Ramaria barenthalensis a new record from western Himalayas, Azad Jammu and Kashmir, Pakistan

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


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.

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 km 2 (Khan 2008). Neelum valley is the largest district of AJK having an area of 13,297 km 2 . 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.
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.
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 welldeveloped. Flesh whitish to brown.

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).

Discussion
In present study, a specimen of genus Ramaria was studied on the basis of morphoanatomical 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.