Journal of Phytology 2011, 3(2): 37-48 ISSN: 2075-6240 Systematics Available Online: www.journal-phytology.com

REGULAR ARTICLE SYSTEMATIC STUDIES IN HERBACEOUS SPP. (REGION: TIRUCHIRAPPALLI DISTRICT IN INDIA) AND A SIMPLE KEY TO AUTHENTICATE 'BHUMYAMALAKI' COMPLEX MEMBERS Dhandayuthapani Kandavel1, Sundaramoorthi Kiruthika Rani2 and Murugaiyan Govindarajan Vinithra2 and Soundarapandian Sekar1* 1Department of Biotechnology, Bharathidasan University, Tiruchirappalli -620024, Tamilnadu, India 2Department of Molecular Biosciences, Bishop Heber College (Autonomous), Tiruchirappalli – 620017, Tamilnadu, India

SUMMARY The taxonomic status of and current systematic position is highlighted. In India, 12 herbaceous of Phyllanthus have been identified and among them herbs referred to as ‘Bhumyamalaki' complex has been extensively used as traditional medicine for various ailments. The medicinally important herb in this group is P. amarus Schum. & Thonn. and is often adulterated with its allied species and hence a simple key to differentiate them is evolved as confusion exists in identification of these herbaceous species due to their similarity and close proximity. P. niruri L. is a native of New World and endemic to America and does not occur in India, although there are many publications in India claiming work on P. niruri L. Those reports are actually pertaining to investigations in ‘niruri complex’ but not on P. niruri L. Herbaceous Phyllanthus species found in Tiruchirappalli district has been recorded and their morphological and anatomical parameters were assessed and a simple key developed. SCAR Analysis for validating the identity of P. amarus is presented for authentication at molecular level. P. debilis, a coastal zone species is first reported from an inland area. Key words: Phyllanthus amarus, Phyllanthus debilis, Phyllanthus maderaspatensis, Phyllanthus virgatus, Phyllanthus fraternus, Phyllanthus urinaria, Phyllanthaceae, Morphology, Taxonomic key, Herbal Authentication

Dhandayuthapani Kandavel et al. Systematic Studies in Herbaceous Phyllanthus Spp. (Region: Tiruchirappalli District in India) and a Simple Key to Authenticate 'Bhumyamalaki' Complex Members. J Phytol 3/2 (2011) 37-48. *Corresponding Author, Email: [email protected] 1. Introduction Systematic studies elucidating deep Fabidae and includes Rosales, , level angiosperm relationships is leaping Cucurbitales, Fagales, Zygophyllales and fast from the past decade due to rapid COM group (Cantino, 2007). COM group advancements in new phylogenetic consists of the orders Celastrales, Oxalidales informations. The clade includes and Malphigiales. Malphigiales is one of 75% of all angiosperm species (remaining the largest orders of flowering , 25% in monocotyledons) and comprises containing 39 families, 716 genera and 15935 several major subclades like , asterids, species which are approximately about 7.8% saxifragales, santalales and caryophyllales of the eudicots. The order is very diverse (Judd and Olmstead, 2004; Soltis et al., 2005; and hard to recognize except with molecular APG II, 2003). Rosids contain 140 families phylogenetic evidence. Molecular clock (70,000 species) and comprises three calculations estimate the origin of stem important groups: Viz., Vitaceae, Eurosids I group Malphigiales at around 100 Mya and Eurosids II (Cantino, 2007; Endress and (million years ago) and the origin of crown Matthews, 2006). Eurosids I is known as group Malphigiales at about 90 Mya Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

(Wanga et al., 2009). Euphorbiaceae sensu Phyllanthaceae is the second largest segregate lato is the largest among Malphigiales and of Euphorbiaceae sensu lato, comprising c. were defined by a few shared 2000 species in 49 genera. Phyllanthaceae morphological characters including includes two subfamilies, Phyllanthoideae unisexual flowers with superior syncarpous and Antidesmatoideae. Phyllanthoideae ovaries, apical-axile placentation and one or consists of four tribes and Phyllanthus belongs two epitropous ovules per locule. to the tribe Phyllantheae. Members of Characters that are usually present include Phyllanthaceae are pantropical and include alternate, stipulate leaves, actinomorphic trees, shrubs, phyllocladous taxa, semi – flowers, and presence of a floral disc, succulents, annual herbs, and even a free pistillode and obturator, as well as a floating species. Vegetatively, most tricarpellate, explosively dehiscing Phyllanthaceae are uniform with pinnate schizocarp leaving a central columella. venation, entire margins and simple The recent classification of angiosperms indumentums. Flowers are small and recognises five lineages of Euphorbiaceae sensu actinomorphic but display great diversity in lato (Figure 1) at family rank: Euphorbiaceae shape, size and number of floral organs sensu stricto, Pandaceae Engl. & Gilg, (Hoffmann et al., 2006; Kathriarachchi et al., Phyllanthaceae Martynov, Picrodendraceae Small 2006). and Putranjivaceae (APG III, 2009).

Fig. 1: Phylogenetic classification of Phyllanthaceae (Hoffman P, Kathriarachchi H and Wurdack K J, 2006)

The genus Phyllanthus L. was first Phyllanthus fraternus Webster, Phyllanthus described by Linnaeus in 1753 and consists debilis Klein ex Wild and Phyllanthus urinaria of ca.833 species (Govaerts et al., 2000) in the L. (Chaudhary and Rao, 2002). Although world and is chiefly distributed in moist these species closely resemble each other humid tropics. In India, it is represented by they also show sufficient characters to ca. 40 species (Henry & Santapau, 1973), maintain them as distinct species. However, although Hooker f. (1887) has recorded 56 ethnomedical uses and some aspects of species from the then British India. In total, pharmacological activities among these 12 species of herbaceous Phyllanthus have species are different (Theerakulpisut et al., been identified in India. They are used since 2008). Confusion exists in identification of ancient times in different systems of these herbaceous species mainly due to medicine, particularly for the treatment of referring them all with a common liver disorders and urinary infection. The vernacular name, their similarity in gross herbs known as ‘Bhumyamalaki’ in Indian morphology, close proximity in growth literature refer to a complex group of habitat and lack of guideline legal system to Phyllanthus amarus Schum. & Thonn., Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48 check the authenticity and quality of the 2. Materials and Methods medicinal plant sold. Tiruchirappalli district is centrally Webster (1955, 1956a, b, 1957, 1958, 1967, located in the state of Tamilnadu, India with 1970 and 1994; Webster and AiryShaw, 1971) a total geographic extent of 4404.12 sq. Km. has worked exhaustively on Phyllanthus and It lies between 78° 10' to 79°5' east longitude has provided detailed taxonomic accounts of and 10°15' and 11°2' north latitude. It lies at West Indian Phyllanthus. He observed that an altitude of 78 m above sea level. It falls true P. niruri L. is a native of New World and under the Cauvery Delta agroclimatic zone. endemic to America and does not occur in The annual mean maximum temperature is India. But there are many publications 37.7oC and the annual mean minimum published even today from India bearing title temperature is 18.9°C. The annual total on P. niruri L. In 1985, Mitra and Jain, after rainfall is 778 mm. The study area was critical examination of Indian materials of thoroughly surveyed for the presence of Phyllanthus revealed that the Phyllanthus niruri Phyllanthus spp. throughout the year from L. described in Flora of British India (Hooker f, time to time to observe the seasonal 1887) is actually a mixture of three closely variation in diversity (if any). They were related but distinct species namely P. amarus identified referring to standard floras Schum. & Thonn., P. debilis Klein ex Wild and (Mathew, 1982, 1983a, b and c, 1988; Gamble P. fraternus Webster. Those reports done in and Fischer, 1915-1936; Nair and Henry, India on P. niruri L. are actually pertaining to 1983; Henry et al., 1987; Henry et al., 1989). investigations in any one member of this For SCAR analysis fresh leaf samples ‘niruri complex’ but not on P. niruri L. The (young leaves) collected from the study present study was conducted to investigate areas in Tiruchirappalli district were used the diversity of Phyllanthus spp. found in for the isolation of genomic DNA (Sharma et Tiruchirappalli district of Tamilnadu, India al., 2003). Forward and Reverse primers and attempt to resolve the nomenclatural were designed (Jain et al., 2008) and were problem persisting in this genus by analysing synthesized by Sigma-Aldrich Chemicals the morphological and anatomical characters Pvt. Ltd., Bangalore. The PCR reaction mix of these plants and evolve a simple key for (25 μl) used in the experiment was: DNA easy identification of the related species. template - 1.72 μl (25 ng); dNTP mix - 0.5 μl Through distinct morphological features, the (200 μM each); Taq DNA polymerase - 0.5 μl identity of P. amarus can be confirmed but as (0.2 unit); PCR buffer - 2.5 μl; Forward an additional proof the validity of Sequence Primer - 1 μl (10 pmol); Reverse Primer - 1 Characterised Amplified Regions (SCAR) μl (10 pmol) and Sterile D. H2O - 17.78 μl. markers developed earlier was assessed (Jain The PCR was carried out using a DNA et al., 2008). PCR-based methods, including engine thermocycler (Eppendorf Pro-S Random Amplified Polymorphic DNA gradient cyler) programmed for the initial (RAPD), have been used for authentication of denaturation at 94oC for 5 min and 40 cycles medicinal plant materials. However, RAPD of 94°C for 1 min, 38oC for 1 min and 72oC markers are difficult to reproduce and are for 2 min, with a final Extension of 72oC for therefore preferentially converted to more 5 min. The amplified products were specific SCAR markers. For building a SCAR separated on 1.8% agarose gel containing 0.5 primer, a RAPD gel has to be run first using μg / ml of ethidium bromide and random primers and then the unique bands photographed with Alphainnotech have to be isolated, cloned, sequenced and alphaimager (USA). The profile was developed as SCAR primers useful for unique analysed using the Alphaview for identification of that species. Use of SCAR Alphaimager systems (version 1.2.0.1) marker for identification of P. emblica L. in its software. commercial samples and in Triphala churna, a Voucher specimens were deposited in multi-component ayurvedic formulation has the Department Herbarium, Department of been done earlier (Dnyaneshwar et al., 2006). Biotechnology, Bharathidasan University for future reference. For anatomical details of Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48 the species, Cross Sections (C.S.) were Four herbaceous species of Phyllanthus obtained free hand with sectioning blades, were found in the study area and are P. then stained in safranine and mounted in amarus Schum. & Thonn., P. debilis Klein ex glycerine. Semi permanent slides, so Willd., P. maderaspatensis L. and P. virgatus prepared, were examined under Nikon PFX Forster f (Figure 2). The SCAR profile showed microscope. The photography was made the characteristic 1150 bp band confirming the with the help of Labomed CXR3, identity of P. amarus and additional bands LaboAmerica, Inc., US, Compound (475 bp, 400bp, 250 bp, 100bp and 50 bp) were microscope with camera. For external also produced (Figure 3). Anatomical features Motic SMZ – 140, China, Stereo comparison between the species and Zoom microscope with DM -143 camera Morphological distinctions of the species was used. revealed the prominent differences (Figures 2, 4 and 5). The morphological and anatomical 3. Results distinctions were listed (Table 1). Table 1: Morphological and Anatomical characters of herbaceous Phyllanthus spp. seen in Tiruchirappalli District

P. amarus Schum. & Character P. debilis Klein ex Wild P. maderaspatensis L. P. virgatus Forster f. Thonn. Vegetative Characters Stem shape Terete Angular Angular Angular Stem surface Hispidulous Glabrous Glabrous Glabrous Cataphyll Three. Triangular- Three. Narrowly Absent Absent lanceolate; acuminate and lanceolate and acuminate turn black at maturity Branchlet Present Present Absent Absent Leaf shape Oblong Narrowly elliptic in Spathulate Oblong-elliptic upper part and cuneate at base Leaf tip Rounded Acute Apiculate Obtuse Reproductive Characters Tepals Five Six Six Six Disc Five Six Six Six segments Stamens Three Three Three Three Filaments Completely connate Connate at the base and Completely connate Free free at tip Style Three. Minutely bifid at Three. Bifid about to the Three. Minutely bifid at Three. Bilobed at tip middle. Appressed to the tip. Free apex. Free ovary Female Disc Star shaped Irregular Rectangular Orbicular Capsule Slightly warty Smooth Smooth Tubercled Surface Ovary Trigonous Trigonous Trigonous Trigonous Chambers Ovules Six Six Six Six Placentation Axile Axile Axile Axile Anatomical Characters Stem hairs Present Absent Absent Absent Shape Circular Angular (pentagonal) Angular (pentagonal) Circular with with protruded bulges at with bulges at the angles protrusion at the the angles poles Epidermis Present Present Present Present Hypodermis Outer 2-3 layers of 4-5 layers of 4 – 5 layers of 3-4 layers of collenchyma and inner 2- chlorenchyma chlorenchyma chlorenchyma 3 layers of chlorenchyma Vascular Conjoint, Collateral, Conjoint, Collateral, Conjoint, Collateral, Conjoint, Collateral, Bundles Open and Endarch. Open and Endarch. Open and Endarch. Open and Endarch. Arranged in a ring Arranged in a ring Arranged in a ring Arranged in a ring Pith Large, central and Large, central and Large, central and Small, centered and parenchymatous parenchymatous parenchymatous parenchymatous Branchlets Epidermis, 2-3 layers of Epidermis, 2-3 layers of Absent Absent chlorenchyma and two chlorenchyma and two conjoint, collateral, open conjoint, collateral, open vascular bundles vascular bundles Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

As evident from the table, P. to the ovary, star shaped female disc and maderaspatensis and P. virgatus can be easily slightly warty capsule……...P. amarus 1. distinguished from other members of 1b. Angular stem with glabrous surface, Phyllanthus by virtue of their unique Cataphyll remain green, leaf shape characters like leaf shape, absence of narrowly elliptic in upper part and cuneate cataphylls, absence of branchlets, etc. The at base with acute apex, Six tepals, Stamen capsule of P. amarus Schum. & Thonn. has connate at the based and free at tip, Style been reported to be smooth hither to bifid about to the middle and appressed to (Chaudhary and Rao, 2002) but the the ovary , irregularly lobed female disc and observation made in this study has revealed smooth capsule……..P. debilis 2. its minutely warty surface nature. P. debilis Klein ex Wild originally thought to be In P. debilis Klein ex Wild the proximal confined to coastal belts (Chaudhary and axils bear male flowers and distal axils bear Rao, 2002; Mitra and Jain, 1985) are female flowers but in P. amarus Schum. & identified in some parts of the study area Thonn. both male and female flowers (inland) in rich distribution. P. debilis Klein occurring together has been observed. This ex Wild collected from the study area floral character can also serve as additional showed characters such as leaf margins parameter for differentiating these two glabrous, female calyx lobes subequal, species, but, the male flower distribution staminal filaments connate at base and along the axils is reported as varying over slightly free at tip, styles distinctly bifid at geographical area (Chaudhary and Rao, tip, female disk irregularly lobed, etc. to 2002) and hence not included in the key. confirm its identity (Figure 4). Mathew The elaborate characters described in earlier (1983) reported the distribution of P. debilis works (Chaudhary and Rao, 2002; Mathew, Klein ex Wild in hills (900 – 1200 m), fallow 1983; Mitra and Jain, 1985; Bagchi et al., fields and river banks. But Chaudhary and 1992) help one to identify the group but Rao (2002) and Mitra and Jain, 1985 in their characters with sharp variation and monographs on taxonomic study of distinction aiding easy examination are herbaceous Phyllanthus species recorded its short-listed and presented as a simple distribution only in coastal regions of West dichotomous key here to correctly Bengal, Orissa, Maharashtra, Andhra distinguish members of the ‘Bhumyamalaki’ Pradesh, Karnataka, Kerala, Tamil Nadu complex: and Andaman and Nicobar islands. However, here we report its luxuriant 1. a. Tepals 5, Stem terete distribution in Sathamangalam Revenue throughout…………..P. amarus 1. Village of Tiruchirappalli district, which is a b. Tepals 6, Stem angular throughout or plain area and far from sea-shore. Based on angular above…………………...2 the morphological characters observed, a 2. a. Leaves hispidulous along margins, simple and easy to follow dichotomous key female flower in lower axils and 1-3 male is presented below to distinguish P. amarus flowers in upper axils ……………………P. Schum. & Thonn. from P. debilis Klein ex urinaria 2. Wild. b. Leaves glabrous along margins, proximal few nodes of branchlets with 2-4 Key for distinguishing P. amarus Schum. male flowers, succeeding distal nodes with & Thonn. and P. Debilis Klein ex Wild solitary female flower…...…….3 1a. Terete stem with hispidulous surface, 3. a. Leaves with acute tip, Staminal Cataphyll turn black at maturity, leaf shape filaments slightly free at tip, styles distinctly oblong with rounded apex, Five tepals, bifid at tip, female tepals sub equal and Stamen completely connate into a column, female disk almost rounded or irregularly Style minutely bifid at tip and not appressed lobed, styles appressed and spreading ……………………….P. debilis 3. Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

b. Leaves with obtuse tip, staminal The characters mentioned in the key are filaments completely connate into a column, arranged sequentially based on their styles minutely bifid at tip, female tepals importance and not like the usual vegetative unequal in size and female disk deeply to reproductive character order. lacinate, styles erect and spreading…………………….P. fraternus 4.

Fig.2 Habit and Uprooted view

Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

Fig. 3: SCAR profile of Phyllanthus amarus Schum. & Thonn. collected from three study areas in Tiruchirappalli district

Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

Fig. 4: Stereo zoom macroscopic view of some vegetative and reproductive parts of Phyllanthus spp. Studied

Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

Fig. 5: Distinguishing features of Phyllanthus debilis Klein ex Wild observed

4. Discussion meteric tonnes (Ved and Goraya, 2008). A study by National Medicinal Plants Almost all of this is sourced from the wild Board (NMPB), Government of India (Ved, or natural populations of the species D. K. and G. S. Goraya, 2008) has revealed (Kuipers, 2003; Ved and Goraya,2008). the total number of medicinal plants traded However due to a high level of in India as 960. Among them, 178 species morphological similarity among the fall under high volume trade or mentioned species (Chaudhary and Rao, consumption category (>100 Million 2002; Ganeshaiah et al., 1998) raw drug Tonnes/Year). P. amarus Schum. & Thonn. samples often contain species admixtures is one among them and a prioritized (Dymock, 1883; Dymock et al., 1893; Kirtikar medicinal plant of National Medicinal and Basu, 1975; Nadkarni, 1954; van Rhede, Plants Board (NMPB) for high demand in 1690). Khatoon et al. (2006) showed that the the domestic and international markets for three species of Phyllanthus (P. amarus, P. cultivation, conservation and development. fraternus and P. maderaspatensis) that are It has become common to add admixtures often mixed together have significantly with morphologically allied and different phytochemistry and only P. amarus geographically co-occurring species to raw was found to contain phyllanthin and drugs (Srirama et al., 2010; Bisset, 1984; hypophyllanthin, the two major compounds Khatoon et al., 2006; Mitra and Kannan,2007; believed to be responsible for the hepato Nair et al., 1983; Sunita, 1992; Ved and protective activity (Calixto et al., 1998). In Goraya, 2008). The annual volume of Tiruchirappalli district P. debilis Klein ex Phyllanthus trade in India is about 2000–5000 Wild is often misidentified as P. amarus Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

Schum. & Thonn. Local herbal vendors and APG III. 2009. An update of the Angiosperm collectors often sell this plant in shandies Phylogeny Group classification for the with the Tamil Vernacular name orders and families of flowering plants: ‘keezhanelli’. Plant collectors pay little APG III. Angiosperm Phylogeny Group. attention to proper identification of the Bot. J. Linn Soc., 161(2):105-121. plants for sale. This lack of accuracy often Bagchi, G., D., G.. N. Srivastava and S. C. leads to an inadequate linkage of the trade Singh, 1992. Distinguishing Features of data with the specific plant species though Medicinal herbaceous species of many of these species may be of high Phyllanthus occurring in Lucknow conservation concern. The SCAR profile district (U.P) India. International Journal obtained here is slightly different from the of Pharmacognosy, 30(3): 161 – 168. earlier work (Jain et al., 2008). The profile Bisset, W. G., 1984. Herbal Drugs and obtained by them showed only a single Phytopharmaceuticals. CRC Press, band of 1150 bp length. But, in this London. investigation along with the characteristic Calixto, J. B., A. R. S. Santos, V. C. Filho and 1150 bp band, additional bands (475 bp, R. A. Yunes, 1998. A review of the plants 400bp, 250 bp, 100bp and 50 bp) were of the genus Phyllanthus: their chemistry, produced. This may be due to difference in pharmacology, and therapeutic PCR stringency levels. Eventhough SCAR potential. Medical Research Reviews, 18, markers (Jain et al., 2008) and DNA barcodes 225–258. (Srirama et al., 2010) are useful for Cantino, P. D., 2007. Towards a distinguishing the Phyllanthus species, they phylogenetic nomenclature of are beyond the reach of a local herb collector. tracheophyta. Taxon. 56: 822 – 846. Hence, we believe simple morphological Chaudhary, L, B and R. R. Rao, 2002. and anatomical character based keys for Taxonomic study of herbaceous species easy identification of herbs existing along of Phyllanthus L. (Euphorbiaceae) in with closely related allied species should be India. Phytotaxonomy 2: 143 – 162. attempted with. Most of the time the Dnyaneshwar, W., C. Preeti, J. Kalpana and taxonomic keys are prepared exhaustively P. Bhushan, 2006. Development and and serves the purpose of scientific Application of RAPD-SCAR Marker for purposes and so keys for layman with Identificationof Phyllanthus emblica minimal technical details should either be LINN. Biol. Pharm. Bull. 29(11): 2313— developed or abridged from elaborate ones. 2316. The key presented here to a large extent can Dymock, W., 1883. The Vegetable Materia be examined with naked eye and does not Medica of Western India. Education need any DNA marker or laboratory. P. Society’s Press, Bombay. amarus can be easily distinguished from its Dymock, W., C. J. H. Warden and D. allies by its unique characteristics of terete Hooper, 1893. Pharmacographia indica: stem and five tepals. A History of the Principal Drugs of the Vegetable Origin. Kegsm Paul, Trench, Acknowledgements Trubner & Co.,Ltd., London, pp. 261– The authors thank University Grants 265. Commission, Government of India, New Endress, P. K and M. L. Matthews, 2006. Delhi for the award of Teacher fellowship Floral structure and systematics in four (Faculty Development Programme) to one orders of rosids including a broad of them (DK). survey of floral mucilage cells. Plant Syst Evol., 260: 223-251. References Gamble, J, S and C. E. C. Fischer, 1915-1936. The Flora of the Presidency of Madras. APG II. 2003. An update of the Angisoperm XI Parts. Adlard & Son, Ltd., London. Phylogeny Group classification for the Ganeshaiah, K, N., R. Ganesan, R. Uma orders and families of flowering plants: Shaanker and C. Meera, 1998. APG II. Bot. J. Linn Soc., 141(4): 399 – 436. Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

Phyllanthus niruri: A Taxonomic Hurdle Kirtikar, K. R and B. D. Basu, 1975. Indian or Hurdled by Taxonomists. Amruth, Medicinal Plants. Bishen Singh August, 3–8. Mahendra Pal Singh, New Connaught Govaerts, R., D. G. Frodin and A. Radcliffe Place, Dehrahun. Smith, 2000. World checklist and Kuipers, S. E., 2003. Trade in medicinal Bibliography of Euphorbiaceae. 4 vols. plants. In: Bodeker G, Bhat K K S, Burley Royal Botanic Gardens, Kew. J, Vantomme P. (Eds.), Medicinal Plants Henry, A, N and H. Santapau H, 1973. for Forest Conservation and Health Care, Dictionary of the flowering plants in FAO, Rome, pp. 45–59. India. CSIR, New Delhi. Matthew, K. M., 1982. Illustrations on the Henry, A, N., G. R. Kumari and V, Chitra, Flora of the Tamil Nadu Carnatic. The 1987. Flora of Tamil Nadu. Vol .II. Rapinat Herbarium. St. Joseph's College, Botanical Survey of India, Department Tiruchirappalli, India. of Environment, Southern Circle, Matthew, K. M., 1983a. The Flora of the Coimbatore, India. Tamil Nadu Carnatic. Part I. Polypetalae. Henry, A, N., V. Chitra and N. P. The Rapinat Herbarium. St. Joseph's Balakrishnan, 1989. Flora of Tamil Nadu. College, Tiruchirappalli, India. Vol III. Botanical Survey of India, Matthew, K. M., 1983b. The Flora of the Department of Environment, Southern Tamil Nadu Carnatic. Part II. Circle, Coimbatore, India. Gamopetalae & Monochlamydeae. The Hoffmann, P., H. Kathriarachchi and K. J. Rapinat Herbarium. St. Joseph's College, Wurdack, 2006. A phylogenetic Tiruchirappalli,India. classification of Phyllanthaceae Matthew, K. M., 1983c. The Flora of the (Malphighiales; Euphorbiaceae sensu Tamil Nadu Carnatic. Part III. lato). Kew Bulletin 61: 37 – 53. Monocotyledones. The Rapinat Hooker, J. D., 1887. Phyllanthus In: J. D. Herbarium. St. Joseph's College, Hooker, Flora of British India, Reeve & Tiruchirappalli, India. Co., London. 5: 285 – 305. Matthew, K. M., 1988. Further Illustrations Jain, N., A. K. Shasany, S. Singh, S. P. S. on the Flora of the Tamil Nadu Carnatic. Khanuja and S. Kumar, 2008. SCAR The Rapinat Herbarium. St. Joseph's markers for correct identification of College, Tiruchirappalli, India. Phyllanthus amarus, P. fraternus, P. debilis Mitra, R, L and S. K. Jain, 1985. Concept of and P. urinaria used in scientific Phyllanthus niruri (Euphorbiaceae) in investigations and dry leaf bulk herb Indian Floras. Bull. Bot. Surv. India. 27: 1- trade. Planta Medica, 74,296–301. 4: 161-176. Judd, W, S and R. G. Olmstead, 2004. A Mitra, S. K and R. Kannan, 2007. A Note on survey of tricolpate (eudicot) phylogeny. unintentional adulterations in ayurvedic Amer. J. Bot. 91: 1627 – 1644. herbs. Ethnobotanical Leaflets, 11, 11–15. Kathriarachchi, H., R. Samuel, P. Hoffmann, Nadkarni, A. K., 1954. Dr. K.M. Nadkarni’s J. Mlinarec, K. J. Wurdack, H. Indian Materia Medica. Popular Book Ralimanana, T. F. Stuessy and M. W. Depot,Bombay. Chase, 2006. Phylogenetics of tribe Nair, N. C and A. N. Henry, 1983. Flora of Phyllantheae (Phyllanthaceae; Tamil Nadu. Vol .I. Botanical Survey of Euphorbiaceae sensu lato) based on India, Department of Environment, NRITS and Plastid MATK DNA Southern Circle, Coimbatore, India. sequence data. American Journal of Nair, V. K., K. R. Yoganarasimhan, K. Botany, 93(4), 637-655. Murthy and T. R. Shantha, 1983. Studies Khatoon, S., V. Rai, A. K. S. Rawat and S. on some South Indian market samples Mehrotra, 2006. Comparative of ayurvedic drugs II. Ancient Science of pharmacognostic studies of three Life, 3,60–66. Phyllanthus species. Journal of Paran, I and R. W. Michelmore, 1993. Ethnopharmacology, 104, 79–86. Development of reliable PCR-based Dhandayuthapani Kandavel et al./J Phytol 3/2 (2011) 37-48

markers linked to downy mildew dominated forests. PNAS, 106 (10), 3853 resistance genes in lettuce. Theoretical – 3858. and applied genetics, 85: 985-993. Webster, G., L., 1955. Studies of the Soltis, D, E., P. S. Soltis, P. K. Endress and M. Euphorbiaceae, Phyllanthoideae I. W. Chase, 2005. Phylogeny and Taxonomic notes on the West Indian Evolution of Angiosperms. Sinauer, species of Phyllanthus. Contr. Gray. Herb. Sunderland, MA. (Harvard University)., 176: 45-63. Srirama, R., U. Senthilkumar, N. Sreejayan, Webster, G, L., 1956a. Studies on the G. Ravikanth, B. R. Gurumurthy, M. B. Euphorbiaceae, Phyllanthoideae II. The Shivanna, M. Sanjappa, K. N. American species of Phyllanthus Ganeshaiah and R. Uma Shaanker, 2010. described by Linnaeus. J. Arnold Arb., 37 Assessing species admixtures in raw (1): 1 – 14. drug trade of Phyllanthus, a hepato- Webster, G, L., 1956b. A monographic study protective plant using molecular tools. J. of the West Indian species of Phyllanthus. of EthnoPharmacol, 130: 208-215. J. Arnold Arb., 37 (2): 91-122. 217-263, Sunita, G.., 1992. Substitute and Adulterant 340-359. Plants. Periodical Experts Book Webster, G, L., 1957. A monographic study Agency,New Delhi. of the West Indian species of Phyllanthus. Theerakulpisut, P., N. Kanawapee, D. J. Arnold Arb., 38: 51-64, 170-198, 295-373. Maensiri, S. Bunnag and P. Webster, G. L., 1958. A monographic study Chantaranothai, 2008. Development of of the West Indian species of Phyllanthus. species-specific SCAR markers for J. Arnold Arb., 39: 49 – 200, 111-212. identification of three medicinal species Webster, G. L., 1967. Genera of of Phyllanthus. Journal of Systematics and Euphorbiaceae in Southern United Evolution, 46 (4): 614–621. States. J. Arnold Arb., 48: 303-361, 363- van Rhede, A., 1690. Horti Muluhorici Purs 430. De&m de Herhis er Diversis Illurum Webster, G. L., 1970. A revision of Specienus, vol. 10. Someren, Amsterdam, Phyllanthus (Euphorbiaceae) in the pp. 29–31. continental . Brittonia., 22: Ved, D, K and G. S. Goraya, 2008. Demand 44-76. and supply of medicinal plants in India. Webster, G. L., 1994. Synopsis of the genus Bishen Singh, Mahendra Pal Singh, and suprageneric taxa of Euphorbiaceae. Dehradun & FRLHT, Bangalore, India. Ann. Missouri Bot. Gard., 81: 33-144. Wanga, H., M. J. Moore, P. S. Soltis, C. D. Webster, G. L and K. H. Airy – Shaw, 1971. Bell, S. F. Brockington, R. Alexandre, C. A provisional synopsis of the New C. Davos, S. R. Latvis M. Manchester Guinea taxa of Phyllanthus and D. E. Soltis, 2009. Rosid radiation (Euphorbiaceae). Kew Bull., 26(1): 85-109. and the rapid rise of angiosperm