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

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.

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 microdonta W.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.

Material and methods

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).

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. Lo) H. S. Lo	Y. Tong 13082342 (IBSC)	Hainan, China	–	KP744224	KP744230	KP744227
Crossandra infundibuliformis (L.) Nees	Y. F. Deng 2007006 (IBSC)	Guangdong, China	–	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 122 (IBSC)	Hainan, China	KP744329	KP744165	KP744246	KP744201
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	–	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	–	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 & C. Y. Wu	Y. F. Deng 18452 (IBSC)	Yunnan, China	KP744339	KP744175	KP744256	KP744211
Pachystachys lutea Nees	Y. F. Deng 20593 (IBSC)	Guangdong, China	KP744340	KP744176	KP744257	KP744212
Peristrophe japonica (Thunberg) Bremekamp	2000 MO-IBSC expedition to Hainan 114 (IBSC)	Hainan, China	KP744341	KP744177	KP744258	KP744213
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 Oliver)	Y. F. Deng 17923 (IBSC)	Yunnan, China	KP744344	KP744180	KP744261	KP744216
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. Smith), Moylan & Y. F. Deng	Y. F. Deng 18215 (IBSC)	Yunnan, China	KP744348	KP744184	KP744265	KP744220
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	–	–
Aphanosperma sinaloensis (Leonard & Gentry) T. F. Daniel	Daniel 4070cv (CAS)	American	EU0874541	EU0875501	EU0810721	EU0875031
Brachystephanus africanus S. Moore	Luke et al. 6704 (US)	Tanzania	DQ3724693	–	DQ3724913	EU087537
Calycacanthus magnusianus K. Schum	Daniel 10072 (CAS)	Sydney	EU0874811	–	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) Vollesen	Daniel 9379 (CAS)	South Africa	EU0874741	EU0875651	EU0811001	EU0875281
Chileranthemum pyramidatum (Lindau) T. F. Daniel	Breedlore & Daniel 70767 (CAS)	Mexico	AF2897972	AF2897522	–	–
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	–	DQ3725033	–
Harpochilus neesianus Mart. ex. Nees	Souza et al. 5413 (CAS)	Brazil	AF2897622	AF2897212	–	–
Henrya insularis Nees ex Benth	Jenkins 89–432 (ARIZ)	Mexico	AF1698432	AF0631252	EU0810711	EU0875071
Herpetacanthus stenophyllus Gomez-Laur. & Grayum	Herrera 3855 (ARIZ)	Costa Rica	AF2897952	AF2897502	–	–
Hoverdenia speciosa Nees	Deniel & Baker 3739 (CAS)	Mexico	AF2897772	AF2897382	EU0810891	EU0875191
Kalbreyeriella rostellata Lindau	McDade 1007 (DUKE)	Colombia	DQ3724733	–	DQ3724983	–
Kudoacanthus albonervosus Hosok	W. C. Leong 2939 (HAST)	China	EU5288891	EU5289241	EU5289651	EU5290291
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 94–23 (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	–	–
Oreacanthus mannii Benth.	Leeuwenberg 8925 (BR)	Cameroon	DQ3724713	–	DQ3724953	–
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	–	–
Ruttya fruticosa Lindau	Danile s. n. (CAS)	American	AF2898012	AF2897562	–	–
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	–	–
Stenostephanus chiapensis T. F. Daniel	Breedlove & Burns 72688cv (CAS)	American	AF2897922	AF2897472	DQ3725063	–
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 87–307 (ARIZ)	American	AF1698392	AF0631352	–	–
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

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.

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 2.2.6.1 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).

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 μm), 1 μl each primer (0.2 μm), 2.5 μl 10 × buffer, 0.25 μl Taq polymerase (0.2U), and 17.5 μl ddH2O. The PCR cycling conditions were exactly the same for all markers, [96 °C 3 min, (94 °C 1 min, 52 °C or 50 °C 1 min, 72 °C 1 min) × 32 cycles, 72 °C 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.

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.

Results

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 linear-lanceolate; 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).

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.

Discussion

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, Lindau, 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.

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 “Knötchenpollen”, 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 “Knötchenpollen” 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 “Knötchenpollen” 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, Daniel, 1995, Daniel, 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, 1988, Darbyshire et al., 2010, Hu et al., 2011, McDade et al., 2012, Rueangsawang et al., 2012, Rueangsawang et al., 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; Hedrén, 1988, Hedrén, 1989, Graham, 1988, Ensermu et al., 1992, Immelman, 1990, Balkwill et al., 1996, Balkwill and Getiliffe Norris, 1988, Balkwill and Campbell-Young, 1999, Darbyshire, 2008, Greuter and Rankin, 2010, Darbyshire et al., 2011, Rueangsawang 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, 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, Rueangsawang et al., 2012). Hedrén (1988) described the seed surface as “tuberculate” in his paper on the Justicia capensis Thunb. group. Later, Hedrén (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 double-reticulate. 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.

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).

Taxonomic treatment

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 linear-lanceolate; 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, semi-savanna vegetation and relatively few plants. Jinshajiang Valley is rich in endemic taxa and four genera were 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 Professor Wu 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 BotanyChinese Academy of Sciences, 2014, Peng et al., 2014).

(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. A–G, I–M drawn from F. Ducloux 5741 (P) by Yun-Xiao Liu, G–H, N–O 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 in NW 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, IUCN, 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).