**4. Results and discussion**

#### **4.1. Example for basidiocarp identification (problem solving)-**

*Entoloma rhodopolium* is a poisonous species causes gastrointestinal diseases, and muscarine, muscardine, and choline have also been insulated as noxious mediators. It is commonly known as wood pink gill often confused with morphologically similar species *E. sarcopum* (edible). To save someone's life, correct and authentic identification is very much necessary here. Hence, finally phylogenetic investigation of *E. rhodopolium* was accompanied by using RPB2 and ITS sequences, and the result was matched with that of previously described species from Europe making three clades. Based on the taxonomy, a simple proof for the identification technique, PCRRFLP was followed to distinguish between edible *E. sarcopum* and poisonous species which was actual parallel in morphology. The learning can provide assistance to elucidate the classification of complex *E. rhodopolium*-related species, and to take avoiding action from food poisoning [17] (**Figure 5**).-

Similarly, Nawaz et al. [24] carried out a research to identify *Melanoleuca* species from Pakistan. Only morphological parameters cannot help to identify of *Melanoleuca* species [25, 26], and so, their identification mainly depends on phylogenetic analyses [27]. *Melanoleuca dirensis*  is distinct from the other taxa in the subgenus based on the morphoanatomical and phylogenetic characters. Although, the size of the stipe and lageniform cystidia are shared characters between *M. cinereifolia* and *M. dirensis*, *M. dirensis* differs from *M. cinereifolia* in having white lamellae and fusoid-ventricose cheilocystidia, while *M. cinereifolia* bears gray lamellae [25, 27]. *Melanoleuca dirensis,* a new species from Pakistan [24] belonging to above mentioned genus was identified by phylogenetic tree analyses.-

#### **4.2. Example for ectomycorrhizal morphotype identification-**

Ectomycorrhizal association of Basidiomycetes is an important part of any ecosystem for trees- growth which leads toward increase in forestry. Previously, ectomycorrhizal morphotypes-

**Figure 5.** *Entoloma rhodopolium* (copyright) of Michael Kuo (Kuo, M.-(2014, January). *Entoloma rhodopolium*. Retrieved from the MushroomExpert.Com).

were identified by morphotyping methods [28]. No doubt, characters for morphotyping are important for the identification and taxonomic purpose, but some time these characters- mislead in identification [29] due to similar characters in different morphotypes and different characters of same species morphotypes when their host tree is different. Molecular and- phylogenetic analyses resolve this problem. Now mycologists can easily identify mycobiont as well as phycobiont by using such advanced methods. Corresponding author of this chapter has identified many mycobionts from Himalayan range of Pakistan by using molecular- methods [30–34]. Following phylogenetic trees are two examples among these. **Figure 6**  explains ectomycorrhizal morphotypes of *Suillus flavidus*. These morphotypes were isolated- from rhizosphere of conifers from Pakistan and were tried to identify by morphotyping- methods, but ultimate identification was possible only by molecular and phylogenetic analyses [32]. Similarly, **Figure 7** explains mycobiont of another mushroom *Suillus himalayensis,* 

**Figure 6.** Phylogenetic analyses of ectomycorrhizal morphotypes of *Suillus flavidus* [32].

Importance of Molecular and Phylogenetic Analyses for Identification of Basidiomycetes 51 http://dx.doi.org/10.5772/intechopen.80671

**Figure 7.** Phylogenetic place of *Suillus himalayensis* [34].

a new species reported from Pakistan by corresponding author. Its ectomycorrhizal relationship was confirmed when morphotypes were analyzed phylogenetically [34].

#### **5. Problems that need to be addressed**

The absence of sequences at a local level would be a chief hindrance for the recognition of some Basidiomycetes. Robles etal. [35] worked to analyze the scope of factsattainedfrom ITS sequences as taxonomic implements to inspect local woodrotting fungi.- Phylogenetic analyses were made under static and vibrant homologies, but identification of some of these fungi was not attained due to the intricacy of the genera and the deficit of sequences [35].

Another fungus *LeucoCalocybe mongolica* has application in food industry and atmosphere investigation, is a noteworthy unusual wild edible mushroom in Northeast Asia. Its genomic sequence is vital to be studied at genus and species level in taxonomic classification. Beyond that, there is limitation in further study by virtue of the way that transcriptomic and genomic information of *L. mongolica* lacked in the biological information database. For such investigation, the transcriptome information is accomplished by virtue of Illumina paired-end sequencing innovation [36].

For taxonomic identification of Basidiomycetes, the sequence of the ITS region is a superior molecular DNA barcode [37]. As most of the studies so far done to identify the fungal species has used primers (forward and reverse) against this most highly varied region to amplify. Most of the times partial rDNA sequences, including the Internal Transcribed Spacer I5.8SrDNAInternal Transcribed Spacer II, are used, and further phylogenetic assessments are made to see relationships between edible species of the Basidiomycetes. Polymorphism occurred due to insertiondeletion and point mutations throughout the ITS regions and can be clearly distinguished within genera as well as families [38].

#### **5.1. Why practice molecular documents?**

Today, virtually all evolutionary interactions are contingent from molecular sequence data. This is because:-


Morphological lineages are also made where genetic lineages are not possible (e.g., in few fossil records), but they are not reliable as we discern that every now and then the similar morphological mannerism can ascend from manifold independent evolutionary lineages.

#### **5.2. Stages**


#### **5.3. Phylogenetic resources at EMBL-EBI**

EMBLEBI offers a range of tools and resources that are relevant to the field of phylogenetics:-


After performing the first initial BLAST, a phylogenetic tree is produced using different software, for example, different versions of MEGA and SYPRUS (**Figure 8**).-

## **5.4. Explanation of the figure obtained by using MEGA 6 software for molecular characterization and phylogenetic analysis of** *Coprinopsis* **species**

After morphoanatomical characterization of *Coprinopsis* species gathered from plain territories of Pakistan, it was considered for molecular affirmation. Sequence brought about- 1070bp of their ITS region. The sequence was gone intensive BLAST search. Introductory- BLAST investigation indicated 99% match with *C. cinerea* (AB097562). In addition, comparative groupings were likewise incorporated into this phylogeny. The entire informational collection involves 32 nucleotide sequences comprising 701 positions. The phylogenetic tree for- *Coprinopsis* with sequences from Genbank was separated in four clades. *Coprinopsis cinerea*  (BIF S21) falls in Clade I in *Cinerea* section making bunched with other *C. cinerea* species of different countries.-

 **Figure 8.** Phylogenetic analysis of Coprinus species collected from-Pakistan based on nrITSr-DNA regions. This is the original phylogenetic tree made by one of the author of this chapter.
