683 MycoKeys

MycoKeys 121: 143-166 (2025) DOI: 10.3897/mycokeys.121.157282

Research Article

Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

Muhammad Hagnawaz'2®, Muhammadah Khalid?, Qirui Li?,© Pingzhu Lu’, Aysha Bibi2®, Arooj Naseer®, Najam ul Sehar Afshan?, Abdul Rehman Niazi?, Abdul Nasir Khalid2®

1 Chongqing Three Gorges Medical College, Wanzhou Chongqing, 404100, China

2 Fungal Biology and Systematics Research Laboratory, Institute of Botany, University of the Punjab, Quaid-e-Azam Campus 54590, Lahore, Pakistan

3 The High Efficacy Application of Natural Medicinal Resources Engineering Centre of Guizhou Province (The Key Laboratory of Optimal Utilization of Natural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, Guizhou, 550004, China

Corresponding author: Pingzhu Lu (1435962566@qq.com)

OPEN Qaccess

This article is part of:

Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi

Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam

Academic editor: Samantha C. Karunarathna Received: 28 April 2025 Accepted: 2 August 2025 Published: 28 August 2025

Citation: Hagnawaz M, Khalid M,

Li Q, Lu P Bibi A, Naseer A, Afshan NulS, Rehman Niazi A, Khalid AN (2025) Morphology and multi-

locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan. MycoKeys 121: 143-166. https://doi.org/10.3897/ mycokeys.121.157282

Copyright: © Muhammad Haqnawaz et al.

This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0).

Abstract

In this study, four new species of the genus Candolleomyces are described from Paki- stan. Based on morphological and multi-locus phylogenetic analyses, we confirmed the distinction of these taxa. The newly identified species are designated as Candolleomyces conicus, C. denticulatus, C. kanhattiensis, and C. swaticus. The distinction of these spe- cies from previously known taxa in the genus was verified through comparisons of mor- phological features and phylogenetic analyses. Three of these species were collected from tropical plains, whereas C. swaticus was found in temperate mountainous regions. This distribution highlights the adaptability of the genus to diverse ecological niches across the country. A key to the Pakistani Candolleomyces species is also provided.

Key words: Agaricales, macrofungal diversity, mushrooms, new taxa, Psathyrellaceae

Introduction

The family Psathyrellaceae Vilgalys, Moncalvo & Redhead was established by Redhead et al. (2001), with Psathyrella (Fr.) Quél. as the designated type genus. Species within the family have a cosmopolitan distribution, typically growing on decaying logs, humus, or soil, as well as woody debris, in woodlands, lawns, or bogs, and can exhibit either broad or specific substrate relationships (Wachter and Melzer 2020; Wang et al. 2022). The family exhibits remarkable diversity, comprising 20 genera worldwide (Wachter and Melzer 2020; Wang et al. 2022). Among these genera, Candolleomyces D. Wacht. & A. Melzer was separated from Psathyrella based on multi-locus phylogenetic analyses and the absence of pleurocystidia (Wachter and Melzer 2020). Based on extensive specimen analy- sis, including morphological and phylogenetic studies, 25 species of Psathyrella have been reassigned to Candolleomyces (Wachter and Melzer 2020). The ge- nus is characterized by small- to large-sized basidiomata; a veil that is present mainly as fibrillose to scaly or granulose; pleurocystidia absent; clamps pres- ent; and a germ pore that is central but often indistinct. Species of this genus are mostly reported from lignicolous, terrestrial, and, rarely, fimicolous habitats.

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Globally, 73 species have been reported (Ahmad et al. 1997; Ahmad et al. 2024; Han et al. 2024; Cao et al. 2025; Hongsanan et al. 2025; Kiran et al. 2025; Li et al. 2025; Nayana and Pradeep 2025a, b). Asia is the richest continent for Can- dolleomyces diversity, with 51 species, followed by 11 from Europe, six from North America, three from Africa, and one from South America (Haqnawaz et al. 2023b; Ahmad et al. 2024; Han et al. 2024; Kiran et al. 2025; Li et al. 2025; Nayana and Pradeep 2025a, b). In Pakistan, 19 species of the genus have been documented (Ahmad et al. 2024; Han et al. 2024; Haqnawaz et al. 2024a, b; Kiran et al. 2025; Li et al. 2025; Nayana and Pradeep 2025a, b). Taxonomically, this group is challenging due to limitations in phylogenetic analyses, such as inaccurate sequences and small numbers of base pair differences among spe- cies, even when multiple regions are used (Yang et al. 2025). This study aims to assess the diversity of macrofungi in general, and specifically of the genus Candolleomyces, to facilitate resolution of the genus’s delimitation.

Materials and methods

Sampling site Fig. 1

Several basidiomata of Candolleomyces were collected from four sites: one in Khyber Pakhtunkhwa (Swat) and three in the Punjab Plains (Khushab, Kot Addu, and Lahore) during the monsoon seasons (July-September) of 2016-2022. The climate of District Swat is moist temperate, with average temperatures ranging from a winter minimum of 4.8 °C to a summer maximum of 33.5 °C and an average annual rainfall of approximately 800 mm. Precipitation occurs mainly in spring and summer, with snowfall at higher elevations. Dominant for- est trees include Abies pindrow (Royle) Spach, Pinus wallichiana A. B. Jackson, and Diospyros kaki (Roxb. ex D. Don) G. Don (Sher et al. 2010). Kanhatti Garden, located in Khushab District, experiences hot summers and severe winters, with an overall temperature range of —1 to 50 °C and an average annual rainfall of 103 mm (Shah and Rahim 2017). The climates of Kot Addu and Lahore are very hot during summer and mild in winter, with maximum recorded temperatures of 51 °C and 48.3 °C, respectively, and average annual rainfalls of 127 mm and 575 mm, respectively (Haqnawaz et al. 2023a). The predominant flora of the Punjab Plains consists of Calotropis procera (Ait.) Ait. f., Dalbergia sissoo Roxb. ex DC., Diospyros malabarica (Desr.) Kostel., Punica granatum Linn., Tamarix aphylla (L.) H. Karst., Saccharum bengalense Retz, S. spontaneum L., Syzygium cumini (L.) Skeels, Vachellia nilotica (L.) PJ.H. Hurter & Mabb, and Zizyphus mauritiana Lam. (Stewart 1972; Haqnawaz et al. 2023a, b).

Morphological characterization

Fresh basidiomata were photographed in situ, and information was recorded (Rathnayaka et al. 2025). The specimens were then taken to the laboratory at the Institute of Botany, University of the Punjab, where their morphological charac- teristics were recorded using the standardized terminology of Vellinga (2001). Color codes were noted according to the Munsell Color Chart (Munsell 1994). All basidiomata were dried either using a hot-air dryer at 30-35 °C or in sunlight,

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Muhammad Hagnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

(a) Khushab _ : (b) Kot Addu ?

(c) Swat (a) Scale: 1:2,200,000* (d) Lahore (b) Scale: 1:1,600,000*

(c) Scale: 1:2,400,000* (d) Scale: 1:95,000* *Scale is in Km

Figure 1. Maps of sampling sites where the novel taxa were found are shown in red.

packed in polythene bags, and deposited at the LAH Herbarium, Institute of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan. Tissues from different parts of the basidiomata were rehydrated in 5% KOH and stained in Congo Red (1% w/v). Microscopic structures were examined using a compound microscope (OLYMPUS BX43, Tokyo, Japan) and measured using Scopelmage 9.0 software, which was connected to the microscope through a digital camera (HDCE-90D). The short form [n/m/p] indicates ‘n’ basidiospores measured from ‘m’ basidiomata of ‘p’ collections. Basidiospore measurements were recorded as (a—) b-c (-d), where a = extreme minimum value; range b-c covers at least 90% of the calculated values; and d = extreme maximum value. ‘LE denotes length, ‘W’ denotes width, ‘av.L denotes average length, and ‘av.W’ denotes average width. ‘Q’ represents the individual spore length/width ratio, while ‘Qav’ represents the average of all Q values (Ge et al. 2010).

Molecular phylogenetic analyses

DNA was extracted from the lamellae of dried basidiomata using a modified 2% CTAB protocol (Bruns 1995). The quality of the extracted DNA was evaluated by 1% (w/v) agarose gel electrophoresis (Voytas 2000). The ITS region of the rDNA was amplified using the ITS1F and ITS4 primers; for the LSU region, the LROR and LRd primers; and for the tef-1a gene, the EF1—983F and EF1-1567R

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Muhammad Hagnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

primers were used (Vilgalys and Hester 1990; White et al. 1990; Gardes and Bruns 1993). PCR conditions described by Gardes and Bruns (1993) were car- ried out with the cycling program as follows: initial denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s; annealing at 54 °C for 1 min; extension at 71 °C for 2 min; and a final extension at 71 °C for 5 min. Successful amplifica- tion of both regions was confirmed by 1% agarose gel electrophoresis using a DNA ladder of 1 Kb and 2 Kb (Usman and Khalid 2020). PCR products for ITS and LSU and DNA products for tef-1a rRNA were then sent to the sequencing company Tsing Ke, China.

The final internal transcribed spacer (ITS), large subunit (LSU) ribosomal RNA, and translation elongation factor 1-alpha (tef-1a) consensus sequences were obtained by assembling both forward and reverse primers using BioEdit v. 7.2.5 (Hall 1999). The reference sequences used for phylogenetic tree con- struction were retrieved from the NCBI GenBank database (Sayers et al. 2024). The BLASTn results of close matches showed a similarity of 95% identity or greater, encompassing all published sequences of the genus Candolleomyces (Haqnawaz et al. 2024a, b; Li et al. 2025). For the phylogenetic analyses, a Clustal W MUSCLE alignment was implemented in BioEdit v. 7.2.5 with manual adjustments. A combined (ITS-LSU-tef-1a) maximum likelihood phylogenetic tree was constructed using RAxML-HPC2 v. 8.2.12 on XSEDE (8.2.10) imple- mented on the CIPRES Science Gateway (Miller et al. 2010). The GTR+GAMMA nucleotide substitution model was used, with 1,000 bootstrap iterations per- formed using rapid bootstrapping. Bayesian inference phylogenetic analyses were performed using MrBayes v. 3.2.2 (Ronquist et al. 2012). The model of evolution was estimated by MrModeltest 2.2 (Nylander 2004). Markov chain Monte Carlo (MCMC) sampling in MrBayes v. 3.2.2 (Ronquist et al. 2012) was used to determine posterior probabilities (PP). Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 1,000" gen- eration. Bootstrap values = 50% and Bayesian PP = 0.90 are indicated on the branches, which were visualized in FigTree v. 1.4.2 (Rambaut 2014). The newly generated sequences were submitted to GenBank, and short descriptions of the species were deposited in MycoBank (Robert et al. 2013).

Results

Phylogenetic analyses Fig. 2, Tables 1, 2

A combined phylogenetic tree was constructed based on ITS, LSU, and tef-1a rRNA sequences, using both maximum likelihood and Bayesian inference meth- ods. All sequences in the dataset had an aligned length of 2,011 nucleotide sites, consisting of 646 from ITS, 864 from LSU, and 501 from tef-1a. Among these, 1,441 were conserved sites, 554 were variable, 289 were parsimony-informative, 264 were singletons, and 50 were undetermined characters. The maximum like- lihood analysis resulted in a final optimized likelihood value of -8939.483906, and the gamma distribution shape parameter was a = 0.7201. The estimated substitution rate matrix was A-C (1.0625), A-G (1.8756), A-T (1.4431), C-G (0.5381), C-T (4.2531), and G-T (1.0000). The tree comprises 69 sequences of the genus Candolleomyces, with two sequences of Psathyrella thujina A.H. Sm.

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LAH38259 Candolleomyces virgatus LAH38260 Candolfeomyces virgatus LAH37634 Candolleomyces amygdaliformis LAH35719 Candolleomyces campanulatus LAH36809 Candolleomyces asiaticus K-M 252085 Candolleomyces kely LAH37840 Candolleomyces parvipileus SFSU DED 8339 Candolleomyces cacao HMJAU37807 Candolleomyces subcacao TBGT(M)18412 Candolleomyces niveofloccosus TBGT(M)19327 Candolleomyces gregalis K-M 252139 Candolleomyces analalavaensis HMJAU37811 Candolfeomyces subsingeri . LAH35327 Candolleamyces iqhbalii A400 MICH AH-14321 Candolleomyces halophilus LAH38108 Candolleomyces umbonatus Sharp20863 Candoifeomyces uteopallidus oao.9a7-‘ GB:LO55-12 Candolleemyces sulcatotuberculosus — HMJAU 37877 Candoteomyces singeri HMJAU37801 Candolleomyces subminutisporus GLM-F 126263 Candolleomyces eurysporus IHI B618 Candolleomyces aberdarensis HKAS123158 Candolleomyces ruhunensis LAH37636 Candolleomyces koladduensis

69/1 90/0.95

ot!

1100/4

100/71

HKAS123162 Candolleomyces typhae 100! MJD10-17 Candolleomyces typhae TBGT(M)18427 Candolleomyces typhae RDE205 Candolleomyces typhae

7100/1

100/41

GMB-W1197 Candolfeomyces hyder

0.03

Figure 2. Phylogenetic tree of the genus Candolleomyces as generated by maximum likelihood (ML) and Bayesian analyses, based on combined ITS, LSU, and tef-1a sequences. Bootstrap values = 50%, based on 1,000 replicates and Bayesian poste- rior probabilities (PP) = 0.90, are shown at the branches. Novel sequences, generated during this study, are shown in bold.

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Muhammad Hagnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

Table 1. Sequences used for phylogenetic analyses of Candolleomyces, including their current names, locality, voucher specimens, and GenBank accession numbers of the ITS, LSU, and tef-1a regions. Sequences generated for this study are shown in bold.

Species Name Locality Voucher ITS LSU tef-la

Candollemyces aberdarensis Kenya IHI B618 MK421517 MK421517 -

C. amygdaliformis T Pakistan LAH37634 PP375293 PP375296 5

C. analalavaensis Madagascar K-M 252139 PQ636530 - -

C. albipes Sao Tomé DED 8340 KX017209 - -

C. albosquamosus T India TBGT(M)18600 0Q676549 - -

C. asiaticus T Pakistan LAH36809 OK392605 0Q802842

C. badhyzensis T Turkmenistan TAA79478 KC992883 -

C. bivelatus Italy MCVE29117 MF325962 - MF521811 C. brevisporus T China HMAS 258919 OR822167 OR822149 OR819986 C. cacao T Sao Tomé SFSUDED8339 KX017210 -

C. campanulatus T Pakistan LAH35719 0Q308881 0Q802837

C. candolleanus Sweden LAS73030 KM030175 KM030175 -

C. cladii-marisci T Italy CLU F302 MK080112 - :

C. conicus T Pakistan LAH38582 PV265173 PV265184 PV357393 C. conicus Pakistan LAH38583 PV265174 PV265185 PV357394 C. crenatus T Pakistan LAH38257 PQ329548 PQ329553 PQ369432 C. crenatus Pakistan LAH38258 PQ329547 PQ329554 PQ369433 C. denticulatus T Pakistan LAH38584 PV265175 PV265186 PV357395 C. denticulatus Pakistan LAH38585 PV265176 PV265187 PV357396 C. gregalis India TBGT(M)19327 PQ415499 - -

C. gyirongicus T China HMAS 287612 PP734613 PP734624 PP729326 C. hydei China GMB-W1197 PV434834 PV434836 -

C. kotadduensis T Pakistan LAH37636 0Q968359 0Q968362 -

C. kanhattiensis T Pakistan LAH38586 PV265177 PV265189 PV357397 C. kanhattiensis Pakistan LAH38587 PV265178 PV265188 PV357398 C. kely China K-M 252085 PQ636531 - -

C. lignicola T China HMAS 258921 OR822169 OR822151 OR819988 C. pakistanicus T Pakistan LAH37829 0Q968356 0Q968363 -

C. efflorescens Sri Lanka Pegler2133 KC992941 ; 5

C. eurysporus T Vietnam GLM-F126263 MT651560 MT651560 -

C. halophilus T Spain MICH AH-14321 MG825900 - -

C. incanus T China BJTC Z777 ON042759 ON042766 ON098509 C. iqbalii T Pakistan LAH35327 0Q968353 0Q968366 -

C. luteopallidus T USA Sharp20863 KC992884 KC992884 -

C. niveofloccosus T India TBGT(M)18412 0Q878345 OR244387 -

C. niveosquamosus T India TBGT(M)18773 PP741631 PP741635 -

C. pabbiensis T Pakistan LAH35326 PP058339 PP058345 -

C. parvipileus T Pakistan LAH37840 0Q968357 OR506278 -

C. pallidosquamulosus Pakistan LAH38243 PP973161 PP973169

C. rubrobrunneus T India TBGT(M)19475 PP741633 PP741636 -

C. ruhunensis T Sri Lanka HKAS123158 ON685315 - -

C. swaticus T Pakistan LAH37132 PV265182 PV265191 -

C. swaticus Pakistan LAH37131 PV265183 PV265190 -

C. shennongjianus T China HMAS 258909 OR822157 OR822139 OR819976 C. shennongdingicus T China HMAS 258918 OR822166 OR822148 OR819985 C. sindhudeltae T Pakistan LAH37632 0Q247908 0Q247912 -

C. singeri China HMJAU 37877 MW301073 MW301091 MW314080 C. subcacao T China HMJAU37807 MW301064 MW301092 MW314081 C. sichuanicus T China HMAS 287616 PP734617 PP734628 PP729330

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Species Name Locality Voucher ITS LSU tef-la

C. subcandolleanus T China BJTC Z239 ON042755 ON042762 ONO098505 C. subminutisporus T China HMJAU37801 MW301066 MW301094 MW314083 C. subsingeri T China HMJAU37811 MG734715 MW301097 MW314085 C. sulcatotuberculosus Germany GB:LO55-12 KJ138422 - -

C. sultanii T Pakistan LAH35714 0Q308835 0Q801565 -

C. thailandensis T Thailand SDBR-CMUNK0443 MZ146874 - -

C. tuberculatus Benin ADK3564 KC992934 - -

C. typhae China HKAS123162 ON692696 - =

C. typhae China MJD10-17 JX077004 - 5

C. typhae India TBGT(M)19427 OP225544 : :

C. typhae China RDE205 PQ415503 - -

C. umbonatus T Pakistan LAH38108 PP058341 PP058347 ;

C. undulatus T Pakistan LAH38255 PQ329546 PQ329551 PQ369428 C. virgatus T Pakistan LAH38259 PQ329543 PQ329549 PQ369430 C. virgatus Pakistan LAH38260 PQ329544 PQ329550 PQ369431 C. yanshanensis T China BJTC Z110 ON042758 ON042765 ON098507 C. sp. 1 Pakistan LAH36628 PV265179 - : C.-sp.2 Pakistan LAH36629 PV265180 : ; C.-sp.3 Pakistan LAH38590 PV265181 - : Psathyrella thujina (Outgroup) USA Smith66720 KC992876 KC992876 - Hausknechtia leucosticta (Outgroup) India CNF 1/6900 ON745618 ON745617 ON746005

Notes: Type specimens are marked with T; the symbol “-” indicates no sequence available in GenBank.

Table 2. Base pair differences between newly described Candolleomyces species and closest sister taxa based on ITS, LSU, and tef-1a sequence comparisons.

New Species Closest Sister Species Region(s) Base Pair Differences

C. conicus C. sindhudeltae ITS + LSU 7 C. undulatus ITS + LSU + tef-1a 14

C. denticulatus C. rubrobrunneus ITS + LSU 6 C. kanhattiensis ITS + LSU + tef-1a 10

C. pabbiensis ITS + LSU 19

C. kanhattiensis C. rubrobrunneus ITS 6 C. pabbiensis ITS + LSU 13

C. swaticus C. candolleanus ITS 11 C. badhyzensis ITS + LSU 18

and Hausknechtia leucosticta (Pat.) Tkaléec, J.Q. Yan, C.F. Nie & C.K. Pradeep as the outgroup. The newly proposed taxa are represented in bold in the final phylogenetic tree (Fig. 2). All four new species are present in three clades: Can- dolleomyces conicus occurs in the blue clade; C. kanhattiensis and C. denticula- tus occur in the green clade; and C. swaticus occurs in the yellow clade. Candolleomyces conicus (LAH38582, type) forms a separate branch from its closest species, C. sindhudeltae Haqnawaz, Niazi & Khalid (LAH37632) and C. un- dulatus Haqnawaz, Niazi & Khalid (_AH38255), with differences of 7 base pairs (ITS and LSU) and 14 base pairs (ITS, LSU, and tef-1a), respectively. Candolleomy- ces denticulatus (LAH38584, type) forms a separate branch from its sister spe- cies C. rubrobrunneus, C. kanhattiensis sp. nov., and C. pabbiensis, with differenc- es of 6 base pairs (ITS and LSU), 10 base pairs (ITS, LSU, and tef-1a), and 19 base pairs (ITS and LSU), respectively. Candolleomyces kanhattiensis (_AH38584, type) forms a distinct branch from its sister species C. rubrobrunneus and C. pabbiensis,

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with differences of 6 base pairs (ITS) and 13 base pairs (ITS and LSU), respective- ly. Candolleomyces swaticus (_LAH37132) forms a separate branch from its sister species C. candolleanus (Fr.) D. Wacht. & A. Melzer (LAS73030) and C. badhyzen- sis (Kalamees) D. Wacht. & A. Melzer (TAA79478), with differences of 11 base pairs (ITS) and 18 base pairs (ITS and LSU), respectively.

Taxonomy

Candolleomyces conicus Haqnawaz, Niazi & Khalid, sp. nov. MycoBank No: 858281 Figs 3,4

Etymology. The species name “conicus” (Latin) refers to the conical pileus.

Holotype. PAKISTAN * Punjab Province: Lahore District, University of the Pun- jab, (31°29'39"N, 74°18'06"E, 227 m a.s.l.), on loamy soil, rich in organic mat- ter, under Dalbergia sissoo, 10 August 2024, Muhammad Haqnawaz, PU-08 (LAH38582). GenBank: PV265173 [ITS], PV265184 [LSU], PV357393 [tef-1a].

Diagnosis. Candolleomyces conicus is different from the closest species, C. sindhudeltae, by its relatively larger (20-90 mm diam.), conical, plane, red- dish gray to light gray pileus with uplifted margins; brownish gray to dull red- dish-brown lamellae, a large stipe (50-130 x 4-8 mm), a sheathing annulus with a wavy fibrillous margin, lecythiform, fusiform, tibiform cheilocystidia, and the pileipellis, a transition between hymeniderm and epithelium.

Description. Pileus 20-90 mm diam., parabolic to campanulate with undulat- ing margin in young stages, conical to campanulate than plane with uplifted and undulating margin when old; with a conical umbo, appressed scales, cracked at center, presence of veil elements; dull orange (7.5 YR 7/3) with grayish brown (7.5YR 6/2) center when young, reddish gray (5R 6/1) to light gray (10 YR 8/2) with dull orange (5YR 6/3) center when old, with light gray (5YR 8/1) squamu- les. Lamellae adnate, narrow to broad, even, with 3-7 tiers of lamellulae, light brownish (5YR 7/1) when-young, brownish gray (5 YR 6/1) to dull reddish brown (2.5 YR 5/3) when mature. Stipe 50-130 x 4-8 mm, central, flexuous, tomen- tose, broad at base and narrow at apex, fibrillose, grayish white (N 8/0) to light gray (7.5 Y 7/2). Annulus sheathing, wavy and fibrillous at the margin.

Basidiospores [200/4/2], (6—)6.5-8.5(—9) x (4-)4.5-5(-6) um, Q = 1.3-1.8, Qav = 1.65, ellipsoid to amygdaliform, thick-walled, smooth, guttulate, central germ pore present, red in water. Basidia (12—)13-21(-22) x (6-)7—9(-10) um, clavate, hyaline in water, thick-walled, smooth, with 2-4 sterigmata. Cheilo- cystidia (11—)12—49(-50) x (10-)11-44(—45) um, utriform, narrowly clavate to broadly clavate, ovoid, globose to subglobose, capitate to lecythiform, cylin- drical, oblong, lageniform, flexuose, fusiform, tibiform, hyaline to olive yellow in water, thick-walled. Pleurocystidia absent. Pileipellis a transition between hymeniderm and epithelium, one-layered, thin-walled, 36-60 x 29-50 um, glo- bose to subglobose and ellipsoid to broadly ellipsoid, clavate to broadly clav- ate, cylindrical cells, hyaline to olive yellow in water. Stipitipellis a cutis, hyphae subregular, branched 6-17 um in diameter, thin-walled, septate, and hyaline to yellow in water. Caulocystidia (18—)19-39(—40) x (9-)10-15(-16) um, utri- form, clavate to broadly clavate, ellipsoid, oblong, hyaline to olive yellow in wa- ter. Clamp connections present in all tissues.

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Muhammad Hagnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

Figure 3. A-F. Basidiomata of Candolleomyces conicus sp. nov. (holotype LAH38582). Scale bars: 20 mm.

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Muhammad Haqnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

Figure 4. Microscopic structures of Candolleomyces conicus sp. nov. (holotype LAH38582). A. Basidia; B. Basidiospores; C. Cheilocystidia; D. Caulocystidia. Scale bars: 10 um (A-F).

Ecology and habitat. Terrestrial, caespitose, on loamy soil rich in organic matter, under the dead trunk of Dalbergia sissoo.

Additional material examined. PAKISTAN * Punjab Province: Kot Addu, Pir- har Gherbi (30°32'48'"N, 70°50'40"E, 136 m a.s.l.), on loamy soil, 11 Aug. 2023, Muhammad Haqnawaz, HQ-210 (LAH38583). GenBank: PV265174 [ITS], PV265185 [LSU], PV357394 [tef-1a].

Notes. According to phylogenetic analyses, the new species Candolleomyces conicus forms a separate clade from its sister species, such as C. sindhudeltae and C. undulatus. Morpho-anatomically, Candolleomyces conicus is different from C. sindhudeltae by having a plane, reddish gray pileus with uplifted mar- gins, dull reddish-brown lamellae, a long stipe (50-130 mm length), lecythiform, and tibiform cheilocystidia. Candolleomyces sindhudeltae has an umbonate, grayish white, scalloped to cracked pileus with a decurved margin, small stipe (20-35 mm length), ovoid-pedunculate cheilocystidia (Haqnawaz et al. 2023b).

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Candolleomyces undulatus has small (10-27 mm), straight shape, light purplish gray pileus with light brownish gray center, 1-3 tiers of lamellulae, light reddish gray lamellae, small stipe (15-46 x 2-4 mm), cylindrical, spherical with broad peduncle of basidia, ovoid, spheropedunculate, and globose caulocystidia (Li et al. 2025).

Candolleomyces denticulatus M. Khalid, Haqnawaz & Afshan, sp. nov. MycoBank No: 858282 Figs 5, 6

Etymology. The species name “denticulatus” (Latin) refers to the toothed Cap margins.

Holotype. PAKISTAN * Punjab Province: Lahore District, University of the Pun- jab, (31°29'39"N, 74°18'06"E, 227 maz.s.l.), on loamy soil, rich in organic matter, under Punica granatum Linn, 15 Aug. 2024, Muhammadah Khalid, Abdul Nasir Khalid, and Muhammad Haqnawaz, PU-10 (LAH38584). GenBank: PV265175 [ITS], PV265186 [LSU], PV357395 [tef-1a].

Diagnosis. Candolleomyces denticulatus differs from C. rubrobruneus in having toothed cap margins, abundant squamules when young, a grayish-white pileus with a pale orange center, grayish-white edges of the lamellae, and cheilocystidia that are globose, ellipsoid, pedunculate, globose, oblong, or lecythiform; at the same time, caulocystidia are tibiiform, lecythiform, oblong, fusiform, and moniliform.

Description. Pileus 20-40 mm diam., parabolic in young stages, convex to plane when old, veil remnants present, dense squamules present in young stag- es, mostly removed when old, margin straight and split in symmetry to toothed, cracked surface, light gray (10 YR 8/1) to grayish white (N 8/0) with dull reddish brown (10 YR 5/4) to pale orange (5 YR 8/3) center, grayish white (N 8/0) squa- mules. Lamellae adnate, narrow, even to rarely eroded, with 3-7 tiers of lamel- lulae, forked, grayish brown (5 YR 6/2) to light brownish gray (5 YR 7/2), edge grayish white (N 8/0). Stipe 20-30 mm, central, equal, flexuous, surface sca- brous, dull reddish brown (10 YR 5/4) to grayish white (N 8/0). Annulus absent.

Basidiospores [150/3/2], (6.0—)6.5-11.0 (-11.6) x (4.2-)4.5-6.0 (-—6.3) ym, Q=1.2-1.7, Qav = 1.56, amygdaliform in side view, ellipsoid in frontal view, smooth, guttulate, thick-walled, light red in water. Basidia (11—-) 11.9-21.1(—21.5) x (7.5- )7.9-9.1 (—9.2) pm, clavate, hyaline to light gray (5Y 7/1) in 5% KOH, thin-walled, smooth, with 2-4 sterigmata, basal clamp connections present. Cheilocystidia 25-42 x 8-20 um, narrowly utriform to broadly utriform, pedunculate, ovoid to conical, globose to subglobose, lecythiform, ellipsoid—pedunculate, narrowly la- geniform, thick-walled, hyaline to yellowish gray (2.5Y 5/1) in 5% KOH. Pleurocys- tidia absent. Pileipellis an epitheloid hymeniderm, thick-walled, cells abundantly globose to subglobose, clavate to broadly clavate, ellipsoid to broadly ellipsoid, rarely utriform, oblong, lecythiform, cylindrical, 20-55 x 15-45 pm, hyaline to light gray (5Y 7/1) in 5% KOH. Stipitipellis made of 4.3—5.4 um diam., subregular hy- phae, rarely branched, hyaline in 5% KOH, thin-walled septate, clamp connections abundant. Caulocystidia 27-64 x 8-18 um, utriform to utriform pedunculate, slightly cylindrical, flexuous, ellipsoid, tibiiform, lecythiform, oblong, capitate, fusi- form, moniliform, hyaline to light gray (5Y 7/1) in 5% KOH, thick-walled.

Ecology and habitat. Gregarious and caespitose, terrestrial, on alluvial soil rich in organic matter.

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r + . 4 4 = i ana ead bal : . a be dS -*

Figure 5. A-D. Basidiomata of Candolleomyces denticulatus sp. nov. (holotype LAH38584). Scale bars: 10 mm.

Additional material examined. PAKISTAN * Punjab Province: Kot Addu, Pirhar Gherbi (30°33'07'"N, 70°50'40"E, 137 m a.s.l.), on loamy soil, 21 Aug. 2024, Mu- hammadah Khalid, Abdul Nasir Khalid, and Muhammad Haqnawaz, KA-103 (LAH38585). GenBank: PV265176 [ITS], PV265187 [LSU], PV357396 [tef-1a].

Notes. Phylogenetically, Candolleomyces rubrobrunneus, C. pabbiensis, and C. kanhattiensis nov. sp. formed a separate branch from the new taxon C. den- ticulatus. Morpho-anatomically, Candolleomyces denticulatus is different from C. rubrobrunneus by having a toothed pileus margin, abundant squamules at the immature stage, grayish white pileus with pale orange center, grayish white edges of lamellae, yellowish gray basidiospores, ellipsoid pedunculate, utriform, oblong, lecythiform, cylindrical cheilocystidia; tibiiform, lecytheform, fusiform, moniliform caulocystidia. In contrast, C. rubrobrunneus has a straight margin, a dark brown pileus with a brownish, pale, fibrillose to floccose veil, grayish-orange to brown edges of lamellae, olive-brown basidiospores, broadly fusiform capi- tate cheilocystidia, and subcapitate apex of caulocystidia (Nayana and Pradeep 2024). However, C. pabbiensis is distinguished from our new taxon by conical to broadly parabolic pileus with straight margins, grayish white lamellae, light brown basidiospores, and plump cheilocystidia with an obtuse to subobtuse

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Figure 6. Microscopic structures of Candolleomyces denticulatus sp. nov. (holotype LAH38584). A. Cheilocystidia; B. Basidiospores; C. Pileipellis; D. Caulocystidia. Scale bars: 10 um (A-D).

apex (Haqnawaz et al. 2024b). Candolleomyces kanhattiensis differs from the new taxon C. denticulatus by having a sub-globose to campanulate pileus with decurved, undulating margins, a shiny stipe with a clavate base, greenish gray ba- sidiospores, and cylindrical, ellipsoid, and globose cheilocystidia (in this study).

Candolleomyces kanhattiensis Bibi, Haqnawaz, Afshan & Khalid, sp. nov. MycoBank No: 858285 Figs 7,8

Etymology. The species name “kanhattiensis” (Latin) refers to the type locality of the species, ‘Kanhatti Garden’, Khushab, Pakistan.

Holotype. PAKISTAN * Punjab Province: Khushab District, Kanhatti Garden (32°25'22"N, 72°14'59"E, 555 maz.s.l.), on loamy soil, rich in organic matter, un- der Phoenix dactylifera, 15 Aug. 2022, Ayesha Bibi, KUN-03 (LAH38586). Gen- Bank: PV265177 [ITS], PV265189 [LSU], PV357397 [tef-1al.

Diagnosis. This species is different from its closest species, Candolleomy- ces rubrobrunneus, by having sub-globose to campanulate, dull orange pileus with decurved margins, shiny stipe with clavate base, light brownish gray basid- iospores, and cylindrical, ellipsoid, and globose cheilocystidia.

Description. Pileus 15-25 mm in diam., sub-globose to campanulate, con- vex, slightly umbonate, decurved with even to undulating margins, smooth sur- face, orange (5YR 7/6) to dull orange (7.5YR 7/4), with distinct dark orange (7.5YR 8/6) center. Lamellae adnate, narrow, even, with 3-7 tiers of lamellulae,

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— nd ' . —; aa itae\ a rr Lis ae ‘ | a” ‘ sR ¥

Figure 7. A-D. Basidiomata of Candolleomyces kanhattiensis sp. nov. (holotype LAH38586). Scale bars: 10 mm.

dull orange (5YR 7/3), entire edge. Stipe 30-65 x 2-5 mm, cylindrical, central, equal, grayish white (N 8/0), shiny and smooth upwards, clavate base, narrow toward pileus. Annulus absent.

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aS) ee:

Figure 8. A-D. Microscopic structures of Candolleomyces kanhattiensis sp. nov. (ho- lotype LAH38586). A. Basidia; B. Basidiospores; C. Cheilocystidia; D. Pileipellis. Scale bars: 10 um (A-D).

Basidiospores [150/3/2], (5.5-) 6-6.5 (-7) x (3.9-) 4-4.2 (-4.5) um, Q = 1.541-1.55, Qav = 1.53, ellipsoid to ovoid, thick-walled, dull reddish in wa- ter, smooth, apiculate, germ-pore absent, inner contents visible. Basidia (18-) 19-22 (-24) x (6-) 7-8.5 (—9) pm, sub-clavate to clavate, cylindrical, hyaline in KOH, thin-walled, smooth, with 2-4 small sterigmata. Cheilocystidia (18-) 19-45 (—46) x (8.5-) 9-16 (-17) um, avl x avw = 34 x 14 um, ovoid, clavate, lageniform, broadly cylindrical, ellipsoid to broadly ellipsoid, utriform, globose to subglobose, thick-walled. Pleurocystidia absent. Pileipellis an irregular epithelium, 15-25 x 8-20 um, made up of oblong, subglobose, ellipsoid to broadly ellipsoid, clavate hyaline to yellowish in water, thin-walled, smooth, up to 39 um broad. Stipitipellis a cutis made up of thin-walled hyphae, 4-12 um diam., smooth, hyaline in KOH, branched, clamp connections present.

Ecology and habitat. Gregarious, rich clayey soils in damp places, some in rotting leaves, compost piles, or dead small sticks of wood.

Additional specimens examined. PAKISTAN * Punjab Province, District Khu- shab, in groups on muddy nutrient-rich soil under Dalbergia sissoo, sometimes on woody debris, 05 Aug. 2023, Ayesha Bibi, KHU-33 (LAH38587). GenBank: PV265178 [ITS], PV265188 [LSU], PV357398 [tef-1a].

Notes. Phylogenetically and morpho-anatomically, Candolleomyces kan- hattiensis is closely related to C. rubrobrunneus, C. denticulatus nov. sp., and

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C. pabbiensis. |t can be distinguished from C. rubrobrunneus by its sub-globose to campanulate, dull orange with dark orange center, decurved, undulating margin of pileus, shiny stipe with clavate base, greenish gray to light brown- ish gray basidiospores, and cylindrical, ellipsoid, and globose cheilocystidia. In contrast, C. rubrobrunneus has a conical, reddish brown to dark brown pileus with straight margins and a fibrillose to floccose surface, a pruinose apex of the stipe, broadly fusiform, capitate, flexuose cheilocystidia, and brownish yellow basidiospores (Nayana and Pradeep 2024). Candolleomyces pabbiensis exhib- its a conical to broadly parabolic pileus with straight margins, grayish white lamellae, light brown basidiospores, and plump cheilocystidia with an obtuse to subobtuse apex (Haqnawaz et al. 2024b). Candolleomyces denticulatus has toothed margins, abundant squamules when young, a grayish white pileus, and reddish gray to yellowish gray basidiospores. Furthermore, the cheilocystidia are conical, and polymorphic caulocystidia are also present (in this study).

Candolleomyces swaticus Naseer, Haqnawaz & Khalid, sp. nov. MycoBank No: 858287 Figs 9, 10

Etymology. The species name “swaticus” (Latin) refers to the type locality, Swat.

Holotype. PAKISTAN * Khyber Pakhtunkhwa Province: Swat (35°04'37'N, 72°24'33'E, 1480 m a.s.l.), on loamy soil, 11 Aug. 2016, Arooj Naseer, A. N. Kha- lid, SK-23 (LAH37132). GenBank: PV265182 [ITS], PV265191 [LSU].

Diagnosis. Candolleomyces swaticus is different from Candolleomyces candolleanus by its large (20-60 mm), light gray with yellow-orange cen- ter, umbonate pileus, dull reddish-brown lamellae with 3-7 tiers, scales on stipe surface, bright brown basidiospores, capitate, lageniform, flexuose, tibiform, moniliform cheilocystidia.

Description. Pileus 20-60 mm diam., campanulate at young stage, convex to plane when old, broadly umbonate, margin straight, split and irregularly wavy, pres- ence of veil elements, smooth dull surface; light gray (10 YR 8/1) with dull yellow orange (10 YR 7/3) center. Lamellae adnexed, narrow to broad, average, with 3-7 tiers of lamellulae, dull reddish brown (2.5 YR 5/4). Stipe 25-55 x 4-8 mm, central, flexuous, tomentose, equal in width, scaly, grayish white (N 8/0). Annulus absent.

Basidiospores [150/3/3], (6—)6.5-7.5(-8) x (4-)4.2-4.8(-5) um, Q = 1.3- 1.8, Qav = 1.65, amygdaliform in side view, ovoid in frontal view, thick-walled, smooth, guttulate, central germ pore present, bright brown in water. Basidia 15-20 x 6.3-10.2 tm, clavate to broadly clavate, hyaline in water, thick-walled, smooth, with 3-4 sterigmata. Cheilocystidia (30—-)31-—49(-50) x (10-)11-19(- 20) um, utriform to slightly lecythiform, capitate, cylindrical to cylindrical with median constriction at upper side, narrowly lageniform to broadly lageniform, flexuose, broadly fusiform, tibiform, moniliform, hyaline to slightly yellowish in water, thick-walled. Pleurocystidia absent. Pileipellis an irregular epithelium, one layer, of thick-walled, 25-75 x 18-70 um, globose to subglobose and ellip- soid to broadly ellipsoid, clavate to broadly clavate, cylindrical cells, hyaline to olive yellow in water. Stipitipellis a cutis, made of hyphae subregular, branched, 6-17 um in diameter, thin-walled, septate, and hyaline to yellow in water. Cau- locystidia not recorded. Clamp connections present in all tissues.

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e- < ¢

Figure 9. A-C. Basidiomata of Candolleomyces swaticus sp. nov. (holotype LAH37132). Scale bars: 20 mm.

Ecology and habitat. Terrestrial, gregarious, under a coniferous tree, on allu- vium soil, during the rainy season.

Additional material examined. PAKISTAN * Khyber Pakhtunkhwa Province: Swat (35°04'37'N, 72°24'33'E, 1480 m a.s.l.), on loamy soil, 15 Aug. 2019, Arooj Naseer, A. N. Khalid, SK-01 (LAH37131). GenBank: PV265183 [ITS], PV265190 [LSU].

Notes. According to phylogenetic analyses, Candolleomyces swaticus is close- ly related to C. candolleanus and C. badhyzensis. Morpho-anatomically, the for- mer is different from the latter by having a campanulate pileus when young, being light gray with a dull yellow-orange center, having absence of hairy material (vs.

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Figure 10. A-D. Microscopic structures of Candolleomyces swaticus sp. nov. (holotype LAH37132). A. Basidia; B. Basidiospores; C. Cheilocystidia; D. Pileipellis. Scale bars: 10 um (A-C); 20 um (D).

C. candolleanus, which is covered with hairy material), having the presence of a broad umbo, and having irregular, wavy cap margins. Candolleomyces candolle- anus has a parabolic pileus at a young stage, a bright brown pileus, and straight to even cap margins (Wachter and Melzer 2020). The next closely related taxon is Candolleomyces badhyzensis, which differs from the new species by a broadly bell- shaped, yellowish-brown pileus and cylindrical to ellipsoid to phaseoliform, pale brown basidiospores (vs. amygdaliform to ovoid, grayish red) (Kalamees 1981).

Key to the Candolleomyces species from Pakistan

1 Annulus preset Ee St ee are, Rae ee ba ee Ra eee eae A eer 2 Annulus absel We scasectes gqnbe sted el ose aea MASE he canter nha PR APM e ania sd UULAM GOO pay axe Pe'eniaten y 2. Pileus width = 50 mm elateta sre okeCotete! wele -eislarctete' sis o(sie-sio'e's'slainie ie hea a e.ere arse viele eieceeeie sie’ ofeelaie vieieisie sieeve oste treleceltrere!s 3

- Pileus width s 50 mm, with uplifted margins, light gray with dull orange cen- ter, long stipe (50-130 x 4-8 mm), capitate to lecythiform cheilocystidia.

C j

paliinevee Mev ate re PE Mel cena tsa pesbelnenaee interes enue emenintanersesdsanieiuemtene sperm n elon nth - Conicus

i Umbo PSS Cll ciscne cttcandenttnastewans Ponsenadtesh Mens pethaate Minne N Lode dard Peuuhireteea deed ddsiate 4 WPT OF AD SEN Bere eee ose UE TLL Nea Rs RR oth nee roa iba a 6

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18

PIlGUS: NOTArGOl al Gey cssefen/dsaverractaadaneddamnmnnaalddoed asupeeretuadderd dave cas fend deve 5 Pileus areolate, bilayered, decurved, and scalloped to cracked cap mar- gins; serrate lamellae, capitate, lageniform, ovoid pedunculate cheilocys-

js | Fe Bis, Rl een tht role, Bin aoe ee Sih d ARE eRe MTSE C. sindhudeltae Paraboloid to campanulate, grayish brown pileus, with appressed pale or- ange squamulose, caespitose base of stipe, absence of caulocystidia ..... PATER tet DS ROME ro. kee oh MoreTTe Deen. CRETE C. pallidosquamulosus White to dirty-white pileus, striations on surface, forked lamellae near margins, presence of Caulocystidia ............. ccc ccesecesseeeeseeees C. sultanii Convex pileus, white to grayish with orange-yellow center, crenate to de- Curved margins Of PilCUS .......... cece ccceecceesccesseeeesseeeesees C. pakistanensis Parabolic to campanulate, light purplish gray pileus with light brownish gray center, eroded lamellae, presence of caulocystidia......... C. undulatus NGC ONESIES, BON cucee cant ct ne oven) ttareenoresiea petteiniene vig anebeen niceent eerie ecstatic ack 8 GG ES 0} | FSET helt peineenretierdll el Pie om. A pected: lah oni hotest fe elencthy Hrevernctet ll 14 NoOt-Caimpanulate PNW S = sss rcscsdabdesa reverses tress qaanet toate Arh decaganttescias Sted dees 9 CalMmpanulare.DNCUS. 4.44492. Anwe distor tees eadealoae Me eA ateaacars mean eereaahedeae as 11 WHITESCOlO Of AGI Os Lo cco aeehon edb heSdpnonenis obheepacedvudeneoobhetpedegmuacediontaoem 10 Reddish gray lamellae, circular, applanate to plano-concave, centrally de- pressed, at center dull reddish brown pileus.....................06 C. granulosis Hemispleuric pileus, covered with saccharine material ...... C. efflorescens Conical to broadly parabolic pileus, covered with light gray squamu- FOSS atte areca es Raa ee en Ee ano ae a ee C. pabbiensis EVeInORregulalCaniMarginsin 2:55 ccna. dco ac, enhances sa AUR aol han doe ld Ree, 12 Striate margins, dull yellow-orange pileus, presence of caulocystidia ........ Po RY eC AR C. campanulatus Light Drownish. Gray; DICUS.< as Sele, Says See eee 0, See er BAe eed ee 13 Bright brown pileus covered with hairy material. ............... C. candolleanus Conical pileus with light gray squamules, amygdaliform to phaseoliform DASICIOS DONES). 2. ro. 5.02: -cccavencodtahectseeesd-sentidaatedenaweanedbahivets C. amygdaliformis Virgate pileus, umbonate, grayish red lamellae.....................06 C. virgatus WING: Pres enitic;..tetsvshetareste arya wale ore dea turds ie old, Baste esa cine, 15 WIM O=AID SONIA sere, <csns raed eevtentacreratlddand draaunpenalddends wtgntt truiehalbard drwwaahet tees tant eat 19 Undulatiig Orirreqular-Ca PATA QU <u. ner sns econ apc dod Sen eddvade os eepadbeemddneedene ors 16 Srenate-Or Silalesca Pt MarGimSy. se xt ct. ce the Mat AEE Redhead SSE 18 Glandular dotted surface of stipe with broad base, sphero-pedunculate CHENGCYV SUM As rece cis erass.antaitcoded neansentreastaones wuaadinnna denver teaassnant C. umbonatus Absence of fibrils on pileus and Shiny Stipe ............. cc cceceeesceeeeeeeeeeees 17 Presence fibrils on pileus, stipe with bulbous base................... C. asiaticus Orange to dull orange, with distinct dark orange center, dull orange lamel- lae, ovoid, ellipsoid, globose cheilocystidia...................... C. kanhattiensis

Light gray with dull yellow orange center, dull reddish brown lamellae, le- cythiform, capitate, broadly fusiform, tibiform, moniliform cheilocystidia.. NS Perce eas dnscnte asics sa Meee phate ich eRe eohsteasTocetbohecomase ace ets ttetee C. swaticus Glandular dotted surface of stipe with broad base, dark reddish to brown lamellae with yellow orange edges, spheropedunculate cheilocystidia,

dark brown baSidiOSpore ..............c.ccccsscceessseceesseeeeesseeeeeeseees C. umbonatus Shiny surface and long (40-65 mm) stipe, light yellowish basidiospores.. MA rhe OLB he, Selena cease ton 25 AA De lean ana tenes MA e de Rattles C. iqbalii

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OPS SUrArO IG TIAL GMI ase ess caaldaneds covuppcrecsaiddas dAraseuures aaslney Aeecnpctatasdsedaroaumnenssadeedades 20 - Decurved margins, light brown to golden brownish pileus, long (55- PASH IAA UHR SUI ©) <tihie Wht aye ede Senet Stent Rak MMe ties Alene hd C. parvipileus 201 - Adnexed Or danate lamellae: cna. neers ccesudereaseuncecsbvetvexsmncenhvanseeteseteeasdhinees 21 - Emarginate lamellae, striations toward the margin of pileus, obovoid pe- dunculate:cheloey Stiga via. cascemsccsadM end eneame maya ahaqetonents C. kotadduensis Zab CLE MATS OSU ALS ALOUD ute. 8200 8c nasronat SV oe bead ire toutes Mibact ws neubieile «suRee Uasaadiions 22 - Split in symmetry to toothed margin, forked, grayish brown lamellae with Ghayi Si Vente SUS 115 :10r 2 cst Aya Oradea ag AE nD C. denticulatus 22 Fimbriate, 1-5 tiers of lamellulae, marginate base of stipe, caulocystidia DIC SCM ccccceececenastopancns voce racrratapevcarceeenesarrs potevccvgerersurapssscuiaseneerennss C. crenatus

- 1-2 tiers of lamellulae clavate base of stipe, absence of caulocystidia..... brie Pesto b eS has Ta Bua cice dh oes aaa chen TP eats hehe MPO eTOE LE C. albogranulosus

Discussion

According to phylogenetic analyses and morpho-anatomical characteriza- tion, all taxa—Candolleomyces conicus, C. denticulatus, C. kanhattiensis, and C. swaticus—which are new to science, formed separate branches from their closest species with significant differences. In comparison to each other, all new taxa show unique characters: C. conicus has large basidiomata, a conical cap with an uplifted margin, an areolate pileus, lecythiform to lagen- iform cheilocystidia, and growth around the dead trunk of Dalbergia sissoo; C. denticulatus has toothed cap margins, grayish white edges of lamellae, and growth under Punica granatum; C. kanhattiensis has a subglobose pileus with decurved margins and a shiny stipe with clavate base; and C. swaticus has a distinct broad umbo, a light gray pileus with a yellow-orange center, dull reddish-brown lamellae, and occurrence in mountainous areas under coniferous trees. Candolleomyces conicus, C. denticulatus, and C. swaticus are terrestrial, while C. kanhattiensis is lignicolous. Candolleomyces conicus, C. denticulatus, and C. kanhattiensis are reported from subtropical regions, while C. swaticus is reported from tropical regions.

In a previous study, certain species of Candolleomyces exhibited signifi- cant differences in their ITS regions. Nevertheless, some Candolleomyces species with high similarity (more than 99%) are still treated as separate spe- cies based on distinct morpho-anatomical characters (Bau and Yan 2021). Therefore, some species are identified based only on the ITS region, while others require additional regions (LSU, tef-1a, and B-tub) for identification. The addition of four new species to the fungal flora of Pakistan makes a significant contribution, increasing the total number of species in the coun- try to 23, which highlights the rich diversity of the genus in the region. This discovery not only expands our understanding of the Candolleomyces genus but also emphasizes the vast diversity of fungal species that remain to be explored. It also serves as evidence of mycologists’ ongoing efforts to un- cover the hidden diversity of fungal species and advance our knowledge of the natural world. Consequently, further extensive systematic surveys are imperative to explore, understand, and document the full extent of Candolle- omyces diversity and distribution within the region.

MycoKeys 121: 143-166 (2025), DOI: 10.3897/mycokeys.121.157282 162

Muhammad Hagnawaz et al.: Morphology and multi-locus phylogeny reveal four new Candolleomyces (Psathyrellaceae) species from Pakistan

Acknowledgments

We sincerely thank Dr. Muhammad Usman, Dr. Entaj Tarafder, and Dr. Munaza Kiran for their critical comments during the preparation of the manuscript. We also thank Dr. Kamran Habib for his assistance with phylogenetic analyses and his suggestions to improve the manuscript. Additionally, we are grateful to Mr. Muhammad Luqman for his help during the field trips.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of Al

No use of Al was reported.

Funding

No funding was reported.

Author contributions

All authors have contributed equally.

Author ORCIDs

Arooj Naseer ® https://orcid.org/0000-0002-4458-9043

Data availability

All of the data that support the findings of this study are available in the main text.

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