K B C D E I H F J

Home , Clematis brachiata, Clematis chrysocoma, Clematis cunninghamii, Clematis lasiantha, Clematis ligusticifolia, Clematis linearifolia

Botanical Journal of the Linnean Society, 2016, 182, 825–867. With 2 ﬁgures

Sensitive phylogenetics of Clematis and its position in Ranunculaceae

SAMULI LEHTONEN1,*, MAARTEN J. M. CHRISTENHUSZ2,3 and DANIEL FALCK4 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 1Herbarium, University of Turku, FI-20014 Turku, Finland 2Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 4DS, UK 3Plant Gateway, Hertford, Hertforshire, SG13 7BX, UK 4Paraisten kaupunki, FI-21600 Parainen, Finland

Received 23 December 2015; revised 11 April 2016; accepted for publication 20 July 2016

Ranunculaceae are a nearly cosmopolitan plant family with the highest diversity in northern temperate regions and with relatively few representatives in the tropics. As a result of their position among the early diverging eudicots and their horticultural value, the family is of great phylogenetic and taxonomic interest. Despite this, many genera remain poorly sampled in phylogenetic studies and taxonomic problems persist. In this study, we aim to clarify the infrageneric relationships of Clematis by greatly improving taxon sampling and including most of the relevant subgeneric and sectional types in a simultaneous dynamic optimization of phenotypic and molecular data. We also investigate how well the available data support the hypothesis of phylogenetic relationships in the family. At the family level, all five currently accepted subfamilies are resolved as monophyletic. Our analyses strongly imply that Anemone s.l. is a grade with respect to the Anemoclema + Clematis clade. This questions the recent sinking of well-established genera, including Hepatica, Knowltonia and Pulsatilla, into Anemone.InClematis, 12 clades conceptually matching the proposed sectional division of the genus were found. The taxonomic composition of these clades often disagrees with previous classifications. Phylogenetic relationships between the section-level clades remain highly unstable and poorly supported and, although some patterns are emerging, none of the proposed subgenera is in evidence. The traditionally recognized and horticulturally significant section Viorna is both nomenclaturally invalid and phylogenetically unsupported. Several other commonly used sections are likewise unjustified. Our results provide a phylogenetic background for a natural section-level classification of Clematis. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 182, 825–867

ADDITIONAL KEYWORDS: Anemone – direct optimization – Hepatica – Pulsatilla – sensitivity analysis – total evidence.

INTRODUCTION 59–55 Mya; Bell, Soltis & Soltis, 2010). However, the recently discovered Leefructus Ge Sun, Dilcher, Ranunculaceae (c. 2500 species and 50–60 genera; H.S.Wang, Z.D.Chen, a fossil from 125.8–122.6 Mya Tamura, 1993) are placed in the early branching from Early Cretaceous deposits in China, has been eudicot order Ranunculales, as sister to Berberi- assigned to this family and, if correctly placed, this daceae (Hoot, 1995). Ranunculaceae have a nearly will push back the age estimate and, indeed, may cosmopolitan distribution with the greatest diversity change our understanding of the evolution of eudi- in the northern temperate regions, but they extend cots as a whole (Sun et al., 2011). into the tropics and the Southern Hemisphere, where Ranunculaceae had been variously subdivided on they can be found in various habitats (Tamura, the basis of morphological variation until chromoso- 1993). They are estimated to have diverged c. mal characters became the most important criterion 87–73 Mya (Anderson, Bremer & Friis, 2005) or for classification (Gregory, 1941; Tamura, 1997). 85–65 Mya (Wikstrom,€ Savolainen & Chase, 2003), These classifications have subsequently been revised although younger estimates also exist (e.g. in the light of increasing molecular phylogenetic understanding (Hoot, 1995; Jensen et al., 1995; *Corresponding author. E-mail: samile@utu.fi Johanson, 1995; Kosuge et al., 1995; Ro, Keener &

McPheron, 1997) and, currently, ﬁve subfamilies, (van der Neut & Pfeiffer, 1982). In East Asia, spe- Glaucidioideae, Hydrastidoideae, Coptidoideae, cies such as C. ﬂorida Thunb. and C. patens C.Mor- Ranunculoideae and Thalictroideae, are accepted ren & Decne. have also been in cultivation for (Wang et al., 2009; Cai et al., 2010). Numerous phy- centuries, from where they were introduced to logenetic studies have focused on a particular genus, European and American gardens in the late 18th to e.g. Aconitum L. (e.g. Luo, Zhang & Yang, 2005; Jab- mid-19th century (Christenhusz, 2000; Johnson, bour & Renner, 2011), Actaea L. (Compton, Culham 2001). More than 3500 cultivars have been named & Jury, 1998), Anemone L. (Hoot, Reznicek & Pal- since the mid-19th century (some dating back long mer, 1994; Schuettpelz et al., 2002; Ehrendorfer before that), several of which are now lost to horti-

et al., 2009; Hoot, Meyer & Manning, 2012), Aquile- culture (Clematis on the Web, 2015). Partly because Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 gia L. (Bastida et al., 2010), Caltha L. (Schuettpelz of its popularity, Clematis has attracted attention & Hoot, 2004), Clematis L. (Miikeda et al., 2006; Xie, from amateur and professional botanists alike, Wen & Li, 2011), Delphinium L. (e.g. Jabbour & resulting in various classifications of the genus dif- Renner, 2012), Hamadryas Comm. ex Juss. (Hoot, fering in systematic approaches. Many of these tra- Kramer & Arroyo, 2008), Helleborus L. (Sun, McLe- ditional classifications contradict each other and win & Fay, 2001), Hepatica Mill. (Pfosser et al., have contributed, partly inadvertently and partly 2011), Ranunculus L. (Horandl€ et al., 2005; Paun consciously, to much confusion and nomenclatural et al., 2005; Emadzade et al., 2010; Horandl€ & chaos, which is not unusual for genera of horticul- Emadzade, 2011) and Thalictrum L. (Soza et al., tural importance. 2012), but have mostly utilized different molecular Traditionally, the classification and nomenclature markers, preventing the combination of existing data of Clematis have been unruly, covering different to compile well-sampled family-level analyses. The ranks (genus, subgenus, section, subsection and ser- existing family-level phylogenetic analyses are sur- ies), and the names for these have been used in a prisingly sparse, often have poor taxon sampling and multitude of combinations [see Johnson (2001) for a are based on a single gene only (e.g. Ro et al., 1997; summary of these different traditional classification Cai et al., 2010; Wang et al., 2010), which may com- schemes]. The concept of what constitutes a species promise the current taxonomies, although these (or a taxon at any other rank) has not been entirely issues have recently been addressed by Cossard et al. clear, and in a 19th and early 20th century horticul- (2016). The multigene analyses are congruent with tural taxonomic practice, each observed minute dif- the chromosomal character data, but, instead of sup- ference led to the description of a new taxon. This porting two separate groups, the Thalictrum (T)-type trend was exacerbated by the desirability of differen- chromosome is resolved as plesiomorphic and para- tiating clones by different names and the description phyletic with respect to the Ranunculus (R)-type of horticultural novelties to boost sales, particularly (Hoot, 1995; Wang et al., 2009; Cossard et al., 2016). at a time when cultivar names were not yet in com- It has been established that monospecific Anemo- mon use. The International Code of Nomenclature clema (Franch.) W.T.Wang is sister to Clematis for Cultivated Plants (Brickell et al., 2009) and the (Wang et al., 2009; Zhang, Kong & Yang, 2014), but Clematis cultivar classifications (Snoeijer, 2008; Don- whether this clade is embedded in a paraphyletic ald, 2009) have alleviated this situation somewhat, Anemone s.l. (including Anemone, Hepatica, Pul- with the move towards a cultivar classification sepa- satilla Mill. and several other genera) or is sister to rate from botanical nomenclature applicable to wild monophyletic Anemone s.l. has remained controver- plants and plants of wild origin. sial, some studies suggesting the former (Wang Despite the general interest and apparent taxo- et al., 2009; Pfosser et al., 2011; Cossard et al., 2016) nomic challenges, Clematis has escaped major phylo- and others the latter (Johanson, 1995; Barniske, genetic investigations. Although the first attempt to 2009; Ehrendorfer et al., 2009; Zhang et al., 2014) understand the phylogenetic history of Clematis was position. published in the early 1980s (Ziman, 1981), the first Clematis is one of the larger genera in Ranuncu- truly analytical studies were not performed until laceae with 250–350 species (Tamura, 1995; Wang Brandenburg (2000) used morphological data and & Li, 2005). It is one of the most widespread gen- Slomba, Garey & Essig (2004) investigated just five era of flowering plants, being found on all conti- species in a molecular analysis. Soon after, Miikeda nents except Antarctica, and is most diverse in et al. (2006) sampled 33 species and confirmed that warm-temperate and montane regions. Clematis the previously segregated genera Archiclematis includes some widely used ornamentals with culti- Tamura, Atragene L., Clematopsis Bojer ex Hutch. vation dating back at least to the 16th century in and Naravelia Adans. are all nested in a mono- Europe (Dodonaeus, 1554; Johnson, 2001), and its phyletic Clematis. The generally accepted subgenera medical use in antiquity has been well documented and sections were found to be para- or polyphyletic.

However, their sampling was not sufficient to pro- 2013) using the PhyLoTA browser (Sanderson et al., pose a fully revised infrageneric classification. Xie 2008). Six clusters with best taxonomic coverage et al. (2011) more than doubled the taxonomic cover- (ITS, matK, trnL-trnF, 26S, rbcL, psbA-trnH) were age to c. 75 species, with the aim to reconstruct a downloaded and these data were explored in Sequen- sectional-level phylogeny. They recognized ten clades ceMatrix (Vaidya et al., 2011). All taxa that were that were mostly congruent with the nine clades represented by less than three loci and taxa with the found by Miikeda et al. (2006), and concluded that lowest pairwise distances compared with other, more previous morphology-based infrageneric classifica- completely sampled taxa were excluded from the tions were not supported. A side-by-side comparison dataset. Thus, the dataset was reduced to a computa-

of these two studies shows that, although their tionally more convenient size of < 100 terminal taxa Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 clades are similar with respect to the species placed (Appendix 1). As an outgroup, we used three taxa in them, the relationship of these clades to each from sister family Berberidaceae. other varies wildly, because of the lack of a robust resolution at the deeper nodes. These findings revealed that many of the clades correspond well TAXON SAMPLING AT THE GENUS LEVEL with the traditional sectional divisions of Clematis. The genus-level dataset was compiled from four Nevertheless, most of the previously postulated sub- sources. We used all data from the two relatively genera and many of the sections are apparently well-sampled earlier molecular phylogenetic studies paraphyletic in their current circumscriptions. Poor of Clematis (Miikeda et al., 2006; Xie et al., 2011). support for and apparent instability of deeper nodes, In addition, we downloaded all the other Clematis and the fact that, in both studies, several DNA sequences available in GenBank and, fourth, we regions were excluded because of alignment difficul- produced new sequences from material obtained ties, suggests that their results may be sensitive to specifically for this study (Appendix 2). Unsurpris- assumptions of nucleotide-level homology. Further- ingly, this dataset had large amounts of missing more, the taxon sampling still remains poor and lar- data because of its heterogeneous origin. To clean gely excludes sectional type species, making the up the dataset, we ran a series of preliminary anal- application of existing sectional names to the phylo- yses. For these analyses, we aligned the sequences genetically resolved clades contentious. with default settings in Mafft v7.023b (Katoh & In this study, our first aim is to examine how Standley, 2013) and used the script by Pol & much can actually be concluded about the phyloge- Escapa (2009) in TNT (Goloboff et al., 2008a; Golob- netic relationships in Ranunculaceae based on cur- off, Farris & Nixon, 2008b) to identify unstable rently available data and to investigate the most taxa. The taxa, mostly incompletely sampled, suitable outgroups for a phylogenetic study on that were found to be responsible for a dramatic Clematis. Second, we expand the taxon sampling of reduction in resolution were excluded from further Clematis to include 18 of the 19 type species of analyses. Johnson’s (2001) sections, and 33 of the 36 subsec- Our cleaned, final dataset included 132 ingroup tional types, with only C. section Atragene (L.) DC. and 14 outgroup taxa. Several ingroup taxa were subsection Brachyblasti M.Johns. and C. section represented by more than one specimen, so that the Aspidanthera Spach subsection Papuasicae total number of terminals was 189. Outgroup taxa (Hj.Eichler) M.Johns. lacking representation were selected on the basis of the results obtained because of the difficulty of obtaining material for from the family-level analyses and, in accordance these subsections. We also raise the number of with these results, Anemone quinquefolia L. was sampled Clematis spp. to c. one-third to one-half of used as the ultimate outgroup to root the Clematis the estimated number of species in the genus. We tree. specifically address the robustness of the phylogenetic hypotheses by performing sensitivity analyses and by utilizing both molecular and phenotypic DNA EXTRACTION AND SEQUENCING characters. Total genomic DNA was extracted with the dilution protocol of the Phireâ Plant Direct PCR Kit (Finn- zymes, Espoo, Finland) from fresh or silica-dried leaf material or from seeds. We amplified and sequenced MATERIAL AND METHODS four plastid DNA regions (atpB-rbcL, matK, psbA- TAXON SAMPLING AT THE FAMILY LEVEL trnH-trnQ, rbcL-accD) and the nuclear ribosomal Phylogenetically informative sequence clusters that ITS region. Amplifications were performed using belong to Ranunculaceae were queried from Gen- Phusion Flash PCR Master Mix (Finnzymes) follow- Bank and 194 entries were found (as of 15 February ing the manufacturer’s protocol. For each target

DNA fragment, the PCR program included an initial PHYLOGENETIC ANALYSES ° denaturation of 10 s at 98 C, followed by 30 (atpB- Phylogenetic history is written in the DNA of the rbcL) or 40 amplification cycles (atpB-rbcL and species and, hence, we agree with Wheeler (1996) ° ° ° matK:98C for 1 s, 52 C for 5 s, 72 C for 40 s; psbA- that tree search and search for homology correspon- ° ° ° trnH-trnQ and ITS: 98 C for 1 s, 59 C for 5 s, 72 C dences (i.e. sequence alignment) are logically insepa- ° ° ° for 20 s; rbcL-accD:98C for 1 s, 58 C for 5 s, 72 C rable problems (contra Simmons, 2004; see also ° for 40 s), and a final extension at 72 C for 1 min. We Padial, Grant & Frost, 2014; and references therein used the same primers as Miikeda et al. (2006), for further discussion). The molecular data available except that, for ITS, we used ITS4 and ITS5 (White for this study largely consist of length-variable inter- et al., 1990). PCR products were purified and genic spacer regions. This contributes problems for Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 TM sequenced using BigDye terminator cycling condi- phylogenetic inference at multiple levels: in detecting tions by Macrogen Inc., Seoul, South Korea/Amster- homologous nucleotide correspondences; in dealing dam, the Netherlands (www.macrogen.com). with the phylogenetic information of the observed Chromatograms were read and edited with PhyDE sequence length variation; and in evaluating the € (Muller et al., 2007). impact of all necessary assumptions. We favour a total-evidence approach (Kluge, 1989), as all synapomorphies, whether geno- or phenotypic, contribute to PHENOTYPIC DATA our understanding of phylogenetic relationships. We Phenotypic data were mostly coded from descriptions recognize the importance of indel events in carrying available in the literature (Rasmussen, 1979; John- phylogenetic information and are interested in phylo- son, 2001; Wang & Li, 2005; Wang, 2007; various flo- genetic uncertainty possibly caused by the alterna- ras, see Appendix 3) with some characters verified tive sequence alignments. This goal effectively by studies of living or herbarium specimens. As a precludes the use of current mainstream approaches result, 42 phenotypic characters were coded at the based on similarity alignments and model-based phy- family level and 40 at the genus level. The list of logenetic inference which, usually, incorrectly treat characters and their states is provided in Appendix 3 gaps as unknown bases (see Padial et al., 2014). In and coded data matrices are provided in Appen- our view, hiding the sensitivity of hypotheses does – dices 4 6. Characters coded as continuous (two at not make them stronger. It is both honest to show the family and nine at the genus level, respectively) and instructive to compare uncertainties involved in were analysed as such (Goloboff, Mattoni & Quin- transforming original observations into phylogenetic teros, 2006). To avoid these characters overwhelming hypotheses on the way to a more complete under- the analyses, we weighted them, as suggested by standing of the taxa studied. Under these premises, Koch, Soro & Ramırez (2015), so that the cost from we analyse these data under the parsimony frame- the observed minimum state to the observed maxi- work (Farris, 1983) using computer program POY mum state was roughly one step. The discretely (Wheeler et al., 2015) and applying direct optimiza- coded characters were treated as non-additive, with tion (DO) (Wheeler, 1996) for the genotypic data. the exception of carpel number and fruit dehiscence This method performs sequence alignment at the (characters R22 and R33) in the family dataset and same time as phylogeny estimation (tree alignment chromosome number in both datasets (R41 and C39). contra similarity alignment) and uses gaps as phylo- Chromosome numbers were coded as custom genetically informative transformations. Previous alphabet characters and a specific step-matrix follow- studies have shown that the DNA regions used do ing Lehtonen (2009) was applied to them, in which not support strongly incongruent topologies (Miikeda doubling the chromosome number has an equal cost et al., 2006; Xie et al., 2011) and we followed the to adding a single chromosome (e.g. changes from total-evidence approach and analysed all the data = = = = n 7ton 14 and from n 7ton 8 both equal simultaneously (Kluge, 2004). one step). Under this step matrix, the change from Three sets of analyses were conducted for both the = = n 7ton 11, for example, can take place either family- and genus-level datasets: (1) analyses of phe- via the addition of extra chromosomes notypic data only; (2) analyses of genotypic data = ? = ? ... ? = [n 7 n 8 n 11 (cost 4)] or via poly- only; and (3) combined total-evidence analysis of ploidy and subsequent reduction of chromosomes both. Results from the combined analyses are pre- = ? = ? ... ? = [n 7 n 14 n 11 (cost 4)]. For some ferred and other analyses were run mainly to test taxa, both diploid and polyploid chromosome num- the contribution of different data sources to the final bers have been reported. As it is not possible to results. The analyses were run with either POY apply polymorphic states for custom alphabet charac- 5.1.1. in CSC supercomputer cluster using 32 nodes, ters, these taxa were coded as having the diploid each hosting two eight-core 2.6-GHz Intel Sandy state.

Bridge processors, or with POY 5.1.0. in a 2 9 2.26- transversion cost (2:4:2:1), a regime with higher gap GHz Quad-Core Intel Xeon Macintosh with 32GB of opening cost (3:2:2:1) and its equivalent with double RAM. transversion costs (3:4:2:1), a regime with low gap Some molecular datasets were partitioned before opening and extension cost (3:4:4:1) and, ﬁnally, a the analyses to reduce the computational burden and regime otherwise similar, but again with double cost to increase the number of characters to be resampled for transversions (3:8:4:1). In the total-evidence anal- in dynamic jackkniﬁng (see below). In the family- yses, phenotypic data were given weight equal to the level analyses, matK and 26S were both cut into two highest cost for a single-nucleotide transformation in partitions from a highly conserved region and the the corresponding molecular cost regime, following

ITS marker was divided into three partitions, corre- the recommendation of Schulmeister (2003). The Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 sponding to the internal transcribed spacer 1, the results of the sensitivity analyses were pooled 5.8S ribosomal RNA and the internal transcribed together and visualized using Cladescan (Sanders, spacer 2. At the genus level, matK and ITS were par- 2010). titioned in the same way and the rbcL-accD spacer The POY analyses were initiated by building 200 was divided into three partitions. Thus, a total of ten random addition starting trees with command build and 11 molecular data partitions were used in the (200). These were swapped, evaluating all the trees family-level and genus-level analyses, respectively. within 5% of the best cost with command swap A sensitivity analysis (Wheeler, 1995; Giribet, (threshold:5.0). The resulting trees were subjected to 2003) was performed by repeating all the molecular 20 rounds of ratchet (Nixon, 1999) with command and total-evidence analyses under eight different perturb(transform(static_approx),iterations:20, transformation cost regimes, with different weight- ratchet:(0.25,3)) and treefusing (Goloboff, 1999) with ings for substitutions, gap opening and gap exten- command fuse(iterations:200,swap()). The retention sion. Equal weight parsimony is often supported on index (RI, Farris, 1989) was calculated for pheno- philosophical grounds (e.g. Frost et al., 2001; Grant typic characters under the topologies of phenotypic & Kluge, 2005), but, in practice, is not applicable to and preferred total-evidence analyses using TNT. all transformations (De Laet, 2005, 2015; Giribet & We further evaluated nodal support (sensu Giribet, Wheeler, 2007) and may not be well founded (Golob- 2003) by performing jackknife analyses (Farris et al., off et al., 2008a,b). Transitions are generally consid- 1996). These analyses were conducted under ered to be more homoplasious than transversions dynamic homology by resampling data partitions and and, for this reason, are often given lower weight applying a DO search. One hundred pseudoreplicates (but see Broughton, Stanley & Durrett, 2000). We were created with a removal probability of 37% and applied cost regimes with transversions given the applying search commands build(10), swap(), perturb same or twice the weight of transitions. Gap charac- (iterations:20,rathcet:(0.25,3)). The number of data ters of varying lengths are especially problematic partitions (ten and 11 molecular data partitions in because they may compromise the assumption of the family- and genus-level analyses, respectively) is base-to-base homology. De Laet (2005, 2015) intro- considered to be adequate for resampling analyses, duced a transformation cost regime of a substitution as Simmons, Muller€ & Norton (2010) found no differ- cost 2, gap opening cost 2 + 1 and gap extension cost ence in dynamic jackknife values obtained by resam- 1 (usually referred to as ‘3221’, but, because of differ- pling a dataset divided into either ten or 100 ent usage of gap opening cost in POY5, expressed fragments. Jackknife support values were calculated here as 2:2:2:1) to maximize total sequence homol- under the cost regimes that maximized stability, ogy. Indeed, several studies have supported low gap where stability of a node is calculated by summing extension cost as logically and practically superior to the number of alternative cost regimes supporting equal transformation costs (Aagesen, 2005; Aagesen, each node (e.g. a node is maximally stable when it is Petersen & Seberg, 2005; Pons & Vogler, 2006; recovered under all eight cost regimes). Spagna & Alvarez-Padilla, 2008; Liu et al., 2009). High transformation cost means that the optimization algorithm tries to minimize these events, but RESULTS also means that, whenever hypothesized, these events have a stronger impact on the topology. Given PHYLOGENETIC RELATIONSHIPS AT THE FAMILY LEVEL this background information, we applied an equal In the family-level analyses, c. 6% of the phenotypic cost regime (gap opening = 0: transversion = 1: tran- data and 29% of the molecular data at the fragment sition = 1: gap extension = 1), a cost regime with level were missing. The amount of missing molecular double cost for transversions (0:2:1:1), De Laet (2005) data varied from 5% (ITS2) to 74% (psbA-trnH). We homology maximization cost regime (2:2:2:1), a lack c. 20 generally accepted genera from our analy- regime otherwise similar, but with double ses, most from tribes Ranunculeae and Anemoneae.

All trees produced in this study are available in regimes, Asteropyrum J.R.Drumm. & Hutch. was sis- Nexus format as Supporting information (Data S1). ter to Caltha, and Callianthemum C.A.Mey. was sis- The analysis of phenotypic data resulted in four ter to either Ranunculeae, Anemoneae or the clade equally parsimonious trees of 167.9 steps. In the formed by these tribes. Paraphyly of Anemone s.l. strict consensus tree (Fig. S1), subfamilies Ranuncu- was strongly supported and evident under all studied loideae and Coptidoideae are monophyletic, but Thal- parameter costs. ictroideae are resolved as a grade leading up to the The stability of total-evidence analysis was also rest of the family. At a ﬁner scale, tribes Delphinieae maximized under the same 3:4:2:1 (phenotypic data and Anemoneae are both well resolved and sup- 4) parameter set, but with a topological reorganiza-

ported. Ranunculeae are resolved as paraphyletic tion (Fig. 1). Ranunculoideae were monophyletic Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 with respect to Anemoneae. Likewise, Anemone s.l. under five cost regimes, Ranunculeae and Anemo- is paraphyletic to Anemoclema + Clematis. neae were sister tribes under five cost regimes and The parameter set 3:4:2:1 maximized stability in Nigella was sister to Delphinieae under six cost the analyses of molecular data and the dynamic jack- regimes. Helleborus remained highly unstable and knife support was calculated under this cost regime the sister relationship between Asteropyrum and (Table 1, Fig. S2). Under this cost regime, Thalic- Caltha was less stable than under the molecular troideae were nested in a paraphyletic Ranuncu- analyses. Anemone s.l. remained paraphyletic with loideae, but this unconventional position lacked high support and stability. Under three cost regimes, jackknife support. However, most of the explored the addition of phenotypic data slightly decreased the cost regimes placed Thalictroideae in various posi- stability of the phylogenetic results, whereas stability tions inside Ranunculoideae, and only one resolved was slightly increased under five cost regimes these tribes as sisters. The relationships of the other (Table 1). Average jackknife support was lower in the three subfamilies were constant and well supported. combined analysis compared with the molecular anal- Helleborus and Nigella L. were phylogenetically ysis (65.3% vs. 71.5%). The RI of four phenotypic unstable and placed in various positions depending characters was increased in total-evidence analyses on the cost regime. Under most of the studied cost when compared with the phenotypic data only; these were (R13) flower opening, (R15) perianth phyllotaxy, (R23) carpel fusion and (R27) ovule curvature; the RI Table 1. Parameter sets analysed (gap opening:transversion:transition:gap extension:phenotypic data) with the of a further 17 characters remained the same and the cost and number of the most-parsimonious solutions and RI of 21 characters decreased (Appendix 3). stability value for each parameter regime as obtained in the sensitivity analysis PHYLOGENETICS OF CLEMATIS Equally One terminal (Clematis sp. Wen 9476), an unidenti- optimal Stability fied sample originating from the study of Xie et al. Parameter set Minimum cost solutions value (2011), was excluded from the phenotypic analyses because of a lack of morphological information. Ranunculaceae – molecular/total data Otherwise, c. 10% of the phenotypic data matrix cells 0:1:1:1/0:1:1:1:1 9383/9646 1/1 494/499 were coded as missing information (‘?’). The molecu- 0:2:1:1/0:2:1:1:2 11 491/12 016 1/1 482/491 lar data matrix was compiled for 189 terminal taxa 2:2:2:1/2:2:2:1:3 c18 202/18 991.5 1/1 499/495 and was divided into 11 fragments. At the fragment 2:4:2:1/2:4:2:1:4 23 233/24 249 1/1 477/504 level, c. 36% of the data were missing. The best rep- 3:2:2:1/3:2:2:1:4 18 947/19 972 1/1 501/494 resented fragment was the atpB-rbcL spacer, with 3:4:2:1/3:4:2:1:4 24 224/25 270 1/1 504/520 only 9% of the terminals lacking this fragment in the 3:4:4:1/3:4:4:1:4 31 888/32 884 1/1 478/474 dataset. In contrast, the middle partition of the rbcL- 3:8:4:1/3:8:4:1:8 38 756/40 876 1/1 431/467 accD spacer was the least sampled marker, coded as Clematis – molecular/total data missing for 68% of the terminals. 0:1:1:1/0:1:1:1:1 6666/7163.48 1/1 783/920 Analysis of the phenotypic data resulted in a sin- 0:2:1:1/0:2:1:1:2 7977/8885.86 1/1 847/919 2:2:2:1/2:2:2:1:3 11 503/12 907.13 1/1 849/951 gle most-parsimonious tree with 330.33 steps 2:4:2:1/2:4:2:1:4 14 137/16 018.68 1/1 846/960 (Fig. S3). This tree was completely resolved, but full 3:2:2:1/3:2:2:1:4 12 386/14 255 1/1 834/950 resolution can be expected because of the minute dif- 3:4:2:1/3:4:2:1:4 15 218/17 079.2 1/1 809/958 ferences between taxa in their coded values for con- 3:4:4:1/3:4:4:1:4 17 622/19 498.76 1/1 853/966 tinuous characters. In this situation, it is important 3:8:4:1/3:8:4:1:8 22 557/26 138.12 1/1 715/834 to examine the support values and, especially, their changes caused by the inclusion of continuous Highest stability values are indicated in bold. characters (Goloboff et al., 2006). The majority of

Berberis bealei Epimedium koreanum 98 Dysosma versipellis Glaucidium palmatum Glaucidoideae Hydrastis canadensis Hydrastidoideae Xanthorhiza simplicissima Coptis chinensis Coptidoideae 100 100 100 Coptis japonica

RANUNCULACEAE Leptopyrum fumarioides Thalictroideae 67 Paraquilegia microphylla 99 Paropyrum anemonoides 75 64 Thalictrum dioicum Thalictrum dasycarpum 81 99 Thalictrum javanicum Aquilegia oxysepala 99 Semiaquilegia ecalcarata 93 Semiaquilegia adoxoides 69 Urophysa henryi 100 Dichocarpum dalzielii Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 100 Dichocarpum sutchuenense Enemion raddeanum 99 Isopyrum manshuricum Nigella damascena Aconitum scaposum Ranunculoideae 98 Aconitum septentrionale 52 93 Aconitum racemulosum 100 Aconitum napellus 96 Aconitum pendulum 93 Gymnaconitum gymnandrum 66 Delphinium balansae Delphinium balcanicum Delphinium favargeri Delphinium macropetalum Consolida armeniaca Consolida hellespontica Consolida persica Delphinium barbatum Consolida ajacis 92 Consolida ambigua Delphinium thirkeanum Delphinium anthoroideum Delphinium delavayi Delphinium grandiflorum 97 62 Delphinium exaltatum 63 Delphinium tatsienense Adonis annua Adonis amurensis 92 100 Adonis vernalis 100 Calathodes oxycarpa 100 Calathodes palmata 100 Trollius vaginatus Trollius chosenensis 98 97 Trollius laxus Helleborus foetidus 100 Helleborus multifidus 68 Helleborus thibetanus Asteropyrum cavaleriei Caltha appendiculata 100 Caltha palustris Anemonopsis macrophylla 100 Beesia calthifolia Eranthis hyemalis 100 86 Eranthis stellata Actaea japonica 88 Actaea asiatica 100 Actaea simplex Actaea vaginata Callianthemum taipaicum Halerpestes cymbalaria Oxygraphis glacialis Trautvetteria caroliniensis Myosurus minimus 63 Ranunculus bungei Ranunculus penicillatus Ranunculus trichophyllus Ranunculus recurvatus Ranunculus japonicus 71 Ranunculus cantoniensis Ranunculus polyanthemos Anemone quinquefolia Anemone virginiana Anemone flaccida 98 Anemone hupehensis Pulsatilla patens 93 100 steps 61 100 Pulsatilla cernua Hepatica henryi Hepatica acutilobia 64 64 Hepatica americana Anemoclema glaucifolium Clematis afoliata 99 79 Clematis laurifolia Clematis vitalba 0:1:1:1:1 2:2:2:1:3 3:2:2:1:4 3:4:4:1:4 100 Clematis montana 57 Clematis armandii 58 Clematis hexapetala 0:2:1:1:2 2:4:2:1:4 3:4:2:1:4 3:8:4:1:8 86 96 Clematis terniflora

Figure 1. Sensitivity plots superimposed on the preferred hypothesis of the phylogeny of Ranunculaceae obtained by direct optimization of molecular and phenotypic data under the transformation cost regime 3:4:2:1:4 (gap opening: transversion:transition:gap extension:phenotypic). Numbers below the nodes indicate dynamic jackknife values above 50%. Subfamilies are indicated and Clematis is highlighted.

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 182, 825–867 832 S. LEHTONEN ET AL. nodes lacked jackknife support, but the deepest analysis. The most stable topology was that obtained nodes and the outgroup relationships were generally under the weights 3:4:4:1 (phenotypic data giving supported, including the monophyly of Clematis strong impact with weight 4), the same as with the (99%). However, some of the derived groups also molecular data alone. Despite generally increased received jackknife support above 50%. Searches stability, the backbone of the phylogenetic tree for based only on discretely coded phenotypic characters Clematis still remained highly unstable and unsup- also resulted in a single most-parsimonious tree (269 ported. The section-level clades were largely congru- steps) with high resolution (Fig. S4). These topolo- ent with the molecular results in their species gies generally supported neither the previous mor- composition. Clade A was sister to all the other

phology-based classifications nor the molecular Clematis under four cost regimes (under one of these Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 results obtained here. Four larger groupings were including C. haenkeana C.Presl. otherwise placed congruent with the molecular results: Clades H and elsewhere). Clade G was sister to the remainder of G, and two subclades of Clade L that were placed in the genus under two cost regimes and, only under two independent lineages in the phenotypic analysis two cost regimes, neither one of these clades were in including continuously coded characters. In the phe- that position. Clades D and E were generally notypic analysis excluding continuously coded char- resolved as sisters and Clades J, K and L formed a acters, Clade L was recovered as monophyletic as in highly stable clade. The RI of seven phenotypic char- the molecular analyses, but generally the jackknife acters increased in the total-evidence analysis in support and congruence with molecular results were comparison with the analysis of the phenotypic data lower in comparison with the analysis including the only. These characters were: (C3) pedicel length, continuously coded characters. (C5) achene body length, (C6) achene body length/ Analyses of molecular data resulted in a single width ratio, (C13) presence of cataphylls, (C15) most-parsimonious tree under each studied parame- arrangement of the first non-cotyledonous leaves, ter set (Table 1). The overall tree stability in the sen- (C28) flower sexuality and (C38) pollen aperture sitivity analyses was maximized by parameter set type. The RI of another seven characters remained 3:4:4:1. This topology is presented in Figure S5. All unchanged and the RI of 26 characters decreased studied parameter sets were nearly congruent in (Appendix 3). splitting Anemone s.l. into several clades. These groupings also received high dynamic jackknife support values. Hepatica was resolved as sister to DISCUSSION A. flaccida and Pulsatilla as sister to Knowltonia Salisb. in all analyses. In addition, A. hupehensis RANUNCULACEAE: THE STATE OF PHYLOGENETIC and A. rivularis formed a separate clade. Anemo- UNDERSTANDING clema was a stable and well-supported sister to a We prefer the total-evidence topology in which all monophyletic Clematis.InClematis, 12 stable clades five currently accepted subfamilies were resolved as conceptually matching the section-level classification monophyletic. A number of genera, especially from were recovered. As in previous studies of the genus, the Southern Hemisphere, are missing from our phy- the relationships between these clades remained logenetic analyses, thus rendering it difficult to eval- highly unstable in general and lacked jackknife sup- uate tribal-level classifications and make port. However, one general pattern was that Clade A biogeographical hypotheses. However, most of the was sister to the remainder of the genus under three missing genera are taxonomically uncertain and cost regimes, whereas Clade G was in this position placed in Ranunculeae and Anemoneae. In general, under three other cost regimes, and, under one cost the taxon sampling within genera remains too poor regime, these two clades together formed a sister in our analyses to make strong conclusions about group to other Clematis. Only under one cost regime their monophyly or to revise the taxonomy. Even the neither one of these clades occupied this position. more systematically sampled analysis by Cossard Clematis acerifolia Maxim., C. aethusifolia Turcz. et al. (2016) failed to resolve the relationships and C. otophora Franch. ex Finet & Gag- between tribes. It is evident that our understanding nep. + C. repens Finet & Gagnep. were unstable and of the phylogenetics of Ranunculaceae remains lim- not clearly associated with any of the larger clades. ited. The combined analyses resulted in single most- Both Pfosser et al. (2011) and Ehrendorfer (1995) parsimonious trees (Fig. 2), with improved tree sta- questioned the concept of Anemone s.l. and criticized bility in comparison with the molecular analyses the practice of using Clematis to root Anemone. This alone, under all parameter sets (Table 1). In addi- has come about because, in the early molecular phy- tion, the average jackknife support increased from logenetic analyses of the family (notably Hoot, 1995; 56.9% in the molecular to 63.2% in the combined Johanson, 1995), Clematis was found to be sister to

A Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021

Figure 2. Sensitivity plots superimposed on the preferred hypothesis of the phylogeny of Clematis obtained by direct optimization of molecular and phenotypic data under the transformation cost regime 3:4:4:1:4 (gap opening:transversion:transition:gap extension:phenotypic). Numbers below the nodes indicate dynamic jackknife values above 50%. Section-level clades as recognized in this study are indicated.

Anemone and then seemed to be the most likely can- remarkable that the topological relationships in didate, but genera were poorly represented in these Anemone s.l. recovered in our genus-level tree are early studies. Our results support the notion that fully congruent with a re-rooted Bayesian tree of the Anemone s.l. is a grade leading up to Anemo- much better sampled analysis by Hoot et al. (2012). clema + Clematis. However, our sampling of the rele- A re-evaluation of the available data and phyloge- vant taxa is poor and the same taxa were not netic hypotheses of Anemoneae is urgently needed. included in the family- and genus-level analyses. The topologies obtained using these two datasets are not fully congruent, although the reconstructed relation- GENERAL REMARKS ON THE PHYLOGENETICS OF CLEMATIS ships are generally stable and well supported in both Several major classiﬁcation schemes have been pro- analyses. The incongruence may be largely a result posed for Clematis (e.g. Prantl, 1888; Tamura, 1995; of the different molecular markers used, but it is Johnson, 1997, 2001; Grey-Wilson, 2000; Wang & Li,

B Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021

Figure 2. Continued

2005), of which those by Johnson (1997, 2001) are Table 2. Comparison of the results with previous studies the most congruent with our phylogenetic results. Therefore, we mainly compare our results with his Miikeda classification below (see Table 2). Our results do not et al. Xie et al. support the recognition of subgenera because of poor Clade (2006) (2011) Johnson (2001) support, short branch lengths and lack of morpholog- – ically identifiable units at this level. However, we A Clade IV (Subsection recognize the presence of 12 well-established clades Africanae p.p.) B – Clade VIII p.p. Subsection corresponding, at least conceptually, with the sec- Henryanae tions of previous classifications. Previous authors Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 (+ subsection have further divided some of these sections into sub- Connatae p.p.) sections, and sometimes these have been divided into C Clade 1 Clade VII p.p. Sections Clematis, even narrower formal categories (e.g. Tamura, 1995; Lasiantha, Wang & Li, 2005). We have now reached a level of Tubulosae sampling at which we can propose a preliminary sec- subsection tional classification for Clematis, but we acknowledge Williamsianae that future studies may discover further clades wor- (+ subsection thy of recognition at this rank and that some of our Potaninianae p.p.) clades may prove to be unsupported. Phenotypic D Clade 3 Clade IX Sections Naravelia, synapomorphies for these clades are listed in Naraveliopsis p.p. Table 3. Currently, we are unable to propose a finer E Clade 2 Clades VII Sections scale classification (i.e. subsections) and we doubt p.p. + VIII Archiclematis, that this rank will ever be necessary. During our p.p. + X Connatae p.p., studies, it became clear that a large number of the Pseudanemonae infrageneric names traditionally used in classifica- subsections tions of Clematis are illegitimate in light of the (Africanae p.p.), International Code of Nomenclature (McNeill et al., Fasciculiflorae, 2012). A parallel publication revising the nomencla- Montanae p.p., ture of all the infrageneric names (above the rank of Phlebanthae, species) in Clematis is under preparation and, for Potaninianae p.p. F –– (Subsection Montanae now, we refer to section-level clades by informal p.p.) names only (Clades A–L). G –– Section Cheiropsis Previous molecular phylogenetic studies (Miikeda H Clade 9 Clade II Section Atragene et al., 2006; Xie et al., 2011) concluded that many of I Clade 4 Clade I Sections Meclatis, the morphological characters traditionally considered Bebaeanthera, to be important in Clematis systematics were highly Fruticella homoplasious and not indicative of phylogenetic J Clades Clade III Sections affinity. However, these studies did not evaluate the 7 + 8 Aspidanthera, actual fit of these morphological characters or their Novae-Zeelandiae congruence in the phylogenetic inference with the (Naraveliopsis p.p.) molecular data. On the contrary, our results showed K Clade 5 Clade V Sections Flammula, relatively high RI values for many phenotypic char- Pterocarpa acters, although almost all of them showed some L Clade 6 Clade VI Sections Viticella, homoplasy. The RI of several characters remained Viorna constant or improved in the combined analyses when compared with the analyses of phenotypic data only, Note that most of the section-level names in Johnson suggesting that, through careful re-analysis of char- (2001) are nomenclaturally invalid. p.p., pro parte, section acters, i.e. reciprocal illumination (Hennig, 1966), it names which are written without parentheses indicate may be possible to achieve a much better character that the nomenclatural type is included, whereas section coding and understanding of the morphological evo- names written in parentheses do not include the type. lution in this genus. We specifically tested the sensitivity of our highly variable sequence regions are included in the hypotheses for alternative transformation costs with- analyses and, simultaneously, reveals relationships out removal of data partitions considered to be unre- that appear to be assumption sensitive. Despite the liable a priori. This allows us to pinpoint fact that the phylogenetic hypotheses supported by relationships which appear robust even though the phenotypic data were largely incongruent with

Table 3. Phenotypic synapomorphies supporting section- Table 3. Continued level clades in Clematis on total-evidence topology under parameter costs 3:4:4:1:4 Clade Phenotypic synapomorphies

Clade Phenotypic synapomorphies L C1 Tepal length (mm): 15–20 > 22–25 C9 Growth form: woody > herbaceous A C1 Tepal length (mm): 15–16 > 8–9 C19 Division of lamina: ternate > pinnate C2 Tepal length/width ratio (910): 25–28 > 33–35 C26 Inflorescence architecture: multiflowered C6 Pedicel length (mm): 20 > 15 > one-flowered C18 Leaf texture: herbaceous-papery > C37 Achenes: not rimmed > rimmed leathery-coriaceous Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 C19 Division of lamina: simple-ternate > multicompound C28 Flower sexuality: bisexual > unisexual the molecular hypotheses, particularly in the Clematis B C4 Achene tail (permanent style) length analysis, their inclusion in the sensitivity analyses (mm): 26–27 > 30–40 increased overall phylogenetic stability. This can sug- C6 Pedicel length (mm): 20 > 25–40 gest that the phenotypic data aided DO to resolve C7 Spinule height: 17–20 > 10–11 nodes for which molecular data were unable to provide C11 Foliage: deciduous > evergreen a clear phylogenetic signal. However, it is also possible C27 Flower exposure: erect > nodding that any static dataset, even if phylogenetically ran- C29 Flower opening: flat > urn-shaped dom, has a stabilizing effect in the sensitivity analy- C33 Stamens: glabrous > pubescent ses. It is noteworthy that, although the phenotypic C C1 Tepal length (mm): 15–16 > 10–12 and molecular data were much more congruent in our D C4 Achene tail (permanent style) length family-level analyses, the stabilizing effect of the phe- (mm): 26–27 > 40–50 notypic data was much stronger at the genus level. In C5 Achene body length (mm): 4–5 > 8 both cases, the stability was maximized by the combi- C6 Pedicel length (mm): 17–20 > 53 nation of low gap opening and extension costs with C8 Number of spinules: 13–15 > 16–17 heavily weighted phenotypic transformations. This > C20 Lamina margin: lobed entire suggests that searching the parameter space with low E C4 Achene tail (permanent style) length (mm): gap costs may be fruitful for the sensitivity analyses. – > 26 27 25 We recommend further studies exploring the effects of F C0 Number of tepals: 4 > 5–8 phenotypic data on the sensitivity of the phylogenetic C2 Tepal length/width ratio (910): 25–26 > 17–21 hypotheses under DO. C3 Pedicel length (mm): 30–40 > 55–100 One possible error in our study is uncertainty in C5 Achene body length (mm): 4 > 2–3 species identification. Many of the sequence data C9 Growth form: woody > herbaceous C10 Habit: climbing > not climbing used in this study were taken from GenBank, and it C19 Division of lamina: ternate > simple is well known that misidentifications are common in C31 Tepal indument: pubescent > glabrous large databases (Vilgalys, 2003). In addition, we G C4 Achene tail (permanent style) length (mm): sampled many plants of cultivated origin and, 35–40 > 45–50 despite many being supposedly of natural origin (e.g. C25 Bract connation: free > united botanical garden collections), hybridization may have C37 Achenes: not rimmed > rimmed occurred in cultivation. H C1 Tepal length (mm): 20–23 > 25–40 C2 Tepal length/width ratio (910): 25–26 > 27–30 C3 Pedicel length (mm): 30–40 > 60–100 BIOGEOGRAPHICAL IMPLICATIONS C17 Petioles: free > joined The Himalayas and adjacent southern-central China C19 Division of lamina: ternate > multicompound have traditionally been considered as the centre of C23 Inflorescence position: axillary > terminal origin for Clematis (e.g. Tamura, 1968a, 1995; Bran- C32 Staminodes: absent > present denburg, 2000; Xie et al., 2011). This area is I No phenotypic synapomorphies undoubtedly an important centre of diversity for the J C4 Achene tail (permanent style) length (mm): genus, but our analyses cast some doubt over the – > 26 27 30 hypothesis that it is also the centre of origin of the – > – K C1 Tepal length (mm): 15 20 12 13 genus. Biogeographical scenarios are often built on C18 Leaf texture: herbaceous-papery > the idea that less diverse sister groups occupy areas leathery-coriaceous ancestral for the more diverse sister groups (Heads,

2009). Under this disputable concept, the restricted southern China, Taiwan, Indochina and Malesia. It distribution of Anemoclema in south-western China has several, but not unique, phenotypic synapomor- indeed suggests a Sino-Himalayan origin for Clematis. phies, including evergreen foliage, nodding, urn- Xie et al. (2011) further postulated that the crown shaped ﬂowers, pubescent stamens and several group of Clematis started to diversify during the Late changes in continuously coded characters. The clade Miocene (c. 8 Mya) as a vicariant response to the was resolved in various, mostly derived positions in uplift of the Himalayan mountains and the Tibetan our sensitivity analyses, most frequently as sister to plateau. The split from Anemone s.l. was estimated to Clade C. However, in our preferred tree and under have occurred at 9–44 Mya (Xie et al., 2011), although some other cost regimes in the combined analyses,

the age of the stem lineage remained unestimated the clade appeared in a much deeper phylogenetic Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 because Anemoclema was not sampled. We consider position. both this timeline and these phylogenetic relationships to be problematic under this scenario. Fruit and leaf fossils assigned to Clematis have been reported CLADE C from Eocene–Oligocene deposits of Europe and North Clade C has a broad distribution in the Northern America (Pigg & DeVore, 2005). The actual identity of Hemisphere, expanding into South America. The these fossils remains to be verified, but if they are cor- only phenotypic synapomorphy is the slightly short- rectly identified, they suggest an earlier origin for the ened tepals, but all the species in this clade share a genus. In addition, despite the fact that the backbone woody habit. The clade lacked jackknife support and of the phylogenetic trees remained unstable in our was sensitive to parameter changes in the combined analyses, we found no evidence that the deepest splits analysis, although not as sensitive in the molecular in the genus would have occurred in the Sino-Himala- analyses. The position of the clade in the genus yan region. Instead, our combined analyses variously remains uncertain. placed the Madagascan clade (Clade A), Mediter- This clade includes the type species of the genus, ranean–Himalayan clade (Clade G) or Indo-Malayan C. vitalba L., and therefore is equivalent to section clade (Clade D) as sister to the remaining genus. Clematis. Tamura (1987) included three subsections These splits obviously can be explained by random under this section: Clematis, Dioicae (Prantl) back-and-forth long-distance dispersal events, but Tamura and Pierotianae (Tamura) Tamura. Com- could also indicate a widespread ancestral distribu- pared with later classifications (Tamura, 1995; John- tion around the Tethys Ocean in tropical climes. son, 1997; Grey-Wilson, 2000; Wang & Li, 2005), this Currently, we can only conclude that a better sam- seems to be closest to our results. The subshrub taxa pling of tropical taxa, still largely absent from molec- usually classified in section Tubulosae Decne. are ular datasets, and additional molecular markers are nested in section Clematis (C. tubulosa Turcz., needed in order to obtain a more complete picture of C. heracleifolia DC. and C. stans Siebold & Zucc.). the deeper level phylogenetic relationships and bio- Another surprising addition to the clade is C. japon- geographical patterns in Clematis. ica Thunb., usually classified with C. barbellata Edgew. in section Bebaeanthera Edgew. The Ameri- can taxa (C. denticulata Vell., C. dioica L., C. drum- CLADE A mondii Torr. & A.Gray, C. haenkeana, C. lasiantha This clade consists of species from Madagascar with Nutt., C. ligusticifolia Nutt., C. virginiana L.) all leathery-coriaceous leaves, multicompound lamina, have small white flowers and belong to a derived unisexual flowers and some continuous character subclade with remarkably similar composition to state changes as phenotypic synapomorphies. These subsection Dioicae of Tamura (1987). Clematis character states also occur elsewhere in the genus. lasiantha, the type species of section Lasiantha The monophyly of the clade is stable across the stud- Tamura, is firmly nested in this subclade, suggesting ied cost regimes with a high resampling support. We that it ought to be dissolved. Himalayan C. grata found this clade often among the early diverging lin- Wall. is nested in this subclade with high stability in eages of the genus, as sister to the remainder of the molecular analyses, but not in our combined genus, either alone or in association with some other analyses. species or with Clade D as the next diverging lineage after the first branching Clade G. CLADE D This clade has a tropical distribution in India and CLADE B South East Asia and includes section Naravelia This small clade consists of two species, (Adans.) Baillon, often treated as a separate genus C. leschenaultiana DC. and C. henryi Oliv., from (e.g. de Candolle, 1818; Tamura, 1995; Grey-Wilson,

2000; Fu & Robinson, 2001; Wang & Li, 2005), the CLADE F monotypic section Naraveliocarpa (Tamura) Tamura Clematis acerifolia is a local endemic from China. It and C. loureiriana DC., the type species of section has traditionally been placed in subsection Montanae Naraveliopsis Hand.-Mazz. The current composition Schneider (e.g. Grey-Wilson, 2000; Johnson, 2001; of this clade is congruent with Prantl’s (1888) cir- Wang & Li, 2005), but was found to be a phylogeneti- cumscription of the section Naravelia. Phenotypic cally isolated lineage by Mu & Xie (2011). Our sensi- synapomorphies for the clade include the entire lam- tivity analyses support this latter view and it seems ina margin and several continuously coded character evident that the species needs to be placed in a sepa- changes. Monophyly of the clade is strongly sup- rate section. This is further emphasized by the many ported in this and previous studies (Miikeda et al., phenotypic synapomorphies, including herbaceous Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 2006; Xie et al., 2011), but its phylogenetic positions and not climbing habit, simple lamina, glabrous in these studies is highly variable. In our sensitivity tepals and several changes in continuously coded analyses of the molecular data, this clade was placed characters. among the early diverging groups, associated with Clades A and G under three cost regimes, whereas other cost regimes and the combined analyses gener- CLADE G ally placed clade D in various derived positions in Members of this clade have not been sampled in pre- Clematis. Despite this uncertain position, it is clear vious studies. Clematis cirrhosa L. is Mediterranean that Naravelia cannot be upheld as a distinct genus. and C. napaulensis DC. is from the Himalayas. This small clade has fused bracts, rimmed achenes and a slightly increased achene tail length as phenotypic CLADE E synapomorphies. The fused bracts gave the name to This clade is diverse in temperate East Asia and the section Cheiropsis DC. (from the Greek veιqὸς, hand) Himalayas, from where it also extends into tropical in which most authors (Tamura, 1995; Johnson, Peninsular Malaysia. A subclade with an Afrotropi- 1997, 2001; Grey-Wilson, 2000; Wang & Li, 2005) cal distribution is nested in this clade with a have additionally placed several species lacking such strongly supported sister relationship to C. rehderi- bracts, following de Candolle’s (1818) original, some- ana Craib, a species from western China. The only what tentative, circumscription. These are C. mon- phenotypic synapomorphy for the clade is the tana and its allies, C. fasciculiflora (except Johnson, slightly increased achene tail length. The clade is a 1997, 2001) and C. williamsii A.Gray., all of which mixture of species traditionally classified in numer- clearly do not belong to this clade. Clade G was ous distinct sections or subsections (see Table 2), and recovered under all the cost regimes and in our phe- these species have never been associated before. Nev- notypic analysis, and it also received strong jack- ertheless, the clade is highly stable in our molecular knife support. In the molecular analyses, this clade analyses, although more sensitive in the combined was often placed as sister to the rest of the analyses, albeit the resampling support remained genus, but the total-evidence analyses generally poor. It is noteworthy that most of the species that resolved it in a more derived position in association have been assumed ‘basal’ in the genus by various with Clade H. authors are included in this clade, such as C. alternata Kitam. & Tamura (Tamura, 1955; Johnson, 1997, 2001), C. villosa DC. (Prantl, 1888; Hutchin- CLADE H son, 1920), C. ranunculoides Franch. (Ziman, 1981) Species of this clade are distributed in Eurasia and and C. potaninii Maxim. + C. montana Buch.-Ham. western North America. Previous molecular phylo- ex DC. (Wang & Li, 2005). genetic analyses of Clematis lacked samples of the Several stable and generally well-supported sub- American species. Those that we sampled showed a clades can be recognized in Clade E. These include sister relationship to the Eurasian taxa. The pheno- C. fasciculiflora Franch. with its sister group, which typic synapomorphies of this clade include, in addi- corresponds to subsection Montanae Schneider, the tion to some synapomorphies in continuously coded clade with C. rehderiana as sister to subsection Afri- characters, the joined petioles, multicompound lam- canae M.Johnson (Johnson, 1997, 2001; cf. section ina, terminal inflorescences and presence of stamin- Brachiatae Snoeijer; Snoeijer, 1992) mixed with sec- odes. These states also occur outside of this clade; tion Pseudanemone Prantl, and the two subclades hence, it does not have any unique characteris- associated with C. connata [the type of section Con- tics. The clade is highly stable and also supported natae (Koehne) M.Johns.] and C. lasiandra Maxim. by phenotypic data, but jackknife support was (the type of section Campanella Tamura), respec- not particularly high in the analyses of molecular tively. data.

CLADE I CLADE K This clade is distributed from Greece through Cen- The members of this clade occur from the Mediter- tral Asia and the Himalayas, eastwards to China ranean throughout Asia to Mongolia, Laos and and Korea. The clade is currently lacking phenotypic Japan. This clade has, in addition to slightly short- synapomorphies. We found weak jackknife support ened tepals, leathery-coriaceous leaves as its pheno- in our analyses, but high stability in the sensitivity typic synapomorphy, but not all species in the clade analyses. It unites section Meclatis (Spach) Baill. share this character state, and it has also evolved with most of the species from section Fruticella independently in other lineages. The combined anal- Tamura. It seems that Fruticella ought to be dis- yses indicate a close relationship of this clade with solved as the type species of this section, C. fruticosa Clades J and L. We found no evidence to support the Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Turcz., is nested here with some other members of subsections as classified, for example, by Johnson the section (C. ispahanica Boiss., C. songorica (1997). He included the newly erected monotypic Bunge). Our analyses placed previously unsampled subsection Fasciculiflorae M.Johns. under section C. fruticosa as an early diverging member of this Flammula (Flammula) DC based on the entire leaf- clade. Although it does not share an immediately lets of C. fasciculiflora. Our results are in agreement obvious set of morphological characters with other with the other classifications (Handel-Mazzetti, 1939; species in the clade, except for the yellow colour of Grey-Wilson, 2000; Wang & Li, 2005) in which this its tepals, Shi & Li (2003) reported that it possesses species is placed in or near subsection Montanae polygonal epidermal cells and has stomata on both Schneider (see Clade E). The monotypic section Pte- sides of the leaves, similar to the species placed in rocarpa Tamura, based on C. brachyura Maxim., is section Meclatis. In addition, C. barbellata, the type included in this clade, as already shown in previous species of section Bebaeanthera Edgew., is firmly studies (Miikeda et al., 2006; Xie et al., 2011), and nested in this clade. Clematis pogonandra Maxim., should be synonymized. another species traditionally classified elsewhere, is also a member, as already shown by Xie et al. (2011). CLADE L This clade is widely distributed across the Northern CLADE J Hemisphere. Two horticulturally important sections, The distribution of this clade expands from Taiwan Viticella (Moench) DC and Viorna (Persoon) Baillon, through Malesia to Australia and New Zealand. The are united in this clade, as already shown in earlier slightly increased achene tail length is the only pheno- studies (Miikeda et al., 2006; Xie et al., 2011), typic synapomorphy for the clade, but it is additionally although these studies lacked the type species of characterized by being mostly evergreen and includ- these sections. Despite being widely used, the name ing many unisexual species, both characters which Viorna as an infrageneric rank has been erro- are relatively rare in Clematis. Monophyly of this neously applied to the American C. viorna L. and clade was well supported in Xie et al. (2011) and we its close relatives (e.g. Tamura, 1987; Johnson, found it to be highly stable in the sensitivity analyses. 1997, 2001). Its first use at the sectional rank was Jackknife support remained weak in the molecular by Baillon (1867), who included C. cirrhosa in it, analysis (58%), but was slightly improved in the com- the type species of section Cheiropsis that has bined analysis (62%). The taxa included have tradi- nomenclatural priority, making section Viorna a tionally been classified in different sections: the later synonym of that section. Australian species in Aspidanthera Spach, the species Increased length of tepals, herbaceous habit, pin- from New Zealand in Novae-Zeelandiae M.Johns., nately divided blades, single-flowered inflorescences C. javana DC. in Clematis by Tamura (1995), and rimmed achenes are phenotypic synapomorphies C. tashiroi Maxim. in Naraveliopsis Hand.-Mazz. by for this clade, although these character states also Johnson (1997) and C. akoensis Hayata in subsection occur elsewhere in the genus and some reversals Rectae (Prantl) Schneider by Wang & Li (2005). have occurred in this clade. In addition, most species The position of this clade in the genus remains in the clade have nodding, either campanulate or unstable, but, in the sensitivity analyses, it is nearly urn-shaped, flowers. Support for the monophyly of always associated with Clades L and K. The internal this clade was rather poor in previous studies (Mii- relationships of this clade are stable. Taiwanese spe- keda et al., 2006; Xie et al., 2011) and in our jack- cies C. tashiroi and C. akoensis form a clade in a sis- knife resampling of the molecular data, but the clade ter relationship to the remaining species. The New was constantly resolved under all parameter costs Zealand species form a sister clade to C. ja- and received higher jackknife support in the com- vana + Australian taxa. bined analysis.

This clade consists of a subclade of taxa around representing the entire generic diversity of the fam- C. patens and C. florida in a sister relationship to ily and by the paucity of data from tropical taxa. The the rest of the clade, including, for example, C. viti- monophyly of numerous genera remains untested or cella L. and C. viorna. These two subclades were also poorly established. An often assumed monophyly of a supported by phenotypic data. In the phenotypic broadly defined Anemone s.l. apparently is an arte- analysis excluding continuously coded characters, fact caused by erroneous outgroup selection and poor Clade L was monophyletic, but if continuously coded sampling of non-focal groups. characters were included, the two subclades were not A natural section-level classification is emerging resolved as sisters. Although the subclade containing from the phylogenetic studies of the horticulturally

C. florida is entirely East Asian, the other subclade important genus Clematis. This can be expected to Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 includes, among East Asian species, two species be of value for the breeding of cultivars by hybridiza- extending into Europe (C. viticella and C. integrifolia tion, in addition to the purely scientific interests of L.) and a monophyletic group of North American spe- diversification, biogeography and evolution of the cies. The North American species seem to be closely genus. However, the horticultural ambitions and var- related, given the short branches and poor resolution ious taxonomic opinions and concepts have led to a in this subclade. Clematis viticella is resolved as sis- flood of nomenclaturally invalid names at every ter to the North American species under five cost rank. A thorough nomenclatural revision of all the regimes in the molecular analyses, but this position relevant names is therefore needed before any formal is not supported in the total-evidence analyses. infrageneric classifications can be proposed for the Two synapomorphies possibly shared by Clades K genus. and L are the shortened achene tail and the entire It is generally understood that Clematis originated margins of the first leaves. It seems that the species in the Sino-Himalayan region, from where it purport- in these two clades also have their first non-cotyledo- edly dispersed and reached its modern global distri- nous leaves oppositely arranged. However, these bution, but our results indicate that the clades were resolved as sisters only by one cost biogeographical history of the genus may be more regime in the molecular analyses and by three complex. It now seems possible that the early splits regimes in the total-evidence analyses. in the genus involve not only the Sino-Himalayas, but also the Madagascan, Mediterranean and Indo- malayan regions, all centred around the former INCERTAE SEDIS Tethys Ocean and distributed across tropical–sub- The phylogenetic positions of some species were tropical climates. Distribution in the northern areas found to be highly sensitive to the parameter choice, and in the Americas and continental Africa appears or they did not clearly belong to any of the section- to be of a more recent origin. However, poor support level clades. For instance, East Asian C. aethusifolia, for deeper level nodes and the lack of most tropical was found in a sister relationship to various clades taxa from molecular datasets preclude any formal under different cost regimes. However, in the com- biogeographical analyses at this time. bined analyses, it was often placed in close affinity Phylogenetic studies generally aim at high resolu- with Clade H. We also recognized a small clade con- tion and support. However, we would like to stress sisting of previously unsampled C. otophora and that poorly resolved and unsupported trees are C. repens from China. The phylogenetic association equally, if not more, interesting. Therefore, we advo- of this clade remained unclear in this study. Further cate an approach without filtering the datasets until sampling of the species lacking in our dataset may strongly supported trees emerge from whatever data help to clarify whether these clades deserve to be remain, and we suggest a consideration of the total recognized as sections of their own, or whether they evidence and the alternative scenarios it may sup- more naturally belong to some of those discussed port. above.

ACKNOWLEDGEMENTS CONCLUSIONS This study was financially supported by the Academy Ranunculaceae can be divided into five natural sub- of Finland grant to the first author. The Willi Hen- families, Glaucidioideae (G), Hydrastidoideae (H), nig Society is acknowledged for making TNT freely Coptidoideae (C), Thalictroideae (T) and Ranuncu- available. We are grateful to the collectors who pro- loideae (R), with phylogenetic relationships (G(H(C vided material for this study, in particular Wen-Bin (T + R)))). At lower levels, phylogenetic understand- Yu and Nan Jiang, Ming-Li Zhang and Hong-Xiang ing is hampered by the lack of comparable datasets Zhang, Dr T. Y. Aleck Yang and his assistants, and

© 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 182, 825–867 PHYLOGENETICS OF CLEMATIS 841 to the Royal Botanic Gardens, Kew for providing de Candolle AP. 1818. Ordo primus Ranunculaceae. Regni samples of Clematis. In addition, we would like to Vegetabilis Systema Naturale 1: 127–548. extend our gratitude to Lyndy Broder, Brian Colling- Christenhusz MJM. 2000. De botanische introducties van wood, Ton Hannink, Deborah Hardwick, Lindsey Philipp Franz von Siebold. Dendroflora 37: 4–10. Hatch of Joy Plants Nursery, New Zealand, Mike Clematis on the Web. 2015. Clematis on the Web. Available Miller, Roy Nunn, Rufino Osorio, Helen Poirier, at: clematis.hull.ac.uk/index.cfm (accessed 3 April 2015). Peter Zale, the British Clematis Society, the Herbar- Compton JA, Culham A, Jury SL. 1998. Reclassification of ium of Finnish Museum of Natural History (H) and Actaea to include Cimifuga and Souliea (Ranunculaceae): the Botanical Garden of Turku University. phylogeny inferred from morphology, nrDNA ITS, and cpDNA trnL-F sequence variation. Taxon 47: 593–634. Cossard G, Sannier J, Sauquet H, Damerval C, de Craene Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 LR, Jabbour F, Nadot S. 2016. Subfamilial and tribal relationships of Ranunculaceae: evidence from eight molecular REFERENCES markers. Plant Systematics and Evolution 302: 419–431. Aagesen L. 2005. Direct optimization, affine gap costs, and De Laet J. 2015. Parsimony analysis of unaligned sequence node stability. Molecular Phylogenetics and Evolution 36: data: maximization of homology and minimization of homo- 641–653. plasy, not minimization of operationally defined total cost Aagesen L, Petersen G, Seberg O. 2005. Sequence length or minimization of equally weighted transformations. variation, indel costs, and congruence in sensitivity analy- Cladistics 31: 550–567. sis. Cladistics 21: 15–30. De Laet JE. 2005. Parsimony and the problem of inapplica- Anderson CL, Bremer K, Friis EM. 2005. Dating phyloge- bles in sequence data. In: Albert VA, ed. Parsimony phylo- netically basal eudicots using rbcL sequences and multiple genetics and genomics. New York: Oxford University Press, fossil reference points. American Journal of Botany 92: 148–159. 1737–1748. Dodonaeus R 1554. Cruyde-boeck, in den welcken die ghe- Baillon H 1867. Histoire des plantes, Vol. 1. Paris, London, heele historie, dat es tgheslacht, tfatsoen, naem, natuere, Leipzig: L. Hachette et Cie. cracht ende werckinghe van den cruyden, niet alleen hier te Barniske AM. 2009. Mutational dynamics and phylogenetic lande wassende, maar oock van den anderen vremden in utility of plastid introns and spacers in early branching der medecijnen oorboorlijck. Antwerp: J. van der Loe. eudicots. Dresden: Mathematisch-Naturwissenschaftlichen Donald D. 2009. The International Clematis Register and Fakultat€ der Technischen Universitat€ Dresden. Checklist 2002. Third Supplement. London: The Royal Hor- Bastida JM, Alcantara JM, Rey PJ, Vargas P, Herrera ticultural Society. CM. 2010. Extended phylogeny of Aquilegia: the biogeo- Ehrendorfer F 1995. Evolutionary trends and patterns in graphical and ecological patterns of two simultaneous but the Anemoninae. Plant Systematics and Evolution 9 contrasting radiations. Plant Systematics and Evolution [Suppl.]: 283–293. 284: 171–185. Ehrendorfer F, Ziman SN, Konig€ C, Keener CS, Dutton Bell CD, Soltis DE, Soltis PS. 2010. The age and diversifi- BE, Tsarenko ON, Bulakh EV, Bosßcaiu M, Medail F, cation of the angiosperms re-revisited. American Journal of Kastner€ A. 2009. Taxonomic revision, phylogenetics and Botany 97: 1296–1303. transcontinental distribution of Anemone section Anemone Brandenburg WA. 2000. Meclatis in Clematis: yellow flow- (Ranunculaceae). Botanical Journal of the Linnean Society ering Clematis species. Systematic studies in Clematis L. 160: 312–354. (Ranunculaceae), inclusive of cultonomic aspects. Thesis, Eichler H, Jeanes JA. 2007. Clematis. Flora of Australia Wageningen Universiteit, Wageningen. Online. Canberra: Australian Biological Resources Study. Breitwieser I, Brownsey PJ, Heenan PB, Nelson WA, Available at: http://www.environment.gov.au/biodiversity/abrs/ Wilton AD eds. 2010. Flora of New Zealand Online. Avail- online-resources/flora/main/index.html (accessed 3 April 2015). able at: www.nzflora.info (accessed 3 April 2015). Emadzade K, Lehnebach C, Lockhart P, Horandl€ E. 2010. Brickell CD, Alexander C, David JC, Hetterscheid A molecular phylogeny, morphology and classification of gen- WLA, Leslie AC, Malecot V, Jin X, Cubey JJ; Members era of Ranunculeae (Ranunculaceae). Taxon 59: 809–828. of the Editorial Committee. 2009. International Code of Engell K. 1995. Embryo morphology of the Ranunculaceae. Nomenclature for Cultivated Plants (8th ed.). Scripta Horti- Plant Systematics and Evolution 9: 207–216. culturae 10: 1–184. Essig F. 1991. Seedling morphology in Clematis (Ranuncu- Broughton RE, Stanley SE, Durrett RT. 2000. Quantifica- laceae) and its taxonomic implications. Sida 14: 377–390. tion of homoplasy for nucleotide transitions and transver- Falck D. 2014. Watching them grow: seedling forms in sions and a reexamination of assumptions in weighted Clematis. The Clematis 2014: 136–143. phylogenetic analysis. Systematic Biology 49: 617–627. Farris JS. 1983. The logical basis of phylogenetic analysis. Cai YF, Li SW, Chen M, Jiang MF, Liu Y, Xie YF, Sun In: Platnick NI, Funk VA, eds. Advances in Cladistics II. Q, Jiang HZ, Yin NW, Wang L, Zhang R, Huang CL, New York: Columbia University Press, 7–36. Lei K. 2010. Molecular phylogeny of Ranunculaceae based Farris JS. 1989. The retention index and the rescaled con- on rbcL sequences. Biologia 65: 997–1003. sistency index. Cladistics 5: 417–419.

Farris JS, Albert VA, Kallersj€ o€ M, Lipscomb D, Kluge and related Ranunculeae (Ranunculaceae). International AG. 1996. Parsimony jackknifing outperforms neighbor- Journal of Plant Sciences 169: 433–443. joining. Cladistics 12: 99–124. Hoot SB, Meyer KM, Manning JC. 2012. Phylogeny and eFloras. 2008. Published on the Internet. Available at: reclassification of Anemone (Ranunculaceae), with emphasis http://www.efloras.org (accessed 3 April 2015).Missouri on austral species. Systematic Botany 37: 139–152. Botanical Garden, St. Louis, MO, & Harvard University Hoot SB, Reznicek AA, Palmer JD. 1994. Phylogenetic Herbaria, Cambridge, MA. relationships in Anemone (Ranunculaceae) based on mor- Frost DR, Rodrigues MT, Grant T, Titus TA. 2001. Phy- phology and chloroplast DNA. Systematic Botany 19: 169– logenetics of the lizard genus Tropidurus (Squamata: 200. Tropiduridae: Tropidurinae): direct optimization, descrip- Horandl€ E, Emadzade K. 2011. The evolution and biogeog- tive efficiency, and sensitivity analysis of congruence raphy of alpine species in Ranunculus (Ranunculaceae) – a Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 between molecular data and morphology. Molecular Phylo- global comparison. Taxon 60: 415–426. genetics and Evolution 21: 352–371. Horandl€ E, Paun O, Johansson JT, Lehnebach C, Arm- Fu D, Robinson OR. 2001. Naravelia. In: Wu Z-Y, Raven P, strong T, Chen L, Lockhart P. 2005. Phylogenetic rela- eds. Flora of China, Vol. 6. Beijing: Science Press; St. tionships and evolutionary traits in Ranunculus s.l. Louis, MO: Missouri Botanical Garden Press. (Ranunculaceae) inferred from ITS sequence analysis. Giribet G. 2003. Stability in phylogenetic formulations and Molecular Phylogenetics and Evolution 36: 305–327. its relationship to nodal support. Systematic Biology 52: Hutchinson J. 1920. Clematopsis, a primitive genus of 554–564. Clematideae. Bulletin of Miscellaneous Information, Royal Giribet G, Wheeler WC. 2007. The case for sensitivity: a Botanic Gardens, Kew 1920: 12–22. response to Grant and Kluge. Cladistics 23: 294–296. Jabbour F, Renner SS. 2011. Resurrection of the genus Goloboff PA. 1999. Analyzing large data sets in reasonable Staphisagria J. Hill, sister to all the other Delphinieae times, solutions for composite optima. Cladistics 15: 415– (Ranunculaceae). PhytoKeys 7: 21–26. 428. Jabbour F, Renner SS. 2012. A phylogeny of Delphinieae Goloboff PA, Carpenter JM, Arias JS, Miranda Esqui- (Ranunculaceae) shows that Aconitum is nested within Del- vel DR. 2008a. Weighting against homoplasy improves phinium and that Late Miocene transitions to long life phylogenetic analysis of morphological data sets. Cladistics cycles in the Himalayas and Southwest China coincide with 24: 758–773. bursts in diversification. Molecular Phylogenetics and Evo- Goloboff PA, Farris JS, Nixon KC. 2008b. TNT, a free lution 62: 928–942. program for phylogenetic analysis. Cladistics 24: 774–786. Jensen U, Hoot SB, Johansson JT, Kosuge K. 1995. Sys- Goloboff PA, Mattoni CI, Quinteros AS. 2006. Continuous tematics and phylogeny of the Ranunculaceae – a revised characters analyzed as such. Cladistics 22: 589–601. family concept on the basis of molecular data. Plant Sys- Grant T, Kluge AG. 2005. Stability, sensitivity, science and tematics and Evolution 9 [Suppl.]: 273–280. heurism. Cladistics 21: 597–604. Johanson JT. 1995. A revised chloroplast DNA phylogeny of Gregory WC. 1941. Phylogenetic and cytological studies in the Ranunculaceae. Plant Systematics and Evolution 9 the Ranunculaceae Juss. Transactions of the American [Suppl.]: 253–261. Philosophical Society 31: 443–521. Johnson M. 1997. Slaktet€ Klematis.Sodert€ alje:€ M. Johnsons Grey-Wilson C. 2000. Clematis, the genus. Portland, OR: Plantskola AB. Timber Press. Johnson M. 2001. The genus Clematis.Sodert€ alje:€ Magnus Handel-Mazzetti H. 1939. Plantae sinenses a Dre. H. Smith Johnsons Plantskola AB. annis 1921–1922, 1924 et 1934 lectae. XXXIII. Ranuncu- Katoh K, Standley DM. 2013. MAFFT multiple sequence laceae. Acta Horti Gothoburgensis 13: 37–220. alignment software version 7: improvements in perfor- Heads M. 2009. Globally basal centres of endemism: the Tas- mance and usability. Molecular Biology and Evolution 30: man–Coral Sea region (south-west Pacific), Latin America 772–780. and Madagascar/South Africa. Biological Journal of the Kluge AG. 1989. A concern for evidence and a phylogenetic Linnean Society 96: 222–245. hypothesis of relationships among Epicrates (Boidae, Ser- Hennig W. 1966. Phylogenetic systematics. Urbana, IL: pentes). Systematic Zoology 38: 7–25. University of Illinois Press. Kluge AG. 2004. On total evidence: for the record. Cladistics Hoot SB. 1991. Phylogeny of the Ranunculaceae based on 20: 205–207. epidermal microcharacters and macromorphology. System- Koch NM, Soro IM, Ramırez MJ. 2015. First phylogenetic atic Botany 16: 741–755. analysis of the family Neriidae (Diptera), with a study on Hoot SB. 1995. Phylogeny of the Ranunculaceae based on the issue of scaling continuous characters. Cladistics 31: preliminary atpB, rbcL and 18S nuclear ribosomal DNA 142–165. sequence data. Plant Systematics and Evolution 9 [Suppl.]: Kosuge K, Sawada K, Denda T, Adach J, Watanabe K. 241–251. 1995. Phylogenetic relationships of some genera in the Hoot SB, Kramer J, Arroyo MTK. 2008. Phylogenetic posi- Ranunculaceae based on alcohol dehydrogenase genes. tion of the South American dioecious genus Hamadryas Plant Systematics and Evolution 9 [Suppl.]: 263–271.

Langlet O. 1932. Uber€ Chromosomenverhaltnisse€ und Sys- the London Clay. American Journal of Botany 92: 1650– tematik der Ranunculaceae. Svensk Botanisk Tidskrift 26: 1659. 381–400. Pol D, Escapa IH. 2009. Unstable taxa in cladistic analysis: Lee J, Kim JH, Lee SM, Park SH, Kim J, Ali MA, Lee C, identification and the assessment of relevant characters. Kim G. 2015. Seeds of wild plants of Korea. Available at: Cladistics 25: 515–527. http://www.seedbank.re.kr/ (accessed 3 April 2015). Pons J, Vogler AP. 2006. Size, frequency, and phylogenetic Lehtonen S. 2009. Systematics of the Alismataceae – a mor- signal of multiple-residue indels in sequence alignment of phological evaluation. Aquatic Botany 91: 279–290. introns. Cladistics 22: 144–156. Liu K, Nelesen S, Raghavan S, Linder CR, Warnow T. Prantl K. 1888. Beitrage€ zur Morphologie und Systematik 2009. Barking up the wrong treelength: the impact of gap der Ranunculaceen. Botanische Jahrbucher€ fur System- penalty on alignment and tree accuracy. IEEE/ACM Trans- atik, Pflanzengeschichte und Pflanzengeographie 9: Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 actions on Computational Biology and Bioinformatics 6: 7– 225–273. 21. Rasmussen H. 1979. The genus Knowltonia (Ranuncu- Loconte H, Estes JR. 1989. Phylogenetic systematics of laceae). Opera Botanica 53: 1–44. Berberidaceae and Ranunculaceae (Magnoliidae). System- Ro KE, Keener CS, McPheron BA. 1997. Molecular phy- atic Botany 14: 565–579. logenetic study of the Ranunculaceae: utility of the Luo Y, Zhang FM, Yang QE. 2005. Phylogeny of Aconitum nuclear 26S ribosomal DNA in inferring intrafamilial rela- subgenus Aconitum (Ranunculaceae) inferred from ITS tionships. Molecular Phylogenetics and Evolution 8: sequences. Plant Systematics and Evolution 252: 11–25. 117–127. McNeill J, Barrie FR, Buck WR, Demoulin V, Greuter Sanders JG. 2010. Program note, Cladescan, a program for W, Hawksworth DL, Herendeen PS, Knapp S, Mar- automated phylogenetic sensitivity analysis. Cladistics 26: hold K, Prado J, van Prud’homme RW, Smith GF, 114–116. Wiersema JH, Turland NJ, eds. 2012. International Code Sanderson MJ, Boss D, Chen D, Cranston KA, Wehe A. of Nomenclature for algae, fungi and plants (Melbourne 2008. The PhyLoTA browser: processing GenBank for Code). Regnum Vegetabile 154. Koenigstein: Koeltz Scien- molecular phylogenetics research. Systematic Biology 57: tific Books. 335–346. Miikeda O, Kita K, Handa T, Yukawa T. 2006. Phyloge- Schuettpelz E, Hoot SB. 2004. Phylogeny and biogeogra- netic relationships of Clematis (Ranunculaceae) based on phy of Caltha (Ranunculaceae) based on chloroplast and chloroplast and nuclear DNA sequences. Botanical Journal nuclear DNA sequences. American Journal of Botany 91: of the Linnean Society 152: 153–168. 247–253. Mu L, Xie L. 2011. A preliminary study on the systematic Schuettpelz E, Hoot SB, Samuel R, Ehrendorfer F. position of Clematis acerifolia based on the analyses of ITS 2002. Multiple origins of Southern Hemisphere Anemone and plastid regions. Journal of Beijing Forestry University (Ranunculaceae) based on plastid and nuclear sequence 33: 49–55. data. Plant Systematics and Evolution 231: 143–151. Muller€ J, Muller€ KF, Neinhuis C, Quandt D. 2007. Schulmeister S. 2003. Simultaneous analysis of basal PhyDE – Phylogenetic Data Editor. Available at: http:// Hymenoptera (Insecta): introducing robust-choice sensitiv- www.phyde.de. (accessed 11 March 2013). ity analysis. Biological Journal of the Linnean Society 79: van der Neut A, Pfeiffer E. 1982. Introductie en genee- 245–275. skrachtige werking van Europese soorten van Clematis L. in Shi JH, Li LQ. 2003. Leaf epidermal feature in Clematis botanische literatuur tot omstreeks 1753. Wageningen: (Ranunculaceae) with reference to its systematic signifi- Landbouwniversiteit Wageningen. cance. Acta Botanica Sinica 45: 257–268. Nixon KC. 1999. The parsimony ratchet, a new method for Simmons MP. 2004. Independence of alignment and rapid parsimony analysis. Cladistics 15: 407–414. tree search. Molecular Phylogenetics and Evolution 31: Padial JM, Grant T, Frost DR. 2014. Molecular systemat- 874–879. ics of terraranas (Anura: Brachycephaloidea) with an Simmons MP, Muller€ KF, Norton AP. 2010. Alignment of, assessment of the effects of alignment and optimality crite- and phylogenetic inference from, random sequences: the ria. Zootaxa 3825: 1–132. susceptibility of alternative alignment methods to creating Paun O, Lehnebach C, Johansson JT, Lockhart P, artifactual resolution and support. Molecular Phylogenetics Horandl€ E. 2005. Phylogenetic relationships and biogeog- and Evolution 57: 1004–1016. raphy of Ranunculus and allied genera in the Mediter- Slomba JM, Garey JR, Essig FB. 2004. The actin I intron– ranean and the European alpine system (Ranunculaceae). a phylogenetically informative DNA region in Clematis Taxon 54: 911–930. (Ranunculaceae). Sida 21: 879–886. Pfosser M, Sun BY, Stuessy TF, Jang CG, Guo YP, Tae- Snoeijer W. 1992. A suggested classification for the genus jin K, Hwan KC, Kato H, Sugawara T. 2011. Phylogeny Clematis. The Clematis 1992: 7–20. of Hepatica (Ranunculaceae) and origin of Hepatica maxima Snoeijer W. 2008. Clematis cultivar group classification with Nakai endemic to Ullung Island, Korea. Stapfia 95: 16–27. identifying key and diagrams. Gouda: Wim Snoeijer. Pigg KB, DeVore ML. 2005. Paleoactaea gen. nov. (Ranun- Soza VL, Brunet J, Liston A, Salles Smith P, Di Stilio culaceae) fruits from the Paleogene of North Dakota and VS. 2012. Phylogenetic insights into the correlates of dioecy

in meadow–rues (Thalictrum, Ranunculaceae). Molecular Wheeler WC. 1995. Sequence alignment, parameter sensitiv- Phylogenetics and Evolution 63: 180–192. ity, and the phylogenetic analysis of molecular data. Sys- Spagna JC, Alvarez-Padilla F. 2008. Finding an upper tematic Biology 44: 321–331. limit for gap costs in direct optimization parsimony. Cladis- Wheeler WC. 1996. Optimization alignment: the end of mul- tics 24: 787–801. tiple sequence alignment in phylogenetics? Cladistics 12: Sun G, Dilcher DL, Wang H, Chen Z. 2011. A eudicot from 1–9. the early Cretaceous of China. Nature 471: 625–628. Wheeler WC, Lucaroni N, Hong L, Crowley LM, Varon Sun H, McLewin W, Fay MF. 2001. Molecular phylogeny of A. 2015. POY version 5: phylogenetic analysis using Helleborus (Ranunculaceae), with an emphasis on the East dynamic homology under multiple optimality criteria. Asian–Mediterranean disjunction. Taxon 50: 1001–1018. Cladistics 31: 189–196. Tamura M. 1955. Systema clematidis asiae orientalis. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Science Reports (Osaka University) 4: 43–55. and direct sequencing of fungal ribosomal RNA genes for Tamura M. 1968a. Morphology, ecology and phylogeny of phylogenetics. In: Innis MA, Gelfund DH, Sninsky JJ, the Ranunculaceae VII. Science Reports (Osaka University) White TJ, eds. PCR protocols: a guide to methods and 16: 21–43. applications. San Diego, CA: Academic Press, 315–322. Tamura M. 1968b. A new species from China. Acta Phyto- Wiegand KM. 1895. The structure of the fruit in the order taxonomica et Geobotanica 23: 36–38. Ranunculacœ. Proceedings of the Royal Microscopical Soci- Tamura M. 1987. A classification of genus Clematis. Acta ety 16: 69–100. Phytotaxonomica et Geobotanica 38: 33–44. Wikstrom€ N, Savolainen V, Chase MW. 2004. Angiosperm Tamura M. 1993. Ranunculaceae. In: Kubitzki K, Rohwer divergence times: congruence and incongruence between JG, Bittrich V. eds. The families and genera of vascular fossils and sequence divergence estimates. In: Donoghue plants, Vol. 2. Berlin: Springer-Verlag, 563–583. PCJ, Smith MP, eds. Telling the evolutionary time: molecu- Tamura M. 1995. Clematidinae. In: Hiepko P, ed. Die natur-€ lar clocks and the fossil record. Boca Raton, FL: CRC Press, lichen Pflanzenfamilien. Zwei Aufl 17a (4). Berlin: Duncker 142–165. und Humbolt, 365–388. Xie L, Li LQ. 2012. Variation of pollen morphology, and its Tamura M. 1997. Taxonomic studies of the Ranunculaceae: implications in the phylogeny of Clematis (Ranunculaceae). retrospect and prospect. Memoirs of the School of B.O.S.T. Plant Systematics and Evolution 298: 1437–1453. of Kinki University 2: 69–85. Xie L, Wen J, Li LQ. 2011. Phylogenetic analyses of Clema- Vaidya G, Lohman DJ, Meier R. 2011. SequenceMatrix: tis (Ranunculaceae) based on sequences of nuclear riboso- concatenation software for the fast assembly of multi-gene mal ITS and three plastid regions. Systematic Botany 36: datasets with character set and codon information. Cladis- 907–921. tics 27: 171–180. Yang QE, Gong X, Gu ZJ, Wu QA. 1993. A karyomorpho- Vilgalys R. 2003. Taxonomic misidentification in public logical study of five species in the Ranunculaceae from Yun- DNA databases. New Phytologist 160: 4–5. nan, with a special consideration on systematic positions of Wang W, Hu H, Xiang XG, Yu SX, Chen ZD. 2010. Phylo- Asteropyrum and Calathodes. Acta Botanica Yunnanica 15: genetic placements of Calathodes and Megaleranthis 179–190. (Ranunculaceae): evidence from molecular and morphologi- Yang TYA. 2009. Additional remarks on Ranunculaceae in cal data. Taxon 59: 1712–1720. Taiwan (7). Two new taxa and a new distributional record Wang W, Lu AM, Ren Y, Endress ME, Chen ZD. 2009. of Clematis in Taiwan. Botanical Studies 50: 499–510. Phylogeny and classification of Ranunculales: evidence Yuan Q, Yang QE. 2006a. Cytology, palynology, and taxon- from four molecular loci and morphological data. Perspec- omy of Asteropyrum and four other genera of Ranunculaceae. tives in Plant Ecology, Evolution and Systematics 11: Botanical Journal of the Linnean Society 152: 15–26. 81–110. Yuan Q, Yang QE. 2006b. Tribal relationships of Beesia, Wang WT. 2004. A revision of Clematis sect. Aspidanthera Eranthis and seven other genera of Ranunculaceae: evi- s.l. (Ranunculaceae) (Continued). Acta Phytotaxonomica dence from cytological characters. Botanical Journal of the Sinica 42: 97–135. Linnean Society 150: 267–289. Wang WT. 2007. A revision of Clematis sect. Viticella Zhang Y, Kong HH, Yang QE. 2014. Phylogenetic relation- (Ranunculaceae). Guihaia 27: 1–28. ships and taxonomic status of the monotypic Chinese genus Wang WT, Bartholomew B. 2001. Clematis. In: Wu ZY, Anemoclema (Ranunculaceae). Plant Systematics and Evo- Raven P, eds. Flora of China, Vol. 6. Beijing: Science Press; lution 301: 1335–1344. St. Louis, MO: Missouri Botanical Garden Press. Zhao L, Wang E, Ren Y, Bachelier JB. 2012. Floral Wang WT, Li LQ. 2005. A new system of classification of development in Asteropyrum (Ranunculaceae): implications the genus Clematis (Ranunculaceae). Acta Phytotaxonomica for its systematic position. Annales Botanici Fennici 49: Sinica 43: 431–488. (in Chinese with English abstract). 31–42. Wang ZF, Ren Y. 2008. Ovule morphogenesis in Ranuncu- Ziman SM. 1981. Analysis of the phylogenetic relations in laceae and its systematic significance. Annals of Botany the subtribe Clematidinae (Ranunculaceae). Ukrayins’kyi 101: 447–462. Botanichnyi Zhurnal 48: 4–13.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Data S1. Tree topologies obtained in this study in Nexus format. Figure S1. The strict consensus of the four equally parsimonious trees found in the analysis of the phenotypic data of Ranunculaceae. Numbers below the nodes indicate jackknife values above 50%. The results from the sensitivity analyses of molecular data are plotted on the tree. Figure S2. Sensitivity plots superimposed on the phylogenetic tree for Ranunculaceae obtained by direct opti-

mization of molecular data under the transformation cost regime 3:4:2:1 (gap opening:transversion:transition:- Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 gap extension). Numbers below the nodes indicate dynamic jackknife values above 50%. Figure S3. The most-parsimonious tree found in the analysis of phenotypic data of Clematis. Numbers below the nodes indicate jackknife values above 50%. The results from the sensitivity analyses of molecular data are plotted on the tree. Figure S4. The most-parsimonious tree found in the analysis of phenotypic data of Clematis excluding the continuously coded characters. Numbers below the nodes indicate jackknife values above 50%. The results from the sensitivity analyses of molecular data are plotted on the tree. Figure S5. Sensitivity plots superimposed on the Clematis phylogeny obtained by direct optimization of molecular data under the transformation cost regime 3:4:4:1 (gap opening:transversion:transition:gap extension). Numbers below the nodes indicate dynamic jackknife values above 50%.

Appendix 1. GenBank numbers of the sequences used in the family-level analyses

Taxon 26S ITS matK trnH-psbA rbcL trnL-trnF

Aconitum napellus L. – AF216544 JN895413 FN675819 EU053898 – Aconitum pendulum N.Busch – AY150235 JF331793 ––JF331728 Aconitum racemulosum Franch. AY954473 AY150233 FJ626484 – AY954488 AY150248 Aconitum scaposum Franch. – JF975818 JF953037 JN043766 JF940670 AY150246 Aconitum septentrionale Koelle – AF216552 JF331795 – JF331678 JF331730

Actaea asiatica Hara FJ626436 Z98276 FJ626485 – FJ626575 – Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Actaea japonica Thunb. – FJ525888 AF353578 ––EU186383 Actaea simplex Wormsk. AY954469 GQ351363 AB044753 – EU053908 EU186384 Actaea vaginata (Maxim.) J.Compton FJ626452 Z98274 FJ626507 – FJ626588 – Adonis amurensis Regel & Radde AY954472 AF454927 FJ626486 – EU053900 FJ626534 Adonis annua L. – AY148280 JN895412 – JN891273 AH012590 Adonis vernalis L. U52625 AJ347910 AJ414340 ––AJ413302 Anemoclema glaucifolium FJ626437 KR909768 FJ626487 KR909860 FJ626576 FJ626535 (Franch.) W.T.Wang Anemone ﬂaccida F.Schmidt – AY055391 AB110530 AB117604 AB117001 – Anemone hupehensis (Lemoine) Lemoine FJ626438 HQ440207 FJ626488 – FJ626577 FJ626536 Anemone quinquefolia L. U52619 GU257978 GU257980 ––– Anemone virginiana L. – FJ639898 HQ593171 HQ596597 HQ589955 – Anemonopsis macrophylla AF131289 Z98275 FJ626489 – EU053904 – Siebold & Zucc. Aquilegia oxysepala Trautv. & C.A.Mey FJ626439 JX233769 EU827665 – EF437140 EF437097 Asteropyrum cavaleriei J.R. AY954466 – FJ626490 – AF079453 FJ626537 Drumm. & Hutch. Beesia calthifolia Ulbr. AY954468 AJ496613 FJ626492 – AF079452 – Berberis bealei Fortune FJ626461 FJ424229 KF176554 – FJ449858 FJ626558 Calathodes oxycarpa Sprague – HQ440197 HQ440174 ––HQ440186 Calathodes palmata Hook.f. & Thomson – HQ440198 HQ440175 ––HQ440187 Callianthemum taipaicum W.T.Wang FJ626441 –––FJ626580 FJ626539 Caltha appendiculata Pers. – AY365385 FJ626493 – AF307908 AY365366 Caltha palustris L. FJ626442 AY515398 AY515232 HQ596617 FJ626581 FJ626540 Clematis afoliata J.Buch. – AJ347911 AB110512 AB117586 AB116982 AJ413303 Clematis armandii Franch. GQ305993 FJ572047 – GU732659 FJ449852 – Clematis hexapetala Pall. – JN809681 – GU732678 EU053909 – Clematis laurifolia (Wall. ex – GU732646 AB110526 GU732727 –– Hook. f. & Thomson) M.Johns. Clematis montana Buch.-Ham. ex DC. – FJ424227 – GU732691 FJ449855 – Clematis terniﬂora DC. – JN809682 AB110502 GU732689 GU135147 – Clematis vitalba L. – GU732641 AB110525 AB117599 FR865151 – Consolida ajacis (L.) Schur FJ626443 AF216557 JF331756 ––JF331689 Consolida ambigua (L.) P.W.Ball U52626 AF258682 –––AF258629 & Heywood Consolida armeniaca (Stapf ex – JF331883 JF331757 ––JF331690 Huth) F.C.Schrad. Consolida hellespontica (Boiss.) Chater – JF331889 JF331763 ––JF331697 Consolida persica (Boiss.) Schrodinger€ – JF331897 JF331773 ––JF331708 Coptis chinensis Franch. AY954482 JF423949 AB695566 AB727577 – AB551237 Coptis japonica Makino – AB695605 AB695561 AB727576 – AB551236 Delphinium anthoroideum Boiss. – JF331875 JF331747 – JF331666 JF331680 Delphinium balansae Boiss. & Reut. – JF331931 JF331797 ––JF331732 Delphinium balcanicum Pawł. – JF331949 JF331798 ––JF331733 Delpihium barbatum Bunge – JF331876 JF331748 ––JF331681 Delphinium delavayi Franch. – JF976227 – JN044402 – AF258659

Appendix 1. Continued

Taxon 26S ITS matK trnH-psbA rbcL trnL-trnF

Delphinium exaltatum Aiton U52627 AF258731 –––AF258651 Delphinium favargeri C.Blanche, – JF331968 JF331800 ––JF331735 Molero & Simon Pall. Delphinium grandiﬂorum L. – AF258761 AY515249 ––AY150256 Delphinium macropetalum DC. – JF331996 JF331804 ––JF331739 Delphinium tatsienense Franch. – JF976258 JF953661 JN044436 – JN573591 Delphinum thirkeanum Boiss. – JF331879 JF331753 ––JF331686 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Dichocarpum dalzielii (J.R.Drumm. – EF437115 EF437129 ––EF437098 & Hutch.) W.T.Wang & Hsiao Dichocarpum sutchuenense AY954479 HQ844061 EF437130 ––EF437099 (Franch.) W.T.Wang & Hsiao Dysosma versipellis (Hance) FJ626458 EU592023 DQ478619 – AF079454 EU017369 M.Cheng ex T.S.Ying Enemion raddeanum Regel AY954478 JX233767 AB069846 ––JQ691533 Epimedium koreanum Nakai FJ626459 JX040542 AB069837 – L75869 JN975653 Eranthis hyemalis Salisb. U52633 AY148284 AJ414342 ––JF505891 Eranthis stellata Maxim. AY954467 JF505766 AY515248 ––JF505893 Glaucidium palmatum Siebold & Zucc. AF389267 X83837 AB627930 ––EF437113 Gymnaconitum gymnandrum – AY150238 JF331792 – JF331677 JF331727 (Maxim.) Wei Wang & Z.D.Chen Halerpestes cymbalaria Greene AY954475 AY680196 AY954237 ––FJ626544 Helleborus foetidus L. U52634 AJ347892 AJ414322 ––AJ413283 Helleborus multiﬁdus Vis. – AY515403 AY515246 ––AJ413288 Helleborus thibetanus Franch. AY954470 AJ347904 FJ626500 ––AJ413296 Hepatica acutiloba DC. – AM267285 DQ994677 HQ596595 HQ589952 – Hepatica americana (DC.) Ker.Gawl. U52621 AY055386 AF542590 – EU053901 – Hepatica henryi Steward FJ626445 AM267290 DQ994674 ––FJ626545 Hydrastis canadensis L. AF389268 X83840 AB069849 ––EF437112 Isopyrum manshuricum Kom. FJ626446 JX233768 EF437133 ––JQ691532 Leptopyrum fumarioides Rchb. – AJ347912 EU827652 ––JX573531 Myosurus minimus L. FJ626447 AJ347913 FJ626502 ––AJ413305 Nigella damascena L. FJ626448 EU699446 FJ626504 ––AY150260 Oxygraphis glacialis (Fisch. FJ626449 AY680198 FJ626505 ––FJ626547 ex DC.) Bunge Paraquilegia microphylla FJ626450 JX233771 EF437136 ––EF437105 J.R.Drumm. & Hutch. Paropyrum anemonoides Ulbr. – EF437118 EF437132 ––EF437101 Pulsatilla cernua (Thunb.) AY954477 JN811071 AB110531 AB117605 – FJ626548 Bercht. & J.Presl Pulsatilla patens (L.) Mill. – AM267280 – EU285569 – GQ244564 Ranunculus cantoniensis DC. AY954474 JQ439725 HM565150 ––DQ410736 Ranunculus bungei Steud. FJ626440 – FJ626491 – FJ626579 GU733873 Ranunculus japonicus Thunb. – EU370102 AY954200 AB244028 – DQ410744 Ranunculus penicillatus – AY680070 AY954130 HQ894439 –– (Dumort.) Bab. Ranunculus polyanthemos L. – AY680121 AY954185 ––HM590338 Ranunculus recurvatus Poir. U52631 AY680118 AY954175 HQ596810 –– Ranunculus trichophyllus – AY680067 AY954133 HQ894441 –– Chaix ex. Vil Semiaquilegia adoxoides Makino FJ626451 JX233770 EF437137 ––JX573529 Semiaquilegia ecalcarata AY954481 U75657 EU827654 – AY954495 EF437096 (Maxim.) Sprague & Hutch.

Appendix 1. Continued

Taxon 26S ITS matK trnH-psbA rbcL trnL-trnF

Thalictrum dasycarpum U52614 JX233681 –––JX573451 Fisch. & Ave-Lall. Thalictrum dioicum L. U52611 JX233685 HQ593464 HQ596861 – JX573455 Thalictrum javanicum Blume AY954480 EF437124 DQ478615 ––EF437107 Trautvetteria caroliniensis (Walter) Vail U52630 FJ639909 FJ626508 ––FJ626550 Trollius chosenensis Ohwi AF131285 AY515399 FJ597983 ––HQ440188 (= Megaleranthis saniculifolia Ohwi) Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Trollius laxus Salisb. AY954471 AY148266 FJ626509 ––AH012570 Trollius vaginatus Hand.-Mazz. – HQ440205 HQ440184 ––HQ440195 Urophysa henryi Ulbr. FJ626453 EF437126 EF437139 ––EF437109 Xanthorhiza simplicissima Marshall AF389270 – HE651033 ––EF437111

Sequences produced in this study are marked in bold.

Appendix 2. Genbank numbers of the sequences used in the genus-level analyses

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Anemoclema glaucifolium KR909717 KR909768 KR909860 KR909901 KR909804 – (Franch.) W.T.Wang – JN005 (KUN) Anemone ﬂaccida F.Schmidt – AB115462 AB120212 AB117604 AB116909 AB110530 AB117001 GenBank Anemone hupehensis (Lemoine) AY055418 HQ440207 ––FJ626488 – Lemoine – GenBank Anemone parviﬂora Michx.– FJ639851 FJ639887 ––JN966091 – GenBank Anemone quinquefolia L.– JF810696 GU257978 ––GU257980 – GenBank Anemone rivularis L.– GenBank GU732490 GU732571 GU732652 GU732733 –– Clematis acerifolia Maxim. – GU732491 GU732572 GU732653 GU732734 –– GenBank Clematis aethusifolia Turcz. – GU732492 GU732573 GU732654 GU732735 –– GenBank Clematis afoliata Buchanan – AB115443 AB120193 AB117586 AB116890 AB110512 AB116982 GenBank Clematis akebioides (Maxim.) GU732493 GU732574 GU732655 GU732736 –– H.J.Veitch – GenBank Clematis akoensis Hayata *1 – KR909731 KR909786 ––KR909805 KR909691 TUR601927 Clematis akoensis Hayata *2 – ––– –KR909806 KR909692 TYY21417 (TNM) Clematis albicoma Wherry *1 – KR909751 KR909789 ––KR909807 KR909673 TUR601918 Clematis albicoma Wherry *2 – KR909750 –– –KR909808 KR909674 TUR601953 Clematis alpina (L.) Mill. *1 – GU732494 GU732575 GU732656 GU732737 –– GenBank Clematis alpina (L.) Mill. *2 – GU732495 GU732576 GU732657 GU732738 –– GenBank Clematis alternata Kitam. & AB115440 AB120190 AB117582 AB116887 AB110509 AB116979 Tamura – GenBank

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis apiifolia DC. *1 – AB115430 AB120180 AB117572 AB116877 AB110499 AB116969 GenBank Clematis apiifolia DC. *2 – GU732496 GU732577 GU732658 GU732739 –– GenBank Clematis aristata R.Br. ex KR909759 KR909787 KR909861 – KR909809 KR909698 Ker.Gawl. – TUR601939 Clematis armandii Franch. – GU732497 GU732578 GU732659 GU732740 –– Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 GenBank Clematis baldwinii Torr. & KR909738 KR909796 KR909862 – KR909810 KR909671 A.Gray – TUR601943 Clematis barbellata Edgew. – KR909720 KR909778 KR909863 – KR909811 – Jackson 70 (K) Clematis brachiata Thunb. *1 – GU732498 GU732579 GU732660 GU732741 –– GenBank Clematis brachiata Thunb. *2 – GU732499 GU732580 GU732661 GU732742 –– GenBank Clematis brachiata Thunb. *3 – GU732500 GU732581 GU732662 GU732743 –– GenBank Clematis brachiata Thunb. *4 – GU732501 GU732582 GU732663 GU732744 –– GenBank Clematis brachyura Maxim. – AB115454 AB120204 AB117596 AB116901 AB110522 AB116993 GenBank Clematis brevicaudata DC. – GU732502 GU732583 GU732664 GU732745 –– GenBank Clematis buchananiana DC. – ––KR909864 – KR909812 – TUR601962 Clematis cadmia Buch.-Ham. KR909732 KR909771 KR909865 – KR909813 KR909670 ex Hook.f. & Thomson – TUR601922 Clematis carrizoensis D.Estes – KR909745 –– –KR909814 KR909684 TUR601947 Clematis cf. insidiosa Baill. – GU732563 GU732644 GU732725 GU732806 –– GenBank Clematis chinensis Osbeck – GU732503 GU732584 GU732665 GU732746 –– GenBank Clematis chinensis Osbeck var. KR909726 –– –KR909815 KR909689 tatushanensis T.Y.A.Yang – TYY21701 (TNM) Clematis chrysocoma Franch. *1 – GU732504 GU732585 GU732666 GU732747 –– GenBank Clematis chrysocoma Franch. *2 – GU732505 GU732586 GU732667 GU732748 –– GenBank Clematis chrysocoma Franch. *3 – GU732506 GU732587 GU732668 GU732749 –– GenBank Clematis cirrhosa L. var. KR909763 KR909774 KR909866 – KR909816 – cirrhosa – TUR601933 Clematis cirrhosa L. var. KR909764 KR909775 KR909867 – KR909817 KR909696 balearica (Rich) Willk. – RBG Kew 1973–19713 Clematis columbiana (Nutt.) KR909721 KR909769 KR909868 – KR909818 KR909707 Torr. & A.Gray var. columbiana – TUR601937

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis columbiana var. ––– –KR909820 KR909709 tenuiloba (A.Gray) J.S.Pringle *1 – RBG Kew 1991–463 Clematis columbiana var. KR909722 –– –KR909819 KR909708 tenuiloba (A.Gray) J.S.Pringle *2 – TUR601948 Clematis confusa Grey-Wilson – GU732507 GU732588 GU732669 GU732750 –– Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 GenBank Clematis connata DC. – KR909766 – KR909869 – KR909821 KR909703 TUR601964 Clematis courtoisii Hand.-Mazz. KR909736 – KR909870 – KR909822 KR909666 – TUR601924 Clematis crassifolia Benth. – AB115444 AB120194 AB117585 AB116891 AB110513 AB116983 GenBank Clematis crassifolia Benth. – ––– –KR909823 KR909690 TYY19987 (TNM) Clematis crispa L. *1 – – KR909795 KR909871 – KR909824 KR909680 TUR601936 Clematis crispa L. *2 – GenBank GU732508 GU732589 GU732670 GU732751 –– Clematis cunninghamii Turcz. – KR909756 – KR909872 ––KR909693 TUR601938 Clematis delavayi Franch. *1 – AB115452 AB120202 AB117594 AB116899 AB110520 AB116991 GenBank Clematis delavayi Franch. *2 – GU732509 GU732590 GU732671 GU732752 –– GenBank Clematis delicata H.Eichler ex KR909758 – KR909873 ––– W.T.Wang – TUR601931 Clematis denticulata Vell. – – KR909799 KR909874 – KR909825 KR909716 Chase 567 (K) Clematis dioica L. – – KR909800 KR909875 – KR909826 KR909715 Christenhusz 5579 (BM) Clematis drummondii Torr. & GU732510 GU732591 GU732672 GU732753 –– A.Gray – GenBank Clematis eichleri (Tamura) AB115459 AB120209 AB117601 AB116906 AB110527 AB116998 Tamura – GenBank Clematis elisabethae–carolae KR909754 KR909780 KR909876 ––KR909688 Greuter – TUR601919 Clematis erectisepala L.Xie, GU732511 GU732592 GU732673 GU732754 –– J.H.Shi & L.Q.Li – GenBank Clematis fasciculiflora Franch. – AB115453 AB120203 AB117595 AB116900 AB110521 AB116992 GenBank Clematis finetiana H.Lev. & GU732512 GU732593 GU732674 GU732755 –– Vaniot – GenBank Clematis flammula L. – KR909753 KR909781 KR909877 – KR909827 – TUR601928 Clematis florida Thunb. – AB115436 AB120186 AB117578 AB116883 AB110505 AB116975 GenBank Clematis foetida Raoul – KR909757 – KR909878 ––KR909697 TUR601935 Clematis fremontii S.Watson – KR909752 KR909792 KR909879 – KR909828 KR909681 TUR601952 Clematis fruticosa Turcz. – ––KR909880 – KR909829 KR909712 TUR601945

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis fusca Turcz. *1 – AB115429 AB120179 AB117571 AB116876 AB110535 AB116968 GenBank Clematis fusca Turcz. *2 – GU732513 GU732594 GU732675 GU732756 –– GenBank Clematis gentianoides DC. – AB115460 AB120210 AB117602 AB116907 AB110528 AB116999 GenBank Clematis glycinoides DC. – GU732514 GU732595 GU732676 GU732757 –– Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 GenBank Clematis gracilifolia Rehder & – KR909801 KR909881 – KR909830 – E.H.Wilson – Fliegner et al. 1362 (K) Clematis grata Wall. – KR909728 –– –KR909832 KR909713 TUR601963 Clematis gratopsis W.T.Wang – – FJ424226 – ––– GenBank Clematis haenkeana C.Presl. – ––– –JQ588883 – GenBank Clematis hancockiana Maxim. – KR909735 KR909772 KR909882 – KR909833 KR909668 TUR601920 Clematis henryi Oliv. – KR909730 KR909797 ––KR909834 KR909701 TUR601944 Clematis heracleifolia DC. – GU732515 GU732596 GU732677 GU732758 –– GenBank Clematis hexapetala Pall. *1 – GU732516 GU732597 GU732678 GU732759 –– GenBank Clematis hexapetala Pall. *2 – AB115449 AB120199 AB117591 AB116896 AB110534 AB116988 GenBank Clematis hirsutissima Pursh – KR909748 –– –KR909835 KR909672 TUR601940 Clematis huchouensis Tamura – KR909734 KR909784 KR909883 ––– TUR601926 Clematis ibarensis Baker – KR909767 KR909798 ––KR909836 KR909706 Andriamahay & Rakotoarisoa s.n. (K) Clematis integrifolia L. *1 – GU732517 GU732598 GU732679 GU732760 –– GenBank Clematis integrifolia L. *2 – GU732518 GU732599 GU732680 GU732761 –– GenBank Clematis ispahanica Boiss. – KR909718 KR909777 KR909884 – KR909837 KR909711 TUR601942 Clematis japonica Thunb. – AB115437 AB120187 AB117579 AB116884 AB110506 AB116976 GenBank Clematis javana DC. – GenBank – DQ499136 – ––– Clematis koreana Kom. – ––KR909885 – KR909838 KR909710 Kirkham, Flanagan & Boyce 51 (K) Clematis ladakhiana Grey- KR909719 KR909776 KR909886 – KR909803 – Wilson – TUR601932 Clematis lanuginosa Lindl. & KR909733 KR909770 ––KR909839 KR909669 Paxton – TUR601925 Clematis lasiandra Maxim. *1 – AB115435 AB120185 AB117577 AB116882 AB110504 AB116974 GenBank

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis lasiandra Maxim. *2 – GU732519 GU732600 GU732681 GU732762 –– GenBank Clematis lasiantha Nutt. *1 – AB115450 AB120200 AB117592 AB116897 AB110518 AB116989 GenBank Clematis lasiantha Nutt. *2 – GU732520 GU732601 GU732682 GU732763 –– GenBank Clematis lasiantha Nutt. *3 – GU732521 GU732602 GU732683 GU732764 –– Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 GenBank Clematis laurifolia (Wall. ex AB115458 AB120208 AB117600 AB116905 AB110526 AB116997 Hook.f. & Thomson) M.Johns. *1 – GenBank Clematis laurifolia (Wall. ex GU732565 GU732646 GU732727 GU732808 –– Hook.f. & Thomson) M.Johns. *2 – GenBank Clematis leschenaultiana DC. *1 KR909729 KR909785 KR909887 – KR909840 KR909702 – TUR601917 Clematis leschenaultiana DC. *2 – GU732522 GU732603 GU732684 GU732765 –– GenBank Clematis ligusticifolia Nutt. *1 – AB115451 AB120201 AB117593 AB116898 AB110519 AB116990 GenBank Clematis ligusticifolia Nutt. *2 – GU732523 GU732604 GU732685 GU732766 –– GenBank Clematis ligusticifolia Nutt. *3 – GU732524 GU732605 GU732686 GU732767 –– GenBank Clematis linearifolia Steud. – GU732525 GU732606 GU732687 GU732768 –– GenBank Clematis longicauda Steud. ex KR909725 KR909782 KR909888 – KR909841 KR909705 A.Rich – Mason s.n. (K) Clematis loureiroana DC. – GU732526 GU732607 GU732688 GU732769 –– GenBank Clematis meyeniana Walp. – GU732528 GU732609 GU732690 GU732771 –– GenBank Clematis microphylla DC. – KR909760 – KR909889 – KR909842 KR909694 TUR601956 Clematis montana Buch.-Ham. GU732529 GU732610 GU732691 GU732772 –– ex DC. *1 – GenBank Clematis montana Buch.-Ham. GU732530 GU732611 GU732692 GU732773 –– ex DC. *2 – GenBank Clematis napaulensis DC. – KR909762 KR909773 KR909890 – KR909843 KR909695 TUR601934 Clematis nobilis Nakai – AB115456 AB120206 AB117598 AB116903 AB110524 AB116995 GenBank Clematis ochotensis (Pall.) Poir. – AB115432 AB120182 AB117574 AB116879 AB110501 AB116971 GenBank Clematis ochroleuca Aiton – KR909747 KR909793 KR909891 – KR909844 KR909682 TUR601951 Clematis orientalis L. *1 – AB115446 AB120196 AB117588 AB116893 AB110515 AB116985 GenBank Clematis orientalis L. *2 – GU732531 GU732612 GU732693 GU732774 –– GenBank Clematis orientalis L. *3 – GU732532 GU732613 GU732694 GU732775 –– GenBank

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis otophora Franch. ex KR909723 – KR909892 – KR909845 KR909700 Finet & Gagnep. – TUR601921 Clematis paniculata J.F.Gmel. – KR909755 KR909788 KR909893 – KR909846 – TUR601941 Clematis parviloba Gardner & KR909765 –– –KR909847 KR909704 Champ. – TYY21538 (TNM) Clematis patens C.Morren & AB115434 AB120184 AB117576 AB116881 AB110503 AB116973 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Decne. – GenBank Clematis peterae Hand.-Mazz. *1 – GU732533 GU732614 GU732695 GU732776 –– GenBank Clematis peterae Hand.-Mazz. *2 – GU732534 GU732615 GU732696 GU732777 –– GenBank Clematis phlebantha KR909761 KR909779 ––KR909848 KR909699 L.H.J.Williams – TUR601957 Clematis pierotii Miq. – AB115441 AB120191 AB117583 AB116888 AB110510 AB116980 GenBank Clematis pinnata Maxim. – GU732535 GU732616 GU732697 GU732778 –– GenBank Clematis pitcheri Torr. & A.Gray GU732536 GU732617 GU732698 GU732779 –– – GenBank Clematis pogonandra Maxim. – GU732537 GU732618 GU732699 GU732780 –– GenBank Clematis potaninii Maxim. *1 – AB115448 AB120198 AB117590 AB116895 AB110517 AB116987 GenBank Clematis potaninii Maxim. *2 – GU732538 GU732619 GU732700 GU732781 –– GenBank Clematis pubescens Hugel€ ex GU732539 GU732620 GU732701 GU732782 –– Endl.– GenBank Clematis ranunculoides Franch. – GU732540 GU732621 GU732702 GU732783 –– GenBank Clematis recta L. *1 – GenBank GU732541 GU732622 GU732703 GU732784 –– Clematis recta L. *2 – GenBank GU732542 GU732623 GU732704 GU732785 –– Clematis rehderiana Craib – GU732543 GU732624 GU732705 GU732786 –– GenBank Clematis repens Finet & KR909724 –– –KR909849 – Gagnep. – TUR601946 Clematis reticulata Walter – GU732544 GU732625 GU732706 GU732787 –– GenBank Clematis rutoides W.T.Wang *1 – GU732545 GU732626 GU732707 GU732788 –– GenBank Clematis rutoides W.T.Wang *2 – GU732546 GU732627 GU732708 GU732789 –– GenBank Clematis rutoides W.T.Wang *3 – GU732547 GU732628 GU732709 GU732790 –– GenBank Clematis serratifolia Rehder – AB115455 AB120205 AB117597 AB116902 AB110523 AB116994 GenBank Clematis siamensis J.R.Drumm. GU732548 GU732629 GU732710 GU732791 –– & Craib – GenBank Clematis sibirica Mill. – GU732549 GU732630 GU732711 GU732792 –– GenBank Clematis socialis Kral – KR909749 –– –KR909850 KR909675 TUR601923

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis songorica Bunge *1 – – KC415707 KT001240 ––– GenBank Clematis songorica Bunge *2 – – KC415716 KT001241 ––– GenBank Clematis songorica Bunge *3 – – KC415712 KT001242 ––– GenBank Clematis sp. Birmingham – KR909743 – KR909899 – KR909802 KR909679 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 TUR601929 Clematis sp. Roma – TUR601930 KR909742 – KR909900 – KR909851 KR909687 Clematis sp. Wen9476 – GU732564 GU732645 GU732726 GU732807 –– GenBank Clematis stans Siebold & Zucc. – AB115438 AB120188 AB117580 AB116885 AB110507 AB116977 GenBank Clematis strigillosa Baker – GU732550 GU732631 GU732712 GU732793 –– GenBank Clematis taiwaniana Hayata – GU732551 GU732632 GU732713 GU732794 –– GenBank Clematis tangutica Korsh. *1 – AB115445 AB120195 AB117587 AB116892 AB110514 AB116984 GenBank Clematis tangutica Korsh. *2 – GU732552 GU732633 GU732714 GU732795 –– GenBank Clematis tashiroi Maxim. – AB115442 AB120192 AB117584 AB116889 AB110511 AB116981 GenBank Clematis terniﬂora DC. – AB115433 AB120183 AB117575 AB116880 AB110502 AB116972 GenBank Clematis terniﬂora DC. var. GU732527 GU732608 GU732689 GU732770 –– mandshurica (Rupr.) Ohwi – GenBank Clematis texensis Buckley *1 – AB115447 AB120197 AB117589 AB116894 AB110516 AB116986 GenBank Clematis texensis Buckley *2 – GU732553 GU732634 GU732715 GU732796 –– GenBank Clematis tibetana Kuntze *1 – GU732554 GU732635 GU732716 GU732797 –– GenBank Clematis tibetana Kuntze *2 – GU732555 GU732636 GU732717 GU732798 –– GenBank Clematis trichotoma Nakai – ––KR909894 ––– TUR601961 Clematis tubulosa Turcz.– RBG KR909727 KR909783 KR909895 – KR909852 KR909714 Kew 1984–10 Clematis uncinata Benth. – GU732556 GU732637 GU732718 GU732799 –– GenBank Clematis uncinata Benth. var. AB115439 AB120189 AB117581 AB116886 AB110508 AB116978 ovatifolia (Maxim) Tamura – GenBank Clematis versicolor Small ex. KR909744 –– –KR909853 KR909683 Rydb. – TUR601950 Clematis villosa DC. – GenBank AB115461 AB120211 AB117603 AB116908 AB110529 AB117000 Clematis viorna L. *1 – KR909739 – KR909896 – KR909854 KR909676 TUR601958 Clematis viorna L. *2 – KR909740 KR909790 KR909897 – KR909855 KR909677 TUR601959

Appendix 2. Continued

Taxon atpB-rbcL ITS psbA-trnH-trnQ rpoB-trnC matK rbcL-accD

Clematis viorna L. *3 – KR909741 KR909791 ––KR909856 KR909678 TUR601954 Clematis virginiana L. *1 – GU732557 GU732638 GU732719 GU732800 –– GenBank Clematis virginiana L. *2 – GU732558 GU732639 GU732720 GU732801 –– GenBank Clematis virginiana L. *3 – GU732559 GU732640 GU732721 GU732802 –– Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 GenBank Clematis vitalba L. *1 – AB115457 AB120207 AB117599 AB116904 AB110525 AB116996 GenBank Clematis vitalba L. *2 – GU732560 GU732641 GU732722 GU732803 –– GenBank Clematis vitalba L. *3 – GU732561 GU732642 GU732723 GU732804 –– GenBank Clematis vitalba L. *4 – GU732562 GU732643 GU732724 GU732805 –– GenBank Clematis viticaulis E.S.Steele – KR909746 KR909794 ––KR909857 KR909685 TUR601955 Clematis viticella L. – ––– –KR909858 KR909686 TUR601949 Clematis williamsii A.Gray – AB115431 AB120181 AB117573 AB116878 AB110500 AB116970 GenBank Clematis xiangguiensis KR909737 – KR909898 – KR909859 KR909667 W.T.Wang – TUR601960 Hepatica nobilis Schreb. var. AB115464 AB120214 – AB116911 AB110532 AB117003 japonica Nakai – GenBank Hepatica transsilvanica Fuss – FJ639869 FJ639905 ––DQ994670 – GenBank Knowltonia sp. – GenBank AB115465 AB120215 AB117606 AB116912 AB110533 AB117004 Pulsatilla cernua (Thunb.) GU732566 GU732647 GU732728 GU732809 AB110531 AB117002 Bercht. & J.Presl – GenBank Pulsatilla chinensis (Bunge) GU732569 GU732650 GU732731 GU732812 –– Regel – GenBank Pulsatilla dahurica (Fisch. ex GU732567 GU732648 GU732729 GU732810 –– DC.) Spreng. – GenBank Pulsatilla sp. – GenBank GU732570 GU732651 GU732732 GU732813 –– Pulsatilla turczaninovii Krylov GU732568 GU732649 GU732730 GU732811 –– & Serg. – GenBank

New sequences produced in this study are shown in bold type.

Appendix 3. Phenotypic character descriptions and discussion. Retention index (RI) values were calculated for each phenotypic character with TNT and are given for phenotypic/preferred total- evidence topologies in square brackets at the end of each character description. [RI: 0.78/0.75] Characters used in family-level analysis. Continuously coded characters R0 Number of petals. [RI: 0.67/0.66] R1 Number of tepals. [RI: 0.46/0.38] Discretely coded characters

R2 Habit: (0) perennial; (1) annual. Wang et al. (2009). [RI: 0.69/0.56] Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 R3 Growth form: (0) shrub; (1) liana; (2) herb. Loconte & Estes (1989). [RI: 0.83/0.67] R4 Rhizome: (0) absent; (1) present, non-tuberous; (2) present, tuberous. Coded as non-additive. Wang et al. (2009). [RI: 0.61/0.57] R5 Xylem: (0) V-shaped; (1) non V-shaped. Wang et al. (2009). [RI: 1.00/0.00] R6 Leaf arrangement: (0) alternate; (1) opposite. Wang et al. (2009). [RI: 1.00/1.00] R7 Involucre on peduncle: (0) absent; (1) present. Hoot (1991). [RI: 0.82/0.82] R8 Involucral leaves: (0) contiguous with flower, calyx-like; (1) separate from flower. [RI: 1.00/1.00] R9 Involucral leaves: (0) free; (1) fused. [RI: 1.00/1.00] R10 Stomatal length: (0) < 35 μm; (1) > 35 μm. Hoot (1991). [RI: 1.00/1.00] R11 Inflorescence: (0) solitary or occasionally with one to two additional lateral flowers; (1) spike, raceme, or botryoid; (2) richly branched (panicle or compound inflorescence or spikes, racemes botryoids). Coded as non-additive. Wang et al. (2009). [RI: 0.70/0.60] R12 Flower sexuality: (0) bisexual; (1) unisexual. [RI: 0.50/0.00] R13 Flower opening: (0) erect; (1) nodding. [RI: 0.42/0.50] R14 Floral symmetry: (0) actinomorphic; (1) zygomorphic. Hoot (1991). [RI: 1.00/1.00] R15 Perianth phyllotaxy: (0) whorled; (1) spiral. Wang et al. (2009). [RI: 0.43/0.57] R16 Petals: (0) laminate; (1) tubular. Hoot (1991). [RI: 0.82/0.79] R17 Nectar petals: (0) absent; (1) present. Wang et al. (2009). [RI: 0.63/0.63] R18 Tepals: (0) not spurred and enclosing staminodia; (1) spurred, enclosing staminodia. Hoot (1991). [RI: 1.00/1.00] R19 Tepals: (0) colourful, longer than petals; (1) green and/or shorter than petals; (2) like petals. Coded as non-additive. Hoot (1991). [RI: 0.83/0.74] R20 Number of stamens: (0) 6; (1) 5; (2) more than 10. [RI: 1.00/1.00] R21 Androecium phyllotaxy: (0) whorled; (1) spiral. Wang et al. (2009). [RI: 0.50/0.50] R22 Carpel number: (0) single; (1) less than five; (2) more than five. Coded as additive. [RI: 0.78/0.76] R23 Carpel fusion: (0) not fused; (1) fused to some extent. Hoot (1991). [RI: 0.33/0.50] R24 Permanent style in fruit: (0) glabrous; (1) plumose. [RI: 1.00/0.90] R25 Style: (0) not elongated in fruit; (1) strongly elongated in fruit. [RI: 1.00/0.86] R26 Ovule number: (0) one; (1) more than two. [RI: 0.97/0.94] R27 Ovule curvature: (0) anatropous; (1) hemitropous. Zhao et al. (2012). [RI: 0.78/0.89] R28 Integument number: (0) unitegmic; (1) bitegmic. Loconte & Estes (1989); Wang et al. (2009); Wang & Ren (2008). [RI: 0.93/0.93] R29 Micropyle type: (0) formed from both integuments; (1) formed from inner integument only. Coded inapplicable for taxa with unitegmic ovules. Loconte & Estes (1989); Wang et al. (2009); Wang & Ren (2008). [RI: 0.67/0.67] R30 Embryo size: (0) small; (1) minute. Wiegand (1895); Engell (1995). [RI: 0.85/0.84] R31 Embryo differentiation: (0) weak differentiation; (1) well-differentiated. Wiegand (1895); Engell (1995). [RI: 0.88/0.83] R32 Fruit: (0) dry; (1) fleshy. [RI: 0.67/0.50] R33 Fruit dehiscence: (0) indehiscent; (1) dehiscing along ventral suture; (2) dehiscing along both sutures. Fleshy fruits coded inapplicable. Coded as additive. [RI: 0.91/0.91] R34 Fruit: (0) sessile; (1) stalked. [RI: 0.50/0.33] R35 Pollen aperture type: (0) irregular; (1) colpate; (2) porate. Coded as non-additive. Wang et al. (2009). [RI: 0.63/0.50] R36 Benzylisoquinolines: (0) present; (1) absent. Wang et al. (2009). [RI: 0.91/0.89] R37 Ranunculins: (0) absent; (1) present. Wang et al. (2009). [RI: 0.96/0.96] R38 Diterpene alkaloids: (0) absent; (1) present. Wang et al. (2009). [RI: 1.00/1.00]

Appendix 3. Continued

R39 Ferulic and sinapic acids: (0) absent; (1) present. Wang et al. (2009). [RI: 0.96/0.96] R40 Chromosome type: (0) T-type; (1) R-type. Langlet (1932); Yang et al. (1993); Yuan & Yang (2006a,b); Wang et al. (2009). [RI: 1.00/1.00] R41 Base chromosome number: (0) n = 6; (1) n = 7; (2) n = 8 (3) n = 9; (4) n = 10; (5) n = 12; (6) n = 13; (7) n = 14; (8) n = 16; (9) n = 18. A speciﬁc step matrix was applied for this character. [RI: 0.52/0.43]

Characters used in genus-level analysis. The main body of characters was coded from Johnson (2001) with reference to Wang & Bartholomew (2001). The outer ends of the total range were used for the continuously Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 coded characters for which the ranges differed in the sources. Additional data sources were: Tamura (1968b); Grey-Wilson (2000); Wang (2004, 2007); Eichler & Jeanes (2007); eFloras (2008); Yang (2009); Breitwieser et al. (2010); Lee et al. (2015). [RI: 0.66/0.63]

Continuously coded characters C0 Number of tepals. [RI: 0.74/0.66] C1 Tepal length (mm). [RI: 0.65/0.64] C2 Tepal length/width ratio (910). [RI: 0.47/0.46] C3 Pedicel length (mm). [RI: 0.53/0.54] C4 Achene tail (permanent style) length (mm). [RI: 0.45/0.42] C5 Achene body length (mm). [RI: 0.64/0.65] C6 Achene body length/width ratio (910). [RI: 0.47/0.58] C7 Spinule height. Xie & Li (2012) [RI: 0.78/0.66] C8 Number of spinules. Xie & Li (2012) [RI: 0.59/0.58] Discretely coded characters C9 Growth form: (0) woody; (1) herbaceous. [RI: 0.83/0.73] C10 Habit: (0) climbing; (1) not climbing. [RI: 0.80/0.64] C11 Foliage: (0) deciduous; (1) evergreen. [RI: 0.70/0.61] C12 Basal leaf rosette: (0) present; (1) absent. [RI: 1.00/1.00] C13 Presence of cataphylls: (0) absent; (1) present. Essig (1991); Falck (2014). [RI: 0.89/0.93] C14 Margin of the first non-cotyledon leaf: (0) entire; (1) not entire. Essig (1991); Falck (2014). [RI: 0.91/0.88] C15 Arrangement of the first non-cotyledonous leaves: (0) alternate; (1) opposite. Essig (1991); Falck (2014). [RI: 0.86/0.91] C16 Leaf position at adult stage: (0) alternate; (1) opposite. [RI: 0.92/0.92] C17 Petioles: (0) free; (1) joined. [RI: 0.62/0.46] C18 Leaf texture: (0) herbaceous-papery; (1) leathery-coriaceous. [RI: 0.74/0.56] C19 Division of lamina: (0) simple; (1) ternate; (2) pinnate; (3) multicompound. Coded as non-additive. [RI: 0.78/0.64] C20 Lamina margin: (0) entire; (1) lobed. [RI: 0.92/0.68] C21 Dentation at lamina margin: (0) absent; (1) present. [RI: 0.91/0.82] C22 Tendrils: (0) absent; (1) terminal leaf segment reduced to a tendril; (2) terminal segment and the uppermost lateral segment pair reduced to tendrils; (3) whole leaf reduced to a tendril. Coded as non-additive. [RI: 0.87/0.87] C23 Inflorescence position: (0) terminal; (1) axillary. [RI: 0.87/0.78] C24 Number of bracts: (0) two; (1) three; (2) four. Coded as non-additive. [RI: 1.00/1.00] C25 Bract connation: (0) united; (1) free. [RI: 0.86/0.86] C26 Inflorescence architecture: (0) one-flowered; (1) three-flowered; (2) multiflowered. Coded as non-additive. [RI: 0.81/0.79] C27 Flower exposure: (0) erect; (1) nodding. [RI: 0.95/0.79] C28 Flower sexuality: (0) unisexual; (1) bisexual. [RI: 0.78/0.81] C29 Flower opening: (0) flat; (1) campanulate; (2) urn-shaped. Coded as non-additive. [RI: 0.85/0.70] C30 Tepals: (0) straight; (1) recurved. [RI: 0.86/0.75] C31 Tepal indument: (0) glabrous; (1) pubescent. [RI: 0.60/0.37] C32 Staminodes: (0) absent; (1) present. [RI: 0.81/0.81]

Appendix 3. Continued

C33 Stamens: (0) glabrous; (1) pubescent. [RI: 0.88/0.80] C34 Stamen connectives: (0) not protruding; (1) protruding. [RI: 0.83/0.61] C35 Style elongation: (0) not elongated in fruit; (1) strongly elongated in fruit. [RI: 0.88/0.75] C36 Fruit indument: (0) glabrous; (1) plumose tail; (2) completely hairy; (3) hairy achene with a glabrous tail. Coded as non-additive. [RI: 0.58/0.42] C37 Achenes: (0) not rimmed; (1) rimmed. [RI: 0.77/0.62] C38 Pollen aperture type: (0) tricolpate; (1) pantoporate; (2) polycolpate; (3) eupantocolpate. Coded as non-additive. Xie & Li (2012). [RI: 0.60/0.65] C39 Base chromosome number: (0) n = 7; (1) n = 8; (2) n = 14 (3) n = 16; (4) n = 24. A speciﬁc step matrix was Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 applied to this character. [RI: 0.50/0.50]

Appendix 4. Phenotypic data matrix for family-level analyses

1111111111 2222222222 3333333333 44 Taxa 0 1 23456789 0123456789 0123456789 0123456789 01

Aconitum napellus 2 5 022?00-- 1100111110 2110001011 0101010011 12 Aconitum pendulum 2 5 022?00-- 1100111110 2110001011 0101010011 12 Aconitum racemulosum 2 5 022?00-- 1100111110 2110001011 0101010011 12 Aconitum scaposum 2 5 021?00-- 1100111110 2110001011 0101010011 12 Aconitum septentrionale 2 5 021?00-- 1100111110 2110001011 0101010011 12 Actaea asiatica 2–10 3–5 021?00-- 1100010001 210-001011 101-011000 12 Actaea japonica 2–10 3–5 021?00-- 1200010001 210-001011 101-011000 1? Actaea simplex 2–10 3–5 021?00-- 1100010001 210-001011 101-011000 1? Actaea vaginata 2–10 3–5 021?00-- 1100010001 210-001011 101-011000 1? Adonis amurensis 10 5–8 021?00-- 1000010001 2120000111 0100010001 12 Adonis annua 3–10 5–8 120?00-- 1000010001 2120000111 0100010001 12 Adonis vernalis 10 5–8 021?00-- 1000010001 2120000111 0100010001 12 Anemoclema glaucifolium 0 5 021?a110 110001--0- 212011000- ??000?110? 12 Anemone ﬂaccida 05–6 02100110 100001--0- 212000000- 0100011100 11 Anemone hupehensis 0 5 02100110 120001--0- 212010000- 0100011100 12 Anemone quinquefolia 0 5 02100110 100001--0- 212000000- 0100011100 18 Anemone virginiana 0 5 02100110 100001--0- 212000000- 0100011100 12 Anemonopsis macrophylla 10–13 7–10 021?00-- ?101010102 2110001?11 1101011000 12 Aquilegia oxysepala 5 5 021000-- 0101001101 2110001011 1001010000 01 Asteropyrum cavaleriei 5–8 5 020?00-- ?000010100 2120001011 ??010?0?00 12 Beesia calthifolia 0 5 021?00-- ?c0001--0- 210-001011 ??01111?00 12 Berberis bealei 6 6 001000-- 0201000102 000-001?10 001-10000? ?7 Calathodes oxycarpa 0 5 021?00-- ?00001--0- 2120001??? ??0101?0?? 12 Calathodes palmata 0 5 021?00-- ?00001--0- 2120001??? ??0101?0?? 1? Callianthemum taipaicum 9–13 5–10 021?00-- ?000010101 2120001011 ??000?1000 1? Caltha appendiculata 0 5 021000-- 100001--0- 2120001011 1001010000 15 Caltha palustris 0 5 021000-- 110001--0- 2120001011 1001020000 18 Clematis afoliata 04–5 011010-- 111001--0- 212011000- 01000?1100 12 Clematis armandii 04–6 011010-- 120001--0- 212011000- 0100011100 1? Clematis hexapetala 04–8 021010-- 110001--0- 212011000- 0100011100 12 Clematis laurifolia 8–12 4 011?10-- 1100010001 2120110??? ??0002?1?? 1? Clematis montana 0 4 011010-- 110001--0- 212011000- 0100011100 12 Clematis terniﬂora 0 4 011010-- 120001--0- 212011000- 0100011100 18 Clematis vitalba 0 4 011010-- 120001--0- 212011000- 01000?1100 12 Consolida ajacis 1 5 120?00-- 1100111110 210--01011 0101010011 12 Consolida ambigua 1 5 120?00-- 1100111110 210--01011 0101010011 12

Appendix 4. Continued

1111111111 2222222222 3333333333 44 Taxa 0 1 23456789 0123456789 0123456789 0123456789 01

Consolida armeniaca 1 5 120?00-- 1100111110 210--01011 0101010011 1? Consolida hellespontica 1 5 120?00-- 1100111110 210--01011 0101010011 12 Consolida persica 1 5 120?00-- 1100111110 210--01011 0101010011 11 Coptis chinensis 5–10 5 021?00-- 0200011100 2120001011 1001120000 03 Coptis japonica 5–10 5 021?00-- 02a0011100 2120001011 1001120000 03 Delphinium anthoroideum 1 5 120?00-- 1100111110 210-001011 01010?00?1 1? Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Delphinium balansae 4 5 020000-- 1100111110 2110-01011 0101010011 12 Delphinium balcanicum 4 5 120000-- 1100111110 2110-01011 0101010011 12 Delphinium barbatum 1 5 120?00-- 1100111110 210-001011 01010?00?1 12 Delphinium delavayi 4 5 021000-- 1100111110 2110-01011 0101010011 12 Delphinium exaltatum 4 5 021000-- 1100111110 2110-01011 0101010011 1? Delphinium favargeri 4 5 120000-- 1100111110 2110-01011 0101010011 12 Delphinium grandiﬂorum 4 5 021000-- 1100111110 2110-01011 0101010011 12 Delphinium macropetalum 4 5 120000-- 1100111110 2110-01011 0101010011 12 Delphinium tatsienense 4 5 021000-- 1100111110 2110-01011 0101010011 1? Delphinium thirkeanum 1 5 120?00-- 1100111110 210-001011 01010?00?1 1? Dichocarpum dalzielii 5 5 021?00-- ?101011100 2111001011 ??010100?? 05 Dichocarpum sutchuenense 5 5 021?00-- ?100011100 2111001011 ??010100?? 0? Dysosma versipellis 6 6 022000-- ?b01000001 000-001?10 ??1-1110?? ?0 Enemion raddeanum 0 5 021?00-- ?10001--0- 2110001??? ??01010000 0? Epimedium koreanum 4 8 021000-- ?101001101 000-001?10 ??020100?? ?0 Eranthis hyemalis 4–11 5–8 022?0110 1000011100 2120001?11 1001110000 18 Eranthis stellata 4–11 5–8 022?0110 1000011100 2120001?11 1001110000 18 Glaucidium palmatum 0 4 021100-- ?00000--0- 2111001?11 ?10201100? 04 Gymnaconitum gymnandrum 2 5 120?00-- 1100111110 2120001?11 01010?00?? 12 Helleborus foetidus 5–15 5 021000-- 1201011100 211100100- 1001010100 18 Helleborus multiﬁdus 5–15 5 021000-- 1001011100 211100100- 1001010100 18 Helleborus thibetanus 5–15 5 021000-- 1001011100 211100100- 1001010100 18 Hepatica acutiloba 05–12 02100100 100001--0- 2120000?0- 0100011100 11 Hepatica americana 05–12 02100100 100001--0- 2120000?0- 0100011100 11 Hepatica henryi 0 6 021?0100 100001--0- 2120000?0- 0100011100 17 Hydrastis canadensis 0 3 021100-- 000000--0- 2120001?10 101-0100?? 06 Isopyrum manshuricum 5 5 022?00-- 0200011100 21a0001?11 1001010000 01 Leptopyrum fumarioides 5 5 120?a0-- ?200011100 2120001?0- 1001010000 01 Nigella damascena 5–10 5–25 120?00-- 1000011100 2121001011 01010?0011 10 Oxygraphis glacialis 57–16 020?00-- ?000010101 212000000- ??000?1??? 12 Paraquilegia microphylla 5 5 021?00-- ?100010100 2120001??? ??010100?? 01 Paropyrum anemonoides 5 5 021?a0-- ?200011100 2110001?1? ??0101?0?? 0? Pulsatilla cernua 05–6 021?0111 100101--0- 2120110?0- 0100021100 12 Pulsatilla patens 05–8 02100111 100101--0- 2120110?0- 01000?1100 12 Halerpestes cymbalaria 5–8 5 020?00-- ?000010101 2120000??? ??000111?? 12 Myosurus minimus 5 5 120?00-- ?100010101 2120000?0- 10000?1100 12 Ranunculus bungei 5 5 020?00-- ?000010101 212000010- ??000?1?0? 12 Ranunculus cantoniensis 5 5 120?00-- 1000010101 212000010- 01000?1100 18 Ranunculus japonicus 5 5 021?00-- 1200010101 212000010- 0100011100 11 Ranunculus penicillatus 5 5 02??00-- 1000010101 212000010- 01000?1100 12 Ranunculus polyanthemos 5 5 02??00-- 1000010101 212000010- 0100011100 12 Ranunculus recurvatus 5–6 5 02??00-- 1200010101 212000010- 01000?1100 12 Ranunculus trichophyllus 5 5 02??00-- 1000010101 212000010- 0100011100 18 Semiaquilegia adoxoides 5 5 022?00-- ?101010100 2110001?1? 1001010000 01 Semiaquilegia ecalcarata 5 5 021000-- 0101001101 2110001011 1001010000 01 Thalictrum dasycarpum 04–6 021000-- 021001--0- 2120000011 1000020000 0?

Appendix 4. Continued

1111111111 2222222222 3333333333 44 Taxa 0 1 23456789 0123456789 0123456789 0123456789 01

Thalictrum dioicum 04–6 021000-- 021001--0- 2120000011 1000020000 01 Thalictrum javanicum 0 4 021000-- 020001--0- 2120000011 1000020000 0? Trautvetteria caroliniensis 03–5 021?00-- ?20001--0- 2120000?0- ??01011100 12 Trollius chosenensis 8 5 021?00-- ?000010100 2120001011 1?0101?0?? 12 Trollius laxus 8–17 5–8 021?00-- 1000010100 2120001011 1101010000 12 Trollius vaginatus 8–17 5–8 021?00-- 1000010100 2120001011 1101010000 1? Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Urophysa henryi 5 5 021?00-- ?100010100 2110001??? ??0101?00? 01 Xanthorhiza simplicissima 5 5 001000-- 0200001100 1020001?11 0001020000 09

Missing data are indicated by ‘?’ and inapplicable states by ‘–’. Polymorphic states are represented as follows: a = [01]; b = [012]; c = [12].

Appendix 5. Continuously coded phenotypic characters for the genus-level analyses

Taxa 0 1 2 3 4 5 6 7 8

Anemoclema glaucifolium 516–44 13–15 ? 8 4 27 ? ? Anemone ﬂaccida 4–85–10 17–20 40–70 0 3–416–20 ? ? Anemone hupehensis 520–30 15 30–100 ? 2 20 ? ? Anemone parviﬂora 4–78–20 20–22 40–180 1–32–320? ? Anemone quinquefolia 56–25 15–31 20–60 1 3–515? ? Anemone rivularis 5–10 6–15 15–20 20–120 2 5–816–25 ? ? Clematis acerifolia 5–815–25 17–21 55–100 22–26 3 17–21 10–17 15–17 Clematis aethusifolia 412–20 25–30 30–90 16–27 3–413–15 13–19 18–19 Clematis afoliata 415–25 25–50 10–20 15–22 2–413–20 ? ? Clematis akebioides 416–27 25–27 25–70 30 3 17 10–15 22–24 Clematis akoensis *1 5–620–35 27–40 40–75 40 5–81719–28 9–11 Clematis akoensis *2 5–620–35 27–40 40–75 40 5–81719–28 9–11 Clematis albicoma *1 414–27 27–28 20–50 20–40 3–610–12 13–18 17–18 Clematis albicoma *2 414–27 27–28 20–50 20–40 3–610–12 13–18 17–18 Clematis alpina *1 440–50 33–40 100 30–40 2 13 ? ? Clematis alpina *2 440–50 33–40 100 30–40 2 13 ? ? Clematis alternata 418–22 18–36 25–38 18 ? ? 13–18 15–16 Clematis apiifolia *1 46–825–27 5–12 8–25 4–523–35 20–31 9–12 Clematis apiifolia *2 46–825–27 5–12 8–25 4–523–35 20–31 9–12 Clematis aristata 413–35 50–65 30–90 28–48 3–72022–46 12–14 Clematis armandii 4–612–47 24–60 18–70 16–48 4–519–20 14–29 9–11 Clematis baldwinii 430–40 29–30 20–39 60–80 6–712? ? Clematis barbellata 420–35 20–21 50–110 50 ? ? ? ? Clematis brachiata *1 48–15 ? ? 20–30 3–415? ? Clematis brachiata *2 48–15 ? ? 20–30 3–415? ? Clematis brachiata *3 48–15 ? ? 20–30 3–415? ? Clematis brachiata *4 48–15 ? ? 20–30 3–415? ? Clematis brachyura 4–68–10 25–27 25–50 3–45–714–17 11–27 15–16 Clematis brevicaudata 49–11 28–30 9–13 12–20 3 20 ? ? Clematis buchananiana 420–30 33–40 10–35 35–50 3–515–20 20–25 7–9 Clematis cadmia 5–612–43 12–27 4–71–35–717–18 19–28 19–20 Clematis carrizoensis 418–30 23–26 75–170 30–55 6–910–13 ? ? Clematis cf. insidiosa 4–66–735–60 10–24 25–40 3 15 ? ? Clematis chinensis 47–12 24–35 14–30 18–40 4–51314–21 13–15 var. tatushanensis

Appendix 5. Continued

Taxa 0 1 2 3 4 5 6 7 8

Clematis chinensis 46–20 30–50 14–30 18–40 3–710–18 14–21 13–15 Clematis chrysocoma *1 416–30 15–20 35–85 22–27 4–517–20 13–22 16–19 Clematis chrysocoma *2 416–30 15–20 35–85 22–27 4–517–20 13–22 16–19 Clematis chrysocoma *3 416–30 15–20 35–85 22–27 4–517–20 13–22 16–19 Clematis cirrhosa 420–25 18–20 20–50 50 3–515–20 17–32 18–20 var. balearica Clematis cirrhosa 425–35 17–18 20–50 50 3–512–17 17–32 18–20 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 var. cirrhosa Clematis columbiana 425–60 25–40 60–100 20–55 5 ? ? ? var. columbiana Clematis columbiana 415–50 30–56 50–130 20–45 ? ? ? ? var. tenuiloba *1 Clematis columbiana 415–50 30–56 50–130 20–45 ? ? ? ? var. tenuiloba *2 Clematis confusa 412–20 24–29 15–40 40 4 20 ? ? Clematis connata 416–22 37–53 15–40 40 3–41520–31 10–14 Clematis courtoisii 627–55 20–27 34–68 12–35 4–513–20 19–29 18–20 Clematis crassifolia 412–22 55–60 10–28 24–40 4–6138–14 13–15 Clematis crispa *1 420–50 13–17 ? 20–35 6–91014–42 9–11 Clematis crispa *2 420–50 13–17 ? 20–35 6–91014–42 9–11 Clematis cunninghamii 5–88–15 30–80 7–20 15–30 3–420? ? Clematis delavayi *1 4–68–14 16–18 14–20 25 4 20 8–23 12–13 Clematis delavayi *2 4–68–14 16–18 14–20 25 4 20 8–23 12–13 Clematis delicata 4–520–55 200–275 9–20 100–120 6 35–37 ? ? Clematis denticulata 4–67–13 19–35 10–30 100 5–725? ? Clematis dioica 47–835–27 10–25 45 4 20 16–48 6–7 Clematis drummondii 49–13 ? 15–70 40–100 3–52021–30 6–7 Clematis eichleri ?? ? 35–45 50 8–10 53 ? ? Clematis elisabethae–carolae 4–611–13 26–28 30–50 ? ???? Clematis erectisepala 41025–33 5–10 ? ???? Clematis fasciculiflora 412–20 24–25 5–24 10–16 6–827–37 13–20 11–12 Clematis finetiana 4–610–20 40–50 15–60 15–25 5 33 ? ? Clematis flammula 44–17 20–34 30–150 20–50 5 10 8–10 13–16 Clematis florida 620–50 17–20 37–85 6–83–510–13 ? ? Clematis foetida 4–86–12 24–60 6–15 14–30 2–420–27 19–29 8–9 Clematis fremontii 419–40 31–38 30–60 15–30 5–913? ? Clematis fruticosa 410–24 20 4–18 25 5–617–18 14–25 14–15 Clematis fusca *1 414–30 20–25 2–40 30–40 5–713–14 10–28 18–20 Clematis fusca *2 414–30 20–25 2–40 30–40 5–713–14 10–28 18–20 Clematis gentianoides 4–820–40 ? ? 20–30 4–520–22 ? ? Clematis glycinoides 411–25 83–110 3–20 30–47 3–815–27 30–46 9–10 Clematis gracilifolia 47–32 16–18 20–60 15–22 4–512–13 16–32 17–19 Clematis grata 46–10 25–30 6–15 12–26 2–315–17 ? ? Clematis gratopsis 47–10 20 8–24 23–45 4 20 ? ? Clematis haenkeana 47–10 23–33 ? 65 3–4? ? ? Clematis hancockiana 415–25 30–36 50–66 35–50 5 16 13–20 17–18 Clematis henryi 414–19 24–35 20–40 40 3 25 7–10 16–17 Clematis heracleifolia 415–24 34–50 8–35 25 3–515–18 16–26 6–7 Clematis hexapetala *1 610–25 25–33 5–70 15–30 3–51310–23 18–19 Clematis hexapetala *2 610–25 25–33 5–70 15–30 3–51310–23 18–19 Clematis hirsutissima 425–45 23–48 80–150 40–90 5–615–17 ? ? Clematis huchouensis 414–22 37–47 12–30 8–13 4–610–11 14–18 8–10 Clematis ibarensis 4–58–20 20–50 ? 30–40 ? ? 24–39 8–9

Appendix 5. Continued

Taxa 0 1 2 3 4 5 6 7 8

Clematis integrifolia *1 430–50 33–43 50–200 35–50 5–10 13–17 13–17 20–22 Clematis integrifolia *2 430–50 33–43 50–200 35–50 5–10 13–17 13–17 20–22 Clematis ispahanica 410–13 26–33 40–200 30–40 3–412? ? Clematis japonica 425–30 ? 40–100 30–35 6–84012–17 21–23 Clematis javana 49–15 30 16–20 16–30 3 21–25 ? ? Clematis koreana 417–40 33–34 40–120 30–45 4–517–20 ? ? Clematis ladakhiana 415–25 25–38 6–47 40 2–313–15 ? ? Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Clematis lanuginosa 5–640–70 20 50–100 40–60 4–51016–48 10–12 Clematis lasiandra *1 410–17 20–21 15–35 20–30 3 17 13–18 17–20 Clematis lasiandra *2 410–17 20–21 15–35 20–30 3 17 13–18 17–20 Clematis lasiantha *1 410–21 21–25 30–150 30–55 3–42024–36 8–10 Clematis lasiantha *2 410–21 21–25 30–150 30–55 3–42024–36 8–10 Clematis lasiantha *3 410–21 21–25 30–150 30–55 3–42024–36 8–10 Clematis laurifolia *1 47–918–23 15–40 40–50 8–12 53–69 11–30 23–25 Clematis laurifolia *2 47–918–23 15–40 40–50 8–12 53–69 11–30 23–25 Clematis leschenaultiana *1 416–22 28–32 20–50 30–40 5–645–50 8–11 9–10 Clematis leschenaultiana *2 416–22 28–32 20–50 30–40 5–645–50 8–11 9–10 Clematis ligusticifolia *1 46–13 20–26 5–40 30–35 3–420–30 23–36 7–8 Clematis ligusticifolia *2 46–13 20–26 5–40 30–35 3–420–30 26–36 7–8 Clematis ligusticifolia *3 46–13 20–26 5–40 30–35 3–420–30 26–36 7–8 Clematis linearifolia 415–35 88–150 ? 69–126 5–728–30 ? ? Clematis longicauda 430–50 20–25 ? 120 4–525–40 ? ? Clematis loureiriana 410–20 25 20–80 30–50 6–10 50–60 13–20 16–17 Clematis meyeniana 48–13 33–40 6–16 20–40 5–728–35 11–26 19–20 Clematis microphylla 412–25 ? ? 20–50 4–517–20 20–40 9–11 Clematis montana *1 413–65 13–26 10–200 20–60 4–51317–32 17–18 Clematis montana *2 413–65 13–26 10–200 20–60 4–51317–32 17–18 Clematis napaulensis 412–23 26–30 2–12 45–50 4–615–20 13–22 9–10 Clematis nobilis 432–38 ? ?? ???? Clematis ochotensis 440–55 23–27 70–150 35–45 3–515–17 ? ? Clematis ochroleuca 410–35 25–29 50–180 30–60 3–610–12 ? ? Clematis orientalis *1 412–20 33–40 14–55 25–40 3–418–25 10–16 21–24 Clematis orientalis *2 412–20 33–40 14–55 25–40 3–418–25 10–16 21–24 Clematis orientalis *3 412–20 33–40 14–55 25–40 3–418–25 10–16 21–24 Clematis otophora 418–27 20–23 10–40 35–40 4–5208–12 11–13 Clematis paniculata 6–820–35 23–29 40 30–65 2–42029–49 5–6 Clematis parviloba 410–24 20–25 12–30 20–32 3–515–18 22–31 8–9 Clematis patens 5–835–60 17–23 35–100 30–38 4–511–12 10–34 11–12 Clematis peterae *1 46–10 20–30 7–15 15–33 2–410–16 16–30 8–9 Clematis peterae *2 46–10 20–30 7–15 15–33 2–410–16 16–30 8–9 Clematis phlebantha 5–710–20 20 20–80 25–30 5 20 ? ? Clematis pierotii 48–14 ? 10–30 20 5 25 ? ? Clematis pinnata 412–19 38–40 5–18 ? ? ? 20–31 5–6 Clematis pitcheri 410–40 10–13 70–100 10–30 6–81010–38 14–15 Clematis pogonandra 422–29 29–44 30–100 25 4–5207–11 6–7 Clematis potaninii *1 5–618–38 16–23 30–98 25–32 3–515–16 25–33 12–13 Clematis potaninii *2 5–618–38 16–23 30–98 25–32 3–515–16 25–33 12–13 Clematis pubescens 415–40 38–57 30–90 23–64 5–728–30 ? ? Clematis ranunculoides 47–14 18–23 6–50 15–25 3–418–20 12–17 15–17 Clematis recta *1 4–68–15 21 25–50 15 4–613–15 14–29 15–18 Clematis recta *2 4–68–15 21 25–50 15 4–613–15 14–29 15–18 Clematis rehderiana 414–19 27–28 3–28 20–25 3–412–13 14–18 13–15 Clematis repens 412–25 24–31 25–80 30–55 3–420–21 8–11 23–25

Appendix 5. Continued

Taxa 0 1 2 3 4 5 6 7 8

Clematis reticulata 412–30 24–120 40–150 40–60 5–61311–39 12–14 Clematis rutoides *1 45–933–36 5–24 27–40 3–415? ? Clematis rutoides *2 45–933–36 5–24 27–40 3–415? ? Clematis rutoides *3 45–933–36 5–24 27–40 3–415? ? Clematis serratifolia 415–27 25–30 30–70 30–40 2–313–15 12–16 20–21 Clematis siamensis 415–23 38 15–32 ? ? ? 8–14 15–17 Clematis sibirica 422–50 22–25 70–100 25–45 3–414–15 12–19 17–19 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Clematis socialis 420–25 ? ? 15–25 10 ? ? ? Clematis songorica *1 4–65–15 17 10–80 14–30 3–414–17 13–18 19–20 Clematis songorica *2 4–65–15 17 10–80 14–30 3–414–17 13–18 19–20 Clematis songorica *3 4–65–15 17 10–80 14–30 3–414–17 13–18 19–20 Clematis sp. Birmingham 422–32 40–44 8–15 ? 6–813–15 ? ? Clematis sp. Roma 4? ? ? ? 5–712–17 ? ? Clematis sp. Wen9476 ?? ? ? ? ?? ? ? Clematis stans 413–25 63–65 10–25 15–30 4 16 20–34 8–10 Clematis strigillosa 48–25 17–27 10–30 25–45 3 14–15 ? ? Clematis taiwaniana 46–10 ? ?? ???? Clematis tangutica *1 415–40 25 35–165 50–55 3–515–20 12–15 19–20 Clematis tangutica *2 415–40 25 35–165 50–55 3–515–20 12–15 19–20 Clematis tashiroi 4–618–30 38–45 55–120 40–60 4–518–20 17–32 19–22 Clematis terniﬂora 45–17 25–28 20–60 15–20 4–913–15 13–19 20–21 Clematis terniﬂora 45–17 25–28 40–70 20–35 4–615–16 13–19 20–21 var. mandshurica Clematis texensis *1 415–30 30 50–100 40–70 6–710? ? Clematis texensis *2 415–30 30 50–100 40–70 6–710? ? Clematis tibetana *1 415–35 21–25 20–140 35 3 17 ? ? Clematis tibetana *2 415–35 21–25 20–140 35 3 17 ? ? Clematis trichotoma 4–61224 20–30 20–25 3 12 ? ? Clematis tubulosa 428221–10 14–20 3 17 ? ? Clematis uncinata 46–15 21–30 10–22 15–20 5–736–39 25–34 9–10 var. ovatifolia Clematis uncinata 46–15 21–30 10–22 15–20 5–736–39 25–34 9–10 Clematis versicolor 413–30 26–30 20–50 50–60 6–71012–36 15–17 Clematis villosa 4–617–40 16–17 10–120 50–60 4–10 13–15 24–41 8–10 Clematis viorna *1 415–30 30 20–70 25–60 3–810–13 ? ? Clematis viorna *2 415–30 30 20–70 25–60 3–810–13 ? ? Clematis viorna *3 415–30 30 20–70 25–60 3–810–13 ? ? Clematis virginiana *1 46–14 20–28 10–50 25–50 3–41732–48 6–8 Clematis virginiana *2 46–14 20–28 10–50 25–50 3–41732–48 6–8 Clematis virginiana *3 46–14 20–28 10–50 25–50 3–41732–48 6–8 Clematis vitalba *1 48–12 24–27 10–60 20–25 2–513–20 ? ? Clematis vitalba *2 48–12 24–27 10–60 20–25 2–513–20 ? ? Clematis vitalba *3 48–12 24–27 10–60 20–25 2–513–20 ? ? Clematis vitalba *4 48–12 24–27 10–60 20–25 2–513–20 ? ? Clematis viticaulis 414–25 23–25 10–40 20–35 4–612–13 ? ? Clematis viticella 422–38 ? 100 5–10 6–91012–19 9–12 Clematis williamsii 412–20 14–15 ? 25 5 ? 10–17 17–18 Clematis xiangguiensis 617–30 17–20 32–60 8 4 13 ? ? Hepatica nobilis 6–11 8–14 23–27 ? 0 4 ? ? ? var. japonica Hepatica transsilvanica 6–11 ? ? ?? ???? Knowltonia sp. 8–23 ? ? ?? ???? Pulsatilla cernua 5–618–30 ? 25–60 40 ? ? ? ?

Appendix 5. Continued

Taxa 0 1 2 3 4 5 6 7 8

Pulsatilla chinensis 5–628–44 22–31 25–55 35–65 4 ? ? ? Pulsatilla dahurica 5–62020–40 75 50–60 3 ? ? ? Pulsatilla sp. 5–6? ? ? ? ? ? ? ? Pulsatilla turczaninovii 5–622–42 22–32 15 40–50 4 ? ? ?

Missing data are indicated by ‘?’. Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021

Appendix 6. Phenotypic data matrix, discretely coded characters for genus-level analyses

1111111111 2222222222 3333333333 Taxa 9 0123456789 0123456789 0123456789

Anemoclema glaucifolium 1 100??0a000 1100112011 11001120?1 Anemone ﬂaccida 1 100??01001 1100a12010 0000002020 Anemone hupehensis 1 100?101001 1100112010 0000002001 Anemone parviﬂora 1 100??01001 1100a10010 0100003031 Anemone quinquefolia 1 100?101001 1100110010 0000003003 Anemone rivularis 1 100?101001 1100d12010 0100000031 Clematis acerifolia 1 1010100000 1101012010 0000012001 Clematis aethusifolia 1 001???000e 110a01b112 1101a12101 Clematis afoliata 0 0110100000 003101b001 01001120?1 Clematis akebioides 0 001?1000a2 110101a11d 100101210? Clematis akoensis *1 0 001?10001d 010101b010 010011200? Clematis akoensis *2 0 001?10001d 010101b010 010011200? Clematis albicoma *1 1 1011100000 0000010112 1101112111 Clematis albicoma *2 1 1011100000 0000010112 1101112111 Clematis alpina *1 0 0010?00103 1100010111 01110121?? Clematis alpina *2 0 0010?00103 1100010111 01110121?? Clematis alternata 1 001??01000 110101a112 1101012?0? Clematis apiifolia *1 0 001??0000f 110a012010 0100112001 Clematis apiifolia *2 0 001??0000f 110a012010 0100112001 Clematis aristata 0 011??0001c 010a012000 010011d101 Clematis armandii 0 011?010011 000101b010 000011200? Clematis baldwinii 1 1011010000 a000010112 11011121?1 Clematis barbellata 0 001??001?1 a101012111 11010121?1 Clematis brachiata *1 0 0010100102 110a012010 11010121?? Clematis brachiata *2 0 0010100102 110a012010 11010121?? Clematis brachiata *3 0 0010100102 110a012010 11010121?? Clematis brachiata *4 0 0010100102 110a012010 11010121?? Clematis brachyura 1 101???000c 000a01a010 0100102001 Clematis brevicaudata 0 001???000e 110a012010 01001120?1 Clematis buchananiana 0 0010100002 1101012112 1101112101 Clematis cadmia 1 001?01010e a001010010 010011011? Clematis carrizoensis 1 001???0112 a011010112 10011121?? Clematis cf. insidiosa 0 001???0012 000a012000 01000120?? Clematis chinensis var. tatushanensis 0 001101000e a00a012010 000011210? Clematis chinensis 0 0011010002 000a012010 000011210? Clematis chrysocoma *1 0 101??00001 110101b010 0000012001 Clematis chrysocoma *2 0 101??00001 110101b010 0000012001 Clematis chrysocoma *3 0 101??00001 110101b010 0000012001 Clematis cirrhosa var. balearica 0 001?100001 1101000111 1000012101 Clematis cirrhosa var. cirrhosa 0 001?10000a 1101000111 1000012101

Appendix 6. Continued

1111111111 2222222222 3333333333 Taxa 9 0123456789 0123456789 0123456789

Clematis columbiana var. columbiana 0 001?100103 1100010111 01100120?? Clematis columbiana var. tenuiloba *1 0 101?10010f 1000010111 01100120?? Clematis columbiana var. tenuiloba *2 0 101?10010f 1000010111 01100120?? Clematis confusa 0 001???010d 1101012112 11011120?? Clematis connata 0 001010010d 1101012112 1101112001 Clematis courtoisii 1 001111010e aa01010010 000011101? Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Clematis crassifolia 0 001???001a 000a012010 010001200? Clematis crispa *1 1 001101010d aa10010112 1101112111 Clematis crispa *2 1 001101010d aa10010112 1101112111 Clematis cunninghamii 0 0110100001 aa01012000 01001120?1 Clematis delavayi *1 0 101???0102 a00001a010 000001200? Clematis delavayi *2 0 101???0102 a00001a010 000001200? Clematis delicata 1 011???0??3 a000012000 00000110?? Clematis denticulata 0 001??000?g aa010120a0 01001120?? Clematis dioica 0 001???001d aa010120a0 110001200? Clematis drummondii 0 001??0001d 110101b000 0100012101 Clematis eichleri 0 001???000e 002101201? ?????110?? Clematis elisabethae-carolae 1 111???001a 000a012010 0000?1???? Clematis erectisepala 0 001???000g aa0a012111 000001???? Clematis fasciculiflora 0 011???0011 0001012111 110001100? Clematis finetiana 0 011???001a 000a01b010 00001120?1 Clematis flammula 0 001001001g aa0a012010 000011da01 Clematis florida 0 0a1111000g 0001010010 00101101?1 Clematis foetida 0 011???0011 aa01012000 0101012001 Clematis fremontii 1 1011010010 0000010112 11011121?1 Clematis fruticosa 0 101010011b aa0001a111 1100012001 Clematis fusca *1 1 0011010002 aa1a010112 1101112a01 Clematis fusca *2 1 0011010002 aa1a010112 1101112a01 Clematis gentianoides 0 1110100010 aa0001a000 01001120?1 Clematis glycinoides 0 011???001a aa0a012000 010011200? Clematis gracilifolia 0 001???000d 110101b010 01000110?? Clematis grata 0 001010000g 110a012010 01001120?1 Clematis gratopsis 0 0?1???0002 110a01d010 010001?0?? Clematis haenkeana 0 0?1???00?d aa0a0120a0 01000121?? Clematis hancockiana 1 001???010e a001010010 010011201? Clematis henryi 1 011???0010 010101b112 0101112001 Clematis heracleifolia 0 1010100011 110a01b112 1100012111 Clematis hexapetala *1 1 101001001e a00a01b010 0100112001 Clematis hexapetala *2 1 101001001e a00a01b010 010011200? Clematis hirsutissima 1 1011010002 1000010112 1101112111 Clematis huchouensis 1 001a010002 a00101a111 1100110103 Clematis ibarensis 0 0?1???00?g 010a01a000 0100?1200? Clematis integrifolia *1 1 1011010000 000001b111 1101012101 Clematis integrifolia *2 1 1011010000 000001b111 1101012101 Clematis ispahanica 0 10100001?h aa0a01a010 11001121?1 Clematis japonica 0 001???0001 0101012112 1001111001 Clematis javana 0 0?1???000g 110a012010 01000120?? Clematis koreana 0 001010010f 110001a111 1111012a?? Clematis ladakhiana 0 001010000e 110a01b111 010??120?? Clematis lanuginosa 0 001?01000a 0000010010 0100112011 Clematis lasiandra *1 1 001???010g 110a01b112 1101112101 Clematis lasiandra *2 1 001???010g 110a01b112 1101112101

Appendix 6. Continued

1111111111 2222222222 3333333333 Taxa 9 0123456789 0123456789 0123456789

Clematis lasiantha *1 0 0010?0000d 110101a000 0100012001 Clematis lasiantha *2 0 0010?0000d 110101a000 0100012001 Clematis lasiantha *3 0 0010?0000d 110101a000 0100012001 Clematis laurifolia *1 0 0?1???0012 0121012010 ?11011201? Clematis laurifolia *2 0 0?1???0012 0121012010 ?11011201? Clematis leschenaultiana *1 0 011?10000a 110101d112 110101200? Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Clematis leschenaultiana *2 0 011?10000a 110101d112 110101200? Clematis ligusticifolia *1 0 001010000d 1101012000 0100012101 Clematis ligusticifolia *2 0 001010000d 1101012000 0100012101 Clematis ligusticifolia *3 0 001010000d 1101012000 0100012101 Clematis linearifolia 0 011???0??3 000a012000 000?11???? Clematis longicauda 0 0?1???0001 110101h112 1100012??? Clematis loureiriana 0 011???0101 0001012000 0100112?0? Clematis meyeniana 0 011???001a 000a012010 0100112101 Clematis microphylla 0 011011011e aa0a012000 0100a1d101 Clematis montana *1 0 0010100001 1101012010 0100111101 Clematis montana *2 0 0010100001 1101012010 0100111101 Clematis napaulensis 0 0a10100001 aa01000112 110001210? Clematis nobilis 0 101???0103 1100010111 ??1?01???? Clematis ochotensis 0 001???0103 1100010111 01110120?1 Clematis ochroleuca 1 101???0010 aa00010112 11011121?1 Clematis orientalis *1 a 001??0000e aa01012111 1101112101 Clematis orientalis *2 a 001??0000e aa01012111 1101112101 Clematis orientalis *3 a 001??0000e aa01012111 1101112101 Clematis otophora a 0010100101 010101a112 1001112001 Clematis paniculata 1 0110100011 aa01012000 0000012001 Clematis parviloba 0 001???000g aa0a012010 010011200? Clematis patens 1 001101000b 0000010010 0100112111 Clematis peterae *1 0 001??00002 aa0a012010 010011110? Clematis peterae *2 0 001??00002 aa0a012010 010011110? Clematis phlebantha 0 10101001?2 110001a010 01000121?? Clematis pierotii 0 001?10000f aa0a012010 01001120?? Clematis pinnata 0 001???000d 110a012010 010111??0? Clematis pitcheri 1 0011010002 a00a01b112 1101112111 Clematis pogonandra a 001???0001 0101010112 110111200? Clematis potaninii *1 0 001010000g 110101a010 010011110? Clematis potaninii *2 0 001010000g 110101a010 010011110? Clematis pubescens 0 011???0011 010a012000 01001120?? Clematis ranunculoides 1 101010000c 110a01b111 1101112001 Clematis recta *1 1 101101001d a000012010 0000011101 Clematis recta *2 1 101101001d a000012010 0000011101 Clematis rehderiana 0 001??00002 1101012112 1101012001 Clematis repens a 001???000a 1101010112 110111200? Clematis reticulata 1 0011010012 a01101a112 110111211? Clematis rutoides *1 1 001???0013 aa0a012000 01000121?? Clematis rutoides *2 1 001???0013 aa0a012000 01000121?? Clematis rutoides *3 1 001???0013 aa0a012000 01000121?? Clematis serratifolia 0 001010000e 010101a111 0101112101 Clematis siamensis 0 0?1???0001 010101b112 110101??0? Clematis sibirica 0 001???010f 1100010111 011111200? Clematis socialis 1 101???000b a000010112 11011121?? Clematis songorica *1 0 101???000h aa0a012010 1100112a0?

Appendix 6. Continued

1111111111 2222222222 3333333333 Taxa 9 0123456789 0123456789 0123456789

Clematis songorica *2 0 101???000h aa0a012010 1100112a0? Clematis songorica *3 0 101???000h aa0a012010 1100112a0? Clematis sp. Birmingham 1 0011010102 a01a01a112 11011121?? Clematis sp. Roma 1 0011010002 a01?01??1? ??0??121?? Clematis sp. Wen9476 ? ?????????? ?????????? ?????????? Clematis stans 0 1010100101 1100012112 1101112a11 Downloaded from https://academic.oup.com/botlinnean/article/182/4/825/2724265 by guest on 28 September 2021 Clematis strigillosa 0 0?1???001a 1101012111 01010121?? Clematis taiwaniana 0 0?1???000f 1101012010 01000120?? Clematis tangutica *1 0 001??0000e 110a01a111 1101112001 Clematis tangutica *2 0 001??0000e 110a01a111 1101112001 Clematis tashiroi 0 011???010d 000101b010 010011200? Clematis terniﬂora 0 0010110002 aa0a012010 0100112003 Clematis terniﬂora var. mandshurica 0 0010110002 a00a012010 0100112004 Clematis texensis *1 1 0011010012 a01101ba12 11011121?1 Clematis texensis *2 1 0011010012 a01101ba12 11011121?1 Clematis tibetana *1 0 001???00ae 110a01a111 01011120?? Clematis tibetana *2 0 001???00ae 110a01a111 01011120?? Clematis trichotoma 0 001???000d 1a01011010 11001110?? Clematis tubulosa 0 101?1001a1 1101012a02 11001120?1 Clematis uncinata var. ovatifolia 0 001a000113 a00a012010 000011100? Clematis uncinata 0 001a00011e 000a012010 000011100? Clematis versicolor 1 001???0012 a01101b112 1001112011 Clematis villosa 1 101010000e 1100012111 010101200? Clematis viorna *1 1 0011010002 a01101b112 11011121?1 Clematis viorna *2 1 0011010002 a01101b112 11011121?1 Clematis viorna *3 1 0011010002 a01101b112 11011121?1 Clematis virginiana *1 0 001???0001 0a0101d000 0100112101 Clematis virginiana *2 0 001???0001 0a0101d000 0100112101 Clematis virginiana *3 0 001???0001 0a0101d000 0100112101 Clematis vitalba *1 0 001??00002 aa0a012010 01001120?1 Clematis vitalba *2 0 001??00002 aa0a012010 01001120?1 Clematis vitalba *3 0 001??00002 aa0a012010 01001120?1 Clematis vitalba *4 0 001??00002 aa0a012010 01001120?1 Clematis viticaulis 1 101???0000 a000010112 11011121?1 Clematis viticella 0 001101000g a00a01a111 1000110101 Clematis williamsii 0 001???0001 1101010111 010001200? Clematis xiangguiensis 1 001???000g a001010010 0000012??? Hepatica nobilis var. japonica 1 110???1010 1000110010 0000003000 Hepatica transsilvanica 1 110??01010 1100110010 0?00003032 Knowltonia sp. 1 110???101f 1100112011 000000001? Pulsatilla cernua 1 100?101001 1100100011 0110012011 Pulsatilla chinensis 1 100?101001 1a00100011 0110012001 Pulsatilla dahurica 1 100?101001 1a00100111 0110012001 Pulsatilla sp. 1 100?10100? 1?00100?1? ?1100120?? Pulsatilla turczaninovii 1 100?101003 1100100010 0110012001

Missing data are indicated by ‘?’. Polymorphic states are represented as follows: a = [01]; b = [012]; c = [0123]; d = [12]; e = [23]; f = [13]; g = [123]; h = [02].