Medicinal plants of Dagala region in Bhutan: their diversity, distribution, uses and economic potential

Background The traditional g.so-ba-rig-pa hospitals in Bhutan uses more than 100 polyingredient medicines that are manufactured by the Menjong Sorig Pharmaceuticals (MSP). The MSP has been collecting medicinal plants from Lingzhi region for about 48 years and therefore the ecological pressure on these plants have increased. It is MSP’s top priority to identify an alternative collection site to ease the problem. Therefore, this study was carried out to determine whether Dagala region could potentially be an alternative collection site for MSP. Methods First the multidisciplinary research team generated a tentative plant list by reviewing a body of ancient g.so-ba-rig-pa literature, current formulations, and the MSP medicinal plants inventory documents. Second, the research team visited the study areas in Dagala region for spot identification of medicinal plants. Third, we confirmed our traditional and botanical identification by crosschecking the descriptions with the series of books on traditional texts, Flora of Bhutan, scientific papers on medicinal plants, and the plant databases. Results We have identified 100 species of high altitude medicinal plants from Dagala region. Of these, 24 species grow abundantly, 29 species grow in moderate numbers and 47 species were scarce. More than 85 species belonged to the herbaceous life form and 51 of them are used as a whole plant. A total of 68 species grow in between 4000 and 4999 meter above sea level. These 100 medicinal plants represented 39 different families and 80 genera and the maximum number of plants belonged to the family Asteraceae. Of 60 species that are currently used for formulating medicines at MSP, 16 species have economic importance with potential for commercial collection. Out of seven areas covered by the survey, Kipchen hosted maximum number of medicinal plants (21 species). Conclusions Our survey identified 100 medicinal plants from Dagala region and of these, 16 species has economic potential that could benefit both MSP and Dagala communities. It is feasible to establish an alternative medicinal plants collection center in Dagala Gewog.


Background
Bhutan has a total of 20 Dzongkhags (districts) and 205 Gewogs (blocks). Dagala, also known as Dakarla, is one of the eight Gewogs under Thimphu Dzongkhag with total land coverage of 85 km 2 , five chiwogs (villages), 178 households and 814 inhabitants [1] (Fig. 1). It has huge rangelands and supports livestock including the Yaks. People inhabiting these areas are known as Byjops or Jops and live in the scattered settlements at widely differing altitudes as yak herders or looking after the cattle. Yaks give milk, butter, cheese, buttermilk, chugo (dried hardened cheese consumed as snacks), tough hairy wool, meat and dung. Its tail is traded as a spiritual item, decoration peace and house duster. Its hairy wool is used for weaving clothes, bags, tents, ropes and handicraft items. Its dung serves as manure and is often used as a substitute for firewood. They barter or sell their wool, yak's meat (expensive than normal beef ) and other dairy products for food grains, sugar, tea leaf, clothing and other modern merchandise from the city.
Topographically, Dagala falls within Wang River Basin (WRB) and is known for its beautiful snow-capped mountains, ridges, alpine pastureland (tsamdro), beautiful lakes, rivers and pristine environment that are still intact in its natural forms. It is said that there exist countless mountains that connects Lhasa (Tibet/China) in the north and India in the south [2,3] and these mountains are believed to contain as many as 108 lakes [2]. The lakes supports fish and other lake ecosystems and also serve as the perennial river sources, which attracts rich fauna and flora downstream. There were few unsubstantiated claim about the lush growth of medicinal plants in Dagala region. It is possible that this claim are true as it share similar topography, vegetation and climatic conditions as that of Lingzhi region in the northern part of the country where medicinal plants thrive plentifully. Lingzhi region has been the medicinal plants collection centre for the Ministry of Health (MoH) in Bhutan for more than 48 years. The MoH, under the instruction of the third King of Bhutan, Jigme Dorji Wangchuck, integrated the traditional g.so-ba-rigpa (pronounced as So-wa-rig-pa) medicine with the mainstream modern health care system in 1967 [4,5]. The Bhutanese g.so-ba-rig-pa medicine (BSM) belong to the larger corpus of a scholarly Tibetan medicine, which is in practice wide across the world [6]. In ancient times, g.so-ba-rig-pa medicine was only practiced in Tibet, Bhutan, Nepal (Dolpo and Mustang), Mongolia, and India (Sikkim and Ladakh).
The g.so-ba-rig-pa medicine has undergone constant process of evolution, adaptation, scientific scrutiny and quality control system ever since its integration with modern health care system in 1967 [4,5,[7][8][9][10][11][12][13][14][15][16][17][18]. The BSM has evolved into a more sophisticated system with three functional organizations: a) National Institute of Traditional Medicine -recently transformed into a Faculty of Traditional Medicine (FTM) under the Khesar Gyalpo University of Medical Sciences, b) National Fig. 1 Map of Bhutan and Dagala Gewog (shaded white and labeled with white arrow) [21] Traditional Medicine Hospital -recently converted to a Department of Traditional Medicine DTM) under MoH, and c) Pharmaceutical and Research Unit -currently renamed as Menjong Sorig Pharmaceuticals under MoH. While the FTM provides university level training and education to the students, the DTM oversees the provision of free traditional health care services to the people through 58 traditional medical hospitals/units in the country, which are established alongside modern hospitals and Basic Health Units. After completing the Drungtsho course (5 years duration) and Menpa course (3 years duration) from the FTM, the students are recruited by the Royal Civil Service Commission of Bhutan to serve in the traditional medicine hospitals/units and the Menjong Sorig Pharmaceuticals (MSP).
The MSP manufactures more than 100 different polyingredient medicinal formulations and supplies them to the traditional medicine hospitals and units across the country [19]. The polyingredient medicinal formulations are prepared into different dosage forms as powder, capsules, pills, tablets, ointments and decoctions. The raw materials (mostly medicinal plants) for preparing these formulations are either collected within Bhutan (mostly from Lingzhi region) or imported from India. The medicinal plants, which grow in the higher elevation of alpine mountains (>2000 meters above sea level) including that from Lingzhi region, are known as the High Altitude Medicinal Plants (HAMP) and the others that grow in the temperate and tropical environment are called the Low Altitude Medicinal Plants (LAMP) [20]. Due to persistent collections of HAMP from Lingzhi region for more than 48 years, the pressure on medicinal plants has increased significantly over the recent years. Therefore, identifying an alternative medicinal plants collection site
We have chosen Dagala as our study area for the following reasons: a) MSP is in dire need of an alternative collection centre for HAMP and Dagala presented as a viable alternative sources of medicinal plants. It is located in a close proximity to MSP and share similar agro-climatic features to that of Lingzhi region (current medicinal plants collection sites for MSP), b) there was unsubstantiated/anecdotal claim about the lush medicinal plants growth in the region, c) no medicinal plants survey has been conducted in this region, and d) Dagala Jops are poor and their engagement in the medicinal plants collection program could help them generate cash income. We have covered seven main areas under Dagala Gewog and they are: There is no motor road in all these study sites and takes many days to reach there on foot. From some halt points, the research team had to walk or trek minimum of 1-2 days to find some of the listed medicinal plants.

Study design, survey methods and team
Our study was to survey and identify g.so.ba-rig-pa high altitude medicinal plants that grow in Dagala region. It is a literature-guided observational and plant identification study. We have used the study design described by Wangchuk et al. [20]. Both traditional and botanical identification methods were used during the plant survey. In order to find out what types and numbers of medicinal plants are used in the g.so-ba-rig-pa medicine, we have reviewed the bodies of literature including Shelgong shel-phreng (ancient textbook on g.so-ba-rig-pa medical system) [24], the current Traditional Medicine Formulary of Bhutan [19], and the medicinal plants described by Wangchuk [25] and Ugyen [26]. We have freely listed down all the HAMP that are being described in the literature and that are currently in use at MSP. MSP consider plants that grow above 2000 masl as HAMP (sometime referred to as high elevation medicinal plants (HEMP)). The plant sample size as per se was not an issue in this study, as the inventory included all the medicinal plants known to grow in Dagala region. Local Jops were not interviewed as they lacked knowledge on medicinal plants used by MSP. Second, the field trip was made to the study area for field observations, photographing, herbarium specimen collections and other data collections. The medicinal plants were surveyed using the convenience sampling methods. Using g.so-ba-rig-pa names and g.so-ba-rig-pa plant characterization, we did a spot identification of the medicinal plants while in the field. We have also used few Tibetan textbooks [27,28] to assist our g.so-ba-rig-pa plant identifications. The location of specimen collecting areas and the altitudes were recorded using a hand-held Garmin Etrex GPS-Altimeter unit. Other standard data such as vegetation, habitat description, other medicinal plants present, local plant name, locality name (if known) and species abundance were also recorded at each field site. Herbarium specimens were pressed, prepared and preserved at the Menjong Sorig Pharmaceuticals in Bhutan.
After authenticating and confirming the g.so-ba-rig-pa names, botanical identification of the medicinal plants were carried out in the field and confirmed either at the base-camp or upon returning to MSP based on the herbarium specimens, plant photographs and other recorded field information including life form, parts collected, altitude and the habitat. To help us and confirm our fieldbased botanical identification, we used series of original publications on Flora of Bhutan [29][30][31][32][33][34][35][36][37] and other Himalayan plants publications [38,39] to authenticate our botanical identifications and naming. The plant nomenclature was also confirmed through 'The Plant List' [40], eFloras [41], and TROPICOS [42].
The research team was comprised of one Drungtsho (traditional g.so-ba-rig-pa physician from the National Traditional Medicine Hospital), one Senior Drungtsho Consultant (working as a chief consultant at MSP), one senior researcher (with the background on botany and plant chemistry from MSP), and one Byjop (local inhabitant of Dagala region) who served us as a local guide and informant for the study areas.

Data management, criteria setting and analysis
Traditional method of plant identification is based on physical description and the organoleptic observations such as taste, odor and the color. Each plant species were scored for their status as 'abundant' , 'moderate' and 'rare'. The plants that were found less than 10 counts or citations in the study areas (only covered by our survey) were scored as rare or available in limited number. Those plants with 10-50 counts/citations in the area at the time of the survey were scored as moderately available and those with more than 50 counts were considered abundantly available.
Live specimens were collected and pressed on the way and the halt points. The photographs of live medicinal plants were also taken during the survey. The altitude and name of the places where the medicinal plants grew were recorded in the herbarium sheet or the notebook. The type of habitat for the particular species of a medicinal plant was also observed during the survey. In order to obtain lead information on some medicinal plants, few Yak herders from each halt points were casually asked if they have come across the medicinal plants in their areas by showing the plant photos of our interest.
All the information was recorded in the herbarium sheet or in the field workbook, which remained protected from rain and other damages. The information gathered by the research team during the survey was entered into MS excel sheet, analyzed and then the data were represented using bar graph. The analysis was grouped into five categories: family diversity, plant distribution by locality and altitude, plant status and the parts used. All the medicinal plants identified in the present study were ascribed their Bhutanese g.so-ba-rig-pa name (written in transliteration), botanical name, family, part used and ethnomedical uses. The information was maintained with the Research and Development Section of the MSP and a report was shared with the World Health Organization.

Study limitations
Some faraway places were not covered in this study due to the short data collection timeframe and the budget.
Places like Yakla, where Dactylorhiza hatagirea and Nardostachys grandiflora were reported to grow plentifully could not be covered under this study.

Habitat and diversity of medicinal plants
While Dagala Jops were aware of their places, we found that they were unfamiliar with the medicinal plants that grow in their forests and rangelands. The study areas had a gradient or rising vegetation with conifer forest giving way to receding tree line, rhododendron shrubberies, alpine grasslands and many lakes as we move towards higher elevations of the study areas (Fig. 2). This gradient vegetation supported various fauna and flora. Its conifer forests and Rhododendron shrubberies provides shelters to many edible mushrooms including Sangay Shamu or Matsutake (Tricholoma matsutake), and its alpine meadows are home to Cordyceps sinensiseven though we failed to source them at the time of study in August.
The areas we visited during the study period were found to host diverse range of medicinal plants belonging to at least seven habits or life forms. We have identified a total of 100 species from the seven localities of the Dagala Gewog (Table 1).

Current status and the availability pattern of medicinal plants
The study identified 100 medicinal plants from Dagala Gewog and 60 of them (current sourcing is from Lingzhi) are currently in use at Menjong Sorig Pharmaceuticals. Other 40 species were described in the traditional text. Upon segregation by their availability status as abundance, moderate and rare or less common (criteria described in the methods), 24 species fell within the category of abundance, 29 species under moderate and 47 species under rare or less common group (Fig. 3).
While the less commonly found species are not advisable to collect, those species that are found in abundance and in moderate distribution across the seven study sites can be collected on an annual basis. Of the 24 species that are abundantly found in Dagala Gewog, 16 of them are in current use at Menjong Sorig Pharmaceuticals ( Table 1).

Diversity of life forms or habit of medicinal plants
The 100 medicinal plants that we have identified from the study areas fell within seven habit groups as epiphytic, herb, lichen, parasitic, shrub, tree and vine ( Fig. 4). Majority of the plants (85 species) belonged to the herbaceous life form. Nine plant species belonged to shrub group.
While lichens made 2 % of the medicinal plants that were identified in the study areas, the parasitic, epiphytic, tree, and vine life forms each made up 1 %. Only one tree species, Juniperus pseudo-sabina (Shug-pa) was used in the medicine and this plant is considered one of the most sought-after plants for making incense. The representative plant image of each life form is given in Fig. 5.     Boschniakia himalaica (Orobanchaceae) (Fig. 5e) was identified as a medicinal plant for the first time by our team (senior Drungtsho). The description of this plant can be found in the ancient traditional text but it is not currently used in the BTM formulation. Interestingly, B. himalaica is an achlorophyllic non-photosynthetic parasitic plant that grows on the roots of Rhododendrons. Since it cannot produce its own food due to lack of chlorophyll, the plant depend totally on the host (Rhododendrons) for its nourishment. The plant is similar to Squawroot (Conopholis americana) (also called "cancer root" or "bear cone"), which grows on a parasitic connection to the roots of oak, and also beech host trees that are found throughout eastern part of North America.

Plant parts usage and their availability status
We found that the medicinal plants (100 species), which we identified from Dagala Gewog, are used in the Bhutanese g.so-ba-rig-pa medicines as a whole, stem, seed, root, leaf, fruit, flower, bark, aerial and mixed (for example, flower along with leaf ) (Fig. 6).
Majority of plants are used as a whole (entire plant) with 51 out of 100 (total identified) species falling in this usage category. This agrees with the life forms present in the alpine vegetation where majority of medicinal plants belong to herbaceous life form. Out of 51 species used as a whole, 29 of them belong to the category (status) of rare or less common species (Fig 6). Sixteen species are used for its roots, which is closely followed by flower with 14 species. Aerial, stem and fruits are the least used plant parts with only one species in each usage category. A plant that is used for its fruit falls within our categorization of the plant status as rare or less commonly available medicinal plants in Dagala Gewog.

Frequency of plant family and genera
The 100 medicinal plants that were identified from the study areas belonged to 39 families (Table 2) and 80 genera. Maximum number of plants belonged to the family Asteraceae (12 species) that is closely followed by Ranunculaceae (9 species). Gentianaceae, Rosaceae and Orobanchaceae contained 6 species each, which are closely followed by Lamiaceae, Plantaginaceae and Primulaceae with four species each. Apiaceae, Caryophyllaceae, Ericaceae, Papaveraceae Polygonaceae and Saxifragaceae all contained three species each. While six families contained two plant species each, 19 families were represented by only one species each (Table 2). Among the genera, Gentiana and Pedicularis ranked highest with each of them containing four species, which is closely followed by Aster, Primula, and Rhododendron with three species each. While nine genera contained two species each, all the rest of the genera contained only one plant species each.

Distribution of medicinal plants across elevations (altitudes) of study sites
Dagala Gewog is known for its vast alpine rangelands, lakes and steep mountain ranges with varying elevations and diverse habitat/vegetation (Fig. 2). During the field survey of medicinal plants, the lowest altitude recorded was 2870 m above sea level (masl) at Tshotsum and the highest elevation where the research team visited was 4980 masl at Hammanyi. Out of 100 medicinal plants identified from seven localities, 68 species were found growing in the altitudinal range of 4000-4999 masl (Fig. 7). In between 3000 and 3999 masl, 28 species were identified and only four species were identified from the altitudinal range of 2000-2999 masl. Analyzing the availability status of medicinal plants against each altitudinal ranges; we found that more than half of the species (36 species) identified in between 4000 and 4999 masl were rare or less commonly available in the study areas (Fig. 7). Only 14 species were found in abundance.

Distribution of medicinal plants across seven study sites
A total of seven major local places of Dagala Gewog were covered during the study. Among these seven study sites, Kipchen was found to host maximum number of medicinal plants with 21 species followed by Dabgaythang (18 species) and Chalichung (15 species) (Fig. 8).
While Yumtsho Gewa and Tshotsum hosted same number of species, Hammanyi had 11 species in its area. The least was found in Kheregewa with only 9 species present at the time of study. Among the localities (Fig. 8), Hammanyi and Dabgaythang hosted maximum number of less common or rare species. In Hammanyi locality, 81.8 % of the species that grow there were rare (9 out of 11 species found in the locality). In Dabgaythang locality, 66.7 % of the species that grows there were found rare (12 out of 18 species identified). Together, these two localities accounted for 21 % of the rare medicinal plants that were identified from the whole Dagala Gewog.

Medicinal plants of Dagala and their economic potential
Dagala is known by its formidable mountains, vast rangelands and beautiful lakes that support rivers and fragile mountain ecosystem. Its vegetation and climatic conditions are similar to that of Lingzhi region in the north (near Tibet/China) from where MSP has been sourcing their HAMP for more than 48 years. About 116 species of HAMP have been identified from Lingzhi region [43]. This is the first report of medicinal plants identified from Dagala region in Bhutan and 60 species of the total 100 medicinal plants that we have identified from the area are currently in use (but sourced from Lingzhi in entirety) at the MSP. Out of 60 species, we have considered 29 of them as the less commonly observed species in the study areas. At least 16 species grows abundantly in Dagala Gewog and can be collected by MSP. Other species that are not currently in use at MSP adds to the inventory of already existing HAMP identified from Lingzhi region. These species can be of use in future especially when the Bhutan Traditional Medicine Essential Drug List (BTMEDL) revise and add new formulations to the list.
It is the practice of the health system and the Drug Regulatory Authority in Bhutan to revise the essential drug list after every two years to uphold Good Medicine Dispensing Practices (GMDP), keep updated on the trends and patterns of diseases, and devise appropriate pharmaceutics to combat them. When the revision of BTMEDL are made, the traditional drugs that are not used during the past two years are either deleted or substituted with alternative formulations or completely new formulations are added to the list. Most of the time these changes requires collections of Our study identified 40 medicinal plants that are not used for formulating any of the 100 different current formulations at MSP. However, these inventoried species will serve as the backup or contingency list for future use.
These 16 species that are found in abundance have the economic potential since MSP require them in bulk quantities to prepare g.so-ba-rig-pa medicines. Since g.so-ba-rig-pa is also practiced across the globe, these medicinal plants could be in demand by other countries including India, Nepal, Mongolia, Tibet and Switzerland (PADMA company based on Tibetan medicine). However, the first priority would be to focus on meeting the domestic demand of MSP for these medicinal plants. MSP currently engage yak herders for collecting medicinal plants from Lingzhi. As a result of medicinal plants collection program, the Lingzhip (local inhabitants of Lingzhi region) have improved their socio-economic status and contributed significantly to the realization of country's 'Gross National Happiness' (GNH) indices including preservation of traditional medical knowledge, conservation of environment and socio-economic prosperity [44]. The GNH is a country's holistic development policy propounded by the 4 th King of Bhutan, Jigme Singye Wangchuk and this philosophy stresses on balancing the growth of spiritual well-being and the material gains. It is an internationally well-known development philosophy. Same program could be duplicated for Dagala communities. What makes it even more promising and profitable collection site is that it is closer to MSP as compared to Lingzhi, which take 5-7 days on foot to reach there.

Feasibility of establishing HAMP collection and drying centre in Dagala Gewog
For establishing an alternative collection site or HAMP center, we considered the following factors as critical.  Our survey found that the Dagala region meets all these five critical factors or criteria and could be an economical alternative HAMP collection centre for MSP. We have identified 16 species that grow abundantly in Dagala region. These plants have huge economic potential since MSP requires them in bulk quantities for preparing g.so-ba-rig-pa medicines. MSP could engage yak herders from Dagala Gewog to collect these HAMP on an annual basis. Informally socializing with the Jops during the survey, we came to know that they would be interested to collect the medicinal plants while tending to their regular Yak herding activities. The geographical features of the area are also favorable and easily accessible by the Yak herders. Since Kipchen host the highest number of medicinal plants and takes only 4 h walk from the motor road point in Genekha village, a medicinal plants drying center can be established there. Other places like Dabgaythang (second highest number of medicinal plants found), Hammanyi, Yumtsho Gewa and Chalichung are within 4-5 h walking distances from Kipchen. It is also only one hour drive from the MSP in Thimphu (capital city of Bhutan). Therefore, the cost of collection would be much lesser than Lingzhi. Based on these feasible factors, it is advisable for MSP to establish an alternative drying centre at Kipchen and subsequently provide training to the Yak herders (Dagala communities) on sustainable and good medicinal plants harvesting practices.

Sustainability and management of medicinal plants
The risk factors for the sustainability of medicinal plants in Bhutan have been described elsewhere [45]. Briefly, these risk factors broadly fall into four categories: biological, ecological, social and economic factors. While Lingzhi region has been affected by all these risk factors due to persistent collection practices for more than 48 years, Dagala region provides fresh hope of sustainable supply of medicinal plants for the MSP. However, since biological (includes life form and species resilience) and ecological factors (includes habitat, population density, natural calamities, and pastureland) are inherent in nature, these risk factors are applicable to the medicinal plants that grow in Dagala region. Out of 100 species that we have identified from the study areas, 47 species can be considered rare or perceived to be lesser in number based on their population density observed at the time of the study. The risk is that some of these rare plants can be vulnerable for illegal collection and trade thereby endangering species.
Yak/cattle herding and ecotourism are the economic mainstay of the Dagala Gewog. There is no limit to the population size of the Yaks/cattle that a family can own. Ministry of Agriculture and Forest [22] recorded that as of 2015 the percentage proportion of livestock holdings by Dagala communities included Yaks (70.9 %), poultry (12.7 %), improved cattle (10.9 %) and sheep (5.5 %). During the survey, we found that most of the Dagala family or a household, which we interacted with had minimum of 50 Yaks (Fig. 9). Overgrazing the rangelands appeared obvious to us.
Yaks consume every type of herbage vegetation and are able to feed on very short growth. They have an ability to cover large areas of grazing ground every day and could negotiate narrow footpaths on rocky slopes to consume some herbs and grasses from isolated pockets. The intensity of Yak overgrazing the alpine and temperate rangeland would accelerate the rate of ecological destruction. The pastures around Laya, Lungo, Jarila and Lingshi communities are already affected by the grazing activities of large herds of Yaks joined by flocks of wild blue sheep and marmots [46]. Most of the important HAMP are restricted to delicate, specialized and pockets of habitats. Intensive browsing or overgrazing the pastures could lead to more habitat destruction and shrinkage of the natural population sizes of the valued medicinal plants. A balance between Yaks grazing the pastureland and the growth of medicinal plants must be maintained if the medicinal plants collection program is to be established in Dagala Gewog. A study on Yaks' food chain would shed lights on what medicinal plants are consumed by the Yaks and this could navigate the formulation of strategies to conserve those particular species.

Conclusions and future direction
This study identified 100 species of HAMP that grows in Dagala region. Out of these at least 16 species grows abundantly and has marketing potentials for the Dagala communities. Among the places that this study covered, Kipchen was found to host maximum number of medicinal plants species. As the pressure on HAMP in Lingzhi region has been increasing due to persistent collection for more than 48 years, this finding provide basis for the MSP to establish an alternative collection center with a drying facilities at Kipchen. This place falls in the center of other localities and is not too far away from the motor road of Genekha community. Establishing an alternative HAMP collection centre in Dagala Gewog has multi-pronged benefits. The tangible and immediate MSP could obtain sustainable supply of HAMP to meet the demand of g.so-ba-rig-pa medicine production, c) training on sustainable collection of HAMP (always provided by MSP as a package of collection program) would educate Dagala Jops on the values, protection and preservation of plants, d) establishing this alternative collection center would ease the pressure on Lingzhi HAMP and could enable MSP to collect the plants on a rotational basis, and e) since Dagala region is known for eco-tourism, having the medicinal plants collection centre and the herb garden would enhance the in-flow of eco-tourists especially the botanists and the herbalists. This case study, although conducted locally in Dagala region, has international significance especially for those countries, which practices Tibetan g.so-ba-rig-pa medicine and uses the medicinal plants that we have identified.