11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.69.3356333563Research ArticleBasidiomycotaBiodiversity & ConservationMolecular systematicsTaxonomyAsiaNew species of Pseudosperma (Agaricales, Inocybaceae) from Pakistan revealed by morphology and multi-locus phylogenetic reconstructionSabaMalkarustflora@gmail.com1HaelewatersDannyhttps://orcid.org/0000-0002-6424-0834234PfisterDonald H.https://orcid.org/0000-0002-9018-86462KhalidAbdul Nasirhttps://orcid.org/0000-0002-5635-80315Department of Plant Sciences, Quaid-i-Azam University, Islamabad, 45320, PakistanQuaid-i-Azam UniversityIslamabadPakistanFarlow Herbarium of Cryptogamic Botany, Harvard University, Cambridge, Massachusetts, USAHarvard UniversityCambridgeUnited States of AmericaDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, USAPurdue UniversityWest LafayetteUnited States of AmericaFaculty of Science, University of South Bohemia, České Budějovice, Czech RepublicUniversity of South BohemiaČeské BudějoviceCzech RepublicDepartment of Botany, University of the Punjab, Lahore, PakistanUniversity of the PunjabLahorePakistan
Corresponding author: Malka Saba (rustflora@gmail.com;msaba@qau.edu.pk)
Academic editor: Olivier Raspé
202010072020691311EE0969F-EC49-5A54-BF77-9CCFA62DB5E639506540302201912052020This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
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 Pseudospermabrunneoumbonatum, P.pinophilum and P.triacicularis. These species belong in the genus PseudospermafideMatheny et al. (2019) = Pseudosperma clade fideMatheny (2005) = Inocybe sect. Rimosaes.s.fideLarsson 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 Inospermavinaceobrunneum, Mallocybeerratum, Pseudospermaalboflavellum, Pseudospermafriabile and Pseudospermaneglectum.
Saba M, Haelewaters D, Pfister DH, Khalid AN (2020) New species of Pseudosperma (Agaricales, Inocybaceae) from Pakistan revealed by morphology and multi-locus phylogenetic reconstruction. MycoKeys 69: 1–31. https://doi.org/10.3897/mycokeys.69.33563
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.
InocybesectionRimosae sensu stricto (fideLarsson et al. 2009, = clade PseudospermafideMatheny 2005), traditionally placed in subgenusInosperma (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 (fideMatheny 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 Pseudospermawith affiliation tosect.Rimosae s.s. were collected in the vicinity of pure stands of Pinusroxburghii 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 methodsMorphological 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 ITS–nrLSU–mtSSU dataset of Rimosae s.s. and Inosperma clades (dataset #1); a concatenated ITS–nrLSU–mtSSU 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).
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 ITS–nrLSU–mtSSU dataset of Rimosae s.s. and Inosperma clades, #2 = concatenated ITS–nrLSU–mtSSU 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
Alnicolabohemica
EL71b-03
Sweden
FJ904179
FJ904243
Larsson et al. (2009)
OUT
OUT
Alnicolasalicis
EL71a-03
Sweden
FJ904180
Larsson et al. (2009)
OUT
OUT
Alnicolasubmelinoides
TAA185174
Estonia
AM882885
Ryberg et al. (2008)
OUT
OUT
Conocybesiliginea
LÖ93-04
Sweden
DQ389731
Larsson and Orstadius (2008)
OUT
Crepidotuscalolepis
EL14-08
Sweden
FJ904178
FJ904242
Larsson et al. (2009)
X
Crepidotusmollis
EL45-04
Sweden
AM882996
Ryberg et al. (2008)
X
Inospermaadaequatum
PC2008-0014
Great Britain
FJ904177
FJ904240
Larsson et al. (2009)
X
Inospermaadaequatum
MR00022
Sweden
AM882706
FJ904241
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermabongardii
EL123-04
Sweden
AM882941
FJ904186
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermacf.calamistrata
KHL13071
Costa Rica
AM882948
Ryberg et al. (2008)
X
Inospermacervicolor
SJ04024
Sweden
AM882939
FJ904185
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermacookei
MR00035
Sweden
AM882954
Ryberg et al. (2008)
X
Inospermacookei
EL191-06
Great Britain
FJ904173
FJ904234
Larsson et al. (2009)
X
Inospermacookei
EL70a-03
Sweden
AM882953
Ryberg et al. (2008)
X
Inospermacookei
EL73-05
Sweden
AM882955
Ryberg et al. (2008)
X
Inospermacookei
EL109-04
Sweden
AM882956
FJ904233
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermacf.cookei
EL104-04
Sweden
AM882952
Ryberg et al. (2008)
X
Inospermaerubescens
TAA185164
Estonia
AM882950
Ryberg et al. (2008)
X
Inospermaerubescens
KGN980714
Sweden
AM882951
FJ904239
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermaerubescens
BH910707
Sweden
AM882949
Ryberg et al. (2008)
X
Inospermamaculatum
EL74-05
Sweden
AM882959
Ryberg et al. (2008)
X
Inospermafulvum
EL78-03
Sweden
AM882962
Ryberg et al. (2008)
X
Inospermafulvum
EL166-08
Sweden
FJ904171
FJ904231
Larsson et al. (2009)
X
Inospermafulvum
EL114-06
Sweden
FJ904170
Larsson et al. (2009)
X
Inospermafulvum
SJ05029
Sweden
AM882994
FJ904230
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermafulvum
EL247-06
France
FJ904169
Larsson et al. (2009)
X
Inospermafulvum
PAM01100120
France
FJ904168
Larsson et al. (2009)
X
Inospermafulvum
SJ06007
Sweden
FJ904167
Larsson et al. (2009)
X
Inospermamaculatum
MR00020
Sweden
AM882958
Ryberg et al. (2008)
X
Inospermamaculatum
EL121-04
Sweden
AM882957
FJ904232
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermamaculatum
EL58-03
Sweden
AM882963
Ryberg et al. (2008)
X
Inospermamaculatum
EL126-04
Sweden
AM882964
Ryberg et al. (2008)
X
Inospermamaculatum
EL182-08
Slovenia
FJ904172
Larsson et al. (2009)
X
Inospermaquietiodor
RP980718
Sweden
FJ936169
FJ904238
Larsson et al. (2009)
X
Inospermaquietiodor
LAS97-067
Sweden
AM882974
Ryberg et al. (2008)
X
Inospermaquietiodor
LAS94-023
Sweden
AM882961
Ryberg et al. (2008)
X
Inospermaquietiodor
PAM01091310
France
FJ936168
FJ904237
Larsson et al. (2009)
X
Inospermaquietiodor
EL115-04
Sweden
AM882960
FJ904236
Ryberg et al. (2008), Larsson et al. (2009)
X
Inospermaquietiodor
JV20202
Norway
FJ904174
FJ904235
Larsson et al. (2009)
X
Inospermarhodiolum
PAM00090117
France
FJ904176
Larsson et al. (2009)
X
Inospermarhodiolum
EL223-06
France
FJ904175
Larsson et al. (2009)
X
Inospermasubhirsutum
EL45-05
Norway
FJ904187
Larsson et al. (2009)
X
Inospermavirosum
TBGT753
India
KT329458
Pradeep et al. 2016
X
Inospermavirosum
CAL1383
India
KY549138
K.P. Deepna Latha and P. Manihoman unpubl.
X
Mallocybeagardhii
EL88-04
Sweden
FJ904123
FJ904182
Larsson et al. (2009)
X
Mallocybedulcamara
EL89-06
Sweden
FJ904122
FJ904181
Larsson et al. (2009)
X
Mallocybefulvipes
EL37-05
Norway
AM882858
FJ904184
Ryberg et al. (2008), Larsson et al. (2009)
X
Mallocybeterrigena
EL117-04
Sweden
AM882864
FJ904183
Ryberg et al. (2008), Larsson et al. (2009)
X
Pseudospermaaestivum
BK18089706
USA, Utah
EU600847
Matheny et al. (2009)
X
X
Pseudospermaalboflavellum
TBGT11280
India
KP171058
Pradeep et al. (2016)
X
Pseudospermaarenicola
RC GB99-014
France
FJ904134
FJ904189
Larsson et al. (2009)
X
Pseudospermaarenicola
EL238-06
France
FJ904133
FJ904188
Larsson et al. (2009)
X
Pseudospermabreviterincarnatum
BK18089724
USA, Utah
EU555449
Matheny et al. (2009)
X
Pseudospermabreviterincarnatum
BK28080407
USA, Utah
EU555451
Matheny et al. (2009)
X
Pseudospermabreviterincarnatum
PBM1914
USA, Washington
JQ319677
Kropp et al. (2013)
X
Pseudospermabrunneoumbonatum
MSM#0053
Pakistan
MG742419/MG742420
n/a
This study
X
X
X
Pseudospermabrunneoumbonatum
MSM#00545
Pakistan
MG742421/MG742422
n/a
This study
X
X
X
Pseudospermabulbosissimum
EL51-05
Norway
AM882764
Ryberg et al. (2008)
X
X
X
Pseudospermabulbosissimum
EL66-05
Norway
AM882765
FJ904224
Ryberg et al. (2008), Larsson et al. (2009)
X
X
X
Pseudospermabulbosissimum
EL37-06
Sweden
FJ904161
FJ904223
Larsson et al. (2009)
X
X
X
Pseudospermabulbosissimum
EL75-07
Sweden
FJ904160
FJ904222
Larsson et al. (2009)
X
X
X
Pseudospermabulbosissimum
EL88-06
Sweden
FJ904159
FJ904221
Larsson et al. (2009)
X
X
X
Pseudospermabulbosissimum
EL30-06
Sweden
FJ904158
FJ904220
Larsson et al. (2009)
X
X
X
Pseudospermacercocarpi
BK20069806
USA, Utah
EU600890
Matheny et al. (2009)
X
Pseudospermacercocarpi
BK20069807
USA, Utah
JQ319683
Kropp et al. (2013)
X
Pseudospermadulcamaroides
EL29-08
USA, Montana
FJ904127
Larsson et al. (2009)
X
Pseudospermadulcamaroides
EL112-06
Sweden
FJ904126
FJ904194
Larsson et al. (2009)
X
Pseudospermaflavellum
EL56-08
Sweden
FJ904131
FJ904198
Larsson et al. (2009)
X
Pseudospermaflavellum
EL137-05
Sweden
AM882776
FJ904199
Ryberg et al. (2008), Larsson et al. (2009)
X
Pseudospermaflavellum
LAS89-030
Sweden
AM882775
Ryberg et al. (2008)
X
Pseudospermacf.flavellum
GK080924
Great Britain
FJ904129
FJ904196
Larsson et al. (2009)
X
Pseudospermacf.flavellum
PAM05062502
France
FJ904128
FJ904195
Larsson et al. (2009)
X
Pseudospermacf.flavellum
EL118-05
Finland
AM882782
Ryberg et al. (2008)
X
Pseudospermacf.flavellum
BJ920829
Sweden
AM882774
Ryberg et al. (2008)
X
Pseudospermacf.flavellum
EL90-04
Sweden
AM882773
Ryberg et al. (2008)
X
Pseudospermagriseorubidum
CAL1253
India
KT180327
Deepna Latha and Manimohan (2015)
X
Pseudospermahygrophorus
EL97-06
Sweden
FJ904137
FJ904202
Larsson et al. (2009)
X
Pseudospermakeralense
TBGT12854
India
KP171059
Pradeep et al. (2016)
X
Pseudospermakeralense
TBGT12828
India
KP171060
Pradeep et al. (2016)
X
Pseudospermamelliolens
PAM05052303
France
FJ904148
FJ904211
Larsson et al. (2009)
X
X
X
Pseudospermamelliolens
EL224-06
France
FJ904149
Larsson et al. (2009)
X
X
X
Pseudospermacf.microfastigiatum
EL113-06
Sweden
FJ904156
FJ904217
Larsson et al. (2009)
X
X
X
Pseudospermamimicum
EBJ961997
Sweden
FJ904124
FJ904191
Larsson et al. (2009)
X
Pseudospermamimicum
TK2004-114
Sweden
AM882781
Ryberg et al. (2008)
X
Pseudospermaniveivelatum
BK21089714
USA, Utah
JQ319695
Kropp et al. (2013)
X
X
Pseudospermaniveivelatum
BK27089718
USA, Utah
EU600831
Matheny et al. (2009)
X
X
Pseudospermaniveivelatum
Stz12816
USA, Washington
JQ319696
Kropp et al. (2013)
X
X
Pseudospermaobsoletum
EL17-04
Sweden
AM882769
FJ904204
Ryberg et al. (2008), Larsson et al. (2009)
X
OUT
X
Pseudospermaobsoletum
BJ890915
Sweden
AM882770
Ryberg et al. (2008)
X
OUT
X
Pseudospermaoccidentale
PBM525
USA, Washington
AY038321
Matheny et al. (2002)
X
Pseudospermaoccidentale
BK27089703
USA, Utah
EU600893
Matheny et al. (2009)
X
Pseudospermapakistanense
LAH35285
Pakistan
MG958608
Ullah et al. (2018)
X
Pseudospermapakistanense
LAH35283
Pakistan
MG958609
Ullah et al. (2018)
X
Pseudospermaperlatum
BJ940922
Sweden
AM882772
Ryberg et al. (2008)
X
OUT
X
Pseudospermaperlatum
EL74-04
Sweden
AM882771
FJ904205
Ryberg et al. (2008), Larsson et al. (2009)
X
OUT
X
Pseudospermapinophilum
MSM#0046
Pakistan
MG742414/MG742418
MG742416
This study
X
X
X
Pseudospermapinophilum
MSM#0047
Pakistan
MG742417/MG742415
MK474612
This study
X
X
X
Pseudospermarimosum
AO2008-0250
Great Britain
FJ904147
FJ904210
Larsson et al. (2009)
X
X
X
Pseudospermarimosum
EL118-08
Sweden
FJ904146
FJ904209
Larsson et al. (2009)
X
X
X
Pseudospermarimosum
EL102-04
Sweden
AM882761
Ryberg et al. (2008)
X
X
X
Pseudospermarimosum
EL211-06
France
FJ904145
Larsson et al. (2009)
X
X
X
Pseudospermarimosum
TK97-156
Sweden
AM882844
Ryberg et al. (2008)
X
X
Pseudospermarimosum
PAM03110904
France
FJ904144
FJ904208
Larsson et al. (2009)
X
X
X
Pseudospermarimosum
EL75-05
Sweden
AM882762
FJ904207
Ryberg et al. (2008), Larsson et al. (2009)
X
X
X
Pseudospermarimosum
SJ04007
Sweden
AM882763
Ryberg et al. (2008)
X
X
X
Pseudospermarimosum
PAM06112703
Corsica
FJ904143
FJ904206
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
EL71-04
Sweden
AM882786
FJ904193
Ryberg et al. (2008), Larsson et al. (2009)
X
Pseudospermacf.rimosum
JD2008-0241
Great Britain
FJ904125
FJ904192
Larsson et al. (2009)
X
Pseudospermacf.rimosum
I116-06
Australia
FJ904142
Larsson et al. (2009)
X
Pseudospermacf.rimosum
PAM05061101
France
FJ904155
FJ904216
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
JV26578
Estonia
FJ904154
FJ904215
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
EL127-04
Sweden
AM882768
FJ904219
Ryberg et al. (2008), Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
TAA185135
Estonia
AM882766
Ryberg et al. (2008)
X
X
X
Pseudospermacf.rimosum
JV22619
Estonia
FJ904157
FJ904218
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
PC080925
Great Britain
FJ904153
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
JV8125
Finland
FJ904152
FJ904214
Larsson et al. (2009)
X
X
X
Pseudospermacf.rimosum
EL81-06
Sweden
FJ904135
FJ904190
Larsson et al. (2009)
X
Pseudospermasororium
Kuoljok0512
Sweden
FJ904150
FJ904212
Larsson et al. (2009)
X
X
X
Pseudospermasororium
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
Pseudospermasquamatum
SJ08003
Sweden
FJ904136
FJ904201
Larsson et al. (2009)
X
Pseudospermasquamatum
TK96-109
Sweden
AM882780
Ryberg et al. (2008)
x
Pseudospermasquamatum
SJ85048
Norway
AM882778
Ryberg et al. (2008)
X
Pseudospermasquamatum
PAM05052301
France
FJ904132
FJ904200
Larsson et al. (2009)
X
Pseudospermacf.squamatum
I93-04
Australia
FJ904141
Larsson et al. (2009)
X
Pseudospermacf.squamatum
I113-05
Australia
FJ904140
Larsson et al. (2009)
X
Pseudospermacf.squamatum
SJ92-010
Sweden
AM882785
Ryberg et al. (2008)
X
Pseudospermacf.squamatum
SM92-013
Sweden
AM882783
Ryberg et al. (2008)
X
Pseudospermacf.squamatum
SJ92-017
Sweden
AM882784
Ryberg et al. (2008)
X
Pseudospermacf.squamatum
Stordal18318
Norway
FJ904139
Larsson et al. (2009)
X
Pseudospermacf.squamatum
JV2609
Finland
FJ904138
FJ904203
Larsson et al. (2009)
X
Pseudospermatriaciculare
MSM#0039
Pakistan
MG742423/MG742424
MG742425
This study
X
X
X
Pseudospermatriaciculare
MSM#0041
Pakistan
MG742429/MG742430
MG742431
This study
X
X
X
Pseudospermatriaciculare
MSM#0040
Pakistan
MG742426/MG742427
MG742428
This study
X
X
X
Pseudospermaumbrinellum
JV13699
Finland
FJ904165
FJ904228
Larsson et al. (2009)
X
X
X
Pseudospermaumbrinellum
JV17954
Estonia
FJ904166
FJ904229
Larsson et al. (2009)
X
X
X
Pseudospermaumbrinellum
PC081010
Great Britain
FJ904164
FJ904227
Larsson et al. (2009)
X
X
X
Pseudospermaumbrinellum
PC080816
Great Britain
FJ904163
FJ904226
Larsson et al. (2009)
X
X
X
Pseudospermaumbrinellum
PAM01102912
France
FJ904162
FJ904225
Larsson et al. (2009)
X
X
X
Pseudospermaxanthocephalum
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).
ResultsNucleotide 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 Naucoriabohemica 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, fideLarsson 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, fideLarsson 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.
The best-scoring ML tree (-lnL = 27210.474) of the Rimosae s.s. and Inosperma clades, reconstructed from the concatenated ITS–nrLSU–mtSSU 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.
https://binary.pensoft.net/fig/430573
In all three phylogenetic reconstructions (Figures 1–3), 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 (fideLarsson et al. 2009). Pseudospermabrunneoumbonatum 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.brunneoumbonatum – I. sp. Papua New Guinea is placed between Rimosae subclades A and B (fideLarsson et al. 2009) with maximum support (Figure 1). Pseudospermatriaciculare sp. nov. is retrieved with high support (BS = 95–100) as an independent clade without clear affinities outside of Rimosae s.s. subclade A.
The best-scoring ML tree (-lnL = 9359.879) of Rimosae s.s. subclade A, reconstructed from the concatenated ITS–nrLSU–mtSSU 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.
https://binary.pensoft.net/fig/430574
Our phylogenetic reconstructions (Figures 1–3) 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.
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.
Pseudospermabrunneoumbonatum: 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).
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 Pinusroxburghii (Pinaceae).
Notes.
In all phylogenetic reconstructions (Figures 1–3), 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. Pseudospermaaestivum 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). Pseudospermaperlatum (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.
Pseudospermaneoumbrinellum (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. Pseudospermahimalayense (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 Pinuswallichiana. Pseudospermahimalayense 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. Pseudospermapakistanense (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. Inocybeneglecta 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). Inocybefriabilis, 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 InospermasectionRimosae. These are [all referred to as Inocybe in Kobayashi (2002)]: Inospermaadaequatum (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., Pseudospermaavellaneum (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 Inocybefastigiata (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.
Pseudospermaavellaneum 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 (sensuKobayashi 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.
Comparison of ecological and morphological characters among the three newly described Pakistani species of Pseudosperma and phylogenetically similar species P.rimosum and P.umbrinellum.
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
Pseudospermapinophilum: 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).
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 Pinusroxburghii and P.wallichiana (Pinaceae).
Notes.
Both P.brunneoumbonatum and P.pinophilum are placed in sect.Rimosae s.s. subclade A (Figures 1–3), which corresponds to P.rimosum senso lato, including the several formae and variations described for this species (Larsson et al. 2009). Pseudospermapinophilum 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. Pseudospermasororium 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. Pseudospermahimalayense was found near Pinuswallichiana trees, but an ITS sequence generated from root tips (GenBank acc. no. HG796995) confirmed an ectomycorrhizal association with Quercusincana (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. Pseudospermapakistanense was found in a mixed conifer-dominated forest with some deciduous trees, under Quercusincana (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. Pseudospermaavellaneum has smaller basidiospores and the pileipellis hyphae are almost hyaline (vs. pale brown in P.pinophilum). Pseudospermabisporum 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. Pseudospermamacrospermum 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).
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.
Pseudospermatriaciculare: 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).
From Latin, meaning “three-needled,” with reference to the association with the three-needled pine Pinusroxburghii.
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 Pinusroxburghii (Pinaceae).
Notes.
Pseudospermatriaciculare has been found in association with Pinusroxburghii, the three-needled pine. This new species forms a distinct monophyletic group without clear affinities outside of Rimosae s.s. subclade A (Figures 1–3). 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. Pseudospermatriaciculare 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. Pseudospermaumbrinellum 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).
Pseudospermatriaciculare 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). Pseudospermagriseorubidum 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. Pseudospermakeralense 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. Pseudospermatriaciculare 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. Pseudospermatriaciculare 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). Pseudospermapakistanense 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. Pseudospermayunnanense 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. Pseudospermabisporum 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). Pseudospermatransiens 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.
FungiAgaricalesInocybaceae69B3C893-DC3D-5EA9-990A-AE4D321FDCE7InospermavinaceobrunneumIndex Fungorum No: IF557431(Matheny, Ovrebo & Kudzma) Haelew., Index Fungorum 436: 1 (2020).≡ Inocybevinaceobrunnea 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). Inospermavinaceobrunneum 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).
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).
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).
This combination was made, based on phylogenetic evidence. Pseudospermafriabile 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 (fideMatheny 2005, Matheny and Kudzma 2019).
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 (fideMatheny 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 (sensuHawksworth 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]: Inospermaadaequatum, I.cookei, I.erubescens, I.maculatum, I.quietiodor (Bon) Matheny & Esteve-Rav., I.reisneri (Velen.) Matheny & Esteve-Rav., Pseudospermaarenicola (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 MG742414–MG742431. 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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