GEOGRAPHICAL DISTRIBUTION AND CONSERVATION OF A RARE MEDICINAL PLANT MUNRONIA PINNATA ( WALL . ) THEOB . ( MELIACEAE ) IN SRI LANKA

In the present study, distribution and abundance of Munronia pinnata (Wall.) Theob. in Sri Lanka were explored in 6 provinces, 7 districts, 68 Divisional Secretariat Divisions (DSD) and 395 Grama Niladari (GN) areas. Fifty three GN areas were identified as M. pinnata abundant areas. In 217 GN areas, the plant is found in small scale and in 65 GN areas it was rarely found. M. pinnata was not found in 8 DSDs. Ten new localities were found and three of them were in the wet zone. The highest diversity was found in Monaragala and Matale districts. Populations well adopted for a range of climatic conditions were observed in Madulla, Nilgala, Warakapola, Ritigala and Haldumulla. Monaragala, Wellawaya, Mathurata, Meemure and Kithulpe were identified as unique populations for conservation. Monaragala, Badulla and Matale appear to be the most suitable districts for commercial cultivation of M. pinnata. This is the first record of an extensive systematic survey on the distribution of M. pinnata in Sri Lanka. Introduction The Genus Munronia Wight. (Meliaceae), comprising 13-15 species, is naturally distributed in southern China, Vietnam, Myanmar, Java, Sri Lanka, India, Indonesia and the Philippines (Qi et al., 2003). Out of these, five species of Munronia are restricted to tropical Asia, and subtropical China, up to 1800 m and in Sri Lanka up to 700 m from the mean sea level (Dassanayake et al., 1995; Peng and Bartholomew, 2008). Munronia pinnata (Wall.) Theob. is a rare medicinal plant species (Dassanayake et al., 1995). According to the literature available, plants of M. pinnata with an array of variable phenotypic characters (3, 5, 7, 9 and 11 leaflets types) exist in various locations in Sri Lanka (Jayaweera, 1982; Dassanayake et al., 1995). According to Hooker (1874), M. pinnata was an abundant and widely distributed plant in Sri Lanka in early days. In Chinese and Sri Lankan traditional medicine, Munronia has been used since historic times for many ailments such as tuberculosis, cough, stomach-ache, sores, malaria, recurrent fever, dysentery and purification of blood (Jayaweera, 1982; Qi et al., 2003). Moreover, there are over 32 written recipes including ‘Sudarshana Churna’, ‘Chandraprabha watee’ and ‘Denimba debatu adee kashaya’ in Sri Lankan Ayurvedic *Corresponding author. Email: dharma@iti.lk Herbal Technology Section, Industrial Technology Institute, 363, Bauddhaloka Mawatha, Colombo 7, Sri Lanka. 40 DHARMADASA et al. Pharmacopeia, in which the entire plant of M. pinnata is used as the major ingredient of preparations used for above ailments (Anonymous, 1979). On the other hand M. pinnata is one of the most expensive plant materials (US$ 50-110/kg) used in traditional systems of medicine in Sri Lanka. Further almost all raw material requirements are obtaining from natural habitats due to lack of systematic cultivations, lack of information on cultivation and processing and lack of sufficient planting materials to establish commercial cultivation in Sri Lanka as well as elsewhere. Therefore, there is a tremendous pressure on this rare plant which might lead to extinction due to over exploitation. Recording of existing populations in different locations with their abundance, identifying potential areas and morphotypes for cultivation, recognizing population/s for conservation and sustainable use of this valuable medicinal plant in traditional and Ayurveda medicine seem to be timely important issues. These data will certainly provide information needed to establish cultivations for sustainable use of M. pinnata in Sri Lanka. Information available on the distribution is very old and the most recent record is also more than 20 years old while some evidence are more than 100 years old (Dassanayake et al., 1995). Therefore, attempts were made to investigate the present distribution and the abundance of M. pinnata in different localities in physically accessible areas of the country. Materials and Methods Island wide survey on the distribution: For administrative purposes, the country is divided into nine provinces and 26 districts. Each district has 3-7 Divisional Secretariat Divisions (DSD) and each DSD has many Gramaniladari divisions (GN). The GN division is the smallest administrative division in Sri Lanka. The present study was carried out during 2004-2007. The systematic survey comprises four stages as collecting information from available literature, collecting data from GN divisions using a questionnaire, visiting areas of the country where M. pinnata is available (guided by available literature) and gathering information by personnel communication with traditional practitioners of ayurveda. Collecting information from available literature: A literature survey was carried out on the distribution of M. pinnata in Sri Lanka. Information was collected from literatures and databases, herbarium specimens deposited at Royal Botanical Garden Peradeniya, Sri Lanka and personal communication with personnel involved in traditional medical practices. Collecting data from GN divisions: A systematic survey was carried out covering all DSDs of the country. A questionnaire for this survey was prepared and evaluated by trying out with 4-5 persons before giving the questionnaire to Gramaniladaris. The questionnaire was distributed among traditional ayurvedic doctors, cultivators and collectors of medicinal plants in each of the GN divisions through the government DISTRIBUTION AND CONSERVATION OF MUNRONIA PINNATA 41 administrative officer (“Gramaniladari”) of the area. Completed questioners were collected through the same way and information was compiled. Field visits: Field surveys were carried out by visiting various places, which were selected based on available literature and information collected through the questionnaire survey in different ecological regions of the country. Selected areas for field visits are given in Map 1. Distribution of M. pinnata as found in the present study was compared with data available in the literature (Appendix B) to mark populations for conservation as well as for places for cultivation. Collection and maintenance of different populations: Out of the 16 locations listed in Table 3, plants from 13 locations were collected for the present study. Ten to twenty plants were collected from each location depending on the availability of plants. When there were only a few plants in a particular location, neighboring areas were searched for more plants without disturbing the existing population. Plants collected were brought to Industrial Technology Institute, Sri Lanka and potted in plastic or clay pots filled with a mixture of topsoil 1: compost 2: sand 1. Each sample was labeled using the respective notation and was maintained in the greenhouse for 5 years. Close observations were made during that period on the survival, growth performance, flowering and fruiting of each morphotype under normal day light and temperature 27C ± 2. Collection of ecological data: The altitude, latitude and longitude of each population were measured using Global Positioning System (ETrex Vista Garmin Model). Soil samples were collected from each location using a soil auger to measure the soil pH. The agro-ecological region and rainfall data were adopted from Panabokke and Kannangara (1996). Determination of the stomatal index: Stomatal index was calculated as described by Trease and Evance (2002) with slight modifications. End leaflet pieces of each population (5 × 5 mm) other than from extreme margin and midrib were warmed up in saturated chloral hydrate solution until they become transparent. Subsequently these were strained with 1% safranin in 50% ethanol and were made into temporary mounts using glycerin. Slides were examined under compound light microscope fitted with an eye piece micrometer. Counts were made of the number of epidermal cells and of stomata (two guard cells and ostiole being considered as a single unit) within the square grid. Successive adjacent fields were examined until about 400 cells have been counted. The stomatal index value for each population was calculated using standard formula given by Trease and Evance (2002).


Introduction
The Genus Munronia Wight.(Meliaceae), comprising 13-15 species, is naturally distributed in southern China, Vietnam, Myanmar, Java, Sri Lanka, India, Indonesia and the Philippines (Qi et al., 2003).Out of these, five species of Munronia are restricted to tropical Asia, and subtropical China, up to 1800 m and in Sri Lanka up to 700 m from the mean sea level (Dassanayake et al., 1995;Peng and Bartholomew, 2008).Munronia pinnata (Wall.)Theob. is a rare medicinal plant species (Dassanayake et al., 1995).According to the literature available, plants of M. pinnata with an array of variable phenotypic characters (3,5,7,9 and 11 leaflets types) exist in various locations in Sri Lanka (Jayaweera, 1982;Dassanayake et al., 1995).According to Hooker (1874), M. pinnata was an abundant and widely distributed plant in Sri Lanka in early days.
In Chinese and Sri Lankan traditional medicine, Munronia has been used since historic times for many ailments such as tuberculosis, cough, stomach-ache, sores, malaria, recurrent fever, dysentery and purification of blood (Jayaweera, 1982;Qi et al., 2003).Moreover, there are over 32 written recipes including 'Sudarshana Churna', 'Chandraprabha watee' and 'Denimba debatu adee kashaya' in Sri Lankan Ayurvedic Pharmacopeia, in which the entire plant of M. pinnata is used as the major ingredient of preparations used for above ailments (Anonymous, 1979).On the other hand M. pinnata is one of the most expensive plant materials (US$ 50-110/kg) used in traditional systems of medicine in Sri Lanka.Further almost all raw material requirements are obtaining from natural habitats due to lack of systematic cultivations, lack of information on cultivation and processing and lack of sufficient planting materials to establish commercial cultivation in Sri Lanka as well as elsewhere.Therefore, there is a tremendous pressure on this rare plant which might lead to extinction due to over exploitation.
Recording of existing populations in different locations with their abundance, identifying potential areas and morphotypes for cultivation, recognizing population/s for conservation and sustainable use of this valuable medicinal plant in traditional and Ayurveda medicine seem to be timely important issues.These data will certainly provide information needed to establish cultivations for sustainable use of M. pinnata in Sri Lanka.Information available on the distribution is very old and the most recent record is also more than 20 years old while some evidence are more than 100 years old (Dassanayake et al., 1995).Therefore, attempts were made to investigate the present distribution and the abundance of M. pinnata in different localities in physically accessible areas of the country.

Materials and Methods
Island wide survey on the distribution: For administrative purposes, the country is divided into nine provinces and 26 districts.Each district has 3-7 Divisional Secretariat Divisions (DSD) and each DSD has many Gramaniladari divisions (GN).The GN division is the smallest administrative division in Sri Lanka.The present study was carried out during 2004-2007.The systematic survey comprises four stages as collecting information from available literature, collecting data from GN divisions using a questionnaire, visiting areas of the country where M. pinnata is available (guided by available literature) and gathering information by personnel communication with traditional practitioners of ayurveda.
Collecting information from available literature: A literature survey was carried out on the distribution of M. pinnata in Sri Lanka.Information was collected from literatures and databases, herbarium specimens deposited at Royal Botanical Garden Peradeniya, Sri Lanka and personal communication with personnel involved in traditional medical practices.
Collecting data from GN divisions: A systematic survey was carried out covering all DSDs of the country.A questionnaire for this survey was prepared and evaluated by trying out with 4-5 persons before giving the questionnaire to Gramaniladaris.The questionnaire was distributed among traditional ayurvedic doctors, cultivators and collectors of medicinal plants in each of the GN divisions through the government administrative officer ("Gramaniladari") of the area.Completed questioners were collected through the same way and information was compiled.
Field visits: Field surveys were carried out by visiting various places, which were selected based on available literature and information collected through the questionnaire survey in different ecological regions of the country.Selected areas for field visits are given in Map 1. Distribution of M. pinnata as found in the present study was compared with data available in the literature (Appendix B) to mark populations for conservation as well as for places for cultivation.
Collection and maintenance of different populations: Out of the 16 locations listed in Table 3, plants from 13 locations were collected for the present study.Ten to twenty plants were collected from each location depending on the availability of plants.When there were only a few plants in a particular location, neighboring areas were searched for more plants without disturbing the existing population.Plants collected were brought to Industrial Technology Institute, Sri Lanka and potted in plastic or clay pots filled with a mixture of topsoil 1: compost 2: sand 1.Each sample was labeled using the respective notation and was maintained in the greenhouse for 5 years.Close observations were made during that period on the survival, growth performance, flowering and fruiting of each morphotype under normal day light and temperature 27 o C ± 2.
Collection of ecological data: The altitude, latitude and longitude of each population were measured using Global Positioning System (ETrex Vista Garmin Model).Soil samples were collected from each location using a soil auger to measure the soil pH.The agro-ecological region and rainfall data were adopted from Panabokke and Kannangara (1996).
Determination of the stomatal index: Stomatal index was calculated as described by Trease and Evance (2002) with slight modifications.End leaflet pieces of each population (5 × 5 mm) other than from extreme margin and midrib were warmed up in saturated chloral hydrate solution until they become transparent.Subsequently these were strained with 1% safranin in 50% ethanol and were made into temporary mounts using glycerin.Slides were examined under compound light microscope fitted with an eye piece micrometer.Counts were made of the number of epidermal cells and of stomata (two guard cells and ostiole being considered as a single unit) within the square grid.Successive adjacent fields were examined until about 400 cells have been counted.The stomatal index value for each population was calculated using standard formula given by Trease and Evance (2002).

Stomatal index =
Where S = the number of stomata in a given area of leaf, E= the number of epidermal cells (including trichomes) in the same area of leaf.

S × 100 E + S
Data analysis: The range of each variable/character was sub-divided and ranked, and then a numerical value was given to each level (Table 1).Using these numerical values, a data table (Table 2) for cluster analysis was prepared.Cluster analysis was done by using SPSS Version 10.Clusters were generated following Unweighted Pair Group Method with Arithmetic Means (UPGMA), which is an agglomerative clustering method.

Results and Discussion
Island wide survey carried out using a questionnaire revealed that out of the 68 DSDs considered, M. pinnata could be naturally found in only 38 DSD divisions in Sri Lanka.M. pinnata was abundant in 395 GN divisions.In 217 GN Divisions it was found in small scale and in 65 it was found very rarely.The 38 DSDs are shown in Appendix A and list of places where M. pinnata had been recorded in literature as cited in the Handbook of Flora of Ceylon (Dassanayake et al., 1995)    morphotypes was found in Monaragala district, which comprises of four populations (two types of 3-leaflets, 7-leaflets and 9/11-leaflets types).
Normal growth was observed in all populations under greenhouse conditions.Flowering was rare and even when occurred, no fruiting was observed in five out of 13 populations under greenhouse conditions (Table 4).Flowering is one of the phenological processes influenced by external environmental factors especially temperature.Therefore difficulty observed in flowering in populations of KP and MR is quite acceptable as they were collected from Nuwaraeliya district which is in the hill country of Sri Lanka where the average temperature is around 20 o C. Furthermore, day length fluctuation is also higher in this area than that of the low laying areas of the country, where these plants were acclimatized in greenhouse.It indicates that conservation of these populations demands in situ conservation.If not they have to be grown in greenhouses under carefully controlled conditions.The morphotype collected from Meemure (Matale district) produced some flowers, but did not produce fruits.Since Meemure is isolated and surrounded from huge mountains it has its own microclimatic conditions.Therefore, this population may have adapted to these climatic conditions especially for flowering and fruiting.On the other hand population collected from Ritigala performed well producing flowers and fruits under normal greenhouse condition in Colombo.Although Ritigala is separated from wet and intermediate zones by dry plains, its isolation and high elevation has produced a unique climate with wet and intermediate characteristics.
Hence Ritigala provides platform for 410 taxa of lower and higher plants.It shows that this population could easily be cultivated in areas with wet and intermediate characteristics.Geographical isolation must have restricted RG population to that area.Some populations collected from Monaragala and Matale performed well under climatic conditions of greenhouse in Colombo, while three of them namely, Monaragala, Wellawaya and Meemure did not.These populations bear 7-9 and 11-leaflets and are not common in other areas indicating that they may be genetically adapted to grow in these areas and their restricted distribution is not merely due to geographical isolation.In situ conservation seems to be the best method for these populations, but when ecological conditions were analyzed, these three clustered with the rest of the populations collected from Matale and Monaragala.It shows that there is a possibility for cultivation of these populations in other localities.
According to the analysis of ecological data (Fig. 1), M. pinnata growing in Sri Lanka could be separated into three main clusters such as 1.MD, NU, NG, MG, HM, WW, MM; 2. WP, PW, KPT; 3. RG, KP and MR.This indicates that MD, NU, NG, MG, HM, WW and MM require approximately the same climatic conditions compared to the other populations.These include plants collected from Badulla, Matale and Monaragala districts.This group comprises of populations varying from 3, 5, 7 9 and 11 leaflet types.Flower and fruit setting of MG, WW and MM populations were very unsatisfactory under greenhouse conditions.These findings are very important in the conservation point of view as it shows the possibility of establishing large scale cultivation in areas where these populations do not exist naturally at present.Populations collected from Nuwara Eliya (KP and MR) formed a separate cluster which was collected from hilly areas with a cold climate.Their failure in producing flowers and fruits under low country conditions (Temperature around 27 o C ± 2) shows that they can be cultivated only in the areas with Fig. 1.A dendrogramme of ecological relationship of 13 M. pinnata populations (For abbreviations see Table 3).
similar environmental conditions.In the conservation point of view, they need special attention for survival.Population collected from Ritigala got separated from all three clusters.This is acceptable as the microclimate in this area is very specific and quite different from those of other locations.According to the present study, M. pinnata could be grown within a considerable range of ecological conditions including all three agroecological regions in the country and within a considerable range of altitude (30-1000 m).Moreover, studies on stomatal index of different populations did not show clear correlation with environmental factors or number of leaflets of different populations.Our findings are in agreement with the previous reports (Dassanayake et al., 1995;Peng and Bartholomew, 2008), who pointed out that M. pinnata, was grown up to 700 m from mean sea level in Sri Lanka and up to 1800 m in China.Furthermore, Qi et al. (2003) and Peng and Bartholomew (2008) reported that Munronia species can grow in heights varying from 200 m to 1800 m from mean sea level in China.Findings of the present study are in agreement with the previous work.
In order to conserve the medicinal plants ethnobotanical surveys are very useful (Chellaiah et al., 2006;Bekalo et al., 2009).The present study also highlights his important issue.The present study revealed that this rare and valuable medicinal plant could easily be cultivated in different parts of Sri Lanka under various climatic conditions.This opens up an avenue to establish large growing areas of M. pinnata in places where it has not been reported or cultivated before.This study was unable to find, M. pinnata in some of the localities reported earlier (Dassanayake et al., 1995).Several reasons including urbanization, clearing forests for cultivation, natural disasters such as landslides and over-exploitation might have exerted unfavorable impacts on these populations, making them very rare or extinct in those localities.

Conclusion
This is the first record of an extensive systematic survey on the distribution of M. pinnata in Sri Lanka finding 10 new localities including three in the wet zone.It shows that this plant could be cultivated in the wet zone though it has been previously recorded only from the dry and intermediate zones.Matale, Badulla and Monaragala seem to be the most suitable districts to establish large scale cultivations of M. pinnata.Populations collected from Ritigala (RG) could easily be cultivated even in Colombo.This is quite promising as it was identified as a unique population for conservation with regard to morphology and molecular characters (unpublished data).
Six populations i.e.MD (Madulla), NU (Naula), NG (Nilgala), WP (Warakapola), RG (Ritigala) and HM (Haldummulla), were grown well, under a range of climatic conditions producing large number of flowers and fruits.However the ability to produce flowers and seeds of the morphotypes KP, MR, MG, WW and MM are very low and hence there should be a special conservation plan for them particularly, otherwise they might be extinct from the country soon.

Table 1 . Parameters used in numerical analysis and ranking of their data (the ranks are given in parenthesis).
IM = Mid country intermediate zone; IL= Low country intermediate zone; WL= Low country wet zone; DL= Low country dry zone; RB/ RBE= Reddish brown/ Reddish brown earth; RYP Red yellow podzolic soils.

Table 3 .
is shown in Appendix B. Results of the present study on distribution and abundance of M. pinnata is presented in Different populations collected from different locations are shown in Plate 1. Areas recorded in the present study together with those recorded in literature are presented in Map 1. Presence/absence of flowering and fruiting of 13 populations are presented in Table4.

Table 3 . Distribution and abundance of M. pinnata (Based on the present study)
*Abundance was estimated visually with relevant to the size of the populations A-abundant, B -only a very few plants available; **New localities found in the present study; NC-North central, NW-North western