Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Tsuyoshi Hosoya

Study on fresh collections and type material of Hamatocanthoscypha rotundispora showed that the original description is incomplete and that the variation range in the essential characters is too narrowly defined. In this paper we therefore provide a detailed description, including ascospore morphometry using length, width, length/width ratio and volume. Non-parametric statistics showed significant deviation of normal distribution and high variability of all morphometrical variables, suggesting that use of living material for ascospore morphometrical analysis is more reliable than that of a traditional approach. Geometric criteria for defining the simple spore shape regarding the length/width ratio, polarity and symmetry in straight symmetric spores are proposed. Refractive cytoplasmic globules are described as a new type of inclusions in living cells. Species ecology and biogeography is also discussed.

Synedroid diatoms are morphologically characterized by a putative synapomorphic structure called an ocellulimbus, a terminal pore field whose surface is depressed relative to the rest of the valve surface. Among these diatoms is the genus Hyalosynedra Williams and Round. Characters associated with that genus are bilayered wall construction, densely packed virgae and an asymmetric rimoportula. However, these characters diagnose partially overlapping (incongruent) groups. The apical pore field itself, when present, is sometimes sunken deeply, sometimes weakly and sometimes not at all among these diatoms. Here we make additional morphological observations and add molecular sequence data (nuclear SSU rDNA, rbcL, psbC) to attempt to resolve these apparent conflicts. The molecular phylogeny does not resolve any morphological character without homoplasy, but conversely does resolve taxa which share an asymmetric rimoportula and/or densely packed virgae into a single clade we call the " hyalosynedroid " taxa. This clade contains our representatives of Thalassionematales which had been linked to Hyalosynedra by the bilayered wall structure and the possession by some species of an asymmetric rimoportula. We describe three new genera, and a total of 11 new species. Whereas four species of Hyalosynedra were previously described, we describe three new species of Hyalosynedra, describe the new genus Divergita with three species (one new, one transferred from Synedra and one from Hyalosynedra), and the new genus Stricosus with seven new species. A key to species of Hyalosynedra, Divergita and Stricosus is included.

f u n g a l b i o l o g y 1 1 8 ( 2 0 1 4 ) 1 5 0 e1 6 7 journal homepage: www.elsevier.com/locate/funbio Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses Jae-Gu HANa,1, Tsuyoshi HOSOYAb, Gi-Ho SUNGc, Hyeon-Dong SHINa,* a Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea b Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan c Mushroom Research Division, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 441-707, South Korea article info abstract Article history: Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by Received 30 July 2012 minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafa- Received in revised form milial classification remain ambiguous. This study performed molecular phylogenetic anal- 5 November 2013 yses using multiple genes including the ITS-5.8S rDNA, the D1eD2 region of large subunit of Accepted 19 November 2013 rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objec- Available online 1 December 2013 tive was to evaluate the phylogenetic utility of morphological characters traditionally used in Corresponding Editor: the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and Joseph W. Spatafora its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three Keywords: tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs Arachnopezizeae is rejected as a synapomorphy, since morphologically diversified hairs have originated inde- Evolution pendently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae Hyaloscypheae are hypothesized to have evolved through different evolutionary processes; the true- Lachneae subiculum is likely the product of a single evolutionary origin, while the false-subiculum is Taxonomy hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha. ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Hyaloscyphaceae Nannfeldt is the largest family in Helotiales, comprising of about 933 species in 74 genera (Kirk et al. 2008). It has a cosmopolitan distribution with species functioning as saprobes, or rarely as parasites, on various woody and herbaceous substrates. Hyaloscyphaceae is one of the most morphologically distinct families among the smaller discomycetes with species possessing brightly coloured apothecia that are ornamented with conspicuous hairs around the margin and lower surface (Fig 1). Despite this striking morphology, the family is infrequently collected in the field due to the fruiting bodies often being less than 1 mm in diameter. * Corresponding author. Tel.: þ82 2 3290 3063; fax: þ82 2 921 1715. E-mail address: hdshin@korea.ac.kr (H.-D. Shin). 1 Present address: Mushroom Research Division, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 441-707, South Korea. 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.11.004 Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses When Hyaloscyphaceae was first established, Nannfeldt (1932) subdivided it into three tribes: Arachnopezizeae, Hyaloscypheae, and Lachneae. Arachnopezizeae included species with an apothecium seated on a well-developed subiculum or subiculum-like hyphae; Hyaloscypheae contained species with tiny apothecia, hairs with highly diverse shapes, and mostly cylindric paraphyses; and Lachneae included species with relatively large apothecia, multiseptate granulate hairs, and lanceolate paraphyses. The infrafamilial classification of Hyaloscyphaceae has subsequently undergone several revisions (Dennis 1962; Dharne 1965; Korf 1973, 1978; Raitviir 1970, 1987, 2004; Haines & Dumont 1983; Spooner 1987; ek 1987). The hair morphology, along with features of Svrc the excipular structure, paraphyses, asci, and ascospores, has been regarded as the most important criterion for delimiting the genera and species of Hyaloscyphaceae. However, the generic delimitation and infrafamilial taxonomy remain ambiguous due to the different interpretations of these diagnostic characters. During 1990s, some authors argued that Hyaloscyphaceae was not monophyletic based on the heterogeneity of morphological characters and that some genera within Hyaloscyphaceae were more closely related to taxa in other families of Helotiales (Raitviir & Spooner 1994; Sutton & Hennebert 1994). A cladistic study of Hyaloscyphaceae by Cantrell & Hanlin (1997) suggested that this family is probably monophyletic, but their analysis was not conclusive due to its limited taxon sampling (Hosoya et al. 2010). Based on morphological and molecular phylogenetic data, Raitviir (2004) elevated the tribe Lachneae to the familial rank. Hosoya et al. (2010) assessed the current delimitations of Lachnaceae using a multigene analysis. However, despite these recent molecular phylogenetic studies, the insufficiency of taxon and character sampling has prevented the construction of a natural classification of Hyaloscyphaceae. The taxon sampling performed in the present study included species within all three tribes in Hyaloscyphaceae with a focus on the tribes Arachnopezizeae and Hyaloscypheae since Lachneae has been more intensely addressed in the previous studies (Cantrell & Hanlin 1997; Hosoya et al. 2010). Twentyfive helotialean taxa that are representatives of each family were also included to resolve phylogenetic relationships within the order. The main objectives of the present study were to 1) test the monophyly of Hyaloscyphaceae, 2) elucidate phylogenetic relationships of the genera studied here, and 3) evaluate the phylogenetic informativeness of morphological characters used in classical taxonomy of this family. Material and methods Taxon sampling and data sets The specimens and isolates used in this study were obtained from public herbaria (TNS and KUS) and culture collections (CBS, KACC, and NBRC). Available sequences of Hyaloscyphaceae and representative species of Helotiales were downloaded from the GenBank database. The source of voucher specimens or strains is listed with the GenBank accession numbers in Table 1. 151 Two data sets, INTRA-SET and INTER-SET, were prepared to construct the familial phylogeny. INTRA-SET (Table 1) consisted of various genera of Hyaloscyphaceae, including 7 species of Arachnopezizeae, 43 species of Hyaloscypheae, and 5 species of Lachneae (Nannfeldt 1932). Chlorencoelia torta (Hemiphacidiaceae) was selected as an outgroup taxon, in accordance with the results of a recent phylogenetic study (Wang et al. 2006). INTER-SET (Table 1) comprised representative genera of Hyaloscyphaceae and other helotialean taxa, and was used to test the monophyly of Hyaloscyphaceae and its phylogenetic placement within Helotiales. Thirty-seven species of Hyaloscyphaceae and 25 taxa of Bulgariaceae, Dermateaceae, Helotiaceae, Hemiphacidiaceae, Loramycetaceae, Phacidiaceae, Rutstroemiaceae, Sclerotiniaceae, Vibrisseaceae, and Incertae sedis in Helotiales were included in INTER-SET, with Spathularia flavida (Cudoniaceae, Rhytismatales) included as an outgroup taxon. DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted from cultured mycelia or dried apothecia using the methodology described by Lee & Taylor (1990). The concentration and purity of extracted nucleic acid were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). PCR was performed in 50 ml reactions that comprised 39 ml of sterile water, each primer at 0.4 mM, 1 unit of TaKaRa ExTaq DNA polymerase (TaKaRa, Tokyo, Japan), a dNTP mixture consisting of all four nucleotides with each at a concentration of 2.5 mM, 10 ExTaq buffer containing 20 mM Mg2þ, and approximately 100 ng of template DNA. Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer region of rDNA, including the 5.8S gene (ITS), LR0R and LR5 (Vilgalys & Hester 1990; Moncalvo et al. 1995) for the D1eD2 region of 28S rDNA (LSU), fRPB2-5F and fRPB2-7cR or RPB2-P7R pairs (Liu et al. 1999; Hansen et al. 2005) for the partial RNA polymerase II subunit 2 (RPB2), and mrSSU1 and mrSSU3R (Zoller et al. 1999) for the partial mitochondrial small subunit (mtSSU). The cycling parameters for ITS and LSU comprised an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 2 min, with a final extension at 72 C for 10 min. The conditions used for the RPB2 region were an initial denaturation at 95 C for 2 min, followed by seven cycles of 95 C for 30 s, 56 C for 30 s, and 72 C for 1.5 min, in which the annealing temperature was successively decreased by 1 C per cycle. The remaining 33 cycles were performed with the annealing temperature fixed at 50 C, and were followed by a final extension at 72 C for 10 min. PCR amplification for the mtSSU region was performed with an initial denaturation at 94 C for 3 min, followed by 35 cycles of 94 C for 1 min, 50 C for 1 min, and 72 C for 1 min, and a final extension at 72 C for 7 min. After confirming the amplicons on an agarose/TBE electrophoretic gel (Certified Molecular Biology Agarose, Bio-Rad Laboratories, Spain), the PCR products were purified using a LaboPass PCR kit (COSMO Genetech, Korea) following the manufacturer’s protocols. Sequencing was performed on an automatic sequencer (ABI Prism 377 DNA Sequencer) using the BigDye Cycle Sequencing Kit (version 3.1, Applied 152 J.-G. Han et al. Fig 1 e Morphological and ecological diversity of Hyaloscyphaeae. (A): Brunnipila fuscescens, (B): Urceolella carestiana, (C): Lachnum alnifolium, (D): Hyaloscypha sp., (E): Amicodisca castaneae, (F): Arachnopeziza aurelia, (G): Trichopeziza sulphurea, (H): Arachnopeziza aurata, (I): Urceolella carestiana on rachis of ferns, (J): Trichopeziza sulphurea on herbaceous stems, (K): Calycina herbarum on dead stems, (L): Lachnum alnifolium on fallen leaves of Alnus hirsuta. (M): Microscypha ellisii on previous year’s leaves of Carex sp., (N): Lachnum rachidicola on a rachis of Juglans mandshurica, (O): Arachnopeziza aurata on damp rotting wood, (P): Dasyscyphella nivea on dead twigs, (Q): Lachnum abnorme on fallen branches, (R): Amicodisca castaneae on a chestnut bur, (S): Cistella sp. on a corncob, (T): Hamatocanthoscypha sp. on dead stems of Equisetum hyemale. Species name Amicodisca castaneae J.G. Han, Hosoya & H.D. Shin Amicodisca castaneae J.G. Han, Hosoya & H.D. Shin Arachnopeziza aurata Fuckel Arachnopeziza aurata Fuckel Arachnopeziza aurelia (Pers.) Fuckel Arachnopeziza aurelia (Pers.) Fuckel Specimen (or strain)# KUS-F51377 KUS-F51917 KUS-F52038 NBRC102331/ TNS-F-11212 KUS-F51520 Arachnopeziza delicatula Fuckel Arachnopeziza obtusipila Grelet NBRC102330/ TNS-F-11211 TNS-F-12770 TNS-F-12768 Arachnopeziza obtusipila Grelet TNS-F-12769 Ascocoryne cylichnium (Tul.) Korf Ascocoryne sarcoides (Jacq.) J.W. Groves & D.E. Wilson Brunnipila fuscescens (Pers.) Baral KUS-F52351 e Bulgaria inquinans (Pers.) Fr. CBS 315.71/ KACC45620 CBS 247.62/ KACC45615 KUS-F51458 KUS-F52362 KUS-F52256 KUS-F52240 KUS-F52447 € hn. Calycellina populina (Fuckel) Ho Calycina herbarum (Pers.) Gray Calycina herbarum (Pers.) Gray Chlorencoelia torta (Schwein.) J.R. Dixon Ciboria americana E.J. Durand Ciboria shiraiana (Henn.) Whetzel Cistella albidolutea (Feltgen) Baral Cistella sp. Cudoniella clavus (Alb. & Schwein.) Dennis Cyathicula microspora Velen. ek Dematioscypha dematiicola (Berk. & Broome) Svrc Dermea cerasi (Pers.) Fr. Dicephalospora rufocornea (Berk. & Broome) Spooner Fabrella tsugae (Farl.) Kirschst. ek Hamatocanthoscypha laricionis (Velen.) Svrc ek Hamatocanthoscypha laricionis (Velen.) Svrc KUS-F52678 KUS-F52527 AFTOL-ID 166 M267 NBRC108583/ TNS-F-17834 KUS-F50981 KUS-F52274 e NBRC108586/ TNS-F-13530 NBRC108596/ TNS-F-24336 HMAS71954 GenBank accession# Data-set ITS LSU RPB2 mtSSU Intra-set Inter-set Chestnut burr/Wonju, Korea/16 IX 2006 Chestnut burr/Goesan, Korea/5 X 2007 Twigs/Hongcheon, Korea/28 IV 2008 Broad leaf tree log/Kyoto, Japan/27 IV 2003 JN033389 JN033411 JN033393 JN033436 JN086692 JN086714 JN086696 AB546936 JN086843 JN086860 JN086847 JN086881 JN086766 JN086786 JN086770 JN086806 C C C C e C C e Fallen leaves, branches, and fruits of Quercus acutissima/Cheongju, Korea/11 IV 2007 Wood/Mito, Ibaraki, Japan/19 IV 2003 JN033409 JN086712 e JN086785 C e JN033435 AB546937 JN086880 JN086805 C C Wood/Iwamizawa, Hokkaido, Japan/25 IX 2004 Branches of Quercus crispula/Aomori, Japan/24 V 2006 Log, Betula ermanii/Hakkoda, Aomori, Japan/25 V 2006 Wood/Hoengseong, Korea/23 IX 2008 (From GenBank) JN033433 JN033445 JN086736 JN086746 JN086877 JN086890 JN086802 JN086815 C C e C JN033446 JN086747 JN086891 JN086816 C e JN033406 AY789388 JN086709 AJ406399 e e JN086782 e e e C C Leaves of Quercus sp./Yangpyeong, Korea/27 IV 2008 Quercus robur/Bern, Switzerland/15 II 1970 JN033392 JN086695 JN086846 JN086769 C C JN033386 JN086689 JN086841 e e C Lyon, France JN033382 JN086685 JN086837 JN086762 C C Stems of Boehmeria sp./Jinju, Korea/28 X 2006 Herbaceous stems/Wonju, Korea/24 IX 2008 Wood/Wonju, Korea/15 VIII 2008 Chestnut bur/Chuncheon, Korea/5 VIII 2008 Buried fruits of Morus bombycis/Wonju, Korea/29 IV 2009 Sheath of Carex sp./Muju, Korea/19 V 2010 corncob/Muju, Korea/1 VII 2009 (From GenBank) Frøslev, Sweden (from GenBank) Wood/Sugadaira, Nagano, Japan/17 IX 2005 JN033390 JN033407 JN033400 JN033399 JN033430 JN086693 JN086710 JN086703 JN086702 JN086733 JN086844 e JN086854 JN086853 JN086873 JN086767 JN086783 JN086776 e e C C C e e C e C C C JN033429 JN033419 DQ491502 EU940165 JN033438 JN086732 JN086722 DQ470944 EU940088 JN086739 JN086872 JN086864 e e JN086883 JN086798 e e EU940240 JN086808 C C e e C C C C C C Bark of Prunus sp./Taean, Korea/31 XII 2005 Branches/Gongju, Korea/21 VIII 2008 JN033387 JN033401 JN086690 JN086704 e JN086855 e JN086777 e e C C (From GenBank) Needles of Cryptomeria japonica/Yakushima, Kagoshima, Japan/22 X 2005 Coniferous wood/Odawara, Kanagawa, Japan/11 II 2009 China (from GenBank) U92304 JN033441 AF356694 JN086742 e JN086886 e JN086811 e C C C JN033455 JN086755 JN086905 JN086830 C e AY789297 AY789296 e e e C (continued on next page) 153 Heyderia abietis (Fr.) Link KUS-F52031 Substrate/collection site/collection date Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses Table 1 e Information of sequence data used in the present study. 154 Table 1 e (continued ) Species name Hyalopeziza leuconica Raitv. Hyalopeziza nectrioides (Rehm) Raschle Hyalopeziza pygmaea (Mouton) Huhtinen Hyalopeziza pygmaea (Mouton) Huhtinen KUS-F52474 CBS 597.77/ KACC45613 KUS-F51564 Substrate/collection site/collection date GenBank accession# Data-set ITS LSU RPB2 mtSSU Intra-set Inter-set Wood/Inje, Korea/12 V 2009 Alnus viridis/Aletschwald, Switzerland/19 IX 1973 JN033416 JN033381 JN086719 JN086684 e JN086836 e JN086761 C C C C Leaves of Quercus sp./Pyeongchang, Korea/10 V 2007 Leaves/Towada, Aomori, Japan/25 V 2006 JN033410 JN086713 e e C e JN033448 JN086748 JN086894 JN086819 C C Wood/Takeda, Oita, Japan/9 X 2005 JN033439 JN086740 JN086884 JN086809 C e Twig/Ueda, Nagano, Japan/28 IV 2006 JN033442 JN086743 JN086887 JN086812 C C Bamboo/Towada, Aomori, Japan/26 V 2006 JN033449 JN086749 JN086895 JN086820 C e Wood/Shimoda, Shizuoka, Japan/26 VII 2004 JN033431 JN086734 JN086874 JN086799 C e Wood/Ryugasaki, Ibaraki, Japan/6 VII 2002 JN033437 JN086738 JN086882 JN086807 C e Daigo, Ibaraki, Japan JN033456 JN086756 JN086906 JN086832 C e albohyalina var. spiralis (Velen.) KUS-F52652 Wood/Yangpyeong, Korea/28 X 2009 JN033426 JN086729 JN086870 JN086795 C C albohyalina var. spiralis (Velen.) NBRC108585/ TNS-F-17909 KUS-F52070 TNS-F-11209 Wood/Kikuchi, Kumamoto, Japan/10 X 2005 JN033440 JN086741 JN086885 JN086810 C e Wood/Cheolwon, Korea/16 V 2008 Wood of Cryptomeria japonica/Kiryu, Gunma, Japan/22 III 2002 Scotland, UK (from GenBank) Wood/Inje, Korea/23 VII 2009 JN033394 AB546942 JN086697 AB546943 JN086848 JN086879 JN086771 JN086804 C C C e EU940228 JN033423 EU940152 JN086726 e JN086867 EU940292 JN086793 C C e e NBRC108593/ TNS-F-18073 M233, TUR172135 M171 Wood/Kandatsu, Niigata, Japan/18 VI 2006 JN033451 N086751 JN086897 JN086822 C e Scotland, UK/25 VIII 2005 (from GenBank) Finland (from GenBank) EU940230 EU940194 EU940154 EU940118 e e EU940294 EU940266 C C e C M339 Finland (from GenBank) EU940226 EU940150 e EU940290 C e NBRC106631/ TNS-F-17333 NBRC108580/ TNS-F-17335 NBRC108581/ TNS-F-17350 NBRC108592/ TNS-F-17694 Wood/Tomakomai, Hokkaido, Japan/23 IX 2004 AB546939 AB546938 JN086875 JN086800 C e Wood/Tomakomai, Hokkaido, Japan/23 IX 2004 JN033432 JN086735 JN086876 JN086801 C e Wood/Iwamizawa, Hokkaido, Japan/25 IX 2004 JN033434 JN086737 JN086878 JN086803 C e Wood/Yuzawa, Niigata, Japan/18 VI 2006 JN033450 JN086750 JN086896 JN086821 C e Hyalopeziza sp. Hyalopeziza sp. albohyalina var. albohyalina (P. Karst.) albohyalina var. albohyalina (P. Karst.) aureliella (Nyl.) Huhtinen aureliella (Nyl.) Huhtinen Hyaloscypha aureliella (Nyl.) Huhtinen Hyaloscypha leuconica var. bulbopilosa (Feltgen) Huhtinen Hyaloscypha leuconica var. bulbopilosa (Feltgen) Huhtinen Hyaloscypha fuckelii Nannf. Hyaloscypha hepaticicola (Grelet & Croz.) Baral, Huhtinen & J.R. De Sloover Hyaloscypha hepaticicola (Grelet & Croz.) Baral, Huhtinen & J.R. De Sloover Hyaloscypha sp. Hyaloscypha sp. Hyaloscypha sp. Hyaloscypha sp. M234, TUR172136 KUS-F52573 J.-G. Han et al. albohyalina var. monodictys Hosoya & NBRC108590/ TNS-F-17940 NBRC108584/ TNS-F-17879 NBRC108587/ TNS-F-17975 NBRC108591/ TNS-F-18048 NBRC108579/ TNS-F-17137 NBRC108582/ TNS-F-11213 TNS-F-5013 Hyalopeziza sp. Hyaloscypha Boud. Hyaloscypha Boud. Hyaloscypha Huhtinen Hyaloscypha Huhtinen Hyaloscypha Huhtinen Hyaloscypha Hyaloscypha Specimen (or strain)# Hyaloscypha vitreola (P. Karst.) Boud. Hyaloscypha vitreola (P. Karst.) Boud. Hymenoscyphus caudatus (P. Karst.) Dennis NBRC108595/ TNS-F-31287 CBS 127.91/ KACC45484 M39, TUR164914 KUS-F52291 Hymenoscyphus fructigenus (Bull.) Gray Hyphodiscus hyaloscyphoides Hosoya, J.G. Han & G.H. Sung Hyphodiscus hymeniophilus (P. Karst.) Baral M159, TUR164966 TNS-F-13588 Hyphodiscus hymeniophilus (P. Karst.) Baral TNS-F-31802 Hyphodiscus otanii Hosoya TNS-F-7099 Hyphodiscus sp. Hyphodiscus theiodeus (Cooke & Ellis) W.Y. Zhuang KUS-F52558 TNS-F-31803 Lachnum abnorme (Mont.) J.H. Haines & Dumont Lachnum virgineum (Batsch) P. Karst. Lanzia huangshanica W.Y. Zhuang KUS-F52080 TNS-F-16583 KUS-F52405 Loramyces macrosporus Ingold & B. Chapm. CBS 235.53/ KACC45617 KUS-F52489 Microscypha ellisii Dennis Microscypha ellisii Dennis Microscypha sp. Microscypha sp. TNS-F-31801 Pezicula carpinea (Pers.) Tul. ex Fuckel KUS-F52663 KUS-F52625 NBRC108589/ TNS-F-18016 KUS-F52303 KUS-F52533 KUS-F52181 KUS-F52307 TNS-F-38901 CBS 478.97/ KACC45619 CBS 100302/ KACC45226 KUS-F51029 Phacidium coniferarum (G.G. Hahn) DiCosmo, Nag Raj & W.B. Kendr. Phialina lachnobrachyoides (Raitv.) Huhtinen CBS 322.53/ KACC45618 KUS-F52183 Phialina lachnobrachyoides (Raitv.) Huhtinen KUS-F52576 Microscypha sp. Microscypha sp. Mollisia ventosa P. Karst. Mollisina uncinata Arendh. & R. Sharma Mollisina uncinata Arendh. & R. Sharma Neobulgaria pura (Pers.) Petr. ek Olla millepunctata (Lib.) Svrc Wood/Nishi-Murayama, Yamagata, Japan/30 VI 2007 Sorbus aucuparia/Kaarina, Finland/20 X 1986 JN033454 JN086754 JN086900 JN086825 C e JN033378 JN086681 JN086834 JN086758 C e Finland (from GenBank) Leaves of Zelkova serrata/Seosan, Korea/27 VIII 2008 Finland (from GenBank) Betula ermanii wood/Hakkoda, Aomori, Japan/25 V 2006 Decaying coniferous wood/Wada, Nagano, Japan/ 13 II 1992 Unidentified wood/Iizuna, Nagano, Japan/5 X 1992 Unidentified wood/Senboku, Akita, Japan/16 V 1995 Twigs/Hongcheon, Korea/16 VII 2009 Unidentified decaying wood/Towadako, Aomori, Japan/13 VI 2000 Wood/Daejeon, Korea/24 V 2008 Wood/Yamakita, Kanagawa, Japan/2 VII 2005 Petioles of Castanopsis cuspidata var. sieboldii/ Seogwipo, Korea/5 XI 2008 Equisetum limosum/Cumbria, UK EU940231 JN033402 EU940155 JN086705 e JN086856 EU940295 JN086778 C e C C EU940233 AB546944 EU940157 AB546945 e JN086892 EU940297 JN086817 e C C e AB546948 AB546946 JN086901 JN086826 C e AB546951 AB546950 JN086904 JN086829 C C AB546949 AB546947 JN086902 JN086827 C e JN033421 AB546953 JN086724 AB546952 JN086866 JN086903 JN086791 JN086828 C C e C JN033395 AB481268 JN033408 JN086698 AB481306 JN086711 JN086849 AB481343 e JN086772 JN086831 JN086784 C C e C C C JN033383 JN086686 JN086838 JN086763 e C Sheath of Carex cf. shimidzensis/Yangpyeong, Korea/3 VI 2009 Sheath of Carex sp./Hongcheon, Korea/29 IV 2010 Fern/Jeju, Korea/2 IX 2009 Fern/Hinuki, Iwate, Japan/23 V 2006 JN033418 JN086721 JN086863 JN086789 C e JN033428 JN033425 JN033444 JN086731 JN086728 JN086745 e JN086869 JN086889 JN086797 JN086794 JN086814 C C C e e e Fern/Guri, Korea/3 IX 2008 Fern/Guri, Korea/11 VII 2009 Wood/Yangpyeong, Korea/5 VII 2008 leaves of Quercus sp./Guri, Korea/3 IX 2009 Quercus leaves/Furano, Hokkaido, Japan Log with moss/New York, USA/28 IX 1996 JN033403 JN033420 JN033397 JN033404 JN033457 JN033385 JN086706 JN086723 JN086700 JN086707 JN086757 JN086688 JN086857 JN086865 JN086851 JN086858 JN086907 JN086840 JN086779 JN086790 JN086774 JN086780 JN086833 e C C e C C e C e C C e C Epifagus virginianus/New York, USA/23 IX 1997 JN033380 JN086683 JN086835 JN086760 C C Barks of Carpinus laxiflora/Seogwipo, Korea/8 V 2006 Picea abies, dead bark/Sunnfjord, Norway JN033388 JN086691 JN086842 JN086765 e C JN033384 JN086687 JN086839 JN086764 e C Leaves of Betula platyphylla var. japonica/ Pyeongchang, Korea/17 VII 2008 Leaves, Tilia sp./Inje, Korea/23 VII 2009 JN033412 JN086715 JN086861 e C e JN033424 JN086727 JN086868 e C C 155 (continued on next page) Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses Hyaloscypha sp. 156 Table 1 e (continued ) Species name Polydesmia pruinosa (Gerd. ex Berk. & Broome) Boud. Proliferodiscus sp. Proliferodiscus sp. Psilachnum sp. Specimen (or strain)# GenBank accession# Data-set ITS LSU RPB2 mtSSU Intra-set Inter-set TNS-F-12764 Pyrenomycetes/Ueda, Nagano, Japan/25 IX 2006 JN033453 JN086753 JN086899 JN086824 C C KACC45526/KUSF52660 NBRC108594/ TNS-F-17436 KUS-F52448 Twigs/Hongcheon, Korea/29 IV 2010 JN033427 JN086730 JN086871 JN086796 C C Wood/Riku-mura, Gunma, Japan/3 VII 2006 JN033452 JN086752 JN086898 JN086823 C e Leaves of Philadelphus schrenckii/Wonju, Korea/29 IV 2009 Leaves of Staphylea bumalda/Hongcheon, Korea/3 VI 2008 Leaves of Magnolia sieboldii/Wonju, Korea/16 IV 2009 Sheath of Carex sp./Danyang, Korea/28 IV 2009 (From GenBank) (From GenBank) Soil/Pyeongchang, Korea/21 IX 2009 Herbaceous stem/Pocheon, Korea/29 VII 2008 Wood/Hoengseong, Korea/20 V 2009 Rachis of Thelypteris nipponica/Hinuki, Iwate, Japan/23 V 2006 Geranium sylvaticum/Hautes Alpes, France/25 VII 1975 Cercidiphyllum japonicum leaf/Towadako, Aomori, Japan/20 V 2006 Leaves of Acer pseudosieboldianum/Yangpyeong, Korea/27 IV 2008 Rachis of Juglans mandshurica/Hongcheon, Korea/ 16 VII 2009 Log in a stream/North Carolina, USA (from GenBank)/18 VI 1993 JN033415 JN086718 e JN086788 C e JN033396 JN086699 JN086850 JN086773 C e JN033413 JN086716 e e e C JN033414 AY789408 AF455526 JN033405 JN033398 JN033417 JN033443 JN086717 AY789407 AY789347 JN086708 JN086701 JN086720 JN086744 JN086862 e e JN086859 JN086852 e JN086888 JN086787 e e JN086781 JN086775 e JN086813 C e e e C C C C C C C C C e JN033379 JN086682 e JN086759 C C JN033447 AB546954 JN086893 JN086818 C C JN033391 JN086694 JN086845 JN086768 C e JN033422 JN086725 e JN086792 e C AY789403 AY789402 e e e C Psilachnum staphyleae J.G. Han, M.J. Park & H.D. Shin Pyrenopeziza sp. KUS-F52105 Rodwayella citrinula (P. Karst.) Spooner Scleromitrula shiraiana (Henn.) S. Imai Sclerotinia sclerotiorum (Lib.) de Bary Spathularia flavida Pers. Trichopeziza sulphurea (Pers.) Fuckel Trichopezizella sp. Urceolella carestiana (Rabenh.) Dennis Venturiocistella japonica Hosoya, Y. Harada & Y. Otani Venturiocistella sp. KUS-F52443 Hirayama062001 FH-WZ0067 KUS-F52331 KUS-F52218 KUS-F52478 NBRC108588/ TNS-F-18014 CBS 608.77/ KACC45486 NBRC106633/ TNS-F-18030 KUS-F52028 nchez Vibrissea filisporia (Bonord.) Korf & A. Sa KUS-F52561 Vibrissea truncorum (Alb. & Schwein.) Fr. CUP-62562 Urceolella crispula (P. Karst.) Boud. Substrate/collection site/collection date KUS-F52417 A dash ( ) indicates no data for this field. Abbreviations for Herbaria and culture collections are as follows: CBS e Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; HMAS e Herbarium of Mycology, Academia Sinica, Beijing, China; KACC e Korean Agricultural Culture Collection, Suwon, Korea; KUS e Korea University Herbarium, Seoul, Korea; NBRC e NITE Biological Resource Center, Kisarazu, Japan; TNS e National Museum of Nature and Science, Tsukuba, Japan; TUR e Herbarium, University of Turku, Finland. J.-G. Han et al. Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses 157 Table 2 e Primers used in this study. Sequences (50 /30 ) Direction Purpose Reference GAY GAY MGW GAT CAY TTY GG CCC ATR GCT TGY TTR CCC AT TCC GTA GGT GAA CCT GCG G TCC TCC GCT TAT TGA TAT GC ACC CGC TGA ACT TAA GC TCC TGA GGG AAA CTT CG AGC AGT GAG GAA TAT TGG TC ATG TGG CAC GTC TAT AGC CC CCC ATS GCY TGY TTA CCC AT TCY TCY TCY TCI GCR TC TGY CCI GCI GAR ACI CCH GAR GG Forward Reverse Forward Reverse Forward Reverse Forward Reverse Reverse Reverse Forward PCR PCR PCR, sequencing PCR, sequencing PCR, sequencing PCR, sequencing PCR, sequencing PCR, sequencing PCR Sequencing Sequencing Liu et al. 1999 Liu et al. 1999 White et al. 1990 White et al. 1990 Moncalvo et al. 1995 Vilgalys & Hester 1990 Zoller et al. 1999 Zoller et al. 1999 Hansen et al. 2005 Reeb et al. 2004 Reeb et al. 2004 Primer name fRPB2-5F fRPB2-7cR ITS1 ITS4 LR0R LR5 mrSSU1 mrSSU3R RPB2-P7R RPB2 1554R RPB2 980F Follow the international nomenclature for degenerate positions: R ¼ G or A, K ¼ G or T, S ¼ G or C, W ¼ A or T, M ¼ A or C, Y ¼ T or C, B ¼ G, T, or C, H ¼ A, T, or C, and N ¼ G, A, T, or C. Biosystems, Foster City, CA, USA) with the same primers as those used for the amplifications. For the RPB2 region, RPB2 980F and RPB2 1554R (Reeb et al. 2004) were also used as sequencing primers. All of the primers used in the present study are listed in Table 2. The ITS rDNA and partial LSU rDNA sequences were 444e948 and 526e1294 bp in length, respectively. The partial RPB2 and partial mtSSU sequences were 720e1214 and 666e1202 bp in length, respectively. In total, 233 new sequences were generated for this study, while 55 sequences were retrieved from GenBank (Table 1). Sequence alignment After assembling contigs using SeqMan Pro version 7.1.0 (DNASTAR, Lasergene, Madison, WI, USA), the sequence data of each gene partition were aligned separately using MAFFT version 6 (Katoh et al. 2002). The ITS-5.8S rDNA, the D1eD2 region of rDNA, and the mitochondrial SSU regions were aligned using the QeINSei algorithm (Katoh & Toh 2008), which considers the secondary structural information of RNA. The RPB2 gene coding region was aligned using the GeINSei algorithm (Katoh et al. 2005), which is an iterative refinement method that uses global pairwise alignment information. Ambiguously aligned positions were identified with the aid of Gblocks version 0.91b (Castresana 2000) using default parameter settings, and manually adjusted using BioEdit version 7.0.5.2 (Hall 1999) whenever necessary. Phylogenetic analyses To test for phylogenetic conflict among gene partitions, separate RAxML analyses for each gene partition were performed (Wiens 1998) as described below. Individual gene trees were reciprocally compared for conflict among nodes with a criterion of >70 % bootstrap value. Maximum parsimony (MP) analysis was carried out using PAUP* version 4.0b10 (Swofford 2002). An MP heuristic search was performed with 5000 replications, each with 500 random sequence additions, branch swapping by tree bisection-reconnection (TBR), and Maxtree set at 20 000. Gaps were treated as missing data, and all nucleotide substitutions were equally weighted and unordered. The consistency index (CI) and retention index (RI) were calculated for all parsimonious trees (Kluge & Farris 1969; Farris 1989). The relative robustness of the individual branches was Table 3 e Summary of sequence data matrix used in MP analyses. Gene partition INTRASET INTERSET ITS-5.8S D1eD2 LSU RPB2 mtSSU Combined ITS-5.8S D1eD2 LSU RPB2 mtSSU Combined Included taxa 77 77 64 71 77 60 60 41 43 60 Total length Ambiguously Variable Informative of alignment aligned positions sites sites 574 551 752 1537 3414 653 788 1131 1596 4168 94 7 45 879 1025 173 251 412 965 1801 320 174 436 720 1650 389 278 648 832 2147 Combined data constituted by ITS rDNA, D1eD2 region of LSU rDNA, RPB2, and mtSSU. a CI: consistency index. b RI: retention index. c MAXTREES set to 5000 in heuristic search. 263 117 378 548 1306 291 186 559 467 1503 Tree Number length of MP trees 1314 587 3531 818 6439 1653 855 3459 790 7037 238 357 5000c 5000c 6 288 418 5000c 5000c 1 CIa RIb 0.3128 0.3101 0.1994 0.3912 0.2511 0.2680 0.2772 0.2102 0.3506 0.2393 0.6991 0.7158 0.5677 0.7425 0.6262 0.4775 0.5318 0.3599 0.5119 0.4022 158 J.-G. Han et al. Fig 2 e Phylogenetic tree inferred from maximum-likelihood (ML) of four genes combined sequence data (INTRA-SET) of the complete ITS region (ITS1, 5.8S rDNA, and ITS2), D1eD2 region of large subunit (LSU) of rDNA, RPB2 protein-coding gene, and mitochondrial small subunit (mtSSU). Bootstrap values greater than 50 % from the ML and MP analyses, and posterior probabilities greater than 90 % generated from the Bayesian analysis are indicated above the branches in the order of ML BS, MP BS, and Bayesian PP. Thickened branches mean ML BS ‡80 %. Sequence data obtained from the GenBank database were marked with an asterisk (*). The number of nucleotide changes among taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. The general appearance of apothecial hair was depicted at the right of corresponding clade. Figured taxa (from top to bottom) are Lachnum virgineum, Urceolella carestiana, Cistella sp., Arachnopeziza aurata, Dematioscypha dematiicola, Hyaloscypha albohyalina, Hyalopeziza leuconica, Venturiocistella japonica, Phialina lachnobrachyoides, Calycellina sp., and Microscypha sp. Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses 159 Fig 3 e Phylogenetic tree inferred from maximum-likelihood (ML) of four genes combined sequence data (INTER-SET) of the complete ITS region (ITS1, 5.8S rDNA, and ITS2), D1eD2 region of large subunit (LSU) rDNA, RPB2 protein-coding gene, and mitochondrial small subunit (mtSSU). Bootstrap values greater than 50 % from the ML and MP analyses, and posterior probabilities greater than 90 % generated from the Bayesian analysis are indicated above the branches in the order of ML BS, MP BS, and Bayesian PP. Thickened branches mean ML BS ‡80 %. Sequence data obtained from the GenBank were marked with an asterisk (*). The number of nucleotide changes among taxa is represented by branch length and the scale bar equals the number of nucleotide substitutions per site. estimated by bootstrapping (BS) using 10 000 replicates with 10 heuristic searches using the same parameters as in the MP analysis. Maximum-likelihood (ML) inference was conducted using RAxML version 7.2.6 with the evolutionary best-fit model of nucleotide substitution selected based on the result of jModelTest version 0.1.1 (Stamatakis 2006; Posada 2008). The general time-reversible model with gamma-distributed substitution rates and a separate proportion of invariant sites 160 for each gene (GTR þ I þ G) was chosen for all of the partitions, except for the mtSSU gene partition of INTRA-SET and the ITS5.8S rDNA gene partition of INTER-SET, for which the GTR model without invariance option (GTR þ G) was selected. Bayesian inference was implemented in MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003). To test the convergence of the log-likelihood in Bayesian analyses, four incrementally heated simultaneous Markov chains were run for one million generations, sampling a tree every 100th generation, of which the first 2000 trees were identified as part of the burn-in and removed from subsequent analyses. MrBayes was used to compute a 50 % majority-rule consensus of the remaining trees to obtain estimates for the posterior probabilities (PP). Branch lengths were calculated as mean values over the sampled trees. This analysis was repeated four times in order to test the reproducibility of the results, starting with random trees and default parameter values. We regarded the threshold for the presence of statistically significant nodal support to be 70 % BS in MP and ML analyses and a Bayesian PP of 0.9. Trees were visualized by the TreeView program version 1.6.6 (Page 1996) or CLC sequence viewer 6.4 (CLC bio, Cambridge, MA, USA). Results Combinability Separate phylogenetic analyses of ITS rDNA, D1-D2 LSU rDNA, RPB2, and mtSSU indicated that branches supported by BS >70 % were not in conflict among the trees inferred from each gene partition (Supplementary Fig 1). The gene partitions were therefore deemed combinable and so were concatenated into a combined alignment. Characteristics of gene partitions Seventy-six sequences of 55 species in 24 genera of Hyaloscyphaceae were incorporated in INTRA-SET. The combined data set included 3414 nucleotide positions, of which 1764 were invariable and 1306 were parsimony-informative characters. The final analyses excluded the following 1025 ambiguously aligned nucleotide sites: 19e86 and 134e159 of ITS-5.8S rDNA; 575e581 of D1eD2 28S rDNA; 1126e1170 of RPB2; and 1878e2,202, 2235e2,365, 2404e2,429, 2769e2,809, 2962e3,133, and 3231e3414 of mtSSU. INTER-SET comprised 34 species of Hyaloscyphaceae and 25 additional species of Helotiales. The total length of the concatenated alignment was 4168 bp, of which 2021 bp were invariable and 1503 bp were parsimonyinformative. The final analyses excluded the following 1801 ambiguously aligned sites: 1e59, 115e179, 442e451, and 615e653 of ITS-5.8S rDNA; 654e681 and 1219e1441 of D1eD2 28S rDNA; 1442e1787 and 2507e2572 of RPB2; and 2573e3,084, 3472e3,664, and 3816e4075 of mtSSU. At least two gene characters were complete in the combined alignment for all of the analysed taxa. The final alignments were deposited in TreeBASE (http://www.treebase.org), where they are available under submission accession ID 12034. Table 3 provides summarized information about the combined and separate sequence matrix from the two data sets. J.-G. Han et al. Multigene phylogeny based on INTRA-SET INTRA-SET (Table 1), which was constructed with 76 sequences from 55 species in 24 genera, was designed to investigate the phylogenetic relationships among hyaloscyphaceous genera. Based on the concatenated sequence data of ITS rDNA, D1eD2 28S rDNA, RPB2, and mtSSU, ML analysis yielded an optimal tree with a best log-likelihood of 43 620.78. Six equally most-parsimonious trees of 6439 steps were produced, with a CI of 0.2511 and an RI of 0.6262. Bayesian analysis generated a 50 % majority-rule consensus tree with a log-likelihood (harmonic mean) of 43 640.44. Since the overall topologies inferred by MP, ML, and Bayesian inferences were congruent, the RAxML tree is shown in Fig 2 along with the ML BS, MP BS, and Bayesian PP values presented above the corresponding branches. The phylogenetic tree inferred from INTRA-SET (Fig 2) identified ten strongly supported terminal clades, which are described as follows. Clade 1 included the genera Microscypha ek (ML BS ¼ 100 %, Syd. & P. Syd. and Hamatocanthoscypha Svrc MP BS ¼ 81 %, and PP ¼ 1.00). Clades 2 and 3 were strongly supported (ML BS ¼ 100 %, MP BS ¼ 100 %, and 1.00 PP); the former comprised species of Calycina Nees ex Gray and Calycellina € hn., while the latter contained species of Mollisina Ho € hn. ex Weese and Phialina Ho € hn. Clades 1e3 formed a larger Ho monophyletic group with strong nodal support (ML BS ¼ 99 %, MP BS ¼ 70 %, and PP ¼ 1.00). Clade 4 included Hyphodiscus Kirschst., Venturiocistella Raitv., and Hyalopeziza pygmaea (ML BS ¼ 94 %, MP BS ¼ 91 %, and PP ¼ 1.00), while species of Hyalopeziza Fuckel and Olla Velen. were included in Clade 5 (ML BS ¼ 100 %, MP BS ¼ 100 %, and PP ¼ 1.00). Clade 6 contained Hyaloscypha spp. (ML BS ¼ 100 %, MP BS ¼ 81 %, and PP ¼ 1.00) with Hyaloscypha albohyalina var. albohyalina placed as the earliest diverging lineage. Clade 7 included Amicodisca ek with Dematioscypha Svrc ek (ML BS ¼ 100 %, MP Svrc BS ¼ 100 %, and PP ¼ 1.00). Clades 5e7 formed a monophyletic group with strong nodal support (ML BS ¼ 81 %, MP BS ¼ 75 %, and PP ¼ 1.00). Species of Arachnopeziza Fuckel were included in Clade 8 (ML BS ¼ 100 % BS, MP BS ¼ 100 %, and 1.00 PP). Clade l., Psilachnum Ho € hn., Rod9 contained species of Cistella Que wayella Spooner, and Urceolella Boud with strong ML and Bayesian support (ML BS ¼ 93 % and PP ¼ 1.00). Clade 10 included taxa in the Lachneae tribe, with Brunnipila Baral, Lachnum Retz., and Proliferodiscus J.H. Haines & Dumont forming a strongly supported subclade (Subclade 10A; ML BS ¼ 100 %, MP BS ¼ 98 %, and PP ¼ 1.00), while Trichopeziza Fuckel and Trichopezizella Dennis ex Raitv. formed another subclade (Subclade 10B) with strong support by ML BS (98 %) and Bayesian PP (1.00), and moderate support by MP BS (69 %). Multigene phylogeny based on INTER-SET INTER-SET (Table 1, Fig 3) included representative species of Hyaloscyphaceae and various helotialean taxa in order to understand the phylogenetic positions of the genera of Hyaloscyphaceae within Helotiales. We included 34 taxa representing Hyaloscyphaceae, and 25 helotialean species representing Bulgariaceae (Bulgaria Fr.), Dermateaceae (Dermea Fr., Mollisia (Fr.) P. Karst., Pezicula Tul. & C. Tul., and Pyrenopeziza Fuckel), Helotiaceae (Cudoniella Sacc., Cyathicula De Not., Hymenoscyphus Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses Gray, and Neobulgaria Petr.), Hemiphacidiaceae (Chlorencoelia J.R. Dixon, Fabrella Kirschst., and Heyderia Link), Loramycetaceae (Loramyces W. Weston), Phacidiaceae (Phacidium Fr.), Rutstroemiaceae (Dicephalospora Spooner and Lanzia Sacc.), Sclerotiniaceae (Ciboria Fuckel, Scleromitrula S. Imai, and Sclerotinia Fuckel), Vibrisseaceae (Vibrissea Fr.), and Incertae sedis (Ascocoryne J.W. Groves & D.E. Wilson). The ML tree (loglikelihood ¼ 53 450.73) was inferred from the four-gene combined data set (Fig 3). MP analyses resulted in a mostparsimonious tree of 7037 steps with a CI of 0.2393 and an RI of 0.4022, and Bayesian analyses resulted in a 50 % majorityrule consensus tree with a log-likelihood (harmonic mean) of 53 444.61. The nodal support in the ML tree is indicated by the ML BS, MP BS, and Bayesian PP values presented above the corresponding nodes. Ten terminal clades delineated in INTRA-SET were also resolved in the INTER-SET tree, although the overall nodal support was weaker than that of the INTRASET tree. In the INTER-SET tree, the monophyly of Hyaloscyphaceae was not supported and the included hyaloscyphaceous genera were intermixed among other helotialean taxa. Clades 1e3 were resolved as monophyletic clades with strong support (ML BS ¼ 100 %, MP BS ¼ 100 %, and PP ¼ 1.00). The close relationship among Clades 1e3 was supported by ML BS (80 %) and PP (1.00). Hyphodiscus and Venturiocistella spp. as well as Hyalopeziza pygmaea were grouped in Clade 4 with strong support (ML BS ¼ 100 %, MP BS ¼ 93 %, and PP ¼ 1.00). Clade 5, comprising Hyalopeziza and Olla spp., was strongly supported by ML BS (¼100 %) and PP (¼1.00), and moderately supported by MP BS (¼62 %). Clade 6 (ML BS ¼ 97 %, MP BS ¼ 94 %, and PP ¼ 1.00) included all sampled Hyaloscypha spp. Amicodisca and Dematioscypha spp. were included in Clade 7 with a strong support (ML BS ¼ 100 %, MP BS ¼ 100 %, and 1.00 PP). Clade 8 represents a monophyletic Arachnopeziza (ML BS ¼ 100 %, MP BS ¼ 100 %, and PP ¼ 1.00). Species in Clade 9 were not resolved in the INTER-SET tree; Cistella, Psilachnum, Rodwayella, and Urceolella spp. were included in separate clades in the MP tree (data not shown). Species of Lachneae formed a robust clade (Clade 10) containing two strongly supported subclades (ML BS ¼ 99 % and PP ¼ 1.00). Species of Helotiales except for Phacidiaceae and Bulgariaceae were included in a single clade with strong support (ML BS ¼ 98 %, MP BS ¼ 80 %, and PP ¼ 1.00). Species of Hemiphacidiaceae, Rutstroemiaceae, Sclerotiniaceae, and Vibrisseaceae formed monophyletic clades, with ML BS ¼ 86e100 %, MP BS ¼ 65e100 %, and PP ¼ 0.97e1.00. Species of Dermateaceae were separated into Dermea-Pezicula and Mollisia-Pyrenopeziza clades. There was strong support for close relationships between Hemiphacidiaceae and Sclerotiniaceae (ML BS ¼ 100 %, MP BS ¼ 85 %, and PP ¼ 1.00) and between Helotiaceae and Rutstroemiaceae (ML BS ¼ 98 %, MP BS ¼ 75 %, and PP ¼ 1.00). A clade containing Vibrisseaceae, Loramycetaceae, Mollisia, and Pyrenopeziza was strongly supported (ML BS ¼ 99 % and PP ¼ 1.00). Taxonomy In addition to the type variety Hyaloscypha albohyalina var. albohyalina, two more varieties were incorporated in the present study: H. albohyalina var. spiralis (exhibiting simple ascal bases 161 and producing the Pseudaegerita anamorph) and H. albohyalina var. monodictys (exhibiting characteristic resin on hairs and the production of the Monodictys anamorph). In the resulting tree (Fig 2, Clade 6), H. albohyalina var. albohyalina was resolved as the earliest diverging lineage of the genus. The remaining two varieties were nested within the core clade of Hyaloscypha and H. albohyalina var. monodictys showed a sister relationship to all core members of the genus. The phylogenetic separations of H. albohyalina with the morphological differences indicate that these two varieties should be raised to the specieslevel. We therefore propose new names as follows: Hyaloscypha spiralis (Velen.) J.G. Han, Hosoya, H.D. Shin, comb. nov. MycoBank no.: MB803062. Basionym: Chrysothallus spiralis Velen., Monogr. Discom. Bohem. (Prague): 269 (1934) (Lectotype e Czechoslovakia, Bohemia, kov, Mnichovice, Hubac Corylus avellana, 29.XII.1928 (PRM 150477)). Velenovsky Synonym: Hyaloscypha albohyalina var. spiralis (Velen.) Huhtinen, Karstenia 29: 99 (1989). Hyaloscypha monodictys (Hosoya & Huhtinen) J.G. Han, Hosoya, H.D. Shin, comb. nov. MycoBank no.: MB803063. Basionym: Hyaloscypha albohyalina var. monodictys Hosoya & Huhtinen, Mycoscience 43: 405 (2002) (Holotype e Japan, Honshu Ibaraki Prefecture, Kuji-gun, Daigo-machi, Fukuroda waterfall, on coniferous wood, 29.X.1994 (TUR 157227)). Hyalopeziza pygmaea has been considered an unusual Hyalopeziza species due to its unique feature such as rough protrusions on glassy-walled hairs. Furthermore, the paraphyses are gradually transformed into a hair-like appearance, with clavate shapes as well as glassy and granulate walls. In the examination of Huhtinen (1987), hair warts became detached, deformed, or dissolved in lactic acid, cotton-blue, and congo-red mountants in some specimens of H. pygmaea. As shown in the phylogenies (Figs 2 and 3), H. pygmaea was not grouped with Hyalopeziza spp. (Clade 5), suggesting that this taxon has been misclassified (Huhtinen 1987; Hosoya & Otani 1997). Although H. pygmaea was nested within Hyphodiscus spp. (Clade 4), its peculiar characteristics are not consistent with the morphological circumscription of that genus. Therefore, we propose a new monotypic genus for H. pygmaea based on its distinct morphological characteristics and molecular phylogenetic evidence. Hyphopeziza J.G. Han, Hosoya & H.D. Shin, gen. nov. MycoBank no.: MB803064. Etym.: The name refers to the features of the genus intermediate between those of Hyalopeziza and Hyphodiscus. Type species (designated here): Trichopeziza pygmaea Mouton Bull. Soc. R. Bot. Belg. 36(C.R.): 19 (1897). Apothecia superficial, gregarious, broadly sessile; receptacle cupulate at first, becoming discoid, externally covered with white to grayish hairs; disc 0.5e1 mm in diameter, flat to slightly convex, somewhat pruinose, gray when fresh becoming white when dry; ectal excipulum composed of thinwalled globose to isodiametric cells, hyaline to pale brown, cell walls thicken toward the base; hairs cylindric-conical to lageniform, entirely coarsely warty, hyaline, aseptate, occasionally 1-septate near the base, with glassy wall that is Non-glassy Multi-celled Granulate/ smooth retained even after potassium hydroxide (KOH) pretreatment; asci arising from croziers, clavate to cylindric-clavate, hyaline, apex conical, apical pore blued in Melzer’s reagent; ascospores biseriate, elliptic-clavate to cuneiform or ellipsoid, hyaline, aseptate, aguttulate, smooth; paraphyses cylindric, hyaline, apex clavate or lanceolate, commonly aseptate, less frequently 1-septate near the base, forked or not, apical parts turning coarsely warty and glassy like hairs. Non-glassy Hyphopeziza pygmaea (Mouton) J.G. Han, Hosoya, H.D. Shin, comb. nov. MycoBank no.: MB803065. Basionym: Trichopeziza pygmaea Mouton Bull. Soc. R. Bot. Belg. 36: 19 (1897) (Holotype e Belgium, Beaufays, on leaves of Carpinus, Mouton 473 (BR)). Synonyms: Hyalopeziza pygmaea (Mouton) Huhtinen, Mycotaxon 29: 279 (1987). Hyaloscypha pygmaea (Mouton) Boud., Hist. Class. Discom. Eur. (Paris): 127 (1907). ek, Cesk Mykol. 39: 217 Unguicularia pygmaea (Mouton) Svrc a (1985). Hyaloscypha subtilis var. drupacea Velen., Monogr. Discom. Bohem. (Prague): 273 (1934). Discussion Is Hyaloscyphaceae sensu lato monophyletic? Non-glassy a Following the infrafamilial classification suggested by Nannfeldt (1932). b The glassiness of hairs is retained after KOH treatment. c The glassiness of hairs is dissolved after KOH treatment. Non-glassy Few-celled Smooth Non-glassy Non-glassy/glassy Glassy (KOHstableb) (KOH-stableb) Non-glassy Non-glassy Few-celled Smooth/(partly) granulate Non-glassy/glassy (KOH-solublec) Few-celled Granulate Few-celled Smooth Few-celled Granulate Few-celled Smooth Lageniform Hyaline Cylindric Hyaline Cylindric Hyaline Hairs Cylindric Hyaline Cylindric With yellowish inclusions Few-celled Smooth Cylindric/conical With yellowish inclusions Few-celled Smooth Few-celled Granulate Absence/presence (false-subiculum) Cylindric Hyaline/brownish Subiculum Absence Absence Absence Absence Absence Absence Arachnopezizeae Arachnopezizeae/ Hyaloscypheae/Lachneae Absence Presence (true- Absence/presence (false-subiculum) subiculum) Cylindric/ conical Cylindric Cylindric Hyaline/ brownish Hyaline Hyaline Hyaloscypheae Hyaloscypheae Hyaloscypheae Hyaloscypheae Tribea Hyaloscypheae Hyaloscypheae Hyaloscypheae Arachnopeziza Amicodisca Dematioscypha Hyaloscypha Hyalopeziza Olla Hyalopeziza Hyphodiscus Hyphopeziza Venturiocistella Mollisina Phialina Hamatocantho- Calycellina Calycina scypha Microscypha Included genera 3 2 1 Clade# Table 4 e Conserved morphological traits in each clade. 4 5 6 7 8 Cistella Psilachnum Rodwayella Urceolella 9 Belonidium Lachnum Proliferodiscus Trichopeziza Trichopezizella Lachneae J.-G. Han et al. 10 162 In the classification system of Hyaloscyphaceae, the presence of hairs has been considered as one of the most important characters for the generic level of delimitation. The number of genera in Hyaloscyphaceae has drastically increased from 13 in 1932 (Nannfeldt 1932) to 74 in 2008 (Kirk et al. 2008). It is currently the largest family within the order Helotiales (Kirk et al. 2008); however, its monophyly has frequently been questioned because of its extremely diverse morphologies, especially in terms of the apothecial hairs (Raitviir & Spooner 1994; Sutton & Hennebert 1994). INTRA-SET was designed to infer strongly supported clades of hyaloscyphaceous taxa with taxon sampling based on its current classification (Wang et al. 2006). However, the resulting tree indicated that the backbone nodes were not strongly supported (Fig 2). In the trees based on INTER-SET, Bulgariaceae and Phacidiaceae were resolved as the earliest diverging lineages, and species of Hyaloscyphaceae were intermixed with other helotialean taxa (Fig 3). The monophyly of Hyaloscyphaceae was not strongly supported, which contrasts with the results of previous studies (Cantrell & Hanlin 1997; Abeln et al. 2000). In the present study, we employed intensive taxon and gene sampling to obtain reliable phylogenetic results for the natural classification of Hyaloscyphaceae. Below we discuss the phylogenetic relationships of clades with nodal support in Hyaloscyphaceae, and reinterpret the traditional classification of this family to provide a fundamental basis for its taxonomic revision in the future. Arachnopezizeae A distinct character of Arachnopezizeae is the presence of a subiculum around the apothecia, and this tribe is thus considered Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses to be a discrete group in Hyaloscyphaceae (Raitviir 1970, 1987). To better understand the phylogenetic relationship of Arachnopezizeae, we included the following four genera in the present analyses: Arachnopeziza, Polydesmia, Proliferodiscus, and Rodwayella. The resulting trees (Figs 2 and 3) demonstrated that the tribe Arachnopezizeae is polyphyletic, but its phylogenetic position within the family could not be determined since most of the basal nodes were not supported statistically. Korf (1978) elevated the Nannfeldt’s tribe Arachnopezizeae (1932) to the subfamilial rank as Arachnopezizoideae containing two tribes: Arachnopezizeae and Polydesmieae. According to the circumscription of Korf (1978), Arachnopezizeae comprises Arachnopeziza and Tapesina Lambotte (¼ Velutaria Fuckel), and Polydesmieae includes Eriopezia (Sacc.) Rehm, Parachnopeziza Korf, Polydesmia Korf, and Proliferodiscus. While Arachnopezizeae is characterized by a mycelial pad on which the apothecia are seated (‘true-subiculum’), members of the Polydesmieae possess a pad that serves as anchoring hyphae to attach the apothecia onto the substrate (‘false-subiculum’). Clade 8 included Arachnopeziza spp. in the inferred phylogenies with strong nodal support (Figs 2 and 3). Although the monotypic genus Tapesina was not included in the present study, the present phylogenetic analyses inferred that tribe Arachnopezizeae sensu Korf is monophyletic, indicating that the true-subiculum is hypothesized to have a single evolutionary origin. In contrast, species of Polydesmieae were placed in multiple clades in our analyses; Rodwayella was resolved as an early diverging member of Clade 9 and Proliferodiscus were grouped with lachnoid species in Clade 10 with strong support. The false-subiculum is inferred as having evolved convergently. The genus Rodwayella is characterized by its excipular structure composed of parallel or interwoven hyphae, ascospores constricted at the septa, and apothecia developed on a false-subiculum (Spooner & Dennis 1985). Although Rodwayella spp. do not exhibit well-differentiated hairs, they have been considered members of Hyaloscyphaceae due to the similarity of their ascal structures (Spooner 1987). On the other hand, the phylogenetic placement of Proliferodiscus spp. in Clade 10 is consistent with the previous findings that Proliferodiscus belongs to Lachnoideae instead of Arachnopezizoideae (Cantrell & Hanlin 1997). Polydesmia spp. produce small apothecia with pruinose hymenium developed on subiculum-like hyphae. Polydesmia pruinosa was not grouped with any major clades labelled in our analyses (Figs 2 and 3). This is consistent with the long-standing debate about the taxonomic placement of Polydesmia because it is characterized by its large ascospores and paraphyses that are repeatedly dichotomously branched near the apices (¼ propoloid paraphyses), which are atypical in Hyaloscyphaceae (Dennis 1960, 1968; Korf 1973, 1978). Lachneae The tribe Lachneae consists of Lachnum and allied genera and is characterized by its comparatively large apothecia with conspicuous hairs and lanceolate paraphyses. Although earlier molecular studies indicated that Lachneae is monophyletic (Cantrell & Hanlin 1997; Abeln et al. 2000), Hosoya et al. (2010) demonstrated that Lasiobelonium, Trichopeziza, and 163 Trichopezizella do not group with the core Lachneae clade, which is characterized by granulated hairs. In contrast, smooth-walled hairs are characteristic of members that were not placed in the core clade. Hosoya et al. (2010) proposed that Lachnaceae be restricted to genera with granulate hairs. In the present analyses, Lachneae formed a monophyletic lineage in Clade 10 (Figs 2 and 3). This clade was subdivided into two subclades (i.e., Subclades 10A and 10B): Subclade 10A included species with granulate hairs, whereas Subclade 10B included those with smooth-walled hairs. Our results are consistent with both the original familial concept of Raitviir (2004) and the reduced concept of Hosoya et al. (2010), since both Clade 10 (corresponding to Lachneae sensu Raitviir) and Subclade 10A (corresponding to Lachneae sensu Hosoya) are monophyletic. However, it is noticeable that the monophyly of Subclade 10A was always strongly supported, while that of Clade 10 was not statistically significant (MP BS ¼ 50 %) in the INTER-SET tree. The genus Lachnum includes approximately 250 species and is one of the most diverse genera within Hyaloscyphaceae (Kirk et al. 2008). Lachnum has been regarded as a heterogeneous group by numerous researchers (e.g. Baral & Krieglsteiner 1985; Haines 1989; Raitviir 1970, 1987; Spooner 1987). For example, Haines & Dumont (1984) emphasized the ascospore morphology in a taxonomic revision of Lachnum. However, it is difficult to delimit the genus based only on the size of the ascospore (short vs. long ascospores). The present analyses incorporated Lachnum virgineum and Lachnum abnorme, which produce ellipsoid spores and long-cylindric spores, respectively. These two species did not form a monophyletic clade in the inferred trees (Figs 2 and 3), suggesting that this genus is not monophyletic. However, further analyses with more intensive taxon sampling are necessary to achieve an accurate taxonomic revision. Hyaloscypheae Hyaloscypheae includes genera with tiny apothecia, mostly cylindric paraphyses, and hairs with highly diverse shapes. The monophyly of the tribe was not supported in the inferred phylogenies (Figs 2 and 3), with taxa distributed across eight clades (Clades 1e7 and 9). Below we present our phylogenetic results for terminal clades to allow reinterpretation of their morphology within the phylogenetic framework. Hyaloscypha clade (Clade 6 in Figs 2 and 3) Hyaloscypha is characterized by small apothecia, prismatic ectal excipulum, lageniform to conical hairs, and filiform paraphyses. All of the previously reported anamorphs of Hyaloscypha have a holoblastic morphology (Huhtinen 1989; Hosoya & Huhtinen 2002). Hyaloscypha is a monophyletic group in our phylogenies (Clade 6, Figs 2 and 3). Although Hyaloscypha subgenus Eupezizella was proposed to include lignicolous Hyaloscypha spp. with blunt hairs normally encrusted with abundant resinous exudates and lacking dextrinoid reactions (Huhtinen 1989), this subgeneric division is not supported by our results. Among the taxa examined in this study, Hyaloscypha aureliella can be classified in Eupezizella, which is placed as an early diverging member of the core Hyaloscypha clade (Clade 6, Fig 2), indicating that the subgeneric 164 division is not supported. Clade 6 formed a sister relationship with Clade 7 that includes Amicodisca and Dematioscypha. Species of Hyaloscypha, Amicodisca, and Dematioscypha exhibit morphologically similar conical to cylindric hairs. Hyalopeziza clade (Clade 5 in Figs 2 and 3) Hyalopeziza is a representative genus of the glassy-haired Hyaloscyphaceae family. The glassy-haired species of Hyaloscyphaceae have been recognized by their abnormal external appearance and histochemical alteration of hairs upon exposure to KOH, as the glassiness of these species can be either retained or dissolved after the treatment (Raschle 1977; Korf & Kohn 1980). Hyalopeziza possesses cylindric hairs externally encrusted with glassy walls. Urceolella is morphologically similar, but does not retain the hair glassiness after the treatment of KOH. In our phylogenies (Figs 2 and 3), Hyalopeziza was placed in Clade 5 while Urceolella was placed within Clade 9. Although Mollisina uncinata and Hyphopeziza pygmaea exhibit glassywalled hairs, they were nested within Clades 3 and 4, respectively. These results indicate that the glassy-haired Hyaloscyphaceae is not a monophyletic group. Furthermore, taxa (i.e., M. uncinata and Urceolella spp.) whose hair glassiness is lost after KOH treatment do not form a monophyletic group (Figs 2 and 3), indicating that KOH-soluble hair glassiness is not an important criterion at the subfamilial level of Hyaloscyphaceae. In addition to Hyalopeziza, Clade 5 includes Olla millepunctata Velen., which is a type species of the genus. Olla is distinguished from Hyalopeziza by its hair apices that are broadly rounded and turn reddish in Melzer’s and Lugol’s reagents (Raitviir 2004; Huhtinen et al. 2008). We suggest that Olla should be synonymized with Hyalopeziza due to the strong support found for Clade 5. J.-G. Han et al. The hairs of Calycellina spp. also exhibit yellowish inclusions. However, Calycellina populina CBS 247.62 was placed in the strongly supported Calycina-Calycellina clade (Clade 2) in the present analysis (Figs 2 and 3). In addition to Calycellina, Clade 2 also included members of Calycina, and small apothecia with hyphoid hairs and prismatic excipulum are synapomorphic characters for this clade. The familial placement of Calycina and Calycellina has been historically debated because their hairs are much shorter and indistinct compared with those of other € hnel 1918; Nannfeldt 1932; Seaver Hyaloscyphaceae genera (Ho 1934, 1951; White 1943; Dennis 1956; Dumont 1972; Lowen & Dumont 1984). Among the helotialean species (Fig 3), Clade 2 represented the closest phylogenetic relatives to the hyaloscyphaceous groups (Clades 1, 3, and 4) with significant ML BS and PP support, indicating that Calycina and Calycellina should be included in the current circumscription of Hyaloscyphaceae. Hyphodiscus clade (Clade 4 in Figs 2 and 3) The genus Hyphodiscus comprises fungicolous or lignicolous species having gelatinous ectal excipulum, short hairs with coarse warts, and small unicellular ascospores (Zhuang 1988; Hosoya 2002; Untereiner et al. 2006). Since the presence of a gelatinous matrix in the ectal excipulum is more common in Helotiaceae and a Phialophora-like anamorph (Catenulifera) has never been reported for other species of Hyaloscyphaceae, the taxonomic position of the genus has frequently been questioned (Hosoya 2002; Untereiner et al. 2006; Hosoya et al. 2011). Hyphodiscus spp. grouped with Hyphopeziza pygmaea and Venturiocistella spp. with strong nodal support (Clade 4, Fig 2), which is consistent with the shared morphological traits of few-celled hairs covered with coarse granules and a gelatinous excipulum. Cistella clade (Clade 9 in Figs 2 and 3) Microscypha-Hamatocanthoscypha, Calycina-Calycellina, and Mollisina-Phialina clades (Clades 1e3 in Figs 2 and 3) Microscypha is morphologically similar to Hamatocanthoscypha in exhibiting cylindric, few-celled, smooth-walled hairs with blunt apices. However, hairs are apically hooked or helicoid in Hamatocanthoscypha spp. Microscypha and Hamatocanthoscypha were grouped in Clade 1 with strong nodal support (Figs 2 and 3). The monophyly of Microscypha was strongly supported. Although Hamatocanthoscypha was strongly supported in the present study, further taxon sampling is necessary to confidently address the monophyly of this genus. In our results the Microscypha-Hamatocanthoscypha clade (Clade 1) formed a sister-group relationship with members of the Mollisina-Phialina and Calycina-Calycellina clades (Clades 1 and 2, respectively). In the Mollisina-Phialina clade, Mollisina and Phialina formed a monophyletic group that is labelled as Clade 3 in Figs 2 and 3. Both of these genera exhibit entirely smoothwalled hairs that taper upward, and they have yellowish inclusions in both the hairs and paraphyses. Mollisina is characterized by glassy-walled finger-like protrusions that extend from both hairs and excipular cells. Hyalopeziza digitipila Huhtinen and Urceolella equiseti (Huhtinen) Huhtinen also produce similar branched hairs (Huhtinen 1987). Although these two species were not examined in the present study, the phylogenetic placements of Hyalopeziza in Clade 5 and Urceolella in Clade 9 provide evidence for the hypothesis that the hairbranching characteristic does not have a single origin. Clade 9 included Cistella, Psilachnum, Rodwayella, and Urceolella spp. In the INTRA-SET tree this clade received significant statistical support except for MP BS (Fig 2), whereas in the INTER-SET tree it was supported only by PP (Fig 3). Consistent with this result, species in Clade 9 do not exhibit any obvious shared morphological characteristics. Cistella spp. have small apothecia with few-celled, apically granulate clavate hairs and cylindric paraphyses. A close relationship between Cistella and Lachnum has been suggested due to their morphological similarities (Raitviir 1970; Spooner 1987). However, our results indicated that Cistella spp. (Clade 9) are distantly related to lachnoid groups (Clade 10), as shown in Figs 2 and 3. Psilachnum is morphologically similar to Microscypha in having entirely smoothwalled cylindric hairs. Except for the nongranulate hairs, its microstructures are morphologically similar to those of Lachnum. However, the inferred tree (Fig 2) did not reflect this morphological similarity since Microscypha was assigned to Clade 1 and Lachnum was assigned to Clade 10. Hyaloscyphaceae sensu stricto and evaluation of taxonomic criteria used in the classification of Hyaloscyphaceae Based on the premise that Hyaloscyphaceae is monophyletic, most mycologists have considered the presence of hairs to be a shared ancestral characteristic of this family. The results of the present study did not support the monophyly of Hyaloscyphaceae. In addition, some species of Chlorociboria Seaver Phylogenetic reassessment of Hyaloscyphaceae based on multigene analyses € hn. (Rutstroemiaceae) in other (Helotiaceae) and Lambertella Ho families produce hair-like projections on the ectal excipulum (Korf & Zhuang 1985; Johnston & Park 2005). The outermost excipular cells are hypothesized to have originated independently during the evolutionary history, and the presence of hairs is not a synapomorphic trait of Hyaloscyphaceae. Therefore, it is necessary to reconsider the presence of hairs as being a critical taxonomic criterion delimiting this family. Hyaloscyphaceae is considered as a heterogeneous assemblage of genera (Raitviir & Spooner 1994; Sutton & Hennebert 1994), and it is necessary to define Hyaloscyphaceae sensu stricto within a phylogenetic framework. The core clade of Hyaloscyphaceae, which included the type genus Hyaloscypha, appeared as a monophyletic group in Clade 6 (Figs 2 and 3). This clade was closely related to Clades 5 and 7, with significant nodal support in the INTRA-SET analyses (Fig 2). However, Hyaloscypha spp. formed an independent lineage in the INTER-SET analyses, while the relationships to Clades 5 and 7 were not resolved (Fig 3). Therefore, Hyaloscyphaceae sensu stricto is restricted here to the genus Hyaloscypha, although future analyses are likely to identify other associated taxa. Hyaloscyphaceous species with common hair characteristics tended to group into the same clades in the inferred phylogenies (Fig 2). For example, Clades 1e3 grouped species with completely smooth-walled hairs. Species exhibiting yellowish inclusions in their hairs constituted Clades 2 and 3, while those without coloured inclusions were nested in a basal clade (Clade 1). Species in Clade 3 have cylindric to conical hairs with elongated apices. Clade 4 included species having few-celled short hairs covered with warts. Species with glassy-walled smooth hairs that are stable in KOH formed a monophyletic group (Clade 5). Clade 6 contained Hyaloscypha spp. exhibiting lageniform hairs. Species with long-cylindric multiseptate hairs (generally longer than 50 mm) were placed in Clade 10. These results indicate that the diverse morphologies of hairs are still useful taxonomic criteria at both the genera and species-levels of Hyaloscyphaceae sensu lato. The pattern of ascal development (from croziers or simple septa) is constant within the taxa, but its value as a taxonomic criterion remains unclear (Huhtinen 1989). Our data set included H. albohyalina var. albohyalina and H. albohyalina var. spiralis, which have been distinguished by their different ascal bases. In the present analyses (Figs 2 and 3) they were nested within Clade 6, but placed separately in the basal clade and the core clade of Hyaloscypha, respectively, suggesting that the ascal structure should be regarded as a diagnostic character for these taxa. The presence of the subiculum is the primary criterion that defines the tribe Arachnopezizeae. However, some authors have speculated that the subiculum is a feature that has arisen multiple times since it is also found in other groups of discomycetes, such as Pyronema Carus (Pyronemataceae, Pezizales) and Tapesia (Pers.) Fuckel (Dermateaceae, Helotiales) (Cantrell & Hanlin 1997). Our results support the homoplasious nature of this character because the species of Arachnopezizeae were not resolved as monophyletic (Figs 2 and 3; Clades 8e10). Among the included species, Arachnopeziza spp. that produce a true-subiculum formed a monophyletic group (Clade 8). It is therefore hypothesized that the truesubiculum has a single evolutionary origin, while the false- 165 subiculum has evolved independently for the functional purpose of anchoring the apothecia onto substrates. The morphological features of Hyaloscyphaceae that are consistent with clades are summarized in Table 4. Conclusions The presence of apothecial hairs has been used as the criterion to delimit Hyaloscyphaceae, and it is possible that this has resulted in the inadvertent inclusion of phylogenetically unrelated species with hair-like structures in this family. Our phylogenetic analyses of Hyaloscyphaceae inferred from multiple genes showed that this group is heterogeneous. Among the three tribes suggested by Nannfeldt (1932) (i.e., Arachnopezizeae, Hyaloscypheae, and Lachneae), only Lachneae formed a monophyletic lineage, while Arachnopezizeae and Hyaloscypheae were resolved as polyphyletic. These results support the recent decision to raise Lachneae to the familial rank (Raitviir 2004; Hosoya et al. 2010), and indicates that Arachnopezizeae and Hyaloscypheae should be taxonomically recircumscribed. Only the species of Arachnopezizeae with a true-subiculum formed a monophyletic clade, but those with a false-subiculum were not. The new monotypic genus Hyphopeziza is proposed based on its characteristics being intermediate between those of Hyalopeziza and Hyphodiscus. In addition, Hyaloscypha spiralis and Hyaloscypha monodictys are proposed as new combinations. The phylogenetic analyses performed in this study made it possible to evaluate the morphological characters used in the classification of Hyaloscyphaceae. The presence of hairs appears to be a homoplasious character, but the unique morphologies of hairs are often used in genus- and species-level classifications. While the presence of the subiculum was found to be homoplasious, the true-subiculum is inferred as having a single evolutionary origin. Since the monophyly of Hyaloscyphaceae sensu lato is not supported, this group should be recircumscribed by defining Hyaloscyphaceae sensu strict. The type genus Hyaloscypha formed a monophyletic lineage, but there is no hyaloscyphaceous genus exhibiting a close relationship with significant nodal support. Thus, we tentatively propose that Hyaloscyphaceae sensu stricto is restricted to a single genus, Hyaloscypha. Further phylogenetic analyses involving more taxa and genes are necessary to resolve the backbone of the trees in order to produce a better classification system of Hyaloscyphaceae sensu lato and to understand the evolutionary history of its morphology and ecology. Acknowledgement This work was supported by grants from the BioGreen 21 Program (no. 20080401034028) and the Next-Generation BioGreen 21 Program (no. PJ008154 and PJ009508), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2013.11.004. 166 references Abeln ECA, de Pagter MA, Verkley GJM, 2000. Phylogeny of Pezicula, Dermea and Neofabraea inferred from partial sequences of the nuclear ribosomal RNA gene cluster. Mycologia 92: 685e693. Baral HO, Krieglsteiner GJ, 1985. 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