Wuacanthus (Acanthaceae), a new Chinese endemic genus segregated from Justicia (Acanthaceae)
Yunfei Denga, ChunmingGao Pengb, Nianhe Xiaa, Hua Pengc     
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, Faculty of 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
Abstract: A new genus, Wuacanthus Y.F. Deng, N.H. Xia & H. Peng (Acanthaceae), is described from the Hengduan Mountains, China. Wuacanthus is based on Wuacanthus microdontus (W.W.Sm.) Y.F. Deng, N.H. Xia & H. Peng, originally published in Justicia and then moved to Mananthes. The new genus is characterized by its shrub habit, strongly 2-lipped corolla, the 2-lobed upper lip, 3-lobed lower lip, 2 stamens, bithecous anthers, parallel thecae with two spurs at the base, 2 ovules in each locule, and the 4-seeded capsule. Phylogenetic analyses show that the new genus belongs to the Pseuderanthemum lineage in tribe Justicieae. Wuacanthus is closely related to Pseuderanthemum but differs from the latter by its shorter corolla tube and two minute spurs at the base of each anther-theca. W. microdontus is assessed with the status EN B2ab (iii) based on the IUCN Red List Categories and Criteria.
Keywords: Acanthaceae    Jinshajiang Valley    Justicia    Justicieae    Pseuderanthemum lineage    Wuacanthus    
1. Introduction

Acanthaceae is a pantropical family consisting of about 230 genera and 4000 species( Mabberley, 2008 ; Hu et al., 2011). Recent phylogenetic studies of Acanthaceae have resulted in the recognition of four subfamilies, viz., Nelsonioideae, Thunbergiodeae, Avicennioideae and Acanthoideae. Acanthoideae differs from the other three subfamilies in having reticulata and is further divided into seven tribes, Acantheae, Ruellieae, Justicieae, Barlerieae, Neurantheae, Andrographideae and Whitifieldieae( Reveal, 2012). Four lineages were recognized by McDade et al.(2000) in Justicieae, viz., the Justicia lineage, Tetramerium lineage, Isoglossinae and Pseuderanthemum lineage.

Justicia L. is the largest genus in the family Acanthaceae and belongs to tribe Justicieae. It comprises approximately 600 species and is distributed in the tropical and subtropical regions of the world( Graham, 1988 ; Mabberley, 2008 ; Hu et al., 2011). Two divergent trends were suggested by previous studies of the genus, resulting either in the recognition of a large number of small segregate genera or in the adoption of a very broad definition of Justicia . In the former, Bremekamp(1948) separated Justicia s.l. into several genera and his treatment was followed by many authors(e.g., Hu and Tsui, 2002). In contrast, Graham(1988) adopted a very broad definition of Justicia and divided the genus into 16 sections and 7 subsections. Graham's treatment was followed by the most recent works( Hu et al., 2011 ; Wood, 2014). The genus Justicia is characterized by several characters, including the 2-lipped corolla with the bilobed upper lip and trilobed lower lip, two bithecous stamens, usually one thecae above the other, and the lower one with a spur at the base( Graham, 1988 , Hu et al., 2011). However, recent molecular evidence has indicated that 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, Justicia microdontaW.W. Smith(1918) is quite different in that each anther-theca has two spurs at the base, whereas the lower anther-theca has but one spur in other Chinese Justicia . The number of spurs of each theca seems to be an important character for delimitating generic boundary in the Acanthaceae. For example, three genera in tribe Ruellieae were found to have two spurs at the base of each theca. Diceratotheca is a genus described from Thailand in recent years( Wood et al., 2012). Stenothyrsus C.B. Clarke was described from the Malaysian state of Perak( Clarke, 1908). The recent studies indicated that Chinese species of Echinacanthus differ from the type of Echinacanthus, Echinacanthus attenuatus Wall., in having two spurs at the base of each theca and represent a new undescribed genus(Nees von Esenbeck, 1832 ; Lo and Fang, 1985 ; Hu and Tsui, 2002 ; Deng et al., 2010 ; Hu et al., 2011 ; Tripp et al., 2013). Thus, it is also necessary to re-evaluate the phylogenetic and taxonomic position of J. microdonta W. W. Smith.

In the present study, we determine the phylogenetic position of J. microdonta based on morphological data, especially the characters of anther, pollen and seed examined using scanning electron microscope(SEM), and molecular data.

2. Material and methods 2.1. Plant material

Samples of pollen grains and seeds of J. microdonta were removed from Deng Yunfei et al . 25860 collected from Butuo Xian, Sichuan Province, China during our field investigations in October, 2014 . Voucher specimens were deposited with the Herbarium of the South China Botanical Garden, Chinese Academy of Sciences(IBSC)and the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences(KUN).

In order to determine phylogenetic relationships in Justicieae using molecular markers, we sampled 35 taxa in 18 genera. Leaf material was dried in silica gel, and specimens from our field work were deposited at IBSC. To complete the dataset, additional DNA sequences of 39 species in 39 genera were obtained from GenBank(Table 1).

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).
TaxonVoucher specimensVoucher locationGenBank
Asystasia gangetica (L.) T. AndersonY.F.Deng 1836 (IBSC)Yunnan, ChinaKP744316KP744151KP744233KP744187
Asystasiella neesiana (Wallich) NeesY. F. Deng 1915 (iBSc)Hunan, ChinaKP744317KP744152KP744234KP744188
Clinacanthus nutans (N. L. Burman) L.Y. F. Deng 2007002 (IBSC)Guangdong, ChinaKP744318KP744153KP744235KP744189
Cosmianthemum viriduliflorum (C. Y. Wu & H. S.
Lo) H. S. Lo
Y. Tong 13082342 (IBSC)Hainan, China-KP744224KP744230KP744227
Crossandra infundibuliformis (L.) NeesY. F. Deng 2007006 (IBSC)Guangdong, China-KP744154KP744236KP744190
Dicliptera chinensis (L.) JussieuY. F. Deng 18025 (IBSC)Guangdong, ChinaKP744319KP744155KP744237KP744191
Eranthemum tetragonum A. Diereich ex NeesY. F. Deng 18140 (IBSC)Yunnan, ChinaKP744320KP744156KP744238KP744192
Hygrophila ringens (L.) R. Brown exSteudelY. F. Deng 19358 (IBSC)Guangxi, ChinaKP744321KP744157KP744239KP744193
Hypoestes phyllostachya BakerY. F. Deng 200711 (IBSC)Guangdong, ChinaKP744322KP744158KP744240KP744194
Isoglossa collina (T. Anderson) B. HansenY. G. Wei 06450 (IBSC)Guangxi, ChinaKP744323KP744159KP744241KP744195
Justicia acutangula H. S. Lo & D. FangY. G. Wei 06262 (IBSC)Guangxi, ChinaKP744324KP744160KP744242KP744196
Justicia adhatoda L.Y. F. Deng18220 (IBSC)Yunnan, ChinaKP744325KP744161KP744243KP744197
Justicia bentonica LinnaeusY. F. Deng 17927 (IBSC)Guangdong, ChinaKP744326KP744162KP744244KP744198
Justicia brandegeeana Wasshausen & L.B. SmithY. F. Deng 20591 (IBSC)Guangdong, ChinaKP744327KP744163KP744245KP744199
Justicia demissa N.H. Xia & Y. F. Deng2000 MO-IBSC expedition to
Hainan 122 (IBSc)
Hainan, ChinaKP744329KP744165KP744246KP744201
Justicia gendaruss N. L. Burm.Y. F. Deng 17547 (iBSC)Hainan, ChinaKP744338KP744174KP744255KP744210
Justicia grossa C. B. ClarkeQ. L. Wang s. n. (IBSC)Hainan, ChinaKP744328KP744164-KP744200
Justicia latiflora HemsleyY. F. Deng 19854 (IBSC)Hunan, ChinaKP744330KP744166KP744247KP744202
Justicia leptostachya Hemsl.N. H. Xia s. n.(IBSC)Guangdong, ChinaKP744331KP744167KP744248KP744203
Justicia microdonta W. W. SmithY. F. Deng 25860 (IBSC)Sichuan, ChinaKP744315KP744150KP744232KP744186
Justicia microdonta W. W. SmithY. F. Deng 25796 (IBSC)Sichuan, ChinaKP744222KP744223KP744229KP744226
Justicia patentiflora HemsleyY. F. Deng 18515 (iBSc)Yunnan, ChinaKP744332KP744168KP744249KP744204
Justicia procumbens L.Y. F. Deng 18044 (iBSc)Yunnan, ChinaKP744333KP744169KP744250KP744205
Justicia pseudospicata H. S. Lo & D. FangY. F. Deng 19922 (IBSC)Guangxi, ChinaKP744334KP744170KP744251KP744206
Justicia quadrifaria (Nees) T. AndersonY. F. Deng 19148 (IBSC)Hunan, ChinaKP744335KP744171KP744252KP744207
Justicia thunbergioidesWood et al. 22799 (OXF)BoliviaKP744336KP744172KP744253KP744208
Justicia vagabunda BenoistY.F.Deng21099 (IBSC)Yunnan, China-KP744225KP744231KP744228
Justicia ventricosa Wallich ex HookerY.F.Deng 17534 (iBSc)Hainan, ChinaKP744337KP744173KP744254KP744209
Leptostachya wallichii NeesY. F. Deng 20851 (IBSC)Sichuan, ChinaKP744349KP744185KP744266KP744221
Mackaya tapingensis (W.W. Sm.) Y. F. Deng
& C. Y. Wu
Y. F. Deng 18452 (IBSC)Yunnan, ChinaKP744339KP744175KP744256KP744211
Pachystachys lutea NeesY. F. Deng 20593 (IBSC)Guangdong, ChinaKP744340KP744176KP744257KP744212
Peristrophe japonica (Thunberg) Bremekamp2000 MO-IBSC expedition to
Hainan 114(IBSc)
Hainan, ChinaKP744341KP744177KP744258KP744213
Pseuderanthemum graciliflorumY. F. Deng 17910 (IBSC)Yunnan, ChinaKP744342KP744178KP744259KP744214
Pseuderanthemum latifolium (Vahl) B. HansenY. F. Deng17909 (IBSC)Yunnan, ChinaKP744343KP744179KP744260KP744215
Pseuderanthemum polyanthum (C. B. Clarke ex
Y. F. Deng 17923 (IBSC)Yunnan, ChinaKP744344KP744180KP744261KP744216
Rhinacanthus nasutus (L.) KurzY.F.Deng 17820 (IBSC)Yunnan, ChinaKP744345KP744181KP744262KP744217
Rungia chinensis BenthamY. F. Deng 19254 (IBSC)Guangdong, ChinaKP744346KP744182KP744263KP744218
Rungia mina H. S. LoY. F. Deng 18217 (iBSc)Yunnan, ChinaKP744347KP744183KP744264KP744219
Strobilanthes fluviatilis (C. B. Clarke ex W. W.
Smith), Moylan & Y. F. Deng
Y. F. Deng 18215 (iBSc)Yunnan, ChinaKP744348KP744184KP744265KP744220
Angkalanthus oligophylla Balf.MillerM10292 (UPS)YemenEU0874781EU0875671EU0811051EU0875331
Anisacanthus brasiliensis LindauSilva 2333 (US)BrazilEU0874571EU0875521EU0810761EU0875091
Anisotes madagascariensis BenoistDaniel & Butterwick 6736 (CAS)MadagascarAF2897722AF2897332--
Aphanosperma sinaloensis (Leonard & Gentry)
T. F. Daniel
Daniel 4070cv (CAS)AmericanEU0874541EU0875501EU0810721EU0875031
Brachystephanus africanus S. MooreLuke et al. 6704 (US)TanzaniaDQ3724693-DQ3724913EU087537
Calycacanthus magnusianus K. SchumDaniel 10072 (CAS)SydneyEU0874811-EU0811081EU0875361
Carlowrightia haplocarpa Robinson & greenmManktelow 715 (UPS)MexicoEU0874501EU0875481EU0810671EU0875001
Chalarothyrsus amplexicaulis LindauDaniel & Bartholomew 4842gh (CAS)AmericanAF2897802AF2897402EU0810741EU0875051
Chorisochora transvaalensis (A. Meeuse)
Daniel 9379 (CAS)South AfricaEU0874741EU0875651EU0811001EU0875281
Chileranthemum pyramidatum (Lindau) T. F.
Breedlore & Daniel 70767 (CAS)MexicoAF2897972AF2897522--
Chlamydocardia buettneri LindauAccession No. 95-0034-44 (BR)BelgiumEU0874801EU0875691EU0811071EU0875351
Ecbolium syringifolium (Vahl) VollesenDaniel & Butterwick 6733 (CAS)MadagascarAF2897862AF2897432DQ3724803EU0875291
Forcipella sp.Daniel et al. (PH)MadagascarEU5288871EU5289201EU5289581EU5290211
Fittonia albivenis (Lindl. Ex Veitch) BrummittMcDade 1178 (ARIZ)AmericanAF2897812AF2897412EU0810791EU0875131
Gypsacanthus nelsonii E. J. Lott, V. Jaram. & RzedDaniel 8357 (CAS)MexicoAF2897792AF2897392EU0810701EU0875061
Habracanthus charien LeonardWood 4547 (US)ColombiaDQ3724763-DQ3725033-
Harpochilus neesianus Mart. ex. NeesSouza et al. 5413(CAS)BrazilAF2897622AF2897212--
Henrya insularis Nees ex BenthJenkins 89-432 (ARIZ)MexicoAF1698432AF0631252EU0810711EU0875071
Herpetacanthus stenophyllus Gomez-Laur. &
Herrera 3855 (ARIZ)Costa RicaAF2897952AF2897502--
Hoverdenia speciosa NeesDeniel & Baker 3739 (CAS)MexicoAF2897772AF2897382EU0810891EU0875191
Kalbreyeriella rostellata LindauMcDade 1007 (DUKE)ColombiaDQ3724733-DQ3724983-
Kudoacanthus albonervosus HosokW. C. Leong 2939 (HAST)ChinaEU5288891EU5289241EU5289651EU5290291
Megalochlamys revoluta (Lindau) VollesenMaDade & Balkwill 1264 (!)South AfricaEU0874731EU0875641EU0810991EU0875271
Megaskepasma erythrochlamys LindauMcDade 253 (DUKE)Costa RicaAF1698402AF0631262EU5289801EU5290411
Metarungia galpinii (Baden) C. BadenDeniel 9322 (CAS)South AfricaAF2897762AF2897372EU5289841EU5290461
Mexacanthus mcvaughii T. F. DanielVan Devender 94-23 (CAS)MexicoEU0874331EU0875391EU0810471EU0874841
Mirandea hyssopus (Nees) T. F. DanielDiaz B. and Carranza 7498 (CAS)MexicoEU0874591EU0875551EU0810941EU0875121
Odontonema tubiforme (Bertol) KuntzeMcDade 1182 (ARIZ)AmericanAF1697482AF0631272DQ0592973DQ0592153
Oplonia microphylla (Lam.) StearnOrnduff 7814cv (CAS)AmericanAF2897982AF2897532--
Oreacanthus mannii Benth.Leeuwenberg 8925 (BR)CameroonDQ3724713-DQ3724953-
Poikilacanthus macranthus LindauHaber 707 (MO)Costa RicaAF1698382AF0670662EU5289941EU5290541
Populina richardii Baill.Kerardren 1671 (P)MadagascarEU0874771EU0875661EU0811041EU0875321
Ptyssiglottis pubisepala (Lindau) B. HansenDaniel 6630 (CAS)Papua New GuineaAF2897872AF2897442DQ3724833EU5290551
Razisea spicata OerstHammel 7974 (DUKE)Costa RicaAF1698482AF0631312DQ3725023EU5290561
Ruspolia seticalyx (C. B. Clarke) Milne-RedhDaniel & Butterwick 6635 (CAS)AmericanAF2898002AF2897552--
Ruttya fruticosa LindauDanile s. n. (CAS)AmericanAF2898012AF2897562--
Schaueria calicotricha (Link & Otto) NeesFoote s. n. (CAS)AmericanAF2897822EU0875561EU0810801EU0875141
Spathacanthus parviflorus LeonardDaniel et al. 8403 (CAS)MexicoAF2898032AF2897572--
Stenostephanus chiapensis T. F. DanielBreedlove & Burns 72688cv (CAS)AmericanAF2897922AF2897472DQ3725063-
Streblacanthus cordatus LindauDaniel et al. 8203 (CAS)PanamaAF2897842AF2897422EU0810841EU0875171
Justicia caudata A. GrayFaivre 64 (ARIZ)MexicoAF1698372AF0631342EU5289641EU5290281
Justicia longii HilsenbVan Devender 87-307 (ARIZ)AmericanAF1698392AF0631352--
Tetramerium nervosum NeesMcDade & Jenkins 1154 (ARIZ)AmericanAF1698472AF0631332EU0810581EU0874931
Trichaulax mwasumbii VollesenMwasumbi 14238 (CAS)TanzaniaEU0874711EU0875621EU0810971EU0875251
Yeatesia mabryi Hilsenb.Deniel & Baker 3698 (CAS)AmericanEU0874601EU0875541EU0810781EU0875111
2.2. Androecium observation

The corolla samples were opened and observed under light microscope and the number of stamens and staminodes were observed immediately. The anther samples were taken from the flower and observed under SEM.

2.3. Pollen and seed morphology observation

Pollen grains were acetolysed following the modified method of Erdtman(1969) . The sampled pollen grains and seeds were mounted on stubs and coated with gold for 110 s in JFC-1600 sputter coaters(JEOL Ltd, Tokyo, Japan). A JSM-6360LV scanning electron microscope(SEM)(JEOL Ltd, Tokyo, Japan)was used for detailed examination. Measurements of polar axis(P)and equatorial diameter(E)were made on digital SEM images(20-30 pollen grains)with JEOL's Smile View software(JEOL Ltd, Tokyo, Japan). Shape classes of pollen were determined following Erdtman(1969) . The terminology for pollen descriptions follows Punt et al.(2007) , while that for seeds follows Graham(1988) and Liu et al.(2004) .

2.4. DNA amplification

Total genomic DNA was extracted from silica gel-dried leaves following the method of Doyle and Doyle(1990) . Four DNA fragments(trnS-G, trnL-F, rps16 , ITS)were amplified and sequenced. Each gene was amplified by polymerase chain reaction(PCR)using a total volume of 25 μl containing 1 μl DNA(100 ng/ml), 2.5 μl dNTP(250 mm), 1 μl each primer(0.2 mm), 2.5 μl 10 × buffer, 0.25 μl Taq polymerase(0.2U), and 17.5 μl ddH 2 O. The PCR cycling conditions were exactly the same for all markers, [96 ℃ 3 min, (94 ℃ 1 min, 52 ℃ or 50 ℃ 1 min, 72 ℃ 1 min)× 32 cycles, 72 ℃ 10 min]. The PCR products were purified on a column of sephadex and sequence reactions were read on an ABI 3730 or ABI 3730xl genetic analyzer.

2.5. Phylogenetic analyses

Sequences for each region were edited by SeqMen(Lasergene 7), aligned with ClustalX( Thompson et al., 1997), and then adjusted by EditPlus manually. All sequences obtained in this study have been deposited in Genbank(Table 1). Maximum parsimony(MP)and Bayesian inference(BI)analyses were used on the individual and combined datasets. MP analyses were run with Paup * v.4.0b10( Swofford, 2003). Heuristic search was performed with 1000 replicates of random addition sequence, tree bisection-reconnection(TBR)branch-swapping, retaining up to a single most parsimonious tree at each replicate. Strict consensus trees were constructed from the most parsimonious trees. Bootstrap analyses(BP)were used to examine the support for individual clades( Felsenstein, 1985). Bootstrap proportions >70% are considered well supported( Hillis and Bull, 1993). Bayesian analyses were performed using MrBayes version 3.1.2( Ronquist and Huelsenbeck, 2003). These were produced using Modeltest 3.7( Posada and Crandall, 1998)by Hierarchical likelihood ratio test(hLRTs)and Akaike information criterion(AIC)( Posada and Buckley, 2004). The Bayesian analyses started from random trees sampling on tree every 100th generation with four incremental heated chains. The Markov chain Monte Carlo(MCMC)algorithm was run for 2000000-4000000 generations for each dataset. The first 1000 trees corresponding to the "burn-in” period were discarded, and the remaining trees were used to construct a majority-rule consensus tree. We consider posterior probabilities(PP)> 0.95 to indicate significant probability for each clade.

3. Results 3.1. Morphological characters

J. microdonta is subshrub(Fig. 1: B)with the following characteristics: leaves opposite, glabrous on both surfaces; inflorescence a terminal spike with 2-4 flowers per node(Fig. 1: C); bracts linearlanceolate; bracteoles 2, similar to bracts; calyx 5-lobed almost to base, lobes linear-lanceolate(Fig. 1: E); corolla 2-lipped, lower lip 3-lobed, upper lip 2-lobed, tube short, limb longer than tube(Fig. 1: D); stamens 2(Fig. 1: F), filaments pilose, anther bithecate, thecae oblong, each with two white spurs at base(Fig. 1: G); staminodes 2(Fig. 1: F); ovary with 2 ovules in each locule(Fig. 1: H), style pilose; capsule 4-seeded, stalked at the base(Fig. 1 : H).

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 .

The morphological observations showed that J. microdonta has an androecium of two stamens and two staminodes(Fig. 1: F), with parallel bithecate anthers, with two very small spurs at the base of each theca(Fig. 1: G).

Pollen grains of J. microdonta are oblate-spheroidal(P/E = 1.15), 37.8(31-34)× 33.0(26-38)μm, triangular in polar view, ellipsoidal in equatorial view, tricolporate, with 2 pseudocolpi per mesocolpium that are shorter than the colpi, perforate exine, and verrucate ornamentation(Fig. 2: A-D).

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.

Seeds of J. microdonta are broadly ovoid, compressed, 3.85-5.56 × 3.32-4.17 × 1.2-1.89 mm, with favulariate testa folded with granules, and reticulate(Fig. 2: E-F).

3.2. Phylogenetic analyses

The tree topologies produced with maximum parsimony(MP)and Bayesian inference(BI)were similar, and Fig. 2 shows the maximum parsimony tree with the addition of bootstrap support values.

The MP tree formed four main clades(Fig. 3). The result is in accordance with the previous work of McDade et al.(2000) , who recognized four lineages in Justicieae, i.e., the Justicia lineage, Tetramerium lineage, Isoglossinae and Pseuderanthemum lineage. Species of Justicia were separated into three different lineages:(1)the majority of Justicia is accommodated in the Justicia lineage and nested with Dicliptera , Peristrophe , Hypoestes, Codonacanthus, Rhinacanthus, Poikilacanthus, Harpochilus, Megaskepasma, Anisotes, Rungia and Metarungia ;(2)Justicia grossa is included in "Tetramerium" lineage and is closely related to Clinacanthus (PP = 1.00); the Justicia lineage is strongly supported and is a sister group to the "Tetramerium" lineage(PP = 1.00);(3)J. microdonta resolved with the "Pseuderanthemum"lineage, forming a sister taxon with Pseuderanthemum (PP = 1.00).

Fig. 3 Maximum parsimony tree based on nuclear ITS and plastid trnL-K, trnS-G and rps16 regions. Numbers above branches indicate bootstrap percentages.
4. Discussion 4.1. Polyphyly of Justicia

Justicia is the largest genus in the family Acanthaceae and comprises about 600 species distributed in the tropical and subtropical regions of the world( Graham, 1988 ; Daniel, 1998 ; Mabberley, 2008 ; Hu et al., 2011). Species placed in Justicia have the 2-lipped corolla, two stamens, superposed anther-thecae, the upper one muticous and lower one spurred or not. As with earlier studies( McDade et al., 2000 , 2008), our results show that the genus Justicia is not monophyletic. All sampled taxa of Justicia by McDade et al.(2000 , 2008)were nested in the Justicia lineage. In our analyses, at least seven clades are recognized and all taxa belong to Justicia lineage, except J. microdonta and J. grossa . Morphologically, Justicia is one of the most diverse genera in Acanthaceae. Characters of the anther and pollen are important for delimiting generic boundaries. In Justicia , the two thecae vary from being superposed to parallel and spurred or not at the base. Pollen morphology is one of the important characters for defining generic boundary in the family Acanthaceae( Lindau, 1893 , 1895 ; Bremekamp, 1944 ; Daniel, 1998). Pollen grains of Justicia vary from tricolporate to bicolporate and the pollen have been divided into ten major types by Graham(1988) . The seeds of Justicia vary in shape from spherical to discoid, and the ornamentation of seed surfaces is also variable. Graham(1988) divided the seeds of Justicia into eleven types based on the testa morphology. These characters of androecium, pollen and seeds indicate that the genus Justicia may not be monophyletic. Justicia microdonra and J. grossa were nested in the Pseuderanthemum and Tetramerium lineages, respectively. Both differ from other species of Justicia by both anther-theca being spurred at the base. J. grossa belongs to Justicia sect. Grossa( Hansen, 1987). Justicia sect. Grossa consists of three species and is distributed in S. China, Vietnam, Laos, Thailand and Peninsular Malaysia( Hansen, 1987 , Hu et al., 2011). It might represent another undescribed new genus and will be treated after further studies because only one taxon was sampled in the present study. Because of the limited taxon sampling for Justicia , further studies on generic delimitation will be necessary.

4.2. Morphological evidence

Palynologically, the genus Justicia is one of the most diverse genera of Acanthaceae( Lindau, 1895 ; Raj, 1961 ; Graham, 1988 ; Daniel, 1998 ; Hu et al, . 2005 ; Rueangsawang et al., 2013). Lindau(1895) recognized eleven pollen types in the family Acanthaceae and classified the pollen of Justicia as being "Knöotchenpollen", that is, characterized by having two or three pores and an aperture area, with 1-3 rows of insulae on each side of the pore. Graham(1988) recognized ten major pollen types in the genus Justicia. Her types 1-9 belong to the "Knöotchenpollen" category of Lindau(1895) and type 1 is accordance with "Spangenpollen" in being tricolporate with six pseudocolpi( Lindau, 1895 ; Raj, 1961 ; Bremekamp, 1965). Rueangsawang et al.(2013) separated Thai Justicia species into two groups based on pollen morphology based on whether pollen grains had pseudocolpi. One group has the tricolporate pollen grain with six pseudocolpi, which is the same as the Spangenpollen of Lindau(1895) . Another group has bi- to tricolporate pollen grains, equivalent to the "Knöotchenpollen" of Lindau(1895) . "Spangenpollen" was also recognized as a typical character of subtribe Odontoneminae( Lindau, 1895 ; Raj, 1961 ; Bremekamp, 1965). The ornamentation of "Spangenpollen" in Justicia observed by Graham(1988) and Rueangsawang et al.(2013) is microreticulate or reticulate with granules in muri. Pollen grains of J. microdonta were tricolporate with six pseudocolpi and belong to the "Spangenpollen", but differ from other species in having "Spangenpollen" with verrucate ornamentation. The pollen of J. microdonta resemble those of most genera in subtribe Odontoneminae as shown by previous studies( Lindau, 1895 ; Raj, 1961 ; Bremekamp, 1965 ; Stern, 1971 ; Ensermu et al., 1992 ; Daniel, 1993 , 1995 , 1998 ; Scotland and Vollesen, 2000 ; Hu et al., 2005 ; Deng and Wu, 2009). The pollen morphology implied a close relationship between Justicia and subtribe Odontoneminae.

Traditionally, the genus Justicia was recognized by having simple spicate or compound inflorescences, solitary or in sessile clusters, a tubular and bilabiate corolla, two stamens and bithecate and basally spurred anthers(Graham, 1998 ; Darbyshire et al., 2010 ; Hu et al., 2011 ; McDade et al., 2012 ; Rueangsawang et al., 2012 , 2013). The characters of two stamens and two staminodes suggest that Justicia micrantha might be removed from Justicia and more related to the subtribe Odontoneminae ( Lindau, 1895 ; Bremekamp, 1965).

Morphology of the testa cells of the seeds of Acanthaceae is variable and has been used in circumscribing taxa at different ranks( Kippist, 1842 ; Schaffnit, 1906 ; Oehm, 1932 ; Bremekamp, 1944 ;Hedréen, 1988 , 1989 ; Graham, 1988 ; Ensermu et al., 1992 ; Immelman, 1990 ; Balkwill et al., 1996 ; Balkwill and Getliffie Norris, 1988 ; Balkwill and Campbelg-Young, 1999 ; Darbyshire, 2008 ; Rodriguez Greuter and Rankin , 2010 ; Darbyshire et al., 2011 ; Ruengsawang et al., 2012 ; Tripp et al., 2013 ; Kiel and McDade, 2014 ; Al-Hakimi and Latiff, 2015). For example, seeds of Ruellieae have spiral hygroscopic mucilaginous hairs( Kippist, 1842 ; Greuter and Rankin Rodriguez, 2010 ; Duan, 2013). Clarke(1885) described the seeds of Justicia as ovoid and strongly compressed or compressed, and the testa being tubercular, scaly or glochidiate-subspinescent. Balkwill and Getliffie Norris(1988) re-appraised tribal and subtribal limits of the Acanthaceae of southern Africa and considered that seed coats lacking hairs were definitive of the Justicieae and some genera. However, some species of Justicia with hairy seeds were included in later studies( Graham, 1988 ; Ruengsawang et al., 2012). Hedréen(1988) described the seed surface as "tuberculate” in his paper on the Justicia capensis Thunb. group. Later, Hedréen(1989) proposed six testa types in Justicia sect. Harnieria from tropical Africa. Graham(1988) recognized three distinct groups and some 20 seed types using a low-power binocular microscope, and noted that the testa is an important character for distinguishing the sections. Ensermu(1990) observed 12 species of Justicia sect. Anellia from Ethiopia and described the testa of all species as doublereticulate. Immelman(1990) described the seed surface of southern African Justicia and pointed out that seed surface characteristics can be used for delimiting some sections in Justicia . Ruengsawang et al.(2012) observed 30 species of Justicia from Thailand and recognized five types and four subtypes. The seed of J. microdonta resembles some species classified as Type 1 of Graham(1988) and Ruengsawang et al.(2012) , but differs by the reticulate testa sculpture being favulariate folded with granules. However, the seed testa sculpture of J. microdonta is more similar to that of some species of Pseuderanthemum ( Duan, 2013). Seed morphology implied a close relationship between J. microdonta and Pseuderanthemum .

4.3. The new genus Wuacanthus and its systematic position

Our results support the exclusion of J. microdonta from Justicia and its affinity with the Pseuderanthemum lineage, which is defined as the subtribe Odontoneminae by Bremekamp(1965) . The Pseuderanthemum lineage differs from another three lineages of McDade et al.(2000) by having four stamens or two stamens and two staminodes, and was recognized as a basal lineage in Justicieae( McDade et al., 2000). Morphologically, Wuacanthus differs from other Justicia species in having two stamens and two staminodes, which identify it with the Pseuderanthemum lineage. Spur number at the base of each theca seems to be a very useful character for delimiting basic taxonomic boundaries in the family Acanthaceae. Some sister genera are mainly differentiated based on the number of spurs. Chroesthes is separated from Lepidagathismainly by the spurred anther thecae, which are multicous in Lepidagathis ( Hansen, 1983). Three genera in tribe Ruellieae were found to have two spurs at the base of each theca. Diceratotheca J.R.I.Wood et al. is distributed in Thailand( Wood et al., 2012). Stenothyrsus C.B. Clarke was described from the Malaysian state of Perak( Clarke, 1908). Recent studies have shown that Chinese species of Echinacanthus differ from the type of Echinacanthus, E. attenuatus Wall., in having two spurs at the base of each theca and represent another undescribed genus( Nees von Esenbeck, 1832 ; Lo and Fang, 1985 ; Hu and Tsui, 2002 ; Hu et al., 2011 ; Tripp et al., 2013). J. microdonta differs from other genera in the Pseuderanthemum lineage and can be recognized as an independent genus, Wuacanthus , within the Pseuderanthemum lineage.

There are two main clades in the Pseuderanthemum lineage, the Pseuderanthemum and Asystasia clades. The Pseuderanthemum clade is characterized by two stamens and two staminodes and includes Wuacanthus, Pseuderanthemum, Ruspolia, Ruttya, Chileranthemum, Odontonema, Oplonia and Mackaya . The Asystasia clade is characterized by four stamens without staminodes and includes Asistasia and Asystasiella . Wuacanthus formed sister groups with Pseuderanthemum in our studies, and share the characters of the flowers arranged in terminal thyrses, 2-lipped limbs, erect 2-lobed upper lip, reflexed 3-lobed lower lip, two stamens and two staminodes, basally solid 4-seeded capsules., Wuacanthus and Pseuderanthemum formed sister groups with Ruspolia and Ruttya , but differ from the monothecate condition of the latter two genera in having bithecate anthers. Wuacanthus differs from Pseuderanthemum by the shorter corolla tube and bi-spurred theca base of the anther. In Pseuderanthemum , the corolla is salverform with a long slender tube that is not or scarcely apically enlarged, and the antherthecae are multicous at the base( Hu et al., 2011).

5. Taxonomic treatment

Wuacanthus Y.F. Deng, N.H. Xia & H. Peng, gen. nov. - Type: Wuacanthus microdontus (W.W.Sm.)Y.F. Deng, N.H. Xia & H. Peng.

Shrubs. Leaves opposite, petiolate, blade margin entire, penninerved. Spikes terminal, with 2-4 flowers per node; bracts linearlanceolate; bracteoles 2, similar to bracts. Calyx 5-lobed almost to base; lobes linear-lanceolate. Corolla 2-lipped, upper lip 2-lobed, lower lip 3-lobed; tube short, limb longer than tube. Stamens 2, included; filaments pilose; anther bithecate, thecae oblong, each with two white spurs at the base. Staminodes 2, minute. Ovary glabrous, 2 ovules in each locule; style densely pilose. Capsule 4- seeded, stalked at the base. Seeds reddish brown, ovoid.

Distribution . Monospecific; endemic to the Jinshajiang Valley, China, occurring in Sichuan and Yunnan Provinces. Jinshajiang Valley is located at the basal zone of the Hengduan Mountain Region, which is situated within the Indo-Burma hotspot, one of 25 biodiversity hotspots defined by Myers et al.(2000) . About 13 genera of seed plants are endemic to the Hengduan Mountain Region, including Acanthochlamys P.C. Kao, Dipoma Franch., Metanemone W.T. Wang, Salweenia E.G. Baker, Sinadoxa C.Y. Wu et al., Smithiorchis T. Tang & F.T.Wang, etc.( Wu, 1988 ; Li and Li, 1993 ; Wu and Wu, 1996). Jinshanjiang Valley has a special and vulnerable ecosystem characterized by aridity, high temperatures, semisavanna vegetation and relatively few plants. Jinshajiang Valley is rich in endemic taxa and four generawere previously recognized as endemic there, viz., Anemoclema (Franch.)W.T. Wang, Nouelia Franch., Musella(Franch.)C.Y.Wu & H.W. Li, and Trailliaedoxa W.W. Sm. & G. Forrest( Jin et al., 1994 ; Guan et al., 2013).

Etymology. The generic name honors ProfessorWu Zhengyi(Wu Chengyih)(1916-2013). Prof. Wu has devoted over 70 years to the study of the flora and vegetation of China. In 2007, he was awarded the State Preeminent Science and Technology Award of China for his outstanding achievements in the fields of systematic botany and plant geography, as well as plant diversity, conservation, and sustainable use of plant resources( Peng et al., 2013 ; Deng et al., 2013). Previously, three generic names, Baijiania A.M. Lu & J.Q. Li( Cucurtibitaceae, Li, 1993), Sinobaijiania C. Jeffrey & W.J. de Wilde(Cucurtibitaceae, Jeffrey and de Wilde, 2006) and Zhengyia T. Deng, D.G. Zhang & H. Sun( Urticaceae, Deng et al., 2013), and nineteen species names were published in honor of Prof. Wu Zhengyi(Kunming Institute of Botany, Chinese Academy of Sciences, 2013 ; Peng et al., 2014).

Wuacanthus microdontus (W.W.Sm.)Y.F. Deng, N.H. Xia & H. Peng, comb. nov .≡Justicia microdonta W.W. Sm. in Not. Bot. Gard. Edinb. 10: 183. 1918. ≡ Mananthes microdonta (W.W. Sm.)C.Y.Wu & C.C. Hu in C. C. Hu, Fl. Reipubl. Popularis Sin. 70: 296. 2002. - Type: CHINA. Yunnan : mountains of the Chungtien Plateau [27°30′N], July 1914, G. Forrest 12822(Lectotype E! here designated; isolectotype K!). Fig. 4 .

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).

Shrubs to 2 m tall, much branched. Branches quadrangular, sparsely pilose. Petiole 1-2 cm; leaf blade papery, ovate, (2-)3-5.5 × 1.5-2.5 cm, abaxially light green and pilose along midvein, adaxially green and pilose especially along veins, base broadly cuneate and decurrent onto petiole, margin entire, apex shortly acuminate to acute, lateral veins 5-8 pairs. Spikes terminal, 5-7(-10)cm, unbranched, nodes distant, with 2-4 flowers per node; peduncle short; rachis densely pilose; bracts linear-lanceolate, 3-5 cm; bracteoles similar to bracts. Calyx ca. 7 mm, 5-lobed almost to base; lobes linear-lanceolate, 5-6 mm, pilose along the midveins. Corolla white, with purplish-red dots on lower lip inside, 1-1.3 cm, outside somewhat pubescent to glabrescent; tube short; limb longer than tube; lower lip 4-5 mm, 3-lobed, lobes pilose inside and ciliate on margin; upper lip 2-lobed. Stamens 2, included; filaments pilose, purple; anther bithecate, thecae oblong, ca. 2 mm long, each with two white spurs at base. Staminodes 2, minute. Ovary glabrous; style densely pilose. Capsule ca. 2 cm, 4- seeded. Seeds reddish brown, circular in outline, 3-4 mm in diameter, favulariate folded with granules, reticulate.

Distribution and habitat . Endemic to the Hengduan Mountains, China, occurring inNW Yunnan and SW Sichuan(Fig. 5). It grows in thickets at rocky sites in dry valleys at 690-1500 m.

Fig. 5 Distribution of Wuacanthus microdontaus (W.W. Sm.) Y.F. Deng, N.H. Xia & H. Peng (solid circles).

Conservation status . This species is only known from nine collections from seven localities. Amongst these, three collections from Yunnan Province were collected more than 100 years ago. Before the authors re-collected the species at the two localities of Jinyang and Butuo in Sichuan Province in 2014, others were collected more than 30 years ago. Only several collections were collected from a few localities but it would potentially occur in the Jinshajiang Valley. Apart from habitat destruction, no other specific threats are known. The provisional conservation status of the species could therefore be considered as Endangered EN B2ab(iii), based on the IUCN Red List Categories and Criteria( IUCN, 2012 , 2014). On the other hand, for the present, it may be better to consider this as Data Deficient(DD)because there have been no specific field searches for the population of the species.

Phenology . It was observed flowering from July to October and fruiting from October to next February.

Typification . In his protologue, Smith(1918) cited two collections(G. Forrest 12822 and 13162 )and both of them are syntypes according to Art. 9.5 of the ICN( McNeill et al., 2012). Both are in accordance with the original description and can be candidates for being lectotype. Here we choose G. Forrest 12822 as the lectotype.

Additional specimens examined . CHINA. Sichuan : Butuo Xian, Jiaojihe Qu, Dadiba, hilltop, pathside, in the thickets, fl. white, Aug. 20, 1959, Chuan Jing Liang 5636(KUN); Butuo Xian, Niujiaowan Xiang, bank of Xixihe, 690 m, 11 October 2014, Y.F. Deng with H. Peng, Y. Tong & Y.P. Chen 25860 (IBSC, KUN); Ganluo Xian, near Suxiong Qu, 800 m, Sept. 24, 1976, unknown collector 14291 (CDBI); Jinyang xian, Tiandiba Zhen, 810 m, in the thicket, ravine, 9 October 2014, Y.F. Deng with H. Peng, Y. Tong & Y.P. Chen 25796 (IBSC, KUN); Jinyang Xian, Tiandiba Zhen, Jinshajiang Valley, 800 m, Oct. 26, 1985, H. Li 249 (KUN); Jinyang Xian, on the way from Lugao to Pailai, 1100 m, on slope, roadside, Aug. 19, 1964, T.P. Zhu et al 295 (CDBI). Yunnan : Deqên Xian, Yangtze Valley at Paung-tzu-la [28°12′N], 7000 ft, Aug 1914, G. Forrest 13162 (E, K); Yulong Naxizu Zizhixian, Baoshan Xiang, Shitoucheng, October 2014, Z.K. Wu s.n .(IBSC); Qiaojia Xian, Siao Fa Ka dans la region de Kiao Kia, 4 Febuary 1908, F. Ducloux 5741 (P).


This study was supported by the National Natural Science Foundation of China(grant nos. 30670124,31270247,31470302,31400184,31670191,31110103911)and Science and Technology BasicWork(grant no. 2013FY112100).We sincerely thank Mrs. Yun- Xiao Liu(IBSC)and Mr. Ding-Han Cui(IBSC)for preparing the line drawings; Dr. Sheng-Xiang Yu for preparing the distribution map; Prof. Wong Khoon Meng(SING)for improving the manuscript; Dr. Yihua Tong(IBSC)and Ms. Ya-Ping Chen(KUN)for their assistance with field work; and the curators of the herbaria CDBI,E,IBSC,KUN and P for facilitating access to their collections.

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