Traditional knowledge of wild edible plants with special emphasis on medicinal uses in Southern Shan State, Myanmar

Background Myanmar is one of the hotspots of biodiversity and is a rapidly developing country. Performing floristic research in Myanmar is an urgent issue, and ethnobotanical studies of wild edible plants (WEPs) will provide new information on natural plant resources. Method Ethnobotanical data were collected in three villages with different historical backgrounds in Southern Shan State, Myanmar. A total of 19 key informants were interviewed, and specimens were collected in the fields with the participation of key informants in June–July 2015. Group discussions were organized during 2016 and 2017 to reinforce the information on use of WEPs. DNA barcoding was used to facilitate species identification. Results A total of 83 species from 44 families of angiosperms were recorded as WEPs. Most of the species were used as wild vegetables (47 species), followed by fruits and nuts (31 species). Eighteen WEPs were consumed as medicinal foods. Differences in use of WEPs between the communities of the villages were observed. The age class of 30–39 years was more familiar with the environments where they could collect WEPs and had more knowledge of WEPs than did the older groups. The use of Elaeocarpus floribundus as an edible oil is a very interesting tradition. Conclusion WEPs play an important role in the livelihood of local communities. The indigenous society has maintained traditional knowledge of the WEPs. Historical background, land use system and surrounding vegetation could have effects on the variation in the traditional uses of WEPs. Increasing awareness of the importance of WEPs will encourage the conservation of traditional knowledge of indigenous populations.


Background
Wild edible plants (WEPs) are defined as plant species collected in the wild to be consumed as food or drink. Although important nutrients for humans are available from WEPs, it was argued that the use of WEPs is decreasing in urban-style cooking [1,2]. Being an important source of energy and micronutrients, WEPs can increase the diversification of human diets [3,4]. WEPs were also important food sources during famine, when the normal food supply was disrupted [2]. Currently, humans focus on a limited number of plant species for staple food, neglecting the importance and usefulness of WEPs. This restriction can lead to global food shortage and loss of the knowledge about WEPs [5,6]. Documentation of WEPs is important for the identification of food sources from the surrounding environment, and WEPs are serving as gene pools for genetic improvement of crops to achieve higher productivity, disease resistance and compatibility with global climate change [7]. WEPs also have the potential to be developed into new crops through domestication [8]. Moreover, the nutritional and medicinal properties of WEPs are increasingly recognized [9][10][11][12]. The traditional uses of plant resources and the lifestyles of rural communities have changed in accordance with the switch from subsistence farming/hunting and gathering to profit-oriented agricultural systems [9]. The local food tradition is a kind of cultural expression, and a loss of traditional knowledge of WEPs implies a loss of cultural identity [13].
Myanmar is one of the hotspots of biodiversity [14,15] and is a rapidly developing country. Describing the flora of Myanmar is an urgent issue, and ethnobotanical studies will provide new information on natural plant resources [16,17]. A total of 135 ethnic groups are officially recognized in Myanmar [18]. These indigenous people regard natural resources as essential to their culture and well-being [19], but they are not truly interested in the conservation of natural resources. Furthermore, local communities are rarely allowed to participate in decision-making processes concerning the impact of ecosystem changes, and their dependence on plant resources is not adequately considered in formulating the strategies of rural development [20]. In Myanmar, quite a few ethnobotanical studies have been carried out mostly emphasizing medicinal and financial value [21][22][23][24].
Field research was carried out in three villages, each with different historical backgrounds, namely Eden, Myin Ka, and Pin-sein-pin, located in the western part of Southern Shan State (Table 1 and Fig. 1). Southern Shan State is situated on the Shan Plateau, which rises to the east from the central basin of Myanmar and occupies the eastern half of the country. The average elevation of the plateau is approximately 900 m. The average annual precipitation is between 1900 mm and 2000 mm, and the average daily temperature is 22°C [25]. The ethnic groups residing in the study area are Danu, Taung-yoe, Shan, Bamar, Pao, Kayan and Kayin. The majority of people in the villages of Myin Ka and Pin-sein-pin are Danu and Taung-yoe. The major ethnic group in Eden village is Kayan. According to the folklore of the villagers, the old village of Myin Ka was established during the Bagan Era (9th-13th centuries) one mile away from the current village location. In the late nineteenth century, the old village was destroyed because of ethnic conflicts in the area and was moved to the current location. The villagers of Eden migrated from Kaya State because of civil war in their former area and established the village in 1990.
This study focused on the local communities in Southern Shan State to document their traditional knowledge of and practices involving WEPs. In the present study, a DNA barcoding technique was used to facilitate species identification. DNA barcoding is a microgenomic identification system in which a short, standard DNA region that is universally present in the target lineages is analyzed [26,27]. With the help of DNA barcoding techniques, species identification can be facilitated in the ethnobotanical studies  [28,29] and floristic studies of unexploited tropical regions [30]. A total of 83 species from 44 families of angiosperms have been documented as WEPs in the study area. The indigenous societies, who have been living in the area for a long time, could have a considerable amount of traditional knowledge of WEPs.

Interviews and collection of plant specimens
First, meetings were organized with villagers, and the objectives of the research were explained. Consent was obtained before carrying out the ethnobotanical survey. The ethical guidelines of the International Society of Ethnobiology were strictly obeyed [31]. The ethnobotanical data were first collected from 19 key informants who were selected for their reputation of being specialists in the use of WEPs. The information on WEPs was gathered from interviews and fieldwork. The ages of the key informants ranged from 30 to 57 years, and one informant was female. All key informants were farmers. The field surveys were carried out in June-July 2015, and voucher specimens and the leaves for DNA analysis were collected. During the field surveys, the vernacular name, growth habitat and folk use of each species were recorded. Interviews and field sampling were carried out with one individual informant at a time to avoid the incorporation of the knowledge from other villagers. Voucher specimens were deposited in the herbaria of the Makino Botanical Garden (MBK), Japan, and the Forest Research Institute (RAF), Myanmar.
After gathering ethnobotanical data from the key informants, group discussions were organized again in 2106 and 2017. In addition to the 19 key informants, 23 other informants were invited for the group discussion. These 23 informants were selected for their willingness to participate in this study, ability to work together in the field, and reputation for being knowledgeable on the use of WEPs. Therefore, a total of 42 villagers participated in this project, including 5 female informants. A total of 13, 12 and 17 informants from the villages of Eden, Myin Ka and Pin-sein-pin, respectively, participated in the group discussion. The age of informants ranged from 23 to 82 years, and the group included 37 farmers, one small shop owner, one forester and three herbalists. We also gathered information on WEPs with a group of informants by using the "walk-in-the-wood" method [32].

DNA barcoding and species identification
Total DNA was extracted from dried leaf tissue by the CTAB method. Approximately 20 mg of dried leaf tissue was ground in a 2 mL microcentrifuge tube using Tis-sueLyser (Qiagen, Hilden, Germany). The sample was incubated with 2xCTAB solution at 65°C for 30 min. After chloroform (chloroform:isoamylalcohol = 24:1) treatment for 30 min, the mixture was centrifuged for 10 min at 3000 rpm. The aqueous layer was transferred to a new 1.5 mL microcentrifuge tube, and then the DNA was precipitated with ethanol. The pellet was dissolved with TE buffer. PCR amplification of the rbcL region followed the procedure of the CBOL Plant Working Group [33]. rbcLa_F and rbcLa_R were used as primers. For the PCR, GoTaq Green Master Mix (Promega, Madison, WI, USA) was used. Amplification was performed in a TaKaRa PCR Thermal Cycler Dice Touch (Takara, Kusatsu, Japan) programmed for one cycle of 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and one cycle of 72°C for 5 min. The PCR products were resolved by electrophoresis in 1.0% agarose gels stained with GelGreen (Wako, Osaka, Japan). DNA fragments were recovered using the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cycle sequencing was performed using a Big-Dye Terminator v3.1 Cycle Sequencing Kit and an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
The Basic Local Alignment Search Tool (BLAST) program was used as a tool to search for similar DNA sequences from the database of the National Center for Biotechnological Information (NCBI). Among the compared sequences, the species with the most similar sequences were used to guide species identification. The final identification of specimens was determined by cross referencing with specimens housed at MBK, the type specimen images available on the JSTORE Global Plants website (https://plants.jstor.org), and the online herbarium catalog of the Royal Botanic Gardens, Kew (http://apps.kew.org/ herbcat/navigator.do). The plant descriptions in the Flora of China and Flora of Thailand were consulted. The scientific names of species were assigned in accordance with The Plant List (http://www.theplantlist.org). The uses of WEPs and medicinal plants were classified into categories according to the standards developed by the Royal Botanic Gardens, Kew [34].

Diversity of WEPs in the study area
A total of 153 specimens were collected as WEPs from the three villages in this study. Of these, 138 specimens were identified to the species level and consisted of 83 species. Araceae, Fabaceae and Moraceae were the major families with the largest number of species (5 species each). Zingiberaceae, Asteraceae, Myrtaceae and Rutaceae included four species. Ethnobotanical data such as vernacular names, collection sites and folk uses are listed in Table 2. Among the 83 species, most are indigenous in Myanmar, but some of them originated outside of Asia: Annona cherimola, Casimiroa cf. edulis, Coffea arabica and Psidium guajava escaped from plantations; and Alternanthera philoxeroides, Bassia scoparia, Bidens biternata, Crassocephalum rubens, Marah macrocarpa, Oxalis latifolia, Physalis angulata, Physalis pubescens and Solanum torvum are naturalized.
In total, 34, 57 and 47 species were used as WEPs in the villages of Eden, Myin Ka and Pin-sein-pin, respectively (Fig. 2). Although most of the WEPs were used with a single vernacular name within a village, eight species had two or three local names. Physalis angulata and Leea indica were used without local names in Eden village. Two species of Amorphophallus and three species of Syzygium were used with the same local name in all three villages. Among the 39 WEPs used in more than one village, eight species were used with different local names. Phyllanthus emblica had different local names in all three villages.
A total of 47 species from 25 families were used as vegetables. All five species in Araceae were used as wild vegetables. Based on the interviews with villagers, the most preferred species for use as wild vegetables were Phyllanthus emblica, Rotheca serrata and Docynia indica. P. emblica and R. serrata were also used as medicinal plants in the study area. In addition to collecting it from the wild, the villagers planted P. emblica in their home gardens for their own consumption.
A total of 31 species from 23 families were used for their fruits and nuts. Species in Myrtaceae, Moraceae and Sapindaceae were frequently used as fruit plants. The most preferred fruit plants were Docynia indica, Ficus semicordata, F. auriculata, Myrica esculenta and Archidendron jiringa. In addition to collecting it from the wild, small-scale planting of Docynia indica was observed not only for fruits but also for fuel wood. The whole plants of Oxalis latifolia were eaten by children as a snack in Myin Ka village. This species is a perennial weed originating from Central and South America [35] and is growing in home compounds of the village. From the other two villages, no species were reported as a snack for children.    Four species from three families were used as spices and condiments in traditional curry. The dry powder of the rhizome from Curcuma cf. amada was used in various traditional dishes. Freshly pounded rhizome of C. cf. amada was also used as a spice. Leaves of Cinnamomum tamala, Laurus cf. nobilis and Zanthoxylum armatum were used in traditional curry for flavor. Z. armatum is also used as a spice in Yunnan, the province of China nearest to Myanmar [36].
The different methods of preparation of vegetables for traditional dishes are as follows: (i) Hin, the most common traditional food, is prepared by boiling the vegetable with edible oil, fermented fish and salt into a semiliquid dish; (ii) Akyaw is prepared by frying the vegetable with salt, spices, chili, and fermented fish; (iii) Athote is prepared by mixing the raw or boiled vegetable with pounded fried groundnut, salt and chili; (iv) Atoe is a raw salad eaten with fermented fish or with fermented bean; and (v) Hincho is a soup prepared by boiling the vegetable with fermented fish and salt.
Most of the WEPs in this study were used directly as vegetable dishes without any pretreatments, although it is known that some plant species, such as those in Arisaema and Celastrus, are toxic. Only the tubers of Amorphophallus purpurascens and A. cf. muelleri were boiled, peeled and pounded before preparing traditional dishes. Most of the fruits, which were consumed as a snack food, were eaten fresh without any preparation. The fresh fruits of Ficus racemosa were preserved by soaking them in salty water. The petiole of Colocasia esculenta and the fruits of F. semicordata were pickled and consumed as a fermented food similar to pickles.

Use of WEPs as medicine
A total of 18 WEPs from 15 families were used as medicine (Table 3). Among these species, 11 were consumed as food for medicinal uses, and seven species were prepared as a main ingredient for traditional medicine. Dichrocephala integrifolia, Plantago major and Laggera alata were rarely used as food in the study area; however, these species were traditionally consumed for medicinal use. In the study area, the shoots and leaves of D.  integrifolia were prepared as soup and consumed as a postpartum tonic for mothers. This species is also reported as a valuable medicinal plant for the treatment of Alzheimer's disease [37]. The young leaves of P. major were traditionally prepared as salad and consumed for hypertension and food poisoning. It is reported that the extract from P. major has antifungal activity [38]. The shoots of L. alata were fried with eggs and consumed to treat swelling in the body. Isochlorogenic acid A from L. alata is reported as a potential candidate antihepatitis B drug [39].

Species identification
We applied the DNA barcoding technique as a guide for species identification. The chloroplast rbcL sequences of 71 species (85.5% of 83 identified species) were successfully sequenced. The sequences of 53 species (71.6% of sequenced species) yielded a single haplotype from a particular species from the BLAST analysis, whereas the remaining sequences yielded two or more haplotypes from closely related genera. As many of the collected specimens were sterile plants, the DNA barcoding technique facilitated the identification of the WEPs in this study.

Notable uses of WEPs
Lasia spinosa and Cheilocostus speciosus were important seasonal foods for the cash income of the local people. The villagers collected these two species from the wild seasonally and sold them in the local markets. Because customers prefer these two species as wild plants, the villagers do not cultivate them for markets (Fig. 3a). Tender leaves of Acacia concinna were used as vegetables, but the decoction of fruits of Acacia concinna is traditionally and popularly used as a shampoo in Myanmar. The stems and tubers of Amorphophallus purpurascens and Amorphophallus cf. muelleri were used as vegetables, and the sliced tubers were preserved after being sun dried. The tubers of these species were boiled, peeled and pounded to make konjac, referred to as 'Wa-u' in Myanmar (Fig. 3b). Boiled seeds of Archidendron jiringa are a traditional food in the area, and the seeds are also a source of income for the villagers. Although the boiled seeds are consumed widely in the tropics, they have also been found to have some toxicological effects on the heart, kidney, liver and pancreas [40]. The seeds of Elaeocarpus floribundus were used to extract vegetable oil in Myin Ka village. The vegetable oil was used locally, but it was not produced at a commercial scale. To the best of our knowledge, there are no reports on the use of seed oil of E. floribundus, although its fruits are eaten raw as a wild edible fruit in South Asia [41]. It is interesting that the extracts of leaves of E. floribundus showed significant activities against CEM-SS cancer cells [42].
It is reported that Rotheca serrata is mainly used as a medicinal plant in other areas [43]. However, R. serrata was mainly consumed as a wild vegetable in the study area and was used for digestive system disorders. The young leaves of Celastrus paniculatus were prepared in a traditional soup and consumed as a dietetic food. The food is not used for a particular ailment. However, the villagers believe that the soup is good for health. The use of this species was recorded in a traditional song expressing that the plant is highly recommended for traditional soup. The fruits of C. paniculatus are consumed orally as a vermifuge by the locals living around Popa Mountain Park, Myanmar [44]. It was reported that the extract from C. paniculatus can inhibit the growth of breast cancer cells [45].

WEPs used in each village
The differences in the use of WEPs between the communities of three villages have been observed. The differences among ethnic groups could be the main reason for using different local names for the same species. The three villages are situated in different townships, and there are no social relationships among the villages. This is also another reason for using different local names.
The village of Myin Ka used a larger number of WEPs than the other two villages. Myin Ka village has a longer history than the other two villages and could have inherited much more traditional knowledge of WEPs. In spite of the same major ethnic group (Danu people) and similar vegetation being found in the villages of Myin Ka and Pin-sein-pin, 33 out of 71 WEP species were used in both of the villages, but the remaining 38 species were not common between the two villages. These two communities have different historical backgrounds, and there were no social relationships between the two villages. These differences may be a cause of different uses of the plant resources.
The village of Eden has fewer shared species with the other two villages. Most of the species documented in this study from the families Ebenaceae, Elaeagnaceae, Elaeocarpaceae, Fagaceae, Lamiaceae, Lauraceae, Myricaceae, Rosaceae, Rubiaceae and Rutaceae were used in the villages of Myin Ka and Pin-sein-pin, but not in the village of Eden. However, most species from the families Burseraceae, Lythraceae and Sapindaceae were used only in Eden village. The elevation of Eden is approximately 350 m above sea level, while that of Myin Ka and Pin-sein-pin is more than 1000 m. The forests around the village of Eden are mostly deciduous forests, while those around the villages of Myin Ka and Pin-sein-pin are evergreen forests. These differences could be the causes of variation in plants used by the local communities. Moreover, the village of Eden is newly established by Kayan people who migrated from Kayah State, which is different from the villages of Myin Ka and Pin-sin-pin, which were established by Danu people in Shan State. The migrant community may be less familiar with the plant resources in the new environment. In addition, the villagers of Eden practice shifting cultivation for banana plantations. Shifting cultivation could have detrimental effects on the environment, and the availability of WEPs could be diminished.
The average number of species recognized by a single key informant was different among the three villages ( Table 4). The key informants from Myin Ka village reported a greater number of species (an average of 18.0 species per key informant) than did those from the villages of Eden and Pin-sein-pin (an average of 10.2 and 12.1 species, respectively). The amount of knowledge on WEPs was also different between the age classes of key informants in the study area. Usually, the older generations transfer the farming activities and knowledge of WEPs to the younger generations in the study area. However, the Fig. 3 Use of WEPs in the study area. a wild Lasia spinosa and Cheilocostus speciosus at a local roadside market. Informed consent was obtained for the use of the photograph. b sun-dried slices of Amorphophallus purpurascens for preservation (left) and 'Wa-u' (right) age class of 30-39 years reported more WEPs than did the older groups. The interviews revealed that the age class of 30-39 years was much more familiar with the environments where they could collect WEPs. The sharing of work between family members was revealed, and the age class of 30-39 years took more responsibilities in the collection of WEPs. The older generations might face the inability to recall the knowledge of gathering WEPs. The abundance of knowledge of WEPs in younger age classes and a decrease in the knowledge exhibited by the older groups have been reported previously [46,47].
The number of species reported as WEPs by more than two key informants in the villages of Eden, Myin Ka and Pin-sein-pin was 8 (33.3% of all species reported by all key informants in Eden), 29 (56.9%) and 15 (40.5%) species, respectively. This result indicates more share knowledge of WEPs within the community of Myin Ka. Myin Ka village has 120 ha of common land for watershed conservation, and all villagers of Myin Ka village have access to the common land for collection of WEPs. In contrast, most of the land area around Pin-sein-pin village is privately owned. In the case of Eden village, shifting cultivation is practiced in the reserve forests, where the villagers do not have the legal right of land ownership. Thus, land use and the land ownership system could have effects on the sharing of knowledge of WEPs among the villagers.

Conclusion
The rich diversity and traditional knowledge of WEPs have been documented in this study. This study indicates that WEPs play an important role in the livelihood of local communities. Historical background, land use system and surrounding vegetation could have effects on the variation in the traditional uses of WEPs. The indigenous societies, especially that in the village of Myin Ka, maintained a considerable amount of traditional knowledge of WEPs. In addition to their food value, other uses of WEPs, such as their medicinal uses, make them more important in the livelihood of local people. WEPs are important not only for increasing the diversity of local food consumption but also for generating income in the local communities. Increasing awareness of the importance of WEPs will encourage the conservation of traditional knowledge of indigenous populations. Further investigations on the nutritional value and pharmacological activities of WEPs will add more value to the traditional knowledge.