Taxonomic Study of the subtribe Andrographinae, tribe Ruellieae, family Acanthaceae in Thailand Winai Somprasong1/ Srunya Vajrodaya2/ Kongkanda Chayamarit3/ ABSTRACT The Andrographinae is one of the famous subtribes in the Tribe Ruelliae (Acanthaceae). The most famous genera in this subtribe are Andrographis Wall ex Nees, Graphandra Imlay, Gymnostachyum Nees and Phlogacanthus Nees. Their large diversity was found in various tropical countries including Thailand. However, thereis no revision of their diversity in Thailand for decades. Thus, this study aimed to collect and revise the species list for the Flora of Thailand. Data was collected from relevant previous documents, herbarium specimen supplied from 5 herbaria, and field survey across country during October 2009 to September 2010. Morphological characters including pollen morphology were determined and recorded.Twenty-three species of the Andrographinae were found in this study viz. Andrographis (2), Grapahndra (1), Gymnostachyum (8), and Phlogacanthus (12). The differences of morphological characters among them were discussed and the checklist of the subtribe was provided. Keywords : Subtribe Andrographinae, Acanthaceae 1/ 2/ 3/ 4/ Graduate School, Kasetsart University, 50 Phahon Yothin Rd., Chatuchak, Bangkok 10900 THAILAND, Department of Agriculture, Phahon Yothin Rd., Chatuchak, Bangkok 10900 THAILAND Faculty of Science, Kasetsart University, 50 Phahonyothin Rd., Chatuchak, Bangkok 10900 THAILAND Department of National Parks, Wildlife and Plants, Phahon Yothin Rd., Chatuchak, Bangkok 10900 THAILAND Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 85 Introduction The Family Acanthaceae is a large pantropical family of about 229 genera and 3,450 species in the world (Mabberley, 1997). Most of them are herbs or shrubs, including twining forms, some are spiny. There were about 40 genera and over 230 species in Thailand and the majority are confined all over the country (Hansen, 1985). At present, the studies on the family Acanthaceae are diagnosed through the details of corolla aestivation, palynological and phylogenetic data to serve the classification of this family. This family was treated by Scotland & Vollesen (2000), which was classified it into three subfamilies, i.e. Nelsonioideae, Thunbergioideae and Acanthoideae. The last one is the largest sub family which composts of two tribes, i.e. Acantheae and Ruellieae. The tribe Ruellieae was classified into 4 subtribes, i.e. Ruelliinae (48 genera), Andrographinae (8 genera), Justiciinae (103 genera) and Barleriinae (9 genera). All of them are famous for medicinal purposes, especially both subtribe Barleriinae and Andrographinae. The famous genera in the subtribe Andrographinae are as follows : Barleriinae (2 genera : Barleria L. and Lepidagathis 86 Willd.) and Andrographinae (2 genera : Andrographis Wall. ex Nees and Phlogacanthus Nees) (Hansen, 1985). The subtribe Andrographinae is an interesting acanthus group to be revised for this research, due to lacking the clear-cut taxonomic data and also they are wellknown as the Thai medicinal plants used by traditional herbalist. Some well-known species used as medicinal purposes in minority groups of Thailand are Phlogananthus curviflorus Nees and P. pulcherimus T. Anders. This study aim to collect, revise, do a check list to serve as an information to the Flora of Thailand and agricultural purposes by emphasizing on botanical literature, herbarium study, field survey, specimen collection, morphological study and to conduct a taxonomic treatment for this genus and enumerate the number of species and construct the keys to genera. As mentioned, this research is fruitful for the taxonomic works for the Flora of Thailand in the future. Materials and Methods 1. Taxonomic Study Taxonomic study was carried out by diagnosing through details of corolla วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 aestivation, cystolith, retinaula, number of stamen and seed data followed Benoist (1936), Cramer (1998) and Scotland & Vollesen (2000). The literature and herbarium specimens of the subtribe Andrographinae were examined at Thai and foreign herbaria as follows: Bangkok Herbarium, Department of Agriculture (BK), Forest Herbarium, National Park, Wildlife and Plant Conservation Department (BKF), Queen Sirikit Botanic Garden Herbarium, The Botanical Organization, Ministry of Natural Resources and Environment (QBG), the Biological Department Hebarium, Chiang Mai University (CMU), Prince of Songkhla University Herbarium (PSU), Royal Botanic Gardens, Kew (K). Field surveys and specimen collections were conducted throughout the country by choosing the three represented conserved area from each region, such as north, north-eastern, eastern, south-western, eastern, central, south-eastern and peninsular Thailand. Measurement of vegetative and reproductive features of dried and spirit specimens collected by the author, and herbarium specimens deposited in the above Thai and foreign herbaria are carried out by using a hand ruler or scale calibrated in cm and mm under the stereomicroscope for the characters of indumentum, leaves and flowers. Illustrations and photographs are provided for clarification. The herbarium specimens together with the voucher specimens are identified by taxonomic literature and the comparison with the type specimens. Preparation the keys to subtribe, genera and species based on the significant morphological characters are constructed to prove their taxonomic units. The study on the subtribe Andrographinae, family Acanthaceae in Thailand during October, 2009 to September, 2010. 2. Pollen morphology Selected pollen of some species were collected to be fixed in 3 % gluteraldehyde in phosphate buffer and 1% osmium tetroxide. (Hopwood and Milne, 1991). The SEM study was conducted by dropleting of pollen-ethanol sputter-coated with a platinum. Observation and measurements was conducted by using scanning electron microscopy (SEM). Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 87 Results and Discussion 1. Taxonomic Study The classification of Acanthaceae and taxonomic characters among this subtribe (4 genera) were listed in Table 1. Among this 4 genera, 23 taxa were enumerated in Thailand. Two taxa were belong to Andrographis, whereas had 1, 8 and 12 taxa were classified to the genera Graphandra, Gymnostachyum and Phlogacanthus respectively (Table 2). Andrographis Wall. Herbs or very small shrubs, erect or procumbent. Leaves opposite, simple, entire. Inflorescence: axillary and terminal racemes, lax and panicled, or dense, often 1-sided; bracts small; bracteoles 2 or absent. Calyx small, divided almost to the base into 5 sepals. Corolla small, obscurely or definitely bilabiate, with a short tubular base; lobes imbricated in the bud. Stamens 2, inderted near the mouth of the corolla tube; filaments ciliate or not; anthers included or exserted, bilocular, the cells equal or slightly unequal, parallel, muticous, bearded at the base; pollen “Daubenpollen” (Lindau) – grains ellipsoid, subglobose or obscurely trigonous, finely pitted, germ – pores 3 in 88 short and broad grooves. Ovary bilocular, 6-12 ovulate; disc fleshy; style minutely bifid at the apex. Capsule linear or oblong-elliptic, compressed at right angles to the septum. Seeds subglobose, glabrous, pitted. (Figure 1A) There are about twenty species from India, and tropical Asia. Only two species are found in Thailand. Graphandra J.B. Imlay Small herb, procumbent. Leaves opposite, small, entire, sassile or subsessile. Inflorescence terminal and axillary; small simple bracteates spikes; flowers alternate; bracts opposite, more or less imbricated, larger than the calyx; bracteoles equaling or a little longer than the calyx. Calyx of 4 sepals free to the base. Corolla tube almost as long as the limb; limb bilabiate, the upper lip narrow, slightly concave, entire or subentire, the lower lip oblong, 3-lobed; lobes imbricated in the bud. Stamens 2, affixed to the corolla tube by a thin membrane, free in the mouth of the tube; anther cells 2 equal, parallel, oblong, minutely mucronate at the base, hairy but not bearded at the base; pollen “Daubenpollen” (Lindau) – almost globose, finely pitted, 3 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 Table 1 Plant characters of 4 genera in the sub-tribe Andrographinae Taxa Habit Inflorescence Inflorescense calyx type/bract corolla Andrographis Wall. ex Nees Annual herb raceme/bract small contracted Graphandra J.B. Imlay Annual herb spike/bract larger than calyx Gymnostachyum Nees Annual herb spike Small, calyx lobe 4, deeply lobed small, calyx bilabiate/ lobe 5, corolla tube long Corolla lobe corolla 5 shallowly sub-equally lobed 5-lobed, corolla tube ventricose, curved towards the throat/ straight Phlogacanthus Nees Annual / perennial herb panicle germ – pores in 3 very small grooves. Ovary bilocular, 12 – ovulate; style minutely bifid at the apex. Capsule oblong, scarcely flattened, not stalked, 12 – seeded from the base. Seeds flattened, smooth, almost glabrous. (Figure 1B) Only one species is listed in Thailand Gymnostachyum Nees Herbs or under shrubs, erect, often sub-acauline. Leaves cauline or almost radical, opposite, equal, entire or corolla sub-equally 5-lobed corolla sub-equally 5-lobed stamen ovule 2 6-12 2 ,adnate 12 to corolla tube 2 2 fruit/seed linear/ seed flattened/ 6-12 seeded oblong/ 12seeded 6 ovules/ cylindrical/8-20 many seeded ovules 10-16 cylindrical/1016 seeded or many seeds subentire. Inflorescence terminal and axillary, simple, branched or panicled; flowers. Flowers opposite or alternate, solitary, clustered or cymose, sessile or subsessile, forming simple spikes or terminal lax panicles, sometimes unilateral; bracts and 2 bracteoles very call. Calyx 5- lobed almost to the base; sepals subequal, the posterior sometimes smaller than the others. Corolla tube slender straight or slightly curved, narrow at the base then ridening slightly Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 89 Table 2 List of taxa in the subtribe Andrographinae, family Acanthaceae in Thailand. Taxa Andrographis laxiflora Lindau A. paniculata Nees Graphandra procumbens Imlay Gymnostachyum canescens T.Anderson Gymnostachyum coriaceum Imlay Gymnostachyum gracile Bremek. Gymnostachyum hirtum Ridl. Gymnostachyum leptostachyum Nees. Gymnostachyum signatum (Benoist) Imlay Gymnostachyum trilobum Ridl. Gymnostachyum venustum T. Anderson Phlogacanthus asperulus Nees Phlogacanthus colaniae Bebiust & Benoist Phlogacanthus curviflorus Nees. Phlogacanthus murtonii Craib Phlogacanthus paniculatus (T.Anderson) Imlay Phlogacanthus pauciflorus Imlay Phlogacanthus pedunculatus Imlay Phlogacanthus pulcherrimus T. Anderson Phlogacanthus racemosus Bremek. Phlogacanthus rectiflorus Imlay Phlogacanthus thyrsiflorus Nees. Phlogacanthus vitellinus T. Anderson Specimen Investigation Document BK BKF CMU PSU K 1 2 3 4 5 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x 6 x x x x Remarks: BK = Bangkok Herbarium, Thailand BKF = The Forest Herbarium, Thailand CMU = Chiang Mai University Herbarium, Thailand; PSU = Prince of Songkla University Herbarium , Thailand; K = Botanic Gardens, Kew Herbarium, UK (1) = Clarke (1885); (2) = Benoist (1935); (3) = Imlay (1939); (4) = Bremekamp (1965); (5) = Cramer (1998); (6) = The Forest Herbarium (2001) upwards; limb spreading, bilabiate; upper lip shortly 2-lobed or emarginated; lower lip shortly 3-lobed, the lobes imbricated in the bud. Stamens 2, inserted below the middle of the corolla tube; anthers included or sub-excert, bilocular; loculi oblong, muticous or very shortly 90 mucronata at the base; pollen “Daubenpollen” (Lindau) – grains ellipsoid and finely pitted, “Daubenpollen” (Lindau) – grains ellipsoid and finely pitted, with 3 short and broad grooves pointed at both ends and Uned with 2 rows of minute papillae, containing the 3 germ – pores. วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 Every bilocular, 8 – 30 – ovulate; disc conspicuous, rather fleshy; style minutely bifid at the apex. Capsule linear, terete or cotusely 4 – sided, seed – bearing from the base, each valve 3 – grooved seeds many, obliquely ovate, flattened, covered with hygroscopic bairs; ejaculatora hard curved. (Figure 1C-1F) There are about forty species from tropical Asia and Malaysia. Nine species are enumerated from Thailand. Phlogacanthus Nees Broad; midrib conspicuous above, prominent below; lateral nerves 6-8 pairs, conspicuous above, prominent below; A B C D E F Figure 1 Plant forms in some species of Andrographis, Graphandra and Gymnostachyum (A) Andrographis laxiflora, (B) Graphandra procumbens (C) Gymnostachyum leptostachyum, (D) G. signatum (E) G. trilobum (F): G. venustrum Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 91 margin subentire, glabrous; petiole 1-1.5 cm long, glabrous. Inflorescence a narrow terminal panicle, 2-6 cm long, 1-2 cm broad; upper flowers solitary, opposite, the lower flowers in small shortly peduncled cymes; pedicels ca 3 mm long or less; peduncles and pedicels shortly pubescent; bracts at the base of the pedicel, lanceolate, acuminate, shortly pubescent, 3-4 mm long; bracteoles 1.5 mm long, linear, shortly pubescent, affixed about the middle of pedicel. Calyx shortly pubescent outside, 4.5-5 mm long; sepals lanceolate, acuminate, united at the base for ca 1.25 mm, shortly pubescent inside towards the apex, minutely ciliate, 3nerved. Corolla pink with yellow patch on lower lip (ex Kerr), or red, densely shortly pubescent outside, many hairs glandular, 0.8-1.2 cm long; tube narrow at the base, then suddenly curved and inflated, glabrous inside except for an incomplete ring of short hairs near the throat; limb shortly obscurely bilabiate; lobes 3-5 mm B A D C E F Figure 2 Plant forms in some species of Phlogacanthus (A) Phlogacanthus curviflorus, (B) P. pauciflorus, (C) P. pedunculatus, (D) P. pulcherrimus, (E) P. rectiflorus, (F.) P. vitellinus 92 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 long, ovate or ovate-oblong, acute, puberulous inside towards the margin. Stamens 2 fertile and 2 minute rudiments inserted 5 mm from the base of the corolla; filaments 6mm. long, curved, shortly hairy at the base; anthers exserted, oblong 2.5 mm long, glandular at the back. Ovary glabrous. 8-ovulate; style 9 mm long, glabrous. Capsule 1.7 cm long, glabrous, 8-seeded from the base. (Figure 2) There are about twenty species from tropical India, and IndoChina. Twelve species are found in Thailand. 2. Distribution of subtribe Andrographinae, family Acanthaceae in Thailand The field survey of 4 genera were reported with distribution map in 8 floristic regions of Thailand, North, Northeastern, Eastern, South-western, Eastern, Central, South-eastern and peninsular Thailand. The results showed that both of Andrographis laxiflora (Blume) Lindau and A. paniculata (Burm.) Wall ex Nees were found in North, South-east and Central. A. laxiflora was found in East and South – West (Figure 3A). Gymnostachyum canescens (Nees) T. Anders. was found in North and South-west. G. coriaceum Imlay was found in north-west. G. decurrens Stapf and G. hirtum Ridl. were found in South-west (Figure 2B). G. leptostachyum Nees were found in Northeast and South-east. G. signatum (Benoist) Imlay and G. trilobatum Ridl. were found in South-east and peninsular. G. venustum (Wall.) T. Anders. was found in South-west (Figure 3C). Phlogacanthus asperulus was found in north. P. colaniae Benoist was found in South-east. P. curviflorus (Wall.) Nees was found in North and South-west. P. murtonii Craib was found in South-east region. (Figure 3D). P. paniculatus (T. Anders.) J.B. Imlay was found in North and North-east. P. pauciflorus J.B. Imlay was found in North. P. pedunculatus J.B. Imlay was found in North and North-west. P. pulcherrimus T. Anders. was found in East and peninsular (Figure 3E). P. racemosus Brem. was found in North. P. rectiflorus J.B. Imlay and P. thyrsiflorus Nees were found in peninsular. P. vitellinus T. Anders. was found in South-east (Figure 3F). Graphandra procumbens J.B. Imlay was found as only one species at Tha Uthen District, Nakhon Phanom Province in North-east. Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 93 Andrograhis Wall. Andrograhis Wall. Gymnostachyum Nees (1) Gymnostachyum Nees (1) Gymnostachyum Nees (2) Gymnostachyum Nees (2) Andrograhis Wall. Gymnostachyum Nees (1) Gymnostachyum Nees (2) Phlogacanthus Nees (1) Phlogacanthus Nees (1) Phlogacanthus Nees (2) Phlogacanthus Nees (2) Phlogacanthus Nees (3) Phlogacanthus Nees (3) Figure 3 Distribution maps of subtribe Andrograpinae in Thailand (A) Andrograhis Wall. (B) Gymnostachyum Nees 1. (C) Gymnostachyum Nees 2 (D) Phlogacanthus Nees 1 (E) Phlogacanthus Nees 2 (F) Phlogacanthus Nees 3 94 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 2. Pollen Morphology The pollen types within subtribe Andrographinae has the evidence for the discovery of taxa. As in the study of Scotland and Vollesen (2000) it may be that pollen morphology is the most accurately utilized and informative at fairly low taxonomic levels, and best used in studies of individual genera and relationships between small groups of genera. The preliminary results showed that the shape and ornamentation were recognized the differences among genera. The pollen types from the selected species of 3 genera (Andrographis, Gymnostachyum and Phlogacanthus) are investigated (Figure 4) as follows: Andrographis laxiflora : tricolporate, distinct aperture margins (Figure 4A). Gymnostachyum leptostachyum: tricolporate, open reticulate tectum with scattered granules (Figure 4B). G. signatum: tricolporate, open reticulate tectum (Figure 4C). G. trilobum: tricolporate, open reticulate tectum (Figure 4D). G. venustrum: tricolporate, open reticulate tectum (Figure 4E). Phlogacanthus curviflorus: tricolporate, distinct aperture margins (Figure 4F). P. pauciflorus: tricolporate, distinct aperture margins (Figure 4G). P. pedunculatus: tricolporate, distinct aperture margins (Figure 4H). P. pulcherrimus: tricolporate, distinct aperture margins (Figure 4I). P. rectiflorus: tricolporate, distinct aperture margins (Figure 4J). P. vitellinus: tricolporate, distinct aperture margins (Figure 4K). Conclusions The overview characters of the subtribe Andrographinae comprise corolla imbricate or ascending cochlear aestivation; stamen 2; ovules 6 – many, capsule extending to the base, bearing 6 ovules – many; pollen tricolporate with distinct aperture margin. The differences in morphological characters among them were the ones that bear the corolla sub-equally 5-lobed; sepal 4 or 5 (Andrographis, Grapahndra, Phlogacanthus) and the others with the corolla bilabiate; sepal 5 (Gymnostachyum). The number of the subtribe Andrographinae were enumerated into 23 species, Andrographis (2), Grapahndra (1), Thai Agricultural Research Journal Vol. 33 No. 1 January - April 2015 95 A B C D E F G H I J K Figure 4 Pollen types, (A) Andrographis laxiflora; (B) Gymnostachyum leptostachyum; (C) G. signatum; (D) G. trilobatum; (E) G. venustum; (F) Phlogacanthus curviflorus (G) P. pauciflorus; (H) P. pedunculatus; (I) P. pulcherrimus; (J) P. rectiflorus; (K) P. vitellinus 96 วารสารวิชาการเกษตร ปีที่ 33 ฉบับที่ 1 มกราคม - เมษายน 2558 Gymnostachyum (8), and Phlogacanthus (12) Acknowledgements I am grateful to express my thanks to Agricultural Research and Development Agency (Public Organization) (ARDA) for the research Fund, to my supervisor, Assoc. Prof. S. Vajrodaya for her keen advice and to Dr. K. Chayamarit, former director of the Forest Herbarium, Department of Natonal Parks, Wildlife and Plants, Thailand for the encouragement and assistance. Furthermore, I would like to thank my colleagues, such as Dr. Pramote Triboun, Miss Panarat Thongphoem, Mr. Pakorn Thipayasri, Mr. Phatarawee Phromnat, Mr. Nopparat Toonmal and Mr. Samak Rattanathip for their help in field work and available document preparation. REFERENCES Benoist, R. 1936. Acanthacees. Pages 610-772. In : Fl. Gen. de L’IndoChine., Paris. Bremekamp, C.E.B. 1965. Acanthaceae. Pages 544-593. In: Flora of Java Vol. II. N.V.P. Noordhoff, Groningen, The Netherlands. Clarke, C.B. 1885. Acanthaceae. Pages 387-558. In: Fl. Brit. Ind. Vol. IV. L. Reeve & Co. Kent, UK. Cramer, L.H. 1998. Acanthaceae Pages 1140. In: A Revised Handbook to the Flora of Ceylon Vol. XII. A.A. Balkema, Rotterdam, The Netherlands. Hansen, B. 1985. Studies on the Acanthaceae of Thailand. Fl. Malesiana Bull. 38: 173-178. Harris, R. 1991. Electron Microscopy in Biology A Practical Approach. Oxford University Press, Oxford. 308 p. Imlay, J.B. 1939. The Taxonomy of The Siamese Acanthaceae. Ph.D. Thesis. 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