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 pinophilum Saba & Khalid sp. nov. Figure 5 Diagnosis. Characterised by the pale to light yellow equal stipe, basidiospores (8.2-)9.4-15.8 x 6.3-8 µm and an ecological association with Pinus . Figure 5. Pseudosperma pinophilum : A Basidiomata of holotype collection (FH 00304582) B-E microscopic characters: B basidia C cheilocystidia D basidiospores E pileipellis. Scale bars: 1 cm ( A ), 10 µm ( B, D ), 30 µm ( C, E ). Types. Holotype : Pakistan, Prov. Khyber Pakhtunkhwa, Abbottabad, Shimla, 14 Sep 2012, leg. M. Saba & A.N. Khalid; MSM#0046 (FH 00304582); GenBank accession nos. MG742414 (ITS), MG742418 (nrLSU), MG742416 (mtSSU). Paratype : Pakistan, Prov. Khyber Pakhtunkhwa, Shangla, Yakh Tangay, under Pinus wallichiana , 2 Sep 2013, leg. M. Saba & A.N. Khalid; MSM#0047 (LAH 310049); GenBank accession nos. MG742417 (ITS), MG742415 (nrLSU), MK474612 (mtSSU). Etymology. From Greek, referring to an association with pine species. Description. Pileus 16-31 mm in diam., convex, broadly convex or plane with an acute umbo; margin straight or flaring to deflexed; surface dry, dull, rimose, cracked towards centre, strong brown throughout (5YR4/6 to 5YR4/8) with dark brown umbo. Lamellae regular, adnexed to sinuate, close, white when young, light olivaceous at maturity; edges even. Stipe 54-70 mm, central, equal, longitudinally fibrillose, white with pale greenish-yellow (10Y9/4) or light yellow (5Y9/6) tinge or olivaceous tinge; veil not observed. Context white. Odour not distinctive. Basidiospores (8.2-)9.4-15.8 x 6.3-8.0 µm [x = 13.5 x 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 x (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 x 10-20 µm , numerous, clavate or cylindrical, hyaline to pale brown in KOH, thin-walled. Caulocystidia not observed. Pileipellis a cutis of repent hyphae, hyphae cylindrical, 4-12 µm wide, thin-walled, pale brown in KOH, septate. Lamellar trama of parallel hyphae, 5-11 µm wide; subhymenium of compact hyphae, 3-6 µm wide. Stipitipellis cylindrical hyphae, 5-12 µm wide, hyaline in mass in KOH; all structures inamyloid. Clamp connections present. Habit and habitat. Occurring in September, solitary or in groups, scattered on the forest floor in stands of Pinus roxburghii and P. wallichiana ( Pinaceae ). Notes. Both P. brunneoumbonatum and P. pinophilum are placed in sect. Rimosae s.s. subclade A (Figures 1 - 3 ), which corresponds to P. rimosum senso lato, including the several formae and variations described for this species ( Larsson et al. 2009 ). Pseudosperma pinophilum clusters with P. cf. rimosum (isolates JV1825 and PC080925). The pale yellow to light yellow tinged, equal stipe in P. pinophilum is very different compared to the white (rarely tinged with ochre), sub-bulbous stipe typical for P. rimosum . Moreover, P. pinophilum has broader basidiospores ((8.2-)9.4-15.8 x 6.3-8.0 µm ) compared to P. rimosum (9-11(-13) x 4.5-6.0 µm ). Also P. brunneoumbonatum has broader - and generally larger - basidiospores (10.3-15.3(-16.7) x 6.6-9.9 µm ) compared to P. rimosum . Pseudosperma sororium is relatively closely related to P. pinophilum and can be differentiated in having different pileus colouration (greyish-brown to pinkish-grey or pale pinkish-beige) and measurement of basidiospores (10-12.5 x 5.5-6.0 µm ) ( Kauffman 1926 ). Two more species of Pseudosperma are known from Pakistan; both P. himalayense and P. pakistanense were described, based on material collected in Pakistan. Pseudosperma himalayense was found near Pinus wallichiana trees, but an ITS sequence generated from root tips (GenBank acc. no. HG796995) confirmed an ectomycorrhizal association with Quercus incana ( Liu et al. 2018 ). It can be distinguished from P. pinophilum by the pale yellowish to camel brown, fibrillose pileus; longer cheilocystidia (43-60 µm vs. 25-47 µm ); and much thicker pileipellis. In addition, P. himalayense was resolved as sister to P. cf. microfastigiatum ( Kuehner ) Matheny & Esteve-Rav. in Liu et al.'s (2018) ITS phylogeny. Pseudosperma pakistanense was found in a mixed conifer-dominated forest with some deciduous trees, under Quercus incana ( Ullah et al. 2018 ). This species can be differentiated from the new species by the presence of pleurocystidia, the smaller stipe (50 mm vs. 54-70 mm in P. pinophilum ) and its phylogenetic position ( Ullah et al. 2018 ). In our nrLSU phylogeny, P. pakistanense was retrieved as sister to P. alboflavellum (C.K. Pradeep & Matheny) Haelew. (Figure 3 ). The Japanese species in sect. Rimosae without sequence data from Kobayashi (2002) , P. avellaneum , P. bisporum , P. macrospermum and P. transiens , are also different from P. pinophilum in their morphology. Pseudosperma avellaneum has smaller basidiospores and the pileipellis hyphae are almost hyaline (vs. pale brown in P. pinophilum ). Pseudosperma bisporum has lamellae with serrate edges, its stipe is much shorter (17-26 vs. 54-70 mm in P. pinophilum ), the basidia are 2-sterigmate, the cheilocystidia are usually shorter (max. 31 µm in length) and the pileipellis hyphae are smaller in diameter. Pseudosperma macrospermum has a smaller pileus diameter, a shorter stipe, narrower basidia, usually shorter cheilocystidia and pileipellis hyphae that are smaller in diameter. Finally, both the basidiospores (4.8-6.5 vs. 6.3-8.0 µm in P. pinophilum ) and basidia (8.8-9.5 vs. (9-)11-14 µm in P. pinophilum ) of P. transiens are narrower. In addition, the cheilocystidia of P. pinophilum are hyaline to pale brown in KOH, whereas in P. transiens , they are "rarely filled with yellowish brown contents" ( Kobayashi 2002 ).