Li & al. • Phylogenetic position of Wenchengia TAXON 61 (2) • April 2012: 392–401 Phylogenetic position of Wenchengia (Lamiaceae): A taxonomically enigmatic and critically endangered genus 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 Plant Resources Conservation and Sustainable Utilization, South China 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 Botany, the Chinese Academy of Sciences, Kunming 650204, Yunnan, 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 Scutellarioideae, or suggested to be related to Lamioideae or Ajugoideae. 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 Holmskioldia, Tinnea, and Scutellaria 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 species 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 leaves, racemose phylogenetics at the familial (Wagstaff & Olmstead, 1997; Wag- inflorescences, 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, stamens, 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 leaf 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 Ajuga 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 family 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 shrubs 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 (Achyrospermum Blume, 0151) from Hainan Island, and transplanted in the greenhouse Chelonopsis Miq., Comanthosphace S. Moore, Leucosceptrum of South China Botanical Garden (SCBG). Smith, Rostrinucula Kudo) in lacking a sclerenchyma region Flowers 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 plants and treated following tion of Wenchengia. the protocol described by