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New species of Pseudosperma (Agaricales, Inocybaceae) from Pakistan revealed by morphology and multi-locus phylogenetic reconstruction
expand article infoMalka Saba, Danny Haelewaters§|, Donald H. Pfister, Abdul Nasir Khalid#
‡ Quaid-i-Azam University, Islamabad, Pakistan
§ Purdue University, West Lafayette, United States of America
| University of South Bohemia, České Budějovice, Czech Republic
¶ Harvard University, Cambridge, United States of America
# University of the Punjab, Lahore, Pakistan
Open Access

Abstract

During fungal surveys between 2012 and 2014 in pine-dominated forests of the western Himalayas in Pakistan, several collections of Pseudosperma (Agaricales, Inocybaceae) were made. These were documented, based on morphological and molecular data. During this work, three new species came to light, which are here formally described as Pseudosperma brunneoumbonatum, P. pinophilum and P. triacicularis. These species belong in the genus Pseudosperma fide Matheny et al. (2019) = Pseudosperma clade fide Matheny (2005) = Inocybe sect. Rimosae s.s. fide Larsson et al. (2009). Macro- and micro-morphological descriptions, illustrations and molecular phylogenetic reconstructions of the studied taxa are provided. The new species are differentiated from their close relatives by basidiospore size and colouration of basidiomata. Molecular phylogenetic relationships are inferred using ITS (ITS1–5.8S–ITS2), nrLSU and mtSSU sequence data. All three newly-described taxa likely share an ectomycorrhizal association with trees in the genus Pinus. In addition, five names are recombined in Inosperma, Mallocybe and Pseudosperma. These are Inosperma vinaceobrunneum, Mallocybe erratum, Pseudosperma alboflavellum, Pseudosperma friabile and Pseudosperma neglectum.

Keywords

Ectomycorrhizal fungi, molecular systematics, phylogeny, Pinus roxburghii, southern Asia, taxonomy

Introduction

Inocybe (Fr.) Fr. (Agaricales, Inocybaceae) in the broad sense (sensu lato) is a highly diverse, ectomycorrhizal genus comprising about 735 known species worldwide (Ullah et al. 2018). Inocybe has a widespread distribution and is found commonly in temperate areas and, to a lesser extent, in the tropics (Matheny et al. 2009, Bougher et al. 2012, Matheny et al. 2012). Multi-locus phylogenies of the Inocybaceae by Matheny et al. (2002, 2009) and Matheny (2005) have confirmed that the family is monophyletic. Matheny (2005, 2009) recognised seven major clades within the Inocybaceae; clade names were given with a suggestion to recognise each informally at the generic rank within the family.

Inocybe section Rimosae sensu stricto (fide Larsson et al. 2009, = clade Pseudosperma fide Matheny 2005), traditionally placed in subgenus Inosperma (Kuyper 1986, Kobayashi 2002), is one of the seven major clades in the Inocybaceae. Species of this clade are typically characterised by a rimose pileus surface; furfuraceous to furfuraceous-fibrillose stipe; absence of metuloids and pleurocystidia; smooth, elliptical to indistinctly phaseoliform basidiospores; and cylindrical to clavate cheilocystidia. Unlike species in clades Mallocybe and Inosperma (fide Matheny 2005) and the genera Auritella Matheny & Bougher and Tubariomyces Esteve-Rav. & Matheny, all of which also lack pleurocystidia, the basidia of species in the Pseudosperma clade are hyaline and not necropigmented. The Nothocybe clade is represented by only one species, I. distincta K.P.D. Latha & Manim. This species also lacks pleurocystidia and can be differentiated based on molecular phylogenetic data (Latha et al. 2016). Some lineages in the Pseudosperma clade are composed of multiple cryptic species (Ryberg et al. 2008) and they form ectomycorrhizal associations with a broad range of host trees, both gymnosperms and angiosperms (Kuyper 1986, Stangl 1989, Jacobsson 2008).

Based on a six-locus phylogeny of the family Inocybaceae, Matheny et al. (2019) formally proposed genus names for the different clades: Inocybe sensu stricto, Inosperma (Kühner) Matheny & Esteve-Rav. (elevated from subgenus-level), Mallocybe (Kuyper) Matheny, Vizzini & Esteve-Rav. (elevated from subgenus-level), Nothocybe Matheny & K.P.D. Latha and Pseudosperma Matheny & Esteve-Rav., in addition to Auritella and Tubariomyces that were previously described. The authors decided to provide a formal generic system to name the different clades, because this allows better communication and provides the taxonomic precision needed for conservation issues and identification of biodiversity hot spots.

During an investigation of ectomycorrhizal fungi associated with pine species in Pakistan, three species of Pseudosperma with affiliation to sect. Rimosae s.s. were collected in the vicinity of pure stands of Pinus roxburghii Sarg. and P. wallichiana A.B. Jacks. The species were documented, based on morphological and molecular phylogenetic data. In this paper, we describe these taxa as new species, P. brunneoumbonatum, P. pinophilum and P. triaciculare. This is the first study in which a combination of morphological and multi-locus phylogenetic data was used to describe species of Inocybe sensu lato in sect. Rimosae s.s. – now genus Pseudosperma – from Pakistan.

Material and methods

Morphological studies

Basidiomata were collected, described and photographed in the field. Colours were compared to the Munsell Soil Color 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 in Congo red. Micromorphological analysis, photographs and measurements were made, using an Olympus BX40 light microscope with Olympus XC50 digital camera and Microsuite special edition software 3.1 (Soft imaging solutions GmbH). Thirty 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 LAH (University of the Punjab Herbarium, Lahore) and FH (Farlow Herbarium, Harvard University).

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 include the complete ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal RNA gene (hereafter ITS), the first ca. 900 bp of the nuclear 28S rRNA gene (nrLSU) and the mitochondrial small subunit rRNA gene (mtSSU).

Primers used for amplification were: ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for ITS; LR0R and LR5 for nrLSU (Vilgalys and Hester 1990); and MS1 and MS2 for mtSSU (White et al. 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. 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. For mtSSU: 95 °C for 10 min; then 30 cycles of 95 °C for 30 sec, 52 °C for 30 sec and 72 °C for 40 sec; and 72 °C for 7 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 and 16S 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 v3.1 Cycle Kit (Life Technologies, Carlsbad, California). Sequencing was carried out using the same primers as those used for PCR.

Sequence alignment and phylogenetic analysis

Sequences were manually edited and assembled in BioEdit v7.2.6 (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. Three data matrices for phylogenetic inferences were prepared: a concatenated ITSnrLSUmtSSU dataset of Rimosae s.s. and Inosperma clades (dataset #1); a concatenated ITSnrLSUmtSSU dataset of Rimosae s.s. subclade A (dataset #2); and an extended nrLSU dataset of Rimosae s.s. subclade A (dataset #3). We applied the clade names used by Larsson et al. (2009) in the methods and results sections to maintain consistency and clarity.

Sequences were downloaded from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The majority of sequences were generated in the studies of Larsson et al. (2009) and Ryberg et al. (2008), complemented by nrLSU sequences from more recent papers and our newly-generated sequences (details and references in Table 1). Sequences were aligned by locus (ITS+nrLSU, mtSSU) using Muscle v3.7 (Edgar 2004), available in the Cipres Science Gateway (Miller et al. 2010). Ambiguously-aligned regions were detected and removed using trimAl v1.3 (Capella-Gutiérrez et al. 2009), with the following parameters: 60% gap threshold, 50% minimal coverage. The ITS1, 5.8S, ITS2 and nrLSU loci were extracted from the aligned ITS+nrLSU dataset. This allowed us to select substitution models for each region, which is important because there are different rates of evolution within and amongst these components and rDNA loci (e.g. Hillis and Dixon 1991, discussion in Haelewaters et al. 2018).

Table 1.

Isolates used in phylogenetic analyses, with geographic origin and GenBank accession numbers. Accession numbers of sequences generated during this study are in boldface. Explanation of datasets: #1 = concatenated ITSnrLSUmtSSU dataset of Rimosae s.s. and Inosperma clades, #2 = concatenated ITSnrLSUmtSSU dataset of Rimosae s.s. subclade A, #3 = extended nrLSU dataset of Rimosae s.s. subclade A (dataset #3). X under #1, #2, #3 = sequence(s) were used in the respective dataset. OUT = outgroup.

Species Isolate Geographic origin GenBank Reference(s) Dataset
ITS/nrLSU mtSSU #1 #2 #3
Alnicola bohemica EL71b-03 Sweden FJ904179 FJ904243 Larsson et al. (2009) OUT OUT
Alnicola salicis EL71a-03 Sweden FJ904180 Larsson et al. (2009) OUT OUT
Alnicola submelinoides TAA185174 Estonia AM882885 Ryberg et al. (2008) OUT OUT
Conocybe siliginea LÖ93-04 Sweden DQ389731 Larsson and Orstadius (2008) OUT
Crepidotus calolepis EL14-08 Sweden FJ904178 FJ904242 Larsson et al. (2009) X
Crepidotus mollis EL45-04 Sweden AM882996 Ryberg et al. (2008) X
Inosperma adaequatum PC2008-0014 Great Britain FJ904177 FJ904240 Larsson et al. (2009) X
Inosperma adaequatum MR00022 Sweden AM882706 FJ904241 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma bongardii EL123-04 Sweden AM882941 FJ904186 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma cf. calamistrata KHL13071 Costa Rica AM882948 Ryberg et al. (2008) X
Inosperma cervicolor SJ04024 Sweden AM882939 FJ904185 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma cookei MR00035 Sweden AM882954 Ryberg et al. (2008) X
Inosperma cookei EL191-06 Great Britain FJ904173 FJ904234 Larsson et al. (2009) X
Inosperma cookei EL70a-03 Sweden AM882953 Ryberg et al. (2008) X
Inosperma cookei EL73-05 Sweden AM882955 Ryberg et al. (2008) X
Inosperma cookei EL109-04 Sweden AM882956 FJ904233 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma cf. cookei EL104-04 Sweden AM882952 Ryberg et al. (2008) X
Inosperma erubescens TAA185164 Estonia AM882950 Ryberg et al. (2008) X
Inosperma erubescens KGN980714 Sweden AM882951 FJ904239 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma erubescens BH910707 Sweden AM882949 Ryberg et al. (2008) X
Inosperma maculatum EL74-05 Sweden AM882959 Ryberg et al. (2008) X
Inosperma fulvum EL78-03 Sweden AM882962 Ryberg et al. (2008) X
Inosperma fulvum EL166-08 Sweden FJ904171 FJ904231 Larsson et al. (2009) X
Inosperma fulvum EL114-06 Sweden FJ904170 Larsson et al. (2009) X
Inosperma fulvum SJ05029 Sweden AM882994 FJ904230 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma fulvum EL247-06 France FJ904169 Larsson et al. (2009) X
Inosperma fulvum PAM01100120 France FJ904168 Larsson et al. (2009) X
Inosperma fulvum SJ06007 Sweden FJ904167 Larsson et al. (2009) X
Inosperma maculatum MR00020 Sweden AM882958 Ryberg et al. (2008) X
Inosperma maculatum EL121-04 Sweden AM882957 FJ904232 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma maculatum EL58-03 Sweden AM882963 Ryberg et al. (2008) X
Inosperma maculatum EL126-04 Sweden AM882964 Ryberg et al. (2008) X
Inosperma maculatum EL182-08 Slovenia FJ904172 Larsson et al. (2009) X
Inosperma quietiodor RP980718 Sweden FJ936169 FJ904238 Larsson et al. (2009) X
Inosperma quietiodor LAS97-067 Sweden AM882974 Ryberg et al. (2008) X
Inosperma quietiodor LAS94-023 Sweden AM882961 Ryberg et al. (2008) X
Inosperma quietiodor PAM01091310 France FJ936168 FJ904237 Larsson et al. (2009) X
Inosperma quietiodor EL115-04 Sweden AM882960 FJ904236 Ryberg et al. (2008), Larsson et al. (2009) X
Inosperma quietiodor JV20202 Norway FJ904174 FJ904235 Larsson et al. (2009) X
Inosperma rhodiolum PAM00090117 France FJ904176 Larsson et al. (2009) X
Inosperma rhodiolum EL223-06 France FJ904175 Larsson et al. (2009) X
Inosperma subhirsutum EL45-05 Norway FJ904187 Larsson et al. (2009) X
Inosperma virosum TBGT753 India KT329458 Pradeep et al. 2016 X
Inosperma virosum CAL1383 India KY549138 K.P. Deepna Latha and P. Manihoman unpubl. X
Mallocybe agardhii EL88-04 Sweden FJ904123 FJ904182 Larsson et al. (2009) X
Mallocybe dulcamara EL89-06 Sweden FJ904122 FJ904181 Larsson et al. (2009) X
Mallocybe fulvipes EL37-05 Norway AM882858 FJ904184 Ryberg et al. (2008), Larsson et al. (2009) X
Mallocybe terrigena EL117-04 Sweden AM882864 FJ904183 Ryberg et al. (2008), Larsson et al. (2009) X
Pseudosperma aestivum BK18089706 USA, Utah EU600847 Matheny et al. (2009) X X
Pseudosperma alboflavellum TBGT11280 India KP171058 Pradeep et al. (2016) X
Pseudosperma arenicola RC GB99-014 France FJ904134 FJ904189 Larsson et al. (2009) X
Pseudosperma arenicola EL238-06 France FJ904133 FJ904188 Larsson et al. (2009) X
Pseudosperma breviterincarnatum BK18089724 USA, Utah EU555449 Matheny et al. (2009) X
Pseudosperma breviterincarnatum BK28080407 USA, Utah EU555451 Matheny et al. (2009) X
Pseudosperma breviterincarnatum PBM1914 USA, Washington JQ319677 Kropp et al. (2013) X
Pseudosperma brunneoumbonatum MSM#0053 Pakistan MG742419/MG742420 n/a This study X X X
Pseudosperma brunneoumbonatum MSM#00545 Pakistan MG742421/MG742422 n/a This study X X X
Pseudosperma bulbosissimum EL51-05 Norway AM882764 Ryberg et al. (2008) X X X
Pseudosperma bulbosissimum EL66-05 Norway AM882765 FJ904224 Ryberg et al. (2008), Larsson et al. (2009) X X X
Pseudosperma bulbosissimum EL37-06 Sweden FJ904161 FJ904223 Larsson et al. (2009) X X X
Pseudosperma bulbosissimum EL75-07 Sweden FJ904160 FJ904222 Larsson et al. (2009) X X X
Pseudosperma bulbosissimum EL88-06 Sweden FJ904159 FJ904221 Larsson et al. (2009) X X X
Pseudosperma bulbosissimum EL30-06 Sweden FJ904158 FJ904220 Larsson et al. (2009) X X X
Pseudosperma cercocarpi BK20069806 USA, Utah EU600890 Matheny et al. (2009) X
Pseudosperma cercocarpi BK20069807 USA, Utah JQ319683 Kropp et al. (2013) X
Pseudosperma dulcamaroides EL29-08 USA, Montana FJ904127 Larsson et al. (2009) X
Pseudosperma dulcamaroides EL112-06 Sweden FJ904126 FJ904194 Larsson et al. (2009) X
Pseudosperma flavellum EL56-08 Sweden FJ904131 FJ904198 Larsson et al. (2009) X
Pseudosperma flavellum EL137-05 Sweden AM882776 FJ904199 Ryberg et al. (2008), Larsson et al. (2009) X
Pseudosperma flavellum LAS89-030 Sweden AM882775 Ryberg et al. (2008) X
Pseudosperma cf. flavellum GK080924 Great Britain FJ904129 FJ904196 Larsson et al. (2009) X
Pseudosperma cf. flavellum PAM05062502 France FJ904128 FJ904195 Larsson et al. (2009) X
Pseudosperma cf. flavellum EL118-05 Finland AM882782 Ryberg et al. (2008) X
Pseudosperma cf. flavellum BJ920829 Sweden AM882774 Ryberg et al. (2008) X
Pseudosperma cf. flavellum EL90-04 Sweden AM882773 Ryberg et al. (2008) X
Pseudosperma griseorubidum CAL1253 India KT180327 Deepna Latha and Manimohan (2015) X
Pseudosperma hygrophorus EL97-06 Sweden FJ904137 FJ904202 Larsson et al. (2009) X
Pseudosperma keralense TBGT12854 India KP171059 Pradeep et al. (2016) X
Pseudosperma keralense TBGT12828 India KP171060 Pradeep et al. (2016) X
Pseudosperma melliolens PAM05052303 France FJ904148 FJ904211 Larsson et al. (2009) X X X
Pseudosperma melliolens EL224-06 France FJ904149 Larsson et al. (2009) X X X
Pseudosperma cf. microfastigiatum EL113-06 Sweden FJ904156 FJ904217 Larsson et al. (2009) X X X
Pseudosperma mimicum EBJ961997 Sweden FJ904124 FJ904191 Larsson et al. (2009) X
Pseudosperma mimicum TK2004-114 Sweden AM882781 Ryberg et al. (2008) X
Pseudosperma niveivelatum BK21089714 USA, Utah JQ319695 Kropp et al. (2013) X X
Pseudosperma niveivelatum BK27089718 USA, Utah EU600831 Matheny et al. (2009) X X
Pseudosperma niveivelatum Stz12816 USA, Washington JQ319696 Kropp et al. (2013) X X
Pseudosperma obsoletum EL17-04 Sweden AM882769 FJ904204 Ryberg et al. (2008), Larsson et al. (2009) X OUT X
Pseudosperma obsoletum BJ890915 Sweden AM882770 Ryberg et al. (2008) X OUT X
Pseudosperma occidentale PBM525 USA, Washington AY038321 Matheny et al. (2002) X
Pseudosperma occidentale BK27089703 USA, Utah EU600893 Matheny et al. (2009) X
Pseudosperma pakistanense LAH35285 Pakistan MG958608 Ullah et al. (2018) X
Pseudosperma pakistanense LAH35283 Pakistan MG958609 Ullah et al. (2018) X
Pseudosperma perlatum BJ940922 Sweden AM882772 Ryberg et al. (2008) X OUT X
Pseudosperma perlatum EL74-04 Sweden AM882771 FJ904205 Ryberg et al. (2008), Larsson et al. (2009) X OUT X
Pseudosperma pinophilum MSM#0046 Pakistan MG742414/MG742418 MG742416 This study X X X
Pseudosperma pinophilum MSM#0047 Pakistan MG742417/MG742415 MK474612 This study X X X
Pseudosperma rimosum AO2008-0250 Great Britain FJ904147 FJ904210 Larsson et al. (2009) X X X
Pseudosperma rimosum EL118-08 Sweden FJ904146 FJ904209 Larsson et al. (2009) X X X
Pseudosperma rimosum EL102-04 Sweden AM882761 Ryberg et al. (2008) X X X
Pseudosperma rimosum EL211-06 France FJ904145 Larsson et al. (2009) X X X
Pseudosperma rimosum TK97-156 Sweden AM882844 Ryberg et al. (2008) X X
Pseudosperma rimosum PAM03110904 France FJ904144 FJ904208 Larsson et al. (2009) X X X
Pseudosperma rimosum EL75-05 Sweden AM882762 FJ904207 Ryberg et al. (2008), Larsson et al. (2009) X X X
Pseudosperma rimosum SJ04007 Sweden AM882763 Ryberg et al. (2008) X X X
Pseudosperma rimosum PAM06112703 Corsica FJ904143 FJ904206 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum EL71-04 Sweden AM882786 FJ904193 Ryberg et al. (2008), Larsson et al. (2009) X
Pseudosperma cf. rimosum JD2008-0241 Great Britain FJ904125 FJ904192 Larsson et al. (2009) X
Pseudosperma cf. rimosum I116-06 Australia FJ904142 Larsson et al. (2009) X
Pseudosperma cf. rimosum PAM05061101 France FJ904155 FJ904216 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum JV26578 Estonia FJ904154 FJ904215 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum EL127-04 Sweden AM882768 FJ904219 Ryberg et al. (2008), Larsson et al. (2009) X X X
Pseudosperma cf. rimosum TAA185135 Estonia AM882766 Ryberg et al. (2008) X X X
Pseudosperma cf. rimosum JV22619 Estonia FJ904157 FJ904218 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum PC080925 Great Britain FJ904153 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum JV8125 Finland FJ904152 FJ904214 Larsson et al. (2009) X X X
Pseudosperma cf. rimosum EL81-06 Sweden FJ904135 FJ904190 Larsson et al. (2009) X
Pseudosperma sororium Kuoljok0512 Sweden FJ904150 FJ904212 Larsson et al. (2009) X X X
Pseudosperma sororium JV15200 Sweden FJ904151 FJ904213 Larsson et al. (2009) X X X
Pseudosperma sp. TR138_05 Papua New Guinea JN975009 Ryberg and Matheny (2012) X X X
Pseudosperma sp. TR133_05 Papua New Guinea JQ319709 Kropp et al. (2013) X X X
Pseudosperma sp. TR104_05 Papua New Guinea JN975011 Ryberg and Matheny (2012) X X X
Pseudosperma squamatum SJ08003 Sweden FJ904136 FJ904201 Larsson et al. (2009) X
Pseudosperma squamatum TK96-109 Sweden AM882780 Ryberg et al. (2008) x
Pseudosperma squamatum SJ85048 Norway AM882778 Ryberg et al. (2008) X
Pseudosperma squamatum PAM05052301 France FJ904132 FJ904200 Larsson et al. (2009) X
Pseudosperma cf. squamatum I93-04 Australia FJ904141 Larsson et al. (2009) X
Pseudosperma cf. squamatum I113-05 Australia FJ904140 Larsson et al. (2009) X
Pseudosperma cf. squamatum SJ92-010 Sweden AM882785 Ryberg et al. (2008) X
Pseudosperma cf. squamatum SM92-013 Sweden AM882783 Ryberg et al. (2008) X
Pseudosperma cf. squamatum SJ92-017 Sweden AM882784 Ryberg et al. (2008) X
Pseudosperma cf. squamatum Stordal18318 Norway FJ904139 Larsson et al. (2009) X
Pseudosperma cf. squamatum JV2609 Finland FJ904138 FJ904203 Larsson et al. (2009) X
Pseudosperma triaciculare MSM#0039 Pakistan MG742423/MG742424 MG742425 This study X X X
Pseudosperma triaciculare MSM#0041 Pakistan MG742429/MG742430 MG742431 This study X X X
Pseudosperma triaciculare MSM#0040 Pakistan MG742426/MG742427 MG742428 This study X X X
Pseudosperma umbrinellum JV13699 Finland FJ904165 FJ904228 Larsson et al. (2009) X X X
Pseudosperma umbrinellum JV17954 Estonia FJ904166 FJ904229 Larsson et al. (2009) X X X
Pseudosperma umbrinellum PC081010 Great Britain FJ904164 FJ904227 Larsson et al. (2009) X X X
Pseudosperma umbrinellum PC080816 Great Britain FJ904163 FJ904226 Larsson et al. (2009) X X X
Pseudosperma umbrinellum PAM01102912 France FJ904162 FJ904225 Larsson et al. (2009) X X X
Pseudosperma xanthocephalum PAM00100606 France FJ904130 FJ904197 Larsson et al. (2009) X

The data for each locus were concatenated in MEGA7 (Kumar et al. 2016) to create matrices of 2537 bp with sequence data for 123 isolates in the Rimosae s.s. and Inosperma dataset (#1); and of 2561 bp for 50 isolates in the Rimosae s.s. subclade A dataset (#2). The nrLSU dataset (#3) consisted of 1383 bp for 62 isolates belonging to Rimosae s.s. subclade A. Alignments generated during this study are available for download in NEXUS format from the figshare online repository (https://doi.org/10.6084/m9.figshare.c.4701338). Nucleotide substitution models were selected for each locus (ITS1, 5.8S, ITS2, nrLSU, mtSSU) using jModelTest2 (Darriba et al. 2012) by considering the Akaike Information Criterion (AIC). For both concatenated datasets #1 and #2, models were selected for ITS1, 5.8S, ITS2, nrLSU and mtSSU; for dataset #3, the best model was selected for nrLSU. Maximum likelihood was inferred for each dataset under partitioned models using IQ-tree (Nguyen et al. 2015, Chernomor et al. 2016). Ultrafast bootstrapping was done with 1000 replicates (Hoang et al. 2017).

Results

Nucleotide alignment datasets and phylogenetic inferences

Concatenated dataset #1 consisted of 2537 characters, of which 1448 were constant and 841 were parsimony-informative. A total of 123 isolates were included, of which Naucoria bohemica Velen., N. salicis P.D. Orton and N. submelinoides (Kühner) Maire (Agaricales, Hymenogastraceae) served as outgroup taxa. The following models were selected by jModelTest2 (AIC): TIM2+I+G (ITS1, -lnL = 6194.8143), TPM2+I (5.8S, -lnL = 445.7026), GTR+G (ITS2, -lnL = 4445.9240), TIM3+I+G (nrLSU, -lnL = 10227.1599) and TVM+I+G (mtSSU, -lnL = 4034.3342). Concatenated dataset #2 consisted of 2561 characters, of which 2026 were constant and 399 were parsimony-informative. A total of 50 isolates were included, of which P. obsoletum (Romagn.) Matheny & Esteve-Rav. and P. perlatum (Cooke) Matheny & EsteveRav. (Rimosae s.s. subclade B, fide Larsson et al. 2009) served as outgroup taxa. The following models were selected by jModelTest2 (AIC): TPM2uf+G (ITS1, -lnL = 2070.5127), TrNef (5.8S, -lnL = 261.9437), TPM1uf+I+G (ITS2, -lnL = 1683.9167), TrN+I+G (nrLSU, -lnL = 4608.2667) and TIM2+G (mtSSU, -lnL = 1758.7165). Finally, dataset #3 consisted of 1383 characters, of which 1091 were constant and 205 were parsimony-informative. A total of 67 isolates were included, again with N. bohemica, N. salicis and N. submelinoides as outgroup taxa. For this single-locus dataset, the TrN+I+G model gave the best-scoring tree (nrLSU, -lnL = 5708.4547).

Six strongly supported clades (referred to as subclades A to F, fide Larsson et al. 2009) and two additional clades with maximum support were recovered in the ML analysis of the Rimosae s.s. and Inosperma clades (dataset #1, Figure 1). A strongly supported clade with 35 sequences corresponds with Rimosae s.s. subclade A and includes the following species: P. bulbosissimum (Kühner) Matheny & Esteve-Rav., P. melliolens (Kühner) Matheny & Esteve-Rav., P. pinophilum sp. nov., P. rimosum (Bull.) Matheny & Esteve-Rav. (s.s.), P. sororium (Kauffman) Matheny & Esteve-Rav. and P. umbrinellum (Bres.) Matheny & Esteve-Rav. In addition, numerous taxa on single branches and less-supported clades are recovered.

Figure 1. 

The best-scoring ML tree (-lnL = 27210.474) of the Rimosae s.s. and Inosperma clades, reconstructed from the concatenated ITSnrLSUmtSSU dataset. ML bootstraps (if ≥ 70) are presented above or in front of the branch leading to each node. Thick branches have maximum support (ML BS = 100). Subclade designations within sect. Rimosae s.s. follow Larsson et al. (2009) in the strict sense. Newly-described species are in boldface.

In all three phylogenetic reconstructions (Figures 13), there is high support (BS = 81–100) for the grouping of P. pinophilum sp. nov. with P. cf. rimosum from Europe (isolates JV8125 and PC080925). This clade is deeply nested in Rimosae s.s. subclade A (fide Larsson et al. 2009). Pseudosperma brunneoumbonatum sp. nov. is retrieved as sister to an undescribed species from Papua New Guinea (isolates TR104_05 and TR133_05) with high support (BS = 96–100). In both datasets #2 and #3, this clade, again, is deeply nested in Rimosae s.s. subclade A. In dataset #1, however, the clade P. brunneoumbonatumI. sp. Papua New Guinea is placed between Rimosae subclades A and B (fide Larsson et al. 2009) with maximum support (Figure 1). Pseudosperma triaciculare sp. nov. is retrieved with high support (BS = 95–100) as an independent clade without clear affinities outside of Rimosae s.s. subclade A.

Figure 2. 

The best-scoring ML tree (-lnL = 9359.879) of Rimosae s.s. subclade A, reconstructed from the concatenated ITSnrLSUmtSSU dataset. ML bootstraps (if ≥ 70) are presented above or in front of the branch leading to each node. Thick branches have maximum support (ML BS = 100). Well-supported clades that represent described species within Rimosae s.s. subclade A are named. Newly-described species are in boldface.

Our phylogenetic reconstructions (Figures 13) indicate that several undescribed species occur in Rimosae s.s. subclade A (see Discussion). All ML analyses recovered two new Pakistani species, P. triaciculare and P. pinophilum, as strongly-supported lineages nested within this subclade, whereas a third species, P. brunneoumbonatum, forms a strongly-supported clade outside of what is currently recognised as subclade A. These three new taxa from Pakistan can be distinguished, based on molecular phylogenetic data, as well as morphology and ecology.

Figure 3. 

The best-scoring ML tree (-lnL = 5704.951) of Rimosae s.s. subclade A, complemented with recently-described species within sect. Rimosae s.s., reconstructed from the nrLSU dataset. ML bootstraps (if ≥ 70) are presented above or in front of the branch leading to each node. Thick branches have maximum support (ML BS = 100). Newly-described species are in boldface.

Taxonomy

Pseudosperma brunneoumbonatum Saba & Khalid, sp. nov.

MycoBank No: 822655
Figure 4

Diagnosis

Characterised by the dark brown umbo and basidiospores 10.3–15.3(–16.7) × 6.6–9.9 µm and an ecological association with Pinus.

Figure 4. 

Pseudosperma brunneoumbonatum: A Basidiomata of holotype collection (LAH 310032) B–E microscopic characters: B basidia C cheilocystidia D basidiospores E pileipellis. Scale bars: 1 cm (A), 10 µm (B), 30 µm (C, E), 20 µm (D).

Types

Holotype : Pakistan, Prov. Khyber Pakhtunkhwa, Abbottabad, Shimla, 14 Sep 2012, leg. M. Saba & A.N. Khalid; MSM#0053 (LAH 310032); GenBank accession nos. MG742419 (ITS), MG742420 (nrLSU). Paratype: ibid., 6 Aug. 2014; MSM#00545 (LAH 31003); GenBank accession nos. MG742421 (ITS), MG742422 (nrLSU).

Etymology

From Latin, referring to dark brown colour of the umbo.

Description

Pileus 20–38 mm in diam., plane to broadly convex with an acute umbo; margin straight or flaring to deflexed; surface dry, dull, strongly rimose, cracked towards centre but disc smooth and unbroken; strong brown (5YR4/8), disc/umbo deep brown (5YR2/6). Lamellae regular, adnexed to sinuate, close, pale orange yellow (10YR8/4) or pale yellow (5Y9/4), becoming yellowish-brown with age, concolorous with stipe; edges even; lamelullae one tier; edges white and fimbrirate. Stipe 22–40 mm, central to slightly eccentric, equal, recurved squamulose, longitudinally fibrillose, pale yellow (5Y9/4) or light yellowish-brown (10YR7/4), veil not observed. Odour spermatic. Context white, lacking any colour changes where cut or bruised.

Basidiospores 10.3–15.3(–16.7) × 6.6–9.9 µm [x = 12.5 × 7.5 µm, Q = 1.2–1.96], smooth, phaseoliform or ellipsoid, thin-walled, pale brown to reddish-brown in KOH, apiculus present or absent, apex obtuse. Basidia 27–39 × 10.6–16 µm, clavate with refractive contents, primarily 4-sterigmate, less often 2-sterigmate, thin-walled, hyaline in KOH; sterigmata 3–6 µm long. Pleurocystidia absent. Cheilocystidia 24–35 × 14–29 µm, numerous, clavate, some catenate, hyaline to pale brown, thin-walled. Caulocystidia clavate or cylindrical, similar to cheilocystidia, infrequent. Pileipellis a cutis, hyphae cylindrical, 5–9 µm wide, thin-walled, pale brown in KOH, some with encrustations, septate. Lamellar trama of parallel hyphae, 5–10 µm wide; subhymenium of compact hyphae, 3–6 µm wide. Stipitipellis cylindrical hyphae, hyaline in mass in KOH. All structures inamyloid. Clamp connections present.

Habit and habitat

Occurring in August and September, solitary or in groups, scattered on the forest floor in stands of Pinus roxburghii (Pinaceae).

Notes

In all phylogenetic reconstructions (Figures 13), P. brunneoumbonatum sp. nov. is sister to Pseudosperma sp. (isolates TR104_05 and TR133_05). This undescribed species from high-elevations in Papua New Guinea is associated with Castanopsis (Fagaceae). Of the north temperate species, P. brunneoumbonatum is phylogenetically most closely related to P. umbrinellum (Figure 3, Table 2). In terms of morphology, P. brunneoumbonatum differs from P. umbrinellum by its strong brown pileus with an acute umbo (hazel to cinnamon brown) and somewhat larger basidiospores (measuring 10–13 × 5.5–6.5 μm in P. umbrinellum). Other related North American taxa are P. aestivum (Kropp, Matheny & Hutchison) Matheny & Esteve-Rav. and P. niveivelatum (D.E. Stuntz ex Kropp, Matheny & Hutchison) Matheny & Esteve-Rav. Pseudosperma aestivum can be separated by larger basidiomata and different pileus colouration (yellowish to pale yellow with yellow-brown centre), whereas P. niveivelatum has a white stipe and a non-rimose pileus with different colouration (covered with abundant white velipellis) (Kropp et al. 2013). Pseudosperma perlatum (Cooke) Matheny & Esteve-Rav. superficially resembles P. brunneoumbonatum. However, the slightly larger basidiospores, pale orange yellow stipe and a presumed association with Pinus distinguish the new species from P. perlatum, which is an associate of deciduous trees (Vauras and Huhtinen 1986). It differs from I. rimosum in having broader basidiospores.

Pseudosperma neoumbrinellum (T. Bau & Y.G. Fan) Matheny & Esteve-Rav. is an Asian species (described from China) with similar basidioma size and colouration (Bau and Fan 2018). The basidiospores of P. brunneoumbonatum, however, are remarkably larger. Pseudosperma himalayense (Razaq, Khalid & Kobayashi) Matheny & Esteve-Rav. was recently described from Pakistan (Liu et al. 2018) and is similar to P. brunneoumbonatum in having similar pileus size. This species was found at different localities in the western Himalayas, but always near Pinus wallichiana. Pseudosperma himalayense has a much longer stipe (50–80 mm vs. max. 40 mm in P. brunneoumbonatum); white to pale yellow, olive yellow or light brown pileus; and somewhat smaller basidiospores. Pseudosperma pakistanense (Z. Ullah, S. Jabeen, H. Ahmad & A.N. Khalid) Matheny & Esteve-Rav., another species described from Pakistan, can be differentiated by the presence of pleurocystidia, somewhat smaller basidiospores and phylogenetic placement (Ullah et al. 2018, Figure 3).

The following two species have not yet been recombined in Pseudosperma. However, phylogenetic evidence undoubtedly places both I. neglecta E. Horak, Matheny & Desjardin and I. friabilis Matheny & Kudzma in the newly-recognised genus Pseudosperma (Horak et al. 2015, Matheny and Kudzma 2019). The new combinations are presented at the end of the taxonomy section. Inocybe neglecta from Thailand was described in the Pseudosperma clade by Horak et al. (2015). While it also lacks pleurocystidia and has a strong brown umbonate pileus, it is different from P. brunneoumbonatum by the smaller pileus (12–18 mm vs. 20–38 mm) and smaller and differently-shaped basidiospores. In addition, I. neglecta is only known from the type locality, growing in a tropical montane forest dominated by Lithocarpus Blume and Castanopsis (D. Don) Spach (both in Fagaceae). Inocybe friabilis, described from North America in the Pseudosperma clade, resembles P. brunneoumbonatum by lacking pleurocystidia and having a similarly coloured pileus. However, I. friabilis has smaller basidiospores, is associated with Quercus and Carya and has an eastern United States distribution.

In The taxonomic studies of the genus Inocybe, Kobayashi (2002) discussed 136 species, of which 13 (including four varieties and three formae) in subgenus Inosperma section Rimosae. These are [all referred to as Inocybe in Kobayashi (2002)]: Inosperma adaequatum (Britzelm.) Matheny & Esteve-Rav., I. aureostipes (Kobayasi) Matheny & Esteve-Rav., I. cookei (Bres.) Matheny & Esteve-Rav., I. erubescens (A. Blytt) Matheny & Esteve-Rav. [as its synonym I. patouillardii Bres.], I. maculatum (Boud.) Matheny & Esteve-Rav., Pseudosperma avellaneum (Kobayasi) Matheny & Esteve-Rav., P. bisporum (Hongo) Matheny & Esteve-Rav., P. flavellum (P. Karst.) Matheny & Esteve-Rav., P. macrospermum (Hongo) Matheny & Esteve-Rav., P. rimosum [as its synonym Inocybe fastigiata (Schaeff.) Quél.], P. squamatum (J.E. Lange) Matheny & Esteve-Rav., P. transiens (Takah. Kobay.) Matheny & Esteve-Rav. and P. umbrinellum. Since no sequence data are available for P. avellaneum, P. bisporum, P. macrospermum and P. transiens, we will compare their morphology with the newly-proposed Pakistani species.

Pseudosperma avellaneum has a pale greyish ochraceous pileus, its basidiospores are smaller and its cheilocystidia are distinctly narrower (width 9.5–14.5 vs. 14–29 μm) compared to P. brunneoumbonatum. As the only species in sect. Rimosae (sensu Kobayashi 2002), P. bisporum is 2-sterigmate. In addition, this species has a generally shorter stipe (17–26 vs. 22–40 mm in P. brunneoumbonatum), the edges of its lamellae are serrate (with small teeth as a saw) and, again, the cheilocystidia are narrower (width 10.0–13.8 vs. 14–29 μm in P. brunneoumbonatum). Another Japanese species, P. macrospermum, is morphologically different in the following characters: the stipe has a bulbous base, the basidia are shorter and narrower and its pileus is much smaller in diameter. Finally, P. transiens has a much longer stipe, its basidia are always narrower (up to 9.5 μm wide) and its cheilocystidia are both longer and narrower ((29–)38–52 × 9.5–13.8 μm) compared to P. brunneoumbonatum.

Table 2.

Comparison of ecological and morphological characters among the three newly described Pakistani species of Pseudosperma and phylogenetically similar species P. rimosum and P. umbrinellum.

Species P. brunneoumbonatum P. pinophilum P. triacicularis P. rimosum P. umbrinellum
Host association(s) Pinus Pinus Pinus Abies, Alnus, Betula, Carpinus, Cedrus, Corylus, Fagus, Larix, Picea, Pinus, Populus, Quercus, Salix, Tilia Helianthemum, Pinus, Populus, Quercus
Pileus color Strong brown (5YR4/8), disc/umbo deep brown (5YR2/6) Strong brown throughout (5YR4/6 to 5YR4/8), with dark brown umbo Brownish orange (5YR5/8) to fulvous Highly variable, from pale to ochraceous yellow brown to dark brown, usually darkest around center; sometimes very conspicuous and bright yellow; sometimes blackish brown Hazel to cinnamon brown, warm yellowish to reddish brown caps with a dark center and contrasting strongly rimose and lighter periphery
Umbo Acute Acute Acute to subacute or obtuse Acute Blunt
Velipellis Absent Absent Present Absent Absent
Basidiospores 10.3–15.3(–16.7) × 6.6–9.9 µm (8.2–)9.4–15.8 × 6.3–8 µm (7.7–)8.9–12.5 × 6.1–7.7 µm 9.5–12.5 × 6.0–7.0 µm 10.0–13.0 × 5.5–6.5 µm
Reference(s) This paper This paper This paper Kuyper (1986), Larsson et al. (2009) Kuyper (1986), Larsson et al. (2009)

Pseudosperma pinophilum Saba & Khalid, sp. nov.

MycoBank No: 822656
Figure 5

Diagnosis

Characterised by the pale to light yellow equal stipe, basidiospores (8.2–)9.4–15.8 × 6.3–8 µm and an ecological association with Pinus.

Figure 5. 

Pseudosperma pinophilum: A Basidiomata of holotype collection (FH 00304582) B–E microscopic characters: B basidia C cheilocystidia D basidiospores E pileipellis. Scale bars: 1 cm (A), 10 µm (B, D), 30 µm (C, E).

Types

Holotype : Pakistan, Prov. Khyber Pakhtunkhwa, Abbottabad, Shimla, 14 Sep 2012, leg. M. Saba & A.N. Khalid; MSM#0046 (FH 00304582); GenBank accession nos. MG742414 (ITS), MG742418 (nrLSU), MG742416 (mtSSU). Paratype: Pakistan, Prov. Khyber Pakhtunkhwa, Shangla, Yakh Tangay, under Pinus wallichiana, 2 Sep 2013, leg. M. Saba & A.N. Khalid; MSM#0047 (LAH 310049); GenBank accession nos. MG742417 (ITS), MG742415 (nrLSU), MK474612 (mtSSU).

Etymology

From Greek, referring to an association with pine species.

Description

Pileus 16–31 mm in diam., convex, broadly convex or plane with an acute umbo; margin straight or flaring to deflexed; surface dry, dull, rimose, cracked towards centre, strong brown throughout (5YR4/6 to 5YR4/8) with dark brown umbo. Lamellae regular, adnexed to sinuate, close, white when young, light olivaceous at maturity; edges even. Stipe 54–70 mm, central, equal, longitudinally fibrillose, white with pale greenish-yellow (10Y9/4) or light yellow (5Y9/6) tinge or olivaceous tinge; veil not observed. Context white. Odour not distinctive.

Basidiospores (8.2–)9.4–15.8 × 6.3–8.0 µm [x = 13.5 × 7.6 µm, Q = 1.4–1.9], smooth, phaseoliform or ellipsoid, thin-walled, pale brown to golden brown in KOH, apiculus small and not distinctive, apex obutse. Basidia 21–40 × (9–)11–14 µm, clavate with refractive contents, primarily 4-sterigmate, less often 2-sterigmate, thin-walled, hyaline in KOH; sterigmata 2.5–4.0 µm long. Pleurocystidia absent. Cheilocystidia 25–47 × 10–20 µm, numerous, clavate or cylindrical, hyaline to pale brown in KOH, thin-walled. Caulocystidia not observed. Pileipellis a cutis of repent hyphae, hyphae cylindrical, 4–12 µm wide, thin-walled, pale brown in KOH, septate. Lamellar trama of parallel hyphae, 5–11 µm wide; subhymenium of compact hyphae, 3–6 µm wide. Stipitipellis cylindrical hyphae, 5–12 µm wide, hyaline in mass in KOH; all structures inamyloid. Clamp connections present.

Habit and habitat

Occurring in September, solitary or in groups, scattered on the forest floor in stands of Pinus roxburghii and P. wallichiana (Pinaceae).

Notes

Both P. brunneoumbonatum and P. pinophilum are placed in sect. Rimosae s.s. subclade A (Figures 13), which corresponds to P. rimosum senso lato, including the several formae and variations described for this species (Larsson et al. 2009). Pseudosperma pinophilum clusters with P. cf. rimosum (isolates JV1825 and PC080925). The pale yellow to light yellow tinged, equal stipe in P. pinophilum is very different compared to the white (rarely tinged with ochre), sub-bulbous stipe typical for P. rimosum. Moreover, P. pinophilum has broader basidiospores ((8.2–)9.4–15.8 × 6.3–8.0 µm) compared to P. rimosum (9–11(–13) × 4.5–6.0 µm). Also P. brunneoumbonatum has broader – and generally larger – basidiospores (10.3–15.3(–16.7) × 6.6–9.9 µm) compared to P. rimosum. Pseudosperma sororium is relatively closely related to P. pinophilum and can be differentiated in having different pileus colouration (greyish-brown to pinkish-grey or pale pinkish-beige) and measurement of basidiospores (10–12.5 × 5.5–6.0 µm) (Kauffman 1926).

Two more species of Pseudosperma are known from Pakistan; both P. himalayense and P. pakistanense were described, based on material collected in Pakistan. Pseudosperma himalayense was found near Pinus wallichiana trees, but an ITS sequence generated from root tips (GenBank acc. no. HG796995) confirmed an ectomycorrhizal association with Quercus incana (Liu et al. 2018). It can be distinguished from P. pinophilum by the pale yellowish to camel brown, fibrillose pileus; longer cheilocystidia (43–60 µm vs. 25–47 µm); and much thicker pileipellis. In addition, P. himalayense was resolved as sister to P. cf. microfastigiatum (Kühner) Matheny & Esteve-Rav. in Liu et al.’s (2018) ITS phylogeny. Pseudosperma pakistanense was found in a mixed conifer-dominated forest with some deciduous trees, under Quercus incana (Ullah et al. 2018). This species can be differentiated from the new species by the presence of pleurocystidia, the smaller stipe (50 mm vs. 54–70 mm in P. pinophilum) and its phylogenetic position (Ullah et al. 2018). In our nrLSU phylogeny, P. pakistanense was retrieved as sister to P. alboflavellum (C.K. Pradeep & Matheny) Haelew. (Figure 3).

The Japanese species in sect. Rimosae without sequence data from Kobayashi (2002), P. avellaneum, P. bisporum, P. macrospermum and P. transiens, are also different from P. pinophilum in their morphology. Pseudosperma avellaneum has smaller basidiospores and the pileipellis hyphae are almost hyaline (vs. pale brown in P. pinophilum). Pseudosperma bisporum has lamellae with serrate edges, its stipe is much shorter (17–26 vs. 54–70 mm in P. pinophilum), the basidia are 2-sterigmate, the cheilocystidia are usually shorter (max. 31 µm in length) and the pileipellis hyphae are smaller in diameter. Pseudosperma macrospermum has a smaller pileus diameter, a shorter stipe, narrower basidia, usually shorter cheilocystidia and pileipellis hyphae that are smaller in diameter. Finally, both the basidiospores (4.8–6.5 vs. 6.3–8.0 µm in P. pinophilum) and basidia (8.8–9.5 vs. (9–)11–14 µm in P. pinophilum) of P. transiens are narrower. In addition, the cheilocystidia of P. pinophilum are hyaline to pale brown in KOH, whereas in P. transiens, they are “rarely filled with yellowish brown contents” (Kobayashi 2002).

Pseudosperma triaciculare Saba & Khalid, sp. nov.

MycoBank No: 822657
Figure 6

Diagnosis

Characterised by the acutely umbonate brownish-orange to fulvous pileus, the presence of a pale velipellis coating on the pileus, septate cheilocystidia and an ecological association with Pinus.

Figure 6. 

Pseudosperma triaciculare: A Basidiomata of paratype collection (FH 00304561) B–F microscopic characters: B Basidia C cheilocystidia D caulocystidia E basidiospores F pileipellis. Scale bars: 1 cm (A), 10 µm (B, E), 30 µm (C, D, F).

Types

Holotype : Pakistan, Prov. Khyber Pakhtunkhwa, Mansehra, Batrasi, under Pinus roxburghii, 3 Aug 2014, leg. M. Saba & A.N. Khalid; MSM#0039 (LAH 310054); GenBank accession nos. MG742423 (ITS), MG742424 (nrLSU), MG742425 (mtSSU). Paratypes: ibid., 3 Aug 2014; MSM#0040 (LAH 310055); GenBank accession nos. MG742426 (ITS), MG742427 (nrLSU), MG742428 (mtSSU). Ibid., 3 Aug 2014; MSM#0041 (LAH 310056); GenBank accession nos. MG742429 (ITS), MG742430 (nrLSU), MG742431 (mtSSU). Pakistan, Prov. Khyber Pakhtunkhwa, Abbottabad, Shimla, 14 Sep 2012, leg. M. Saba & A.N. Khalid; MSM#0038 (FH 00304561).

Etymology

From Latin, meaning “three-needled,” with reference to the association with the three-needled pine Pinus roxburghii.

Description

Pileus 12–29 mm in diam., conical when young, plane to convex at maturity, with acute to subacute or obtuse umbo; margin radially rimose, straight or flaring to uplifted; surface dry, dull, colour brownish-orange (5YR5/8) to fulvous, presence of a pale velipellis coating over the disc. Lamellae regular, adnexed to sinuate, close, pale orange yellow (10YR8/4), edges even; two tiers of lamelullae. Stipe 19–60 mm, central, equal, fibrillose, white with pale orange yellow tinge (10YR8/4). Odour mild, not diagnostic.

Basidiospores (7.7–)8.9–12.5 × 6.1–7.7 µm [x = 10.2 × 6.9 µm, Q = 1.64–2.2], smooth, mostly elliptic, thin-walled, yellowish-brown in KOH, apiculus present small and indistinctive. Basidia 24–36 × (9–)10–13 µm, clavate to broadly clavate with refractive contents, 4-sterigmate, thin-walled, hyaline in KOH; sterigmata 2.5–4.0 µm long. Pleurocystidia absent. Cheilocystidia cylindrical to clavate, septate, some with sub-capitate apices, terminal cells 23–54 × 9–16 µm, non-encrusted, hyaline, thin-walled. Caulocystidia 36–98 × 7–14 µm, cylindrical, non-encrusted, hyphoid, thin-walled. Pileipellis a cutis, hyphae cylindrical, 6–12 µm wide, thin-walled, golden brown or yellowish-brown in KOH, without encrustations, septate. Lamellar trama of parallel hyphae, 6–12 µm wide; subhymenium of compact hyphae, 3–6 µm wide. Stipitipellis cylindrical hyphae, 2–12 µm wide, hyaline in mass in KOH; all structures inamyloid. Clamp connections present.

Habit and habitat

Occurring in August to September, solitary or in groups, scattered on the forest floor in stands of Pinus roxburghii (Pinaceae).

Notes

Pseudosperma triaciculare has been found in association with Pinus roxburghii, the three-needled pine. This new species forms a distinct monophyletic group without clear affinities outside of Rimosae s.s. subclade A (Figures 13). Some of the unique features of this species are the umbonate brownish-orange to pale orange yellow pileus; cylindrical to clavate cheilocystidia; and cylindrical, non-encrusted, hyphoid caulocystidia. Allied species include P. brunneoumbonatum, P. griseorubidum (K.P.D. Latha & Manim.) Matheny & Esteve-Rav., P. keralense [synonym I. rimulosa C.K. Pradeep & Matheny] and P. umbrinellum. Pseudosperma triaciculare shares the same presumed Pinus association and shape of basidiomata with P. brunneoumbonatum, but can be distinguished by its brownish-orange pileus and smaller basidiospores. Pseudosperma umbrinellum is differentiated from P. triaciculare by the presence of an obtuse umbo (acute in P. triaciculare), yellowish- or reddish-brown pileus (brownish-orange in P. triaciculare), somewhat narrower basidiospores (5.5–6.5 µm vs. 6.1–7.7 µm) and a broad host range, including species in Cistaceae, Fagaceae, Pinaceae and Salicaceae (Larsson et al. 2009).

Pseudosperma triaciculare is most closely related to P. griseorubidum and P. keralense, described recently from tropical India (Latha and Manimohan 2015, Pradeep et al. 2016, Figure 3). Pseudosperma griseorubidum can be differentiated by its pileus, which is greyish-red and rarely with an umbo. In addition, P. griseorubidum is associated with members of Dipterocarpaceae (Latha and Manimohan 2015). The differences between P. keralense and P. triaciculare are more subtle. Pseudosperma keralense can be separated based on the following features: its lamellae have serrate edges and its basidiospores are narrower on average (6.1 vs. 6.9 µm in P. triaciculare). It is also phylogenetically clearly different; the ITS sequence of the holotype collection (GenBank acc. no. KM924523) is 84.11% identical to the holotype of P. triaciculare, whereas the LSU (KM924518) is 95.13% identical.

Other similar Asian species include P. himalayense, P. neoumbrinellum, P. pakistanense and P. yunnanense (T. Bau & Y.G. Fan) Matheny & Esteve-Rav. Pseudosperma triaciculare resembles P. neoumbrinellum in its pileus and basidiospores. However, it is easily differentiated by the characteristic brownish-orange to fulvous colouration of its pileus, whereas the pileus of P. neoumbrinellum is chocolate to dark brown in colour (Bau and Fan 2018). In addition, the shape and size of caulocystidia in these two species are very different: 20–48 × 10–17 µm in P. neoumbrinellum vs. 36–98 × 7–14 µm in P. triaciculare. Pseudosperma triaciculare is different from the recently-described P. himalayense from Pakistan (Liu et al. 2018) by the presence of a velipellis and a shorter stipe (16–60 vs. 50–80 µm). Pseudosperma pakistanense is separated from P. triaciculare by the absence of velipellar hyphae (unless the authors referred to the velipellis by their description of “[pileus] sometimes peeling off in the form of fine threads”), presence of pleurocystidia and a generally wider stipitipellis lacking caulocystidia (Ullah et al. 2018). Finally, P. yunnanense, described from China, also has velipellar hyphae, but its basidiomata are much larger in size (pileus 30–60 mm in diam., stipe 60–70 mm) and it lacks caulocystidia (Bau and Fan 2018). We did not include P. yunnanense in our phylogenetic analyses, but blasted the ITS sequence of the holotype collection (GenBank acc. no. MH047250) against P. triaciculare, resulting in 89.09% identity. Pseudosperma yunnanense is phylogenetically most similar to P. perlatum.

Finally, P. avellaneum, P. bisporum, P. macrospermum and P. transiens from Kobayashi’s (2002) morphological Inocybe treatment are all different from P. triaciculare. Of all four, P. avellaneum is probably most difficult to separate from the new species: its pileus is pale greyish-ochraceous, the stipe is less slender and – this seems the best character for separating both species – no caulocystidia were observed. Pseudosperma bisporum has lamellae with serrate edges, 2-sterigmate basidia and pileipellis hyphae that are smaller in diameter. In addition, again, no caulocystidia were observed in this species. Compared to P. triaciculare, the basidiospores of P. macrospermum are longer (10.5–)14.0–15.5(–18.3) vs. (7.7–)8.9–12.5) µm, its basidia are narrower (8.8–9.5(–12.5) vs. (9–)10–13 µm) and its cheilocystidia are wider (16–18 vs. 9–16 µm). Pseudosperma transiens has basidiospores (4.8–6.5 vs. 6.1–7.7 µm) and basidia (8.8–9.5 vs. (9–)10–13 µm) that are both narrower than those in P. triaciculare. In addition, the pileus of P. transiens is coloured brown to dark brown, whereas P. triaciculare has a brownish-orange to fulvous pileus.

New combinations

During our studies of Inocybe sensu lato, we came across species of Inocybe that had not been recombined in the appropriate genera after Matheny et al. (2019) proposed a new generic system. Five names are recombined in Inosperma, Mallocybe and Pseudosperma.

Inosperma vinaceobrunneum (Matheny, Ovrebo & Kudzma) Haelew., Index Fungorum 436: 1 (2020).

Inocybe vinaceobrunnea Matheny, Matheny and Kudzma, J. Torrey Bot. Soc. 146(3): 227 (2019). [Basionym]

Note

This combination was made, based on a four-locus phylogeny (ITS, nrLSU, rpb1, rpb2). Inosperma vinaceobrunneum was retrieved in a clade with two other species (I. rodiolum (Bres.) Matheny & Esteve-Rav. and an undescribed species), sister to I. adaequatum (Matheny and Kudzma 2019).

Mallocybe erratum (E. Horak, Matheny & Desjardin) Haelew., comb. nov.

Inocybe errata E. Horak, Matheny & Desjardin, Phytotaxa 230(3): 210 (2015). [Basionym]

Note

This combination is based on phylogenetic evidence of the holotype (Horak et al. 2015). Based on both nrLSU-alone and nrLSU–rpb1–rpb2 datasets, it is placed deep in Mallocybe. It is highly supported as a sister species to an undescribed Zambia species (“I. microdulcamara” nom. prov.), both sister to M. heimii (Bon) Matheny & Esteve-Rav. (Matheny et al. 2009, Horak et al. 2015).

Pseudosperma alboflavellum (C.K. Pradeep & Matheny) Haelew., Index Fungorum 436: 1 (2020).

Inocybe alboflavella C.K. Pradeep & Matheny, Pradeep et al., Mycol. Progr. 15: 13 (2016). [Basionym]

Note

This combination was made, based on phylogenetic placement of the isotype (Pradeep et al. 2016, this study). In our nrLSU phylogeny, it was retrieved as a sister species to P. pakistanense with high support (Figure 3).

Pseudosperma friabile (Matheny & Kudzma) Haelew., Index Fungorum 436: 1 (2020).

Inocybe friabilis Matheny & Kudzma, J. Torrey Bot. Soc. 146(3): 226 (2019). [Basionym]

Note

This combination was made, based on phylogenetic evidence. Pseudosperma friabile is most closely related to P. gracilissimum (Matheny & Bougher) Matheny & Esteve-Rav. and P. keralense (K.P.D. Latha & Manim.) Matheny & Esteve-Rav., deep in the Pseudosperma clade (fide Matheny 2005, Matheny and Kudzma 2019).

Pseudosperma neglectum (E. Horak, Matheny & Desjardin) Haelew., comb. nov.

Inocybe neglecta E. Horak, Matheny & Desjardin, Phytotaxa 230(3): 208 (2015). [Basionym]

Note

The combination of I. neglecta in genus Pseudosperma is made, based on phylogenetic evidence. Horak et al. (2015) presented the phylogenetic reconstruction of an nrLSU dataset and found high statistical support for the Pseudosperma clade (fide Matheny 2005) including P. neglectum. While P. neglectum was retrieved as sister to the remaining members of the Pseudosperma clade, there was no support for this relationship. The same result was also found by Kropp et al. (2013). In addition, blasting the ITS sequence of the holotype (GenBank acc. no. EU600829) against sequences from type materials, resulted in P. occidentale (Kropp, Matheny & Hutchison) Matheny & Esteve-Rav. and P. illudens (Matheny, Bougher & G.M. Gates) Matheny & Esteve-Rav. with the highest percentages of identity (96.46% and 96.28%, respectively).

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 Köppen-Geiger 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 (BWh, Peel et al. 2007). Pakistan has a very rich flora; in an ongoing effort to write 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 are 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 Pseudosperma is poorly known in Pakistan, with only three species that were known before this study: P. himalayense, P. rimosum and P. pakistanense (Ahmad et al. 1997, Liu et al. 2018, Ullah et al. 2018).

In his dissertation about smooth-spored species of Inocybe from Europe, Kuyper (1986) presented a key to species of sect. Rimosae. He included 12 species [all as Inocybe]: Inosperma adaequatum, I. cookei, I. erubescens, I. maculatum, I. quietiodor (Bon) Matheny & Esteve-Rav., I. reisneri (Velen.) Matheny & Esteve-Rav., Pseudosperma arenicola (R. Heim) Matheny & Esteve-Rav., P. flavellum, P. mimicum (Massee) Matheny & Esteve-Rav., P. rimosum (sensu lato), P. squamatum and I. vinosistipitatum (Grund & D.E. Stuntz) Matheny & Esteve-Rav. Kuyper (1986) followed a conservative approach for P. rimosum – citing 31 species and varieties as synonyms and allowing considerable morphological plasticity and broad ecological amplitude. Larsson et al. (2009) followed a less conservative approach and recognised P. obsoletum, P. perlatum and P. umbrinellum as separate species in their identification key of Maculata and Rimosae s.s. clades in north-western Europe. These three species were amongst the synonymies of P. rimosum as treated by Kuyper (1986). Following both keys, our newly described taxa are most similar to P. rimosum and P. umbrinellum (Table 2). From our phylogenetic analyses, it is obvious that both P. rimosum and P. umbrinellum are separated from our Pakistani species. Other, more recently described taxa of Pseudosperma are also differentiated from the newly-proposed species, based on morphology, molecular phylogeny and geographic distribution.

Our phylogenetic analyses revealed that several undescribed species or collections that have not yet been properly identified occur in Rimosae s.s. subclade A (Larsson et al. 2009, Kropp et al. 2012). These are represented by singleton clades and clades including tentatively (cf.) or unidentified isolates. For example, isolates TR104_05 and TR133_05 represent an undescribed species from Papua New Guinea. In addition, isolates JV1825, PC080925, JV22619 and TAA185135 were identified as P. cf. rimosum, but represent at least two different species, either undescribed or previously described, but without available DNA sequence data. The isolate JV26578, which forms a singleton clade with unresolved position in our phylogenetic analyses, was also identified as P. cf. rimosum, but this identification is again inaccurate. We agree with Larsson et al. (2009) that more taxa need be sampled before the diversity and evolutionary relationships in this section can be fully understood.

Data availability

All holotype and paratype collections of the new species are deposited at LAH and FH. The sequences generated during this study are deposited in NCBI GenBank under accession numbers MG742414MG742431. The sequence alignments generated in the present study are available from figshare (https://doi.org/10.6084/m9.figshare.c.4701338).

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 the International Research Support Initiative Program (IRSIP). We thank P. Brandon Matheny (University of Tennessee-Knoxville, USA), Olivier Raspé (Botanic Garden Meise, Belgium) and Martin Ryberg (Uppsala University, Sweden) for critically reviewing the manuscript. Finally, we acknowledge the efforts of Meike Piepenbring and Carola Glatthorn (Goethe-Universität Frankfurt, Germany) to provide us with necessary literature during the COVID-19 pandemic and subsequent lockdown.

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