Project: Gbpi/Ierp-Nmhs/15-16/07/08

PROJECT: GBPI/IERP-NMHS/15-16/07/08

SPECIES DIVERSITY OF CORDYCEPS FOR THEIR PHARMACOLOGICAL EFFECTS ON SELECTIVE PHYSIOLOGICAL PARAMETERS AND ARTIFICIAL CULTURE OF SELECTED SPECIES

SECOND ANNUAL REPORT (April 2017-March 18)

Submitted to

G B PANT NATIONAL INSTITUTE OF HIMALAYAN ENVIRONMENT & SUSTAINABLE DEVELOPMENT, KOSI-KATARMAL, ALMORA

Submitted by

CHANDRA SINGH NEGI, DEPARTMENT OF ZOOLOGY, L S M GOVERNMENT POSTGRADUATE COLLEGE, PITHORAGARH- 262 501 (UTTARAKHAND)

CONTENTS

CHAPTER 1: LITERATURE REVIEW (UPDATED AND RELATED TO THE PROJECT OBJECTIVES)

CHAPTER 2: HEPIALIDAE (FAMILY OF THITARODES) AND THITARODES (THE PRINCIPAL HOSTS OF CORDYCEPS)

CHAPTER 3: EXPLORING OTHER MOTH DIVERSITY

CHAPTER 4: LIVESTOCK GRAZING PRESSURE VIS-A-VIS STUDY OF BROAD SOIL PARAMETERS

CHAPTER 5: VEGETATION CHARACTERISTIC DIFFERENCES ACROSS THREE ALPINE MEADOW SITES DIFFERING IN MAGNITUDE OF GRAZING PRESSURE

CHAPTER 6: CULTURE OF BROWN AND YELLOW SPECIES OF CORDYCEPS

REFERENCES

CHAPTER 1: LITERATURE REVIEW (Updated and related to the project objectives)

A. INTRODUCTION The Latin etymology of Cordyceps sinensis is as follows: cord means “club,” ceps means “head,” and sinensis means “Chinese.” The mushroom is also called the “caterpillar fungus” on account of its origin, and, less frequently, “winter worm, summer grass” because the ancient Chinese believed that the fungus was an animal in winter and a vegetable in summertime. Around 1850, Japanese herbalists began importing the mushroom from China and named it tochukaso, a Japanese translation of “winter worm, summer plant.” The mushroom is sometimes called the “club-head fungus,” a direct translation of its Latin name. The common name used in China today is dong chongxia cao (chong cao for short). In Tibet, it is called Yartsa Gunbu, which means “winter worm, summer grass.” There are over 680 documented varieties of Cordyceps mushroom, of which Cordyceps sinensis is but one. Many Cordyceps fungi grow by feeding on insect larvae and sometimes on mature insects. Cordyceps mushrooms grow on just about every category of insect- crickets, cockroaches, bees, centipedes, black beetles, and ants, to name a few. For example, Cordyceps curculionum attacks the body of ants and rides the ants high into the trees to disperse its spores.

The first written record of the Cordyceps mushroom comes from China, in the year A.D. 620, at the time of the Tang Dynasty (A.D. 618–A.D. 907), bringing substance to the once intangible allegorical narrative, which spoke of a creature, whose annual existence alluded to a transformation from animal to plant in summer, and then again from plant to animal in winter. The earliest known documentation of Yartsa Gunbu is by Nyamnyi Dorje, a Tibetan physician and lama who lived from 1439 to 1475. His text, titled “An Ocean of Aphrodisiacal Qualities,” describes the value of the mushroom as a sexual tonic. By contrast, the first record in China seems to be more than 200 years later in Wang Ang’s 1694 compendium of material medica, Ben Cao Bei Yao (Grace Yue, pers. comm. 2005). Tibetan scholars wrote of the healing animal=plant through the fifteenth to eighteenth centuries, and, in 1757, the earliest objective and scientifically reliable depiction of the Cordyceps mushroom was written by Wu-Yiluo in Ben Cao Congxin (‘‘New Compilation of Materia Medica’’), during the Qing Dynasty.

According to Gawä Dorje (1995), another name for Cordyceps sinensis in Tibetan medicine is tsa daji (Wylie 1959: tswa da byid). A medicine known as daji (not tsa daji, however) is mentioned even earlier in the fundamental Tibetan medical text Gyü Zhi (also known as the “Four Tantras;” Wylie 1959: rgyud bzhi), composed between the 8th and 11th centuries and frequently republished (e.g., Yutog 2002). However, the identity of daji as Cordyceps remains controversial. It is recommended as a general tonic, for boosting the immune system and virility, and is prescribed, usually in conjunction with other medicines, for kidney, lung, and heart problems, as well as for Hepatitis B; nowadays it is also frequently mentioned as improving eyesight.

Kobayashi (1941) listed 137 species as valid members of the genus Cordyceps and of these 125 were recorded as being parasitic on insects (with one or two species growing on sub-terranean insects). However, at present 200 species of genus Cordyceps are known (Dube 1983). Von Tubeuf

3 experimented with one of the species, Cordyceps militaris, for controlling insect pests, but without success (McEwen 1963). Some of the known species of Cordyceps are Cordyceps acicularis, C. canadenensis, C. forquignoni, C. ophioglossoides Link. and C. sinensis Berk. Yar tsa Gumba, the more common name for the Cordyceps sinensis- is an entomophilus fungus belonging to order Hypocreales and family ophioclavicipataceae, is unique black and blade-shaped and is found primarily at alpine regions, from 3200m to 4000m amsl. The fungus is parasitic, growing on and deriving nutrients from several species of caterpillar, although primarily those belonging to the genus Hepialus, a moth species belonging to the order Lepidoptera and the family Hepialidae, represented by 60 genera and 587 species (Nielsen et al. 2000).

Thirty-three species of Cordyceps sensu lato have been recognized in the Tibetan Plateau and Himalayan region (Zang and Kinjo 1998), but only a few are collected for their medicinal properties. The best known and most commonly used is Ophiocordyceps sinensis, but Zang and Kinjo have described several distinct, closely related species (C. gansuensis K. Zhang, C. Wang & M. Yan, C. kangdingensis M. Zang & Kinjo, and C. nepalensis M. Zang & Kinjo) that in the past have been mistaken for O. sinensis. A recent molecular study by Sung et al. (2007) concluded that Cordyceps should be divided into three genera, with C. sinensis belonging to the largest of the three, Ophiocordyceps. It remains to be seen if this new designation will be accepted, because C. sinensis is an economically important species; there is ample precedent for retaining its better known name, as used here.

Though the Tibetan name for C. sinensis is Yartsa Gunbu (“summer-grass, winter-worm”), it is commonly referred to simply as bu (“worm” or “insect”). The full name describes the main life stages of Cordyceps sinensis: its infected caterpillar host passes the winter (gun) in the soil as a “worm” (bu, pronounced “boo” as in “book”); then in early summer (yar) the mushroom or “grass” (tsa) emerges above ground. The word tsa is usually translated as grass, but can also be used to denote some mushrooms. Cordyceps sinensis parasitizes various grass root boring Thitarodes (Hepialus) caterpillars, which hatch as “ghost moths” when not pre-empted by Cordyceps. Thirty of the nearly 40 species of Thitarodes known from the Tibetan Plateau can be infected by Cordyceps sinensis (Chen et al. 2000). Many of the regional differences in size of the Cordyceps mushroom are probably caused by size differences in the host species. While the normal reproductive cycle for Thitarodes species takes up to five years, most of the life cycle is lived as a caterpillar; the whitish adult moth lives for only a few days in order to mate.

The genus Hepialus constitutes of around 51 species, out of which about 40 species have been identified as being the host of Cordyceps sinensis (Li et al. 2001), with the most commonly recognized species being Hepialus armoricanus, H. oblifurcus, and H. varians as being the ascribed host species of C. sinensis in the Kumaun hills of Central Himalaya. One must, however, take note of the fact that actual factual scientific data on the exact host of Cordyceps sinensis in Central Himalayas, is at present altogether lacking. Hepialus spp. generally feed upon the roots and tubers of their host plant species (during their larval stages) and upon the leaves and floral parts of the same plant species in their adult stages (Shen et al. 1990), and hence the present need of phytosociology to ascertain the associated floral species in the present case of study in Kumaun, Central Himalayas, becomes vital.

There are currently more than 680 documented species of Cordyceps, found on all six inhabited continents and in many climatic zones and habitats, and feeding off a range of hosts, including plants, insects, arachnids, and even other fungi, such as truffles. These figures are subject to rapid change, as what we know of this genus, and the life cycles of its constituents expand. As studies of related species continue, it has become increasingly apparent that the potential medicinal benefits of C. sinensis are, in fact, not related to only one species, and hence the need for more and more exploration of the different species, of which the potential is explicitly there in the present landscape. Of the many different varieties of Cordyceps, those presently being cultivated for medicinal purposes and use in health supplements include C. sinensis, C. militaris, C. sobolifera, C. subsessilus, C. ophioglossoides, and others.

Origin of scientific name of O. sinensis Westwood (1842) misidentified O. sinensis as Clavaria entomorhiza (Dicks.) Westwood. Clavaria entomorhiza was reported by Dickson (1785) from England as Sphaeria entomorhiza Dicks. and was well known at that time. The following year, Clavaria entomorhiza of Westwood (1842) was identified as a new fungal species by Berkeley (1843) and named Sphaeria sinensis Berk. Berkeley described S. sinensis in Latin as “fusca, stipite cylindraceo deorsum subincrassato; capitulo cylindrico cum stipite confluente apiculato; apiculo sterili” which means “it is dark, the stem is cylindrical, somewhat thicker downwards, the head is cylindrical and pointed, confluent with the stem and the tip being sterile”. Identification was based on specimens bought and sent by Reeves to the Linnaean Society of England from Guangzhou, then known as Canton, the capital city of Southeast Chinese Province of Guangdong (Westwood 1842; Berkeley 1843). At that time, the mushroom fungus to be brought to Guangzhou from the western Provinces of China such as Tibet and Sichuan for sale. The specimens sent by Reeves are said to have been collected from western Sichuan (Zang et al. 1990), but the locality was mentioned only as China (Berkeley 1843). The specimens have been preserved in Royal Botanical Garden, Kew, UK (K) (Berkeley 1843; Massee 1895; Zang and Kinjo 1996). Some of the O. sinensis specimens maintained in Kew have also been collected from Nepal and Bhutan (Dr. Begoña Aguirre-Hudson, 2010, personal communication).

It is well known that Saccardo (1878, 1883) listed the scientific name from Sphaeria sinensis to Cordyceps sinensis (Berk.) Sacc. However, Berkeley (1857) had already named it as C. sinensis, while revising Cordyceps diversity of the United States, but he did not justify the name change. Later, Soubeiran and Thiersant (1874) and Bretschneider (1881) also named it as C. sinensis, but did not give any reference nor did they justify the new name. Saccardo (1878) listed C. sinensis and simply referred to Berkeley (1843), but did not provide any further description. Saccardo (1883), however, gave the full description of Berkeley (1843) in Latin along with its synonym. Not only that, he grouped C. sinensis as an imperfectly identified Cordyceps species due to lack of information on micromorphological characters. In the true sense, Berkeley’s (1843) specimens do not serve to identify C. sinensis, as his specimens are immature (Massee 1895; Zang and Kinjo 1996). The taxonomy of C. sinensis has been generally conceived as problematic and controversial, based on type specimens (Chen et al. 1999; Kang et al. 2000; Kinjo and Zang 2001; McKenna et al. 2002; Chen et al. 2004). Some authors have even gone so far as to think that there is no type specimen of C. sinensis or even wonder what C. sinensis actually is. An appropriate epitype should be designated to stabilize the use of the name (Hyde and Zhang 2008).

Based on molecular phylogenetic study, Sung et al. (2007) separated the mega genus Cordyceps into four genera, viz. Cordyceps (40 spp.), Ophiocordyceps (146 spp.), Metacordyceps (6 spp.) and Elaphocordyceps (21 spp.), while the remaining 175 spp. were left in the Cordyceps group. As a result, C. sinensis was transferred to Ophiocordyceps, hence renamed as O. sinensis. There are more than 350 types of so called Cordyceps or its substitutes in terms of their medicinal values have been found worldwide today, such as Cordyceps militaris (L.) Link (the most commonly used substitute), C. martialis Speg. C. hawkesii Gray, C. liangshanensis Zang, Liu et Hu, sp. nov., C. barnesii Thwaites, C. cicadicola, C. gracilis (Grav.) Dur. et Mont., C. ramose Teng, C. ophioglossoides (Ehrh. Fr) Link and C. gunnii (Berk.) Berk, etc.

Mycological data Kingdom - Fungi Phylum - Ascomycota Class - Ascomycetes Order - Hypocreales Family - Ophioclavicipitaceae Genus - Ophiocordyceps Species - Ophiocordyceps sinensis (Berk.) Sacc. Basionym - Sphaeria sinensis Berk. Anamorphs - Cephalosporium donqchongxiacao, C. sinensis, Chrysosporium sinensis, Hirsutella sinensis, Mortierella hepiali, Paecilomyces hepiali, Scytalidium sp., Scytalidium hepiali, Tolypocladium sinensis, and others English names - Cordyceps mushroom, caterpillar fungus Japanese names - Totsu kasu, tochukasu Chinese names - Hia tsao tong tchong, dongchongxiacao [chongcao], Literal translation - ‘winter worm, summer plant’

Life cycle and distribution of O. sinensis Ophiocordyceps sinensis has an unusual life cycle in nature. In late autumn, the fungus infects underground larvae of ghost moths within the family Hepialidae. It is not known how the fungus infects the caterpillar; perhaps the caterpillar ingests a fungal spore, perhaps a fungal hypha penetrates a spiracle of the insect, or perhaps an ascospore or a conidium adhering to the insect surface germinates and directly penetrates the insect’s cuticle. After entering the caterpillar’s body, the fungus grows vegetatively and ﬁlls the caterpillar with threadlike hyphae. Even infected by the fungus, the larvae can still move from positions 5(10)–25(35) cm to positions 2–5 cm beneath the soil surface before dying with the head upward. The fungus then grows out from the dead host (usually from the head) and forms a small stroma bud before soil freezing in winter (Buenz et al. 2005; Li et al. 2006).

In the following spring, the stroma bud grows upward, emerging above the soil surface and forming a stalked fruiting body (a sexual, perithecial stroma) (Pu and Li 1996; Buenz et al. 2005). Thread-like ascospores are released from the Perithecia and can presumably infect new caterpillars. In addition to the ascospores, O. sinensis can also produce asexual conidia. The anamorph of the fungus is widely recognized as Hirsutella sinensis (Liu et al. 1989). The conidia are

6 produced by conidiogenous cells formed on vegetative hyphae or germinated ascospores. Growth and reproduction of O. sinensis in nature requires a host insect, but the fungus has a wide host range that includes more than 50 species within the family Hepialidae (Lepidoptera) (Wang and Yao 2011). Generally, the life cycle of O. sinensis insect hosts requires 2 or 3 years. The larval stages live underground and feed on roots of plants of more than 19 angiosperm families in alpine meadows on the Tibetan Plateau (Zhu et al. 2004).

In late autumn, the fungal spores release the fungal mycelia, which then infect the caterpillar. By early summer of the following year, the fungal infestation has killed the caterpillar and the fruiting body can be seen protruding from the caterpillar's head, and since it resembles a grass sprout, the vernacular name-Yar tsa Gumba means roughly "winter insect, summer grass." The infected larval stage of the caterpillar is pink or brownish in colour and measures 2.8 to 5.0 cms in length, with 3 pairs of prolegs and 4 prominent pairs of abdominal legs (plate 1). In the Himalayas, it is found mainly in the higher altitudes of Nepal, Sikkim, Bhutan, and Arunachal Pradesh and has recently been discovered from the Kumaon Himalayas, where it is known as Keera Ghaas (insect-grass). Presently various places like Chiplakot in the confluence of Darma and Choudas valleys, Sumdum, Philam, Bon, Baling, Dugtu and Daantu in Darma Valley and Ralam Dhura, Panchachuli base, Nagnidhura and Namik in Dharchula-Munsiari region of district Pithoragarh, Uttarakhand are known for its occurrence (Negi 2003, 2009, Negi et al. 2006).

Kendrick (1992) reports that Cordyceps fungi have developed a special adaptation to improve their chances of reproductive success. Since reproduction is dependent on a very specific host, each spore fragments into 100 or more part-spores so that each fungal fructification produces 32 million propagules, thus increasing the odds of landing on a larva. Li et al. (1999) report that Cordyceps sinensis produces 30-60 propagules, and that usually 15 days pass between spore dispersion, their breaking up into propagules and larval infection. The propagules usually attach themselves to the larval state of the insect, but they also can attach themselves to mature moths as well. Apparently, the larvae are forced by the fungus to move into its final position before being immobilized, since the fungus needs proximity to the surface to grow its fruiting body (stroma) above ground. Not infected larvae will not hibernate close to the surface.

The mycelium- a cotton-like mesh composed of white threads (hyphae) - develops inside the body of the insect, first feeding on less vital parts, until it has taken over the complete organism filling the caterpillar with its hyphae. After the insect is completely mummified and emptied of nutrients, leaving behind the larval exoskeleton filled with the Cordyceps mycelium, the fungus will develop a fruiting body out of the head above the eyes, where the larvae have a horn-like protuberance in early spring. The 5-10 cm long, brown, club-shaped fruiting body grows above ground to have its propagules dispersed by the wind in order to find a new host insect. The stroma is nearly twice as long as the caterpillar when fresh. It takes several weeks for the spores to mature. As the insect is the sole source of food for the fungus, the size of its stroma is dependent on the size of the host caterpillar (Arora 1986). Fruiting of Cordyceps sinensis, according to collectors in Lithang, is enhanced by high precipitation. In general, Cordyceps is not found in areas where precipitation is below 300 mm/annum.

The distribution of this fungus is very limited probably because it is psychrophilic and must parasitize belowground larvae of ghost moths. Chinese Cordyceps reported outside of “High Asia” were erroneously identiﬁed and refer to other Cordyceps species, e.g. Cordyceps militaris, Cordyceps gunnii (Shrestha et al. 2010; Yang et al. 2010). Although the distribution of natural Chinese Cordyceps is limited and its production has decreased (Chen, Zhong et al. 2010), the annual yield of this natural resource in China has been as high as 100–200 tons in recent years (Ma 2010; Zhang, Li et al. 2010). Among the ﬁve provinces, Qinghai has the largest production (about 100 tons per year), and Yushu and Guoluo contributed 80% of the total yield of this province (Cai and Sun 2010). In Tibet, the total yield is about 40 tons, with 80% contributed by Nagqu and Qamdo (Xu et al. 2010).

Parasitism of host insects by O. sinensis and its ecological consequence Ophiocordyceps sinensis infects insect larvae in soil. A ﬁeld investigation revealed that the highest infection occurred when 4th to 5th instar larvae were shedding old cuticles and forming new cuticles (Yang et al. 1989). In nature, this occurs from early to mid-August and overlaps with the time when O. sinensis releases sexual ascospores (Yang et al. 1989). Larvae at less advanced stages are seldom infected probably due to their limited movement and food intake, and those at more advanced stages are seldom infected probably owing to increased resistance. When larvae are infected, they become less active in 6 -10 days and die in 15-25 days; infected larvae, however, often move to positions 2–5 cm beneath the soil surface before dying, which facilitates the growth of the fruiting body and its emergence from the soil in the next year (Yang et al. 1989). A small stroma bud often grows from the head of infected larvae before the soil freezes in winter. Even though rare, the locals have often observed larvae with small stroma buds that were alive and able to move.

O. sinensis hyphal bodies were detected in the hemocoels of 10–20% of the 3rd to 8th instar larvae that were collected in nature (Li et al. 1998). Mycelia of O. sinensis enter the hemocoel of the larva and then fragment into fusiform hyphae that multiply by yeast-type budding. After plasmogamy between hyphal fragments occurs, the mycelia of O. sinensis ﬁll the hemocoel (Zeng et al. 2006). Two cuticle-degrading serine protease genes (csp1 and csp2) from O. sinensis have been cloned and found to be involved in the degradation of host cuticle proteins (Zhang et al. 2008). Various factors can inﬂuence the infection process. Loose soil structure and precipitation facilitate the movement of O. sinensis spores into soil and thereby increase infection (Yang et al. 1989). Conditions of generally low temperatures with substantial differences between day and night temperatures can increase the vertical movements of larvae, thus increase the probability that they encounter O. sinensis spores and become infected (Yang et al. 1989).

The grasslands providing habitat for Thitarodes ghost moths and thus for Cordyceps sinensis consist predominantly of sedges (Kobresia spp.), which can cover up to 80–90 per cent of the subalpine grasslands (Wu 1997). Thitarodes moths prefer to feed on young roots of plant species of the families Polygonaceae, Fabaceae, Cyperaceae (including Kobresia), Poaceae, and Liliaceae (Chen et al. 2000). Omnipresent grazing by livestock keeps the grass and herbs short, making the small fruiting bodies much easier to find during the collecting season. Tibetan collectors report that fruiting of Cordyceps sinensis is enhanced by unusually high precipitation during the preceding monsoon season or in the form of winter snowfall, especially when followed by mild temperatures

8 in the spring. However, if snow cover remains for longer than a few days during the harvest season, Cordyceps fruiting bodies decay and much of the harvest is lost. C. sinensis occurs at altitudes of 3,200 to 4,800 m, but only where average annual precipitation is at least 350 mm and usually more than 400 mm. In the arid reaches of the north-western Tibetan Plateau, where annual precipitation is below 300 mm, C. sinensis is apparently absent.

Difficulties faced in phylogenetic analysis of the genus Cordyceps Cordyceps is a genus of perithecial ascomycetes (Phylum Ascomycota) classified in the Clavicipitaceae, a family supported by molecular phylogenetic analyses as a monophyletic group derived from the order Hypocreales (Artjariyasripong et al., 2001; Rehner and Samuels, 1995; Spatafora and Blackwell, 1993; Suh et al., 1998). Cordyceps species are parasites of insects or fungi, often exhibiting a high degree of host specificity; as a result of this host specificity, the anamorphic forms of some species (e.g., Beauvaria bassiana) are widely used as insect biocontrol agents (Huang et al., 2002). According to molecular phylogenetic analyses, Cordyceps does not represent a single evolutionary lineage; instead, Cordyceps appears to represent several lineages within the Clavicipitaceae (Ito and Hirano, 1997; Artjariyasripong et al., 2001; Sung et al., 2001; Zare et al., 2000). Similarly, the Cordyceps species associated with Lepidopteran hosts do not represent a monophyletic group (Nikoh and Fukatsu, 2000; Park et al., 2001; Sung et al., 2001). Furthermore, there appears to be a high degree of genetic variation within the sinensis species (Chen and Hseu, 1999). These levels of variation create a significant challenge in verifying samples for analysis of Cordyceps sinensis.

The most signiﬁcant challenge working with Cordyceps sinensis is the lack of a well-deﬁned mechanism to identify sample material. Many ascomycetes, including species of Cordyceps, have both an asexual (anamorphic) and sexual (teleomorphic) form. Cordyceps species have been shown in mycological culture studies to be associated with a number of anamorphic genera including Paecilomyces, Beauvaria, Metarhizium, Verticillium, and Tolypocladium (Hodge et al., 1996; Huang et al., 2002; Nikoh and Fukatsu, 2000; Nikoh and Fukatsu, 2001; Obornik et al., 2001; Sung et al., 2001; Zare et al., 2000), and phylogenetically related to several species of yeast-like endosymbionts of insects (Suh et al., 2001). Determining the anamorphic state of Cordyceps sinensis has posed difficulty for researchers; as a result, 22 names in 13 anamorph genera have previously been associated with Cordyceps sinensis (Jiang and Yao, 2002). However, recent molecular evidence (Chen et al., 2001; Chen et al., 2002; Liu et al., 2001) and generation of the anamorphic state from germinated Cordyceps sinensis ascospores (Liu et al., 2001) supports Hirsutella sinensis Liu et al. as being the correct anamorph of Cordyceps sinensis. Many anamorphic fungi, including Hirsutella sinensis, can be cultured under laboratory conditions. Because Cordyceps sinensis is considered to be declining in the wild due to overharvest (Liu et al., 2003), culture of the anamorphic state may therefore be a means of producing material for research and medicinal preparations in the face of a shortage of teleomorph material.

Several Cordyceps species have been described that appear morphologically similar to Cordyceps sinensis, including Cordyceps nepalensis M. Zang & Kinjo, Cordyceps multiaxialis M. Zang & Kinjo, Cordyceps gansuensis Zhang et al., and Cordyceps crassispora Zang et al. However, these species may simply represent morphological variants of Cordyceps sinensis (Kinjo and Zang, 2001; Liu et

9 al., 2001). Whether these morphotypes correspond to differences in pharmacological activity has not been determined. Other Cordyceps species (e.g., Cordyceps militaris) have been shown to have some of the same medicinal properties as Cordyceps sinensis (Wu et al., 2000), but appear to be less highly regarded by consumers and practitioners.

Previous genetic analyses of multiple Cordyceps sinensis populations have examined ribosomal DNA (rDNA) sequence diversity (Chen and Hseu, 1999; Chen et al., 2001; Chen et al., 2002; Kinjo and Zang, 2001; Liu et al., 2001) and patterns of genetic variability exhibited by randomly ampliﬁed polymorphic DNA (RAPD) markers (Chen et al., 1999; Cheng et al., 1998). Variability in ribosomal ITS sequences and 18S ribosomal DNA restriction fragment length polymorphism (RFLP) patterns has been shown to be informative for differentiating Cordyceps sinensis from other Cordyceps species and from market counterfeits (Chen et al., 1999; Chen et al., 2001; Chen et al., 2002; Kinjo and Zang, 2001; Liu et al., 2001), and for generating Cordyceps sinensis-speciﬁc DNA probes (Chen et al., 2002); however, rDNA sequence variation is too low to allow accurate genotyping at the strain level (Chen et al., 2001; Kinjo and Zang, 2001; Liu et al., 2001).

B. THERAPEUTIC APPLICATIONS According to Zhu et al. 1998, Cordyceps, the "miracle ingredient," the power within Cordyceps was discovered 1,500 years ago in Tibetan mountain pastures. About 1,000 years later, the Emperor's physicians in the Ming Dynasty learned about this Tibetan wonder and used this knowledge with their own wisdom to develop powerful and potent uses of this wonder substance known as Cordyceps. Practitioners of Chinese traditional medicine believes that Cordyceps sinensis has a sweetish taste and a warm character; it enters through the kidney and king channels and enhances lung functioning, bolsters the kidney's yang energy, relieves coughing, poor vitality, impotence, spermatorrhea, asthma, aching back and knees, and general debility caused by long term illness and lessens sputum. It is recorded in the historical annals of Swechuan and Materia Medica that Cordyceps sinensis is classed as a warm tonic agent, and is highly effective at replenishing the essence and strengthening the body. This apart, in TCM, Cordyceps has been used to treat conditions including respiration and pulmonary diseases, renal, liver, and cardiovascular diseases, hypo sexuality, and hyperlipidemia. It is also used in the treatment of immune disorders and as an adjunct to modern cancer therapies (chemotherapy, radiation treatment, etc.) (Zhou et al. 1998). Many also believe it to be a medicine for the treatment for impotence, acting as an aphrodisiac in both men and women. Cordyceps is often prescribed for the elderly to ease general aches and pains. Practitioners of TCM also recommend the regular use of Cordyceps to strengthen the body’s resistance to infections, such as colds and flues, and to generally improve the homeostasis of the patient. Modern science is attempting to confirm the efficacy of Cordyceps for most of its traditional uses; however, most medical studies regarding its efficacy remain incomplete.

Ng and Wang (2005) review the chemical constuents and pharmacological properes. The chemical constuents include (a) cordycepin (3̕-deoxyadenosine) and its derivatives, (b) ergosterol, (c) polysaccharides, (d) a glycoprotein and (e) peptides containing a-aminoisobutyric acid. The activities ascribed to the fungus are anti-tumour, antimetastatic, immunomodulatory, antioxidant, anti-inflammatory, insecticidal, anti-microbial, hypolipidaemic, hypoglycaemic, anti-aging, neuroprotective and renoprotective effects: So, a vast a range of properties from a narrow spread

10 of compounds. Polysaccharides account for the anti-inflammatory, antioxidant, anti-tumour, antimetastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects. Cordycepin contributes to the anti-tumour, insecticidal and anti-bacterial activity. Ergosterol (a universal fungal compound) exhibits anti-tumour and immunomodulatory activity.

Cordycepin, 3α-deoxyadenosine, is a derivative of the nucleoside adenosine differing from the latter by the absence of oxygen in the 30 position of its ribose entity. Initially, it was extracted from Cordyceps; however, it is now produced synthetically. Some enzymes do not discriminate between adenosine and so it can participate in certain reactions. For example, it can be incorporated into RNA molecules causing premature termination of its synthesis. It is classified as an anticancer compound. It is interesting that cordycepin, a compound originally isolated from C. militaris as much as 60 years ago (Cunningham et al. 1950), is known to exert Cytotoxic effects through nucleic acid methylation (Kredich 1980).

Over 30 bioactivities, such as immune-modulatory, antitumour, anti-inflammatory, and antioxidant activities, have been reported for wild Cordyceps sp. as well as for its mycelia, and culture supernatants. These bioactivities derive from over 20 bioactive ingredients, mainly extracellular polysaccharides, intracellular polysaccharides, cordycepin, adenosine, mannitol, and sterols, among several others such as cordymin, myriocin, melanin, lovastatin, GABA (γ-amino butyric acid), and cordysinins. The caterpillar fungus Ophiocordyceps sinensis is one of the entomogenous ascomycetes and parasitizes the larva of lepidoptera to form the well-known ‘Yar tsa Gumba’. Although natural O. sinensis have significant pharmaceutical effects, the commercial cultivation of this fungus on larvae of moth to produce fruiting body has not yet been successful.

Table 2: Fungi that have been isolated from natural Chinese Cordyceps and developed into health products (Guo et al. 2003, Ke 2005) Fungus Product Note Hirsutella sinensis Bailing capsule The accepted anamorph of O. sinensis; approved for =Cephalosporium production as one of the state-level new drugs in 1988 dongchongxiacae in China; described in the Chinese pharmacopoeia =Hirsutella hepiali since 2005. =Synnematium sinense Paecilomyces hepiali Jinshuibao capsule Approved as one of the state-level new drugs in 1987 in China; described in the Chinese pharmacopoeia since 2000 Mortierella hepiali Zhiling capsule Approved for production in 1985; described in the Chinese pharmacopoeia since 2010 Cephalosporium Ningxinbao capsule Approved for production in 1985 sinensis Gliocladium roseum Xinganbao capsule Approved for production in 1987 Paecilomyces sinensis Chongcaojun capsule Production has been prohibited since 2001

1. Immunomodulating effects It is now well established that Cordyceps derived polysaccharides exert a significant potentiation of immune function (Wasser 2002). This is thought to be one of the major mechanisms of

11 antitumour activity in Cordyceps. Among the multiple polysaccharides produced by C. sinensis, beta-d-glucans are one class of polymers that have been shown to increase both innate and cell- mediated immune response. These polysaccharides increase the production of such cytokines as TNF-α, interleukins, interferons, NO, and antibodies by the activated immune cells. This activation of immune response may be triggered by polysaccharide binding to specific receptors on the surface of the immune systems cells, called the CR3 receptor (Smith et al. 2002). They are also thought to be involved in cell-to-cell communications, perhaps acting as messenger molecules.

There is an extensive body of research looking at the immune enhancement and immune suppression properties of various species of Cordyceps. This bi-directional regulation of immune function, which can be either up-regulated or down-regulated, is termed immunomodulation, and seems to come from the same mechanism in the body. When Cordyceps is given to a patient in an immune-deficient state, such as cancer, hepatitis or HIV infection, the number and activity of the white blood cells increase. Conversely, if the same Cordyceps is given to someone in a hyper- immune state such as is found in Lupus, Lymphoma or Rheumatoid arthritis, the number and activity of the white blood cells drop, while the red blood cells often increase in number. How can the same compound be both an immune stimulant in some patients and yet act in other patients as an immune depressant? The mechanism appears to be in the differentiation phase of blood cell production. The blood cells are all produced in the bone marrow, primarily in the long bones of the legs. They leave the bone marrow as immature cells, and travel to other organs where they mature into specific types of blood cells such as red blood cells, T-cells, natural killer cells and others. It would appear that Cordyceps exerts some influence over the differentiation mechanism that signals the body where to direct these immature cells for maturation. This mechanism is clearly shown in studies looking at the way Cordyceps affects leukemia cells maturing (Chen et al 1997). This immunomodulation-at-the-differentiation level is like nature’s smart bomb against disease. The body gets the signal it needs to mount an effective response to a disease state, whether the problem is too great an immune response or not enough.

While the drug cyclosporin has allowed some advances in medicine, facilitating the transplant of organs, there has been a drawback to its use. The high toxicity of cyclosporin has caused many patients suffer from serious kidney damage, related to the use of the drug. In 1995, a study was undertaken in China in which 69 kidney-transplant patients were given either cyclosporin alone or in conjunction with C. sinensis, at 3g/day. After 15 days it was clearly evident that the group receiving C. sinensis in addition to cyclosporin had a much lower incidence of kidney damage than the group receiving only cyclosporin, as measured by the levels of urinary NAG, serum creatinine, and blood urea nitrate (Xu et al. 1995).

It has been reported that cordycepin is intracellularly converted into its 5΄-mono-, di- and triphosphates that inhibit the activity of ribose-phosphate pyrophosphokinase and 5- phosphoribosyl-1-pyrophosphate amidotransferase in the de novo biosynthesis of purines (Klenow 1963, Overgaard-Hansen 1964, Chassy and Suhadolnik 1969, Rottman and Guarino 1964). Furthermore, the phosphate esteriﬁed cordycepin inhibits the synthesis of nucleic acids, the polyadenylation of mRNA and the release of mRNA from nuclei to cytoplasm (Cory et al. 1965, Rich et al. 1965, Siev et al. 1969, Rizzo et al. 1972, Penman et al. 1970, Rose et al. 1977, Kletzein

1980). Many studies based on these reports have shown the signiﬁcant promise of cordycepin and its analogues as anti-fungal, anti-tumor and anti-viral agents (Sugar and McCaffrey 1998, Ahn et al. 2000, Koc et al. 1996, Kodama et al. 2000, Richardson et al. 1975, Chapekar and Glazer 1983, Muller et al. 1991). More recently, cordycepin has also been shown to regulate the production of interleukins in T lymphocytes (Zhou et al. 2002).

It is well established that numerous fungal derived simple- and protein-bound polysaccharides exert a significant potentiation of immune function (Wasser 2002). This is thought to be one of the major mechanisms of antitumor action by Cordyceps. Among the multiple polysaccharides produced by Cordyceps, beta-d-glucans are one class of these polymers that have been shown to increase both innate and cell-mediated immune response. These polysaccharides increase the production of such cytokines as TNF-α, interleukins, and interferons, NO, and antibodies by the activated immune cells. This activation of immune response may be triggered by polysaccharide binding to specific receptors on the surface of the immune systems cells, called the CR3 receptor. (Smith et al 2002). They are also thought to be involved in cell-to-cell communications, perhaps by acting as messenger molecules.

Many clinical studies, conducted in China and Japan with cancer patients (Wang et al 2001) with whom Cordyceps was used, have yielded positive results. In one study of fifty patients with lung cancer, who were administered Cordyceps at 6 grams per day in conjunction with chemotherapy, tumours were reduced in size in 46 per cent of patients studied. A trial involving cancer patients with several different types of tumours found that Cordyceps taken over a two-month period at 6 g per day, improved subjective symptoms in the majority of patients. White blood cell counts were kept high while tumor size was significantly reduced in approximately half of the patients (Zhou et al 1998).

There is evidence of another mechanism at play in the Cordyceps antitumor response besides the well-known immune modulation triggered by the polysaccharide compounds. This other mechanism has to do with the structure of at least some of the altered nucleosides found in Cordyceps, exemplified by the compound cordycepin [3´deoxyadenosine]. This is a molecule almost identical to normal adenosine, with the exception that it is lacking an oxygen atom on the ribose portion of the molecule at the 3´ position. The same lack of this 3´ oxygen can be seen in other Cordyceps compounds as well, such as Dideoxyadenosine, (Didanosine™, Videx™). The lack of oxygen at this particular position is thought to be important in a very specific way. The structure of DNA depends on this oxygen to create the bond between adjacent nucleosides. This bond is between the 3´ and the 5´ positions on the ribose portions of the nucleosides, effectively forming the ‘ladder structure’ that holds the DNA together. In the replication of any cell, the first step is the separation of the DNA molecule down the middle, like unzipping, between the pairs of complimentary nucleosides.

The next step is the insertion, one at a time, of new compliment nucleosides. These form hydrogen bonds between the complement pairs, and form phosphate-sugar bonds between the 3´ and 5´ position at the outside edge of the molecule, which is the ribose portion. This, in essence, is the structure that holds the DNA together. The synthesis of the new DNA molecules proceeds

13 apace, with the sequential insertion of new compliment nucleosides one at a time into the newly forming DNA molecule, until the original strand of DNA is replicated twice, each of these strands being exact copies of the original and forming the genetic code for a new generation of cells. That is, this synthesis continues to proceed with the insertion of each new nucleoside, unless a 3´ deoxyadenosine (cordycepin) molecule is pulled in. When this happens, there is no oxygen present at that vital position to form the 3´-5´ bond, and the replication of the new DNA molecule stops. Once the DNA synthesis stops, the cell cannot continue to divide and no new cell is formed. In normal mammalian cells, this insertion of the deoxygenated adenosine is of little importance, as healthy cells have an inherent DNA repair mechanism. When this sort of error occurs, the altered nucleoside (the cordycepin) is removed from the string of nucleosides, and a new segment of adenosine is inserted. However, by their very nature, cancer cells have lost this DNA repair mechanism. (If they could correct their DNA errors, they would not be cancer cells).

Most bacteria and all viruses (including the HIV virus) lack this DNA repair mechanism. When we look at the rate at which cancer cells replicate, it is clear how this mechanism could exert a significant antitumor response. For example, normal healthy breast tissue cells have an average life span of about 10 days, after which they reproduce and a new cell is formed. But breast cancer cells multiply much quicker than healthy cells. They reproduce themselves on average every 20 minutes. This means that the breast cancer cells are replicating about 750 times faster than the surrounding healthy tissue. If the cordycepin were equally toxic to both types of cells, it would be killing off the cancer cells 750 times faster than the healthy cells. But because of that DNA repair mechanism in the healthy cells, cordycepin appears not to interfere with the healthy cell replication, and the tumor-cell kill rate is actually much higher than the 750-to-1 ratio. The same sort of DNA interruption mechanism is responsible for the antitumor effects of some other chemotherapy agents as well. This same mechanism of DNA synthesis inhibition is probably the responsible mechanism for the anti-viral effects seen with cordycepin as well. See the following illustrations for a structural analysis of this mechanism (Holliday 2004b, Liu and Zheng, 1993).

2. Effect on kidneys Nephrosis is a kidney disease, always considered to be serious, which is harmful to the health of the urinary system. It can recur easily and acutely, while it is difficult to cure. The medical treatment for the disease mainly uses glucocorticoids and an immunodepressant. In DCXC, there are about 19 amino acids, alcoholic components, nucleotides, trace elements and vitamins as previously stated. The effects in protecting the kidney are mainly presented as three aspects: (1) a therapeutic effect on toxic kidney injury; (2) protecting against chronic renal function failure; (3) reversing the effect of glomerulonephritis in an animal model (Shi 2005).

Traditional views of the Cordyceps mushroom held that its consumption strengthened the kidneys. Studies have shown that much of Cordyceps’ kidney enhancing potential stems from its ability to increase 17-hydroxy-corticosteroid and 17-ketosteroid levels in the body (Zhou et al. 1998). Chronic renal failure is a serious disease, one often affecting the elderly. In a study among 51 patients suffering from chronic renal failure, it was found that the administration of 3-5 g/day of C. sinensis significantly improved both the kidney function and overall immune function of treated patients, compared to the untreated control group (Guan et al. 1992). Patients with chronic renal

14 failure or reduced kidney function often suffer from hypertension, proteinuria, and anaemia. In a study with such patients, it was found that after one month on C. sinensis, a 15 per cent reduction in blood pressure was observed. Urinary protein was also reduced. Additionally, increases in super oxide dismutase (SOD) were seen. The increase in SOD, coupled with an observed decrease in serum lipoperoxide, suggests an increase in the oxygen free radical scavenging capacity, resulting in reduced oxidative cellular damage (Jiang and Gao 1995).

Simultaneous administration of CS with gentamycin protects the proximal tubular cells from gentamycin toxicity. After the establishment of Kanamycin nephrotoxic ARF, CS treatment causes an earlier recovery from ARF. The possible mechanisms of CS on ARF include protection of tubular cell sodium pump activity, attenuation of tubular cell lysosome over function stimulated by phagocytosis of aminoglycoside, and reduction of tubular cell lipid peroxidation in response to toxic injury (Zhen et al 1992). The protective effect of CS on aminoglycoside nephrotoxicity in elderly patients has also been observed. Patient groups receiving amikacin sulphate for 6 days were administered with CS or placebo for 7 days. CS treated group developed less prominent nephrotoxicity as determined by less urinary NAGase and β-microglobulin (Bao et al 1994). In one of the human clinical study, 57 patients with gentamycin-induced kidney damage were either treated with 4.5 g of Cordyceps per day or by other, more conventional methods. After six days, the group that received Cordyceps had recovered 89 per cent of their normal kidney function, while the control group had recovered only 45 per cent of normal kidney function. The time-to recovery was also significantly shorter in the Cordyceps group when compared with that of the control group (Zhou et al. 1998).

CS also stimulates tubular epithelial cell growth (Tian et al. 1991). Primary cultured rat tubular epithelium was used to investigate the effect of CS on cellular proliferation and metabolism. Incorporation of 3H-TdR into DNA is increased indicating that CS promotes DNA synthesis and cell proliferation. In association with the beneficial effect to reduce aminoglycoside nephrotoxity in vivo, CS may enhance the regeneration of injured tubular cells. A recent hypothesis suggests that the pathogenesis of immunoglobulin A nephropathy (IgAN) involves the deposition of nephritogenic IgA immune complexes (IgAIC) in the kidney that stimulates resting mesangial cells to release cytokines and growth factors. These cytokines and growth factors cause mesangial cell proliferation and release matrix, chemical mediators that lead to the glomerular injury. In cultured human mesangial cells (HMC) stimulation with IL-1 plus IL-6 causes mesangial cell proliferation and increases production of chemical mediators and superoxide anion. Active fractions and a natural product (CS-H1-A) from CS inhibit the activated human mesangial cell proliferation in this model (Lin et al 1999).

3. Hypoglycemic effect Another area of particular interest is the effect of Cordyceps on the blood glucose metabolism system. Cordyceps has been tested on animals and humans to investigate its potential as an agent in blood sugar regulation. In experiments, using diabetic animal models created by treatment of mice with either alloxan or streptozotocin (STZ), blood glucose was significantly reduced by the extract, as compared to the placebo control group (p < 0.05 to < 0.01). The authors suspected that the hypoglycemic effects were mediated by component polysaccharides that were identified by

15 paper chromatography. The polysaccharides consist of five major monosaccharides: L-arabinose, L-xylose, D-mannose, d-galactose, and D-glucose. In fact, CS-F30, a purified polysaccharide isolated from a fermented mycelial Cordyceps product, exhibited potent hypoglycemic activity after oral administration in mice. The hypoglycemic activity was even more potent when the product was administered by injection (Intraperitoneal or intravenous) (Kiho et al 1993). It was further demonstrated that CS-F30 could markedly increase the activity of hepatic glucokinase, hexokinase, and glucose-6-phsphate dehydrogenase.

In one randomized trial, 95 per cent of patients treated with 3 g/day of C. sinensis saw improvement in their blood sugar profiles, while the control group showed only 54 per cent improving with treatment by other methods (Guo and Zhang 1995). In animal studies, isolated polysaccharides have been shown to improve blood glucose metabolism and increase insulin sensitivity in normal animals (Zhao et al. 2002), to lower blood sugar levels in genetically diabetic animals (Kiho et al. 2000), and to positively affect blood sugar metabolism in animals with chemically induced diabetes (Hsu and Lo 2002). The common thread throughout all these trials is the increase in insulin sensitivity and hepatic glucose regulating enzymes, glucokinase and hexokinase. In our own study, the antihyperglycemic potential of the crude extract extracted in 50 per cent ethanol, in sucrose loaded rat model resulted in 27.3 decline in sugar content (Negi 2009).

4. Effects on lung Chinese medicine has characterized C. sinensis as a guardian of respiratory health for more than a thousand years. There have been trials on humans, using Cordyceps to treat many respiratory illnesses, including asthma, COPD (chronic obstructive pulmonary disease), and bronchitis, either alone or as an adjunct to standard antibiotic therapy, and in many studies that have been conducted, it appears to be useful for all of these conditions (Zhu and Rippe 2004, Donohue 1996, Manfreda et al. 1989, Han 1995, Qiuo and Ma 1993, Qu et al. 1995, Zheng et al. 1985, Zheng and Deng 1995). Much of its reputation for protecting the lungs, again, is believed to stem from its ability to promote enhanced oxygen utilization efficacy. In environments lacking sufficient oxygen, mice treated with Cordyceps were able to survive up to three times longer than those left untreated, demonstrating a more efficient utilization of the available oxygen. This provides support for Cordyceps’ long history of use in preventing and treating altitude sickness (Zhu and Rippe 2004). Extracts of C. sinensis have been shown to inhibit tracheal contractions, especially important in asthma patients, as it allows for increased airflow to the lungs. In addition, its anti- inflammatory properties may prove to bring further relief to asthma patients, whose airways become obstructed, because of an allergic reaction resulting in the swelling of the bronchial pathways (Hobbs 1995, Zhou et al. 1998, Halpern 1999). In an unpublished clinical trial conducted at the Beijing Medical University involving 50 asthma patients, symptoms among the group treated with Cordyceps were reduced by 81.3 per cent, within an average of five days; while among those treated with conventional antihistamines, the symptom reduction averaged only 61.1 per cent, and took an average of nine days for symptoms to subside (Donohue 1996, Halpern 1999).

Much of its reputation for protecting the lungs, again, is believed to stem from its ability to promote enhanced oxygen utilization efficacy. In environments lacking sufficient oxygen, mice treated with Cordyceps were able to survive up to three times longer than those left untreated, demonstrating a more efficient utilization of the available oxygen. This provides support for Cordyceps’ long history of use in preventing and treating altitude sickness (Zhu and Rippe 2001). Such efficacy alludes to the use of Cordyceps as an effective treatment for bronchitis, asthma, and COPD. Extracts of C. sinensis have been shown to inhibit tracheal contractions, especially important in asthma patients, as it allows for increased airflow to the lungs. In addition, its anti- inflammatory properties may prove to bring further relief to asthma patients, whose airways become obstructed, because of an allergic reaction resulting in the swelling of the bronchial pathways (Hobbs 1995, Zhou et al. 1998, Halpern 1999).

5. Protection of liver Animal tests and clinical research data show that Cordyceps has a protective effect in liver patients, including those with viral hepatitis A, chronic hepatitis B, chronic hepatitis C, hepatic fibrosis, etc. It enhances organic cell immunological function, reverts HBeAg -positive to HBeAg- negative, improves liver function, inhibits hepatic fibrosis, and so on (Liu and Shen 2003). In recent years, significant progress has been made in the prevention of liver disease. For instance, to find an effective drug to cure patients with chronic hepatitis B, Gong et al (2000) have treated 25 patients with C. sinensis. T lymphocyte subsets (CD4, CD2), hyaluronic acid (HC) and precollagen type III (PC III), were observed before and after treatment. After 3 months of treatment, CD4 and CD4/CD2 ratio increased significantly (P < 0.05), while HA and PC III decreased significantly (P < 0.05) compared with the control. These results suggested that beneficial effects might be obtained by using C. sinensis to adjust the T lymphocyte subset levels and to treat hepatic fibrosis in patient with chronic hepatitis B (Gong et al 2000).

Bioactive components of Cordyceps for liver protection are mostly CPs. The CPs can improve the immunological functions of organic cells, removing harmful components and thus reducing the injury to liver cells. The effects of CPs in protecting the liver were presented as follows (Chen and Jia 2005): (1) protective effect on immune liver injury; (2) effect on patients with chronic hepatitis B; (3) effect on patients with hepatocirrhosis after hepatitis (Qiu et al 1995); (4) protective effect on liver fibrosis (Gong et al 2000). The key to treating liver disease is to inhibit and clear the virus from the liver of patients, regulate the immunological function of the organism, and reverse the developing process of liver fibrosis, etc. C. sinensis and its related products obviously act to hasten macrophage phagocytosis, and enhance the immune function of organisms and possess a strong capacity to clear the virus (Liu ad Zhou 2001).

Cordyceps is commonly used as an adjunct in the treatment of chronic hepatitis B and C. In one study, Cordyceps extract was used in combination with several other medicinal mushroom extracts as an adjunct to lamivudine for the treatment of hepatitis B. Lamivudine is a common antiviral drug used in the treatment of hepatitis. In this study, the group receiving the Cordyceps and other medicinal mushroom extracts had a much better outcome in a shorter period of time than the control group who received only the lamivudine. (Wang et al 2002).

6. Protection of other organs C. sinensis also has obvious effects on other organ systems (Guo and Guo 2000, Xu 2006, Zhang and Yuan 1997). For example, on the central nervous system, C. sinensis has sedative, anticonvulsant and cooling effects. On the respiratory system, C. sinensis has a significant relaxant role in the bronchi, markedly increases adrenaline secretion from the adrenal glands and also has a role in tracheal contraction caused by histamine; it also has an antitussive, expectorant and anti- asthmatic action and prevents pulmonary emphysema. On the endocrine system, C. sinensis has effects as a male hormone; CP can increase plasma corticosterone levels, etc.

Table 3: Major pharmacological functions of Ophiocordyceps sinensis S. No Broad category Reported function 1. Antioxidant Antioxidant and anti-lipid peroxidation activity; suppresses LDL oxidation (Li et al. 2001; Liu et al. 1991). 2. Hepatic function Stimulation of energy metabolism; activation of Kupffer cell function (water-soluble fraction); reduction of post-hepatitis cirrhosis (Wang and Shiao 2000); CordyMax Cs-4 raises ATP in the liver of mice (Manabe et al. 1996, 2000; Dai et al. 2001). 3. Renal function Reduction of aminoglycoside-induced nephrotoxicity, haematuria and proteinuria in experimental IgA nephropathy (IgAN): low MW sterols (Wang and Shiao 2000); Improves kidney function and reduces damage caused by nephrotoxic chemicals. Possible modulation of T-cell mediated immune function (Li et al. 1996). Cordycepic acid has diuretic, antitussive, and anti-free radical activities (Jennings et al. 1998). 4. Endocrine and Stimulation of corticosterone production by cultured rat adrenal cells: steroid system water-soluble fraction (Wang and Shiao 2000; Wang et al. 1998); Increases 17-ketosteroid and 17-hydroxycorticosteroid, increases weight of sexual organs, plasma cortisol and testosterone in rats (Yang 1985; Liu et al. 1997). 5. Cardiovascular Inhibition of platelet aggregation (adenosine and other related function nucleosides), reduction of aconitine, BaCl2 and ouabain-induced arrhythmia: low MW metabolites (Wang and Shiao 2000); Reduced mean arterial pressure, possibly by stimulating release of NO and endothelium- derived hyperpolarizing factor (Chiu et al. 1998; Francia et al. 1999). 6. Anticancer Inhibits tumour growth; antimetastasis of liver Lewis lung carcinoma and activities B16 melanoma cells in mice; inhibits cultured human glomerular mesangial cell induced by LDL (Liu et al. 1997; Chiu et al. 1998; Francia et al. 1999; Nakamura et al. 1999; Zhao-Long et al. 2000). Antitumour function via immunopotentiation and cytokine production: polysaccharides (Wang and Shiao 2000). 7. Immunomodulation Immunopotentiation (polysaccharides); immunosuppressant: cyclosporine-like metabolites and others (Liu et al. 1991); Cordycepin is effective against antibiotic-resistant bacteria (Yang 1985; Chiu et al. 1998; Gong et al. 1990; Lin et al. 1999). 8. Hypoglycemic Polysaccharides lower serum and plasma glucose in STZ- induced (Wang activity and Shiao 2000) and epinephrine-induced hyperglycemic mice (Kiho et al. 1993, 1999).

9. Hepatitis B May adjust protein metabolism and correct inversion of albumin and globulin (Zhao-Long et al. 2000). 10. Improved physical May increase energy by improving utilization of oxygen. Cordycepic acid performance and relaxes bronchia and strengthens adrenal glands (Dai et al. 2001; Zhang et quality of life al. 1997). 11. Anti-aging agent Jinshuibao capsules treat and relieve symptoms of senile deficiency syndrome by inhibiting the formation of monoamine oxidase (Zhang et al. 1997; Song 1992).

Table 4: Pharmacological studies conducted on the efficacy of Cordyceps or its mycelia extracts. Activity Experimenta Experimental dosage Result Reference studied l model Hepatic Mice Cs extract (200mg/kg/day) for increase in [ATP]/ Wang and function 3 weeks in vivo [Pi ]ratio Shiao 2000 Water extract of Cs-4 (0.2 or Zhu et al. 0.4g/ kg) orally for 7 days 1998 Renal Human Cs extract 3-5 gram per day improvement in Holliday and function beings kidney function Cleaver 2004 Rat Cs-4 (0.8g/kg) for 7 days Increases in Zhu et al. accumulated 24 hr 1998 Urine N-acetyl-Beta- D-Glucosaminidase (NAG) Antitumor Mice Cs extract (CI &CI-A) Show antitumor Holliday and activities 1-10mg/kg/day activities Cleaver 2005 Exopolysaccharide fraction Metastasis of B16 Zhong et al. (EPSF) of C.sinensis120mg/Kg) melanoma cells to 2009 Lungs and liver (30,60 &120mg/Kg) was significantly inhabited by EPSF Immuno- Human Cyclosporine + Cs 3g/day Within 15 days Holliday and modulating beings improvement is Cleaver 2005 effects noted in kidney transplant patients Mice 0.1mg/L polysaccharide Production of tumor Zhong et al. prepared from C. sinensis necrosis factor alpha 2009 (TNF-alpha), interleukin (IL)-6&10 Hypoglycemic Human and 3 gram/day of C. sinensis Regulation of blood Holliday and Effect Animals sugar Cleaver 2008 Rats 250 & 500 mg/kg per day for Significantly reduced Lo and Wasser 17 days orally fasting blood glucose 2011 by 27 and 24 % from base lines

Traditional recipes and perceived uses of Cordyceps Du Haled, in his historical account of China in 1736, gave the following account of Cordyceps (cited in Pegler et al. 1994): “You must take five grams of this root entire to the very end, stuff the belly of a tame duck with it, and boil it over a gentle fire; when it is boiled take it out of the duck again, the virtue of which will have entered entirely into the flesh of the duck; eat of this morning and night for eight or ten days together. I accordingly made the experiment when I immediately found my appetite return, and my strength restored…”

Traditionally, it has been found that most local folk/traditional healers use Cordyceps to treat 21 ailments, including erectile dysfunction, female aphrodisiac, malignant tumours, bronchial asthma, bronchitis, diabetes, cough and cold, jaundice, alcoholic hepatitis, among others. The traditional uses of Cordyceps among the Bai, Naxi, Lisu and Tibetan people living in the mountainous Northern Yunnan province include improving eyesight, in the treatment of calcium deficiency (specific to children), diabetes and associated nephropathy, and indigestion (specific to children), to speed up the labour or parturition, and to strengthen the immune system.

In some parts of Nepal, C. sinensis is powdered and combined with the rhizome of Dactylorhiza hatagirea for consumption (Adhikari 2000). It is also used as tonic for yak and sheep. A combination is made with D. hatagirea (D. Don), honey and cow’s milk for tonic and aphrodisiac (Lama et al. 2001). It is widely used as a tonic and aphrodisiac in Thak areas, Mustang. It is taken as a whole orally in combination with honey and cow’s milk (Satyaal 2006). As a tonic and for the purpose of sexual stimulant, people of both sexes normally use a daily combined dose of one dried C. sinensis with half liter of milk and two teaspoons of ghee for a week. Sometimes only a C. sinensis with a cup of milk is also used. Local users believed that if this practice is continued until recovery, every disease could be cure.

The Mykots make a yogurt out of Cordyceps. They milk the yaks, skim the fat from the milk, and soak dried Cordyceps in the milk overnight. In the morning, the milk turns to yogurt. “Yogurt is typically made from Lactobacillus bacteria, which coagulates the proteins. But Lactobacillus is not present in the yogurt that the Mykot make from Cordyceps. Some kind of enzymatic action keeps it from happening. Perhaps Cordyceps yogurt presents an opportunity for a new health food product.” Ancient texts describe a couple of unusual ways to take Cordyceps. One recipe called for the mushroom to be soaked in yellow wine to make a tonic for the relief of pain in the groin and knees. Another described preparing Cordyceps in the belly of a male duck. People suffering from cancer or fatigue were instructed to stuff 8.5 grams of a whole Cordyceps mushroom, with the caterpillar casing still attached, into the belly of a newly killed duck, and boil the duck over a slow fire. After the duck had been boiled, the patient was to remove the Cordyceps and eat the duck meat for 8 to 10 days until healthy.

In another practice, one piece of C. sinensis is put in one cup of local homemade alcohol and left for half hour and drunk in the morning or evening as a tonic. Hot water could be used instead of alcohol. Rich traders and shopkeepers of Dunei and Juphal area in Dolpa keep 5-6 pieces of powdered Cordyceps sinensis and a piece of dried musk (approx 0.5 g) in alcohol (1000 ml) for six months. After six months they drink the product as normal alcohol. They have expressed that such

20 preparations are highly tasteful and good as sexual stimulant and tonic. Regarding the stage of Cordyceps sinensis, young or immature ones (thick and golden larvae with short and light brown aerial fungal part) are good for taste and their values than matured one (with long and dark brown fungal part and light in weight). According to their view main part of Cordyceps is caterpillar part but not an aerial fungal part.

It has been reported that in Dharchula, the locals consume Cordyceps along with local brew, Chyakti. The mode of intake is that Cordyceps is left in the local drink for a period lasting for few days, and then consumed (Garbyal et al. 2004). But I have reservations, for the simple reason that a specimen costing for hundreds of rupees is presently not being consumed or utilized, but are meant exclusively for sell, ultimately destined for markets in Tibet. I am yet to find a single person who routinely consumes Yartsa Gumba! What Garbyal et al. 2004 mentions are simply the possession of knowledge as to the mode of intake, and not a routine practice, or even if yes, then a rarity and definitely confined to those who can afford to! Interestingly, Jiang (1994) ascribes different healing properties of Yartsa Gunbu, as per its mode of preparation (table 5).

Table 5: Dietary uses of Cordyceps in the treatment of the disorders in traditional medicinal dishes (Jiang 1994). S.No. Medicinal dish preparation Use for 1. Cooked with an old duck For patients with cancer, asthenia, or after severe illness 2. Cooked with hen For hyposexuality (especially emission) 3. Cooked with black-bone hen For asthenia (especially Qi-Yin asthenia) 4. Cooked with lean pork For fatigue, male impotence, and kidney asthenia 5. Cooked with sparrow For anti-aging or senescence 6. Cooked with quail For fatigue, poor appetite, kidney asthenia and tuberculosis 7. Cooked with steamed turtle For male and female hyposexuality 8. Cooked with baked abalone Fr chronic bronchitis, COPD, tuberculosis, arteriosclerosis, cataracts, etc.

Wild Cordyceps is usually consumed as part of an elaborate soup or stew. The following recipe comes from the kitchens of the Ming emperors. i. Chicken soup with Astragalus, ginseng, Cordyceps, and dates 1-2 tbsp sesame oil, organic cold-pressed 3-4 slices ginger root, fresh 1 medium brown onion, sliced 1-2 cups root vegetables of your choice (carrot, turnip, rutabaga, daikon), chopped 2-3 chicken legs (or other parts if you like), skinless and hormone-free 1 tbsp dark miso paste 1 tsp white pepper (more or less to taste) ii. Other formulation 2–3 oz Astragalus root (Huang Qi)

1 oz Chinese red ginseng root (Ren Shen) 1 oz American ginseng root (Xi Yang Shen) 5–6 pieces Cordyceps fungus (Dong Chong Xia Cao) 3 pieces Dioscorea yam root (Shan Yao) 1–2 pieces aged tangerine peel (Chen Pi) 3–4 pieces Chinese red date (Da Zao) 2–3 Indian green Cardamom pods or Chinese Cardamom (Sha Ren) 3–4 pieces Poria fungus (Fu Ling)

Fry the sliced brown onion and thinly sliced ginger root in the sesame oil. When slightly browned, add as much chicken as you like. Vegetarians may substitute tofu or tempeh at this stage. Sauté for 5 minutes longer and then add the root vegetables and herbs with enough water to reach 2-3 inches above the ingredients. Bring to a boil and reduce to medium-low. Cook for 45 minutes to 1 hour in a heavy pot with a tight lid. Ten minutes before finishing, add the miso paste (after mixing it in a little water) and the pepper and let it simmer to perfection. Salt can be added if the miso is not salty enough. Those on a low-fat diet can reduce the oil to 1 tsp, but generally fat is not the issue for those eating this soup. iii. Cordyceps Duck Here is a traditional Chinese recipe for preparing Cordyceps Duck: Cordyceps sinensis (12 grams) 1 duck (750 grams) White wine (dash) Scallions (2 tablespoons) Chicken stock (1 quart) Ginger (1 tablespoon)

Soak the Cordyceps in lukewarm water until it is soft. Meanwhile, boil the duck thoroughly. Place the duck in a new pot along with the cooking wine, scallions, soup stock, and ginger. Add salt. Seal the pot tightly and steam for three hours. When done, remove the ginger and scallions. Add pepper.

Table 6: Ethno pharmacological use of O. sinensis S.No. Ethno pharmacological mode of intake use for 1. Improving eyesight In case of the Bai and Naxi tribe, dried Yartsa Gunbu is (specifically for the old grounded and the powder is mixed with raw eggs, which is age) then steamed and taken orally or at times even stewed with chicken. In yet another version, the Lisu people first dry the Yartsa Gunbu over the fire, which is then chewed with hot water or grain alcohol. Additionally, the inhabitants of the Deqin County (TAR), dried Yartsa Gunbu is grounded and mixed with butter, which is then prepared as typical butter tea; sometimes the same is boiled in water and then steeped

for a day before being prepared as tea. 2. Treatment of calcium In case of the Bai and Naxi tribe, dried Yartsa Gunbu is taken deficiency (specifically in with hot water. Very often the Yartsa Gunbu is dried over a case of children) fire. 3. Indigestion (specifically In case of the Bai and Naxi tribe, powder of dried Yartsa in case of children) Gunbu mixed with pork is steamed and taken orally 4. Diabetes and In case of the Bai and Naxi tribe, the dried Yartsa Gunbu is nephropathy taken raw 5. Strengthening immune In case of the Bai and Naxi tribe, the Yartsa Gunbu is steeped system in grain alcohol for a day before being taken as such 6. Hyper-tension and Dried Yartsa Gunbu is taken with hot water. rheumatism 7. Initiation of labour pain Dried Yartsa Gunbu stewed with pork is taken by the women or in parturition 8. As a tonic In case of the Lisu people (TAR), dried Yartsa Gunbu is grounded and then mixed with chicken soup. However, the inhabitants of the Deqin County (TAR), first stew the Yartsa Gunbu with chicken, which is then taken with the chicken soup. In fact, the most common prescribed mode of intake remains stuffing the chicken with Yartsa Gunbu, which is then steamed, Or more simply Yartsa Gunbu is steeped in grain alcohol before being consumed.

Types of diseases to be treated and methods of application The indigenous Bai, Naxi, Lisu, and Tibetan people’s traditional ethnopharmacological use of O. sinensis, separately. The Bai and Naxi people in Mt. Laojunshan use O. sinensis for improving eyesight, treating calcium deficiency, indigenous, diabetes, nephropathy, and strengthening the immune system to prevent disease. The Lisu of Weixi Lisu AC and Tibetan people in Deqin County use O. sinensis as a general tonic or for improving eyesight. The Tibetans in Shangri-La county use O. sinensis for treating hypertension and rheumatism, blindness caused by rheumatism, and for speeding up labour parturition. Different diseases are treated with different methods. For example, for improving eyesight, different peoples have different applicative methods. The Bai and Naxi peoples living on Mt. Laogunshan often first grind O. sinensis, mix the powder with raw eggs, and then steam the mixture to take it orally; sometimes they also stew it with chicken. The Lisu people always first dry it over the fire and then chew it with water or grain alcohol. Tibetan people may grind it and mix it with butter, then drink it just like their typical butter tea; sometimes they boil it in water and let it steep for one day before drinking the tea and eating the caterpillar fungus. The people of Dolpa in Nepal use O. sinensis as a tonic and sexual stimulant (Devkota 2006).

C. CLINICAL STUDIES

1. Improvement of physical performance and quality of life

The effects of Cordyceps extracts on the energy state of mouse liver were examined using in vivo serial 31P NMR spectroscopy. After mice were given water extracts of Cs-4 (0.2-0.4 g/Kg) orally for 4 days, the ratio of adenosine triphosphate (ATP): Inorganic phosphate (Pi) in the liver was significantly increased by an average of 45-55 per cent, as compared with the placebo control group. The elevated ATP: Pi ratios returned to the baseline levels after Cs-4 treatment was discontinued.

In addition to the promotion of higher bioenergy levels by Cordyceps, researchers examined oxygen consumption by mice and their ability to survive after Cs-4 therapy in a hypoxic environment, to elucidate the effects of Cs-4 on oxygen utilization efficiency (Lou et al. 1986). Under conditions of stimulation of oxygen consumption by a sub-cutaneous injection of isoprenaline (300 micro g/kg), Cs-4 extract significantly reduced the oxygen consumption by the mice by 41 per cent to 49 per cent within 10 minutes and by 30 to 36 per cent in the second 10 minutes, as compared to the controls (Lou et al 1986). In a low-oxygen environment, the mice lived 2 to 3 times longer after the Cs-4 treatment. The Cs-4 induced reduction of oxygen consumption and the prolonged survival of treated animals in a hypoxic environment indicated a more efficient use of oxygen to support essential physiological activities of organs/ tissues and greater tolerance to hypoxia-induced acidosis than that of controls.

These above results suggest that natural Cordyceps and its mycelial fermentation products may improve bio-energetic status by improving an internal balance mechanism by which test animals are able to make more efficient utilization of oxygen under economy of energy consumption. This effect may allow animals to manage efficiently inadequate oxygen supply and a basic energy requirement for essential physiological activities, and to promote greater tolerance to hypoxia- induced acidosis than controls. Whether these properties may to some extent account for the apparent overall enhancement of physical capabilities and endurance and anti-fatigue effects found in humans using natural Cordyceps, or its fermentation products as a dietary supplement, is currently the focus of on-going multi-disciplinary research.

2. Antisenescence and oxygen-free radical scavenging activity There is evidence to show that during aging in humans and animals, a considerable accumulation of an excess of oxygen-free radicals occur, which results in oxidative damage to cells and their intracellular organelles, including age- and illness-associated damage to the energy-producing mitochondria. As one mechanism, an aging- or disease-related dramatic reduction in the oxygen free radical scavenging activity of superoxide dismutase (SOD) is believed to be responsible for excessive cellular oxidative damage. The Antisenescence effects of Cordyceps in relation to an ability to activate SOD and scavenge oxygen free radicals were studied in humans and animals.

In placebo-controlled clinical trials (Cao and Wen 1993, Zhang et al 1995), Cs-4 was administered orally to elderly patients with asthenia. In association with the majority showing clinical, subjective improvement (table 7 below), there was a concomitant significant increase in red blood cells (RBC). Associated with an increase of SOD, concentrations of plasma malondialdehyde (MDA), a measure of lipoperoxide (an oxygen free radical species) were significantly decreased in Cs-4 treated elderly patients (Zhang et al 1995).

In addition to the free radical hypothesis, another concern in relation to the aging process is catalytic increase in monoamine oxidase-B (MAO-B), which particularly occurs after age 45 (Sparks et al 1991). This enzyme resides on the membrane of mitochondria. Increases in the catalytic function of MAO-B that occur with aging and in some aging-related mental diseases result in an altered metabolism of dopamine and other monoamine neurotransmitters. Researchers suspect that inhibition of MAO-B activity by Cordyceps may benefit the elderly by increasing brain concentrations of numerous amines. When tested in brain homogenates of mice or rats in vitro, MAO-B activity was significantly inhibited (She 1991). These results were found after treating brain homogenates of rats and mice with extract of Cs-4 or other mycelial fermentation products at a ratio of 1:10.

3. Effects on blood lipid metabolism and atherosclerosis When normal rats were treated with Cs-4 for 1 week, serum total cholesterol (TC) and triglycerides (TG) were significantly reduced (Xu and Zhang 1985). Low-density lipoprotein-cholesterol (LDL-c) and very low-density lipoprotein-cholesterol (vLDL-c) were decreased, while high density lipoprotein cholesterol (HDL-c) was increased. As a result, the ratio of HDL-c: TC showed a very significant increase. Under experimental conditions of stress in rats, intragastric perfusion of Cs-4 produced significant decreases in TC, TG, and LDL-c and an increase in the ratio of HDL-c: TC (Xu and Zhang 1985).

The mechanism of cholesterol-lowering effects of Cs-4 has also been studied. When the concentrated product of Cs-4, CsB-851 (200 mg/kg) was tested in mice, 14C incorporation into newly synthesized cholesterol was significantly reduced by about half (Li et al 1992). This indicated an in vivo inhibition of endogenous cholesterol biosynthesis. It was further demonstrated in this study that CsB-851 (800 mg/kg per day for 3 days) did not change faecal discharge of 3H-neutral cholesterol given by mouth, suggesting that there were no changes in the absorption and excretion of cholesterol via the gastrointestinal tract.

Table 7: Percentage changes in blood lipids in patients with hyperlipidemia after Cs-4 treatment. n Cs-4 dose month TC TG HDL-c ß-Lipoprotein Reference 273 3 g/day 2 -17.7 % -9.2 % +27.2 % Shao et al 1990 62 3 g/day 1 -16 % -26 % + 30 % Qin et al 1995 20 3 g/day 2 -21 % -21 % - 12 % Che and Lin 1996 32 3 g/day 2 -9.7 % -20.5 % +26.7 % Tong 1990

4. Clinical use of Cordyceps to improve respiratory functions Several studies have demonstrated an improvement in clinical symptoms of respiratory diseases after the administration of a Cordyceps-containing medication. For example, the vast majority of patients with various respiratory diseases, such as chronic bronchitis, bronchial asthma, or coronary pulmonale, reported a significant clinical improvement after Cs-4 treatment (Han 1995). In another clinical trial (Qu et al 1995), Cs-4 treated patients with various respiratory diseases, with or without antibiotics, were as improved as a positive control group treated with a known, effective drug, Bai-Ling capsule (Swnnematium sinense). In other studies, Cs-4 treatment of patients suffering from chronic bronchitis or bronchitis with asthma resulted in very high rates of

25 clinical improvement, versus the representative control groups (Zheng and Deng 1995, Yang 1995, Zheng et al 1985). The clinical improvement rates were significantly greater than negative controls (al p < 0.01), or as effective as positive controls in different studies.

Table 8: Summary of clinical effect of Cs-4 in the treatment of the respiratory diseases Cs-4 Weeks of n Clinically improved References dosage treatment n Percentage improvement* 3 g/day 2 to 12 100 92 92 Han 1995 3 g/day 4 30 26 87 Qu et al 1995 3 g/day 4 20 17 85 Zheng and Deng 1995 4.5 g/day 4 27 21 78 Yang 1995 4.5 g/day 4 20 18 90 Zheng et al 1985

5. Effects on the cardiovascular system Although Cordyceps is mainly used to treat respiratory and renal diseases, it has been used extensively in treating cardiovascular diseases. For instance, the treatment of arrhythmias with Cordyceps is reported to have a rate of efficacy ranging from 75 per cent to 88 per cent (Tang and Jiang 1994, Li et al 1985, Liu et al 1990, Xu and Zheng 1994, Yan et al 1992a). In a 3-month, open- label clinical trial, Cs-4 was used to treat 38 elderly patients with varying degrees of intractable arrhythmias caused by varying forms of heart disease (Tang and Jiang 1994). Among them, the majority diagnosed with super ventricular arrhythmia experienced complete or partial recovery of their ECG with clinical improvement. Among patients suffering from ventricular arrhythmia or complete blockage of the right branch, the majority gained complete or partial recovery of their ECG. The investigators concluded that Cs-4 appears not only to be effective for tachyarrhythmia, but also for Brady arrhythmia, and that the longer the therapy, the better the clinical improvement. A randomized, double-blind, placebo-controlled trial (Xu and Zheng 1994) demonstrated that treatment using another cultured mycelial Cordyceps product, known as Ningxinbao capsule (Cephalosporium sinensis), appeared very effective in completely to partially improving ECG in patients with atrial or ventricular premature beats, as compared to placebo controls. The total effective rate for the treatment group as significantly higher compared to the control group.

Animal studies, in vivo or with isolated organs, have attempted to explore the underlying mechanisms of the cardiovascular benefits of Cordyceps. Pharmacological activities of natural Cordyceps and its mycelial fermentation products on the cardiovascular system examined on preclinical animal and isolated issues are being summarized below; i. Dilation of the arteries and improvement of nutritional blood supply to organs and the extremities- Dilation of arteries by natural Cordyceps or its fermentation products has been documented, which includes dilation of aorta (Tsunoo et al 1995), of coronary arteries, cerebrovascular, and peripheral arteries (Feng et al 1987). ii. Reducing the heart rate- Natural Cordyceps and its mycelial fermentation products, such as Cs-4, have been found to be effective in reducing the heart rate (Feng at al 1987, Lou et al 1986).

iii. Antiarrhythmic effects- Cs-4 ethanol extract was studied for its antiarrhythmic effects in animals with experimental arrhythmias (Lou et al 1986). Cs-4 (0.5 g/kg) prolonged the induction period of arrhythmia (n=10, p < 0.01) onset induced by administration of aconitine (0.25-0.33 g/kg) and shortened the duration of the arrhythmias (p < 0.001) when compared with the control rats treated with placebo (n = 11). Majority of the arrhythmias occurring in the Cs-4 treated rats were atrial arrhythmias and bigeminal or trigeminal arrhythmias, and were generally much less severe, in contrast to those occurring in the control group.

Table 9: Summary of clinical efficacy of Cordyceps in the treatment of arrhythmias Dose Treatment n Improved Reference (g/kg) (week) n % CS-4 3 1 38 31 81.6 Tang and Jiang 1994 Cephalosporium sinensis 1.5 2 200 149 74.5 Lie et al 1985 Cephalosporium sinensis 3 4 37 30 81.1 Liu et al 1990 Cephalosporium sinensis 3 2 32 25 78.1 Xu and Zheng 1994 Cephalosporium sinensis 1.5 12 50 44 88.0 Yan et al. 1992a

C. sinensis extracts were tested on myocardial ischaemia/ reperfusion injury in rat isolated hearts. Xu et al. (2000) reported that the alcoholic extracts had a protective effect on myocardial injury induced by Adriamycin in rats. The mechanism was suggested to be that mannitol, amino acids and polysaccharides in the extracts played an important role; all the substances had the effect of nourishing the myocardium and enhancing its anti-injury capacity (Xu et al 2000). With the same experimental methods, Gu et al. (2002) reported that Lung fit (C. sinensis compounds) could be beneficial for myocardial ischemia/reperfusion injury as well. On the other hand, C. sinensis has an inhibitory effect on arrhythmias induced by aconitine, barium chloride and adrenaline, and can increase nutritional myocardial blood flow, thereby improving myocardial ischemia (Guo and Yang 1999). Considering antihypertensive effects, Wu et al. (2001, 2005), observed the effect of C. sinensis on blood pressure in renal hypertensive rats. The results showed that the renal hypertension could be prevented significantly by treatment with C. sinensis since the cardiac hypertrophy and vascular remodelling were reversed. Therefore, they came to the conclusion that C. sinensis has a curative role in renal hypertension (Wu et al 2001, 2005). In short, C. sinensis has many effects on the cardiovascular system, such as having a negative frequency, reducing myocardial oxygen consumption, improving myocardial ischemia, anti-platelet aggregation and anti-arrhythmic effects, etc.

In studies of patients suffering from chronic heart failure, the long-term administration of Cordyceps, in conjunction with conventional treatments-digoxin, hydrochlorothiazide, dopamine, and dobutamine- promoted an increase in the overall quality of life. This included general physical condition, mental health, sexual drive, and cardiac function, compared to the control group.

6. Cordyceps and human organ transplants While the drug Cyclosporin has allowed some miraculous advances in medicine in that it makes it possible to transplant organs, there has been a drawback in its use. The toxicity of Cyclosporin is

27 high and many patients suffer from serious kidney damage related to the use of Cyclosporin. In 1995, a study was undertaken in China, where 69 kidney transplant patients were given either Cyclosporin alone, or in conjunction with Cordyceps sinensis at 3 grams per day. After 15 days it was clearly evident that the group receiving Cordyceps sinensis in addition to the Cyclosporin had a much lower incidence of kidney damage then the group receiving only the Cyclosporin, as measure by the levels of urinary NAG, serum creatinine and blood urea nitrate (Xu et al 1995).

Many drugs used in the prevention or treatment of diseases and the amelioration of symptoms can suppress the immune system to a considerable extent. Some examples of these are the steroid drugs such as Prednisone, and many (if not all) of the chemotherapy agents used in combating cancer, such as Cyclophosphamide and 5-FU. In a study of Cordyceps on lymphomic mice to see if it would increase their life span, it was shown that the mice lived significantly longer with the addition of 50 mg/kg/day orally of a hot water extract of Cordyceps sinensis. Furthermore, a group of mice in this test were being treated with cyclophosphamide, which drastically suppressed their immune function. The group of mice receiving the Cordyceps along with the cyclophosphamide had no significant reduction in immune function, but rather their immune function return to normal as measured by the IgM and IgG response as well as macrophage activity (Yamaguchi et al 1990).

7. In the treatment of male/female sexual dysfunction Cordyceps has been used for centuries in traditional Chinese medicine to treat male and female sexual dysfunction, such as hypolibidinism and impotence. Preclinical data on the effects of C. sinensis on mice showed sex-steroid-like effects (Hobbs 1995, Mizuno 1999). Human clinical trials have demonstrated similarly the effectiveness of Cordyceps in combating decreased sex-drive and virility (Zhu and Rippe 2004, Guo 1986). In general, clinical trials have been conducted using 3–4.5 g of C. sinensis per day, except in cases of severe liver disease, where the dosage has usually been higher, in the range of 6–9 g/day (Hobbs 1995, Mizuno 1999). In yet another clinical study, involving 189 male and female patients with decreased libido and desire showed improvement of symptoms in 66 per cent of cases. A double-blind study conducted by the Institute of Materia Medica in Beijing showed an 86 per cent improvement in female libido and desire. The most dramatic physical proof came from a fertility study involving 22 males which showed that, after eight weeks of taking a Cordyceps supplement, their sperm count increased by 33 per cent, their incidence of sperm malformations decreased by 29 per cent and their sperm survival rate increased by 79 per cent (Miller 2009). The presence of amino acids, vitamins, zinc, and other trace elements found in Cordyceps are hypothesized to account for increased sperm survival rates, as demonstrated in clinical and preclinical studies (Guo, 1986).

The effect of a water-soluble extract of CS on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells has been demonstrated (Wang et al. 1998). The corticosterone production by adrenal cells, determined by RIA, is increased by CS treatment. The stimulatory effect can be seen 1 hr after CS treatment and maintained for up to 24 hr. The CS- induced steroidogenesis is different from that for ACTH, since intracellular cAMP level is not increased in CS-treated cells. Combined treatment with calphostin C, a PKC inhibitor, blocks the

28 effect of CS on steroidogenesis, suggesting that activation of PKC is responsible for the CS-induced steroidogenesis (Wang et al. 1998).

8. Effect on physical stamina The best-known medicinal action of Cordyceps is in the increase of physical stamina. In 1993, the Chinese National Games brought this mushroom to the attention of the world's sporting authorities. A group of nine women athletes who had been taking Cordyceps shattered nine world records (Anonymous 1997). Clinical research has shown that Cordyceps use increased cellular bioenergy-ATP (adenosine triphosphate)-by as much as 55 per cent. Increased synthesis of ATP and faster energy recovery has been reported (Dai et al. 2001, Manabe et al. 1996). It would seem that Cordyceps improves the internal balance mechanism, thus making the utilization of oxygen more efficient. These properties may account for the overall physical enhancement, the extra endurance and the anti-fatigue effects that are seen in humans using Cordyceps. Cordyceps was shown to improve significantly the maximum amount of oxygen these people were able to assimilate. Chinese studies of cardiovascular illnesses have shown that ethanol extracts of Cordyceps mycelia and Cordyceps fermentation solutions caused a change in the biological action that allowed for an increase in cellular oxygen absorption by up to 40 per cent. Various studies have shown that Cordyceps sinensis improved the flow of blood in the body by relaxing the smooth muscles of the blood vessels and allowing them to expand, and also enhanced the functioning of the heart and lungs. Cordyceps therefore prevents or reduces the contraction of blood vessels which interferes with blood flow in the legs-the main cause of tired legs (Miller 2009).

9. In possible treatment of cancer Although Cs has been mainly used in traditional medicine to protect and strengthen the lung and kidney, it has shown remarkable antitumour activities in several in vitro and in vivo studies (Yamaguchi et al., 1990; Bok et al., 1999; Huang et al., 2000). Cs has also been recommended for cancer prevention and treatment or as an adjuvant drug of cancer chemotherapy (Zhu et al., 1998; Ji 1999). In most cases, the antitumour effects of Cs and its beneficial effect to cancer therapy have been attributed to its health-protecting and immuno-modulatory functions. Recently, a sterol compound H1-A isolated from Cs has been shown to inhibit autoimmune disease in MRL lpr/lpr mice, and promote apoptosis and to suppress the proliferation of human mesangial cells (Yang et al., 1999, 2002). Several clinical studies with cancer patients have been conducted in China and Japan, using a therapeutic dose of 6.0 grams of Cordyceps per day for over a two months period. In one study with 50 lung cancer patients who were administered Cordyceps in conjunction with chemotherapy, tumours reduced in size in 46 per cent of patients.

The crude methanolic extract of CS fruiting bodies inhibits the growth of tumour cell lines, namely K562, Vero, Wish, Calu-1, and Raji cells (Kuo et al. 1994). After further fractionation by using silica gel column chromatography, two fractions (namely CS-36-39 and CS-48-51) significantly inhibit the growth of these tumour cells. The inhibitory activities are not due to the polysaccharides, which have been eliminated in the extraction and chromatographic separation. These two fractions do not contain cordycepin, either. This suggests that low molecular-weight tumour cell growth inhibitors, other than cordycepin and polysaccharides, are present in CS. Antitumour activity of a warm water extract of CS (ECS) against murine tumour cell lines has been observed (Yoshida et al.

1989). Two antitumour sterols, namely 5α, 8α-epidioxy-24(R)-methylcholesta-6, 22-dien-3β-D- glucopyranoside and 5, 6-epoxy-24(R)-methylcholesta-7, 22-dien-3β-ol have been isolated from the methanolic extract of cultured mycelia of CS (Bok et al. 1999). The glycosylated form of ergosterol peroxide at 10 μg/mL inhibits the proliferation of K562, Jurkat, WM-1341, HL-60 and RPMI-8226 tumour cell lines more potently than its aglycone, 5α, 8α-epidioxy-24(R)- methylcholesta-6, 22-dien-3β-ol.

The water extract of CS (WECS) inhibits spontaneous liver metastasis of Lewis lung carcinoma (LLC) and B16 melanoma cells in syngeneic mice (C57BL/6J) (Nakamura et al. 1999). Animals were given a s.c. injection of LLC and B16 cells and sacrificed 20 and 26 days after tumour inoculation, respectively. WECS was administered daily (p.o. 100 mg/kg bw) in the experiment of LLC and in a dose of 100 or 200 mg/kg bw. In the experiment of B16 from one week before tumour inoculation, the relative liver weight of tumour-inoculated mice increases due to tumour metastasis. CS treatment reduces the liver weight in both LLC and B16 experiments indicating that CS has an antimetastatic activity that is not due to cordycepin. Inhibitory effects of Cordyceps on carcinogenesis of the fore stomach in mice (Liu 1984) and antitumour activities of CS and cultured Cordyceps mycelia (Du 1986) on Lewis lung cancer of mice (Zhang 1987) have also been demonstrated. Antitumour and immuno-stimulating activities were observed in the treatment of mice inoculated with Sarcoma 180 tumour cells, when treated with an ethanol extract of C. sinensis (Shin et al. 2003).

The bioactive components in Cordyceps that have antitumour activity are mainly the polysaccharides, sterols and adenosine. The antitumour action of CP has been proved extensively (Yuan et al. 2005, Miyazaki et al. 1977, Ohno et al. 1986, Cheng et al. 2006) and that of sterols and adenosine is a hot research topic in Cordyceps (Jagger et al. 1961, Huang et al. 2007). New antitumour compounds have been continually isolated, and the effects of Cordyceps extract on tumour growth have been evaluated (Bok et al. 1999, Ohta et al. 2007). For instance, Zhang et al. (2005) compared the antitumour effects and chemical components of the extracts of cultivated C. sinensis with natural C. sinensis. The Cordyceps was extracted with petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH) or water. The cytotoxicity of all the extracts was observed with MTT assay on a breast cancer cell line (MCF-7), a mouse melanoma cell line (B16), a human premyelocytic leukaemia cell line (HL-60) and a human hepatocellular carcinoma cell line (Hep G2). The chemical constituents in the extracts were analysed by HPLC. The results showed that all of the extracts from the Cordyceps mushroom had much stronger cytotoxicity on the B16 cell line and the EtOAc extract had the most potent activity; the chemical components of all the extracts were analysed and ergosterol and adenosine were found to be potential compounds (Zhang et al. 2005).

We still do not know the mechanism by which Cordyceps inhibits the growth of various cancer cells; inhibition might occur by one of several means, summarized as follows: (1) enhancing immunological function and non-specific immunity (Sun et al. 2002); (2) selectively inhibiting RNA synthesis, thereby affecting the protein synthesis (Wu et al. 2005); (3) restricting the sprouting of blood vessels (Yoo et al. 2004); (4) inducing tumour cell apoptosis (Koc et al. 1996); (5) regulation of signal pathways (Fuller et al. 1987); (6) antioxidation and anti-free radical activity (Li et al.

2003); (7) anti-mutation effect; (8) interfering with the replication of tumour-inducing viruses (Li et al. 2003); inducing nucleic acid methylation (Ji et al. 2005).

10. Apoptosis Apoptosis, or programmed cell death, is an essential event in organism development (Hidalgo and French- Constant, 2003; Vaux and Korsmeyer, 1999) and homeostasis (Kucharczak et al., 2003, Cory and Adams, 2002); however, it is becoming clear that numerous disorders such as stroke (Zheng et al., 2003), myocardial infarction (Krijnen et al., 2002), and HIV (Buenz and Badley, in press) incorporate apoptosis in their etiology and pathogenesis. There are numerous events that can induce a cell to undergo apoptosis (Nagata, 1997) and since the implication of apoptosis in various disease states as an effector mechanism, the ability to inhibit apoptosis has emerged as an important potential therapy. Interestingly, while inducing apoptosis has already proven an efficient method to treat cancer (Hu and Kavanagh, 2003), the ability to inhibit apoptosis in a clinical setting is just starting to be explored (Liston et al., 2003). The potential for a cell to undergo apoptosis exists in a balance between endogenous factors characteristic of apoptosis induction, such as Bax (Degli Esposti and Dive, 2003; Scorrano and Korsmeyer, 2003), and factors characteristic of apoptosis inhibition, such as Bcl-2 (Cory et al., 2003; Gross et al., 1999). Once a cell receives sufficient pro-apoptotic stimuli, or lack of anti-apoptotic stimuli, the effector caspase, a family of cysteine aspartate proteases, are activated (Fischer et al., 2003). Cells undergoing apoptosis experience a cascade of events that ultimately result in phenotypic changes such as phosphotydylserine expression on the outer cell leaﬂet (Maderna and Godson, 2003), nuclear condensation, and DNA fragmentation (Nagata et al., 2003)

Many disorders (e.g. stroke, myocardial infarction and HIV) incorporate apoptosis in their etiology and pathogenesis. The ability to inhibit apoptosis has emerged as an important potential therapy (e.g. cancer). There are reports of CS extracts inhibiting and inducing apoptosis (Shahed et al., 2001); this is not contradictory but allows a foundation to determine the molecular mechanism of activity. Indeed, Cordyceps may contain compounds that inhibit apoptosis; however, conflicting evidence has been obtained. The fungus can scavenge reactive oxygen species by inhibiting malondialdehyde formation by SIN-1, the peroxynitrite generator, which has been confirmed by xanthine oxidase, hemolysis, and lipid peroxidation assays.

Furthermore, an isolated extract of CS, H1-A (this is reported as a pure compound (see above), inhibited induced apoptosis, by permeabilizing the cell membrane and up regulating nitric oxide synthase (Trubiani et al., 2003). On the other hand, extracts of CS did not inhibit hydrogen peroxide-induced apoptosis (Buenz et al., 2004); in reactive oxygen species model (Fauconneau et al., 2002). Alternatively, CS down-regulated apoptotic genes and modulated apoptosis in a rat kidney ischemia reperfusion model. Reported anti-apoptotic effects of CS include in (a) the mouse (anti-cytotoxic activity, anti-oxidant activity, cell proliferation inhibition), (b) cell culture (anti- proliferation activity, cell proliferation inhibition, hemolysis inhibitory activity, lipid peroxide formation inhibition, radical scavenging effect), (c) human cells (proliferation inhibition, natural killer cell inhibition, tumour necrosis factor inhibition) and (d) the rat (gene expression inhibition).

Furthermore, decreases in Fas, Fas ligand and tumour necrosis factor expression and decreased caspase-3 activity have been demonstrated (Shahed et al., 2001). CS inhibited TNF-a expression (Kuo et al., 1996). However, when apoptosis was initiated via a Fas agonist antibody (CH- 11) (Alderson et al., 1994), aqueous and alcohol extracts of CS did not rescue cells induced by Fas receptor ligation (Buenz et al., 2004). Furthermore, cell cycle arrest and/or inhibition of proliferation yield cells resistant to apoptosis. Papers concerning proliferation of leukemic U937 cells (Chen et al., 1997) and glomerular mesangial cells inhibition (Zhao-Long et al., 2000; Lin et al., 1999) may be explained by conferring an apoptotic resistance to cells: the alteration may involve p53 (Fridman and Lowe, 2003) or NF-jB (Karin et al., 2004).

Overall, aqueous, and organic extracts of CS have the ability to inhibit apoptosis. Cells pre- incubated with CS extracts were equally sensitive to hydrogen peroxide and Fas-mediated apoptosis. Thus, the putative antioxidant and anti-apoptotic properties of CS were insufficient to rescue cells from apoptosis in vitro (Buenz et al., 2004). Furthermore, cancer chemotherapeutics have involved the ability to induce apoptosis. Hence the polysaccharide fraction (H1-A) induced apoptosis by inhibiting (a) phosphorylation of Bcl-2 and Bcl-xL and (b) apoptosis induced by dimethyl sulfoxide (Yang et al., 2003). These data require to be confirmed by further work (Buenz et al., 2005). Finally, direct cytotoxic activity may be a factor (Nakamura et al., 1999a, b; Kuo et al., 1994; Sato, 1989; Buenz et al., 2005).

Currently, there are reports of Cordyceps sinensis extracts both inhibiting (Table 1) and inducing apoptosis (Table 2). Most of these reports reﬂect a phenomenon level of observation, such as treatment with Cordyceps sinensis resulting in decreased caspase-3 activity (Shahed et al., 2001). While these reports do not examine the mechanism of action, they do facilitate a necessary foundation to allow examination of the molecular mechanism.

Table 10: Reported anti-apoptotic effects of Cordyceps sinensis Reported functions Model Concentration Reference Anti-cytotoxic activity Mouse 150 mg/kg Yu et al. 1993 Antioxidant activity Mouse 50 mg/kg Yamaguchi et al. 2000b Anti-proliferation activity Cell culture 100 µg/ml Zhao and Xia 2000 Cell proliferation inhibition Cell culture 10 µg/ml Chen et al. 1997 Cell proliferation inhibition Human adult 71 µg/ml Kuo et al. 1996 Cell proliferation inhibition Human adult 40 µg/ml Lin et al. 1999 Cell proliferation inhibition Mouse 1.0 % of diet Lin et al. 1999 Gene expression inhibition Rat 0.5 ml per animal Shahed et al. 2001 Hemolysis inhibitory activity Cell culture 1.5 mg/ml Li et al. 2001 Lipid peroxide formation Cell culture 5.0 mg/ml Li et al. 2001 inhibition Natural killer cell inhibition Human adult 12.9 µg/ml Kuo et al. 1996 Radical scavenging effect Cell culture 5.0 mg/ml Shahed et al. 2001 Radical scavenging effect Cell culture 0.08 mg/ml Li et al. 2001 Tumor necrosis factor inhibition Human adult 2.7 µg/ml Kuo et al. 1996

Anti-tumour activity Mouse Not stated Zang et al. 1985 Anti-tumour activity Mouse 5.0 g/kg Xu et al. 1992 Cell proliferation stimulation Cell culture 10 µg/ml Chen et al. 1997 Cytotoxic activity Cell culture 2.0 µg/ml Kuo et al. 1994 Cytotoxic activity Cell culture 500 µg/ml Sato 1989 Cytotoxic activity Cell culture 10 µg/ml Nakamura et al. 1999 Metastasis inhibition Mouse 100 mg/kg Nakamura et al. 1999

11. Erythropoiesis and hemopoiesis CS stimulates erythropoiesis in mouse bone marrow (Li et al 1993). A preparation of CS (CS-Cr) stimulates proliferation of erythroid progenitor cells (CFU-E and BFU-E) in LACA mouse marrow in vivo and in vitro. The numbers of CFU-E and BFU-E increase after a 5-day treatment with CS-Cr (100, 150 and 200 mg/kg). Higher doses (> 150 mg/kg) result in a reduction of the peak of CFU-E and BFU-E. Ara-C increases in the percentage of CFU-E and BFU-E cells in S-phase after CS-Cr treatment. Pre-treatment of mice with CS-Cr protects CFU-E and BFU-E against harringtonine (a cytotoxic agent). Addition of CS-Cr (150-200 μg/ml) to culture system stimulates the generation of CFU-E and BFU-E in vitro. The stimulatory action of CS-Cr on fibroblast colony-forming units (CFU- F) proliferation has been observed in vivo and in vitro (Li et al 1993). Platelet hemopoiesis and ultra-structure changes in mice treated with natural CS and cultured mycelia have also been observed (Chen 1987).

Oral administration of (50 mg/kg) of CS-OHEP, a crude polysaccharide preparation obtained from 5 per cent sodium hydroxide extraction, reduces plasma glucose without affecting plasma insulin level in normal mice. A neutral polysaccharide (CS-F30; MW about 45 kDa) exhibited higher hypoglycemic activity than CS-OHEP by i.p. injection. CS-F30 is composed of galactose, glucose, and mannose in molar percent of 62:28:10. CS-F30 exhibits a hypoglycemic activity in genetic diabetic mice after i.p. administration. It also quickly reduces plasma glucose in normal and STZ- induced diabetic mice after i.v. administration. Administration of CS-F30 to normal mice increases hepatic glucokinase, hexokinase and glucose-6-phosphate dehydrogenase activities and reduces hepatic glycogen content. CS-F30 also lowers the plasma triacylglycerol and cholesterol levels in mice (Kiho et al. 1996).

A study using murine models verified that oral administration of a hot water extract of C. sinensis consequently resulted in the activation of macrophages, thereby increasing the production of GM- CSF and IL-6, which act on the systemic immune system (Koh et al. 2002). In a study of mice subcutaneously implanted with lymphoma cells, oral administration of an extract of C. sinensis led to a decrease in tumour size and a prolonged survival time (Yamaguchi et al. 1990). Furthermore, mice treated with cyclophosphamide, which suppresses immune function, also treated with the same hot water extract saw their immune function return to normal, as measured by the IgM and IgG response and macrophage activity (Yamaguchi et al. 1990). The median survival time of the treated mice compared to untreated controls was over 300 per cent, and the lack of activity of the extract against EAC cells grown in vitro indicated that the antitumour effect in the mice may be mediated through immune enhancing activity, rather than directly (Yoshida et al. 1989).

D. CHEMICAL CONSTITUENTS AND PRINCIPAL BIO-ACTIVE COMPOUNDS ISOLATED

Cordyceps contains a broad range of compounds, which are considered nutritional (Hobbs 1995, Holliday et al. 2004). It contains all of the essential amino acids, vitamins E and K, and the water- soluble vitamins B1, B2, and B12. In addition, it contains many sugars, including mono-, di-, and oligosaccharides, and many complex polysaccharides, proteins, sterols, nucleosides, and trace elements (K, Na, Ca, Mg, Fe, Cu, Mn, Zn, Pi, Se, Al, Si, Ni, Sr, Ti, Cr, Ga, V, and Zr). Chemically 25 to 32 per cent of the herb consists of crude protein Cordycepin [3α-deoxyadenosine (C10H13O3N6)] and Cordycepic acid (D-mannitol). It is relatively non-toxic and is thus generally not prescribed as medicine or drug but as a nutritive supplement/additive to be cooked with meat (Huang 1999).

Potentially bioactive constituents o The molecular weight of cordycepin, C10H13N5O3, is 251. Its melting point is 230-231 C, and maximum absorption is at 259 nm. It can be dissolved in saline, warm alcohol or methanol, but not in benzene, ether or chloroform, so many researchers in laboratories used sterilized saline and phosphate-buffered saline (PBS) as solvent (Li and Jiang 2005). The chemical constituents of C. sinensis were first studied in 1957, when a crystalline substance was isolated and the structure was imprecisely concluded to be 1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid (Chatterjee et al 1957). Subsequently, the structure of the crystalline substance named ‘cordycepic acid’ was identified by Sprecher and Sprinson (1963) as D-mannitol. Mannitol is a major bio-product with important biological activity.

Cordycepic acid, an isomer of quinic acid, is one of the main active medicinal components. The chemical constituents of C. sinensis were first studied in 1957, when a crystalline substance was isolated and the structure was imprecisely concluded to be 1,3,4,5-tetrahydroxycyclohexane-1- carboxylic acid (Chatterjee et al. 1957). Subsequently, the structure of the crystalline substance named ‘cordycepic acid’ was identified by Sprecher and Sprinson (1963) as D-mannitol. Mannitol is a major bio-product with important biological activity, and exists in the wild in the roots, stems and leaves of plants, while more is found in edible fungus, carrot and lichens. In general, the content of cordycepic acid in DCXC is 7-29%, differing in the various growing stages of the fruiting bodies (Jiang 1987).

Chemically, mannitol is an alcohol and a sugar, or a polyol; it is similar to xylitol and sorbitol. However, mannitol has a tendency to lose hydrogen ions in aqueous solution, which causes the solution to become acidic. For this reason, it is not uncommon to add a substance to adjust its pH, such as sodium bicarbonate. The chemical formula of mannitol is C6H14O6, its molecular weight is 182, its melting point is 166oC, its relative density is 1.489 (20oC) and its boiling point is 290-295oC (467 kPa). Mannitol, as a functional polyol with notable properties, has been widely used in the medicine and food industries. The content of mannitol varies with the original habitat. In general, there is about 29-85 mg/g in Cordyceps fruiting bodies; the mannitol content in the mycelia of Cordyceps is higher than in fruiting bodies (Xu 1997).

Cordycepin and cordycepic acid were the initial bioactive compounds first isolated from C. militaris. Ng and Wang (2005) review the chemical constituents and pharmacological properties.

The chemical constituents include (a) cordycepin (30-deoxyadenosine) and its derivatives, (b) ergosterol, (c) polysaccharides, (d) a glycoprotein and (e) peptides containing a-aminoisobutyric acid. The activities ascribed to the fungus are anti-tumour, antimetastatic, immunomodulatory, anti-oxidant, anti-inflammatory, insecticidal, anti-microbial, hypolipidemic, hypoglycemic, anti- aging, neuroprotective and renoprotective effects: So, a vast a range of properties from a narrow spread of compounds. Polysaccharides account for the anti-inflammatory, antioxidant, antitumour, anti-metastatic, immunomodulatory, hypoglycemic, steroidogenic and hypolipidemic effects. Cordycepin contributes to the anti-tumour, insecticidal and anti-bacterial activity. Ergosterol (a universal fungal compound) exhibits anti-tumour and immunomodulatory activity.

Cordycepin, 3α-deoxyadenosine, is a derivative of the nucleoside adenosine differing from the latter by the absence of oxygen in the 30 position of its ribose entity. As such is may be quite common. Initially, it was extracted from Cordyceps; however, it is now produced synthetically. Some enzymes do not discriminate between adenosine and so it can participate in certain reactions. For example, it can be incorporated into RNA molecules causing premature termination of its synthesis. It is classified as an anticancer compound. The data suggests that the inhibitory effect of cordycepin might be associated with the down-regulation of Ca2+ and the elevation of cAMP/cGMP production. This result has obvious significance for prevention of thrombus formation. Finally, cordycepin inhibited the growth of B16 melanoma cells inoculated subcutaneously into right murine footpads (Yoshikawa et al. 2004).

Chen and Chu (1996) announced the characterization of cordycepin and 2α-deoxyadenosine, using nuclear magnetic resonance (NMR) and infrared spectroscopy (IR) in an extract of C. sinensis. Other components found included various saccharides and polysaccharides, including cyclofurans, which are cyclic rings of five-carbon sugars (whose function is yet unknown), beta-glucans, beta- mannans, cross linked beta-mannan polymers, and complex polysaccharides consisting of both five- and six-carbon sugars joined together in branching chains, employing both alpha and beta- bonds. Many other nucleosides have been found in Cordyceps, including uridine, several disnct structures of deoxyuridines, adenosine, 2̕,3̕-dideoxyadenosine, hydroxyethyladenosine, cordycepin triphosphate, guanidine, deoxyguanidine, and altered and deoxygenated nucleosides, which were not found anywhere else in nature. Of particular note are various immunosuppressive compounds found in Cordyceps, including cyclosporine, a constituent of the species C. subsessilis (anamorph: Tolypocladium infalatum) (Segelken 2002).

Other immunosuppressant compounds have also been found in Isaria sinclairii, a species closely related to Cordyceps (Mizuno 1999). In brief, the six basic classes of compounds are; 2. The polysaccharides, which comprise most of the medicinal compounds, are soluble in hot water and not in alcohol. The immuno-stimulant type of action so well known in mushrooms is from this class of compounds. In the fungal kingdom, and particularly in Cordyceps, polysaccharides are perhaps the best known and understood of the medicinally active compounds (Ukai et al. 1983, Wasser 2002). Research has shown that these polysaccharides are effective in regulating blood sugar (Kiho et al. 1993), and have antimetastatic and antitumour effects (Nakamura et al. 1999).

3. Nucleotides (including adenosine, uridine and guanosine) are effective components in Cordyceps mushrooms. Investigations show that guanosine has the highest content of all in natural and artificial Cordyceps (Li et al 2001). The content of nucleotides in artificial Cordyceps is obviously higher than in natural Cordyceps. This may be related to the rapid metabolism of artificial cultures. A remarkable variation in the adenosine content is found in artificial Cordyceps, with the highest concentration being over six times that of the lowest. The content of adenosine in freshly acquired natural Cordyceps is too low to quantity, but the content of the nucleotide in a preparation stored for a long time is higher. This shows that nucleotides in natural Cordyceps derive from degradation of the nucleoside during the storage process. Subsequent research found that dampness and warmth can significantly increase the nucleotide content in Cordyceps. At a temperature of 40oC and relative humidity of 75 per cent, Cordyceps were stored for 10 days, when the content of nucleotides increased 1–10 fold, while in artificial Cordyceps adenosine, uridine and guanosine showed no obvious change in content. This suggests that the nucleotides in natural and artificial Cordyceps are to some extent different (Wei et al 2003).

4. Crude protein and amino acids- Previous papers have reported that the content of crude protein was in the range 29.1-33 per cent (Hsu et al 2002, Xu 1997, Crisan and Sands 1978). The protein was composed of 18 amino acids, including aspartic acid, threonine, serine, glutamate, proline, glycine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, cystine, cysteine and tryptophan. The content of amino acids after hydrolysis is mostly reported as 20-25 per cent, the lowest being 5.53 per cent, the highest being 39.22 per cent. The highest contents are glutamate, arginine and aspartic acid, and the major pharmacological components are arginine, glutamate, tryptophan and tyrosine (Ji et al 1999). The content of amino acids in the commercial preparation of Cordyceps is significantly higher than in the mycelia of C. sinensis, which is similar to the content in the fruit body of C. sinensis.

5. Fatty acids and metal elements- Fatty acids are composed of carbon, hydrogen and oxygen and are the major component of lipids, phospholipids and glycolipids; they can be classified as saturated or unsaturated fatty acids. In Cordyceps, the unsaturated fatty acid content reaches 57.84 per cent, including Cl6:1, Cl7:1, Cl8:l and Cl8:2. The linoleic acid content is the highest at 38.44 per cent, followed by the oleic acid, which is 17.94 per cent. The saturated fatty acid content is 42.16 per cent, including Cl4, Cl5, Cl6, Cl7, Cl8, C20 and C22. The palmitic acid and the octadeca acid contents are the highest, and are 21.86 per cent and 15.78 per cent, respectively. Unsaturated fatty acid is an effective physiologically active component, which has the unique function of decreasing blood lipids and protecting against cardiovascular disease.

6. Ergosterol- Ergosterol is a sterol unique to fungi and is an important precursor of vitamin D2, which has important medicinal value. The content of free ergosterol from natural and artificial Cordyceps has been compared. The result showed that the content of free ergosterol in different natural Cordyceps varies significantly, but is evidently higher than in the mycelia of Cordyceps. The content of ergosterol in the artificial fruit body of C. militaris

is very high, only lower than that of natural Cordyceps in Tibet, and higher than that in Qinghai and Sichuan. Li et al. (2001) reported that the content of ergosterol in the mycelia of Cordyceps was 1.44 mg/g, much lower than that in the fruit bodies of Cordyceps (10.68 mg/g).

7. Peptides (i) Cyclodipeptides- A cyclodipeptide named Cordycedipeptide A, a natural compound has been isolated from the liquid culture of the Cordyceps sinensis, and which exhibited cytotoxic activity against L-929, A375, and Hela cells. (ii) Cordymin- The peptide Cordymin from Cordyceps sinensis exhibiting neuroprotective effect in the ischemic brain due to inhibited inflammation and increased anti-oxidant activity related to lesion pathogenesis can be used as a potential preventive agent against cerebral ischemia-reperfusion injury. (iii) Myriocin- Myriocin (also known as the anti-biotic ISP-I) is a new type of immune inhibitor extracted from C. sinensis, and is shown to significantly inhibit the upregulated expression of cyclin D1 induced by high concentration of glucose, restoring the expression of cyclin D1. (iv) Melanin- The anti-oxidant activity of melanin, derived from a black pigment, was isolated from the fermentation broth of C. sinensis, and showed much stronger scavenging abilities for 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferrous ion chelation compared to the mycelial water extract. (v) Lovastatin, γ-amino butyric acid and ergothioneine- Lovastatin, GABA, and ergothioneine are secondary metabolites of fungal growth. Chen et al indicated that mycelia of C. sinensis contained 1365 mg/kg of Lovastatin, 220.5 mg/kg of GABA, and detectable ergothioneine and had different hypolipidemia, hypotension, and anti-oxidant activities, and (vi) Cordysinins- Five Cordysinins A-E, from the mycelia of C. sinensis have been identified and have been shown to have anti-inflammatory activities.

Table 11: Summary of markers currently used for quality control of Cordyceps and associated activities (after Li et al 2006). Compound Pharmacological activities Comments type Nucleosides Anti-tumour activities; Ca2+ Nucleosides, especially adenosine, are antagonist; the release of various usually used as markers, and the neurotransmitters presynaptically profiles can be applied for and anticonvulsant activity; authentication of Cordyceps stimulate axon growth in vitro and in the adult central nerve system Polysaccharides Anti-oxidation, immunopotentiation, Represents the most biological anti-tumour, and hypoglycemic properties of Cordyceps, and less used activity; anti-inflammatory activity as marker for quality control. Thus, it and suppress the humoral immunity should be developed in mice Ergosterol and Cytotoxic activity, anti-viral activity, Ergosterol can be used as marker for its analogs and anti-arrhythmia effect; suppress quality control

the activated human mesangial cells and alleviate immunoglobulin A nephropathy (Berger’s disease) Mannitol Diuretic, anti-tussive and anti-free It sometimes is used as marker for radical activities quality control Peptides Anti-tumour and immuno A potential marker for quality control potentiation activities

E. ARTIFICIAL CULTURE

Many workers have reported their significant contributions towards in vitro culture of C. sinensis, (Song et al., 1988; Shiling et al., 1988; Shunzhi et al., 1993; Li et al., 2004; Chen et al., 1992; Chen et al., 1993; Chen et al., 1994; Xiao et al., 1997; Yu, 2004; Yong, 2004; Wu, 2006; Chunru & Peng, 2004; Holliday et al., 2004; Holliday et al., 2005; Dong and Yao, 2005). Artificially planting C. sinensis has been studying since 1980. Anamorph of C. sinensis is identified as Hirsutella sinensis and is separated by Liu X. J. et al., (Yong, 2004) and the series of cultivating experiments have been done. Stroma can develop in medium host. During recent year’s in vitro isolation of C. sinensis and C. militaris was done on Potato dextrose agar medium successfully (Kim and Yun, 2005; Kim et al., 2003; Wongsa et al., 2005).

There are two methods used today in the cultivation of Cordyceps. The method primarily used in China is known as Liquid Culture or Fermentation, in which a small bit of Cordyceps tissue is inoculated into a sterilized liquid medium. It grows in this liquid environment very rapidly and is usually ready for harvest in about 5 days. The Cordyceps mycelium is harvested by filtering out from the liquid broth, after which it is dried and ground to a fine powder. After it is dried, it can be used as is, or further processed by extracting it with hot water or some other solvent, and the resulting extract either supplied as a liquid or again dried and powdered. The majority of Cordyceps available on the market is liquid cultured in this way. This results in a fairly good product, since this is a very economical method for large-scale production and the ease of controlling the growth parameters in large sealed tanks of liquid results in a very consistent product with very little variation in quality from batch to batch.

However, there is a major drawback to the fermented Cordyceps; which is the loss of the extra- cellular compounds which Cordyceps produces. When the mycelium is filtered out of the culture broth and the residual liquid discarded, all of the bioactive extra-cellular compounds produced throughout the growth process are lost. These are many of the unique secondary metabolites produced by Cordyceps that have some of the most potent medicinal effects. Consider for a moment: In the fungal kingdom nearly everything of biological importance happens outside of the cell wall. This has to be so, since the fungi have no mouths. Probably as much as 90% of the bioactive compounds of interest that are produced by the Cordyceps are in the liquid that is discarded after the mycelium is harvested. In the wild collected Cordyceps, the caterpillar body, which is harvested along with the fruit body, is fully mummified with the Cordyceps mycelium. But more importantly, it acts as a natural reservoir for those entire exuded extra-cellular compounds that were produced. The compounds, which were exuded outside of the mycelium, still remain in

38 the caterpillar body. That is probably the main reason why wild collected Cordyceps is thought to be more potent than cultivated Cordyceps. It is the presence of these bioactive extra-cellular compounds, which were lost in the harvesting process of the liquid-cultivated type.

There is a second method of Cordyceps cultivation practiced, called the solid-substrate method or biomass method. In this type of cultivation, the Cordyceps is inoculated onto some type of sterilized solid nutrient source, usually a cereal grain or mixture of grains. It grows much more slowly on solid material than it does in liquid, but eventually the growth of the mycelium consumes most or all of the substrate and is ready for harvest. At this point, the entire contents of the growing container are harvested and dried; the mycelium, the residual substrate and the entire compliment of all the extra-cellular compounds which were produced throughout the entire growth process. In this way it is possible to capture these unique compounds, which are naturally lost when cultivated by fermentation technique. The quality potential would seem to be much greater when cultivated under solid substrate method verses liquid fermentation method. When grown this way the quality is high indeed, often exceeding the potency of wild Cordyceps by a factor of five times, (Holliday et al., 2004).

Medium for maintaining stock cultures The usual nutrient agar mixtures used in maintaining mycological cultures can almost all be used for maintaining Cordyceps cultures. It is worth noting that Cordyceps will usually adapt quickly to a new medium, which is both a blessing and a curse. A blessing in that almost any medium used to propagate the culture will support growth, but a curse in that the organism rapidly develops a preference to this new medium, probably through the generation of enzymes specific to the character of that particular medium. This will result in the culture losing its growth vigour for a time when transferred to another medium. This also leads to early culture senescence when the culture is repeatedly grown generation after generation on the same medium. The easy way around this issue is to constantly challenge the culture by transferring it onto a new medium with each successive generation. In this way, the culture is forced to maintain a broader spectrum of digestive enzymes to deal with the varying food sources, much as happens in nature. It also helps quite a bit to introduce some of the final substrate upon which the Cordyceps is to be grown into the maintenance medium. Some of the nutrient agar mediums that have been found to support good mycelial growth are:  Standard Malt Extract Agar  Standard Potato Dextrose Agar  Cat food Agar (10 g dry cat food and 20 g agar per L water)  Dog food Agar (10 g dry dogwood and 20 g agar per L water)  Oval tine Agar (10 g Oval tine drink mix, 20 g agar per L water)

A review of the different methods for artificial culture- both liquid as well as solid culture is presented in tables 11 & 12, below.

Table 12: Different methods used for artificial culturing of Cordyceps sinensis and related species of Cordyceps are given below Culture method Requirement reference Mycelium of C. militaris was grown in 750 ml Erlenmeyer flasks on a G/L, Glucose-30g, Peptone-2g, KH2PO4-1g, Puchkova et al shaker at 180 rpm and static condition on glucose-peptone medium. K2HPO4-1g, MgSO4.7H2O- 0.25g, Corn 2010, Smirnov et al extract 2g, deionized water -1L. 2009 Liquid culture media/submerged cultivation of O. sinensis at 1.25% Glucose,1.25% Sucrose, 0.02% Yue et al 2013 temperature 24oC and 92 h peptone, 0.0625% yeast powder, 0.025% KH2PO4, 0.0125% MgSO4.7H2O, 0.0025% vitamin B1, neutral pH. Liquid culture media: 20% potato, 0.08% beef extract, 0.2% Shang et al 2011 o At temperature 23 C and with neutral pH 130r/min. peptone, 0.15% KH2PO4 ,0.15% MgSO4.7H2O, 1.5% Sucrose, 2.5% Glucose. Solid state cultivation of C. militaris: Nutrient solution was packed 25g organic rice in 25 ml nutrient solution Wu et al.2013 into culture bottles sealed & autoclaved at 121oC for 2 hrs. After (20g/l glucose and was obtained from the cooling seed inoculated and bottles were covered with plastic film ultra-filtration supplemented with 20g/l and incubated for 7 days at 20oC in dark. When hyphae grow in the corn steep powder (CSP), 0.5 g/l bottom than plastic film was pricked with sterilized needle to monopotassium phosphate, 0.5 g/l ventilate and provide fresh air. When colour of hyphae changes magnesium sulphate. turned orange placed it under incandescent light (300 lux) to induce fruiting body. Submerged liquid fermentation of Cordyceps militaris pH adjusted at 20.0g/L glucose (filtrate), monopotassium Wu et al. 2013 6 followed by autoclaving. The flask culture experiments were phosphate 0.5g/l, dipotassium phosphate performed in 250-ml flasks containing 100ml of medium after 0.5g/l, magnesium sulphate 0.5g/l, ferrous inoculating with 5 % (v/v) of the seed culture. The culture was sulphate heptahydrate 0.1g/l and organic incubated at 25oC on a rotary shaker incubated at 150 rpm for 15 nitrogen source CSP 5g/l or 10g/l or YE days. 5g/l and 10g/l C. militaris 14014 purchased from the China Centre of Industrial 20.0g/l glucose, 3.0g/l KH2PO4, 1.5g/l Tang et al 2015 o Culture Collection was stored at 4 C. The strain was inoculated on MgSO4.7H2O, and small amount of Vitamin potato-agar-dextrose (PDA) slants or plate containing the materials B1. mentioned in next column. The slants and plates were then incubated at 25oC for 7 days.

The liquid medium was composed of chemicals or materials which 40g/l glucose, 10g/l tryptone, 6g/l yeast are mentioned in next column. Approximately 50 ml of the medium extract, 0.5 g/l MgSO4.7H2O, 0.5 g/l was placed in a 250 ml flask and sterilized K2HPO4.3H2O, 0.5g/l KH2PO4. The cultures were raised from the stipe and stroma portion of Distilled water, 0.1% mercuric chloride Pathania et al. Healthy, sun-dried and fresh specimens. The specimens were first solution, PDA (potato dextrose agar) 2015, Wu et al. washed with distilled water and then the tissue from the stipe and medium. (39 g PDA + 1000 ml distilled 2013, Barseghyan stroma portion were cut with the help of a sterilized Blade. The bits water) et al. 2011 of tissue (2-3 mm) were taken up with sterilized forceps and dipped in 0.1% mercuric chloride Solution for 5-10 seconds. Now the tissue was placed on Filter paper to remove the excess moisture. The small bits of Cordyceps tissues were then transferred aseptically into the Petri plates containing potato-dextrose agar (PDA) medium with the help of a sterilized forceps. These were then Incubated at 25oC for at least 8-10 days and pH is 7.5. The actively growing mycelial colonies were sub cultured to obtain pure cultures. The fermentation medium, experiments were conducted in 250 ml Glucose 40g/l, peptone 15g/L, KH2PO4 0.5 Hung et al 2009 Erlenmeyer flasks containing 45 ml medium. Flasks were inoculated g/l, K2HPO4 0.5g/L and MgSO4.7H2O 0.5g/l with 5 ml of seed culture and then incubated at four different temperatures (15, 20, 25 and 30oC) in the dark and Static conditions for 15 days.

Solid-state Fermentation: Fruiting medium of C. militaris was Brown rice 20g, 32 ml nutritional solution Chi Wen et al 2014 prepared by mixing 20 g of rice (or other Substrates) and 32 ml of (20g/L Sucrose, 10g/l peptone, 0.1 g/l nutritional solution in a 300 mL cylindrical glass bottle (8 cm in MgSO4.7H2O and 0.1 g/l K2HPO4 with diameter and 12 cm in height) and then sealed with plastic and were 1000ml distilled water. autoclaved for 30 min at 121oC. The medium was cooled to room temperature and inoculated with 5 mL seed culture and incubated at 20oC for 12 d and was given dark treatment for promoting vegetative growth. Primordial of fruiting bodies began to form at 12- 15 d after lowering the incubation temperature to 16oC at night (darkness) with culture temperature maintained at 23oC during the day (the white light maintained at 500 lx) and relative humidity (RH) at 90%-95%. While the temperature was maintained at 23oC and RH at 80%-90%, sufficient air exchanges were used to maintain CO2 levels. Illumination with 300 lx intensity did not exceed 12 hours per day. The culture developed into 5-9 cm long fruiting bodies within 50-60 d following Inoculation. Potato dextrose broth and basal growth medium and pH 5.5 at 30oC PDA, Peptone 2.0 g, yeast extract 2.0 g, Ramesh et al 2014 for a period of 10, 20, 30 and 40 d were used. KH2PO4 1.0g, MgSO4.7H2O 0.6 g, and 1000 ml distilled water. Liquid medium: The culture was initially grown on PMP agar Requirement for PMP medium: Potatoes Mani et al 2015. medium in a Petri dish, and then transferred to liquid medium 5%, dextrose 1%, malt extract 1% and By punching out 5 mm culture disc from the agar plate with a peptone 0.1% sterilized cork borer. The culture was first Incubated for 5 days at 20°C on a rotary shaker (150 rev/min) in darkness, and then incubated without Shaking at 20°C in luminance with 12 h shifts for next 15 days Solid media were inoculated with 5 ml of POL medium culture and Requirement of POL medium: (NH4)2SO4 o incubated for 30 days at 20 C with 70-80% moisture with 12 h 0.5%, MgSO4 0.02%, K2HPO4 0.1%, yeast luminance shifts extract 0.2%, peptone 0.1% and glucose 4%

Liquid cultures: Cordyceps isolate EFCC C-783 was grown in cotton- 70g unmilled brown rice, 30g silkworm Sung et al 2006. plugged, 1100 ml Plastic bottles on rice/silkworm medium, an pupal tissue and 100 ml water. autoclaved mix of 70 g unmilled brown rice, 30 g silkworm pupal tissue, and 100 ml water. The inoculated rice/silkworm cultures were incubated at 24oC under 1000 lux of continuous fluorescent light. After 7-10 d, primordial of stromata became apparent and continued to grow. After these primordial were 5-10 mm tall, the bottles were transferred to 20oC, 80-90% RH, and 1000 lux of illumination under standard fluorescent lamps. In order to maintain a high local relative humidity for the cultures, the outsides of the culture bottles were repeatedly sprayed with sterilized water for several days after transfer to these cooler growing conditions. Brown rice as a medium: A mixture of 50-60 g of brown rice, 10-20 g 50-60g brown rice, 10-20g of pupa of Kim et al 2010 of pupa and 50-60 ml of distilled water in 1,000ml polypropylene silkworm, and 50-60 ml of distilled water (PP) bottle were found to be the ideal conditions for Fruiting body in 1000ml polypropylene (PP) bottle. production of C. cardinalis. The temperature maintained at 25oC and pH is 6. Under continuous white light conditions, fruiting bodies were formed vigorously whereas under complete dark conditions very few stromata were produced The optimized medium: The media contains 30 g/l sucrose, 4 g/l 30 g/l sucrose, 4 g/l beef extract, 10 mg/l Singh et al 2014. beef extract, 10 mg/l folic acid, 1 mg/l calcium chloride, 500 mg/l folic acid, 1 mg/l calcium chloride, 500 zinc chloride, along with 24g/l PD broth. pH at 5.5, temperature 10⁰C mg/l zinc chloride, along with 24g/l PD and agitation at 200 rpm was maintained for 30 days. Dry weight broth. was obtained same as mentioned in media used and culture condition. Liquid media for C. pruinose. The optimum temperature for mycelia Potato Starch 2%, sucrose 2.5%, soya bean Dong et al 2016 growth of C. sinensis is 15-20oC. 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K2HPO4 0.1%, and MgSO4.5H2O 0.05% PDA medium: (PDA broth 2.4% and agar 2%) in a Petri dish and then PDA Broth 2.4%, & Agar 2% Kim and Yun 2005 transferred to the seed culture medium

Liquid seed cultures were prepared in a 250 ml flask containing 50 Glucose 10g/l, Yeast extract 3g/l, Malt ml of YM medium at 25oC on rotary shaker incubator at 150rev min-1 extract 3g/l, Meat Peptone 5g/l. for 4 days. The main flask culture experiment was performed in 250ml flask containing 50ml of YM after inoculating with 4%(v/v) of the seed culture.

Table 13: List of Solid and Liquid media used for the cultivation of C. Militaris (source: Mani et al 2015) Media Composition BBS medium Brown rice paste 53%, beer-wort 6% and soybean meal juice 42% Brown rice medium Brown rice 60% and silkworm pupae powder 10% Dog feed medium Pedigree 8% and agar 1.5% Rice pupae medium Rice 50%, pupae 1.7%, peptone 0.68%, KH2PO4 0.075% and MgSO4.7H2O 0.06% Soybean medium Potatoes 5%, (NH4)2SO4 0.5%, MgSO4 0.02%, K2HPO4 0.1%, malt extract 1%, peptone 0.1%, glucose 5%, chitin 1%, agar 1.5% and a monolayer of germinated soybean

CHAPTER 2: HEPIALIDAE (FAMILY OF THITARODES) AND THITARODES (the principal hosts of Cordyceps)

A. HEPIALIDAE

The family mainly includes Ghost or Swift Moths. It comprises of about 300 species which are widely distributed but best represented in Australia (Tindale 1932-42) and New Zealand (Dumbleton 1966). It is a peculiarly isolated group and although primitive in many features of the internal and external anatomy. The species of this family vary tremendously in size, some might aptly be described as minute, while others span as much as seven inches from tip to tip of their wings. The smaller species have a wing-expanse of at least 25 mm, while the gigantic species attain the wing expanse of about 180 mm (Davies and Richards 1977). The best-known species are brown or ashy gray in colour, with the wings marked with silvery white spots. In colour they are usually sober, but some of the exotic species may be gorgeous.

All the Hepialids are difficult to capture, since their flight is low and rapid. It is said that these moths fly near the earth, and only in the evening after sunset, hiding under some low plants, or clinging to the stalk of an herb during the day. Some of them fly with extreme rapidity, with an irregular mazy flight, and have, therefore, been named swifts by collectors. In the Hepialidae, the posterior lobe of the forewing is a slender, finger like organ, which is stiffened by a branch of the third anal vein, and which projects beneath the costal margin of the hind wing. As the greater part of the inner margin of the forewing overlaps the hind wing, the hind wing is held between the two. This is the type of posterior lobe of the forewing to which the term jugum is applied (Comstock 1984).

The Gold Swift Hepialus hectus is much smaller insect, also found near bracken. The Ghost Moth, Hepialus pumuli is the commonest of all. The larvae feed upon the roots of grasses, dock and ragroot. The larvae are eruciform and furnished with sixteen legs, they feed upon wood or bark, and are found at the roots or within the stems of plants. They usually under metamorphosis in their burrows, or within loose cocoons, in the case of those that feed outside. The pupae are usually elongate and active and are armed with spines, toothed ridges and cutting plates on the abdominal segments, which are special adaptations for making their way to the surface. The 2nd to 6th abdominal segments are free in the female and the 7th also in the male (Packard 1895). The male is an insect of exceptional colour, being white above in consequence of a dense formation of imperfect scales, while the female is of brownish tints, which is usual in swift moths. In the month of June, male selects a spot where he is conspicuous, and hovers persistently there for a period of about twenty minutes in the twilight; his colour has a silvery- white, glistering appearance, so that the insect is really conspicuous notwithstanding the advanced hour. Females may be detected hovering in somewhat similar manner, but are not conspicuous like the male, their colour being obscure; while so hovering they are ovipositing, dropping the eggs on the grass. Females that have not been fertilized move very differently, and dash about in erratic manner till they see a male, they then by buzzing near or colliding with him. The larvae mostly live underground or in the wood, the pupae, which are very elongate, are active (Ealand 1984, Imms 1973, Metcalf and Flint 1990).

This family is represented by two genera (Comstock 1984). (i) Hepialus- The genus includes smaller species, which range in wing-expanse from 25 to 55 mm. In Hepialus the apices of the

45 forewing are more rounded and (ii) Sthenopis- This genus includes larger species. In these the apices of the forewings are more pointed, and in some species are sub-falcate.

Morphology The family Hepialidae is considered to be very primitive with a number of structural differences to other moths including very short antennae and lack of a functional proboscis or frenulum (Kristensen 1999). Like other Exoporia the sperm is transferred to the egg by an external channel between the "Ostium" and the ovipore. Other non-ditrysian moths have a common cloaca. The moths are "homoneurous" with similar forewings and hind wings and are sometimes included as 'honorary' members of the Macrolepidoptera, though archaic they are. Strictly speaking they are phylogenetically too basal and constitute Microlepidoptera, although hepialids range from very small moths to a wingspan record of 250 mm in Zelotypia (Nielsen et al. 2000). Many species display strong sexual dimorphism with males smaller but more boldly marked than females; or at high elevation, females of Pharmacis and Aoraia show "brachypterous" wing reduction (Sattler 1991).

Distribution 37 species of insects, belonging to 4 genera of Hepialidae (Lepidoptera) - Hepialus, Forkalus, Hepialiscus and Bipectilus, are found to be the host of Chinese insect herb Cordyceps sinensis (now presently referred to as Ophiocordyceps sinensis). Among these species, Hepialus armoricanus, H. oblifurcus, H. deqinensis, H. baimaensis and H. yulongensis are the most commonly ascribed hosts. The geographical distribution of Hepialid moths has obvious regional and geographical band characteristic. Except for Hepialus armoricanus, which is distributed extensively, the others show narrow distribution. In the Oriental and Neotropical regions hepialids have diversified in rainforest environments but this not apparently the case in the Afro- tropics (Nielsen et al. 2000). Hepialids mostly have low dispersive powers and do not occur on Oceanic islands with the exception of Phassodes on Fiji and Western Samoa and a few species in Japan and Kurile Islands.

Behaviour Swift moths are crepuscular and some species form leks (Kristensen, 1999). In most genera, males fly swiftly to virgin females attracted by the pheromones being secreted by them. In other genera, virgin females "assemble" upwind to displaying males (Mallet 1984), which emit a musky pheromone from scales on the metathoracic tibiae. In such cases of sex role reversal, there may be visual cues also: males of the European Ghost Swift are possibly the most frequently noticed species, being white, ghostly and conspicuous when forming a lek at dusk (Andersson et al. 1998). Sometimes they hover singly as if suspended from a thread or flying in a figure of eight motion. The chemical structures of some pheromones have been analyzed (Szhulz et al. 1990).

Biology Larvae are subterranean root feeders on a variety of herbaceous plants (Yang et al. 1996). Larvae in the Tibetan Plateau and the Himalaya are host to the entomophagous fungal parasite Ophiocordyceps sinensis that is used as a traditional Chinese medicine (Maczey et al. 2011, Wang and Yao 2011, Zou et al. 2012). Larvae of Thitarodes pui feed on humus and the roots of a wide range of alpine plants, particularly Ranunculus brotherusii, Cyananthus macrocalyx, Juncus leucanthus and Veronica ciliata. Development is completed in three to four years and adults

46 emerge during the day at any time between sunrise and sunset from late June to mid-July (Zou et al. 2012).

The female does not lay its eggs in a specific location but scatters them while in flight, sometimes in huge numbers (29,000 were recorded from a single female Trictena, which is presumably a world record for the Lepidopterans, Tindale 1932). The maggot-like larvae feed in a variety of ways. Probably all Exoporia have concealed larvae, making silken tunnels in all manner of substrates. Some species feed on leaf litter, fungi (Mallet 1984), mosses, decaying vegetation, ferns, gymnosperms and a wide span of monocot and dicot plants (Grehan 1989). There is very little evidence of host plant specialization; even though the South African species Leto venus is restricted to the tree Virgilia capensis, a rare phenomenon of "ecological monophagy" (Nielsen et al. 2000). Most feed underground on fine roots, at least in early instars and some then feed internally in tunnels in the stem or trunk of their host plants. The pupa has rows of dorsal spines on the abdominal segments as in other lower members of the Heteroneura (Kristensen 1999). Hepialidae constitute by far the most diverse group of the infra order, exporia. There are 56 genera and at least 540 valid species of these primitive moths recorded worldwide till date. Among the 56 genera the genus Hepialus comprises approximately 173 species, out of which 54 species have been recently placed in new genus Thitarodes.

B. THITARODES

Thitarodes is a genus of moths of the family Hepialidae. In English Thitarodes is known as "ghost moth". The majority are restricted to the Tibetan Plateau. Often in Chinese entomological nomenclature Thitarode is still referred to as Hepialus, although the name was changed back in 1968. Furthermore, some authors use incorrectly the term "bat moth" which is a bad translation of the Chinese term for ghost moth. Many larvae of this genus are the hosts to the parasitic fungus Ophiocordyceps sinensis. The fungus-insect complex is known as Caterpillar fungus or by its original Tibetan name Yartsa Gunbu. It was first used in Traditional Tibetan Medicine, but is now highly prized by practitioners of Traditional Chinese Medicine (TCM), wherein it is referred to as Dong Chong Xiacao or in short Chong Cao. The males of many species have metatibial androconia that are sometimes prominent and almost extend the length of the abdomen. Species that share the presence of a basal spine on the male valve may represent a monophyletic group as proposed for the original four-member species by Viette (1968).

Collection and Identification Larval `mummies’, practically solid with mycelium, usually together with an attached fruiting body are collected, dried and used widely as a notably expensive component in traditional Chinese medicine. The Hepialidae insect Hepialus armoricanus Oberthur was first recognized as host insect of Cordyceps spp. by Zhu (1965). The altitudinal range of distribution of C. sinensis and its host insect in Nepal was 4500±300 by Aryal et al. (2008) and 3700±500 in India by Arif and Kumar (2003). The grasslands providing habitat for Hepialus ghost moths and thus for C. sinensis consist predominantly of sedges (Kobresia spp.).

Collection methods of Hepialus moth has briefly been explored by Maczey et al. (2010) in Bhutan. The author summarized that unsuccessful light trapping during earlier in the season (early June 2005, mid-June 2007), suggested that the flight period of adult Thitarodes does not start before the last days in June and apparently coincides with the height of the monsoon

47 characterized by frequent rainfall. It has been reported that members of the genus Hepialus are often not easily attracted by light (Zhu et al. 2004). The true diversity of the genus is not easy to establish, as in number of cases existing descriptions and illustrations are inadequate for unequivocal identification. According to recent Chinese publications Hepialidae lists several additional species, while not referring to several others species originally described from China. In a number of cases the existing descriptions and relative inaccessibility of type material does not allow these taxa to be properly compared with each other. In their revision of Chinese Hepialids, Zhu and Wang (1985) regarded the differences in male genitalia as described by Viette (1968) as too insignificant for generic character.

Thitarode species collected- preliminary investigations The adult moths collected were pinned and stretched on a spreading board and the moths were identified on the basis of external characteristics, i.e. its mode of flight, mouth parts, wing venation, antenna type and shape of thorax and abdomen. Identification of collected specimens was further confirmed by Peter Smetacek, Naturalist, Bhimtal (Uttarakhand). The genus Thitarodes was established by Viette (1968), based on the presence of an acute process protruding from the base of the valve on the male genitalia and Hepialiscus group by vein R4 branching from R4+R5. Viette (1968) designated Hepialus armoricanus Oberthur, 1909 as the type species and included three new species from Nepal: Thitarodes danieli, T. eberti and T. dierli. Further species were described by Robinson et al. (1995), Ueda (1996, 2000) and Maczey et al. (2010), but the genus Thitarodes was not adopted by Chinese scientists who assigned new congeneric Chinese species to the genus Hepialus Fabricius (Chu & Wang 1985, Chu et al. 2004). In 2000, Nielsen et al. (2000) transferred these ‘Hepialus’ species to Thitarodes, with a total of 51 species (Zou et al 2011).

Systematic position of Thitarodes

Kingdom: Animalia

Phylum: Arthropoda

Class: Insecta

Order: Lepidoptera

Family: Hepialidae Genus: Thitarodes Viette 1968 Description of the Male- Description is based on holotype (figure below)

Table 14: Description of the taxonomical features of the only Thitarode collected till date Features Description studied Size Wingspan: 32-35 mm; forewing length: 16-17 mm; hind wing length: 13-14 mm; body length: 14-15 mm Antennae Rust brown with segments 4 to 9 dorsally darkened to blackish brown. Body Head and thorax blackish brown covered densely with rust coloured hairs turning ochraceous posteriorly; prothorax and mesothorax interspersed with dark brown hairs; abdomen brown covered in ochraceous yellow hairs. Forewings Fore wings 16 mm long. Mainly rust coloured with extensive grey-white and black markings. Costa straight, cross vein Rs- M1 reaching Rs3/ Rs4 at the furcation; postmedial line from apex to M3; posterior margin slightly concave between Cu and A1, Sc unbranched, R straight, Rs1 and Rs2 stalked, cross vein Rs – M1 reaching Rs3/Rs4 at the furcation. The principal M is branched into M1, M2, and M3. Post medial line from apex to M3. Cu straight; CuA1 and CuA2 stalked Hind wing Hind wing 14 mm long. Uniformly grey, slightly translucent; fringe between R1 and Cu2 ochraceous yellow and blackish brown; wing venation similar to forewing but CuP reaching margin; A weak, not reaching margin Costa straight, posterior margin slightly concave between Cu and A1, Sc unbranched; R straight, Rs1 and Rs2 stalked, cross vein Rs – M1 reaching Rs3/Rs4 at the furcation. The principal M is branched into M1, M2, and M3. Post medial line from apex to M3. CuA is straight and stalked into CuA1 and CuA2 Legs All legs densely setose with ochraceous and dark brown hairs, lacking spurs; hind tibia slightly broadened and dorsally densely covered with ochraceous 5 to 6 mm long scent-brushes i. Forelegs Coxa is simple thin plate like structure. Anterior end of coxa there is a long cylindrical Femur, which joint with the Tibia. Tibia consist a small outgrowth epiphysis. After Tibia there is segmented Tarsus, consist 5 segments. Last segment of Tarsus consists a hook like aperture claw. In male foreleg many hairy projection bristles are present. ii. Hind leg Pattern of hind leg is similar to the fore leg. Difference only seen in Tibia, long hairy pencil is present in hind leg. Genitalia In male genitalia is present in eight abdominal segments. Pseudoteguminal process is sickle – shaped, apex pointed, middle region broader than apex, inferior region narrower than the middle and inner margin circular. Both of

teguminal consist of minute teeth process. Valva moderate and gradually broadened apically, the superior part is rounded, middle is slightly lobe and posterior part flattened. Basal acute process produced horizontally, then upturn. Succus is broad and flattened appeared like ‘W’

Habitat Ecology of Thitarodes Most species occur in alpine or high latitude meadows and grasslands, but lowland forest habitats are also inhabited (Maczey et al. 2011). The high concentration of species in the Tibetan plateau may be the result of this region’s high geomorphic and ecological diversity (Zou et al. 2012). The insect herb moths live in the place below alpine snow line and upper 3000 meters of altitude. Its distribution upper limit is 5080 meters. Most of the larva are found in alpine meadow-humic soil. Some are in alpine frigid desert soil, a little are found in brown conifer forest soil, too. The larvae live in tunnel dug beneath the soil surface and feed on tender root or rhizome of Polygonum, Astragalus, Salix, Arenaria, Rhododendron, and have greater cold tolerance. Larvae are easier to be parasitized by fungus Ophiocordyceps sinensis during second exuviation time that lasts in and around mid-July to August. The rate of parasitism, during this interim is however, reportedly very low, about 2.6 - 16.1 per cent. In contrast, the fourth and fifth instar larvae are infected more easily, and the infected rate is about 90 per cent.

Effect of atmospheric conditions on Thitarodes Hepialus pupae are usually harvested from early May to early July each year, while the adults emerge out of their cocoon from mid-June to late-July; usually between 4:00 PM to 8:00 PM. Adults usually mate on the same, after the emergence, or at times next day; and have two mating peaks at 6:30 and 8:00 PM. Oviposition immediately follows the mating and begins 20 minutes after the mating. The average number of eggs laid by each female is around 400. The larval stage consists invariably of six instar stages. The broad physical variables associated with the Ophiocordyceps and its host insect, Thitarode is being listed below in table 15, below.

Table 15: Niche requirements for Thitarodes 1. Atmospheric temperature 20-21oC 2. Soil temperature 9-10oC 3. Rainfall 90-100 mm 4. Relative humidity 75-85 per cent 5. Soil moisture 40-50 per cent 6. pH 7.4 ~ 7.5 7. Soil salinity 70 g/kg

Host plant and feeding habit of larvae and adult As the dominant host for Ophiocordyceps sinensis, members of the ghost moth genus Hepialus (Thitarodes) play a significant role in the ecological system at the heart of harvest and trade of O. sinensis. Larval maturity depends on species and environmental conditions (Chen et al. 2002, Yin et al. 2004). The phytophagous caterpillars of Hepialus sp. in Laspa were mostly subterranean root-feeders (Koranga pers. commu.), but invariably also thrive upon the aerial parts like stem and leaf of many alpine herbs (Yang et al. 1996). A total of 15 species representing 10 families were recorded for host plant species of Hepialus sp. in Laspa; with the family Rosaceae and Cyperaceae being represented by 3 species each, compositae represented by two, followed by

Ranunculaceae, Poaceae, Polygonaceae, Asteraceae, Scrophulariaceae, Ericaceae and Campanulaceae, each being represented by single species (Koranga pers. commu.).

The feeding habit of caterpillars depends upon host range of plants and its tunneling behaviour on soil (Wagner 1989). The tunneling reaches up to 1.5 feet below the soil, where mostly live as first or second instar larvae. So, the feeding of these instars depends upon the length of root tissues, mostly of Potentilla argyrophylla, Bistorta vivipara, Pedicularis rhinanthoides and Cyananthus lobatus. In middle layer of tunnel, the third and fourth instar stages dwell, while in the upper layer dwells the mature fifth instar larvae. According to Canon et al. (2009) working in Bhutan, they never observed the late instar larvae of Hepialus sp. on dwelling on the surface at any time of the year.

CHAPTER 3: EXPLORING OTHER MOTH DIVERSITY A number of moths were collected both during the harvesting season as well as in the month of July, which according to our earlier field observation, were likely to yield the specimens. This time round three additional sites were explored for the collection of the moths, apart from the three sites where study on effect of livestock grazing pressure is being conducted, i.e. Balmiya top, Rukhiyan and Jhanthari. However, due to inclement weather conditions, as well as because of administrative-implemented ban on issue of passes to these study sites, collection of the moths unfortunately did not yield any of the Thitarode species. However, we did collect a number of moths, which still need identification, for which help has been sought from experts in Zoological Survey of India, Kolkata. the identification work is still being carried out, which remains difficult since we have only dispatched the plates of the collected specimens to the expert. However, since Cordyceps (including Ophiocordyceps sinensis) is known to infect a number of moth species, apart from the Thitarodes, identification of the moths remains vital for subsequent/future studies on the likelihood ascertaining other host species of moths for Cordyceps. In addition, other offshoot of the present study (even though it does not cover the enshrined objectives of the present study) is the creation of inventory of moth diversity in the alpine sites, which have not been explored fully. The present study thus will definitely yield a number of moth species, either (i) discovered for the first time from this region, or might even (ii) yield entirely new species.

Genera to be studied or explored 1. Bipectilus Chu and Wang 1985 Characterized as one of the most distinctive and peculiar hepialid genera, Bipectilus differs from all other Eurasian and Oriental genera in having bipectinate antennae and highly specialized male genitalia.

2. Hepialiscus Hampson (1893) group A small genus of six species, with H. nepalensis (Walker, 1856) distributed along the Himalaya, H. htayaungi in southern Myanmar (Grehan & Mielke 2016), an unnamed species in northern Myanmar and Thailand (C. Mielke, pers. comm.), and three species in Taiwan (Ueda 1988). The taxon H. nepalensis subsumes four other names from the Himalayan region (Tindale 1942; Nielsen et al. 2000), but because the type specimen was never dissected before the abdomen was lost the precise identification of specimens from this region remains problematic. The only reference to larval biology is a brief note by Hampson (1893) that the larvae feed on the roots of grasses and other plants. The characters listed for Hepialiscus by Ueda (1988) were not characterized as unique for the genus. The presence of a Tuberculate plate (pinnaculm) anterior to the spiracle on the 2nd to 6th abdominal segments would, however, appear to distinguish it from Bipectilus, but not Endoclita. No work on Hepialiscus has been carried out, even though 6 species have been reported from the Himalaya.

3. Thitarodes Viette 1968 Originally proposed with four species, Thitarodes was subsequently extended to include a further 53 species (Ueda 1996, 2000, Nielsen et al. 2000). Viette (1968) drew attention to the presence of a basal spine for the species originally included, but at least 6 species included by Nielsen et al. (2000) lack the basal sine (as illustrated by Chu and Wang 1985),

52 and a basal spine occurs in some other genera such as the monotypic Mexico-Central American Schausiana Viette 1950. Thitarodes is distinguished from the Hepialiscus group by vein R4 branching from R4+R5, and from other sympatric genera by the male genitalia. Larvae are sub-terranean root feeders in alpine meadows or pastures (Yang et al. 1996, Maczey et al. 2010).

4. Palpifer Hampson (1893) Larvae are root borers (Kalshoven 1965, Robinson et al. 1995). It comprises of nine species and is in need of taxonomic revision (Robinson et al. 1995). Monophyly for some or all species may be indicated by the uniquely shared presence of a small dark spot along the posterior forewing margin basal to vein CuA2, a white spot at the base of the forewing discal cell on or near vein M3, and an orange-brown fringe on some or much of the outer and posterior hind wing margins.

A widespread, but poorly documented genus of 11 species scattered across parts of India and eastern Asia. The five Indian species are all from northern India. The moths have a dark brown body and forewings that are rounded at the apex. The male has an oval shaped scent gland at the base of the forewing (illustrated for P. sexnotatus (Moore 1879) by Tindale 1942) although its presence in all species has not yet been confirmed. The only record of Palpifer larval biology is for P. sordida Snellen, 1900 from Java that was characterized by Kalshoven (1965) as a root borer ‘at some depth’ in tubers of Dioscorea, Alocasia, and Amosphophallus. Moths were reared in January-February and July-October, and two moths were observed to emerge at 16:00hr. A mated female produced eggs at the end of October that hatched 30 days later. Thirty-five larvae placed directly on Dioscorea tubers developed over 6–8 weeks resulting in one adult. Larval tunnels encircle the tubers and stems of Dioscorea and Alocasia. Kalshoven (1965) also referred to root feeding on Colocasia antiquorum by larvae of P. sexnotatus (= P. hopponis Matsumura, 1931) in Taiwan causing host mortality and suggested that monocots were the principal hosts of Palpifer.

The probable site for the collection of Palpifer would be the Ranthi-Jumma, where the Palpifer probably infects the potato tuber. Potato tubers could be studied for signs of tunnels, and probable inhabiting larva.

5. Endoclita Felder (1874) This genus was first established by Felder & Felder (1874) but its member species were for a long time widely referred to as Phassus Walker, 1856 (the name also including Mexican and central American species). Tindale (1941, 1942) substantiated the priority of Endoclita for Asian species while Phassus was restricted to the New World species (a group that may yet prove to be closely related). Some Indian species have also been referred to as Sahyadrassus as proposed by Tindale (1941) for those species lacking the forewing vein Sc1, but this feature also applies to other genera. There is currently no clear evidence of monophyly for Sahyadrassus which was subsumed under Endoclita by Nielsen et al. (2000). Moths are generally various shades of brown with more or less developed transverse markings, with the exception of E. viridis (Swinhoe 1892) of southern India, which is unique in having featureless pale yellowish-green forewings. Although the generic limits of Endoclita are not well defined, it is probable that it represents a monophyletic group as the described external female genitalia all have a prominent dorsal extension to the central lobe of the

53 lamella antevaginalis (Grehan & Mielke 2016). Individual species are critically in need of further taxonomic work and it is probable that there remain undescribed species within and beyond India. The genus has not yet been characterized by one or more features to support its Monophyly. Larvae are wood borers or trees and shrubs (Grehan 1989).

A brief inventory of the collected moths is presented below; Table 16: Plates of the collected moths (diversity)

CHAPTER 4: LIVESTOCK GRAZING PRESSURE VIS-A-VIS STUDY OF BROAD SOIL PARAMETERS

Introduction Overgrazing means grazing land with livestock in such numbers as to adversely affect the growth, quality or species composition of vegetation on that land to a significant degree (Statutory Instrument 1996). Overgrazing can mean different things to the grazer and the range manager. For the grazer, it implies that the pasture can no longer carry as many animals as before, or that its productivity has declined so that the performance of the animal either in terms of live weight gain or offspring reproduction has worsened. The fact that overgrazing is not a function of animal numbers, but rather a function of time, has to be emphasized. Overgrazing occurs when animals are kept in a paddock too long or brought back too soon, the latter means that a plant is grazed before it has recovered from a previous grazing (Pratt 2002). Domestic livestock are creatures of habit. They show the following behaviour characteristics: 1. Loitering at water access points, especially in shaded riparian areas, 2. Limited use of upper slopes and higher elevations, 3. Preference for particular vegetation types, 4. Preference for previously grazed areas.

However, in several countries, the movement of a herd on a certain pasture is not strictly controlled, which means that animals can freely graze and utilize the pasture (Molnár and Jávor 1997). The results of poor distribution are paradoxical, overgrazing and over resting occur in the same pasture (BCMF 2002). Further, botanical composition of the pasture is influenced by the joint effect of several environmental factors. In an experiment, Jones and Bunch (1995) found that the spread of a specific plant species was more affected by the annual precipitation than by the presence of animals. Grazing animals also have an effect on the botanical composition by trampling and selective grazing. Furthermore, animal faeces and urine change the element content of soil and plants. Species composition is also influenced by the time of the year that a pasture is grazed. As per Hyder et al. (1975), repeated heavy grazing during any particular month in the growing season had approximately three times higher effect on key species as did grazing during the months when plants were senescent.

Moreover, high grazing pressure decreases plant density. However, this may not decrease the total plant production of a given community, because the roots of other plants may simply occupy that space in the soil. These other plant species are often less productive and less palatable, often weedy forbs and brush, which would result in decreased animal productivity (PAI 2004). According to Pratt (2002), it is important to notice that weeds do not make the land unhealthy; they appear because the land is unhealthy. Over grazing has very often been associated with appearance of bare soil, primarily in such sites as camps for rest, water and salt sources. These sites are very often marked by the presence grazing tolerant or unpalatable species, not native and not typical of the land, such as Polygonum aviculare and Chenopodium album, etc.

Grassland soils usually have extreme physical and chemical properties as well. Soil microorganisms play a significant role in developing soil fertility. The dominant

58 characteristics influencing the existence and activity of soil microbes are soil water content and storing capacity, texture, size and rate of pores (Kátai 1994). However, treading may decrease habitable pore space and increase soil bulk density, which negatively affect soil microbes (Kátai 1998).

Sheep night penning, a form of heavy grazing, has developed into a successful method of removing the native vegetation and establishing a new pasture. Results show that high sheep density for a short duration removes almost all of the above-ground natural vegetation, but does not significantly affect the soil bulk density, the penetration resistance, and the air permeability. Jiang et al. (1996) also found that sheep night penning combined with grazing has eliminated the natural vegetation containing shrubs. The removal of natural vegetation is caused by the fact that the concentrations of ammonium-N and nitrate-N in the soil were high enough to be toxic to plant roots during and after sheep night penning (Zhang et al. 2001).

Livestock grazing is a composite disturbance that encompasses the effects of defoliation, trampling, defecation and urination (Wilson 1990, Abensperg-Traun et al. 1996, Yates and Hobbs 1997a). The results show that livestock grazing has an impact not only on vegetation structure and composition but also on soil surface condition, soil chemical, physical and hydrological properties, and near ground and soil microclimate. These effects may have important consequences for restoration of plant species diversity and community structure.

Livestock grazing also had a major impact on soil surface structure and hydrology. The higher soil bulk density and increased soil penetration resistance in heavily grazed woodlands reflect reduced pore volume and may be due to the loss of perennial vegetation cover and soil cryptogams, and the impact of sheep trampling. Perennial vegetation cover is important for two reasons: (i) there is a positive feedback from plants to the soil in the form of organic materials for decomposition and nutrient cycling, and (ii) vegetation cover protects the soil surface from raindrop splash and surface sealing effects. Both of these factors result in the soil having improved physical properties. Soils with higher organic matter content usually contain more stable aggregates and undergo less slaking in water (Tisdall and Oades 1982, Greene and Tongway 1989, Greene 1992). The lower concentrations of soil organic carbon in heavily grazed remnants probably contribute to soil aggregates being less stable and more susceptible to breaking down under raindrop splash. Sheep trampling, when soils are wet, may also break down soil aggregates; soil pressures from sheep hooves might be as much as 200 kPa (Willatt and Pullar 1983).

Plant traits, growth forms and grazing resistance The alpine vegetation is characterized by low-stature woody species, tussocks of graminoids and rosette-forming, perennial herbs (Körner 2003). Plants with these traits show varying responses to herbivory depending on their level of grazing resistance, in terms of different avoidance- and tolerance strategies (Skarpe and Hester 2008). One of the most common plant-responses to herbivory is an increase of lateral shoots. This happens when grazers eat the top-shoot of the plants and remove the apical meristem, which reduces the apical dominance (Haukioja and Koricheva 2000). Basal meristems, which are less exposed to herbivory, is thus a beneficial tolerance-strategy found in grasses (Skarpe and Hester 2008).

This functional trait gives grasses an advantage in grazed environments as compared to e.g. herbs. A large regrowth capacity is also a good tolerance strategy because that makes plants able to quickly respond to removed biomass (Strauss and Agarwal 1999). Grasses and sedges are in general the most grazing tolerant plants (Bowns and Bagley 1986, Mysterud 2006), and may even increase in response to increased grazing pressure (Bowns and Bagley 1986). Shrubs and woody species allocate most of their energy in developing woody tissue, mainly for transport or storage. This can function as an adaptation for alpine- or browsed environments (Körner 2003), for light-competition or as herbivory avoidance (Skarpe and Hester 2008). Woody plants live longer than herbaceous plants and investment in woody structures, rather than reproduction, may thus be more beneficial for them than for short- lived plants (Haukioja and Koricheva 2000). Storing energy in woody structures can function as a good grazing-escape strategy because these structures are less palatable and nutritious than herbaceous structures (Skarpe and Hester 2008). Herbaceous plants are thus highly selected among sheep, which are selective feeders with a preference for herbs (Bowns and Bagley 1986), and especially tall herbs with a low C: N ratio in leaves (Evju et al. 2009). Herbs are however less grazing tolerant than graminoids (Bowns and Bagley 1986), and are therefore expected to decrease in abundance under high sheep densities.

Herbivores regulate the structure of plant communities through selective foraging, trampling, urination and defecation (Pastor and Naiman 1992, Jefferies et al. 1994, Hobbs 1996, Augustine and McNaughton 1998), resulting in alteration of resource availability, changing of the physical environment, and changing of inter- and intraspecific interactions. High densities of herbivores may alter the plant species composition in the area towards less palatable species (Diaz et al. 2007). To which extent grazing affects plant communities depends however on the herbivore density (Austrheim et al. 2008a, Mysterud 2006), productivity (Eskelinen et al. 2012, Harrison and Bardgett 2008) and evolutionary history of grazing (Milchunas et al. 1988), as well as the time aspect (Olofsson 2006). Different ecosystems may therefore show varying responses to enhanced grazing, and high densities of large herbivores may even be beneficial for many plants. Plants of low stature growth are among those plants that may perform better in grazed environments (Diaz et al. 2007), as a response to less shading, when the total biomass and vegetation height is reduced (Vittoz et al. 2009). Their ability to colonize new sites also increases as grazing creates vegetation gaps (Olofsson et al. 2005). This gives plants of low-stature growth advantages in grazed environments.

Tolerance and resistance denote two main strategies for plants to cope with herbivores, collected under the umbrella term defence, which is defined as “any trait that confer a fitness benefit to the plant in presence of herbivores” (cf. Strauss and Agarwal, 1999). Tolerant species are able to regrow and/or reproduce after a grazing event. They are characterized by having high relative growth rate, high photosynthetic rate, high branching/tillering rates, high leaf production, short leaf lifespan or low apical meristem (e.g. McIntyre et al., 1999, Strauss and Agarwal, 1999). Resistant species, on the other hand, avoid grazing through low digestibility, content of secondary compounds, morphological attributes such as thorns or spines, or through temporal or spatial unavailability (e.g. low stature; Coley et al., 1985, Seldal et al., 1994, Strauss and Agarwal, 1999). How herbivores change the relative abundance of selected and resistant species is, however, often difficult to predict, but has large impact on long-term ecosystem productivity, as selected species

60 are more easily decomposed (Coley et al., 1985, Grime et al., 1996, Cornelissen et al., 1999), promoting positive feedback of nutrient cycling in the system (Frank and Groffman, 1998, Frank et al., 2000).

Studies of effects of grazing in alpine and arctic habitats are abundant. However, how herbivores affect vegetation has mainly been addressed using small-scale enclosure experiments (e.g. Moen and Oksanen, 1998, Olofsson, 2001, Grellmann, 2002), or by comparing areas with contrasting herbivore densities (e.g. Olofsson, 2006, Brathen et al., 2007, Ims et al., 2007). The effect of different densities of herbivores on plants has rarely been examined experimentally on large spatial scales (Bullock et al., 2001). Using spatial contrasts of ‘highly grazed’ and ‘less grazed’ areas might, however, conceal non-linear responses of plant species to increasing grazing pressure (Westoby, 1999). The ability of plants to express tolerance or resistance, or the magnitude of defence offered by these strategies, are not constants, but will depend upon both herbivore selectivity (Augustine and McNaughton, 1998) and the frequency and intensity of grazing (del-Val and Crawley, 2005), factors that are all partly dependent upon herbivore density. Tolerance may be a viable strategy if grazing frequency and intensity are low enough to ensure that the plant has resources available for regrowth, and the opportunity for regrowth is also linked to environmental conditions such as soil nutrients and moisture (Hobbs, 1996). Furthermore, at high herbivore densities, high-quality forage may become scarce, and less-preferred resistant species may be grazed to such an extent that their abundance decline (Augustine and McNaughton, 1998). Consequently, the same species can respond both positively and negatively to increased grazing pressure (Stohlgren et al., 1999, Pakeman, 2004).

Mechanisms behind grazing response seem, however, to depend upon season of grazing. Bullock et al. (2001) found that under summer grazing, grazing response was positively related to gap colonizing ability, whereas under winter and spring grazing, species which were selected by sheep increased in abundance. Furthermore, the relationship between grazing response and plant traits is also dependent on-site productivity (Pakeman, 2004) and regional climatic conditions (Vesk and Westoby, 2001, Vesk et al., 2004, de Bello et al., 2005). Grazing can affect different stages of the life cycle of a plant species differently. Seedlings are expected to be more susceptible to grazing than adult plants (Crawley, 1997). Large individuals are, however, often selected over small by the herbivores (e.g. Ehrlen, 1995b, Knight, 2004). Grazing can reduce individual survival (Ehrlen, 1995b) or growth (Knight, 2004). Grazing can also reduce seed output in the population, through direct consumption of inflorescences (Hunt, 2001a, b, Lennartsson and Oostermeijer, 2001, Brys et al., 2004), or through a general size-reduction of the plants (Bastrenta, 1991, Ehrlen, 1995a, Knight, 2004).

Indirectly, however, large herbivores could positively affect individual plants, through increasing light availability by removal of tall neighbours, prevent accumulation of litter or creating vegetation gaps that facilitate germination and seedling establishment (e.g. Bullock et al., 1994, Lennartsson and Oostermeijer, 2001, Brys et al., 2004, Ehrlen et al. 2005). A tall, herbivore selected species could be predicted to respond negatively to high levels of grazing and be favoured by cessation of grazing. In contrast, a low stature species that avoids grazing by large herbivores could be expected to be favoured by high levels of grazing but

61 respond negatively to cessation of grazing as increased growth of tall neighbours would lead to reduced light availability.

The alpine ecosystem as a study system Alpine vegetation is characterized by the absence of trees and by dominance of plants of generally low stature (Billings, 1973, Korner, 1995), which represent few types of growth forms: low stature or prostrate shrubs, graminoids and perennial herbs (Billings and Mooney, 1968). Of these, herbs contribute the least to biomass, but the most to species richness (Korner, 1995). Due to a large variety of microhabitats, caused mainly by variation in depth and duration of snow cover, the structural and functional diversity among alpine plants is considered to be high (Korner, 2003, Austrheim et al., 2005, Choler, 2005). The majorities of alpine plants are long-lived and possess means for clonal reproduction (Billings and Mooney, 1968, Billings, 1974). For some species life spans up to 300 years have been estimated (Morris and Doak 1998, Forbis and Doak 2004).

Sexual reproduction is generally assumed to be of less importance, both because it is predicted that clonal species allocate less resources to sexual reproduction (Eriksson, 1989, Callaghan et al., 1992), and because of low temperatures that could limit pollination (Totland, 1997) and seed maturation (Stephenson 1981). Several studies show, however, that seedlings are indeed abundant in alpine vegetation (e.g. Chambers, 1995, Welling and 5 Laine, 2000, 2002, Welling et al., 2004) and that establishment rates of seedlings are as high as in temperate regions (Forbis 2003). As relates to the livestock density in alpine grasslands in the present landscape, the livestock is dominated by sheep, followed by goats; both species are highly selective herbivores, in general preferring herbs (Bowns and Bagley, 1986) over graminoids and shrubs (Nedkvitne et al., 1995). In ecosystems with lower productivity, as is represented by alpine meadows, it has been found that even lower densities of sheep will be expected to have stronger impacts on vegetation (Austrheim et al., 2007). Furthermore, Hui and Jackson (2005) demonstrated that livestock grazing can increase carbon and nitrogen allocation to the below-ground biomass, enhancing the carbon input to the soil and nitrogen conservation and leading to the accumulation of soil organic carbon.

Livestock foraging not just alters the above-ground community structure, but also affect the below-ground conditions (e.g., soil properties and root biomass), although the root biomass responses to livestock grazing can be ambiguous, i.e. no changes, increases or is a function of grazing intensity (Milchunas and Lauenroth, 1993; Turner et al. 1993; Frank, 2002); however other studies suggest that the below-ground biomass and R:S ratio significantly increased with increasing grazing pressure. This latter aspect of increasing biomass allocation to the roots is an important adaptive response of plants to grazing and is favourable for grassland ecosystem restoration, e.g., alpine meadows. The increases in carbon and nitrogen may contribute to the grazing-induced increases in root biomass and plant residues because the roots and plant residues are important carbon and nitrogen sinks in grasslands (Gao et al. 2009). Lastly, the urine and dung of livestock may accelerate nitrogen cycling in grassland ecosystems (McNaughton et al. 1997).

Preliminary results of the effect of grazing pressure

(i) Soil texture Soil texture across the three different sites invariable is dominated by silt, followed by sand, gravel and clay (in negligible quantity). However, the proportion of silt varies significantly across the three sites, with Balmiya top being represented by silt percentage varying between 63.83 to 68.5 per cent, while Rukhiyan and Janthri have proportionately less percentage of silt, being marked by silt percentage between 60.2 to 60.5, and 51.8 to 63.2 percent, respectively. In contrast, the percentage of sand is relatively more in Janthri, between 30.66 to 38.4 per cent, while in Balmiya, it is between 26.8 to 30.2 per cent, and with Rukhiyan represented by 27.2 to 28.1 per cent of sand. Gravel percentage is markedly more in Rukhiyan study site, i.e. between 8 to 11.8 per cent, followed by Janthri between 5.87 and 9.79 per cent, and Balmiya top being represented by 4.21 to 5.92 per cent of gravel. Percentage of gravel remains markedly insignificant, of worth as relates to soil physical properties. The soil in all the three sites would thus be defined as loam-sandy or sandy-loam. The proportion of the soil texture across the different depths (0-10 cm, 10-20 cm, an 20-30 cms) do not differ very markedly, across all the three sites.

The proportion of the clay, which remains less than 0.5 per cent, in invariably all the sites, aided by relatively greater proportion of the sand and silt define the extent of water retention, and thus soil moisture or water holding capacity. In other words, greater proportion of sand (between 30.66 to 38.46 per cent) in Janthri would relate to lesser water holding capacity or moisture content, as compared with Rukhiyan (sand percentage between 27.2 and 28.1) or of Balmiya top (sand percentage between 26.85 and 30.2).

(ii) Bulk density Interestingly, the bulk density of the soils in all the three study sites does not vary significantly, irrespective of the mark difference in the proportion of sand (principally). In other words, bulk density, as mark of soil compactness (invariably on account of the livestock grazing pressure, or resultant of anthropogenic pressure during the interim of the harvesting season, is equal throughout the three sites. In brief, this important variable to ascertain the intensity of the grazing pressure remains null, and of inconsequence, OR we may require more soil samples distributed temporally and as well as space to ascertain if the grazing pressure (or anthropogenic pressure) does impact upon the bulk density of the soil.

(iii) Moisture content Moisture content invariably relates to the soil texture and reflects upon the greater proportion of sand in Rukhiyan and Janthri, with proportionately lesser moisture content (between 70- 80 percent) as compared with Balmiya top, with moisture content between 86 and 91 per cent.

0.48 0.47 ) 3 0.46

(g/cm 0.45 0.44 0.43 0.42 Bulk density 0.41 0.4 0.39 Balmiya top Rukhyan Jantri Study sites

Figure 2: Bulk density of the soil profiles of the study sites (for the month of August)

120

100

60 0-10 cm

40 10-20 cm 20-30 cm Moisture contentMoisture (%) 20

0 Balmiya top Rukhyan Jantri Study sites

Figure 3: Moisture content of the soil profiles of the study sites (for the month of August)

(iv) Soil pH Soil pH in all the three study sites remains around 5 (between 5 and 5.5). This is one of the important variables that determines the availability of the host larva and consequently of the prized specimen- the Ophiocordyceps sinensis. The pH, however show marked variation, considering the soil samples taken in two intervals, the month of June and August. The difference in pH are marked considering that the pH value difference of 0.5 equal to 5 times increase or decrease in pH; and also considering the fact that usual arrival of the shepherds with their livestock to the alpine meadows (the study sites) is towards the mid-July aided by the fact that these livestock stay in the pens for a duration of 1 month; the soil pH reading of the samples collected in the month of August should be marked by a difference (as compared to those of samples collected in the month of June (devoid of livestock presence). Notwithstanding this fact, the only site with marked change in the pH remains the Balmiya

Top, with pH between 4.99 and 5.08 in the month of June, and between 5.21 and 5.48 in the month of August. This change in the pH correlates well with the grazing pressure, since Balmiya top harbours greater livestock grazing pressure than the rest of the two sites.

5.6 5.5 5.4 5.3 5.2 5.1 0-10 cm

pH of soil 5 10-20 cm 4.9 20-30 cm 4.8 4.7 4.6 Balmiya top Rukhyan Jantri Study sites

Figure 4: Soil pH across the soil profiles in three different study sites (for the month of June)

(v) Organic carbon content Organic carbon content shows strong correlation with the intensity of the grazing pressure, with Balmiya top experiencing greater livestock grazing pressure concomitantly exhibiting greater organic carbon content; with proportionate decline in the two sites- Rukhiyan and Janthri, respectively. The organic carbon content in Balmiya top lying between 3.17 and 4.41, with Rukhiyan and Janthri sequentially showing the organic carbon content between 2.73 – 3.45, and 2.68- 2.97, respectively, for the month of June. The figures for the month of August (experiencing greater livestock grazing pressure) show a marked increase in the over-all organic carbon content, with Balmiya top exhibiting organic carbon content between 3.68- 4.52; Rukhiyan- 3.24- 4.03; and Janthri with the figures between 3.06- 3.97. Invariably in all the study sites, the total organic carbon decreases across the soil profiles, with maximum in the top soil profile (0-10 cms) and least exhibited by soil profile (20-30 cm). For example, the organic carbon content for the soil profiles 0-10, 10-20, and 20-30 cms in Balmiya top for the month of August, are 4.52, 4.46, and 3.68, respectively. Similar trend is exhibited by other sites, too.

5 4.5 4 3.5 3 2.5 0-10 cm 2 10-20 cm 1.5 rganic carbon (%)

O 20-30 cm 1 0.5 0 Balmiya top Rukhyan Jantri Study sites

Figure 5: Organic carbon (in percentage value) across the soil profile in three different study sites (for the month of August)

(vi) Total organic matter Total organic matter content reciprocates the strong correlation of the organic carbon content with the intensity of the grazing pressure, with Balmiya top again exhibiting proportionately greater total organic matter- ranging between 5.53 – 7.55; followed by Rukhiyan and Janthri sequentially, with figures between 4.65 – 5.91 and 3.93 – 5.07, respectively, for the month of June. The figures for the month of August similarly correlate well with increased livestock grazing pressure, with the three sites- Balmiya, Rukhiyan and Janthri, exhibiting the total organic matter content between 6.3 -7.73, 5.54 – 6.88, and 5.26 – 6.81, respectively. As would be expected; invariably in all the study sites, the total organic matter, too decreases across the soil profiles, with maximum in the top soil profile (0-10 cms) and least exhibited by soil profile (20-30 cm). For example, the total organic matter content for the soil profiles 0-10, 10-20, and 20-30 cms in Balmiya top for the month of August, are 7.73, 7.67, and 6.3, respectively. Similar trend is exhibited by other sites, too.

9 8 7 6 5 0-10 cm 4 3 10-20 cm 2 20-30 cm

Total organic matter (%) 1 0 Balmiya top Rukhyan Jantri Study sites

Figure 6: Total organic matter (in percentage value) across the soil profile in three different study sites (for the month of August)

(vii) Total nitrogen The total nitrogen again content shows strong correlation with the intensity of the grazing pressure, with Balmiya top experiencing greater livestock grazing pressure concomitantly exhibiting greater total nitrogen content; with proportionate decline in the two sites- Rukhiyan and Janthri, respectively. The total nitrogen content in Balmiya top lie between 0.56 – 0.75, with Rukhiyan and Janthri sequentially showing the total nitrogen content between 0.47 – 0.58, and 0.30 – 0.46, respectively, for the month of June. However, the figures for the month of August (experiencing greater livestock grazing pressure) show a marked decline in the over-all total nitrogen content, with Balmiya top exhibiting total nitrogen between 0.32 – 0.43; Rukhiyan- 0.22 – 0.38; and Janthri experiencing the least grazing pressure, with figures between 0.29 – 0.37, lying in between the two study sites.

Invariably, as in the case of the organic carbon content, the total nitrogen content, too decreases across the soil profiles, with maximum in the top soil profile (0-10 cms) and least exhibited by soil profile (20-30 cm). For example, the total nitrogen content for the soil profiles 0-10, 10-20, and 20-30 cms in Balmiya top for the month of August, are 0.43, 0.37, and 0.32, respectively. Similar trend is exhibited by other sites, too.

What this above fact amounts to is that any factor, inclusive of the uprooting of the Yartsa Gunbu by the harvesters, or disturbance caused by the livestock will result in loss of the top soil and resultant changes in the carbon-nitrogen ratio as a result of loss of rich organic carbon layer due to soil erosion, and with declined total nitrogen content as a result of livestock grazing, as is depicted by the figures for the month of June and August.

0.9 0.8 0.7 0.6 0.5 0-10 cm

Nitrogen (%) 0.4 10-20 cm 0.3

Total 20-30 cm 0.2 0.1 0 Balmiya top Rukhyan Jantri Soil profile of three different study sites

Figure 7: Total nitrogen (in percentage value) across the soil profile in three different study sites (for the month of June)

(viii) Available nitrogen

The available nitrogen, as expected show similar trends as exhibited by total nitrogen content; and thus, shows strong correlation with the intensity of the grazing pressure, with Balmiya top experiencing greater livestock grazing pressure concomitantly exhibiting greater available nitrogen content; with proportionate decline in the two sites- Rukhiyan and Janthri, respectively. The figures for the month of August (experiencing greater livestock grazing pressure) show a marked decline in the over-all available nitrogen content, with Balmiya top exhibiting available nitrogen between 148.78-172.47; Rukhiyan- 110.79-129.26; and Janthri experiencing the least grazing pressure, with figures between 108.36- 127.87. Invariably, as in the case of the organic carbon content, the available nitrogen content, too decreases across the soil profiles, with maximum in the top soil profile (0-10 cms) and least exhibited by soil profile (20-30 cm). For example, the total nitrogen content for the soil profiles 0-10, 10-20, and 20-30 cms in Balmiya top for the month of June, are 270.04, 206.97, and 158.13, respectively. Similar trend is exhibited by remaining two sites, too.

(ix) Total potassium The total potassium shows an inverse relationship with the intensity of the grazing pressure, i.e. lesser the intensity of the grazing pressure (i.e. in Janthri) relatively more the concentration of the total potassium content. The figures for the month of August that primarily relates with the actual intensity of the livestock grazing pressure, immediately after the return of the last harvester of Yartsa Gunbu, are: Balmiya top between 0.90 – 0.97, with Rukhiyan and Janthri sequentially exhibiting between 0.96- 1.06 and 1.04- 1.11. Invariably, as in the case of the organic carbon content, the total potassium content, too decreases across the soil profiles, with maximum in the top soil profile (0-10 cms) and least exhibited by soil profile (20-30 cm). However, the figure for the Janthri site remains more or less constant (month of August).

350

300 250 (Kg/ Ha) 200 0-10 cm 150 10-20 cm 100 20-30 cm 50 Available Nitrogen 0 Balmiya top Rukhyan Jantri Soil profile of three different study sites

Figure 8: Available nitrogen (in kg/hac value) across the soil profile in three different study sites (for the month of June)

1.4

1.2

0.8 0-10 cm 0.6 10-20 cm

tal Potassium (%) 0.4 20-30 cm To 0.2

0 Balmiya top Rukhyan Jantri Study sites

Figure 9: Total potassium (in percentage value) across the soil profile in three different study sites (for the month of August)

(x) Available potassium The available potassium, in contrast to total potassium however do not exhibit any relationship with the intensity of the grazing pressure, as well as across the soil profiles, with different sites though exhibiting a decline in available potassium concentration across the immediate two soil profiles 0-10 cm and 10-20 cm. However, the soil profile 20-30 cm, again exhibit an increased concentration of the available potassium. Interestingly, in Janthri (with least grazing pressure), the available potassium concentration in fact show a constant decreasing trend with soil depth; the figures being 227.39, 193.39 and 152.66 for the soil depths 0-10, 10-20 and 20-30 cm, respectively.

350 300 250

um (kg/ha) 200 0-10 cm 150 10-20 cm 100 20-30 cm 50 Available Potassi 0 Balmiya top Rukhyan Jantri Study sites

Figure 10: Available potassium (in kg/ha) across the soil profile in three different study sites (for the month of August)

(xi) Total phosphorus The total phosphorus does not exhibit any relationship with the intensity of the grazing pressure, as well as across the soil profiles, with different sites though exhibiting a decline in available potassium concentration across the immediate two soil profiles 0-10 cm and 10-20 cm. However, the soil profile 20-30 cm, again exhibit an increased concentration of the available potassium. It seems that the effect of the grazing pressure on the total phosphorus content do vary across the upper two soil profile, but the exhibited variance is minor, of any significance, what’s so ever. On the hindsight, it seems that we need to further divide each individual site further into several segments differing in intensity of grazing pressure. The results would then vary substantially, and more informative. 0.25

0.2

0.15 0-10 cm 0.1 10-20 cm 20-30 cm Total phosphorus (%) 0.05

0 Balmiya top Rukhyan Jantri Study sites

Figure 11: Total phosphorus (in percentage value) across the soil profile in three different study sites (for the month of August)

(xii) Available phosphorus The results of the available phosphorus content, in general replicate the results of the total phosphorus content, both in term of the relative intensity of the grazing pressure, as well as concentration across the soil profiles.

(xiii) Exchangeable sodium The results of the exchangeable sodium, in general replicate the results of the total phosphorus content, both in term of the relative intensity of the grazing pressure, as well as concentration across the soil profiles, in general. However, the concentration of the exchangeable sodium in Janthri site (with least or non-existent grazing pressure) does exhibit a declining trend across the soil profiles, with the figures being 25.3, 22.7 and 12.8 across the soil profiles 0-10, 10-20 and 20-30 cms. Interestingly for both the months of June and August, the concentration of the exchangeable sodium does show a declining trend in the first two soil profiles (i.e. from 0 cm to 20 cms), in invariably all the study sites, except Rukhiyan (with moderate grazing pressure), which show an increase in concentration with increase in depth.

350

300

250

200 0-10 cm 150

phosphorus(mg/Ha) 10-20 cm 100 20-30 cm

Available 50

0 Balmiya top Rukhyan Jantri Study sites

Figure 12: Available phosphorus (in mg/ha) across the soil profile in three different study sites (for the month of August) 45 40 35 30 25 0-10 cm 20 15 10-20 cm 10 20-30 cm 5 Exchangeable sodium (ppm) 0 Balmiya top Rukhyan Jantri Study sites

Figure 13: Exchangeable sodium (in ppm) across the soil profile in three different study sites (for the month of August)

(xiv) Exchangeable calcium The results for the exchangeable calcium for the month of June (when there is no immediate presence of grazing pressure in all the three study sites) remains inconsequent of any derivable value, except in Janthri, which exhibits a declining trend in concentration of the exchangeable calcium with soil depth. The figures being 17.1, 16.6 and 12.0, for the soil profiles 0-10, 10-20 and 20-30 cms. However, the results for the month of August (when there is very presence of the livestock grazing pressure) are very conspicuous. There is direct and inverse relationship of exchangeable calcium with the intensity of the grazing pressure, as exhibited by the sharp decline in concentration of the exchangeable calcium with intensity of grazing pressure; greater in Balmiya top (maximum intensity of grazing pressure), followed by Rukhiyan (with moderate grazing pressure), across the soil profiles.

(xv) Exchangeable magnesium The results for the exchangeable magnesium for the month of June (when there is no immediate presence of grazing pressure in all the three study sites) remains inconsequent of any derivable value, except in Janthri, which exhibits a declining trend in concentration of the exchangeable magnesium with soil depth. However, the results for the month of August (when there is very presence of the livestock grazing pressure) are very conspicuous. There is direct and inverse relationship of exchangeable magnesium with the intensity of the grazing pressure, as exhibited by the sharp decline in concentration of the exchangeable magnesium with intensity of grazing pressure; greater in Balmiya top (maximum intensity of grazing pressure), followed by Rukhiyan (with moderate grazing pressure), and Janthri, across the soil profiles. 45 40 35 30 25 0-10 cm 20 10-20 cm 15 10 20-30 cm 5

Exchangeable calcium (meq/l ) 0 Balmiya top Rukhyan Jantri Study sites

Figure 14: Exchangeable calcium (in meq/l) across the soil profile in three different study sites (for the month of August)

eq /l) 20

15 0-10 cm 10 10-20 cm 5 20-30 cm

Exchangeable magnesium (m Balmiya top Rukhyan Jantri -5 Study sites

Figure 15: Exchangeable magnesium (in meq/l) across the soil profile in three different study sites (for the month of August)

No doubt, we need to further divide each of the study sites into segments that differ in the intensity of the grazing pressure and collect samples of soil from these segments. This will yield more reliable information as relates to the effect of the grazing pressure on soil parameters. Grazing intensity is to be calculated in term of the total area grazed with total population size of the sheep; i.e. number per hectare. In other words, while the number of sheep population might be large in one site; the total area too may be large, and hence the grazing intensity could be less than suppose another site with relatively smaller size of sheep population, but also with less grazing area. .

Table 1: Soil parameters studies across the three different sites differing in magnitude of grazing intensity Parameter Study sites (Month of June) Study sites (Month of August) studied Balmiya Top Rukhiyan Janthri Balmiya Top Rukhiyan Janthri 1* 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Soil Gravel 4.21 5.92 5.70 8.01 11.86 10.44 5.87 9.57 9.79 texture Sand 26.85 29.21 30.20 27.49 27.19 28.10 30.66 38.46 32.14 (in %) Silt 68.52 64.66 63.83 64.1 60.65 60.62 63.21 51.8 57.87 Clay 0.42 0.21 0.27 0.40 0.30 0.84 0.26 0.17 0.20 Bulk density 0.32 0.35 0.40 0.49 0.28 0.44 0.49 0.28 0.44 0.40 0.35 0.52 0.51 0.45 0.54 0.45 0.44 0.44 Mean 0.35 ± 0.023 Mean 0.4 ± 0.063 Mean 0.4 ± 0.063 Mean 0.42 ± 0.05 Mean 0.5 ± 0.02 Mean 0.44 ± 0.002 Moisture 82.22 95.43 85.86 58.13 81.9 89.75 79.92 74.78 69.27 91.13 86 89.90 78.68 70.01 75.58 79.59 79.68 80.09 content ±2.1 ±0.86 ±0.43 ±0.21 ±5.24 ±0.54 ±0.87 ±2.81 ±6.1 ±3.04 ±4.72 ±2.49 ±12.83 ±11.7 ±12.49 ±2.24 ±2.14 ±1.57 pH 4.99± 5.05 5.08 5.09 5.20 5.25 5.12 5.31 5.42 5.21 5.48 5.32 5.15 5.20 5.16 5.28 5.30 5.31 0.062 ±0.03 ±0.03 ±0.05 ±0.02 ±0.03 ±0.03 ±0.03 ±0.02 ±0.13 ±0.1 ±0.13 ±0.005 ±0.02 ±0.019 ±0.04 ±0.03 ±0.015 Organic carbon 4.13 4.41 3.17 3.44 3.45 2.73 2.97 2.68 2.31 4.52 4.46 3.68 4.03 3.84 3.24 3.97 3.7 3.06 ±0.18 ±0.12 ±0.1 ±0.26 ±0.18 ±0.23 ±0.30 ±0.3 ±0.47 ±0.21 ±0.7 ±0.11 ±0.25 ±0.21 ±0.14 ±0.13 ±0.27 ±0.32 Total organic 7.05 7.55 5.53 5.88 5.91 4.65 5.07 4.58 3.93 7.73 7.67 6.3 6.88 6.56 5.54 6.81 6.32 5.26 matter ±0.31 ±0.22 ±0.18 ±0.46 ±0.32 ±0.41 ±0.51 ±0.52 ±0.8 ±0.37 ±0.12 ±0.19 ±0.43 ±0.37 ±0.25 ±0.23 ±0.47 ±0.53 Total nitrogen 0.75 0.66 0.56 0.58 0.50 0.47 0.46 0.37 0.30 0.43 0.37 0.32 0.38 0.34 0.22 0.37 0.31 0.29 ±0.05 ±0.03 ±0.03 ±0.03 ±0.02 ±0.02 ±0.04 ±0.03 ±0.02 ±0.04 ±0.04 ±0.028 ±0.022 ±0.04 ±0.38 ±0.02 ±0.01 ±0.02 Available 270.0 207 158.9 226.1 139.7 137.6 185.0 166.2 116.7 160.6 148.8 172.47 129.26 114.9 110.79 127.9 110.1 108.36 nitrogen ±13.0 ±8.45 ±3.69 ±27.8 ±2.4 ±2.24 ±10.7 ±4.66 ±10.8 ±13.9 ±12.6 ±21.17 ±9.37 ±4.95 ±10.36 ±8.55 ±3.52 ±5.93 Total potassium 0.49 0.65 0.72 1.04 1.23 1.20 0.82 0.95 0.73 0.97 0.97 0.90 1.06 0.97 0.96 1.11 1.04 1.11 ±0.16 ±0.14 ±0.2 ±0.12 ±0.07 ±0.05 ±0.07 ±0.11 ±0.07 ±0.43 ±0.5 ±0.33 ±0.03 ±0.02 ±0.4 ±0.46 ±0.02 ±0.4 Available 180.1 136.6 141.2 146.3 164.6 163.1 127.1 125.6 129.1 271.6 190.8 193.13 173.58 210.3 137.00 227.0 193.4 152.66 potassium ±7.48 ±4.0 ±5.51 ±6.59 ±5.05 ±5.4 ±9.98 ±7.12 ±7.2 ±52.5 ±8.21 ±38.85 ±18.18 ±40.9 ±13.22 ±20.7 ±7.22 ±17.14 Total 0.25 0.23 0.25 0.40 0.36 0.40 0.143 0.147 0.130 0.19 0.19 0.19 0.16 0.15 0.16 0.14 0.13 0.13 phosphorus ±0.04 ±0.03 ±0.04 ±0.02 ±0.01 ±0.01 ±0.01 ±0.01 ±0.01 ±0.00 ±0.00 ±0.002 ±0.002 ±0.03 ±0.036 ±0.00 ±0.00 ±0.003 Available 328.8 333.5 290.8 281.2 286 295.7 306.4 230.8 228 234.4 246.4 241.6 214.4 245.6 234.6 152.8 116.8 127.6 phosphorus ±25 ±31.5 ±16.0 ±39.2 ±42.8 ±40.3 ±11.3 ±7.74 ±2.68 ±17.9 ±17.3 ±12.08 ±26.16 ±13.6 ±31.12 ±29.3 ±18.6 ±17.33 Exchangeable 11.35 9.63 9.24 11.94 8.18 9.05 25.32 22.71 12.85 32.9 24.68 21.37 16.01 22.95 26.03 9.98 8.99 13.66 ±1.82 ±2.25 ±1.82 ±2.56 ±1.79 ±3.68 ±1.34 ±2.89 ±3.12 ±6.18 ±2.44 ±4.5 ±0.44 ±6.05 ±3.72 ±1.59 ±1.56 ±4.06

74 sodium Exchangeable 11.48 5.58 11.45 9.60 10.03 10.28 17.11 16.66 12.0 29.77 10.66 11.33 16.66 8.0 7.66 26.44 31.11 19.77 calcium ±2.39 ±1.06 ±2.4 ±1.63 ±3.09 ±2.94 ±3.9 ±2.1 ±3.17 ±9.27 ±1.41 ±4.1 ±3.66 ±2.1 ±2.64 ±5.47 ±4.13 ±3.87 Exchangeable 0.13 4.61 -1.17 2.65 2.53 2.30 8.22 16.0 5.11 5.33 2.66 2.22 5.77 4.66 -0.33 16.22 14.0 2.22 magnesium ±1.8 ±2.55 ±2.32 ±2.26 ±3.38 ±1.86 ±5.86 ±4 ±5.57 ±3.95 ±2.08 ±4.98 ±2.61 ±2.84 ±1.91 ±7.67 ±5.98 ±7.1 * Soil profiles: 1- 0-10 cm, 2- 10-20 cm, and 3- 20-30 cm

CHAPTER 5: VEGETATION CHARACTERISTIC DIFFERENCES ACROSS THREE ALPINE MEADOW SITES DIFFERING IN MAGNITUDE OF GRAZING PRESSURE

Introduction Alpine vegetation is one of the main sources of food for the livestock’s which lives in alpine meadows. In other word it is the vegetation that attracts the livestock toward them. Alpine vegetation is characterised by the absence of trees and by dominance of plants of generally low stature (Billings 1973, Korner 1995). Grazing is a common land use practice in grasslands that influences soil properties (Augustine and Frank 2001, Klumpp et al. 2009, Xie et al. 2014, Costa et al. 2015) and plant community characteristics (Zhu et al. 2012, Tarhouni et al. 2015, Álvarez-Martínez et al. 2016). With increasing human population, the demands on grasslands are increasing. Intensive livestock grazing pressure results in bringing about not just soil degradation, but is also known to bring about reduction in plant species diversity (Álvarez-Martínez et al. 2016; Angassa, 2014), net primary productivity and vegetation cover (Buttolph and Coppock, 2004; Cingolani et al. 2005; Pulido et al. 2016) and changing soil structure and soil nutrients (Gass and Binkley, 2011; Jiang et al. 2011; Mcsherry and Ritchie, 2013; Fivez et al. 2014; Lu et al. 2015; Palacio et al. 2014, Cerdà and Lavee 1999, Sarah and Zonana, 2015).

So, seeking a rational grassland management regime is an urgent issue for professionals, herders and the government to achieve sustainable animal production and to maintain the health of the grassland ecosystem (Conant et al. 2001, Watkinson and Ormerod 2001). However, grazing exclusion could also result in loss of plant density and species richness in high-productivity grassland (Oba et al. 2001), which may result from greater competition for canopy resources, for example, light (Borer et al. 2014) or/and nutrient availability (Van der Wal et al. 2004). Species diversity plays an important role in maintaining ecosystem resilience, and thus it is necessary to graze at a moderate stocking rate for restoring and maintaining a high level of biodiversity and ecosystem function (Bai et al. 2004, 2007; Tilman et al. 2006; Shang et al. 2008; Wu et al. 2009; Cong et al. 2014; Qian et al. 2014). Grazing intensity, in addition to bringing about above-mentioned facts, is also responsible for bring about above-ground change in flora composition with non-palatable species replacing the palatable species.

Lastly, even though studies of effects of grazing in alpine habitats are abundant, but these mostly concern the studies undertaken with one patch of grazing site being kept fenced (as control), and others differing in magnitude of grazing intensities in terms of the livestock population size being allowed to graze. Such experiments cannot be replicated in the present study, where the prime objective remains to ascertain the effect of the grazing pressure on the yield of the Yartsa Gunbu (Ophiocordyceps sinensis) for the simple fact that ‘control site’ as one would envision cannot be practically possible since the harvesters of Yartsa Gunbu would not leave the small patch left untouched, however small the control site would be. The present study thus attempts to study the effect of grazing intensity, in terms of the livestock population and the duration of their stay, on the likely yield of Yartsa Gunbu vis-a-vis changes in the above-ground species profile, ad likely impact of the livestock on the soil physical and chemical nature. The present paper however deals with the phytosociology undertaken in three different sites selected on the basis of the grazing intensity of livestock population, comprising wholly of goats and sheep.

Study area The study was carried out in three alpine meadow sites in Munsiari region of Pithoragarh district of Uttarakhand, Western Himalaya known for exploitation of Yartsa Gunbu. The study sites located at altitude above 3200 meters amsl, and between N=30o088’46’’ & E=080o17.935’. The study sites differ in magnitude of grazing pressure, with Balmiya top experiencing maximum grazing pressure, followed by Rukhiyan with moderate grazing pressure, and Janthri with none or insignificant grazing pressure. Selection of the study sites was carried out by prior interviews with the local shepherds to ascertain the intensity of the grazing pressure (i.e., number of the livestock population and the duration of their stay in the alpine meadows being selected for study). All the study sites are separated apart, on average by 2.5 km. The study was carried out in the month of June, with above ground temperature between 7-8oC. Average humidity around 50%, which varies between a minimum of 26% increasing to 83%.

Methodology Each of the study sites were divided into 5 quadrates measuring 5 x 5 meters, taking into account the topographical features, and slope aspects. Within each larger 5 x 5m quadrate three sub quadrates of measuring 1x1m for phytosociology of the herbs was undertaken, as per Ralhan et al. 1982. Phytosociological studies attempted all herbaceous species. Evaluation of the plant species richness and relative abundance- the two basic parameters to assess biodiversity changes across the different sites differing in intensity of grazing pressure was undertaken, as was the species diversity.

Result and Discussion (i) Diversity Diversity is a combination of two factors - the number of species present, referred to as species richness and the distribution of individuals among the species, referred to as evenness or equitability. The diversity in three alpine meadows was determined with the Shannon–Wiener index. The sampling process and the adjusting of the quadrates are randomly sampled so that the all species of plants from a community are included in the sample. The Shannon-Wiener Index (H’) of Balmiya top; Rukhiyan and Janthri are 1.69; 0.042; 2.12 and the evenness (J) of the three sites are 0.53; 0.079 and 0.66. The β-diversity between Balmiya top and Rukhiyan was 0.78; for Rukhiyan and Janthri-1.21, while between Balmiya top and Janthri, it was 1.31. Small differences in the Beta diversity between the two sites- Rukhiyan and Janthri and between Balmiya top and Janthri indicate that the growth forms among different stands respond in similar fashion (Adhikari et al. 1991), while low value of beta diversity as in case of study sites Balmiya top and Rukhiyan indicate that the species composition does not vary significantly across the two study sites.

Table 1: Describe the β-Diversity of study site (A, B and C) Indices of diversity Study sites Balmiya top Rukhiyan Janthri Shannon-Wiener Index (H’) 1.69 2.21 2.12 Evenness (J) 0.53 0.66 0.66 β-Diversity Balmiya top and Rukhiyan and Balmiya top Rukhiyan Janthri and Janthri 0.82 0.84 0.93

(ii) Phytosociology The three different Alpine meadows of Munsiari have different species of herbaceous plants. A total of 31 species were recorded in which 22 species are palatable and 9 are unpalatable. The different species found in three study sites are Nomocharis species, Caltha palustris, Geum elatum, Viola biflora, Cortia depressa, Anemone tetrasepala, Polystachyum stimulens, Hypericum montanium, Aconitum violacium, Persicarya polystchya, Polyganum chinensis, Aruncus diocus, ligularia amplexicaulis, Aquilegia pubiflora, Corydalis cashmerianum, Euphorbia cognate, Anemone tetrasepala, Kobresia nepalensis, Potentilla fulgens, Persicaria amplexicaule, Cyprus sp., Clintonia udensis, Nardostachys grandiflora, Delphinium brunonianum, Anaphalis cuneifolia, A. margarita, Selinum candollii, Angelica archangelica, Megacarpaea polyandra, Dactylorhiza hatagirea, Primula denticulata.

The number of species in three sampling sites indicates that these meadows are comparatively species rich. Out of the three sites, Rukhiyan (B) has high species richness followed by Janthri and Balmiya top. It was also found that the number of unpalatable species was high in Rukhiyan followed by Janthri and Balmiya top. The above-mentioned species encountered in study areas are grouped on the bases of palatability and unpalatability and are listed in the following table. 3. The plants density has been recorded as 3657778/hac. in Balmiya top (A), 9005926 /hac. in Rukhiyan (B) and 2075185/hac. in Janthri (C). In variably in all the three study sites, the dominant species remains Danthonia cachmeriana. This species apart, in study site Balmiya top (A) and Janthri (C) Viola biflora is the dominant species followed by Polyganum chinensis and then Cortia depressa only in Balmiya top (A) whereas in Rukhiyan (B) dominant species was Polyganum chinensis followed by Viola biflora. But in Janthri (C) Cortia depressa is the second dominant after Viola biflora followed by Euphorbia cognate (Tables 2-4).

(iii) Palatable versus non-palatable species Grazing by livestock leads to profound changes in the over-all above ground species diversity, with palatable species being replaced by more resistant non-palatable species. In fact, number and abundance of the non-palatable species vis-a-vis palatable ones remains a sure sign of intensity of grazing pressure. In the present study, which was confined for a period of just one month, the number of non-palatable species in fact is more in Rukhiyan site (08 spp.), followed by Balmiya top (05 spp.) and lastly Janthri (04 spp., table 5).

Table 2: Phytosociology in the study site Balmiya top S. No Species name Frequency (%) Abundance A/F ratio Density/ha Relative Freq. Relative Density Relative IVI Basal area 1. Nomocharis species 40.74 22.90 0.56 93333.33 0.031 0.025 0.0017 0.057 2. Caltha palustris 100 6.66 0.06 61111.11 0.076 0.016 0.0039 0.095 3. Geum elatum 96.29 13.22 0.13 132222.22 0.074 0.036 0.0087 0.118 4. Viola biflora 100 155.74 1.55 1557407.40 0.076 0.425 0.0008 0.501 5. Cortia depressa 100 62.25 0.62 622592.59 0.076 0.170 0.0169 0.262 6. Anemone tetrasepala 96.29 10.61 0.11 98518.51 0.074 0.026 0.0008 0.100 7. Polystachyum stimulens 59.025 2.62 0.044 13703.70 0.045 0.003 0.0002 0.048 8. Hypericum montanium 70.37 5.57 0.07 39259.25 0.054 0.010 0.0026 0.066 9. Sp. (?) 96.29 17.42 0.18 167777.77 0.074 0.045 0.0001 0.119 10. Aconitum violacium 18.51 2.6 0.14 4444.44 0.014 0.001 0.0003 0.015 11. Persicarya polystchya 51.85 6.42 0.12 33333.33 0.039 0.009 0.0004 0.048 12. Polyganum chinenns 44.44 87.41 1.96 388518.51 0.034 0.106 0.0002 0.140 13. Aruncus diocus 14.81 12.5 0.84 18518.52 0.011 0.005 0.0001 0.016 14. ligularia amplexicaulis 40.74 2 0.04 8518.51 0.031 0.002 0.0008 0.033 15. Euphorbia cognata 48.14 8.53 0.17 41111.11 0.037 0.011 0.0004 0.048 16. Anemone tetrasepala 74.07 4.7 0.06 34814.81 0.056 0.009 0.0008 0.065 17. Danthonium cachmeriana 55.55 7.26 0.13 44074.07 0.042 0.012 0.80 0.854 18. Primula denticulata 22.22 2.83 0.12 6296.29 0.017 0.001 0.15 0.168 19. Potentilla fulgens 48.14 11.30 0.23 54444.44 0.037 0.014 0.0013 0.052 20. Persicaria amplexicaule 74.07 27.25 0.36 201851.85 0.056 0.055 0.0041 0.151 21. Cyprus?? 33.33 8.11 0.24 27037.03 0.025 0.007 0.00015 0.032 22. Clintonia udensis 7.40 8.5 1.14 7037.03 0.005 0.001 0.0008 0.006 23. Nardostachys grandiflora 7.40 2.5 0.3 1851.85 0.005 0.000 0.0003 0.005 Total 1299.675 488.9 3657778

Table 3: Phytosociology in Rukhiyan study site S. No Species name Frequency (%) Abundance A/F ratio Density/ha Relative Freq. Relative Density Relative Basal area IVI 1. Nomocharis species 14.81 10.5 0.70 15555.55 0.012 0.012 0.0017 0.025 2. Caltha palustris 77.77 10.85 0.13 84444.44 0.066 0.066 0.0039 0.135 3. Geum elatum 100 7.96 0.07 80000.00 0.085 0.085 0.0087 0.178 4. Viola biflora 100 78.59 0.78 7875925.92 0.085 0.085 0.0008 0.170 5. Cortia depressa 51.85 23.64 0.45 122592.59 0.044 0.044 0.0169 0.104 6. Anemone tetrasepala 37.03 8.2 0.22 30370.37 0.031 0.031 0.0008 0.062 7. Polystachyum stimulens 66.66 4.05 0.06 24444.44 0.056 0.056 0.0002 0.112 8. Hypericum montanium 88.88 6.83 0.07 62592.59 0.075 0.075 0.0026 0.152 9. Sp. (?) 70.37 10.73 0.15 73333.33 0.060 0.060 0.0001 0.120 10. Aconitum violacium 11.11 1.33 0.11 1481.48 0.009 0.009 0.0003 0.018 11. Persicarya polystchya 81.48 6.86 0.08 52592.59 0.069 0.069 0.0004 0.138 12. Polyganum chinenns 18.51 111.8 6.0 207037.03 0.015 0.015 0.0002 0.030 13. Aruncus diocus 11.11 4.33 0.38 4814.81 0.009 0.009 0.0001 0.018 14. ligularia amplexicaulis 66.66 4.27 0.06 28518.51 0.056 0.056 0.0008 0.112 15. Corydalis cashmerianum 70.37 4.15 0.05 29259.25 0.060 0.060 0.00008 0.120 16. Euphoria cognata 3.70 2 0.54 740.74 0.003 0.003 0.0004 0.006 17. Anemone tetrasepala 66.66 4.5 0.06 30000.00 0.056 0.056 0.0008 0.112 18. Danthonium cachmeriana 40.74 5.36 0.13 14444.44 0.034 0.034 0.80 0.868 19. Potentilla fulgens 7.40 4 0.54 2962.96 0.006 0.006 0.0013 0.013 20. Cyprus?? 48.14 14.07 0.29 66666.66 0.041 0.041 0.00015 0.082 21. Clintonia udensis 14.81 7.5 0.50 10740.74 0.012 0.012 0.0008 0.024 22. Nardostachys grandiflora 22.22 16.66 0.74 37777.77 0.018 0.018 0.0003 0.036 23. Sp. (?) 14.81 63.75 4.30 94444.44 0.012 0.012 0.0019 0.025 24. Delphinium brunonianum 22.22 6.16 0.27 13703.70 0.018 0.018 0.0001 0.036 25. Anaphalis cuneifolia 7.40 6 0.81 4444.44 0.006 0.006 0.0004 0.012 26. Selinum candollii 22.22 11.83 0.53 22592.59 0.018 0.018 0.0008 0.036 27. Angelica archangelica 29.62 4.37 0.14 12962.96 0.025 0.025 0.00013 0.050

28. Megacarpaea polyandra 3.70 9 2.43 1481.48 0.003 0.003 0.00001 0.006 Total 9005926

Table 4: Phytosociology in Janthri study site S.No. Species name Frequency (%) Abundance A/F ratio Density/ha Relative Freq. Relative Density Relative Basal area IVI 1. Nomocharis species 59.25 12 0.28 71111.11 0.051 0.034 0.0017 0.086 2. Caltha palustris 100 11.5 0.12 115555.55 0.087 0.055 0.0039 0.145 3. Geum elatum 96.29 11.7 0.12 109629.62 0.084 0.052 0.0087 0.144 4. Viola biflora 100 63.8 0.64 631481.48 0.087 0.30 0.0008 0.387 5. Cortia depressa 100 40.1 0.40 544444.44 0.087 0.26 0.0169 0.363 6. Anemone tetrasepala 85.18 9.7 0.11 82962.96 0.074 0.039 0.0008 0.113 7. Polystachyum stimulens 55.55 5.2 0.09 29259.25 0.048 0.014 0.0002 0.062 8. Hypericum montanium 85.18 12 0.14 102222.22 0.074 0.049 0.0026 0.125 9. Sp. (?) 92.59 10.1 0.10 96666.66 0.080 0.046 0.0001 0.126 10. Aconitum violacium 25.92 5 0.19 12962.96 0.022 0.006 0.0003 0.028 11. Persicarya polystchya 29.62 4.5 0.15 13703.70 0.025 0.006 0.0004 0.031 12. Polyganum chinenns 7.40 2 0.27 1481.48 0.006 0.0007 0.0002 0.006 13. Aruncus diocus 7.40 3.5 0.47 2592.59 0.006 0.0012 0.0001 0.007 14. ligularia amplexicaulis 70.37 18.3 0.26 59629.62 0.061 0.028 0.0008 0.089 15. Aquilegia pubiflora 3.70 1 0.27 3703.70 0.003 0.0017 0.00004 0.004 16. Corydalis cashmerianum 29.62 2.5 0.08 7407.40 0.025 0.0035 0.00008 0.028 17. Euphorbia cognata 18.51 28 1.51 51851.85 0.061 0.024 0.0004 0.085 18. Anemone tetrasepala 48.14 4.07 0.08 20000.00 0.042 0.009 0.0008 0.051 19. Danthonium cachmeriana 44.44 6.1 0.13 27407.40 0.038 0.013 0.80 0.851 20. Primula denticulata 3.70 3 0.81 1111.11 0.003 0.00053 0.15 0.153 21. Potentilla fulgens 3.70 17 4.59 6296.29 0.003 0.0030 0.0013 0.007 22. Cyprus sp. 48.14 9.15 0.19 44074.07 0.042 0.021 0.00015 0.063 23. Clintonia udensis 7.40 4.5 0.60 3333.33 0.006 0.0016 0.0008 0.008 24. Nardostachys grandiflora 22.22 16.3 0.7 36296.29 0.019 0.017 0.0003 0.036 Total 2075185

As per the observations made, the highly degraded, i.e. over-grazed sites within the larger landscape sites are encroached by Rumex nepalensis (syn. Shyama). This species in fact can be cited as an indicator species of over-grazed sites. The present study does reflect upon the negative impacts of the grazing pressure; even though as relates to the number of the unpalatable versus palatable species, it would necessitate that the number of quadrates per study sites need be increased so that maximum species could be made inclusive, which in turn would be more reflective of the grazing intensity.

Table: Root: shoot ratio of the above-ground vegetation in three different study sites differing in magnitude of grazing intensity S.No. Species June August 1* 2 3 1 2 3 A. Non-palatable species 1. Aconitum violacium 3.93 3.93 2.83 1.44 1.4 0.56 2. Anaphalis cuneifolia - 5.90 - - 0.56 - 3. Angelica archangelica - 2.79 - - 0.72 0.21 4. Clintonia udensis 0.88 0.88 0.81 0.71 0.47 0.3 5. Dactylorhiza hatagirea - - - - 2.96 - 6. Delphinium brunonianum - 13.47 - - - - 7. Ligularia amplexicaulis 0.74 0.74 0.54 3.2 3.2 1.91 8. Megacarpaea polyandra - 5.73 - - 0.17 - 9. Nardostachys grandiflora 2.95 2.95 0.45 0.43 0.7 0.58 10. Primula denticulata 1.01 9.8 0.97 - - - 11. Raun 11.71 11.71 10.90 0.14 0.14 0.13 12. Selinum candollii - 4.28 - - 0.79 - B. Palatable species 13. Anemone tetrasepala 5.73 5.73 3.65 0.70 0.67 0.59 14. Anemone sp. 2.72 4.63 2.26 - - - 15. Aquilegia pubiflora - - 3.39 2.63 2.56 2.13 16. Aruncus diocus 4.61 4.61 3.85 0.20 0.19 0.17 17. Caltha palustris 13.47 13.47 10.53 0.41 0.33 0.29 18. Cortia depressa 2.79 2.79 2.53 0.29 0.27 0.21 19. Corydalis cachmeriana - 1.68 0.85 4.92 2.61 1.38 20. Cyperus? 3.95 3.95 2.65 0.55 0.49 0.45 21. Dhub ghaas 5.41 5.41 1.21 0.5 0.46 0.35 22. Euphorbia cognata 1.68 2.72 0.79 4.11 4.11 2.66 23. Geum elatum 5.9 5.9 5.13 0.34 0.32 0.29 24. Hypericum montanium 1.06 1.06 0.92 0.76 0.76 0.6 25. Kobresia nepalensis 4.63 1.01 3.70 0.23 0.21 0.2 26. Nomocharis species 13.57 13.57 8.96 - - - 27. Persicarya amplexicaule 4.5 - 01 0.5 - - 28. Persicarya polystachya 0.27 0.27 - 1.53 1.28 1.08 29. Polyganum chinensis 0.83 0.83 0.6 0.21 0.09 0.08 30. Potentilla fulgens 9.8 4.5 6.52 0.23 0.23 0.17 31. Bindi ghaas - 13.57 - - - - 32. Viola biflora 4.28 4.28 1.57 0.87 0.79 0.56 * Palatability of the above-ground species was ascertained from the anwals- the traditional shepherds. Study sites: 1- Balmiya Top, 2. Rukhiyan, and 3. Janthri

CHAPTER 6: CULTURE OF BROWN AND YELLOW SPECIES OF CORDYCEPS

Preliminary trials Protocol for preparation of the culture media Three different culture media were prepared for the culture of fungal mycelia, and two different species (though not yet identified, but given ‘brown’ and ‘yellow’ names) were tried. The three media differ in composition. The following steps are used for media preparation. Step 1: Take 25 g of brown rice. Step 2: Mix 30 ml of nutritional solution in rice. Step 3: Autoclave for 30 min at 121oC. Step 4: Freshly prepared media is then maintain at pH 5.6, by addition of 1N HCl or 1N NaOH solution. Step 5: Cool the media in laminar flow at room temperature. Step 6: Inoculate the seed (mycelia taken from infected caterpillar) in sterilized Petri dish in laminar flow with care. Here with care means inoculation must be done near the burner so that no microbial contamination of the culture media takes place. Step 7: After due inoculation, the Petri dish or the conical flask are sealed with parafilm. Step 8: Place the Petri dish/conical flasks in B.O.D incubator at 18oC and humidity 60 %. Step 9: Observe the mycelia growth every day.

Peptone media Malt extract Corn steep powder Sucrose = 20 g/ L Malt extract = 25 g / L Organic rice =25 g Peptone = 10 g/ L Soluble starch = 10 g/ L Nutrient solution = 30 ml Magnesium sulphate Agar = 15 g / L Nutrient solution consists of (MgSO4.7H2O) = 0.5 g/L Distilled water = 1000 ml Glucose= 20 g/ L Di- potassium ortho pH =5.6 Corn steep powder = 20 g /L phosphate (K2HPO4) = 0.5 g/ Di-potassium L orthophosphates (K2HPO4) = Distilled water = 1000 ml 0.5 g/ L pH = 5.6 Magnesium sulphate (MgSO4 .7H2O) = 0.5 g /L Distilled water = 1000 ml pH= 5.6

Observation Mycelial growth was observed on 5th day of inoculation. Subsequently the temperature of the BOD was raised to 25oC for initiating the growth of the fruiting bodies, in terms of the change in colour of the mycelia. Even though colour change did take place, fruiting bodies did not develop. This was primarily ascribed to lack of very basic facilities to carry out culture experiments, as also probably due to lack of circulating air as well as humidity, which though were both present and maintained in the BOD, the culture flask was however sealed, to really effect the progress of the experiment. Other hit-and-trials will be required for the growth of the fruiting bodies which would need the following steps in future: (i) Raising the temperature or the humidity, or both immediately after the observation of the colour change (ii) Raising the luminosity, which invariably is absent in the present set up

References

Aarons, S.R., Hosseini, H.M., Dorling, L. and Gourley, C.J.P. 2004. Dung decomposition in temperate dairy pastures. II. Contribution to plant-available soil phosphorus. Aust. J. Soil Res. 42: 115-123.

Abensperg-Traun, M., Smith, G. T., Arnold, G. W. and Steven, D. E. 1996. The effects of habitat fragmentation on animal communities in remnants of gimlet Eucalyptus salubris woodland in the Western Australian wheat belt. 1. Arthropods. J. Appl. Ecol. 33: 1281–301.

Abril, A. and Bucher, E.H. 1999. The effects of overgrazing on soil microbial community and fertility in the Chaco dry savannas of Argentina. Applied Soil Ecology 12 (2): 159-167.

Abril, A. and Bucher, E.H. 2001. Overgrazing and soil carbon dynamics in the western Chaco of Argentina. Applied Soil Ecology 16 (3): 243-249.

Adamson, D. A. and Fox, M. D. 1982. Change in Australasian vegetation since European settlement. In Smith, J.M.B. (Ed), A History of Australasian Vegetation), McGraw-Hill, Sydney, pp. 109–46.

Adhikari, B.S., Rikhari, H.C, Rawat, Y.S. and Singh, S.P. 1991. High altitude forest composition, Diversity and profile structure in a part of Kumaon Himalaya. Trop. Ecol. 32: 86-97.

Adler, P. B., R. Salguero-Go´ mez, A. Compagnoni, J. S. Hsu, J. Ray-Mukherjee, C. Mbeau-Ache, and M. Franco. 2014. Functional traits explain variation in plant life history strategies. Proceedings of the National Academy of Sciences USA 111: 740-745.

Adugna, A. and Abegaz, A. 2015. Effects of soil depth on the dynamics of selected soil properties among the highlands resources of Northeast Wollega, Ethiopia: are these sign of degradation? Solid Earth Discuss. 7: 2011-2035.

Agrawal, A.A. and Fishbein, M. 2006. Plant defense syndromes. Ecology 87: S132-S149.

Ahn, Y.J., Park, S.J., Lee, S.G., Shin, S.C. and Choi, D.H. 2000. Cordycepin: selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp. J Agric Food Chem 48: 2744-2748.

Aiken, G.E. 1990. Plant and animal responses to a complex grass-legume mixture under different grazing intensities. Dissertation Abstracts International 51 (3): 1045.

Albon, S.D. and Langvatn, R. 1992. Plant phenology and the benefits of migration in a temperate ungulate. Oikos 65: 502-513

Alconada, M., O.E. Ansin, R.S. Lavado, V.A. Deregibus, G. Rubio, and F.H. Gutiérrez Boem. 1993. Effect of water retention of run-off water and grazing on soil and on vegetation of a temperate humid grassland. Agric. Water Manage. 23:233–246.

Alderson, M.R., Tough, T.W., Braddy, S., Davis-Smith, T., Roux, E., Schooley, K., Miller, R.E. and Lynch, D.H. 1994. Regulation of apoptosis and T cell activation by Fas-specific mAb. Int. Immunol. 6: 1799-1806.

Altesor, A., G. Piñeiro, F. Lezama, R.B. Jackson, M. Sarasola, and J.M. Paruelo. 2006. Ecosystem changes associated with grazing in sub-humid South American grasslands. J. Veg. Sci. 17:323–332.

Alvarez-Martínez J, Gómez-Villar A, Lasanta T. 2016. The uses of goats grazing to restore pastures invaded by shrubs and avoid desertification: a preliminary case study in the Spanish Cantabrian Mountains. Land Degradation and Development 27: 3-13.

Amezaga, I., Mendarte, S., Albizu, I., Besga, G., Garbisu, C. and Onaindia, M. 2004. Grazing Intensity, Aspect, and Slope Effects on Limestone Grassland Structure. J Range Manage 57: 606-612.

Anderson, P. and Radford, E. 1994. Changes in vegetation following reduction in grazing pressure on the National Trust’s Kinder Estate, Peak District, Derbyshire, England. Biological Conservation 69: 55-63.

Anderson, R.C. 1994. Height of white-flowered trillium (Trillium grandiflorum) as an index of deer browsing intensity. Ecological Applications 4: 104-109.

Anderson, V.J. and Briske, D.D. 1995. Herbivore-induced species replacement in grasslands – is it driven by herbivory tolerance or avoidance. Ecol Appl 5: 1014-1024.

Andersson, M. and Jonasson, S. 1986. Rodent cycles in relation to food resources on an alpine heath. Oikos 46: 93-106.

Andersson, S., Rydell, J. and Svensson, M.G.E. 1998. Light, predation and the lekking behavior of the ghost swift Hepialus humuli (L.) (Lepidoptera, Hepialidae), Proceedings of the Royal Society B: Biological Sciences 265: 1345-1351.

Andren, H., Delin, A. and Seiler, A. 1997. Population response to landscape changes depends on specialization to different landscape elements. Oikos 80: 193-196.

Angassa, A. 2014. Effects of grazing intensity and bush encroachment on herbaceous species and rangeland condition in Southern Ethiopia. Land Degradation and Development 25: 438-451.

Arianoutsou-Faraggitaki, M. 1985. Desertification by overgrazing in Greece: the case of Lesvos Island. Journal of Arid Environments 9 (3): 237-242.

Arif, M. and Kumar, N. 2003. Medicinal insect and insect fungus relationship in high altitude areas of Kumaun hills in Central Himalayas. J. Exp. Zool. India 6 (1): 45-55.

Arora, D. 1986. Mushrooms Demystified. Ten Speed Press, Berkeley.

Artjariyasripong, S., Mitchell, J.I., Hywel-Jones, N.L. and Jones, E.B.G. 2001. Relationship of the genus Cordyceps and related genera, based on parsimony and spectral analysis of partial 18S and 28S ribosomal gene sequences. Mycoscience 42: 503-517.

Aryal, A., Dutta, I.C., Dhungel, S.K. and Pyakurel, A. 2008. Parasitic fungal on moth’s larvae: Yarsagumba (Cordyceps sinensis), ecology and local economic contribution in Nepal, Biodiversity Research and Training Forum (BRTF), Nepal.

Augustine, D.J. and Frank, D.A. 2001. Effects of migratory grazers on spatial heterogeneity of soil nitrogen properties in a grassland ecosystem. Ecology 82: 3149-3162.

Augustine, D.J. and McNaughton, S.J. 1998. Ungulate effects on the functional species composition of plant communities: herbivore selectivity and plant tolerance. Journal of Wildlife Management 62: 1165-1183.

Austrheim, G. and Eriksson, O. 2001. Plant species diversity and grazing in the Scandinavian mountains - patterns and processes at different spatial scales. Ecography 24(6): 683-695.

Austrheim, G., Evju, M. and Mysterud, A. 2005. Herb abundance and life-history traits in two contrasting alpine habitats in southern Norway. Plant Ecology 179(2): 217-229.

Austrheim, G., Mysterud, A., Hassel, K., Evju, M. and Økland, R.H. 2007. Interactions between sheep, rodents, graminoids and bryophytes in an oceanic alpine ecosystem of low productivity. Écoscience 14: 178-187.

Austrheim, G., Mysterud, A., Pedersen, B., Halvorsen, R., Hassel, K. and Evju, M. 2008. Large scale experimental effects of three levels of sheep densities on an alpine ecosystem. Oikos 117(6): 837-846.

Austrheim, G., Mysterud, A., Pedersen, B., Okland, R. H., Hassel, K. and Evju, M. 2008b. Large scale experimental effects of three levels of sheep densities on an alpine ecosystem. Oikos 117: 837-846.

B€utof, A., von Riedmatten, L.R., Dormann, C.F., Scherer-Lorenzen, M., Welk, E. and Bruelheide, H. 2012. The responses of grassland plants to experimentally simulated climate change depend on land use and region. Global Change Biology 18: 127-137.

Bai, Y.F., Wu, J.G., Pan, Q.M., Huang, J.H., Wang, Q., F., Li., Buyantuyev, A. and Han, X.G. 2007. Positive linear relationship between productivity and diversity: evidence from the Eurasian Steppe. Journal of Applied Ecology 44: 1023-1034.

Bailey, D.W. and Rittenhouse, L.R. 1989. Management of cattle distribution. Rangelands 11: 159-161.

Bailey, D.W. and Welling, G.R. 1999. Modification of cattle grazing distribution with dehydrated molasses supplement. Journal of Range Management 52: 575-582.

Bailey, D.W., Gross, J.E., Laca, E.A., Rittenhouse, L.R., Coughenour, M.B., Swift, D.M. and Sims, P.L. 1996. Mechanisms that result in large herbivore grazing distribution patterns. Journal of Range Management 49: 386-400.

Bailey, D.W., Welling, G.R. and Miller, E.T. 2001. Cattle use of foothills rangeland near dehydrated molasses supplement. Journal of Range Management 54: 338-347.

Bao, Z.D., Wu, Z.G. and Zheng, F. 1994. Amelioration of aminoglycoside nephrotoxicity by Cordyceps Barseghyan sinensis in old patients. Chung-Kuo Chung Hsi i Chieh Ho Tsa Chih. 14: 271-273.

Bardgett, R.D., and D.A. Wardle. 2003. Herbivore-mediated linkages between aboveground and belowground communities. Ecology 84:2258–2268.

Barlow, H.S. 1982. An introduction to the moths of South East Asia. The Malayan Nature Society, Kuala Lumpur, E. W. Classey Ltd., Faringdon.

Barrett, C.G. 1895. Lepidoptera of the British Isles Vol. II, Reeve, London, pp. 151-168.

Bastrenta, B. 1991. Effect of sheep grazing on the demography of Anthyllis vulneraria in Southern France. Journal of Ecology 79: 275-284.

Baur, B. et al. 2006. Effects of abandonment of subalpine hay meadows on plant and invertebrate diversity in Transylvania, Romania. Biological Conservation 132: 261-273.

Baur, B. et al. 2007. Intensified grazing affects endemic plant and gastropod diversity in alpine grasslands of the Southern Carpathians, Romania. Biologia 62: 438-445.

BCMF (British Columbia, Ministry of Forests) 2002. Considering tools for remediation. Rangeland Health Brochure 4. British Columbia, Canada, pp. 1-22.

Bhuyan, S.I., Tripathi, O.P. and Khan, M.L. 2013. Soil nutrients status in prominent agro-ecosystems of East Siang district, Arunachal Pradesh. Int. J. Environ. Sci. Te. 3: 1957-1968.

Billings, W.D. 1973. Arctic and alpine vegetation: similarities, differences, and susceptibility to disturbance. BioScience 23: 697-704.

Billings, W.D. 1974. Adaptations and origins of alpine plants. Arctic and Alpine Research 6: 129-142.

Billings, W.D. 1987. Constraints to plant growth, reproduction, and establishment in Arctic environments. Arc Alp Res 19: 357-365

Billings, W.D. and Mooney, H.A. 1968. The ecology of arctic and alpine plants. Biological Reviews of the Cambridge Philosophical Society 43: 481-529.

Binns, T. 1990. Is Desertification a Myth? Geography 75:106-113

Biondini, M.E., Patton, D. and Nyren, P.E. 1998. Grazing intensity and ecosystem processes in northern mixed - grass prairie, USA. Ecological Applications 8: 469-479.

Bisigato, A.J., Laphitz, R.M.L. and Carrera, A.L. 2008. Non-linear relationships between grazing pressure and conservation of soil resources in Patagonian Monte shrublands. Journal of Arid Environments 72: 1464-1475.

Bjork, R. and Molau, U. 2007. Ecology of alpine snowbeds and the impact of global change. Arct. Antarct. Alp. Res. 39: 34-43.

Bliss, L.C. 1971. Arctic and alpine plant life cycles. Ann Rev Ecol Syst 2:405-438.

Bok, J.W., Lermer, L., Chilton, J., Klingeman, H.G. and Towers, G.H. 1999. Anti-tumor sterols from the mycelia of Cordyceps sinensis. Phytochemistry 51(7): 891-898.

Borer, E.T., Seabloom, E., Gruner, D., Harpole, W.S., Hillebrand, H., Lind, E., Adler, P., Alberti, J., Anderson, T.M., Bakker, J., Biederman, L., Blumenthal, D., Brown, C., Brudvig, L., Buckley, Y., Cadotte, M., Chu, C., Cleland, E., Crawley, M., Daleo, P., Damschen, E., Davies, K., DeCrappeo, N., Du, G., Firn, J., Hautier, Y., Heckman, R., Hector, A., HilleRisLambers, J., Iribarne, O., Klein, J.A., LaPierre, K., Li, W., MacDougall, A., Melbourne, B., Moore, J., Mortensen, B., O’Halloran, L., Orrock, J., Pascual, J.S., Prober, S., Pyke, D., Risch, A., Schuetz, M., Smith, M., Stevens, C., Sullivan, L., Williams, R., Wragg, P., Wright, J., Yang, L., McCulley, R.L., Mitchell, C.E. 2014. Herbivores and nutrients control grassland plant diversity via light limitation. Nature 508: 517-520.

Bowns, J.E. and Bagley, C.F. 1986. Vegetation responses to long-term sheep grazing on mountain ranges. Journal of Range Management 39(5): 431-434.

Brady, N.C. and Weil, R.R. 1999. The Nature and Properties of Soils, 12th ed., Prentice Hall, New Jersey.

Brandt, J.S., Haynes, M.A., Kuemmerle, T., Waller, D.M. and Radeloff, V.C. 2013. Regime shift on the roof of the world: Alpine meadows converting to shrublands in the southern Himalayas. Biological Conservation 158: 116- 127.

Brathen, K.A. and Oksanen, J. 2001. Reindeer reduce biomass of preferred plant species. J Veg Sci 12:473-480.

Bråthen, K.A., Ims, R.A., Yoccoz, N.G., Fauchald, P., Tveraa, T. and Hausner, V.H. 2007. Induced shift in ecosystem productivity? Extensive scale effects of abundant large herbivores. Ecosystems 10: 773-789.

Braunack, M.V. and Walker, J. 1985. Recovery of some surface soil properties of ecological interest after sheep grazing in semi-arid woodland. Aust. J. Ecol. 10: 451–60.

Bret-Harte, M.S., et al. 2001. Developmental plasticity allows Betula nana to dominate tundra subjected to an altered environment. Ecology 82(1): 18-32.

Brizuela, M.A. and Cid, M.S. 1993. Initial signs of overgrazing in a heterogeneous pasture under continuous grazing by sheep. Revista Argentina de Produccion Animal 13 (1): 61-70.

Brower, B. 1991. Sherpa of Khumbu: People, Livestock and Landscape, Oxford University Press, Delhi.

Brunstrom, R. 1976. Vegetation and sheep grazing in the Kinder-Bleaklow SSSI, Derbyshire and Yorkshire. MSc dissertation, University of London.

Brys, R., Jacquemyn, H., Endels, P., De Blust, G. and Hermy, M. 2004. The effects of grassland management on plant performance and demography in the perennial herb Primula veris. Journal of Applied Ecology 41: 1080- 1091.

Buckler, W. 1887. The larvae of the British Butterflies and moths Vol. II, Royal Society, London, pp. 1-175.

Buenz, E.J., Bauer, B.A., Osmundson, T.W. and Motley, T.J. 2005. The traditional Chinese medicine Cordyceps sinensis and its effects on apoptotic homeostasis. J. Ethnopharmacol. 96: 19-29.

Buenz, E.J., Weaver, J.G., Bauer, B.A., Chalpin, S.D. and Badley, A.D. 2004. Cordyceps sinensis extracts do not prevent Fas-receptor and hydrogen peroxide-induced T-cell apoptosis. J. Ethnopharmacol. 90: 57-62.

Buhler, C. and B. Schmid 2001. The influence of management regime and altitude on the population structure of Succisa pratensis: Implications for vegetation monitoring. Journal of Applied Ecology 38: 689-698

Bullock, J.M. 2000. Gaps and seedling colonization. In Fenner, M. (Ed), Seeds. The ecology of regeneration in plant communities, 2nd edition, CABI Publishing, Wallingford, pp 375-395

Bullock, J.M., Clear Hill, B. and Silvertown, J. 1994. Demography of Cirsium vulgare in a grazing experiment. Journal of Ecology 82: 101-111.

Bullock, J.M., Franklin, J., Stevenson, M.J., Silvertown, J., Coulson, S.J., Gregory, S.J. and Tofts, R. 2001. A plant trait analysis of responses to grazing in a long-term experiment. Journal of Applied Ecology 38: 253-267.

Bullock, J.M., Hill, B.C., Silvertown, J. and Sutton, M. 1995. Gap colonization as a source of grassland community change: effects of gap size and grazing on the rate and mode of colonization by different species. Oikos 72:273-282.

Burke, I.C., C.M. Yonker, W.J. Parton, C.V. Cole, K. Flach, and D.S. Schimel. 1989. Texture, climate and, cultivation effects on soil organic matter content in U.S. grassland soils. Soil Sci. Soc. Am. J. 53:800–805.

Burke, I.C., Laurenroth, W.K. and Milchunas, D.G. 1997. Biogeochemistry of managed grasslands in central North America. In: Paul, E.A., Paustian, K., Elliott, E.T., Cole, C.V. (Eds.), Soil Organic Matter in Temperate Agroecosystems: Long-term Experiments in North America, CRC Press, Boca Raton, FL, pp. 85-102.

Buttolph, L.P. and Coppock, D.L. 2004. Influence of deferred grazing on vegetation dynamics and livestock productivity in an Andean pastoral system. Journal of Applied Ecology 41: 664-674.

Cai, P.Y. and Sun, S.Y. 2010. Preliminary analysis on Ophiocordyceps sinensis resource sustainable development and utilization in Qinghai Province. Chin J Grassland. 32(Supp.):6-9

Cale, P. and Hobbs, R. J. 1991. Condition of roadside vegetation in relation to nutrient status. In Saunders, D.A. and Hobbs, R.J. (Eds), Nature Conservation 2: The Role of Corridors (Eds), Surrey Beatty and Sons, Chipping Norton, pp. 353–62.

Callaghan, T.V., Carlsson, B.A. and Tyler, N.J.C. 1989. Historical records of climate related growth in Cassiope tetragona from the Arctic. Journal of Ecology 77: 823-837.

Callaghan, T.V., Carlsson, B.A., Jonsdottir, I.S., Svensson, B.M. and Jonasson, S. 1992. Clonal plants and environmental change: introduction to the proceedings and summary. Oikos 63: 341-347.

Cannon, P.F., Hywel-Jones, N.L., Maczey, N., Norbu, L., Tashi Samdup, T. and Lhendup, P. 2009. Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan. Biodiversity and Conservation 18 (9): 2263-2281.

Cao, Z. and Wen, Y. 1993. Therapeutic effect analysis of JinShuiBao capsule in treatment of 33 elderly senescent XuZheng patients. J. Applied Traditional Chinese Medicine 1: 32-33.

Carr, S.A. 1990. A study of overgrazing and soil erosion in the Coledale Valley. BA dissertation, Newcastle University.

Casimir, M.J. and Rao, A. 1985. Vertical control in the Western Himalaya: Some notes on the pastoral ecology of the nomadic Bakarwal of Jammu and Kashmir. Mountain Research and Development 5:221-232.

Cebrian, J. 1999. Patterns in the fate of production in plant communities. Am. Nat. 154:449–468.

Cerdà, A. and Lavee, H. 1999. The effect of grazing on soil and water losses under arid and mediterranean climates: Implications for desertification. Pirineos (153-154): 159-174.

Chambers, J.C. 1995. Disturbance, life-history strategies, and seed fates in alpine herb field communities. American Journal of Botany 82: 421-433.

Chaneton, E.J., and R.S. Lavado. 1996. Soil nutrients and salinity after long-term grazing exclusion in a Flooding Pampa grassland. J. Range Manage. 49:182–187

Chaneton, E.J., Lemcoff, J.H. and Lavado, R.S. 1996. Nitrogen and phosphorus cycling in grazed and ungrazed plots in a temperate subhumid grassland in Argentina. Journal of Applied Ecology 33: 291-302.

Chapekar, M.S. and Glazer, R.I. 1983. Cordycepin analog of (A2΄p) An evidence that it funcons as a product of cordycepin. Biochem Biophys Res Commun 115: 137-143.

Chassy, B.M. and Suhadolnik, R.J. 1969. Nucleoside antibiotics IV. Metabolic fate of adenosine and cordycepin by Cordyceps militaris during cordycepin biosynthesis. Biochim Biophys Acta 182: 307-315.

Chatterjee, R., Srinivasan, K.S. and Maiti, P.C. 1957. Cordyceps sinensis (Berkeley) Saccardo: structure of cordycepic acid. J Am Pharm Assoc. 46: 114-118.

Chen, D.D., Zhang, S.H., Dong, S.K., Wang, X.T. and Du, G.Z. 2010. Effect of land-use on soil nutrients and microbial biomass of an alpine region on the northeastern Tibetan plateau, China. Land Degradation and Development 21: 446-452.

Chen, D.M. 1987. Platelet hemopoiesis and ultrastructure observations in mice treated with natural Cordyceps sinensis and its cultured mycelia. Bull. Chin. Materia Medica. 12: 47-49.

Chen, H.X. and Jia, X.B. 2005. Research progress on pharmacological actions of Cordyceps polysaccharides from Cordyceps militaris. J Jiangsu Univ (Med) 15: 74-77.

Chen, J., Yamamura, Y., Hori, Y., Shiyomi, M., Yasuda, T., Zhou, H.K., Li, Y.N. and Tang, Y.H. 2008. Small-scale species richness and its spatial variation in an alpine meadow on the Qinghai-Tibet Plateau. Ecological Research 23: 657-663.

Chen, S.J., Yin, D.H., Li, L., Zha, X., Shuen, J.H. and Zhama, C. 2000. Resources and Distribution of Cordyceps sinensis in Naqu Tibet. Zhong Yao Cai 2311:673-675. [Chinese, English abstract]

Chen, S.J., Yin, D.H., Li, L., Zhou, X.L. and Za, X. 2001. Studies on anamorph of Cordyceps sinensis (Berk.) from Naqu, Tibet. China J Chin Mat Med. 26: 453-454.

Chen, S.J., Yin, D.H., Zhong, G.Y. and Huang, T.F. 2002. Study on the biology of adult parasite of Cordyceps sinensis, Hepialus biruensis. China J Chin Mat Med. 27: 893-895.

Chen, S.Z. and Chu, J.Z. 1996. NMR and IR studies on the characterization of cordycepin and 20- deoxyadenosine. Zhongguo Kangshengsu Zaxhi 21: 9-12.

Chen, T., Tang, C. and Mao, C. 1973. A preliminary study on the biology of the swift moth Hepialus. Acta Entomologica Sinica 16: 198-202.

Chen, Y.J., Shiao, M.S., Lee, S.S. and Wang, S.Y. 1997. Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells. Life Sciences 60: 2349-2359.

Chen, Y.J., Zhang, Y.P., Yang, Y.X. and Yang, D. 1999. Genetic diversity and taxonomic implication of Cordyceps sinensis as revealed by RAPD markers. Biochem Gen. 37: 201-213.

Chen, Y.Q., Hu, B., Xu, F., Zhang, W.M., Zhou, H. and Qu, L.H. 2004. Genetic variation of Cordyceps sinensis, a fruit body- producing entomopathogenic species from different geographical regions in China. FEMS Microbiol Lett. 230: 153-158.

Chiu, J.H., Ju, C.H., Wu, L.H., Lui, W.Y., Wu, C.W., Shiao, M.S. and Hong, C.Y. 1998. Cordyceps sinensis increases the expression of major histocompatibility complex class II antigens on human hepatoma cell line HA22T/VGH cells. American Journal of Chinese Medicine 26(2): 159-170.

Choler, P. 2005. Consistent shifts in alpine plant traits along a mesotopographical gradient. Arctic, Antarctic, and Alpine Research 37: 444-453.

Choquenot, D. 1991. Density-dependent growth, body condition, and demography in feral donkeys: Testing the food hypothesis. Ecology 72: 805-813.

Chu, H.F. and Wang, L. 1985. ‘Insect-herb’ versus hepialids with descriptions of new genera and new species of Chinese Hepialidae. Sinozoologia 3: 121-134.

Cingolani, A.M., Cabido, M.R., Renison, D. and Viviana, S.N. 2003. Combined effects of environment and grazing on vegetation structure in Argentine granite grasslands. Journal of Vegetation Science 14: 223-232.

Cingolani, A.M., Noy-Meir, I. and Diaz, S. 2005a. Grazing effects on rangeland diversity: A synthesis of contemporary models. Ecological Applications 15: 757-773.

Cingolani, A.M., Posse, G. and Collantes, M.B. 2005. Plant functional traits, herbivore selectivity and response to sheep grazing in Patagonian steppe grasslands. Journal of Applied Ecology 42: 50-59.

Clary, W.P. and W.C. Leininger 2000. Stubble height as a tool for management of riparian areas. Journal of Range Management 53: 562-573.

Coldea, G. 2003. The alpine flora and vegetation of the south eastern Carpathians. In Nagy, L., Grabherr, G., Körner, C. and Thompson, D. B. A. (Eds), Alpine Biodiversity in Europe, Springer, Berlin, pp. 65–73

Coley, P.D., Bryant, J.P. and Chapin, F.S. 1985. Resource availability and plant anti-herbivore defense. Science 230: 895-899.

Common, I.F.B. 1970. Lepidoptera (moth and butterflies). In Waterhouse, D.F. (Ed), The Insects of Australia, Melbourne University Press, Melbourne, pp. 765-866.

Comstock, J.H. 1984. An Introduction to Entomology, 9th ed., Satish book enterprise, bookseller and publishers, Moti Katra, Agra, pp. 1064.

Conant, R.T., Paustian, K. and Elliott, E.T. 2001. Grassland management and conversion into grassland: effects on soil carbon. Ecological Applications 11: 343-355.

Cong, W.F., van Ruijven, J., Mommer, L., De Deyn, G.B., Berendse, F. and Hoffland, E. 2014. Plant species richness promotes soil carbon and nitrogen stocks in grasslands without legumes. Journal of Ecology 102: 1163-1170.

Cook, C.W. 1966. Factors affecting utilization of mountain slopes by cattle. Journal of Range Management 19: 200-204.

Cook, C.W. 1967. Increased capacity through better distribution on mountain ranges. Utah Sci. 28: 39–42.

Coomes, D.A. and Grubb, P.J. 2003. Colonization, tolerance, competition and seed-size variation within functional groups. Trends in Ecology and Evolution 18: 283-291.

Cornelissen, J.H.C., Perez-Harguindeguy, N., Diaz, S., Grime, J.P., Marzano, B., Cabido, M., Vendramini, F. and Cerabolini, B. 1999. Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two continents. New phytologist 143: 191-200.

Cory, J.G., Suhadolnik, R.J., Resnick, B. and Rich, M.A. 1965. Incorporaon of cordycepin (3΄-deoxyadenosine) into ribonucleic acid and deoxyribonucleic acid of human tumor cells. Biochim Biophys Acta 103: 646-653.

Cory, S., Huang, D. and Adams, J. 2003. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22: 8590-8607.

Costa, C., Papatheodorou, E.M., Monokrousos, N., Stamou, G.P. 2015. Spatial variability of soil organic C, inorganic N and extractable P in a Mediterranean grazed area. Land Degradation and Development 26: 103- 109.

Cowlishaw, S.J. 1969. The carrying capacity of pastures. Journal of the British Grassland Society 24: 207-214.

Crawley, M.J. 1997. Plant ecology, Blackwell, Oxford.

Crawley, M.J., Albon, S.D., Bazely, D.R., Milner, J.M., Pilkington, J.G. and Tuke, A.L. 2004. Vegetation and sheep population dynamics. In Clutton-Brock, T.H. and Pemberton, J.M. (Eds), Soay Sheep: Dynamics and Selection in an Island Population, Cambridge University Press, Cambridge.

Cremene, C. et al. 2005. Alterations of steppe-like grasslands in Eastern Europe: a threat to regional biodiversity hotspots. Conservation Biology 19: 1606–1618.

Dahlin, A.S., U. Emanuelsson, and J.H. McAdam. 2005. Nutrient management in low input grazing-based systems of meat production. Soil Use Manage. 21:122–131.

Dai, G.W., Bao, T., Xu, C., Cooper, R. and Zhu, J.S. 2001. Cordyceps Cs- 4 improves steady-state bio-energy status in mouse liver. J Altern Complement Med. 7 (3): 231-240. de Bello, F., Lepš, J. and Sebastia, M-T. 2005. Predictive value of plant traits to grazing along a climatic gradient in the Mediterranean. J Appl Ecol 42: 824-833.

DeGabriel, J.L., Albon, S.D., Fielding, D.A., Riach, D.J., Westaway, S. and Irvine, R.J. 2011. The presence of sheep leads to increases in plant diversity and reductions in the impact of deer on heather. Journal of Applied Ecology 48(5): 1269-1277. del-Val, E. and Crawley, M.J. 2005. Are grazing increaser species better tolerators than decreasers? An experimental assessment of defoliation tolerance in eight British grassland species. Journal of Ecology 93: 1005-1016. Demment, M.W. and Van Soest, P.J. 1985. A nutritional explanation for body-size patterns of ruminant and non-ruminant herbivores. Am Nat 125: 641-672.

Devasahayam, S., Premkumar, T. and Abdulla Koya, K.M. 1987. Record of Sahyadrassus malabaricus (Moore) damaging Gliricidia maculate, a standard of black pepper Piper nigrum in Kerala. Entomology 12: 391-396.

Dhanarajan, G. 1976. Some observations of the teak collar ring borer- Endoclita gmelina (Lepidoptera: Hepialidae) in North-western Malaysia. Malays. For. 39: 214-223.

Diaz, A. 2000. Can plant palatability trials be used to predict the effect of rabbit grazing on the flora of ex- arable land? Agric Ecosyst Environ 78: 249-259.

Diaz, S., et al. 2007. Plant trait responses to grazing - a global synthesis. Global Change Biology 13(2): 313-341.

Diaz, S., Lavorel, S., McIntyre, S., Falczuk, V., Casanoves, F., Milchunas, D.G., Skarpe, C., Rusch, G., Sternberg, M., Noy-Meir, I., Landsberg, J., Zhang, W., Clark, H. and Campbell, B.D. 2007. Plant trait responses to grazing − a global synthesis. Glob Change Biol 13: 313-341.

Diaz, S., Noy-Meir, I. and Cabido, M. 2001. Can grazing response of herbaceous plants be predicted from simple vegetative traits? J Appl Ecol 38: 497-508.

Doll, U. 1991. C-14 translocation to the below ground subsystem in a temperate humid grassland (Argentina). p. 350–358. in B. McMichael, and H. Persson (ed.) Plant roots and their environment. Elsevier, Amsterdam.

Dong, C. and Yao, Y. 2005. Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture. Journal of Applied Microbiology, 99: 483-492.

Donohue, J.F. 1996. Recent advances in the treatment of asthma. Curr. Opin. Pulmonary Med. 2: 1–6.

Dorji, T., Moe, S.R., Klein, J.A. and Totland, Ø. 2014. Plant species richness, evenness, and composition along environmental gradients in an alpine meadow grazing ecosystem in central Tibet, China. Arctic, Antarctic, and Alpine Research 46: 308-326.

Dormaar, J.F. and Willms, W.D. 1990. Effects of grazing and cultivation on some chemical properties of soils in the mixed prairie. Journal of Range Management 43: 456-460.

Dormaar, J.F., B.W. Adams, and W.D. Willms. 1994. Effect of grazing and abandoned cultivation on a Stipa-Bou- teloua community. J. Range Manage. 47:28–32.

Dormann, C.F. and Woodin, S.J. 2002. Climate change in the Arctic: using plant functional types in a Meta- analysis of field experiments. Funct. Ecol. 16: 4-17.

Douma, J.C., Witte, J.P.M., Aerts, R., Bartholomeus, R.P., Ordonez Barragan, J.C., Olde Venterink, H., Wassen, M.J. and Bodegom, P.M. 2012. Towards a functional basis for predicting vegetation patterns; incorporating plant traits in habitat distribution models. Ecography 35: 294-305.

Drewry, J.J. 2006. Natural recovery of soil physical properties from treading damage of pastoral soils in New Zealand and Australia: A review. Agric. Ecosyst. Environ. 114:159–169.

Dube, H.C. 1983. An Introduction to Fungi, Vikas Publishing House Pvt. Ltd., New Delhi, pp.616.

Dubuis, A., Rossier, L., Pottier, J., Pellissier, L., Vittoz, P. and Guisan, A. 2013. Predicting current and future spatial community patterns of plant functional traits. Ecography 36: 1158-1168.

Dugdale, J.S. 1975. Alpine moths. In Knox, R. (Ed), New Zealand’s Nature Heritage Vol. 4, Hamlyn, Hong Kong, pp. 1526-1530.

Dumbleton, L.J. 1945. Contribution to the ecology of Oxycanus Walk. The New Zealand Jr. of Science and Technology 27: 114-128.

Dumbleton, L.J. 1966. Genitalia, classification and zoogeography of the New Zealand Hepialidae (Lepidoptera). New Zealand Jr. of Science 9: 920-981.

Duru, M., Theau, J.P. and Cruz, P. 2012. Functional diversity of species-rich managed grasslands in response to fertility, defoliation and temperature. Basic and Applied Ecology 13: 20-31.

Ealand, G.A. 1984. Insect life, IB & PSS, New Delhi, pp. 340.

Edwards, C.A. 1964. The bionomics of swift moths 1- the ghost swift moth, Hepialus humuli (L.). Bulletin of Entomological Research 55: 147-160.

Ehrlen, J. 1995a. Demography of the perennial herb Lathyrus vernus. II. Herbivory and plant population dynamics. Journal of Ecology 83: 297-308.

Ehrlen, J. 1995b. Demography of the perennial herb Lathyrus vernus. I. Herbivory and individual performance. Journal of Ecology 83: 287-295.

Ehrlén, J., Syrjänen, K., Leimu, R., García, M. B. and Lehtilä, K. 2005. Land use and population growth of Primula veris: an experimental demographic approach. Journal of Applied Ecology 42 317-326.

Ehrlen, J., Syrjanen, K., Leimu, R., Garcia, M.B. and Lehtilä, K. 2005. Land use and population growth of Primula veris: an experimental demographic approach. Journal of Applied Ecology 42: 317-326.

Eldridge, D. J. and Koen T. B. 1993. Run-off and sediment yield from a semi-arid woodland in eastern Australia. II. Variation in some soil hydrological properties along a gradient in soil surface condition. Rangel. J. 15: 234– 246.

Ellis, J.E. and Swift, D.M. 1988. Stability of African pastoral ecosystems: Alternate paradigms and implications for development. Journal of Range Management 41:450-459.

Eriksson, O. 1989. Seedling dynamics and life histories in clonal plants. Oikos 55: 231-238.

Eskelinen, A., Harrison, S. and Tuomi, M. 2012. Plant traits mediate consumer and nutrient control on plant community productivity and diversity. Ecology 93(12): 2705-2718.

Esson, M.J. 1970. Time-lapse photography for the observation of porina caterpillar behaviour. Proceedings of the 23rd Weed and pest control conference, pp. 200-204.

Evans, J.W. 1941. Tasmanian grassgrubs. Tasmanian department of Agriculture Bulletin, pp. 22.

Evans, R. 1977. Overgrazing and soil erosion on hill pastures with particular reference to the Peak District. Journal of the British Grassland Society 32: 65-76.

Evans, R. 1990. Soils at risk of accelerated erosion in England and Wales. Soil Use and Management 6: 125-131.

Evans, R. 1992. Sheep: their impacts on some central Lake District fells. Report for Friends of the Lake District.

Evans, R. 1996. Some impacts of overgrazing by reindeer in Finnmark, Norway. Rangifer 16 (1): 3-19.

Evju, M. 2009. Sheep grazing and dynamics of alpine plants in a landscape-scale experiment, PhD thesis, University of Oslo, Oslo.

Evju, M., Austrheim, G., Halvorsen, R. and Mysterud, A. 2009. Grazing responses in herbs in relation to herbivore selectivity and plant traits in an alpine ecosystem. Oecologia 161(1): 77-85.

Evju, M., Mysterud, A., Austrheim, G. and Økland, R.H. 2006. Selecting herb species and traits as indicators of sheep grazing pressure in a Norwegian alpine habitat. Ecoscience 13: 459-468.

Eyayu, M., Heluf, G., Tekalign, M. and Mohammed, A. 2009. Effects of land use change on selected soil properties in the Tara Gedam catchment and adjacent agro-ecosystem, Northwest Ethiopia. Ethiopian Journal of Natural Resources 11: 35-65.

Fan, Y., Hou, X., Shi, H. and Shi, S. 2013. Effects of grazing and fencing on carbon and nitrogen reserves in plants and soils of alpine meadow in the three Headwater Resource Regions. Russian Journal of Ecology 44: 80- 88.

Farrell, J.A.K. 1972. Observations on the soil-inhabiting insect populations of improved pasture in Nelson province, with particular reference to Costelytra zealandica (White) (Col. Scarabaeidae). New Zealand Jr. of Agricultural Research 15: 878-892.

Feng, M.G., Zhou, Q.G., Feng, G.M. and Shuai, J.P. 1987. Vascular dilation by fermented mycelia of Cordyceps sinensis in anesthetized dogs. J. Chinese Materia Medica 12 (12): 745-749.

Fischer, U., Janicke, R.U. and Schulze-Osthoff, K. 2003. Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death and Differentiation 10: 76-100.

Fleet, H. 1986. The concise natural history of New Zealand, Auckland, Heinmann.

Forbis, T.A. 2003. Seedling demography in an alpine environment. American Journal of Botany 90: 1197-1206.

Forbis, T.A. and Doak, D.F. 2004. Seedling establishment and life history trade-offs in alpine plants. American Journal of Botany 91: 1147-1153.

Francia, C., Rapior, S., Coutecuisse, R. and Siroux, Y.Y. 1999. Current research findings on the effects of selected Mushrooms on Cardiovascular diseases. Int. J. Medicinal mushrooms 1: 169-172.

Frank, A.B. 2002. Carbon dioxide fluxes over a grazed prairie and seeded pasture in the Northern Great Plains. Environment Pollution 116: 397-403.

Frank, A.B., Tanaka, D.L., Hofmann, I. and Follet, R.F. 1995. Soil carbon and nitrogen of northern Great Plains grassland as influenced by long –term grazing. Journal of Range Management 48: 470-474.

Frank, D.A. and Groffman, P.M. 1998. Ungulate vs. landscape control of soil C and N processes in grasslands of Yellowstone national park. Ecology 79: 2229-2241.

Frank, D.A., Gehring, C.A., Machut, L. and Phillips, M. 2003. Soil community composition and the regulation of grazed temperate grassland. Oecologia 137: 603-609.

Frank, D.A., Groffman, P.M., Evans, R.D. and Tracy, B.F. 2000. Ungulate stimulation of nitrogen cycling and retention in Yellowstone Park grasslands. Oecologia 123: 116-121.

Fridman, J.S. and Lowe, S.W. 2003. Control of apoptosis by p53. Oncogene 22: 9030-9040.

Fu, G., Zhang, X., Yu, C., Shi, P., Zhou, Y., Li, Y., Yang, P. and Shen, Z. 2014. Response of soil respiration to grazing in an alpine meadow at three elevations in Tibet. The Scientific World Journal 2014: 265142.

Fuls, E.R. 1992. Ecosystem modification created by patch-overgrazing in semi-arid grassland. Journal of Arid Environments 23 (1): 59-69.

Galop, D., Houet, T., Mazier, F., Leroux, G. and Rius, D. 2011. Grazing activities and biodiversity history in the Pyrenees: new insights on high altitude ecosystems in the framework of a human–environment observatory. PAGES News 19: 53-55.

Gao, Q., Wan, Y., Xu, H., Li, Y., Jiangcun, W. and Borjigidai, A. 2010. Alpine grassland degradation index and its response to recent climate variability in Northern Tibet, China. Quaternary International 226: 143-150.

Gao, Y.H., Schuman, M., Chen, H., Wu, N. and Luo, P. 2009. Impacts of grazing intensity on soil carbon and nitrogen in an alpine meadow on the eastern Tibetan Plateau. Journal of Food Agriculture Environment 7: 749- 754.

Garibaldi, L.A., M. Semmartin, and E.J. Chaneton. 2007. Grazing-induced changes in plant composition affect litter quality and nutrient cycling in flooding Pampa grasslands. Oecologia 151:650–652.

Gass, T.M. and Binkley, D. 2011. Soil nutrient losses in an altered ecosystem are associated with native ungulate grazing. Journal of Applied Ecology 48: 952–960.

Gifford, G.F., and R.H. Hawkins. 1978. Hydrologic impact of grazing on infiltration. Water Resour. Res. 14:305– 313.

Goldney, D. C. and Bowie, J. S. 1990. Some management implications for the conservation of vegetation remnants and associated fauna in the central western region of New South Wales. Proc. Ecol. Soc. Aust. 16: 427–440.

Goldney, D., Bauer, J., Bryant, H., Hodgkins, D. and Watson, G. 1995. Winning battles but losing the war: the education marketing imperative. In Saunders, D.A., Craig, J. and Mattiske, L. (Eds), Nature Conservation 4: The Role of Networks, Surrey Beatty & Sons, Chipping Norton, NSW, pp. 547–588.

Golodets, C., and B. Boeken. 2006. Moderate sheep grazing in semiarid shrubland alters small-scale soil surface structure and patch properties. Catena 65:285–291.

Gong, H.Y., et al. 2000. Effects of Cordyceps sinensis on T lymphocyte subsets and hepatofibrosis in patients with chronic hepatitis B. Bull Hunan Med Univ. 25: 248-250.

Graetz, R. D. and Tongway, D. J. 1986. Influence of grazing management on vegetation, soil structure and nutrient distribution and the infiltration of applied rainfall in a semi-arid chenopod shrub land. Aust. J. Ecol. 11: 347–360.

Grant, S.A., Bolton, G.R. and Torvell, L. 1985. The response of blanket bog vegetation to controlled grazing by sheep. Journal of Applied Ecology 22: 739-775.

Green, R. H. 1993. Application of repeated measures designs in environmental impact and monitoring studies. Aust. J. Ecol. 18: 81–98.

Greene, R. S. B. 1992. Soil physical properties of three geomorphic zones in a semi-arid mulga woodland. Aust. J. Soil Res. 30: 55–69.

Greene, R. S. B. and Tongway, D. J. 1989. The significance of (surface) physical and chemical properties in determining soil surface condition of red earths in rangelands. Aust. J. Soil Res. 27: 213–225.

Greenwood, K.L., and B.M. McKenzie. 2001. Grazing effects on soil physical properties and the consequences for pastures: A review. Aust. J. Exp. Agric. 41:1231–1250

Grehan, J.R. 1979a. Notes on the biogeography and phylogeny of eastern Asian ghost moths (Lepidoptera: Hepialidae). Bulletin of the Buffalo Society of Natural Sciences 40: 59-74.

Grehan, J.R. 1979b. Larvae of Aenetus virescens (Lepidoptera: Hepialidae) in decaying wood. New Zealand Jr. of Zoology 6: 583-586.

Grehan, J.R. 1981. Morphological changes in the three-phase development of Aenetus virescens larvae (Lepidoptera: Hepialidae). New Zealand Jr. of Zoology 8: 505-514.

Grehan, J.R. 1987. Life cycle of the wood borer Aenetus virescens (Lepidoptera: Hepialidae). New Zealand Journal of Zoology 14: 209-217.

Grehan, J.R. 1988a. Evolution of arboreal tunneling by larvae of Aenetus (Lepidoptera: Hepialidae). New Zealand Jr. of Zoology 14: 441-462.

Grehan, J.R. 1988b. Fungal and vascular plant polysaccharide digestion by larvae of Aenetus virescens (Lepidoptera: Hepialidae). New Zealand Entomologist 11: 57-67.

Grehan, J.R. 1989. Larval feeding habits of the Hepialidae (Lepidoptera). Journal of Natural History 23(4): 803- 824.

Grehan, J.R. 2012. Morphological evidence for phylogenetic relationships within the Hepialidae (Lepidoptera: Exoporia). Bulletin of the Buffalo Society of Natural Sciences 42: 33-62.

Grellmann, D. 2002. Plant responses to fertilization and exclusion of grazers on an arctic tundra heath. Oikos 98: 190-204.

Grime, J.P. 1973. Competitive exclusion in herbaceous vegetation. Nature 242: 344-347.

Grime, J.P., Cornelissen, J.H.C., Thompson, K. and Hodgson, J.G. 1996. Evidence of a causal connection between anti-herbivore defence and the decomposition rate of leaves. Oikos 77: 489-494.

Gross, A., McDonnell, J.M. and Korsmeyer, S.J. 1999. BCL-2 family members and the mitochondria in apoptosis. Genes and Development 13: 1899-1911.

Grubb, P. J. 1977. The maintenance of species-richness in plant communities: the importance of regeneration niche. Biological Reviews 52: 107-145.

Gu, Z.L. et al. 2002. The protective effect of lung fit on myocardial ischemia/reperfusion injury in isolated rat heart. Chin J Hemorheol. 12: 279-282.

Guo, H.P. and Yang, Z.M. 1999. Study progress in pharmacologic action of Cordyceps sinensis. Chin Trad Herb Drugs 30: 231-233.

Hafner, S., Unteregelsbacher, S., Seeber, E., Lena, B., Xu, X., Li, X., Guggenberger, G., Miehe, G. and Kuzyakov, Y. 2012. Effect of grazing on carbon stocks and assimilate partitioning in a Tibetan montane pasture revealed 13 by CO2 pulse labeling. Global Change Biology 18: 528-538.

Hall, F.C. and Bryant, L. 1995. Herbaceous Stubble Height as a Warning of Impending Cattle Grazing Damage to Riparian Areas. US Department of Agriculture Forest Service General Technical Report PNW-GTR-362, Portland, Oregon.

Hall, R. 2008. Soil Essentials: Managing your Farm’s Primary Asset, Landlinks Press, Collingwood.

Halpern, G.M. 1999. Cordyceps: China’s healing mushroom, Avery Publishing Group, New York, pp. 63-70.

Hamblin, A. P. 1984. The effect of tillage on soil surface properties and the water balance of a xeralfic alfisol. Soil Tillage Res. 4: 543–559.

Han, S.R. 1995. Experiences in treating patients of chronic bronchitis and pulmonary diseases with Cs-4 capsule (JinShuiBao). Admin. Tradit. Chin. Med. 5 (Suppl.): 33-34.

Hardy, R.J. 1973. The biology of Fraus simulans Walker (Lepidoptera: Hepialidae). Jr. of the Australian Entomological Society 12: 113-120.

Hardy, R.J. 1974. Biology and pest status of Oxycanus fuscomaculatus Walker (Lepidoptera: Hepialidae). Jr. of the Australian Entomological Society 13: 317-328.

Harrison, K.A. and Bardgett, R.D. 2008. Impacts of grazing and browsing by large herbivores on soils and soil biological properties. In Gordon, I.J. and Prins, H.H.T. (Eds), The Ecology of Browsing and Grazing, Berlin Heidelberg: Springer-Verlag, pp. 201-213.

Hart, R.H., Bissio, J., Samuel, M.J. and Waggoner, J.W. 1993. Grazing systems, pasture size, and cattle grazing behavior and gains. Journal of Range Management 46: 81–87.

Haukioja, E. and Koricheva, J. 2000. Tolerance to herbivory in woody vs. herbaceous plants. Evolutionary Ecology 14(4-6): 551-562.

Haynes, R.J., and P.H. Williams. 1993. Nutrient cycling and soil fertility in the grazed pasture ecosystem. Adv. Agron. 49:119–199.

Heads, M.J. 1984. Principia Botanica: Croizat’s contribution to Botany. Tuatara 27: 26-48.

Heath, J. 1976. Hepialidae. In Heath, J. (Ed), The moths and butterflies of Great Britain and Ireland, Blackwell Scientific Publications Ltd., and The Curwen Press Ltd., pp. 166-170.

Hebblewhite, M., Merril, E. and McDermid, G. 2008. A multi-scale test of the forage maturation hypothesis in a partially migratory ungulate population. Ecol Monogr 78: 141-166.

Henderson, D.C., B.H. Ellert, and M.A. Naeth. 2004. Grazing and soil carbon along a gradient of Alberta rangelands. Rangeland Ecol. Manag. 57:402–410.

Hidalgo, A. and Ffrench-Constant, C. 2003. The control of cell number during central nervous system development in ﬂies and mice. Mechanisms of Development 120: 1311-1325.

Hilker, T., Natsagdorj, E., Waring, R.H., Lyapustin, A. and Wang, Y. 2014. Satellite observed widespread decline in Mongolian grasslands largely due to overgrazing. Global Change Biology 20: 418-428.

Hirota, M., Tang, Y., Hu, Q., Kato, T., Hirata, S., Mo, W., Gao, G. and Mariko, S. 2005. The potential importance of grazing to the fluxes of carbon dioxide and methane in an alpine wetland on the Qinghai-Tibetan Plateau. Atmospheric Environment 39: 5255-5259.

Hobbs CH. 1995. Medicinal Mushrooms: An Exploration of Tradition, Healing, and Culture. Santa Cruz, CA: Botanica Press, pp. 251.

Hobbs, N.T. 1996. Modification of ecosystems by ungulates. Journal of Wildlife Management 60(4): 695-713.

Hodge, K.T., Krasnoff, S.B. and Humber, R.A. 1996. Tolypocladium inﬂatumis the anamorph of Cordyceps subsessilis. Mycologia 88: 715-719.

Holechek, J.L., Gomez, H., Molinar, F. and Galt, D. 1999. Grazing studies: what we've learned. Rangelands 21: 12-16.

Holliday J, Cleaver P, Loomis-Powers, M, Patel D. 2004. Analysis of quality and techniques for hybridization of medicinal fungus Cordyceps sinensis. International Journal of Medicinal Mushrooms 6: 147-160.

Holliday, J. 2004b. Investigation into mechanism of action with Aloha Medicinals Inc. hybrid Cordyceps altered nucleosides. Unpublished research, 2002-2004.

Holliday, J. and Cleaver, M. 2008. Medicinal value of the caterpillar fungi species of the genus Cordyceps (fr.) Link (Ascomycetes) - A review. Inter. Jr. Medicinal Mushrooms 10 (3): 219-234.

Holliday, J., Cleaver, M. and Wasser, S.P. 2005. Cordyceps. In Coates, P.M., Blackman, M.R., Cragg, G., Levine, M., Moss, J. and White, J. (Eds), Encyclopedia of dietary supplements, Marcel Dekker, New York, pp. 1-13.

Hooper, J.F., Workman, J.P., Grumbles, J.B. and Cook, C.W. 1969. Improved livestock grazing distribution with fertilizer: a preliminary economic evaluation. Journal of Range Management 22: 108-110.

Howling, G. 1996. Temperate Woodlands: Their Conservation Status in Australia, WWF Australia, Sydney.

Hsu, T.H. and Lo, H.C. 2002. Biological activity of Cordyceps (Fr.) Link species (Ascomycetes) derived from a natural source and from fermented mycelia on diabetes in STZ-induced rats. Int. J. Med. Mushroom 4: 111-125.

Hu, W. and Kavanagh, J.J. 2003. Anticancer therapy targeting the apoptotic pathway. The Lancet Oncology 4: 721-729.

Huang, B., Li, C.R., Li, Z.G., Fan, M.Z. and Li, Z.Z. 2002. Molecular identiﬁcation of the teleomorph of Beauveria bassiana. Mycotaxon 81: 229-236.

Huang, B.M., Chuang, Y.M., Chen, C.F. and Leu, S.F. 2000. Effects of extracted Cordyceps sinensis on steroidogenesis in MA-10 mouse Leydig tumor cells. Biological and Pharmaceutical Bulletin 23: 1532-1535.

Hubbell, S. 2001. The unified neutral theory of biodiversity and biogeography, Princeton University Press, Princeton, New Jersey, USA.

Hudson, G.V. 1885. Life history of Charagia virescens. Entomologist 18: 30-36.

Hui, D. and Jackson, R.B. 2005. Geographic and inter-annual variability in biomass partitioning in grassland ecosystems: A synthesis of field data. New Phytologist 169: 85-93.

Hung, L.T., Keawsompong, S., Hanh, V.T., Sivichai, S. and Hywel-Jones, N.L. 2009. Effect of temperature on cordycepin production in Cordyceps militaris. Thai Jr. of Agricultural Science 42(4): 219-225.

Hunt, L.P. 2001a. Heterogeneous grazing causes local extinction of edible perennial shrubs: a matrix analysis. Journal of Applied Ecology 38: 238-252.

Hunt, L.P. 2001b. Low seed availability may limit recruitment in grazed Atriplex vesicaria and contribute to its local extinction. Plant Ecology 157: 53-67.

Hyder, D.N., Bement, R.E., Remmenga, E.E. and Hervey, D.F. 1975. Ecological responses of native plants and guidelines for management of shortgrass range, United States Department of Agriculture-Agricultural Res. Service, Tech. Bulletin Number 1503, US Government Printing Office, Washington, DC. pp. 87.

Imms, A.D. 1973. A General Textbook of Entomology, ELBS publication, pp. 886.

Ims, R.A., Yoccoz, N.G., Brathen, K.A., Fauchald, P., Tveraa, T. and Hausner, V. 2007. Can reindeer overabundance cause a trophic cascade? Ecosystems 10: 607-622.

Ioras, F. 2003. Trends in Romanian biodiversity conservation policy. Biodiversity and Conservation 12: 9-23.

Ito, Y. and Hirano, T. 1997. The determination of the partial 18S ribosomal DNA sequences of Cordyceps species. Letters in Applied Microbiology 25: 239-242.

Ives, J.D. and Messerli, B. 1989. The Himalayan Dilemma: Reconciling Development and Conservation, Rutledge, New York.

Jefferies, R.L., Klein, D.R. and Shaver, G.R. 1994. Vertebrate herbivores and northern plant communities: reciprocal influences and responses. Oikos 71: 193-206.

Jennings, D.B., Ehrenshaft, M., Pharr, D.M. and Williamson, J.D. 1998. Role for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense. Proc Natl Acad Sci., USA 95: 15129-15133.

Ji, P.Y., et al. 2005. On activity and mechanism of anti-tumor of Cordyceps militaris. J Beihua Univ. (Nat Sci) 6: 324-329.

Ji, Y.B. 1999. Pharmacological actions and applications of anticancer traditional Chinese medicines (150. Cordyceps sinensis (Berk) Sacc). Heilongjiang Ke-Xue-Ji-Shu Chu-Ban-She, Ha’erbin, China: 494-501. [In Chinese]

Jiang, P. 1987. Pharmacology constituent and function of Cordyceps sinensis. J Westnorthern Med. 2: 43-44.

Jiang, W.L., Wa, Q.R. and Liu, G.Y. 1996. Study on the effects of improving natural grassland with sheep night penning: 1. Sheep night time, intensity and herbage mixture. Acta Pratacultural Sinica 5:17-25.

Jiang, Y. and Yao, Y.J. 2002. Names related to Cordyceps sinensis anamorph. Mycotaxon 84: 245-254.

Jones, R.M. and Bunch, G.A. 1995. Yield and population dynamics of Chamaecrista rotundifolia cv. Wynn in coastal south-eastern Queensland as affected by stocking rate and rainfall. Tropical Grasslands 29 (2): 65-73.

Jónsdóttir, I.S. 1991. Effects of grazing on tiller size and population dynamics in a clonal sedge (Carex bigelowii). Oikos 62: 177-188.

Joshi, S.P. and Raizada, A. 1990. Carrying capacity of an alpine grazing land of Garhwal Himalaya. Range Manage Agrofor. 11: 141-150.

Joshi, S.P. and Srivastava, M.M. 1988. Effect of grazing on species composition, diversity and productivity in a high-altitude pasture in Garhwal Himalaya. Int J Ecol Environ Sci. 14:221-227.

Joshi, S.P., Raizada, A. and Srivastava, M.M. 1988a. Net primary productivity of an alpine pasture in Garhwal Himalaya. Trop Ecol. 29:15-20.

Joshi, S.P., Srivastava, M.M. and Raizada, A. 1988b. Alpine plant communities of a high-altitude grassland in the Garhwal Himalaya. In Singh, P., Pathak, P.S. (Eds), Rangeland Resources and Management: Proceedings of the National Rangeland Symposium, 1987 Nov 9–12; Jhansi, India. Jhansi (India): Range Management Society of India, pp. 162-167.

Joshi, S.P., Srivastava, M.M. and Raizada, A. 1991. Biomass and net productivity under a deferred grazing pattern in a Himalayan high-altitude grassland. Range Manage Agrofor. 12(1):1-13.

Joubert, P.C. 1975. The ‘Ghost moth’ (Dalaca rufescens Hampson) in Natal (Lepidoptera: Hepialidae). Technical communication 130, Department of Agricultural Technical Services.

Kala, C.P. 2004. Community composition, species diversity, and secondary succession in grazed and ungrazed alpine meadows of West Himalaya, India. Int J Fieldwork Stud. 2: 1.

Kalembasa, S.J. and Jenkinson, D.S. 1973. A comparative study of titrimetric and gravimetric methods for the determination of organic carbon in soil. Journal of Science of Food and Agriculture 24: 1085-1090.

Kalshoven, L.G.E. 1965. Notes on some injurious Lepidoptera from Java. Tijdschrift voor Entomologie 108: 73- 93.

Kang, J.C., Liang, Z.Q., Liu, A.Y. and Kong, R.Y.C. 2000. Molecular evidence of polymorphism in Cordyceps based on 5.8S rRNA and ITS2 sequences. Mycosystema 19: 492-497.

Kátai, J. 1994. Javítóanyagok hatása a gyep talajára. DGYN 12. Legeltetéses Állattartás, Debrecen 229-247.

100

Kátai, J. 1998. Relationships between the physical, chemical and microbiological characteristics on a grassland th experiment. Proc. of the 17 General Meeting of the EGF, Debr., pp. 77-81.

Kauffman, J.B. and Krueger, W.C. 1984. Livestock impacts on riparian ecosystems and streamside management implications. Journal of Range Management 37: 430-438.

Kaul, M.C. 1998. Transhumance and customary pastoral rights in Himachal Pradesh: Claiming the high pastures for Gaddis. Mountain Research and Development 18: 5-17.

Kaur, R., Joshi, V. and Joshi, S.P. 2010. Impact of degradation on biodiversity status and management of an alpine meadow within Govind Wildlife Sanctuary and National Park, Uttarkashi, India. International Journal of Biodiversity Science, Ecosystem Services and Management 6 (3-4): 146-156.

Kausrud, K., Mysterud, A., Rekdal, Y., Holand, O. and Austrheim, G. 2006. Density dependence in foraging behaviour of sheep on alpine pastures: effects of scale. Journal of Zoology 270: 63-71.

Ke, C.K. 2005. Studies on Cordyceps sinensis in China, China Science and Technology Press, Beijing

Kendrick, B. 1992. The Fifth Kingdom, Focus Publishing, Newburyport, Ma.

Kiełtyk, P. and Mirek, Z. 2015. Importance of molehill disturbances for invasion by Bunias orientalis in meadows and pastures. Acta Oecologica 64: 29-34.

Kiho, T., Hui, J., Yamane, A. and Ukai, S. 1993. Polysaccharides in fungi. XXXII. Hypoglycemic activity and chemical properties of a polysaccharide from the cultural mycelium of Cordyceps sinensis. Biol. Pharm. Bull. 16 (12): 1291-1293.

Kiho, T., Yamane, A., Hui, J., Usui, S. and Ukai, S. 1996. Polysaccharide in fungi. XXXVI. Hypoglycemic activity of a polysaccharide (CS-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Biol. Pharm. Bull. 19: 294-296.

Kiho, T., Yamane, A., Hui, J., Usui, S. and Ukai, S. 2000. Hypoglycemic activity of a polysaccharide (CSF30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Phytother. Res. 4 (8): 647-649.

Kile, G.A., Hardy, R.J. and Turnbull, C.R.A. 1979. The association between Abantiades latipennis (Lepidoptera, family Hepialidae) and Eucalyptus oblique and E. regnans in Tasmania. Jr. of the Australian Entomologica Society 18: 1-17.

Kim, H.O. and Yun, J.W. 2005. A comparative study of the production of exopolysaccharides by two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures. J. Appl. Microbiol. 99: 728-738.

Kim, K.M., Kwon, Y.G., Chung, H.T., Yun, Y.G., Pae, H.O., Han, J.A., Ha, K.S., Kim, T.W. and Kim, Y.M. 2003. Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppressing NF-κB activation. Toxicol. Appl. Pharmacol. 190: 1-8.

Kim, S.Y., Shrestha, B., Gi-Ho Sung, G.H., Han, S.K and Sung, J.M. 2010. Optimum conditions for artificial fruiting body formation of Cordyceps cardinalis. Mycobiology 38(2): 133-136.

Kinjo, N. and Zang, M. 2001. Morphological and phylogenetic studies on Cordyceps sinensis distributed in Southwestern China. Mycoscience 42: 567-574.

Kirkpatrick, J. and Gilfedder, L. 1995. Maintaining integrity compared with maintaining rare and threatened taxa in remnant bushland in subhumid Tasmania. Biol. Conserv. 74: 1–8.

101

Klein, J., Yeh, E., Bump, J., Nyima, Y. and Hopping, K. 2011. Coordinating environmental protection and climate change adaptation policy in resource-dependent communities: a case study from the Tibetan Plateau. In Ford, J.D.F.A.L.B. (Ed.), Climate Change Adaptation in Developed Nations, Springer.

Klenow, H. 1963. Formation of the mono-, di- and triphosphate of cordycepin in Ehrlich ascites-tumor cells in vitro. Biochim Biophys Acta 76: 347-353.

Kletzien, R.F. 1980. Nucleocytoplasmic transport of RNA. The eﬀect of 3΄-deoxyadenosine triphosphate on RNA release from isolated nuclei. Biochem J 192: 753-759.

Klimešova, J., Latzel, V., de Bello, F. and van Groenendael, J.M. 2008. Plant functional traits in studies of vegetation changes in response to grazing and mowing: towards a use of more specific traits. Preslia 80: 245- 253.

Klumpp, K., Fontaine, S., Attard, E., Le Roux, X., Gleixner, G. and Soussana, J.F. 2009. Grazing triggers soil carbon loss by altering plant roots and their control on soil microbial community. Journal of Ecology 97: 876- 885.

Knight, T.M. 2004. The effect of herbivory and pollen limitation on a declining population of Trillium grandiflorum. Ecological Applications 14: 915-928.

Kobayashi, Y. 1941. The genus Cordyceps and its allies, Science reports of the Tokyo Bunrika Daigaku 84: 53- 260.

Koc, Y., et al. 1996. Induction of apoptosis by cordycepin in ADA inhibited TdT-positive leukemia cells. Leukemia 10: 1019-1024.

Kodama, E.N., McCaffrey, R.P., Yusa, K. and Mitsuya, H. 2000. Antileukemic activity and mechanism of action of cordycepin against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells. Biochem Pharmacol 59: 273-281.

Koerner, S.E. and Collins, S.L. 2014. Interactive effects of grazing, drought, and fire on grassland plant communities in North America and South Africa. Ecology 95: 98-109.

Koh, J.H., et al. 2002. Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured medium of Cordyceps sinensis. Biosci Biotechnol Biochem 66: 407-411.

Korkanc, S.Y., Ozyuvaci, N. and Hizal, A. 2008. Impacts of land use conversion on soil properties and soil erodibility. J. Environ. Biol. 29: 363-370.

Korner, C. 1995. Alpine plant diversity: A global survey and functional interpretations: Patterns, cause and ecosystem consequence. In Chapin, F.S. and Korner, C. (Eds) Arctic and alpine biodiversity, Springer, Berlin, pp. 45-62.

Korner, C. 1995. Alpine plant diversity: A global survey and functional interpretations. In Chapin, F.S. and Korner, C. (Eds), Arctic and alpine biodiversity: patterns, causes and ecosystem consequences, Springer, Berlin, pp. 45-62.

Körner, C. 2003. Alpine plant life: functional plant ecology of high mountain ecosystems, Springer-Verlag, Berlin Heidelberg.

Kristensen, N.P. 1978. A new family of Hepialoidea from South America, with remarks on the phylogeny of the sub-order Exoporia (Lepidoptera). Entomologica Germanica 4: 272-294.

Kristensen, N.P. 1984. Studies on the morphology and systematics of primitive Lepidoptera (Insecta). Steenstrupia 10: 141-191.

102

Kristensen, N.P. 1999. Handbook of Zoology Vol IV part 35. Lepidoptera, Moths and Butterflies Vol. 1: Evolution, systematics, and Biogeography, Walter de Gruyter, Berlin.

Kristensen, N.P. 1999. The non-Glossatan Moths. In Kristensen, N.P. (Ed.), Lepidoptera, Moths and Butterflies, Vol. 1: Evolution, Systematics, and Biogeography, Walter de Gruyter, New York, pp. 41-62.

Kucharczak, J., Simmons, M., Fan, Y. and Gelinas, C. 2003. To be, or not to be: NF-kappa B is the answer-role of Rel/NF-kappa B in the regulation of apoptosis. Oncogene 22: 8961-8982.

Kuo, Y.C., Lin, C.Y., Tsai, W.J., Wu, C.L., Chen, C.F. and Shiao, M.S. 1994. Growth inhibitors against tumor cells in Cordyceps sinensis other than cordycepin and polysaccharides. Cancer Invest. 12: 611-615.

Kuo, Y.C., Tsai, W.J., Shiao, M.S., Chen, C.F. and Lin, C.Y. 1996. Cordyceps sinensis as an immunomodulatory agent. American Journal of Chinese Medicine 24: 111-125.

Kursar, T.A., Dexter, K.G., Lokvam, J., Pennington, R.T., Richardson, J.E., Weber, M.G., Murakami, E.T., Drake, C., McGregor, R. and Coley, P.D. 2009. The evolution of anti-herbivore defences and their contribution to species tree genus Inga. Proc. Natl. Acad. Sci. USA 106: 18073-18078.

Kvamme, M. 1988. Pollen analytical studies of mountain summer-farming in western Norway. In Birks, H.H., Birks, H.J.B., Kaland, P.E. and Moe, D. (Eds), The Cultural landscape: past present and future, Cambridge University Press, Cambridge, pp. 349-367.

Lagocki, H.F.R. 1978. Surface soil stability and controlled by a drainage criterion. p. 233–237. In W.W Emerson, R.D. Bond, and A.R. Dexter (ed.) Modification of soil structure. John Wiley & Sons, Hoboken.

Lantzke, N. 1992. Soils of the Northam Advisory District, 1 The Zone of Ancient Drainage. Department of Agriculture, Western Australia.

Large, A.R.G. and Hamilton, A.C. 1991. The distribution, extent and causes of peat loss in central and northwest Ireland. Applied Geography 11: 309-326.

Laughlin, D.C., Joshi, C., van Bodegom, P.M., Bastow, Z.A. and Fule´, P.Z. 2012. A predictive model of community assembly that incorporates intraspecific trait variation. Ecology Letters 15: 1291-1299.

Lavado, R.S., and M.A. Taboada. 1987. Soil salinization as an effect of grazing in a native grassland soil in the flooding Pampa of Argentina. Soil Use Manage. 4:143–148.

Lavado, R.S., Sierra, J.O. and Hashimoto, P.N. 1996. Impact of grazing on soil nutrients in a Pampean grassland. Journal of Range Management 49: 452-457.

Lavorel, S., McIntyre, S., Landsberg, J. and Forbes, T.D.A. 1997. Plant functional classifications: from general groups to specific groups based on response to disturbance. TREE 12: 474-478.

Laycock, W.A. 1983. Evaluation of management as a factor in the success of grazing systems. USDA Forest Serv. Gen. Tech. Rep. INT-157.

Leader-Williams, N. 1988. Reindeer on South Georgia. Cambridge University Press, Cambridge.

Lennartsson, T. and Oostermeijer, J.G.B. 2001. Demographic variation and population viability in Gentianella campestris: effects of grassland management and environmental stochasticity. Journal of Ecology 89: 451-463.

Li LS, Zheng F, Liu ZH (1996). Experimental study on effect of Cordyceps sinensis in ameliorating amino glycoside induced nephrotoxicity. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih. 16 (12): 733-737.

Li SP, Li P, Dong TT, Tsim KW (2001). Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia. Phytomedicine 8 (3): 207-212.

103

Li, B.S. 1993. The Alpine Timberline of Tibet. In Alden J., Mastrantonio J.L. and Odum S. (Eds.), Forest Development in Cold Climates, Plenum Press, NYC, pp. 511-527.

Li, C., X. Hao, M. Zhao, G. Han, and W.D. Willms. 2008. Influence of historic sheep grazing on vegetation and soil properties of a desert steppe in Inner Mongolia. Agric. Ecosyst. Environ. 128:109–116

Li, C.B., Tong, X.D., Bai, J. and Fan, S.D. 2004. Artificial stromata production of Cordyceps militaris. Journal of Dalian National University 6(5): 29-31.

Li, J. and Jiang, H.Y. 2005. Progression on study of Cordycepin. US Chin Health Hyg J. 8: 27-30.

Li, L.S., Zheng, F. and Liu, Z.H. 1996. Experimental study on effect of Cordyceps sinensis in ameliorating amino glycoside induced nephrotoxicity. Chung Kuo Chung Hsi I Chieh Ho Tsa Chih. 16 (12): 733-737.

Li, P.Z., Liu, B.H., Wang, W.P., Yu, H.S., Sun, L.Q., Xu, G.N., Song, Z.Y. and Huang, K.W. 1985. Analysis of therapeutic effects of Ningxinbao on treatment of 200 patients with arrhythmia. Zhongchengyao Yanjiu 7: 24- 25.]

Li, S.P. and Tsim, K.W.K. 2004. The biological and pharmacological properties of Cordyceps sinensis, a traditional Chinese medicine that has broad clinical applications. In Packer, L., Ong, C.N. and Halliwell, B. (Eds), Herbal and traditional medicine: molecular aspects of health, Marcel Dekker, New York, pp. 657-684.

Li, S.P., et al. 2001. Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia. Phytomedicine 8: 207-212.

Li, S.P., et al. 2003. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-induced injury. Life Sci. 73: 2503-2513.

Li, S.P., Zhao, K.J., Ji, Z.N., Song, Z.H., Dong, T.T.X., Lo, C.K., Cheung, J.K.H. and Tsim, K.W.K. 2003. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-induced injury. Life Sci. 73: 2503-2513.

Li, W., Tian, F.P., Ren, Z.W., Huang, H.Z. and Zhang, Z.N. 2013a. Effects of grazing and fertilization on the relationship between species abundance and functional traits in an alpine meadow community on the Tibetan Plateau. Nordic Journal of Botany 31: 247-255.

Lin CY, Ku FM, Kuo YC, Chen CF, Chen WP, Chen A, Shiao MS (1999). Inhibition of activated human mesangial cell proliferation by the natural product of Cordyceps sinensis (H1-A): an implication for treatment of IgA mesangial nephropathy. J. Lab. Clin. Med. 133 (1): 55-63.

LingHao, L., ZuoZhong, Ch., QiBing, Q., XianHua, L., YongHong, L., Li, L.H., Chen, Z.Z., Wang, O.B. and Liu, X.H. 1997. Changes in soil carbon storage due to over-grazing in Leymus chinensis steppe in the Xilin River Basin of Inner Mongolia. Journal of Environmental Sciences 9 (4): 486-490.

Liston, P., Fong, W. and Korneluk, R. 2003. The inhibitors of apoptosis: there is more to life than Bcl2. Oncogene 22, 8568-8580.

Liu, B. 1984. The Chinese medical fungi, Shanxi People’s Press, Taiyuan.

Liu, J.L. and Zhou, Y.C. 2001. Effect of Cordyceps taii on macrophage phagocytosis and uviofast protection. Nat Prod Res Dev. 13: 20-22.

Liu, J.M., Zhong, Y.R., Yang, Z., Cui, S.L. and Wang, F.H. 1989. Chemical constituents of Cordyceps militaris (L.) Link. China J. Chin. Materia Medica. 14: 608-609.

104

Liu, R.X., Sun, M. and Zhou, H.Y. 1990. Clinical analysis of therapeutic efficacy of Cordyceps sinensis (Berk.) Sacc. in premature beats. Bull. Hunan Medical University 15 (4): 384-386.

Liu, X.J., Guo, Y.L., Yu, Y.X. and Zeng, W. 1989. Isolation and identification of the anamorphic state of Cordyceps sinensis (Berk.) Sacc. Acta Mycol Sin. 8: 35-40.

Liu, Y., Wu, C. and Li, C. 1991. Anti-oxidation of Paecilomyces sinensis (Cordyceps sinensis) S. Pnov. Zhongguo Zhong Yao Za Zhi. 16 (4): 240-242.

Longhi, F., Pardini, A., Tullio, V.G., di Tullio, V.G., Eldridge, D. and Freudenberger, D. 1999. Biodiversity and productivity modifications in the Dhofar rangelands (Southern Sultanate of Oman) due to overgrazing. People and rangelands: building the future. Proceedings of the VI International Rangeland Congress Queensland, Australia, pp. 664-665.

Lopez-Marsico, L., Altesor, A., Oyarzabal, M., Baldassini, P. and Paruelo, J.M. 2015. Grazing increases belowground biomass and net primary production in temperate grassland. Plant and Soil 392: 155-162.

Loreau, M. 2010. Linking biodiversity and ecosystems: towards a unifying ecological theory. Philosophical Transactions of the Royal Society B 365: 49-60.

Lou, Y., Liao, X. and Lu, Y. 1986. Cardiovascular pharmacological studies of ethanol extracts of Cordyceps mycelia and Cordyceps fermentation solution. Chinese Traditional and Herbal Drugs 17 (5): 17-21 & 209-213.

Lu, X., Kelsey, K.C., Yan, Y., Sun, J., Wang, X., Cheng, G. and Neff, J.C. 2017. Effects of grazing on ecosystem structure and function of alpine grasslands in Qinghai–Tibetan Plateau: a synthesis. Ecosphere 8(1): ecs01656. 10.1002/ecs2.1656

Ludwig, J. A., Tongway, D. J. and Marsden, S. G. 1994. A flow filter model for simulating the conservation of limited resources in spatially heterogeneous, semi-arid landscapes. Pac. Conserv. Biol. 1: 209–213.

Ludwig, J. A., Tongway, D. J., Freudenberger, D., Noble, J. and Hogkinson, K. 1997. Landscape Ecology, Function and Management Principles from Australia’s Rangelands, CSIRO Australia, Melbourne.

Ludwig, J.A. and Tongway, D.J. 1995. Spatial organisation of landscapes and its function in semi-arid woodlands, Australia. Landscape Ecol. 10: 51–63.

Ludwig, J.A. and Tongway, D.J. 1996. Rehabilitation of semiarid landscapes in Australia. 2. Restoring vegetation patches. Restor. Ecol. 4: 398–406.

Lunt, I. D. 1991. Management of remnant lowland grasslands and grassy woodlands for nature conservation: a review. Vic. Natur. 108: 57–66.

Ma, D.T., Guo, Y.X., Hou, F.J., Zhai, X.Y., Wang, W., Tian, M., Wang, C.Z. and Yan, X.B. 2014. Plant genetic diversity and grazing management on the Qinghai–Tibetan Plateau: a case study of a dominant native wheatgrass (Elymus nutans). Biochemical Systematics and Ecology 56: 16-23.

Ma, Y.X. 2010. Ophiocordyceps sinensis resource and its management in China. In Ophiocordyceps sinensis resources and environment, Lanzhou University Press, Lanzhou, pp. 3-6.

MacDonald, D., et al. 2000. Agricultural abandonment in mountain areas of Europe: Environmental consequences and policy response. Journal of Environmental Management 59(1): 47-69.

Maczey, N., Dhendup, K., Cannon, P.F., Hywel-Jones, N.L. and Rai, T.B. 2010. Thitarodes namnai sp. nov. and T. caligophilus sp. nov. (Lepidoptera: Hepialidae), hosts of the economically important entomopathogenic fungus Ophiocordyceps sinensis in Bhutan. Zootaxa 2412: 42-52.

105

Maczey, N., Zhang, F. and Cannon, P.F. 2011. Ecology of Thitarodes spp., hosts of the economically important entomopathogenic fungus Ophiocordyceps sinensis in Bhutan. Chinese Journal of Grassland 31: 109-121.

Maderna, P. and Godson, C. 2003. Phagocytosis of apoptotic cells and the resolution of inﬂammation. Biochimica et Biophysica Acta 1639: 141-151.

Main, B. Y. 1992. Social history and impact on landscape (with Trayning: case history of a shire by D. Couper). In Hobbs, R.J. and Saunders, D.A. (Eds), Reintegrating Fragmented Landscapes Towards Sustainable Production and Nature Conservation, Springer-Verlag, New York, pp. 23–65.

Mallet, J. 1984. Sex roles in the ghost moth Hepialus humuli (L.) with a review of mating in the Hepialidae (Lepidoptera). Zoological Journal of the Linnaean Society 79: 67-82.

Manabe, N., Azuma, Y., Sugimoto, M., Uchio, K., Miyamoto, M., Taketomo, N., Tsuchita, H. and Miyamoto, H. 2000. Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice. Br J Nutr. 83 (2): 197-204.

Manabe, N., Sugimoto, M., Azuma, Y., Taketomo, N., Yamashita, A., Tsuboi, H., Tsunoo, A., Kinjo, N., Nian-Lai, H. and Miyamoto, H. 1996. Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism in the mouse. Jpn J Pharmacol. 70 (1): 85-88.

Manfreda, J., Mao, Y. and Litven, W. 1989. Morbidity and mortality from chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. S19-S26: 140.

Mani, A., Patel, J., Kalam, S., Singh. and Sandhu, S.S. 2015. Evaluation of mycelia and exopolysaccharide production from Cordyceps militaris. Int. Journal of Applied Sciences and Engineering Research 5(4): 609-619.

Manley, J.T., Schuman, G.E., Reeder, J.D. and Hart, R.H. 1995. Rangeland soil carbon and nitrogen responses to grazing. Journal of Soil Conservation 50: 294-298.

Marrs, R.H., A. Rizand, and A.F. Harrison. 1989. The effects of removing sheep grazing on soil chemistry, above- ground nutrient distribution, and selected aspects of soil fertility in long-term experiments at Moor House National Nature Reserve. J. Appl. Ecol. 26:647–661.

Martínez, L.J., and J.A. Zinck. 2004. Temporal variations of soil compaction and deterioration of soil quality in pasture areas of Colombian Amazonia. Soil Tillage Res. 75:3–17.

Massee, G. 1895. A revision of the genus Cordyceps. Ann Bot. 9: 1-44.

Mathews, B.W., Sollenberger, I.E., Nair, V.D. and Staples, C.R. 1994. Impact of grazing management on soil nitrogen, phosphorus, potassium, and sulphur distribution. Journal of Environmental Quality 23: 1006-1013.

Maurset, M.U. 2015. Long-term Effects of Shifts in Grazing Pressure on Alpine Plant Species along an Elevational Gradient, Ph. D thesis, Norwegian University of Science and Technology, NTNU, Trondheim

McAlister, J.J., Smith, B.J. and Sanchez, B. 1998. Forest clearance: impact of land use change on fertility status of soils from the Sao Francisco Area of Niteroi, Brazil. Land Degrad. Dev. 9: 425-440.

McArthur, W. M. 1991. Reference Soils of South-Western Australia. Department of Agriculture, Western Australia, Perth.

McDougald, N.K., Frost, W.E. and Jones, D.E. 1989. Use of supplemental feeding locations to manage cattle use on riparian areas of hardwood rangelands. Proc. California Riparian Systems Conference, Davis, USDA, pp. 124–126.

McEwen, F.L. 1963. Cordyceps Infections. In Steinhaus, E.A. (Ed.), Insect Pathology. An Advanced treatise, Vol. II, Academic Press, New York, pp. 581.

106

McGill, B.J., Enquist, B.J., Weiher, E. and Westoby, M. 2006. Rebuilding community ecology from functional traits. Trends Ecol. Evol. 21: 178-185.

McGranahan, D.A., Engle, D.M., Fuhlendorf, S.D., Winter, S.J., Miller, J.R. and Debinski, D.M. 2012. Spatial heterogeneity across five rangelands managed with pyric-herbivory. Journal of Applied Ecology 49: 903-910.

McIntyre, S. and Lavorel, S. 1994. How environmental and disturbance factors influence species composition in temperate Australian grasslands. J. Veg. Sci. 5: 373–384.

McIntyre, S., Lavorel, S., Landsberg, J. and Forbes, T.D.A. 1999. Disturbance response in vegetation - towards a global perspective on functional traits. Journal of Vegetation Science 10: 621-630.

McKenna, D.J., Jones, K. and Hughes, K. 2002. Botanical medicines: the desk reference for major herbal supplements, 2nd Ed, Haworth Herbal Press, New York, pp. 1138.

McNaughton, S.J. 1983. Compensatory plant growth as a response to herbivory. Oikos 40: 329-336.

McNaughton, S.J. 1985. Ecology of a grazing ecosystem: the Serengeti. Ecological Monographs 55: 259-294.

McNaughton, S.J., Banyikwa, F.F. and McNaughton, M.M. 1997. Promotion of the cycling of diet-enhancing nutrients by African grazers. Science 278: 1798-1800.

McNaughton, S.J., Ruess, R.W. and Seagle, S.W. 1988. Large mammals and process dynamics in African ecosystems. Bioscience 38: 794-800.

Mcsherry, M.E. and Ritchie, M.E. 2013. Effects of grazing on grassland soil carbon: a global review. Plant and Soil 346: 331-347.

Mcsherry, M.E. and Ritchie, M.E. 2013. Effects of grazing on grassland soil carbon: a global review. Global Change Biology 19: 1347-1357.

Mehra, B.S. and Mathur, P.K. 2001. Livestock Grazing in the Great Himalayan National Park Conservation Area- -A Landscape Level Assessment. Himalaya, the Journal of the Association for Nepal and Himalayan Studies 21(2): 89-96.

Metcalf, C.L. and Flint, W.P. 1990. Fundamentals of Insect Life, L.P. Publication, New Delhi, pp. 581.

Metzger, K.L., Coughenour, M.B., Reich, R.M. and Boone, R.B. 2005. Effects of seasonal grazing on plant species diversity and vegetation structure in a semi-arid ecosystem. Journal of Arid Environments 61: 147-160.

Miehe, G. et al. 2011. Plant communities of central Tibetan pastures in the Alpine Steppe/Kobresia pygmaea ecotone. Journal of Arid Environments 75: 711-723.

Milchunas, D.G. and Lauenroth, W.K. 1993. Quantitative effects of grazing on vegetation and soils over a global range of environments. Ecological Monographs 63: 327-366.

Milchunas, D.G., Lauenroth, W.K. and Burke, I.C. 1998. Livestock grazing: animal and plant biodiversity of shortgrass steppe and the relationship to ecosystem function. Oikos 83: 63-74.

Milchunas, D.G., Sala, O.E. and Lauenroth, W.K. 1988. A generalized model of the effects of grazing by large herbivores on grassland community structure. American Naturalist 132: 87-106.

Miller, D. 1984. Common insects in New Zealand. Revised by A.K. Walker, Wellington, Auckland, Sydney, and Melbourne, Reed.

Miller, R.A. 2009. The Cordyceps sinensis- Medicinal mushroom. Nexus magazine, Apr-May issue: 23-28.

107

Miller, R.H. and Keeney, D.R. 1982. Methods of soil analysis, 2nd eds. Part 2. Chemical and Microbiological Properties, 2nd ed., ASA, SSSA, Madison, WI, pp. 1-129.

Milyanovskii, E.S. and Mitrofanov, P.I. 1952. The Caucasian hepialid- a new pest of grape vines in Abkhasiya. Entomologicheskoe Obozrenie 32: 82-85.

Mitter, C. and Brooks, D.R. 1983. Phylogenetic aspects of coevolution. In Futuyama, D.J. and Slatkin, M. (Eds), Coevolution, Sunderland, Massachusetts: Sinauer Associates Inc., pp. 65-98.

Miyazaki, T., et al. 1977. Studies on fungal polysaccharides XX. Galactomannan of Cordyceps sinensis. Chem Pharm Bull 25: 3325-3328.

Mizuno, T. 1999. Medicinal effects and utilization of Cordyceps (Fr.) Link (Ascomycetes) and Isaria Fr. (Mitosporic fungi) Chinese caterpillar fungi, ‘‘Tochukaso’’ (review). Int. J. Med. Mushrooms 1: 251-162.

Mobak, R., Mysterud, A., Loe, L.E., Holand, O. and Austrheim, G. 2008. Density dependent and temporal variability in habitat selection by a large herbivore: an experimental approach. Oikos in press.

Moeed, A. and Meads, M.J. 1983. Invertebrate fauna of four tree species in Orongorongo Valley, New Zealand, as revealed by trunk traps. New Zealand Jr. of Ecology 6: 39-53.

Moen, J. and Oksanen, L. 1998. Long-term exclusion of folivorous mammals in two arctic-alpine plant communities: a test of the hypothesis of exploitation ecosystems. Oikos 82: 333-346.

Mohamed Saleem, M.A. 1998. Nutrient balance pattern in African livestock systems. Agric. Ecosyst. Environ. 71:241–254.

Moles, R. 1992. Trampling damage to vegetation and soil cover within the Burren National Park, Mullack Mar, Co. Clare. Irish Geography 25: 129-137.

Molnár, G. and Jávor, A. 1997. Néhány európai ország gyakorlata a juhok környezetvédelmi hasznosításában. Tiszántúli Mezőgazdasági Napok, Karcag, pp. 382.

Montane, F., Rovira, P. and Casals, P. 2007. Shrub encroachment into mesic mountain grasslands in the Iberian Peninsula: effects of plant quality and temperature on soil C and N stocks. Global Biogeochem. Cycl. 21.

Moore, R. M. 1970. South-eastern temperate woodlands and grasslands. In Moore, R.M. (Ed), Australian Grasslands, Australian National University Press, Canberra, pp. 169–90.

Morgan, P.C. 2005. Soil Erosion and Conservation, 3rd ed., Blackwell Publishing, Malden.

Morris, W.F. and Doak, D.F. 1998. Life-history of a long-lived gynodioecious plant, Silene acaulis (Carophyllaceae), inferred from size-based population projection matrices. American Journal of Botany 85: 784-793.

Muller, W.E., Weiler, B.E., Charubala, R., Pﬂeiderer, W., Leserman, L., Sobol, R.W., et al. 1991. Cordycepin analogues of 2΄, 5΄-oligoadenylate inhibits human immunodeﬁciency virus infection via inhibition of reverse transcriptase. Biochemistry 30: 2027-2033.

Mysterud, A. 2006. The concept of overgrazing and its role in management of large herbivores. Wildlife Biology 12(2): 129-141.

Nagata, S. 1997. Apoptosis by death factor. Cell 88: 355-365.

Nagata, S., Nagase, H., Kawane, K., Mukae, N. and Fukuyama, H. 2003. Degradation of chromosomal DNA during apoptosis. Cell Death and Differentiation 10: 108-116.

108

Nagy, L. et al. (Eds) 2003. Alpine Biodiversity in Europe. Springer, Berlin, pp. 477.

Nakamura, K., Yamaguchi, Y., Kagota, S., Kwon, Y.M., Shinzuka, K. and Kunitomo, M. 1999. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice. Japanese Journal of Pharmacology 79: 335-341.

Naveh, Z. and Whittaker, R.H. 1980. Structural and floristic diversity of shrublands and woodlands in Northern Israel and other Mediterranean areas. Vegetatio 41: 171-190

Nedkvitne, J.J., Garmo, T.H. and Staaland, H. 1995. Beitedyr i kulturlandskap, Landbruksforlaget, Oslo.

Neff, J.C., Reynolds, R.L., Belnap, J. and Lamothe, P. 2005. Multi-decadal impacts of grazing on soil physical and biogeochemical properties in southeast Utah. Ecological Applications 15: 87-95.

Negi, C.S. 2003. Yar Tsa Gumba. Down To Earth, October 15, 49-51.

Negi, C.S. 2009. Habitat ecology, biochemical analysis and pharmacological tests of crude extracts of Yar tsa Gumba (Cordyceps sinensis Berk). The Botanica 57: 71-79.

Negi, C.S., Koranga, P.R. and Ghinga, H.S. 2006. Yar tsa Gumba (Cordyceps sinensis): A call for its sustainable exploitation. Int. Jr. Sus. Dev. & World Ecol. 13(6): 165-172.

Negi, P.S., Koranga, P.S. and Ahmed, Z. 2009. Biodiversity of Cordyceps in the Himalayan Hills of Uttarakhand, India. Presentation given at IMMC 5, September, Nantong.

Ng, T.B. and Wang, H.X. 2005. Pharmacological actions of Cordyceps, a prized folk medicine. J. Pharm. Pharmacol. 57: 1509-1519.

Nielsen ES, Robinson GS, Wagner DL. 2000. Ghost-moths of the world: a global inventory and bibliography of the Exoporia (Mnesarchaeoidea and Hepialoidea) (Lepidoptera). Journal of Natural History 34 (6): 823-878.

Nielsen, E.S. 1985. Primitive (non-ditrysian) Lepidoptera of the Andes: diversity, distribution, biology and phylogenetic relationships. Jr. of Research on Lepidoptera 1: 1-16.

Nielsen, E.S. 1988. The peculiar Asian ghost moth genus Bipectilus Chu and Wang: taxonomy and systematic position (Lepidoptera: Hepialidae s. str.). Systematic Entomology 13: 171-195.

Nielsen, E.S. and Robinson, G.S. 1983. Ghost moths of southern South America (Lepidoptera: Hepialidae). Entomonograph 4: 1-192.

Nielsen, E.S. and Scoble, M.J. 1986. Afrotheora, a new genus of primitive Hepialidae from Africa (Lepidoptera: Hepialidae). Entomologica Scandinavica 17: 29-54.

Nielsen, E.S., Robinson, G.S. and Wagner, D.L. 2000. Ghost-moths of the world: A global inventory and bibliography of the Exoporia (Mnesarchaeoidea and Hepialoidea) (Lepidoptera). Jr. of Natural History 34: 823- 878.

Niering, W.A. 1987. Vegetation dynamics (succession and climax) in relation to plant community management. Conservation Biology 1:287-295.

Nikoh, N. and Fukatsu, T. 2000. Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Molecular Biology and Evolution 17: 629-638.

Nikoh, N. and Fukatsu, T. 2001. Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps. Molecular Biology and Evolution 18: 1631-1642.

109

Niu, K., Choler, P., Zhao, B. and Du, G. 2009. The allometry of reproductive biomass in response to land use in Tibetan alpine grasslands. Functional Ecology 23: 274-283.

Niu, K., Zhang, S., Zhao, B. and Du, G. 2010. Linking grazing response of species abundance to functional traits in the Tibetan alpine meadow. Plant and Soil 330: 215-223.

Nulsen, R.A. 1992. Changes in soil properties. In Hobbs, R.J. and Saunders, D.A. (Eds), Reintegrating Fragmented Landscapes Towards Sustainable Production and Nature Conservation, Springer-Verlag, New York, pp. 107-145.

Oba, G., Vetaas, O.R. and Stenseth, N.C. 2001. Relationships between biomass and plant species richness in arid-zone grazing lands. Journal of Applied Ecology 38: 836-845.

Obornik, M., Jirku, M. and Dolezel, D. 2001. Phylogeny of mitosporic entomopathogenic fungi: is the genus Paecilomyces polyphyletic? Canadian Journal of Microbiology 47: 813-819.

Oesterheld, M., C.M. DiBella, and H. Kerdiles. 1998. Relation between NOAA-AVHRR satellite data and stocking rate of rangelands. Ecol. Appl. 8:207–212.

Oesterheld, M., J. Loreti, M. Semmartin, and J.M. Paruelo. 1999. Grazing, fire, and climate effects on primary productivity of grasslands and savannas. p. 287–306. In L.R. Walker (ed.) Ecosystems of disturbed ground. Ecosystems of the world 16. Elsevier, Amsterdam.

Ohta, Y., et al. 2007. In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans. J Agric Food Chem 55: 10194-10199.

Oksanen, L. and Ranta, E. 1992. Plant strategies along mountain vegetation gradients: a test of two theories. J Veg Sci 3: 175-186.

Oldemann, L.R., Hakkeling, R.T.A. and Sombroek, W.C. 1991. World Map of the Status of Human-induced Soil Degradation: An Explanatory Note, 2nd revised edn. International Soil Reference and Information Centre, Nairobi/United Nations Environment Programme, Wageningen.

Olofsson, J. 2001. Influence of herbivory and abiotic factors on the distribution of tall forbs along a productivity gradient: a transplantation experiment. Oikos 94: 351-357.

Olofsson, J. 2006. Short- and long-term effects of changes in reindeer grazing pressure on tundra heath vegetation. Journal of Ecology 94(2): 431-440

Olofsson, J., Hulme, P.E., Oksanen, L. and Suominen, O. 2004. Importance of large and small mammalian herbivores for the plant community structure in the forest tundra ecotone. Oikos 106: 324-334.

Olofsson, J., Hulme, P.E., Oksanen, L. and Suominen, O. 2005. Effects of mammalian herbivores on revegetation of disturbed areas in the forest-tundra ecotone in northern Fennoscandia. Landscape Ecology 20(3): 351-359.

Olofsson, J., Kitti, H., Rautiainen, P., Stark, S. and Oksanen, L. 2001. Effects of summer grazing by reindeer on composition of vegetation, productivity and nitrogen cycling. Ecography 24: 13-24.

Olofsson, J., Oksanen, L., Callaghan, T., Hulme, P.E., Oksanen, T. and Suominen, O. 2009. Herbivores inhibit climate-driven shrub expansion on the tundra. Global Change Biology 15(11): 2681-2693.

Overgaard-Hansen, K. 1964. The inhibition of 5-phosphoribosyl-1-pyrophosphate formation by cordycepin triphosphate in extracts of Ehrlich ascites tumor cells. Biochim Biophys Acta 80: 504-507.

Packard, A.S. 1895. On a new classification of the Lepidoptera. Am. Nat. 29: 636-647.

110

PAI (Plant-Animal Interactions) 2004. (Colorado State University Cooperative Extension) [Online article]. Retrieved November 10 http://www.coopext.colostate.edu/SEA/Tim/plant-animal.htm

Painter, E.L. and Detling, J.K. 1981. Effects of defoliation on net photosynthesis and regrowth of western wheatgrass. Journal of Range Management 34: 68-70.

Pakeman, R.J. 2004. Consistency of plant species and trait responses to grazing along a productivity gradient: a multi-site analysis. Journal of Ecology 92: 893-905.

Pandey, S. and Wells, W.P. 1997. Ecodevelopment Planning at India's Great Himalayan National Park for Biodiversity Conservation and Participatory Rural Development. Biodiversity and Conservation 6: 1277- 1292.

Pangtey, Y.P.S., Rawat, R.S., Bankoti, N.S. and Samant, S.S. 1990. Phenology of high-altitude plants of Kumaon in Central Himalayas, India. Int J Biometeorol. 34: 122-127.

Park, J.E., Kim, G.Y., Park, H.S., Nam, B.H., An, W.G., Cha, J.H., Lee, T.H. and Lee, J.D. 2001. Phylogenetic analysis of caterpillar fungi by comparing ITS 1-5.8S-ITS 2 ribosomal DNA sequences. Mycobiology 29: 121-131.

Parton, W.J., D.S. Schimel, C.V. Cole, and D.S. Ojima. 1987. Analysis of factors controlling soil organic matter levels in Great Plains grasslands. Soil Sci. Soc. Am. J. 51:1173–1179.

Pastor, J. and Naiman, R. J. 1992. Selective foraging and ecosystem processes in boreal forests. American Naturalist 139: 690-705.

Pathania, P., Joshi, M. and Sagar, A.2015. Morphological, Physiological and Molecular studies on wildly collected Cordyceps militaris from North West Himalayas, India. European Journal of Biotechnology and Bioscience 3 (1): 53-62.

Paton, D., Nunez, J., Munoz, A. and Tovar, J. 1997. Analysis of overgrazing in Mediterranean grasslands grazed by Retinto cattle using bioindicator plants. Archivos de Zootecnia 46 (176): 357-365.

Paulsamy, S., Lakshmanachary, A.S. and Manian, S. 1987. Effects of overgrazing on the phytosociology of a tropical grassland ecosystem. Indian Journal of Range Management 8 (2): 103-107.

Penman, S., Rosbash, M. and Penman, M. 1970. Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. Proc Natl Acad Sci USA 67: 1878-1885.

Perez-Harguindeguy, N., Diaz, S., Vendramini, F., Cornelissen, J.H.C., Gurvich, D.E. and Cabido, M. 2003. Leaf traits and herbivore selection in the field and in cafeteria experiments. Austral Ecology 28: 642-650.

Perju, T. and Ghizdavu, I. 1977. Contributii la cunoasterea biologiei si ecologiei omizii hameiului, Triodia sylvina L. (Hepialidae: Lepidoptera). Serie Biologie Animalia 29: 81-85.

Perrott, D.C.F. 1974. Porina moth, Wiseana species, Life cycle, DSIR Information Series No 105/1.

Perroux, K. M. and White, I. 1988. Designs for disk permeameters. Soil Sci. Soc. Am. J. 52: 1205–1215.

Petit, N.E., Froend, R.H. and Ladd, P.G. 1995. Grazing in remnant woodland vegetation: changes in species composition and life form groups. J. Veg. Sci. 6: 121–130.

Phillimore, P. 1982. Marriage and Social Organisation among Pastoralists of the Dhaula Dhar, Western Himalaya, Ph.D. Thesis, University of Durham, Durham.

Pielou, E.C. 1966. Species diversity and pattern diversity in the study of ecological succession. J Theor Biol. 10: 370-383.

111

Pietola, L., R. Horn, and M. Yli-Halla. 2005. Effects of trampling by cattle on the hydraulic and mechanical properties of soil. Soil Tillage Res. 82:99–108.

Pottinger, R.P. 1980. Porina: Biology, damage and control. Aglink, Ministry of Agriculture and Fisheries (New Zealand).

Powell, J.A. 1980. Evolution of larva feeding preferences in Microlepidoptera. Annual Review of Entomology 25: 133-159.

Pratt, D. 2002. Stop Overgrazing, Beef. Minneapolis 38 (12): 22.

Prober, S. M. 1996. Conservation of grassy white box woodlands: rangewide floristic variation and implications for reserve design. Aust. J. Bot. 44: 57–77.

Prober, S. M. and Thiele, K. R. 1993. The ecology and genetics of remnant grassy white box woodlands in relation to their conservation. Vic. Natur. 110: 30–36.

Prober, S. M. and Thiele, K. R. 1995. Conservation of the grassy white box Woodlands: Contributions of size and disturbance to floristic composition and diversity of remnants. Aust. J. Bot. 43: 349–366.

Proffitt, A. P. B., Bendotti, S., Howell, M. R. and Eastham, J. 1993. The effect of sheep trampling and grazing on soil physical properties and pasture growth for a red brown earth. Aust. J. Agric. Res. 44: 317–331.

Proulx, M. and Mazumder, A. 1998. Reversal of grazing impact on plant species richness in nutrient-poor vs. nutrient-rich ecosystems. Ecology 79: 2581-2592.

Pu, Z.L. and Li, Z.Z. 1996. Insect Mycology, Anhui Science and Technology Press, Aihui.

Pucheta, E., Bonamici, I., Cabido, M. and Diaz, S. 2004. Below-ground biomass and productivity of a grazed site and a neighbouring ungrazed enclosure in a grassland in central Argentina. Austral Ecology 29: 201-208.

Puchkova, T.A., Babitskaya, V.G., Shcherba, V.V., Bisko, N.A and Poyedinok, N.L. 2010. Polysaccharides of medicinal caterpillar fungus Cordyceps militaris (L: Fr) Link (ascomycetes): Production and composition. Inter. Jr. Medicinal Mushrooms 12(4): 419-425.

Pulido, M., Schnabel, S., Lozano-Parra, J. and González, F. 2016. The impact of heavy grazing on soil quality and pasture production in rangelands of SW Spain. Land Degradation and Development. DOI:10.1002/ldr.2501.

Qian, J., Wang, Z., Liu, Z. and Busso, C.A. 2014. Belowground bud bank responses to grazing intensity in the Inner-Mongolia steppe, China. Land Degradation and Development. DOI:10.1002/ldr.2300.

Qiuo, Y.L. and Ma, X.C. 1993. Treatment of 32 tussive asthma patients with JinShuiBao. Chin. J. Integr. Tradit. Western Med. 13 (11): 660.

Qu, Z.Y., Song, K., Cai, W.L. and Tang, J. 1995. Evaluation of therapeutic effects of JinShuiBao capsule for treatment of respiratory disease. J. Admin. Tradit. Chin. Med. 5 (Suppl.): 29-30.

Quail, A. 1902. On Charagia virescens Dbld. Transactions and proceedings of the New Zealand Institute 35: 249-255.

Ralhan, P.K., Saxena, A.K. and Singh, J.J. 1982. Analysis of forest vegetation at and around Nainital in Kumaon Himalaya. Proc. Indian National Science Academy B 48: 121-137.

Ram, J. and Arya, P. 1991. Plant forms and vegetation analysis of an alpine meadow of Central Himalaya. Proc Indian Natl Sci Acad. 57: 311-318.

112

Ram, J., Singh, J.S. and Singh, S.P. 1989. Plant biomass, species diversity and net primary production in a Central Himalayan high-altitude grassland. J Ecol. 77: 456-468.

Ram, J., Singh, S.P. and Singh, J.S. 1988. Community level phenology of grassland above treeline in Central Himalaya, India. Arct Alp Res. 20: 325-332.

Ramesh, V., Karunakaran, C and Rajendran, A. 2014. Optimization of submerged culture conditions for mycelia biomass production with enhanced antibacterial activity of the medicinal macro fungus Xylaria sp. Strain R006 against drug resistant bacterial pathogens. Current Research in Environmental & Applied Mycology 4 (1): 88-98.

Raunkiaer, C. 1934. The lifeforms of plants and stastistical plant geography, the collected papers of C. Raunkiaer, Clarendon Press, Oxford (UK), pp. 639.

Rawat, G.S. 1998. Temperate and alpine grasslands of the Himalaya: ecology and conservation. Parks 8(3): 27- 36.

Rawat, G.S. and Pangtey, Y.P.S. 1987. Floristic structure of snowline vegetation in Central Himalaya, India. Arct Alp Res. 19: 195-201.

Rawat, G.S. and Uniyal, V.K. 1993. Pastoralism and plant conservation: The Valley of Flowers dilemma. Environmental Conservation 20: 164-167.

Rawlins, J.E. 1984. Mycophagy in Lepidoptera. In Wheeler, Q. and Blackwell, M. (Eds), Insect-fungus relationships, Columbia University Press, New York, pp. 382-423.

Read, J.L. 2002. Experimental trial of Australian arid zone reptiles as early warning indicators of overgrazing by cattle. Austral-Ecology 27 (1): 55-66.

Reeder, J.D., Schuman, G.E., Morgan, J.A. and Lecain, D.R. 2004. Response of organic and inorganic carbon and nitrogen to long-term grazing of the shortgrass steppe. Environmental Management 33: 485-495.

Rees, M., Condit, R., Crawley, M., Pacala, S. and Tilman, D. 2001. Long-term studies of vegetation dynamics. Science 293: 650-655.

Rehner, S.A. and Samuels, G.J. 1995. Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs. Canadian Journal of Botany 73: S816-S823.

Rich, M.A., Meyers, P., Weinbaum, G., Cory, J.G. and Suhadolnik, R.J. 1965. Inhibition of human tumor cells by cordycepin. Biochim Biophys Acta 95: 194-204.

Richardson, L.S., Ting, R.C., Gallo, R.C. and Wu, A.M. 1975. Effect of cordycepin on the replication of type-c RNA tumor viruses. Int J Cancer 15: 451-456.

Rikhari, H.C., Negi, G.C.S., Pant, G.B., Rana, B.S. and Singh, S.P. 1992. Phytomass and primary productivity in several communities of a Central Himalayan Alpine Meadow, India. Arctic and Alpine Research 24: 344- 351.

Ritchie, M.E., D. Tilman, and J.M.H. Knops. 1998. Herbivore effects on plant and nitrogen dynamics in oak savanna. Ecology 79:165–177.

Rizzo, A.J., Kelly, C. and Webb, T.E. 1972. Effect of cordycepin on ribosome formation and enzyme induction in rat liver. Can J Biochem 50: 1010-1015.

Robinson, D. and Traill, B. 1996. Conserving woodland birds in the wheat and sheep belts of southern Australia. RAOU Conservation Statement 10 (Supplement to Wingspan 6).

Rose, K.M., Bell, L.E. and Jacob, S.T. 1977. Selective inhibition of initial polyadenylation in isolated nuclei by low levels of cordycepin 5΄-triphosphate. Biochim Biophys Acta 475: 548-552.

113

Rostagno, C.M. 1989. Infiltration and sediment production as affected by soil surface conditions in a shrubland of Patagonia, Argentina. J. Range Manage. 42:382–385.

Rottman, F. and Guarino, A.J. 1964. The inhibition of phosphoribosyl-pyrophosphate amidotransferase activity by cordycepin mono phosphate. Biochim Biophys Acta 89: 465-472.

Saberwal, V. 1999. Pastoral Politics: Shepherds, Bureaucrats, and Conservation in the Western Himalaya, Oxford University Press, New Delhi.

Sala, O.E., M. Oesterheld, A. Soriano, and R.J.C. León. 1986. Grazing effect upon plant community structure in subhumid grasslands of Argentina. Vegetatio 67:27–32.

Sala, O.E., W.K. Lauenroth, and I.C. Burke. 1996. Carbon budgets of temperate grasslands and the effects of global change. p. 101–119. In A.I. Breymeyer, D.O. Hall, J.M. Melillo, and G.I. Ågren (ed.) Global change: Effects on coniferous forests and grasslands. John Wiley & Sons, Hoboken.

Samant, J. 1992. Human impact on the conservation of biological diversity: Case studies from wildlife sanctuaries of Western Ghats. In Singh, K.P. and Singh, J.S. (Ed), Tropical Ecosystems: Ecology and Management, Wiley Eastern Limited, New Delhi.

Sarah, P. and Zonana, M. 2015. Livestock redistribute runoff and sediments in semi-arid rangeland areas. Solid Earth 6: 433-443.

Sato, A. 1989. Studies on anti-tumor activity of crude drugs. I. The effects of aqueous extracts of some crude drugs in short-term screening test. Yakugaku Zasshi. Journal of the Pharmaceutical Society of Japan 109: 407- 423.

Sattler, K. 1991. A review of wing reduction in Lepidoptera. Bulletin of the British Museum of Natural History (Entomology) 60: 243-288.

Scheffer, M. 2009. Critical Transitions in Nature and Society, Princeton University Press, Princeton, NJ.

Scholefield, D., P.M. Patto, and D.M. Hall. 1985. Laboratory research on the compressibility of four topsoils from grassland. Soil Tillage Res. 6:1–16.

Schulz, S., Francke, W., König, W.A., Schurig, V., Mori, K., Kittmann, R. and Schneider, D. 1990. Pheromone of swift moth, Hepialus hecta L. (Lepidoptera: Hepialidae). Journal of Chemical Ecology 16(12): 3511-3521.

Schuman, G.E., Reeder, J.D., Manley, J.T., Hart, R.H. and Manley, W.A. 1999. Impact of grazing management on the carbon and nitrogen balance of a mixed grass rangeland. Ecological Applications 19: 65-71.

Scorrano, L. and Korsmeyer, S.J. 2003. Mechanisms of Cytochrome C release by proapoptotic BCL-2 family members. Biochemical and Biophysical Research Communications 304: 437-444.

Scougall, S. A., Majer, J. D. and Hobbs, R. J. 1993. Edge effects in grazed and ungrazed Western Australian wheat belt remnants in relation to ecosystem reconstruction. In Saunders, D.A., Hobbs, R.J. and Ehrlich, P.R. (Eds), Nature Conservation 3: Reconstruction of Fragmented Ecosystems, Global and Regional Perspectives, Surrey Beatty and Sons, Chipping Norton, New South Wales, pp. 163–178.

Segelken, R. 2002. Cyclosporine mold’s ‘‘sexual state’’ found in New York forest Cornell students’ discovery could target additional sources of nature-based pharmaceuticals. Cornell Univ. Sci. News.

Seldal, T., Andersen, K.-J. and Högstedt, G. 1994. Grazing-induces proteinase inhibitors: a possible cause for lemming population cycles. Oikos 70: 3-11.

Semmartin, M., Oyarzabal, M., Loreti, J. and Oesterheld, M. 2007. Controls of primary productivity and nutrient cycling in a temperate grassland with year-round production. Austral Ecology 32: 416-428.

114

Semmartin, M.M.R.A., R.A. Distel, A.S. Moretto, and C.M. Ghersa. 2004. Litter quality and nutrient cycling affected by grazing-induced species replacements along a precipitation gradient. Oikos 107:148–161.

Shahed, A.R., Kim, S.I. and Shoskes, D.A. 2001. Down-regulation of apoptotic and inflammatory genes by Cordyceps sinensis extract in rat kidney following ischemia/reperfusion. Transplant. Proc. 33: 2986-2987.

Shang L, Chen J, Li J, Zhang W. 2011. An exopolysaccharide from cultivated Cordyceps sinensis and its effects on cytokine expressions of immunocytes. Applied Microbiology and Biotechnology 163: 669-678.

Shang, Z. and Long, R. 2007. Formation causes and recovery of the “Black Soil Type” degraded alpine grassland in Qinghai–Tibetan Plateau. Frontiers of Agriculture in China 1: 197-202.

Shang, Z.H., Ma, Y.S., Long, R.J. and Ding, LM. 2008. Effect of fencing, artificial seeding and abandonment on vegetation composition and dynamics of ‘black soil land’ in the headwaters of the Yangtze and the Yellow Rivers of the Qinghai–Tibetan Plateau. Land Degradation and Development 19: 554–563.

Shannon, C.E. and Wiener, W. 1963. The mathematical theory of communication, University of Illinois Press, Urbana (IL), pp. 117.

Sharma, K.D., Walling, D.E. and Probst, J.L. 1997. Assessing the impact of overgrazing on soil erosion in arid regions at a range of spatial scales. Human impact on erosion and sedimentation. Proceedings of an International Symposium of the Fifth Scientific Assembly of the International Association of Hydrological Sci. (IAHS), Rabat, Morocco, 1997, pp. 119-123.

Sharpley, A., and B. Moyer. 2000. Phosphorus forms in manure and compost and their release during simulated rainfall. J. Environ. Qual. 29:1462–1469.

She, M. 1991. Effects of Cordyceps mycelia on brain monoamine oxidase B. Chinese Traditional Herbal Drugs 22 (5): 218-219.

Shen, F., Yang, D., Yang, Y., Dong, D., Sinaduji, L. and Yu, R. 01990. Observations on the feeding habits of Hepialus baimaensis Liang. Insect Knowledge 27: 36-37.

Shen, F.R. and Zhou, Y.S. 1997. Two new species of the genus Hepialus from Yunnan, China (Lepidoptera: Hepialidae). Acta Entomologica Sinica 40 (2): 198-201. [In Chinese with English abstract]

Shi, X.M., Li, X.G., Li, C.T., Zhao, Y., Shang, Z.H. and Ma, Q. 2013. Grazing exclusion decreases soil organic C storage at an alpine grassland of the Qinghai–Tibetan Plateau. Ecological Engineering 57: 183-187.

Shipley, B., Vile, D. and Garnier, E. 2006. From plant traits to plant communities: a statistical mechanistic approach to biodiversity. Science 314: 812-814.

Siddique, M.D., Noor-E-Alam, M.M., Islam, M.D., Halim, A., Kamaruzzaman, M.D., Sultana, J. and Karim, D. 2014. Mapping of Site-Specific Soil Spatial Variability by Geostatistical Technique for Textural Fractions in a Terrace Soil of Bangladesh. Journal of Bioscience and Agriculture Research 1: 8-16.

Siev, M., Weinberg, R. and Penman, S. 1969. The selective interruption of nucleolar RNA synthesis in Hela cells by cordycepin. J Cell Biol 41: 510-520.

Simpson, E.H. 1949. The measurement of diversity. Nature 163: 688.

Singh, S., Ranjan, S., Negi, P.S and Arif, M. 2014. Optimization of nutritional necessities for in vitro culture of Ophiocordyceps sinensis. International Jr. of Science and Research 5(3): 1523-1528.

Sivertsen, D. 1993. Conservation of remnant vegetation in the box and ironbark lands of New South Wales. Vic. Natur. 110: 24–29.

115

Skarpe, C. and Hester, A. 2008. Plant Traits, Browsing and Grazing Herbivores, and Vegetation Dynamics. In I.J. Gordon and H.H.T. Prins (Eds), The Ecology of Browsing and Grazing, Berlin Heidelberg: Springer-Verlag, pp. 217-247.

Skovlin, J.M. 1957. Range riding- the key to range management. Journal of Range Management 10: 269–271.

Smirnov, D.A., Babitskaya, V.G., Puchkova, T.A., Shcherba, V.V., Bisko, N.A and Poyedinok, N.L. 2009. Some biologically active substances from a mycelia biomass of medicinal caterpillar fungus Cordyceps sinensis (Berk) Sacc. (Ascomycetes). Inter. Jr. Medicinal Mushrooms 11(1): 69-76.

Song, C.H., Jeon, Y.J., Yang, B.K., Ra, K.S. and Sung, J.M. 1998. Anti-complementary activity of exopolymers produced from submerged mycelial cultures of higher fungi with particular reference to Cordyceps militaris. Journal of Microbiology and Biotechnology 8: 536-539.

Sparks, D.L., Woeltz, V.M. and Markesbery, W.R. 1991. Alterations in brain monoamine oxidase activity in aging, Alzheimer’s disease, and Pick’s disease. Arch. Neurol. 48 (7): 718-721.

Spatafora, J.W. and Blackwell, M. 1993. Molecular systematics of unitunicate perithecial ascomycetes: the Clavicipitales-Hypocreales connection. Mycologia 85: 912-922.

Speed, J.D.M., Austrheim, G., Hester, A.J. and Mysterud, A. 2013b. The Response of Alpine Salix Shrubs to Long-Term Browsing Varies with Elevation and Herbivore Density. Arctic Antarctic and Alpine Research 45(4): 584-593.

Sprecher, M. and Sprinson, D.B. 1963. A reinvestigation of the structure of ‘cordycepic acid’. J Org Chem 28: 2490-2491.

Srivastava, A.K. and Kumar, S. 1995. The disturbed mountain ecosystem. A case study of Salani village in Kumaon Himalaya. Indian Forester 121: 1-2.

Statutory Instrument 1996. The Beef Special Premium Regulations 1996. No. 3241. Queen’s Printer of Acts of Parliament, United Kingdom, pp. 1-15.

Stavi, I., Lavee, H., Ungar, E.D. and Sarah, P. 2009. Eco-geomorphic feedbacks in semi-arid rangelands: a review. Pedosphere 19: 217-229.

Stephensen, A.G. 1981. Flower and fruit abortion: proximate causes and ultimate consequences. Annual Review of Ecology and Systematics 12: 253-279.

Stewart, A. and Frank, D. 2008. Short sampling intervals reveal very rapid root turnover in temperate grassland. Oecologia 157: 453-458.

Stohlgren, T.J., Schell, L.D. and Heuvel, B.V. 1999. How grazing and soil quality affect native and exotic plant diversity in Rocky Mountain grasslands. Ecological Applications 9: 45-64.

Strauss, S.Y. and Agrawal, A.A. 1999. The ecology and evolution of plant tolerance to herbivory. Trends in Ecology and Evolution 14(5): 179-185.

Su, Y.Z., Li, Y.L., Cui, Z.Y and Zhao, W.Z. 2005. Influences of continuous grazing and livestock exclusion on soil properties in a degraded sandy grassland, Inner Mongolia, northern China. Catena 59: 267-278.

Suh, S.O., Noda, H. and Blackwell, M. 2001. Insect symbiosis: derivation of yeast-like endosymbionts within an entomopathogenic ﬁlamentous lineage. Molecular Biology and Evolution 18: 995-1000.

Suh, S.O., Spatafora, J.W., Ochiel, G.R.S., Evans, H.C. and Blackwell, M. 1998. Molecular phylogenetic study of a termite pathogen Cordycepioideus bisporusogia. Mycologia 90: 611-617.

116

Sun, D.S., Wesche, K., Chen, D.D., Zhang, S.H., Wu, G.L., Du, G.Z. and Comerford, N.B. 2011. Grazing depresses soil carbon storage through changing plant biomass and composition in a Tibetan alpine meadow. Plant, Soil and Environment 57: 271-278.

Sun, J., Cheng, G.W. and Li, W.P. 2013a. Meta-analysis of relationships between environmental factors and aboveground biomass in the alpine grassland on the Tibetan Plateau. Biogeosciences 10: 1707-1715. Sun, J., Cheng, G.W., Li, W.P., Sha, Y.K. and Yang, Y.C. 2013. On the Variation of NDVI with the Principal Climatic Elements in the Tibetan Plateau. Remote Sensing 5: 1894-1911.

Sun, J., Wang, X., Cheng, G., Wu, J., Hong, J. and Niu, S. 2014. Effects of Grazing Regimes on Plant Traits and Soil Nutrients in an Alpine Steppe, Northern Tibetan Plateau. PLOS One 9 (9): e108821

Sun, Y. et al. 2002. Experimental study on fruiting bodies of Cordyceps militaris in inhibiting the growth of carcinoma in mice with hepatocarcinoma and promoting the activity of IL-2 and NK cells. Chin Pharm. 11: 39- 40.

Sundriyal, R.C. 1995. Grassland forage production and management in the Himalaya: A review. Journal of Hill Research 8: 135- 150.

Sundriyal, R.C. and Joshi, A.P. 1990. Effect of grazing on standing crop, productivity and efficiency of energy capture in and alpine grassland ecosystem at Tungnath (Garhwal Himalaya), India. Trop Ecol. 31(2): 84-97.

Sundriyal, R.C. and Joshi, A.P. 1991. Annual nutrient budget for an alpine grassland in the Garhwal Himalaya. J Veg Sci. 2: 21-26.

Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JJ, Shrestha B and Spatafora, J.W. 2007. Phylogenetic classification of Cordyceps and the Clavicipitaceous Fungi. Studies in Mycology 57: 5-59.

Sung, G.H., Spatafora, J.W., Zare, R., Hodge, K.T. and Gams, W. 2001. A revision of Verticillium sect. Prostrata, II. Phylogenetic analyses of SSU and LSU nuclear rDNA sequences from anamorphs and teleomorphs of the Clavicipitaceae. Nova Hedwigia 72: 311-328.

Sung, J.M., Lee, J.O., Humber, R.A., Sung, G.H., and Shrestha, B. 2006. Cordyceps bassiana and Production of Stromata in vitro Showing Beauveria Anamorph in Korea. Mycobiology 34(1): 1-6.

Taboada, M.A., and R.S. Lavado. 1988. Grazing effects of the bulk density in a Natraquoll of Argentina. J. Range Manage. 41:502–505.

Taboada, M.A., and R.S. Lavado. 1993. Influence of trampling on soil porosity under alternate dry and ponded conditions. Soil Use Manage. 9:139–143.

Taboada, M.A., R.S. Lavado, H. Svartz, and A.M.L. Segat. 1999. Structural stability changes in a grazed grassland Natraquoll of the Flooding Pampa of Argentina. Wetlands 19:50–55.

Taboada, M.A., Rubio, G. and Chaneton, E.J. 2011. Grazing impacts on soil physical, chemical, and ecological properties in forage production systems. In Hatfield, J.L. and Sauer, T.J. (Eds), Soil Management: Building a Stable Base for Agriculture, American Society of Agronomy and Soil Science Society of America, Guilford Road, Madison, WI, USA, pp. 301-319.

Tallis, J.H. and Yalden, D.W. 1983. Peak District Moorland Restoration Project: Phase 2 Report. RP-vegetation Trials. Peak Park Joint Planning Board, Bakewell.

Tang J.P., Qian, Z.Q. and Zhu L. 2015, Two-step shake-static fermentation to enhance cordycepin production by Cordyceps militaris. Chemical Engineering Transactions 46: 19-24.

117

Tang, L.W. and Jiang, X.Y. 1994. Clinical observation of fermented Cordyceps (JinShuiBao capsule) in treating 38 elderly patients with intractable arrhythmia. Practical J. Integrating Chinese with Western Medicine 7 (B8-9): 532.

Tarhouni, M., Ben Hmida, W. and Neffati, M. 2015. Long-term changes in plant life forms as a consequence of grazing exclusion under arid climatic conditions. Land Degradation and Development. DOI:10.1002/ldr.2407.

Thomas, T.M. 1965. Sheet erosion induced by sheep in the Pumlumon (Plynlimon) area, mid-Wales. Rates of Erosion and Weathering in the British Isles, Institute of British Geographers, Bristol, pp. 11-14.

Thuiller, W., Lavorel, S., Araujo, M.B., Sykes, M.T. and Prentice, I.C. 2005. Climate change threats to plant diversity in Europe. Proc. Nat. Acad. Sci. U.S.A. 102: 8245-8250.

Tilman, D. 1988. Plant strategies and the dynamics and structure of plant communities, Princeton University Press, Princeton, New Jersey, USA.

Tilman, D., Reich, P.B. and Knops, J.M. 2006. Biodiversity and ecosystem stability in a decade-long grassland experiment. Nature 441: 629-632.

Tindale, N.B. 1932. Revision of the Australian ghost moths (Lepidoptera Homoneura, family Hepialidae), Part 1. Records of the South Australian Museum 4: 497-536.

Tindale, N.B. 1981. The origin of the Lepidoptera relative to Australia. In Keast, A. (Ed), Ecological biogeography of Australia, The Hague, Boston, London, Junk, pp. 356-377.

Tisdall, J. M. and Oades, J. M. 1982. Organic matter and waterstable aggregates in soils. J. Soil Sci. 33: 141–163.

Tobi, D.R., Grehan, J.R. and Parker, B.L. 1993. Review of the ecological and economic significance of forest Hepialidae (Insecta: Lepidoptera). Forest Ecology and Management 56: 1-12.

Tongway, D.J. 1991. Functional analysis of degraded rangelands as a means of defining appropriate restoration techniques. In Gaston, A., Kerrick, M. Le Houerou, H. (Eds), Proceedings of Fourth International Rangeland Congress, Association Francaise de Pastoralisme, Offset 2000, Montpellier, France, pp. 166–168.

Tongway, D.J. 1994. Rangeland Soil Condition Assessment Manual. CSIRO, Melbourne, Australia, Canberra.

Tongway, D.J. and Ludwig, J.A. 1990. Vegetation and soil patterning in the semi-arid mulga lands of Eastern Australia. Aust. J. Ecol. 15: 23–34.

Tongway, D.J. and Ludwig, J.A. 1993. Rehabilitation of mine sites and pastoral lands: the ecosystem function approach. In di Russo, M. (Ed.), Proceedings of the Goldfields International Conference on Arid Land care, Mileno di Russo, Hamilton Hill, Western Australia, pp. 51–57.

Tongway, D.J. and Ludwig, J.A. 1994. Small-scale resource heterogeneity in semi-arid landscapes. Pac. Conserv. Biol. 1: 201–208.

Tongway, D.J. and Ludwig, J.A. 1996. Rehabilitation of semiarid landscapes in Australia. 1. Restoring Productive Soil Patches. Restor. Ecol. 4: 388–397.

Török, K. and West, N.E. 1996. The effect of overgrazing on the species composition of different Hungarian grassland communities. In: Rangelands in a sustainable biosphere, Proceedings of the Fifth International Rangeland Congress, Salt Lake City, USA, pp. 565-566.

Totland, Ø. 1997. Limits on reproduction in alpine Ranunculus acris. Canadian Journal of Botany 75: 137-144.

Tremont, R. M. and McIntyre, S. 1994. Natural grassy vegetation and native forbs in temperate Australia: structure, dynamics and life histories. Aust. J. Bot. 42: 641–658.

118

Trubiani, O., Salvolini, E., Staffolani, R., Di Primio, R. and Mazzanti, L. 2003. DMSO modiﬁes structural and functional properties of RPMI-8402 cells by promoting programmed cell death. International Journal of Immunopathology and Pharmacology 16: 253-259.

Turner, C.L., Seastedt, T.R. and Dyer, M.I. 1993. Maximization of aboveground grassland production: the role of defoliation frequency, intensity, and history. Ecological Applications 3: 175-186.

Ueda, K. 1988. New species of the genus Hepialiscus Hampson (Lepidoptera, Hepialidae) from Taiwan. Bulletin of the Kitakyushu Museum of Natural History 8: 39-54.

Ueda, K. 1996. New species of Thitarodes Viette (Lepidoptera: Hepialidae) from Japan. Bulletin of the Kitakyushu Museum of Natural History 15: 35-41.

Ueda, K. 2000. Hepialidae. Japan Heterocerist’s Journal 16 (supplement 1): 70-93.

Ukai, S., Kiho, T., Hara, C., Morita, M., Goto, A., Imaizumi, N. and Hasegawa, Y. 1983. Polysaccharides in fungi. XIII. Antitumor activity of various polysaccharides isolated from Dictyophora indusiata, Ganoderma japonicum, Cordyceps cicadae, Auricularia auriculajudae, and Auricularia species. Chem. Pharm. Bull. 31: 741-744.

Unteregelsbacher, S., Hafner, H., Guggenberger, G., Miehe, G., Xu, X., Liu, J. and Kuzyakov, Y. 2012. Response of long-, medium- and short-term processes of the carbon budget to overgrazing-induced crusts in the Tibetan Plateau. Biogeochemistry 111: 187-201.

Valentine, J.F. 1990. Grazing Management, Academic Press, San Diego, California.

Valentine, K.A. 1947. Distance from water as a factor in grazing capacity of rangeland. J. Forest 45: 749–754. van der Wal, R. 2006. Do herbivores cause habitat degradation or vegetation state transition? Evidence from the tundra. Oikos 114: 177-186.

Van der Wal, R., Bardgett, R.D., Harrison, K.A. and Stien, A. 2004. Vertebrate herbivores and ecosystem control: cascading effects of faeces on tundra ecosystems. Ecography 27: 242-252.

Van Haveren, B.P. 1983. Soil bulk density as influenced by grazing intensity and soil type on a shortgrass prairie site. J. Range Manage. 36:586–588.

Vandvik, V. 2004. Gap dynamics in perennial subalpine grasslands: trends and processes change during secondary succession. J Ecol 92: 86-96.

Vaux, D.L. and Korsmeyer, S.J. 1999. Cell death in development. Cell 96: 245-254.

Vermeire, L.T., Wester, D.B., Mitchell, R.B. and Fuhlendorf, S.D. 2005. Fire and grazing effects on wind erosion, soil water content, and soil temperature. Journal of Environment Quality 34: 1559-1565.

Vesk, P.A. and Westoby, M. 2001. Predicting plant species' responses to grazing. Journal of Applied Ecology 38: 897-909.

Vesk, P.A., Leishman, M.R. and Westoby, M. 2004. Simple traits do not predict grazing response in Australian dry shrublands and woodlands. Journal of Applied Ecology 41: 22-31.

Viette, P. 1948. Lepidopteres homoneures. In Lechevalier, P. (Ed), Faune de France 49, Paris, pp. 2-83.

Viette, P. 1968. Contribution a l’etude des Hepialidae (36e note). Lepidoptera Hepialidae du Nepal. Fosch Unternehmens Nepal Himalaya 3: 128-133.

119

Villalba, J.J. and Provenza, F.D. 1999. Effects of food structure and nutritional quality and animal nutritional state on intake behaviour and food preferences of sheep. Appl Anim Behav Sci 63: 145-163.

Villamil, M.B., Amiotti, N.M. and Peinemann, N. 1997. Physical fertility loss of soils in the southern Caldenal region, (Argentina), by overgrazing. Ciencia-del-Suelo 15 (2): 102-104.

Villamil, M.B., Amiotti, N.M. and Peinemann, N. 2001. Soil degradation related to overgrazing in the semi-arid Southern Caldenal area of Argentina. Soil-Science 166 (7): 441-452.

Villn€as, A., Norkko, J., Hietanen, S., Josefson, A.B., Lukkari, K. and Norkko, A. 2013. The role of recurrent disturbances for ecosystem multifunctionality. Ecology 94: 2275-2287.

Vittoz, P., Randin, C., Dutoit, A., Bonnet, F. and Hegg, O. 2009. Low impact of climate change on subalpine grasslands in the Swiss Northern Alps. Global Change Biology 15(1): 209-220.

Wada, N., Narita, K., Furukawa, A. and Kumar, S. 1995. Impact of overgrazing on seed predation by rodents in the Thar desert, northwestern India. Ecological-Research 10 (2): 217-221.

Wagner, D.L. 1985. The biosystematics of Hepialus F. s. lato with special emphasis on the californicus-hectoides species group, Ph. D thesis, University of California, Berkeley.

Wagner, D.L. 1989. Rearing Hepialid moths (Lepidoptera). Pan Pacific entomology 65(4): 391-396.

Walker, M. and Wahren, C, 2006. Plant community responses to experimental warming across the tundra biome. Proc. Nat. Acad. Sci. U.S.A. 103: 1342-1346.

Walkley, A. and Black, I.A. 1934. An examination of Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci. 37: 29-38.

Waller, J.B. 1966. Sexing of pupae of Wiseana cervinata (Walker) Hepialidae. New Zealand Entomologist 3: 5.

Wan, S.Q., Luo, Y.Q. and Wallace, L.L. 2002. Changes in microclimate induced by experimental warming and clipping in tall grass prairie. Global Change Biology 8: 754-768.

Wang, G.X., Cheng, G.D., Shen, Y.P. and Qian, J. 2003. Influence of land cover changes on the physical and chemical properties of alpine meadow soil. Chinese Science Bulletin 48: 118-124.

Wang, J., Sha, L., Li, J. and Feng, Z. 2008. CO2 efflux under different grazing managements on subalpine meadows of Shangri-La, Northwest Yunnan Province, China. Acta Ecologica Sinica 28: 3574-3583.

Wang, S. and Shiao, M. 2000. Pharmacological functions of Chinese medicinal fungus Cordyceps sinensis and related species. Jr. of Food and Drug Analysis 8(4): 248-257.

Wang, X., Yan, Y. and Cao, Y. 2012. Impact of historic grazing on steppe soils on the northern Tibetan Plateau. Plant and Soil 354: 173-183.

Wang, X.L. and Yao, Y.J. 2011. Host insect species of Cordyceps sinensis: A review. ZooKeys 127: 43-59.

Wardle, D.A., and R.D. Bardgett. 2004. Human-induced changes in large herbivorous mammal density: The consequences for decomposers. Front. Ecol. Environ. 2:145–153.

Warren, A. and Khogali, M. 1992. Assessment of Desertification and Drought in the Sudano-Sahelion Region IW-/99, United Nations Sudano-Sahelion Office.

Warren, S.D., M.B. Nevill, W.H. Blackburn, and N.E. Garza. 1986. Soil response to trampling under intensive rotation grazing. Soil Sci. Soc. Am. J. 50:1336–1340

120

Wasser, S.P. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60 (3): 258-274.

Watkinson, A.R. and Ormerod, S.J. 2001. Grasslands, grazing and biodiversity: editors’ introduction. Journal of Applied Ecology 38: 233-237.

Weiher, E., van der Werf, A., Thompson, K., Roderick, M., Garnier, E. and Eriksson, O. 1999. Challenging Theophrastus: A common core list of plant traits for functional ecology. J Veg Sci 10: 609-620.

Welling, P. and Laine, K. 2000. Characteristics of the seedling flora in alpine vegetation, subarctic Finland, I. Seedling densities in 15 plant communities. Annales Botanici Fennici 37: 69-76.

Welling, P. and Laine, K. 2002. Regeneration by seeds in alpine meadow and heath vegetation in sub-arctic Finland. Journal of Vegetation Science 13: 217-226.

Welling, P., Tolvanen, A. and Laine, K. 2004. The alpine soil seed bank in relation to field seedlings and standing vegetation in subarctic Finland. Arctic, Antarctic, and Alpine Research 36: 229-238.

Westoby, M. 1989. Selective forces exerted by vertebrate herbivores on plants. Trends in Ecology and Evolution 4: 115-117.

Westoby, M. 1999. The LHS strategy scheme in relation to grazing and fire. In Eldridge, D. and Freudenberger, D. (Eds), VIth International Rangeland Congress, International Rangeland Congress, Townsville, pp. 893-896.

Westwood, W.W. 1842. Proceedings of learned societies. Entomological Society, March 1st, 1841. Ann Mag Nat Hist. 8: 217-222.

Whisenant, S.G., Thurow, T.L. and Maranz, S.J. 1995. Initiating autogenic restoration on shallow semi-arid sites. Restor. Ecol. 3: 61–67.

Whitlow, R. 1988. Soil erosion and conservation policy in Zimbabwe. Land Use and Policy 5, pp. 419-433.

Wiesmeier, M., Kreyling, O., Steffens, M., Schoenbach, P., Wan, H., Gierus, M., Taube, F., K€olbl, A. and K€ogel- Knabner, I. 2012. Short-term degradation of semiarid grasslands-results from a controlled grazing experiment in Northern China. Journal of Plant Nutrition and Soil Science 175: 434-442.

Willatt S.T. and Pullar, D.M. 1983. Changes in soil physical properties under grazed pastures. Aust. J. Soil Res. 22: 343-348.

Wilson, A. D. 1990. The effect of grazing on Australian ecosystems. Proc. Ecol. Soc. Aust. 16: 235–244.

Wilson, A.D. and MacLoad, N.D. 1991. Overgrazing: present or absent? Journal of Range Management 44 (5): 475-482.

Wipf, S. and Rixen, C. 2010. A review of snow manipulation experiments in Arctic and alpine tundra ecosystems. Polar Res. 29: 95-109.

Woldeamlak, B. and Stroosnijder, L. 2003. Effects of agro-ecological land succession on soil properties in the Chemoga watershed, Blue Nile Basin, Ethiopia. Geoderma 111: 85-89.

Woldu, Z., and Saleem, M.A.M. 2000. Grazing induced biodiversity in the highland ecozone of East Africa. Agriculture, Ecosystems and Environment 79: 43-52.

Wongsa, P., Tasanatal, K., Watts, P. and Jones-Hywel, N. 2005. Isolation and in vitro cultivation of the insect pathogenic fungus Cordyceps unilateralis. Polym. 85: 753-758.

121

Wu, F., Chen, Y., Chang, S. and Shih, l. 2013. Cultivation of medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes), and production of cordycepin using the spent medium from levan fermentation. Inter. Jr. Medicinal Mushroom 15(4): 393-405.

Wu, G.L., Du, G.Z., Liu, Z.H. and Thirgood, S. 2009. Effect of fencing and grazing on a Kobresia-dominated meadow in the Qinghai-Tibetan Plateau. Plant and Soil 319: 115-126.

Wu, G.L., Shang, Z.H., Zhu, Y.J., Ding, L.M. and Wang, D. 2015. Species abundance-seed size patterns within a plant community affected by grazing disturbance. Ecological Applications 25: 848-855.

Wu, G.Q., et al. 2005. Study progress in research tumour with aweto. Guiding J TCM 11: 80-82.

Wu, J., Shen, Z., Shi, P., Zhou, Y. and Zhang, X. 2014b. Effects of grazing exclusion on plant functional group diversity of alpine grasslands along a precipitation gradient on the northern Tibetan Plateau. Arctic Antarctic and Alpine Research 46: 419-429.

Wu, J., Zhang, X., Shen, Z., Shi, P., Yu, C. and Chen, B. 2014a. Effects of livestock exclusion and climate change on aboveground biomass accumulation in alpine pastures across the Northern Tibetan Plateau. Chinese Science Bulletin 59: 4332-4340.

Wu, N., Liu, J. and Yan, Z. 2004. Grazing intensity on the plant diversity of alpine meadow in the eastern Tibetan plateau. Rangifer 15: 9-15.

Wu, Z., Hang, S., Zhou, K., Hua, P. and Li, D. 2007. Origin and differentiation of endemism in the flora of China. Fron. Biol. China 2: 125-143.

Wylie, T.V. 1959. A Standard System of Tibetan Transcription. Harvard Journal of Asiatic Studies 2:261-267.

Xie, Z., Le Roux, X., Wang, C.P., Gu, Z.K., An, M., Nan, H.Y., Chen, B.Z., Li, F., Liu, Y.J., Du, G.Z., Feng, H.Y. and Ma, X.J. 2014. Identifying response groups of soil nitrifiers and denitrifiers to grazing and associated soil environmental drivers in Tibetan alpine meadows. Soil Biology and Biochemistry 77: 89-99.

Xiong, D., Shi, P., Sun, Y., Wu, J. and Zhang, X. 2014. Effect of grazing exclusion on plant productivity and soil carbon, nitrogen storage in alpine meadows in Northern Tibet, China. Chinese Geographical Science 24: 488- 498.

Xu, C.D. 2006. Advances of researches on Cordyceps sinensis. J Fungal Res. 4: 60-64.

Xu, F., Huang, J.B., Jiang, L., Xu, J. and Mi, J. 1995. Amelioration of cyclosporin nephrotoxicity by Cordyceps sinensis

Xu, J. and Wilkes, A. 2004. Biodiversity impact analysis in northwest Yunnan, southwest China. Biodivers. Conserv. 13: 959-983.

Xu, J.M. and Zheng, H.J. 1994. Treating 64 patients with arrhythmia by Ningxinbao capsule: A randomized, double-blind observation. Shanghai J. Traditional Chinese Medicine 4: 4-5.

Xu, J.S. and Zhang, B.Q. 1985. Preliminary study on effects and mechanisms of fermented Cordyceps on blood lipids under different conditions, Collections on the Basic Medicinal and Clinical Studies of Submerged Culture Cordyceps sinensis, Jiangxi TCM/IMM, Nanchang, Beijing, China, pp. 40-44.

Xu, J.T. 1997. China Medicinal and Edible Fungi, Beijing Medical University and China Xiehe Medical University Press, Beijing, pp.997.

Yamaguchi, N., Yoshida, J., Ren, L.J., Chen, H., Miyazawa, Y., Fujii, Y., Huang, Y.X., Takamuta, S., Suzuki, S., Koshimura, S. and Zeng, F.D. 1990. Augmentation of various immune reactivities of tumor-bearing hosts with an extract of Cordyceps sinensis. Biotherapy 2: 199-205.

122

Yamaguchi, Y., Kagota, S., Nakamura, K., Shinozuka, K. and Kunitomo, M. 2000a. Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis. Phytotherapy Research: PTR 14: 647-649.

Yan, J.P., Bao, Z.X., Lu, C.H., Peng, X.W., Duan, J.K. and Wang, Z.P. 1992a. Analysis of therapeutic effects of XinGanBao capsule on treatment of 50 children with arrhythmia. Chinese J. Integrated Traditional Western Medicine 12 (11): 680-681.

Yang L.Y. 1985. Treatment of sexual hypo function with Cordyceps sinensis. Jiangxi Zhongyiyao. 2: 546-547.

Yang, D., Li, C., Shu, C. and Yang, Y. 1996. Studies on the Chinese species of the genus Hepialus and their geographical distribution. Acta Entomologica Sinica 39: 413-422.

Yang, D.R., Li, C.D. and Shen, F.R. 1992a. Three new species of the genus Hepialus from Yunnan and Xizang, China (Lepidoptera: Hepialidae). Zoological Research 13 (3): 245-250. [In Chinese with English abstract]

Yang, D.R., Li, C.D., Shu, C. and Yang, Y.X. 1996. Studies on the Chinese species of the genus Hepialus and their geographical distribution. Acta Entomolog Sinica 39: 413-422.

Yang, D.R., Peng, Y.Q., Chen, J.Y., Cao, Y.Q. and Yang, P. 2010. The distribution pattern of Cordyceps sinensis in China and the response to environmental changes. Chin J Grassland 32(Supl.): 22-27.

Yang, Z., Guo, H., Zhang, J. and Du, G. 2013. Stochastic and deterministic processes together determine alpine meadow plant community composition on the Tibetan Plateau. Oecologia 171: 495-504.

Yates, C. J. 1995. Factors Limiting the Recruitment of Eucalyptus salmonophloia F. Muell. (Salmon gum). PhD Thesis, Murdoch University, Perth, Western Australia.

Yates, C. J. and Hobbs, R. J. 1997a. Temperate eucalypt woodlands: a review of their status, processes threatening their persistence and techniques for restoration. Aust. J. Bot. 45: 949–973.

Yates, C. J. and Hobbs, R. J. 1997b. Woodland restoration in the Western Australian Wheatbelt. A conceptual framework using a state and transition model. Rest. Ecol. 5: 28–35.

Yates, C.J. 1995. Factors Limiting the Recruitment of Eucalyptus salmonophloia F. Muell. (Salmon gum). PhD Thesis, Murdoch University, Perth, Western Australia.

Yates, C.J. and Hobbs, R.J. 1997a. Temperate eucalypt woodlands: a review of their status, processes threatening their persistence and techniques for restoration. Aust. J. Bot. 45: 949–973.

Yates, C.J., Norton, D.A. and Hobbs, R.J. 2000. Grazing effects on plant cover, soil and microclimate in fragmented woodlands in south-western Australia: implications for restoration. Austral Ecology 25: 36-47.

Yi, S., Wu, N., Luo, P., Wang, Q., Shi, F., Sun, G. and Ma, J. 2007. Changes in livestock migration patterns in a Tibetan-style agropastoral system. Mount. Res. Dev. 27: 138-145.

Yifru, A. and Yaye, B. 2011. Effects of land use on soil organic carbon and nitrogen in soils of Bale, Southwestern Ethiopia. Tropical and Subtropical Agroecosystems 14: 229-235.

Yin, D.H., Chen, S.J. and Li, L. 2004. Study on the biological character of the host of Cordyceps sinensis, Hepialus biru in Tibet. Special Wild Econ Animal Plant Res. (2): 1-5. [In Chinese]

Ying, J., Mao. X., Mao, Q., Zong, Y. and Wen, H. 1987. Icons of Medicinal Mushroom from China, Science Press, Beijing, pp. 151-155.

Yoo, H.S. et al. 2004. Effects of Cordyceps militaris extract on angiogenesis and tumor growth. Acta Pharm Sin. 25: 657-665.

123

Yoshida, J., Takamura, S., Yamaguchi, N., Ren, L.J., Chen, H., Koshimura, S. and Suzuki, S. 1989. Antitumor activity of an extract of Cordyceps sinensis (Berk.) Sacc. against murine tumor cell lines. Jpn. J. Exp. Med. 59: 157-161.

Yu, H.Y., Luedeling, E. and Xu, J.C. 2010. Winter and spring warming result in delayed spring phenology on the Tibetan Plateau. Proceedings of the National Academy of Sciences of the United States of America 107: 22151- 22156.

Yuan, J.G. et al. 2005. Studies on the components and pharmacological action of polysaccharide from Cordyceps sinensis. Food Drug 7: 45-48.

Yue, K., Ye, M. and Lin, X. 2013. The artificial cultivation of medicinal caterpillar fungus, Ophiocordyceps sinensis (Ascomycetes): A review. Inter. Jr. Medicinal Mushrooms 15(5): 425-434.

Zang, M. and Kinjo N. 1998. Notes on the alpine Cordyceps of China and nearby nations. Mycotaxon 66: 215- 229.

Zang, M. and Kinjo, N. 1996. Type study on the Cordyceps sinensis. Acta Bot Yunn. 18: 205-208.

Zang, M., Yang, D.R. and Li, C.D. 1990. A new taxon in the genus Cordyceps from China. Mycotaxon. 37: 57-62.

Zare, R., Gams, W. and Culham, A. 2000. A revision of Verticillium sect. Prostrata, I. Phylogenetic studies using ITS sequences. Nova Hedwigia 71: 465-480.

Zhang, C.K., Yuan, S.R. 1997. Recent advance in studies on immuno pharmacological activities of Cordyceps sinensis and its submerged cultured mycelia. J Capital UnivMed Sci. 18: 37-38.

Zhang, J.T. 2006. Grassland degradation and our strategies: a case from Shanxi Province, China. Rangelands 28: 37-43.

Zhang, Q.X. et al. 2005. Comparison of antitumor effect of extracts in cultivated Cordyceps sinensis fungus HK-1 and natural Cordyceps sinensis. Chin Tradit Herbal Drugs 36: 1346-1349. Z hang, S.L. 1987. Lewis lung cancer of mice treated with Cordyceps sinensis and its artificial cultured mycelia. Bull. Chin. Materia Medica 12: 53-54.

Zhang, W., Yang, J., Chen, J., Hou, Y. and Han, X. 2005. Immunomodulatory and antitumour effects of an exopolysaccharide fraction from cultivated Cordyceps sinensis (Chinese caterpillar fungus) on tumour-bearing mice. Biotechnol Appl Biochem. 42: 9-15.

Zhang, Y.J., Jiang, W.L. and Ren, J.Z. 2001. Effects of sheep night penning on soil nitrogen and plant growth. New Zealand Journal of Agricultural Research 44: 151-157.

Zhou, H., Tang, Y., Zhao, X. and Zhou, L. 2006. Long-term grazing alters species composition and biomass of a shrub meadow on the Qinghai-Tibet Plateau. Pakistan Journal of Botany 38: 1055-1069.

Zhou, H., Zhao, X., Tang, Y., Gu, S. and Zhou, L. 2005. Alpine grassland degradation and its control in the source region of the Yangtze and Yellow Rivers, China. Grassland Science 51: 191-203.

Zhou, J.S., Halpern, G. and Jones, K. 1998. The scientific rediscovery of an ancient Chinese Herbal Medicine: Cordyceps sinensis. J. Alternat. Complement. Med. 4: 429-457.

Zhu, J., Halpern, G.M. and Jones, K. 1998. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis. J. Altern. Compl. Med. 4 (3): 289-303.

124

Zhu, H., Wang, D., Wang, L., Bai, Y.F., Fang, J.Y. and Liu, J. 2012. The effects of large herbivore grazing on meadow steppe plant and insect diversity. Journal of Applied Ecology 49: 1075–1083.

Zhu, H.F. 1965. Hepialus armoricanus Oberthur, host insect of Cordyceps sinensis. Acta Entomologica Sinica 14: 620-621.

Zhu, H.F. and Wang, L.Y. 1985. Cordyceps sinensis and Hepialidae. Sinozoologia 3: 121-134.

Zhu, H.F., Wang, L.Y. and Han, H.X. 2004a. Lepidoptera: Hepialidae, Epiplemidae, Insecta, Vol. 38, Fauna Sinica, Science Press, Beijing, China, 29 pp. Zhu, H.F., Wang, L.Y. and Han, H.X. 2004b. Fauna Sinica: Insecta, Volume 38: Lepidoptera Hepialidae Epiplemidae, Science Press, Beijing

Zhu, J.S. and Rippe, J. 2004. Proceedings of the American Physiological Society’s (APS) Annual Scientific Conference, Experimental Biology, Washington, DC, April 17-21.

Zhu, J.S., Halpern, G.M. and Jones, K. 1998. The scientific rediscovery of an ancient Chinese herbal medicine: Cordyceps sinensis. Journal of Alternative and Complementary Medicine 4: 289-303 (I), 429-457 (II).

Zhu, Z.H., Lundholm, J., Li, Y. and Wang, X. 2008. Response of Polygonum viviparum species and community level to long-term livestock grazing in alpine shrub meadow in Qinghai-Tibet Plateau. Journal of Integrative Plant Biology 50: 659-672.

Zou, J., Zhao, L., Xu, S., Xu, X., Chen, D., Li, Q., Zhao, N., Luo, C. and Zhao, X. 2014. Field CO2 pulse labeling reveals differential partitioning patterns of photo-assimilated carbon in response to livestock enclosure in a Kobresia meadow. Biogeosciences 11: 4381-4391.

Zou, Z.W, Li, J. and Zhang, G. 2012. Biology and Life Cycle of Thitarodes pui (Lepidoptera, Hepialidae), a Host of the Caterpillar Fungus Cordyceps sinensis in the Tibetan Plateau. In Pourali, K. and Raad, V.N. (ed.), Larvae Morphology, Biology and Life Cycle, Nova Science Publishers, New York, pp. 137-152.

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