Phytotaxa 403 (1): 025–038 https://www.mapress.com/j/pt/ Copyright © 2019 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.403.1.2 Three new species of Incrucipulum (Lachnaceae, Helotiales, Ascomycota) from Japan YUKITO TOCHIHARA1,2* & TSUYOSHI HOSOYA2 1 Department of Biological Sciences, Graduate Scholl of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0003, Japan 2 Department of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, Ibaraki, 305-0005, Japan * Corresponding author: tochi@kahaku.go.jp Abstract Three new species of Incrucipulum were described from Japan: I. foliicola and I. pseudosulphurellum on Myrica gale subsp. tomentosa and I. hakonechloae-macrae on Hakonechloa macra. Disposition to Incrucipulum was justified by molecular phylogenetic analysis based on ITS-5.8S, LSU and RPB2 regions, and monophyly of Incrucipulum was also confirmed. Some apomorphic characters of Incrucipulum were identified. By addition of three new species, the genus Incrucipulum now contains 13 species. Key words: Incrucipulum foliicola, Incrucipulum hakonechloae-macrae, Incrucipulum pseudosulphurellum, mycobiota, taxonomy Introduction The genus Incrucipulum Baral (1985: 71) belongs to the family Lachnaceae Raitviir (Helotiales, Leotiomycetes, Ascomycota). Members of Incrucipulum were separated from wider genus Dasyscyphus Nees ex Gray (1821: 670) (=currently recognized congeneric with Lachnum Retzius (1769: 255) (Hosoya et al. 2010)) based on their thickwalled (0.5–1.3 μm), cubic ectal excipular cells with densely granulated surface (Baral & Krieglsteiner 1985) as distinguishing characters. Thin to thick-walled hairs with crystallized and thick-walled apices were also mentioned as common characters (Baral & Krieglsteiner 1985). The structure of ectal excipulum was noted also by Le Gal (1939) for I. ciliare (Schrader 1799: 63) Baral (1985: 72) and I. capitatum (Peck 1878: 60) Baral (1985: 72) but had not been adopted as a genus-level taxonomic character at that time. Now the generic concept of Incrucipulum is widely accepted. Four species (I. capitatum, I. ciliare [type species], I. sulphurellum (Peck 1878: 59) Baral (1985: 72) and I. virtembergense (Matheis 1977: 240) Baral (1985: 73)) were originally included (Baral & Krieglsteiner 1985). Six species were later added: I. densiseptatum (Raitviir & R. Galán (in Galán & Raitviir 1994: 464)) Raitviir (2006a: 45), I. longispineum (Hosoya & Issh. Tanaka (in Tanaka & Hosoya 2001: 598)) Sasagawa & Hosoya (in Hosoya et al. 2010: 175), I. radiatum (Issh. Tanaka & Hosoya 2001: 606) Sasagawa & Hosoya (in Hosoya et al. 2010: 175), I. saccardoi (Raitviir & Sacconi (in Raitviir 1991: 166)) Raitviir (2006a: 47), I. sinegoricum (Raitviir 1985: 3) Raitviir (2006b: 140) and I. uralense (Chlebicki 2002: 84) Chlebicki & Suková (in Chlebicki 2005: 109) (Baral & Krieglsteiner 1985, Index Fungorum 2018, Raitviir 2006b). Species of Incrucipulum occur on various substrates such as fallen twigs, culms and leaves and have strong selectivity to host species and their parts. In the process of elucidating Japanese lachnaceous mycobiota, four undocumented species of Incrucipulum, including three undescribed species and one new record from Japan, were collected. Molecular phylogenetic analysis was conducted to confirm the validity of inclusion of the three new species into Incrucipulum and identify apomorphic characters in Incrucipulum. Accepted by Ruvishika Jayawardena: 17 Apr. 2019; published: 9 May 2019 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 25 Material and methods Collection and Isolation Five Incrucipulum specimens of three new species and I. capitatum were collected from four localities in Japan. Specimens were air-dried naturally for one week in 20 °C and deposited in the mycological herbarium of the National Museum of Nature and Science (TNS). Multi-spored isolates were obtained by collecting discharged ascospores on potato dextrose agar (PDA, Nissui, Tokyo) and kept on PDA slants at 4 °C. Isolates were numbered beginning with ‘FC-’ and will be deposited to NITE National Bioresource Center (NBRC). Morphological examination External appearance of apothecia was examined using SZ61 stereoscopic microscope (Olympus, Tokyo, Japan) and microphotographs were taken using DS-L4 (Nikon, Tokyo, Japan). To examine microscopic characters, dried apothecia were detached from the substrate and rinsed in 70% ethanol followed by rehydration in ion-exchanged water for six hours, and then observed in cotton blue dissolved in lactic acid (CB/LA) in squash mount using BX51 microscope equipped with Nomarski interference contrast device (Olympus). Microphotographs were taken using DS-L3 with the camera head DS-Fi2 (Nikon). Line drawings were prepared using drawing device U-DA (Olympus) equipped with BX51. Ascal apex iodine reaction was checked by Melzer’s reagent. DNA extraction Two mL of 2% malt extract broth was inoculated with isolates and cultivated at room temperature for two weeks. The cultivated mycelia were frozen at −80 °C for two hours, dried by a Bulk Tray Dryer (Labconco, Kansas, USA) and then crushed using TissueLyser (Quiagen, Hilden, Germany). Powdered fungal samples were incubated in cetyltrimethylammonium bromide buffer (CTAB buffer, 2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA) at 65 °C for one hour, and protein was removed using the mixture of chloroform/isoamylalcohol (24:1). The solution was purified by 6M sodium iodine buffer (6 M NaI, 50 mM Tris-HCl pH 7.4, 10 mM EDTA, 0.1 M Na2SO3) (Hosaka & Castellano 2008) with GLASSMILK (Funakoshi, Tokyo, Japan) and washed by ethanol/buffer solution (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM NaCl, 50% EtOH). Purified DNA was eluted and dissolved in Tris-EDTA buffer (TE buffer, 10 mM Tris-HCl, 1 mM EDTA). When isolates were not available, DNA was extracted directly from a fresh apothecium using DNA extraction buffer (SDS 0.35 mM, Proteinase K 0.1mg/ml, Tris-HCl 10 mM, MgCl2 1.5 mM, KCl 50 mM, dH2O to adjust to total volume 50 mL). Apothecium was put in a 1.5 mL Eppendorf tube and smashed manually using 1.5 mL pellet pestle (Fisher Scientific, Hampton, USA). 50 μl DNA extraction buffer was added and incubated for 90 min. at 40 °C followed by 10 min. incubation at 90 °C. After centrifuged, the supernatant was preserved as extracted DNA. Extracted DNA samples were deposited to Molecular Biodiversity Research Center in the National Museum of Nature and Science (Tsukuba, Ibaraki, Japan) and available for molecular phylogenetic researches. Polymerase chain reaction (PCR) and sequencing For phylogenetic analysis, the internal transcribed spacer region of nuclear ribosomal DNA containing partial ITS15.8S-ITS2 (ITS-5.8S), the partial large subunits nuclear ribosomal RNA gene (LSU) and the section ‘6–7’ of the second largest subunit of the nuclear RNA polymerase II gene (RPB2) were used, because analysis using only ITS5.8S did not make robust trees in inter- and intra-genus level (Hosoya et al. 2010). Extracted DNA was amplified by PCR using EmeraldAmp PCR Master Mix (Takara, Kusatsu, Japan). For ITS-5.8S, the forward primer ITS1F (CTT GGT CAT TTA GAG GAA GTA A) (Gardes & Bruns 1993) and the reverse primer ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990) were used. For LSU, the forward primer LR0R (ACC CGC TGA ACT TAA GC) and the reverse primer LR5 (TCC TGA GGG AAA CTT CG) (Vilgalys & Hester 1990) were used. For RPB2, the forward primer RPB2-P6F (TGG GGW YTS GTM TGY CCT GC) and the reverse primer RPB2-P7R (CCC ATS GCY TGY TTA CCC AT) (Liu et al. 1999) were used. For ITS-5.8S and LSU, 10 μl PCR reaction were performed in a following protocol: initial denaturation for 3 min at 95 °C, 30 cycles of 94 °C for 35 s, 51 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were purified using ExoProStar Sequencing Clean-up kit (Illumina, San Diego, USA). For RPB2, 20 μl PCR reaction solution was used in a following protocol: initial denaturation for 3 min at 95 °C, followed by 25 cycles of 95 °C for 45 s, 52 °C for 40 s and 74 °C for 2 min, then another 25 cycles of 95 °C for 50 s, 52 °C for 45 s and 74 °C for 2 min 5 s, and a final extension at 10 min for 72 °C. Appropriate length of amplified DNA in 26 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA the agarose gel were checked using the LED transilluminator (Fujifilm Wako, Tokyo, Japan) and cut out using a spatula and then purified using MonoFas DNA Purification Kit I (GL Sciences, Tokyo, Japan) following the manufacturer’s instruction. Cycle sequence reaction was conducted with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA) using the same PCR primers mentioned above. Sequencing was carried out using Applied Biosystems 3500xL Genetic Analyzer. Sequences obtained in the present study were assembled using ATGC Ver. 7 (Genetyx, Tokyo, Japan), and then registered to DNA Data Bank of Japan (DDBJ) database synchronized with the GenBank. For TNS-F-81441, DNA was also extracted directly from an apothecium in addition to DNA extraction from an isolate (FC-6879), because the isolate turned out to be contaminated by bacteria. Sequences derived from the isolate and the apothecia were examined separately. Phylogenetic analysis For species of Incrucipulum, all available sequences obtained by Hosoya et al. (2010) and newly obtained sequences were assorted, and 12 OTUs composed of seven species were obtained (Table 1). For other lachnaceous members, one or two species were selected from five major lachnaceous genera (Albotricha Raitviir (1970: 40), Brunnipila Baral (1985: 49), Erioscyphella Kirschstein (1938: 384), Lachnellula P. Karsten (1884: 138), Lachnum (sensu stricto)), since monophyly of these genera were already suggested (Hosoya et al. 2010, Perić & Baral 2014). Species of Capitotricha (Raitviir 1970: 88) Baral (1985: 60), Neodasyscypha Suková & Spooner (in Suková 2005: 163) and Proliferodiscus J.H. Haines & Dumont (in Haines 1983: 536) not included in Hosoya et al. (2010) were also added. Since Lachnum varians (Rehm 1900: 94) M.P. Sharma (1986: 411) was presumed as a separate lineage from Lachnum (sensu stricto) (Hosoya et al. 2010, Guatimosim et al. 2016), L. varians was also included. Two species of Dasyscyphella Tranzschel (1899: 11), whose generic affiliation is still unresolved, were incorporated. Species of Lasiobelonium Ellis & Everhart (1897: 136), Trichopeziza Fuckel (1870: 295) and Trichopezizella Dennis ex Raitviir (in Raitviir 1969: 68) were not included because they were indicated to be phylogenetically apart from other lachnaceous genera (Hosoya et al. 2010). Hyaloscypha spiralis (Velenovský 1934) J.G. Han, Hosoya & H.D. Shin (in Han et al. 2014: 161), Urceolella carestiana (Rabenhorst 1866: 189) Dennis (1963: 335) and Hymenoscyphus varicosporoides Tubaki (1966: 346) were selected as outgroup taxa based on molecular phylogenetic assessment of Hyaloscyphaceae (Han et al. 2014). In total, 27 lachnaceous OTUs of 22 species and three outgroup OTUs were gathered (Table 1). Sequences of 30 OTUs were initially aligned by MAFFT 7 (Katoh 2013) and adjusted manually for each region. Molecular phylogenetic analysis of 30 OTUs was executed based on Maximum likelihood (ML; Felsenstein 1981) and Bayesian inference (BI) analyses. The combined sequence datasets were divided into five data partitions (ITS5.8S, LSU and each codon position of RPB2). The optimal model was estimated using Kakusan 4 (Tanabe 2011) with default settings. In the ML analysis, the separate model was selected based on Akaike’s information criterion (AIC). ML tree search and ML bootstrap analysis (Felsenstein 1985) with 1,000 bootstrap replications were performed using RAxML v.8.2.X (Stamatakis 2016). In the BI analysis, proportional model was selected based on Bayesian information criterion (BIC). Four chains of Metropolis Coupled Markov chain Monte Carlo (MCMCMC) analysis resulted in 5 million generations and sampled every 100 generations using MrBayes v.3.2 (Ronquist et al. 2012). The average standard deviation of split frequencies (ASDSF) was verified to become <0.01 and each run was checked to become convergent using Tracer v1.7.1 (Rambaut 2018a). Bayesian Posterior probabilities (BI PPs) were used to evaluate reliability and 50% majority rule consensus tree was adopted. Trees were illustrated using FigTree v1.4.4 (Rambaut 2018b) based on ML analysis and ML bootstrap value (BS) and BI PPs were plotted on each branch. The data matrix was deposited in TreeBASE with accession ID S24265 (available from http://purl.org/phylo/treebase/phylows/study/TB2:S24265). THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN Phytotaxa 403 (1) © 2019 Magnolia Press • 27 28 • Phytotaxa 403 (1) © 2019 Magnolia Press TABLE 1. Taxa investigated in the molecular phylogenetic analysis. ‘-’ appears when DNA was extracted not from an isolate but directly from an apothecium. Specimens and sequences of Incrucipulum newly obtained in this study were shown in bold. Taxon Specimen no. Locality Substrate Collecting Isolate / GenBank/DDBJ accession no. ITS LSU RPB2 date Culture no. TNS-F-16740 Japan, Nagano, Sugadaira Culm of bamboo 6/17/2006 NBRC AB481234 LC438571 AB481354 Albotricha acutipila (P. Karst.) Raitv. 104380 5/18/2005 NBRC AB481235 LC424943 AB481340 Albotricha albotestacea (Desm.) Raitv. TNS-F-16497 Japan, Nagano, Sugadaira Culm of Miscanthus sinensis 101346 TNS-F-16635 Japan, Gunma, Agatsuma Unidentified leaf 4/27/2006 NBRC AB481255 LC424945 AB481348 Brunnipila fuscescens (Pers.) Baral 104365 TNS-F-65670 Switzerland, Graubünden, Filisur Twig of Prunus spinosa 6/6/2016 FC-6101 LC424834 LC424942 LC425011 Capitotricha bicolor (Bull.) Baral TNS-F-65752 Switzerland, Verwaltungskreis, 6/4/2016 FC-6075 LC438560 LC438573 LC440395 Capitotricha rubi (Bres.) Baral Twig of Rubus idaeus Saicourt TNS-F-16439 Japan, Kanagawa, Yamakita 4/17/2005 NBRC AB481239 LC424947 AB481331 Dasyscyphella longistipitata Hosoya Cupule of Fagus crenata 101335 TNS-F-16527 Japan, Gunma, Agatsuma Unidentified wood 5/21/2005 NBRC AB481242 LC438577 AB481336 Dasyscyphella montana Raitv. 102336 TNS-F-80478 Japan, Shizuoka, Oyama Unidentified wood 6/26/2017 NBRC LC424837 LC424949 LC425009 Erioscyphella abnormis (Mont.) Baral 113934 Incrucipulum capitatum (Peck) Baral TNS-F-81420 Japan, Hokkaido, Sapporo LC424838 LC424954 LC438592 6/17/2018 NBRC Leaf of Quercus crispula TOCHIHARA & HOSOYA 7/26/2018 113938 NBRC 113940 NBRC 113941 NBRC 113942 - LC438569 LC438586 LC438599 8/12/2018 - LC438564 LC438581 LC438594 7/29/2006 NBRC 102347 FC-6822 Incrucipulum ciliare (Schrad.) Baral Incrucipulum ciliare (Schrad.) Baral TNS-F-81514 Japan, Saitama, Ogano TNS-F-81516 Japan, Saitama, Ogano Leaf of Quercus crispula Leaf of Castanea crenata 8/12/2018 8/12/2018 Incrucipulum ciliare (Schrad.) Baral Leaf of Quercus crispula 8/18/2018 Incrucipulum foliicola Tochihara TNS-F-81520 Japan, Shizuoka, Shizuoka, Umegashima TNS-F-81508 Japan, Hokkaido, Nemuro 7/20/2018 Incrucipulum foliicola Tochihara TNS-F-81526 Japan, Hokkaido, Oshamambe Incrucipulum hakonechloae-macrae Tochihara Incrucipulum longispineum (Hosoya & Issh. Tanaka) Sasagawa & Hosoya Incrucipulum pseudosulphurellum Tochihara TNS-F-81512 Japan, Saitama, Ogano TNS-F-17632 Japan, Miyagi, Sendai Leaf of Myrica gale var. tomentosa Leaf of Myrica gale var. tomentosa Leaf and culm of Hakonechloa macra Leaf of Lyonia ovalifolia TNS-F-81441 Japan, Hokkaido, Oshamambe Twig of Myrica gale var. 7/26/2018 LC438568 LC438565 LC438585 LC438582 LC438598 LC438595 LC438566 LC438583 LC438596 LC438567 LC438584 LC438597 AB481256 LC438579 AB481362 LC438563 LC438588 LC438601 tomentosa ...Continued on next page THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN TABLE 1. (Continued) Taxon Incrucipulum pseudosulphurellum Tochihara Incrucipulum radiatum (Issh. Tanaka & Hosoya) Sasagawa Incrucipulum radiatum (Issh. Tanaka & Hosoya) Sasagawa & Hosoya Lachnellula calyciformis (Batsch) Dharne Specimen no. Locality TNS-F-81441 Japan, Hokkaido, Oshamambe Twig of Myrica gale var. tomentosa Japan, Nagano, Sugadaira Leaf of Fagus crenata Japan, Niigata, Tokamachi Leaf of Fagus crenata Japan, Hokkaido, Engaru Twig of Abies TNS-F-16769 TNS-F-36248 TNS-F-81248 Substrate sachalinensis Twig of Larix kaempfer Unidentified leaf Phytotaxa 403 (1) © 2019 Magnolia Press • 29 Lachnellula suecica (de Bary ex Fuckel) Nannf. Lachnum soppittii (Massee) Raitv. TNS-F-16529 Japan, Nagano, Sugadaira TNS-F-16551 Japan, Ibaraki, Mt. Tsukuba Lachnum varians (Rehm) Spooner TNS-F-17631 Lachnum virgineum (Batsch) P. Karst. TNS-F-16583 Japan, Kagoshima, Yakushima Stem of unidentified fern Japan, Kanagawa, Yamakita Unidentified wood Neodasyscypha cerina (Pers.) Spooner TNS-F-65625 Proliferodiscus alboviridis (Sacc.) Spooner TNS-F-17436 Switzerland, Verwaltungskreis, Saicourt Japan, Ibaraki, Tsukuba Botanical Garden Japan, Kumamoto, Kikuchi TNS-F-17909 Hyaloscypha spiralis (Velen.) J.G. Han, Hosoya & H.D. Shin (outgroup) TNS-F-16472 Japan, Ibaraki, Kasumigaura Hymenoscyphus varicosporoides Tubaki (outgroup) TNS-F-18014 Japan, Iwate, Hanamaki Urceolella carestiana (Rabenh.) Dennis (outgroup) Newly described species as well as specimen number and sequences are shown in bold. a) The duplicate sequence is registered to DDBJ as ‘AB705235’. Collecting date Isolate / Culture no. 7/26/2018 - LC438570 LC438587 LC438600 9/24/2006 NBRC 104385 NBRC 113933 NBRC AB481261 LC438572 AB481359 LC438559 LC438580 LC438593 LC438561 LC438574 LC438590 AB481248 LC424944 AB481341 AB481266 LC438578 AB481344 AB481267 LC438576 AB481330 AB481268a) AB926119 AB481343 LC424836 LC424948 LC425013 LC438558 LC424950 LC438589 LC438602 LC438604 LC438606 AB926052 LC424952 AB481329 LC438603 LC438605 LC438607 9/20/2010 7/12/2017 5/21/2005 5/28/2005 10/23/2005 7/2/2005 Twig of Crataegus sp. Unidentified wood 6/8/2016 Unidentified wood 10/10/2005 Unidentified wood 5/5/2005 Stem of Thelypteris nipponica 5/23/2006 7/8/2006 113935 NBRC 101348 NBRC 104361 NBRC 102332 NBRC 104358 FC-6068 NBRC 108594 NBRC 108585 NBRC 104355 NBRC 108588 GenBank/DDBJ accession no. ITS LSU RPB2 Taxonomy Incrucipulum foliicola Tochihara, sp. nov. (Figs. 1, 2) MycoBank no.:—MB829358 Ecology:—Saprotrophic on dead leaves of Myrica gale subsp. tomentosa (C.DC.) E. Murray. Abundant on the fallen leaves accumulated on the wetlands. Holotype:—JAPAN. Hokkaido: Yamakoshi-gun, Oshamanbe-cho, Shizukari wetland, 5 m, 42.573644° N, 140.430907° E, 26 June 2018, on dead leaves of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81526!). FIGURE 1. Incrucipulum foliicola. A: Apothecia scattered on the substrate. B: Fresh apothecium. C: Dried apothecium. D: Ascus. E: Ascal bases with croziers. F: Paraphysis. G: Hairs with cruciate crystals like ‘cross shuriken’. H: Ascospores. I: Ectal excipular cells with granulate surface. TNS-F-81508 (A, C–I), TNS-F-81526 (B). Scale bars = 1 cm (A), 0.5 mm (B, C), 10 μm (D–I). Paratype:—JAPAN. Hokkaido: Nemuro-city, Cape Ochiishi wetland, 47 m, 43.166311° N, 145.512608° E, 20 July 2018, on dead leaves of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81508!). Etymology:—Referring to the part of substrates. Description:—Apothecia scattered on the substrate, superficial, cup-shaped, 0.1–0.4 mm in diameter when fresh and dry, having long and slender stipes, up to 0.6 mm high, externally covered with hairs, lemon yellow throughout when fresh and dry. Disc concave, concolourous with external receptacle. Ectal excipulum textura prismatica composed of hyaline cubic cells with granulated surface arranged in parallel rows, thick-walled; wall up to 2 μm thick. Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical, up to 90 × 3–5 μm, densely septate, hyaline, granulated throughout, thick-walled; wall up to 2 μm thick; individual cells 2.5–10 μm long; apex blunt or a little bit swollen, equipped with one cross-shaped crystal like “cross shuriken” ca. 10 μm across or one tetrahedral crystal detached easily in squash mounts. Asci 27–37 × 2.5–4 μm, 8-spored, cylindrical-clavate with slightly protruding apices; apical pore blue in Melzer’s reagent without 3% KOH pretreatment; croziers present at the basal septa. Ascospores 5–8 × 1.2–1.5 μm, long ellipsoid to narrowly fusiform, aseptate, containing some small lipid bodies. Paraphyses straight, lanceolate, septate, up to 4 μm wide, exceeding the asci up to 25 μm. 30 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA FIGURE 2. Incrucipulum foliicola (TNS-F-81508). A: Apothecium. B: Ascospores. C: Hair with a crystal. D: Ectal excipular cells. E: Paraphyses. F: Asci. Notes:—Incrucipulum foliicola, I. pseudosulphurellum and I. sulphurellum occur on Myrica gale L. (including M. gale. subsp. tomentosa) and share tetrahedral or cross-shaped crystals of hairs (Figs. 1G, 2C) as a common character. Incrucipulum foliicola much resembles I. sulphurellum which differs in the absence of croziers and parts of host, based on the original description (Peck 1878) and redescription of Haines (1989). Incrucipulum foliicola differs from I. pseudosulphurellum in short stipes, the presence of croziers, hyaline ectal excipular cells and parts of host. Considering that all Incrucipulum species and other lachnaceous species have strong selectivity to the parts of host and that foliicolous species never occur on twigs and other parts, I. foliicola should be treated as a new species. Incrucipulum foliicola is currently known from two wetlands in Hokkaido. Incrucipulum hakonechloae-macrae Tochihara, sp. nov. (Figs. 3, 4) MycoBank no.:—MB829308 Ecology:—Saprotrophic on fallen and damp leaves, culms and other leaf-like parts of Hakonechloa macra (Munro) Honda stuck to the ground. Holotype:—JAPAN. Saitama Pref.: Chichibu-gun, Ogano-machi, Mt. Futagoyama, 1008 m, 36.068342° N, 138.867364° E, 12 August 2018, on dead leaves and culms of Hakonechloa macra, Y. Tochihara (TNS-F-81512!). Etymology:—Referring to the host plant Hakonechloa macra. Description:—Apothecia scattered on the substrate, superficial, cup-shaped, 0.2–0.4(–0.5) mm in diameter, externally covered with short, white, capitate hairs bearing drops of dew when moist, stipitate, up to 0.2–0.5 mm high when fresh and dry; stipe white, sometimes brown in the lower part. Disc concave, lemon yellow to orange when fresh and dry. Ectal excipulum textura prismatica composed of hyaline cubic cells like stone pavings, 2.5–15 × 5–13 μm, containing yellow oil drops, thick-walled; wall up to 3 μm thick with granulate surface; cells arranged in parallel rows. Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical, sometimes with rounded apices, 38–90 × 3–8 μm, 2–5-septate, hyaline, completely covered by granules, thin to relatively thickwalled; wall up to 1.5 μm thick; apices blunt or a little bit swelled, bearing amber crystal caps detached easily in squash mounts. Asci 46–63(–75) × 6–10 μm, 8-spored, cylindrical-clavate; apical pore blue in Melzer’s reagent without 3% KOH pretreatment; croziers present at the basal septa. Ascospores 8–14 × 2.5–5 μm, fusiform, aseptate or rarely 1-septate, usually packed by gelatinous sheath, containing some large lipid bodies conspicuous in CB/LA mount. Paraphyses straight, cylindrical to narrowly lanceolate, rarely swollen at the apices, sometimes branched near the bases, septate, up to 3 μm wide, containing hyaline to orange oil drops, exceeding the asci up to 7.5 μm. THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN Phytotaxa 403 (1) © 2019 Magnolia Press • 31 FIGURE 3. Incrucipulum hakonechloae-macrae (TNS-F-81512). A: The host Hakonechloa macra and its litter in the collection site of I. hakonechloae-macrae. B: Scattered apothecia of I. hakonechloae-macrae occurring on the host leaf. C, D: Fresh apothecia. E: Ascus. F: Ascus arising from a crozier. G, H: Paraphysis. I: Hair arising from ectal excipular cells with granulate surface. J: Ascospores packed by gelatinous sheath. K: Ascospore with a septum. L: Ascospores containing large lipid bodies. M: Thick-walled ectal excipular cells containing orange oil drops. Scale bars = 1 cm (B), 0.5 mm (C, D), 10 μm (E–M). FIGURE 4. Incrucipulum hakonechloae-macrae (TNS-F-81512). A: Apothecium. B: Ascospores. C: Ectal excipular cells. D: Paraphyses. E: Hairs with crystals. F: Asci. 32 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA Notes:—Incrucipulum hakonechloae-macrae is distinguishable from other species of Incrucipulum in yellow to orange disc (Fig. 3C) and ascospores containing very large oil drops and packed by gel sheath (Figs. 3J–3L). Ascospores did not germinate in PDA at all. Some spores germinated in water agar or corn meal agar, but no further extension of hyphae was observed. Incrucipulum hakonechloae-macrae was collected only from Hakonechloa macra, an endemic grass in the central Honshu, Japan (Katoh & Ebihara 2011). Incrucipulum hakonechloae-macrae is possibly also endemic to the Honshu Island corresponding to the host distribution, because most of lachnaceous species have strong host selectivity. Incrucipulum pseudosulphurellum Tochihara, sp. nov. (Figs. 5, 6) MycoBank no.:—MB829362 Ecology:—Saprotrophic on fallen twigs of Myrica gale subsp. tomentosa. Holotype:—JAPAN. Hokkaido: Yamakoshi-gun, Oshamanbe-cho, Shizukari wetland, 5 m, 42.573644° N, 140.430907° E, 26 June 2018, on dead twigs of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81441!). Etymology:—Referring to the similarity to I. sulphurellum. FIGURE 5. Incrucipulum pseudosulphurellum (TNS-F-81441). A, B: Fresh apothecia. C, D: Dried apothecium. E: Ascus. F: Ascus with an irregularly branched base. G: Immature ascus with a crozier-like structure (arrow head). H: Paraphysis. I: Hair with a tetrahedral crystal. J: Hair with an irregular-shaped crystal. K: Ascospores. L: Hyaline ectal excipular cells. M: Subhyaline ectal excipular cells. N: Surface of ectal excipular cells with granules (arrow heads). Scale bars = 0.5 cm (A–D), 10 μm (E–N). Description:—Apothecia scattered, superficial, 0.4–0.8 mm in diameter, having short and dark brown stipes, up to 0.5 mm high, white to somewhat dull yellow on the upper part and thin to dark brown in the lower part, densely covered by white, capitate hairs. Disc concave, pure white to orange when fresh and deep orange when dry. Ectal excipulum textura prismatica to textura angularis, 4–21 × 3–15 μm, hyaline in the upper part and subhyaline to brown in the lower part, slightly thick-walled; wall up to 2 μm thick; surface smooth or rarely equipped with sparse granules. Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical with slightly rounded apices, up to 130 × 4.5–7.5 μm, completely granulate, multiseptate, arising from rounded basal cells; hair cells 5–25 μm long, thin to slightly thick-walled; wall up to 1.5 μm thick; apical cells thick-walled; wall up to 2 μm thick; apices bearing various shapes of crystals easily detached by squash mounts, i.e. tetrahedral ones, cross-shaped THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN Phytotaxa 403 (1) © 2019 Magnolia Press • 33 ones like ‘cross shuriken’ or amber crystal caps. Asci 33–44(–52) × 3.5–7.5 μm, 8-spored, cylindrical-clavate; pore blue in Melzer’s reagent without 3% KOH pretreatment; croziers absent, but base sometimes irregularly branched and forming ‘fake-croziers’. Ascospores 6.3–10 × 1.5–2.2 μm, oblong to narrowly fusiform, aseptate. Paraphyses straight, lanceolate, up to 5.5 μm wide, exceeding the asci by 25 μm. Notes:—Incrucipulum pseudosulphurellum resembles I. foliicola and I. sulphurellum, but differs in short and dark brown stipes (Figs. 5A–5D), brownish ectal excipulum rarely equipped with granules (Figs. 5L–5M). Little granules on ectal excipular cells is an atypical character of Incrucipulum, but other characters agrees with generic definition. Incrucipulum pseudosulphurellum is currently known only from the type locality, but probably widely distributed corresponding to the distribution of host plants. Wooly appearance and short stipes of this species are similar to Capitotricha bicolor (Bulliard 1789: 410) Baral (1985: 60) macroscopically but differs in smaller asci and lacking orange oil drops in paraphyses. FIGURE 6. Incrucipulum pseudosulphurellum (TNS-F-81441). A: Apothecium. B: Ascospores. C: Ectal excipular cells. D: Paraphyses. E: Hairs with and without crystals. F: Asci. Results and Discussion In the molecular phylogenetic analysis, we obtained sequence matrices composed of 350 bases of ITS-5.8S, 808 bases of LSU and 665 bases of RPB2. The optimal model was selected as follow; GTR model with a discrete gamma distribution (GTR+G) for ML analysis, and K80+G for ITS, GTR+G for LSU, SYM+G for RPB2 first and third codon position, and HKY85+G for RPB2 second codon position for BI analysis. In the BI analysis, the first 10% of the generated trees in the cold chain were discarded as the burn-in, because ASDSF became <0.01 and model parameters converged when 500,000 sample trees generated. ML tree with BI PPs calculated by the remaining trees was shown (Fig. 7). All seven species of Incrucipulum formed a strongly supported clade and robust phylogenetic inner relationship of Incrucipulum was revealed. Inclusion of the three new species into Incrucipulum was justified and monophyly of Incrucipulum was also confirmed. 34 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA FIGURE 7. Phylogenetic tree of Incrucipulum and other lachnaceous species constructed by RAxML based on combined sequences of ITS (350 bases), LSU (808 bases) and RPB2 (665 bases). ML bootstrap values (ML BS; before the slash marks) and BI posterior probabilities (BI PPs; after the slash marks) were shown in each branch. ‘-’ represents ML BS are under 50% or BI PPs are under 0.90, or a branch did not appear in BI analysis. ‘䠆’ represents BI PPs are 1.00. Bolded branches show ML BS are 90% or more and BI PPs are 0.95 or more in Incrucipulum clade. Sequences of Incrucipulum species newly described in this study were shown in bold. Within Incrucipulum, a strongly supported clade excluding I. foliicola and I. pseudosulphurellum was recognized. Within this clade another strongly supported clade (Clade A), composed of I. ciliare (type species) and other three species of Incrucipulum, was recognized. The species in Clade A all had cylindrical to narrowly lanceolate paraphyses while others had lanceolate paraphyses. Since lanceolate paraphyses are shared by most of lachnaceous genera and Clade A is terminal in Incrucipulum, cylindrical to narrowly lanceolate paraphyses are presumed to be apomorphic and evolved multiple times. Species of Clade A were also characterized by large (>10 μm long) and conspicuously fusiform ascospores and longer asci (mainly > 40 μm long), while others have small (<10 μm long) and oblong to narrowly fusiform ascospores and smaller asci (mainly < 40 μm long). Fusiform ascospores and larger asci are also presumed to be apomorphic in Incrucipulum, as has been suggested in the morphological data of Tanaka & Hosoya (2001). In the present research, I. capitatum was newly collected from Japan. Eight species of Incrucipulum are currently known from Japan. Five species, I. foliicola, I. hakonechloae-macrae, I. longispineum, I. pseudosulphurellum and I. radiatum, have been known only from Japan (Katsumoto 2010, Otani 1967, Raitviir 1977, Tanaka & Hosoya 2001, this study). Considering that lachnaceous species tend to show strong host specificity, it is presumed that species of Incrucipulum were diversified in the Far East corresponding to characteristic vascular plant flora, such as tall herb grasslands and bamboo grove (Raitviir 1979). With the addition of three new species, Incrucipulum now contains 13 species. In addition to the currently known members, Lachnum radovii Svrček (1984: 201), L. roseum (Rehm 1881: 41) Rehm (1893: 882) and L. soppitii (Massee 1895: 330) Raitviir (1986: 2) were mentioned as potential members of Incrucipulum (Baral & Krieglsteiner 1985). Lachnum soppitii is phylogenetically apart from the genus Incrucipulum. (Fig. 7). Based on the description and the illustration of Raitviir (1977) Lachnum lespedezae (Raitviir 1977: 691) Raitviir (1986: 2) is also a potential member due to cylindrical paraphyses, thick-walled hairs and thick-walled ectal excipulum like stone pavings, but the presence or absence of granules on ectal excipulum of the type was not documented. We hesitate to propose their new combinations in this study because type studies and phylogenetic analyses (excluding L. soppitii) have not been done. THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN Phytotaxa 403 (1) © 2019 Magnolia Press • 35 Key to species of Incrucipulum 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. - Having obvious lanceolate paraphyses ...............................................................................................................................................2 Having cylindrical to narrowly lanceolate paraphyses .......................................................................................................................6 Having hairs with >6 septa .................................................................................................................................................................3 Having hairs with ≤6 septa ............................................................................................................................................... I. capitatum Occurring on Myrica gale ..................................................................................................................................................................4 Occurring on dead culms of grasses .......................................................................................................................... I. densiseptatum Occurring on dead leaves .................................................................................................................................................... I. foliicola Occurring on dead twigs .....................................................................................................................................................................5 Having hyaline ectal excipulum .................................................................................................................................. I. sulphurellum Having brownish ectal excipulum .................................................................................................................... I. pseudosulphurellum Occurring on dead leaves of ericaceous plants...................................................................................................................................7 Occurring on plants except for Ericaceae ...........................................................................................................................................8 Croziers present at the basal septa of asci ...................................................................................................................I. longispineum Croziers absent at the basal septa of asci..................................................................................................................I. virtembergense Occurring on dead stems of tall herbaceous plants .......................................................................................................I. sinegoricum Occurring on other plants ...................................................................................................................................................................9 Croziers present at the basal septa of asci ........................................................................................................................................10 Croziers absent at the basal septa of asci..........................................................................................................................................11 Occurring on dead leaves of Fagus ....................................................................................................................................I. radiatum Occurring on dead leaves or culms of grasses...............................................................................................I. hakonechloae-macrae Spores <12 μm long ..........................................................................................................................................................I. saccardoi Spores ≥12 μm long .........................................................................................................................................................................12 Occurring mainly on dead leaves of fagaceous plants ........................................................................................................... I. ciliare Occurring on dead leaves of Dryas octopetala ...................................................................................................................I. uralense Acknowledgments We are very thankful to Dr. Hidehisa Koba at Obirin University for identifying a host grass, Hakonechloa macra and Dr. Kentaro Hosaka & Dr. Yasuhide Nakamura at National Museum of Nature and Science for helpful discussion. References Baral, H.O. & Krieglsteiner, G.J. (1985) Bausteine zu einer Askomyzeten-Flora der Bundersrepublik Deutschland. In: Süddeutchland gefundene inoperculte Diskomyceten mit taxonomischen, ökologischen, chorologischen Hinweisen und einer Farbtafel. Beiheften zur Zeitschrift für Mykologie 6: 1–160. Bulliard, J.B.F. (1789) Herbier de la France, 9. Chez l’auteur, Didot, Debure, Belin, Paris, 600 pp. https://doi.org/10.5962/bhl.title.5365 Chlebicki, A. (2002) Biogeographic relationships between fungi and selected glacial relict plants. The use of host-fungus data as an aid to plant geography on the basis of material from Europe, Greenland and northern Asia. Monographiae Botanicae 90: 1–230. https://doi.org/10.5586/mb.2002.001 Chlebicki, A. (2005) On three foliicolous Helotiales on Dryas. Mycotaxon 93: 105–113. Ellis, J.B. & Everhart, B.M. (1897) New species of fungi from various localities. Bulletin of the Torrey Botanical Club 24: 125–137. https://doi.org/10.2307/2477878 Dennis, R.W.G. (1963) A redisposition of some fungi ascribed to the Hyaloscyphaceae. Kew Bulletin 17 (2): 319–379. https://doi.org/10.2307/4118967 Felsenstein, J. (1981) Evolutionary trees from DNA sequences: A maximum likelihood approach. Journal of Molecular Evolution 17 (6): 368–376. https://doi.org/10.1007/BF01734359 Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. https://doi.org/10.2307/2408678 Fuckel, L. (1870) Symbolae mycologicae. Beiträge zur Kenntniss der Rheinischen Pilze. Jahrbücher des Nassauischen Vereins für Naturkunde 23–24: 1–459. https://doi.org/10.5962/bhl.title.47117 Galán, A. & Raitviir, A. (1994) Some new or interesting species of the Hyaloscyphaceae from Spain. Nova Hedwigia 58 (3–4): 453–473. 36 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA Gardes, M. & Bruns, T.D. (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology 2 (2): 113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x. Gray, S.F. (1821) A natural arrangement of British plants, 1. Baldwin, Cradock and Joy, London, 824 pp. https://doi.org/10.5962/bhl.title.43804 Guatimosim, E., Schwartsburd, P.B., Crous, P.W. & Barreto, R.W. (2016) Novel fungi from an ancient niche: lachnoid and chalara-like fungi on ferns. Mycological Progress 15: 1239–1267. https://doi.org/10.1007/s11557-016-1232-6 Haines, J.H. (1983) Studies in the Hyaloscyphaceae II: Proliferodiscus, a new genus of Arachnopezizoideae. Mycologia 75 (3): 535–543. https://doi.org/10.2307/3792695 Haines, J.H. (1989) Studies in the Hyaloscyphaceae V: Species described by C.H. Peck. Mycotaxon 35 (2): 317–352. Han, J.G., Hosoya, T., Sung, G.H. & Shin, H.D. (2014) Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses. Fungal Biology 118: 150–167. https://doi.org/10.1016/j.funbio.2013.11.004 Hosaka, K. & Castellano, M.A. (2008) Molecular Phylogenetics of Geastrales with Special Emphasis on the Position of Sclerogaster. Bulletin of the National Science Museum. Series B, Botany 34 (4): 161–173. Hosoya, T., Sasagawa, R., Hosaka, K. Sung, G.-H., Hirayama, Y., Yamaguchi, K., Toyama, K. & Kakishima, M. (2010) Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material. Mycoscience 51: 170–181. https://doi.org/10.1007/s10267-009-0023-1 Index Fungorum (2018) Available from: http://www.indexfungorum.org (accessed 1 December 2018) Katoh, M. & Ebihara, A. (2011) Endemic Plants of Japan. Tokai University Press, Hadano, 528 pp. [in Japanese] Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30 (4): 772–780. https://doi.org/10.1093/molbev/mst010 Karsten, P.A. (1884) Fungi rariores Fennici atque nonnulli Sibirici a Dr. Edw. Wainio lecti. Meddelanden af Societas pro Fauna et Flora Fennica 11: 136–147. Katsumoto, K. (2010) List of fungi recorded in Japan. The Kanto Branch of the Mycological Society of Japan, Funabashi, 1177 pp. [in Japanese] Kirschstein, W. (1938) Über neue, seltene und kritische Ascomyceten und Fungi imperfecti. I. Annales Mycologici 36 (5–6): 367–400. Le Gal, M. (1939) Florule mycologique des Bois de la Grange et de l’Etoile, Discomycetes. Revue de Mycologie 4: 25–63. Liu, Y.J., Whelen, S. & Hall, B.D. (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Massee, G.E. (1895) British Fungus-Flora. 4. George Bell & Sons, London, 522 pp. Matheis, W. (1977) Über einige Dasyscyphus-Arten auf Blättern von Vaccinium. Sydowia 29 (1–6): 237–244. Otani, Y. (1967) Notes on some cup fungi of the Hyaloscyphaceae collected in Hokkaido, Japan. Transactions of the Mycological Society of Japan 8: 33–42. Peck, C.H. (1878) Report of the Botanist (1877). Annual Report on the New York State Museum of Natural History 31: 19–60. Perić, B. & Baral, H.O. (2014) Erioscyphella curvispora spec. nov. from Montenegro. Mycologia Montenegrina 17: 89–104. Rabenhorst, L. (1866) Notizblatt für kryptogamische studien, nebst Repertorium für kryptogamische. Literatur. Hedwigia 5 (12): 177–192. Raitviír, A. (1969) Discomycetes of Middle Asia. I. Descriptions of some new Helotiales. Eesti NSV Teaduste Akadeemia Toimetised 18: 66–69. Raitviir, A. (1970) Synopsis of the Hyaloscyphaceae. Scripta Mycologica 1: 1–115. Raitviir, A. (1977) Some new species of Dasyscyphus. Kew Bulletin 31 (3): 691–693. https://doi.org/10.2307/4119419 Raitviir, A. (1979) Analysis of the flora of the hyaloscyphaceous fungi in the Far East. In: Harkevich, S.S. (Ed.) XIV Pacific Science Congress. USSR Khabarovsk, August 1979. Kommittee H, Pacific Science Association, Moscow, pp. 36–37. Raitviir, A. (1985) Macromycetes of Kamchatka II. Hyaloscyphaceae. Folia Cryptogamica Estonica 17: 2–4. Raitviir, A. (1986) Some new combinations into the genus Lachnum Retz. Folia Cryptogamica Estonica 20: 1–11. Raitviir, A. (1991) Some Interesting Hyaloscyphaceae from North Italy. 2. Mycologia Helvetica 4: 161–168. Raitviir, A. (2006a) Rare or noteworthy Helotiales. Fungi Non Delineati 31: 1–55. Raitviir, A. (2006b) Discomycetes from the Moneron Island. In: Raitviir, A. & Bogacheva, A. (Eds.) Flora and fauna of Moneron Island. Dalinauka, Vladivostok, pp. 138–145. [in Russian] Rambaut, A. (2018a) Tracer. Molecular evolution, phylogenetics and epidemiology, Edinburgh. Available from: http://beast.community/ THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN Phytotaxa 403 (1) © 2019 Magnolia Press • 37 tracer (accessed 16 December 2018) Rambaut, A. (2018b) FigTree. Molecular evolution, phylogenetics and epidemiology, Edinburgh. Available from: http://tree.bio.ed.ac. uk/software/figtree/ (accessed 16 December 2018) Rehm, H. (1881) Ascomyceten in getrockneten Exemplaren herausgegeben. Berichte des Naturhistorischen Vereins Augsburg 26: 1–132. Rehm, H. (1893) Ascomyceten: Hysteriaceen und Discomyceten. In: Fischer, A., Hauck, F., Limpricht, G., Luerssen, Ch., Migula, W., Rehm, H., Richter, P. & Winter., G. (Eds.) Rabenhorst‘s Kryptogamen-Flora von Deutschland, Oesterreich und der Schweiz, Pilze, edition 2, 1 (3). Verlag von Eduard Kummer, Leipzig, pp. 1–56. Rehm, H. (1900) Beiträge zur Pilzflora von Südamerika. VIII. Discomycetes. Hedwigia 39: 80–99. Retzius, A.J. (1769) Anmärkningar vid Skänes Ört-Historie. Kungliga Svenska Vetenskapsakademiens Handlingar Serie 1 30: 243–255. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029 Schrader, H.A. (1799) Plantae cryptogamicae novae, rariores aut minus cognitae. Journal für die Botanik 2: 55–80. Sharma, M.P. (1986) Indian Hyaloscyphaceae. Nova Hedwigia 43 (3–4): 381–422. Suková, M. (2005) A revision of selected material of lignicolous species of Brunnipila, Capitotricha, Dasyscyphella and Neodasyscypha from the Czech Republic. Czech Mycology 57 (1–2): 139–172. Stamatakis, A. (2016) The RAxML v8.2.X Manual. Heidelberg Institute for Theoretical Studies. Available from: http://sco.h-its.org/ exelixis/web/software/raxml/#documentation (accessed 1 September 2018). Svrček, M. (1984) New or less known Discomycetes. XIII. Česká Mykologie 38 (4): 197–202. Tanabe, A.S. (2011) Kakusan 4 and Aminosan: two programs for comparing nonpartitioned, proportional, and separate models for combined molecular phylogenetic analyses of multilocus sequence data. Molecular Ecology Resources 11: 914–921. https://doi.org/10.1111/j.1755-0998.2011.03021.x Tanaka, I. & Hosoya, T. (2001) Hyaloscyphaceae in Japan (4): New records of the genus Lachnum. Mycoscience 42: 597–609. https://doi.org/10.1007/BF02460959 Tranzschel, W.A. (1899) Zwei neue europaeische Ascomycetengattungen. Beiblatt zur Hedwigia 38: 10–12. Tubaki, K. (1966) An undescribed species of Hymenoscyphus, a perfect stage of Varicosporium. Transactions of the British Mycological Society 49 (2): 345–349. https://doi.org/10.1016/S0007-1536(66)80069-4 Velenovský, J. (1934) Monographia Discomycetum Bohemiae, Czechoslovakia, Prague, 436 pp. Vilgalys, R. & Hester, M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky. J.J. & White, T.J. (Eds.) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp. 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 38 • Phytotaxa 403 (1) © 2019 Magnolia Press TOCHIHARA & HOSOYA