10.6092/issn.2531-7342/18381
This study concerns a novel white–spored Agaricales collected from Darjeeling Hills (India) during the monsoon. The new species, Tricholomopsis darjeelingensis, is described based on its morphological and molecular data. It is characterized by its caespitose fruiting body, long and slender stipe, occasionally eccentric, pileus covered with dense violet fibrils, 2 – 4 spored basidia, thin–walled caespitose cheilocystidia and basidiospores globose to broadly ellipsoid, 2.9 – 4.72 – 5.9 × 2.9 – 3.93 – 4.4 μm.
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A new species of <em>Tricholomopsis</em> (Agaricales) from Darjeeling Hills, India
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Zhao RL, Li GJ, Sánchez–Ramírez S, Stata M, Yang ZL, Wu G, Dai YC, He SH, Cui BK, Zhou JL, Wu F, He MQ, Moncalvo JM, Hyde KD (2017) A six–gene phylogenetic overview of Basidiomycota and allied phyla with estimated divergence times of higher taxa and a phyloproteomics perspective. Fungal Diversity 84:43–74. https://doi.org/10.1007/s13225–017–0381–5
A new species of <em>Tricholomopsis</em> (Agaricales) from Darjeeling Hills, India
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 144-153
                Journal homepage: https://italianmycology.unibo.it/

Research article

                      A new species of Tricholomopsis (Agaricales)
                             from Darjeeling Hills, India

                        Alisha Thapa1, Juna Tamang1,2, Krishnendu Acharya1
1
  Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata
700 019, India
2
  Department of Botany, Bangabasi Evening College,19, Raj Kumar Chakraborty Ln, Baithakkhana, Kolkata, West Bengal
700009, India

Corresponding author e-mail: krish_paper@yahoo.com




Article info
Received 3/11/2023; accepted 24/11/2023
https://doi.org/10.6092/issn.2531-7342/18381

Abstract
This study concerns a novel white–spored Agaricales collected from Darjeeling Hills (India) during the monsoon.
The new species, Tricholomopsis darjeelingensis, is described based on its morphological and molecular data. It is
characterized by its caespitose fruiting body, long and slender stipe, occasionally eccentric, pileus covered with
dense violet fibrils, 2 – 4 spored basidia, thin–walled caespitose cheilocystidia and basidiospores globose to broadly
ellipsoid, 2.9 – 4.72 – 5.9 × 2.9 – 3.93 – 4.4 μm.

Keywords
ITS, LSU, novel species, phylogeny, taxonomy



Introduction
Tricholomopsis Singer (Basidiomycota, Agaricales) is a genus that can be recognized by its bright, mostly
yellow basidiomata, with adnate to sinuate yellow lamellae, a fibrillose to squamulose dry pileus with red
or yellow tones, white spore print, central stipe without a ring, smooth inamyloid spores, presence of large
cheilocystidia, a pileipellis as a trichoderm or cutis type, and the presence of clamp connections (Singer,
1986; Olariaga et al., 2015; Hosen et al., 2020). Most Tricholomopsis species are known to be living on
wood (Smith, 1960) generally growing primarily on conifer, rarely on hardwood, but also a handful of
species have been documented to be living on bamboo or on soil (Dennis, 1951; Hongo, 1960; Singer,
1986). The Index Fungorum as of the date (7/07/2023) shows records of 78 species of Tricholomopsis,
among them 49 taxa have been validated (www.indexfungorum.org). The taxonomy of this genus is still
to be resolved, and the distribution of almost all of the species is not fully known except for Tricholomopsis
rutilans (Schaeff. ex Fr.) Singer (Holec et al., 2019). But for T. rutilans the situation is trickier for its
species complex (Olariaga et al., 2015). So far, three species of Tricholomopsis have been recognized in
India, viz., T. rutilans from Darjeeling (Sarwal, 1984), T. crocobapha (Berk. & Br.) Pegler from Himachal
Pradesh (Rai et al., 2007) and T. sulphureoides (Peck) Singer from North-Western Himalaya (Lakhanpal,
2014).

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
Thapa et al.                                                       Italian Journal of Mycology 52 (2023): 144-153

https://doi.org/10.6092/issn.2531-7342/18381



   Here we provide complete information on a novel species of Tricholomopsis collected from the Darjeeling
District of West Bengal which has been done based on morphological features and phylogenetic analyses
based on nuclear ribosomal internal transcribed spacer (nrITS) and nuclear ribosomal large subunit
(nrLSU) sequences.

Materials and methods
Specimen collection
The study material was collected in Darjeeling Hills during the rainy season in the year 2022. Photographs,
chemical reactions, and other essential field characters were well documented on the spot. The specimen
was carefully dried and preserved referring to Pradhan et al. (2015) and was deposited to Calcutta
University Herbarium (holotype, CUH AM791; isotype, CUH AM872 and CUH AM911).

DNA extraction, PCR amplification, DNA sequencing
E.Z.N.A.® Fungal DNA Kit (Norcross, Georgia) was used for genomic DNA extraction from the dried
specimen. The protocol provided by the manufacturer of the fungal DNA kit was preferred. Two rDNA
regions viz., nrITS and nrLSU, were amplified with the primer pairs ITS1/ITS4 (White et al., 1990) and
LROR/LR3 (Vilgalys and Hester, 1990) respectively. PCR protocol followed was: 94ºC for 4min, followed
by 30 cycles of 94ºC for 30 s, 55ºC for 30 s, 72ºC for 1 min, and a final extension of 72ºC for 5 min.
PCR products were purified using the QIAquick® Gel Extraction Kit (QIAGEN, Germany). Sequencing
was performed on ABI3730xl DNA Analyzer (Applied Biosystems, USA). For manual editing of the
newly generated sequences of Tricholomopsis darjeelingensis, BioEdit v.7.0.5 (Hall 1999) software was used
and the sequences were subjected to BLAST search (https://blast.ncbi.nlm.nih.gov). Newly generated
sequences of T. darjeelingensis were deposited in GenBank (ITS, OQ913610; LSU, OQ861109).

Sequence alignment and phylogenetic analyses
The final dataset of sequences including T. darjeelingensis and those acquired from GenBank (Table 1)
were used for phylogenetic analyses. The sequences of Pleurocybella porrigens (Pers.) Singer, Phyllotopsis sp.
and Pluteus romelli (Britzelm.) Lapl. were designated as outgroup taxa referring to Hosen et al. (2020).
The datasets comprised 37 ITS and 29 LSU sequences of Tricholomopsis species. In the final dataset, ITS
and LSU sequences were aligned individually in Mega v.7.0 (Kumar et al., 2016) and then concatenated
for further analyses using MEGA X (Kumar et al., 2018). The best substitution model (GTR+I+G) was
determined using jModelTest2 on XSEDE using the CIPRES web portal (www.phylo.org). Maximum
likelihood (ML) and Bayesian Inference (BI) were executed using raxmlGUI 2.0 (Edler et al., 2020) and
MrBayes v.3.2.7 (Ronquist et al., 2012) respectively to analyze the combined (ITS+LSU) dataset. Vishal
et al. (2021) followed for Bayesian Metropolis–Coupled Markov chain Monte Carlo (MCMC) analyses.
The values below 70% and 0.70 of Maximum Likelihood bootstrap (MLBS) and Bayesian posterior
probabilities (pp) accordingly are not included in the phylogenetic tree (Fig. 1).

Morphological studies
Morphological details of the studied material are established based on the field notes and color photographs
of the specimen. Methuen Handbook of color was referred to for shade codes (Kornerup and Wanscher,
1978). Free-hand sections were prepared from dried specimens and mounted in 5% KOH (potassium
hydroxide), stained with Congo red, and observed under a compound microscope for the microscopical
details. Melzer’s reagent was used to check the amyloidity of the basidiospores. The abbreviation used for
basidiospores are: ‘Q’ for the length/width ratio and ‘Qm’ for the mean quotient of all spores.




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Table 1 - Lists of Tricholomopsis species used in phylogenetic analyses with locality, GenBank accession numbers, voucher
numbers, and references.
 Species                    Country           Accession      Accession       Voucher no.            References
                                              n. ITS         n. LSU
 Tricholomopsis aff. rutilans Germany         KP058981                       TUB11582               Olariaga et al. 2015
 T. aff. rutilans           Sweden            KP058984       KP058985        UPS–F–646220           Olariaga et al. 2015
 T. aurea                   Africa            MF100960       MF100993        SFSUDED8327            Desjardin et al. 2017
 T. aurea                   Africa            MF100961       MF100994        SFSUBAP618             Desjardin et al. 2017
 T. badinensis              Slovakia          LS992163                       PRM946195              Holec 2019
 T. badinensis              Slovakia          LS992164                       PRM899423              Holec 2019
 T. darjeelingensis         India             OQ913610       [OQ861109] CUHAM 119                   This study
 T. decora                  Slovakia          HE649942                       PRM 899160             Holec et al. 2012
 T. decora                  Czech Republic    FN554891                       PRM 898238             Holec et al. 2009
 T. decora                  Czech Republic    FN554890                       PRM 882317             Holec et al. 2011
 T. flammula                Spain             KP058973       KP058974        ARAN–Fungi 00322 Olariaga et al. 2015
 T. flammula                Sweden            KP058975       KP058976        S–F156625              Olariaga et al. 2015
 T. galeata                 China             MW871732 MW871622              BJTC FM1107            Mao et al. 2021
 T. galeata                 China             MW871782 MW871630              BJTC FM1229            Mao et al. 2021
 T. ornaticeps              New Zealand       KY010816       KY010825        PDD:57441              Cooper et al. 2017
 T. ornaticeps              New Zealand       KY010817       KY010826        PDD:72909              Cooper et al. 2017
 T. ornaticeps              New Zealand       KY010824       KY010828        PDD:102769             Cooper et al. 2017
 T. ornaticeps              New Zealand       KY010820       KY010827        PDD:82501              Cooper et al. 2017
 T. osiliensis              Poland            LT984726       LT978511        PRM:946096             Holec 2019
 T. osiliensis              China             LT716068       KY418884        ZRL20151760            Zhao et al. 2017
 T. pallidolutea            China             MW871614 MW871640              HSA398                 Mao et al. 2021
 T. pallidolutea            China             MW871749 MW871631              BJTC FM1184            Mao et al. 2021
 T. pteridicola             Spain             KP058988       KP058989        ARAN–Fungi 00121 Olariaga et al. 2015
 T. pteridicola             Spain             KP058986       KP058987        ARAN–Fungi 320         Olariaga et al. 2015
 T. rubroaurantiaca         China             MN912496       MN912493        GDGM74616              Hosen et al. 2020
 T. rubroaurantiaca         China             MN912494       MN912491        GDGM61454              Hosen et al. 2020
 T. rutilans                Czech Republic    HE649946                       PRM:899460             Holec et al. 2012
 T. rutilans                Sweden            KP058977       KP058978        UPS–F–646219           Olariaga et al. 2015
 T. scabra                  New Zealand       KY010821       KY010831        PDD:102100             Cooper et al. 2017
 T. scabra                  New Zealand       KY010819       KY010830        PDD:81264              Cooper et al. 2017
 T. scabra                  New Zealand       KY010818       KY010829        PDD:78235              Cooper et al. 2017
 T. scabra                  New Zealand       KY010823       KY010832        PDD:102579             Cooper et al. 2017
 T. sulphureoides           China             MW871623 MW867238              HSA 277                Mao et al. 2021
 T. yunnanensis             China             MZ470249                       HKAS28976              Liu et al. 2022
 T. yunnanensis             China             MZ470247       OL614950        KUN–HKAS48887          Liu et al. 2021
 T. yunnanensis             China             OK393697       OL614949        KUN–HKAS80034          Liu et al. 2021
 T. yunnanensis             China             MZ470248       OL614951        HKAS93555              Liu et al. 2021
 Pleurocybella porrigens    Canada            KC581309                       UBCF23773              Direct submission
 Phyllotopsis sp.                             DQ404382       AY684161        AFTOLID773             Direct submission
 Pluteus romelli            USA               AY854065       AY634279        AFTOLID625             Direct submission


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Results
Phylogenetic analyses
The combined ITS and LSU dataset contained 40 taxa and 3880 nucleotides including the gaps. Since
the phylogenetic trees generated from ML and BI analyses show the same topology, only the tree resulting
from ML analysis is shown. Our collected specimen Tricholomopsis darjeelingensis (voucher number: CUH
AM791) formed an independent lineage with MLBS of 73% and PP of 0.74 rather than clustering with
other species of the genus (Fig. 1). However, T. darjeelingensis shows neighbour homologues with T. aff.
rutilans (Germany) and T. pteridicola Olariaga, Laskibar & Holec (Spain), but T. aff. rutilans is found to
be undescribed, T. pteridicola has distinctive morphological characters, and both taxa are easily separated
from our specimen.




                     Fig. 1 Phylogenetic tree generated from Maximum Likelihood (ML) analyses
                     based on concatenated ITS and LSU sequence datasets. Maximum likelihood
                     bootstrap and Bayesian posterior probabilities support values greater than 70%
                     and 0.70, respectively are indicated on the nodes. The highlighted region in the
                     tree refers to the species selected as an outgroup.

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Taxonomy
Tricholomopsis darjeelingensis A. Thapa & K. Acharya, sp. nov                             Figs 2, 3
MycoBank no.: MB 848465
    Diagnosis: Tricholomopsis darjeelingensis is characterized by its caespitose fruit body, long and slender
stipe, sometimes eccentric, pileus covered with dense fibrils which are violet in color, 2 – 4 spored basidia,
thin–walled caespitose cheilocystidia, and its terrestrial nature. Basidiospores globose to broadly ellipsoid,
2.9 – 4.72 – 5.9 × 2.9 – 3.93 – 4.4 μm.




               Fig. 2 (a-b) Fresh basidiomes of Tricholomopsis darjeelingensis (c) basidiospores (d-e) basidia (f-h)
               cheilocystidia (i) pileipellis. Scale bars a-b = 10 mm; c-i = 10 μm.

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   Type: INDIA. West Bengal, Darjeeling, Old Military Road, 27°00’04.9”N 88°15’42.4”E, 17 Jul 2022,
col. A. Thapa, J. Tamang (CUH AM791, holotype; CUH AM872, CUH AM911, isotype).
   Etymology: referring to the place of collection of the specimen.

   Basidiomata are small to large-sized. Pileus 18 – 75 mm in diameter, broadly convex, margin straight,
moist, slightly depressed at the center, light lilac (15A5) to greyish violet (15D6) towards the center,
surface pubescent, margin non–striate, incurved, context up to 3 mm wide, white (2A1), unchanging
with bruising. Lamellae up to 3 mm broad, adnate to sinuate, yellowish white (4A2), crowded, edge
serrate, lamellulae with 6 – 7 tiers. Stipe 59 – 72 × 7.6 – 9 mm, central to slightly eccentric, surface similar
to pileus, with dense hairs, cylindrical, slightly curved, compressed, hollow, fleshy, concolorous to pileus.
Odor and taste are not distinctive. Spore-print white.




               Fig. 3 Tricholomopsis darjeelingensis A. Thapa & K. Acharya, sp. Nov (a) basidiospores (b) basidia (c)
               cheilocystidia.




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    Basidiospores 2.9 – 4.72 – 5.9 × 2.9 – 3.93 – 4.4 μm, Q = 1 – 1.67, Qm = 1.2, globose to broadly
ellipsoid, rarely oblong, slightly thick-walled, smooth, hyaline, inamyloid, guttate, with an apicule up
to 0.3 μm long. Basidia 23.5 – 35.3 × 5.6 – 5.9 μm, 2- to 4-spored, clavate, sterigmata up to 8.2
μm long, smooth, hyaline, thin–walled, with basal clamp, guttate. Pleurocystidia absent. Lamellae edge
sterile. Cheilocystidia 41.2–117.6 × 13.5–35.3 μm, abundant, caespitose, clavate to ovoid, and sometimes
fusoid, hyaline, smooth, thin-walled, with a basal clamp. Hymenophoral trama regular, composed of long,
cylindrical hyphae, up to 26.5 μm wide, thin-walled. Pileipellis is a cutis layer, composed of inflated
hyphae up to 12.35 μm broad. Stipitipellis a cutis, composed of parallel, cylindrical hyphae, 2.9 – 14.1
μm broad. Caulocystidia absent. Clamp connections up to 3 μm wide present in all tissues.
    Habit and habitat: Caespitose, terrestrial in a forest dominated by Pinus sp., Quercus sp., and Betula sp.

Discussion
Tricholomopsis darjeelingensis is characterized by its small to large basidiomes, covered with dense violet
hairs, stipe concolorous to the pileus, globose-broadly ellipsoid basidiospores, 2 – 4 spored basidia, and
abundant cheilocystidia. In the field, our specimen might be confused with T. rutilans, T. pteridicola, T.
yunnanensis (M. Zang) Li R. Liu, Yan C. Li & Zhu L. Yang, and T. galeata L. Fan & N. Mao, but all of
those species differ from our specimen by having few distinguishable macro and microscopic characters
such as size and color of the pileus, absence or presence of the cystidia, etc. as compared in Table 2.
   Though three species of Tricholomopsis have been reported from different regions of India, none of
them have shown the phylogenetic analyses in past works. Thus, this study shows the first phylogenetic
approach from India on the genus Tricholomopsis. The morphological details indicated our specimen T.
darjeelingensis to be a novel species, which is well supported by molecular evidence.

Dichotomous key to species of Tricholomopsis reported from India

1. Pileus covered with dense minute purple/violet fibrils.……………………………….……….…..2
1b. Pileus glabrous or with sparse yellow/buff fibrils..……...…………………...……….…………...3
2. Caulocystidia present...…………………………………….……………………………..T. rutilans
2b. Caulocystidia absent...……………………………………………...………...…T. darjeelingensis
3. Pileipellis trichoderm–type, pleurocystidia abundant………………………….…..T. sulphureoides
3b. Pileipellis cutis–type, pleurocystidia absent………………………………….……..T. crocobapha


Acknowledgments
The authors are very grateful for the facilities provided by the Department of Botany, University of
Calcutta. They are also indebted to the Government of West Bengal Directorate of Forests, and the West
Bengal Biodiversity Board for their help. A. Thapa (UGCRef.No: 600/CSIR –UGC NET JUNE 2017)
duly acknowledges the University Grant Commission (UGC) for the funding.




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Table. 2 Comparative table of characters of look–alike species of Tricholomopsis with T. darjeelingensis. References: (a) Liu et al. 2022; (b) Olariaga et al. 2015; (c) Mao et al. 2021; (d) Cooper
et al. 2016; (e) This study.
 Characters                Pileus              Stipe             Lamellae         Basidiospores        Basidia           Pleurocystidia      Cheilocystidia      Caulocystidia Pileipellis
 T. darjeelingensis (e)    18–75 mm,           72×9 mm,          Adnate to        2.94–5.9×2.9–        23.52–            Absent              41.16–              Absent           Cutis–type
                           covered with        central to        sinuate          4.4 μm, globose      35.28×5.58–                           117.6×13.52–
                           liliac to violet    eccentric,                         to broadly           5.88 μm, 2–4                          35.28 μm,
                           hairs               covered with                       ellipsoid            spored                                caespitose,
                                               violet hairs                                                                                  clavate to ovoid
 T. rutilans (d)           20–160 mm,          25–85×3–25        Adnate or        6.5 μm×4.9 μm        35–45×6.0–        Absent/sparse       40–120×12–25        Present (20–     Cutis with
                           covered with        mm, covered       sinuate          broadly ellipsoid    8.0 μm, 4–                            μm, clavate,        80×4.0 μm)       transitions to a
                           purplish–reddish    with purplish–                     to ellipsoid         spored                                with brown,                          trichoderm
                           hairs               reddish hairs                                                                                 intracellular
                                                                                                                                             pigment
 T. galeata (c)            50–130 mm,        35–120×12–          –                5–7×4–5 μm,          30–40×6–8         Absent              55–105×13–23        Absent           Cutis–type
                           covered with red 20 mm,                                broadly ellipsoid    μm, 2–4                               μm, mostly
                           to purple reddish glabrous                             to ellipsoid         spored                                narrowly clavate
                           hairs                                                                                                             to lageniform–
                                                                                                                                             fusiform
 T. pteridicola (b)        7–28(60) mm,        14–35(70) ×       Adnate–sub       5.5–7×4–5.5          25–35×5.5–8       40–62×4–6.5         53–98×10.5–18       Absent           Cutis–type
                           covered with        3–5(12) mm,       decurrent        μm, subglobose       μm, 2–4           μm, cylindrical     μm, mostly
                           purple-red hairs    covered with                       to broadly           spored            to narrowly         clavate
                                               purple red                         ellipsoid                              clavate
                                               hairs
 T. yunnanensis (a)        41–95 mm,           7–13×2.1–5        Adnexed          5–7.5×4–6 μm,        22–29×6–8         30–50×5–7 μm, 60–195×11–39              Absent           Palisadoderm
                           covered with        cm, pale                           subglobose to        μm, 4–spored      sub fusiform or   μm, fusiform
                           red-brown hairs     lemon–yellow,                      broadly ellipsoid                      cylindrical to
                                               covered with                                                              narrowly clavate,
                                               red–violet                                                                often slightly
                                               hairs                                                                     flexuous




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