Introduction

Morels (Morchella spp.) are edible macro-fungi, well known due to their proteins, fibers, minerals, and bioactive contents [1, 2]. They are characterized by a distinct honeycomb-like hymenium on the hollow fruiting body and typically fruit in the spring (at least in the temperate zone), except for some autumn species [3]. Morels are important because of their scientific and economic worth, in addition to their nutritional and therapeutic qualities [4-6]. Morels occur in a variety of environments worldwide. In Asian countries, mature fruiting bodies of morel are used in traditional herbal medicines [7]. Morels are a great source of immunostimulants and anticancer agents and are used extensively in soups and cookeries because of their distinct flavor and high nutritional content [8].

Taxonomically, Morchella is a polymorphic and problematic genus within the Pezizales. Some authors have identified more than 50 species within the genus Morchella, whereas others consider only three species [9]. Consequently, the genus is currently being studied using integrative scientific techniques that combine molecular data with morphological, ecological, and distributional information [10]. Recent research has shown that many morel species are endemic, frequently restricted to certain regions or continents, in contrast to earlier theories that the genus is made up of a small number of worldwide species. All currently known morel species are included in three major categories: the Rufobrunnea clade (sect. Rufobrunnea), the Esculenta clade (sect. Morchella), and the Elata clade (sect. Distantes) [11]. Numerous aspects of morel biology, such as their reproductive methods, interspecific hybridization, nuclear and mitochondrial genome analysis, transcriptome analysis, physiological and biological traits, trophic modes, and the dynamics of the microbial community related to morel cultivation, have recently yielded significant new insights [3]. Morels were used to cure a range of stomach issues in China, as documented in the renowned pharmacological treatise “Compendium of Materia Medica,” penned by Li Shizhen during the Ming Dynasty. Some of the most significant fungi in the Himalayan range from an economic, sociological, and ethnomycological standpoint are morels, also known as “Guchhi” in the Indian market [12].

Therefore, molecular methods were used in addition to morphological characterization to support the traditional taxonomy of fungi. The best methods for identifying mushrooms to begin phylogenetic investigations have been found to be sequencing the ITS area, which is composed of the 5.8 s rRNA gene, along with, SSU, LSU, RNA polymerase, and RAPD-PCR study [8]. Molecular phylogeny has confirmed more than 70 species of the genus Morchella worldwide [13]. Prominent and taxonomically important features of Morchella include the color and configuration of the ridges, pits, discoloration, bruising, the attachment of the cap to the stem (sinus), and the length of the stem. Important microscopic characters are spore size, shape, and ornamentation, the paraphyses, the ectal esxcipulum, and the stipe hairs [14-16]. Monophyletic status for Morchellaceae and the genus Morchella has been confirmed by O’Donnell et al. [17] using nrDNA. Phylogenetic studies, including sequencing of Internal Transcribed Spacer (ITS1 & ITS4) regions, small subunit-coding sequences (SSU), large subunit-coding sequences (LSU), and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) are suitable techniques for morel identification [18-20].

In Pakistan, only five species of Morchella have been confirmed by DNA sequencing: Morchella pakistanica [21], Morchella pulchella [22], Morchella crassipes, Morchella spongiola, and M. elata [23]. In the past, different Morchella species, namely M. crassipes (Vent.), Morchella esculenta (L.) Pers., M. elata, and Morchella semilibera DC. have been reported solely based on morphological identifications [24, 25]. In-depth research on the genetic diversity and identification of morels growing in the western Himalayan region of Kashmir Forest has not been done. The major objective of the present research was to identify the morels from the Western Himalayan region AJK, by using a morphoanatomical and phylogenetic approach. In this study, we report and describe M. conica, M. costata, M. elata, M. deliciosa, and M. tridentina for the first time from the state of Jammu and Kashmir based on their morpho-anatomical and molecular bases.

Materials and Methods

DNA Analysis and Phylogeny

DNA extractions were performed from dried fruiting bodies using 2% CTAB buffer with slight modifications [29]. DNA extraction was confirmed through Gel Electrophoresis using 1% Agarose gel for 30 min at 70V [30]. Further amplification of nrDNA and the internal transcribed spacer region (ITS) by PCR using a primer combination. ITS1F (5, -CTTGGTCATTTAGAGGAAGTAA-3,), and ITS4 (5,-TCC TCCGCT TAT TGA TAT GC-3,) were proposed by Janowski et al. [31]. Both the forward and reverse sequences were used to construct the final sequence using BioEdit ver. 7.2.5 [32]. Most similar sequences were retrieved from the National Center for Biotechnology Information (NCBI) to compare the sequences generated in the study [33] by using the Basic Local Alignment Search Tool (BLAST). All the sequences were included to construct the final phylogram [34-36]. Multiple sequences were aligned using the online MUSCLE alignment tool (http://www.ebi.ac.uk/Tools/msa/muscle) [37]. The phylogram was constructed several times in RAxML-HPC2 using the XSEDE tool (8.2.10) at 1000 bootstrapping to get the best bipartition results.

Fig. 1.

Map of study area and collection sites (Neelum Valley, AJK).

Results

Morpho-anatomical features of Morchella species recorded during the study are given below.

Taxonomical Description and Phylogenetic Analysis of M. elata

Pileus: 6.1-7.3 cm long, 4.9-6 cm wide, globular, elongated to cylindrical, and sometimes slightly pointed cap. The main ribs of the pileus are thin, parallel, and connected by narrow, transverse, and oblique ridges. Elongate, irregular, deep, and tetragonal pits are present between the ribs. The stalk is usually inflated into a bulbous base, 2-8 cm thick and 6-8.8 cm long. The color was noted as brown to reddish-purple. The taste is pleasant. Ascospores (1/1/40). 26.5-33×12-16 µm, broadly ellipsoidal, and smooth with homogeneous contents, hyaline in 5% potassium hydroxide. Asci 198.7-210×10- 15.5 µm; 8 spores cylindrical to narrow long base, hyaline in 5% KOH. Paraphyses: 4.8-6.1 µmwide, 125-167.3 µm long, filamentous, and septate, narrow to clavate at the base; hyaline in 5% potassium hydroxide.

Table 1.

List of Morels species with collection sites, collection date, and accession numbers.

Sr. No	Name of species	Habitat	Collection site	Altitude (m)	Date of collection	Gene Bank Accession Numbers
1.	Morchella conica Krombh., Naturgetr. Abbild. Beschr.	Gregarious on soil, under Pinus wallichiana, and at decayed hardwood and organic matter.	Azad Jammu and Kashmir Dawarian (Western Himalayan part)	1996	12-04-2017	MT950315
2.	Morchella costata Pers.	Growing under Cedrus deodara and Quercus incana, Sporophores in groups and scattered, late April-June	Azad Jammu and Kashmir Dawarian and Surgon	1981, 2205	24-05-2018, 25-05-2018	Under Process
3.	Morchella deliciosa Fr.	Fruiting bodies grow under conifer forests and Quercus trees during April-June.	Azad Jammu and Kashmir Dawarian	1959	24-05-2018	MW558089
4.	Morchella elata Fr.	Scattered and alone, grow under mixed conifer forests during April-May	Azad Jammu and Kashmir Dawarian, Arang Kel (Western Himalayan part)	1936, 2034	12-04-2017, 08-05-2017	MT584846 & MT977069
5.	Morchella tridentina Bres.	Mycorrhizal, alone, scattered, or gregariously under hardwoods, specifically under Pinus wallichiana, during late spring	Azad Jammu and Kashmir, Surgon and Dawarian	1898, 1956	28-04-2017, 12-05-2018	MT584841 & MT957957

Fig. 2.

(A) A fruiting body of M. conica T-18, (B) ascospores, (C) paraphyses, (D) the fruiting bodies of M. elata T-02 & T-09, (E) Asci with ascospores, (F) Excipulum, (G) the fruiting body of M. deliciosa T12, (H) Ascospores, (I) Ascus with ascospores, Scale Bars; A = 4.5 cm, B = 12 µm, C = 8.8 µm, D = 11.2 µm, (J) the fruiting bodies of M. costata T04, (K) Asci with ascospores, (L) Excipulum, Scale Bars; A = 5 cm, B = 4.3 cm, C = 12µm, D = 11.6 µm, (M) the fruiting bodies of M. tridentina T-05 and T-06, (N) Asci, (O) paraphyses.

The molecular amplification of internal transcribed regions (ITS1 & ITS4) of the samples T-02 and T-09 had primers that produced about 680 bp and 745 bp sequences, respectively. The ITS sequence match of the sequences generated in the study (MT584846, MT977069) has shown 100% query coverage and similarity with M. elata (GQ228471). Clade M. elata with a 100% bootstrap value contains (T-02: MT584846 and T-09: MT977069) along with its closet matching sequences (DQ257329, GQ228469, and KX809734). Sequences used in the construction of the phylogenetic tree showed maximum similarities with the sequences generated in this study (Fig. 2 D, E, and F).

Discussion

All the studied Morchella species from the sampling sites in the District Neelum (34°37’ N and 73°56’ E) were found at an elevation between 1900 to 2300 m in temperate coniferous forests. The temperate forests of Neelum valley are spread over a larger area in western Himalayas and well-known hub for biodiversity [39]. These temperate forests are consisting of dominant plant species i.e., Cedrus deodara, Abies pindrow, Pinus wallichiana, Aesculus indica, Acer caesium, Prunus padus, Parrotiopsis jacquemontiana, Viburnum grandiflorum, Picea smithiana, Taxus wallichiana, Quercus incana, and Berberis lyceum [40, 41]. These forests provide a suitable habitat for the diversity of fungi including morel species. The state of Azad Jammu and Kashmir is blessed with various geographical regions and environmental conditions. Morels can be found in a range of forest types and exhibit symbiotic and saprotrophic mycelial dynamics [7].

Fig. 3.

A Phylogenetic tree separated the Morels into distinct clades based on molecular data.

In the present study, five species of Morchella have been described for the first time in the State of Jammu and Kashmir. Among these, M. elata has ascospores, 26.5-33×12-16 µm, broadly ellipsoidal with asci up to 198.7 µm, width up to 16.5 µm, cylindrical to narrow long base. It has slight differences from the M. elata studied by Ali et al. [23] in the Hindu Kush region of Pakistan. The specimens studied by Ali et al. [23] have a large-sized ascoma 6-9 cm higher than large-size hymenophores. The black morel of Kashmir has slight differences in morpho-anatomical characteristics from the previously described Morchella species by Kanwal et al. [42] from India. The difference observed could be due to variations in geographic, environmental, and edaphic characteristics, soil organic matter, and soil fertility. More variations occur among the same species of Morchella in a geographically isolated population [43]. M. crassipes, M. esculenta, and M. deliciosa produces white or yellow fruiting bodies, while M. angusticeps, M. eleta, and M. conica produce black fruiting bodies [42]. Various recent studies used molecular approaches for the correct identification of Morels because, due to slight differences in soil properties and environmental conditions, the morphology of Morels significantly varied among individuals of the same species [44]. Many morel species, including M. rotunda, M. esculenta, M. elata, M. deliciosa, M. semilibera, and M. conica have been described from various parts of Pakistan based on their morphological features [45]. On the other hand, M. pulchella has been reported based on molecular methods [46]. In this study, M. tridentina is characterized by its cap, 5.6-9.8×3.1-6 cm, wide, conical, brownish grey to light brown and whitish in color; ascospores, 18.5-25x12-18 µm; elliptical and ovoid; smooth; and asci, 16.5-29 µm wide, up to 250-315 µm long (Table 1). The results of the present study agree with the previous study on M. tridentina by Loizides et al. [13].

The taxonomic status of M. tridentina and its synonyms have been confirmed through combined morpho-anatomical and phylogenetic studies. The name M. tridentina was first proposed by Machuca [47] from Chile, and it is a poorly known name because it doesn’t appear in any influential works of the twentieth century [48, 49]. Their material, studied from different regions, was analyzed by Richard et al. [50], Machuca et al. [51], and Clowez [52] through DNA sequence analysis. All the sequences submitted in the GenBank database named M. frustrata and M. elatoides are like M. tridentina. These taxa are synonyms of M. tridentina [50, 13]. Compared with the previous study on M. tridentina, morph-anatomical and molecular features [13] affirm this taxon is the first time reported in the western Himalayan region of the State of Jammu and Kashmir.

Previously, Morchella pulchella and M. pakistanica have been added to the fungi of Pakistan, identified based on a combined morpho-molecular approach [22, 23]. M. elata, M. crassipes, and M. spongiola are Morchella spp., which are the first time identified (molecular and micromorphological) species from Pakistan by Ali et al. [13]. Based on our morphology study in M. costata, ascospores are 7-7.5×3.1-4.8 µm broadly rounded to ellipsoid, with asci 66.7-79.5×11.2- 13.9 µm cylindrical, having a blunt, narrow base. In M. deliciosa, ascospores are 10.2-15.5 µm×8.5-11 µm, ellipsoidal, smooth, having asci 242.3-258.3 µm×63 -82.6 µm, narrow long up to 298 µm, wide up to 11.8µm, and cylindrical (Table 2). Based on our morphology study in M. costata, ascospores are 7-7.5×3.1-4.8 µm broadly rounded to ellipsoid, with asci 66.7-79.5×11.2- 13.9 µm cylindrical, having a blunt, narrow base. In M. deliciosa, ascospores are 10.2-15.5 µm×8.5-11 µm, ellipsoidal, smooth, having asci 242.3-258.3×63-82.6 narrow long up to 298µm, wide up to 11.8µm, and cylindrical (Table 2).

The phylogenetic tree (Fig. 3) separated the black morels M. elata, M. conica, and M. costata from M. tridentina and M. deliciosa into distinct clades. Verpa bohemica was used as an out-group taxon. A phylogenetic tree constructed based on ITS sequence analysis confirms the position of studied morels with their nearest match species in the database. The findings of the present study are also supported by previous studies, i.e., [3, 53-57]. The identification of Morchella species through morphological and anatomical characters using microscopic and phylogenetic techniques results in the addition of these morels in the western Himalayan region of Kashmir. Now, the number of Morchella species confirmed through molecular tools in the State of Jammu and Kashmir has increased to five.

The current research is the first attempt to observe the morphological and anatomical characters of the genus Morchella using microscopic and phylogenetic techniques from the state of Jammu and Kashmir. Variations among species have been observed in both qualitative and quantitative characters. Variations in these characters play a significant role in solving problems in the classification of species within the genus. The Morchella genus is still unexplored in terms of pharmaceutics. Correct identification of morel species is important to use in the isolation of bioactive contents to develop drugs in the future.

Conclusions

The temperate forests of Western Himalaya are hub of biodiversity as well as rich habitats for the morels. Different field visits were made to collect morels from various unexplored regions of the District Neelum from 2017 to 2021. All the species of morels were collected during the spring season. The collected species of the genus Morchella were identified by investigating morphological and anatomical features using microscopic and phylogenetic techniques. Significant variations were observed among the investigated species of Morchella, and these variations play a significant role in the correct identification of species in fungal taxonomy by using advanced molecular approaches. The highest value of Pileus length and width was observed in M. costata (6.9- 12×4.5-5.4 cm) while the lowest value was observed in M. conica (2.5-4×3.9-4.8 cm). The largest stipe length was calculated in M. elata (6-8.8 cm), whereas the lowest value was calculated in M. deliciosa (2-3.5 cm). The accurate identification of Morels species is very crucial for the isolation of bioactive compounds, and these compounds play a vital role in drug discovery. So, we recommend a comprehensive exploration of Morels in the Western Himalayan Forest by using advanced molecular approaches and the isolation of bioactive compounds because this genus is still unexplored in the context of pharmaceuticals.