Two new species of Curcuma (Zingiberaceae) used as cobra-bite antidotes

Journal of Systematics and Evolution 46 (1): 80–88 (2008) (formerly Acta Phytotaxonomica Sinica) doi: 10.3724/SP.J.1002.2008.07003 http://www.plantsystematics.com Two new species of Curcuma (Zingiberaceae) used as cobra-bite antidotes Arunrat CHAVEERACH* 1Runglawan SUDMOON 2Tawatchai TANEE Piya MOKKAMUL 1Nison SATTAYASAI 3Jintana SATTAYASAI (Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand) (Faculty of Environment and Resource Studies, Mahasarakham University, Mahasarakham 44000, Thailand) (Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand) Abstract Two new species of Curcuma, C sattayasaii A Chaveerach & R Sudmoon and C zedoaroides A Chaveerach & T Tanee with rhizomes traditionally used for many decades as cobra-bite antidotes are described and illustrated Curcuma sattayasaii is similar to C longa L., but differs in rhizome horizontally branching on ground; coma bracts pinkish-white or pinkish-pale green; corolla pale yellow with orange tip; labellum pale orange with an orange central band; anther crest very short, broadly ovate, wider than long Curcuma zedoaroides is similar to C zedoaria (Christm.) Roscoe, but differs in rhizome branching pattern; the protruding secondary rhizomes curved down; blades oblong to oblong-lanceolate; peduncle glabrous; fertile and coma bracts glabrous; corolla lobes pale yellow to white, lateral lobe ovate, dorsal lobe broadly ovate The new taxa have been found in a village of Khon Kaen Province, Northeastern Thailand Key words Curcuma, Curcuma sattayasaii A Chaveerach & R Sudmoon, Curcuma zedoaroides A Chaveerach & T Tanee, cobra-bite antidote, new species, Khon Kaen, Thailand The genus Curcuma was established in 1753 by Linnaeus Members of the genus are represented by 60 species, and consist of 34 species in Thailand (Larsen & Saksuwan, 2006) In 1950, species were reported from the Malay Peninsula by Holttum for The Gardens’ Bulletin of Singapore In 1966, Sirirugsa reported 23 species and unknown from Thailand Twelve species were reported by Wu and Larsen (2000) in Flora of China with key to species and species descriptions In 2006, Sabu listed 20 species from South India in his book, Zingiberaceae and Costaceae of South India Many species have economic value because of their volatile oils, showy flowers and are used as spices, medicines, dyes, foods, perfumes, tonics, and as tropical ornamentals For medicinal properties, many species of Curcuma have been used world-wide since ancient times to today (Sirirugsa, 1996; Padua et al., 1999) In addition to helping cure some common diseases, Curcuma species also show some medicinal properties for the treatment of snake bites (Ratanabanangkoon et al., 1993) and as anti-tumor (Baatout et al., 2004) Since usefulness varies with plant species, taking the right plant is very important Some of the Curcuma species are well characterized, but others are difficult to distinguish The overall appearance of ——————————— Received: 16 January 2007 Accepted: 28 March 2007 * Author for correspondence Tel.: 66-4334-2908 ext 209; Fax: 66-43364169; E-mail: many species is very similar as they differ in small morphological details (Sirirugsa, 1996; Padua et al., 1999) There are two Curcuma species found at Ban Khok Sa-Nga (the village in which native peoples feed king cobra and many kinds of snakes), Khon Kaen province in northeastern Thailand The native traditional healers have been using the rhizomes as cobra-bite antidotes for a long time The chemical compounds of the plants are very useful for modern medicinal treatment Therefore, scientific identification of the plants is critical Morphological characters of these two plants are similar to C longa L and C zedoaria (Christm.) Roscoe, respectively However, specimens studied, A Chaveerach 609 (BK63812), P F von Siebold s.n (LEIDEN), D Ullrich & A T Gwee GW11 (SING); A Chaveerach 614 (BK63813), P C Yii & S P Runi S60630 (KEP), S P Runi et al S67409 (KEP), J Rantai et al S67359 (KEP) show that there are some morphological differences The results of this research support that they are different species Botanical illustrations and descriptions of them are given As mentioned by Sirirugsa (1996) and Padua et al (1999), the overall appearance of many species is very similar as they differ in small morphological details, DNA fingerprinting can be used to support the morphological data It is obtained from a number of PCR-based DNA markers which have been widely CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes used to investigate population genetics, including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) techniques Each of these methods has many advantages and limitations ISSR markers are extremely variable and have proven to be sensitive enough to differentiate cultivars and natural populations (Wolfe et al., 1998) DNA markers provide a powerful tool for the understanding of genetic evolution and the breeding of crops, especially for identification of cultivars and species (Prevost & Wilkinson, 1999) and phylogenetic evolution (Blair et al., 1999) ISSR markers which use SSR primers to amplify the genomic sequence between two simple sequence repeats (ISSRs or microsatellite) have generated many polymorphic DNA markers in several agricultural crops (Prevost & Wilkinson, 1999) The similarity of genetics (S) can be used to measure the relatedness of samples (Nybom & Hall, 1991; Welsh et al., 1991) The method of depicting the results of a phenetic analysis is by way of a branching diagram called a dendrogram produced by cluster analysis which is a method for grouping of operational taxonomic units (OTUs), for instance, morphological characters and DNA banding patterns The result is a branching diagram that connects all of the OTUs and OTUs cluster at levels corresponding to their degree of similarity By selecting an appropriate range of S to represent a given level in the taxonomic hierarchy, the taxonomist may then recognize species, genera, etc Plant groups for which all OTUs have similarities between 85%–100% might be recognized as part of the same species, while a 65% criterion might be used for genera However, the ultimate interpretation of the dendrogram is dependent upon the taxonomist’s knowledge of the operational taxonomic units (Rost et al., 1984) In this research we used molecular data as OTUs for dendrogram construction The obtained similarity coefficient values, together with containing cobra-bite antidotes, can be used to support morphological data for plant identification 81 Waan Phaya Ngu Tuamia), and C aeruginosa Roxb as outgroup in the analysis were taken from areas and neighboring villages about 30–40 km from the first for DNA extraction 1.2 DNA extraction Total genomic DNA was extracted using a modified CTAB procedure (Porebski et al., 1997) The approximate DNA concentration was determined using a spectrophotometer (UV-160A, Shimadzu) 1.3 PCR with ISSR primers Amplification was carried out in a 25 µL reaction consisting of reaction buffer, mmol/L of MgCl2, 0.2 mmol/L each of dNTPs, 0.5 µmol/L of primer, 1.25 units of DNA polymerase (Invitrogen), and ng of DNA template The 16 out of 36 ISSR primers that succeed in amplifying clear bands are as follows: (AG)7AA, (AG)7AAC, (AG)7AAG, (AG)8T, (AG)8C, (GA)6CC, (GA)6GG, (CA)6AC, (CA)6GT, (CA)6AG, (CA)9A, (GT)6CC, CCCC(GT)6, (CAC)3GC, (GAG)3 GC, (GACA)4 The reaction mixture was incubated at 94 ˚C for and the amplification was performed with the following thermal cycles: 35 cycles of denaturation at 94 ˚C for min, annealing at annealing temperature of each primer ((AG)7AA at 52 ˚C, (AG)7AAC at 55 ˚C, (AG)7AAG at 55 ˚C, (AG)8T at 55 ˚C, (AG)8C at 58 ˚C, (GA)6CC at 52 ˚C, (GA)6GG at 52 ˚C, (CA)6AC at 49 ˚C, (CA)6GT at 49 ˚C, (CA)6AG at 49 ˚C, (CA)9A at 58 ˚C, (GT)6CC at 52 ˚C, CCCC(GT)6 at 60 ˚C, (CAC)3GC at 45 ˚C, (GAG)3GC at 45 ˚C, (GACA)4 at 55 ˚C) for min, extension at 72 ˚C for min, and a final extension for at 72 ˚C All amplification reactions were repeated at least two times using a PCR machine (Gene Amp PCR system 9700, Applied Biosystems) Amplified products were detected by agarose gel electrophoresis in TAE buffer (0.4 mol/L Tris, 0.114% acetic acid, mmol/L EDTA pH 8.0) and visualized by ethidium bromide staining 1.4 Data analysis Data points are the presence (1)/absence (0) of each distinct band across all samples for the same primer These 0/1 data are then subjected to the DNA Fingerprinting II (Bio Rad) Pearson correlation program for dendrogram construction for cluster analysis Material and methods 1.1 Plant materials Two individuals of each studied plant, Curcuma longa, C cf longa (C sattayasaii A Chaveerach & R Sudmoon, local name: Waan Phaya Ngu Tuaphu), C zedoaria (Christm.) Roscoe, C cf zedoaria (C zedoaroides A Chaveerach & T Tanee, local name: Results and discussion 2.1 Molecular analysis Molecular analysis was performed to obtain more evidence to characterize the plants as different taxonomic units PCR using ISSR primers was selected since it shows good results in identification of many 82 Journal of Systematics and Evolution Vol 46 No plants (Wolfe & Liston, 1998; Yamagishi et al., 2002) The 16 ISSR primers generated a total of 947 bands with sizes ranging from 200 to 2000 base pairs In all ten individuals studied, there are species-specific profiles (Fig 1) Banding markers of all individuals studied were obtained, including the outgroup species Curcuma aeruginosa (Fig 2) The dendrogram (Fig 3) indicates that there are actually five species within the ten studied individuals Similarity values (Table 1) of C longa and C sattayasaii range from 58.9% to 66.2%, whereas those of C zedoaria and C zedoaroides range from 55.1% to 61.4% In this research, C aeruginosa was included since it is in the same genus but shows different morphological characters from the two pairs of plants All known samples of Curcuma species are separated from each other with the S values less than 75% Fig ISSR fingerprints of all studied plants with primer (AG)8C, showing species-specific profile 2008 Fig Dendrogram constructed from 16-ISSR primers (Fingerprinting II, Bio Rad), illustrating the possible relationship of studied plants Numerical on each branch is a cophenetic correlation value (Table 1, Fig 3) The results indicate suitability of the PCR procedure Moreover, C longa and C sattayasaii, as well as C zedoaria and C zedoaroides, are well separated by the dendrogram Therefore, C sattayasaii and C zedoaroides are assigned to the genus Curcuma but are considered different species, although they look almost alike 2.2 Pharmacological properties The pharmacological properties of the species are also different Curcuma sattayasaii and C zedoaroides have anti-cobra venom activity whereas C longa and C zedoaria not or show very low activity (data not shown), so villagers can not use them as cobra-bite antidotes Although the mentioned characters and activity may vary due to environmental factors, season and soil type, the differences were still observed in plants taken from the same place at the same period of time The anti-venom properties of these plants will be reported in an upcoming publication Taxonomic treatment Fig ISSR markers (arrows) of ten studied individuals generated from primer (GAG)3GC Curcuma sattayasaii A Chaveerach & R Sudmoon (Vernacular name: Waan Phaya Ngu Tuaphu), sp nov Fig 4; Fig 6: A, B Species nova haec est similis C longae L., a qua rhizomate ramos rectos in terra emittente, bracteis veternis subroseo-albis vel viridi-roseis claris, foliis flavis claris cum cuspidibus flavo-luteis, labello luteolo, ejus fascia media lutea, antherae crista curta late ovata latiore quam longiore differt Thailand Khon Kaen: Nam Phong district, Ban Khok Sa-Nga (the King Cobra village), alt 200 m, A Chaveerach 609 (BK63812), 2001-07-20 (holotype, BK; isotype, BKF) Terrestrial rhizomatous herb Rhizome branched, CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes 83 Table The percentage of similarity values of each studied pair Curcuma longa C longa C sattayasaii C sattayasaii C zedoaria C zedoaria C zedoaroides C zedoaroides C aeruginosa C aeruginosa 100 94.4 58.9 63.9 64.9 73.1 57.4 58.7 73.4 66.7 100 62.1 66.2 66.0 74.0 60.6 62.4 74.0 67.3 100 77.9 61.9 57.6 56.3 59.4 40.5 37.7 100 46.6 47.4 58.8 62.3 50.7 50.8 main rhizome conical, 8.0–10.0 cm long and 1.5–2.5 cm in diameter, secondary rhizome cylinder, 1.0–1.5 cm in diameter, horizontal, externally brownishyellow, internally deep orange-yellow, fragrant Root fibrous Pseudostem up to 70–100 cm tall Leaves annual, 5–6, leaf sheaths 30–50 cm long, green, glabrous Petiole slender, 15.0–35.0 cm long Ligule very short, 2–3 mm long, membranous, apex emarginate Leaf blades oblong to oblong-lanceolate, both sides glabrous, 30–45×7–13 cm, base cuneate, apex acute to acuminate Inflorescences terminal, 16–17 cm long, peduncles 30–31 cm long, glabrous Bracts imbricate; fertile bracts 12–17, broadly ovate, 4.0–5.0 ×3.2–3.5 cm, saccate, pale green to pale yellow with greenish tip, apex acute, basal adnated ca 1/3 of the length, both sides glabrous, 2–3 flowered per bract, coma bracts 4–7, elliptic, 5.8–7.0×3.0–3.5 cm, pinkish-white or pinkish-pale green, base saccate, basal adnated ca 1/5 of the length Bracteoles elliptic, 2.5–3.0×1.8–2.2 cm, white, glabrous Calyx tubular, membranous, pubescent, 0.6–0.8×0.2–0.3 cm, apex irregularly trilobed, lobe apex rounded, slit down on one side ca mm long Corolla tube 2.0–2.3 cm long, pale yellow to white, 3-lobed; dorsal lobe broadly ovate, 1.7–2.0×1.5–1.7 cm, pale yellow to white, saccate, apex hooded and mucronate 2–3 mm long, glabrous, lateral lobe ovate, 1.7–2.2×1.0–1.2 cm, apex round, pale yellow to white, glabrous Labellum obovate, light orange with a median orange band from the base to apex, 2.2–2.5×1.8–2.0 cm, apex slightly trilobed with distal part of median lobe bilobed Lateral staminodes oblong, 0.8–0.9×0.5–0.6 cm, light orange, apex round Stamen 1, filament flat, ca 1.0 cm long, base adnated to lateral staminodes Anther versatile, 4–5×2–3 mm, long spurs basal, dorsally pubescent Anther crest very short, broadly ovate, wider than long, 3.0×1.5–2 mm, white, glabrous Ovary inferior, pubescent, 3-locular, axillary placentation Style 1, filiform, ca cm long beyond the ovary, stigma cup-shaped, 4-lobed, ostiole ciliated Epigy- 100 88.2 61.6 59.9 59.1 50.0 100 55.1 61.4 62.9 54.0 100 89.4 62.5 56.8 100 62.5 56.1 100 91.3 100 nous glands 2, narrowly oblong, mm long, light yellow Fruit not found Flowering from July to September Curcuma sattayasaii is similar to C longa, but differs in the following characters: rhizome horizontally branching on ground; coma bracts pinkish-white or pinkish-pale green; corolla pale yellow with orange tip; labellum pale orange with an orange central band; anther crest very short, broadly ovate, wider than long Distribution: This species has been cultivated on open areas of home gardens with moist clay soil and under the shade of trees at an altitude of 200 m at Ban Khok Sa Nga and a few neighboring villages, Nam Phong district, Khon Kaen Province in northeastern Thailand Notes: Curcuma sattayasaii is similar to C longa in the following characters: rhizome branched, internal yellow; leaf blades green, oblong or elliptic; inflorescences terminal, labellum with central band, obovate The vernacular name of this new species is “Waan Phaya Ngu Tuaphu” according to the traditional healers Several members of the Zingiberaceae and those of Amaryllidaceae, Liliaceae, Araceae, Orchidaceae and Iridaceae, form an ethnobotanically important plant group known in Thai as “waan” This term refers to the underground storage roots or stem The word “Phaya Ngu” in Thai means “king cobra”, while the word “Tuaphu” means “male”, so in this case it means “male king cobra ginger” The specific epithet of this new species is named following the family name of Assoc Prof Dr Nison Sattayasai and Assoc Prof Dr Jintana Sattayasai who have previously studied its active ingredient including quality, quantity, activity for anti-venom property and structure From their observation and hypothesis, these two new species are different species from the two most similar species, C longa and C zedoaria This is the beginning of our molecular research and analyses supporting the morphology of these plants 84 Journal of Systematics and Evolution Vol 46 No 2008 Fig Curcuma sattayasaii A Chaveerach & R Sudmoon A, rhizome with lower part of pseudostem; B, an inflorescence developed on pseudostem; C, upper part of an inflorescence; D, fertile bract; E, coma bract; F, bracteole; G, side view of a flower; H, front view of a flower with calyx tube and ovary; I, calyx tube with ovary; J, long section of a flower; K, dorsal lobe; L, lateral lobe; M, upper part of a dorsal lobe (side view); N, upper part of a lateral lobe (side view); O, labellum; P, anther basal with long spurs, stigma and style (front view); Q, anther basal with long spurs, anther crest, stigma and style (side view); R, stigma; S, ovary with epigenous glands Drawing by Piya Mokkamul CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes 85 Fig Curcuma zedoaroides A Chaveerach & T Tanee A, rhizome with an inflorescence; B, upper part of an inflorescence; C, fertile bract; D, coma bract; E, bracteole; F, side view of a flower with calyx tube and ovary; G, front view of a flower; H, calyx tube with ovary; I, dorsal lobe; J, lateral lobe; K, upper part of a dorsal lobe (side view); L, upper part of a lateral lobe (side view); M, lateral staminodes; N, labellum; O, front view of an anther basal with long spurs, stigma and style; P, side view of an anther; Q, stigma; R, ovary with epigenous glands Drawing by Piya Mokkamul 86 Journal of Systematics and Evolution Vol 46 No 2008 Fig A, B Curcuma sattayasaii A Chaveerach & R Sudmoon A, An inflorescence developed on pseudostem B, Rhizomes C, D, C zedoaroides A Chaveerach & T Tanee C, Inflorescences; D, Rhizomes Photographs by Tawatchai Tanee Curcuma zedoaroides A Chaveerach & T Tanee (Vernacular name: Waan Phaya Ngu Tuamia), sp nov Fig 5; Fig 6: C, D Species nova haec est similis C zedoariae (Christm.) Roscoe, a qua rhizomatibus secundariis e rhizomatis primarii gemmis axillaribus nascentibus supra ea secundaria positis vel ex eis leviter remotis ad terram curvatis, foliorum laminis oblongis vel oblongo-lanceolatis, pedunculo glabro, bracteis fertilibus utrinque glabris, bractais comae glabris, corollae lobis luteolis vel albis, eis lateralibus ovatis, eis dorsalibus late ovatis distinguitur Thailand Khon Kaen: Nam Phong district, Ban Khok Sa-Nga (the King Cobra village), alt 200 m, A Chaveerach 614 (BK63813), 2001-07-20 (holotype, BK; isotype, BKF) Terrestrial rhizomatous herb, perennial Pseudostems up to 70–100 cm tall, roots fibrous, some expanded to ovoid or conical tubers at the apex Rhizomes branched; primary rhizome erect, broadly conical, 10.0–12.0×3.0–5.0 cm, secondary rhizome cylinder, 8.0–10.0×2.5–3.0 cm, developed from axillary bud on primary rhizome, growing downwards, external pale greenish and silver glance, internal pale yellow in dry season and pale green to yellow in rainy season, fragrant Leaves annual, 5–7, leaf CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes sheaths 25–40 cm long, green, glabrous Ligules very short, 2–3 mm long, apex shallowly bilobed Petioles slender, 8.0–12.0 cm long or more Leaf blades oblong to oblong-lanceolate, 70–100×15–18 cm, upper surface glabrous, green with a reddish-purple patch along the midrib, lower surface hairy Inflorescences developed on sides of primary rhizomes, erect, 15.0–18.0 cm long; peduncles 25.0–28.0 cm long, basal with 4–5 sheaths, elliptic, 3.5–6.5×2.0–2.3 cm, apex emarginated and mucro ca mm long beyond the tip, green, both sides glabrous Bracts imbricate, fertile bracts15–18, broadly ovate, 4.2–5.0×3.2–3.5 cm, saccate, green, both sides glabrous, base connated ca 1/3 of the length, apex round or acute with purple margin, 3–8 flowered per bract, coma bract 4–7, ovate-oblong, 5.8–7.0×3.2–3.5 cm, base connated ca 1/5 of the length, apex obtuse, dark pink, both sides glabrous; bracteoles elliptic-ovate, ca 1.0 cm long, apex rounded, membranous, white, both sides glabrous Calyx tubular, 0.5–0.7×0.2–0.3 cm, membranous, pubescent, apex irregularly trilobed, lobe apex rounded, split down on one side 1.0–2.0 mm Corolla tube 2.0–2.3 cm long, pale yellow to white, 3-lobed, pale yellow to white; dorsal lobe broadly ovate, 1.7–2.0×1.5–1.7 cm, saccate, apex hooded and mucronate 0.2–0.3 cm long, outer side sparsely pubescent, inner side glabrous, lateral lobes ovate, 1.8–2.1×1.0–1.2 cm, both sides glabrous, apex rounded; labellum obovate, 2.0–2.2×1.6–1.8 cm, pale yellow with a dark yellow band at the middle along the base to apex, apex slightly trilobed Lateral staminodes oblong, 1.0–1.2×0.5–0.6 cm, apex rounded, pale yellow Stamen 1, filament flat, 0.8–1.0 cm long, base adnated to lateral staminodes, anther versatile, 0.4–0.5×0.2–0.3 cm, long spurs basal, dorsally pubescent Ovary inferior, ca 0.2 cm long, 3-locular, axillary placentation, pubescent Style 1, filiform, 0.5–0.7 cm long Stigma cup-shaped, slightly 4-lobed, ostiole ciliated; epigenous glands 2, mm long, narrowly oblong, light yellow, apex acute Fruit not found Flowering from March to May Curcuma zedoaroides is similar to C zedoaria, but differs in the following characters: Rhizome branching pattern with secondary rhizomes developed from axillary bud on primary rhizome in series and the other ones developed on above or slightly deviate from the previously old ones; the protruding secondary rhizomes curved down; blades oblong to oblong-lanceolate; peduncle glabrous; fertile bracts glabrous on both sides; coma bracts glabrous; corolla lobes pale yellow to white, lateral lobe ovate, dorsal lobe broadly ovate 87 Distribution: This species has been found cultivated in open areas of home gardens with moist clay soil and under the shade of trees at an altitude of 200 m, in home gardens of Ban Khok Sa Nga and a few neighboring villages, Nam Phong district, Khon Kaen province in Northeastern Thailand Note: This species is similar to C zedoaria in the following characters: fibrous root ends enlarged ca cm in diameter; rhizome erect, branched, external pale yellow, silver glance, internal pale yellow; leaf blades dark green with dark purplish along mid nerve, upper surface glabrous, lower surface glabrous or hairy; inflorescence developed on rhizome often appearing before the leaves, calyx irregularly-trilobed, corolla, staminode and labellum yellow, labellum with dark yellow band “Waan Phaya Ngu Tuamia” means “female king cobra ginger” in Thai folk taxonomy because “Tuamia” means “female” The specific epithet of the last one is named for its close relation to C zedoaria References Baatout S, Derraji H, Jacquet P, Ooms D, Michaux A, Mergeay M 2004 Effect of Curcuma on radiation induced apoptosis in human cancer cells International Journal of Oncology 24: 321–329 Blair MW, Panaud O, McCouch SR 1999 Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.) 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CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes 83 Table The percentage of similarity values of each studied pair Curcuma longa C longa C sattayasaii C sattayasaii C zedoaria... CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes 85 Fig Curcuma zedoaroides A Chaveerach & T Tanee A, rhizome with an inflorescence; B, upper part of an inflorescence;... internal pale yellow in dry season and pale green to yellow in rainy season, fragrant Leaves annual, 5–7, leaf CHAVEERACH et al.: Two new species of Curcuma used as cobra-bite antidotes sheaths 25–40