Termitomyces srilankensis sp. nov. (Lyophyllaceae, Agaricales), a new species from Sri Lanka

Abstract

New species of Termitomyces from wet-zone of Sri Lanka

Full text

MycolObs - Mycological Observations
Vol. 6: 47-53 2023-08-03
Article received 8 February 2023 (pre-reviewed), accepted 27 February 2023
Ediriweera AN, Voto P, Karunarathna SC, Dilshan BC (2023). Termitomyces srilankensis sp. nov. (Lyophyllaceae,
Agaricales), a new species from Sri Lanka. MycolObs 6:47−53
Termitomyces srilankensis sp. nov. (Lyophyllaceae, Agaricales), a new species from Sri Lanka
Aseni Navoda Ediriweera
1,2,3,a
, Pietro Voto
4,b*
, Samantha Chandranath Karunarathna
5,c
, Bulathsinhalage Chamath
Dilshan
6,d
1
Centre for Mountain Futures, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
2
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
CIFOR-ICRAF China Program, World Agroforestry Centre, 132 Lanhei Road, Kunming 650201, China
4
Via Garibaldi 173, I-45010 Villadose (RO), Italy
5
Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Re-source and Food
Engineering, Qujing Normal University, Qujing 655011, China;
6
Department of Biosystems Technology, Faculty of Technology, University of Ruhuna, Sri Lanka
a
aseniediriweera@gmail.com; https://orcid.org/0009-0004-5876-7615
b
pietrovoto@libero.it; https://orcid.org/0000-0003-1922-1324
c
samanthakarunarathna@gmail.com
d
99chamathdilshan@gmail.com
*Corresponding author: pietrovoto@libero.it
Abstract: Based on morphological and phylogenetic support, the new agaric
species Termitomyces srilankensis is described from samples collected from
the wet zone in Sri Lanka. The species is described with pictures of
basidiomes and main micro-characters while molecular identification was
used to verify the new species in comparison with the closest taxa. The new
species is characterised by broad, convex to applanate pileus with tiny pointed
perforatorium, crowded lamellulae of 3 tiers, and ellipsoid basidiospores. A
molecular phylogenetic analysis based on ITS sequencing data verifies and
separates T. srilankensis from T. fuliginosus, T. globulus and T. heimii.
INTRODUCTION
The genus Termitomyces R. Heim, type species Termitomyces striatus (Beeli) R. Heim, belongs to the family
Lyophyllaceae Jülich which comprises 19 genera (Wijayawardene et al. 2022) with Lyophyllum P. Karst as the
type. Taxa of this family are commonly characterized by siderophilous granule-filled basidia except the genus
Ossicaulis Readhead & Ginns and most species of the genus Clitocybe (Fr.) Staude (Hofstetter et al. 2002; Singer
1986; Clémençon 1974, 1978, 1984; Kuhner 1938). Its member species have been recorded as saprobes or
plant parasites and are mostly distributed in north-temperate and arctic areas (Bellanger 2015; Vesterholt &
Ludwig 2012; Singer 1986). Some species of Termitomyces are well known for their edibility.
Species of Termitomyces form an obligate symbiotic or mutualistic association with the fungus-feeding
termites (Aanen et al. 2002; Aanen & Eggleton 2005). The fruiting bodies of Termitomyces are the main source
of food for fungus-growing termites of family Macrotermitinae which are exclusively found in Africa and
Southeast Asia (Aanen et al. 2002; Aanen 2006). Kirk et al. (2008) reported approximately 30 taxa of
Termitomyces and 102 taxa are listed in the Index Fungorum (2023).
Apart from being popular as a seasonal culinary delicacy, species of Termitomyces are known to contain
medicinal properties such as antioxidants, immunomodulators, antitumorals and antimicrobials that are used
to treat neurodegenerative disorders (Teke et al. 2018; Hsieh & Ju 2018).
MATERIALS AND METHODS
Sampling Site
The specimen was collected from Kegalle, Sabaragamuwa province located in the wet zone of Sri Lanka
which receives a mean annual rainfall of over 2,500 mm, with a strong contribution from the south-western
Key words:
Agaricales
Basidiomycetes
Lyophyllaceae
Molecular
phylogeny
saprotrophic
taxonomy
tropical

Ediriweera, Voto, Karunarathna, Dilshan
48
monsoons. The mean annual temperature is in the range of 28–33°C while the relative humidity is in the range
of 85–91%. The specimens were collected mostly from home gardens and bear lands.
Sample Collection
Basidiomata were collected in 2020 and 2021 and photographed with a XD5 digital camera while they were
in the natural habitat and after being separated from the habitat, using a Canon XD5 digital camera. Collected
basidiomata were cleaned as much as possible to remove soil or attached debris. Specimens were wrapped
separately in aluminum foil to prevent spore contamination and damage. The specimens were taken to the
laboratory for further studies.
Morphological Studies
Macro-morphological characteristics such as size, shape, and structure of the pileus and stipe were
recorded. The colour terminology used for macro-morphological identification followed Kornerup & Wanscher
(1978). The specimens were dried with a portable dryer at 40°C for 24–48 hours and sealed in zip-lock plastic
bags containing silica gel as a desiccant to control humidity. All the herbarium specimens were deposited in the
Fungarium of the University of Ruhuna (FUOR), Sri Lanka and duplicated in the Herbarium of Kunming Institute
of Botany, Chinese Academy of Sciences, Kunming, China (HKAS). Micro-morphological observations of dry
specimens were carried out with free hand sections. Slides were prepared with distilled water. In addition,
3‒5% KOH, Congo red, and 3‒5% NH4(aq) were used to investigate some morphological characters where
necessary. Morphological characters were observed and photographed using a compound light microscope
(Nikon Model Eclipse Ci–s) attached to a Canon 550D digital camera. The measurements were taken with the
Tarosoft Image Frame Work program, while images used for figures were processed with Adobe Photoshop CS3
(Version 15.0.0, Adobe®, San Jose, CA, USA) extended version 10.0 (Adobe Systems, San Jose, CA, USA).
DNA Extraction, PCR Amplification, and Sequencing
The genomic DNA of dried specimen was extracted from dried samples using a Biospin Fungus Genomic
DNA Extraction Kit (Bioer Technology Co., Ltd., Hangzhou, P.R. China). The nuclear ribosomal internal
transcribed spacer (nrITS) and large subunit (nrLSU) regions were amplified using the primer pair ITS5/ITS4 and
LROR/LR5 (Vilgalys 1990; White 1990). The amplification process was carried out for total volume of 25 µL
comprising 1.0 µL of template DNA, 9.5 µL of double-distilled water, 1.0 µL of each primer, and 12.5 µL of 2×
Power Taq PCR Master Mix. The latter consisted of a premixed, ready to use solution that included 0.1 Units/
μLTaq DNA polymerase, 500 µm of dNTP mixture each (dATP, dCTP, dGTP, and dTTP), 20 mM of Tris–HCl pH
8.3, 100 mMKCl, 3 mM of MgCl2, stabilizer, and enhancer. During the polymerase chain reaction (PCR), each
sample underwent 35 cycles according to the following settings: denaturation (95°C, 30 s), annealing (52°C, 30
s), extension (72°C, 1 min), and final extension (72°C, 10 min). Amplified products were confirmed on a 1%
agarose gel electrophoresis stained with ethidium bromide. The amplified PCR fragments were sent to a
commercial sequencing provider (Beijing Bai Mai Hui Kang Biological Engineering Technology Co., P.R. China).
The nucleotide sequence data were deposited in GenBank.
Sequence Alignment and Phylogenetic Analyses
The obtained sequences were checked and assembled using BioEdit 7.0.9.0 (Hall 1999) and compared with
those available in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) on the BLAST algorithm.
Taxon information applied in the molecular work is listed in Table 1. The ITS dataset comprises 35 sequences,
including 33 Termitomyces sequences from GenBank which include the type species of the genus, T. striatus.
Lyophyllum connatum and Lyophyllum infumatum were chosen as the outgroup taxa for ITS phylogenetic trees.
The ITS sequence data were analyzed using maximum likelihood (ML), and Bayesian analyses. The
reconstruction of ML analysis was performed using raxmlGUI v.0.9b2 with the model GTRGAMMA. A Bayesian
analysis was conducted with MrBayes v. 3.1.2 (GTR+I+G model) to valuate posterior probabilities (PP) by
Markov chain Monte Carlo sampling (BMCMC). Sequences for each strain were aligned using Clustal X
(Thompson et al. 1997). Ambiguously aligned regions were excluded from all analyses. Trees were inferred
using the heuristic search option with TBR branch swapping and 1000 random sequence additions. The final
tree (Fig. 2) was displayed in FigTree v1.4.0 (Rambaut 2012) and then copied to Microsoft PowerPoint 2013 and
converted to jpeg files using Adobe Photoshop CS3 Extended 10.0 (Adobe Systems, San Jose, CA, USA).

Termitomyces srilankensis
sp.
Fig. 1 (previous page).
Morpholog
Mature basidiome; (d-
h) Basidios
bars: (a
-
c) = 3 cm, (d, h) = 3
µm, (i
TAXONOMY
Termitomyces srilankensis
A.N. Edir
Typus: Sri Lanka, Sabaragamuwa Provinc
a
high moisture level, abundant with t
Ediriweera, Herb. FUOR0016AGS, GenBa
Isotype: HKAS123147.
Etymology: The specific epithet
derives f
Diagnosis
: This taxon is characterized
yellowish and grey, a stipe that is ta
lacrymoid spores of (10.41) 10.75–
11.44
utriform cheilocystidia of (21.12) 22.
pleurocystidia of (36. 89) 38.56 –
39.94 (
sp. nov. (
Lyophyllaceae, Agaricales
), a new species
49
ology of
Termitomyces srilankae
(HKAS123147/FU
idiospores; (i
-l) Basidia; (m-o) Cheilocystidia; (p-r
) P
m, (i
-
r
) = 5
µm. All images by
A.N. Ediriweera
.
Ediriweera, P. Voto, S.C. Karunarathna & H. Kular
ovince, Kegalle District, ectomycorrhizal on humu
ith termites and termite nests, 2
nd
August 202
enBank accession number ON685313, Index Fung
ives from “Sri Lanka”, where the species was first
ized by a 9
–
13 cm broad, weakly radially fibri
is tapering downwards to an abrupt rooting b
1.44 (11.67) × (4.12) 4.18
–
5.12 (5.45) µm, clava
22.18
–23.48 (23.98) × (6.08) 6.21 -
6.92
.94 (40.04) × (26.89) 27.17
– 28.19 (28.87) µm.
ecies from Sri Lanka
7/FUOR0016AGS): (a
-c)
) Pleurocystidia.
Scale
Kularathne, sp. nov. (Fig. 1)
umus soil mixed with clay with
2021, H. Kularathne and A.N.
Fungorum number: IF 553494.
first detected.
fibrillose pileus with tones of
ing base,
subamygdaliform to
clavate to ellipsoid or ellipsoid
-
92 (7.04) µm, and pyriform

Ediriweera, Voto, Karunarathna, Dilshan
50
Description
Basidiomata large.
Pileus 9 – 13 cm across, when young convex and cuspidate, at maturity broadly convex to applanate with a tiny
pointed perforatorium, margin at maturity straight to only slightly incurved, moderately indented and radially
split, surface yellowish grey (4B2) to sand colour (4B3) with a light yellow (4A4) center, almost smooth with
radial white fibrils.
Lamellae free, 2 – 4 mm wide, white (1A1) when young and pale yellow (1A3) at maturity, very crowded with
intermingled lamellulae of 3 tiers of 1.2, 3.2 and 4 cm, edge smooth, equal.
Stipe 9.0 – 11.0 × 1.0 – 1.2 cm, central, cylindrical but in the lower portion tapering down to a thin abruptly
rooting base 2.5 – 3.5 cm long, white (1A1) to pale yellow (1A3), apex fibrillose and squamulose, elsewhere
smooth and glabrous, longitudinally striate, stuffed.
Context fleshy, moderately thick, 1.5 – 2.5 mm.
Basidiospores (n=40) (10.41) 10.75 – 11.44 (11.67) × (4.12) 4.18 – 5.12 (5.45) µm, on average 11.08 × 4.63 µm,
Q = 1.6 – 1.9, in front view elliptic, in side view subamygdaliform to lacrymoid, thin-walled, smooth, hyaline
with a yellowish to golden colour content.
Basidia (19.73) 21.33 – 22.02 – (23.8) × (6.1) 6.14 – 7.38 (7.78) µm, on average 23.11 × 6.77 µm, 4-spored,
sterigmata 2 – 3.5 µm long, clavate to sub cylindraceous, thin walled, smooth, hyaline to sub hyaline.
Cheilocystidia (21.12) 22.18 – 23.48 (23.98) × (6.08) 6.21 – 6.92 (7.04) µm, on average 22.94 × 6.38 µm, clavate
to ellipsoid or ellipsoid-utriform, thin-walled, hyaline to sub-hyaline.
Pleurocystidia (36. 89) 38.56 – 39.94 (40.04) × (26.89) 27.17 – 28.19 (28.87) µm, on average 39.12 × 27.77 µm,
pyriform, thin-walled, hyaline.
Ecology, Habit and Habitat: Saprotrophic, solitary, in humus soil mixed with clay with high moisture level,
abundant with termites and termite nests or mounds above and below the surface soil layer.
NOTES
Termitomyces species grow on clayey soils associated with termites. Our new taxon also was collected from
clay-mixed humus soil where huge networks of clefts formed by termites were present under the uppermost
layer of soil during heavy rains. A differentiated shape both of the spore side view and of cheilocystidia
represents some of the most peculiar features in the descriptive dataset of our new taxon which otherwise
shares several morphological characters with other congeneric species. It is closely related to some unspecified
Termitomyces species on a molecular basis with 99.55 % to 99.86 % identity in the BLAST search. Besides, our
collection formed a distinct clade with the three following taxa of Termitomyces, originally described from
tropical Africa, in the phylogenetic analysis with high bootstrap support (100 % MLBS) (Figure 4.17, Clade A): T.
fuliginosus R. Heim (MRNo215), T. globulus R. Heim & Gooss. (BUMR03) and T. eurrhizus (Berk.) R. Heim. (WHX-
2015). These three vouchers are phylogenetically closely related to our new strain (FUOR0016AGS). In the base
pair (bp) comparison of ITS sequences data revealed that there are1.68%, 5.35% and 8.71% bp differences
between our strain and the strains of T. fuliginosus, T. globulus and T. eurrhizus respectively.
Termitomyces fuliginosus, differs from T. srilankensis by having a cap-shaped or bell-shaped pileus at both
young and mature stages with an incurved margin at maturity, and smaller and ellipsoid basidiospores 7.3 × 4.5
µm on average. The voucher MRNo215, identified as T. fuliginosus and reported from Thailand, which appears
in our phylogram with a sufficiently high identity percentage (98.32%), is unpublished and therefore not
comparable.
Termitomyces globulus differs from our new species by having a larger pileus of 8 – 21 cm with a reddish-
brown central area and a finely striate margin, smaller and ellipsoid basidiospores of 6 – 9 × 3.5 – 6 µm, larger
cheilocystidia of 20 – 60 × 16 – 25 µm, and pleurocystidia of 35 – 71 × 17 – 28 µm (Heim 1951; Pegler &
Vanhaecke
1994).
In addition, Termitomyces eurrhizus differs by having a pileus with a brown to reddish-brown surface with a
straight margin, smaller basidiospores of 6 – 9 × 4 – 6 µm, larger cheilocystidia of 13 – 55 × 8 – 33 µm and
pleurocystidia of 18 – 69 × 10 – 35 µm (Wei et al. 2009; Heim 1942; Pegler & Vanhaecke
1994).

Termitomyces srilankensis sp. nov. (Lyophyllaceae, Agaricales), a new species from Sri Lanka
51
Table 1
. Fungal taxa, Voucher numbers and GenBank accession
numbers of the sequences
used in the phylogenetic analyses of ITS
Termitomyces heimii Natarajan, represented in the sister clade to Clade A in our phylogram, was described
from the adjacent South India, it differs from T. srilankensis by a smaller pileus up to 10 cm broad even at
maturity, convex to plano-convex and with an incurved margin, a thick annulus, much larger basidiospores
(19.5 – 21 × 5.5 – 7 µm,), 2- or 4-spored basidia, rare pleurocystidia and absence of cheilocystidia (Natarajan
1979).
Pegler & Vanhaecke
(1994) reports two species from Sri Lanka: T. eurhizus and T. microcarpus (Berk. &
Broome) R. Heim.
Combined morphological characters and phylogenetic analyses support the description of our collection as
a new species, T. srilankensis, in Termitomyces.
Taxa names Collection ID ITS
Lyophyllum connatum (Schumach.) Singer SR-32 HE819396
L. infumatum (Bres.) Kühner MCVE 10152 JF908334
Termitomyces acriumbonatus Usman & Khalid LAH36363_MU106 MT179688
T. clypeatus R. Heim MU19-50 FJ147329
T. clypeatus MU25-49 HQ702547
T. cylindricus S.C. He MRNo170 LC068786
T. cylindricus INDO18 MH651799
T. entolomoides R. Heim BUMR06 MK743955
T. eurrhizus (Berk.) R. Heim WHX-2015 KU179194
T. fragilis L. Ye, Karun, J.C. Xu, K.D. Hyde &
Mortimer HKAS:88906 paratype KY214477
T. fragilis HKAS:88909 paratype KY214476
T. fragilis HKAS:88912 type KY214475
T. fuliginosus R. Heim MRNo215 LC068788
T. globulus R. Heim &Gooss.-Font. BUMR03 MK743956
T. heimii Natarajan TERM055 MN160309
T. heimii UOC MAT MT01 KP943503
T. intermediusHar. Takah. &Taneyama GDGM46325 MF488973
T. intermedius GDGM46311 MF488972
T. medius R. Heim & Grassé BUMR07 MK743976
T. Medius CUH:AM080 KJ768983
T. microcarpus (Berk. & Broome) R. Heim UOC KAUNP MK04 KP780436
T. microcarpus MU195-46 HM230661
T. radicatus Natarajan MRNo173 LC068787
T. sheikhupurensis Izhar, Khalid & H. Bashir LAH36413 paratype MT192218
T. sheikhupurensis LAH 35710 type NR_172179
T. sp. HKAS 117638 MZ869839
T. sp. HKAS 117639 MZ869840
T. sp. HKAS 117640 MZ869843
T. sp. HKAS 117641 MZ869844
T. sp. OS2 AF321375
T. srilankensis sp.nov. FUOR0016AGS ON685313
T. striatus (Beeli) R. Heim TERM048 MN160302
T. striatus TERM049 MN160303
T. striatus TERM051 MN160305
T. umkowaan (Cooke &Massee) D.A. Reid HUH-SH5 KJ703245

Ediriweera, Voto, Karunarathna, Dilshan
52
Fig. 2
(below)
.
Phylogram generated from maximum likelihood analysis based on RAxML analysis based on
the ITS sequence data including 35 strains. Bootstarp support values for maximum likelihood (ML, left),
higher than 70%, and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are provided. The
tree is rooted with
Lyophyllum connatum
and
L
yophyllum
infumatum
. The new record is
printed
in blue
.

Termitomyces srilankensis sp. nov. (Lyophyllaceae, Agaricales), a new species from Sri Lanka
53
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