New species of Pseudosperma (Agaricales, Inocybaceae) from Pakistan revealed by morphology and multi-locus phylogenetic reconstruction Author Saba, Malka Department of Plant Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan rustflora@gmail.com Author Haelewaters, Danny Farlow Herbarium of Cryptogamic Botany, Harvard University, Cambridge, Massachusetts, USA & Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, USA & Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic https://orcid.org/0000-0002-6424-0834 Author Pfister, Donald H. Farlow Herbarium of Cryptogamic Botany, Harvard University, Cambridge, Massachusetts, USA https://orcid.org/0000-0002-9018-8646 Author Khalid, Abdul Nasir Department of Botany, University of the Punjab, Lahore, Pakistan text MycoKeys 2020 69 1 31 http://dx.doi.org/10.3897/mycokeys.69.33563 journal article http://dx.doi.org/10.3897/mycokeys.69.33563 1314-4049-69-1 1EE0969FEC495A54BF779CCFA62DB5E6 Pseudosperma brunneoumbonatum Saba & Khalid sp. nov. Figure 4 Diagnosis. Characterised by the dark brown umbo and basidiospores 10.3-15.3(-16.7) x 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) x 6.6-9.9 µm [x = 12.5 x 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 x 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 x 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 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 x 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.) Quel .], 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 x 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) x 6.6-9.9 µm	(8.2-)9.4-15.8 x 6.3-8 µm	(7.7-)8.9-12.5 x 6.1-7.7 µm	9.5-12.5 x 6.0-7.0 µm	10.0-13.0 x 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)