Research Article

Asterophora lycoperdoides (Bull.) Ditmar, a New Generic Record from Sub-Himalayan Forests of Pakistan

Annum Razzaq1*, Zia Ullah2, Arooj Naseer1 and Abdul Nasir Khalid1

1Institute of Botany, University of the Punjab, Lahore, Pakistan; 2Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan.

Received | December 01, 2023; Accepted | December 20, 2023; Published | December 23, 2023

*Correspondence | Annum Razzaq, Institute of Botany, University of the Punjab, Lahore, Pakistan; Email: annumrazzaq@gmail.com

Citation | Razzaq, A., Z. Ullah, A. Naseer and A.N. Khalid. 2023. Asterophora lycoperdoides (Bull.) Ditmar, a new generic record from Sub-Himalayan Forests of Pakistan. Biologia (Lahore), 69(2): 61-65.

DOI | https://dx.doi.org/10.17582/journal.Biologia/2023/69.2.61.65

Keywords | Bhurban, ITS, Lyophyllaceae, Punjab, Taxonomy

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The genus Asterophora Ditmar (Lyophyllaceae) is commonly known as fungicolous fungi or mycoparasitic fungi. These fungicolous fungi are diversified, trophic and ecological groups of organisms associated with other fungi such as symbionts, saprotrophs and even neutrals (Sun et al., 2019). The species of Asterophora mostly grow on basidiomata of other fungi (common hosts include the species of family Russulaceae, notably genus Lactarius Pers. and Russula Pers.). Besides its unique habitat, the genus can easily be distinguished by the production of chlamydospores (Redhead and Seifert, 2001). According to an estimation, approximately 18 species of Asterophora have been described from all over the world so far (http://www.indexfungorum.org/).

Asterophora lycoperdoides (Bull.) Ditmar is one of the well-documented species of this genus and is mainly recognized by its habitat (on mushrooms); small, whitish, globose to pulvinate pileus that become brown and powdery by the formation of stellate chlamydospores and poorly formed gills. These chlamydospores are very large (often greater than 20 µm long), pigmented and profusely produced early, hence they are conspicuous and dominant (Redhead and Seifert, 2001). It is found in gregarious clusters on rotting Russula (especially the R. dissimulans-R. nigricans complex) and Lactarius spp.

The moist temperate forests of the Himalayan region of Pakistan are recognized as one of the twenty-five biodiversity hotspots (Myers et al., 2000). These forests are characterized by their lush vegetation and evergreen cover, host a diverse array of plant species, thus indicating an anticipated high diversity of fungal species. During a macrofungal survey conducted in various parts of the Himalayan forests in Bhurban, Punjab, Pakistan, a rare species Asterophora lycoperdoides was identified as thriving on decaying Russula species. This study provides a concise diagnosis, a detailed description and employs phylogenetic analyses based on ITS sequence data.

Materials and Methods

Sampling and morpho-anatomical characterization

A mycological investigation was carried out between August 2020 and September 2021 in the Himalayan temperate forests of Bhurban. These forests have high humidity and a wide range of elevations from 1800 to 2420 meters above sea level. Precipitation occurs throughout the entire year in the area, with an average rainfall of approximately 1800 mm (30 years period: World Weather Information Service, 2018). The vegetation in this region primarily consists of Pinus wallichiana B. Jacks, Quercus dilata Royle, and Cedrus deodara (Roxb. ex D. Don) G.Don. These conditions create an ideal environment for the proliferation of mycorrhizal fungi (Hameed et al., 2012).

Fresh basidiomata of the specimen were collected from two distinct locations (Golf club and Kashmiri Bazar) of Bhurban, (Coordinates: 33°56’26.24N to 73°26’59.48E; 1828 m a.s.l) Punjab, Pakistan. All the environmental factors like habitat, location and type of soil were noted and images were taken. The standardized color system given by Munsell’s (1975) was used to give color codes and Vellinga (2001) terminology was followed. A fan heater at 40–50°C was used to dry the samples which were later deposited in the LAH Herbarium, Institute of Botany, University of the Punjab, Lahore, Pakistan.

For the anatomical research, 5% KOH (w/v) was used for slide preparation. 1% aqueous solution of Congo Red and Melzer’s reagent was using the microscopic procedures of Liang et al. (2018). The anatomical characters were observed using a light microscope through an HDCE-X5 camera under 400X and oil immersion 1000X magnification. The measurements are described in description as (a)b–c(d), where ‘a’ is the lowest reading, ‘b–c’ are as a minimum 90% of readings, and ‘d’ is the highest reading, whereas ‘Q’ is the length to width ratio of basidiospore (Bas, 1969; Yu et al., 2020); and av. L × av. W describes the average length and average width.

DNA extraction, PCR and sequencing

DNA was extracted from dried herbarium specimens using a 2% CTAB procedure (Bruns, 1995), with minor changes given by Zhao et al. (2011). For the qualitative investigation of extracted DNA, gel electrophoresis was carried out by means of 1% agarose gel (Voytas, 2000). A thermocycler was used to amplify the extracted DNA using polymerase chain reaction. For molecular analysis, ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) primer pairs were used to amplify the Internal Transcribed Spacer (ITS) region (White et al., 1990; Gardes and Bruns, 1993). PCR was carried out in a thermocycler (Perkin-Elmer, Applied Biosystems) using a 25 μl reaction mixture following the conditions of Yan and Bau (2018). Bidirectional sequencing was done using the same pairs of primers. All the new sequences were deposited in GenBank (ITS OP160530; OP160531; OP537167; MG822871).

Phylogenetic analysis

Consensus sequences consisting of 610-650 bps were generated using BioEdit software version 7.0.4.1 (Hall, 1999). These consensus sequences were then subjected to BLAST searches on the NCBI (National Center for Biotechnology Information) platform (https://www.ncbi.nlm.nih.gov/). The closely related Asterophora sequences were retrieved from GenBank and were aligned using the online MUSCLE tool at EMBL-EBI (http://www.ebi.ac.uk/). For phylogenetic analysis of ITS dataset, Maximum likelihood (ML) bootstrap analyses were performed in MEGA11 that involved the utilization of the ultrafast bootstrap approximation, employing 1000 replicates.

Results and Discussion

Phylogenetic analysis

The aligned ITS dataset comprises 22 nucleotide sequences, which were subjected to BLAST searching against GenBank. The dataset includes sequences from Entoloma sericeum Quél. and E. sericeonitidum P.D. Orton Arnolds, serving as outgroup references (Bellanger et al. 2015). The ITS sequences of our collection showed 99.67 % similarity with sequences of Asterophora lycoperdoides (KP192566 from France, OM809276 and MZ159455 from the USA, LN714522 from Czech Republic). The newly generated sequences clustered in a clade with Asterophora lycoperdoides specimens that is in sister relationship with Asterophora mirabilis but with no support. The branch representing the Asterophora lycoperdoides specimens show the strong bootstrap value of 100%. Newly generated sequences are highlighted with a bullet (Figure 1).

 

Taxonomy

Asterophora lycoperdoides (Bull.) Ditmar. 1809: Pileus 3-25 mm, pin head size at initial stage, hemispherical convex at young stage, becoming broadly convex to plano convex at maturity; incurved margins; at first surface smooth, dry, non-striate, non-fibrillose rapidly becoming rough, powdery and fibrillose; whitish at young stage forming a prominent and bright appearance over decaying brown host, brown patches appeared on the surface of pileus and stipe with time and ultimately covered with brown or cinnamon colored mass by asexual spores (chlamydospores) formation. Hymenophore usually less developed or completely lacking; 0.3-2mm deep; when present normal, moderately crowded to distant, adnate to decurrent; white to pale or cream, concolorous with pileus at young basidiomata becoming brown or cinnamon colored by the formation of chlamydospores; edge even or entire. Stipe long and thin, 5‒60 × 4‒18 mm; central; cylindrical, often curved; whitish at young stage becoming pale brown at maturity; surface dry, dull, concolorous with pileus, with longitudinally appressed fibrils. Context white when young becoming cream in the pileus, stipe, and over the lamellae. Veil absent. Odor insignificant. Taste not recorded. Basidiospores very sparse and hard to obtain; chlamydospores cover the lamellae at mature stages. Chlamydospores or secondary spores abundant in the lamellar and pileal trama and even numerous on the host tissue, (12.3‒) 12.9–15.6 (‒15.9) × (11.3‒) 11.9–14.2 (‒14.4) µm, avI × avW = 14.3 × 13.2 µm; globose, thick-walled, starburst shape, hyaline to brown; containing granular contents, thick, conical and large projections 3‒6 µm; sometime obtuse or truncate apex (Figure 2). Stiptipellis a cutis of parallel cylindrical hyphae, 4‒18 μm, hyaline to pale yellow.

 

Material examined

Pakistan, Punjab province, Bhurban town, 1828 m asl; September 7, 2020, Annum Razzaq GB108 (LAH38040), GenBank (ITS; OP160530); September 10, 2020, Annum Razzaq GB111 (LAH38041), Golf club, 1828m asl; August 10, 2019, Annum Razzaq GC36 (LAH38042), GenBank (ITS; OP160530); clustered on pileus of a decaying Russula sp. under coniferous tree of sub-himalayan temperate forests.

The Asterophora is an uncommon genus and has received limited attention in available reports. Russula and Lactarius are the most common the host of this genus and produce edible fruiting bodies. These are frequently gathered from natural forests without a comprehensive understanding of the potential long-term consequences of such harvesting. Both Russula and Lactarius are ectomycorrhizal and serve as a food source for the local tribal communities.

In this study, a mycoparasitic mushroom, Asterophora lycoperdoides is described as a new generic record for Pakistan on morpho-anatomical and phylogenetic bases. The proper identification of Asterophora species is often difficult to perform as the taxon has rudimentary lamellae and does not produce basidiospores. However, the reproduction takes place through the formation of enormous stellate chlamydospores in the upper pileal trama, hence, giving the brown powdery appearance to the basidiomata (May and Fuhrer, 1995; Redhead and Seifert, 2001). The Pakistani collections of A. lycoperdoides show similar morphological features; like the shape and size of pileus and chlamydospores to the A. lycoperdoides described by Sharma et al. (2007) from India. The nrITS sequences of the present collections (GB111, GB108, GC36) phylogenetically cluster with A. lycoperdoides KP192566 (France), OM809276, MZ159455 (USA), LN714522 (Czech Republic) indicating the cosmopolitan distribution of this species. Previously, the genus has already been described from North America, Europe, Asia, North Africa and Papua New Guinea (Corner, 1966; Horak, 1980; Singer, 1986). However, no species of this genus has ever been described from Pakistan.

Conclusions and Recommendations

The forests of Pakistan hold substantial and yet unexplored mushroom biodiversity. Continued efforts in characterizing mycorrhizal genera are crucial for comprehending their significance as fungal symbionts. This understanding is essential for future management strategies aimed at ensuring ecosystem sustainability and productivity. In this context, this report marks the initial generic documentation of the species A. lycoperdoides in the temperate forests of Pakistan, contributing to the expanding mycoflora knowledge of the region.

Acknowledgement

We are grateful to Dr. Francis Brearley, Assistant professor, School of Science and The Environment, Manchester Metropolitan University, UK for his critical comments and suggestions to improve the manuscript.

Novelty Statement

By exploring the diversity of macrofungi, their phylogenetic relationship can be identified and will provide a baseline data to use macrofungi in applied sciences research.

Author’s Contribution

Annum Razzaq: Data curation, conceptualization, methodology, writing original draft, editing.

Zia Ullah: Collection, DNA extraction, PCR, sequencing.

Arooj Naseer: Supervision of formal analysis, supervision.

Abdul Nasir Khalid: Supervision, review and editing.

Conflict of interest

The authors have declared no conflict of interest.

References

Bas, C., 1969. Morphology and subdivision of Amanita and a monograph of its section Lepidella. Persoonia, 5(4): 96-97.

Bellanger, J.M., Moreau, P.A., Corriol, G., Bidaud, A., Chalange, R., Dudova, Z. and Richard, F., 2015. Plunging hands into the mushroom jar: A phylogenetic framework for Lyophyllaceae (Agaricales, Basidiomycota). Genetica, 143(2): 169-194. https://doi.org/10.1007/s10709-015-9823-8

Bruns, T.D., 1995. Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil, 170(1): 63-73. https://doi.org/10.1007/BF02183055

Corner, E.J.H., 1966. A monograph of canthareiloid fungi. A monograph of Canthareiloid fungi.

Gardes, M. and Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes–application to the identification of mycorrhizae and rusts. Mol. Ecol., 2(2): 113-118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x

Hall, T.A., 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser., 41: 95-98.

Hameed, M., Nawaz, T., Ashraf, M., Ahmad, F., Ahmad, K.S., Ahmad, M.S.A., Raza, S.H., Hussain, M. and Ahmad, I., 2012. Floral biodiversity and conservation status of the Himalayan foothill region, Punjab. Pak. J. Bot., 44: 143-149.

Horak, E., 1980. New and remarkable hymenomycetes from tropical forests in Indonesia (Java) and Australasia. In Sydowia; Annales mycologici.

Liang, J.F., Yu, F., Lu, J.K., Wang, S.K. and Song, J., 2018. Morphological and molecular evidence for two new species in Lepiota from China. Mycologia. 110(3): 494-501. https://doi.org/10.1080/00275514.2018.1464333

May, T.A. and Fuhrer, B.A., 1995. Nyctalis mirabilis (Fungi:Agaricales), a new species from Australia. Muelleria, 8: 385-390. https://doi.org/10.5962/p.198467

Munsell, A.H., 1975. Munsell soil color charts. Baltimore, MD, USA.

Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A.B. and Kent, J., 2000. Biodiversity hotspots for conservation priorities. Nature, 403: 853-858. https://doi.org/10.1038/35002501

Redhead, S.A. and Seifert, K.A., 2001. Asterophora Ditmar ex Link 1809 versus Nyctalis Fries 1825, and the status of Ugola Adanson 1763.Taxon, 50(1): 243-268. https://doi.org/10.2307/1224526

Sharma, R., Rajak, R.C. and Pandey, A.K., 2007. New Indian record of a rare fungus: Asterophora lycoperdoides. Biodiversity, 8(1): 21-26. https://doi.org/10.1080/14888386.2007.9712819

Singer, R., 1986. The Agaricales in modern taxonomy. 4th ed. Koeltz Scientific Books. Koenigstein.

Sun, J.Z., Liu, X.Z., McKenzie, E.H., Jeewon, R., Liu, J.K.J., Zhang, X.L. and Hyde, K.D., 2019. Fungicolous fungi: Terminology, diversity, distribution, evolution, and species checklist. Fungal Diver., 95(1): 337-430. https://doi.org/10.1007/s13225-019-00422-9

Vellinga, E.C., 2001. Agaricaceae. In: (eds. Noordeloos, M.E., Kuyper, T.W. and Vellinga, E.C.) Flora Agaricina Neerlandica Vol. 5. A.A. Balkema Publishers, Rotterdam.

Voytas, D., 2000. Agarose gel electrophoresis. Curr. Protocols Mol. Biol., 51(1): 2-5. https://doi.org/10.1002/0471142727.mb0205as51

White, T.J., Bruns, T., Lee, S. and Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: (eds. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J.) PCR protocols: A guide to methods and applications. Academic Press, New York, NY, USA. pp. 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

World Weather Information Service, 2018. https://worldweather.wmo.int/en/home.html

Yan, J.Q. and Bau, T., 2018. The Northeast Chinese species of Psathyrella (Agaricales, Psathyrellaceae). MycoKeys, 33: 85–102. https://doi.org/10.3897/mycokeys.33.24704

Yu, W.J, Chang, C., Qin, L.W., Zeng, N.K., Wang, S.X. and Fan, Y.G., 2020. Pseudosperma citrinostipes (Inocybaceae), a new species associated with Keteleeria from southwestern China. Phytotaxa, 450(1): 8–16. https://doi.org/10.11646/phytotaxa.450.1.2

Zhao, R.L., Karunarathna, S.C., Raspe, O., Parra, L.A., Guinberteau, J., Moinard, M., De Kesel, A., Barroso, G., Courtecuisse, R., Hyde, K.D., Guelly, A.K., Desjardin, D.E. and Callac, P., 2011. Major clades in tropical Agaricus. Fungal Divers., 51(1): 279-296. https://doi.org/10.1007/s13225-011-0136-7