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Research Article
Ramaria barenthalensis a new record from western Himalayas, Azad Jammu and Kashmir, Pakistan
expand article infoTariq Saiff Ullah‡§, Syeda Sadiqa Firdous|, Wayne Thomas Shier, Abdul Nasir Khalid#
‡ University of Kotli, Kotli, Pakistan
§ University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan
| Women University of Azad Jammu & Kashmir Bagh, Bagh, Pakistan
¶ University of Minnesota, Minneapolis, United States of America
# University of the Punjab, Lahore, Pakistan

Corresponding author: Tariq Saiff Ullah ( tariq.saiff@uokajk.edu.pk )

Academic editor: Lorenzo Peruzzi
© 2022 Tariq Saiff Ullah, Syeda Sadiqa Firdous, Wayne Thomas Shier, Abdul Nasir Khalid.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Ullah TS, Firdous SS, Shier WT, Khalid AN (2022) Ramaria barenthalensis a new record from western Himalayas, Azad Jammu and Kashmir, Pakistan. Italian Botanist 14: 133-143. https://doi.org/10.3897/italianbotanist.14.96790
Open Access

Abstract

The genus Ramaria is globally distributed, inhabiting different substrates. In the present study, samples of R. barenthalensis were collected during a fungal survey from Neelum valley AJK, Pakistan. Identification was carried out through combined morpho-anatomical and molecular data from nrITS region, which confirmed the identification as R. barenthalensis. The taxon is a new record for fungi of AJK, Pakistan.

Keywords

Coral fungi, ITS, Mushrooms of Kashmir, Neelum valley, nrDNA, Ramaria

Introduction

Ramaria species are cosmopolitan in distribution and grow on living and dead hardwood, tree trunks, partially decomposed organic matter, and under conifers as mycorrhizal species (Marr and Stuntz 1973; Kuo 2009; Dorjey et al. 2016). They are commonly known as coralloid fungi, due to colored and much branched basidiocarps. They grow in very diverse habitats and form a mycelial mat in soil beneath the sporocarps (Kumar and Gautam 2017). This genus comprises more than 200 species distributed worldwide (Ghosh et al. 2021). Ramaria flava (Schaeff.) Quél. and R. stricta (Pers.) Quél., are common edible coralloid fungi (Krupodorova and Sevindik 2020). Ramaria species have been identified through scanning electron microscopy and molecular techniques by Martin et al. (2020). Internal transcribed spacer (ITS) restriction length polymorphism was observed among Ramaria species and used as a diagnostic tool for characterization (Nouhra et al. 2005). The regions of Azad Jammu & Kashmir are enriched in terms of macro-fungal diversity.

Ramaria abietina (Pers.) Quél., R. aurea (Schaeff.) Quél., R. apiculata (Fr.) Donk, R. botrytis (Pers.) Bourdot, R. flava (Schaeff.) Quél., R. flavescentoides Hanif & Khalid, R. formosa (Pers.) Quél., R. pallida (Schaeff.) Ricken, R. soluta (P. Karst.) Corner and R. stricta (Pers.) Quél. have previously been reported from Pakistan (Ahmad et al. 1997; Khalid 2022). Previously, only Ramaria stricta (Pers.) Quél. has been reported from AJK, based on morpho-anatomical characters (Gardezi 2005). In current study, R. barenthalensis is reported as new record from Neelum valley and AJK, Pakistan to increase the knowledge about its distribution and phylogeny.

Materials and methods

Sampling sites description

The study area state of Azad Jammu and Kashmir lies between longitude 73–75°E and latitude 33–36°N and comprises an area of 13,297 km2 (Khan 2008). Neelum valley is the largest district of AJK having an area of 13,297 km2. The climate is temperate (Average -2.0 °C) with very cold winter, moderate summers (Average temp. 37.0 °C) and average rainfall 165 cm, annually. This area is mostly hilly, and covered with dense forests of conifers. Soil is loamy to sandy loam, helping in better growth of forests. The main vegetation of the area includes Pinus wallichiana A.B.Jacks., Abies pindrow (Royle ex D.Don) Royle, Cedrus deodara (Roxb.) G.Don, Aesculus indica (Wall. ex Cambess.) Hook. and shrubs like Viburnum grandiflorum Wall. ex DC., Indigofera heterantha Wall. ex Brandis and Betula jacquemontii Spach.

Collection and micro-morphological characterization

Fresh basidiomata were collected from selected sites in Neelum valley, AJK, Pakistan, during 2019–2020 through consecutive field surveys (Fig. 1). Photography with proper tags and field notes of collected specimens were made during field visits in fresh conditions. Specimens were air-dried dried and preserved in polythene zipper bags for further studies. All studied specimens were deposited in the LAH Herbarium of Institute of Botany, University of the Punjab, Lahore.

Figure 1. 

Map of collection site Neelum valley, AJK, Pakistan.

Slides were mounted in 5% KOH (w/v) and 1% Congo red to study anatomical features, examined by using a light microscope (MX4300H, Japan) at 100× magnification: size and shape of basidiospores, basidia, cystidia and other structures. For basidiospores and other structures at least 50 measurements were made and fungal specimen names with authorities were retrieved from Index Fungorum (http://www.indexfungorum.org).

DNA extraction, sequencing and phylogenetic analysis

DNA was extracted from dried samples using a modified CTAB method as proposed by Bruns (1995). Amplification of the ITS nrDNA region was carried out using universal primer sequences (ITS1/ITS4), ITS1F (5’-CTT GGT CAT TTA GAG GAA GTA A-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) (White et al. 1990; Gardes and Bruns 1993). PCR products were sent to TsingKe, China for sequencing. The newly generated sequence was deposited in GenBank under accession number ON209680.

Consensus sequence was generated using the molecular tool BioEdit ver. 7.2.5 (Hall 1999). Most similar sequences were retrieved from NCBI using BLAST (https://www.ncbi.nlm.nih.gov/guide/) for the construction of a phylogenetic tree. Online MUSCLE tool (https://www.ebi.ac.uk/Tools/msa/muscle) was used for alignment of all the sequences. Maximum likehood analysis was performed on CIPRES (Miller et al. 2010) using the RAxML-HPC2 with 1000 bootstrap replicates (Stamatakis 2014). Figtree ver. 1.4.2. software was used for phylogenetic tree visualization and exported to Adobe illustrator for final editing.

Results

Taxonomy

Ramaria barenthalensis Franchi & M.Marchetti, Riv. Micol. 61: 199 (2019)

Figure 2

Description

Basidiomata 5–12 cm in height, 4–9 cm wide with a well-developed branched mycelium. Branches vertically oriented, furcated, elongated to flattened, smooth, light brown when young and dark brown at maturity. Base variable, sometimes reduced, and well-developed, whitish to brown. Flesh whitish to light brown and soft. Odor is not distinctive and pleasant. Basidiospores [50/5/2] 6.5–9 × 3–4.6 µm, elliptical to ovoid, roughened. Basidia 45–59 × 7.5–9 µm, sterigmate. Cheilocystidia 46–62 × 7–8.5 µm, elongated. Clamp-connections are present. Trama hyphae are thick-walled with clamps. Hyphae in basal tomentum are smooth, with a tapering base (Fig. 2). Comparisons in morpho-anatomical characteristics with other described species of Ramaria are also given (Table 2).

Figure 2. 

A Basidiomata of Ramaria barenthalensis (TS-40) B basidia and basidiole C–H basidiospores I clamp connections J trama hyphae. Scale bars: 3.6 cm (A); 12 µm (B); 6.7 µm (C–H); 13 µm (I); 12.8 µm (J).

Table 1.
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ITS dataset (Fig. 3) used in the current study on genus Ramaria. Our newly generated sequence shown in boldface.

Species Strain Location Substrate GenBank Accession Number
Ramaria sp. SD125.4 USA Fir forest DQ365646
Ramaria sp. OSC 81622 USA Fir forest EU652343
R. flavobrunnesens AY102864 USA Fir forest AY102864
R. barenthalensis AMB17386 Spain mixed forest MK493039
R. barenthalensis AMB17381 Spain Abies and Piecea forest MK493038
R. barenthalensis T40 Kashmir Dead hardwood of conifers ON209680
R. edwinii ALV11173 Spain Abies and Piecea MK493034
Ramaria sp. RAM2 Srinagar J&K Abies forest MH930937
Ramaria sp. OSC 73311 USA Abies and Piecea mix wood forest KP658154
Ramaria sp. OSC 144044 USA Abies and Piecea mix wood forest KF206335
Ramaria sp. MHM312 Mexico NA EU569259
Ramaria sp. NVE 367 Amazonia NA KF937356
Ramaria sp. OSC 115803 USA NA KP658139
Ramaria sp. DB215-08/5 USA NA KT968608
R. flava AMB n. 17484 Italy Abies and Piecea MK581224
R. flava AMB 17393 Italy Abies and Piecea MK493035
R. flava AMB n. 17481 Italy Mix forest type MK557953
R. flava MA-Fungi 48072 Spain Abies and Piecea AJ408367
R. flava ZT Myc 55613 Italy NA KY626146
R. pseudoflava AMB 17390 Italy NA MK493045
R. pseudoflava AMB 17392 Italy NA MK493046
R. pseudoflava AMB 17391 Italy NA MK493044
R. flava MA-Fungi 48061 Spain Abies and Piecea AJ408364
Ramaria sp. OSC 61837 USA Mix forest DQ365602
Ramaria sp. OSC 134657 USA Mix forest JX310403
R. magnipes WTU-F-063057 USA Mix forest MK169351
R. magnipes WTU 063057 Italy Fir forest MK493040
R. gracilis OSC 134659 USA Abies and Piecea mix wood forest JX310399
R. gracilis OSC 112168 USA Abies and Piecea mix wood forest KY354745
Hysterangium crassirahis OSC 4860 USA Abies and Piecea mix wood forest MN809540
Table 2.
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Morphoanatomical comparisions of different Ramaria species with R. barenthalensis.

Ramaria abietina R. subaurantiaca R. stricta R. flava R. flaccida R. flavescens R. apiculata R. formosa R. barenthalensis
Basidiomata Basidiomata up to 12 cm high, 4.5–8 cm in width, erect, branched, tips pointed with glabrous surface. Basidiomata have densely crowded branches and sometimes with a fused base. Orange to brownish. Mature Fruit bodies branched, 4–12 cm high, 4–8 cm wide. Branches with tapered and pointed ends, mostly erect and slender. Basidiomata 10.5–20 cm in length with a width of 7–15 cm having numerous densely crowded branches. Mycelium, multi branched, slender and less or more week. Basidiomata branched and large up to 18.8 cm; clamps are present. Basidiomata much branched 4–9 cm tall, fan shaped, rounded, tips. Basidiomata up to 20 cm high and multi branched mycelium. 5–12 cm high, 4–9 cm wide with a well-developed branched mycelium, Vertically oriented, furcated, elongated to flattened, smooth.
Color Light pink to pinkish buff, pale yellow to light brown tips, darkened when bruised. A persistent light brown to light yellow color from early stage of development to final stage of maturity. Surface glabrous, pinkish-buff, tips of branches pale-yellow, buff brown when bruised. Brown to light yellow more dark to ochraceous with age. tips of branches tan to gold, no color change on bruising. Branches yellowish-brown when young, pale to pinkish brown at maturity. Branches Brown-light to yellow brown. Branches Light brown. The flesh is white to yellowish. Light brown when young and dark brown at maturity. Clamp connections present.
odor Slightly aromatic and bitter Aromatic Pleasant Pleasant Not known Not known Pleasant Distinctive
Stipe Sometime less developed and reduced. Up to 1–2 cm in length, 0.5–1.5 cm wide, color changes to light-brown on bruising. 1–2.5 cm in length, 0.5–1.5 cm in width, sometimes absent. When present, pallid and tomentose with a tough context, light-brown on bruising. Reduced and sometime developed up to 2 cm, flesh whitish to light brown. Stipe, 50–80 mm long with a width of 40–50 mm. Base of stipe, whitish, pale-yellow to reddish brown on bruising. Medium stipe 1.5–2 cm high and whitish. Stipe large, 2.5–6.6 cm with a width of 1.5 2 cm, whitish. Stipe large, 1.5–4 cm with a width of 1–1.5 cm, whitish. Stipe large, 2–4.5 cm with a width of 1.5–18 cm, light brown-whitish. Stipe variable in length and width, sometimes reduced to well-developed. Flesh whitish to brown.
Spores 6.5–9.5 µm in diameter, ellipsoidal, slightly warted. Spores 7–8.5 µm, ellipsoidal. Elliptical, rough, rusty-yellowish 7.5–10.5 × 3.5–4.5 µm, clamp connections present. pale elliptical and roughened, 10.5–17.5 × 4–6.5 µm. Pale elliptical less to more roughened, 9–14.5 × 3–4.5 µm. Thick walled, minute and rough outer surface, cyanophilous warts, ellipsoidal, tips squared, 7–10×3.5–5 µm. less to more roughened, with cyanophilous warts; ellipsoidal with a squared tip; 7–10×4–5 µm. slightly roughened, with cyanophilous warts; ellipsoidal and squared tip; 7.5–9×3–3.5 µm. 6.5–9 × 3–4.6 µm, elliptical to ovoid, roughened.
Edibility Edible Edible Edible on choice Edible Edible on choice sometimes laxative Edible Not known Not known Edible when young
Taste Pleasant Taste mild to pleasant bitter Taste mild to pleasant Pleasant Pleasant Taste mild to pleasant Bitter/mild Pleasant
Ecology Solitary and gregarious, on hardwood and tree trunks of conifers. Solitary and in small groups on hardwood and logs, soil and decomposed organic matter. Growing in groups, under conifers and mixed vegetation, on dead wood and on ground. Solitary and gregarious. Fruiting season varies from late summer to autumn. Grow on the ground. In mixed forest and hardwoods. Late autumn. Solitary to gregarious, on decomposed hardwood in association with rotten conifer needles. Gregarious, on soil with decomposed organic matter and conifer needles. Gregarious, on soil with decomposed organic matter and conifer needles. Gregarious and sometime alone, on fallen tree turns of coniferous and other mixed vegetation during late summer.
References (Petersen 1969; Sultan et al. 1997) (Petersen 1989; Ahmad et al. 1997) (Petersen and Olexia 1967; Petersen 1969; Ahmad et al. 1997) (Petersen 1969; Ahmad et al. 1997) (Ahmad et al. 1997) (Ahmad et al. 1997) (Ahmad et al. 1997) (Kuo 2005) Present study
Basidiomata of different species of Ramaria
Ramaria abietina R. subaurantiaca R. stricta R. flava R. flaccida R. flavescens R. apiculata R. formosa R. barenthalensis

Ecology

In mixed coniferous forest, associated with Abies pindrow and Betula jacquemontii, solitary or scattered and gregarious, common in the temperate zone of AJK, Pakistan.

Material examined

Pakistan, Neelum valley (Azad Jammu and Kashmir) collected from a mixed coniferous forest on decayed hardwood, in late summers, 1524 m a.s.l, August 2020, Tariq Saiff Ullah, TS-40.

Phylogeny

The newly generated ITS sequence of Pakistani Ramaria (T40) specimen, yielded a fragment of 702 base pairs. In initial BLAST search results, our sequence showed 99.33% similarity with R. barenthalensis MK493039, with 98% query cover, and 0.0 E value. The phylogram comprised 30 sequences and Hysterangium crassirh Zeller & C.W. Dodge was chosen as an out-group taxon. The final aligned dataset consist of 421 conserved sites, 256 were variable, 167 were informative and 86 were singletons. Our sequence Ramaria barenthalensis (T40) grouped with R. barenthalensis (MK493038 and MK493029) with strong bootstrap value (Fig. 3).

Figure 3. 

Molecular Phylogenetic tree of Ramaria barenthalensis (ON209680) by maximum likehood method based on ITS sequences. The sequence generated in this study represents in bold.

Discussion

In present study, a specimen of genus Ramaria was studied on the basis of morpho-anatomical and molecular approach. Our study is consistent with the original specimen description given by Franchi and Marchetti (2019). In phylogram, our specimen grouped in the same clade with R. barenthalensis (MK493038 and MK493039) with a strong bootstrap value.

The analyzed sample of R. barenthalensis (T-40) has similar morphological features, but with slight differences in basidiomata to other previously descibed species of Ramaria (Agerer et al. 1996). Few species of genus Ramaria: R. apiculata, R. flava (Schaeff.) Quél., R. flavescens (Schaeff.) R.H.Petersen, R. flavescentoides, R. formosa, R. pallida (Schaeff.) Ricken, and R. subaurantiaca Corner, have been reported from the regions of Jammu and Kashmir, in India and Pakistan based on morphological features (Ahmad et al. 1997; Nasim et al. 2008; Sharma et al. 2015; Hanif et al. 2019). Morpho-anatomical features are insufficient to identify a mushroom species. Fifteen species of Ramaria were identified and characterized by Martin et al. (2020) using combined morphological and molecular data. In conclusion, R. barenthalensis is recorded as a new coralloid fungus to the state of Jammu and Kashmir, Pakistan based on morpho-anatomical and molecular characterization. This fungal species could be used as a source of food and medicines after biochemical characterization.

Acknowledgements

The first author is thankful to Javaid Qayum Swati and Muhammad Usman Mughal for their help during field and lab work, respectively.

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