Macrofungal diversity in the Western Ghats , Kerala , India : members of Russulaceae

Acknowledgements: The author wishes to thank MoEF, Government of India, New Delhi, for financial assistance for carrying out the research. Field support provided by the staff of Kerala Forest Department and technical assistance by Mr. P.M. Sumesh, Mr. P. Rajesh and K.B. Anila are also acknowledged. Macrofungi are an important component of the forest ecosystems and play a major role in ecosystem dynamics, such as litter decomposition, nutrient cycling and nutrient transport. Most macrofungi are saprobes, a few are ectomycorrhizal and some are pathogens of woody plants, insects and fungi. Many of them are edible, while a few are hallucinogenic, medicinal, toxic or poisonous. Macrofungal diversity, as with all other subsects of biodiversity, exhibits distinct patterns in both space and time. Such fungal diversity patterns are, to a large extent, unexplored. Recently, systematic studies were undertaken by the authors to understand the ecology, diversity, distribution, taxonomy and economic potential of macrofungi in different forest ecosystems in the Western Ghats. The study reveals that the Western Ghats is endowed with a remarkably rich macro-fungal flora. Moist-deciduous and semi-evergreen forests support a maximum number of macrofungi followed by evergreen and shola forests. Macro-fungal assemblage, species abundance and frequency were very characteristic of shola forests and Myristica swamps. Terricolous, humicolous and lignicolous macro-fungi form the major group. A total of 166 genera and 550 species falling in 51 families belonging to Basidiomycota and Ascomycota were encountered and of these, a large number of genera and species are reported for the first time from India (Mohanan 2011). In the present paper, taxonomical, ecological and diversity details of members of Russulaceae, viz., 13 species of Russula and two species of Lactarius are dealt with. Abstract: A macrofungal biodiversity inventory carried out in different forest ecosystems viz., west coast tropical evergreen forests, west coast tropical semi-evergreen forests, south Indian moist deciduous forests, southern subtropical broadleaved hill forests, southern montane wet temperate forests (shola forests), southern tropical dry deciduous forests, grasslands, Myristica swamp forests, and forest plantations falling in different forest divisions in the Western Ghats, Kerala employing opportunistic as well as fixed-size plot sampling methods from 2006–2011 yielded several rare and hitherto unrecorded macrofungi. In Russulaceae 15 species of macrofungi belonging to the genera Russula and Lactarius were recorded. Of these, 12 species of Russula viz. Russula aciculocystis, R. adusta, R. atropurpurea, R. cinerella, R. congoana, R. delicula, R. hygrophytica, R. luteotacta, R. mariae, R. martinica, R. michiganensis and R. periglypta and white coloured latex exuding Lactarius nebulosus are new records for the Western Ghats. All the Russulaceae members exhibit an ectomycorrhizal association with tree species like Hopea ponga, H. parviflora, Myristica malabarica, Vateria indica, Calophyllum apetalaum, among others.

Macrofungi are an important component of the forest ecosystems and play a major role in ecosystem dynamics, such as litter decomposition, nutrient cycling and nutrient transport.Most macrofungi are saprobes, a few are ectomycorrhizal and some are pathogens of woody plants, insects and fungi.Many of them are edible, while a few are hallucinogenic, medicinal, toxic or poisonous.Macrofungal diversity, as with all other subsects of biodiversity, exhibits distinct patterns in both space and time.Such fungal diversity patterns are, to a large extent, unexplored.Recently, systematic studies were undertaken by the authors to understand the ecology, diversity, distribution, taxonomy and economic potential of macrofungi in different forest ecosystems in the Western Ghats.The study reveals that the Western Ghats is endowed with a remarkably rich macro-fungal flora.Moist-deciduous and semi-evergreen forests support a maximum number of macrofungi followed by evergreen and shola forests.Macro-fungal assemblage, species abundance and frequency were very characteristic of shola forests and Myristica swamps.Terricolous, humicolous and lignicolous macro-fungi form the major group.A total of 166 genera and 550 species falling in 51 families belonging to Basidiomycota and Ascomycota were encountered and of these, a large number of genera and species are reported for the first time from India (Mohanan 2011).In the present paper, taxonomical, ecological and diversity details of members of Russulaceae, viz., 13 species of Russula and two species of Lactarius are dealt with.

Materials and Methods
Study areas were selected in the major types of forests, such as, western coast tropical evergreen forests, western coast tropical semi-evergreen forests, southern Indian moist deciduous forests, southern subtropical broadleaved hill forests, southern montane wet temperate forests (shola forests), southern tropical dry deciduous forests, grasslands, Myristica swamp forests, and forest plantations falling in different forest divisions in the Western Ghats of Kerala.Opportunistic sampling of macrofungi was carried out from as many habitats in the areas as possible.Fixed-size plot sampling (three sample plots of 100x100 m in four locations, namely, Nilambur (moist deciduous forests), Iringole (semievergreen forests), Nelliampathy (evergreen forests), Mannavan Shola (shola forest) was also carried out to yield quantitative data.Sampling was carried out during both south-west and north-east monsoons and preand post-monsoon periods.These activities included: collecting sporocarps of macrofungi at the field site, labelling them, taking photographs, chemical spot tests, setting up spore prints, recording macro-morphological data in the illustrated data sheet, writing morphological descriptions, details on substratum, ectomycorrhizal status, host plants associated, processing the specimens for recording microscopic characters and identification of the specimens up to species level.Quantitative data on macrofungi were also recorded from the sample plots periodically for biodiversity analyses.Colours and chemical reactions of tissues and spores in water, KOH, NH 4 OH, FeSO 4 , HCl, melzer's reagent, etc. were studied and used for identification of the taxa.Digital photomicrographs of the fungal structures were also made.The recent taxonomic concept based on the emerging information on molecular phylogenetic data has been followed and taxa have been rearranged accordingly (Kirk et al. 2008).

Results
In the present study, 13 species of Russula and two species of Lactarius were collected and identified up to the species level from different forest ecosystems of the Western Ghats, Kerala.Taxonomic details, distribution and ectomycorrhizal status, for each species are given below.Basidiome small to medium sized.Pileus 4-6.5 cm dia., hemispherical or convex with incurved margin when young, soon broadly convex to plane, finally plano-depressed; surface vivid red, violet brown (11E7) at centre, sticky when wet, sulcate striate at the margin, cuticle separable one-half to two-thirds way to disk, exposing the white or pale pinkish context below.Lamellae decurrent, close to sub-distant, forked near stipe, lamellulae rare, ventricose, up to 4mm broad at mid radius, white when young soon pale buff; edge minutely fimbriate.Context white, unchanging.Stipe 2.5-5 cm x 8-12 mm, central, equal or tapered towards base, stuffed becoming hollow; surface white to pale pink, smooth.Annulus absent.Odour agreeable.
Russula aciculocystis is distributed in semi-evergreen to evergreen forests and occurs solitary or scattered on soil under Hopea ponga, H. parviflora, Myristica malabarica, Vateria indica, and Calophyllum apetalaum forming an ectomycorrhizal association.Basidiome medium to large sized.Pileus 4-8 cm dia., with a deep depression at centre at maturity; margin inrolled when young, becoming uplifted when fully expanded; surface dull white becoming grayishwhite then dark brown and finally black on handling or ageing, non-striate.Lamellae adnate, white turning black on handling, more or less crowded with lamellulae of different lengths, sometimes bifurcated close to the stipe; edge smooth, entire, stipe 3-6 cm x 1-2 cm, central equal, solid; surface chalky white, immediately turning black when handled.Odour not distinctive.Context white, discolouring blackish on exposure, heteromerous with nests of sphaerocytes.
Russula adusta is distributed in semi-evergreen to evergreen forests and occurs solitary or scattered on soil under Hopea ponga, H. parviflora, Myristica malabarica, Vateria indica, and Diospyros malabarica forming an ectomycorrhizal association.This species is characterized by blackening all the parts of the basidiome with age.Basidiome small to medium sized.Pileus 3-8 cm dia., subglobose to convex, finally depressed, often lobed; surface bluish-red (12A7) to vivid red (11B6), yellowishred at the centre, weakly viscid when moist, shiny, smooth, not striate.Lamellae adnexed to adnate, white to pale pinkish, moderately crowded, with lamellulae, occasionally furcated.Stipe 2-5.5 cm x 8-16 mm, firm, cylindric, solid surface white with pinkish tints or grayish when moist.Context thick and firm white containing groups of large sphaerocytes; taste more or less acrid.
Russula congoana is distributed in moist-deciduous to evergreen forests and occurs solitary or scattered on soil under Hopea ponga, H. parviflora, Myristica malabarica, Vateria indica, and Diospyros malabarica forming an ectomycorrhizal association.This is a very striking species easily recognized by the pastel red pileus and the heavy ornamentation of the spores.This species is edible.Basidiome small to medium sized.Pileus 3-6 cm dia., convex becoming uplifted and infundibuliform at maturity; margin inrolled when young, becoming uplifted when fully expanded; surface yellowish-grey (3B2) with grayish-orange (5B3) tints, viscid when wet, otherwise dry, non-striate.Lamellae subdecurrent, white, up to 4mm broad, often forked at or near the stipe, interveinose, white to pale buff, close, without lamellulae; edge smooth, entire.
Russula hygrophytica is distributed in semi-evergreen to evergreen forests and sacred groves of the state.This species occurs solitary or scattered in large groups on soil often forms a ring under Vateria indica, Hopea parviflora stands.Basidiome small to medium sized.Pileus 3-6 cm dia., fleshy, convex then expanded with a central depression; surface uniformly ivory (4B3) to grayish-white, areolately cracked forming patches of ivory squamules on a off white ground, entire at the disk; margin radially plicatostriate for two-thirds of radius from the margin and cracking along radial striae to expose underlying white context below, gelatinized under wet weather.Lamellae adnexed to subdecurrent, white, up to 4mm wide, crowded, lacking lamellulae.Context white, up to 4mm wide, heteromerous, with thin-walled, hyaline hyphae, 2-6 µm wide, intermixed with sphaerocytes, 16-24 µm.Stipe 2-5 cm x 5-15 mm, central cylindric, equal, solid becoming stuffed; surface white, smooth.Annulus absent.Odour not distinctive.Taste slightly acrid.

Russula leelavathyi
Spores 6-7.5 x 4.5-6.5 µm, subglobose to broadly ovoid, hyaline with an ornamentation of coarse, amyloid, verrucae and scattered fine connectives forming a partial reticulum.Basidia clavate, 45-60 x 9-11 µm, 4-spored.Lamella-edge sterile; cheilocystidia 33-35 x 10-12 µm, clavate fusoid, lageniform, often with acutely pointed or mucronate apex.Macrocystidia 70-90 x 5.5-7.5 µm, ventricose, fusoid to acuminate, thin-walled, with granular contents, numerous both on sides and edges of the lamellae.Hymenophoral trama heteromerous composed of thin-walled, hyaline hyphae, intermixed with sphaerocytes.Pileipellis distinctly two layered; an upper epithelial layer and a lower loosely interwoven gelatinized layer.Stipitipellis with abundant caulocystidia, 20-40 x 7-12 µm, similar to cheilocystidia Russula leelavathyi is distributed in moist-deciduous to evergreen forests and occurs solitary or scattered in large groups on soil under Hopea ponga, H. parviflora, Vateria indica, and Diospyros malabarica forming an ectomycorrhizal association.The furcated lamellae together with lack of lamellulae are characteristic features of this species.This species is closely related to R. moyersoeni Buyck, in the overall habit and pigmentation but can be distinguished by virtue of the white pileus, smaller spores with a finer ornament, and lack of pileal macrocystidia and presence of caulocystidia.Basidiome small to medium sized.Pileus 5-6 cm dia., fleshy, convex with a broad depression at the centre; surface greyish-red (9B6) to red (9B7) sometimes with white tints at places, sticky when wet, translucent striate at the margin, pellicle peels off easily up to mid radius; margin crenate, incised.Lamellae adnexed, appearing free when mature, pale yellow, crowded with out lamellulae, rarely bifurcated.Context white, unchanging, brittle.Stipe 3-4.5 cm x 5-9 mm, central, slightly broader at the base, stuffed; surface white, smooth with a faint red colour at apex and middle.Annulus absent.Odour agreeable.Spore-print pale yellow in mass.
Russula martinica is distributed in moist-deciduous to evergreen forests and sacred groves of the state.This species occurs solitary or scattered in large groups on soil under Hopea parviflora stands.
Lactarius nebulosus is widely distributed in semievergreen to evergreen forests of the Western Ghats, Kerala, and occurs solitary or scattered in small clusters on soil.

Discussion
The family Russulaceae contains 1243 species (Kirk et al. 2008).The family is dominated by the two well-known genera Russula and Lactarius.Members are widespread from the Arctic and Antarctic regions through to the tropics, both in the old and new world.The genera with gasteroid or hypogeous gasteroid basidiomes have traditionally been separated from the agaricoid genera.However, recent molecular analysis has shown that the derived gasteroid form is relatively recent and appears to justify an integration of the gasteroid taxa in the agaricoid genera, Russula and Lactarius (Binder & Bresinsky 2002).Further, as more tropical taxa are discovered the generic boundaries in the family are eroded.The preliminary study indicates the strong likelihood that Lactarius evolved from Russula, that the two genera may be very closely related in the Russula brevipes-Lactarius piperatus area, and the previous suggestions that the associated gasteroid genera belong in the family are correct (Miller et al. 2001;Eberhardt 2002;Binder & Bresinsky 2002;Miller & Buyck 2002;Moncalvo et al. 2002).Presence of large spherical cells, 'sphaerocysts' in stipe is an important characteristic feature distinguishing the members of Russulaceae from other mushrooms.In Russula and Lactarius, the stipe breaks like the flesh of an apple, whilst in most other families it only breaks into fibres.
The genus Russula sometimes known as 'brittle gills', comprises about 750 species, the majority of which are quite difficult to identify.They have splitting gills and do not exude a milky substance at cut surfaces, contrary to the genus Lactarius.There are several edible species, while a few are toxic, such as red caped species like R. emetica, R. sordonia, R. nobilis, etc.Most of the species form an ectomycorrhizal association with native trees.So far, only two species, Russula indica and R. leelavathyi have earlier been reported from Kerala (Sathe & Daniel 1980;Vrinda et al. 1997).Of the 13 species of Russula recorded herein 12 species, namely, Russula aciculocystis, R. adusta, R. atropurpurea, R. cinerella, R. congoana, R. delicula, R. hygrophytica, R. luteotacta, R. mariae, R. martinica, R. michiganensis and R. periglypta are new records for Kerala.
The genus Lactarius or 'milk caps' is a large genus of ectomycorrhizal fungi, characterized by caps and stalks that exude a milky latex when cut; this liquid may be white, yellow, orange, red, or lilac, and may develop its final colour only after exposure to air.Most of the species form an ectomycorrhizal association with native tree species.A few are edible.So far, only Lactarius ignifluus, a salmon coloured latex exuding species has earlier been reported from Kerala (Pradeep et al. 2002).Lactarius nebulosus, a white coloured latex exuding species, recorded herein is a new record for the Western Ghats.