Molecular identification and phylogeny of different macrofungi in Mt. Bangkay, Cuyapo, Nueva Ecija, Philippines based on its NRDNA region.
Macrofungi may also be called "mushrooms", that are either edible or non-edible. Many mushrooms were used worldwide as nutritious foods and classified as having high medicinal value . The diversity of mushroom species in the Philippines is relatively abundant; Asia was roughly estimated to have about 10,000 to 25,000 mushroom species . Previous publication on the listing and molecular identification of mushrooms in the Philippines focused on the mushrooms found in the ancestral domain and resettlement areas of Aetas in three provinces of Luzon .
In the Philippines, mushrooms has been considered important resources as foods and utilized as medicine by the indigenous communities. So far, the biodiversity of the mushrooms in the Philippines however, has not been done extensively, as well as its identification using molecular approach. The Philippines was considered to be one of the mega diverse regions in the world in terms of biodiversity richness  and proper identification of the mushroom species in this region of the archipelago was deemed important. Recently, direct sequencing of PCR products using the universal molecular markers for barcoding was found to be powerful tool for the identification of organisms, specifically sequencing the repetitive nuclear DNA of mushrooms [5, 6, 7, 8].
At present, some of the reported species of mushrooms or macrofungi in other part of Asia has been recorded and documented using morphological description. In Burma, 176 species of macrofungi has been recorded , there are 778 species recorded in India , 275 species in China , while there are 60 species records in Malaysia . In the Philippines, there are eight species of macrofungi were found and recorded in Puncan, Carranglan, Nueva Ecija, Philippines . Recently, records of the macrofungi in the Philippines includes the 76 species previously found in Central Luzon using morphological information and the phylogenetic studies of seven wild edible macrofungi . In spite of these, studies on the molecular identification and phylogenetic aspect of the mushrooms in the Philippines have been limited.
Moreover, identification of the identity of wild macrofungi using morphological description has been found problematic considering the complexity of the morphology of other species of mushroom. Thus, the taxonomical classification mushroom species must combine with molecular information to resolved issues in their respective identity and nomenclature. The molecular data based on phylogenetic analysis of macrofungi may provide important knowledge and information on their identity, diversity and extinction. The nuclear ribosomal internal transcribed spacer (ITS) region was found to be the universal DNA barcode marker for fungi . This to provide listing of the collected mushrooms together with the phylogenetic analysis inferred from the internal transcribed spacer region of the macrofungi found in Mt. Bangkay, Cuyapo, and Nueva Ecija, Philippines.
MATERIALS AND METHODS
Collection and preservation of macrofungi:
Macrofungi were collected at Mt. Bangkay, Cuyapo, and Nueva Ecija, Philippines on the month of August 2015. Collected samples were wrapped in wax paper, placed in paper bags with proper labeling and brought to the laboratory for preliminary identification using morphological information, a portion of fruiting bodies were preserved in 2X CTAB buffer at room temperature.
DNA isolation, PCR and sequencing:
The total genomic DNA was extracted from fruiting bodies using CTAB method (with modifications) according to Murray and Thompson . About 100 mg of fresh mushroom tissues was directly crushed using mortar and pestle with liquid nitrogen and placed in 2.0 mL tube. Seven hundred fifty (750) [micro]l of 2x CTAB buffer pre-warmed to 65[degrees]C with 50 pl 20% SDS was added and homogenized thoroughly by vortexing and incubated in a water bath at 65[degrees]C for 45 minutes. Sample was cooled briefly prior to the addition of 500 pl of chloroform and vortexed for 5 seconds followed by 30 minutes centrifugation at 10,000 rpm. To precipitate the DNA 600 [micro]l of ice cold isopropanol was added. The mixture was incubated overnight at -20[degrees]C. The DNA was pelleted by centrifugation for 10 minutes at 10,000 rpm and washed twice with 500 [micro]l of 70% ethanol. Alcohol was discarded and drained dry by inverting the tubes in a paper towel for about 10 minutes. The DNA pellet was dissolved in 50[micro]l of TE buffer with RNAse and incubated at room temperature for 3-4 hours. Genomic DNA was stored at 4[degrees]C until use. DNA was diluted 1:100 using TE buffer with RNAse. The nrDNA ITS region was then amplified using the ITS 3R sequence (5'-ATCGATGAAGAACACAG-3') and ITS4 sequence (5'TCCTCCGCTTATTGATAGC-3'). Reactions were carried out in 0.2 mL PCR tubes of 50 [micro]L containing 32.80 [micro]L of distilled water, 2.5 [micro]L of forward primer (10 [micro]M), 2.5 [micro]L of reverse primer (10 [micro]M), 5 [micro]L of dNTP's (25 mM), 5 ng (4 [micro]L) of DNA template, 5 [micro]L 10X buffer and .02 [micro]L of Taq polymerase. ITS primer have been observed to amplify the ITS region plus a small portion of the 18S and 25S rRNA genes from DNA of fungi. PCR with 35 cycles was performed fitted with a heated lid using the following temperature profile: 94[degrees]C for 5 min, followed by 30 cycles of 94[degrees]C for 30 sec, 55[degrees]C for 45 sec, and 72[degrees]C for 30 sec, with final extension step of 72[degrees]C for 7 min. Amplification products of 1 [micro]L and the 1 kb DNA ladder was subjected for electrophoresis for 30 minutes at 100 V in 1.0 % agarose gel prepared in 1X TAE stained with gel red and was viewed under gel photo documentation system (BioRad), Once the expected size of amplified fragments have been confirmed. The PCR products were sent to FistBase Malaysia Laboratory for clean-up and sequencing procedure.
BLAST analysis and phylogeny matrix using Phylogeny fr.  including the software default parameters such as MUSCLE for sequence alignment, PhyML for phylogeny and TreeDyne for tree rendering were used.
The amplified sequences of the ITS region ranges from 262 to 335 bps (Table 1). The sequences were subjected for phylogeny analysis. The BLAST analysis summary of the different samples using the nuclear internal transcribed spacer sequence fragment was presented in Table 1.
A total of 28 sequences including the ITS sequence of the Sample_ID_1 were used in phylogeny analysis (Figure 1). Phylogeny tree supported by bootstrap value revealed that Sample_ID_1 was Microporus sp. (KJ612044.1) with 91% identity and has 90% bootstrap value using the 335 bp amplified fragment from the sample (Figure 1).
The Sample_ID _2 was identified as Ganoderma lucidum (HM053453.1) supported with 99% identity using the 325 bp length of the amplified fragment and provide 76% bootstrap value from 14 related sequences (Figure 2).
The Sample_ID_3 was identified as Meripilus giganteus (GQ355959.1) having 99% identity with 97% coverage based on the 324 bp sequence of the sample and gives 99% bootstrap value using 28 related sequences (Figure 3).
On the other hand, Sample_ID_4 was identified as Xylaria papulis (GU300100.1) with 100% identity using 272 bp sequences and found to have 99% bootstrap value using the 28 related sequences (Figure 4).
Lastly, the Sample_ID_5 was identified as identified as Leucoagaricus cepaestipes (CU85321.1). with 83% bootstrap value as shown in phylogeny tree using 11 sequences (Figure 5) with 99% identity using the 329 bp sequence confirmed the preliminary identification based on morphological description.
The ITS nrDNA sequences gives clear resolution for the identification of mushroom species collected in the area supported by the lineages and grouping of the members belong to the same family and percentage identity of the sequences. All of the sequences used for the analyses were downloaded from NCBI.
We have identified and confirmed five species of macrofungi found and collected in Mt. Bangkay, Cuyapo, Nueva Ecija, Philippines during the collection time. In the past, classification and identification of macrofungi using the morphological characteristics, with the advent of biotechnology and its modern application using gene sequence such as ITS markers, LSU nuclear DNA, mitochondrial SSU rDNA sequences, ribosomal DNA locus among the few have been extensively used for phylogeny analysis. The elucidation of phylogenetic relationship of Phelinus and its related genera were made possible using the nuclear internal transcribed spacer (ITS) region and mitochondrial small subunit ribosomal DNA sequences . The phylogenetic study of Cataperyrinium sp. and its related genus Placidiopsis were based on the large subunit (nuLSU) sequences . Moreover, the taxonomy and classification of Amanita were resolved in the previous study using also the nuLSU . These related studies shows that molecular approach provides the possible identification of unresolved species using morphological data.
The use of ITS nrDNA sequence provides the identification of five species with regards to a set of species established during BLAST analysis. The Microporus sp. was under the class basidiomycetes and considered as important macrofungi because it was found to have active compound known as ergosterol peroxide . There has been not much studies on the distribution of Microporus sp., although there are records of Microporus xanthopus (Fr.) Kunze considered as wild mushroom reported in Akoko, Ondo State, Nigeria and characterized using morphological description . Moreover, Microporus xanthopus (Fr.) Kunze was collected at Phuphan National park, Sakon Nakhon province in Thailand and identified using molecular technique .
Ganoderma lucidum (Curtis) P. Karst. 1881, under class basidiomycetes was also considered as important medicinal mushroom, extract from this has been used for the prevention and treatment of various diseases including cancer . There are 29 isolates of G. lucidum complex from temperate and subtropical that were undergone parsimony analysis from nucleotide sequence of ITS region and from large ribosomal subunit gene. It was found out that the extensive convergence or parallelism of morphological characters has occurred during the evolution of Ganoderma . However, remarkable morphological difference may occur with little divergence time. Moreover, G. lucidum in North America and in Asia are not conspecific with European G. lucidum. The sister group of European G. lucidum is an Argentinean taxon labeled G. oerstedii. North American G. lucidum is related to a Formosan isolate identified as G. boninense. G. tsugae is absent from Taiwan and probably also from Japan and China. Because of its complexity correct naming and classification using molecular approach of several species of G. lucidum still to be investigated.
Evidence of the existence of Meripilus giganteus (Pers.) P. Karst. 1882, was noted North America, has been studied using the species microscopic characteristics. Early North American records of M. giganteus are sparse. M. giganteus and its closely allied species represented by Grifola sumstinei Murr. and G. lentrifondosa Murr. In North America, the fungus is widely known as Polyporusgiganteus (Pers.: Fr.) Fr. . It has a large a multi-capped fruiting body, as well as its pore surface that quickly darkens black when bruised or injured. Interestingly, there has been no report yet on the molecular identification of M. giganteus in the Philippines.
The Xylaria papulis Lloyd, was under class ascomycetes, first illustrated and described by Van der Gucht ; the species was collected and identified using morphology at Papua New Guinea. So far, the only species of Xylaria described was the Xylaria atroglobosa from China and was considered as new species . Moreover, Okane et al.,  used the molecular approach in the identification of Xylaria species particularly using the 28S rDNA D1/D2 sequences and thus, revealing 21 xylariaceous species inhabiting tropical foliage of Khao Yai Nationa Park, Thailand. Presently, there has been limited data illustrating the phylogenetic analysis of the Leucoagaricus species and its related species using molecular approach, the Leoucoagaricus species was under class basidiomycetes. In the present study, the taxonomic classification of the sample and its identification was highly resolved using the ITS nrDNA sequences. The Leocoagaricus cepaestipes was found in the different clustered as with that of Lepiota species and other Leocoagaricus species. It has been suggested that accelerating the identification and phylogeny of this species may give better understanding on its proper taxonomy using molecular data.
In summary, five species of mushroom were collected at Mt. Bangkay, Cuyapo Nueva Ecija, Philippines and were subjected for molecular identification. The collected samples were identified using the ITS nrDNA sequences revealing the phylogenetic relationship. In general, phylogenetic relationship among taxa showed high resolutions that are informative on the identification of the collected samples. Furthermore, additional collection of the species and using molecular approach as basis for identification of mushroom and may provide concrete data and information on the occurrence of mushroom species in Mt. Bangkay, Cuyapo, Nueva Ecija, Philippines.
This work was supported by the Philippine Council for Health Research and Development (PCHRD) under the Tuklas Lunas Project of the Department of Science and Technology (DOST), Philippines.
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(1) Jesusa Q. Undan, (1) Danny O. Alfonso, (1,2) Rich Milton Dulay, (1,2) Angeles M. De Leon, (1,2) Sofronio P. Kalaw, (1,2,3) Jerwin R. Undan and (1,2) Renato G. Reyes
(1) Tuklas Lunas Center, Central Luzon State University, Science City of Munoz, Nueva Ecija 3120 Philippines
(2) Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija 3120 Philippines
(3) Molecular Biology and Biotechnology Laboratory, College of Arts and Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija 3120 Philippines
Address For Correspondence:
Jerwin R. Undan, Molecular Biology and Biotechnology Laboratory, College of Arts and Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija 3120 Philippines.
Received 12 August 2016; Accepted 17 December 2016; Available online 22 December 2016
Caption: Fig. 1: Phylogenetic relationship of Microporus sp. inferred from the ITS nrDNA gene sequences.
Caption: Fig. 2: Phylogenetic relationship of Ganoderma lucidum inferred from the ITS nrDNA gene sequences.
Caption: Fig. 3: Phylogenetic relationship of Meripilus giganteus inferred from the ITS nrDNA gene sequences.
Caption: Fig. 4: Phylogenetic relationship of Xylaria papulis inferred from the ITS nrDNA gene sequences.
Caption: Fig. 5: Phylogenetic relationship of Leocoagaricus cepaestipes inferred from the ITS nrDNA gene sequences.
Table 1: Blast analysis summary of the different samples using the nuclear internal transcribed spacer sequence fragment. Sample Code Sequenced Highest % Identity Length Coverage (Accession No.) Blasted (bp) Sample ID 1 335 91% 91% (KJ612044.1) Sample ID 2 325 100% 99% (HM053453.1) Sample ID 3 324 97% 99% (GQ355959.1) Sample ID 4 272 100% 100% (GU300100.1) Sample ID 5 329 96% 99% (CU85321.1) Sample Code Identified Name of Samples Sample ID 1 Microporus sp. Sample ID 2 Ganoderma lucidum Sample ID 3 Meripilus giganteus Sample ID 4 Xylaria papulis Sample ID 5 Leucoagaricus cepaestipes