5 Genetic Resources of Phyllanthus in Southern India Identification of Geographic and Genetic Hot Spots and Its Implication for Conservation G. Ravikanth, R. Srirama, U. Senthilkumar, K. N. Ganeshaiah, and R. Uma Shaanker CONTENTS 5.1 5.2 Introduction .................................................................................................... 98 Distribution of Phyllanthus Species ............................................................. 104 5.2.1 Identiication of Geographic Hot Spots of Phyllanthus in South India: Contours of Species Richness ................................................ 104 5.3 Identiication of Genetic Hot Spots of Economically Important Phyllanthus Species ...................................................................................... 107 5.3.1 Phyllanthus emblica ......................................................................... 107 5.3.2 Phyllanthus amarus .......................................................................... 108 5.4 Impact of Harvesting on The Genetic Variability of P. emblica .................. 108 5.5 Phyllanthus: Taxonomic Incongruities, Species Adulteration, and DNA Bar Coding.................................................................................................... 110 5.6 Implications for Utilization and Conservation ............................................. 111 Acknowledgments.................................................................................................. 112 References .............................................................................................................. 113 97 98 5.1 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications INTRODUCTION The genus Phyllanthus (family: Phyllanthaceae) is one of the most important groups of plants traded as a raw herbal drug in India (Ved and Goraya, 2008). Plants of this genus have been used in traditional medicine for a variety of uses, including as an antipyretic, laxative, tonic, antibacterial, antioxidative, immunomodulatory, antiviral, antiatherosclerotic, and antineoplasic (Unander et al., 1991, 1995; Calixto et al., 1998). In India, Phyllanthus is used as a common folk remedy for the treatment of jaundice and hepatitis. The genus is also used as a general tonic and to treat weakness in infants (Unander et al., 1991). A number of taxa are cultivated for their leshy edible fruits and for preparation of herbal drugs. Among the Phyllanthus species in India, P. amarus, P. debilis, P. fraternus, P. urinaria, P. kozhikodianus, P. maderaspatensis, P. emblica, and P. indoischeri are widely used as herbal medicines, and some of these species are also cultivated in southern India (Table 5.1). Phyllanthus amarus, a predominant species occurring in southern India, has been shown to suppress the growth and replication of hepatitis B virus (Venkateswaran et al., 1987; Thyagarajan et al., 1988; Yeh et al., 1993; Jayaram and Thyagarajan, 1996; Lee et al., 1996; Paranjape, 2001). A few species, such as P. amarus, P. fraternus, and P. debilis, have been reported to be extensively used for curing jaundice; P. urinaria has been recommended for curing urinary tract diseases (Table 5.1; Jain et al., 2008). Phyllanthin and hypophyllanthin, both present in P. amarus, have been shown to protect hepatocytes against carbon tetrachloride (CCl4) and galactosamineinduced cytotoxicity in rats (Syamasundar et al., 1985). Phyllanthus emblica is another medicinally important species widely distributed across the Indian subcontinent. It is commonly called the Indian gooseberry. Traditionally, it has been used to treat digestive disorders, constipation, fever, cough, and asthma and to stimulate hair growth. Extracts of P. emblica have been shown to possess several pharmacological actions, such as analgesic, anti-inlammatory, antioxidant, and chemoprotective, (Calixto et al., 1998; Vormisto et al., 1997; Khopde et al., 2001). The fruits contain diterpenes; triterpenes; lupeol; lavonoids such as kaempherol-3-O-fl-D-glucoside and quercetin-3-O-fl-D-glucoside; polyphenols such as emblicanin A and B; punigluconin and pedunculagin, and other molecules (Calixto et al., 1998; Bhattacharya et al., 1999; Summanen, 1999; Ghosal et al., 1996). Phyllanthus emblica fruits are used for preparations of pickles, jams, and juices. The fruits are also used by the cosmetic, hair dye and shampoo industries (Ganesan and Shetty, 2004). The annual volume of Phyllanthus trade in India is estimated to be about 2,000– 5,000 metric tonnes of herbaceous material and about 16,000–18,000 metric tonnes of fruits (Ved and Goraya, 2008). Several species are also exported in powder form for the extraction of a number of phytochemicals or for use in the preparation of traditional formulations in the treatment of liver disorders (Kamble et al., 2008). Because of its multifarious use and demand, Phyllanthus species form an important nontimber forest product resource. Most of the material for trade is sourced from the wild by forest-dwelling communities, and only a small percentage is obtained from cultivation (Ved and Goraya, 2008). Because of the often-indiscriminate harvesting, 99 Genetic Resources of Phyllanthus in Southern India TABLE 5.1 Phyllanthus L. Species in India and Their Pharmacological Activities Sl No. Phyllanthus Species Habit Status Bioactivity References 1 P. acidus (L.) Skeels Tree Cultivated 2 P. airyshawii Brunel & Roux P. ajmerianus L.B. Chaudhary & R.R. Rao P. amarus Schumach. Herb Rare Immunomodulatory effect against gastrointestinal disorders No reports Herb Endemic No reports Herb Common P. anamalayanus (Gamble) G.L. Webster P. andamanicus N.P. Balakr. & N.G. Nair P. arbuscula (Sw.) J.F. Gmelin P. baeobotryoides Wall. P. baillonianus Muell.Arg. P. beddomei Gamble P. brevipes Hook.f. Shrub Endemic Antioxidant, Abhyankar et al., 2010; anticancer, Adeneye and Benebo, nephroprotective 2008; activity, Chirdchupunseree and hepatoprotective Pramyothin, 2010; activity, antibacterial Eldeen et al., 2010; activity, Faremi et al., 2008; antiinlammatory, Kassuya et al., 2006; antiallodynic, Kiemer et al., 2003; antitumor Kumar and Kuttan, 2005; Naaz et al., 2007; Narendranathan et al., 1997; Notka et al., 2003; Notka et al., 2004; Rajeshkumar and Kuttan, 2000; Rajeshkumar et al., 2002; Raphael and Kuttan, 2003 No reports Shrub Endemic No reports Shrub Cultivated No reports Shrub Rare No reports Shrub Endemic No reports Shrub Shrub Endemic Endemic No reports No reports 3 4 5 6 7 8 9 10 11 Kundu et al., 2009 (continued) 100 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications TABLE 5.1 (continued) Phyllanthus L. Species in India and Their Pharmacological Activities Sl No. 12 13 14 15 16 17 18 19 20 21 22 23 24 Phyllanthus Species Habit Status P. chandrabosei Govaets & Radcl.-Sm. P. clarkei Hook.f. P. columnaris Muell. Arg. P. debilis Klein ex Willd. P. emblica L. Shrub Endemic No reports Bioactivity Shrub Shrub Very rare Rare No reports No reports Herb No reports Tree Common in plains Common P. imbriatus (Wight) Muell. Arg. P. fraternus G.L. Webster Shrub Endemic Herb Uncommon Hepatoprotective activity P. gageanus (Gamble) M. Mohanan P. glaucus Wall. Shrub Endemic No reports Shrub No reports P. gomphocarpus Hook.f. P. grifithii Muell. Arg. P. heyneanus Muell. Arg. P. indoischeri Bennet Shrub Common in northeast Rare Shrub Endemic No reports Shrub Endemic No reports Tree Endemic No reports References Antioxidant, Al-Rehaily et al., 2002; antisecretory, Bandyopadhyay et al., antiulcer, and 2000; Jose et al., 2001; cytoprotective Liu et al., 2008; Luo et properties; antitumor, al., 2009; Mathur et al., free radical 1996; Nosál’ová et al., scavenging activity, 2003; Perianayagam et hypolipidaemic al., 2004; Pramyothin et activity, antitussive, al., 2006; Reddy et al., antipyretic, analgesic, 2009; Sai Ram et al., hepatoprotective 2002; Sharma et al., activity 2009; Sultana et al., 2008 No reports Gopi and Setty, 2010; Padma and Setty, 1999; Sailaja and Setty, 2006; Sebastian and Setty, 1999 No reports (continued) 101 Genetic Resources of Phyllanthus in Southern India TABLE 5.1 (continued) Phyllanthus L. Species in India and Their Pharmacological Activities Sl No. Phyllanthus Species Habit Status 25 P. juniperinoides Muell. Arg. Shrub No reports 26 P. leschenaultii Muell. Arg. P. macraei Muell. Arg. P. macrocalyx Muell. Arg P. maderaspatensis L. Shrub Endemic to peninsular India Very rare Shrub Endemic No reports Shrub Endemic No reports Herb Common P. megacarpus (Gamble) Kumari &Chandrab. P. myrtifolius (Wight) Muell. Arg. Shrub Endemic Hepatoprotective activity No reports Shrub Cultivated P. narayanaswamii Gamble P. parvifolius Buch.-Ham.ex D.Don P. pendulus Roxb. P. pinnatus (Wight) G.L Webster P. polyphyllus Willd. Herb Endemic Antibacterial, Eldeen et al., 2010 antioxidant, anti-HIV activity No reports Shrub Rare No reports Shrub Shrub Endemic Uncommon No reports No reports Shrub Common in peninsular India Rare Anti-inlammatory activity Rare No reports Cultivated Antitumor, antioxidant, anti-HIV activity Antioxidant, antidiabetic Hepatoprotective activity 27 28 29 30 31 32 33 34 35 36 37 39 P. praetervisus Muell. Shrub Arg. P. pseudoparvifolius Shrub R.L. Mitra & Sanjappa P. pulcher Wall. Shrub 40 P. reticulatus Poir. Shrub 41 P. rheedei Wight Herb 42 P. roeperianus Wall. Shrub 38 Very common Rare in peninsular India Rare Bioactivity References No reports Asha et al., 2004; Asha et al., 2007 Rao et al., 2006 No reports Stanslas et al., 2008; Eldeen et al., 2010 Eldeen et al., 2010; Kumar et al., 2008 Suresh and Asha, 2008 No reports (continued) 102 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications TABLE 5.1 (continued) Phyllanthus L. Species in India and Their Pharmacological Activities Sl No. Phyllanthus Species Habit 43 P. rotundifolius Klein ex Willd. Herb 44 P. sanjappae Chakrab. & M. Gangop. P. scabrifolius Hook.f. P. sikkimensis Muell. Arg. P. simplex Retz Status Bioactivity No reports Shrub Common in coastal areas Endemic Herb Endemic No reports Shrub Very rare No reports Herb Common Shrub Endemic 49 50 P. singampattianus (Sebastine & A.N Henry) Kumari & Chandrab. P. talbotii Sedgw. P. tenellus Roxb. Antidiabetic, antioxidant activity No reports Shrub Herb Endemic Naturalized 51 52 P. tetrandrus Roxb. P. urinaria L. Shrub Herb Rare Common 53 P. wightianus Muell. Arg. Shrub Endemic 45 46 47 48 References No reports Shabeer et al., 2009 No reports Immunomodulatory Ignácio et al., 2001 effect against microbial activity No reports Antibacterial, Eldeen et al., 2010; antioxidant, anti-HIV Chudapongse et al., activity, 2010; Fang et al., 2008; hepatoprotective Hau et al., 2009; Huang activity, antiet al., 2003; Huang et inlammatory, al., 2004; Huang et al., anticancer, antitumor, 2006; Lai et al., 2008; antiangiogenic, Lin et al., 2008; Yang et chemopreventive al., 2005; Yang et al., agent for peptic ulcer, 2007 anti-HSV No reports many of the species face the risk of local or regional extinction of their populations (Uma Shaanker et al., 2002, Ravikanth et al., 2009). In this chapter, we briely review the status of Phyllanthus resources in southern India with the overall aim of understanding the spatial distribution of the species as well as its genetic diversity. This information is critical in designing strategies for the long-term utilization and conservation of Phyllanthus genetic resources. 103 Genetic Resources of Phyllanthus in Southern India TABLE 5.2 Phyllanthus Species Traded in India and Molecular Regions that Have Been Sequenced Along with Their Accession Numbers Sl No. 1 Phyllanthus Species (Trade Name) P. amarus (Bhumiamla) 2 P. debilis (Bhumiamla) 3 P. fraternus (Bhumiamla) P. maderaspatensis (Kanocha) 4 5 P. reticulatus (Buinowla) 6 P. urinaria (Lal-BhuinAnvalah) 7 P. virgatus (Niruri) Molecular Regions ITS psbA-trnH GenBank Accession No. References Pruesapan et al., 2008 Srirama et al., 2010 ITS psbA-trnH rbcL matK, trnK EU623557.1 GU598561–65, GU598577 EU643742 FJ235474.1 FJ235356 FJ235310.1 EU861193.1 AY765265.1 AY936686 AY936591 FJ235265 FJ235311 FJ235357 FJ235475 GU598567–68 GU598566, 69 EU876847 GU598536–38 AY936609 AY936707 AY765290 AY936629 FJ235270 FJ235316 FJ235362 FJ235480 GU598539–40 AY765305 AY936736 AY936637 ITS atpB ndhF phyC atpB matK trnK, matK AY765268 GU598573–74 FJ235485 FJ235367 FJ235321 FJ235275 AY936639 Lee et al., 2006 Srirama et al., 2010 Unpublished Kathriarachchi et al., 2006 Kathriarachchi et al., 2006 Kawakita and Kato, 2009 Kawakita and Kato, 2009 (continued) ndhF phyC atpB trnL rbcL trnK, matK psbA-trnH ndhF phyC atpB matK trnK ITS ITS psbA-trnH ITS trnK psbA-trnH psbA-trnH ndhF phyC atpB matK Pruesapan et al., 2008 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Unpublished Lee et al., 2006 Srirama et al., 2010 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kathriarachchi et al., 2006 Kathriarachchi et al., 2006 Srirama et al., 2010 Unpublished Srirama et al., 2010 Kathriarachchi et al., 2006 Kathriarachchi et al., 2006 Kawakita and Kato, 2009 Srirama et al., 2010 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kathriarachchi et al., 2006 Kawakita and Kato, 2009 Lee et al., 2006 Lee et al., 2006 Kathriarachchi et al., 2006 Kathriarachchi et al., 2006 104 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications TABLE 5.2 (continued) Phyllanthus Species Traded in India and Molecular Regions that Have Been Sequenced Along with Their Accession Numbers Sl No. Phyllanthus Species (Trade Name) 8 P. emblica (Amla) 9 P. indoischeri (Amla/Ittu nelli) 5.2 Molecular Regions ITS rbcL psbA-trnH ndhF phyC atpB trnK, matK ITS rbcL trnL matK psbA-trnH trnL GenBank Accession No. AY936738 GU441778 EU643743 FJ847837 GU441788 AY936689 AY936594 FJ235297 FJ235343 FJ235461 GU598547 GU598558–60 GU930706 Reference Kawakita and Kato, 2009 Kawakita and Kato, 2009 Srirama et al., 2010 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kawakita and Kato, 2009 Kathriarachchi et al., 2006 Kathriarachchi et al., 2006 Unpublished Unpublished Pruesapan et al., 2008 Srirama et al., 2010 Unpublished DISTRIBUTION OF PHYLLANTHUS SPECIES Phyllanthus is one of the most species-rich genera of the family Phyllanthaceae, comprising over 800 species worldwide (Calixto et al., 1998; Govaerts et al., 2000; Wurdack et al., 2004). The genus is subdivided into 11 subgenera: Isocladus, Kirganelia, Cicca, Emblica, Conani, Gomphidium, Phyllanthodendron, Xylophylla, Botryanthus, Ericocus, and Phyllanthus. Plants of this genus are characterized by diverse growth forms, including shrubs, trees, and annual or biennial herbs and are distributed throughout the tropical and subtropical regions of both hemispheres. A few species of Phyllanthus are notorious weeds and occur in four continents (America, Africa, Asia, and Australia). India has 53 species of Phyllanthus, of which 23 species are endemic (Balakrishnan and Chakrabarty, 2007). These are distributed throughout the Indian subcontinent, with higher densities in the southern region. As many as 17 species are endemic to peninsular India, 2 species to the Andaman and Nicobar Islands, and others restricted to central and northeastern India (Balakrishnan and Chakrabarty, 2007). 5.2.1 IdentIfIcatIon of GeoGraphIc hot SpotS of PHYLLANTHUS In South IndIa: contourS of SpecIeS rIchneSS One of the key strategies to the sustainable management of any natural resource is to obtain spatially explicit information on its distribution. Spatially explicit distribution maps of the species could facilitate the management and utilization of these resources, tracking the dynamics of the resources over time; aid in preparing Genetic Resources of Phyllanthus in Southern India 105 germplasm collections; and help in assigning conservation value and priorities. Using geographic information systems, Ganeshaiah and Uma Shaanker (2003) developed a spatially explicit distribution map of all herbaceous species of Phyllanthus in India (Figure 5.1). Secondary data of occurrence of the species were obtained from various sources, like loras, herbaria, books, and other published sources, and then digitized. Species richness of Phyllanthus was summated on grids (1° latitude × 1° longitude), and contours of species richness were plotted (Figure 5.1). Among the 53 species of Phyllanthus, 37 are shrubs, 13 are herbs, and 3 are trees (Balakrishnan and Chakrabarty, 2007). The 13 herbaceous species of Phyllanthus are primarily concentrated in the states of Tamil Nadu, Kerala, Karnataka, Maharashtra, and Andhra Pradesh. A few species of Phyllanthus, such as P. amarus, are distributed throughout the country. Two herbs (i.e., P. rheedii and P. kozhikodianus) are endemic to peninsular India and Sri Lanka. Similarly, P. scabrifolius is endemic to Maharashtra and northern Karnataka. Phyllanthus ajmerianus is endemic to FIGURE 5.1 See color insert. Hypsographic view of Phyllanthus species richness in India. The data on the distribution of the species were obtained from diverse sources (monographs, etc.), and the latitude and longitude were assigned for each record and mapped. The density of the species in each grid of the size 10 km × 10 km was computed and the contours for the density obtained. Based on the contour data, the three-dimensional view was constructed using suitable GIS software. 106 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications Phyllanthus emblica (1) 4 to 4 (1) 3 to 3 2 to 2 (98) 1 to 1 (295) FIGURE 5.2 Resource map of Phyllanthus emblica. The map was developed using data collected from primary sources. Each pixel is the size of 6.25 km × 6.25 km. The lighter shades indicate higher densities. The numbers in brackets indicate the number of records at each pixel. Rajasthan. Among the shrubs, about 7 species are endemic to India. Phyllanthus indoischeri is the only tree species endemic to the Deccan Plateau (Ganesan, 2003; Balakrishnan and Chakrabarty, 2007). The distribution of P. indoischeri overlaps with P. emblica, while P. emblica is distributed throughout the Deccan peninsular region and some parts of central India (Figure 5.2), P. indoischeri is restricted to southern India (Ganesan and Shetty, 2004). These contour maps depicting the relative richness of the species on the Indian landscape clearly are extremely useful both in guiding conservation strategies and in planning the sustained utilization of the resources (Ravikanth et al., 2001, 2002). Besides the species richness maps described, Ganeshaiah and U. Shaanker, 2007 also developed species-speciic maps, especially for those in trade. For example, speciic maps have been developed for P. emblica, one of the most important species in trade (Figure 5.2). These maps, with grids resolution of 6.25 × 6.25 km, provide Genetic Resources of Phyllanthus in Southern India 107 precise spatial information on the resource stock of the species. Further, when overlaid with other parameters, such as demand levels and production data (supply), the species-speciic maps can be used to develop a user-friendly resource management system that can advise on a variety of issues, including the optimal levels of harvesting, rotation schedules of harvesting over the distributional range of the species, and inally providing a dynamic inventory of the resource (Uma Shaanker et al., 2004). 5.3 IDENTIFICATION OF GENETIC HOT SPOTS OF ECONOMICALLY IMPORTANT PHYLLANTHUS SPECIES One of the major limitations in planning the effective utilization and conservation of genetic resources of medicinal plant species is the lack of critical information on the spatial distribution of genetic variability of the species. Spatially explicit analysis of the genetic variability of the species could aid in (1) identiication of genetic hot spots of the species, (2) appropriately designing germplasm collections, and (3) deciding on what and where to conserve. Unfortunately for many medicinally important species, there is a severe dearth of information with respect to the spatial distribution of variability. Here, we briely review attempts that have been carried out to identify the genetic hot spots for the two most important medicinal plants in the genus Phyllanthus. 5.3.1 PHYLLANTHUS EMBLICA Phyllanthus emblica L. constitutes one of the important medicinal plants in the Phyllanthus genus. Phyllanthus emblica is a medium-size tree, and all its parts are used for various medicinal applications. It is widely distributed in the deciduous forests in southern India. The tree is one of the most important nontimber forest product species and is a source of livelihood for scores of forest-dwelling communities in India (Ganesan and Shetty, 2004). In recent years, because of the increase in the demand for herbal products, there has been intense extraction of the fruits. Destructive harvesting practices could have a detrimental effect on the populations of P. emblica (Sinha and Bawa, 2002). This consequently can reduce the regeneration of the species, leading to a loss of genetic variability. Uma Shaanker and Ganeshaiah (1997) assessed the genetic diversity of P. emblica populations in southern India spread across three states, namely, Karnataka, Tamil Nadu, and Kerala. These populations are geographically isolated and represent diverse biogeographic strata from the three states. Based on isozyme analysis of six enzyme systems, the genetic variability of seven populations was assessed. Populations in southern Kerala had the highest allelic diversity as well as allele richness compared to the other populations (Uma Shaanker and Ganeshaiah, 1997). The relative abundance of most of the alleles was also found to be high in the Kerala populations. Based on these genetic parameters, Uma Shaanker and Ganeshaiah (1997) argued that the populations in Kerala could represent a potential hot spot of genetic variability of P. emblica. 108 5.3.2 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications PHYLLANTHUS AMARUS Phyllanthus amarus is traditionally used in the treatment of bile and urinary conditions, hepatitis, lu, cold, jaundice, liver cancer, tuberculosis, diabetes, hypertension, pains, and other maladies (Amaechina and Omogbai, 2007). Phyllanthus amarus is reported to contain lignans such as phyllanthin and hypophyllanthin, alkaloids, and lavonoids such as quercetin (Santos et al., 1995). Phyllanthus amarus is traded as a raw herbal drug and exported for various medicinal formulations for the treatment of liver disorders (Kamble et al., 2008). However, most of the raw trade of P. amarus involves widespread collection of the individuals from the wild. Jain et al. (2003) assessed the molecular diversity of P. amarus across India using RAPD (random ampliied polymorphic DNA) markers. The genetic variability was assessed across 33 locations covering the states of Tamil Nadu, Karnataka, Maharashtra, Gujarat, Assam, West Bengal, Tripura, Uttar Pradesh, Punjab, and Haryana. Intrapopulation variation was larger in accessions from southern India compared to other parts of the country (Jain et al., 2003). In another study, Murthy (2004) assessed the population genetic variability of P. amarus in three southern states of India (Karnataka, Tamil Nadu, and Kerala) and a union territory (Pondicherry). The population genetic parameters were assessed using 10 intersequence simple repeat (ISSR) primers. Kerala populations had the highest percentage of polymorphic loci, while the Karnataka populations had the least. The highest diversity was also found to be in the populations collected from Kerala. There was a clear genetic differentiation of populations based on their sites of origin. In summary, studies of both P. emblica and P. amarus suggested that Kerala populations in southern India are genetically most diverse. It is likely that species radiated and diverged from this region into the rest of the country. 5.4 IMPACT OF HARVESTING ON THE GENETIC VARIABILITY OF P. EMBLICA Phyllanthus species are among the most highly traded medicinal plant species in the country. Fruits of the species (such as P. emblica and P. indoischeri) and whole plants (in case of herbaceous species such as P. amarus, P. debilis, etc.) are extracted mostly from their natural populations. In recent years, because of resurgence of the herbal market globally, there has been an even further upsurge in the extraction of several medicinal plant species, including Phyllanthus, from their natural populations (Ved and Goraya, 2008; Ravikanth et al., 2009). In most cases, harvesting of the medicinal plants goes on unabated with few regulations on the extent and the nature of harvest. Consequently, such high extraction pressures could have a severe impact on the demographic and genetic proile of the populations. For example, a seemingly low-impact use, such as harvesting of Phyllanthus emblica fruits, may have a high long-term effect on populations, either because of the effect on seedling recruitment or because fruit collection involves pruning of branches or sometimes even tree felling (Figure 5.3; Padmini et al., 2001; Uma Shaanker et al., 1996). Among the possible impacts of harvesting, that on the genetic variability and structure of populations has been the least studied (Uma Shaanker et al., 2001; 109 Genetic Resources of Phyllanthus in Southern India 90 80 Percentage 70 60 50 40 30 20 10 0 Control Mild Disturbance levels High FIGURE 5.3 Percentage seedlings and saplings (<10 cm dbh (diameter at breast height) at the various disturbance levels at Biligiri Rangaswamy Temple Wildlife Sanctuary (dark columns) and Mudumalai Wildlife Sanctuary (light columns). (Adapted from Padmini, S., Nageswara Rao, M., Ganeshaiah, K.N., and Uma Shaanker, R. 2001. J. Tropical Forest Sci. 13: 297–310.) Young et al., 2000). Yet, as is well realized, the impact at the level of the gene is most important. Genetic impoverishment of populations due to indiscriminate and unregulated harvesting can lead to a spiraling of effects, including low regeneration, small population sizes, inbreeding and loss of genetic diversity through genetic drift, and inally the local extinction of populations. Surprisingly, except for a few studies investigating the effect of logging on the genetic diversity of trees, hardly any studies have addressed the impact of other anthropogenic pressures on the genetic diversity of trees (Burchert et al., 1997; Wickneswari and Boyle, 2000; Young et al., 2000). Padmini et al. (2001) and Uma Shaanker et al. (2001) examined the impact of harvesting of fruits of P. emblica at two forest sites in southern India along a gradient of harvesting pressures. Using isozyme markers, they examined if populations experiencing greater harvesting pressures actually differed in their genetic diversity and structure compared to populations that were less harvested or not harvested at all. Their results suggested that adult genetic diversity (as measured by percentage heterozygosity) was not affected by harvesting pressure. However, the overall genetic structure of the populations was signiicantly altered due to harvesting pressure; there was a signiicant genetic differentiation between the harvested and nonharvested populations. Alleles were differentially sensitive to harvesting pressures; frequency of a few alleles was adversely affected in the harvested population (Figure 5.4). While this might suggest that there could be a selective harvesting of plants and their fruits (e.g., of plants known to produce greater levels of ascorbic acid), Padmini et al. (2001) did not observe any evidence of selection at harvesting by local collectors. Besides these changes at the genetic level, Padmini et al. (2001) 110 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications 0.4 BR Hills Mudumalai PGI 2 Control – Highly Disturbed 0.3 PGI 2 6 PGDH2 0.2 0.1 I DH3 I DH1 6 PGDH1 0.0 6 PGDH2 –0.0 I DH3 –0.2 –0.3 I DH2 I DH1 I DH2 6 PGDH1 PGI 1 PGI 1 –0.4 –0.2 –0.1 0.0 0.1 0.2 0.3 Control – Mildly Disturbed FIGURE 5.4 Relation between differences in allele frequencies between control and mildly disturbed as well as control and highly disturbed populations of P. emblica from Biligiri Rangaswamy Temple (BRT) Wildlife Sanctuary and Mudumalai Wildlife Sanctuary (MWS). PGI, phosphogluco isomerase; IDH, isocitrate dehydrogenase; PGDH, 6-phosoglouconate dehydrogenase. (Adapted from Padmini, S., Nageswara Rao, M., Ganeshaiah, K.N., and Uma Shaanker, R. 2001. J. Tropical Forest Sci. 13: 297–310.) reported a signiicant reduction in the net regeneration of stems in the harvested compared to the unharvested populations. In summary, these results form one of the few quantitative examinations of the impacts of harvesting Phyllanthus fruits on their regeneration and genetic diversity. Both these impacts—a reduced regeneration and a change in the genetic structure of the population—can lead to a cascade of events that might endanger the local populations. The challenge lies in devising ways and methods that mitigate the harvesting-induced loss. For example, it should be possible to model the change in demographic features and genetic diversity as a function of harvesting intensities and advise on the optimal levels of harvesting. Further, the study could offer possibilities of evolving management protocols for the genetic enrichment of harvested populations to maintain the long-term productivity and adaptability of the species. 5.5 PHYLLANTHUS: TAXONOMIC INCONGRUITIES, SPECIES ADULTERATION, AND DNA BAR CODING Phyllanthus species trade in India is mired by two problems: (1) taxonomic confusion among closely related species (Elvin-Lewis et al., 2002; Ganeshaiah et al., 1998) Genetic Resources of Phyllanthus in Southern India 111 and (2) the fact that many species in trade share a common vernacular name (Srirama et al., 2010). Consequently, it is not uncommon to ind substantial species admixtures in trade samples (Dymock, 1883; Dymock et al., 1893; Kirtikar and Basu, 1975; Nadkarni, 1954; van Rhede, 1690; Ganeshaiah et al., 1998; Srirama et al., 2010; Khatoon et al., 2006). For example, although P. amarus is a predominant species in trade, it is often found mixed with several other Phyllanthus species, including P. fraternus and P. maderaspatensis (Khatoon et al., 2006; Ved and Goraya, 2008). Species admixtures may have signiicant implications on the quality and eficacy of the eventual phytomedicine made from these mixtures (Song et al., 2009). Khatoon et al. (2006) found signiicant differences in the metabolite proile between P. fraternus and P. maderaspatensis both occurring as admixtures in P. amarus trade samples. Phyllanthus amarus was the only species found to contain phyllanthin and hypophyllanthin, the two major compounds believed to be responsible for the hepatoprotective activity (Calixto et al., 1998). The admixtures of different species could lead to diluting the eficacy of the herbal drug. Using both morphotaxonomical keys and molecular techniques, Srirama et al. (2010) addressed species admixtures in southern India. Analyzing Phyllanthus raw herbal samples from 25 different shops from three southern Indian states (i.e., Kerala, Tamil Nadu, and Karnataka), they showed the presence of six different species of Phyllanthus (P. amarus, P. debilis, P. fraternus, P. urinaria, P. maderaspatensis, and P. kozhikodianus). Of the shops, 76% had P. amarus as the predominant species. The species identities were conirmed using species-speciic DNA bar codes developed using the chloroplast psbAtrnH. These bar codes could be used as a diagnostic key to authenticate Phyllanthus species in trade as well as to identify species admixtures (Table 5.2). 5.6 IMPLICATIONS FOR UTILIZATION AND CONSERVATION The genus Phyllanthus forms one of the most important nontimber forest products. In fact, a large number of forest-dwelling and forest fringe communities depend on P. emblica and P. indoischeri. A number of studies have reported that unsustainable and destructive harvesting would have an adverse impact on the regeneration of Phyllanthus species (Murali et al., 1996; Uma Shaanker et al., 1996; Sinha and Bawa, 2002; Ganesan, 2003). Studies by Padmini et al. (2001) have shown that the genetic resources are also adversely affected due to overharvesting. Thus, unless adequate measures are taken to protect the existing populations, the genetic resources of these species will be irreversibly lost. In recent years, there have been efforts to comprehensively address the sustainable utilization and conservation of Phyllanthus genetic resources. Domestication of the Phyllanthus species and maintaining in situ gardens would help in the long-term conservation of the economically important species. In particular, Uma Shaanker and Ganeshaiah (1997) proposed a new model, namely, the forest gene bank model, to address the long-term conservation of Phyllanthus species. In this model, a set of source (gene or allele donor) populations is identiied from which genetic resources (through either pollen or seed) are donated to recipient “sink” populations (Figure 5.5). Sites that can serve as a “source” and those that serve as a “sink” to receive the gene pool have to be identiied. The choice of the source and sink populations is guided by several criteria, including the extent 112 Phyllanthus Species: Scientiic Evaluation and Medicinal Applications KARNATAKA Source Population B.R.T–HILLS THENMALAI " Transfer of seeds or pollen KOLLI HILLS Source Population TAMIL NADU Sink Population-Forest Gene Bank KERALA Transfer of seeds/pollen Source Population PECHIPARAI FIGURE 5.5 See color insert. Genetic diversity map of Phyllanthus emblica. The relative size of the circle indicates the levels of gene diversity. The arrows indicate the possible low of genes either through seeds or pollen to the sink population from the source population (see text for further explanation). (Adapted from Uma Shaanker, R., and Ganeshaiah, K.N. 1997. Curr. Sci. 73: 163–168.) and amount of genetic variation existing in the populations, the occurrence of rare alleles, threats to the population, levels of protection, extent and area of distribution etc. The sink population should ideally be located in the protected area, should be comprised of a higher population size, and should have a broad genetic base (Uma Shaanker et al., 2002). This model combines the virtues of both in situ and ex situ conservation techniques as this allows for the genetic diversity to “evolve” as it would in any other natural habitat of the species (Uma Shaanker et al., 2002). The forest gene bank model allows infusion of genetic material from source to sink population. This infusion encourages gene low among the small and fragmented populations, leading to the overall genetic enrichment of the species. Such genetically enriched populations could serve as repositories of the entire spectrum of genetic variability of the species. In the case of Phyllanthus emblica, Uma Shaanker and Ganeshaiah (1997) proposed that southern Indian populations (in Kerala) can be regarded as the sink into which genetic resources from other parts of southern India can be translocated. Alternatively, the populations in the state of Karnataka, which are located in a wildlife sanctuary (B.R. Hills) could be made into a sink, population and genes/alleles from other parts of southern India can be translocated (Figure 5.5). This would ensure the conservation and maintenance of the global gene pool of P. emblica. 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The in vitro activity of geraniin and 1,3,4,6-tetraO-galloyl-[beta]-d-glucose isolated from Phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection. J. Ethnopharmacol. 110: 555–558. Yeh, S.F., Hong, C.Y., Huang, Y.L., et al. 1993. Effect of an extract from Phyllanthus amarus on hepatitis B surface antigen gene expression in human hepatoma cells. Antiviral Res. 20: 185–192. Young, A., Boshier, D., and Boyle, T. (Eds.). Forest Conservation genetics: Principles and practice. CSIRO Publishing, Collingwood, Australia. FIGURE 5.1 Hypsographic view of Phyllanthus species richness in India. The data on the distribution of the species were obtained from diverse sources (monographs, etc.), and the latitude and longitude were assigned for each record and mapped. The density of the species in each grid of the size 10 km × 10 km was computed and the contours for the density obtained. Based on the contour data, the three-dimensional view was constructed using suitable GIS software. Source Population Transfer of seeds or pollen Source Population Sink Population- Forest Gene bank Transfer of seeds/pollen Source Population FIGURE 5.5 Genetic diversity map of Phyllanthus emblica. The relative size of the circle indicates the levels of gene diversity. The arrows indicate the possible low of genes either through seeds or pollen to the sink population from the source population (see text for further explanation). (Adapted from Uma Shaanker, R., and Ganeshaiah, K.N. 1997. Curr. Sci. 73: 163–168.)