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Li & al. • Phylogenetic position of Wenchengia TAXON 61 (2) • April 2012: 392–401

Phylogenetic position of Wenchengia (): A taxonomically enigmatic and critically endangered

Bo Li,1,7 Weixiang Xu,2 Tieyao Tu,1 Zhongsheng Wang,2 Richard G. Olmstead,3 Hua Peng,4 Javier Francisco-Ortega,5 Philip D. Cantino6 & Dianxiang Zhang1

1 Key Laboratory of Resources Conservation and Sustainable Utilization, South Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650, Guangdong, China 2 Laboratory of Forest Ecology and Global Change, School of Life Science, Nanjing University, Nanjing 210093, Jiangsu, China 3 Department of Biology and Burke Museum, University of Washington, Box 355325, Seattle, Washington 98195-5325, U.S.A. 4 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of , the Chinese Academy of Sciences, Kunming 650204, , China 5 Department of Biological Sciences, Florida International University, and Fairchild Tropical Botanic Garden, Miami, Florida 33199, U.S.A. 6 Department of Environmental and Plant Biology, Ohio University, Athens, Ohio 45701-2979, U.S.A. 7 Graduate University of the Chinese Academy of Sciences, Beijing 100049, China Author for correspondence: Dianxiang Zhang, [email protected]

Abstract The phylogenetic position of the monotypic genus Wenchengia has long been controversial. It variously has been assigned to a monotypic subfamily Wenchengioideae, treated as a member of subfamily , or suggested to be related to or . The recent rediscovery of Wenchengia alternifolia from its type locality provided an opportunity to infer its phylogenetic position using evidence from two plastid DNA regions (rbcL, ndhF), morphological data, and anatomical and cytological characters. Both the molecular data and a combination of the molecular and the morphological data suggested a close relationship of the genus to a clade consisting of , , and representing Scutellarioideae. This relationship is also supported by a striking similarity in gross morphological, anatomical, and cytologi- cal characters between Wenchengia and other genera of Scutellarioideae, suggesting that it should be more appropriate to treat the genus as a member of Scutellarioideae, rather than assigning it to a monotypic subfamily.

Keywords cytology; molecular systematics; morphology; ndhF; rbcL; Scutellarioideae

Supplementary Material The alignment file for ndhF is available in the Supplementary Data section of the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

INTRODUCTION are similar to Ajugoideae, and the peculiar stalked nutlets are unique to Wenchengia. Considering its morphological unique- Wenchengia alternifolia C.Y. Wu & S. Chow (Lamiaceae), ness, Wu & Chow (1965) eventually established a monotypic the sole member of the monotypic genus Wenchengia C.Y. Wu subfamily, Wenchengioideae, to accommodate it. The subfamil- & S. Chow is endemic to the island of Hainan. The genus and ial status has been adopted by some authors (Wu & Li, 1977; Li was established on the basis of two collections (four & Hedge, 1994; Takhtajan, 2009), but Cantino in Harley & al. sheets) from the 1930s (Wu & Chow, 1965), and until now is (2004) placed Wenchengia in subfamily Scutellarioideae. known from mainly these two collections. Recently, significant progress has been made in Lamiaceae The genus is characterized by alternate , racemose phylogenetics at the familial (Wagstaff & Olmstead, 1997; Wag- , and a unique type of nutlet attachment described staff & al., 1998; Ryding, 2007), subfamilial and tribal (Kauff- as vascular funicles and slender stalks. Based on a thorough mann & Wink, 1994; Wagstaff & al., 1995; Conn & al., 2009; morphological comparison with other subfamilies of Lamiac- Scheen & al., 2010; Bendiksby & al., 2011), and generic levels eae, the original authors suggested it to be most closely related (Lindqvist & Albert, 2002; Huang & al., 2008; Bramley & al., to Scutellarioideae because they have similar corollas, , 2009). Wenchengia, however, was not included in these stud- and tuberculate nutlets. However, the same authors also pointed ies, and progress in evaluation of its phylogenetic position was out that Wenchengia differs from Scutellarioideae in lacking a slow until two duplicate specimens of the original collections gynophore and in having a straight embryo, alternate leaves, studied by Wu & Chow (1965) were discovered in the herbarium a shallowly lobed ovary and subterminal style, and nutlets at- of the Arnold Arboretum (A, Harvard University). Using the tached to the receptacle by means of slender stalks. The first Harvard specimens, Cantino & Abu-Asab (1993) found simi- two of these traits are common in Lamiaceae, but the alternate larities in pollen morphology and epidermal anatomy be- leaves are rare; the shallowly lobed ovary and subterminal style tween Wenchengia and some members of Ajugoideae, including

392 TAXON 61 (2) • April 2012: 392–401 Li & al. • Phylogenetic position of Wenchengia

Cymaria Benth., Acrymia Prain, and L. Their cladistic of the endemic species have been the subject of molecular phy- analysis of morphological data suggested that Wenchengia be- logenetic studies. However none of these phylogenies included longs in or near subfamily Ajugoideae, but a position in subfam- any endemic genera, therefore our phylogenetic study is the ily Scutellarioideae was only one step less parsimonious. Based first one to focus exclusively on a genus restricted to Hainan. on the evidence available at the time, they recommended that Our aim was not only to reveal the phylogenetic position of the genus be treated as incertae sedis. Wenchengia within the mint but to investigate to what Ryding (1996), on the other hand, investigated the anatomy extent the endemic flora of Hainan harbors unique phylogenetic of the single mature nutlet in the Harvard specimens. He found lineages that are sister to or nested within larger clades. the pericarp ultrastructure of Wenchengia to be most similar to that of Tinnea Kotschy ex Hook. f., Renschia Vatke, and Scutellaria L. by having the following characters: tubercles MATERIALS AND METHODS formed by thickening of the mesocarp due to enlarged meso- carp cells and presence of additional cell layers; fibers in the Study site and studied population. — The rediscovered mesocarp; and absence of a sclerenchyma region below the me- population of Wenchengia alternifolia is located in a valley socarp. In Cantino’s (1992) morphological matrix, characters in Wanning, Hainan province of China at an altitude of ca. of the pericarp surface structure and fleshiness of the nutlets 200–300 m. It is growing in very dense rainforest, along moist were included, but the characters of pericarp ultrastructure stone cracks and cliffs, distributed along mountain streams and were not, although they later proved to be of systematic sig- falls for about 1 km. It is accompanied mainly by some Rubia- nificance in the family (Ryding, 1995). Accordingly, Ryding ceae and Euphorbiaceae near streams and some ferns (1996) noted that if the sclerenchyma and fiber characters of and few grasses along cliffs. The valley is periodically flooded nutlets were added to Cantino’s (1992) matrix and the cladistic after heavy rains in the rainy season during July to November. analysis were rerun, a position of Wenchengia as sister group Morphological and anatomical studies. — Gross mor- to Cantino’s (1992) Scutellaria-Tinnea-Renschia clade would phological data were derived mainly from observations of the be two steps more parsimonious than a position among Ajugoi- rediscovered population along with specimens deposited in deae. Although Ryding’s (1996) new pericarp ultrastructural the herbarium of South China Botanical Garden, the Chinese data supported a close relationship between Wenchengia and Academy of Sciences (IBSC). Living and dried specimens of Scutellarioideae as circumscribed by Cantino & al. (1992), Wenchengia for morphological and cytological observations Ryding (1996) noted that Wenchengia also resembles some were collected in April and November 2010 (Voucher: B. Li members of subfamily Lamioideae ( Blume, 0151) from Hainan Island, and transplanted in the greenhouse Miq., S. Moore, of South China Botanical Garden (SCBG). Smith, Kudo) in lacking a sclerenchyma region and fruits for micromorphological observations and in having fibers in the mesocarp. Thus, Ryding (1996) were fixed in FAA solution (formalin : acetic acid : alcohol = suggested that Wenchengia should continue to be considered 18 : 1 : 1), dissected under a Zeiss stereoscope, and dehydrated as incertae sedis in Lamiaceae. through an ethanol series. Samples were critical-point-dried The distinctive morphology of Wenchengia suggests that with CO2, mounted on cupreous stubs, sputter-coated with the genus may have a long history of independent evolution gold-palladium, and then examined using a JSE-6300LV scan- and some of its similarities with other Lamiaceae may be due ning electron microscope (SEM). to convergence. However, lack of material for DNA extraction Calyces of Wenchengia alternifolia for anatomical inves- has until now made it difficult to resolve the phylogenetic posi- tigation were taken from living and treated following tion of Wenchengia. the protocol described by Ryding (2007). The morphological In 2010, we rediscovered a single population of about terminology of calyces and the method for estimation of the two 40 living plants of W. alternifolia on Hainan Island during a quantitative characters PX (percentage of the area of the calyx field survey. From our field experience, we believe that until ad- that contains fibers and similar xylem cells) and PXT (percent- ditional extant populations are found, Wenchengia falls within age of the area that contains fibers and similar xylem cells the Critically Endangered category (CR, based on criteria B2a) belonging to tertiary venation) follow Ryding (2007) as well. of the Red List guidelines of the World Conservation Union Cytological studies. — Mitotic chromosomes were investi- (IUCN, 2001). This new finding of Wenchengia provided us a gated using root tips of living plants. Actively growing root tips unique opportunity to reevaluate its phylogenetic position using were pretreated with saturated santochlor solution for about 3 h evidence from molecular as well as more morphological, ana- at 4°C, washed with distilled water, fixed in fresh Carnoy’s Fluid tomical, and cytological data. The results of a multidisciplinary (ethanol : acetic acid = 3 : 1) for 2 h at 4°C, washed with distilled investigation illuminating the relationships of this enigmatic water three times, and later hydrolyzed in 1 mol/L hydrochloric genus are herein presented. acid at 60°C for 5–6 min. After being rinsed in distilled water Hainan Island is the second-largest island of China with three times, root tips were stained in Carbol Fuchsin solution for approximately 397 endemic species and seven endemic genera. more than 30 min and then squashed on the slide for light mi- It harbors the most pristine tropical ecosystems of the country croscopy. The chromosomes of at least ten metaphase cells were (Francisco-Ortega & al., 2010a, b; Liu & Yang, 2011). In a previ- counted and the measurements of three cells were completed. ous study, Francisco-Ortega & al. (2010b) showed that only 24 The karyotype classification of Stebbins (1971) was followed.

393 Li & al. • Phylogenetic position of Wenchengia TAXON 61 (2) • April 2012: 392–401

Taxon sampling and molecular data. — Since our overall as simple indels determined by the program Gapcoder (Young goal was to reveal the position of Wenchengia relative to large & Healy, 2003). Heuristic search was conducted using 1000 clades such as Scutellarioideae and Lamioideae, we sought random addition sequence replicates, tree-bisection-reconnec- markers that help resolve relationships among subfamilies as tion (TBR) branch swapping, MulTrees in effect, and steepest well as genera of Lamiaceae. Because the plastid genes ndhF descent off. Internal branch support (BS) was estimated with and rbcL have been widely used to infer the phylogeny of Lami- 500 bootstrap replicates, and 100 random sequences additions. aceae at the generic and subfamilial level (Kaufmann & Wink, Frequency values greater than or equal to 65% were plotted 1994; Wagstaff & Olmstead, 1997; Wagstaff & al., 1998), we onto the consensus of the most parsimonious trees. employed these two genes to explore the phylogenetic position Bayesian inference (BI) analyses were carried out using of Wenchengia. We sequenced the two genes for six samples MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003). An evo- and obtained 67 and 37 accessions for rbcL and ndhF from lutionary model of the best substitution types (Nst) and rate GenBank published in previous studies (Olmstead & al., 1992, distribution models (rates) for the combined DNA dataset was 2009; Kaufmann & Wink, 1994, 1996; Olmstead & Reeves, determined by the Akaike information criterion (AIC) using 1995; Wagstaff & al., 1995, 1998; Wagstaff & Olm­stead, 1997; ModelTest v.3.7 (Posada & Crandall, 1998) with the hier- Lindqvist & Albert, 2002; Walker & al., 2004). The dataset archical likelihood ratio tests. Based on the model identified includes 67 species of Lamiaceae, representing all seven sub- (TVM + I + G), Markov chain Monte Carlo (MCMC) analyses families recognized by Harley & al. (2004), and six outgroup were performed with a random starting tree and four chains (one species of Plantaginaceae, Scrophulariaceae and . cold, three heated). The chain of the MCMC was sampled every Most sequences of the two genes were generated from the 100th generation from the cold chain. We stopped the MCMC same samples except those of , and after 5,000,000 generations because the value of average stan- , which were pooled from different species. Generic dard deviation was below 0.01, suggesting that the tree samples names, rather than species names, were used to represent the from the two simultaneous runs became increasingly similar. pooled sequences in the phylogenetic tree. Voucher information For the calculation of the Bayesian posterior probabilities (PP), and GenBank accession numbers are provided in the Appendix. the burn-in period was the first 25% of the sampled generations DNA extraction, amplification, and sequencing. — Total as determined by the program Tracer v.1.4 (Rambaut & Drum- genomic DNA was extracted from silica-gel-dried leaves with mond, 2007). The 50% majority-rule consensus tree for the PP a modified CTAB procedure (Doyle & Doyle, 1987). The ndhF was generated by PAUP* v.4.0b10 (Swofford, 2002). Only PP region was amplified using the modified primer pair ndhF1 greater than 90% were marked onto the consensus trees. and ndhF2112R (Olmstead & Reeves, 1995). PCR reactions were made using 2.5 μL sample DNA, 0.5 μL Taq DNA poly- merase, 5 μL 10× reaction buffer, 2.5 μL MgCl2, 8 μL dNTPs, RESULTS 1 μL of each primer in a final reaction volume of 50 μL. The PCR program was as follows: an initial template denaturation Morphology and anatomy. — Examination of the living at 94°C for 5 min, 35 cycles of 1 min denaturation at 94°C, plants corroborated most features described by Wu & Chow 2 min primer annealing at 52°C, 1.5 min extension at 72°C, (1965) and additional descriptions by Cantino & Abu-Asab with a final extension of 8 min at 72°C. The rbcL region was (1993). Our examination based on living individuals presents a amplified using the primers described by Olmstead & al. more detailed description and illustration of the genus, expand- (1992). Reaction volumes and PCR procedures were identical ing the work of Wu & Chow (1965) and Cantino & Abu-Asab to those given above. Sequencing was done by the Invitrogen (1993). sequencing service (Invitrogen, Commercial sequencing facil- Growth form. – Wenchengia alternifolia individuals are ity, Guangzhou, China). subshrubs ranging in height from 15–40 cm with glabrous, Phylogenetic analysis. — Sequencher v.4.5 (Gene Codes woody, solid stems. The lower parts of the stems are gray or Corporation, 2005) was used to evaluate chromatograms for slightly greenish, with many slightly elevated reniform leaf base confirmation and to edit contiguous sequences. All the scars; the young parts are fleshy, leafy, and densely hirtellous DNA sequences were initially aligned by Clustal X v.1.83 (Fig. 1A). The protologue and the collector’s notes stated the (Thompson & al., 1997) and adjusted manually in BioEdit Se- height of the plant but gave no details about its growth form. In quence Alignment Editor v.7.0.0 (Hall, 1999). Since the current the population we observed, individual plants do not have a sin- subfamilial backbone of Lamiaceae was outlined successfully gle taproot but several well-developed, woody to slightly fleshy based on the combined phylogenetic analyses of ndhF and rbcL roots and a short, stout, woody stem base bearing 3–25 slender, sequences (Wagstaff & al., 1998), we accordingly conducted leafy shoots (Fig. 1B). These shoots are flexible and rarely our analysis using a combination of the two genes. We also branch above the point where they radiate from the stem base. analyzed a combination of the DNA dataset and a subset of the Most of the branches are prostrate or ascending, bending into morphological matrix. The morphological matrix is based on inflorescences, but some are much more flexible, trail along Cantino & Abu-Asab’s (1993) matrix with minor modification. stone cracks and take roots from leafy nodes to form clones. Maximum parsimony (MP) analyses were conducted using Leaves. – The alternate leaves are chartaceous and flexible PAUP* v.4.0b10 (Swofford, 2002) with all characters unordered with a cuneate-decurrent base, obtuse or somewhat acuminate and equally weighted, gaps treated either as missing data, or apex, and hirtellous 1.0–2.5 cm long (Fig. 1A). The leaf

394 TAXON 61 (2) • April 2012: 392–401 Li & al. • Phylogenetic position of Wenchengia

Fig. 1. Wenchengia alternifolia. A, habit; B, roots; C, white flowers; D, infructescence with dormant buds (arrows); E, nutlets hanging outside the calyx through calyx on flexible funicles (arrows).

395 Li & al. • Phylogenetic position of Wenchengia TAXON 61 (2) • April 2012: 392–401

blades are oblanceolate or obovate-oblanceolate, 5.5–8.5 cm is arcuate, bearded at the middle inside, and 1.6–2.4 cm long, long and 2.0–4.0 cm wide, adax ially glossy dark green with with a narrow, bent base and a gradually dilated broad throat slightly white vein traces and abaxially greenish-white; the ranging in width from 6.0 to 8.5 mm; the up per lip is 2-lobed, surfaces are slightly hirtellous on both s ides or almost glabrous 4.5–5.0 mm wide, and slightly concave; the lower lip is 3-lobed, above, with shallowly undulate margins; the primary veins are 5.0–6.0 mm wide, subelliptic, and spreading (Fig. 1A, C). convex below and flat above with 4.0–6.0 secondary veins, the The calyces are mostly purple, hirtellous outside, fun- lateral veins anastomosing near the margin. The young stems, nelform, 6.0–8.0 mm long, and 2-lipped with five shallow pe tioles, lower surfaces and margins of the young leaves are teeth (Fig. 2G); the upper lip is formed by three equal, deltoid always purple and hirtellous (Fig. 1A). It is noteworthy that the teeth; the lower lip is formed by two dilated and coalescent leaves of Wenchengia alternifolia are adapted to drought and teeth, nearly truncate, more than twice as long as the upper lip become desiccated for several weeks during the dry season but (Fig. 1D); the calyces have 5 primary veins, 14 secondary veins, rapidly rehydrate with the onset of the rainy season. and many reticulate tertiary veins which are most abundant . – T h e of Wenchengia alternifolia in the distal part and more or less parallel in the proximal to are 10.0–25.0 cm long, solitary, and terminal on branches . Wu central part (Fig. 2G). The PX and PXT estimations are ap- & Chow (1965) described the flowers as spirally arranged, but proximately 18 and 8, respectively. we observed that although the flowers are spirally arranged The four stamens are arcuate, slightly exserted from and in bud, they twist and face the same direction during anthesis adnate to the corolla tube near the mouth, and underlying and (Fig. 1A, C). Sometimes, the flowers are oppositely arranged sligh tly shorter than the upper lip (Fig. 1A, C). The po sterior in bud (Fig. 2B). Although the inflorescences were inferred as pair of stamens is as long as the anterior pair, contrary to Wu annual (Wu & Chow, 1965), they may actually persist through & Chow (1965) who described the posterior pair as longer. The several growing seasons since there are dormant flower buds filaments bear capitate glandular hairs (Fig. 2D); the anthers are near the apex of each infructescence (Fig. 1D). ovoid, dark purple, glandular-ciliate, 2-celled, and conspicu- Flowers. – The flo wers are mostly pink, bluish-purple, ously divaricate due to a deltoid thickening of the connective or rarely white (Fig. 1C). The corolla is obliquely tubular- (Fig. 2E). campanulate, 2-lipped, 2.0–3.0 cm long, and sparsely covered The ovary is terete, densely puberulent and glandular on by capitate glandular and nonglandular hairs; the corolla tube the apical half, and lobed for a quarter of its length; the ovules

Fig. . Morphology of Wenchengia alternifolia. A, nutlets and their attachments; B, oppositely arranged flowers in buds; C, flower bud with one and two tiny bracteoles; D, filament with capitate glandular hairs and anther with capitate glands; E, deltoid thickened connective; F, nutlet with five longitudinal ribs, capitate glands, and pubescence on distal side; G, calyx (opened). — Scales: A–C = 1 mm; D = 200 μm; E = 50 μm; F = 500 μm; G = 5 mm.

396 TAXON 61 (2) • April 2012: 392–401 Li & al. • Phylogenetic position of Wenchengia

are anatropous; the style is subterminal, glabrous and arcu- Phylogenetic analyses. — When gaps were treated as miss- ate; the stigmas are not as equally rhombic as described and ing data, the combined matrix of the two plastid genes comprises illustrated in the original publication (Wu & Chow, 1965), but 3509 characters (1402 from rbcL and 2107 from ndhF), of which instead are unequally 2-cleft, subulate with the anterior lobe 1257 are variable (35.8%) and 792 are parsimony-informative conspicuously arcuate and much longer than the posterior; a (22.6%), and generated 592 equally most parsimonious trees disc is poorly developed. with a tree length of 3320 steps, a consistency index (CI) of The pedicels are purple, hirtellous, 1.6–2.2 mm long in 0.52, a CI excluding uninformative characters of 0.34, and a flower and to 3.5–5.0 mm in fruit. The are purple, linear- retention index (RI) of 0.65. When gaps were treated as simple lanceolate, hirtellous, and as long as the pedicels (Fig. 1D). indels, the combined dataset comprises 23 more characters, of Though Wu & Chow (1965) described that bracteoles are which 8 are parsimony-informative, and generated 600 equally absent, Cantino & Abu-Asab (1993) found that there are two most parsimonious trees with a tree length of 3347 steps, a CI linear-lanceolate bracteoles in some plants. In fact, the bracte- of 0.52, a CI excluding uninformative characters of 0.34, and a oles are present in every young flower (Fig. 2C) but are early RI of 0.65. The strict consensus tree (Fig. 4) generated from the deciduous in most individuals. latter dataset is topologically congruent with that from the former Fruits. – The schizocarpic fruits comprise four dry nutlets dataset but better-resolved at some nodes. attached in pairs to the receptacle by two slender, bifurcating The topology of the tree inferred from the two plastid stalks (Fig. 2A). Each stalk has a broadened point that gives regions using Bayesian inference is congruent with those us- rise to the two white funicles, each one connecting to a nutlet. ing parsimony methods. Monophyly of Lamiaceae is strongly The funicles are reflexed downwards, then gradually bend- supported by our analysis (BS = 100, PP = 100). Within La- ing upwards again and very flexible so that the nutlets hung miaceae, six large clades are well-supported and correspond outside through calyx crack (Fig. 1E). The nutlets are broadly to subfamilies Symphorematoideae, Ajugoideae (BS = 100, PP obovoid, 1.6–2.1 mm long, 1.5–1.8 mm wide, dorsiventrally = 100), (BS = 100, PP = 100), flattened, and apically tuberculate and pubescent. Sometimes (BS = 100, PP = 100), Lamioideae (BS = 87, PP = 100), and the lower two nutlets do not develop (Fig. 2A). The exocarp is Scutellarioideae (BS = 87, PP = 100) sensu Harley & al. (2004); dark brownish, thin, minutely colliculate, and covered with sensu Harley & al. (2004) fall into two separate five longitudinal ribs on the distal side (Fig. 2F). The areole clades that have an unresolved relationship to each other and is on the proximal side of the nutlet, 0.5–0.7 mm high, ca. to the other subfamilies. Wenchengia alternifolia is sister to 0.30–0.35 times as long as the nutlet. The embryos are spatu- the Holmskioldia-Tinnea-Scutellaria clade forming subfamily late and erect with the radicle directed downward and fleshy Scutellarioideae (labeled in Fig. 4). A combination of the DNA cotyledons. sequence data and the morphological data generated an MP Cytology. — The chromosome number in mitotic meta- tree with less resolution. This may be caused by homoplasy of phase cells was counted to be 2n = 36. The chromosomes are some morphological characters. However, this tree is congruent very small, ranging from 0.91 to 1.98 μm (Fig. 3A). The karyo- with the molecular tree with regard to the moderate to well- type formula is 2n = 22m + 12sm + 2m (SAT) (Fig. 3B). Karyo- supported clades (BS ≥ 65%), and the position of Wenchengia type asymmetry is of type 2B. does not change.

Fig. 3. Mitotic metaphase of Wenchengia alternifolia. A, Micrograph of metaphase chromosomes; B, karyotype of mitotic metaphase chromo- somes. — Scales = 10 μm.

397 Li & al. • Phylogenetic position of Wenchengia TAXON 61 (2) • April 2012: 392–401

Congea tomentosa Symphorematoideae 100 hystrix 80 100 Gmelina hainanensis 100 microphylla 100 Premna puberula Viticoideae 100 domingensis 100 100 agnus-castus 100 Vitex negundo 100 rotundifolia 100 100 Prostanthera nivea Prostantheroideae 100 Westringia mexicana hederacea 78 moldavica 100 montana 100 pyrenaicum Rosmarinus officinalis tuberosa vulgaris calycina lanata farinacea Nepetoideae Salvia divinorum rotundifolia 98 100 menthaefolia 87 100 alsinoides 100 laevigatum 100 Perovs kiaabrotanoides 100 86 officinalis 65 100 canadensis stauntonii barbatus 100 Ajuga decumbens 100 Ajuga reptans

100 Pseudocaryopteris bicolor Lamiaceae 78 100 100 incana 100 100 dichotom Ajugoideae 100 100 Clerodendrum 100 100 coulteri 100 Oncinocalyx betchei 100 100 parvifolium 100 fruticans 100 Scutellaria orienta 100 Scutellaria altissima Scutellaria galericulata 91 Scutellaria alpina 92 89 Scutellaria bolanderi 98 Scutellarioideae 100 Scutellaria mociniana 100 95 Scutellaria indica 87 92 Tinnea zambesiaca 100 Holmskioldia sanguinea Wenchengia alernifolia macrantha 72 Stachydeae sylvatica 100 100 haplostachya 90 79 macrophylla 100 100 100 rugosa majus 82 virginia Lamioideae 100 81 purpureum 92 Lamium garganicum 80 87 98 92 100 100 tomentosa vulgare 67 oppositifolia Pogostemon 73 Stachytarpheta dichotoma 65 100 98 Rhaphithamnus spinosus 100 Verbena bonariens volubilis Outgroup 100 Myoporum mauritianum Antirrhinum majus

Fig. . Strict consensus of 600 most parsimonious trees obtained from an MP analysis of the combined ndhF and rbcL dataset when gaps were treated as simple indels (L = 3347, CI = 0.52, RI = 0.65). The topology of the strict consensus tree of the BI analysis was congruent with the MP tree. Bootstrap values ≥ 65% in the MP analysis and posterior probabilities > 90% in the BI analysis are indicated above and below branches, respectively. Subfamily classification follows Harley & al. (2004).

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DISCUSSION the calyces of the two genera at young stages are comparable because they are both 5-toothed. Wenchengia as a member of subfamily Scutellarioideae. Furthermore, Wenchengia and the rest of Scutellarioideae — The topology from the combined plastid regions is consistent have an abundance of calyx fibers. Ryding (2007) found that with previous studies with respect to major groups and well- the amount of fibers in calyces is particularly high in sub- supported relationships (Wagstaff & Olmstead, 1997; Wagstaff families Scutellarioideae and Lamioideae. Wenchengia also has & al., 1998). Results from the MP and BI analyses show that relatively high amounts of calyx fibers, and the PX and PXT Wenchengia emerges as sister to the Holmskioldia-Tinnea- estimations for Wenchengia are close to those for Holmskioldia Scutellaria clade, and inclusion of Wenchengia in subfamily (Ryding, 2007; 18 and 8 respectively versus 18 and 11). Scheen Scutellarioideae is strongly supported (BS = 87, PP = 100). & al. (2010) found that the relatively basal clades of Lamioideae We recommend that Wenchengia be included in Scutellarioi- do not have large amounts of calyx fibers, and these authors deae, rather than recognized as an independent subfamily, even concluded that high amounts of calyx fibers have evolved in- though either classification would be consistent with the phy- dependently in these two subfamilies. Consequently, the high logeny. Morphological, anatomical, and cytological characters amounts of calyx fibers in Scutellarioideae appear to be a syn­ further support its placement in Scutellarioideae. apomorphy of this subfamily even though the same feature Morphological similarities between Wenchengia and evolved independently in Lamioideae. other members of subfamily Scutellarioideae. — Inflores- Nutlets. – Wu & Chow (1965) and Cantino & Abu-Asab cence. – The terminal with spirally arranged flowers (1993) noticed that the nutlet tubercles in Wenchengia super- of Wenchengia was originally described as unique in Lamia- ficially resemble those in Holmskioldia, Renschia, and Scutel- ceae (Wu & Chow, 1965). However, racemose inflorescences laria, and Ryding (1996) reported the tubercles of Wenchengia, are also found in Renschia (Vollesen, 1975), Scutellaria (Li Renschia, and Scutellaria to share similar ultrastructure & Hedge, 1994; Paton & Cantino in Harley & al., 2004), and (Holmskioldia was not included in his study). Thus, the tuber- some species of Tinnea (Vollesen, 1975). Inflorescence struc- culate pericarp shared by Wenchengia, Renschia, most species ture in Lamiaceae is fundamentally cymose, with the cymes of Scutellaria, and presumably Holmskioldia, is probably a often clustered to form thyrses of various shapes, heads, or synapomorphy of the scutellarioid clade. In the case of Tin- infrequently (by reduction of cymes to single flowers) racemes nea, there are elongate and usually plumose projections of the (Harley & al., 2004). In Scutellarioideae, most species have pericarp (Cantino in Harley & al., 2004) which are probably racemes; only Holmskioldia Retz. (Atkins, 1996) and some homologous with the tubercles in these other genera. Based on species of Tinnea (Vollesen, 1975) have inflorescences in which our DNA-based phylogeny, the presence of tuberculate or elon- the cymes are not reduced to single flowers. Although Wu gate outgrowths on the nutlets is apparently a synapomorphy of & Chow (1965) described Wenchengia as lacking bracteoles, Scutellarioideae. This feature is rare in Lamiaceae as a whole. Cantino & Abu-Asab (1993) found that the flowers of one Cytology. – In Scutellarioideae, Holmskioldia also has specimen they studied (Lau 28220) had one to two bracteoles. 2n = 36 chromosomes ranging in length from 0.50–1.66 μm Following our observations, every young bud of W. alterni­ (Choudhary & Roy, 1982, 1983), which are very similar to folia has a central flower and two lateral bracteoles. Thus, the those of Wenchengia. racemose inflorescences of Wenchengia appear to represent Relationship between Wenchengia and , Acrymia, reduced cymes with one bract, one flower, and two reduced and Ajuga. — Cantino & Abu-Asab (1993) found the pollen bracteoles, as also seen in Renschia and some species of Tin- suprareticulate sculpturing and leaf epidermal characters of nea and Scutellaria. The spiral arrangement of the flowers in Wenchengia to be most similar to those of some members of Wenchengia is a derived state (Cantino & Abu-Asab, 1993) that Acrymia, Cymaria, and some species of Ajuga. The pollen of is shared by some species of Scutellaria (e.g., S. discolor Wall. these taxa has suprareticulate sculpturing with low, rounded ex Benth.—Li & Hedge, 1994) and Tinnea (e.g., T. coerulea muri and small, irregular-shaped lumina containing few per- Gürke—Vollesen, 1975). forations, and the leaves have diallelocytic stomata. Holmski- Corolla tube. – The corolla tubes of Wenchengia, Holm- oldia also has similar pollen sculpturing (Cantino & Abu-Asab, skioldia, Renschia, and most species of Scutellaria are very 1993) but lacks diallelocytic stomata (Cantino, 1990). However, similar in having a tubular-campanulate shape, narrow base Cantino & Abu-Asab (1993) also pointed out that Cymaria and (especially geniculate in Wenchengia and some species of Acrymia differ from Wenchengia in having axillary, dichoto- Scutellaria), mostly downcurved middle (almost straight in mously branching cymes and confluent anther thecae. In the Renschia) and gradually dilated broad throat, and all are much cladistic analysis of Cantino & Abu-Asab (1993), Wenchengia longer than the calyx tube. grouped with Cymaria, Acrymia, and Ajuga, but because trees Calyx. – Wu & Chow (1965) surmised that the 19-veined in which Wenchengia grouped with Scutellaria, Tinnea, and calyx of Wenchengia is rare in Lamiaceae and that the strongly Renschia were only one step less parsimonious, these authors accrescent and coherent lower calyx teeth are unique. However, recommended treating Wenchengia as incertae sedis. number of calyx veins of Holmskioldia varies from 16 to 18, Cymaria and Acrymia were included in subfamily Ajugoi- and sometimes is also 19. The funnelform calyx with strongly deae by Cantino & al. (1992), but treated as incertae sedis by accrescent and coalescent lower teeth of Wenchengia is differ- Cantino in Harley & al. (2004). However, both Acrymia and ent from the 5-rounded-lobed calyx of Holmskioldia. However, Cymaria emerge as basal branches in the broader Lamioideae

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clade by molecular phylogenetic analyses (Cymaria—Scheen mints: re-examining generic limits of Vitex and relations (Lamia- & al., 2010; Bendiksby & al., 2011; Acrymia—Bendiksby & al., ceae) in South East Asia. Taxon 58: 500–510. Cantino, P.D. 1990. The phylogenetic significance of stomata and 2011; Olmstead, unpub.). Thus these three genera represent trichomes in the Labiatae and Verbenaceae. J. Arnold Arbor. basal branches in the inclusive clade comprising Lamioideae 71: 323–370. and Scutellarioideae. Acrymia, Cymaria, and Wenchengia Cantino, P.D. 1992. Evidence for a polyphyletic origin of the Lamiac- are small or monotypic genera occurring in some islands and eae. Ann. Missouri Bot. Gard. 79: 361–379. coastal areas of Southeastern Asia, and all have some distinct Cantino, P.D. & Abu-Asab, M.S. 1993. A new look at the enigmatic morphological characters. A further study on the phylogeny of genus Wenchengia (Labiatae). Taxon 42: 339–344. Cantino, P.D., Harley, R.M. & Wagstaff, S. J. 1992. Genera of La- Lamiaceae including these genera may be particularly helpful biatae: Status and classification. Pp. 511–522 in: Harley, R.M. & for understanding whether they have similar histories of ori- Reynolds, T. (eds.) Advances in labiate science. Kew: Royal Bo- gin and diversification. Additionally, the similarities between tanic Gardens. Wenchengia and Ajuga in pollen morphology and leaf epider- Cho, Y., Chung, M. & Palmer, J.D. 1999. Complicated history of the mal anatomy are likely the result of parallel evolution because cox1 group I intron in the mitochondria of angiosperms; hori- they are placed in different clades in our strict consensus tree. zontal transfers, vertical transmissions, and losses of the intron. Unpublished. Implications for evolution of endemics on Hainan Is- Choudhary, S.S. & Roy, R.P. 1982. Meiotic studies and taxonomic con- land. — Including Wenchengia, all seven endemic genera of siderations in some taxa of Verbenaceae. Cytologia 47: 771–777. seed-plants recorded on Hainan Island are monotypic. One Choudhary, S.S. & Roy, R.P. 1983. Karyological studies and trend of likely interpretation for such low number of species may be speciation in some members of Verbenaceae. Cytologia 48: 735–740. that the lineage is too young to have diversified. However, the Conn, B.J., Streiber, N., Brown, E.A., Heywood, M.J. & Olmstead, placement of Wenchengia as sister to the remainder of Scutel- R.G. 2009. Infrageneric phylogeny of Chloantheae (Lamiaceae) based on chloroplast ndhF and nuclear ITS sequence data. Austral. larioideae suggests that it is one of the earliest branches in the Syst. Bot. 22: 243–256. subfamily. As the only living genus of Scutellarioideae endemic Doyle, J.J. & Doyle, J.D. 1987. A rapid DNA isolation procedure for to Hainan Island, Wenchengia may thus represent a relict origi- small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15. nating from the earliest diversification of the subfamily. An Forest, F., Grenyer, R., Rouget, M., Davies, T.J., Cowling, R.M., alternative explanation could be that the comparatively small Faith, D.P., Balmford, A., Manning, J.C., Proches, S., Van der Bank, M., Reeves, G., Hedderson, T.A. & Savolainen, V. 2007. size and homogeneity of habitats in Hainan Island have not Preserving the evolutionary potential of floras in biodiversity provided opportunity for species differentiation for the clade. hotspots. Nature 445: 757–760. Although our study provides the first known evidence for a Francisco-Ortega, J., Wang, F.G., Wang, Z.S., Xing, F.W., Liu, H., Hainan Island endemic having a unique phylogenetic position Xu., H., Xu, W.X., Luo, Y.B., Song, X.Q., Gale, S., Boufford, within a large plant family, we cannot rule out that future phy- D.E., Maunder, M. & An, S.Q. 2010a. Endemic seed plant species logenetic studies of other endemic groups will discover distinct from Hainan Island: A checklist. Bot. Rev. (Lancaster) 76: 295–345. Francisco-Ortega, J., Wang, Z.S., Wang, F.G., Xing, F.W., Liu, H., evolutionary histories. Xu., H., Xu, W.X., Luo, Y.B., Song, X.Q., Gale, S., Boufford, D.E., Maunder, M. & An, S.Q. 2010b. Seed plant endemism on Hainan Island: A framework for conservation actions. Bot. Rev. ACKNOWLEDGEMENTS (Lancaster) 76: 246–376. Gene Codes Corporation. 2005. Sequencher, version 4.5. Ann Arbor, Michigan: Gene Codes Corporation. http://genecodes.com/. The authors are grateful to Mr. Zhong-Hui Ma and Zhu-Qiu Song Hall, T.A. 1999. BioEdit: A user-friendly biological sequence alignment for field assistance in April and November 2010 respectively, to Ms. editor and analysis program for Windows 95/98/NT. Nucl. Acids Xiao-Ying Hu for technical assistance with SEM observation, and to Symp. Ser. 41: 95– 98. Drs. Wei Gong, Pei-Wu Xie and Miss Xing Guo for technical assistance Harley, R.M., Atkins, S., Budantsey, A.L., Cantino, P.D., Conn, with the molecular and phylogenetic analyses. This work was supported B.J., Grayer, R., Harley, M.M., Kok, R. de, Krestovskaja, T., by National Natural Science Foundation of China (Grant Nos. 30970182, Morales, R., Paton, A.J., Ryding, O. & Upson, T. 2004. Labiatae. Pp. 167–275 in: Kubitzki, K. & Kadereit, J.W. (eds.), Families and 31170184) and the Chinese Academy of Sciences Knowledge Innovation genera of vascular plants, vol. 7, Flowering plants: Dicotyledons; Program (Grant No. KSCX2-EW-Z-6-6). This study is contribution (except Acanthaceae including Avicenniaceae). Springer, number 222 from the Tropical Biology Program of Florida International Berlin. University. Huang, M.J., Crawford, D.J., Freudenstein, J.V. & Cantino, P.D. 2008. Systematics of Trichostema (Lamiaceae): Evidence from ITS, ndhF, and morphology. Syst. Bot. 33: 437– 446. IUCN. 2001. IUCN red list categories and criteria, version 3.1. Gland, LITERATURE CITED Switzerland: IUCN. http://www.redlist.org. Kaufmann, M. & Wink, M. 1994. Molecular systematics of the Nepe- Albert, V.A., Williams, S.E. & Chase, M.W. 1992. Carnivorous plants: toideae (Lamiaceae): Phylogenetic implications from rbcL gene Phylogeny and structural evolution. Science 257: 1491–1495. sequences. Biosci. Rep. 49: 635–645. Atkins, S. 1996. Holmskioldia sanguinea Labiatae, formerly Verbena- Kaufmann, M. & Wink, M. 1996. Phylogenetic relationships between ceae. Bot. Mag. 13: 79–81. some members of the subfamily lamioideae inferred from nucleo- Bendiksby, M., Thorbek, L., Scheen, A.C., Lindqvist, C. & Ryding, tide sequences of the root gene. Bot. Acta 109: 139–148. O. 2011. An updated phylogeny and classification of Lamiaceae Li, H.W. & Hedge, I.C. 1994. Wenchengia. Pp. 21–22 in: Li, H.W. & subfamily Lamioideae. Taxon 60: 471–484. Hedge, I.C. (eds.) Flora of China, vol. 17. Beijing: Science Press; Bramley, G.L., Forest, F. & de Kok, R.P.J. 2009. Troublesome tropical St. Louis: Missouri Botanical Garden Press.

400 TAXON 61 (2) • April 2012: 392–401 Li & al. • Phylogenetic position of Wenchengia

Lindqvist, C. & Albert, V.A. 2002. Origin of the Hawaiian endemic Scheen, A.C., Bendiksby, M., Ryding, O., Mathiesen, C., Albert, mints within North American (Lamiaceae). Amer. J. Bot. V.A. & Lindqvist, C. 2010. Molecular phylogenetics, character 89: 1709–1724. evolution, and suprageneric classification of Lamioideae (Lamia- Liu, Y. & Yang, Q.E. 2011. Hainanecio, a new genus of the Sene- ceae). Ann. Missouri Bot. Gard. 97: 191–217. cioneae, Asteraceae from China. Bot. Stud. 52: 115–120. Stebbins, G.L. 1971. Chromosome evolution in higher plants. London: Marx, H.E., O’Leary, N., Yuan, Y., Lu-Irving, P., Tank, D., Mul- Arnold. gura, M.E. & Olmstead, R.G. 2010. A molecular phylogeny and Swofford, D.L. 2002. PAUP*: Phylogenetic analysis using parsimony classification of Verbenaceae. Amer. J. Bot. 97: 1647–1663. (*and other methods), version 4.0b10. Sunderland, Massachusetts: Olmstead, R.G. & Reeves, P.A. 1995. Evidence for the polyphyly of the Sinauer. Scrophulariaceae, based on chloroplast rbcL and ndhF sequences. Takhtajan, A. 2009. Flowering plants, 2nd ed. Berlin: Springer. Ann. Missouri Bot. Gard. 82: 176–193. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Hig- Olmstead, R.G., Bremer, B., Scott, K.M. & Palmer, J.D. 1993. A gins, D.G. 1997. The Clustal X windows interface: Flexible strate- parsimony analysis of the Asteridae sensu lato based on rbcL se- gies for multiple sequence alignment aided by quality analysis quences. Ann. Missouri Bot. Gard. 80: 700–722. tools. Nucl. Acids Res. 25: 4876–4882. Olmstead, R.G., Kim, K.J., Jansen, R.K. & Wagstaff, S.J. 2000. The Vollesen, K. 1975. A taxonomic revision of the genera Tinnea and Ren- phylogeny of the Asteridae sensu lato based on chloroplast ndhF schia (Lamiaceae, Ajugoideae). Bot. Tidsskr. 70: 1–63. gene sequences. Molec. Phylogenet. Evol. 16: 96–112. Wagstaff, S.J. & Olmstead, R.G. 1997. Phylogeny of Lamiaceae and Olmstead, R.G., Michaels, H.J., Scott, K.M. & Palmer, J.D. 1992. Verbenaceae inferred from rbcL sequences. Syst. Bot. 22: 165–179. Monophyly of the Asteridae and identification of their major lin- Wagstaff, S.J., Hickerson, L., Spangler, R., Reeves, P.A. & Ol- eages inferred from DNA sequences of rbcL. Ann. Missouri Bot. mstead, R.G. 1998. Phylogeny of Lamiaceae s.l. inferred from Gard. 79: 249–265. cpDNA sequences. Pl. Syst. Evol. 209: 265–274. Olmstead, R.G., Zjhra, M.L., Lohmann, L.G., Grose, S.O. & Wagstaff, S.J., Olmstead, R.G. & Cantino, P.D. 1995. A phylogenetic Eckert, A.J. 2009. A molecular phylogeny and classification of analysis of restriction site variation in subfamily Nepetoideae (La- Bignoniaceae. Amer. J. Bot. 96: 1731–1743. miaceae). Amer. J. Bot. 82: 886–892. Posada, D. & Crandall, K.A. 1998. Modeltest: Testing the model Walker, J.B., Sytsma, K.J., Treutlein, J. & Wink, M. 2004. Salvia of DNA substitution. Bioinformatics 14: 817–818. (Lamiaceae) is not monophyletic: Implications for the systemat- Rambout, A. & Drummond, A.J. 2007. Tracer, version 1.4. http:// ics, radiation, and ecological specializations of Salvia and beast.bio.ed.ac.uk/Tracer. . Amer. J. Bot. 91: 1115–1125. Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian phyloge- Wu, C.Y. & Chow, S. 1965. Duo taxa nova Labiatarum. Acta Phytotax. netic inference under mixed models. Bioinformatics 19: 1572–1574. Sin. 10: 249–255. [in Chinese] Ryding, O. 1995. Pericarp structure and phylogeny of the Lamiaceae- Wu, C.Y. & Li, H.W. 1977. Wenchengia. Pp. 96–100 in: Wu, C.Y. & Verbenaceae complex. Pl. Syst. Evol. 198: 101–141. Li, X.W. (eds.), Flora Reipublicae Popularis Sinicae, vol. 65(2). Ryding, O. 1996. Pericarp structure and phylogenetic position of the Beijing: Science Press. genus Wenchengia (Lamiaceae). Bot. Jahrb. Syst. 118: 153–158. Young, N.D. & Healy, J. 2003. GapCoder automates the use of in- Ryding, O. 2007. Amount of calyx fibers in Lamiaceae, relation to del characters in phylogenetic analysis. B. M. C. Bioinf. 4: 6, doi: calyx structure, phylogeny and ecology. Pl. Syst. Evol. 268: 45–58. 10.1186/1471-2105-4-6.

Appendix. Taxa, vouchers, and GenBank accessions for species sampled. Sequences obtained from previously published works are indicated by superscripts: a, Kaufmann & Wink, 1994; b, Wagstaff & Olmstead, 1997; c, Olmstead & Reeves, 1995; d, Olmstead & al., 1992; e, Wagstaff & al., 1998; f, Olmstead & al., 1993; g, Lindqvist & Albert, 2002; h, Forest & al., 2007; i, Walker & al., 2004; j, Olmstead & al., 2009; k, Conn & al., 2009; l, Albert & al., 1992; m, Cho & al., 1999; n, Olmstead & al., 2000; o, Marx & al., 2010. Gene abbreviations are as follows: R = rbcL, N = ndhF. Markers noted # are sequences not available. Agastache mexicana (Kunth) Lint & Epling, R- Z37382a, N- #; Ajuga decumbens Thunb., China, Li 0191 (IBSC), R- JQ322527, N- JQ322521; Ajuga reptans L., R- U32163b, N-L36391c; Antirrhinum majus L., R- L11688d, N- L36392c; K. Koch, R- Z37461a, N- #; Caryopteris incana (Thunb. ex Houtt.) Miq., R- U28869b, N- U78681e; Clerodendrum fragrans Vent., R- L11689d, N- #; Clerodendrum chinense (Osbeck) Mabb., R- #; N- U78683e; Colebrookea oppositifolia Sm., R- U78712e, N- U78688e; L., R- Z37386a, N- #; tomentosa Roxb., R- U28870b, N- U78689e; Dracocephalum moldavica L., R- Z37389a, N- #; Elsholtzia stauntonii Benth., R- U28872b, N- U78690e; Glechoma hederacea L., R- L14292f, N- U78691e; Gmelina hainanensis Oliv., China, Li 0176 (IBSC), R- JQ322524, N- JQ322518; Gmelina hystrix Schult. ex Kurz, R- U28873b, N- U78692e; Haplostachys haplostachya H. St. John, R- AF501987g, N- #; Horminum pyrenaicum L., R- Z37392a, N- #; Holmskioldia sanguinea Retz., R- U28874b, N- U78693e; Lamium garganicum L., R- Z37401a, N- #; Lamium purpureum L., R- U75702b, N- U78694e; Lavandula lanata Boiss., R- Z37405a, N- #; Leonotis leonurus (L.) R. Br., R- AM234998h, N- #; Lepechinia calycina (Benth.) Epling ex Munz, R- AY570386i, N- #; Marrubium vulgare L., R- U28875b, N- U78695e; Melissa officinalis L., R- Z37414a, N- #; Mentha rotundifolia Huds., R- Z37417a, N- U78696e; Monarda menthaefolia S.A.Graham., R- Z37419a, N- #; Myoporum mauritianum Knowlet, R- L36445c, N- L36403c; Nepeta tuberosa L., R- Z37423a, N- #; Ocimum basilicum L., R- Z37424a, N- #; Oncinocalyx betchei F. Muell., R- U31458b, N- U78685e; Origanum laevigatum Boiss., R- Z37426a, N- #; Otostegia tomentosa A. Rich., R- AF501988g, N- #; Perovskia abrotanoides Kar., R- Z37428a, N- #; Petitia domingensis Jacq., R- U28878b, N- U78697e; Petrea volubilis L., R- U28880b, N- FJ887872j; Phyllostegia macrophylla (Gaudich.) Benth., R- AF501991g, N- #; (L.) Benth., R- L14405f, N- L36407c; Plectranthus barbatus Andrews, R- U28882b, N- U78698e; Pogostemon patchouli Pellet., R- L14406f, N- #; Pogostemon cablin (Blanco) Benth., R- #; N- U78699e; Prasium majus L., R- U31459b, N- U78700e; Premna microphylla Turcz., R- U28883b, N- U78701e; Premna puberula Pamp., China, Li 0159 (IBSC), R- JQ322526, N- JQ322519; Prostanthera nivea A. Cunn. ex Benth., R- Z37430a, N- #; Prostanthera rotun- difolia R. Br., R- L14008f, N- GQ381199k; Prunella vulgaris L., R- Z37433a, N- #; Pseudocaryopteris bicolor (Roxb. ex Hardw.) P.D. Cantino, R- U78711e, N- U78680e; Rhaphithamnus spinosus (Juss.) Moldenke, R- U32160b, N- L36409c; Rosmarinus officinalis L., R- Z37394a, N- #; Salvia divinorum Epling & Játiva, R- L14407f, N- U78703e; Salvia farinacea Benth., R- AY570415i, N- #; Satureja montana L., R- Z37454a, N- #; Scutellaria alpina L., R- Z37457a, N- #; Scutellaria altissima L., R- Z37458a, N- #; Scutellaria bolanderi A. Gray, R- L01954l, N- U78704e; Scutellaria galericulata L., R- Z37459a, N- #; Scutellaria indica L., China, Li 0188 (IBSC), R- JQ322529, N- JQ322522; Scutellaria mociniana Benth., R- AJ247612m, N- #; L., R- Z37460a, N- #; L., R- Z37464a, N- #; Stachytarpheta dichotoma (Ruiz & Pav.) Vahl, R- U32161b, N- L36414c; Stenogyne rugosa Benth., R- AF502026g, N- #; A. Gray, R- U78714e, N- AF130147n; Teucridium parvifolium Hook. f., R- U78715e, N- U78684e; L., R- L14411f, N- U78686e; Thymus alsinoides Formánek, R- Z37470a, N- #; Tinnea zambesiaca Baker, R- U28886b, N- U78709e; L., R- U28887b, N- U78682e; Verbena bonariensis L., R- L14412f, N- HM216782o; Vitex agnus-castus L., R- U78716e, N- U78707e; Vitex negundo L., China, Li 0126 (IBSC), R- JQ322525, N- JQ322520; Wenchengia alternifolia C.Y. Wu & S. Chow, China, Li 0151 (IBSC), R- JQ322528, N- JQ322523; Westringia rosmariniformis Sm., R- Z37474a, N- #; (Willd.) Druce, R- #; N- GQ381196k.

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