Journal of
Threatened Taxa | www.threatenedtaxa.org | 26 September 2018 | 10(10):
12413–12417
The
gilled mushroom Amanita spissacea (Amanitaceae):
a new report for India
Hmar Lalrinawmi 1,
John Zothanzama 2, Benjamin W. Held 3,
Josiah M.C. Vabeikhokhei 4, Zohmangaiha 5 & Robert A. Blanchette 6
1,2,4,5 Department of
Environmental Sciences, Mizoram University, Tanhril,
Mizoram 796004, India
3,6 University of Minnesota, 495 Borlaug Hall, 1991 Upper
Buford Circle, St. Paul, MN 55108, United States
1 hmarlalrinawmi@gmail.com, 2
john_zza@yahoo.co.in (corresponding author), 3
bheld@umn.edu, 4 mcjosiahmathipy@gmail.com,
5 xohmaa1990@gmail.com, 6 robertb@umn.edu
doi: https://doi.org/10.11609/jott.3471.10.10.12413-12417
Editor: R.K. Verma, Tropical Forest
Research Institute, Jabalpur, India. Date
of publication: 26 September 2018 (online & print)
Manuscript details: Ms # 3471 |
Received 28 April 2017 | Final received 02 August 2018 | Finally accepted 23
August 2018
Citation: Lalrinawmi, H., John Zothanzama,
Benjamin Held, Josiah M.C. Vabeikhokhei, Zohmangaiha & Robert A. Blanchette
(2018). The gilled mushroom Amanita spissacea
(Amanitaceae): a new report for India. Journal of Threatened
Taxa 10(10): 12413–12417; https://doi.org/10.11609/jott.3471.10.10.12413-12417
Copyright: © Lalrinawmi et al. 2018. Creative Commons Attribution 4.0
International License. JoTT allows
unrestricted use of this article in any medium, reproduction and distribution
by providing adequate credit to the authors and the source of publication.
Funding: UGC-MZU (Non-NET) Fellowship.
Competing interests: The authors declare no competing interests.
Acknowledgements:
The authors thank Dr. Karlyn Eckman and collaborators involved in the joint cooperatve research projects between Mizoram University,
India and the University of Minnesota, USA. The authors also thank Dr. Surajit De Mandal, Research
Associate, Department of Biotechnology, Mizoram
University.
Abstract: Mizoram is
regarded as one of the biodiversity hotspots of the World owing to the diverse
group of flora and fauna documented here.
Information regarding the macrofungi, however,
is very limited. For this reason, a
systematic study of mushrooms from Mizoram was undertaken and during the field
survey, Amanita spissacea was collected and
identified. This is the first report of
this mushroom from India. This species
was identified on the basis of its morphological and microscopic characteristics
as well as molecular characterization of the ITS region of rDNA. Phylogenetic analysis also confirmed that A.
spissacea was a distinct species from A. fritillaria, A. sepiacea, A. citrina and other closely related species Amanita section
Valideae.
Keywords: Macrofungi, Mizoram,
phylogeny, taxonomy.
Mizoram
lies in northeastern India sharing its borders with Assam, Manipur and Tripura
and has international borders with Bangladesh and Myanmar. It covers a geographical area of 21,081km2
and lies between 21.966–24.5830N and 91.250–92.4830E. The Tropic of Cancer passes through the state
at 23.5000N (Mizoram Remote Sensing Application Centre 2009).
Amanita Pers., is a well known mushroom genus with global distribution
comprising both edible and poisonous species which are usually mycorrhizal symbionts with
plants. The genus Amanita Pers., contains about 500 species worldwide (Kirk
et al. 2008), and for some time, only 66 species were reported from India
(Bhatt et al. 2003; Semwal et al. 2005, 2007; Vrinda et al. 2005).
Recently, a number of reports have been added to the list from several
researchers (Singh & Kaur 2016; Bhatt et al.
2017) with the latest report of 80 species of Amanitaceae
being listed including 73 species of Amanita reported from different
parts of India (Verma & Pandro
2018).
During
the course of macro-fungal foray to different parts of Mizoram, Amanita spissacea S. Imai was collected and identified. This species is described and illustrated for
the first time from India.
Materials and Methods
Study Area
Collections
of mushrooms growing on soil was done at Mizoram University Campus which is
located in the Western side at a distance of about 15km away from the state
capital, Aizawl, just below Tanhril
Village. The Mizoram University Campus
is about 980 acres in area and lies between 23.756–23.7260N &
92.644–92.6730E. The
elevation ranges from 330–880 m.
Morphological study
Macro-morphological
descriptions were based on field notes and color photographs of the macrofungi.
Micro-morphological data was obtained from the dried specimens with the
aid of a light microscope after sectioning and staining with cotton-blue. Spore prints were taken by placing the fresh
specimen on a microslide. Descriptions of spore shapes are based on the
study reported by Bas (1969).
Phylogenic study
DNA
isolation, amplification and sequencing: Molecular methods were
performed following Zothanzama et al. (2016), where
DNA was extracted using a CTAB method, followed by amplification of the
internal transcribed spacer region (ITS) of the rDNA
and sequenced with both primers (ITS1F and ITS4B).
PCR
amplification: PCR reactions were setup in 0.2ml centrifuge
tubes that contained 12.5µl GoTaq Green Mastermix (Promega, Madison, WI),
9.5µl nuclease free water, 0.5µl bovine serum albumin (BSA), 1µl forward primer
(5µM), 1µl reverse primer (5µM) and 1µl of fungal DNA template for a total
reaction volume of 25.5µl. PCR was
performed using primers ITS1-F (5’-CTT GGT CAT TTA GAG GAA GTA A-3’) ITS4-B
(5’-CAG GAG ACT TGT ACA CGG TCC AG-3’) (White et al. 1990) with the
following parameters; 94C for 5 minutes, followed by 35 cycles of 94C for 1
minute, 52C for 1 minute and 72C for 1 minute with a final extension step of
72C. PCR amplicons
were verified by electrophoresis on a 1% agarose gel
with SYBR green and visualized on a Gel Documentation System. Sequencing was
performed using both primers by using Sanger sequencing using a ABI 3730xl DNA sequencer. Consensus sequences for contigs were trimmed and aligned using Bioedit
sequence alignment editor. Sequences
were then compared to those in GenBank database using
the BLASTn (Altschul et al.
1990) search tool for similarities and submitted to Genbank.
Phylogenetic
analysis: The ITS dataset was aligned with the MAFFT v7.222 (Katoh et al. 2002) and jModelTest
2.1.10 (Darriba et al. 2012) was used to determine
the appropriate model for Bayesian analysis (HKY85). Phylogenetic analysis inferred from ITS
sequences was performed using MrBayes 3.2.6 (Huelsenbeck & Ronquist 2001).
1.1 x 106 MCMC generations were used with a sampling frequency every
200 generations and the first 10% of sampled trees were discarded as burn-in.
Results
Amanita spissacea S. Imai
(Fig. 1 & Image 1)
Specimens
examined: EVS/SF/0012, 27.v.2014, India, Mizoram, Aizawl,
Mizoram University Campus and EVS/SF/0165, 01.vi.2016 (Image 2).
Basidiomata: Small
to medium. 4–9 cm in diam., convex to plano-convex, grayish-brown in color, volva remnants on pileus as scattered felted to crust like patches, margin
non-striated, non-appendeculate, incurved. Lamellae-sometimes forked, lamellulae- of several length. Stipe: 8–14 cm long, 0.5–1
cm wide, tapering toward apex, stuffed, white to grayish-brown with brown
scales. Annulus
membranous, grayish brown, apical. Bulbous base upto 1.5cm long, 1–3 cm thick, glabrous with dark brown
spots. The upper part of the
bulbous base of the stipe is covered with dark grey volval
remnants in 2–5 dotted rings. Context - white and thin. Sporeprint: White. Spores: 7-9.8 x 6.8 - 8.5μm[Q=1.02,1.15]and are globose to subglobose, sometimes rarely broadly ellipsoid, amyloid, colourless, hyaline, thin walled and smooth. Basidia: Clavate, 35–45 × 8–11 μm, four spored, sterigmata 3.2–4.6 x
0.8–1.8μm. Clamp connection absent. Lamellae edge cell: clavate,
35–45 × 7.5–9 μm.
Habitat:
Solitary
to scattered or gregarious on ground in a broad-leaved sub-tropical forest.
Molecular Phylogenetic analysis
The
molecular phylogenetic analysis shown in figure (Fig. 2) involved 17 nucleotide
sequences. The tree with the highest log
likelihood (-2938.15) is shown. In the
phylogenic analysis, the specimen of Amanita spissacea
from Mizoram (MZ10-KY940266, MZJZR1-MG706138) is indicated in bold and
clustered with Amanita spissacea from Belgium
(KY747469), Republic of Korea (KM052550, KM052546) and Japan (AB015683).
Discussion
In this
study, we identified this species based on morphological, microscopic and
molecular characteristics. This is the first report of Amanita spissacea from India.
Results from sectioning of the fruiting body and observations of basidiospores indicated this Amanita species was
most closely related to A. spissacea. Species identification based on morphological
characteristics is difficult to differentiate from other closely related
species such as A. fritillaria, A. sepiacea, A. citrina and
others.
The
present species has been reported and described for the first time by Imai
(1933) and Gilbert (1940) as Amplariella spissacea. The
macro and microscopic features of the present species well matched with the
description given by Imai (1933) who described Amanita spissacea
as pileus with 6–10 cm, convex, then extended,
dark chestnut, warted, white flesh, stalk 10–15 cm
long, firm, bulbous base, covered with dark margin, scaly, membranous ring. Spores globose, 7–8 µm, hyaline, apiculate.
Amanita
spissacea is also closely related to Amanita
fritillaria (Yang et al. 2001) and Amanita sepiacea (Imai, 1933). The macro and microscopic
details are mostly identical but both Amanita fritillaria
and Amanita sepiacea have spores broadly
ellipsoid to ellipsoid, occasionally subglobose or
ellipsoid, rarely globose and the upper part of the
bulbous base of the stipe of Amanita fritillaria
covered with dark grey volval remnants is only 2–4
rings while the former is 2–5 dotted rings.
Moreover, the macroscopic feature of Amanita sepiacea
is bigger in size as compared to Amanita spissacea
with cap 6–15 cm diam., stipe 10–18 cm long, 1–2.5 cm thick and basal bulb
1.5–5.0 cm.
Sequencing
of the ITS region of rRNA and phylogenetic analysis
further showed that the Mizoram sample matched GenBank
accession Amanita spissacea from Belgium
(KY747469), Republic of Korea (KM052550, KM052546) and Japan (AB015683) in a
well-supported clade with A. fritillaria
forming a sister clade. These results
hence confirmed that the specimen of Amanita from Mizoram
(MZ10-KY940266, MZJZR1-MG706138) is Amanita spissacea,
a distinct species and separate from A. frittilaria,
A. sepiacea, A.citrina and
other previous reported Amanita species.
Mizoram
is one of the northeastern states of India which is
rich in mushroom flora. Like many other Amanita
species, A. spissacea has been reported to be
poisonous in China (Zhishu et al. 1993) and recent
mushroom poisonings in Mizoram State (Zothanzama
& Lalrinawmi 2015) are prompting efforts to
identify mushrooms in this region that are poisonous. This report identifies this poisonous
mushroom in India and confirms that it is a distinct species from other Amanita
species. Limited information is
available concerning the wild mushrooms found in Mizoram and further studies
are needed to assess and document the wide variety of wild mushrooms that can
be found in this region.
References
Altschul, S.F.,
W. Gish, W. Miller, E.W. Myers & D.J. Lipman
(1990). Basic local alignment search tool. Journal
of Molecular Biology 215(3): 403–410; https://doi.org/10.1016/S0022-2836(05)80360-2
Bas, C. (1969). Morphology
and subdivision of Amanita and a monograph of its section Lepidella. Persoonia 5(3): 285–579.
Bhatt, R.P., R.E. Tulloss, K.C. Semwal, V.K. Bhatt,
J.M. Moncalvo & S.L. Stephenson (2003). Amanitaceae reported from India. A
critically annotated checklist. Mycotaxon 88: 249–270.
Bhatt, R.P., T. Mehmood, P. Uniyal & U. Singh
(2017). Six new records of genus Amanita (Amanitaceae) from Uttarakhand,
India. Current Research in Environmental & Applied Mycology
7(3): 161–182
Darriba, D.,
G.L. Taboada, R. Doallo
& D. Posada (2012). jModelTest
2: more models, new heuristics and parallel computing. Nat Methods 9:
772–772; https://doi.org/10.1038/nmeth.2109
Gilbert, E.J. (1940). Iconographia mycologica, Amanitaceae. Iconographia Mycologica
27: 1–198.
Huelsenbeck
J.P. & F. Ronquist (2001).
MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:
754–755; https://doi.org/10.1093/bioinformatics/17.8.754
Imai,
S. (1933). Studies on the Agaricaceae of Japan I. Volvate
Agarics in Hokkaido. Botanical Magazine (Tokyo)
47: 423–432.
Katoh, K.,
K. Misawa, K. Kuma & T. Miyata (2002). MAFFT:
a novel method for rapid multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Research 30: 3059–3066; https://doi.org/10.1093/nar/gkf436
Kirk, P.M., P.F. Cannon, D.W.
Minter & J.A. Stalpers (2008). Ainsworth
Bisby’s Dictionary of Fungi. 10th
Edition. CABI, Wallingfod, UK.
Mizoram Remote Sensing
Application Centre (2009). Natural Resources Atlas
of Mizoram. MIRSAC Newsletter 2(1): 1–4 < http://mirsac.nic.in/images/MIRSAC_News_July2010.pdf>
Semwal, K.C.,
R.P. Bhatt & R.C. Upadhyay (2005). The genus Amanita from Garhwal
Himalayas region of India. Mushroom Research 14 (2): 50–55.
Semwal, K.C.,
R.E. Tulloss, R.P. Bhatt, S.L. Stephenson & R.C. Upadhyay (2007). New records of Amanita
from Garhwal Himalaya, India-Amanita section Amanita.
Mycotaxon 101: 331–348.
Singh,
Y. & M. Kaur (2016). Two species of genus Amanita from India. World Journal of Pharmacy and Pharmaceutical Sciences 5(5):
1054–1062.
Verma,
R.K. & V. Pandro (2018). Diversity
and distribution of amanitaceous mushrooms in India,
two new reports from Sal forest of central India. Indian Journal of Tropical Biodiversity 26(1): 42–54.
Vrinda, K.B.,
C.K. Pradeep & S.S. Kumar (2005).
Occurrence of a lesser known edible Amanita in
the Western Ghats of Kerala. Mushroom Research 14(1): 5–8.
White,
T.J., T. Bruns, S. Lee & J.W. Taylor (1990). Amplification
and direct sequencing of fungal ribosomal RNA genes for phylogenetics,
pp. 315–322. In: Innis, M.A., D.H. Gelfand,
H.H. Sninsky & T.J. White (eds.). PCR
Protocols: A Guide to Methods and Applications. New York Academic Press
Inc.
Yang,
Z.L., T.H. Li & X.L. Wu (2001). Revision of Amanita collections
made from Hainan, Southern China. Fungal Diversity 6:
149–165.
Zhishu, B.,
Z. Guoyang & L. Taihui
(1993). The Macrofungus Flora
of China’s Guangdong Province. The
Chinese University Press, Hong Kong.
Zothanzama,
J. & H. Lalrinawmi (2015). Wild
edible mushrooms of Mizoram: an overview of knowledge and potential, pp.
171–181. In: Eckman, K. & L. Ralte
(eds.). Integrated Land Use Management in the Eastern
Himalayas Vol - 1. Akansha Publishing House.
New Delhi.
Zothanzama, J.,
R.A. Blanchette, S. Redford, B. Held, Zohmangaiha & J.M.C. Vabeikhokhei
(2016). Using molecular characterization as a tool for
identification of fungi from Mizoram, India, pp. 165–177. In: Eckman, K. & L. Ralte (eds.).
Integrated Land Use Management in the Eastern Himalayas.
Vol - 2. Akansha Publishing House.
New Delhi.