INTRODUCTION AND REVIEW OF LITERATURE 1.1 Background The kingdom fungi (singular: fungus) are a distinct, humongous and diverse group of eukaryotic organisms distributed worldwide favoring varied habitats. This includes yeast, rusts, smuts, molds, mildews and mushrooms (Hawksworth. 2001; Kirk et al., 2008). These are placed distinctly apart from the animal and plant kingdom because of unique biochemical, morphological and molecular characteristics (Burns. 2006). Globally, about 1.5 to 5.1 million species of fungi has been reported, among which only 5% have been classified. According to early taxonomic studies in the 18th and 19th century, fungi were classified broadly on the basis of morphological and physiological characters (Zhou et al., 2010; Petersen and Knudsen, 2015). Recently, molecular genetics have paved new avenues for modern classification of fungi and surpassed the conventional classification based on phenotypic characters or used together to resolve the taxonomic issues. Phylogenetic analysis has placed these organisms in a distinct kingdom known as fungi (Hawksworth 2006; Ruggiero et al., 2015). The kingdom is further classified in single subkingdom which is comprised of seven phyla (Hawksworth 2006; Ruggiero et al., 2015). Among all the groups, Ascomycetes (Morchellaceae) was focused in the current project due to its nutritional, myco-chemical, nutraceutical, socio economical and complexity of their molecular characteristic. 1.2 Ascomycetes Ascomycetes constitute a major taxonomic group within the true fungi (Eumycota) where the majority of the Morchella species collected from the northern and lower plain irrigated areas viz., Morchella crassipes, Morchella elata, M. pulchella, M. eohespera, M. galilaea and M. eximia. Ascomycetes constitute largest group of sac fungi having 64000 recognized species that were reported from various eco-geographical and ecosystem origin (Kirk et al., 2001). They were divided into various orders and classes. According to classification of Schoch et al. (2009), these classes were: Sordariomycetes, Laboulbeniomycetes, Dothidiomycetes, Leottiiomycetes, lecanoromycetes, eurotiomycetes, Orbiliomycetes, Schizosaccharomycetes, Arthoniomycetes, Pezizomycetes, Saccharomycetes, noelectomycetes and Pneumocystidiomycets. The basic classification of a subclass of ascomycetes that’s divided into three order Gymnomycota, Oomycota and Eamycota. The Oomycota further divide in to two c lasses, Phycomycetous and Zygomycetes. Similarly, Emycota divided into two classes, Deuteromycetes (ascomycetes) and Mycophycophata (Hawksworth. 2006). The basic classification of a subclass of ascomycetes that’s euascomycetes has been much censured due to its complex nature. As in the case of classification systems, a single morphological character was used to separate the euascomycetes (Lumbsch et al., 2005). Numerous of these characters, however, have been found less helpful in phylogeny than originally perceived (Ariyawansa et al., 2015). Moreover, besides these, molecular and phylogenetic research based on 18SrDNA sequences phase shown the three major groups in Ascomycota: a). Euascomycetes, a class of filamentous fungi (FM) which are characterized by the formation of asci in a fruiting body (ascoma); b). Hemiascomycetes (HM), encompassing best of the yeasts characterized by the lack of mutually ascogenous hyphae (AH) and fruiting body formation; c). Archiascomycetes, with Taphrina, fission yeast Pneumocystis and related taxa, which are vastly capricious in biochemical and morphological characters (Berbee and Taylor, 1992; Bruns et al., 1992; Tehler et al., 2000). 1.3 Distribution, Occurrence and Socio-economic aspects Ascomycetes species thrive in various habitats and ecosystems with high distribution in aquatic, moist and terrestrial land. Although, these fungi are typically saprobes but also live as parasitic, mutualistic and pathogenic mode of life. Moreover, about 40 % of ascomycota are found in association with algae in lichens contributing around 8 % of our land Earth. These fungi are found enormously in industrial, natural and agricultural environments and have been extracted from harsh climate habitats such as from deep-sea wood (Kohlmeyer. 1977), earth 24 | P a g e sediments and from depths of many rocks in the Antarctica continent (Raghukumar et al., 2004; Selbmann et al., 2005). Globally, wild Morchella species (spp) are greatly valued for being edible mushrooms (Ascomycetes). Morchella spp. are known with different indigenous names for instance Morchel (Germany), Morielje (Netherlands), Morilla (Mexico and Spain), Morille (France), Murkla (Sweden), Olote (Mexico), Pallohuhtasieni (Finland), Pique (Chile), Pumpalka, (Bulgaria), Rundmorkel (Norway), Smardz (Poland), Smrž obecný (Cheez), Spongioli (ancient Grace), Yangdujun (China), Zbírciog (Romania) and Guchi (urdu/Pakistani) (Rolfe and Rolfe 1925; Korhonen. 1986; Chandra. 1989; Kreisel. 2005; Hamayun et al., 2006). Morchella spp. are widely distributed in different countries for instance USA, Pakistan, India, China, Israel and Turkey (Goldway et al., 2000; Pilz et al., 2007; Barseghyan and Wasser, 2008: Masaphy et al., 2009; Michael et al., 2016; Taskin et al., 2015). Various species of Morchella have shown their varied natural habitats viz., Coniferous, tropics, subtropics, temperate, temperate rain and alpine forest. These species are also found in Rocky Mountains, orchid, hardwood, sugar cane and grass fields (Table 1.1) (Korhonen 1986; Guzman. 1998; Hamayun et al., 2003; Kellner et al., 2004, Liu, 2005; Negi. 2006; Pilz et al., 2007; Kuo. 2008; Kuo et al., 2013; Richard et al., 2015; Badshah et al., 2015; Badshah et al., 2018). M. elata, M. conica, M. crassipes M. esculenta has a great socio economic importance globally (Pilz et al., 2007; Matocec et al., 2016). The huge trader countries i.e., Republic of China, United States of America, Israel, sub-continent, and Turkey (Mediterranean and Aegean) export Morchella to various parts of Europe (Pilz et al., 2007). Morchella (morels) are a key source of income for people living near forests and in villages. They are collected for beneficial subsistence, recreational and commercial harvests purposes (Anderson et al., 2002; Roman and Boa, 2004; Farlane et al., 2005). Morchella is among the world’s most valuable and highly prized edible fungi due to their impressively gratifying flavor and sporadic fruiting season (Lakhanpal et al., 2010; Taşkın et al., 2011; Enshasy et al., 2013; Badshah et al., 2015). Commercially export of Morchella species is common in US (Ower et al., 1986), the rising demand has integrated cultivation in a particular indoor facility using advanced technology. Although, initial efforts have been made at growing wild edible Morchella in the different conduction in Yunnan Province (Zhao et al., 2009). In Pakistan, the Morchella species collection comprises over 5000 families in the region (Saqib et al., 2011). A sum of 14,000-28000kg Morchella species from the Swat (Pakistan) 25 | P a g e was founded to export annually to other area of Pakistan as well as in international markets. It raised significant revenue (US 643000$/year) in Swat valley. These collectors use to obtain the lowest money in trade chain of mushrooms due to incremental addition value by traders and sometimes by discrimination of the middle suppliers and merchants (Sher and Shah, 2014). 26 | P a g e Table.1.1. Global distribution of Morchella species in natural habitat STATES No.# of Common species Habitat Citation species Alaska 1 Morchella tomentosa Temperate rainforest Kuo. 2008 Chez 1 M. semilibera Hardwood, Forest Pilz et al., slope 2007 China 1 M. bicostata Deciduous tress Liu. 2005 Finland 2 M. esculenta. M. elata Forest, orchid, Korhonen. 1986 Germany 3 M.esculenta, M. Rocky mountain range Kellner et crassipes. M.spongiola al., 2004 India 5 M. semilibera, M. Subtropical forest, Negi. 2006 crassipes, M. conica, Himalayans M. esculenta, M. elata Israel 4 M.esculenta, M. Tropic subtropics vulgaris M. conica, M. Barseghyan elata, and Wasser, 2008. Mexico 2 M. rufobrunnea, Subtropical forest Guzman. M. guatemalensis 1998 Newfoundland 2 M. eohespera, Mountains National Voitk et al., M. laurentiana Park 2016 North 3 M. americana, M. Coniferous forests Kuo et al., America prava , M. importuna boreal, cold temperate 2013; Richard et al., 2015. Norway 2 M. elata Norway spruce forest Pilz et al., 2007 27 | P a g e Pakistan 3 M. esculenta, Alpine forest, Hamayun et M. crassipes, sugarcane and grasses al.,2003; M. pulchella field Badshah et al., 2015; Badshah et al., 2018. Sweden 3 Morchella species Burned soil Pilz et al., 2007 Turkey 5- 49 M. mediterraneensis, Pine forest Taşkın et al., M.fakeness,M. 2015. conifericola M. magnispora M. galilaea USA 3 M. prava, M. temperate or boreal Richard et americana, and M. forests, al., 2015 importuna 1.4 Morchella: Taxonomy and Systematics Morchella belongs to ascomycetes. Members of this genus have 8-ascospores enclosed in a special sac-like structure called ascus. There are as many as 60 Morchella species/taxa reported globally (Loizides et al., 2016). Additionally, due to a complex taxonomy and lack of sequence data at NCBI (molecular data), the identification of Morchella has been conflicting (Richard et al., 2015). The molecular analyses none the less have suggested that Morchella has its place to family Morchellaceae and order Pezizales, as a separate genus. However, the number of species within the genus remains unclear. The European mycologists have identified three