Advances in Environmental Biology, 10(12) December 2016, Pages: 153-158

AENSI Journals

Advances in Environmental Biology

ISSN-1995-0756 EISSN-1998-1066

Journal home page: http://www.aensiweb.com/AEB/

Molecular identification and phylogeny of some wild

microscopic fungi from selected areas of Jaen, Nueva
Ecija, Philippines
1Ageo Boy V. lopez, 2John Dave C. Aquino, 2Jesusa Q. Undan, 3Kristine Grace D. Waing and 3,4Jerwin
R. Undan
1
Undergraduate Student in Biology, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120
Philippines.
2
Graduate Student in Biology, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120
Philippines.
3
Faculty, Department of Biology, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz, Nueva Ecija 3120
Philippines.
4
Molecular Biology and Biotechnology Laboratory, College of Arts and Sciences, Central Luzon State University, Science City of Muñoz,
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 Muñoz, Nueva Ecija 3120 Philippines

This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Received 12 August 2016; Accepted 17 December 2016; Available online 31 December 2016

ABSTRACT
Background: The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) region of the genomic DNA of the collected wild
macroscopic fungi from selected areas in Jaen, Nueva Ecija, Philippines was amplified and used for nucleotide sequence homology analysis
using the Basic Local Alignment Search Tool (Nucleotide BLAST). Using the rDNA-ITS sequences of the collected macrofungi, the 7 wild
macroscopic were identified. There are 3 macrofungi identified as Lentinus swartzii (GU207276.1) with 100% identity, 1 species was
identified as Lentinus squarrosulus (KT207470.1) with 99% identity, 1 species was identified as Panaeolus foenisecii (KR867660.1) with
100%, 1 species was identified as Coprinellus aureogranulatus (GQ249274.1) with 100% identity and 1 species was identified as
Schizophyllum commune (KR706163.1) with 100 % identity. All of the collected species were identified up to species level. The molecular
identification strategy in this study was proven accurate for identifying mushroom. This study in the molecular identification of collected
species was the first report in the Philippines and the sequences may provide additional information in the molecular taxonomy of the
mushroom and thus important in its domestication and characterization as reference for further research and exploration of its benefits.

KEYWORDS: rDNA-ITS, BLAST, macroscopic fungi, taxonomy

INTRODUCTION

Macrofungi have been part of the fungal diversity for around 300 million years and they are known to be
among the largest of group of fungi that attracted the attention of naturalists before microscopes were invented
[1].
Fungi or also known as "mushroom" is defined as “macrofungus” with a distinctive fruiting body, it can be
hypogeous or epigeous, large enough to be seen by the naked eye and usually pick up by hands. Most
macrofungus or mushroom species are under the Basidiomycota and Ascomycota, the two phyla under the
Kingdom Fungi [2]. Mushrooms are important source of food and utilize to gain income in both developing and

Copyright © 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication).
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developed countries. Furthermore, mushrooms are great recyclers, decomposers and bioremediators; and
therefore play a significant role in the ecosystem. With these, mushrooms which happened to be in the
wilderness, growing on fallen logs, decomposing piles of straws, lawns, meadows and gardens were now
intentionally cultivated for consumption [3].
Mushrooms can be roughly divided into four categories: (1) edible mushrooms (2) medicinal mushrooms,
(3) poisonous mushrooms, and (4) those in a miscellaneous category, which includes a large number of
mushrooms whose properties remain less well defined. Certainly, this approach of classifying mushrooms is not
absolute. Studies on identification and characterization of more mushrooms under the miscellaneous category
must be given attention for research for their possible benefits to mankind.
Ecologically, mushrooms can be classified into three groups: the saprophytes, the parasites and the
symbiotic species (which includes Mycorrhiza sp.). There are only a few parasitic mushrooms that are known
while most of the cultivated gourmet mushrooms are saprophytic fungi. These species have a symbiotic
relationship with some vegetation, particularly trees, i.e. there is a relationship of mutual need.
Moreover, indigenous community are utilizing mushroom for the treatment of different type of diseases and
also as an aphrodisiac and tonic. Different types of edible mushrooms are cultivated on large scale for
commercial use and many more species of mushrooms that grow in the wild, which has much nutritional and
medicinal value. The Philippines as a tropical country has very rich mycological resources and Filipinos are
known to be mushroom eaters. Unfortunately these mycological resources are not yet known and fully utilized
[4].
The study on the molecular identification of wild mushrooms was an important activity to identify those
unknown mycological resources that the Philippines has. Through the advent of molecular marker technology, it
is now possible to determine fungi based on their molecular data. The molecular markers, PCR (Polymerase
Chain Reaction) and non-PCR based are widely used for mushroom identification and characterization [5].
Recently, The Internal Transcribed Spacer (ITS) of nuclear DNA has been proposed as the official bar coding
marker for molecular identification of fungi [6].
The ITS regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in
distinguishing fungal species. The objective of this study was to identify the wild macroscopic fungi found in
selected areas of Jaen Nueva Ecija, Philippines using the amplified rDNA-ITS region collected mushroom. The
morphology of the mushroom was also examined for verification and confirmation. The study also determined
the phylogenetic relationships of the different mushrooms collected.

MATERIALS AND METHODS

Mushroom collection:
The mushrooms were collected at three different areas in the municipality of Jaen Nueva Ecija. The areas
were Barangay Dampulan, Barangay Langla and Barangay Putlod, Jaen, Nueva Ecija, Philippines. Fruiting
bodies of wild mushroom were collected during the summer season (April-May) of 2015. The samples were
either carefully handpicked or with the use of knife. The global coordinates of the sampling sites were recorded
with the use of Global Positioning System (GPS). Specimens were place in a paper bag with tag of location and
other important information. The samples were stored at the at the Molecular Biology and Biotechnology
Laboratory, Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University,
Science City Munoz, Nueva Ecija. The samples were cleaned using fine brush, documented with picture and
standard measurements were taken. The specimens were preserved with 95% ethanol and coded until use for
DNA extraction.

DNA Extraction:
Approximately 10g of the fruiting body preferably from the gills was collected and placed in 2ml tubes for
genomic DNA extraction. Tissue homogenization was carried-out by grinding specimens in liquid nitrogen
using mortar and pestle. Total DNA was extracted using the cethyl-trimethyl ammonium bromide (CTAB)
method based on Murray and Thompson (1980) [7] with minor modification. Pre-warmed 750μl 2X CTAB
buffer and 50 μl of 20% sodium dodecyl sulfate (SDS) were added to the homogenized specimens. The mixture
was thoroughly mixed in a vortex shaker and incubated in a water bath at 65°C for 3 1 hour, after that 750μl of
chloroform was added and thoroughly mixed using vortex. Tubes containing specimens were centrifuged for 30
min at 10,000 rpm.
The aqueous phase was transferred into a new 1.5ml tube, and 600 μl of cold isopropanol were added and
incubated at -20°C overnight. Specimens were centrifuged for 10 min at 10,000 rpm, and then decant the
isopropanol. The DNA pellet was washed with 70% ethanol and centrifuged twice for 3 min at 10,000rpm. After
draining the ethanol and air-drying, the pellet was dissolved in 50μl TE buffer and incubated at room
temperature for 2 to 3 h until the pellets dissolve completely. To check the DNA quality, 1 μl of the DNA mixed
with 1μl loading dye was loaded into 1% agarose gel containing l μl of gel red (GelRed TM Nucleic acid,
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Advances in Environmental Biology, 10(12) December 2016, Pages: 153-158

Biotium). Electrophoresis was carried out at 100 V for 30 minutes using Endoro TM Gel XL, Labnet International
Inc. The gel was viewed in EnduroTM GDS the for gel imaging. Quantification of DNA was done using
QuantusTM Fluorometer (Promega). The genomic DNA was diluted 1:100 by means of sterilized distilled water.
A volume of 1μl of diluted DNA was used for PCR analysis.

PCR amplification, sequencing and phylogeny analysis:

The polymerase chain reaction was performed using the following component: 2.5l of 10X PCR buffer,
1.5l of MgCl2 (25mM), 1.25 l of dNTP mix (25mM each), 1.13l of forward primer [5'-
GTAAGTGCCGAATTCGAAATAACTGAATGGGA-3' (10pm/l)], 1.13l of reverse primer [5'-
AGGGCTGTGGGATCCTCATTCAGGTCTATCACCTC-3' (10pm/l)], .09 l of Taq polymerase and 15.4l
of DNase- and RNase-free water, and 2.0l of DNA. The PCR was performed in a DNA Thermal Cycler
(Applied Biosystem 2720) programmed as follows: 5 min at 94°C (initial denaturation) followed by 35 cycles
of 30 sec at 94°C (denaturation), 30 sec at 56°C (annealing), 45 s at 72°C (extension), and 1 cycle of 10 min at
72°C (final extension). The final step was held at 10°C. The PCR products were stored at 4°C. PCR products
(1 μL) was loaded in the gel with loading dye and gel red along with 1kb DNA ladder (KAPA TM Universal
Ladder). The gel was run at 100 volts for 30 minutes and the amplified products were visualized in the UV
trans-illuminator and photographed in Gel Doc system (EnduroTM GDS Gel Documentation System). The PCR
samples were quantified using Fluorometer (Promega) and were sent to 1stBASE Laboratory at Malaysia for
PCR clean up and sequencing.
After sequencing, the sequences were viewed using 4peaks (nucleobytes.com) and was used for homology
analysis using blastn a default parameter for nucleotide sequence homology from Basic Local Alignment Search
Tool (BLAST). Phylogenetic analysis was performed using advance mode of Phylogeny fr. [8] using MUSCLE
for sequence multiple alignment, PhyML for the construction of phylogeny tree and TreeDyn for tree rendering.

Results:
Molecular identification of macrofungi:
There were seven wild "mushrooms" collected in the three areas. Two were collected at Dampulan (Lat N
15° 20' 28" Long N 120° 54' 14"), two were collected from Langla (Lat N 15° 19' 26" Long N 120° 55' 04") and
three from Putlod (Lat N 15° 25' 5" Long N 120° 53' 39") (Table 1).

Table 1: Code of the sample collected and the position of the collection using Global Positioning System (GPS).
Sample Code Collection area Latitude Longitude
1. SP1 Dampulan N 15 20' 28'' N 120 54' 14"
2. SP2
3. SP3 Langla N 15 19' 26'' N 120 55' 04"
4. SP4
5. SP5 Putlod N 15 25' 05'' N 120 53' 39"
6. SP6
7. SP7

The different samples were collected from varying habitat and environment as presented in Table 2. The
fruiting body of SP1 was found in weedy backyard on a compost area, the SP2 and SP3 were found in weedy
backyard on a decayed branch of tree, the SP4 was found in highland and composted area. The SP5 were found
in small groove decaying wood. The SP6 were collected in wide level highland. The partial nucleotide sequence
of the 7 samples were obtained and analyzed for Basic Local Alignment Search Tool (BLAST) search program
for homology analysis and sample identity query. All of the samples were identified up to species level. The
sample code SP1, SP4 and SP5 were all identified as Lentinus swartzii with 99%, 100% and 99% sequence
similarity and with a GenBank Accession number GU207276.1, respectively. The SP2 was identified as S.
commune with 100% identity (KR706163). The SP3 was identified as Coprinus aureogranulatus with a
sequence similarity of 100% (GQ249274.1). Lastly, the SP6 was identified as Lentinus squarrosulus with 99%
identity (KR183767.1). Lastly, SP7 was identified as Panaeolina foenisecii with 100% identity (KR867660.1)
(Table3).

Table 2: Identities of the specimen after BLAST analysis with the maximum identity, GenBank accession number and the taxon.
Sample Code Species identification Maximum GenBank Taxon
identity Accession
Number
1. SP1 Lentinus swartzii 99% GU207276.1 Fungi, Basidiomycota,
Agaricomycetes, Polypolares,
Polyporacea, Lentinus,

L. swartzii

2. SP2 Schizophyllum commune 100% KR706163.1 Fungi, Basidiomycota,
Agaricomycetes, Agaricales,
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Schizophyllaceae,
Schizophyllum, S. Commune
3. SP3 Coprinellus aureogranulatus 100% GQ249274.1 Fungi, Basidiomycota,
Agaricomycetes, Agaricales,
Psathyrellaceae, Coprinellus, C.
granulates

4. SP4 Lentinus swartzii 100% GU207276.1 Fungi, Basidiomycota,
Agaricomycetes, Polypolares,
Polyporacea, Lentinus,

L. swartzii

5. SP5 Lentinus swartzii 99% GU207276.1 Fungi, Basidiomycota,
Agaricomycetes, Polypolares,
Polyporacea, Lentinus,

L. swartzii

6. SP6 Lentinus squarrosulus 99% KR183767.1 Fungi, Basidiomycota,
Agaricomycetes, Polypolares,
Polyporacea, Lentinus,

L. squarrosulus
7. SP7 Panaeolus foenisecii 100% KR867660.1 Fungi, Basidiomycota,
Agaricomycetes, Bolbitiaceae,
Polyporacea, Panaeolous,
P. foenisecii

Phylogenetic analysis:
The phylogenetic tree was derived from 23 nucleotide sequences closely related from the samples used in
this study using ITS partial sequences (Figure 1). The phylogenetic tree was composed of four main branches
that roughly correspond to four groups or clades. The first group consisted of SP2 [Schizophyllum commune
(KR706163.1)] together with Auricularia polytrichia (FJ617293.1) with a percentage bootstrap support value of
100%. These species belongs to division Basidiomycota and both were commonly found at fallen branch or
logs. Both species are sessile type of mushroom. The second group consists of the SP7 [Panaeolus foenisecii
(KR867660.1)], together with other closely related species belong to Panaeolus species, the P. fimicola
(JF908514.1), P. cyanescens (EU834287.1) and P. retirugis (FJ478119.1) with a bootstrap support value of
99%. This group of species usually has a smooth solid cup and black gills underneath. The third group formed
the cluster of species belong to Coprinellus species (100% bootstrap support value) where in the SP3
[Corinellus aureogranulatus (GQ249274.1)] belong. The other Coprinellus species in this group are; C.
micaceus (JX160060.1), C. radians (JN943117.1), and C. xanthrothrix (JN159578.1). Most of the Coprinellus
species were commonly known as sink cup mushrooms because they dissolve into black ink in maturity. The
SP1, SP4, SP5 [L. swartzii (GU207276.1)] and SP6 [L. squarrosulus (KR183767.1) belong to the fourth group
composed of Lentinus species with percentage support value of 99%. Lentinus species have white color with
brownish shade and become yellowish when mature and most of them had funnel shaped cup and produced
white spore print.

Discussion:
The Schizophyllum commune Fr. like the L. squarrosulus and L. swartzii are edible mushrooms and also
known to have medicinal properties. The molecular data for Schizophyllum commune was still limited and was
just reported recently affecting otitis externa in human [9] other than its benefits, S. commune was a known
pathogen. The molecular data of Panaeolus foenisecii (Pers.) J. Schroet was first reported in Sri Lanka,
collected from elephant dung in dry zone forest reserves [10]. Panaeolus species was known as umbrella
mushrooms because of their umbrella cup like structures. Most of them have grayish black gills and the cups
quickly crumble when handled.
The differences between them can be distinguished by their specific details in their structures and
appearance. The C. aureogranulatus was first reported as Coprinus aureogranulatus [11] and the taxon was
further characterized and verified using molecular approach [12]. This species was found at Papua New Guinea
in 1996 [13]. C. aureogranulatus is a species of mushroom in the Psathyrellaceae family and was later
transferred to Coprinellus in 2001 [12]. The Lentinus Fr. is belongs to genus of decaying-wood Agaricomycetes,
the species found to have tough basidiocarps, with hyaline spores and decurrent lamellae. Previous studies on
the molecular taxonomy of this species still limited and still the application of the generic name Lentinus has
been controversial [14][15][16][17]. Due to different classification scheme, its was the molecular data that have
been strongly proposed and with the support of phylogenetic analysis in order to settle nomenclature issues
surrounding its taxonomic classification. The species of Lentinus species play an important role in the
environment and locally found in diverse ecosystem, from boreal to tropical regions [18][15]. The identification
of mushroom in the Philippines using molecular data using ITS region of Shizophyllum commune, Coprinus
aureogranulatus, Panaeolus foenisecii, Lentinus swartzii and Lentinus squarrosulus are important information
for its taxonomy, distribution and phylogeny studies.
157 Ageo Boy V. lopez et al, 2016
Advances in Environmental Biology, 10(12) December 2016, Pages: 153-158

100% I

99%

II
81%

100%
III
100%

99%

IV

Fig. 1: Constructed phylogeny tree based on PhyML method using the partial rDNA-ITS sequence of the
species and other closely related species available from NCBI.

Conclusion:
The study collected and characterized 7 samples from three areas in Jaen, Nueva Ecija, Philippines. The
molecular approach using the rDNA-ITS region firmly verify the different macrofungi in the selected areas. The
samples were separated into four distinct clades with other closely related species in the Basidiomycota. The
complex diversity of macrofungi in nature possesses difficulty in terms of its taxonomic identity using its
morphology alone. With the advent of advance molecular tools and technique, specimens can now be verify and
identify properly and may overpass the limitation of using classical taxonomic classification. Thus, the
molecular identification are very important to elucidate the proper identity of the species exist in our
environment and therefore must fully utilized to identify other species of macrofungi in the different areas in the
Philippines.

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