Molecular identification of European genus Strobilurus from Hindukush region of Pakistan

Molecular identification of European genus Strobilurus from Hindukush region of Pakistan | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 February 2025 V1 Latest version Share on Molecular identification of European genus Strobilurus from Hindukush region of Pakistan Authors : Shahab Ali , Tauseef Ullah 0000-0002-0420-9739 [email protected] , Muhammad Fiaz , Abdul Khalid , Shujaul Khan , and Yu Zhongdong Authors Info & Affiliations https://doi.org/10.22541/au.173894771.19803959/v1 303 views 163 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The genus Strobilurus is prevalent in the Himalayan temperate forests of Pakistan, with sporocarps collected from the Hindukush region. Extensive field surveys spanning five years (2018-2022) were conducted in these locales, resulting in multiple collections of the present specimen. Identification of the specimen was accomplished through comprehensive morphological, anatomical, and molecular analyses. The primary macro- and micro-morphological characteristics of the Pakistani collection include a flat pileus with a rimose margin, varying from yellow to dark-yellow in color, free lamellae, strigos, smooth and reddish stipe, ovoid to ellipsoid basidiospores with a rounded apex, guttulate basidia bearing 2-3 sharp sterigmata, fusoid-ampullaceous cheilocystidia, fusoid-ventricose pleurocystidia, conical and thick-walled hyaline pilipellis, and branched and gutulllated stipitipellis. Morphological, molecular, and phylogenetic analyses collectively confirm the specimen’s identity as Strobilurus albipilatus, marking a new record for Pakistan. Molecular identification of European genus Strobilurus from Hindukush region of Pakistan Abstract The genus Strobilurus is prevalent in the Himalayan temperate forests of Pakistan, with sporocarps collected from the Hindukush region. Extensive field surveys spanning five years (2018-2022) were conducted in these locales, resulting in multiple collections of the present specimen. Identification of the specimen was accomplished through comprehensive morphological, anatomical, and molecular analyses. The primary macro- and micro-morphological characteristics of the Pakistani collection include a flat pileus with a rimose margin, varying from yellow to dark-yellow in color, free lamellae, strigos, smooth and reddish stipe, ovoid to ellipsoid basidiospores with a rounded apex, guttulate basidia bearing 2-3 sharp sterigmata, fusoid-ampullaceous cheilocystidia, fusoid-ventricose pleurocystidia, conical and thick-walled hyaline pilipellis, and branched and gutulllated stipitipellis. Morphological, molecular, and phylogenetic analyses collectively confirm the specimen’s identity as Strobilurus albipilatus , marking a new record for Pakistan. Keywords: Hindukush, molecular identification, mushrooms, phylogenetic tree, taxonomy The genus Strobilurus was erected by Singer (1962) that lies within Physalacriaceae family. Fungal specimen of this genus mostly founds in North America, Europe and Asia and blooms in subtropical or temperate habitat. Fruiting bodies of Strobilurus generally raised on seed shells of Liquidambar H. T. Chang and Magnolia L. plants and cones or branches of family Pinaceae members. Recent studies show that basidiomes are also associated with woody angiospermic plants like Betula L. species. Most taxa are considered substrate-specific, while only a small number may exhibit substrate generalize. Testing whether substrate specificity has led to diversification in the genus Strobilurus might be worthwhile if Strobilurus species are substrate specific (Douhan et al. 2008, Rochet et al. 2011). Key features of this genus include a collybioid fruiting body, a tomentose base, prominent nonamyloid basidiospores, distinct caulocystidia, pleurocystidia, pileocystidia, and cheilocystidia, as well as simple-septate hyphae. They are typically found inhabiting Magnolia (L.) L. plants or cones of the Pinaceae family (Singer 1943). Initially, this genus was represented by four species viz. Strobilurus conigenoides (Ellis) Sing., S. escullentus (Wulf. apud Jacq. ex Fr.) Sing., S. tenacellus (Pers. ex Fr.) Sing. and S. stephanocystis (Hora) Sing. Over time, the species count within this genus has increased to 11, with new species discovered in various locations worldwide (Terashima et al . 2016, Singh et al . 2023). In recent European literature, three species of Strobilurus i.e. S. esculentus , S. conigenus , and S. tenacellus are well known and the differences between them have been clearly defined by Gulden (1996) and Wells and Kempton (1971). In North America, there seems to be little recent interest in the genus and in older literature, references and descriptions of species that may belongs in Strobilurus are confusing. To that end, they are deeply indebted to Dr. R. A. Maas Geesteranus of Leiden, Dr. Gro Gulden of Oslo, Dr. L. R. Hesler of Tennessee, and Dr. A. H. Smith of Michigan for providing the necessary specimens for examination. As a result of that study, they recognize six species in North America in addition to the three species of Europe. The species in Strobilurus form a remarkably natural group (Redhead 1980, Desjardin 2000, Katsumoto 2010, Terashima et al. 2016). In previous phylogenetic studies, Strobilurus was found to be a member of the Physalacriaceae with Xerula or Marasmius epiphyllous as its sister group (Hao et al. 2014, Qin et al. 2018). Their concept of Strobilurus albipilatus was based on Smith 1938(). According to Smith, type study in which he illustrated acute to subacute cystidia a collection on cone (Smith 1938). Smith’s illustration of the cheilocystidia is in agreement with Peck’s description but more importantly, Smith illustrated the other cystidia from type specimens as well. The pleuro- and pileocystidia of Strobilurus albipilatus are also illustrated as obtuse but the caulocystidia are illustrated as tapering to an acute or subacute apex and with somewhat thickened walls. Previously, only two species of Strobilurus including S. ohshimae and S. luchuensis were reported from East Asia (Hongo 1955). Literature study shows that a very little work has been done on Strobilurus genus in Asia. Therefore, the present study was purely based on to investigate the Strobilurus genus in subtropical and temperate forest of Hindu Kush Mountain range of Pakistan. These forests are found in Swat, Lower Dir and Upper Dir and covered by coniferous trees. The typical conifers are; Pinus wallichaina A. B. jacks., Abies pindrow (Royel ex. D.Don) Royel, Cedrus deodara (Roxb ex D.Don) G.Don, Picea smithiana (Wall.) Boiss., and Taxus wallichaina Zucc. Fungal flora constitutes an integral component of these forests, playing a pivotal role in ecosystem dynamics and stability (Mueller & Schmit, 2007). This study, based on field data, morphology, and molecular analysis, marks the first report of Strobilurs albipilatus in south Asia, specifically in Pakistan. This species has been already reported from North America. Material and methods Site characteristics and sampling Extensive field surveys were performed during 2018–2022 in Kumrat valley, District Dirr, Pakistan. The climate zone of Kumrat valley is alpine in nature. Milde cool summer is common in the area with an average temperature of 20 ºC to 25 ºC. Extreme temperature has been recorded in winters, falling between -4 ºC to -10 ºC. The average annual rain fall in the area was recorded 1572 mm. The sporocarps of mushrooms were collected multiple time from the area. The collected samples were photographed in their natural ecological habitat and tagged properly with necessary information such as date of collection, host, and information of locality. After collection, samples were dried with the help of an electric fan heater to minimize the moisture content. For anatomical and molecular analysis sample was preserved and packed properly in a polythene zipper bag. Macro- and micro-morphological evaluation Mushrooms were identified with morphological, anatomical, and molecular tools. Basic macro-morphological features of mature sporocarp including, gills attachment, number of lemellulae, stipe (length, width, and shape), pileus structure and diameter, the base of the stipe, color (of the pileus, stipe, and gills), etc. were noted during collection time and compared with the existence species of Strobilurus genus. Hand lens was used for viewing the details of different morphological features. Microscopic elements including hyphae, basidiospores, basidia, cheilocystidia, pleurocystidia, pilipellis, and stipitipellis of the present species were examined using a light microscope (OLYMPUS CX41) at magnifications of 40X and 100X. The material was mounted in lactophenol and 5–10% KOH medium. Color reactions of the fungal tissue were observed using Melzer’s reagent, Congo red, and lactic acid. Line drawings or illustrations of microscopic characters were prepared with the assistance of a Lucida (Ernst Leitz, Wetzlar, Germany) attached to a light microscope. Characterization of microscopic features are shown in figure 1. Genomic DNA extraction DNA was extracted from dried basidiocarp using 2% CTAB method following Gardes and Bruns protocol with minor changes (1993). A small piece of gills was taken, crushed in CTAB solution (300 µl), incubated on 65 ºC for 45 minutes, added 300 µl of isoamyl alcohol chloroform and shacked for 5 second, centrifuged on maximum speed of 13200 RMP for 25 minutes. After that, supernatant containing DNA was kept in separate tube and add (166 µl) of ice-cold isopropanol, gently mixed the solution and store at on 20 ºC for overnight. On next day, solution was centrifuged for 20 minutes at maximum speed of 13200 RMP, drained out the supernatant DNA and keep the DNA pallet, added 200 µl of 70% ethanol and vertex for 5 second, centrifuge for 5 minutes at maximum speed 1300 RMP. Details of the various steps including PCR amplification are shown in figure 2 following Ullah et al. (2023). PCR amplification and sequence analysis Amplification parameters were denatured at 94 °C for 4 minutes followed by 35 cycles of 45 seconds at 94 °C, 45 seconds at 54 °C and 1 minutes 30 seconds at 72 °C, and a final extension at 72 °C for 2 minutes. After purification of PCR products, bidirectional sequencing was done by Macrogen. All the DNA sequences obtained were submitted to BLAST and used to query the nucleotide collection using default setting in GeneBank (Altschul et al. 1990). These sequences along with the published sequences were downloaded from the DNA database and aligned using clustalW and MUSCLE alignment software (Thompson et al. 1997, Altschul et al. 1997) and consequently improved manually with bioedit (Hall 1999). Unclear insertion/deletion (indels) was excluded from the matrix and Gaps were treated as missing data. Phylogenetic analyses The final aligned dataset was exposed to phylogeny. Phylogenetic analyses were performed in MAGA6 software Tamura et al. 2013(). The evolutionary history was concluded with the maximum likelihood methods based on the Tamura-Nei model (Tamura & Nei 1993). The evolutionary history of the examined species is represented by the bootstrap consensus tree assumed from 1000 replicates (Felsenstein 1985). A possible outgroup was selected for the tree for rooting purposes. The present divergence of the examined taxa was calculated by comparing the sequence pairs about the phylogeny reconstructed by MegAlign (DNASTAR) (Kimura 1980). Details of the sequences used in phylogenetic tree are shown in table 1. Results Molecular phylogenetic analysis Sequences of ITS and ITS4 of DNA from 1 specimen of genus strobilurus which seems morphological different were contested with those in gene bank via basic local alignment search tool (BLAST) searches. They were identified up to species level. Strobilurus sp (SK 010: MG871208 ) shows 100% similarity with 89% Query coverage to S. albipilatus (GQ892818), 99.45% similarity with 89% Query coverage to S. albipilatus (GQ892805), 98.77 % similarity with 89% Query coverage to S. albipilatus (GQ892806), with 89% Query coverage, 99.54% similarity with 89% Query coverage to S. albipilatus (GQ892809) respectively. The complete ITS dataset, comprising 22 sequences of closely related species submitted to the gene bank, was aligned using MEGA6 software. The final aligned data set were used for the phylogenetic analysis. The phylogram for the Strobilurus species is representing by 6 clades viz. S. albipilatus, S. esculentus, S. tenacellus, S. conigenoide, and S. trullisatus while Xerula pudens forming the root ML tree. Clade S. albipilatus with 55% bootstrap value contains S. albipilatus (SK010: MG871208 ) along with its closest match viz. S. albipilatus (GQ892818, GQ892805, GQ892806, GQ892809). Sequences from Pakistan show maximum similarity among them and consequently fall in the same clade (figure 3). Taxonomic descriptions Strobilurus albipilatus (Peck) Wells & Kempton (Figure 4 & 5) MycoBank No: 18610 Diagnosis The diagnostic features of Strobilurus albipilatus are; plane and medium size pileus; free lamellae, rimos margin, light yellow (5Y 9/6 #FFE395) to brilliant yellow (5Y 9/10 #FFE259) color; light greenish yellow (7.5Y 9/6 #F9E594) to greenish yellow (7.5Y 7/4 #BDAE7F) color stipe; thick-walled, ovoid to ellipsoid and round apex basidiospores; thin-walled, clavate to subfusoid, guttiulated basidia; fusoid- ampullaceous cheilocystidia; fusoid-ventricose pleurocystidia. Description Pileus 2–4 cm broad, fleshy, convex-plane, initially umbonate then becomes depressed at maturity; dry and smooth surface, lacking fibrillose scales, wholly homogeneous color but darker at disc, lack of distinct odor; cracked and rimos margin, epigeous, brilliant yellow (2.5Y 9/10 #FFDC62) to greenish yellow (7.5Y 9/6 #F9E594). Lamellae thin, free , distant, intercalated, lamellulae regular, two-three tiers of, light-yellow color (5Y 9/6 #FFE395). Stipe up to 6 cm long and 0.3–0.5 cm wide, central, solid and smooth, cylindrical, strigos; redicating, light greenish yellow (7.5Y 9/6 #F9E594) to greenish yellow (7.5Y 7/4 #BDAE7F) color; annulus absent. Basidiospores 7.5–11.3 × 9.4–15.6 µm, ovoid to ellipsoid, hyaline in KOH, reddish-brown in Melzer reagent, with germ pore present on rounded apex, apiculus not much prominent, 1–1.5 µm high. Basidia 6–7 × 21–29 µm , sharp sterigmata up to 2–3 µm long, color less, hyaline, thin-walled, clavate to subfusoid, guttulated. Cheilocystidia 10–13 × 60–64 µm, thick-walled, fusoid-ampullaceous, present rarely, hyaline in KOH. Pleurocystedia 9–14 × 50–60 µm, thick-walled, fusoid-ventricose, hyaline in KOH. Pileipellis terminal element 13.3–24 × 2.8–4.2 µm, septate, conical, thick walled, hyaline in KOH. Stipitipellis terminal element 23.9–62.1 ×4.2–9 µm, septate, branched, gutulllated, hyaline in KOH. Habitat & Distribution Solitary or gregarious, submerged on pinecones or on fallen needles of Pinus wallichaina ; Seyasan, 2038 m.a.s.l, Hindukush region of Pakistan, March 2019, (SK010: MG871208, Shahab Ali, (LAH35311). Specimen Examined Pakistan, Khyber Pakhtunkhwa, District Dir, Kumrat valley, mixed pine forest at 72º12ʹ35ʺN, 35º32ʹ31ʺN, elev. 2038 m, 20 March 2019, Shahab Ali ( LAH35311 ). Genbank accesion: ITS = MG871208. Discussion Before the present study, no species-specific phylogenetic analysis had been conducted on Strobilurus genus in south Asia, specifically in Pakistan . In this study, phylogenetic analyses were carried out to examine its genetic relationship with closely related species of Strobilurus . A total of 22 Strobilurus sequences, including Xerula_pudens (used as an outgroup), along with previously submitted sequences in the gene bank, were included in our analysis. Based on the phylogenetic tree, the Pakistani species (SK010: MG871208) falls within the S. albipilatus clade, alongside its closest matches: S. albipilatus (GQ892818), (GQ892805), (GQ892806), and (GQ892809). These findings are supported by the results of other researchers (Qin et al. 2018). The Pakistani species is similar with that of reported by Redhead (1980). Pileus 5–24 mm wide, convex to plane marginally translucent–striate when moist varying considerably in color, adnate to adnexed white lamellae varying from distant to crowded, lamellulae two to three tiers, stipe up to 4 cm long up to 0.2 cm wide, equal, densely pruinose, stuffed, becoming hollow with age. Pileipellis a hymeniform layer of clavate, sphaeropedunculate, or globose hyaline, smooth cells, 20–45 µm x 7.5–24 µm, cheilocystidia and pleurocystidia abundant or rarely sparse, prominently projecting, capitate–cylindrical to lecythiform or obtusely ventricose, 40–70 µm x 7.5–1 µm, hyaline, scantily to heavily capped by resin; walls thin or pronounced. Basidia four-spored, clavate, simple–septate, 17–25 µm x 2–4.5 µm. Basidiospores 3–7.5 µm x 1.5–3.5 µm, ellipsoid to sub–lacrymanoid, smooth, thin–walled, hyaline, white in mass, nonamyloid (Redhead 1980). The species is also similar to that of reported by Wells and Kempton (1971). Their concept of S. albipilatus was based on Smith (1938). The species studied by Smith was found on cone having acute to subacute cystidia. From Peck’s original description, we know it has a brown pileus, a pallid stipe, and that it grows from buried pinecones. Peck also stated that the cheilocystidia gradually taper toward the apex which sometimes ends in a knob or abrupt enlargement and concluded that the Strobilurus is distinct genus from others (Peck 1879). Both, anatomical and macromorphological features of specimen studied by Peak are completely overlap with Pakistani collection. Our findings indicate that the current sample, S . albipilatus , was collected from a pinecone. This characteristic mode of occurrence has also been reported in Europe and North America by Singer (1962). Based on an in–depth study of morphological features, molecular analysis, and habitat study, we conclude that the present species is S. albipilatus , which is new to south Asia, particularly to Pakistan. Conclusions The Hindukush semi-moist temperate forest of Pakistan serves as a hotspot of mushroom diversity. Despite its significance, this region remains relatively unexplored, with minimal research conducted on mushrooms. In the current study, we conducted fieldwork in the Hindukush semi–moist temperate forest, focusing on mushroom specimens. Among the collected samples, we identified specimens belonging to the species Strobilurus albipilatus through morpho–anatomical traits and phylogenetic analyses. Our findings indicate that these specimens represent a new record for South Asia, specifically Pakistan. This investigation sheds light on the abundant mushroom diversity present in the Hindukush semi–moist temperate forest of Pakistan. Acknowledgement This paper is the part of my PhD work and the authors would like to extend their sincere appreciation to the local community for assisting in the filed data. Conflict of interest The author has no conflict of interest. Data availability statment No supplementary data are available for this paper References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. 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Conocybe karakensis sp. nov.(Bolbitiaceae, Agaricales) from Pakistan. Phytotaxa. 584(3):135–148. doi:10.11646/phytotaxa.584.3.1. Wells VL, Kempton PE. 1971. Studies on the fleshy fungi of Alaska. V. The genus Strobilurus with notes on extralimital species. Mycologia. 63(2):370–379. doi:10.1080/00275514.1971.12019050. List of Figures captions Figure 1. Microscopic features characterizations of Strobilurs albipilatus. Figure 2. DNA extraction and molecular characterizations of Strobilurs albipilatus. Figure 3. Phylogenetic tree constructed through MEGA6 software using Maximum Likelihood method. Tree shows six distinct clades in which Pakistani collection fall in S. albipilatus section. Species collected from Hindukush region of Pakistan have been labelled with a box (♦). Figure 4. Field photography of Strobilurus albipilatus. Scale bars = 2 cm. Photos by Shahab Ali. Figure 5. Anatomical features of Strobilurs albipilatus. A. Basidiospore, B. Basidia, C. Cheilocystidia, D. Pleurocystidia, E. Pilipallous terminal element, F. Stipitipellis elements. Scale bars = 5 µm. List of tables Table 1. Details of the sequences used in phylogenetic tree. Supplementary Material File (image1.emf) Download 17.46 KB File (list of figures file.docx) Download 1.17 MB File (list of tables file.docx) Download 13.85 KB Information & Authors Information Version history V1 Version 1 07 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords hindukush molecular identification mushrooms phylogenetic tree taxonomy Authors Affiliations Shahab Ali Quaid-i-Azam University View all articles by this author Tauseef Ullah 0000-0002-0420-9739 [email protected] Quaid-i-Azam University View all articles by this author Muhammad Fiaz Hazara University View all articles by this author Abdul Khalid University of the Punjab View all articles by this author Shujaul Khan Quaid-i-Azam University View all articles by this author Yu Zhongdong Northwest A&F University View all articles by this author Metrics & Citations Metrics Article Usage 303 views 163 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shahab Ali, Tauseef Ullah, Muhammad Fiaz, et al. Molecular identification of European genus Strobilurus from Hindukush region of Pakistan. Authorea . 07 February 2025. 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