New species of Pseudosperma (Agaricales, Inocybaceae) from Pakistan revealed by morphology and multi-locus phylogenetic reconstruction

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 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.


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 , Bougher et al. 2012. Multi-locus phylogenies of the Inocybaceae by Matheny et al. (2002Matheny et al. ( , 2009) and Matheny (2005) have confirmed that the family is monophyletic. Matheny (2005Matheny ( , 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,  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.

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 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 andDixon 1991, discussion in Haelewaters et al. 2018).
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 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 (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. brunneoumbonatum -I. 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.
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.  Etymology. From Latin, referring to dark brown colour of the umbo.
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 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. 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.

This paper
This paper This paper Kuyper (1986), Larsson et al. (2009) Kuyper (1986, Larsson et al. (2009) 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 andKudzma 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) 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.
Habit and habitat. Occurring in September, solitary or in groups, scattered on the forest floor in stands of Pinus roxburghii and P. wallichiana (Pinaceae).
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).
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 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. 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 andManimohan 2015, Pradeep et al. 2016, Figure 3). Pseudosperma griseorubidum can be differentiated by its pileus, which is greyishred 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.

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, 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, Jabeen et al. 2016, Farooqi et al. 2017, Razaq and Shahzad 2017, Naseer et al. 2018, Ullah et al. 2018, Song et al. 2019. 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) 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).