Two new varieties of Russula Pers. (Basidiomycota: Russulaceae) from Sal forests of Shiwaliks, India

: This paper deals with two new varieties of Russula species, R. camarophylla var. reticulospora var. nov. and R. aurea var. minuta var. nov. These were collected from the Shiwalik range of northwestern India, in association with Shorea robusta . Russula aurea var. minuta differs from R. aurea in having small sized sporophores, dentate to wavy gill edges with golden or yellow deposition instead of smooth and much smaller spores. Whereas, mushroom R. camarophylla var . reticulospora is close to Russula camarophylla except for the larger carpophores that have white cream pileus surface and larger spores. In basidiospores warts are connected to form mostly complete reticulum instead of mostly isolated warts reported in Russula camarophylla . In view of the presence of some unique varied features in the presently examined collections two new varieties of Russula has been proposed.


INTRODUCTION
Genus Russula is one of the dominant basidiomycetous fungi genus which grow in a wide variety of habitats. These are mostly found in mycorrhizal association with variety of plants including trees (Corrales et al. 2016). Studies on taxonomy and diversity of genus Russula are inviting more attention now a days primarily because of their importance in human welfare, ecosystem functioning and stability. These macro-fungi are important source of food, medicine, nutraceuticals and also play a pivotal role in ecosystem strengthening and maintenance as mycorrhizal associates (Manoharachary et al. 2005). It is reported that Russula spp. can form EcM with many temperate and tropical plant families, including Leguminosae, Fagaceae, Cistaceae, Dipterocarpaceae, Salicaceae, Betulaceae, Nothofagaceae, Myrtaceae, and Pinaceae (Tedersoo et al. 2010;Wang et al. 2017). The compounds derived from these mushrooms are reported to boost up immune system and avert diseases thereby improving human health (Wasser 2002). Different species of Russula are known to possess anti-inflammatory, antiviral, antibacterial, antiparasitic, antioxidant, hepatoprotective, anticancer, and antidiabetic properties (Wasser 2011).
To date, approximately 1,100 Russula species have been reported worldwide (Kirk 2014) and distributed across a wide range of habitats from the tropics to arctic zones (Riviére et al. 2007;Ba et al. 2012). Russula is one of the dominant ectomycorrhizal genera in Indian Himalaya (Saini & Atri 1984, 1989Atri & Saini 1986;Atri et al. 1994;Kumar & Atri 2016Sharma et al. 2016) and is represented by ca. 158 taxa from India . While investigating the EcM diversity of Sal forest, two varieties of R. camarophylla Romagn. and R. aurea Pers. were documented, which upon investigation were found to be new to science based on detailed macro-and micro-morphological examination. In the present study sporocarps and their EcM colonised roots were collected by tracing the hyphal or rhizomorphs connections in association with Shorea robusta from pure Sal forests. These species are fully illustrated and described in this paper.

Study area
Area selected for the present investigation is Sal forests of Shiwalik mountain range of northwestern India (Figure 1), which represent the geologically lowest and youngest mountain range of Himalaya. The study area is located between 30.316N, 78.032E. Elevation range of the area is 400-1500 m and vegetation of the area is typical of tropical moist deciduous type (Champion & Seth 1968).

Sampling, identification and characterization
Sporocarps were collected from different localities of pure Sal forests, during the rainy season of 2013-2015. Macromorphological features were recorded from fresh collections in the field and colour codes used are that of Kornerup & Wanscher (1978). After noting down morphological characters on the field key (Atri et al. 2005) some pieces of sporocarps from cap and stipe were preserved in liquid preservative (25 ml rectified alcohol (95%) + 5 ml formalin (37%) + 70 ml distilled water) for studying the microscopic characters. By adopting the standard procedures spore deposit was taken after bringing the specimens to the temporary laboratory setup. Sporocarps were air dried at 40-45 0 C in a drier specially designed for drying mushroom specimens (Atri et al. 2005) which were finally packed in a cellophane paper packet for permanent preservation in Punjabi University Herbarium under PUN. The cross section of pileus and longitudinal section of stipe were stained in congo red for examination, drawn under a compound microscope and photographed under digital microscope (Leica DM4000 B LED). Observation of basidia, cystidia, and elements of pileipellis and stipitipellis were recorded for further use in taxonomic categorization. Melzer's reagent was used to observe the amyloidy in basidiospore ornamentation. The microscopic details were worked out as per standard methodology (Singer 1986;Atri et al. 2000Atri et al. , 2017.

Scanning electron microscopy
Scanning electron microscopic (SEM) studies of basidiospores were carried out with JSM6610LV GEOL scanning electron microscope. For SEM examination basidiospores from spore print and lamellae tissue were mounted on a double-sided adhesive tape pasted on a metallic specimen holder or stub. The material was scanned at different magnification ranging 3,000-15,000 X in high vacuum mode to observe pattern of spore ornamentation.

Russula aurea Pers. var. minuta var. nov.
(Image 1a-h, Figure 2A-G) MycoBank number: MB834095 Diagnosis: Russula aurea Pers. var. minuta var. nov. is characterised by small golden to brightly yellow pileus with more darker brownish-yellow centre; dentate to wavy gill edges with golden deposition instead of smooth; sour taste , much smaller spores size and presence of pilocystidia.
Etymology: The variety name is based on the smaller size of sporophore and basidiospore as compared to Russula aurea. Holoype

Taxonomic description
Sporophores 2.0-2.5 cm in height. Pileus 1.3-2.0 cm broad, convex to hemispherical when young, flattened depressed at maturity; centre umbonate when young, golden (6C7) to brightly yellow with more darker brownish-yellow centre; margin regular to slightly irregular, nonsplitting at maturity, moist, unchanging, apex depressed at maturity with slight umbo; cuticle half peeling; flesh 0.1 cm thick in the centre, almost absent along the margin, white (1A1), changes to light brown on bruising and cutting, brittle; taste sour, odour mild. Lamellae adnexed to slightly adnate, equal, moderately broad (2-3 mm), crowded (12-16 gills/cm), white with golden edges; gill edges not smooth, eroded or wavy.
Stipe central, 1.5-2.0 cm in length, 0.3-0.5 cm broad, cylindrical to slightly tapering downward, white (1A1) in the upper half, yellowish to pale white in the lower half, unchanging, first solid, than hollow, smooth. Spore deposit deep ochre.
Habitat: Sporophores directly attached to the roots at the base of Shorea robusta tree. Remarks: The overall diagnostic characters of the presently examined collection are in agreement with Russula aurea (Rayner 1970;Romagnesi 1967;Das & Marstad 2014) except that the carpophores are much smaller in size (2.0-2.5 cm instead of 4-9 cm), gill edges not smooth (dentate to wavy with golden or yellow deposition instead of smooth), much smaller spores size (5-7.5 × 4-6 µm instead of 7.5-10 × 6-8 µm) and presence of pilocystidia which are absent in case of Russula aurea. In view of the presence of some unique varied features in the presently examined collections in comparison to R. aurea, a new variety minuta has been named.
Remarks: The external and internal characters of the presently examined collection are in agreement with Russula camarophylla (Romagnesi 1968) except that the carpophores are larger in size with cream white pileus surface and larger spore size (6.5-9 × 5-7.5 µm instead of 5-6.2 (7) × 3.6-4.8 µm). In basidiospores warts are connected to form mostly complete reticulum instead of mostly isolated warts in case of Russula camarophylla as documented in literature.

DISCUSSION
During the present study, R. aurea var. minuta and R. camarophylla var. reticulospora were found forming direct organic connection with Shorea robusta. The overall diagnostic characters of the presently examined collections of R. aurea var. minuta are in agreement with Russula aurea Pers. which is commonly known as the gilded brittle gill or golden Russula and is an uncommon species of mushroom found in deciduous woodland forests. Its specific epithet aurea has been derived from the Latin word aurum, which means golden. Unlike many red-capped members of the genus, Russula aurea is edible and mild-tasting and is easily characterised in the field by its golden pileus, free to adnexed broad fairly distant golden gills, cylindrical smooth light yellow stipe and brittle yellow flesh. Mostly it is reported to grow solitary or scattered forming mycorrhizal association with pine trees (Romagnesi 1967;Rayner 1970;Das & Marstad 2014). Russula aurea var. minuta differs from R. aurea except in having small sized sporophores, dentate to wavy gill edges with golden or yellow deposition instead of smooth, much smaller spores and presence of pilocystidia which are absent in case of Russula aurea. In view of this a new variety Russula aurea var. minuta has been proposed.
Russula aurora probably appears to be morphologically closest species to this undescribed taxon from which it differs in having fairly crowded pale cream lamellae with abundant forkation near the stipe, mild taste, and absence of dermatocystidia in pileipellis and low warted spores (0.25-0.5 µm) with few connections (Romagnesi 1967). Another close taxon is Russula aurantiaca which differs from R. aurea var. minuta in having usually brick-orange, copper to carmine coloured cap, widely spaced rather thick bright yellow ochre strongly interveined lamellae and presence of mild to slightly acrid taste (Romagnesi 1967). Earlier Russula aurea was known as R. aurata and under this name it was documented from different localities of northwestern Himalaya from coniferous and angiospermic forest (Saini & Atri 1984, 1989Atri & Saini 1986;Atri et al. 1994). The present collection is found in pure Sal forest in close vicinity to Shorea robusta tree from Uttarakhand.
Russula camarophylla, a rare western Mediterranean European representative of section Archaeinae is characterized by its camarophylloid habit, pale ochre or creamish sporophores with distant lamellae, very hard and compact flesh, hygrophoroid basidia and tiny spores with barely visible ornamentation (Romagnesi 1968). The present collection of R. camarophylla var. reticulospora is close to Russula camarophylla (Romagnesi 1968) except that the carpophores are larger in size with white cream pileus surface and larger spore size. In basidiospores warts are connected to form mostly complete reticulum instead of mostly isolated warts reported in case of Russula camarophylla (Romagnesi 1968). In view of this a new variety Russula camarophylla var. reticulospora has been proposed. The apical swelling of hyphal terminations in the pileipellis is an important feature that is very common within Russula camarophylla (Buyck et al. 2003) and presently examined collection. R. camarophylla var. reticulospora also resembles the recently described www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.