TAXON 65 (1) • February 2016: 123–136 Chen & al. • Systematic placement of Ombrocharis ()

Resolving the phylogenetic position of Ombrocharis (Lamiaceae), with reference to the molecular phylogeny of Elsholtzieae Ya-Ping Chen,1,2 Bryan T. Drew,3 Bo Li,4 Douglas E. Soltis,5,6 Pamela S. Soltis6 & Chun-Lei Xiang1 1 Key Laboratory for Diversity and Biogeography of East , Kunming Institute of , Chinese Academy of Sciences, Kunming 650201, Yunnan, 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Biology, University of at Kearney, Kearney, Nebraska 68849, U.S.A. 4 Laboratory of Subtropical Biodiversity, College of Agriculture, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China 5 Department of Biology, University of , Gainesville, Florida 32611, U.S.A. 6 Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, U.S.A. Author for correspondence: Chun-Lei Xiang, [email protected] ORCID CLX, http://orcid.org/0000-0001-8775-6967

DOI http://dx.doi.org/10.12705/651.8

Abstract Ombrocharis is the only incertae sedis within Lamiaceae that has not been included in a published molecular phylogenetic study. Here, we adopt a two-step approach to resolve the phylogenetic placement of the genus. Initially, the subfamilial affiliation of Ombrocharis was determined based on a combined ndhF and rbcL dataset covering all seven sub- families of Lamiaceae. Results show that Ombrocharis is a member of , a placement that is also supported by its hexacolpate pollen grains. In the second set of analyses, two nrDNA (ITS, ETS) and four cpDNA (ycf1, rps15-ycf1, trnL-F, rpl32-trnL) markers were used to explore the position of Ombrocharis within Nepetoideae. Our results demonstrate that Ombrocharis and another monotypic genus, Perillula, form a clade that is sister to the remaining genera of tribe Elsholtzieae. Ombrocharis and other taxa within Elsholtzieae share divergent stamens, a weakly 2-lipped corolla, and an asymmetric disc with an elongate anterior lobe, but it is unclear whether these features are apomorphic. This study represents both the most comprehensive phylogenetic analysis of Elsholtzieae to date and the only study to include all genera of Elsholtzieae. The monophyly of Elsholtzieae (including Ombrocharis) is well supported, and there is weak support for Elsholtzieae as sister to the rest of Nepetoideae. Additionally, our results do not support the merging of Keiskea with , and may be polyphyletic.

Keywords Elsholtzia; Elsholtzieae; molecular phylogenetics; Nepetoideae; Ombrocharis; Perillula

Supplementary Material Electronic Supplement (Figs. S1–S6) and alignment files are available in the Supplementary Data section of the online version of this article at http://ingentaconnect.com/content/iapt/tax

INTRODUCTION Harley & al. (2004), viz., , Lami­oideae, Nepetoideae Prostantheroideae, , Symphorematoideae, and Major advancements in both molecular phylogenetic and Vitic­oideae. Meanwhile, ten genera—Acrymia Prain, Calli- morphological studies (Abu-Asab & Cantino, 1992; Cantino, carpa L., Cymaria Benth., Garrettia H.R.Fletcher, Holocheila 1992a, b; Wagstaff & Olmstead, 1997; Wagstaff & al., 1998) (Kudô) S.Chow, Hymenopyramis Wall. ex Griff., Ombrocharis have convincingly demonstrated that the Lamiaceae (mint Hand.-Mazz., Peronema Jack, Petraeovitex Oliv., and Tectona family) is polyphyletic as traditionally circumscribed (e.g., L.f.—were treated as incertae sedis with respect to subfamily Bentham, 1876; Briquet, 1895–1897). In an expansive morpho- (Harley & al., 2004). logical analysis, Cantino (1992a, b) redefined the family based During the past two decades, numerous phylogenetic largely on the classification of Junell (1934), and an expanded studies have been undertaken within Lamiaceae at various Lamiaceae was adopted after incorporating some former mem- taxonomic levels (e.g., Kaufmann & Wink, 1994; Wagstaff bers of into Lamiaceae s.str. This newly circum- & al., 1995, 1998; Wagstaff & Olmstead, 1997; Lindqvist & scribed Lamiaceae s.l. has been widely accepted (see APG III, Albert, 2002; Edwards & al., 2006; Oliveira & al., 2007; Conn 2009). According to the most recent classification of the family & al., 2009; Scheen & al., 2010; Bendiksby & al., 2011; Drew & (Harley & al., 2004), Lamiaceae comprises ca. 7200 Sytsma 2012; Salmaki & al., 2013; Xiang & al., 2013). As a re- in about 235 genera and is the sixth-largest family of - sult, the positions of some formerly unplaced genera have been ing . Lamiaceae were divided into seven subfamilies by resolved or discussed. For example, Holocheila was suggested

Received: 16 Jul 2015 | returned for (first) revision: 3 Oct 2015 | (last) revision received: 21 Oct 2015 | accepted: 25 Oct 2015 || publication date(s): online fast track, 19 Feb 2016; in print and online issues, 8 Mar 2016 || © International Association for Plant (IAPT) 2016

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to be a member of tribe Pogostemoneae in subfamily Lami- Manchester & al., 2009). It was first described by H. Handel- oideae (Chen & al., 2014), while Acrymia and Cymaria were Mazzetti (1936) on the basis of his own collection (H. Handel- recovered as sister to Lamioideae (Bendiksby & al., 2011; Chen Mazzetti 12394) in 1918 from Mt. Yunshan in Hunan. Handel- & al., 2014). In their study on the Vitex L. group, Bramley & Mazzetti placed the genus within subtribe Lamiinae of al. (2009) found that Peronema, Petraeovitex, and Hymeno- subfamily Lamioideae (“Stachyoideae”) sensu Briquet (1895– pyramis form a clade sister to the Gmelina L. and Premna L. 1897) and distinguished it from other genera of Lamiaceae by group. More recently, Chen & al. (2014) showed that Garrettia, virtue of the following suite of characters: forming Hymenopyramis, Peronema, and Petraeovitex may form a clade woody tubers, 6-flowered verticillasters in terminal racemoid sister to the Lamioideae-Acrymia-Cymaria-Scutellarioideae thyrse, 11-veined calyx, and slightly exserted stamens (Fig. 1). clade (but note that this latter clade was not included in Bramley The type specimens of O. dulcis were, until recently, the only & al. (2009), thus these two hypotheses are not necessarily known collections, and resultantly, studies on the genus since in conflict). Two other genera, Tectona and , have the original 1936 publication have been lacking. Based on the been included in several studies (Wagstaff & Olmstead, 1997; description of the external morphology in the protologue, Wu & Wagstaff & al., 1998; Bramley & al., 2009; Scheen & al., 2010; Li (1977) assigned Ombrocharis to subtribe Lamiinae of tribe Bendiksby & al., 2011; Drew & Sytsma, 2012) but have not Lamieae in subfamily Lamioideae sensu Briquet (1895–1897). been consistently placed. Currently, Ombrocharis is the only However, Cantino & Sanders (1986), based on the 3-celled remaining genus of incertae sedis from Harley & al. (2004) pollen of O. dulcis, suggested it might belong to subfamily that has not been included in a molecular phylogenetic study, Nepetoideae sensu Erdtman (1945), despite other morphologi- thus its systematic placement remains unclear. cal characters being more typical of Lamioideae sensu Erdtman The monotypic Ombrocharis, represented by O. dulcis (1945). Cantino & Sanders (1986), working with a paucity of Hand.-Mazz., is narrowly endemic to western Hunan Prov- material, also stressed that their conclusion should remain ten- ince in central China (Li & Hedge, 1994; Wu & al., 2007; tative until additional material could be examined. In the most

Fig. 1. Morphology of Ombrocharis dulcis. A, Plants; B, with fibrous roots and woody tubers; C, Inflorescence; D & E, Frontal and lateral views of flower; F, Frontal view of calyx; G, Disc and gynoecium. — Scale bar: G, 500 μm.

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recent classification of Lamiaceae, Harley & al. (2004) treated rbcL, five ndhF), while other data were taken from our previous Ombrocharis as incertae sedis within the family and suggested studies (Li & al., 2012; Chen & al., 2014) or downloaded from that further studies were needed to determine the relationships GenBank (see Appendix 1). We used the phylogenetic results of the genus. In the online synoptical classification of Lamia- from this first set of analyses as a basis for a more focused les (Olmstead, 2012), the genus was treated as incertae sedis second round of analyses. within Nepetoideae. In the second set of analyses, we further explored the During a field trip to Mt. Yunshan in Hunan in 2013, we placement of Ombrocharis within subfamily Nepetoideae. discovered a single population of Ombrocharis dulcis con- We employed four cpDNA markers (partial ycf1, rps15-ycf1 taining about 30 plants. The rediscovery of the population at spacer, trnL-F, rpl32-trnL) and two nuclear ribosomal DNA the type locality allowed us to evaluate the phylogenetic posi- (nrDNA) loci—the internal and external transcribed spacer tion of Ombrocharis using both molecular and morphological regions (ITS, ETS). These markers have previously been found evidence. to be informative in phylogenetic studies of tribe and subfamily Nepetoideae (Drew & Sytsma, 2011, 2012). This ingroup sampling comprised 55 accessions and represented 52 MATERIALS AND METHODS taxa from 34 genera of Nepetoideae. In the classification of Lamiaceae by Harley & al. (2004), Nepetoideae was divided Field collections. — The rediscovered population of Om- into three tribes: Elsholtzieae, Mentheae, and Ocimeae. Be- brocharis dulcis was located in a moist valley on Mt. Yunshan cause Ombrocharis showed a close relationship to genera of of Wugang City, Hunan Province, central China, at an altitude tribe Elsholtzieae according to the results from the first set of of ca. 1250 m. Fresh were collected and dried by silica analyses, we expanded the sampling to include all six genera gel. Voucher specimens (C.L. Xiang & al. 857) are deposited of Elsholtzieae (24 accessions in total) in our second round in KUN. We also transplanted four individuals from the wild of analyses. Based on Drew & Sytsma (2012), 23 accessions to the greenhouse in Kunming Botanical Garden. from 20 genera of Mentheae were sampled, representing all Anatomical studies. — Microfeatures of 20 pollen grains five subtribes. Within tribe Ocimeae, seven subtribes are cur- and 15 mericarps of Ombrocharis dulcis were studied using rently recognized (Harley & al., 2004; Zhong & al., 2010); scanning electron microscopy (SEM). Samples were all taken we selected one accession for each subtribe. Callicarpa and from our specimens (C.L. Xiang & al. 857) collected from Mt. Tectona were chosen as outgroups. For the second set of analy- Yunshan and were directly mounted onto stubs and coated with ses, we generated 154 original sequences and downloaded the gold. Micromorphological observations were conducted using a remaining data from GenBank (see Appendix 1). Hitachi-S4800 scanning electron microscope (Hitachi, Tokyo, Because only one population of Ombrocharis dulcis is Japan) with 10 kV voltage. Terminology of morphological char- known from the wild, we obtained sequences for the eight acteristics of pollen grains followed Erdtman (1952) and Hesse DNA markers from five individuals. However, no variation was & al. (2009), while description for mericarps followed Salmaki found among the five individuals. Therefore, we only included & al. (2008) and Moon & al. (2009). Mericarps were also col- a single accession of O. dulcis in the final analysis. lected from the wild for cytological study, but unfortunately, Generic circumscription largely follows Harley & al. the seeds failed to germinate. (2004) with the exception of Keiskea Miq., which was treated Taxon sampling and genetic markers. — Our initial analy- as a of Collinsonia L. by Harley & al. (2004), and ses aimed to ascertain the subfamilial assignment of Ombro- leucophylloides (Ramamoorthy & al.) B.T.Drew charis within Lamiaceae. The taxon sampling and choice of & al., which was placed in Neoeplingia Ramamoorthy & al. markers were largely in accord with the previous framework by Harley & al. (2004) but re-named by Drew & al. (2014). of Li & al. (2012) and Chen & al. (2014), with the major dif- Voucher information for accessions with original sequences ferences being our outgroup sampling and the inclusion here and GenBank accession numbers for all sequences used in the of three genera of incertae sedis, Ombrocharis, Tectona, and current study are listed in the Appendix 1. Callicarpa. Based on results from a recent -wide DNA extraction, amplification, and sequencing. — Total study by Refulio-Rodriguez & Olmstead (2014), we selected genomic DNA was extracted from silica-gel-dried material single accessions from each of the five most closely related using the modified CTAB method of Doyle & Doyle (1987), families of Lamiaceae as outgroups: reptans N.E.Br. then dissolved in TE buffer and kept at −20°C for polymerase (), Paulownia tomentosa Steud. (Paulowniaceae), chain reaction (PCR) amplification. Pedicularis groenlandica Retz. (Orobanchaceae), Phryma For the PCR amplification of rbcL, primers Z1F and leptostachya L. (Phrymaceae), and Rehmannia elata N.E.Br. Z1351R (Soltis & al., 1992) were used. The PCR reaction mix- (Rehmanniaceae). Sixty-three accessions from 53 genera of tures and program for rbcL followed Chen & al. (2014). The Lamiaceae were chosen for this first-step analysis, represent- ndhF gene was amplified using primers ndhF1 and ndhF2112R ing all seven subfamilies recognized by Harley & al. (2004), as (Olmstead & Reeves, 1995), while PCR protocols followed well as eight genera treated by Harley & al. (2004) as incertae those of Li & al. (2012). sedis with respect to subfamily (all such genera of Lamiaceae For ycf1, only a fragment of ca. 1000 bp at the 3′-end of except Acrymia and Garrettia). Eight sequences for the two the gene was amplified using the primers 4597f (5′-TTGATT​ plastid DNA (cpDNA) markers were newly sequenced (three GGATGGGAATGAATG-3′) and 5778r (5′-AGATGTATCC​

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TTAASATACYGAAACG-3′); for amplification of the rps15- (http://www.phylo.org/; Miller & al., 2010). A partitioned model ycf1 spacer, the primers rps15r and 5711f given in Drew (-q) was selected, and 1000 bootstrap iterations (-# | -N) were & Sytsma (2011) were used. The universal primers c and f conducted, with other parameters using the default settings. (Taberlet & al., 1991) were used to amplify the trnL-F region. Partitioned BI analyses were carried out using MrBayes PCR amplifications of rpl32-trnL used the primers trnL(UAG) v.3.2.2 (Ronquist, 2012) on the CIPRES Gateway (Miller & al., and rpL32-F provided in Shaw & al. (2007). The PCR program 2010). Models for each partition were selected among models used to amplify these four regions was: 4 min at 94°C; 35 analyzed by MrBayes using Bayesian model choice criteria (nst cycles with 30 s at 94°C, 90 s at 50°C, and 2.5 min at 72°C; = mixed rates = gamma) rather than specifying best-fit models and 7 min at 72°C. using a priori testing procedures (Huelsenbeck & al., 2004; ITS was amplified using either primer pairs ITSA and ITSB Ronquist & al., 2011). Markov Chain Monte Carlo (MCMC) (Blattner, 1999), or ITS4 and ITS5 (White & al., 1990); ETS analysis was executed for 20 million generations with four was amplified with primers ETS-B (Beardsley & Olmstead, chains, each starting with a random tree and sampling one 2002) and 18S-IGS (Baldwin & Markos, 1998). Amplifica- tree every 1000th generation. The temperature parameter was tion of the two markers was performed as follows: an initial lowered to 0.05 to achieve convergence. To avoid the issue of denaturation at 95°C for 5 min followed by 35 cycles of 45 s branch length overestimation (Brown & al., 2010; Marshall, denaturation (95°C), 45 s annealing (57°C), and 50 s extension 2010), we employed a uniform compound Dirichlet prior for (72°C), ending with a final extension at 72°C for 7 min. branch lengths with the default command “brlenspr = uncon- The PCR mixtures for all loci contained 2.0 μl of 10× reac- strained: gammadir (1, 0.1, 1, 1)”. Convergence of runs was ac- tion buffer, 2.0 μl of dNTP mixture, 1.5 μl of MgCl2, 1 μl of cepted when the average standard deviation of split frequencies bovine serum albumin (BSA, 20 mg/ml), 1 μl of each primer (ASDSF) dropped below 0.01. Tracer v.1.6.0 (Rambaut & al., (10 μM; Sangon Biotechnology, Shanghai, China), 0.3 μl Taq 2014) was used to inspect the convergence of model parameters polymerase (2.5 U/μl; Tiangen Biotech, Beijing, China), 2.0 μl and check whether the values of effective sample size (ESS) of unquantified template DNA, and deionized water added to were ≥ 200. The first 25% of the resulting trees were discarded a final volume of 25 μl. as burn-in, and the posterior probabilities (PP) were then calcu- PCR products were checked on 1% TAE agarose gels, lated using the remaining trees to construct a 50% majority-rule and purified using a QIAquick PCR purification kit (BioTeke, consensus tree. Optimal models for the various markers were Beijing, China) following the manufacturer’s instructions. also selected using the Akaike information criterion (AIC) and Sequencing reactions were performed with the dideoxy chain jModelTest v.2 (Guindon & Gascuel, 2003; Darriba & al., 2012) termination method running on an ABI-PRISM-3730 auto- on the CIPRES Gateway (Miller & al., 2010). The corresponding mated sequencer (Sangon Biotechnology). Sequencing prim- parameters were used for BI analyses, and the following results ers for each DNA marker were the same as the PCR primers were compared with those from the analyses described above. described above. Gaps were treated as missing data for all loci initially, Sequence alignment and phylogenetic analyses. — Se- while for the ycf1, rps15-ycf1, trnL-F, and rpl32-trnL datasets quences were assembled and edited using Sequencher v.4.1.4 prior to MP and partitioned BI analyses, gaps were also coded (Gene Codes, Ann Arbor, Michigan, U.S.A.). Alignment was using the simple gap coding of Simmons & Ochoterena (2000) initially performed with MUSCLE (Edgar, 2004), as imple- in SeqState v.1.4.1 (Müller, 2005). The command “coding = mented in MEGA v.6.0 (Tamura & al., 2013), and then adjusted variable rates = gamma” was used for the model of coded gaps manually in PhyDE v.0.9971 (Müller & al., 2010). Ambiguously in the partitioned BI analyses. aligned regions (e.g., characters of uncertain homology among All resulting trees with nodal support values were visual- taxa) in the ITS, ETS, rps15-ycf1 spacer, trnL-F, and rpl32-trnL ized in TreeGraph v.2 (Stover & Muller, 2010). datasets were deleted before phylogenetic reconstruction. Evaluation of marker incongruence. — Incongruences Phylogenetic analyses were conducted using maximum among different datasets (cpDNA regions, nrDNA regions, parsimony (MP), maximum likelihood (ML), and Bayesian combined cpDNA dataset, combined nrDNA dataset) were ex- inference (BI) approaches. plored through visual comparison of tree topologies and sup- MP analyses were conducted using PAUP* v.4.0b10 port values. Hard incongruence was defined as BS ≥ 80% and/ (Swofford, 2003) with the following settings: heuristic search or PP ≥ 0.95 (Pelser & al., 2010). option, tree-bisection-reconnection (TBR) branch swapping with 1000 random sequence addition replicates, 10 trees saved per replicate, all characters weighed equally and unordered. RESULTS A strict consensus tree was summarized from all the most- parsimonious trees (MPTs) retained. Bootstrap support (BS) Supplementary description of Ombrocharis. — Our ob- values were calculated from 10,000 rapid bootstrap replicates servation of living plants in the wild largely corroborates the with each comprising 10 random sequence addition replicates, description of Ombrocharis by H. Handel-Mazzetti (1936). with only one tree saved per replicate. However, we found that the calyx is 5-lobed (3/2) with the Partitioned ML analyses were implemented with RAxML- posterior lip 3-lobed and the anterior lip 2-lobed (Fig. 1F), as HPC2 v.8.1.11 (Stamatakis, 2014) on the Cyberinfrastructure revised in Harley & al. (2004) but contrary to the descrip- for Phylogenetic Research (CIPRES) Science Gateway v.3.3 tion of Ombrocharis (Handel-Mazzetti, 1936). Furthermore,

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the corolla was described as pale violet by Handel-Mazzetti Mericarp micromorphology of Ombrocharis dulcis (1936), while all we observed in the wild were white is shown in Fig. 2C and D. The mean length (L) = 1.910 ± (Fig. 1D, E). 0.121 mm, and the mean width (W) = 1.154 ± 0.089 mm. The Pollen grains of Ombrocharis dulcis are hexacolpate (Fig. shape of the mericarp is obvoid (L / W = 1.663 ± 0.158). The 2A), and the exine ornamentation is bireticulate with perfora- surface sculpturing is colliculate. tions on the ridges (Fig. 2B). The polar axis (P) = 54.353 ± Sequence characterization. — Properties for different 3.500 μm, and the equatorial axis (E) = 46.118 ± 2.315 μm. The datasets are summarized in Table 1. In the first set of analy- pollen shape is subspheroidal (prolate spheroidal to subprolate; ses, the aligned rbcL and ndhF datasets were 1394 bp and 2129 P / E = 1.179 ± 0.061). bp, respectively. In the second set of analyses, the combined

Table 1. Properties of datasets used in this study and resulting tree statistics. No. of ALIG NVC NPIC Tree NCE DNA dataset taxa [bp] [bp] [bp] NMPT length CI RI BFSM [bp] NCG rbcL 63 1394 338 (24%) 207 (15%) 24 868 0.463 0.667 TVM + I + Γ – – ndhF 55 2129 959 (45%) 620 (29%) 6 2788 0.504 0.617 GTR + I + Γ – – Combined rbcL + ndhF 68 3523 1297 (37%) 827 (23%) 422 3688 0.490 0.625 – – – ITS 56 621 306 (49%) 229 (37%) 4 1225 0.429 0.643 GTR + I + Γ 68 – ETS 53 427 322 (75%) 265 (62%) 16 1516 0.387 0.636 GTR + I + Γ 30 – Combined nrDNA 57 1048 628 (60%) 494 (47%) 16 2762 0.403 0.634 – 98 – ycf1 55 1058 468 (44%) 302 (29%) 739 883 0.673 0.845 TVM + Γ – 33 rps15-ycf1 56 659 264 (40%) 162 (25%) 3598 516 0.688 0.837 TVM + Γ 92 66 trnL-F 57 972 260 (27%) 159 (16%) 828 426 0.765 0.896 TVM + Γ 48 82 rpl32-trnL 56 951 435 (46%) 269 (28%) 786 923 0.651 0.805 GTR + Γ 144 119 Combined cpDNA 57 3640 1427 (39%) 892 (25%) 24 2767 0.678 0.837 – 284 300 Abbreviated column headers are as follows: ALIG, alignment (determined after excluding ambiguously aligned characters); NVC, number of variable characters; NPIC, number of parsimony-informative characters; NMPT, number of most-parsimony trees; CI, consistency index; RI, retention index; BFSM, best-fit substitution model; NCE, number of characters excluded; NCG, number of coded gaps (gaps were coded after the ambiguously aligned characters had been excluded)

Fig. 2. Scanning electron micrographs of pollen grain and mericarp of Ombrocharis dulcis. A, Equatorial and polar views of whole pollen; B, Exine surface of pollen; C, Whole mericarp; D, Exine sculpture of mericarp. — Scale bars: A, 9 μm; B, 15 μm; C, 300 μm; D, 30 μm.

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Fig. 3. The Bayesian 50% majority-rule consen- Haplostachys haplostachya sus phylogram based on concatenated ndhF and */*/* Phyllostegia macrophylla rbcL dataset. Support values marked above the 0.93 /-/63 Stenogyne rugosa branches follow the order BI-PP/MP-BS/ML-BS 0.87 /-/51 Prasium majus 0.51/-/- */87/87 macrantha (“-” indicates support values of less than 50% Lamioideae and “ * ” indicates a support value of 100%). Sub- sylvatica Physostegia virginiana familial classification of Lamiaceae is based on */71/94 Lamium garganicum Harley & al. (2004). Taxa marked with an “” */70/91 0.60 /-/68 Lamium purpureum were treated as incertae sedis by Harley & al. */98/99 Leonotis leonurus */-/75 */68/91 (2004). Scale bar denotes the expected number of Otostegia tomentosa substitutions per site in Bayesian analysis. 0.97 /-/70 Marrubium vulgare Pogostemon sp. */86/98 Colebrookea oppositifolia 0.98 /67/68 Holocheila longipedunculata ★

Cymaria dichotoma ★ Scutellarioideae

*/69/90 0.99 /71/69 alpina */67/95 Scutellaria orientalis

*/94/99 Scutellaria bolanderi */97/99 Scutellaria indica */89/98 */72/95 Tinnea zambesiaca Wenchengia alternifolia

*/*/* Hymenopyramis cana ★ 0.81/56/92 Petraeovitex wolfei ★ Peronema canescens ★

0.85 /52/81 */99/* Caryopteris incana */*/* Trichostema dichotomum 0.99 /72/92 Pseudocaryopteris bicolor Lamiaceae */*/* decumbens */84/* Ajuga reptans Ajugoideae */*/* Clerodendrum sp. */-/92 */*/* coulteri

*/*/* Oncinocalyx betchei */*/* Teucridium parvifolium Teucrium fruticans

*/*/* Gmelina hainanensis

*/81/97 Gmelina hystrix */*/* Premna microphylla Premna puberula 0.62 /-/- */*/* Callicarpa dichotoma ★

0.67 /-/- Callicarpa mollis ★

*/*/* Prostanthera rotundifolia Westringia sp. Prostantheroideae

*/*/* Vitex agnus-castus */*/* Vitex negundo Viticoideae 0.76 /-/- Petitia domingensis Congea tomentosa Symphorematoideae Tectona grandis ★ */*/* rotundifolia 0.70 /51/- fistulosa */98/* Thymus alsinoides */86/99 Origanum vulgare

0.78 /70/91 Glechoma hederacea

*/*/* Nepeta sp. Nepetoideae

*/91/* divinorum */86/99 Salvia farinacea Rosmarinus officinalis */*/* */85/95 Collinsonia punctata */*/* Elsholtzia stauntonii

Ombrocharis dulcis ★

*/98/* Ocimum basilicum */95/* Plectranthus barbatus Lavandula dentata

0.86 /-/66 Pedicularis groenlandica 0.97 /-/82 Rehmannia elata Paulownia tomentosa Outgroup Mazus reptans Phryma leptostachya

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nrDNA matrix contained 1048 positions (621 bp for ITS, 427 bp combined cpDNA (ycf1, rps15-ycf1, trnL-F, rpl32-trnL) data- for ETS) while the combined cpDNA dataset was 3640 bp set, and combined nrDNA (ITS, ETS) dataset, with and without (1058 bp for ycf1, 659 bp for rps15-ycf1, 972 bp for trnL-F, gap coding, using Bayesian model choice or a priori model 951 bp for rpl32-trnL) after excluding ambiguously aligned selection, were largely identical in topology (Figs. 3–5; Electr. characters, and not including coded gap characters. Suppl.: Figs. S1–S6; some results not shown). Thus, only the Phylogenetic analysis. — All phylogenies obtained from 50% majority-rule consensus trees resulting from non-gap- MP, ML, and BI analyses of the combined rbcL + ndhF dataset, coded Bayesian analysis of the combined rbcL and ndhF dataset

0.87 /-/62 Cunila incana Fig. 4. The Bayesian 50% Hedeoma multiflora */*/99 majority-rule consensus tree Clinopodium ashei */*/* based on combined cpDNA (ycf1, Monarda citriodora 0.93 /76/89 rps15-ycf1, trnL-F, rpl32-trnL) Bystropogon origanifolius */*/* dataset without gap coding. Micromeria lanata Satureja thymbra Support values displayed on the branches follow the order BI-PP/ 0.98 /-/72 */*/* Dracocephalum parviflorum

*/*/* Hyssopus officinalis MP-BS/ML-BS (“-” indicates Mentheae */*/* Glechoma hederacea support values of less than 50% */*/* 0.86 /-/57 Meehania urticifolia and “ * ” indicates a support value Nepeta cataria of 100%). Multiple accessions of */*/* Cleonia lusitanica */*/* the same species are numbered Prunella vulgaris */92/99 according to Appendix 1. Tribal Horminum pyrenaicum names of Nepetoideae follow */*/* lucidus var. maackianus Harley & al. (2004). Scale bar */*/* Lycopus uniflorus denotes the expected number of */70/95 Lepechinia lamiifolia */74/92 Lepechinia leucophylloides substitutions per site in Bayesian Melissa axillaris analysis. */*/* */*/* Salvia przewalskii */*/98 Salvia yunnanensis 0.86 /63/76 Rosmarinus officinalis

0.74 /52/- Hyptis laniflora Ocimum basilicum 0.97 /79/77 Nepetoideae Isodon dawoensis */*/* Plectranthus sp. Ocimeae */*/* Hanceola sinensis

0.54 /54/53 Lavandula angustifolia Siphocranion macranthum

0.83 /63/72 Elsholtzia capituligera */99/* Elsholtzia communis */*/* Elsholtzia pilosa

*/*/* Elsholtzia blanda Elsholtzia rugulosa */*/* */*/* */*/* Elsholtzia bodinieri

*/75/90 Elsholtzia heterophylla

*/97/97 0.93 /81/94 */*/* Elsholtzia kachinensis Elsholtzia fruticosa Elsholtzieae 0.66 /63/76 */*/* Elsholtzia densa Elsholtzia eriostachya

*/*/* Elsholtzia flava Elsholtzia penduliflora

*/*/* 0.99 /63/99 Keiskea japonica 1

*/*/* Keiskea japonica 2

*/95/98 frutescens 1 */*/* Perilla frutescens 2

*/*/* dianthera */*/* */*/* Mosla sp.

*/*/* Collinsonia punctata

*/87/* Perillula reptans 1 */*/* Perillula reptans 2 Ombrocharis dulcis Callicarpa giraldii Tectona grandis Outgroup

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from the first set of analyses (Fig. 3) and the combined cpDNA 2014). Lamiaceae are strongly supported as monophyletic (BI- dataset (Fig. 4) and the combined nrDNA dataset (Fig. 5) from PP = 1.00/MP-BS = 100%/ML-BS = 100%; all values follow the second set of analyses are presented, with bootstrap sup- this order hereafter), although relationships within the family port values from MP and ML analyses (without gap coding) are not well resolved. Ombrocharis is sister to a well-supported indicated. clade (1.00/85%/95%) formed by Collinsonia and Elsholtzia The phylogenetic framework of Lamiaceae (Fig. 3) result- Willd., and the robustly supported Ombrocharis-Collinsonia- ing from the concatenated rbcL and ndhF dataset is largely Elsholtzia clade (1.00/100%/100%) is nested within the subfam- consistent with previous studies (Li & al., 2012; Chen & al., ily Nepetoideae (1.00/100%/100%).

Fig. 5. The Bayesian 50% majority-rule con- 0.61/-/- Clinopodium ashei sensus phylogram based on combined nrDNA 0.70 /-/64 Hedeoma multiflora (ITS, ETS) dataset without gap coding. Support */96/96 Cunila incana values displayed on the branches follow the order */93/* Monarda citriodora 0.72 /-/61 BI-PP/MP-BS/ML-BS (“-” indicates support val- Bystropogon origanifolius */*/* ues of less than 50% and “ * ” indicates a support Micromeria lanata value of 100%). Multiple accessions of the same Satureja thymbra 0.78 /-/50 Dracocephalum parviflorum species are numbered according to Appendix 1. */98/* */66/84 Hyssopus officinalis

Tribal names of Nepetoideae follow Harley & al. Mentheae */99/98 Glechoma hederacea */93/97 (2004). Scale bar denotes the expected number of Meehania urticifolia substitutions per site in Bayesian analysis. */62/83 Nepeta cataria

*/96/99 Cleonia lusitanica 0.81/70/69 Prunella vulgaris Horminum pyrenaicum */-/88 */*/* Lycopus lucidus var. maackianus Lycopus uniflorus

*/*/* Salvia przewalskii */86/96 Salvia yunnanensis 0.83 /67/73 Rosmarinus officinalis

*/99/* Lepechinia lamiifolia 0.62 /67/71 Lepechinia leucophylloides Melissa axillaris 0.92 /-/67 0.93 /70/70 Ocimum basilicum Plectranthus sp. Nepetoideae 0.70 /-/53 Hanceola sinensis 0.86 /65/69 Hyptis laniflora Ocimeae 0.94 /58/78 Isodon dawoensis */71/89 Lavandula angustifolia Siphocranion macranthum

0.99 /73/86 Elsholtzia communis */90/97 Elsholtzia pilosa */*/* Elsholtzia capituligera

0.98 /66/86 Elsholtzia blanda Elsholtzia rugulosa */*/* */94/* Elsholtzia ciliata */83/* 0.64 /72/- Elsholtzia kachinensis */*/* 0.97 /-/70 Elsholtzia fruticosa */*/* Elsholtzia bodinieri

Elsholtzia heterophylla Elsholtzieae

*/83/97 Elsholtzia densa Elsholtzia eriostachya 0.93 /-/- */*/* Mosla dianthera

0.97 /78/81 Mosla sp.

*/*/* Perilla frutescens 1 */98/99 Perilla frutescens 2

0.92 /-/- */*/* Keiskea japonica 1 */85/93 Keiskea japonica 2

*/*/* Collinsonia canadensis

*/60/84 Collinsonia punctata */*/* Elsholtzia flava Elsholtzia penduliflora Ombrocharis dulcis */*/* Perillula reptans 1 Perillula reptans 2 Callicarpa giraldii Outgroup Tectona grandis

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In the second set of analyses, which aimed to investigate 2-celled stage) and subfamily Nepetoideae (pollen usually hexa- the position of Ombrocharis within Nepetoideae, we ex- colpate and 3-celled when shed). Although Lamioideae sensu panded sampling within the subfamily, especially for the tribe Erdtman (1945) was later shown to be polyphyletic (Cantino, Elsholtzieae, and analyses of two nrDNA and four cpDNA 1992a, b), Nepetoideae sensu Erdtman (1945) has been consis- datasets were conducted. The combined cpDNA dataset yields tently shown to be monophyletic in numerous studies (Cantino a better resolved phylogeny (Fig. 4) than the combined nrDNA & Sanders, 1986; Kaufmann & Wink, 1994; Wagstaff & al., dataset (Fig. 5), probably due to the larger number of vari- 1995, 1998; Wagstaff & Olmstead, 1997; Paton & al., 2004) able sites (1427 vs. 628) and/or the greater consistency (CI = and is one of the best supported and most easily diagnosable 0.678 vs. CI = 0.403) in the cpDNA dataset than in the nrDNA of the seven subfamilies of Harley & al. (2004). As the largest dataset. In both datasets, three clades are recovered within subfamily in Lamiaceae (containing about one-third of the gen- the monophyletic and well-supported subfamily Nepetoideae era and half of the species), Nepetoideae are united by several (Fig. 4: 1.00/100%/100%; Fig. 5: 1.00/83%/100%). These clades synapomorphies, including hexacolpate and trinucleate pollen correspond to tribe Mentheae (Fig. 4: 1.00/100%/100%; Fig. 5: (the most distinctive and easily observed feature), presence of 1.00/86%/96%), tribe Ocimeae (Fig. 4: 1.00/100%/100%; Fig. 5: rosmarinic acid, and an investing embryo (Cantino & Sand- 1.00/71%/89%), and tribe Elsholtzieae (Fig. 4: 1.00/100%/100%; ers, 1986; Harley & al., 2004). Based on limited material from Fig. 5: 1.00/60%/84%). The cpDNA phylogeny (Fig. 4) shows that herbarium specimens, Cantino & Sanders (1986) found that Ombrocharis is sister to Perillula Maxim. (1.00/100%/100%), Ombrocharis had 3-celled pollen. Although the number of colpi and that these two monotypic genera form a clade sister to the was not observed, they hypothesized that Ombrocharis belongs remainder of tribe Elsholtzieae (1.00/100%/100%). Ombrocha- to subfamily Nepetoideae. Our SEM results show that Ombro- ris and the two accessions of Perillula form a trichotomy in the charis indeed has hexacolpate pollen grains (Fig. 2A), further nrDNA phylogeny (Fig. 5), while the sister-group relationship corroborating the placement of the genus within Nepetoideae. between the Ombrocharis-Perillula clade (1.00/100%/100%) Ombrocharis is a member of tribe Elsholtzieae and sister and the rest of Elsholtzieae (0.92/-/-) are weakly supported . to Perillula. — Ombrocharis grouped closely with Collinsonia Incongruence among datasets. — Visual comparison of and Elsholtzia in the first set of analyses (Fig. 3), indicating a the resulting topologies based on supporting values revealed close relationship between Ombrocharis and tribe Elsholtzieae. few well-supported discrepancies except for the combined We included all six genera of Elsholtzieae in the second set of nrDNA (ITS, ETS) and combined cpDNA (ycf1, rps15-ycf1, analyses, the first phylogenetic analysis to do so. Our results trnL-F, rpl32-trnL) datasets. Two clades that received strong (Figs. 4, 5) demonstrate that Ombrocharis is closely related to support but were in conflict in different datasets were identi- Perillula and the two genera form a clade sister to the rest of fied: subtribe Salviinae (represented here by Lepechinia Willd., Elsholtzieae. Notably, the branch lengths of the two genera are Melissa L., Salvia L., and Rosmarinus L.) was monophyletic in both short, suggesting low sequence variation between them. the cpDNA tree (Fig. 4: 1.00/100%/ 100%), but was paraphyletic After careful examination and comparison of the morpho- in the nrDNA phylogeny (Fig. 5) with Salvia and Rosmarinus logical traits, we found that Ombrocharis shares some features forming a clade (0.83/67%/73%) sister to all other subtribes with other genera of tribe Elsholtzieae. One shared feature is of tribe Mentheae (1.00/-/88%) while Lepechinia and Melissa divergent stamens included in, or long exerted from the corolla. formed a clade sister to the rest of Mentheae including the Another shared character state is the weakly 2-lipped corolla Salvia-Rosmarinus clade (0.62/67%/71%). Keiskea, Perilla L., with short and ± porrect lobes and short corolla tube. Corol- and Mosla (Benth.) Buch.-Ham. ex Maxim. formed a strongly las are 4–5-lobed with the posterior lip entire or shallowly supported clade in the cpDNA (Fig. 4: 1.00/100%/100%) and 2-lobed, and the anterior lip 3-lobed with lobes subequal (e.g., nrDNA trees (Fig. 5: 1.00/98%/99%). However, in the cpDNA Ombrocharis, Perillula, and Perilla) or the median lobe much phylogeny (Fig. 4), Mosla was sister to the Keiskea-Perilla clade larger and distinct (especially in Collinsonia). Furthermore, (1.00/100%/100%), while in the nrDNA tree (Fig. 5), Keiskea Ombrocharis has an asymmetric disc with a somewhat elon- was sister to the Mosla-Perilla clade (0.97/78%/81%). Because of gate anterior lobe (Fig. 1G), which is shared with the rest of the discrepancies between the cpDNA and nrDNA datasets, we Elsholtzieae. Although some of these features can also be found did not combine the two matrices in the second set of analyses. in tribe Mentheae and tribe Ocimeae, the particular combina- tion of characters further supports the molecular phylogenetic analysis results that suggest Ombrocharis is a part of tribe DISCUSSION Elsholtzieae. However, it is unclear at this time whether any of these characters is actually apomorphic. Ombrocharis is part of subfamily Nepetoideae. — Our Perillula is monotypic with the single species P. reptans analyses based on the combined rbcL and ndhF dataset provide Maxim. endemic to Japan (Murata & Yamazaki, 1993). Only strong evidence that Ombrocharis is a member of subfamily a few studies (e.g., Zhou & al., 1997; Funamoto & Ogawa, Nepetoideae (Fig. 3). This hypothesis is also supported by pol- 2014) have included the genus since its initial description by len morphology, as previously suggested by Cantino & Sanders Maximowicz (1875), and it has not been included in a molecular (1986). Erdtman (1945) proposed that Lamiaceae be divided phylogenetic publication prior to this study. In addition to their into two subfamilies based on pollen characters, viz., subfam- sister relationship in the cpDNA tree (Fig. 4), Ombrocharis and ily Lami­oideae (with tricolpate pollen grains that shed at the Perillula also bear morphological similarities. For example,

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the two genera are both characterized by short, tuber-like rhi- Generic relationships within tribe Elsholtzieae. — Elsholtz- zomes; 6-flowered verticillasters in terminal racemoid thyrses; ieae was formally validated by Sanders & Cantino (1984) and a campanulate, 11-veined, 2-lipped, and 5-lobed (3/2) calyx; a comprises six genera (Collinsonia, Elsholtzia, Keiskea, Mosla, campanulate, slightly bilabiate, and 5-lobed (2/3) corolla; lon- Perilla, Perillula) in the classification of Cantino & al. (1992). ger and included anterior stamens; and ± parallel and separate In the molecular phylogenetic study of Nepetoideae by Wagstaff thecae with a small connective (Maximowicz, 1875; Wu & Li, & al. (1995), tribe Elsholtzieae was represented by Elsholtzia, 1977; Murata & Yamazaki, 1993). We also compared our pol- Collinsonia, and Perilla and formed a well-supported clade. len grain and mericarp SEM results for Ombrocharis (Fig. 2) Based on the morphological similarities between Collinsonia with those of Zhou & al. (1997: figs. 23, 24, 45, 46) for Peril- and Keiskea, Harley & al. (2003, 2004) treated Keiskea as a lula. Both genera have prolate pollen grains with bireticulate synonym of Collinsonia. Peirson & al. (2003) explored the sculpturing and perforations on the ridges, and compressed phylogeny of Elsholtzieae using the nrITS region and showed obovoid and smooth mericarps (because the mericarp sculpture that Collinsonia was sister to the clade formed by Keiskea and of Perillula was not clearly shown in Zhou & al. 1997, we were Perilla, while Elsholtzia was sister to all three genera. Thus, unable to compare that feature for the two genera). The main they did not support the incorporation of Keiskea into Col- differences seem to lie in the stem and leaf morphology. In linsonia. None of these studies included Mosla or Perillula. Ombrocharis, the stems are erect, and the leaves are oblong- This study is the most comprehensive published phylo- ovate and 4–12 cm long, while in Perillula, the stems are de- genetic analysis of tribe Elsholtzieae. Representatives of all cumbent below and ascendent to erect above, and the leaves seven genera were sampled, and we included Mosla and the are ovate and 1.5–4 cm long. These characters per se do not two monotypic genera Ombrocharis and Perillula, none of warrant generic division, especially given that generic limits which had been included in previous molecular phylogenetic are arbitrary. Furthermore, considering that our study includes analyses. We did not adopt the treatment of Keiskea in Harley only one accession of Ombrocharis dulcis from a single popula- & al. (2003, 2004) but retained its generic status as in Cantino tion, and cytological data for the species are still unavailable, & al. (1992). we consider it premature to merge the two genera until more In the nrDNA phylogeny (Fig. 5), relationships within thorough investigations are conducted. the moderately supported tribe Elsholtzieae (1.00/60%/84%) Tribal relationships within subfamily Nepetoideae. — are poorly resolved; while in the cpDNA tree (Fig. 4), tribe Cantino & al. (1992) adopted the circumscription of the mono- Elsholtzieae is a strongly supported group (1.00/100%/100%) phyletic Nepetoideae sensu Erdtman (1945) and further pro- and is composed of three clades. Ombrocharis and Perillula posed a new tribal subdivision for the subfamily based on form a clade (Figs. 4 and 5: 1.00/100%/100%), as discussed morphological and molecular studies (Cantino, 1992a, b; above. The two genera share terminal racemoid thyrses formed Wagstaff, 1992). Four tribes were recognized: Elsholtzieae, by 6-flowered verticillasters (Fig. 1C), as well as short sta- Lavanduleae, Mentheae, and Ocimeae. Early molecular phylo- mens attached to the middle of the corolla tube and slightly genetic studies (Kaufmann & Wink, 1994; Wagstaff & al., 1995) exserted from the tube, features that differ from other genera partly or largely supported this classification, and it was adopted of Elsholtzieae. by Harley & al. (2004), but with Lavanduleae subsumed within The second well-supported clade (Fig. 4: 1.00/100%/100%; Ocimeae. Although the three tribes of Harley & al. (2004) are Fig. 5: 1.00/85%/93%) consists of Collinsonia, Mosla, Perilla, well-supported, relationships between the three tribes remain and Keiskea. A shared character state for the four genera that unresolved. Previous studies have either found Ocimeae to might or might not be apomorphic is the occurrence of 2-flow- be sister to the Mentheae-Elsholtzieae clade (Wagstaff & al., ered (rarely 6-flowered) verticillasters arranged into a raceme- 1995) or Mentheae to be sister to the Ocimeae-Elsholtzieae clade like inflorescence or panicle. In the cpDNA tree (Fig. 4), (Wagstaff & al., 1998; Paton & al., 2004; Drew & Sytsma, 2012). Keiskea and Perilla are sister groups, with Mosla sister to this All of these results were weakly supported, and none of the clade, while Collinsonia is sister to the Mosla-Keiskea-Perilla studies contained broad sampling within all three tribes. clade. In contrast, Keiskea is sister to a clade comprising Mosla In our analyses of the cpDNA and nrDNA datasets (Figs. and Perilla in the nrDNA phylogeny (Fig. 5). The two conflict- 4, 5), all three tribes of Nepetoideae received moderate to robust ing topologies each received strong support (see Results). It support values. In contrast to the other studies noted above, our appears that the cpDNA and nrDNA gene regions used here results show that Mentheae and Ocimeae group together, but show partially different histories for the three genera (Mosla, with weak support (Fig. 4: 0.86/63%/76%; Fig. 5: 0.92/-/67%); Keiskea, Perilla), possibly resulting from chloroplast capture Elsholtzieae is recovered as the sister to the Mentheae-Ocimeae​ events and/or the influence of incomplete lineage sorting. Mor- clade. Morphologically, both Elsholtzieae and Mentheae are phologically, Mosla is more similar to Perilla in the strongly characterized by ascending or divergent stamens, which are reticulate-foveolate mericarp and exine ornamentation of the often declinate in Ocimeae. However, corollas of taxa from pollen grains (Zhou & al., 1997) and the included stamens, but Elsholtzieae are weakly 2-lipped, while usually distinctly they differ from each other in the number of fertile stamens, 2-lipped in the other two tribes. Because the sampling within which is four in Perilla (and Keiskea) versus two in Mosla, with tribe Mentheae and Ocimeae was not extensive in our analyses, the anterior pair reduced. further studies of tribal relationships within subfamily Nepe- Regarding the relationship between the eastern North toideae are still needed. American Collinsonia and eastern Asian Keiskea, our results

132 Version of Record TAXON 65 (1) • February 2016: 123–136 Chen & al. • Systematic placement of Ombrocharis (Lamiaceae)

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Appendix 1. Sequence information for all samples used in present study. For newly generated sequences, both voucher information and GenBank accession numbers are listed and indicated with an asterisk, while for sequences downloaded from GenBank, only their accession numbers are provided.

Taxon, voucher, accession number of ndhF, rbcL. “–” missing data, “ * ” newly generated sequence.

Ajuga decumbens Thunb.: JQ322521, JQ322527; Ajuga reptans L.: L36391, U32163; Betonica macrantha K.Koch: –, Z37461; Callicarpa dichotoma Raeusch.: L36395, L14393; Callicarpa mollis Siebold & Zucc.: AY310134, HQ384868; Caryopteris incana (Thunb. ex Houtt.) Miq.: U78681, U28869; Clerodendrum sp.: U78683, L11689; Colebrookea oppositifolia Sm.: U78688, U78712; Collinsonia punctata Elliott: U.S.A., Florida, C.L. Xiang 1187 (KUN), KT210252*, KT210257*; Congea tomentosa Roxb.: U78689, U28870; Cymaria dichotoma Benth.: KF509863, KF509870; Elsholtzia stauntonii Benth.: U78690, U28872; Glechoma hederacea L.: U78691, L14292; Gmelina hainanensis Oliv.: JQ322518, JQ322524; Gmelina hystrix Schult. ex Kurz: U78692, U28873; Haplostachys haplostachya H.St.John: –, AF501987; Holocheila longipedunculata S.Chow: KF509860, KF509867; Hymenopyramis cana Craib: KF509865, –; Lamium garganicum L.: –, Z37401; Lamium purpureum L.: U78694, U75702; Lavandula dentata L.: China, Yunnan, Y.P. Chen s.n. (KUN), KT210253*, –; Leonotis leonurus (L.) R.Br.: –, AM234998; Marrubium vulgare L.: U78695, U28875; Mazus reptans N.E.Br.: HQ384817, HQ384872; Mentha rotundifolia Huds.: U78696, Z37417; Monarda fistulosa L.: –, Z37419; Nepeta sp.: KT176846, Z37423; Ocimum basilicum L.: China, Yunnan, Y.P. Chen s.n. (KUN), KT210254*, –; Ombrocharis dulcis Hand.-Mazz.: China, Hunan, C.L. Xiang & al. 961 (KUN), KT210255*, KT210258*; Oncinocalyx betchei F.Muell.: U78685, U31458; Origanum vulgare L.: JX880022, JX880022; Otostegia tomentosa A.Rich.: –, AF501988; Paulownia tomentosa Steud.: L36406, L36447; Pedicularis groen- landica Retz.: HQ384818, HQ384873; Peronema canescens Jack: KF509866, –; Petitia domingensis Jacq.: U78697, U28878; Petraeovitex wolfei J.Sinclair: China, Yunnan, L. Jiang & al. s.n. (KUN), KT210256*, KT210259*; Phyllostegia macrophylla (Gaudich.) Benth.: –, AF501991; Phryma leptostachya L.: AJ429118, U28881; Physostegia virginiana (L.) Benth.: L36407, L14405; Plectranthus barbatus Andrews: U78698, –; Pogostemon sp.: U78699, L14406; Prasium majus L.: U78700, U31459; Premna microphylla Turcz.: U78701, U28883; Premna puberula Pamp.: JQ322519, JQ322526; Prostanthera rotundifolia R.Br.: U78702, L14008; Pseudocaryopteris bicolor (Roxb. ex Hardw.) P.D.Cantino: U78680, U78711; Rehmannia elata N.E.Br.: HQ384820, HQ384874; Ros- marinus officinalis L.: NC_027259, Z37435; Salvia divinorum Epling & Játiva: U78703, L14407; Salvia farinacea Benth.: –, AY570415; Scutellaria alpina L.: –, Z37457; Scutellaria bolanderi A.Gray: U78704, L01954; Scutellaria indica L.: JQ322522, JQ322529; Scutellaria orientalis L.: –, Z37460; Stachys sylvatica L.: –, Z37464; Stenogyne rugosa Benth.: –, AF502026; Tectona grandis L.f.: U78705, U28884; Tetraclea coulteri A.Gray: AF130147, U78714; Teucridium parvifolium Hook.f.: U78684, U78715; Teucrium fruticans L.: U78686, L14411; Thymus alsinoides Formánek: –, Z37470; Tinnea zambesiaca Baker: U78709, U28886; Trichostema dichotomum L.: U78682, U28887; Verbena bonariensis L.: HM216782, L14412; Vitex agnus-castus L.: U78707, U78716; Vitex negundo L.: JQ322520, JQ322525; Wenchengia alternifolia C.Y.Wu & S.Chow: JQ322523, JQ322528; Westringia sp.: GQ381196, Z37474.

Version of Record 135 Chen & al. • Systematic placement of Ombrocharis (Lamiaceae) TAXON 65 (1) • February 2016: 123–136

Appendix 1. Continued.

Taxon, voucher, accession number of ITS, ETS, ycf1, rps15-ycf1, trnL-F, rpl32-trnL. “–” missing data, “ * ” newly generated sequence.

Bystropogon origanifolius L’Hér.: JQ669078, JQ669147, JQ669222, JQ669222, JQ669023, JQ669277; Callicarpa giraldii Hesse ex Rehder: China, Gansu, E.D. Liu & al. 3054 (KUN), FJ593347, KT210228*, KT210360*, KT210309*, FJ593410, KT210284*; Cleonia lusitanica L.: DQ667309, –, JF289006, JF289006, DQ667495, –; Clinopodium ashei (Weath.) Small: DQ667237, JQ669150, JF289008, JF289008, DQ667437, JQ669284; Collinsonia canadensis L.: DQ667248, JQ669157, JF289010, JF289010, JF301364, JQ669291; Collinsonia punctata Elliott: U.S.A., Florida, C.L. Xiang 1187 (KUN), KT210241*, KT210214*, KT210349*, KT210298*, KT210325*, KT210273*; Cunila incana Benth.: DQ667316, JQ669160, JF289012, JF289012, DQ667504, JQ669295; Dracocephalum parviflorum Nutt.: JQ669097, JQ669168, –, –, JQ669038, JQ669304; Elsholtzia blanda Benth.: China, Yunnan, C.L. Xiang & al. 560 (KUN), KT210237*, KT210210*, KT210345*, KT210294*, KT210321*, KT210269*; Elsholtzia bodinieri Vaniot: China, Yunnan, C.L. Xiang & al. 508 (KUN), KT210238*, KT210211*, KT210346*, KT210295*, KT210322*, KT210270*; Elsholtzia capituligera C.Y.Wu: China, Yunnan, E.D. Liu & al. 3364 (KUN), KT210235*, KT210207*, KT210342*, KT210291*, KT210318*, KT210266*; Elsholtzia ciliata (Thunb.) Hyl.: JQ669098, JQ669170, JF289017, JF289017, JF301367, JQ669306; Elsholtzia communis (Collett & Hemsl.) Diels: China, Yunnan, C.L. Xiang & al. 538 (KUN), KT210239*, KT210212*, KT210347*, KT210296*, KT210323*, KT210271*; Elsholtzia densa Benth.: China, Tibet, J.Y. Xiang 09-314 (KUN), KT210231*, KT210203*, KT210338*, KT210287*, KT210314*, KT210262*; Elsholtzia erio- stachya Benth.: China, Tibet, E.D. Liu & al. 3291 (KUN), –, KT210208*, KT210343*, KT210292*, KT210319*, KT210267*; Elsholtzia flava Benth.: China, Yunnan, Y.P. Chen XS-03 (KUN), KT210233*, KT210205*, KT210340*, KT210289*, KT210316*, KT210264*; Elsholtzia fruticosa Rehder: China, Sichuan, W. Fang & al. fw11239 (KUN), KT210232*, KT210204*, KT210339*, KT210288*, KT210315*, KT210263*; Elsholtzia heterophylla Diels: China, Yunnan, H.J. Dong D.Y. 0112 (KUN), KT210236*, KT210209*, KT210344*, KT210293*, KT210320*, KT210268*; Elsholtzia kachinensis Prain: China, Yunnan, C.L. Xiang & al. 534 (KUN), KT210240*, KT210213*, KT210348*, KT210297*, KT210324*, KT210272*; Elsholtzia penduliflora W.W.Sm.: China, Yunnan, C.L. Xiang & H.J. Dong 111 (KUN), KT210234*, KT210206*, KT210341*, KT210290*, KT210317*, KT210265*; Elsholtzia pilosa Benth.: China, Tibet, G.X. Hu & al. 1209064 (KUN), KT210230*, KT210202*, KT210337*, KT210286*, KT210313*, KT210261*; Elsholtzia rugulosa Hemsl.: China, Yunnan, J. Xu & al. s.n. (KUN), KT210229*, KT210201*, KT210336*, KT210285*, KT210312*, KT210260*; Glechoma hederacea L.: JQ669099, JQ669171, JF289018, JF289018, JF301368, JQ669307; Hanceola sinensis (Hemsl.) Kudô: China, Sichuan, E.D. Liu & al. 3041 (KUN), FJ593353, KT210227*, KT210361*, KT210310*, FJ593416, KF855683; Hedeoma multiflora Benth.: JQ669101, JQ669175, JQ669241, JQ669241, JQ669042, JQ669312; Horminum pyrenaicum L.: DQ667257, JF301314, JF289022, JF289022, GU381477, JQ669315; Hyptis laniflora Benth.: JF301548, JF304259, JF289024, JF289024, JF301370, JQ669317; Hyssopus officinalis L.: JQ669106, JQ669180, JF289023, JF289023, JF301371, JQ669318; Isodon dawoensis (Hand.-Mazz.) H.Hara: China, Sichuan, E.D. Liu & al. 3231 (KUN), KF855429, KT210225*, JF289025, JF289025, JF301372, JQ669319; Keiskea japonica Miq. 1: Japan, Kanagawa, T. Funamoto KJ01 (KUN), KT210244*, KT210217*, KT210352*, KT210301*, KT210328*, KT210276*; Keiskea japonica Miq. 2: Japan, Shizuoka, T. Funamoto KJ02 (KUN), KT210245*, KT210218*, KT210353*, KT210302*, KT210329*, KT210277*; Lavandula angustifolia Mill.: EF437225, –, –, JF289028, AY570457, JQ669323; Lepechinia lamiifolia (Benth.) Epling: JF301348, JF301320, JF289034, JF289034, JF301379, JQ669325; Lepechinia leucophylloides (Ramamoorthy, Hiriart & Medrano) B.T.Drew, Cacho & Sytsma: JF301354, JF301327, JF289047, JF289047, JF301390, JQ669348; Lycopus lucidus var. maackianus Maxim. ex Herder: JQ669110, JQ669184, JQ669247, JQ669247, JQ669048, JQ669329; Lycopus uniflorus Michx.: DQ667302, JQ669185, JF289040, JF289040, DQ667488, JQ669330; Meehania urtici- folia (Miq.) Makino: JQ669113, JQ669188, JF289041, JF289041, JF301385, JQ669333; Melissa axillaris (Benth.) Bakh.f.: JQ669114, JQ669189, JQ669250, JQ669250, JQ669051, JQ669334; Micromeria lanata (C.Sm. ex Link) Benth.: JQ669120, JQ669196, JQ669256, JQ669256, JQ669057, JQ669342; Monarda citriodora Cerv. ex Lag.: JQ669124, JQ669200, JF289045, JF289045, JF301388, JQ669346; Mosla dianthera (Buch.-Ham. ex Roxb.) Maxim.: China, Zheji- ang, C.L. Xiang s.n. (KUN), KT210242*, KT210215*, KT210350*, KT210299*, KT210326*, KT210274*; Mosla sp.: China, Guangxi, H. Peng & al. HP10022 (KUN), KT210243*, KT210216*, KT210351*, KT210300*, KT210327*, KT210275*; Nepeta cataria L.: JQ669126, JQ669202, JF289048, JF289048, JF301391, JQ669349; Ocimum basilicum L.: China, Yunnan, Y.P. Chen s.n. (KUN), DQ667240, KT210226*, JF289049, JF289049, AY570462, JQ669350; Ombrocharis dulcis Hand.-Mazz.: China, Hunan, C.L. Xiang & al. 961 (KUN), KT210250*, KT210223*, KT210358*, KT210307*, KT210334*, KT210282*; Perilla frutescens (L.) Britton 1: China, Jiangxi, G.X. Hu & F. Zhao 135 (KUN), KT210246*, KT210219*, KT210354*, KT210303*, KT210330*, KT210278*; Perilla frutescens (L.) Britton 2: China, Hunan, G.X. Hu & F. Zhao 187 (KUN), KT210247*, KT210220*, KT210355*, KT210304*, KT210331*, KT210279*; Perillula reptans Maxim. 1: Japan, Tokushima, T. Funamoto PR01 (KUN), KT210248*, KT210221*, KT210356*, KT210305*, KT210332*, KT210280*; Perillula reptans Maxim. 2: Japan, Tokushima, T. Funamoto PR02 (KUN), KT210249*, KT210222*, KT210357*, KT210306*, KT210333*, KT210281*; Plectranthus sp.: KM877374, –, JF289052, JF289052, JF301393, JQ669354; Prunella vulgaris L.: JQ669130, JQ669206, JF289055, JF289055, DQ667508, JQ669358; Rosmarinus officinalis L.: DQ667241, JF301329, JF289058, JF289058, AY570465, JQ669364; Salvia przewalskii Maxim.: DQ667254, JF301339, JF289068, JF289068, DQ667443, JQ669372; Salvia yunnanensis C.H.Wright: China, Yunnan, G.X. Hu & al. QT001 (KUN), KT210251*, KT210224*, KT210359*, KT210308*, KT210335*, KT210283*; Satureja thymbra L.: JQ669136, JQ669212, JQ669267, JQ669267, JQ669068, JQ669375; Siphocranion macranthum (Hook.f.) C.Y.Wu: China, Sichuan, E.D. Liu & al. 3033 (KUN), FJ593406, JF304200, KT210362*, KT210311*, FJ593464, KF855684; Tectona grandis L.f.: FM163255, –, NC020098, NC020098, NC020098, NC020098.

136 Version of Record