TAXON 65 (1) • February 2016: 123–136 Chen & al. • Systematic placement of Ombrocharis (Lamiaceae) Resolving the phylogenetic position of Ombrocharis (Lamiaceae), with reference to the molecular phylogeny of tribe 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 Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Biology, University of Nebraska 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 Florida, 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 genus 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 Nepetoideae, 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 Collinsonia, and Elsholtzia 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., Ajugoideae, Lami oideae, Nepetoideae Prostantheroideae, Scutellarioideae, Symphorematoideae, and Major advancements in both molecular phylogenetic and Vitic oideae. 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 Verbenaceae 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 species Sytsma 2012; Salmaki & al., 2013; Xiang & al., 2013). As a re- in about 235 genera and is the sixth-largest family of flower- sult, the positions of some formerly unplaced genera have been ing plants. 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 Taxonomy (IAPT) 2016 Version of Record 123 Chen & al. • Systematic placement of Ombrocharis (Lamiaceae) TAXON 65 (1) • February 2016: 123–136 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: rhizomes 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 Callicarpa, 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, Rhizome 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. 124 Version of Record TAXON 65 (1) • February 2016: 123–136 Chen & al. • Systematic placement of Ombrocharis (Lamiaceae) 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 Mentheae and subfamily Nepetoideae (Drew & Sytsma, 2011, 2012). This ingroup