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Research Article
New species of Mallocybe (Agaricales, Inocybaceae) from Pakistan, based on morphological and molecular evidence
expand article infoMalka Saba‡, Abdul Nasir Khalid§, Samina Sarwar|
‡ Quaid-i-Azam University, Islamabad, Pakistan
§ University of the Punjab, Lahore, Pakistan
| Lahore College for Women University, Lahore, Pakistan

Corresponding author: Malka Saba ( rustflora@gmail.com )

Academic editor: María P. Martín
© 2023 Malka Saba, Abdul Nasir Khalid, Samina Sarwar.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Saba M, Khalid AN, Sarwar S (2023) New species of Mallocybe (Agaricales, Inocybaceae) from Pakistan, based on morphological and molecular evidence. MycoKeys 99: 171-186. https://doi.org/10.3897/mycokeys.99.86844
Open Access

Abstract

Within the family Inocybaceae, many species of Mallocybe have been reported, but there are only a few reports of this genus from Pakistan. In this study, six collections of Mallocybe were studied by morphological and phylogenetic methods. Phylogenetic analyses, based on sequence data from two different loci (ITS and LSU) using Maximum Likelihood and Maximum Parsimony methods, have been performed to infer species relationships within Mallocybe. Results indicated that these six collections encompass two new species of Mallocybe i.e. M. pakistanica and M. pinicola, from Pakistan. Their detailed morphological descriptions and illustrations are also provided. In addition, comparison with morphologically closely-related taxa is also discussed. Previously, only two species of this genus have been recorded from Pakistan and, with this addition, the total number of reported taxa of Mallocybe has been raised to four from Pakistan. A key to the described taxa of Mallocybe from Pakistan is also provided.

Key words

Asia, molecular systematics, phylogeny, Pinaceae

Introduction

The Inocybaceae Jülich is a monophyletic family encompassing ectomycorrhizal fungi with worldwide distribution (Matheny et al. 2006) and is estimated to contain 1050 species (Matheny and Kudzma 2019). Mallocybe (Kuyper) Matheny, Vizzini & Esteve-Rav was first described as a subgenus of Inocybe (Fr.) Fr. (1863:346), but recently, Matheny et al. (2019) elevated Mallocybe to one of seven genera in Inocybaceae.

Macroscopically, Mallocybe species are recognised by a fibrous or scaly often flattened pileus, a short stipe, ochre, brown or red brown colouration, a cortina, adnate lamellae and absence of a spermatic odour. Microscopically, distinctive characters of this genus include; smooth spores, absence of pleurocystidia, thin-walled without crystals cheilocystidia and necropigment in basidia of fresh and dried specimens (Kuyper 1986; Stangl 1989; Jacobsson 2008; Matheny et al. 2019). This genus is monophyletic and about 58 species of Mallocybe have been recorded in Index Fungorum (www.indexfungorum.org) from different regions of Africa, Asia, Australia, Europe, New Zealand and North America (Matheny et al. 2019; Saba and Khalid 2020; Aignon et al. 2021; Mao et al. 2022). However, only two species of this genus are yet known from Pakistan (Ahmad et al. 1997; Saba and Khalid 2020). During the exploration of ectomycorrhizal fungi of Pakistan, we encountered several species of Mallocybe which have been described using morphological and molecular analysis (Saba 2016). Amongst them, Mallocybe velutina has already been described (Saba and Khalid 2020), while M. pakistanica and M. pinicola are herein described as new species. Both of these species were found in association with pines in Pakistan.

Materials and methods

Morphological studies

During an investigation of ectomycorrhizal fungi associated with pine species in Pakistan, basidiomata were collected, described and photographed from the selected sampling sites in the field. Colours were compared to the Munsell Soil Colour Charts (1975) guide. Collections were dried using a food dehydrator (at 39 °C for 7–9 hours). Microscopic characters were observed in the laboratory using hand-cut sections of basidiomata mounted in a 5% aqueous solution of potassium hydroxide (KOH) and of Congo red. Micromorphological analysis and measurements were made, using an Olympus B×40 light microscope with Olympus XC50 digital camera and Microsuite special edition software 3.1 (Soft imaging solutions GmbH). Thirty-five basidiospores were measured from each collection cited. Measurements include the range with extremes provided in parentheses. Q values (length/width ratios) and mean values (average basidiospore length and width) are also provided. Line drawings were made with a Leitz camera Lucida (Wetzlar, Germany). Collections of the newly-described species are deposited at ISL (Quaid-i-Azam University, Islamabad, Pakistan).

DNA extraction, PCR amplification and DNA sequencing

Genomic DNA was extracted from a 20 mg piece of dried tissue by a modified CTAB method (Lee et al. 1988). Loci examined during this study included the complete ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal RNA gene (hereafter ITS) and the first ca. 900 bp of the nuclear 28S rRNA gene (nrLSU).

Primers used for amplification were: ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for ITS and LR0R and LR5 for nrLSU (Vilgalys and Hester 1990). The amplification reaction mixture contained 2.5 µl Econo buffer, 0.5 µl dNTPs, 1.25 µl each primer, 0.125 µl Econo Taq, 14.375 µl of deionised water and 5 µl of template DNA. The thermal profile of PCR for ITS was initial denaturation at 94 °C for 1 min.; then 35 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min and extension at 72 °C for 1 min; and final extension at 72 °C for 8 min. For nrLSU: 94 °C for 2 min; then 40 cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1:30 min; and 72 °C for 5 min.

PCR products were run on 1% agarose gel, stained with ethidium bromide and bands were visualised under a UV transilluminator. Amplified PCR products of the ITS region were sent for purification and bidirectional sequencing to Macrogen (Republic of Korea). PCR products of 28S were purified using QIAquick PCR purification kit (Qiagen, Stanford, California) as per manufacturer’s guidelines and sequencing reactions were performed using the Big Dye Terminator v.3.1 Cycle Kit (Life Technologies, Carlsbad, California). Sequencing was carried out using the same primers as those used for PCR.

Sequence alignment

Sequences were manually edited and assembled in BioEdit v.7.2.6 (Hall 1999). Generated ITS sequences were trimmed with the conserved motifs 5’–CATTA– and –GACCT–3’ (Dentinger et al. 2011) and the alignment portion between these motifs was included in subsequent analyses. BLASTn searches were performed in NCBI GenBank.

BLASTn Results

In the BLASTn search, based on ITS sequences, Mallocybe pakistanica had the highest sequence identity (94.76%) with Mallocybe megalospora (Stangl & Bresinsky) Matheny & Esteve-Rav. HQ604786 (unpublished sequence). Mallocybe pinicola had the highest sequence identity (93.86%) with type sequence of Mallocybe siciliana (Brugaletta, Consiglio & M. Marchetti) Brugaletta, Consiglio & M. Marchetti NR_164583 (Brugaletta et al. 2017). In the BLASTn search, based on LSU sequences, Mallocybe pakistanica had the highest sequence identity (98.23%) with Mallocybe sp. BK 6-June-97-24 (MN178541) which is not yet published. Mallocybe pinicola had the highest sequence identity (98.90%) with Inocybe aff. malenconii (MN178539).

Phylogenetic analysis

Closely-related sequences were retrieved from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/), following Vauras and Larsson (2011) and Saba and Khalid (2020). We also added sequences from GenBank of the close hit after initial BLAST to construct the phylogeny using the combined ITS+nrLSU dataset. Finally, we included M. arthrocystis (Kühner) Matheny & Esteve-Rav. as outgroup, following Vauras and Larsson (2011) and Saba and Khalid (2020).

To estimate the placement and phylogenetic relationships of the new species, Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses of the concatenated ITS+nrLSU datasets were conducted. MP analysis was performed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BS) analysis with 1,000 replicates (Felsenstein 1985). Descriptive tree statistics, tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each maximum parsimonious tree generated. Maximum Likelihood (ML) was performed using RAxML 8.0.14 with a general time-reversible (GTR) model of site substitution including estimation of Gamma-distributed rate heterogeneity (+G) and a proportion of invariant sites (+I) on Abe through the CIPRES Science Gateway (www.phylo.org; Miller et al. 2010). Branch support for ML analysis was determined by 1,000 bootstrap replicates (Hillis and Bull 1993). Obtained trees were visualised in FigTree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Bootstrap values (BS) ≥ 70% were considered significant. The ITS+nrLSU alignment was deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S26552).

Results

Phylogenetic inferences

In this study, twelve novel sequences of two genes i.e. ITS and LSU were newly generated from our collections. Combined dataset I (ITS+LSU) contained forty-two sequences from eighteen taxa (Table 1), including six novel sequences from our collections. The length of the aligned dataset was 1,765 bp of which 1,303 characters are constant, 413 are variable and parsimony-uninformative and 300 parsimony-informative. All characters are of unord type and have equal weight. One equally parsimonious tree (TL = 802, CI = 0.678, RI = 0.818, RC = 0.555, HI = 0.322) was derived from the MP analysis. The topologies of ML and MP phylogenetic trees obtained using this dataset were practically the same; therefore, only the tree inferred from the ML analyses is shown (Fig. 1). Both ML and MP bootstraps strongly support the placement of the new species within Mallocybe. The Mallocybe species formed a monophyletic lineage with strong support (MLB = 99%). The sequences of our six collections formed two independent clades, which were respectively recognised and described as two new species: Mallocybe pakistanica and Mallocybe pinicola. M. pakistanica was sister to Mallocybe sp. BK 6-June-97-24 (MN178541) with high supports, implying that they are closely related to each other. Another species M. pinicola clustered with Mallocybe siciliana NR_164583 (Brugaletta et al. 2017) and M. subtomentosa (MN178521) with strong support (MPB = 89%). These two new taxa from Pakistan can be distinguished, based on molecular phylogenetic data, as well as morphology and ecology.

Table 1.
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Taxa of Mallocybe included in the molecular phylogenetic analyses.

Species Specimen voucher/Isolate Country Accession numbers Reference
ITS nrLSU
I. dulcamara EL59-05 Norway GU980643 GU980643 Cripps et al. (2010)
I. dulcamara CLC 1333 USA GU980635 GU980635 Cripps et al. (2010)
M. agardhii AB980912 Denmark HM209790 HM209790 Vauras and Larsson (2011)
M. arenaria EL25008 France FN550937 FN550937 Ryberg et al. (2010)
M. arthrocystis EL9207 Sweden FN550941 FN550941 Ryberg et al. (2010)
M. cf. squarrosoannulata CLC1566 Not given GU980606 GU980606 Cripps et al. (2010)
M. cf. squarrosoannulata EL120-08 Not given GU980607 GU980607 Cripps et al. (2010)
M. fulvipes EL99-07 Sweden GU980600 GU980600 Cripps et al. (2010)
M. fuscomarginata EL10906 Sweden FN550940 FN550940 Ryberg et al. (2010)
M. fuscomarginata BJ890718 Sweden GU980656 GU980656 Cripps et al. (2010)
M. granulosa SJ84030 Not given KR029725 KR029725 Ariyawansa et al. (2015)
M. granulosa EL138-09 Not given KR029727 KR029727 Ariyawansa et al. (2015)
M. granulosa EL138-09 Sweden KR029727 KR029727 Ariyawansa et al. (2015)
M. granulosa SJ84030 Sweden KR029725 KR029725 Ariyawansa et al. (2015)
M. gymnocarpa SJ980707 Sweden AM882866 AM882866 Ryberg et al. (2008)
M. heimii JV 14932F (WTU) USA – AY380379 Matheny (2005)
M. latispora EL190-08 Not given KR029724 KR029724 Ariyawansa et al. (2015)
M. leucoblema SM2324 Sweden GU980630 GU980630 Cripps et al. (2010)
M. leucoblema JV2898 Finland HM209789 HM209789 Vauras and Larsson (2011)
M. leucoloma EL41-07 Sweden GU980622 GU980622 Cripps et al. (2010)
M. leucoloma Ohenoja 880810 Svalbard HM209786 HM209786 Vauras and Larsson (2011)
M. malenconii JV23101 Finland HM209787 HM209787 Vauras and Larsson (2011)
M. malenconii PAM98941302 France HM209788 HM209788 Vauras and Larsson (2011)
M. myriadophylla EL121-08 Sweden HM209792 HM209792 Vauras and Larsson (2011)
M. myriadophylla JV19678 Finland HM209793 HM209793 Vauras and Larsson (2011)
M. myriadophylla JV5968 Finland HM209794 HM209794 Vauras and Larsson (2011)
M. myriadophylla JV19652 Finland HM209791 HM209791 Vauras and Larsson (2011)
M. pakistanica MSM#0061 Pakistan OK360951 OK392118 This paper
M. pakistanica MSM#00132 Pakistan OK360952 OK392119 This paper
M. pakistanica MSM#0201 Pakistan OK360953 OK392120 This paper
M. pinicola MSM#0060 Pakistan OK360954 OK392121 This paper
M. pinicola MSM#00131 Pakistan OK360955 OK392122 This paper
M. pinicola MSM#0200 Pakistan OK360956 OK392123 This paper
M. substraminipes K70-148 USA GU980601 GU980601 Cripps et al. (2010)
M. substraminipes EL12-08 USA GU980601 GU980601 Cripps et al. (2010)
M. terrigena EL24-08 USA GU980648 GU980648 Cripps et al. (2010)
M. terrigena EL11704 Sweden AM882864 AM882864 Ryberg et al. (2008)
M. tomentosula TENN:071837 USA MG773814 MG773814 Unpublished
M. velutina MSM # 0048 Pakistan MK990129 MK999927 Saba and Khalid (2020)
M. velutina MSM # 0049 Pakistan MK990130 MK999928 Saba and Khalid (2020)
M. velutina MSM # 00050 Pakistan MK990131 MK999929 Saba and Khalid (2020)
Figure 1. 

Phylogeny of Mallocybe pakistanica, M. pinicola and related taxa produced from Maximum Likelihood (ML) inference using combined dataset of ITS and nrLSU sequences. Numbers on branches are ML/MP bootstrap values (only ≥ 70). New sequences reported in this study are blue coloured.

Taxonomy

Mallocybe pakistanica Saba & Khalid, sp. nov.

MycoBank No: MycoBank No: MB843490
Figs 2, 3

Diagnosis

Most similar to Mallocybe myriadophylla described from north-western Europe, but differs by the absence of a crowded lamellae, different pileal colouration and somewhat larger basidiospores. Phylogenetically separated from other species of Mallocybe due to unique ITS and LSU sequences.

Types

. Holotype: PAKISTAN, Prov. Khyber Pakhtunkhwa, Mansehra, Chattar Plain, under Pinus wallichiana A. B. Jacks., 22 September 2013, leg. M. Saba & A.N. Khalid; MSM#0061 (ISL-F002); GenBank accession nos. OK360951 (ITS), OK392118 (nrLSU). Paratype: Pakistan, Prov. Khyber Pakhtunkhwa, Mansehra, Chattar Plain, under Pinus wallichiana, 2 September 2015, leg. M. Saba & A.N. Khalid; MSM#00132 (ISL-F003); GenBank accession nos. OK360952 (ITS), OK392119 (nrLSU). Sep 2021, MSM#0201, (ISL-F004); GenBank accession nos. OK360953 (ITS), OK392120 (nrLSU).

Etymology

Referring to the country where it was discovered.

Description

Pileus 19–24 mm diam., plane; margin deflexed in mature basidiomata, not splitting; surface dull, scaly, floccose, light brown (7.5YR6/4) or pale yellowish-brown (10YR7/4). Lamellae adnexed, subdistant, margin entire, regular, concolorous, moderate brown (7.5YR3/4) or strong brown (5YR4/6), one to two tiers of lamellulae or variable. Stipe 31–35 mm, central, equal, floccose, pale yellowish-brown (5YR8/8) or pale yellow (2.5Y9/4), cortina zone not seen; annulus absent. Context pale brown, tough, up to 2 mm thick. Odour not distinctive, somewhat fungoid. Taste not recorded.

Basidiospores 7.4–13.2 × 5–6.6 µm [x = 10.8 × 5.9 µm, Q = 1.3–2.2], ellipsoid, ovoid, thin-walled, pale brown with yellowish contents in KOH. Basidia with yellowish necropigment, 25.7–36 × 6.8–10.3 µm, clavate, usually four-spored, thin-walled, hyaline in KOH; sterigmata 3.6–5.3 µm. Pleurocystidia absent. Cheilocystidia 14.8–31 × 9–15.8 µm, cylindrical, hyaline, in chains. Caulocystidia 38–43.6 × 5.5–7.0 µm, hyphal, yellowish-brown in KOH with clamp connections at base, thin-walled, abundant at the apex of stipe. Pileipellis hyphae cylindrical, pale brown in mass in KOH, 5–12 µm, thin-walled. Stipitipellis hyphae cylindrical, 6–10 µm, yellowish or olivaceous in KOH. All structures inamyloid. Clamp connections present.

Habitat

Occurring in September, solitary, scattered on the forest floor in stands of Pinus wallichiana (Pinaceae).

Known distribution

Currently known from Western Himalayas, Pakistan.

Notes

Mallocybe pakistanica can be characterised by small to medium-sized basidiomata, pale yellowish-brown or light brown pileus, ellipsoid basidiospores and catenate cheilocystidia (in chains). Based on the phylogenetic analysis (Fig. 1), conducted using the combined dataset ITS+LSU, indicate that the closet taxon is Mallocybe sp. BK 6-June-97-24 (MN178541) which is not yet published (Personal communications with P. Brandon Matheny). Mallocybe myriadophylla (Vauras & E. Larss.) Matheny & Esteve-Rav. is another closely related taxon which can be differentiated by different colouration of the pileus (when young pale brownish-grey, then grey brown, pale brown to brown, centre often darkest yellow-brown), presence of crowded lamellae and somewhat smaller phaseoliform basidiospores ((7.3–) 7.9–9.6 (–10.6) × (4.5–) 4.7–5.5 (–5.7) μm) (Vauras and Larsson 2011).

Another closely-related species in the adjacent clade is Mallocybe tomentosula Matheny & Esteve-Rav., in Matheny, Hobbs & Esteve-Raventós which morphologically can be differentiated by the presence of a superior cortinate ring-zone, slightly smaller size of basidiospores and by its occurrence in eastern North America. Both ML and MP phylogenetic analyses also clearly support the identity of this new taxon as independent monophyletic clade.

Figure 2. 

Holotypes A Mallocybe pakistanica (MSM#0061) B Mallocybe pinicola (MSM#0060). Scale bars: 10 mm (A, B).

Figure 3. 

Mallocybe pakistanica (holotype) A basidia B basidiospores C cheilocystidia D pileipellis E caulocystidia. Scale bars: 10 µm (A); 5 µm (B); 15 µm (C); 25 µm (D); 15 µm (E).

Mallocybe pinicola Saba & Khalid, sp. nov.

MycoBank No: MycoBank No: MB843491
Figs 2, 4

Diagnosis

Most similar to M. siciliana and M. subtomentosa, but differs by the combination of pileal colour, absence of umbo, size of basidiospores, pyriform to broadly clavate, catenate cheilocystidia and an ecological association with Pines. Phylogenetically separated from other species of Mallocybe due to unique ITS and LSU sequences.

Types

Holotype : PAKISTAN, Prov. Khyber Pakhtunkhwa, Mansehra, Chattar Plain, under Pinus wallichiana, 22 September 2013, leg. M. Saba & A.N. Khalid; MSM#0060, (ISL-F005); GenBank accession nos. OK360954 (ITS), OK392121 (nrLSU). Paratype: Paratype: Pakistan, Prov. Khyber Pakhtunkhwa, Mansehra, Chattar Plain, under Pinus wallichiana, 2 September 2015 leg. M. Saba & A.N. Khalid; MSM#00131(ISL-F006); GenBank accession nos. OK360955 (ITS), OK392122 (nrLSU). Sep 2021, MSM#0200, (ISL-F007); GenBank accession nos. OK360956 (ITS), OK392123 (nrLSU).

Etymology

Referring to its exclusive association with Pinus.

Description

Pileus 24.9–27 mm diam., plan with slight depression in centre; margin straight or flaring, not splitting; surface dull, scaly, light orange (5YR8/8) or ochre-yellowish, central disc brownish-orange (5YR5/8). Lamellae adnexed, subdistant, margin eroded, strong brown (5YR4/6) or (5YR4/8). Stipe 31–35.6 mm, central, equal, floccose or pruinose near base, light orange (5YR8/8) or moderate orange (5YR7/8), cortina zone present; annulus absent. Context pale yellow to pale brown, tough, up to 3 mm thick. Odour faint not strong. Taste not recorded.

Basidiospores (6.8–) 7.5–11 × 5–7 µm [x = 9.5 × 6.0 µm, Q = 1.1–1.8], ovoid, ellipsoid or phaseoliform, thin-walled, pale brown or golden brown in KOH. Basidia with yellowish necropigment, 27–42.4 × (5.4–) 8–12 µm, clavate, attenuated below, two to four-spored, thin-walled, hyaline in KOH; sterigmata 2.8–5.6 µm. Pleurocystidia absent. Cheilocystidia 11.8–36.5 × 11–15 µm, hyaline, pyriform to broadly clavate, in chains. Caulocystidia 22–70 × (6.3–) 7.7–14 µm, hyphal, yellowish-brown in KOH with clamp connections at base, thin-walled. Pileipellis hyphae cylindrical, hyaline singly or pale brown in mass in KOH, 5–11.3 µm, thin-walled, pileal hyphal endings 23.6–70 × 7.7–13 µm. Stipitipellis hyphae cylindrical, 5–10 µm, yellowish or olivaceous in KOH. All structures inamyloid. Clamp connections present.

Habitat

Occurring in September, solitary, scattered on the forest floor in stands of Pinus wallichiana (Pinaceae).

Known distribution

Currently known from Western Himalayas, Pakistan.

Notes

Mallocybe pinicola is characterised by light orange or ochre-yellowish, medium-sized pileus, absence of umbo, ovoid, ellipsoid or phaseoliform basidiospores, pyriform to broadly clavate, catenate cheilocystidia and its distribution in pine (conifer) forests. Based on the phylogenetic analysis (Fig. 1), constructed using the combined dataset of ITS and LSU, M. pinicola clustered with M. siciliana and M. subtomentosa. M. siciliana was originally described from Europe (Italy) by Brugaletta et al. (2017). It is similar to M. siciliana in having similar colour and size of pileus. However, it can be differentiated from M. siciliana by the entire absence of umbo and presence of larger basidiospores ((6.8–) 7.5–11 × 5–7 µm vs. 6.7–9 × 4.4–5.7 µm). Moreover, Mallocybe siciliana is described from forests having Salix species (S. pedicellata and S. alba), Platunus orientalis, Tamarix gallica and Hypericum hircium, while M. pinnata has been described from pure pine (Pinus wallichiana) forests.

Another closely-related taxa is Mallocybe subtomentosa which was originally described from the United States of America (Rouse’s Point). It resembles M. pinicola in having the entire absence of umbo, nearly similar spore size and shape of basidiospores (8–10 × 5–6 μm and ellipsoid basidiospores in M. subtomentosa). However, the presence of dark brown and minutely hairy to tomentose pileus, absence of cystidia and gregarious or subcaespitose habit in M. subtomentosa make the present species distinct from latter (Massee 1904).

Moreover, phylogenetic analysis (ML and MP), conducted using combined dataset of ITS + LSU, showed the clear separation of our species from these two closely-related taxa and all the sequences of our species clustered together with strong statistical support (99%) forming a monophyletic clade.

Figure 4. 

Mallocybe pinicola (holotype) A basidia B basidiospores C cheilocystidia D caulocystidia E pileipellis. Scale bars: 10 µm (A); 5 µm (B); 15 µm (C); 20 µm (D, E).

Discussion

Pakistan is located in southern Asia. This country is geographically diverse, ranging from the mountainous northern part, where the Himalayas meet their westernmost end, to the southern part with the coastal area along the Arabian Sea. Following the KöppenGeiger classification system for climate, 20 types can be found in Pakistan – including four arid, six temperate, eight cold and even two polar (Beck et al. 2018). Note that, despite this diversity in climate types, most of the country has a hot desert climate (Peel et al. 2007). Pakistan has a very rich flora; in an ongoing effort to compile the Flora of Pakistan, S.I. Ali and colleagues identified 5,521 plant species in 1,572 genera thus far (Ali 2008). When keeping the ratio between vascular plants and fungi (1:6) in mind (sensu Hawksworth 1991), this number of plants only hints at the true potential of in-depth mycological studies in Pakistan, which has been traditionally under-explored.

The multiple geographic features, different climates and plant species richness in Pakistan are suggestive of a high diversity of fungal species. In recent years, many papers have been published, describing new species from different fungal groups collected in Pakistan (e.g. Razaq et al. 2012; Nawaz et al. 2013; Thongklang et al. 2014; Qasim et al. 2015a, 2015b; Sarwar et al. 2015; Hussain et al. 2016, 2017, 2018; Jabeen et al. 2016; Farooqi et al. 2017; Naseer et al. 2018; Ullah et al. 2018; Saba et al. 2019a, 2019b; Kiran et al. 2020). Thirty-five species of Inocybe sensu lato have been reported from Pakistan (Ahmad et al. 1997; Ilyas et al. 2013; Saba et al. 2015; Jabeen et al. 2016; Farooqi et al. 2017; Razaq and Shahzad 2017; Naseer et al. 2018; Ullah et al. 2018; Song et al. 2019; this study). The genus Mallocybe is poorly known in Pakistan, with only two species that were known before this study: M. leucoblema (Kühner) Matheny & EsteveRav. and M. velutina Saba & Khalid (Ahmad et al. 1997; Saba and Khalid 2020).

In the combined ITS and LSU phylogenetic analysis, the new species described in this study occupy independent positions. From our morphological analysis, it is obvious that both Mallocybe pakistanica and M. pinicola are separated from other closely-related Mallocybe species. With the contribution of this research work, the number of known taxa of this genus has been raised to sixty worldwide, with four from Pakistan. However, a considerable number of taxa have yet to be formally described and the number of the species will likely increase as more collections are studied from under-explored localities. A key to Mallocybe species reported from Pakistan is provided below;

1 Basidiomata medium to large, pileus robust, ≥ 30 μm diam., cortina white M. leucoblema
– Basidiomata small to medium, pileus ≤ 30 μm diam., cortina brown 2
2 Pileus surface velutinous, cheilocystidia clavate or cylindrical M. velutina
– Pileus surface scaly, cheilocystidia articulated 3
3 Pileus light orange, moderate orange or brownish-orange, evenly coloured, plan, basidiospores longer and narrower M. pakistanica
– Pileus light orange or ochre-yellowish with central disc slightly depressed and brownish-orange, basidiospores smaller and broader M. pinicola

Acknowledgements

We are highly indebted to the Higher Education Commission (HEC), Islamabad, Pakistan, for funding this project under Phase II, Batch I, Indigenous PhD fellowships programme for 5000 scholars and through International Research Support Initiative Program (IRSIP).

Additional information

Conflict of interest

No conflict of interest was declared.

Ethical statement

No ethical statement was reported.

Funding

Higher Education Commission, Pakistan.

Author contributions

Malka Saba: conceptualization, writing - original draft and review and editing, data curation, formal analysis, investigation, methodology and visualization. Abdul Nasir Khalid: project administration, resources and supervision. Samina Sarwar: writing - review and editing, formal analysis.

Author ORCIDs

Malka Saba https://orcid.org/0000-0001-7673-2345

Abdul Nasir Khalid https://orcid.org/0000-0002-5635-8031

Samina Sarwar https://orcid.org/0000-0002-6377-4498

Data availability

All holotype and paratype collections of the new species are deposited at ISL. The sequences generated during this study are deposited in NCBI GenBank under accession numbers OK360951–OK360956 (ITS) and OK392118–OK392123 (LSU). The ITS þ nrLSU alignment is deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S26552).

References

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