Morpho-anatomical and Phylogenetic studies of

Russula species from Narkanda, India

Munruchi Kaur
Punjabi University
Amandeep Kaur
Punjabi University
Naseema Aqbar Wani (  [email protected] )
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir https://orcid.org/0000-
0002-2421-1609

Research Article

Keywords:

Posted Date: November 26th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-2275176/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Background
The genus Russula is considered one of the most abundant and widely distributed ectomycorrhizal
agaric genera and the Indian subcontinent is no exception to it. The members of this family are
cosmopolitan and form an ectomycorrhizal association with a wide range of angiosperm and
gymnosperm trees.

Methods and Results

Four new records of genus Russula viz., Russula pauriensis var. caulocystidii var. nov., Russula
globispora, Russula cuprea and Russula laeta were collected from Narkanda, Himachal Pardesh, India.
These were described and illustrated on the basis of morpho-anatomical characters as well as
phylogentic analysis of internal transcribed spacer (ITS) ribosomal DNA sequences. On the basis of gross
taxonomic characterization Russula pauriensis var. caulocystidii var. nov. is proposed which is further
confirmed through molecular taxonomy. Russula laeta, Russula globispora, Russula cuprea and Russula
laeta is reported for the first time from India while Russula amethystina is a rereport.

Conclusion
Current environmental challenges of global warming and climate change will affect the regeneration and
growth patterns of delicate fungi that require specific microclimate. Present study is a step towards
deciphering macrofungal diversity, especially in the biodiversity-rich forests of Narkanda, to enable
diversity planning, management and conservation.

Introduction
Narkanda is a small, serene town situated at 31.2578º N and 77. 4602º E with an altitudinal range of
1,762–3400 m in the North-West Himalayas. Members of the family Russulaceae are characterized by
their fleshy and often brightly coloured fruiting bodies with prominent lamellae, a heteromerous context
traversed by conducting hyphae, warted, light-colored amyloid basidiospores which exhibit various types
of ornamentations. Genus Russula Pers. belongs to family Russulaceae Lotsy which is one of the largest
family among the 12 families acknowledged under order Russulales Kreisel ex P.M. Kirk, P.F. Cannon &
J.C. David of phylum Basidiomycota. The Genus Russula Pers. is one of the most challenging genus to
identify mushrooms up to species level. Almost all the species of genus Russula show ectomycorrhizal
association growing in different habitats like tropical and temperate forests. This genus is characterized
by their fleshy carpophores with colourful caps which varies from white, yellow, brown and tan through
orange, pink, red and purple to blue, mauve and green etc. gills attached to stipe and are sometimes
forked, lamellate hymenophore with amyloid ornamented warty spores forming complete to incomplete
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reticulum, the cap and stem flesh consists nests of sphaerocysts, usually in clusters causing brittleness
to fruit bodies and exannulate stipe without volva.

From all over the world 2,056 taxa of genus Russula have been reported [1] while the latest record of
Mycobank shows 2740 legitimate species [2]. There are 161 Russula species reported so far from India
[3, 4, 5]. During present research, two new records of Russula species have been reported from Narkanda,
India.

Materials & Methods

Morpho-anatomical studies
The carpophores were collected from various habitats during extensive fungal forays to the different
localities of Narkanda and adjoining areas along an altitudinal gradient in different vegetational zones
during the late monsoon season in mid-October 2018. The various aspects of material and methods with
respect to collection, macroscopic examination, drying and preservation of material, chemical color
reactions, microscopic details and identification, etc. are discussed below:

Collection Of The Material

A clean, fresh and healthy mushroom was collected and to note down their external characters in field
itself. Field notes including details regarding locality, altitude, collection number, date of collection, soil
type, habitat, association with forest type were noted down on the field key along with photographs taken
with the aid of digital camera [6]. The color terminology was used for recording the color of various parts
of the carpophore [7].

Drying And Preservation

The specimens were preserved in a liquid preservative (25ml of rectified alcohol + 5ml of formaldehyde +
70 ml of water) for further microscopic investigations while the major portion of collection was hot air
dried [8]. The collection examined during the present study was deposited in the herbarium of Botany
Department, Punjabi University, Patiala under PUN.

Chemical Color Reaction

Melzer’s reagent (1.5gm Potassium iodine + 0.5gm Iodine + 20ml Distilled Water + 22gm chloral hydrate).
The basidiospores of the examined collection shows negative reaction with Melzer’s reagent and the
spore wall remains inamyloid.

Congo red (2g Congo Red + 100ml Distilled water). This chemical is used for staining the various parts so
that camera lucida drawings become easy and clear for microscopic observations.

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Microscopic observations

It includes the microscopic study of various parts of the fruiting body viz. gills, basidiospores, pileus,
stipe, etc. were noted and the size of each part of carpophore was measured with the aid of ocular
micrometer under oil immersion. Ornamentation type in case of Russula have been noted down [9].
Drawings of the worked out taxa through camera lucida with respect to their microscopic details have
been drawn under oil immersion. For the nomenclature of the taxa, the rules and regulations followed are
as given in the International Code of Botanical Nomenclature [10].

Molecular Characterization
During collection of mushrooms, a small portion of the cap was preserved in 1X TAE buffer (10ml 50X
TAE + 490ml distilled water {50X TAE = 24.2g tris base + 57.1 ml glacial acetic acid + 100 ml of 500 mM
EDTA (pH 8.0) solution + distilled water to make final volume 1 liter}) for molecular studies. For isolation
of genomic DNA protocol given by QIAGEN DNasey Plant Mini Kit was followed. Further, the internal
transcribed spacer (ITS) regions of the rDNA were amplified through PCR by using universal primers ITS1
and ITS4. The amplification reactions were performed in a DNA Thermal Cycler. For thermal cycling
conditions method with slight modifications were employed [11]. For nucleotide sequencing the pure PCR
products were sent to the ‘Ist Base’ (Singapore) through its distributor ‘Agile Lifescience Technologies
India Pvt. Ltd.’. The final gene sequences were submitted at NCBI to get accession numbers: MW882089
and MW882091 for the ITS sequences of Russula pauriensis var. caulocystidii var. nov. and R. laeta,
respectively. The ITS sequences obtained were compared to GeneBank database using the nucleotide
BLAST search method at NCBI.

Taking the data set as a base, our phylogenetic analysis involved a total of 17 and 18 nucleotide
sequences subsequently for both taxa. All the positions containing gaps and missing data were
eliminated and were aligned using muscle alignment tool. The final data set included the species
examined (Russula pauriensis var. caulocystidii var. nov., R. laeta, Russula globispora, Russula cuprea
and Russula amethystina) and the alignments contains total of 1658, 788, 683, 761 and 659 positions,
respectively. The evolutionary distance trees among isolates and related taxa were determined by using
maximum likelihood method based on Tamura-Nei model [12]. One thousand bootstrap replicates were
analysed to obtain the nodal support values. The trees with the highest log likelihood were shown.
Evolutionary analyses were conducted in MEGA 11 [13].

Results And Discussion

Taxonomic observations & Morphological characterization of Russula species

Russula pauriensis A. Ghosh, K. Das & Buyck, Cryptogamie, Mycologie 38 (3): 392 var. caulocystidii var.
nov.:

Mycobank Number 822859 Fig. 1–6

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Genbank Number MW 882089

Entomology The variety is named after the presence of bunches of regular tufts of caulocystidia.

Basidiocarps 8.0-9.5 cm in height. Pileus 6.0-6.2 cm in diameter, reflexed with depressed centre, umbo
absent, surface grayish magenta (13A4) to dark purple (14F6) towards apex with greenish and yellow
tings towards margins; dry; scaly, appressed fibrillose scales; glabrous; feebly striate; margins irregular,
splitting at maturity; cuticle half peeling; flesh up to 0.6 cm broad, creamish white, unchanging. Pileal veil
absent. Odour mild. Lamellae up to 0.8 cm broad, adnexed, equal, close, creamish white with yellowish
white (1A2) tinge, forked near the attachment to stipe, unchanging; gill edges smooth. Stipe excentric, 7.0-
7.4 cm long, 2.1 cm towards apex, 1.8 cm at centre and 1.8 cm at base, slightly tapering downward
towards the base, creamish white with reddish lilac (14B5) tinges; solid; surface fibrillose; unchanging;
exannulate.

Basidiospores (6.4) 7.47–8.3 × 6.6–7.47µm; Q = 0.96 (1.13)-1.1 µm; globose to sub-globose; warty, warts
0.8–1.6 µm high, isolated, catenulate, incomplete reticulum; amyloid; apiculate, apiculus 2.4–3.6 µm
long. Basidia 38.4–65.6 × 11.2–12.8 µm, clavate, granular, tetra-sterigmate, sterigmata 4.0–8.0 µm,
granular, pointed. Pleurocystidia 65.6–120.0 × 9.6–14.5 µm, fusiform with long tubular neck, blunt and
acute apices, double walled, thick walled, granular, tip is filled with thick granulation, deeply embedded,
abundant. Cheilocystidia 28.8–57.6 × 6.4–12.8 µm, fusoid ventricose with blunt and acute apices, thick
walled, granular. Hymenophoral trama regular. Gill edges sterile. Pileus cuticle hyphal, ixocutis, made up
of 4.8–8.8 µm broad, loosely arranged, septate, horizontally tangled hyphae giving rise to regular turf of
broad, granular, septate hyphae, terminal hyphal cells subulate, ellipsoid to subfusoid; context hyphal
made up of 3.2–8.0 µm broad, septate, granular hyphae intermixed with 4.8–52.8 µm, irregular, hyaline
cells. Stipe cuticle hyphal, non-gelatinized, 3.2–7.2 µm broad, granular hyphae intermixed with 4.0–12.8
µm, granular, irregular cells, giving rise to regular tufts of bunches of caulocystidia; context cellular, made
up of 14.4–40.0 µm broad, hyaline, irregular cells. Caulocystidia 56.0–72.8 × 5.6–8.0 µm, fusoid
ventricose with blunt and acute apices, thick walled, densely granular. Clamp connections absent.

Chemical Reactions KOH: Dry pileus surface turns reddish orange in KOH.

Collection examined Narkanda, Kotgarh road (2084m) 31̊ 17’0ʺN − 77̊ 27’15ʺE, growing scattered on
humicolous soil in mixed forest during late monsoon season, 14th October 2018, Amandeep Kaur, PUN
11187.

Result Morpho-antomically the present collection matches well with the description given for Russula
pauriensis [14]. This collection differs as there is present a regular tuft of bunches of caulocystidia on the
stipe cuticle which are all together lacking in R. pauriensis or its allied species like R. mukteshwarica [15].
Thus, new variety is being proposed as Russula pauriensis var. caulocystidii. R. pauriensis is recorded
from study area for the first time and a new var. is proposed. Morpho-anatomical characters matches well
with molecular characterization of Russula pauriensis. Molecular analysis gave 98% query cover and
98.94% percent identification with Russula pauriensis.
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Phylogen The ITS sequences (Fig. 1) derived from MW882089 (Genbank number) i.e. the holotype for
Russula pauriensis var. caulocystidii var. nov. was fixed among the other species of sub sect. Amoeninae
and retrieved with 37% bootstrap value as a sister species to R. pauriensis (Genbank accession number:
MF53185) from India. The tree shows the highest log likelihood (-3714.64). The extralimital taxa i.e.
Cortinarius furvolaesus was chosen as an out group.

Russula laeta F.H. Møller & Jul. Schäff., Bull. Soc. Mycol. France: 231, t. 62 (1934)

Mycobank Number 305383 Fig. 7–12

Genbank Number MW 882091

Basidiocarp up to 6.7 cm in height. Pileus up to 5.8 cm in diameter, convex, umbo absent, surface
brownish red (8C7) with purplish and pastel red (8A5) tinges; dry; glabrous; margins regular, splitting at
maturity; cuticle half peeling ; flesh up to 0.4 cm broad, white (1A1), unchanging. Pileal veil absent. Odour
fruity. Lamellae up to 0.5 cm broad, adnate, unequal, crowded, pale orange (6A3), unchanging; gill edges
smooth. Stipe central, up to 6.1 cm long, 1.6 cm towards apex, 1.7 cm at centre and 2.2 cm at base, equal
in diameter with distinctly bulbous base, white (1A1) with yellowish white (1A2) tinge at base; hollow;
smooth; unchanging; exannulate.

Basidiospores 7.47–9.13 × 6.64–7.47µm; Q = 1.12–1.2 µm; globose to sub-globose; warty, warts 0.8–2.6
µm high, isolated, blunt, catenulate, incomplete reticulum; amyloid; apiculate, apiculus 1.6–3.2 µm long.
Basidia 33.6–48.0 × 9.6–14.4 µm, clavate, granular, tetra-sterigmate, sterigmata 4.8-8.0 µm, granular,
pointed. Pleurocystidia 52.0–70.4 × 8.0–12.8 µm, clavate, broadly clavate to sub-cylindrical, blunt and
acute apices, granular. Cheilocystidia 40.0–54.4 × 8.0–12.8 µm, clavate with inflated to beaked tips,
granular. Hymenophoral trama cellular. Gill edges sterile. Pileus cuticle gelatinized, gelatinization up to 85
cm broad, epicutis made up of 3.2–4.98 µm broad dermatocystidia, hairy, filamentous, loosely arranged,
encrustated, granular, septate hyphae, context heteromerous, made up of 3.2–6.4 µm broad, septate,
granular hyphae intermixed with 4.8–22.4 µm, irregular, granular cells. Stipe cuticle hyphal, gelatinized,
made up of 2.4–6.4 µm broad, granular, septate hyphae intermixed with 3.2–8.0 µm, granular, irregular
cells, giving rise to caulocystidia; context cellular, made up of 3.2–40.0 µm broad, hyaline, irregular cells
forming spherocystis intermixed with 3.2–6.4 µm broad, septate, granular hyphae. Caulocystidia 38.4–
56.0 × 6.4–11.2 µm, sub-cylindical to fusoid ventricose with blunt and acute apices, densely granular,
septate. Clamp connections absent.

Collection examined Narkanda (2636m) 31̊ 15’6ʺN − 77̊ 28’20ʺE, growing solitary on moss in pure Cedrus
deodara forest during late monsoon season, October 13, 2018, Amandeep Kaur, PUN 11186.

Results This collection falls very close to Russula laeta in its morpho-anatomical details as well as
molecular analysis. Although according to [15] it seems to be very similar to R. velenoveskyi but the cap
in this species does not have any purplish or copper tinges as are present in above described species and
R. laeta. Also the size of the basidiospores and dermatocystidia of the present collection are in an

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arrangement with R. laeta. This species has been not earlier reported from India. So, it is the first report of
this species from India. The molecular analysis was done for futher conformity which gives 100% query
cover and 98.73% of percentage identification with R. laeta.

Phylogeny The sequences derived from our second species (Fig. 12), R. laeta (Genbank accession
number: MW882091) is placed among the other species of subsect. Integrae and clustered with
unidentified R. species (Genbank accession number: MK389376) from Pakistan showing 50% bootstrap
value and formed a sister clade to R. laeta (Genbank accession number: MK386826 and MK389377). The
tree displays the highest log likelihood (-2427.83). The extralimital taxa i.e. Russula rajendrae was chosen
as an out group.

Russula amethystina Quѐl., Comptes Rendus de l' Association Franҫaise pour l' avancement des Sciences
26 (2): 450, 4:13 (1898)

Mycobank Number 174401 Fig. 13–16

Genbank MW882093

Basidiocarp up to 8.0 cm in height. Pileus up to 5.5 cm in diameter, convex; umbo absent; surface dark
brown (6F5) with grayish brown (6D3) and pale yellow (1A3) tinges towards center; scales absent;
margins regular, spilitting at maturity, dry; cuticle half peeling; flesh up to 0.4 cm thick, yellowish white
(3A2), unchanging; odour mild. Pileal veil absent. Lamellae up to 0.8 cm, moderately broad, adnate,
subdistant, equal, pale orange (5A3); gill edges smooth. Stipe central, up to 6.2 cm long, up to 1.5 cm at
apex, up to 2.4 cm at center and 3.0 cm in diameter at base, obclavate; white (1A1); smooth; hollow;
exannulate.

Basidiospores 8.0–11.2 × 6.4–9.6 µm; Q = 1.25–1.16 µm; sub-globose, warty, warts 0.8–1.6 µm, warts 2–
3 connected, incomplete reticulum; amyloid; apiculate, apiculus 2.4–4.8 µm long, supra-hilar pledge
present near apiculus. Basidia 36.8–60.8 × 9.6–17.6 µm, clavate to broadly clavate, granular, bi -
tetrasterigmate, sterigmata up to 3.2–8.0 µm long, hyaline to granular, pointed. Pleurocystidia 54.4–89.6
× 11.2–16.0 µm, clavate to fusoid ventricose with blunt to beaked tips, granular, protruding out of the
basidial layer and deeply placed. Cheilocystidia 32.0–48.0 × 6.4–9.6 µm, clavate with rounded and blunt
tips, granular. Hymenophoral trama hyphal as well as cellular. Gill edges fertile, heteromerous. Pileus
cuticle hyphal intermixed with pileocystidia, made up of 3.2–6.4 µm broad, highly septate, horizontally
tangled hyphae, granular, intermixed with 3.2–4.8 µm broad, irregular, granular cells; context hyphal made
up of 3.2–12.0 µm broad, septate, granular hyphae intermixed with 3.2–32.0 µm, irregular, hyaline to
granular cells. Oleiferous hyphae are present. Pileocystidia 27.2–46.4 × 4.8–6.4 µm, clavate with
rounded, blunt apices, granular. Stipe cuticle hyphal, made up of 3.2–6.4 µm, longitudinally tangled,
granular, septate hyphae giving rise to caulocystidia intermixed with 3.2–8.0 µm broad, irregular, granular
cells; context hyphal, made up of 3.2–8.0 µm broad, longitudinally tangled, septate, granular hyphae
intermixed with 3.2–19.5 µm broad, irregular, hyaline to granular cells. Oleiferous hyphae are present.

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Caulocystidia 14.4–30.4 × 4.8–7.2 µm, clavate to fusoid ventricose with round, blunt apex, granular.
Clamp connections absent.

Collection examined Hattu Peak road (2945 m) 31̊ 15’6ʺN- 77̊ 29’ 19ʺE, growing solitary on Cedrus
deodara needles in mixed forest during late monsoon season, 13th October 2018, Amandeep Kaur, PUN
11183.

Edibility Edible, flesh eating insect was reported.

Results All the morphological and anatomical characters matches well with the details given for Russula
amethystina [15, 16]. The present collection is almost similar to its closely allied R. turci Bres., but due to
the presence of sub isolated warts forming incomplete reticulum and presence of yellow tinges on the
pileus due to persistent rainfall it is placed under R. amethystine. [16] also differentiate the two allied
species on the basis of odour which is weaker in R. amethysina whereas more clear and limited to the
base of stem in R. turci. The present collection differs from those in possessing much larger spore size
[15, 16]. Morpho-anatomical characters matches well with molecular characterization of Russula
amethystina. Earlier this species is reported for the first time from India by [17]. Now, it is rereported from
India.

Phylogeny The ITS sequences from R. amethystina (Genbank accession number: MW882093) (Fig. 16),
are grouped with other species in the subsection Amethystinae and placed with an unidentified R.
amethystina from Pakistan (Genbank accession number: KT953612), forming a sister clade to R. cuprea
(Genbank accession number: KT953616) with an 75% bootstrap value. The tree displays the highest log
likelihood (-4124.55). Lactifluus rajendrae was used as an extralimital taxon.

Russula globispora (J. Blum) Bon, Documents Mycologiques 17 (65): 55 (1986)

Mycobank Number 129550 Fig. 17–22

Genbank Number MW882090

Basidiocarp up to 5.2 cm in height. Pileus up to 4.8 cm in diameter, applanate; umbo absent; surface
grayish orange (6B3) with brownish orange (6C5) tinges towards margins; scales absent; margins regular,
spilitting at maturity, moist; cuticle half peeling; flesh up to 0.4 cm thick, white (1A1), unchanging; odour
fragrant. Pileal veil absent. Lamellae up to 0.6 cm moderately broad, adnexed forked towards stipe,
lamellulae rare, close, equal, orange white (6A2); gill edges smooth. Stipe excentric, up to 4.2 cm long, up
to 1.8 cm at apex, up to 1.5 cm at center and 1.7 cm in diameter at base, obclavate; white (1A1) with pale
orange tinges at base; smooth; solid; exannulate.

Basidiospores 8.0–12.8 × 7.2–10.4 µm; Q = 1.1 − 1.2 µm; sub-globose, warty, warts 0.8–1.6 µm, warts
isolated, rarely connected, incomplete reticulum; amyloid; apiculate, apiculus 3.2–4.8 µm long. Basidia
24.0–60.8 × 9.6–16.0 µm, clavate to broadly clavate, hyaline to granular, bi - tetrasterigmate, sterigmata
up to 0.8 − 6.4 µm long, hyaline, pointed. Pleurocystidia 72.0–97.6 × 10.4–17.6 µm, clavate to broadly
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clavate with blunt to beaked tips, granular, protruding out of the basidial layer and deeply placed,
abundant. Cheilocystidia 35.2–49.6 × 8.8–12.8 µm, clavate to broadly clavate with rounded, beaked,
blunt and capitate tips, granular. Hymenophoral trama hyphal as well as cellular. Gill edges fertile,
heteromerous. Pileus cuticle hyphal intermixed with pileocystidia, geletinized, ixocutis, made up of 2.4–
8.0 µm broad, highly septate, horizontally tangled, branched hyphae, granular, intermixed with 2.4–4.8 µm
broad, irregular, granular cells; context hyphal made up of 3.2–8.0 µm broad, septate, granular hyphae
intermixed with 4.8–41.6 µm, irregular, hyaline to granular cells. Oleiferous hyphae are present in cuticle
as well as context. Pileocystidia 40.0–96.0 × 6.4–11.2 µm, clavate to broadly clavate to sub clavate with
blunt apices, granular. Stipe cuticle hyphal, made up of 3.2–9.6 µm, longitudinally tangled, granular,
septate hyphae giving rise to caulocystidia intermixed with 3.2–12.8 µm broad, irregular, granular cells;
context hyphal, made up of 3.2–10.4 µm broad, longitudinally tangled, septate, granular hyphae
intermixed with 4.0–25.6 µm broad, irregular, hyaline to granular cells. Caulocystidia 32.0–48.0 × 6.4–
11.2 µm, clavate to fusoid ventricose with round, blunt apex, hyaline to granular. Clamp connections
absent.

Collection examined Narkanda (2044 m) 31̊ 17’3ʺN- 77̊ 27’ 17ʺE, growing solitary on leaf litter in mixed
forest during late monsoon season, 14th October 2018, Amandeep Kaur, PUN 11185.

Results All the morpho-anatomical characters matches well with the description given for Russula
globispora [15, 18]. The present collection is featured by the presence of grayish orange (6B3) with
brownish orange (6C5) tinges towards margins, lamellae forked towards stipe, basidiospores isolated to
rarely connected (mostly 2 warts connected), presence of pileocystidia in pileus cuticle along with highly
septate projecting hyphae with intermixed oleiferous hyphae described by [15]. Morpho-anatomical
characters matches well with molecular characterization of Russula globispora. This species has been
not earlier reported from India. So, it is the first report of this species from India. The molecular analysis
was done for futher conformity which gives 95% query cover and 96.31% of percentage identification
with R. globispora.

Phylogeny The sequences derived from our second species (Fig. 22), R. globispora (Genbank accession
number: MW882090) is placed among the other species of subsect. Urentes and clustered with
unidentified R. species (Genbank accession number: MG386702) from Slovakia showing 53% bootstrap
value and formed a sister clade to uncultured Russula (Genbank accession number: LC367737). The tree
displays the highest log likelihood (-1575.26). The extralimital taxa i.e. Cortinarius bubulus was chosen
as an out group.

Russula cuprea Krombh., Naturgetreue Abbildungen und Beschreibungen der Schwӓmme 9:11, t. 66:1–3
(1845)

Mycobank Number 251691 Fig. 23–26

Genbank Number MW882092

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Basidiocarp up to 5.2 cm in height. Pileus up to 4.7 cm in diameter, applanate; umbo absent; surface pale
red (10B3) to violet brown (10E4) with light green tinges and pale yellow center; scales absent; margins
regular, spilitting at maturity, striated; dry; cuticle fully peeling; flesh up to 0.3 cm thick, white (1A1),
unchanging; odour mild. Pileal veil absent. Lamellae up to 0.6 cm broad, adnexed, crowded, equal, pale
orange (5A2), interveined bottom; gill edges smooth. Stipe central, up to 3.9 cm long, up to 1.0 cm at
apex, up to 1.1 cm at center and 1.3 cm in diameter at base, almost equal in diameter throughout with
slightly bulbous base; white (1A1), turns yellowish on handling; smooth; hollow; exannulate.

Basidiospores 8.3–10.79 × 6.64–9.13 µm; Q = 1.25 − 1.18µm; globose to broadly ellipsoid, warty, warts
0.8–3.32 µm, warts isolated, incomplete reticulum, pladge present; amyloid; apiculate, apiculus 1.66–
3.32 µm long. Basidia 41.6–56.0 × 12.8–17.6 µm, clavate to broadly clavate, densely granular,
tetrasterigmate, sterigmata upto 5.6–9.6 µm long, granular, pointed. Pleurocystidia 62.4–92.8 × 9.6–14.4
µm, clavate with long tubular neck with captate to beaked to flamed tips, granular, protruding out of the
basidial layer and deeply placed, abundant. Cheilocystidia 43.2–64.0 × 6.4–11.2 µm, clavate to
subcylindrical, mucronate having inflated tips, granular. Hymenophoral trama cellular. Gill edges fertile,
heteromerous. Pileus cuticle hyphal intermixed with pileocystidia, geletinized, ixocutis, made up of 3.2–
6.4 µm broad, thin hairy, septate, horizontally tangled hyphae, granular, intermixed with 3.2–6.4 µm broad,
irregular, granular cells, gelatinization up to 120 µm thick; context hyphal made up of 3.2–8.0 µm broad,
septate, granular hyphae intermixed with 4.8–32.0 µm, irregular, hyaline to granular cells. Pileocystidia
51.2–97.6 × 8.0–12.8 µm, long cylindrical to obtuse to pyriform with blunt tips, granular. Stipe cuticle
hyphal, made up of 2.4–4.8 µm, longitudinally tangled, granular, septate hyphae giving rise to
caulocystidia intermixed with 4.0–8.0 µm broad, irregular, granular cells; context hyphal, made up of 3.2–
8.0 µm broad, longitudinally tangled, septate, branched, granular hyphae intermixed with 4.0–33.6 µm
broad, irregular, hyaline to granular cells. Caulocystidia 16.0–41.6 × 4.8–12.8 µm, clavate to fusoid
ventricose with round, blunt apex, granular. Clamp connections absent.

Collection examined Narkanda (2619m) 31̊ 15’6ʺN- 77̊ 28’ 21ʺE, growing solitary near Cedrus deodar on
humicolous soil in coniferous forest during late monsoon season, 13th October 2018, Amandeep Kaur,
PUN 11184.

Results The morpho-anatomical characters goes well with the description given for Russula cuprea [15,
16]. The present collection is featured by the presence of pale red to violet brown surface with light green
tinges and pale yellow center, stipe white turns yellow on handling, pileus having thin hairy turf of hyphae
with embeded long cylindrical to obtuse blunt tiped elements which are similar in our species and are
described as pileocysidia as described by Romagnesi. This species has been not earlier reported from
India. So, it is the first report of this species from India. Morpho-anatomical characters matches well with
molecular characterization of Russula cuprea. This species has been not earlier reported from India.
Hence, this species is the first record from India. The molecular analysis was done for futher conformity
which gives 95% query cover and 99.66% of percentage identification with R. cuprea.

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Phylogeny The ITS sequences (Fig. 26), from R. cuprea (Genbank accession number: MW882092), are
placed with the other species of the subsection Urentes and grouped with unidentified R. sp. from China
(Genbank accession number: KX441137) and formed a sister clade to R. cuprea (Genbank accession
number: KF850403) showing 82% bootstrap value. In the tree, the highest log likelihood is shown
(-2013.85). Lactarius alboscrobiculatus, an extralimital taxon, was selected.

Discussion
The present study was conducted on the basis of both morpho-anatomical and molecular analysis of
Russula (order Russulales) species. Based on comparision between morphological and molecular results
from R. pauriensis var. caulocystidii var. nov., Russula cuprea, Russula globispora, Russula amethystina
and Russula laeta were considered in this study. The taxa had been determined at the species level on the
basis of morpho-anatomical characters which were further confirmed by molecular studies as
identification of Russula is an easy task but their identification up to species level is quite difficult due to
their morphological similarities..

Declarations
Declarations
Ethical approval:

This article does not contain any studies with human participants or animals performed by any of the
authors.

Compliance with ethical standards

This study was not funded by any of the funding agency.

Consent to publish:

All authors hereby declare their consent to publish.

Conflict of interest:

Munruchi Kaur declares that she has no conflict of interest, Amandeep Kaur declares that she has no
conflict of interest and Naseema Aqbar Wani declares that she has no conflict of interest.

Author contributions:
Munruchi Kaur, Amandeep Kaur and Naseema Aqbar Wani developed the idea of the manuscript.
Naseema Aqbar Wani and Amandeep Kaur screened the literature and prepared the required draft.
Munruchi Kaur and Amandeep Kaur carried out manuscript writing. Naseema Aqbar Wani carried out
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necessary editing of the manuscript. All the authors approved final draft of the manuscript for
submission.

Acknowledgements
The authors would like to show special thanks of gratitude to the Head of Botany Department, Punjabi
university, Patiala for laboratory facilities and DBT, for financial assistance.

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Contact no.: +917006155232

Keywords Basidiomycota. Mycoflora. Narkanda. Phylogeny. Russula. Taxonomy

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