Orchidaceae, the orchid family, is a morphologically diverse and geographically widespread family of monocots in the order Orchidales. They are found in a variety of ecological conditions and generally occur in four forms based on their habitat (i.e. terrestrial, epiphytic, lithophytic and saprophytic). Among these, the terrestrial species accounts for approximately one-third of the family [1] [2] ; this species tends to live in small isolated populations placing them at risk of extinction [2] .

Orchidaceaeis believed to be the second largest family of flowering plants after Asteraceae, with between 21,950 and 26,049 currently accepted species, grouped into 880 genera. The largest genera are Bulbophyllum (2000 species), Epidendrum (1500 species), Dendrobium (1400 species) and Pleurothallis (1000 species) (World Checklist of Selected Families, 2013). Nepal harbors 437 species of orchid belonging to 104 genera [3] .

Dactylorhiza Necker ex Nevski (Orchidaceae) is a genus of about 75 species, distributed throughout the world with 58 species in Europe and North Africa [4] . The greatest species richness is found in north-western Europe, though only nine species of Dactylorhiza are endemic. The distribution of Dactylorhiza covers most of Europe, temperate Asia, North Africa, Japan, the Aleutian Islands and northern parts of North America. Dactylorhiza is a very challenging genus as it consistently presents taxonomic difficulties [5] [6] . Only one species of Dactylorhiza hatagirea is extended throughout Nepal from an altitudinal range of 2800 m to 3900 m [7] in terrestrial habitat [8] . Apart from the Nepal Himalaya, the species is distributed in India, Bhutan, China, Mongolia, Pakistan and Russia [7] . It is found in grassland slopes or among shrubs, in the upper belt of the temperate to alpine climate zone.

The use of orchids as medicine has a very long history. Chinese were the first to use them as an herbal medicine [9] . The presence of phytochemicals such as alkaloids, flavonoids, and glycosides has made orchids valuable as medicine. Although the Convention on International Trade in Endangered Species of wild fauna and flora (CITES) Nepal has enlisted orchid in its Appendix II where the exploitation, collection and trade are banned, the use of orchids as a medicine for local purposes, as well as its collection and trade through illegal channels [7] , has led to pressure on its natural habitat [10] [11] .

The threat to the species generally arises due to its panoply of uses. Dactylorhiza hatagirea is one of the highest valued medicinal orchids extensively used in Ayurveda. The tubers of this species yield high quality “Selep” which is used extensively for its aphrodisiac properties and sexual stimulant in traditional medicine in different parts of Himalaya [12] - [15] . Apart from this, the tuber is used as farinaceous food and nerve tonic [12] [13] . The paste is also considered nutritive and used in treating weakness in children and women [15] . Root powder is spread on wounds to control bleeding [11] [13] . In addition, the plant possesses antibacterial properties and the whole plant is used in curing various ailments caused by bacteria; the aerial part is effective to inhibit Escherichia coli while the rhizome is effective in treating a disease caused by Shigella flexinerai [16] .

The abundance and distribution ranges of orchid species have undergone dramatic declines in recent decades [17] . Habitat loss and fragmentation are considered important factors in the decline of Dactylorhiza [1] [17] [18] : the smaller population sizes and greater degree of population isolation are strongly associated with the general decline and extinction risk of the population as a whole [19] [20] .

Thus, the species is threatened and various anthropogenic factors presently confine the species to small, isolated populations. In the light of these circumstances, this study aims to 1) sample the present ecological data of the species and regress them with different anthropogenic factors; and 2) identify the threats to the species in the Manaslu Conservation Area.

Dactylorhiza hatagirea showed high density in the open slope in alpine region whereas its availability in the open slope of sub-alpine region was minimum and from conservation point of view, such species are more important. Species with specific habitat requirements have greater possibility of extinction than the species with broad habitat range [23] . Majority of plant taxa of endangered and rare categories grow in Nepal Himalaya are under the pressure of exploitation and destruction of their natural habitats [24] [25] . Similarly, orchid species are facing the greatest threat due to human encroachment, habitat loss, forest destruction and degradation [10] [11] .

The density of D. hatagirea varied with different management practice. We found the population density to be 2.182 individuals·m⁻² whereas [18] [26] [27] and [28] as 1.0 - 4.2 individual·m⁻², 1.13 - 2.19 individuals·m⁻², 0.37 - 18.0 individuals·m⁻² and 0.4 individual·m⁻² respectively. The variation in the above studies mentioned different degree of grazing and trampling as a major cause to decrease the density of D. hatagirea. Similarly in this study as well, different degree of grazing and trampling resulted in low density of targeted species.

Moreover, trampling by livestock was also noted as a major factor for depletion of its population in natural habitat. In relation to this observation, [9] [15] [24] and [29] highlighted the over-collection and grazing pressure as the means of disturbances mostly responsible for low density and continuous decline of plant population. Although based on the report published by [30] , local user are responsible for managing locally available resource such as forest, grassland, alternate source of energy and local tourism for enhancing their livelihood opportunity but due to mismanage grazing, the population of D. hatagirea is been decline. Moreover, [31] also reported the decline in species richness of vascular plants with respect to grazing.

Similarly, [18] also observed that frequent extraction and increase in grazing pressure were mainly responsible for low population status of D. hatagirea in Central Himalaya. Transhumance pastoralism and increasing livestock number in the Himalaya have negative impact on abundance of the species due to mismanage grazing [32] . Similar threat has been experienced in the study area where we found the livestock were grazing in D. Hatagirea potential area. Moreover, from the questionnaire survey it was found that the nomads in the study area practiced transhumance in flowering and fruiting season. Overgrazing and trampling by the livestock result to destroy the aerial parts. Mismanage grazing and periodic movement of livestock in the study area has resulted in the destruction of habitat which further results in low population density of D. hatagirea.

The distribution in fragmented habitats and the wide variation in frequency of occurrence of D. hatagirea suggest a high potentiality of the species to grow over large areas, but due to increase anthropogenic activities it seems unable to form large continuous distribution boundaries [2] . Lacking endosperm, the seeds of D. hata- girea can germinate with only symbiotic fungi in natural conditions. Due to over disturbances and habitat fragmentation it increases mating opportunity between closely related individuals and finally results in loss of genetic diversity [2] [33] .

The present study reveals that the density of D. Hatagirea in protected areas is under continuous threats. The relative density, relative frequency and abundance (2.02%, 7.48% and 2.667 individual·m⁻², respectively) indicates that the associate species play a significant role in its distribution. The major associated species were Anemone demissa, Bistorta macrophylla, Chesneya nubigena, Morina nepalensis, Morina polyphylla etc. Dactylorhiza hatagirea is listed in Appendix II of CITES, and its collection and trade are prohibited, however, there is a decrease in the availability of D. hatagirea, due to increase in pressure from grazing and trampling. Lack of awareness, harvesting for personal use, lack of conservation plan in the study area may collectively account to decline D. hatagirea.

We would like to acknowledge Rufford Small Grant for Nature Conservation for financial support and Idea wild for equipments support to conduct this study. Furthermore we would like to thank Manaslu Conservation Area Project, National Trust for Nature Conservation for providing research permission in the study area. We would also like to thank local communities who shared their valuable knowledge and information during the field work. Sincere thanks goes to staff and students of Buddha Secondary School, Phillim (Sirdibas) and the members of the Samagaun Youth Club. Last but not least, we would like to thank Mr. Daniel Tucker for English edition and proof read of this article.