a clade with Philesiaceae + Ripogonaceae, whereas others (Rudall et al., 2000; Patterson & Givnish, 2002; Fay et al., 2006) found the family positioned as sister

to Liliaceae. Interfamilial relationships in Liliales are, however, beyond the scope of this paper, but considering that Ripogonaceae and Philesiaceae are Southern Hemisphere plants with a mostly climbing and ‘woody’ habit, whereas Liliaceae are non-climbing, herbaceous, Northern Hemisphere plants, we suggest that Smilacaceae, with its near-cosmopolitan distribution, both climbing and erect stems and both woody and herbaceous habits, may be viewed as a pivotal family with the potential to shed light on the evolutionary history and northward migration of Liliales from southern ancestors (Vinnersten & Bremer, 2001). For this reason, it is important for systematists to understand better the phylogenetics and distribution patterns of Smilacaceae. The only published phylogenetic study of Smilacaceae, by Cameron & Fu (2006), was based exclusively on data from the nuclear ribosomal internal transcribed spacer (nrITS) region (including partial 18S, ITS1, 5.8S, ITS2, partial 26S) and showed relatively low levels of resolution; the majority of deeper nodes in the phylogenetic reconstruction received little or no statistical support. This incomplete evolutionary picture served as the impetus

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548

PHYLOGENETICS OF SMILACACEAE for us here to resolve more clearly the phylogenetic relationships with this family. Although > 350 species have been described in the family, recent floristic and monographic studies of parts of Smilacaceae (Andreata, 1980, 1995; Chen & Koyama, 2000; Cameron & Fu, 2006; FerrufinoAcosta, 2010; Li, 2012; Govaerts, 2013) have indicated that > 40% of the species can be treated as synonyms, leaving only c. 210 species currently recognized in the family. These are classified in two genera, Smilax L and Heterosmilax Kunth, the latter of which is a small genus of c. 12 species exclusively from east and south-east Asia that differs from Smilax mainly in the presence of a connate perianth. Smilax is a much larger genus and has been classified into subgeneric taxa. Koyama consulted earlier studies (Kunth, 1850; De Candolle & De Candolle, 1878; Hayata, 1912; Gagnepain, Humbert & Lecomte, 1922) and developed his own interest in the family (Koyama, 1960, 1963, 1975a, 1984). He proposed eight sections under Smilax: China Koyama; Coilanthus A.DC.; Nemexia Raf.; Macranthae Kunth; Nervomarginatae Koyama; Pleiosmilax (Seem.) A.DC.; Smilax (L.) Koyama and Vaginatae Koyama. These were largely based on floral morphology, inflorescence architecture and habit (herbaceous vs. woody). He also divided Heterosmilax into section Polyandrea Gagnep. and section Heterosmilax based on stamen number. When that system of classification is compared with the results of the ITS trees of Cameron & Fu (2006), it is evident that some of the sections under Smilax are almost certainly not monophyletic [Cameron & Fu (2006: fig. 1), e.g. sections China, Macranthae and Coilanthus]. More surprising to them was the fact that the topology indicated that Heterosmilax is polyphyletic and embedded in Smilax, thereby favouring a more restricted circumscription of the family. Considering that these relationships were weakly or not supported by bootstrap analysis, our goal here is also to test the monophyly of Smilax sections and the monophyly and genus status of Heterosmilax further. Moreover, tracking the evolutionary history of the representative characters of these sections and the embedded Heterosmilax is also included in our interests here. We are also interested in the historical biogeography of Smilacaceae, which was first considered by Norton (1916) and then by Mangaly (1968), who suggested a south-east Asian origin for Smilax by studying comparative morphology and the fossil record. Norton assumed some Smilax spp. migrated into North America via an Atlantic land bridge, whereas others arrived via a Bering land bridge or Pacific island hopping. Schaefer & Schoenfelder (2009) more recently found S. rotundifolia L. (North America), S. excelsa L. (Caucasus), S. canariensis Willd. and S. azorica H.Schaef. & P.Schönfelder (Macaronesia) to

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be a monophyletic group based on molecular data, which showed a North America–Macaronesia–West Asia disjunction pattern. Fu et al. (2005) studied herbaceous Smilax (section Nemexia) and showed an East Asian–western and eastern North American disjunction pattern with a probable East Asian origin. Li et al. (2011) noted a strong New World vs. Old World pattern among species of Smilacaceae. However, the species sampling included in most of these studies was limited and/or resulted in conspicuous polytomies among major lineages. This leaves the interpretation of historical biogeographic distributions for Smilacaceae still mostly in the shadows and emphasizes the need for a well-sampled, robust phylogenetic reconstruction at the family level. Hence, in this paper we build upon previous work by adding additional molecular evidence to the nrITS data matrix, specifically from the plastid genome (matK and rpl16 intron), and by greatly expanding the scope of taxon sampling to cover all previous taxonomic sections of Smilax and consider the full geographical range of the family. Our aims are to: (1) reconstruct a more highly resolved and supported phylogenetic hypothesis for Smilacaceae; (2) test the monophyly of Koyama’s infrageneric sections and generic status of Heterosmilax; (3) consider the evolution of morphological characters through ancestral characters state reconstruction analysis; and (4) highlight and confirm the distribution pattern underlying the phylogenetic pattern.

MATERIAL AND METHODS TAXON SAMPLING We sampled 119 taxa of Smilax and six taxa of Heterosmilax to represent Smilacaceae. Taxonomically, the sampling covered all sections proposed by Koyama (1960, 1975b, c) and others. Geographically, we sampled widely across the entire distribution area (Fig. 1) of Smilacaceae, but with a primary focus on Asia and the Americas (two diversification centres), although only five species from South America were sampled. Lapageria rosea Ruiz & Pav. and Philesia buxiflora Lam. ex Poir. from Chile (Philesiaceae) and Ripogonum scandens J.R.Forst & G.Forst and R. elseyanum F.Muell (Ripogonaceae) from New Zealand and Australia, were selected as outgroup taxa based on the close morphological and molecular relationship of Philesiaceae + Ripogonaceae, with Smilacaceae as recovered in broad, family-level analyses of Liliales and other monocots (Fay et al., 2006; Givnish et al., 2006). The majority of data used in this study are new DNA sequences of specimens collected from 1991 to 2011. A full list of taxa examined with corresponding GenBank accession numbers and

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voucher information are provided in the Supporting Information (Table S1).

MOLECULAR

METHODS

Complete nuclear ITS and plastid matK gene and rpl16 intron sequences were targeted for amplification and sequencing based on their suitability to address interspecific phylogenetic questions. Total genomic DNAs were extracted from silica-dried tissues using various methods, including a modified cetyl trimethylammonium bromide (CTAB) extraction method of Doyle & Doyle (1987). In this case, the aqueous phase was extracted with 24:1 chloroform/ isoamyl alcohol, and DNA was resuspended in Trisethlenediaminetetracetic acid (TE) buffer (pH 8.0) following isopropyl alcohol precipitation. Amplification and sequencing of the ITS region and rpl16 intron followed Cameron & Fu (2006) and Fu et al. (2005). Amplification of the plastid matK gene was accomplished using designed primers ‘M3’: GCAAC AATACTTCCTATATCCGCTTCT and ‘M4’: GAACTC TTCTAATAATCCCGAACCTAA. Polymerase chain reactions (PCR) for matK were first denatured at 94 °C for 6 min prior to the start of PCR cycles, then amplified for 35 cycles of 1 min at 94 °C, 1.5 min at 53 °C, 2 min at 72 °C, and one final cycle of 12 min at 72 °C. Forward and reverse sequences were assembled using Geneious Pro v4.8.5 (Drummond et al., 2009). Alignments were performed in MAFFT web server (multiple alignment program for amino acid or nucleotide sequences, available at http://align.bmr .kyushu-u.ac.jp/mafft/online/server/) (Katoh et al., 2005), then the matrix was adjusted manually in Geneious Pro v4.8.5. Matrices for analyses in this study were deposited to TREEBASE.

PHYLOGENETIC

ANALYSES

Phylogenetic analyses were conducted using maximum parsimony (MP), Bayesian inference (BI) optimality criteria and maximum likelihood (ML). All three data sets were first analyszed separately. Then, matK and rpl16 data sets were combined to construct a plastid gene tree. Finally, after observing no obvious cases of hard incongruence, all three loci were combined and analysed together. Maximum parsimony analyses were conducted using PAUP* version 4.0b10 (Swofford, 2003). Equally most-parsimonious trees were found by executing a heuristic search of 1000 random addition replicates using tree bisection– reconnection (TBR) branch swapping, but keeping only five trees per replicate in order to discover possible ‘islands’ of maximum parsimony (Maddison, 1991). For the combined three data sets the mostparsimonious trees were obtained by executing

PAUPRat (Sikes & Lewis, 2001), which implements the parsimony ratchet algorithm (Nixon, 1999) to efficiently obtain the tree with minimum steps from a large data set. Support values for the relationships discovered by analysis of each matrix were calculated by performing bootstrap analyses of 1000 heuristic search replicates using the TBR branching swapping algorithm with 100 random additions per replicate. For Bayesian analysis, the substitution model for each separated and combined analyses was determined with jModelTest (Posada, 2008) under the Akaike information criterion (AIC). In each case, the optimal model was the general time-reversible model, with rate heterogeneity modelled by assuming that some sites are invariable and that the rate of evolution at other sites is modelled using a discrete approximation to a gamma distribution (GTR+I + G). Bayesian analyses were conducted with MrBayes 3.1.2 parallel version (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) on the CIPRES cluster (Miller, Pfeiffer & Schwartz, 2010). Two independent runs of four Markov chains each starting with a random tree were processed in ten threads simultaneously for 50 million generations, sampling trees at every 1000th generation. The first 12 500 sampled trees (25%) were discarded as burn-in samples. Maximum likelihood (Felsenstein, 1973) tree searches and ML bootstrap searches for the individual and combined data sets were performed using RAxML V.7.2.8 (Stamatakis, Hoover & Rougemont, 2008) on the CIPRES cluster (Miller et al., 2010). The same GTR+I + G model was applied for each analysis. ML bootstrap values were estimated from 1000 bootstrap replicates.

ANCESTRAL

STATE RECONSTRUCTION

Based on previously published morphological studies (Koyama, 1960; Chen et al., 2006a; Kong et al., 2007) and a palynology study (Chen et al., 2006b), six representative characters were chosen to reconstruct ancestral character states for these species of Smilacaceae: (1) inflorescence pattern; (2) perianth morphology; (3) prickles on stem; (4) habit; (5) aerial shoot; and (6) pollen types. Inflorescences of Smilacaceae were classified into six types: (1) bearing single umbel, no scale or prophyll on peduncle; (2) bearing multiple umbels, shoot independent from axil; (3) raceme formed by single flowers; (4) umbels forming a spike without scale on peduncle; (5) one or two umbels, peduncle with scale and prophyll; and (6) more than three umbels, peduncle with scale and prophylla. Perianth morphology was classified into four types: (1) perianth free, petals reflexed; (2) perianth free, petals erect or incurved; (3) perianth free, petals spreading; and (4) perianth connate into a

© 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548

PHYLOGENETICS OF SMILACACEAE tube. Stem characters were coded as (1) unarmed or (2) armed. Habit includes (1) climbing vine and (2) erect herb. Aerial shoot was characterized by (1) persistent and lignified or (2) annually senescent and herbaceous. Pollen morphology characters were coded as five types according to Chen et al. (2006b): (1) ornamentation spinulate; (2) ornamentation wartlike; (3) ornamentation block-like; (4) ornamentation spinulate + wart-like; (5) ornamentation reticulate or microreticulate. Ancestral reconstruction analyses of each character were conducted in Mesquite v 2.7.5 (Maddison & Maddison, 2011) under the unordered parsimony model. Results from six individual characters were then combined together and incorporated into the phylogenetic tree for the combined nuclear and plastid loci.

RESULTS PHYLOGENETIC

RELATIONSHIPS

The ITS matrix contains 897 characters, of which 316 (35.2%) are variable and 230 (25.6%) are potentially parsimony informative. The matK matrix contains 1271 characters, of which 212 (16.5%) are variable and 156 (12.3%) are parsimony informative. The rpl16 intron-aligned matrix is 1258 characters long, of which 361 (28.6%) are variable and 237 (18.8%) are potentially parsimony informative. Combining the two plastid loci results in a matrix of 2529 characters, of which 573 (22.6%) are variable and 393 (15.5%) are parsimony informative. Finally, the combined threelocus matrix contains 3426 characters, of which 889 (25.9%) are variable and 623 (18%) are potentially parsimony informative. For parsimony analyses, a strict consensus tree for ITS was obtained from 73 719 equally parsimonious trees [tree length = 659, consistency index (CI) = 0.583, retention index (RI) = 0.823]; a strict consensus tree for the combined plastid loci was obtained from 56 892 equally parsimonious trees (tree length = 888, CI = 0.713, RI = 0.868); and a strict consensus tree for all three loci combined was obtained from 292 most parsimonious trees (tree length = 1551, CI 0.656, RI 0.847). The MP/BI/ML analyses yielded mostly congruent topologies for the individual and combined data sets. The three tree topologies (based on all loci combined, ITS alone and the two plastid loci, respectively) are presented as Figure 2 and in the Supporting Information (Figs S1 and S2), respectively. The most obvious difference between the ITS tree and that for the two plastid loci combined relates to the position of S. aspera L. Rather than being sister to all Smilacaceae, plastid data alone place this enigmatic species sister to the American B clade, albeit with no statistical support (see also Supporting Information, Figs S1 and S2). The com-

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bined tree from all three loci (Fig. 2) shows that Smilacaceae are divided into four major clades as depicted in red (clade A: Smilax aspera L.), brown (clade B: mostly American species), green (clade C: prickleless herbs, non-climbing species and Heterosmilax, mostly Asian species) and blue (clade D: Asian– Australian–African woody species). The monophyly of these four clades is well supported and the sister relationship between clades C and D is fully supported, but the placement of clade A (S. aspera) and Clade B is poorly supported. For the remainder of this paper, we use the combined gene tree (Fig. 2) to present more detailed results and discussion for selected clades. Clade A contains a single species, S. aspera (the only species distributed in both hemispheres in the family), for which we sampled six accessions from India-Himalayas, the Mediterranean region and east Africa. Its position as a sister clade to the rest of the family is poorly supported (support value: 0.81/–/–). Note that our samples from the Himalayas (G3), western Yunnan, China (L63) and Kenya (H31) are monophyletic and sister to a subclade of accessions from southern Europe (PD) and the Canary Islands (T1). We subdivide the American B clade into several subclades (clade B1 + B2 and clade B3–B5). Clade B2 contains species with a unique morphology of stems with needle-like prickles. This character is evident in S. californica A.Gray and S. hispida Raf. from western and central North America (NA), S. moranensis M.Martens & Galeotti and S. jalapensis Schltdl. from Mexico and Central America (CA) and S. sieboldii Miq. and S. scobinicaulis C.H.Wright from East Asia. This lineage is strongly supported as monophyletic [posterior probability (PP) 1.00/bootstrap (BS) 100], with a curious intercontinental disjunct distribution. It is sister to the B1 clade, which consists of S. fluminensis Steud. from Paraguay (SA) and the Hawaiian endemic species S. melastomifolia Sm. Clade B3 includes three unidentified samples from Brazil (Fw135, Fw137, Fw144), S. schomburgkiana Kunth from Venezuela, S. coriacea Spreng. from Cuba, Fw126 from Jamaica and S. havanensis Jacq., distributed from the southern tip of Florida into the Caribbean. Clade B4 consists of all remaining eastern North American species (S. smallii Morong., S. glauca Walter, S. rotundifolia etc.), plus S. regelii Killip & C.V.Morton from the Caribbean, S. velutina Killip & C.V.Morton from Mexico and S. vanilliodora F.W.Apt from Costa Rica, but also contains S. canariensis and S. azorica from Macaronesia and S. excelsa from Turkey. It is noticeable that S. excelsa and S. azorica form a monophyletic group (PP 1.00/ BS 100) with the North American species S. walteri Pursh and S. rotundifolia.

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Figure 2. Phylogram of the best maximum likelihood tree. Statistical support (Bayesian posterior probability ≥ 0.80/ maximum likelihood bootstrap value ≥ 50/maximum parsimony bootstrap value ≥ 50) are indicated on the branches, ‘*’ denotes a fully supported node. The branch colours correspond to the four major clades identified as A, B, C, D. Coloured bars to the right of the tree indicate the sectional classification of Koyama (1960, 1975b, c and 1984). To the right of these are grey and black bars highlighting the Old World and New World distributions, respectively. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548

PHYLOGENETICS OF SMILACACEAE Clade C (PP 1.00/BS 100) is essentially an Old World lineage, with the exception of North American herbaceous species in C1 [i.e. section Nemexia: S. jamesii G.A.Wallace, S. lasioneura Hook., S. hugeri (Small) J.B.Norton, S. herbacea L., S. biltmoreana (Small) J.B.Norton and S. pulverulanta Michx. from North America, S. riparia A.DC. and S. nipponica Miq. from eastern Asia]. Also in clade C we find two species of section Pleiosmilax from New Caledonia and Fiji (C2), which are successively sister (no statistical support) to species from section Nervomarginatae (clade C3), species of Heterosmilax (clade C4) and species from section Vaginatae (clade C5). This latter group (PP 1.00/BS 99) includes one widespread species, S. stans Maxim., distributed in northern South Asia to eastern Asia, which is sister to six others (S. aberrans Gagnep., S. emeiensis J.M.Xu, S. menispermoidea A.DC., S. retroflexa (F.T.Wang & Tang) S.C.Chen, S. trachypoda J.B.Norton and S. tsinchengshanensis F.T.Wang), all native to south-western China and the Indo-Himalayan region. Clade D is an Old World clade of 52 woody, mostly climbing species, most of which are native to temperate and tropical Asia. However, S. australis R.Br. from Australia and S. anceps Willd. from Africa fall here and are intermixed with Asian species (clade D7). We also find that S. purpurata G.Forst. and S. glyciphylla Sm. from New Caledonia and eastern Australia formed a clade with Asian species, but in clade D2. Species from Smilax section China from eastern Asia, a polyploid complex that includes S. china L., S. china var. kuru Sakag. ex Yaman, S. biflora Sieb. ex Miq., S. davidiana A.DC., S. nantoensis Koyama and S. trinervula Miq., share a common ancestor with clade D3 (PP 0.99/BS 99). However, the other species of section China in Asia (S. outanscianensis Pamp., S. polycolea Warb. ex Diels, S. ferox Wall. ex Kunth, S. lebrunii H.Lév., S. chingii F.T.Wang & Tang and S. megalantha C.H.Wright) are distantly related in clade D6. The monophyletic clade D7 (PP 0.99 and BS 54) contains most of the species from section Macranthae, which are characterized by the presence of a paniculate inflorescence, including the Asian, African and Australian species mentioned earlier.

MONOPHYLY

OF

KOYAMA’S

SECTIONS AND ANCESTRAL

CHARACTER STATE RECONSTRUCTION

Our Smilacaceae phylogram does not support monophyly of each of Koyama’s eight sections (Fig. 2). Section China was split between New World clade B and Old World clade D and is further divided into clade D3 and D6 in clade D; section Coilanthus was confined to clade D2, but S. elegantissima Gagnep. from section Smilax is embedded. Section Macranthae

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was spread between clades D4 + D5 and D7. Species from section Nemexia appear in clade C1 and the paraphyletic C2, intermixed with four species from section Coilanthus and section Pleiosmilax. Section Nervomarginatae is composed of species from clades C3 and D1. Except for S. elegantissima in clade D2, clade A (S. aspera) is also included in this section. Clade C5 and S. myrtillus A.DC. in clade D4 comprises section Vaginatae. The results of unordered parsimonious ancestral states inference are shown in Figure 3. They indicate that the common ancestor of Smilacaceae might have been a woody vine that bore a single umbel on a peduncle without a scale or prophyll, but with a free perianth of petals normally spread, stem with prickles and spinulate pollen. This combination of characters defines the extant species of Smilax section China, which are distributed in the Old World (clades D3 and D6) and New World (clade B) (Figs 2 and 3). The character pattern of the common ancestor of the New World clade (clade B) is the same as the whole family, while the Old World clade (clade C + D, Fig. 3) differs in absence of prickles on the stem.

DISCUSSION GENERIC

CIRCUMSCRIPTION OF

SMILACACEAE

In our previous study of phylogenetics of Smilacaceae based on ITS (Cameron & Fu, 2006), Heterosmilax was found to be embedded in Smilax and nonmonophyletic because of the position of one species, H. chinensis F.T.Wang. Re-examination of the voucher specimen for that DNA, and re-sequencing new material, has confirmed that what had been labelled H. chinensis was, in fact, a misidentification of S. glabra Roxb. Taking this into account, Heterosmilax is monophyletic, but still embedded in Smilax; H. chinensis is actually sister to the other Heterosmilax spp. based on this study (Fig. 2, clade C4). Moreover, the pollen morphology also showed the pollen type of Heterosmilax is the same as that in S. nervomarginata (Chen et al., 2006b), which is in a sister clade (Fig. 2, clade C3) here. Hence, we agree with Judd’s suggestion that Heterosmilax would better be treated as a group under Smilax, namely as Smilax section Heterosmilax (Kunth) Judd. It is clear that Smilacaceae now only includes one large genus, Smilax, with c. 210 species. Seemann (1868) established another genus in the family, Pleiosmilax [which Koyama (1960) treated as a section under Smilax], to which we must devote at least some discussion. Pleiosmilax was based on the Hawaiian endemic Smilax melastomifolia (= S. sandwicensis Kunth) and recognized because this species possesses more than six stamens in its male flowers.

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Figure 3. Ancestral character reconstructions based on six morphological features important to the systematics of Smilacaceae: (1) inflorescence types 1–6; (2) perianth types 1–4; (3) stem types 1–2; (4) life form 1 type 1–2; (5) life form 2 types 1–2; (6) pollen types 1–5. (See ANCESTRAL STATE RECONSTRUCTION in MATERIAL AND METHODS for detailed type description of each morphological feature.) Circles at each node and taxon terminal sequentially consistent with these 1–6 morphological features. Grey circles indicate that the feature is not known. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548

PHYLOGENETICS OF SMILACACEAE De Candolle & De Candolle (1878) also noted the anomalous stamen number and placed this species into Smilax as a separate section including three species (S. melastomifolia, S. vitiensis A.DC. and S. orbiculata Labill., the last two from Oceania). We found no evidence to support the recognition of these species as a distinct group. Instead S. vitiensis from Fiji is placed in subclade C2, whereas S. melastomifolia from Hawaii is part of subclade B1. Both species possess 12–18 stamens per male flower, and this example of convergent evolution may be explained by a selective advantage for attracting restricted pollinators after the plants became established on oceanic islands by probable long-distance dispersal. As a side note, some Heterosmilax spp., defined as section Polyandrae Gagnep. (Koyama, 1984), also have more than six stamens. Dahlgren & Clifford (1982), in their book of comparative study of monocots, indicated that flowers with more than six stamens are rare in Liliiflorae, so the reproductive biology of these odd flowers is worthy of further study.

PHYLOGENETIC

RELATIONSHIPS WITHIN

SMILAX

Smilax aspera The results of our study strongly support the division of Smilacaceae into four main lineages, which will be discussed individually. Smilax aspera represents clade A. It is the type species of the genus, widely distributed across the Mediterranean, Macaronesia, east tropical Africa and even disjunct to India, Sri Lanka, and then eastward stretching to Yunnan, China, where it stops just at the intersection of the historical collision between the Indian and Asian continents (Fig. 1, red lined regions). The morphological characters of S. aspera that distinguish it from other Smilax are the spines on the margins and midrib of the coriaceous leaf blades and a spike-like inflorescence with one- to six-flowered, sessile umbels. This type of inflorescence was defined as the unique morphological feature characterizing section Smilax by Koyama (1960). Wang et al. (1978) later included S. elegantissima from Yunnan, China, which also has a spike-like inflorescence, but our results show clearly that this species belongs to another lineage (clade D). The inflorescence architecture of S. aspera is not an autapomorphy for this species. Details of relationships in the S. aspera clade show that the Asian accessions of S. aspera (L63 and G3) are sister to the sample (H31) from east tropical Africa, whereas the Mediterranean samples (including one from the Canary Islands) are a clade sister to them. The blackcap (Sylvia atricapilla L.), a small, migratory, passerine bird, is known to be a main consumer of S. aspera berries (Herrera, 1981). It breeds in temperate Europe and western Asia, but

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overwinters in southern Europe to tropical Africa. Its flight patterns may help to explain a mechanism for dispersal of S. aspera seeds between Europe, Asia and Africa. A number of taxonomic varieties (Grenier & Godron, 1855; De Candolle & De Candolle, 1878) based on leaf shape have been published by European taxonomists, but these characters can often be found in populations in one area, sometimes even within one population. Also, we examined a large number of S. aspera specimens in the field across its range, but could not find any significant morphological divergence between the three main distribution regions. However, in our phylogram (Fig. 2), the divergence between Mediterranean and African–Asian samples showed a degree of divergence comparable with other species groups in the family (clade B2, C5 and D3); it may suggest that this widespread species has undergone a cryptic speciation process. New World clade Clade B comprises all of the New World woody Smilax spp., but also includes S. excelsa from Turkey, S. azorica and S. canariensis from Macaronesia and two species with needle-like spines from eastern Asia (S. scobinicaulis and S. sieboldii). Most of the species in this clade were classified into section China. (Fig. 2, blue bars). A pair of these Old World species, S. excelsa and S. azorica, clustered with North American S. walteri and S. rotundifolia in clade B5. Morphologically, these species are somewhat similar to S. rotundifolia, marked by its quadrangular new branches, and the mature berries of S. excelsa are also red, as in S. walteri. Norton (1916) correctly stated, in part, that ‘S. rotundifolia and the related S. walteri have their nearest relatives in the Azores, Canary Isles, Mediterranean region, Western Asia, Turkestan and western India’. Indeed, S. excelsa is endemic to the Black Sea and Caspian Sea region from Greece and Bulgaria to Iran. Other than S. excelsa, Norton also assumed that S. ferox from western India and S. canariensis from the Azores and Canary Isles should be included in this group. Our results show that this is not the case for S. ferox as it clustered with other Asian species in our tree (S. lebrunii, S. chingii etc. in Fig. 2, clade D6). As for S. canariensis, Schaefer & Schoenfelder (2009) documented a close relationships between S. rotundifolia, S. excelsa and S. canariensis based on ITS, rbcL, matK, trnL intron and trnL-F intergenic spacer sequences, but with a low taxon sampling and without statistical support. They also suggested that S. azorica from the Azores should be treated as a separate taxon from S. canariensis (distributed both in Madeira and Canary Isles). In our family level study, the molecular trees show a clear North American, Azores and Western Asia disjunction of S. rotundifolia, S. walteri, S. azorica and S. excelsa

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(see clade B5), but that S. canariensis is unrelated to them. It is part of another North American– Macaronesia disjunction with S. laurifolia L and S. smallii, both mainly distributed in the southeastern USA (see Fig. 2, clade B4). The other Old World exception for membership in the otherwise New World B clade, is a pair of East Asian species, S. sieboldii and S. scobinicaulis, which are members of the S. hispida group (clade B2). This group is sister to South American S. fluminensis and Hawaiian S. melastomifolia, further highlighting the disjunct nature of this subclade. The S. hispida group defined here includes S. hispida (= S. tamnoides L.) from eastern North America, S. californica confined to northern California and southern Oregon, S. moranensis and S. jalapensis from Mexico and Asian S. sieboldii and S. scobinicaulis. This species group is marked by its long, slender, blackish and needle-like spines on the stems, which are exceptional in the family. The leaves of these species sometime have minutely serrulate margins. Smilax sieboldii is distributed in eastern China, but stretches east to Korea and Japan, and south to Taiwan. S. scobinicaulis extends its range westward in China, to Yunnan Province. In North America, S. californica is narrowly confined to pine and mixed evergreen forest in California and Oregon, whereas S. hispida grows in nearly all states of the eastern USA. S. moranensis and S. jalapensis are known from Mexico, south to Guatemala. Others (Norton, 1916; Wallace, 1983; Cameron & Fu, 2006) had already recognized this group as one worthy of further biogeographic consideration. They suggested an eastward migration from Asia into North America. However, our molecular study shows that this species group is well embedded in the New World clade, and with the widely distributed South American S. flumingensis and Hawaiian endemic S. melastomifolia sister to it, thereby indicating an alternative hypothesis of westward dispersal from the Americas, which is further supported by a largescale phylogeographic study of this species group (Zhao et al., 2013). The remaining species in the New World clade (clade B) are divided into two groups: one from Central and South America and another mostly from North America, with the exceptions already discussed earlier. We point out the fact that S. havanensis from the Caribbean and extreme south Florida is a member of clade B3, the Central and South America lineage, which suggests this species, like so many others, dispersed from the mainland. Norton (1916) felt that S. bona-nox from North America might be related to S. aspera given their similar leaf and spine morphology, but our phylogenetic reconstruction (Fig. 2) shows that S. bona-nox clearly shared a

common ancestor with other North American species. Nearly all members of this lineage bear a single umbel, prickles on the stem and vining habit, a combination of characters consistent with the ancestral character state reconstruction result from the Mesquite analysis (Fig. 3, clade B). Old World clades The Old World, especially Asia, claims c. 65% of the species in the whole family and these are members of two large sister clades (clades C and D). Species in clade C are characterized by herbaceous or semiherbaceous stems, leaves with extremely fine reticulations and flowers with spreading petals. Exceptional in this group are the species of Smilax section Nemexia, which are considered ‘herbaceous’ in the sense that they die back to underground storage organs each year. Our data confirm the results of earlier researchers, including Fu et al. (2005), who used DNA data, and Mangaly (1968), who examined chromosomes, that this group of species is monophyletic and exhibits the classic East Asian–western North American–eastern North America phytogeographic disjunction. Members of subclades C2 and C3 show characters intermediate between herbaceous Smilax (clade C1) and Heterosmilax (clade C4). For example, Li et al. (2011) described S. ligneoriparia C.X.Fu & P.Li as a new transitional species pivotal to understanding the evolution of the non-climbing, herbaceous habit in Smilax. Another unusual species from this lineage, S. pottingeri Prain, is worthy of discussion because of its controversial taxonomical history. Koyama (1975b) treated this species as a member of section Nemexia (herbaceous Smilax), but Wang et al. (1978) established a new combination, Heterosmilax pottingeri (Prain) F.T.Wang & Tang, for it. Years later, Chen & Koyama (2000) moved it back into Smilax. Our results (see Figs 2 and 3) confirm that it is a Smilax, but one that is sister to all species of the C clade other than the herbaceous C1 subclade. Its biology should be examined in greater detail. A final group to consider in the C clade are the elements of subclade C5, which show a number of putative synapomorphies, including semi-herbaceous habit, erect non-climbing stems and vestigial tendrils. All of them inhabit high mountain habitats between 1000 and 3500 m. Their short stature and nonclimbing lifestyle may be an adaptation for survival in these extremely cold, high-elevation habitats. The final lineage to consider is clade D, which includes species with either single umbels or panicles of umbels. Distribution of the first diverging group (clade D1), a natural group without thorns, is confined to south-western China and the IndoHimalayan region. These species are sister to the rest

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PHYLOGENETICS OF SMILACACEAE of the clade classified in non-monophyletic sections China, Macranthae and Coilanthus. Species from clade D1, with those of clade C3, were previously placed under Koyama’s section Nervomarginatae. Five species in subclade D2: S. glabra, S. corbularia, S. glyciphylla, S. elegantissima and S. purpurata, were expected to cluster as they are from section Coilanthus. The leaf morphology of this group is variable, but all species have upright to incurved petals, climbing stems and no prickles on the stem. The remainder of the Old World D clade includes intermixed species of sections China and Macranthea. The only character in common between the two sections is that most of their species have a woody climbing stem with prickles. De Candolle & De Candolle (1878) called them Eusmilax. We found one monophyletic widespread species complex (D3: the S. china complex) embedded in this clade. This group includes S. china with a wide distribution, S. davidiana, S. trinvervula, S. china var. kuru (endemic in Ryukyu), S. biflora (endemic to south Japan and north Ryukyu) and S. nantoensis (endemic to Nantou, Taiwan). Morphologically these taxa are easy to distinguish, but genetically they are closely related. Chromosome data for S. china shows that some elements of this complex are polyploid, from 2n = 32 (diploid), 2n = 60 and 64 (tetraploid) and 2n = 96 (hexaploid) (Nakajima, 1937; Hsu, 1971; Fu et al., 1995; Kong et al., 2007). However, the other four species are exclusively diploid 2n = 32, as demonstrated by Kong et al. (2007). Smilax lanceifolia of clade D5 is one of the most widespread species in Asia. Koyama (1960) and Wang et al. (1978) recorded five varieties in the species. Our genetic data for three accessions also showed high levels of variation and are paraphyletic because of the derived position of sympatric and morphologically similar S. chapaensis Gagnep. The taxonomy of this species should be considered further, especially at the population level. Finally, in subclade D7 we highlight a disjunct group of tropical species, all of which are from section Macranthea (characterized by paniculate inflorescences). An Australian species, S. australis, a New Guinean species, S. leucophylla, and an Indonesian species, S. setosa, form a well-supported clade. These are related to S. anceps from Africa and S. prolifera Wall. from Sri Lanka. The closest relatives of these species are vigorous climbers, mostly from southern and south-eastern Asia: S. blumei A.DC., S. zeylanica L., S. ocreata A.DC., S. ovalifolia Roxb., S. bracteata C.Presl, S. perfoliata Lour., S. lunglingensis F.T.Wang & Tang and S. aspericaulis Wall. These tropical species usually have exceptionally large leaves and vigorous stems and climb high in the rainforests.

CHARACTER

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EVOLUTION, BIOGEOGRAPHIC PATTERNS

AND SYSTEMATIC IMPLICATIONS

Although our molecular phylogenetic results have shown that nearly all currently recognized sections of Smilax based on morphology are not monophyletic, we can still consider the distribution of key morphological features in the context of the phylogenetic reconstruction. The ancestral character state of Smilacaceae was inherited by clade B and independently evolved in the ancestor of clade D. It is the most widely distributed (Americas and East Asia) and frequent (32%) combination of features found in the extant species, possibly indicating that species with this morphology are generally more able to adapt to differing environments. The primary difference between the ancestral character patterns of clade B and clade C + D is the presence or absence of prickles. Loss of prickles has evolved independently in the family several times, for example in S. melastomifolia, S. pumila Walter in clade B, S. glabra, S. darrissi H.Lev., S. austrozhejiangensis Q.Lin in clade D and all species in clade C. The pattern of ancestral characters defining clade C + D is not shared by any extant species in this large Old World clade, but it is shared with the extant S. pumila and S. velutina in clade B. Figure 1 highlights the widespread distribution of Smilax s.l. The red, brown, green and blue lined regions in the figure are drawn in accordance with the four major clades (A, B, C and D) recovered in our phylogenetic analysis and discussed above. The dashed lines in Figure 1 indicate our interpretation that extant Smilacaceae can be assigned to three major geographical realms. All coloured regions out of their realm are species occupying areas that they reached by means of vicariance or long-distance dispersal events (see Discussion above). These three realms exhibit a Nearctic + Neotropic, Palaearctic + Afrotropic and Palaearctic + Indomalaya disjuct pattern of main lineages of extant Smilacaceae. Considering the strong geographical pattern of major lineages in Smilacaceae and the inconsistency of morphological sections with molecular phylogenetic results, we suggest that future systems of classification should include geographical data and that the results of our character evolution analysis in the treatment of subgeneric groups should be used in future monography of Smilacaceae.

CONCLUSIONS

AND FUTURE STUDIES

The analyses reported here have done a great deal to resolve infrafamilial relationships in Smilacaceae. The results indicate that: (1) Heterosmilax should be reduced to a section in Smilax as its recognition at

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generic level renders Smilax non-monophyletic; (2) all eight taxonomical sections under Smilax by Koyama are non-monophyletic; (3) ancestral character states of Smilacaceae still exist in many extant species in the New and Old Worlds; and (4) the family can be divided into four main lineages, and a mostly clear Old World–New World split is evident in the evolution of the family, but with a handful of intercontinental dispersal events. However, relationships among clade A, clade B and clade C + D are poorly supported and there remains a need for better resolution and support in some critical subclades (e.g. monophyly of clade B4 + B5, C2–C5, D3–D7). These factors hindered further analysis of the divergence time and historical biogeography of this family. We are hopeful that progress can be achieved by further increasing species sampling (59% of total in this study), especially in South America (only five in this study) and expanding character sampling across members of the family; for example, using additional low-copy nuclear genes, which have been reported to be useful for resolving infrafamilial relationships. We hope that the phylogenetic reconstruction presented in this paper will serve as a starting point for initiating a more thorough investigation of historical biogeography using ancestral area reconstructions and chronograms of clade divergence in time. An in-depth discussion of the subject is beyond the scope of our present study, but a brief discussion is warranted considering the strong geographical, rather than taxonomic, pattern that has been revealed in our cladograms. Considering the overwhelming task of monographing a genus this large, variable, diverse and widespread, we hope that by recognizing smaller evolutionary units in Smilax, others will be encouraged to focus at a finer level of systematic study on these individual clades in the future.

ACKNOWLEDGEMENTS The authors would like to give special thanks to Akiyo Naike, Deyuan Hong, Hanno Schaefer, Harry Wiriadinata, Joongku Lee, Lianming Gao, Patricia Villena, Petter Weston, Ram P. Chaudhary, Siro Kurita, Tetsuo Ohi-Toma, Timothy Motley and Walter Judd for assistance in sample collection. This study is supported by a key project of the National Science Foundation of China (grant no. 30830011), National Natural Science Foundation of China (grant no. 31110103911), Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences (grant no. KSCX2-EW-Z-1) and Chinese Education Ministry (China Scholarship Council, grant no. 2011632093 and 2009632123).

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Maximum likelihood phylogram and cladogram of Smilacaceae, based on nuclear ribosomal DNA internal transcribed spacer. Figure S2. Maximum likelihood phylogram and cladogram of Smilacaceae, based on combined data of the plastid matK protein coding gene and rpl16 intron. Table S1. Alphabetically arranged species of Smilacaceae, Ripogonaceae and Philesiaceae analysed for this study (with GeneBank accession numbers).

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