Acanthaceae), a New Chinese Endemic Genus Segregated from Justicia (Acanthaceae)

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Plant Diversity xxx (2016) 1e10

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Wuacanthus (Acanthaceae), a new Chinese endemic genus segregated from Justicia (Acanthaceae)

* Yunfei Deng a, , Chunming Gao b, Nianhe Xia a, Hua Peng c a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, People's Republic of China b Shandong Provincial Engineering and Technology Research Center for Wild Plant Resources Development and Application of Yellow River Delta, Facultyof Life Science, Binzhou University, Binzhou, 256603, Shandong, People's Republic of China c Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, People's Republic of China article info abstract

Article history: A new genus, Wuacanthus Y.F. Deng, N.H. Xia & H. Peng (Acanthaceae), is described from the Hengduan Received 30 September 2016 Mountains, China. Wuacanthus is based on Wuacanthus microdontus (W.W.Sm.) Y.F. Deng, N.H. Xia & H. Received in revised form Peng, originally published in Justicia and then moved to Mananthes. The new genus is characterized by its 25 November 2016 shrub habit, strongly 2-lipped corolla, the 2-lobed upper lip, 3-lobed lower lip, 2 stamens, bithecous Accepted 25 November 2016 anthers, parallel thecae with two spurs at the base, 2 ovules in each locule, and the 4-seeded capsule. Available online xxx Phylogenetic analyses show that the new genus belongs to the Pseuderanthemum lineage in tribe Justi- cieae. Wuacanthus is closely related to Pseuderanthemum but differs from the latter by its shorter corolla Keywords: Acanthaceae tube and two minute spurs at the base of each anther-theca. W. microdontus is assessed with the status Jinshajiang Valley EN B2ab (iii) based on the IUCN Red List Categories and Criteria. Justicia Copyright © 2016 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Justicieae Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY- Pseuderanthemum lineage NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Wuacanthus

1. Introduction world (Graham, 1988; Mabberley, 2008; Hu et al., 2011). Two divergent trends were suggested by previous studies of the genus, Acanthaceae is a pantropical family consisting of about 230 resulting either in the recognition of a large number of small genera and 4000 species (Mabberley, 2008; Hu et al., 2011). Recent segregate genera or in the adoption of a very broad definition of phylogenetic studies of Acanthaceae have resulted in the recogni- Justicia. In the former, Bremekamp (1948) separated Justicia s.l. tion of four subfamilies, viz., Nelsonioideae, Thunbergiodeae, Avi- into several genera and his treatment was followed by many au- cennioideae and Acanthoideae. Acanthoideae differs from the other thors (e.g., Hu and Tsui, 2002). In contrast, Graham (1988) adopted three subfamilies in having reticulata and is further divided into a very broad definition of Justicia and divided the genus into 16 seven tribes, Acantheae, Ruellieae, Justicieae, Barlerieae, Neu- sections and 7 subsections. Graham's treatment was followed by rantheae, Andrographideae and Whitifieldieae (Reveal, 2012). Four the most recent works (Hu et al., 2011; Wood, 2014). The genus lineages were recognized by McDade et al. (2000) in Justicieae, viz., Justicia is characterized by several characters, including the 2- the Justicia lineage, Tetramerium lineage, Isoglossinae and Pseu- lipped corolla with the bilobed upper lip and trilobed lower lip, deranthemum lineage. two bithecous stamens, usually one thecae above the other, and Justicia L. is the largest genus in the family Acanthaceae and the lower one with a spur at the base (Graham, 1988, Hu et al., belongs to tribe Justicieae. It comprises approximately 600 species 2011). However, recent molecular evidence has indicated that and is distributed in the tropical and subtropical regions of the Justicia in the broad sense of Graham (1988) is paraphyletic (McDade et al., 2000; Gao, 2010). In China, 43 species were recognized in the recently published volume 19 of the Flora of China (Hu et al., 2011). Among these, * Corresponding author. Justicia microdonta W.W. Smith (1918) is quite different in that each E-mail address: [email protected] (Y. Deng). anther-theca has two spurs at the base, whereas the lower anther- Peer review under responsibility of Editorial Office of Plant Diversity. http://dx.doi.org/10.1016/j.pld.2016.11.010 2468-2659/Copyright © 2016 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Deng, Y., et al., Wuacanthus (Acanthaceae), a new Chinese endemic genus segregated from Justicia (Acanthaceae), Plant Diversity (2016), http://dx.doi.org/10.1016/j.pld.2016.11.010 2 Y. Deng et al. / Plant Diversity xxx (2016) 1e10 theca has but one spur in other Chinese Justicia. The number of a total volume of 25 ml containing 1 ml DNA (100 ng/ml), 2.5 ml spurs of each theca seems to be an important character for dNTP (250 mm), 1 ml each primer (0.2 mm), 2.5 ml10 buffer, delimitating generic boundary in the Acanthaceae. For example, 0.25 ml Taq polymerase (0.2U), and 17.5 ml ddH2O. The PCR cycling three genera in tribe Ruellieae were found to have two spurs at the conditions were exactly the same for all markers, [96 C 3 min, base of each theca. Diceratotheca is a genus described from Thailand (94 C 1 min, 52 Cor50C 1 min, 72 C 1 min) 32 cycles, 72 C in recent years (Wood et al., 2012). Stenothyrsus C.B. Clarke was 10 min]. The PCR products were purified on a column of sephadex described from the Malaysian state of Perak (Clarke, 1908). The and sequence reactions were read on an ABI 3730 or ABI 3730xl recent studies indicated that Chinese species of Echinacanthus genetic analyzer. differ from the type of Echinacanthus, Echinacanthus attenuatus Wall., in having two spurs at the base of each theca and represent a 2.5. Phylogenetic analyses new undescribed genus (Nees von Esenbeck, 1832; Lo and Fang, 1985; Hu and Tsui, 2002; Deng et al., 2010; Hu et al., 2011; Tripp Sequences for each region were edited by SeqMen (Lasergene 7), et al., 2013). Thus, it is also necessary to re-evaluate the phyloge- aligned with ClustalX (Thompson et al., 1997), and then adjusted by netic and taxonomic position of J. microdonta W. W. Smith. EditPlus manually. All sequences obtained in this study have been In the present study, we determine the phylogenetic position of deposited in Genbank (Table 1). Maximum parsimony (MP) and J. microdonta based on morphological data, especially the charac- Bayesian inference (BI) analyses were used on the individual and ters of anther, pollen and seed examined using scanning electron combined datasets. MP analyses were run with Paup* v.4.0b10 microscope (SEM), and molecular data. (Swofford, 2003). Heuristic search was performed with 1000 rep- licates of random addition sequence, tree bisection-reconnection 2. Material and methods (TBR) branch-swapping, retaining up to a single most parsimonious tree at each replicate. Strict consensus trees were constructed 2.1. Plant material from the most parsimonious trees. Bootstrap analyses (BP) were used to examine the support for individual clades (Felsenstein, Samples of pollen grains and seeds of J. microdonta were 1985). Bootstrap proportions >70% are considered well supported removed from Deng Yunfei et al. 25860 collected from Butuo Xian, (Hillis and Bull, 1993). Bayesian analyses were performed using Sichuan Province, China during our field investigations in October, MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003). These 2014. Voucher specimens were deposited with the Herbarium of were produced using Modeltest 3.7 (Posada and Crandall, 1998)by the South China Botanical Garden, Chinese Academy of Sciences Hierarchical likelihood ratio test (hLRTs) and Akaike information (IBSC) and the Herbarium of the Kunming Institute of Botany, criterion (AIC) (Posada and Buckley, 2004). The Bayesian analyses Chinese Academy of Sciences (KUN). started from random trees sampling on tree every 100th generation In order to determine phylogenetic relationships in Justicieae with four incremental heated chains. The Markov chain Monte using molecular markers, we sampled 35 taxa in 18 genera. Leaf Carlo (MCMC) algorithm was run for 2000000e4000000 genera- material was dried in silica gel, and specimens from our field work tions for each dataset. The first 1000 trees corresponding to the were deposited at IBSC. To complete the dataset, additional DNA “burn-in” period were discarded, and the remaining trees were sequences of 39 species in 39 genera were obtained from GenBank used to construct a majority-rule consensus tree. We consider (Table 1). posterior probabilities (PP) > 0.95 to indicate significant probability for each clade. 2.2. Androecium observation

The corolla samples were opened and observed under light 3. Results microscope and the number of stamens and staminodes were observed immediately. The anther samples were taken from the 3.1. Morphological characters flower and observed under SEM. J. microdonta is subshrub (Fig. 1: B) with the following charac- 2.3. Pollen and seed morphology observation teristics: leaves opposite, glabrous on both surfaces; inflorescence a terminal spike with 2e4 flowers per node (Fig. 1: C); bracts linear- Pollen grains were acetolysed following the modified method of lanceolate; bracteoles 2, similar to bracts; calyx 5-lobed almost to Erdtman (1969). The sampled pollen grains and seeds were base, lobes linear-lanceolate (Fig. 1: E); corolla 2-lipped, lower lip 3- mounted on stubs and coated with gold for 110 s in JFC-1600 lobed, upper lip 2-lobed, tube short, limb longer than tube (Fig. 1: sputter coaters (JEOL Ltd, Tokyo, Japan). A JSM-6360LV scanning D); stamens 2 (Fig. 1: F), filaments pilose, anther bithecate, thecae electron microscope (SEM) (JEOL Ltd, Tokyo, Japan) was used for oblong, each with two white spurs at base (Fig. 1: G); staminodes 2 detailed examination. Measurements of polar axis (P) and equato- (Fig. 1: F); ovary with 2 ovules in each locule (Fig. 1: H), style pilose; rial diameter (E) were made on digital SEM images (20e30 pollen capsule 4-seeded, stalked at the base (Fig. 1: H). grains) with JEOL's Smile View 2.2.6.1 software (JEOL Ltd, Tokyo, The morphological observations showed that J. microdonta has Japan). Shape classes of pollen were determined following Erdtman an androecium of two stamens and two staminodes (Fig. 1: F), with (1969). The terminology for pollen descriptions follows Punt et al. parallel bithecate anthers, with two very small spurs at the base of (2007), while that for seeds follows Graham (1988) and Liu et al. each theca (Fig. 1: G). (2004). Pollen grains of J. microdonta are oblate-spheroidal (P/E ¼ 1.15), 37.8 (31e34) 33.0 (26e38) mm, triangular in polar view, ellip- 2.4. DNA amplification soidal in equatorial view, tricolporate, with 2 pseudocolpi per mesocolpium that are shorter than the colpi, perforate exine, and Total genomic DNA was extracted from silica gel-dried leaves verrucate ornamentation (Fig. 2:AeD). following the method of Doyle and Doyle (1990). Four DNA frag- Seeds of J. microdonta are broadly ovoid, compressed, ments (trnS-G, trnL-F, rps16, ITS) were amplified and sequenced. 3.85e5.56 3.32e4.17 1.2e1.89 mm, with favulariate testa fol- Each gene was amplified by polymerase chain reaction (PCR) using ded with granules, and reticulate (Fig. 2:EeF).

Table 1 Voucher information and GenBank accession numbers for taxa used in this study. GenBank accession numbers marked with superscripts have been published (1Daniel et al., 2008; 2McDade et al., 2000; 3Kiel et al., 2006).

Taxon Voucher specimens Voucher location GenBank

ITS trnL-F trnS-G rps16

Asystasia gangetica (L.) T. Anderson Y. F. Deng 1836 (IBSC) Yunnan, China KP744316 KP744151 KP744233 KP744187 Asystasiella neesiana (Wallich) Nees Y. F. Deng 1915 (IBSC) Hunan, China KP744317 KP744152 KP744234 KP744188 Clinacanthus nutans (N. L. Burman) L. Y. F. Deng 2007002 (IBSC) Guangdong, China KP744318 KP744153 KP744235 KP744189 Cosmianthemum viriduliflorum (C. Y. Wu & H. S. Y. Tong 13082342 (IBSC) Hainan, China e KP744224 KP744230 KP744227 Lo) H. S. Lo Crossandra infundibuliformis (L.) Nees Y. F. Deng 2007006 (IBSC) Guangdong, China e KP744154 KP744236 KP744190 Dicliptera chinensis (L.) Jussieu Y. F. Deng 18025 (IBSC) Guangdong, China KP744319 KP744155 KP744237 KP744191 Eranthemum tetragonum A. Diereich ex Nees Y. F. Deng 18140 (IBSC) Yunnan, China KP744320 KP744156 KP744238 KP744192 Hygrophila ringens (L.) R. Brown exSteudel Y. F. Deng 19358 (IBSC) Guangxi, China KP744321 KP744157 KP744239 KP744193 Hypoestes phyllostachya Baker Y. F. Deng 200711 (IBSC) Guangdong, China KP744322 KP744158 KP744240 KP744194 Isoglossa collina (T. Anderson) B. Hansen Y. G. Wei 06450 (IBSC) Guangxi, China KP744323 KP744159 KP744241 KP744195 Justicia acutangula H. S. Lo & D. Fang Y. G. Wei 06262 (IBSC) Guangxi, China KP744324 KP744160 KP744242 KP744196 Justicia adhatoda L. Y. F. Deng18220 (IBSC) Yunnan, China KP744325 KP744161 KP744243 KP744197 Justicia bentonica Linnaeus Y. F. Deng 17927 (IBSC) Guangdong, China KP744326 KP744162 KP744244 KP744198 Justicia brandegeeana Wasshausen & L.B. Smith Y. F. Deng 20591 (IBSC) Guangdong, China KP744327 KP744163 KP744245 KP744199 Justicia demissa N.H. Xia & Y. F. Deng 2000 MO-IBSC expedition to Hainan, China KP744329 KP744165 KP744246 KP744201 Hainan 122 (IBSC) Justicia gendaruss N. L. Burm. Y. F. Deng 17547 (IBSC) Hainan, China KP744338 KP744174 KP744255 KP744210 Justicia grossa C. B. Clarke Q. L. Wang s. n. (IBSC) Hainan, China KP744328 KP744164 e KP744200 Justicia latiflora Hemsley Y. F. Deng 19854 (IBSC) Hunan, China KP744330 KP744166 KP744247 KP744202 Justicia leptostachya Hemsl. N. H. Xia s. n.(IBSC) Guangdong, China KP744331 KP744167 KP744248 KP744203 Justicia microdonta W. W. Smith Y. F. Deng 25860 (IBSC) Sichuan, China KP744315 KP744150 KP744232 KP744186 Justicia microdonta W. W. Smith Y. F. Deng 25796 (IBSC) Sichuan, China KP744222 KP744223 KP744229 KP744226 Justicia patentiflora Hemsley Y. F. Deng 18515 (IBSC) Yunnan, China KP744332 KP744168 KP744249 KP744204 Justicia procumbens L. Y. F. Deng 18044 (IBSC) Yunnan, China KP744333 KP744169 KP744250 KP744205 Justicia pseudospicata H. S. Lo & D. Fang Y. F. Deng 19922 (IBSC) Guangxi, China KP744334 KP744170 KP744251 KP744206 Justicia quadrifaria (Nees) T. Anderson Y. F. Deng 19148 (IBSC) Hunan, China KP744335 KP744171 KP744252 KP744207 Justicia thunbergioides Wood et al. 22799 (OXF) Bolivia KP744336 KP744172 KP744253 KP744208 Justicia vagabunda Benoist Y. F. Deng 21099 (IBSC) Yunnan, China e KP744225 KP744231 KP744228 Justicia ventricosa Wallich ex Hooker Y. F. Deng 17534 (IBSC) Hainan, China KP744337 KP744173 KP744254 KP744209 Leptostachya wallichii Nees Y. F. Deng 20851 (IBSC) Sichuan, China KP744349 KP744185 KP744266 KP744221 Mackaya tapingensis (W.W. Sm.) Y. F. Deng Y. F. Deng 18452 (IBSC) Yunnan, China KP744339 KP744175 KP744256 KP744211 & C. Y. Wu Pachystachys lutea Nees Y. F. Deng 20593 (IBSC) Guangdong, China KP744340 KP744176 KP744257 KP744212 Peristrophe japonica (Thunberg) Bremekamp 2000 MO-IBSC expedition to Hainan, China KP744341 KP744177 KP744258 KP744213 Hainan 114 (IBSC) Pseuderanthemum graciliflorum Y. F. Deng 17910 (IBSC) Yunnan, China KP744342 KP744178 KP744259 KP744214 Pseuderanthemum latifolium (Vahl) B. Hansen Y. F. Deng17909 (IBSC) Yunnan, China KP744343 KP744179 KP744260 KP744215 Pseuderanthemum polyanthum (C. B. Clarke ex Y. F. Deng 17923 (IBSC) Yunnan, China KP744344 KP744180 KP744261 KP744216 Oliver) Rhinacanthus nasutus (L.) Kurz Y. F. Deng 17820 (IBSC) Yunnan, China KP744345 KP744181 KP744262 KP744217 Rungia chinensis Bentham Y. F. Deng 19254 (IBSC) Guangdong, China KP744346 KP744182 KP744263 KP744218 Rungia mina H. S. Lo Y. F. Deng 18217 (IBSC) Yunnan, China KP744347 KP744183 KP744264 KP744219 Strobilanthes fluviatilis (C. B. Clarke ex W. W. Y. F. Deng 18215 (IBSC) Yunnan, China KP744348 KP744184 KP744265 KP744220 Smith), Moylan & Y. F. Deng Angkalanthus oligophylla Balf. Miller M10292 (UPS) Yemen EU0874781 EU0875671 EU0811051 EU0875331 Anisacanthus brasiliensis Lindau Silva 2333 (US) Brazil EU0874571 EU0875521 EU0810761 EU0875091 Anisotes madagascariensis Benoist Daniel & Butterwick 6736 (CAS) Madagascar AF2897722 AF2897332 ee Aphanosperma sinaloensis (Leonard & Gentry) Daniel 4070cv (CAS) American EU0874541 EU0875501 EU0810721 EU0875031 T. F. Daniel Brachystephanus africanus S. Moore Luke et al. 6704 (US) Tanzania DQ3724693 e DQ3724913 EU087537 Calycacanthus magnusianus K. Schum Daniel 10072 (CAS) Sydney EU0874811 e EU0811081 EU0875361 Carlowrightia haplocarpa Robinson & greenm Manktelow 715 (UPS) Mexico EU0874501 EU0875481 EU0810671 EU0875001 Chalarothyrsus amplexicaulis Lindau Daniel & Bartholomew 4842gh (CAS) American AF2897802 AF2897402 EU0810741 EU0875051 Chorisochora transvaalensis (A. Meeuse) Daniel 9379 (CAS) South Africa EU0874741 EU0875651 EU0811001 EU0875281 Vollesen Chileranthemum pyramidatum (Lindau) T. F. Breedlore & Daniel 70767 (CAS) Mexico AF2897972 AF2897522 ee Daniel Chlamydocardia buettneri Lindau Accession No. 95-0034-44 (BR) Belgium EU0874801 EU0875691 EU0811071 EU0875351 Ecbolium syringifolium (Vahl) Vollesen Daniel & Butterwick 6733 (CAS) Madagascar AF2897862 AF2897432 DQ3724803 EU0875291 Forcipella sp. Daniel et al. (PH) Madagascar EU5288871 EU5289201 EU5289581 EU5290211 Fittonia albivenis (Lindl. Ex Veitch) Brummitt McDade 1178 (ARIZ) American AF2897812 AF2897412 EU0810791 EU0875131 Gypsacanthus nelsonii E. J. Lott, V. Jaram. & Rzed Daniel 8357 (CAS) Mexico AF2897792 AF2897392 EU0810701 EU0875061 Habracanthus charien Leonard Wood 4547 (US) Colombia DQ3724763 e DQ3725033 e Harpochilus neesianus Mart. ex. Nees Souza et al. 5413 (CAS) Brazil AF2897622 AF2897212 ee Henrya insularis Nees ex Benth Jenkins 89e432 (ARIZ) Mexico AF1698432 AF0631252 EU0810711 EU0875071 Herpetacanthus stenophyllus Gomez-Laur. & Herrera 3855 (ARIZ) Costa Rica AF2897952 AF2897502 ee Grayum Hoverdenia speciosa Nees Deniel & Baker 3739 (CAS) Mexico AF2897772 AF2897382 EU0810891 EU0875191 Kalbreyeriella rostellata Lindau McDade 1007 (DUKE) Colombia DQ3724733 e DQ3724983 e Kudoacanthus albonervosus Hosok W. C. Leong 2939 (HAST) China EU5288891 EU5289241 EU5289651 EU5290291 (continued on next page)

Table 1 (continued )

Taxon Voucher specimens Voucher location GenBank

ITS trnL-F trnS-G rps16

Megalochlamys revoluta (Lindau) Vollesen MaDade & Balkwill 1264 (J) South Africa EU0874731 EU0875641 EU0810991 EU0875271 Megaskepasma erythrochlamys Lindau McDade 253 (DUKE) Costa Rica AF1698402 AF0631262 EU5289801 EU5290411 Metarungia galpinii (Baden) C. Baden Deniel 9322 (CAS) South Africa AF2897762 AF2897372 EU5289841 EU5290461 Mexacanthus mcvaughii T. F. Daniel Van Devender 94e23 (CAS) Mexico EU0874331 EU0875391 EU0810471 EU0874841 Mirandea hyssopus (Nees) T. F. Daniel Diaz B. and Carranza 7498 (CAS) Mexico EU0874591 EU0875551 EU0810941 EU0875121 Odontonema tubiforme (Bertol) Kuntze McDade 1182 (ARIZ) American AF1697482 AF0631272 DQ0592973 DQ0592153 Oplonia microphylla (Lam.) Stearn Ornduff 7814cv (CAS) American AF2897982 AF2897532 ee Oreacanthus mannii Benth. Leeuwenberg 8925 (BR) Cameroon DQ3724713 e DQ3724953 e Poikilacanthus macranthus Lindau Haber 707 (MO) Costa Rica AF1698382 AF0670662 EU5289941 EU5290541 Populina richardii Baill. Kerardren 1671 (P) Madagascar EU0874771 EU0875661 EU0811041 EU0875321 Ptyssiglottis pubisepala (Lindau) B. Hansen Daniel 6630 (CAS) Papua New Guinea AF2897872 AF2897442 DQ3724833 EU5290551 Razisea spicata Oerst Hammel 7974 (DUKE) Costa Rica AF1698482 AF0631312 DQ3725023 EU5290561 Ruspolia seticalyx (C. B. Clarke) Milne-Redh Daniel & Butterwick 6635 (CAS) American AF2898002 AF2897552 ee Ruttya fruticosa Lindau Danile s. n. (CAS) American AF2898012 AF2897562 ee Schaueria calicotricha (Link & Otto) Nees Foote s. n. (CAS) American AF2897822 EU0875561 EU0810801 EU0875141 Spathacanthus parviﬂorus Leonard Daniel et al. 8403 (CAS) Mexico AF2898032 AF2897572 ee Stenostephanus chiapensis T. F. Daniel Breedlove & Burns 72688cv (CAS) American AF2897922 AF2897472 DQ3725063 e Streblacanthus cordatus Lindau Daniel et al. 8203 (CAS) Panama AF2897842 AF2897422 EU0810841 EU0875171 Justicia caudata A. Gray Faivre 64 (ARIZ) Mexico AF1698372 AF0631342 EU5289641 EU5290281 Justicia longii Hilsenb Van Devender 87e307 (ARIZ) American AF1698392 AF0631352 ee Tetramerium nervosum Nees McDade & Jenkins 1154 (ARIZ) American AF1698472 AF0631332 EU0810581 EU0874931 Trichaulax mwasumbii Vollesen Mwasumbi 14238 (CAS) Tanzania EU0874711 EU0875621 EU0810971 EU0875251 Yeatesia mabryi Hilsenb. Deniel & Baker 3698 (CAS) American EU0874601 EU0875541 EU0810781 EU0875111

Fig. 1. Wuacanthus microdontus (W.W. Sm.) Y.F. Deng, N.H. Xia & H. Peng. A. Habitat; B. Habit; C. Inﬂorescence; D. Flower; E. Calyx and pistil; F. Opened corolla showing androecium; G. Anther base showing spurs; H. Opened capsule. s: stamen; st: staminode; sp: spur.

Fig. 2. Pollen and seed morphology of Wuacanthus microdontus (W.W. Sm.) Y.F. Deng, N.H. Xia & H. Peng. A. Equatorial view of pollen grain; B. Polar view of pollen grain; C. Pollen sexine ornamentation; D. & E. Seed under SEM and LE, respectively; F. Testa sculpture.

3.2. Phylogenetic analyses regions of the world (Graham, 1988; Daniel,1998; Mabberley, 2008; Hu et al., 2011). Species placed in Justicia have the 2-lipped corolla, The tree topologies produced with maximum parsimony (MP) two stamens, superposed anther-thecae, the upper one muticous and Bayesian inference (BI) were similar, and Fig. 2 shows the and lower one spurred or not. As with earlier studies (McDade et al., maximum parsimony tree with the addition of bootstrap support 2000, 2008), our results show that the genus Justicia is not mono- values. phyletic. All sampled taxa of Justicia by McDade et al. (2000, 2008) The MP tree formed four main clades (Fig. 3). The result is in were nested in the Justicia lineage. In our analyses, at least seven accordance with the previous work of McDade et al. (2000), who clades are recognized and all taxa belong to Justicia lineage, except recognized four lineages in Justicieae, i.e., the Justicia lineage, Tet- J. microdonta and J. grossa. Morphologically, Justicia is one of the ramerium lineage, Isoglossinae and Pseuderanthemum lineage. most diverse genera in Acanthaceae. Characters of the anther and Species of Justicia were separated into three different lineages: (1) pollen are important for delimiting generic boundaries. In Justicia, the majority of Justicia is accommodated in the Justicia lineage and the two thecae vary from being superposed to parallel and spurred nested with Dicliptera, Peristrophe, Hypoestes, Codonacanthus, Rhi- or not at the base. Pollen morphology is one of the important nacanthus, Poikilacanthus, Harpochilus, Megaskepasma, Anisotes, characters for deﬁning generic boundary in the family Acanthaceae Rungia and Metarungia; (2) Justicia grossa is included in “Tetrame- (Lindau, 1893, 1895; Bremekamp, 1944; Daniel, 1998). Pollen grains rium” lineage and is closely related to Clinacanthus (PP ¼ 1.00); the of Justicia vary from tricolporate to bicolporate and the pollen have Justicia lineage is strongly supported and is a sister group to the been divided into ten major types by Graham (1988). The seeds of “Tetramerium” lineage (PP ¼ 1.00); (3) J. microdonta resolved with Justicia vary in shape from spherical to discoid, and the ornamen- the “Pseuderanthemum”lineage, forming a sister taxon with Pseu- tation of seed surfaces is also variable. Graham (1988) divided the deranthemum (PP ¼ 1.00). seeds of Justicia into eleven types based on the testa morphology. These characters of androecium, pollen and seeds indicate that the 4. Discussion genus Justicia may not be monophyletic. Justicia microdonra and J. grossa were nested in the Pseuderanthemum and Tetramerium 4.1. Polyphyly of Justicia lineages, respectively. Both differ from other species of Justicia by both anther-theca being spurred at the base. J. grossa belongs to Justicia is the largest genus in the family Acanthaceae and com- Justicia sect. Grossa (Hansen, 1987). Justicia sect. Grossa consists of prises about 600 species distributed in the tropical and subtropical three species and is distributed in S. China, Vietnam, Laos, Thailand

Fig. 3. Maximum parsimony tree based on nuclear ITS and plastid trnL-K, trnS-G and rps16 regions. Numbers above branches indicate bootstrap percentages.

and Peninsular Malaysia (Hansen, 1987, Hu et al., 2011). It might 4.2. Morphological evidence represent another undescribed new genus and will be treated after further studies because only one taxon was sampled in the present Palynologically, the genus Justicia is one of the most diverse study. Because of the limited taxon sampling for Justicia, further genera of Acanthaceae (Lindau, 1895; Raj, 1961; Graham, 1988; studies on generic delimitation will be necessary. Daniel, 1998; Hu et al,. 2005; Rueangsawang et al., 2013). Lindau

(1895) recognized eleven pollen types in the family Acanthaceae et al. (2012) observed 30 species of Justicia from Thailand and and classified the pollen of Justicia as being “Knotchenpollen€ ”, that recognized five types and four subtypes. The seed of J. microdonta is, characterized by having two or three pores and an aperture area, resembles some species classified as Type 1 of Graham (1988) and with 1e3 rows of insulae on each side of the pore. Graham (1988) Ruengsawang et al. (2012), but differs by the reticulate testa recognized ten major pollen types in the genus Justicia. Her types sculpture being favulariate folded with granules. However, the seed 1e9 belong to the “Knotchenpollen€ ” category of Lindau (1895) and testa sculpture of J. microdonta is more similar to that of some type 1 is accordance with “Spangenpollen” in being tricolporate species of Pseuderanthemum (Duan, 2013). Seed morphology with six pseudocolpi (Lindau, 1895; Raj, 1961; Bremekamp, 1965). implied a close relationship between J. microdonta and Rueangsawang et al. (2013) separated Thai Justicia species into two Pseuderanthemum. groups based on pollen morphology based on whether pollen grains had pseudocolpi. One group has the tricolporate pollen grain 4.3. The new genus Wuacanthus and its systematic position with six pseudocolpi, which is the same as the Spangenpollen of Lindau (1895). Another group has bi- to tricolporate pollen grains, Our results support the exclusion of J. microdonta from Justicia equivalent to the “Knotchenpollen€ ” of Lindau (1895). “Spangenpol- and its affinity with the Pseuderanthemum lineage, which is defined len” was also recognized as a typical character of subtribe Odon- as the subtribe Odontoneminae by Bremekamp (1965). The Pseu- toneminae (Lindau, 1895; Raj, 1961; Bremekamp, 1965). The deranthemum lineage differs from another three lineages of ornamentation of “Spangenpollen” in Justicia observed by Graham McDade et al. (2000) by having four stamens or two stamens and (1988) and Rueangsawang et al. (2013) is microreticulate or retic- two staminodes, and was recognized as a basal lineage in Justicieae ulate with granules in muri. Pollen grains of J. microdonta were (McDade et al., 2000). Morphologically, Wuacanthus differs from tricolporate with six pseudocolpi and belong to the “Spangenpol- other Justicia species in having two stamens and two staminodes, len”, but differ from other species in having “Spangenpollen” with which identify it with the Pseuderanthemum lineage. Spur number verrucate ornamentation. The pollen of J. microdonta resemble at the base of each theca seems to be a very useful character for those of most genera in subtribe Odontoneminae as shown by delimiting basic taxonomic boundaries in the family Acanthaceae. previous studies (Lindau, 1895; Raj, 1961; Bremekamp, 1965; Stern, Some sister genera are mainly differentiated based on the number 1971; Ensermu et al., 1992; Daniel, 1993, 1995, 1998; Scotland and of spurs. Chroesthes is separated from Lepidagathismainly by the Vollesen, 2000; Hu et al., 2005; Deng and Wu, 2009). The pollen spurred anther thecae, which are multicous in Lepidagathis morphology implied a close relationship between Justicia and (Hansen, 1983). Three genera in tribe Ruellieae were found to have subtribe Odontoneminae. two spurs at the base of each theca. Diceratotheca J.R.I. Wood et al. is Traditionally, the genus Justicia was recognized by having simple distributed in Thailand (Wood et al., 2012). Stenothyrsus C.B. Clarke spicate or compound inflorescences, solitary or in sessile clusters, a was described from the Malaysian state of Perak (Clarke, 1908). tubular and bilabiate corolla, two stamens and bithecate and Recent studies have shown that Chinese species of Echinacanthus basally spurred anthers (Graham, 1998; Darbyshire et al., 2010; Hu differ from the type of Echinacanthus, E. attenuatus Wall., in having et al., 2011; McDade et al., 2012; Rueangsawang et al., 2012, 2013). two spurs at the base of each theca and represent another unde- The characters of two stamens and two staminodes suggest that scribed genus (Nees von Esenbeck, 1832; Lo and Fang, 1985; Hu and Justicia micrantha might be removed from Justicia and more related Tsui, 2002; Hu et al., 2011; Tripp et al., 2013). J. microdonta differs to the subtribe Odontoneminae (Lindau, 1895; Bremekamp, 1965). from other genera in the Pseuderanthemum lineage and can be Morphology of the testa cells of the seeds of Acanthaceae is recognized as an independent genus, Wuacanthus, within the variable and has been used in circumscribing taxa at different ranks Pseuderanthemum lineage. (Kippist, 1842; Schaffnit,1906; Oehm, 1932; Bremekamp, 1944; There are two main clades in the Pseuderanthemum lineage, the Hedren, 1988, 1989; Graham, 1988; Ensermu et al., 1992; Pseuderanthemum and Asystasia clades. The Pseuderanthemum Immelman, 1990; Balkwill et al., 1996; Balkwill and Getliffie clade is characterized by two stamens and two staminodes and Norris, 1988; Balkwill and Campbelg-Young, 1999; Darbyshire, includes Wuacanthus, Pseuderanthemum, Ruspolia, Ruttya, Chiler- 2008; Greuter and Rankin Rodriguez, 2010; Darbyshire et al., 2011; anthemum, Odontonema, Oplonia and Mackaya. The Asystasia clade Ruengsawang et al., 2012; Tripp et al., 2013; Kiel and McDade, 2014; is characterized by four stamens without staminodes and includes Al-Hakimi and Latiff, 2015). For example, seeds of Ruellieae have Asistasia and Asystasiella. Wuacanthus formed sister groups with spiral hygroscopic mucilaginous hairs (Kippist, 1842; Greuter and Pseuderanthemum in our studies, and share the characters of the Rankin Rodriguez, 2010; Duan, 2013). Clarke (1885) described the flowers arranged in terminal thyrses, 2-lipped limbs, erect 2-lobed seeds of Justicia as ovoid and strongly compressed or compressed, upper lip, reflexed 3-lobed lower lip, two stamens and two stam- and the testa being tubercular, scaly or glochidiate-subspinescent. inodes, basally solid 4-seeded capsules., Wuacanthus and Pseuder- Balkwill and Getliffie Norris (1988) re-appraised tribal and sub- anthemum formed sister groups with Ruspolia and Ruttya, but differ tribal limits of the Acanthaceae of southern Africa and considered from the monothecate condition of the latter two genera in having that seed coats lacking hairs were definitive of the Justicieae and bithecate anthers. Wuacanthus differs from Pseuderanthemum by some genera. However, some species of Justicia with hairy seeds the shorter corolla tube and bi-spurred theca base of the anther. In were included in later studies (Graham, 1988; Ruengsawang et al., Pseuderanthemum, the corolla is salverform with a long slender 2012). Hedren (1988) described the seed surface as “tuberculate” tube that is not or scarcely apically enlarged, and the antherthecae in his paper on the Justicia capensis Thunb. group. Later, Hedren are multicous at the base (Hu et al., 2011). (1989) proposed six testa types in Justicia sect. Harnieria from tropical Africa. Graham (1988) recognized three distinct groups and 5. Taxonomic treatment some 20 seed types using a low-power binocular microscope, and noted that the testa is an important character for distinguishing the Wuacanthus Y.F. Deng, N.H. Xia & H. Peng, gen. nov. e Type: sections. Ensermu (1990) observed 12 species of Justicia sect. Anellia Wuacanthus microdontus (W.W.Sm.) Y.F. Deng, N.H. Xia & H. Peng. from Ethiopia and described the testa of all species as double- reticulate. Immelman (1990) described the seed surface of south- Shrubs. Leaves opposite, petiolate, blade margin entire, penni- ern African Justicia and pointed out that seed surface characteristics nerved. Spikes terminal, with 2e4 flowers per node; bracts linear- can be used for delimiting some sections in Justicia. Ruengsawang lanceolate; bracteoles 2, similar to bracts. Calyx 5-lobed almost to

Fig. 4. Wuacanthus microdontus (W.W. Sm.) Y.F. Deng, N.H. Xia & H. Peng. A. Flowering branch; B. Portion of stem; C. Upper leaf surface; D. Lower leaf surface; E. Bract; F. Bracteole; G. Calyx; H. Opened calyx; I. Flower; J. Opened corolla showing androecium; K. Anther; L. Pistil; M. Stigma; N. Capsule; O. Seed. AeG, IeM drawn from F. Ducloux 5741 (P) by Yun- Xiao Liu, GeH, NeO drawn by Ding-Han Cui from Y.F. Deng et al. 25860 (IBSC). base; lobes linear-lanceolate. Corolla 2-lipped, upper lip 2-lobed, Smithiorchis T. Tang & F.T. Wang, etc. (Wu, 1988; Li and Li, 1993; Wu lower lip 3-lobed; tube short, limb longer than tube. Stamens 2, and Wu, 1996). Jinshanjiang Valley has a special and vulnerable included; filaments pilose; anther bithecate, thecae oblong, each ecosystem characterized by aridity, high temperatures, semi- with two white spurs at the base. Staminodes 2, minute. Ovary savanna vegetation and relatively few plants. Jinshajiang Valley is glabrous, 2 ovules in each locule; style densely pilose. Capsule 4- rich in endemic taxa and four genera were previously recognized as seeded, stalked at the base. Seeds reddish brown, ovoid. endemic there, viz., Anemoclema (Franch.) W.T. Wang, Nouelia Franch., Musella (Franch.) C.Y. Wu & H.W. Li, and Trailliaedoxa W.W. Distribution. Monospecific; endemic to the Jinshajiang Valley, Sm. & G. Forrest (Jin et al., 1994; Guan et al., 2013). China, occurring in Sichuan and Yunnan Provinces. Jinshajiang Etymology. The generic name honors Professor Wu Zhengyi (Wu Valley is located at the basal zone of the Hengduan Mountain Re- Chengyih) (1916e2013). Prof. Wu has devoted over 70 years to the gion, which is situated within the Indo-Burma hotspot, one of 25 study of the flora and vegetation of China. In 2007, he was awarded biodiversity hotspots defined by Myers et al. (2000). About 13 the State Preeminent Science and Technology Award of China for genera of seed plants are endemic to the Hengduan Mountain Re- his outstanding achievements in the fields of systematic botany and gion, including Acanthochlamys P.C. Kao, Dipoma Franch., Meta- plant geography, as well as plant diversity, conservation, and sus- nemone W.T. Wang, Salweenia E.G. Baker, Sinadoxa C.Y. Wu et al., tainable use of plant resources (Peng et al., 2013; Deng et al., 2013).

Previously, three generic names, Baijiania A.M. Lu & J.Q. Li (Cucur- threats are known. The provisional conservation status of the tibitaceae, Li, 1993), Sinobaijiania C. Jeffrey & W.J. de Wilde species could therefore be considered as Endangered EN B2ab (iii), (Cucurtibitaceae, Jeffrey and de Wilde, 2006) and Zhengyia T. Deng, based on the IUCN Red List Categories and Criteria (IUCN, 2012, D.G. Zhang & H. Sun (Urticaceae, Deng et al., 2013), and nineteen 2014). On the other hand, for the present, it may be better to species names were published in honor of Prof. Wu Zhengyi consider this as Data Deficient (DD) because there have been no (Kunming Institute of Botany, Chinese Academy of Sciences, 2013; specific field searches for the population of the species. Peng et al., 2014). Phenology. It was observed flowering from July to October and Wuacanthus microdontus (W.W.Sm.) Y.F. Deng, N.H. Xia & H. fruiting from October to next February. Peng, comb. nov.≡Justicia microdonta W.W. Sm. in Not. Bot. Gard. Typification. In his protologue, Smith (1918) cited two collections Edinb. 10: 183. 1918. ≡ Mananthes microdonta (W.W. Sm.) C.Y. Wu & (G. Forrest 12822 and 13162) and both of them are syntypes ac- C.C. Hu in C. C. Hu, Fl. Reipubl. Popularis Sin. 70: 296. 2002. e Type: cording to Art. 9.5 of the ICN (McNeill et al., 2012). Both are in CHINA. Yunnan: mountains of the Chungtien Plateau [27300N], accordance with the original description and can be candidates for July 1914, G. Forrest 12822 (Lectotype E! here designated; iso- being lectotype. Here we choose G. Forrest 12822 as the lectotype. lectotype K!). Fig. 4. Additional specimens examined. CHINA. Sichuan: Butuo Xian, Jiaojihe Qu, Dadiba, hilltop, pathside, in the thickets, fl. white, Aug. Shrubs to 2 m tall, much branched. Branches quadrangular, 20, 1959, Chuan Jing Liang 5636 (KUN); Butuo Xian, Niujiaowan sparsely pilose. Petiole 1e2 cm; leaf blade papery, ovate, (2e) Xiang, bank of Xixihe, 690 m, 11 October 2014, Y.F. Deng with H. 3e5.5 1.5e2.5 cm, abaxially light green and pilose along midvein, Peng, Y. Tong & Y.P. Chen 25860 (IBSC, KUN); Ganluo Xian, near adaxially green and pilose especially along veins, base broadly Suxiong Qu, 800 m, Sept. 24, 1976, unknown collector 14291 cuneate and decurrent onto petiole, margin entire, apex shortly (CDBI); Jinyang xian, Tiandiba Zhen, 810 m, in the thicket, ravine, acuminate to acute, lateral veins 5e8 pairs. Spikes terminal, 5e7 9 October 2014, Y.F. Deng with H. Peng, Y. Tong & Y.P. Chen 25796 (e10) cm, unbranched, nodes distant, with 2e4 flowers per node; (IBSC, KUN); Jinyang Xian, Tiandiba Zhen, Jinshajiang Valley, peduncle short; rachis densely pilose; bracts linear-lanceolate, 800 m, Oct. 26, 1985, H. Li 249 (KUN); Jinyang Xian, on the way 3e5 cm; bracteoles similar to bracts. Calyx ca. 7 mm, 5-lobed from Lugao to Pailai, 1100 m, on slope, roadside, Aug. 19, 1964, T.P. almost to base; lobes linear-lanceolate, 5e6 mm, pilose along the Zhu et al 295 (CDBI). Yunnan: Deqen^ Xian, Yangtze Valley at midveins. Corolla white, with purplish-red dots on lower lip inside, Paung-tzu-la [28120N], 7000 ft, Aug 1914, G. Forrest 13162 (E, K); 1e1.3 cm, outside somewhat pubescent to glabrescent; tube short; Yulong Naxizu Zizhixian, Baoshan Xiang, Shitoucheng, October limb longer than tube; lower lip 4e5 mm, 3-lobed, lobes pilose 2014, Z.K. Wu s.n. (IBSC); Qiaojia Xian, Siao Fa Ka dans la region de inside and ciliate on margin; upper lip 2-lobed. Stamens 2, Kiao Kia, 4 Febuary 1908, F. Ducloux 5741 (P). included; filaments pilose, purple; anther bithecate, thecae oblong, ca. 2 mm long, each with two white spurs at base. Staminodes 2, Acknowledgements minute. Ovary glabrous; style densely pilose. Capsule ca. 2 cm, 4- seeded. Seeds reddish brown, circular in outline, 3e4mmin This study was supported by the National Natural Science diameter, favulariate folded with granules, reticulate. Foundation of China (grant nos. 30670124, 31270247, 31470302, 31400184, 31670191, 31110103911) and Science and Technology Distribution and habitat. Endemic to the Hengduan Mountains, Basic Work (grant no. 2013FY112100). We sincerely thank Mrs. Yun- China, occurring in NW Yunnan and SW Sichuan (Fig. 5). It grows in Xiao Liu (IBSC) and Mr. Ding-Han Cui (IBSC) for preparing the line thickets at rocky sites in dry valleys at 690e1500 m. drawings; Dr. Sheng-Xiang Yu for preparing the distribution map; Conservation status. This species is only known from nine col- Prof. Wong Khoon Meng (SING) for improving the manuscript; Dr. lections from seven localities. 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