A new attempt to classify the families of the Helotiales Hans-Otto Baral1, Danny Haelewaters2 & Kadri Pärtel3 – 1Blaihofstr. 42, 72074 Tübingen, Germany (e-mail: [email protected]); 2Farlow Herbarium, Harvard University, 22 Divinity Avenue, Cambridge, MA 02138, USA; 3Department of Botany, Institute of Ecology and Earth Sciences, University of Tartu, Chemicum, Ravila 14a, 50411 Tartu, Estonia INTRODUCTION – This study arose from a compilation of the non-lichenized discomycetes for the 13th edition of ‘A. Engler’s Syllabus of Plant due to taxon sampling errors and/or lack of more gene regions. We consider this presentation as a stimulus for further work, which Families’, in which some new families were established and forgotten ones resurrected. It includes recent, partly unpublished results and tries to combine should include more, also protein-coding, gene regions. The families are mostly well represented with high bootstrap support, and also morphological and molecular data. 24 families and some undescribed lineages are here proposed in the Helotiales sensu stricto (excluding Leotiales, Phacidiales, some family associations show high support. Some unexpected results are worth of mention: (1) Roseodiscus formosus does not cluster with and some other orders). Paraphyletic groups are accepted whenever obvious morphological support is noted. A pragmatic approach based mainly on the type R. rhodoleucus; (2) Crocicreas requires restriction to the type C. gramineum, while the large genus Cyathicula is unrelated to them; (3) morphology tries to arrange these 24 families in 4 main groups (A-D). Our phylogenetic analysis hardly represents these groups, although it shows high encoelioid taxa turned out to belong in various families, E. fuckelii in Chaetomellaceae, E. fascicularis in Sclerotiniaceae, E. tiliacea in bootstrap support for most of the proposed families. The lack of support at the basal nodes of the tree indicates limitations of our data, which might be Rutstroemiaceae, E. heteromera and E. fimbriata in Cordieritidaceae, and the type E. furfuracea in Cenangiaceae. A large number of helotialean genera are still unsequenced, and even some sequenced ones cannot presently be placed in any of the presently recognized families. ML phylogenetic analysis based on ITS+LSU rDNA Calloria_neglecta Calloriaceae 100 Psilachnum_sp._KUS-F52448 C 89 Psilachnum_staphyleae ?Psilachnum D Chaetomellaceae 64 Rommelaarsia_sp._HB9684 Mitrulaceae Roseodiscus_subcarneus_DH314a * Roseodiscus ? Ascocorticiaceae Rodwayella_citrinula_KUS-F52443 ”Rodwayella” ? 84 Roseodiscus_lapponicus Roseodiscus ? Roseodiscus_rhodoleucus_HB8488 100 Erysiphe_mayorii_MUMH_3952 INCERTAE SEDIS Erysiphe_quercicola_MUMH_3796 Erysiphales 100 Stamnaria_americana_HB7261 100 Stamnaria_laetissima_FC2732T39244 Stamnaria lineage ? Fam. Ascocorticiaceae J.Schröt. - 3 Stamnaria_sp._NVF3519 * 94 Heyderia_abietis * A genera (3): Ascocorticium (= Acrodontium, 98 Chlorencoelia_torta_KUS-F52256 Cenangiaceae 96 Lambertella_corni_CLX4075 A Laricina), Ascocorticiellum, Ascosorus 100 Rutstroemia_firma_CBS_341.62 Rutstroemiaceae 100 Pycnopeziza_sympodialis Ciboria_americana_F-115887 Fam. Ascodichaenaceae D. 69 Ciboria_amentacea_HB2760 A Chaetomella (Zoellneria) 73 Monilia_laxa_SK278 Sclerotiniaceae Sclerotinia_sclerotiorum_KR1121_1 Hawksw. & Sherwood - 2 genera (4): Ascocorticium 96 Skyttea_gregaria_NY0118113 inamyloid 100 Cordierites_frondosa_HKAS41508 Cordieritidaceae A Ascodichaena, Delpinoina Ionomidotis_fulvotingens Pilidium (Discohainesia) Crocricreas_gramineum_NVF3527 Crocicreas ? Mitrula 99 Calycellina_populina_CBS_247.62 Fam. Chaetomellaceae Baral, P.R. 96 Calycina_herbarum_KUS-F51458 Pezizellaceae D Bisporella_citrina_AFTOL-ID_1301 Johnst. & Rossman - 4 genera (c. 80): Roseodiscus 86 Rodwayella_sessilis_HB9913 Mollisina_uncinata_KUS-F52307 Chaetomella (= Zoellneria), Pilidium (= Ascodichaenaceae Stamnaria-lineage 76 Venturiocistella_japonica_TNS-F18030 Crocicreas 72 Cistella_spicicola Calycina Hyphodiscus_hymeniophilus_MUCL_40275 Hyaloscyphaceae D Discohainesia), Sphaerographium, Synchaetomella Calycina 100 Hamatocanthoscypha_laricionis_TNS_F13530 -type Cistella_acuum Fam. Mitrulaceae Rchb. - 1 genus (5): -type Hymenoscyphus_epiphyllus_HB9911 97 Hymenoscyphus_fructigenus_FC-2375 B Mitrula 95 Bispora_antennata (= Bisporella pallescens) Cudoniella_clavus_AFTOL-ID_166 Helotiaceae 100 Cyathicula_coronatum_F-176601 Stamnaria lineage - 1 genus (c. 7): Cyathicula_cyathideum_NVF3616 Cyathicula_starbaeckii_NVF3620 Stamnaria (= Titaeospora) 99 Trichopeziza_mollissima_FC-2285 Crocicreas 99 Trichopezizella_otanii_FC-2156 D Trichopeziza_sulphurea_KUS-F52218 Lachnaceae Ascodichaena 80 Graddonia_coracina_ILLS_60491 Stamnaria Hymenoscyphus_parasazavae_HB8184 * Roseodiscus rhodoleucus 92 67 Lambertella_tubulosa_3T9D B 100 Hymenoscyphus_cf._imberbis_HB8294 Pezoloma_iodopedis_CUP59415 Helotiaceae Hymenoscyphus_kathiae_HB8616 88 Spirosphaera_floriformis_CBS_402.52 Sclerotiniaceae VBs Hymenoscyphus_rubi-idaei_HB7200 * Sclerotinia-type Rutstroemiaceae Chlorovibrissea_sp._PDD70070 Chlorovibrissea ?C GROUP A - Lachnellula_occidentalis_CBS_160.35 Lachnum_rhytismatis D Sclerotinia-type 100 Lachnum_rachidicola Helotiales Sclerotiniaceous and Dasyscyphella_nivea_BHI-F144 80 Lachnum_soppittii_FC2160 Lachnaceae Lachnum_virgineum_AFTOL-ID_49 Proliferodiscus_inspersus_KUS-52660 encoelioid taxa Incrucipulum_ciliare_HB9922 Brunnipila_fuscescens_KUS-F52031 Neobulgaria_pura 64 98 Ascocoryne_cylichnium_PDD75671 B Calycina-type Gelatinodiscus_flavidus_OSC_65796 (Chloroscypha) Gelatinodiscaceae 98 Xerombrophila_crystallifera Ombrophila_rehnana_HB8634 100 Clathrosporium_intricatum_ICMP14831 Subgroup I - Ionomidotic Torrendiella Rutstroemia 87 Articulospora_atra_CCM_F-01384 ? Hydrocina_chaetocladia_CCM_F-10890 Hydrocina Pyrenopeziza_brassicae_IMI_217176 reaction always absent, VBs partly Heterosphaeria_patella Ploettnerulaceae C Heterosphaeriaceae C Mitrula_paludosa_MBH50636 Mitrulaceae ? present Godronia_cassandra Godroniaceae A Ciboria Heyderia 100 Bryoglossum_gracile_MBH52481 VBs Velutarina Bryoclaviculus_campylopi_PDD101074 ?D Fam. Rutstroemiaceae Holst- 76 Roseodiscus_formosus_DH267b Bryoglossum lineage Cenangiaceae 95 Hyaloscypha_aureliella_M235 - Chlorociboriaceae Rhizoscyphus_ericae_UAMH_6735 Hyaloscyphaceae D Jensen, L.M. Kohn & T. Schumacher Olla_millepunctata 100 Arachnopeziza_aurata_TNS-F11212 Arachnopezizaceae D 6 genera (c. 55): e.g. Lambertella, Lanzia, Arachnopeziza_aurelia_TNS-F11211 Calycina-type Pezoloma_sp._Japan_NBRC103659 Rutstroemia (= Poculum), Torrendiella 95 100 Pezoloma_ciliifera_M283 B 100 Pezoloma_ciliifera_HB7818 Pezoloma lineage Pezoloma_scanicum_SH_06/07 Fam. Sclerotiniaceae Whetzel - 28 Pezoloma_sp._Massachusetts_E17 genera (c. 150): e.g. Botrytis (= Botryotinia), 92 Vibrissea_truncorum_JLF2113 Vibrisseaceae C 68 Mollisia_cinerea_AFTOL-ID_76 Mollisiaceae C 100 Durella_connivens Strossmayeria lineage C Ciborinia, Moellerodiscus, Monilinia, Mycopappus (= Strossmayeria_basitricha_ANM_2055 Loramyces_macrosporus_CBS_235.53 Loramycetaceae C Redheadia), Myriosclerotinia, Piceomphale, 82 Pilidium_concavum_CCTU_1200 100 Chaetomella_oblonga_BPI_843552 Chaetomellaceae ? Scleromitrula, Sclerotinia, Stromatinia Encoelia_fuckelii_GM_2014-12-14 Chlorociboria 100 Chlorociboria_aeruginascens_BHI_F-128b Encoelia 66 Chlorociboria_aeruginosa_UBC_F19715 Chlorociboriaceae A Fam. Cenangiaceae Rehm (= Hysterostegiella Cenangium ferruginosum Medeolaria_farlowii Trochila 85 Dermea_acerina_CBS_161.38 Medeolariales 66 Pezicula_sporulosa_M42 Hemiphacidiaceae Korf) - 15 genera (c. 97 Neofabraea_malicorticis Dermateaceae A Cordieritidaceae Neofabraea_alba 70): e.g. Cenangiopsis, Cenangium, Chlorencoelia, 100 Rhytisma_acerinum Ionomidotic reaction Cudonia_circinans_C319 Rhytismatales Crumenulopsis, Encoelia, Heyderia, Hysterostegiella, 99 Potebniamyces_pyri_AFTOL-ID_744 Bulgaria_inquinans_ZW-Geo52 Phacidiaceae Rhabdocline, Sarcotrochila, Trochila, Velutarina 87 Holwaya_mucida_B_70_0009352 Tympanidaceae 100 Thelebolus_globosus_ANT03-221 Cleistothelebolus_nipigonensis_F.Doveri 100 Geltingia_associata_TU45655 Thelebolales Phacidiales Eleutheromyces_subulatus_CBS_139.90 Helicogoniaceae Gelatinomyces_siamensis_KKUK2 (Myriodiscus) Subgroup II - polyphyletic Claussenomyces_atrovirens_CLA23671 Tympanidaceae 100 Microglossum_olivaceum_FH-DSH97-103 Leotia_lubrica_AFTOL-ID_1 Leotiaceae group comprising ionomidotic taxa 75 Propolis_versicolor_ILLS60497 Marthamyces_desmoschoeni_PRJ_R908 Marthamycetaceae ?Rhytismalates Leotiales 91 Sarcoleotia_globosa_HMAS71956 without VBs 93 Geoglossum_nigritum Geoglossaceae Tuber_albidum_S2877_TUB Fam. Dermateaceae Fr. Ð 3 genera (c. KOH KOH Diplocarpa * = unpublished taxa 70): Dermea, Neofabraea, Pezicula “Encoelia“ fimbriata “Encoelia“ heteromera 0.2 Fam. Cordieritidaceae (Sacc.) Sacc. Ð 14 genera (c. 115): e.g. Ameghiniella, Cordierites, Godroniaceae Table of selected characters of families and lineages here recognized within the Helotiales. Diplocarpa (= Ionomidotis), Llimoniella, Calycina-type ionomidotic Dermateaceae Rhymbocarpus, Skyttea, Unguiculariopsis Fam. Godroniaceae Baral - 4 genera (c. Pezicula-type 45): Ascocalyx (= Bothrodiscus), Atropellis, Godronia Ploettnera-type (= Fuckelia), Gremmeniella (= Brunchorstia) ? Fam. Chlorociboriaceae Baral & P.R. Johnst. - 1 genus (c. 22): Chlorociboria (= Dothiorina) KOH Pezicula Godronia Dermea B. GROUP B – Helotiaceous taxa Fam. Gelatinodiscaceae S.E. Carp. Ð 8 genera (c. 55): e.g. Ascocoryne (= Coryne), Ascotremella, Chloroscypha (= Gelatinodiscus), Neobulgaria, Ombrophila, Xerombrophila

Sclerotiniaceous & encoelioid taxa Fam. Helotiaceae Rehm Ð c. 18 genera (c. 180): e.g. Bisporella (= Bispora), Bryoscyphus, Cudoniella, Cyathicula (= Glarea), Dicephalospora, Gloeotinia (= Endoconidium), Graddonia, Hymenoscyphus, Hymenotorrendiella, Phaeohelotium, Symphyosirinia (= Symphyosirella) Fam. Roesleriaceae Y.J. Yao & Spooner - 2 genera (c. 7): Roesleria, Roeslerina ? Discinella-Pezoloma lineage - 2–3 gen. (c. 15): Discinella (= Geocoryne), Pezoloma (= Articulospora)

Helotiaceous Helotiaceous taxa Pezicula-type Gelatinodiscaceae Discinella-Pezoloma-lineage ~Sclerotinia-type Sclerotinia-type Calycina-type Mollisiaceous taxa taxa Mollisiaceous Chloroscypha Ombrophila Neobulgaria Ascocoryne Calycina-type Discinella VBs Helotiaceae VBs Roesleria Hymenoscy- Hyaloscyph- Hyaloscyph- taxa aceous -ceae phus-type Development: Cor = corticioid; Cs = clavate-stipitate; Pr/Me = apothecioid: opening in the pro- (Pr) or mesohymenial (Me) phase; Cl = cleistothecial; Maz = mazaedium. Stipe: s = sessile; st = stipitate. Hymenoscyphus-type Erumpent: er = erumpent; su = superficial. - Stroma: dark stromatized host tissue present (+) or absent (–). Excipulum: g = globulosa, p = prismatica, i = intricata. Hairs: h = hairs, t = teeth. Crystals: octahedral crystals present (+) or absent (–). Ionomidotic: KOH releasing pigment into medium (ionomidotic reaction), present (+) or absent (–). Paraphysis shape, c = cylindrical to clavate; l = lanceolate. VBs: refractive vacuolar bodies in paraphyses/excipular cells present (+) or absent (–). Amyloidity: iodine reaction of apical ring (Lugol), B = euamyloid (blue), R = hemiamyloid (red, types RB/RR), – = inamyloid. Ring: apical ring type, S = Sclerotinia-type, C = Calycina-type, Pe = Pezicula-type, Pl = Ploettnera-type, H = Hymenoscyphus-type, V = Vibrissea-type. Croziers at the ascus base present (+) or absent (–). Sheath around Pezoloma ascospores, either gelatinous or separating after discharge (app = terminal gelatinous appendages, amyl = amyloid sheath). Lipid: relative lipid content of ascospores, 1 = low, 2 = medium, 3 = high. Asexual Cyathicula Hymenoscyphus state: h = hyphomycetous, pe = penicillate, s = synnematal/sporodochial, a = acervular, p = pycnidial. Conidiogenesis: h = holoblastic, p = phialidic, a = arthric. Lifestyle: s = ± saprobic, p = ± parasitic (or Roesleria mycorrhizal). Desiccation tolerance: xer = desiccation-tolerant (xeric substrate); hyg = desiccation-sensitice (hygric substrate). Hymenotorrendiella

COMMENTS ON SOME CHARACTERS Calloriaceae Ploettnera Drepanopezizaceae Ascus structure: Striking correlations between apical ring types and GROUP C - Mollisiaceous taxa -type Hysteropezizella-lineage molecular data disclosed various misplaced taxa. E.g., species Pezicula-type subgroup I - VBs usually absent previously placed in Torrendiella (Rutstroemiaceae, Sclerotinia-type) had Calycina-type Fam. Heterosphaeriaceae Rehm - 1 genus (7): Heterosphaeria (= Heteropatella) to be transferred to the unrelated new genus Hymenotorrendiella Hysteropezizella lineage - 5Ð7 genera (42): e.g. Cejpia, Coronellaria, Hysteronaevia, (Helotiaceae, Hymenoscyphus-type). Or Bisporella citrina and allies Hysteropezizella Calloria Ploettnera Pseudopeziza Spilopodia (Calycina-type) were transferred to Calycina (Pezizellaceae), whilst the Fam. Calloriaceae Marchand Ð c. 15 genera (c. 110): e.g. Calloria (= Cylindrocolla), type B. pallescens (teleomorph of Bispora antennata, Hymenoscyphus-type) Diplonaevia, Duebenia, Eupropolella, Laetinaevia, Naevala, Naeviopsis, Ploettnera Heterosphaeriaceae Calycina Pyrenopezizaceae clustered in Helotiaceae. Fam. Drepanopezizaceae Bat. & H. Maia Ð c. 10 genera (c. 52): e.g. Blumeriella -type Vacuoles: Presence vs. absence of refractive vacuoles (= VBs, living (= Phloeosporella), Diplocarpon (= Marssonina), Drepanopeziza (= Gloeosporidiella), Leptotrochila (= Coronellaria state!) correlates with phylogenetic lineages, permitting separation at Sporonema), Pseudopeziza, Spilopodia (= Melanodiscus) ?Hysteropezizella the family level: Fam. Ploettnerulaceae Kirschst. (= Pyrenopezizaceae Ruhland ex Velen.) - c. 7 genera (?125): e.g. Dennisiodiscus, Lasiomollisia, Oculimacula (= Helgardia), VBs present VBs absent Pirottaea (= Ploettnerula), Pyrenopeziza (= Cadophora) Heterosphaeria Lasiomollisia Pyrenopeziza Mollisiaceae Ploettnerulaceae Pirottaea Strossmayeria-lineage Pezizellaceae Hyaloscyphaceae Mollisiaceae VBs subgroup II - VBs present Cyathicula (Helotiaceae) Crocicreas (incertae sedis), KOH Vibrisseaceae Fam. Mollisiaceae Rehm - c. 11 genera (?190): e.g. Mollisia (= Belonopsis, Cenangiaceae (major part) Cordieritidaceae Cystodendron, Haglundia, Phaeomollisia, Phialocephala, Tapesia), Niptera (= Nimbomollisia), Loramycetaceae Ionomidotic reaction (IR): The extrusion of KOH-soluble yellow to red- Scutomollisia brown pigment shows rather high correlation with phylogenetic groups. It Fam. Loramycetaceae Dennis ex Digby & Goos - 2 genera (4): Loramyces, Calycina Vibrissea occurs mainly in Cordieritidaceae, Dermea (Dermateaceae), and Obtectodiscus -type nasse -type Strossmayeria Godroniaceae. IR was so far never observed in association with VBs (note Fam. Vibrisseaceae Locq. ex Korf - 3Ð4 genera (c. 35): Leucovibrissea, Pocillum, apicale Vibrissea (= Anavirga) that the yellow extrusion in Mollisia is a colour reaction of VBs, no IR). Strossmayeria lineage - 3 genera (?35): Durella, Gorgoniceps, Strossmayeria (= Obtectodiscus Pseudospiropes) hemiamyloid PHYLOGENETIC ANALYSIS spore appendages ITS and LSU rDNA sequences were generated or downloaded from Mollisia Vibrissea Durella GenBank. Sequences were aligned using MUSCLE 3.7 and further Lachnaceae hand-aligned in Se-Al v2.0a1. Ambiguously aligned regions were GROUP D - Hyaloscyphaceous taxa Hyaloscyphaceae Bryoglossum- removed from the dataset. Phylogenetic inferences were estimated lineage under maximum likelihood (ML) using RAxML 7.2.8 with a GTR+G Fam. Arachnopezizaceae Hosoya, J.G. Han & Baral - 5 genera (c. 30): Arachnopeziza, Arachnoscypha, Austropezia, Eriopezia, model of nucleotide substitution. Rapid bootstrapping was Parachnopeziza implemented with 1000 replicates. The best ML tree (-ln = Fam. Lachnaceae Raitv. - 16 gen. (c. 250): Albotricha, Belonidium, 32060.593754) and bootstrap values ≥ 60% are shown. Our ITS Brunnipila, Capitotricha, Dasyscyphella, Erioscyphella, Incrucipulum, Lachnellula, Cistella +LSU dataset includes 122 taxa; 50 sequences (ITS, LSU) were newly Lachnum, Lasiobelonium, Neodasyscypha, Perrotia, Proliferodiscus, Solenopezia, Trichopezizella Perrotia Capitotricha Amicodisca Urceolella Venturiocistella generated. Trichopeziza, Trichopezizella Fam. Hyaloscyphaceae Nannf. - 26 genera (c. 220): e.g. Pezizellaceae VBs FUTURE DIRECTIONS Amicodisca, Cistella, Dematioscypha (= Haplographium), Echinula, Fuscolachnum, Arachnopezizaceae Bryoglossum Bryoclaviculus Graddonidiscus, Hyalopeziza, Hyaloscypha (= Cheiromycella, Fuscoscypha, Sample more taxa and include more loci in the tree (SSU rDNA, Parorbiliopsis, Rhizoscyphus), Hyphodiscus (= Catenulifera), Incrupila, Olla, RPB2). Protounguicularia, Unguicularia, Unguiculella, Urceolella, Venturiocistella Calycina -type Fam. Pezizellaceae Velen. - 18 genera (c. 170): e.g. Allophylaria, ACKNOWLEDGEMENTS Antinoa, Calycellina (= Chaetochalara, Phialina), Calycina (= Chalara, Gemmina), Calycina Ciliolarina, Hamatocanthoscypha, Micropeziza (= Calloriella), Mollisina, Sarah Verhaeghen is thanked for technical assistance, and Guy -type Phaeoscypha, Psilachnum, Rodwayella (= Remleria), Velutaria (= Tapesina) Calycellina Marson for supplying unpublished sequences. Allophylaria ? Bryoglossum lineage - 2 genera (3): Bryoclaviculus, Bryoglossum Arachnopeziza Calycina Antinoa “Roseodiscus“ formosus