Microfungi associated with Clematis (Ranunculaceae) with an integrated approach to delimiting species boundaries

Abstract

The cosmopolitan plant genus Clematis contains many climbing species that can be found worldwide. The genus occurs in the wild and is grown commercially for horticulture. Microfungi on Clematis were collected from Belgium, China, Italy, Thailand and the UK. They are characterized by morphology and analyses of gene sequence data using an integrated species concept to validate identifications. The study revealed two new families, 12 new genera, 50 new species, 26 new host records with one dimorphic character report, and ten species are transferred to other genera. The new families revealed by multigene phylogeny are Longiostiolaceae and Pseudomassarinaceae in Pleosporales (Dothideomycetes). New genera are Anthodidymella (Didymellaceae), Anthosulcatispora and Parasulcatispora (Sulcatisporaceae), Fusiformispora (Amniculicolaceae), Longispora (Phaeosphaeriaceae), Neobyssosphaeria (Melanommataceae), Neoleptosporella (Chaetosphaeriales, genera incertae sedis), Neostictis (Stictidaceae), Pseudohelminthosporium (Neomassarinaceae), Pseudomassarina (Pseudomassarinaceae), Sclerenchymomyces (Leptosphaeriaceae) and Xenoplectosphaerella (Plectosphaerellaceae). The newly described species are Alloleptosphaeria clematidis, Anthodidymella ranunculacearum, Anthosulcatispora subglobosa, Aquadictyospora clematidis, Brunneofusispora clematidis, Chaetosphaeronema clematidicola, C. clematidis, Chromolaenicola clematidis, Diaporthe clematidina, Dictyocheirospora clematidis, Distoseptispora clematidis, Floricola clematidis, Fusiformispora clematidis, Hermatomyces clematidis, Leptospora clematidis, Longispora clematidis, Massariosphaeria clematidis, Melomastia clematidis, M. fulvicomae, Neobyssosphaeria clematidis, Neoleptosporella clematidis, Neoroussoella clematidis, N. fulvicomae, Neostictis nigricans, Neovaginatispora clematidis, Parasulcatispora clematidis, Parathyridaria clematidis, P. serratifoliae, P. virginianae, Periconia verrucose, Phomatospora uniseriata, Pleopunctum clematidis, Pseudocapulatispora clematidis, Pseudocoleophoma clematidis, Pseudohelminthosporium clematidis, Pseudolophiostoma chiangraiense, P. clematidis, Pseudomassarina clematidis, Ramusculicola clematidis, Sarocladium clematidis, Sclerenchymomyces clematidis, Sigarispora clematidicola, S. clematidis, S. montanae, Sordaria clematidis, Stemphylium clematidis, Wojnowiciella clematidis, Xenodidymella clematidis, Xenomassariosphaeria clematidis and Xenoplectosphaerella clematidis. The following fungi are recorded on Clematis species for the first time: Angustimassarina rosarum, Dendryphion europaeum, Dermatiopleospora mariae, Diaporthe ravennica, D. rudis, Dichotomopilus ramosissimum, Dictyocheirospora xishuangbannaensis, Didymosphaeria rubi-ulmifolii, Fitzroyomyces cyperacearum, Fusarium celtidicola, Leptospora thailandica, Memnoniella oblongispora, Neodidymelliopsis longicolla, Neoeutypella baoshanensis, Neoroussoella heveae, Nigrograna chromolaenae, N. obliqua, Pestalotiopsis verruculosa, Pseudoberkleasmium chiangmaiense, Pseudoophiobolus rosae, Pseudoroussoella chromolaenae, P. elaeicola, Ramusculicola thailandica, Stemphylium vesicarium and Torula chromolaenae. The new combinations are Anthodidymella clematidis (≡ Didymella clematidis), A. vitalbina (≡ Didymella vitalbina), Anthosulcatispora brunnea (≡ Neobambusicola brunnea), Fuscohypha kunmingensis (≡ Plectosphaerella kunmingensis), Magnibotryascoma rubriostiolata (≡ Teichospora rubriostiolata), Pararoussoella mangrovei (≡ Roussoella mangrovei), Pseudoneoconiothyrium euonymi (≡ Roussoella euonymi), Sclerenchymomyces jonesii (≡ Neoleptosphaeria jonesii), Stemphylium rosae (≡ Pleospora rosae), and S. rosae-caninae (≡ Pleospora rosae-caninae). The microfungi on Clematis is distributed in several classes of Ascomycota. The analyses are based on morphological examination of specimens, coupled with phylogenetic sequence data. To the best of our knowledge, the consolidated species concept approach is recommended in validating species.

Table of Contents

Phylum Ascomycota R.H. Whittaker

Subphylum Pezizomycotina Erikss. & K. Winka

Class Dothideomycetes Erikss. & K. Winka

Subclass Pleosporomycetidae Schoch et al.

Pleosporales Luttr. ex M.E. Barr

Amniculicolaceae Zhang et al.

  1. 1.

    Fusiformispora Phukhams. & K.D. Hyde, gen. nov.

  2. 2.

    Fusiformispora clematidis Phukhams., M.V. de Bult & K.D. Hyde, sp. nov.

Amorosiaceae Thambug. & K.D. Hyde

  1. 3.

    Angustimassarina rosarum Tibpromma, Camporesi & K.D. Hyde, new host record

Cyclothyriellaceae Jaklitsch & H. Voglmayr

  1. 4.

    Massariosphaeria clematidis Phukhams., Wanas., Camporesi & K.D. Hyde, sp. nov.

Dictyosporiaceae Boonmee & K.D. Hyde

  1. 5.

    Aquadictyospora clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

  2. 6.

    Dictyocheirospora clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

  3. 7.

    Dictyocheirospora xishuangbannaensis Tibpromma & K.D. Hyde, new host record

  4. 8.

    Pseudocoleophoma clematidis Phukhams. & K.D. Hyde, sp. nov.

Didymellaceae Gruyter, Aveskamp & Verkley

  1. 9.

    Anthodidymella Phukhams., Camporesi & K.D. Hyde, gen. nov.

  2. 10.

    Anthodidymella clematidis (Woudenb., Spiers & Gruyter) Phukhams. & K.D. Hyde, comb. nov.

  3. 11.

    Anthodidymella ranunculacearum Phukhams., Camporesi & K.D. Hyde, sp. nov.

  4. 12.

    Anthodidymella vitalbina (Petr.) Phukhams. & K.D. Hyde, comb. nov.

  5. 13.

    Neodidymelliopsis longicolla Hou, Crous & L. Cai, new host record

  6. 14.

    Xenodidymella clematidis Phukhams., Camporesi & K.D. Hyde, sp. nov.

Didymosphaeriaceae Munk

  1. 15.

    Chromolaenicola clematidis Phukhams. & K.D. Hyde, sp. nov.

  2. 16.

    Didymosphaeria rubi-ulmifolii Ariyaw., Camporesi & K.D. Hyde, new host record

Hermatomycetaceae Locq. ex A. Hashim. & K. Tanaka

  1. 17.

    Hermatomyces clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

Leptosphaeriaceae Barr

  1. 18.

    Alloleptosphaeria clematidis Phukhams. & K.D. Hyde, sp. nov.

  2. 19.

    Sclerenchymomyces Phukhams. & K.D. Hyde, gen. nov.

  3. 20.

    Sclerenchymomyces clematidis Phukhams. & K.D. Hyde, sp. nov.

  4. 21.

    Sclerenchymomyces jonesii (Wanasinghe, Camporesi & K.D. Hyde) Phukhams. & K.D. Hyde, comb. nov.

Longiostiolaceae Phukhams., Doilom & K.D. Hyde

  1. 22.

    Longiostiolaceae Phukhams., Doilom & K.D. Hyde, fam. nov.

Lophiostomataceae Luerss.

  1. 23.

    Neovaginatispora clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

  2. 24.

    Pseudocapulatispora clematidis Phukhams. & K.D. Hyde, sp. nov.

  3. 25.

    Pseudolophiostoma chiangraiense Phukhams. & K.D. Hyde, sp. nov.

  4. 26.

    Pseudolophiostoma clematidis Phukhams. & K.D. Hyde, sp. nov.

  5. 27.

    Sigarispora clematidis Phukhams. & K.D. Hyde, sp. nov.

  6. 28.

    Sigarispora clematidicola Phukhams., Camporesi & K.D. Hyde, sp. nov.

  7. 29.

    Sigarispora montanae Phukhams., Sue & K.D. Hyde, sp. nov.

Melanommataceae Winter

  1. 30.

    Neobyssosphaeria Wanas., Jones & K.D. Hyde, gen. nov.

  2. 31.

    Neobyssosphaeria clematidis Wanas., Phukhams., Jones & K.D. Hyde, sp. nov.

Neomassarinaceae Mapook & K.D. Hyde

  1. 32.

    Pseudohelminthosporium Phukhams. & K.D. Hyde, gen. nov.

  2. 33.

    Pseudohelminthosporium clematidis Phukhams. & K.D. Hyde, sp. nov.

Nigrogranaceae Jaklitsch & H. Voglmayr

  1. 34.

    Nigrograna chromolaenae Mapook & K.D. Hyde, new host record

  2. 35.

    Nigrograna obliqua Jaklitsch & H. Voglmayr, new host record

Occultibambusaceae Dai & K.D. Hyde

  1. 36.

    Brunneofusispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Paradictyoarthriniaceae Doilom, Liu & K.D. Hyde

  1. 37.

    Xenomassariosphaeria clematidis Wanas., Phukhams., Camporesi & K.D. Hyde, sp. nov.

Periconiaceae Nann.

  1. 38.

    Periconia verrucosa Phukhams, Ertz, Gerstmans & K.D. Hyde, sp. nov.

Phaeoseptaceae Boonmee, Thambug. & K.D. Hyde

  1. 39.

    Pleopunctum clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

Phaeosphaeriaceae Barr

  1. 40.

    Chaetosphaeronema clematidicola Phukhams, Ertz, Gerstmans & K.D. Hyde, sp. nov.

  2. 41.

    Chaetosphaeronema clematidis Phukhams, Ertz, Gerstmans & K.D. Hyde, sp. nov.

  3. 42.

    Dermatiopleospora mariae Wanas., Camporesi, Jones & K.D. Hyde, new host record

  4. 43.

    Leptospora clematidis Phukhams., Ertz, Gerstmans, & K.D. Hyde, sp. nov.

  5. 44.

    Leptospora thailandica Phukhams. & K.D. Hyde, new host record

  6. 45.

    Longispora Phukhams. & K.D. Hyde, gen. nov.

  7. 46.

    Longispora clematidis Phukhams. & K.D. Hyde, sp. nov.

  8. 47.

    Pseudoophiobolus rosae Phookamsak, Wanas., Phukhams., Camporesi & K.D. Hyde, new host record

  9. 48.

    Wojnowiciella clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Pleosporaceae Nitschke

  1. 49.

    Stemphylium clematidis Wanas., Camporesi & K.D. Hyde, sp. nov.

  2. 50.

    Stemphylium rosae (Wanas. et al.) Phukhams. & K.D. Hyde, comb. nov.

  3. 51.

    Stemphylium rosae-caninae (Wanas. et al.) Phukhams. & K.D. Hyde, comb. nov.

  4. 52.

    Stemphylium vesicarium (Wallr.) E.G. Simmons, new host record

Pseudoberkleasmiaceae Phukhams. & K.D. Hyde

  1. 53.

    Pseudoberkleasmium chiangmaiense Lu & K.D. Hyde, new host record

Pseudomassarinaceae Phukhams. & K.D. Hyde

  1. 54.

    Pseudomassarinaceae Phukhams. & K.D. Hyde, fam. nov.

  2. 55.

    Pseudomassarina Phukhams. & K.D. Hyde, gen. nov.

  3. 56.

    Pseudomassarina clematidis Phukhams, Camporesi & K.D. Hyde, sp. nov.

Pseudolophiotremataceae Hyde & S. Hongsanan

  1. 57.

    Clematidis italica Tibpromma, Camporesi & K.D. Hyde

Roussoellaceae Liu, Phookamsak, Dai & K.D. Hyde

  1. 58.

    Neoroussoella clematidis Phukhams. & K.D. Hyde, sp. nov.

  2. 59.

    Neoroussoella fulvicomae Phukhams. & K.D. Hyde, sp. nov.

  3. 60.

    Neoroussoella heveae Senwanna, Phookamsak & K.D. Hyde, new host record

  4. 61.

    Pararoussoella mangrovei (Phukhams. & K.D. Hyde) Phukhams. & K.D. Hyde, comb. nov.

  5. 62.

    Pseudoneoconiothyrium euonymi (Crous & Akulov) Phukhams. & K.D. Hyde, comb. nov.

  6. 63.

    Pseudoroussoella chromolaenae Mapook & K.D. Hyde, new host record

  7. 64.

    Pseudoroussoella elaeicola (Konta & K.D. Hyde) Mapook & K.D. Hyde, new host record

Sulcatisporaceae Tanaka & K. Hirayama

  1. 65.

    Anthosulcatispora Phukhams. & K.D. Hyde, gen. nov.

  2. 66.

    Anthosulcatispora brunnea (Chen & C. Norphanphoun) Phukhams. & K.D. Hyde, comb. nov.

  3. 67.

    Anthosulcatispora subglobosa Phukhams. & K.D. Hyde, sp. nov.

  4. 68.

    Parasulcatispora Phukhams. & K.D. Hyde, gen. nov.

  5. 69.

    Parasulcatispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Teichosporaceae Barr

  1. 70.

    Floricola clematidis Phukhams., Camporesi & K.D. Hyde, sp. nov.

  2. 71.

    Magnibotryascoma rubriostiolata (Jaklitsch & Voglmayr) Phukhams., Jones & K.D. Hyde, comb. nov. and new host record

  3. 72.

    Ramusculicola clematidis Phukhams. & K.D. Hyde, sp. nov.

  4. 73.

    Ramusculicola thailandica Thambug. & K.D. Hyde, new host record

Thyridariaceae Tian & K.D. Hyde

  1. 74.

    Parathyridaria clematidis Phukhams., Camporesi & K.D. Hyde, sp. nov.

  2. 75.

    Parathyridaria serratifoliae Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

  3. 76.

    Parathyridaria virginianae Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Torulaceae Corda

  1. 77.

    Dendryphion europaeum Crous & R.K. Schumacher, new host record

  2. 78.

    Torula chromolaenae Li, Phookamsak, Mapook & K.D. Hyde, new host record

Dothideomycetes, family incertae sedis

Dyfrolomycetales Pang, Hyde & E.B.G. Jones

Pleurotremataceae Watson

  1. 79.

    Melomastia clematidis Phukhams., & K.D. Hyde, sp. nov.

  2. 80.

    Melomastia fulvicomae Phukhams., & K.D. Hyde, sp. nov.

Class Lecanoromycetes Erikss. & K. Winka

Subclass Ostropomycetidae Reeb, Lutzoni & Cl. Roux

Ostropales Nannf.

Stictidaceae Fr.

  1. 81.

    Fitzroyomyces cyperacearum Crous, new host record

  2. 82.

    Neostictis Ekanayaka, Camporesi & K.D. Hyde, gen. nov.

  3. 83.

    Neostictis nigricans Ekanayaka, Phukhams., Camporesi & K.D. Hyde, sp. nov.

Class Sordariomycetes Erikss. & K. Winka

Subclass: Sordariomycetidae Erikss. & K. Winka

Chaetosphaeriales Huhndorf, Mill. & F.A. Fernández

Chaetosphaeriales, genera incertae sedis

  1. 84.

    Neoleptosporella Phukhams. & K.D. Hyde, gen. nov.

  2. 85.

    Neoleptosporella clematidis Phukhams., Konta & K.D. Hyde, sp. nov.

Sordariales Chadef. ex Hawksw. & O.E. Erikss.

Chaetomiaceae G. Winter

  1. 86.

    Dichotomopilus ramosissimum (X. Wei Wang & L. Cai) X. Wei Wang & Samson, new host record

Sordariaceae Winter

  1. 87.

    Sordaria clematidis Phukhams. & K.D. Hyde, sp. nov.

Subclass Diaporthomycetidae Senan., Maharachch. & K.D. Hyde

Diaporthales Nannf.

Diaporthaceae Hohn. ex Wehm.

  1. 88.

    Diaporthe clematidina Phukhams., M.V. de Bult & K.D. Hyde, sp. nov.

  2. 89.

    Diaporthe ravennica Thambug., Camporesi & K.D. Hyde, new host record

  3. 90.

    Diaporthe rudis (Fr.) Nitschke, new host record

Phomatosporales Senan., Maharachch. & K.D. Hyde

Phomatosporaceae Senan. & K.D. Hyde

  1. 91.

    Phomatospora uniseriata Phukhams., M.V. de Bult & K.D. Hyde, sp. nov.

Diaporthomycetidae, family incertae sedis

Distoseptisporaceae Hyde & E. McKenzie

  1. 92.

    Distoseptispora clematidis Phukhams., M.V. de Bult & K.D. Hyde, sp. nov.

Subclass Xylariomycetidae Erikss. & W. Winka

Amphisphaeriales Hawksw. & O.E. Erikss

Sporocadaceae Corda

  1. 93.

    Pestalotiopsis verruculosa Maharachch. & K.D. Hyde, new host record

Xylariales Nannf.

Diatrypaceae Nitschke

  1. 94.

    Neoeutypella baoshanensis Raza, Shang, Phookamsak & L. Cai, new host record

Subclass Hypocreomycetidae Erikss. & K. Winka

Glomerellales Chadef. ex Re´blova´ et al.

Plectosphaerellaceae Gams, Summerb. & R. Zare

  1. 95.

    Fuscohypha kunmingensis (Phookamsak, J.F. Li & K.D. Hyde) Jayaward., Phukhams. & K.D. Hyde, comb. nov.

  2. 96.

    Xenoplectosphaerella Jayaward., Phukhams. & K.D. Hyde, gen. nov.

  3. 97.

    Xenoplectosphaerella clematidis Jayaward., Phukhams. & K.D. Hyde, sp. nov.

Hypocreales Lindau

Nectriaceae Tul & C. Tul

  1. 98.

    Fusarium celtidicola Shang, Camporesi & K.D. Hyde, new host record

Sarocladiaceae Lombard

  1. 99.

    Sarocladium clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Stachybotryaceae Lombard & P. Crous

  1. 100.

    Memnoniella oblongispora Lin, McKenzie, Wang & K.D. Hyde, new host record

Introduction

Clematis (Ranunculaceae) is a flowering climber which has become a popular plant in horticulture (Linnaeus 1753; Yuan et al. 2010). The genus contains between 250 and 350 species and hybrids (Grey-Wilson 2000; Lehtonen et al. 2016; He et al. 2019). Clematis is widespread in warm-temperate or montane ecosystems and is native to most areas of China, Europe, Korea, and Russia (Tamura 1956; Ziman and Keener 1989; Yuan and Yang 2020). Clematis vitalba (old man’s beard), the type species of Clematis is an invasive weed broadly distributed in Europe, and also expanding to New Zealand and South America (Ogle et al. 2000; Leuschner and Ellenberg 2017; Redmond and Stout 2018). Clematis vitalba can influence the biodiversity dynamics of native plants (Ogle et al. 2000; Ashton and Lerdau 2008). In Thailand, Clematis species are mostly found in the northern provinces of Chiang Mai, Chiang Rai and Nan, which are mountainous areas with a tropical savanna climate (Tamura 1997, 2000). Many Clematis species are grown as ornamental plants. Clematis species are also used in Traditional Chinese Medicine and some secondary metabolites isolated from Clematis have been tested in vitro, but there are no reports of successful clinical tests or if it is safe to consume the plant parts (Ding et al. 2009; Fu et al. 2010; Feng et al. 2011; Hawaze et al. 2012; Lu et al. 2014; Zhao et al. 2016). Clematis species are herbaceous vines, with opposite compound, bipinnate to tripinnate leaves, and leather-like flowers with feather achenes (Johnson 2001) (Fig. 1). Section-level phylogenetic classification of Clematis by Lehtonen et al. (2016) includes specific characteristics and geographic distribution for each section. The estimated divergence time of the available sequences for Clematis have shown that the stem age was in the Oligocene (25.99 million years ago; Lehtonen et al. 2016).

Fig. 1
figure1

ac Habitats of Clematis species. d Opposite compound with bi-pinnate to tri-pinnate leaves. e Woody climbing stem. f Inflorescence of C. pitcheri.g Inflorescence of C. “Crystal Fountain”. h Inflorescence of C. vitalba.i Achenes of C. vitalba. j Enlarged achenes of C. subumbellata with plumose style

Fungal species associated with Clematis have been documented since the late eighteenth century (Lamarck 1805; Saccardo 1892; Farr and Rossman 2020; Index Fungorum 2020). Index Fungorum and the U.S. National Fungus Collections Fungal Database lists over 500 records, mainly as saprobes, or pathogens that can cause leaf lesions and wilt in Clematis species (Baylis 1954; Braun 1992; Ahn and Shearer 1998; Wanasinghe et al. 2014; Chen et al. 2015; Crous et al. 2019).

In this study, Clematis samples were collected in Belgium, China, Italy, Thailand, and the UK to establish the microfungi associated with this host and to analyze their host-specificity. In addition, fungal isolates were evaluated for their antagonistic activity against selected microorganism (Phukhamsakda et al. 2018; Hyde et al. 2019b; Macabeo et al. 2020). The delineation of new species introduced in this study relies on a polyphasic approach based on morphological traits (MSC), molecular data (PSC), and application of Genealogical Concordance Phylogenetic Species Recognition (GCPSR) (Taylor et al. 2000). GCPSR model relies on performing a pairwise homoplasy index coupled with phylogenetic relatedness in a multi-locus dataset and the interpretation of nucleotide differences (Turner et al. 2013; Quaedvlieg et al. 2014; Jeewon and Hyde 2016). We also compared the morphology of our new collections with documented fungal taxa recorded in public databases and discuss their ecological species concepts.

Materials and methods

Sample collection, morphological study and isolation

Fresh Clematis specimens were collected or received from Belgium, China, Italy, Thailand and England (the UK). Some specimens have single collection because this study mainly focused on the diversity of fungi associated with Clematis. One collection is defined as a sample of fungus that can be identified with a single collecting trip which was used to cast the number of species. Thus, the plant materials were mainly collected and received from a single trip of the aforementioned countries. The specimens were maintained in paper bags for transport to the laboratory. The specimens were examined using a Motic SMZ 168 Series stereo-microscope. Thereafter, vertical free-hand sections were made by a razor blade and placed on a droplet of sterilized water on a glass slide. A Nikon ECLIPSE 80i compound microscope was used to examine the samples and a Canon 600D digital camera fitted to the microscope was used to photograph the samples. Tarosoft (R) Image Frame Work program was used for measurements and photo-plate were made by using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, United States).

Pure cultures were obtained from single ascospores isolation on malt extract agar (MEA: 33.6 g/L, malt extract Difco™) or potato dextrose agar (PDA: 39 g/L, potato dextrose media Difco™) as described by Chomnunti et al. (2014) which were incubated at 16–25 °C with the standard light cycles, 12 h in the light followed by 12 h in the dark for about four up to eight weeks. Asexual reproduction was induced by placing agar squares with mycelia on water agar or MEA placed with additional substances such as sterile pine needles or rice straws. Authentic type specimens are deposited in Mae Fah Luang University (MFLU) herbarium and ex-type living cultures are deposited at the Mae Fah Luang Culture Collection (MFLUCC). Faces of fungi numbers (Jayasiri et al. 2015) and Index Fungorum numbers (2020) are provided.

DNA extraction, amplification and sequencing

The Biospin Fungus Genomic DNA Extraction Kit (BioFlux®) (Hangzhou, P. R. China) and gene extraction kit (Bio Basic Inc., Canada) were used for DNA extraction from mycelium. The fruiting bodies DNA was extracted by using Forensic DNA Kit–D3591-01 (OMEGA bio-tek) following the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to amplify partial gene regions with primer pairs as described in Tibpromma et al. (2018). The PCR amplifications were performed in a total volume of 25 µL solution containing 10–20 ng of DNA template, Easy Taq PCR Super Mix (mixture of Easy Taq TM DNA Polymerase, dNTPs, and optimized buffer) and 10 picomolar forward and reverse primers. Amplification reactions were performed following Phukhamsakda et al. (2016) and Tibpromma et al. (2018). Genomic DNA and PCR amplification products were checked on 1% agarose gel. PCR products were purified as described in the manufacturer’s instructions (EZ-10 PCR Products Purification Kit, Bio basic Canada INC.). Sequences were generated by Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai, P.R. China) and the sequencing service at Helmholtz Centre For Infection Research (HZI, Braunschweig, Germany).

Sequence alignment and phylogenetic analysis

Consensus sequences were assembled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI). Sequences of closely related strains were retrieved using BLAST searches against GenBank (http://www.ncbi.nlm.nih.gov). Sequences were aligned with MAFTT version 7 (Katoh et al. 2019) (http://mafft.cbrc.jp/alignment/server), with minimal adjustment of the ambiguous nucleotides by visual examination and manually corrected in AliView program (Larsson 2014). Leading or trailing gaps exceeding the primer binding site were trimmed from the alignments prior to tree building and alignment gaps were treated as missing data. The concatenation of the multigene datasets was created in Sequence Matrix (Vaidya et al. 2011).

Phylogenetic analyses of the single gene and combined gene were based on maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference posterior probabilities (BYPP). PAUP program was used for MP bootstrap analyses, with 1000 bootstrap replicates using 10 rounds of the heuristic search replicates to estimate the homoplasy yield. The random addition of sequences and subsequent TBR branch swapping during each bootstrap replicate, with each replicate was limited to 1000 rearrangements. Gaps were treated as missing data; all characters were unordered and given equal weight. The statistics for parsimony were described under the phylogenetic legend with the values of Tree Length (TL), Consistency Index (CI), Retention Index (RI), Relative Consistency Index (RC) and Homoplasy Index (HI) calculated for trees generated under different optimality criteria. The best fitting substitution model for each single gene partition and the concatenated data set was determined in MrModeltest 2.3 (Nylander 2004) for Bayesian inference posterior probabilities and ML. Maximum likelihood analyses, including 1000 bootstrap replicates, were performed using the RAxML-HPC2 on XSEDE (8.2.12) in the CIPRES Science Gateway (Stamatakis 2014; Miller et al. 2017). The general time reversible (GTR) model was used for nucleotide substitution with a discrete gamma distribution plus invariant site (GTR + I + G). The bootstrap replicates were summarized onto the best scoring tree (Miller et al. 2017). The Bayesian inference posterior probabilities (PP) distribution (Zhaxybayeva and Gogarten 2002) was estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes 3.2.2 on XSEDE (Ronquist and Huelsenbeck 2003). Six simultaneous Markov chains were run for 1,000,000 to 10,000,000 generations, depending on individual settings for the fungal group. The resulted trees were sampled at one tree every 100th or 1000th generation. The first 10–25% of burn-in phase of the analyses were discarded based on suitable burn-in phases determined by using Tracer version 1.7 (Rambaut et al. 2018). The remaining trees were used to calculate posterior probabilities in the majority-rule consensus (MRC) trees (50%) with critical value for the topological convergence diagnostic set to 0.01.

FigTree v. 1.4 (Rambaut 2014) was used to visualize phylogenetic trees and data files and the phylogram was edited using Adobe Illustrator CS v. 6 (Adobe Systems, USA). All sequences generated in this study were submitted to GenBank. All entries are represented using phylogenetic tree and relevant description.

Genealogical concordance phylogenetic species recognition analysis

The closely related strains that resulted from morphology and phylogeny evidence of recombination were prospectively analyzed using the genetic distances by performing a pairwise homoplasy index test (Φw) (Taylor et al. 2000; Bruen et al. 2006). A pairwise homoplasy index (PHI) test was performed in SplitsTree (version 4.1.4.4) using the Kimura’s two parameter (K2P) models for low genetic distance datasets. LogDet transformation were applied for the average of nucleotide frequencies and splits decomposition graph options (Gu and Li 1996a, b; Taylor et al. 2000; Bruen et al. 2006; Huson and Bryant 2006; Gioan and Paul 2012; Nishimaki and Sato 2019). The standard deviation of split frequencies PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.

Taxonomy

Phylum Ascomycota R.H. Whittaker

The taxa are arranged as in the Outline of Fungi and fungus-like organisms (Wijayawardene et al. 2016, 2020).

Subphylum Pezizomycotina Erikss. & K. Winka

Class Dothideomycetes sensu O.E. Erikss & Winka

For the classification of Dothideomycetes we follow Hyde et al. (2013), Liu et al. (2017) and Hongsanan et al. (2020).

Subclass Pleosporomycetidae C.L. Schoch et al.

Pleosporales Luttrell ex M.E. Barr

Pleosporales is the largest and most diverse order in Dothideomycetes with over 75 families (Hongsanan et al. 2020).

Amniculicolaceae Y. Zhang, C.L. Schoch, J. Fourn., Crous & K.D. Hyde

Amniculicolaceae was introduced for freshwater-associated ascomycetes. This family is characterized by solitary ascomata with a rough black surface. The members usually stain the surface of the substrate purple and have short pedicellate asci, with hyaline or pale brown or brown, 1- to multi-septate or muriform ascospores (Hyde et al. 2013). The family comprises Amniculicola, Murispora, Neomassariosphaeria, Pseudomassariosphaeria and Vargamyces (Zhang et al. 2009; Hyde et al. 2013; Ariyawansa et al. 2015a; Hernández-Restrepo et al. 2017). We introduce a novel saprobic genus, Fusiformispora from Clematis collections in Thailand. Maximum likelihood and Bayesian analyses of the combined dataset (LSU, SSU, ITS, tef1 and rpb2) is shown in Fig. 2.

Fig. 2
figure2figure2

The Bayesian 50% majority-rule consensus phylogram based on combined LSU, SSU, ITS, tef1 and rpb2 sequence data of related families in Pleosporales. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with species of Hysteriales. One hundred and fifty-three strains were included in the DNA analyses which comprised 4394 characters (848 characters for LSU, 1044 characters for SSU, 556 characters for ITS, 910 characters for tef1, and 1036 characters for rpb2, including gap regions). The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The best scoring RAxML tree had a final likelihood value of − 73089.933914. The matrix had 2676 distinct alignment patterns, with 40.81% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.246412, C = 0.245743, G = 0.272077, T = 0.235768; substitution rates AC = 1.617324, AG = 3.695355, AT = 1.662826, CG = 1.183453, CT = 8.283938, GT = 1.000000; gamma distribution shape parameter α = 0.635095. The GTR + I + G model was selected for every partition in Bayesian analysis. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. The type sequences are in bold and the species determined in this study are indicated in blue

Fusiformispora Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557106; Facesoffungi number: FoF 07242, Fig. 3.

Fig. 3
figure3

Fusiformispora clematidis (MFLU 17–1485, holotype). a Appearance of ascoma on host surface. b Close up of ascoma on host substrate. c Vertical section of ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d, jm = 20 µm, ei = 50 µm

Etymology: Genus name reflects the fusiform shape of its ascospores.

Saprobic on decaying wood or herbaceous plant material in terrestrial habitats. Sexual morph: Ascomata on surface of the host, covered by a pseudoclypeus, visible as black spots, solitary, scattered, uniloculate, obpyriform to compressed globose, coriaceous, brown to dark brown, ostiolate. Ostioles central, brown to dark brown, papillate. Peridium multilayered, cells of textura angularis, inner layers comprising thin, hyaline cells. Hamathecium composed of dense, filiform, branched, transverly septate, trabecular pseudoparaphyses anastomosing above asci. Asci 8-spored, bitunicate, fissitunicate, thick-walled, cylindric-clavate, apically rounded, short, with a furcate pedicel, with ocular chamber. Ascospores biseriate, partially overlapping, broad fusiform, tapering towards the acute ends, hyaline, with guttules in each cell, constricted at the septa, smooth-walled, with a thin mucilaginous sheath. Asexual morph: Undetermined.

Type species: Fusiformispora clematidis Phukhams., M.V. de Bult & K.D. Hyde

Notes: Fusiformispora is established as a monotypic genus. In the multi-gene phylogenetic analyses, the isolate MFLUCC 17–2077 formed a basal lineage with other genera in Amniculicolaceae (Fig. 2) with strong support (100% ML/1.00 BYPP). The genus is compatible with the concept of Amniculicolaceae in having compressed globose, coriaceous, brown to dark brown ascomata, ostiolate, with trabeculate, anastomosed pseudoparaphyses (sensu Liew et al. 2000), and fusiform ascospores that are hyaline and septate with mucilaginous appendages (Zhang et al. 2009). Fusiformispora is similar to Amniculicola Zhang & K.D. Hyde, however, the genus differs by having thinner peridium walls with sub-carbonaceous ascomatal type. Amniculicola is an aquatic genus and its species have cylindrical asci and uniseriate arrangement of ascospores, while Fusiformispora has cylindric-clavate asci and biseriate arrangement of ascospores and is from a terrestrial habitat. We therefore, introduce a new genus based on morphological and phylogenetic evidence for a fungal collection on Clematis fulvicoma.

Fusiformispora clematidis Phukhams., M.V. de Bult & K.D. Hyde, sp. nov.

Index Fungorum number: IF557107; Facesoffungi number: FoF 07243, Fig. 3.

Etymology: Epithet reflects the host Clematis.

Holotype: MFLU 17–1485.

Saprobic on dead stems of Clematis fulvicoma.Sexual morph: Ascomata 165–190 × 200–275 μm (\(\bar{x}\)= 175 × 225 μm, n = 5), on surface of host, covered by a pseudoclypeus, visible as black spots, immersed to superficial, solitary, scattered, uniloculate, obpyriform to compressed globose, base flattened, brown to dark brown, partially carbonaceous, rough-walled, with apical ostioles. Ostioles central, 55 × 35 μm, brown to dark brown, papillate, with easily opening by a pore, filled with periphyses. Peridium 10–18 μm wide, multilayered, comprising 4–5 layers of brown to dark brown cells of textura angularis, inner layers comprising thin, hyaline cells. Hamathecium composed of dense, 0.5–1.5 μm wide (\( \bar{x} \)= 1.3 μm, n = 50), filiform, branched, trabeculate pseudoparaphyses, anastomosing above the asci, reaching the ostiole, transversely septate. Asci 86–127 × 18–24 μm (\( \bar{x} \) = 100 × 25 μm, n = 40), 8-spored, bitunicate, fissitunicate, thick-walled, cylindric-clavate, apically rounded, short, with furcate pedicel, ocular chamber clearly visible when immature. Ascospores 24–36 × 5–10 μm (\( \bar{x} \) = 30 × 8 μm, n = 50), biseriate, partially overlapping, broad fusiform, tapering towards the ends, acute at both ends, hyaline, with (1–)3–4 transverse septa, with large guttules in each cell, constricted at the septa, deeply constricted at the median septum, cell above median septum slightly wider than below, smooth-walled, with 4–12 μm wide mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, grey-brown, with reddish brown mixed in the mycelium, dense, colonies circular, flat, umbonate, raised from the agar in the centre, dull, covered with aerial mycelium, white mycelium at the edge; reverse dark brown, dense, circular, with irregular, fimbriate margin, pinkish mycelium radiating outwardly.

Material examined: Thailand, Chiang Rai Province, on dead stems of Clematis fulvicoma Rehder & E.H. Wilson, 20 March 2017, C. Phukhamsakda, CMTH22 (MFLU 17–1485, holotype); ex-type living culture, MFLUCC 17–2077.

Host: Clematis fulvicoma—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214542; SSU: MT226661; ITS: MT310589; tef1: MT394725; rpb2: MT394677.

Notes: In a BLASTn search of GenBank, the LSU sequence of Fusiformispora clematidis MFLUCC 17–2077 showed 96% similarity to Lindgomyces pseudomadisonensis KT 2742 (LC149916), while the ITS sequence had 91% similarity to Vargamyces aquaticus CBS 636.91 (NR_154471). Fusiformispora clematidis is phylogenetically distinct, therefore we introduce the collection as a new species.

Amorosiaceae Thambug. & K.D. Hyde

Amorosiaceae was introduced for Amorosia Mantle & D. Hawksw. and Angustimassarina Thambugala, Tanaka & K.D. Hyde (Thambugala et al. 2015). Amorosiaceae is characterized by immersed or semi-immersed ascomata with a short crest-like papilla, and hyaline ascospores with a mucilaginous sheath. Asexual morphs of this family were described as sporodochia (Mantle et al. 2006; Thambugala et al. 2015). Jayasiri et al. (2019) reported Amorocoelophoma from a decaying pod of Cassia species. Phylogeny combined with morphological observations confirm the placement of Amorocoelophoma as the first coelomycetous species in Amorosiaceae (Fig. 4).

Fig. 4
figure4

The best scoring RAxML tree with a final likelihood value of − 10209.184183 based on combined LSU, SSU, ITS and tef1 sequence data for Amorosiaceae. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with species of Sporormiaceae. The tree from the maximum likelihood analysis had similar topology to the Bayesian 50% majority-rule consensus phylogram. The matrix had 809 distinct alignment patterns with 21.55% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.246053, C = 0.243449, G = 0.269921, T = 0.240577; substitution rates AC = 1.221779, AG = 2.152972, AT = 1.558241, CG = 0.998233, CT = 7.007203, GT = 1.000000; gamma distribution shape parameter α = 2.180328. The species determined in this study is indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than (BS ≥ 50%/BYPP ≥ 0.90)

Angustimassarina Thambug., Kaz. Tanaka & K.D. Hyde

Angustimassarina was described for fungal species that have ascospores resembling Massarina while being narrowly fusiform in shape. The genus has immersed to semi-immersed ascomata, coriaceous, dark brown to black, globose to subglobose, ostiolate, cylindrical to cylindric-clavate asci and fusiform to cylindrical or ellipsoidal-fusiform ascospores surrounded by a mucilaginous sheath. The asexual morph of this genus is hyphomycetous. Twelve species are listed in Index Fungorum for Angustimassarina (Thambugala et al. 2015; Wanasinghe et al. 2018; Hyde et al. 2019a). In this study, Angustimassarina rosarum was isolated from Clematis viticella and identification was based on multigene phylogenetic analysis of LSU, SSU, ITS, and tef1 sequence data (Fig. 4) and its compatible morphology (Fig. 5).

Fig. 5
figure5

Angustimassarina rosarum (MFLU 17–1513). a Appearance of ascomata on host surface. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–h Asci. i–m Ascospores. Scale bars: b = 500 µm, c = 200 µm, df = 50 µm, g, h = 20 µm, im = 10 µm

Angustimassarina rosarum Tibpromma, Camporesi & K.D. Hyde, Fungal Diversity 89: [21] (2018), new host record.

Index Fungorum number: IF553939; Facesoffungi number: FoF 03964, Fig. 5.

Saprobic on dead stems of Clematis viticella. Sexual morph: Ascomata 221–306 × 267–400 µm (\( \bar{x} \) = 265 × 340 µm, n = 5), scattered, gregarious, immersed, coriaceous, dark globose to subglobose, brown to black, ostiolate. Ostioles 57 × 134 µm, central, rounded, papillate, with opening by a pore. Peridium 14–40(–60) µm (\( \bar{x} \) = 20 µm, n = 10), thick at the sides, broad at the apex and thinner at the base, comprising brown to dark brown cells of textura angularis, fusing at the outside with the host tissues. Hamathecium composed of dense, 1.5–2.5 µm wide, septate, long, cellular pseudoparaphyses, embedded in a gelatinous matrix. Asci 77–85 × 10–16 µm (\( \bar{x} \) = 78 × 15 µm, n = 30), 8-spored, bitunicate, fissitunicate, broad cylindrical to cylindrical-clavate, with bulbous pedicel, rounded at the apex, with a minute ocular chamber. Ascospores 17–23 × 4–4.5 µm (\( \bar{x} \)= 20 × 4 µm, n = 15), biseriate, partially overlapping, broad fusiform, hyaline, 1(–3) septate, deeply constricted at the primary septum, widest at the centre and tapering towards the ends, straight, smooth-walled, 1(–3)-guttulate, surrounded by a 5–10 µm wide mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Culture black in the middle, radiating white, dense, circular, umbonate, entries edge, shiny, dull, undulate, radially furrowed, reverse black, radiating outwardly, white.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis viticella L., 13 June 2017, D. Ertz & C. Gerstmans, BRCV1 (MFLU 17–1513); living culture, MFLUCC 17–2155.

Hosts: Clematis viticella, Rosa canina—(Wanasinghe et al. 2018; this study).

Distribution: Belgium, Italy—(Wanasinghe et al. 2018, this study).

GenBank accession numbers: LSU: MT214543; SSU: MT226662; ITS: MT310590; tef1: MT394726; rpb2: MT394678.

Notes: Angustimassarina rosarum (MFLUCC 15–0080) was described from Rosa canina in Italy (Wanasinghe et al. 2018). We compared our collection with A. rosarum and both have similar morphology in terms of ascomata, asci, and ascospores. Angustimassarina rosarum (MFLUCC 15–0080) has globose to subglobose, cylindric-clavate asci (124 × 143 µm), and club-shaped pedicel (70 × 10 µm). It has a minute ocular chamber, with fusiform to ellipsoidal ascospores (19 × 5 µm) and a hyaline, 1 septate at the centre, with two large guttules in each cell. Our collection has larger ascomata (265 × 340 µm), but is similar in overall morphology (Fig. 5). In molecular analysis, the new strain forms a close relationship with A. rosarum (MFLUCC 15–0080). Comparison of the ITS sequence data reveals no significant difference (one base pair difference) between our new collection and A. rosarum (MFLUCC 15–0080). However, the tef1 sequence is not available for A. rosarum (MFLUCC 15–0080) for comparison. Therefore, we introduce a new host record of A. rosarum on Clematis species herein.

Cyclothyriellaceae Jaklitsch & Voglmayr

Cyclothyriellaceae was introduced for Cyclothyriella rubronotata (= Thyridaria rubronotata) and Massariosphaeria phaeospora as revealed by molecular phylogeny (Jaklitsch and Voglmayer 2016). Cyclothyriellaceae is characterized by scattered, immersed-erumpent ascomata, with occasional purple stain on plant tissue, and narrow, anastomosing, trabeculate pseudoparaphyses (sensu Liew et al. 2000). Asci are cylindrical to clavate, bitunicate and 8-spored. Ascospores are brown, ellipsoid to fusoid, with several eusepta. The asexual morph is pycnidial with hyaline to brown conidia (Zhang et al. 2012; Jaklitsch and Voglmayer 2016). We describe a novel species of Massariosphaeria recorded on Clematis vitalba from Italy (Fig. 2).

Massariosphaeria (E. Müll.) Crivelli

Massariosphaeria was introduced for species with reddish brown to brown, multi-septate, phragmosporous to dictyosporous and usually with colouration on the surface of the substrate (Crivelli 1983; Leuchtmann 1984; Zhang et al. 2012). Several studies have proved the polyphyletic placement of Massariosphaeria, classifying them into distinct genera (Zhang et al. 2012; Ariyawansa et al. 2015a; Phukhamsakda et al. 2016). Massariosphaeria has comparable peridium and phragmosporous characters with Chaetomastia (Teichosporaceae) (Barr 1989). However, the characteristic of ascomata position and the ascospores characters of Chaetomastia and Massariosphaeria are distinct. The type species Massariosphaeria phaeospora (CBS 611.86) is currently placed in Cyclothyriellaceae (Fig. 2). Twenty-one epithets are listed under Massariosphaeria in Index Fungorum (2020). Based on phylogenetic analysis including Cyclothyriellaceae, our collection (MFLU 16–0174) clustered with M. phaeospora (CBS 611.86) with strong support (100% MLBS/1.00 BYPP). A new Massariosphaeria species on Clematis vitalba is introduced herein (Fig. 6).

Fig. 6
figure6

Massariosphaeria clematidis (MFLU 16–0174, holotype). a Appearance of ascomata on host surface. b Vertical section of ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–i Asci. j–m Ascospores. n Ascospore in 10% Indian ink. Scale bars: a = 500 µm, b = 200 µm, c = 100 µm, d–i = 50 µm, jn = 20 µm

Massariosphaeria clematidis Phukhams., Wanas., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557108; Facesoffungi number: FoF 07248, Fig. 6.

Etymology: Epithet reflects the host Clematis.

Holotype: MFLU 16–0174.

Saprobic on dead stems of Clematis vitalba.Sexual morph: Ascomata 340–430 × 215–300 μm (\( \bar{x} \) = 280 × 255 μm, n = 5), with only black shiny ostioles present on the surface of host, solitary, scattered, immersed, globose to compressed globose, sub-carbonaceous to coriaceous, dark brown to black, rough-walled, with short hyphae projecting from peridium, ostiolate. Ostioles centrally located, 125–175 × 110–130 μm (\( \bar{x} \) = 140 × 120 μm, n = 10), carbonaceous, papillate, periphysoids. Peridium 22–33 μm wide, composed of 6–8(–12 at apex) layers of dark brown to black cells of textura angularis, inner layer composed of hyaline gelatinous cells. Hamathecium composed of numerous, dense, long, 1.6–3.5 μm wide (\( \bar{x} \) = 2.5 μm, n = 50), filiform, transversely septate, branched, anastomosing, cellular pseudoparaphyses. Asci 150–225 × 20–30 μm (\( \bar{x} \) = 180 × 25 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindric-clavate to broad cylindrical, with furcate pedicel, with ocular chamber visible when immature. Ascospores 35–45 × 10–15 μm (\( \bar{x} \) = 40 × 12 μm, n = 50), biseriate or overlapping, broad fusiform, narrow towards the apex, initially hyaline, becoming yellowish to brown at maturity, 6–8-transversely euseptate, constricted at the septa, third cell from apex usually enlarged, smooth-walled, guttulate and indentations present, surrounded by a 8–20 μm wide, mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 18 °C. Culture from above brown, yellowish towards the edge, dense, circular, flat, dull, fimbriate, radially furrowed, and slightly covered with white aerial mycelium; reverse black with radiating cream mycelium.

Material examined: Italy, Forlì-Cesena Province, Strada San Zeno—Galeata, dead aerial stems of Clematis vitalba L., 7 November 2013, E. Camporesi, IT1509 (MFLU 16–0174, holotype).

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214544; SSU: MT226663; ITS: MT310591.

Notes: Our new collection MFLU 16–0174 from Italy formed a close relationship with the type species of Massariosphaeria, M. phaeospora (CBS 611.86) with strong support (100% ML/1.00 BYPP). The strain is compatible with the concept of Massariosphaeria in having scattered, immersed, papillate ascomata with thick ostiolar wall, dense pseudoparaphyses, cylindro-clavate asci and broad fusiform, reddish brown to brown, multi-septate ascospores with a mucilaginous sheath (Müller 1950; Zhang et al. 2012). MFLU 16–0174 is distinguishable from M. phaeospora by its partial carbonaceous ostioles, narrower, 6–8-transversely euseptate ascospores which are swollen at the third cell. Massariosphaeria vitalbae (≡ Leptosphaeria vitalbae) was described from Clematis vitalba in Switzerland (Ahn and Shearer 1998). Characters of MFLU 16–0174 include a long neck (\( \bar{x} \) = 140 × 120 μm), with sub-carbonaceous to coriaceous peridium types and ascospores enlarged at the third cell. Massariosphaeria vitalbae has sub-parenchymatous cells type with short ostiolar necks and 9–10-septate and ascospores enlarged at the third cell (Müller 1950). Massariosphaeria vitalbae closely resembles Paramassariosphaeria clematidicola, however, fresh collections are required to clarify its taxonomic placement (Wanasinghe et al. 2016b).

In a BLASTn search of GenBank, the LSU sequence of Massariosphaeria clematidis (MFLU 16–0174) was found to be 96% similar to M. phaeospora strain CBS 611.86 (FJ795503). We introduce a novel species of Massariosphaeria, M. clematidis based on the morphological (Fig. 6) and phylogenetic evidence (Fig. 2).

Dictyosporiaceae Boonmee & K.D. Hyde

Dictyosporiaceae was introduced with Dictyosporium as the type genus in Dothideomycetes (Boonmee et al. 2016). The sexual morph of Dictyosporiaceae has immersed to erumpent or superficial, globose to subglobose and dark brown to black ascomata including bitunicate, cylindric-clavate asci with septate and hyaline ascospores with a thick mucilaginous sheath (Tanaka et al. 2015; Boonmee et al. 2016). Most members of Dictyosporiaceae have a hyphomycetous asexual morph with cheirosporous or dictyosporous conidia, pycnidia with coleophoma-like characters and phialidic conidiogenesis cells. Fourteen genera are listed under Dictyosporiaceae (Iturrieta-González et al. 2018; Wijayawardene et al. 2018; Crous et al. 2019). We provide an updated phylogenetic tree of Dictyosporiaceae and propose a new species and a new host record of Aquadictyospora, Dictyocheirospora and Pseudocoleophoma on Clematis (Fig. 7).

Fig. 7
figure7

The best scoring RAxML tree with a final likelihood value of −15336.133569 based on LSU, ITS and tef1 sequence data for Dictyosporiaceae species. The topology and clade stability of the combined DNA analyses was compared to the single gene analyses. The tree is rooted with sequences of Murilentithecium clematidis (MFLUCC 14–0561, MFLUCC 14–0562) in Lentitheciaceae. The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The matrix had 1009 distinct alignment patterns, with 37.31% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.234307, C = 0.254705, G = 0.271061, T = 0.239927; substitution rates AC = 1.414609, AG = 2.411925, AT = 2.055489, CG = 0.612591, CT = 6.466528, GT = 1.000000; gamma distribution shape parameter α = 0.761212. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Aquadictyospora Luo, Hyde & H.Y. Su

Aquadictyospora was introduced to accommodate a dictyosporous taxon on submersed decaying wood, and typified with A. lignicola Z.L. Luo et al. The genus is characterized by sporodochia, superficial, circular or subglobose conidiomata, micronematous conidiophores with monoblastic conidiogenesis cells, and uniformly medium brown dictyosporous conidia with a subglobose, hyaline cell at the basal end (Li et al. 2016). We introduce a second species of Aquadictyospora based on morphology (Fig. 8) and phylogenetic analyses (Fig. 7).

Fig. 8
figure8

Aquadictyospora clematidis (MFLU 17–1488, holotype). a, b Sporodochia on natural substrate. c Vertical section through sporodochium. de Conidia attached to conidiophores. f–k Mature conidia. l Germinated conidia. m, n Culture characteristics on MEA. Scale bars: b = 100 μm, c = 100 μm, dl = 20 μm

Aquadictyospora clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

Index Fungorum number: IF557125; Facesoffungi number: FoF 07250, Fig. 8.

Etymology: Name refers to the host plant, Clematis.

Holotype: MFLU 17–1488.

Saprobic on dead stems of Clematis sikkimensis.Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies 53–97 × 121–213 μm (\( \bar{x} \) = 72 × 153 μm, n = 10), on natural substrate forming sporodochial conidiomata, superficial, compact, scattered, subglobose to oval, dark brown to reddish brown, velvety. Mycelium 2–3 μm wide, immersed, consisting of branched septate hyphae. Conidiophores 6–11 × 2–4 μm (\( \bar{x} \) = 8 × 3 μm, n = 10), micronematous, hyaline to pale brown, smooth. Conidiogenous cells 5–8 × 3–5 μm, holoblastic, monoblastic, solitary, discrete, determinate. Conidia 17–38 × 15–24 μm (\( \bar{x} \) = 32 × 20 μm, n = 50), dictyosporous, compactly depressed, obovoid, appearing broadly rounded in upper half, heavily pigmented at the upper half, smooth, entirely reddish brown, with hyaline mammiform basal cell, smooth basal cell, 5–11 × 4–11 μm (\( \bar{x} \) = 8 × 8 μm, n = 40), not complanate, secession involves splitting of the basal, without appendages.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above brownish beige at the centre, grey radiating outwardly, dense, raised with concave edge, circular, entire edge, umbonate, papillate with fairly fluffy, wrinkled folded, covered with white aerial mycelium; reverse dark brown at the centre, faintly zonate, white mycelium at the edge.

Material examined: Thailand, Nan Province, on dead stem of Clematis sikkimensis (Hook. f. & Thomson) Drumm. ex Burkill, 2 May 2017, C. Phukhamsakda, CMTH26 (MFLU 17–1488, holotype); ex-type living culture, MFLUCC 17–2080.

Host: Clematis sikkimensis—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214545; SSU: MT226664; ITS: MT310592; tef1: MT394727, rpb2: MT394679.

Notes: The species is assigned to Aquadictyospora based on its compatible morphological features such as superficial sporodochia with subglobose to oval conidiomata, micronematous conidiophores with monoblastic conidiogenous cell, and dictyosporous conidia with hyaline, mammiform basal cells (Li et al. 2016). Aquadictyospora clematidis has smaller non-cheiroid conidia (32 × 20 μm) than A. lignicola which has larger cheiroid conidia (50 × 24 μm). Aquadictyospora clematidis was found in a terrestrial habitat while A. lignicola was from submerged substrates (Fig. 8). In a BLASTn search of GenBank, the ITS sequence had 95% similarity while the tef1 sequence had 95% similarity to the type species, A. lignicola. The new strain is introduced as a new species of Aquadictyospora based on polyphasic evidence.

Dictyocheirospora D’souza, Boonmee & K.D. Hyde

Boonmee et al. (2016) introduced Dictyocheirospora (typified by D. rotunda) for an aero-aquatic sporodochial fungus with cheiroid dictyospores. Nineteen species are listed in Index Fungorum (2020) and interestingly, no sexual morph has been described for this genus (Wang et al. 2016; Tibpromma et al. 2018; Hyde et al. 2017, 2019a). We introduce Dictyocheirospora clematidis as a novel species and D. xishuangbannaensis as a new host record from Clematis based on compatible morphological and phylogenetic analysis (Fig. 9).

Fig. 9
figure9

Dictyocheirospora clematidis (MFLU 17–1497, holotype). a, b Sporodochia on natural substrate. c Sporodochium mounted in water. d–i Mature conidia. j Culture characteristics on MEA. Scale bars: b = 500 μm, c = 100 μm, dg = 20 μm, h, i = 10 μm

Dictyocheirospora clematidis Phukhams., D.J. Bhat & K.D. Hyde, sp. nov.

Index Fungorum number: IF557126; Facesoffungi number: FoF 07251, Fig. 9.

Etymology: Name refers to the host plant, Clematis.

Holotype: MFLU 17–1497.

Saprobic on dead stem of Clematis sikkimensis. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies 200–340 μm wide (\( \bar{x} \) = 265 μm, n = 20), on natural substrate forming sporodochial conidiomata, superficial, gregarious, scattered, punctiform, blackish brown, velvety, glistening, orbicular, with abundant sporulation, conidia readily liberated when agitated. Mycelium immersed, composed of brown, smooth, thin-walled, septate hyphae. Conidiophores 15 × 3 μm, micronematous, pale brown, smooth, thin-walled. Conidiogenous cells 5–6 × 3–4 μm, holoblastic, monoblastic, integrated, terminal, determinate, hyaline, smooth-walled. Conidia 42–60 × 15–30 μm (\( \bar{x} \) = 50 × 23 μm, n = 40), solitary, acrogenous, cheiroid, with a basal connecting cell, discharges after mounted in water, cognac brown, consisting of 6–7 rows of cells, individual rows discoid. Conidial arm 34–60 × 7–10 μm (\( \bar{x} \) = 52 × 9 μm, n = 30), digitate, cylindrical, inwardly curved at the tip, arising from a basal cell, 10–12 distosepta, slightly constricted at the septa, with large guttule in each cell.

Culture characters: Colonies on MEA reaching 40 mm diam. after 4 weeks at 25 °C. Cultures from above, black, dense, circular, entire edge, umbonate, papillate with aerial mycelium, wrinkled and folded, narrow fringe of submerged mycelium, covered with grey aerial mycelium; reverse black, white mycelium present at the edge.

Material examined: Thailand, Chiang Rai Province, Doi Tung, on dead stem of Clematis sikkimensis, 2 May 2017, C. Phukhamsakda & M.V. de Bult, CMTHDT06 (MFLU 17–1497, holotype); ex-type living culture, MFLUCC 17–2089.

Host: Clematis sikkimensis—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214546; SSU: MT226665; ITS: MT310593; tef1: MT394728, rpb2: MT394680.

Notes: Dictyocheirospora species are highly diverse especially in tropical regions (Boonmee et al. 2016; Tibpromma et al. 2018; Yang et al. 2018a; Hyde et al. 2019a). Dictyocheirospora clematidis is similar to D. metroxylonis based on characters (Table 1), but the ITS sequence shows 97% similarity (2.8% nucleotide differences) while the tef1 sequence has 96% similarity (4.6% nucleotide differences). Dictyocheirospora clematidis clustered in the same clade as D. taiwanense but it has distinct characters. Dictyocheirospora taiwanense usually has 5 rows of cells in the conidia and they are longer than those of D. clematidis (78 × 18 vs 50 × 23 μm). In the phylogenetic analysis, D. clematidis (MFLUCC 17–2089) clustered with D. metroxylonis (MFLUCC 15–0282) and D. taiwanense (MFLUCC 17–2654) but formed a well-separated clade with good support of 79% in ML and 1.00 in BYPP (Fig. 7).

Table 1 Synopsis of related Dictyocheirospora species and the new species from this study

Dictyocheirospora xishuangbannaensis Tibpromma & K.D. Hyde, Fungal Divers 93:14 (2018), new host record

Index Fungorum number: IF554476; Facesoffungi number: FoF 04485, Fig. 10

Fig. 10
figure10

Dictyocheirospora xishuangbannaensis (MFLU 17–1495). a, b Sporodochia on natural substrate. c Sporodochium mounted in water. d–h Mature conidia. i–j Separated rows of conidium. k Culture characteristics on MEA. Scale bars: b = 200 μm, c = 100 μm, de = 50 μm, fg = 20 μm. hj = 10 μm

Saprobic on dead stem of Clematis sikkimensis. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies 235–423 μm wide (\( \bar{x} \) = 326 μm, n = 20), on natural substrate forming sporodochial conidiomata, superficial, gregarious, scattered, punctiform, blackish brown, velvety, glistening, orbicular, with abundant sporulation, conidia readily liberated when agitated. Mycelium 2 μm wide, immersed, composed of hyaline to pale brown, smooth, thin-walled, septate hyphae. Conidiophores 10–20 × 3–6 μm (\( \bar{x} \) = 12 × 4 μm, n = 10), micronematous, hyaline to light brown, smooth, thin-walled. Conidiogenous cells 3–7 × 2–6 μm (\( \bar{x} \) = 5 × 4 μm, n = 10), holoblastic, integrated, terminal, determinate, hyaline, smooth-walled. Conidia 32–53 × 16–27 μm (\( \bar{x} \) = 46 × 20 μm, n = 50), solitary, acrogenous, cheiroid, oblong or subglobose, with a basal connecting cell, discharges after mounted in water, cognac brown, heavily pigmented upper part, consisting of 6–7 rows of cells, rows digitate. Conidial arm 40–55 × 6–9 μm (\( \bar{x} \) = 46 × 6 μm, n = 30), cylindrical, curved at both ends, arising from a basal cell, reddish brown, heavily pigmented at the upper part, 7–11 distoseptate, slightly constricted at the septa, with large guttule in each cell.

Culture characters: Colonies on MEA reaching 40 mm diam. after 4 weeks at 25 °C. Cultures from above, dark brown, dense, circular, edge entire, umbonate, papillate with white aerial mycelium, wrinkled and folded, narrow fringe of submerged mycelium; reverse dark brown, erose.

Material examined: Thailand, Chiang Rai Province, on dead stem of Clematis sikkimensis, 2 May 2017, C. Phukhamsakda & M.V. de Bult, CMTHDT03 (MFLU 17–1495); living culture, MFLUCC 17–2087.

Hosts: Clematis sikkimensis, Pandanus sp.—(Tibpromma et al. 2018; this study).

Distribution: China, Thailand—(Tibpromma et al. 2018; this study).

GenBank accession numbers: LSU: MT214547; SSU: MT226666; ITS: MT310594; tef1: MT394729.

Notes: Dictyocheirospora xishuangbannaensis was recorded from Pandanus by Tibpromma et al. (2018) and our collection resembles this species morphologically (Fig. 10). The first record of D. xishuangbannaensis was from southern China (Xishuangbanna, Yunnan Province), while our collection was found in the northern part of Thailand (Chiang Rai Province). Northern Thailand and Xishuangbanna both have a tropical climate and share similar weather in both the wet and dry seasons (Cao et al. 2006). It can be hypothesized that D. xishuangbannaensis generally occurs in tropical climates (Boonmee et al. 2016; Tibpromma et al. 2018; Yang et al. 2018a). This is the first record of D. xishuangbannaensis on Clematis species. We also provide sequence data and phylogenetic analyses in Fig. 7.

Pseudocoleophoma Tanaka & K. Hiray.

Pseudocoleophoma is characterized by its immersed to erumpent, ostiolate ascomata, cylindrical to clavate and short pedicellate asci and fusiform, 1-septate, sheathed ascospores. The genus produces coleophoma-like asexual morph with phialidic, doliiform to lageniform conidiogenous cells and cylindrical, hyaline conidia. We introduce a new species in Pseudocoleophoma based on molecular and morphology (Figs. 7, 11).

Fig. 11
figure11

Pseudocoleophoma clematidis (MFLU 16–0280, holotype). a Appearance of conidiomata on Clematis vitalba. b Vertical section through conidioma. c Ostiolar canal. d Section of conidioma wall. eg Conidiogenous cells and conidia. h Conidia. i Culture characteristics on MEA. Scale bars: b = 200 µm, c = 50 µm, d = 20 µm, eh = 5 µm

Pseudocoleophoma clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557127; Facesoffungi number: FoF 07252, Fig. 11.

Etymology: Name refers to the host plant, Clematis from which the fungus was isolated.

Holotype: MFLU 16–0280.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Undetermined. Asexual morph: Conidiomata 130–150 × 100–130 μm (\( \bar{x} \)= 140 × 110 μm, n = 5), pycnidial, solitary, aggregated, uniloculate, immersed, with black shiny ostioles visible, globose to subglobose, coriaceous, subcoriaceous at the outer layers, thick-walled, black to dark brown, ostioles. Ostioles 30 × 50 μm, central, papillate, ovoid. Conidiomatal wall 20–30 μm wide, of equal thickness, multilayered, outer layer composed of 8–10 layers of light brown to brown cells of textura angularis, lined with a thick hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2–4 × 1.5–4 μm (\( \bar{x} \) = 2.5 × 3 μm, n = 30), holoblastic, phialidic, determinate, discrete, cylindrical to subcylindrical, smooth-walled, hyaline, arising from inner layers of conidioma. Conidia 5–8 × 2–4 μm (\( \bar{x} \) = 6 × 4 μm, n = 50), oval, slightly curved towards the ends, aseptate, with 1(–2) guttules in each cell, hyaline when immature, yellowish brown at maturity, smooth-walled.

Culture characters: Colonies on MEA reaching 20 mm diam. after 2 weeks at 16 °C. Cultures from above, cream, dense, circular, umbonate, papillate with fluffy, covered with white aerial mycelium; reverse dark brown at the centre, cream radiating outwardly.

Material examined: Italy, Arezzo Province, Badia Tega—Ortignano Raggiolo, on dead aerial branch of Clematis vitalba, 9 March 2013, E. Camporesi, IT 1110 (MFLU 16–0280, holotype); ex-type living culture, MFLUCC 17–2177.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214548, MT214549; SSU: MT226667; ITS: MT310595, MT310596; tef1: MT394730.

Notes: Based on the multi-gene phylogenetic analyses (Fig. 7), Pseudocoleophoma clematidis strain MFLUCC 17–2177 (Fig. 11) clusters between P. calamagrostidis (KT 3284) and P. typhicola (MFLUCC 16–0123). Pseudocoleophoma clematidis is different from other Pseudocoleophoma species by having pycnidial walls which are flat at the base and yellowish brown conidia (Tanaka et al. 2015; Hyde et al. 2016; Jayasiri et al. 2019, Fig. 11). This study confirmed its placement in Pseudocoleophoma. In a BLASTn search of GenBank, the closest match of the LSU sequence of MFLUCC 17–2177 is P. calamagrostidis (HHUF 30450) with 97% similarity, while the closest match of the ITS sequence is P. polygonicola (HHUF 27558) with 92% similarity.

Didymellaceae Gruyter, Aveskamp & Verkley

Valenzuela-Lopez et al. (2018) accepted 26 genera in Didymellaceae. Didymellaceous taxa frequently occur as plant pathogens, causing drooping and wilting of plant leaves or gummy stem blight leading to death of the plant (Sudisha et al. 2004; Vaghefi et al. 2012; Ahmadpour et al. 2017; Wijayawardene et al. 2017). The combined dataset of LSU, ITS and rpb2 sequences for a multilocus analysis tree revealed distinct lineages in Didymellaceae (Fig. 12). In the present study, a cluster of fungi associated with Clematis species formed a distinct lineage, Anthodidymella, a novel genus in Didymellaceae, a novel species in Xenodidymella, and a new host record in Neodidymelliopsis.

Fig. 12
figure12figure12

Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, and rpb2 sequence data representing Didymellaceae species. Related sequences were taken from Chen et al. (2017) and Valenzuela-Lopez et al. (2018). One hundred and nine strains were included in the combined DNA analyses which comprised 2094 characters (964 characters for LSU, 531 characters for ITS, 599 characters for RPB2, including gap regions). Leptosphaeria conoidea (CBS 616.75) and L. doliolum (CBS 505.75) in Leptosphaeriaceae (Pleosporales) were used as out-group taxa. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The best sorting RaxML tree with a final likelihood value of − 18910.278845 is presented. The matrix had 602 distinct alignment patterns with 6.40% undetermined characters or gaps proportions. Estimated base frequencies were as follows: A = 0.247270, C = 0.227052, G = 0.279182, T = 0.246495; substitution rates AC = 2.142238, AG = 8.227445, AT = 2.335028, CG = 1.021789, CT = 16.735281, GT = 1.000000; gamma distribution shape parameter α = 0.490873. The GTR + I + G model was used for each partition in Bayesian analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Anthodidymella Phukhams., Camporesi & K.D. Hyde, gen. nov.

Index Fungorum number: IF557128; Facesoffungi number: FoF 07255, Fig. 13

Fig. 13
figure13

Anthodidymella ranunculacearum (MFLU 17–1468, holotype). a Appearance of conidiomata on Clematis vitalba. b Close up of conidioma on host substrate. c Vertical section through conidioma. d Ostiolar canal. e Section of partial conidioma wall. fh Conidiogenous cells and conidia (h conidiogenous cells in cotton blue). i Conidia. Scale bars: b = 200 µm, c = 100 µm, d = 20 µm, e = 10 µm, fi = 5 µm

Etymology: Anthos-meaning flower, Anthodidymella refer to species of Didymella that frequently occur on flowering plants.

Saprobic or necrotic on leaf and dead stems of herbaceous plants Sexual morph: Ascomata superficial, solitary or clustered, globose or subglobose to pyriform, with elongated ostioles. Perithecial wall consisting of textura globulosa. Hamathecium composed of numerous, dense, pseudoparaphyses. Asci 8-spored, bitunicate, cylindrical club-shaped pedicel. Ascospores uniseriate or partially overlapping, ovate to obpyriform, 1-septate, hyaline (Woudenberg et al. 2009). Asexual morph: Conidiomata pycnidial, uniloculate, immersed under host epidermis, subglobose to depressed, coriaceous, thin-walled, dark brown to brown, with papillate ostioles. Conidiomatal wall thin layers, pseudoparenchymatous, with brown cells of textura globulosa, lined with a hyaline cell-layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic, determinate, discrete, ampulliform, cylindrical to sub-cylindrical, hyaline. Conidia oblong or oval, rounded ends, hyaline, aseptate or septate, smooth-walled. Chlamydospores absent.

Type species: Anthodidymella ranunculacearum Phukhams., Camporesi & K.D. Hyde

Notes: Anthodidymella is introduced for a strongly supported clade (93% ML/1.00 BYPP, Fig. 12) of Didymella species unit that is associated with Clematis (Aveskamp et al. 2010). Anthodidymella clematidis was described as Phoma clematidina as it clustered with other Phoma clematidina isolates (Woudenberg et al. 2009; Golzar et al. 2011). Phoma clematidina not only causes symptoms on Clematis species, but also is a saprobe on other hosts. It has been used as a control agent of Clematis vitalba in New Zealand (Gourlay et al. 2000). An updated study classified Phoma collections isolated from necrotic leaf tissues of Clematis species into Anthodidymella, Calophoma, and Phoma (Woudenberg et al. 2009; Valenzuela-Lopez et al. 2018, this study, Fig. 12).

Anthodidymella has similar morphology to Didymella in its solitary or clustered, globose ascomata, thin-walled cells of textura globulosa, with ovate to obpyriform, 1 septum, hyaline ascospores without a mucilaginous sheath. The asexual morph is pycnidial with phialidic, determinate, and discrete conidiogenous cells (Chen et al. 2017). However, Anthodidymella have broad-cylindrical asci, obpyriform ascospores while Didymella has oblong asci and broad-fusiform ascospores (Woudenberg et al. 2009; Aveskamp et al. 2010). The asexual morph of Anthodidymella has globose or flask-shaped, phialidic conidiogenous cells with oblong or elongated-oval conidia. The combined dataset of the LSU, ITS, and rpb2 sequences for Didymellaceae revealed a lineage including Anthodidymella clematidis, A. ranunculacearum (type species) and A. vitalbina (Fig. 12).

Anthodidymella clematidis (Woudenb., Spiers & Gruyter) Phukhams. & K.D. Hyde, comb. nov.

Index Fungorum number: IF557129; Facesoffungi number: FoF 07256

Basionym: Didymella clematidis Woudenb., Spiers & Gruyter in Woudenberg et al., Persoonia 22:60 (2009)

Synonym: Phoma clematidina (Thüm.) Boerema, Versl. Medsd. Plziektenk. Dienst Wageningen 153:17 (1979)

Notes: Since the fungus has been introduced before one fungus = one name (Taylor 2011), the isolate CBS 123705 bears two names for its pleomorphic life-cycles The strain was originally described as Phoma clematidina from a necrotic leaf spot of Clematis ligusticifolia and developed both sexual morph and asexual morph characters in pure culture (Woudenberg et al. 2009). Anthodidymella clematidis was initially described as Didymella clematidis for its sexual morph epithet (= Phoma clematidina as asexual name) as it clustered with other Phoma clematidina isolates (Woudenberg et al. 2009; Golzar et al. 2011). The new combination, Anthodidymella clematidis is proposed for Didymella clematidis (CBS 123705).

Host: Clematis ligusticifolia—(Woudenberg et al. 2009).

Distribution: USA—(Woudenberg et al. 2009).

Anthodidymella ranunculacearum Phukhams., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557130; Facesoffungi number: FoF 07257, Fig. 13.

Etymology: The specific epithet reflects the host family, Ranunculaceae.

Holotype: MFLU 17–1468.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Undetermined. Asexual morph: Conidiomata 99–214 × 130–246 μm (\( \bar{x} \)= 142 × 169 μm, n = 5), pycnidial, solitary, sometimes aggregated, uniloculate, immersed under epidermal layer, subglobose to depressed, coriaceous, thin-walled, brown to dark brown, with ostiolate. Ostioles 25 × 42 μm, central, papillate, with pore. Conidiomatal wall 10–28(–36) μm wide, of 2–5 layers, each cell-layer 10 μm wide, light brown to brown cells of textura globulosa, heavily pigmented in the outer layers, lined with a hyaline innermost layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2.5–5 × 1.5–3.5 μm (\( \bar{x} \) = 3.5 × 2 μm, n = 30), phialidic, determinate, discrete, ampulliform, cylindrical to sub-cylindrical, smooth-walled, hyaline, arising from the inner layer of conidioma. Conidia 6–10 × 2–5 μm (\( \bar{x} \) = 6 × 4 μm, n = 50), oblong or oval, slightly curved toward the ends, rounded ends, with 1(–2) guttules in each cell, hyaline, aseptate, smooth-walled.

Culture characters: Colonies on MEA reaching 20 mm diam. after 2 weeks at 25 °C. Cultures; above: greyish brown or dark green, dense, circular, umbonate, papillate, fluffy, covered with aerial mycelium, reverse dark brown.

Material examined: Italy, Forlì-Cesena Province, Valdinoce—Meldola, on dead aerial branch of Clematis vitalba, 3 February 2015, E. Camporesi, IT 2364 (MFLU 17–1536, holotype); ex-type living culture, MFLUCC 17–2184 = MFLUCC 17–2209.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: MFLUCC 17–2184; LSU: MT214550; SSU: MT226668; ITS: MT310597; tef1: MT394731; rpb2: MT394681; act: MT394620. MFLUCC 17–2209; LSU: MT214551; SSU: MT226669; ITS: MT310598; tef1: MT394732; act: MT394621.

Notes: Anthodidymella ranunculacearum (MFLUCC 17–2184) is similar to A. vitalbina (CBS 123707, ex-epitype), a strain recorded from the same host (Woudenberg et al. 2009). However, A. ranunculacearum differs from A. vitalbina in its thicker conidiomatal wall (10–36 vs 5.5–9.5 μm, Fig. 13). In a BLASTn search of GenBank, the ITS sequence had 99.5% similarity (2.25% nucleotide differences), while the act sequence had 91% similarity (77 nucleotide differences in 297 nucleotides). Thus, the new strain is introduced as a new species of Anthodidymella based on guidelines of Jeewon and Hyde (2016). Additionally, A. ranunculacearum is designated as the type species of Anthodidymella based on available material and an ex-type culture.

Anthodidymella vitalbina (Petr.) Phukhams. & K.D. Hyde, comb. nov.

Index Fungorum number: IF557131; Facesoffungi number: FoF 07258.

Basionym: Ascochyta vitalbae Briard & Har. apud Briard, Rev. Mycol. (Toulouse) 13: 17. (1891).

≡ Diplodina vitalbae (Briard & Har.) Allesch., Rabenh. Krypt.-Fl., ed. 2. Pilze 6 (Lief. 69): 683. 1900 (1901).

Synonym: Diplodina clematidina Fautrey & Roum. apud Roum., Rev. Mycol. (Toulouse) 14: 105 (1892).

= Didymella vitalbina Petr, Annls mycol. 38(2/4): 348 (1940).

= Phoma clematidina (Thüm.) Boerema, Versl. Medsd. Plziektenk. Dienst Wageningen, 1978 153: 17 (1979).

Notes: Anthodidymella vitalbina was introduced for Didymella vitalbina (= Phoma clematidina as asexual name) which was reported from a necrotic leaf spot of Clematis species (Woudenberg et al. 2009). Phoma clematidina (strain CBS 123707) was isolated from Clematis vitalba and developed sexual and asexual morphs in culture. The sexual morph is named as Didymella vitalbina Petr. and was chosen as an epitype of D. vitalbina by Woudenberg et al. (2009). In the analyses of combined LSU, ITS, and rpb2 sequence data of Didymellaceae, the ex-epitype strain (CBS 123707) and the related strains clustered with Anthodidymella clematidis. Therefore, we transfer Didymella vitalbina (= Phoma clematidina) to Anthodidymella vitalbina based on phylogenetic relationship of compatible morphology of both morphs. The nomenclature changes are also based on one fungus = one name protocol.

Host: Clematis vitalba—(Woudenberg et al. 2009).

Distribution: Austria, France, Switzerland—(Woudenberg et al. 2009).

Neodidymelliopsis Qian & L. Cai

Neodidymelliopsis was introduced for one section of phoma-like species that reside within Didymellaceae and is typified by N. cannabis (Chen et al. 2017). The genus is characterized by immersed or erumpent subglobose to pyriform, ostiolate ascomata, cylindrical to clavate asci and subovoid to ellipsoidal, hyaline, septate ascospores. The asexual morph is phoma-like with pycnidial conidiomata, a 2–7-layered pseudoparenchymatous pycnidial wall, phialidic conidiogenous cells, and aseptate or occasionally 1-septate conidia (Chen et al. 2015, 2017). We introduce a new host record of N. longicolla from Clematis vitalba in Italy (Fig. 14).

Fig. 14
figure14

Neodidymelliopsis longicolla (MFLU 20–0421). a Appearance of conidiomata on Clematis vitalba. b Vertical section through conidioma. c Section of conidioma wall. df Conidiogenous cells and conidia. g–j Conidia. k Culture characteristics on MEA. Scale bars: b = 100 µm, c = 20 µm, df = 10 µm, gj = 10 µm

Neodidymelliopsis longicolla Hou, Crous & L. Cai, Stud. Mycol. 87: 153 (2017), new host record

Index Fungorum number: IF820006; Facesoffungi number: FoF 07259, Fig. 14.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Undetermined. Asexual morph: Conidiomata 70–95 × 124–134 μm (\( \bar{x} \)= 84 × 130 μm, n = 5), pycnidial, aggregated, uniloculate, superficial or covered by host epidermal layer, subglobose to depressed, cupulate when dried coriaceous, thick-walled, light brown to brown, with papillate ostioles. Ostioles 80 × 38 μm, central, papillate, opening by a pore. Conidiomatal wall 10–17(–27) μm wide, composed of 5–7 layers of light brown to brown cells of textura angularis, heavily pigmented at the outer layers, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2.5–7 × 2–4.5 μm (\( \bar{x} \) = 4.5 × 3.5 μm, n = 30), phialidic, annellidic, determinate, discrete, ampulliform, cylindrical to sub-cylindrical, smooth-walled, hyaline, arising from the inner layers of conidiomata. Conidia 6.5–10 × 2–4.5 μm (\( \bar{x} \) = 8 × 4 μm, n = 50), oblong-elliptical, oval, slightly curved towards the ends, rounded ends, with 1(–2) guttules in each cell, initially aseptate and hyaline, becoming pale brown and 1-septate at maturity, constricted at the septum, wall verrucose.

Culture characters: Colonies on MEA reaching 20 mm diam. after 2 weeks at 25 °C. Cultures from above, cream with white at the centre, medium dense, circular, umbonate, papillate, fluffy, covered with white aerial mycelium; reverse brown white cream at the edge.

Material examined: Italy, Forlì-Cesena Province, Castrocaro Terme, on dead aerial branch of Clematis vitalba, 19 September 2012, E. Camporesi, IT739 (MFLU 20–0421); living culture, MFLUCC 17–2167.

Hosts: Soil in desert, Clematis vitalba—(Chen et al. 2017; this study).

Distribution: Israel, Italy—(Chen et al. 2017; this study).

GenBank accession numbers: LSU: MT214552; SSU: MT226670; ITS: MT310599; tef1: MT394733; rpb2: MT394682.

Notes: A new isolate of Neodidymelliopsis longicolla (MFLUCC 17–2167) was collected from Clematis vitalba in Italy. The new isolate formed a close relationship with the type (100% ML/1.00 BYPP). Neodidymelliopsis longicolla (CBS 382.96) was originally reported from a soil sample in Israel. The characters of the type strain were obtained from a culture on OA medium. The new collection differs slightly from the type material in having a shorter ostioles (Fig. 14).

Xenodidymella Chen & L. Cai

Xenodidymella typified by X. applanata (≡ Didymosphaeria applanata) has Phoma argillacea as the asexual morph (Corlett 1981; Gruyter et al. 2013; Chen et al. 2015). Xenodidymella is distinct from other genera in Didymellaceae in having a thick peridium and ellipsoidal, allantoid or subcylindrical, unicellular conidia. Xenodidymella is reported from Europe and USA and consists of five species (Farr and Rossman 2020; Index Fungorum 2020). We introduce a novel species of Xenodidymella clematidis from Clematis vitalba in Italy and provide phylogenetic and morphological comparisons (Figs. 12, 15).

Fig. 15
figure15

Xenodidymella clematidis (MFLU 16–2288, holotype). a Appearance of conidiomata on Clematis vitalba. b Vertical section through conidioma. c Ostiolar canal. d Section of conidioma wall. e, f Conidiogenous cells and conidia. g Conidia. h Culture characters on MEA. Scale bars: b = 200 µm, c, d = 50 µm, eg = 10 µm

Xenodidymella clematidis Phukhams., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557132; Facesoffungi number: FoF 07260, Fig. 15.

Etymology: Refers to the host genus Clematis.

Holotype: MFLU 16–2288.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Undetermined. Asexual morph: Conidiomata 220–375 × 180–290 μm (\( \bar{x} \)= 280 × 230 μm, n = 5), pycnidial, aggregated, uniloculate, superficial or semi-immersed, subglobose to depressed globose, cupulate, when dried coriaceous, thick-walled, brown to light brown, with ostioles. Ostioles central, papillate, opened-like pore. Conidiomatal wall 10–15(–25 μm at apex) wide, composed of 7–9 layers of light brown to brown cells of textura angularis, heavily pigmented at the outer layers, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2–6(–12) × 2.3–3.3 μm (\( \bar{x} \) = 5 × 3 μm, n = 20), enteroblastic, phialidic, determinate, discrete, ampulliform, smooth-walled, hyaline, arising from the inner layers of conidiomata. Conidia 4–8 × 2–5 μm (\( \bar{x} \) = 6 × 3.5 μm, n = 50), oblong-elliptical, oval, slightly curved towards the ends, rounded ends, hyaline, with 1(–2) guttules in each cell, aseptate, verrucose.

Culture characters: Colonies on MEA reaching 20 mm diam. after 2 weeks at 16 °C. Cultures from above, grey with white aerial mycelium, dense, circular, umbonate, papillate, fluffy, reverse brownish white, cream at the edge.

Material examined: Italy, Forlì-Cesena Province, Monte Fumaiolo, dead aerial branch of Clematis vitalba, 6 August 2016, E. Camporesi, IT3054 (MFLU 16–2288, holotype); ex-type living culture, MFLUCC 16–1365.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214553; ITS: MT310600; act: MT394622.

Notes: In the phylogenetic analysis, Xenodidymella clematidis clustered with X. applanata with strong support (1.00 in BYPP). Xenodidymella clematidis has a long ostiole, with oblong-elliptical or oval conidia, while X. applanata has a short ostiole and ellipsoidal conidia (Gruyter et al. 2002; Chen et al. 2015). Xenodidymella applanata is commonly a pathogen of raspberry (Rubus sp.). Xenodidymella clematidis was a saprobe on Clematis vitalba (Fig. 15). In a BLASTn search of GenBank, the ITS sequence had 98% similarity (11 nucleotides differences out of 488 nucleotides). The new strain is introduced as a new species of Xenodidymella herein.

Didymosphaeriaceae Munk

The latest treatment of Didymosphaeriaceae was by Ariyawansa et al. (2014a). The family is typified with Didymosphaeria. Several genera have been introduced to the family based on morphological and phylogenetic evidence (Tibpromma et al. 2018; Wanasinghe et al. 2018; Wijayawardene et al. 2018). Mapook et al. (2020) introduced Chromolaenicola Mapook & K.D. Hyde into Didymosphaeriaceae. Phylogeny and morphological comparisons revealed a novel species of Chromolaenicola and a new host record of Didymosphaeria rubi-ulmifolii from Clematis (Fig. 16).

Fig. 16
figure16

The best scoring RAxML tree with a final likelihood value of − 12878.030199 based on combined LSU, ITS, SSU, rpb2 and tub sequence data of Didymosphaeriaceae. The tree is rooted with sequences of Montagnula species. Forty-six strains were included in the combined DNA sequence analyses which comprised 3681 characters (860 characters for LSU, 516 characters for ITS, 913 characters for SSU, 935 characters for rpb2, 457 characters for tub, including gap regions). The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The matrix had 764 distinct alignment patterns, with 9.02% undetermined characters or gaps propotions. Estimated base frequencies were as follows: A = 0.239562, C = 0.247759, G = 0.271470, T = 0.241209; substitution rates AC = 1.719408, AG = 2.576378, AT = 1.444379, CG = 0.958169, CT = 9.083623, GT = 1.000000; gamma distribution shape parameter α = 0.621443. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses at the genus level (BS ≥ 70%/BYPP ≥ 0.95)

Chromolaenicola Mapook & K.D. Hyde

Mapook et al. (2020) introduced Chromolaenicola (typified with C. nanensis) for a monophyletic clade of fungi described on Chromolaena odorata. Chromolaenicola is characterized by immersed to semi-immersed and coriaceous ascomata, cylindrical asci, and uniseriate, ellipsoid, muriform ascospores (Mapook et al. 2020). The asexual morph is pycnidial, with enteroblastic, phialidic conidiogenous cells, and oblong or oval to ellipsoid, globose to subglobose conidia (Jayasiri et al. 2019; Mapook et al. 2020). We introduce a new species Chromolaenicola clematidis based on morphological comparison (Fig. 17) and phylogenetic analyses (Fig. 16).

Fig. 17
figure17

Chromolaenicola clematidis (MFLU 17–1483, holotype). a, b Appearance of conidiomata on Clematis subumbellata. c Vertical section through conidioma. d Section of conidioma wall. ef Conidiogenous cells and conidia. gj Conidia. kl Culture characters on MEA. Scale bars: b = 200 µm, c, d = 50 µm, ej = 5 µm

Chromolaenicola clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557133; Facesoffungi number: FoF 07253, Fig. 17.

Etymology: Named after the host genus, Clematis.

Holotype: MFLU 17–1483.

Saprobic on dead stem of Clematis subumbellata.Sexual morph: Undetermined. Asexual morph: Conidiomata 76–145 × 107–128 μm (\( \bar{x} \)= 121 × 117 μm, n = 10), pycnidial, solitary, uniloculate, immersed, globose, coriaceous, thin, brown to light brown, ostiolate. Conidiomatal wall 5–10 μm wide, uniform, wider at apex, composed of 3–5 layers of pale brown to bronze cells of textura angularis, lined with a thin, hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2.6–4.5 × 4–7 μm (\( \bar{x} \) = 3.5 × 5 μm, n = 20), enteroblastic, phialidic, determinate, discrete, truncate, hyaline, smooth, arising from the inner layer of pycnidial wall. Conidia 7–10 × 4.5–7 μm (\( \bar{x} \) = 8.5 × 6 μm, n = 50), broad oblong or oval, rounded ends, hyaline when immature, reddish brown at maturity, 1-septate, with guttule in each cell, rough-walled, verrucose.

Cultural characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, dark brown, radiating outwardly, dense, umbonate, undulate edge, umbonate, papillate, fluffy, covered with white aerial mycelium; reverse dark brown, undulate.

Material examined: Thailand, Chiang Rai Province, Mae Sai District, dead stems of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH19 (MFLU 17–1483, holotype); ex-type living culture, MFLUCC 17–2075.

Host: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214554; SSU: MT226671; ITS: MT310601; rpb2: MT394683.

Notes: In our phylogenetic analysis (Fig. 16), Chromolaenicola clematidis (MFLUCC 17–2075) clustered with C. chiangraiensis Mapook & K.D. Hyde with moderate statistical support (66% ML/0.82 BYPP). A comparison of the ITS sequences showed three nucleotide differences in 516 nucleotides, while the rpb2 showed five nucleotide differences in 935 nucleotides. Chromolaenicola clematidis has smaller conidia than C. chiangraiensis (mean 8.5 × 6 vs 11 × 7.5 µm) with guttules in each cell (Fig. 17, Table 2). The new strain is introduced as a new species of Chromolaenicola based on the guidelines proposed by Jeewon and Hyde (2016).

Table 2 A comparison of Chromolaenicola species discussed in this study

Didymosphaeria Fuckel

Didymosphaeria is characterized by trabeculate pseudoparaphyses (sensu Liew et al. 2000), which anastomose above the cylindrical asci, and uniseriate, 1-septate ascospores (Aptroot 1995; Ariyawansa et al. 2014b). The asexual morph of Didymosphaeria has been suggested to be Ascochyta, fusicladiella-like, Periconia, and phoma-like species but a holomorphic connection has not been proven (Sivanesan 1984; Kirk et al. 2008; Ariyawansa et al. 2014b). Didymosphaeria is typified by D. futilis, however, fresh collections are needed to confirm its phylogenetic placement (Ariyawansa et al. 2014a; Wijayawardene et al. 2018). More than 500 epithets are listed under Didymosphaeria (Index Fungorum 2020), but only seven species were accepted by Aptroot (1995). Only three species of Didymosphaeria have phylogenetic evidence (D. rubi-ulmifolii, D. variabile, and Didymosphaeria sp. (as Paraconiothyrium brasiliense CBS 115.92, CBS 587.84, CBS 122319 and CBS 122320)) (Verkley et al. 2004). In this study, phylogenetic analyses based on a combined dataset of the LSU, ITS, SSU, rpb2, and tub sequences revealed a new host record for D. rubi-ulmifolii on Clematis heracleifolia from China (Fig. 18).

Fig. 18
figure18

Didymosphaeria rubi-ulmifolii (MFLU 17–1535). a, b Appearance of conidiomata on Clematis heracleifolia. c Vertical section through conidioma. d Section of conidioma wall. eh Conidiogenous cells and conidia. il Conidia. Scale bars: a = 500 µm, b = 200 µm, c = 100 µm, d = 20 µm, ek = 5 µm, l = 10 µm

Didymosphaeria rubi-ulmifolii Ariyaw., Camporesi & K.D. Hyde, Phytotaxa 176:111 (2014), new host record

Index Fungorum number: IF808165; Facesoffungi number: FoF 07254, Fig. 18.

Saprobic on dead stem of Clematis heracleifolia.Sexual morph: Ariyawansa et al. (2014a). Asexual morph: Conidiomata 78–160 × 75–244 μm (\( \bar{x} \)= 110 × 120 μm, n = 5), pycnidial, solitary, unilocular or multilocular, scattered, immersed or erumpent, under host epidermis, globose to compressed, brown to dark brown, without ostioles. Pycnidial wall 12–20(−30 μm at apex) wide, composed of 4–5 brown cell layers of textura angularis, inner layer subhyaline, lining bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 1.8–5 × 2–4 μm (\( \bar{x} \)= 3 × 3 μm, n = 20), enteroblastic, phialidic, determinate, smooth-walled, hyaline. Conidia 6–11 × 2.5–4.5 μm (\( \bar{x} \)= 9 × 4 μm, n = 50), ellipsoid, 1 septum, constricted at septum, rounded ends, initially hyaline, pale brown at maturity, with 1–2 guttules, smooth-walled.

Cultural characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 16 °C. Cultures from above, olive brown at the centre, radiating outwardly, medium dense, circular, entire edge, umbonate, papillate, fairly fluffy, covered with white aerial mycelium; reverse dark brown at the centre, faintly zonate slightly present, white mycelium at the edge.

Material examined: China, Dali, on dead terrestrial stem of Clematis heracleifolia DC., 8 May 2016, C. Phukhamsakda, CMCN03 (MFLU 17–1460); living culture, MFLUCC 16–1000.

Hosts: Coffea arabica, Rubus ulmifolius, Clematis heracleifolia—(Verkley et al. 2004; Ariyawansa et al. 2014a; this study).

Distribution: Brazil, China, Italy—(Verkley et al. 2004; Ariyawansa et al. 2014a; this study).

GenBank accession numbers: LSU: MT214555; SSU: MT226672; ITS: MT310602; tef1: MT394734.

Notes: Ariyawansa et al. (2014a) introduced Didymosphaeria rubi-ulmifolii from a collection of Rubus ulmifolius in Italy. The ex-type strain (MFLUCC 14–0023) formed a distinct clade with the type strain of Paraconiothyrium brasiliense (CBS 100299). Based on the multilocus phylogenetic analyses, our strain (MFLUCC 16–1000) formed a close relationship with the other D. rubi-ulmifolii strains with moderate support (Fig. 16), with no significant pairwise differences. Morphological comparison of D. rubi-ulmifolii (MFLUCC 16–1000) with the asexual morph report in D. rubi-ulmifolii (strain CBS 100299) show that they are different in the conidial characters (Fig. 18). Strain MFLUCC 16–1000 is saprobic and has 2-celled conidia while, CBS 100299 has single celled conidia in culture (Verkley et al. 2004). This study is the first record of D. rubi-ulmifolii on Clematis species.

Hermatomycetaceae Locq. ex A. Hashim. & Kaz. Tanaka

Hermatomycetaceae is typified by Hermatomyces and currently only known from asexual morph characters (Tibpromma et al. 2016, 2018; Koukol et al. 2018; Hyde et al. 2019a). We introduce a novel Hermatomyces species based on its distinct morphology with phylogenetic support (Figs. 19, 20).

Fig. 19
figure19

Phylogram generated from maximum parsimony analysis based on combined LSU, ITS, tef1 and rpb2 sequence data. Related sequences are taken from Nuankaew et al. (2019) and retrieved from GenBank. Forty-nine strains were included in the analysis of the combined DNA loci and comprise 3309 characters (826 characters for LSU, 514 characters for ITS, 948 characters for tef1, 1021 characters for rpb2, including gaps). The tree is rooted with Anteaglonium globosum (ANM 925.2) and A. parvulum (MFLUCC 14-0821) in Anteagloniaceae. Maximum parsimony analysis of 471 parsimony informative characters resulted in a most parsimonious tree (CI = 0.674, RI = 0.879, RC = 0.593, HI = 0.326). The best scoring RAxML tree had a final likelihood value of −10433.015131. The matrix had 802 distinct alignment patterns with 28.12% undetermined characters and gaps. Estimated base frequencies were: A = 0.244372, C = 0.264291, G = 0.261114, T = 0.230223; substitution rates AC = 0.858179, AG = 3.949480, AT = 1.122065, CG = 0.761732, CT = 11.028490, GT = 1.000000; gamma distribution shape parameter α = 0.151089. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95). The ex-type strains are in bold and black. The newly generated sequence is in bold and blue

Fig. 20
figure20

The splits graph from the pairwise homoplasy index (PHI) test generated from the concatenated gene set of LSU, ITS, tef1 and rpb2 sequence data of closely related species using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicates significant recombination within the dataset

Hermatomyces Speg.

Hermatomyces tucumanensis is the type species. The genus has sporodochial conidiomata, and muriform, lenticular, hyaline or dematiaceous conidia of one or two types (Spegazzini 1911; Chang 1995; Tibpromma et al. 2016; Hashimoto et al. 2017; Hyde et al. 2019a). Examination of a Clematis collection revealed a novel species Hermatomyces clematidis based on its distinct morphological and phylogenetic relationship from other Hermatomyces. This is the first record of a Hermatomyces species on Clematis (Fig. 21).

Fig. 21
figure21

Hermatomyces clematidis (MFLU 17–1493, holotype). a, b Sporodochia on natural substrate. c Vertical section of sporodochia. d, e Subicular hyphae. f–h Cylindrical conidia. i–l Mature lenticular conidia. m Germinated conidium. n, o Culture characters on MEA. Scale bars: b = 500 μm, c = 100 μm, dm = 20 μm

Hermatomyces clematidis Phukhams., D.J. Bhat & K.D. Hyde, sp. nov.

Index Fungorum number: IF557134; Facesoffungi number: FoF 07244, Fig. 21.

Etymology: Name refers to the host plant, Clematis.

Holotype: MFLU 17–1493.

Saprobic on dead stem of Clematis sikkimensis. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on natural substrate forming sporodochial conidiomata, subiculate, superficial, scattered, circular or oval, blackish brown, velvety, glistening, orbicular, with abundant sporulation, conidia readily liberated when agitated, 410–565 μm wide (\( \bar{x} \) = 490 μm, n = 20). Mycelium 2–4 μm wide, mostly superficial, composed of a loose or compact network of repent, branched, septate, rough-walled, thick-walled, reddish brown to brown hyphae; subicular hyphae short, densely packed. Conidiophores 22–38 × 2–5 μm, micronematous or semi-macronematous, mononematous, cylindrical, erect, verruculose, aseptate, branched, often corresponding to conidiogenous cells, reddish brown to brown. Conidiogenous cells 7–13 × 4–7 μm, holoblastic, monoblastic, solitary, integrated, terminal, determinate, cylindrical or slightly subulate, subsphaerical or ampulliform, reddish brown to brown, sometimes hyaline. Conidia dimorphic solitary, smooth-walled; lenticular conidia: 30–45 × 24–31 μm (\( \bar{x} \) = 40 × 28 μm, n = 30), muriform, smooth, disc-shaped, circular to oval in front view, central cells brown to reddish brown, peripheral cells hyaline to subhyaline, forming a wide and distinct ring, slightly constricted at the septa, inside view composed of one column of 5–6 cells, end cells subhyaline to pale brown, often carrying remnant of conidiogenous cell at base; cylindrical conidia: 29–35 × 12–14 μm (\( \bar{x} \) = 32 × 13 μm, n = 20), straight or flexuous, septate, constricted at the septa, consisting of one or two columns, usually separate at apex, each column with 4–5 transverse septa, obclavate, apical cell acute, basal cells rounded, smooth, hyaline.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, white, dense, circular, margin erose, umbonate, papillate with fairly fluffy, wrinkled, folded, pale orange, covered with cream aerial mycelium; reverse dark brown at the centre, faintly zonate at the edge, greyish orange radiating outwardly.

Material examined: Thailand, Chiang Rai Province, on dead stem of Clematis sikkimensis, 2 May 2017, C. Phukhamsakda & M.V. de Bult, CMTHDT01 (MFLU 17–1493, holotype); ex-type living culture, MFLUCC 17–2085.

Host: Clematis sikkimensis—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214556; SSU: MT226673; ITS: MT310603; tef1: MT394735; rpb2: MT394684.

Notes: Hermatomyces clematidis is introduced as a new species based on its distinct morphology and phylogenetic results of a combined LSU, ITS, tef1, and rpb2 dataset (Fig. 19). Hermatomyces clematidis matches the generic concept in having sporodochial conidiomata, with both lenticular and cylindrical conidia (Doilom et al. 2017; Hashimoto et al. 2017, Fig. 21). Morphological comparison with known Hermatomyces species shows it is similar to H. tucumanensis, however, the conidiophore of H. clematidis are straighter with larger conidia ((22–)27–35 × 18–25 vs 30–45 × 24–31 µm) (Koukol et al. 2018). In the phylogenetic analysis, H. clematidis formed a close relationship with H. trangensis with strong support (100% MP/99% ML/1.00 BYPP). Hermatomyces trangensis is associated with sugar palm in southern Thailand (Nuankaew et al. 2019). Hermatomyces trangensis differs from H. clematidis because it lacks cylindrical conidia. In a BLASTn search of GenBank, the closest match of the LSU sequence of MFLUCC 17–2085 is H. subiculosa (MFLUCC 15–0843) with 97% similarity, while the closest match of the ITS sequence was H. tectonae (MFLUCC 14–1140) (NR_154079) with 97% similarity.

Based on current evidence, phylogenetic results from the concatenated gene loci does not delineate Hermatomyces species, especially the H. sphaericus clade (Fig. 19). Therefore, we applied the GCPSR concept with the H. sphaericus clade. Seventeen isolates of H. sphaericus formed an isolated clade with two species (H. biconisporus and H. pandanicola) forming distant relationship lineage with low statistic support. However, the pairwise homoplasy index showed Φw = 0.009 when the degree of genealogical correlation model was applied between neighbouring strains of the clade (Fig. 20). These results are not congruent with the phylogenetic lineages shown in Fig. 19, which rather indicate that H. biconisporus, H. pandanicola and H. sphaericus should currently be treated as the same species.

Leptosphaeriaceae Barr

Leptosphaeriaceae is typified by Leptosphaeria. Ariyawansa et al. (2015b) illustrated the members of Leptosphaeriaceae and included ten genera. Quaedvlieg et al. (2013) introduced an asexual morph genus, Acicuseptoria Quaedvl., Verkley & Crous to the family, however, a multilocus phylogenetic analysis (Fig. 22) reveals that Acicuseptoria clusters with Paraleptosphaeria. Crous and Groenewald (2017a) introduced Querciphoma isolated from stems and leaves of woody plants in a terrestrial environment. Members of this family usually have a single, papillate, immersed or erumpent, perithecial ascomata, with thick, scleroplectenchymatous or plectenchymatous cells and cylindrical asci with hyaline to brown, transversely septate ascospores (Hyde et al. 2013; Ariyawansa et al. 2015b). The asexual morphs in Leptosphaeriaceae can be coelomycetous or hyphomycetous (Gruyter et al. 2013; Hyde et al. 2013; Crous and Groenewald 2017). We introduce a novel Alloleptosphaeria species and describe a novel genus Sclerenchymomyces based on distinct morphology and phylogenetic support (Fig. 22).

Fig. 22
figure22

The best scoring RAxML tree with a final likelihood value of − 13918.604336 based on combined LSU, SSU, ITS and tef1 sequence data for Leptosphaeriaceae. The tree is rooted with a member of the Didymellaceae. Seventy strains were included in the combined gene sequence analyses, which comprise 3257 characters (831 characters for LSU, 951 characters for SSU, 566 characters for ITS, and 909 characters for tef1, including gap regions). The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The matrix had 649 distinct alignment patterns, with 45.29% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.244507, C = 0.225295, G = 0.271500, T = 0.258698; substitution rates AC = 1.503087, AG = 2.814632, AT = 2.230882, CG = 0.467105, CT = 7.217503, GT = 1.000000; gamma distribution shape parameter α = 0.541381. In our analysis, GTR + I + G model was used for every partition in Bayesian analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (PP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. The supported values from all analyses are BS ≥ 70%/BYPP ≥ 0.95

Alloleptosphaeria Ariyaw., Wanas. & K.D. Hyde

Alloleptosphaeria was introduced as a monotypic genus to accommodate A. italica from Clematis vitalba in Italy (Ariyawansa et al. 2015b). It has immersed ascomata and a thin-walled peridium of reddish brown to dark brown pseudoparenchymatous cells (Ariyawansa et al. 2015b). In our phylogenetic analysis (Fig. 22), Alloleptosphaeria formed a distinct clade basal to Leptosphaeria sensu stricto. We introduce the second species of Alloleptosphaeria from Clematis subumbellata collected in Thailand (Fig. 23).

Fig. 23
figure23

Alloleptosphaeria clematidis (MFLU 17–1479, holotype). a Appearance of ascomata on Clematis subumbellata. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–p Ascospores (p Ascospore in 10% Indian ink). q Germinated ascospore. r, s Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d, fi = 50 µm, e = 20 µm, jp = 10 µm

Alloleptosphaeria clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557109; Facesoffungi number: FoF 07286, Fig. 23.

Etymology: Epithet reflect the host genus Clematis.

Holotype: MFLU 17–1479.

Saprobic on dead stems of Clematis subumbellata. Sexual morph: Ascomata 210–260 × 125–190 μm (\( \bar{x} \)= 237 × 160 μm, n = 5), on surface of the host, covered by a pseudoclypeus, visible as black spots, immersed, solitary, scattered, uniloculate, obpyriform, coriaceous, black to dark brown, rough-walled, with apical ostioles. Ostioles 70–85 × 50–70 μm (\( \bar{x} \)= 80 × 60 μm, n = 5), central, pale brown to dark brown, papillate, opening by a pore, ostioles with periphyses. Peridium 9–17(–25 μm at apex) wide, thin, multilayered, pseudoparenchymatous cell type, comprising 4–5 layers of brown to dark brown cells of textura angularis, inner layers comprising thin, hyaline cells. Hamathecium composed of numerous, 5–3.5 μm wide (\( \bar{x} \)= 2.5 μm, n = 50), dense, filiform branched, anastomosing above asci, reaching the ostioles, transversely septate, cellular pseudoparaphyses. Asci 75–125 × 8–15 μm (\( \bar{x} \) = 105 × 10 μm, n = 40), 8-spored, bitunicate, fissitunicate, cylindrical, apically rounded, short, bulbous pedicel, ocular chamber clearly visible when immature. Ascospores 15–25 × 6–9 μm (\( \bar{x} \) = 18 × 7 μm, n = 50), uniseriate, partially overlapping, broad fusiform, tapering towards the ends, round at both ends, with (1–)4–5 transverse septa, 1-longitudinal septum in each ascospore, initially hyaline, yellowish at maturity, slightly constricted at the septa, deeply constricted at the median septum, cell above median septum slightly wider than that below, verruculose, with guttule in each cell, with thin mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 25 °C. Culture from above dark brown radiating outwardly, wrinkled folded in the middle, dense, circulate, flattened, umbonate, edge irregular, fairly fluffy; reverse black in the middle and dark brown at the edge, orange pigment slightly diffusing into the agar.

Material examined: Thailand, Chiang Rai Province, on dead stems of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH15 (MFLU 17–1479, holotype); ex-type living culture, MFLUCC 17–2071.

Host: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214557; SSU: MT226674; ITS: MT310604; tef1: MT394736; rpb2: MT394685.

Notes: Alloleptosphaeria clematidis is the first report of Alloleptosphaeria from Thailand and the second Alloleptosphaeria species known on Clematis. Alloleptosphaeria clematidis formed a robust clade with A. italica with strong support (97% ML/1.00 BYPP, Fig. 22). Alloleptosphaeria clematidis is compatible with the generic concept of Alloleptosphaeria in having immersed ascomata with a thin-walled peridium of brown to dark brown or reddish brown, pseudoparenchymatous cells (Ariyawansa et al. 2015b). Based on morphology, A. clematidis is distinguished by its cylindrical asci and the presence of 1 longitudinal septum in its yellowish ascospore (Fig. 23). In a BLASTn search of GenBank, the closest match of the ITS region of MFLUCC 17–2071 is 92.5% similarity to Subplenodomus iridicola strain CBS 143395 (NR_159068). Pairwise comparison of the ITS region of MFLUCC 17–2071 with A. italica (MFLUCC 14–0934) shows 97 nucleotide differences from 566 base pairs (17.13%).

Sclerenchymomyces Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557110; Facesoffungi number: FoF 07287, Fig. 24

Fig. 24
figure24

Sclerenchymomyces clematidis (MFLU 16–2492, holotype). a Appearance of conidiomata on Clematis vitalba. b Close up of conidioma on host substrate. c Vertical section through conidiomata. d Ostiole from above. e Section of conidioma wall. fi Conidiogenous cells and conidia. jl Conidia. Scale bars: b = 200 µm, c = 100 µm, d = 20 µm, e = 50 µm, fl = 5 µm

Etymology: Genus name reflects the characteristic of scleroplectenchyma tissue type.

Saprobic on dead or dead branch of herbaceous or woody plants in terrestrial habitats. Sexual morph: Ascomata covered by plant epidermis, located on the surface of host substrate, black, shiny, superficial to semi-immersed, solitary, globose, black, ostiolate. Ostioles central, short, filled with hyaline cells. Peridium composed of blackish to dark brown cells of textura angularis, thick, multilayered, scleroplectenchymatous cells, cells towards the inside lighter. Hamathecium composed of numerous, branched, septate, pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical, short-pedicellate. Ascospores uniseriate, partial overlapping, muriform, broad ellipsoidal, narrowed towards the ends, initially hyaline, becoming brown at maturity, constricted at median septum, guttulate, surrounded by a mucilaginous sheath (Wanasinghe et al. 2016). Asexual morph: Conidiomata pycnidial, solitary, sometimes aggregated, uniloculate or multiloculate, erumpent or superficial on host substrate, with black shiny ostioles, globose to subglobose, coriaceous, dark brown to brown. Ostioles central, papillate, oblong. Conidiomatal wall thick-walled, multilayered, scleroplectenchymatous or pseudoparenchymatous cells, flat at base, composed of textura angularis, lined with a thick hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells enteroblastic, phialidic, determinate, discrete, sub-cylindrical to truncate, smooth-walled, hyaline, arising from the inner layers of conidiomata. Conidia ellipsoid or cylindrical to oblong, rounded at both ends, slightly curved, hyaline when immature, yellowish at maturity, aseptate or septate, guttulate, smooth-walled.

Type species: Sclerenchymomyces clematidis Phukhams. & K.D. Hyde

Notes: Sclerenchymomyces is introduced for a lineage comprising Sclerenchymomyces clematidis and S. jonesii (≡ Neoleptosphaeria jonesii) which received strong support (99% ML/1.00 BYPP, Fig. 22). Based on the multi-gene phylogenetic analyses, Sclerenchymomyces formed a separate lineage to Alloleptosphaeria, Leptosphaeria sensu stricto and Neoleptosphaeria (62% ML/1.00 BYPP). The members of Leptosphaeriaceae are remarkable by their characteristic scleroplectenchymatous or pseudoparenchymatous tissue types (Ariyawansa et al. 2015b). Two isolates of Sclerenchymomyces share similar characters in having black, shiny, superficial to semi-immersed ascomata with a multilayer of scleroplectenchymatous tissue type (Wanasinghe et al. 2016a, Fig. 24). Sclerenchymomyces shares similar pycnidial characters with Leptosphaeria sensu stricto and Neoleptosphaeria in the scleroplectenchymatous or plectenchymatous cell type in the peridium (Ariyawansa et al. 2015b). However, Leptosphaeria species only have transverse septa while Sclerenchymomyces has longitudinal septa in the ascospores (Fig. 24). Sclerenchymomyces has oblong, brown phragmoconidia in nature and phoma-like characters in culture (Gruyter et al. 2013; Wanasinghe et al. 2016). The combined dataset of the LSU, SSU, ITS and tef1 sequences for Leptosphaeriaceae revealed a lineage of Sclerenchymomyces as a new genus from Clematis (Fig. 22).

Sclerenchymomyces clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557111; Facesoffungi number: FoF 07288, Fig. 24.

Etymology: Name refers to the host plant, Clematis.

Holotype: MFLU 16–2492.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Undetermined. Asexual morph: Conidiomata 187–225 × 85–217 μm (\( \bar{x} \)= 210 × 130 μm, n = 10), pycnidial, solitary, sometimes aggregated, uniloculate or multiloculate, erumpent or superficial on host substrate, with black shiny ostioles visible, globose to subglobose, coriaceous, dark brown to brown, ostiolate. Ostioles central, papillate, oblong. Conidiomatal wall 14–30(–35) μm wide, multilayered, scleroplectenchymatous cells, flat at base, outer layer composed of 5–9 layers of light brown to brown cells of textura angularis, lined with a thick hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 3–8 × 1.5–4 μm (\( \bar{x} \) = 5 × 3 μm, n = 30), enteroblastic, phialidic, determinate, discrete, sub-cylindrical to truncate, smooth-walled, hyaline, arising from the inner layers of conidiomata. Conidia 11–18 × 2.5–5 μm (\( \bar{x} \) = 14 × 4 μm, n = 50), broad cylindrical to oblong, rounded at both ends, hyaline when immature, yellowish at maturity, slightly curved, 3-septate, with 1(–2) guttules in each cell, smooth-walled.

Culture characters: Colonies on MEA reaching 40 mm diam. after 4 weeks at 25 °C. Cultures from above, cream-brown, spare mycelia, circular, umbonate, papillate with fluffy, covered with white aerial mycelium; reverse dark brown at the centre, cream radiating outwardly.

Material examined: Italy, Forlì-Cesena Province, near Meldola, on dead aerial branch of Clematis vitalba, 15 November 2013, E. Camporesi, 1518C (MFLU 16–2492, holotype); ex-type living culture, MFLUCC 17–2180.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214558; SSU: MT226675; ITS: MT310605; tef1: MT394737; rpb2: MT394686.

Notes: Sclerenchymomyces clematidis is distinct from S. jonesii in conidial characters (Fig. 24). Sclerenchymomyces clematidis has broad cylindrical to oblong, yellowish conidia with 3 septa and 1(–2) guttules, while S. jonesii has hyaline, aseptate conidia (Wanasinghe et al. 2016a). In a BLASTn search of GenBank, the LSU sequence of S. clematidis (strain MFLUCC 17–2180) was 98.7% similar to S. jonesii (≡ Neoleptosphaeria jonesii), while the ITS sequence showed 97.28% similarity to NR_152375. Pairwise comparison of the ITS sequence reveals nine bases pair differences (1.59%) between S. clematidis and S. jonesii (MFLUCC 16–1442). The tef1 region shows seven bases pair difference between S. clematidis and S. jonesii.

Sclerenchymomyces jonesii (Wanasinghe, Camporesi & K.D. Hyde) Phukhams. & K.D. Hyde, comb. nov.

Basionym: Neoleptosphaeria jonesii Wanasinghe, Camporesi & K.D. Hyde, in Wanasinghe, Camporesi & Hu, Mycosphere 7(9): 1373 (2016)

Index Fungorum number: IF552569; Facesoffungi number: FoF 02716

Notes: Wanasinghe et al. (2016a) introduced Neoleptosphaeria jonesii for a fungus whose morphology and phylogeny are related to Neoleptosphaeria rubefaciens (Gruyter et al. 2013; Wanasinghe et al. 2016a). The analyses of combined LSU, SSU, ITS and tef1 sequence data for Leptosphaeriaceae showed that the ex-type strain of Neoleptosphaeria jonesii (MFLUCC 16–1442) clustered with Sclerenchymomyces clematidis (MFLUCC 17–2180), a fungal strain reported from the same host. Pairwise comparison of the ITS sequence data reveals 85 bases pair difference from 566 (15.9%, including gap region) between N. jonesii (MFLUCC 16–1442) and N. rubefaciens (CBS 223.77 and CBS 387.80). According to phylogeny (Fig. 22) coupled with morphology, we transfer N. jonesii to Sclerenchymomyces.

Host: Clematis vitalba—(Wanasinghe et al. 2016a).

Distribution: Italy—(Wanasinghe et al. 2016a).

Longiostiolaceae Phukhams., Doilom & K.D. Hyde, fam. nov.

Index Fungorum number: IF557086; Facesoffungi number: FoF 07215, Fig. 25.

Fig. 25
figure25

Longiostiolum tectonae (MFLU 15–3532, holotype). a, b Appearance of ascomata on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Cellular pseudoparaphyses. g–i Asci. jl Ascospores. m Culture characters on MEA. n–q Conidiogenous cell. r Conidia. Scale bars: b = 200 µm, c, d = 100 µm, e–l = 50 µm, n = 10 µm, or = 5 µm

Saprobic on dead bark. Sexual morph: Ascomata immersed to semi-immersed, uniloculate, solitary, globose to subglobose, coriaceous, base flattened, ostiolate. Ostioles long, obtuse or dolabriform, central. Peridium thick, comprising several layers of scleroplectenchymatous or pseudoparenchymatous cell types, dark brown to black cells arranged in a textura angularis or textura globosa. Hamathecium composed of numerous, filiform, septate, branched, cellular pseudoparaphyses. Asci 4–8-spored, bitunicate, cylindrical to clavate, pedicellate, with ocular chamber. Ascospores biseriate, partial overlapping, broad fusiform, hyaline, brownish at the mature state, multi-septate, with or without mucilaginous sheath. Asexual morph: coelomycetous-like or hyphomycetous-like structures produced in culture. Conidiomata scattered, globose to subglobose, ostiolate. Conidiomatal wall thick, comprising of multilayered textura angularis, pale brown to brown cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells annellidic, doliiform to ampulliform. Conidia cylindrical, hyaline, bud scars disjunctors at base, multi-septate, smooth, without sheath (Matsumura et al. 2018). Colonies on MEA produce conidia structures on aerial mycelium, subglobose to ellipsoidal, initially hyaline, becoming black, aseptate, smooth-walled.

Type genus: Longiostiolum Doilom, Ariyaw. & K.D. Hyde, in Li et al., Fungal Diversity 78: 55 (2016)

Notes: Longiostiolaceae is introduced to accommodate two ascomycetous genera, Crassiperidium and Longiostiolum. Longiostiolum was introduced by Li et al. (2016) for a fungus associated with Tectona grandis from northern Thailand. Matsumura et al. (2018) reported Crassiperidium from twigs of Fagus crenata in Japan. Crassiperidium and Longiostiolum were associated with wood and share characters in having immersed to semi-immersed, uniloculate ascomata, thick-walled scleroplectenchymatous or pseudoparenchymatous cell, with hyaline ascospores turning brownish at maturity (Li et al. 2016; Matsumura et al. 2018). The asexual morphs of these two genera are different, with Crassiperidium forming pycnidial conidiomata with hyaline conidia, and Longiostiolum forming a hyphomycetous-like structure with globose, black conidia in culture (Fig. 25).

Matsumura et al. (2018) found that Crassiperidium was related to Cyclothyriellaceae (Pleosporales) based on phylogeny of an SSU, LSU and rpb2 sequence dataset. Longiostiolum was assigned as an incertae sedis taxon in Pleosporales based on combined LSU, SSU, rpb2 and tef1 sequence data (Li et al. 2016). Phylogenetic analysis based on the LSU regions of Crassiperidium and Longiostiolum formed a clade together with moderate support (57% ML, data not shown) and sister to Cyclothyriellaceae. The concatenated dataset of LSU, SSU, ITS, tef1 and rpb2 showed Crassiperidium species (C. octosporum and C. quadrisporum) and Longiostiolum tectonae (MFLUCC 12–0562) forming a close relationship with strong support (98% ML/1.00 BYPP) and related to Cyclothyriellaceae (Fig. 2). Therefore, we introduce a new family to accommodate this distinct lineage.

Type genus: Longiostiolum Doilom, Ariyaw. & K.D. Hyde, in Li et al., Fungal Diversity 78: 55 (2016).

Type species: Longiostiolum tectonae Doilom, Bhat & K.D. Hyde, in Li et al., Fungal Diversity 78: 55 (2016).

Genera included: Crassiperidium Matsum. & Kaz. Tanaka (Matsumura et al. 2018); Longiostiolum Doilom, Ariyaw. & K.D. Hyde (Li et al. 2016).

Hosts: Fagus crenata, Tectona grandis—(Li et al. 2016; Matsumura et al. 2018).

Distribution: Japan, Thailand—(Li et al. 2016; Matsumura et al. 2018).

Longiostiolum tectonae Doilom, Bhat & K.D. Hyde, in Li et al., Fungal Diversity 78: 55 (2016)

Index Fungorum number: IF 551900, Facesoffungi number: FoF 01882, Fig. 25.

Notes: Based on the phylogenetic analysis of the combined LSU, SSU, rpb2 and tef1 sequence data, the genus Longiostiolum tectonae (MFLUCC 12–0562) formed a distinct lineage in Pleosporales with no statistical support (Li et al. 2016). In a BLASTn search of GenBank, the closest match of the LSU sequence of MFLUCC 12–0562 is Crassiperidium octosporum (strain MAFF 242971) with 97% similarity, while the ITS sequence showed 86.5% similarity.

Host: Tectona grandis—(Li et al. 2016).

Distribution: Thailand—(Li et al. 2016).

Lophiostomataceae Sacc. [as ‘Lophiostomaceae’]

Lophiostomataceae was accepted by Mugambi and Huhndorf (2009). Members of this family are saprobes in terrestrial and aquatic habitats (Saccardo 1883; Tanaka and Harada 2003; Thambugala et al. 2015; Hashimoto et al. 2018). The family is characterized by carbonaceous ascomata with a slit-like ostiolar neck or opening (Zhang et al. 2009). A revision of Lophiostomataceae by Thambugala et al. (2015) based on multi-locus phylogeny along with the re-examination of holotype specimens revealed the boundaries of the family. Hashimoto et al. (2018) revised the classification based mainly on ascospore appendages and introduced several new genera. Mapook et al. (2020) introduced Pseudocapulatispora Mapook & K.D. Hyde and currently, there are 25 genera in the family (Wijayawardene et al. 2020). A phylogenetic analysis based on a combined LSU, ITS, SSU, tef1 and rpb2 sequence dataset (Figs. 26, 27) of lophiostomataceous taxa on Clematis from Europe and Asia revealed novel species of Neovaginatispora, Pseudocapulatispora, Pseudolophiostoma and Sigarispora, which are introduced with morphological support.

Fig. 26
figure26

The best scoring RAxML tree with a final likelihood value of − 33003.460353 based on combined LSU, ITS, SSU, tef1 and rpb2 sequence data. The tree is rooted with Teichospora rubriostiolata (TR7) and T. trabicola (C134) in Teichosporaceae. One hundred and two strains were included in the combined sequence analyses which comprise 5255 characters (1300 characters for LSU, 921 characters for ITS, 1004 characters for SSU, 992 characters for tef1 and 1038 characters for rpb2, including gaps). The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The matrix had 1938 distinct alignment patterns with 36.36% undetermined characters or gaps proportions. Estimated base frequencies were as follows: A = 0.248970, C = 0.248285, G = 0.267331, T = 0.235414; substitution rates AC = 1.347247, AG = 3.679551, AT = 1.191656, CG = 1.272401, CT = 7.723696, GT = 1.000000; gamma distribution shape parameter α = 0.579807. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses at the genus level (BS ≥ 70%/BYPP ≥ 0.95)

Fig. 27
figure27

The splits graph from the pairwise homoplasy index (PHI) test generated from the concatenated gene set of LSU, ITS, SSU, tef1 and rpb2 sequence data of closely related species of Neovaginatispora (a), Pseudolophiostoma (b) and Sigarispora (c) using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicates significant recombination within the dataset. The strains determined in this study are in bold and blue

Neovaginatispora Hashim., K. Hiray. & Kaz. Tanaka

Hashimoto et al. (2018) reanalysed Lophiostomataceae and segregated Neovaginatispora fuckelii (Sacc.) Hashim., K. Hiray. & Kaz. Tanaka from the type species of Vaginatispora based on phylogenetic analyses. Neovaginatispora is distinct in its thin, sub-carbonaceous peridium of uniform thickness (Thambugala et al. 2015). We introduce a second species of Neovaginatispora from Clematis viticella (Fig. 28).

Fig. 28
figure28

Neovaginatispora clematidis (MFLU 17–1514, holotype). a Appearance of ascomata on host surface. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Culture characteristic on MEA. Scale bars: b = 200 µm, c = 100 µm, d = 50 µm, ei = 20 µm, jn = 10 µm

Neovaginatispora clematidis Phukhams., D. Ertz & C. Gerstmans & K.D. Hyde, sp. nov.

Index Fungorum number: IF557117; Facesoffungi number: FoF 07289, Fig. 28.

Etymology: Name refers to the host plant, Clematis.

Holotype: MFLU 17–1514.

Saprobic on dead stems of Clematis viticella. Sexual morph: Ascomata 145–250 × 108–160 μm (\( \bar{x} \) = 172 × 134 μm, n = 10), solitary, gregarious, semi-immersed to erumpent, globose to compressed, coriaceous, dark brown to black, ostiolate. Ostioles 50–65 × 76–83 μm (\( \bar{x} \) = 58 × 80 μm, n = 5), with a crest-like apex, central, filled with hyaline periphyses. Peridium 13–28 μm wide (\( \bar{x} \) = 19 μm, n= 20), uniform, wider and heavily pigmented at the apex, composed of 5(–6) layers of somewhat flattened, thin-walled cells of textura angularis, cells towards the inside lighter, inner layer composed of thin hyaline gelatinous layer. Hamathecium composed of numerous, dense, 2–3 µm wide, filamentous, branched, septate, pseudoparaphyses, embedded in a gelatinous matrix. Asci 53–105 × 9–12 µm (\( \bar{x} \) = 78 × 11 µm, n = 40), 8-spored, bitunicate, fissitunicate, cylindrical-clavate to clavate, with short, bulbous pedicel, apically rounded, with an ocular chamber. Ascospores 16–19 × 5–7 µm (\( \bar{x} \) = 16 × 6 µm, n = 40), biseriate or partially overlapping, hyaline, broad fusiform with acute ends, tapering towards the ends, 1-euseptate, strongly constricted at the septum, upper cell broader than lower cell, smooth-walled, with two guttules in each cell, with 2 µm wide globose appendages at both ends. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, dark brown, dense, circular, umbonate, rough surface, dull, undulate, radially furrowed, covered with grey aerial mycelium, oil droplets formed in the middle of cultures; reverse black radiating outwardly.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis viticella, 13 June 2017, D. Ertz & C. Gerstmans, BRCV2 (MFLU 17–1514, holotype); ex-type living culture, MFLUCC 17–2156.

Host: Clematis viticella—(This study).

Distribution: Belgium—(This study).

GenBank accession numbers: LSU: MT214559; SSU: MT226676; ITS: MT310606; tef1: MT394738.

Notes: Neovaginatispora clematidis is compatible with the concept of Neovaginatispora in having thin, uniform peridium layers with short and globose appendages at both ends (Thambugala et al. 2015). Neovaginatispora clematidis was found on Clematis viticella in Belgium whereas Neovaginatispora fuckelii has mainly been isolated from herbaceous plant in Japan except the strain MFLUCC 17–2652 that was isolated from Mangifera indica in Taiwan. Neovaginatispora clematidis is somewhat similar to the type specimen of N. fuckelii (CBS 101952), but being distinguishable in its broad fusiform ascospores with a single eusepta (Fig. 28). Neovaginatispora fuckelii and N. clematidis are reported from different continents and closely related by morphology but distinct over the five gene loci phylogeny.

In the multigene phylogenetic analyses, the strain formed a strongly supported clade with N. fuckelii (98% ML/1.00 BYPP, Fig. 26). This clade contains four isolates of N. fuckelii and a single isolate of N. clematidis (Fig. 26). The ITS sequence shows six nucleotide differences while the tef1 sequence has 10 nucleotide differences. In a BLASTn search of the GenBank, the ITS sequence has 98% similarity to Vaginatispora aquatica (MFLUCC 11–0083), while the tef1 sequence has 93.49% similarity to V. scabrispora (LC312583). The GCPSR concept was applied to clade (a) for testing significant recombination between these isolates (Laurence et al. 2014). A pairwise homoplasy index of Φw = 1.0 showed that there is no significant recombination of the gene flow in N. clematidis and N. fuckelii isolates (Fig. 27a). The new species of Neovaginatispora is therefore introduced based on morphological support and phylogenetic analysis.

Pseudocapulatispora Mapook & K.D. Hyde

Pseudocapulatispora was introduced by Mapook et al. (2020) with Pseudocapulatispora longiappendiculata as the type species. The genus is characterized by slit-like ostioles, a peridium of textura prismatica and ascospores with relatively long appendages at the end of capped sheaths. We introduce a second species of Pseudocapulatispora from Clematis subumbellata based on morphology (Fig. 29) and phylogenetic analysis (Fig. 26).

Fig. 29
figure29

Pseudocapulatispora clematidis (MFLU 17–1469, holotype). a Appearance of ascoma on host surface. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Ascospore in cotton blue showing the end chambers. o, p Culture characteristic on MEA. Scale bars: b = 500 µm, c = 200 µm, d–i = 50 µm, jn = 20 µm

Pseudocapulatispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557118; Facesoffungi number: FoF 07290, Fig. 29.

Etymology: The epithet reflects the host, Clematis.

Holotype: MFLU 17–1469.

Saprobic on dead stem of Clematis subumbellata.Sexual morph: Ascomata 197–386 × 160–252 µm (\( \bar{x} \) = 300 × 209 μm, n = 10), solitary, scattered, immersed, with only black shiny ostioles visible, subglobose to compressed, base flattened, coriaceous to carbonaceous at the apex, brown to black, with a developed pseudoclypeus, ostiolate. Ostioles 84–148 × 56–86 μm (\( \bar{x} \) = 107 × 66 μm, n = 5), with a crest-like apex, central, long, elongated and laterally compressed, surrounded by a small blackened pseudoclypeus, with irregular wall, filled with hyaline periphyses. Peridium 10–23(–38 µm at apex) wide (\( \bar{x} \) = 23 µm, n= 20), uniform, wider at the apex, heavily pigmented at the apex, composed of 4(–5) layers of textura prismatica, cells towards the inside lighter, inner layer composed of thin, hyaline gelatinous layer, at the apex fusing and indistinguishable from the host tissues. Hamathecium composed of numerous, dense, 1.5–3 µm wide, filamentous, branched, septate, pseudoparaphyses, situated between and above the asci, embedded in a gelatinous matrix. Asci 68–117 × 12–22 µm (\( \bar{x} \) = 99 × 18 µm, n = 40), 8-spored, bitunicate, fissitunicate, oblong to cylindrical-clavate, with short, furcate pedicel, apically rounded, with an ocular chamber. Ascospores 22–28 × 7–12 µm (\( \bar{x} \) = 25 × 9 µm, n = 50), biseriate or partially overlapping, ellipsoid, tapering towards the ends, acute ends, hyaline, 1-euseptate, strongly constricted at the septum, with guttule in each cell, slightly swollen near median septum, sheath drawn out from both ends to form polar appendages, 14–25 × 3–5 µm (\( \bar{x} \) = 19 × 4 µm, n = 50), end caps visible at the ends of the appendages, with a lateral pad-like structure, up to 4 µm wide at side. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, pale green to yellow brown in the middle, dense, circular, umbonate, surface rough, dull, fimbriate, radially furrowed, covered with yellow aerial mycelia, oil droplets formed in the middle of the culture; reverse cream radiating outwardly, yellow pigment diffusing in the agar.

Material examined: Thailand, Phayao Province, Phu Sang District, dead stems of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH05 (MFLU 17–1469, holotype); ex-type living culture, MFLUCC 17–2063.

Hosts: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU; MT214560; SSU: MT226677; ITS: MT310607; tef1: MT394739; rpb2: MT394687.

Notes: In the phylogeny (Fig. 26), this species clustered with the type species, Pseudocapulatispora longiappendiculata (Mapook et al. 2020). Morphological comparison of P. clematidis and P. longiappendiculata revealed that P. clematidis (Fig. 29) has larger ascomata (300 × 209 vs 240 × 135 µm), with shorter polar appendages (19 × 4 vs 25 × 4.5 µm). The tef1 sequence of Pseudocapulatispora clematidis (MFLU 17–1469) had 95% similarity to P. longiappendiculata (5% nucleotide differences in the tef1 region). Therefore, the new strain is introduced as a new species based on morphology and phylogenetic evidence.

Pseudolophiostoma Thambug., Kaz. Tanaka & K.D. Hyde

Pseudolophiostoma vitigenum is the type species. The genus was introduced for a lophiostomataceous taxon that formed a distinct clade from the type species of Lophiostoma (Hirayama and Tanaka 2011; Thambugala et al. 2015). Five species are listed in Index Fungorum (Index Fungorum 2020). We introduce two new species namely; P. chiangraiensis and P. clematidis, to accommodate species from Thailand which occurred on Clematis species (Figs. 30, 31).

Fig. 30
figure30

Pseudolophiostoma chiangraiense (MFLU 17–1484, holotype). a Appearance of ascoma on host surface. b Close up of ascoma on host substrate. c Vertical section of ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Senescent spores. o Culture characteristics on MEA. Scale bars: b, c = 200 µm, d–i = 50 µm, jn = 10 µm

Fig. 31
figure31

Pseudolophiostoma clematidis (MFLU 17–1489, holotype). a Appearance of ascoma on host surface. b Close-up of ascoma on host substrate. c Vertical section of ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–h Asci. i–l Ascospores. m Germinated ascospore n, o Culture characteristics on MEA. Scale bars: a = 500 µm, b, c = 200 µm, d, e = 100 µm, fh = 50 µm, il = 10 µm

Pseudolophiostoma chiangraiense Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557119; Facesoffungi number: FoF 07291, Fig. 30.

Etymology: The epithet reflects the location where the fungus was collected.

Holotype: MFLU 17–1484.

Saprobic on dead stem of Clematis fulvicoma.Sexual morph: Ascomata 276–293 × 94–294 μm (\( \bar{x} \) = 286 × 239 μm, n = 10), solitary, scattered, sometimes gregarious, immersed, with only black shiny ostioles visible, subglobose or compressed, flattened base, coriaceous, carbonaceous at the apex, dark brown to black, with a well-developed clypeus, indistinguishable from substrate, ostiolate. Ostioles 74–127 × 88–129 μm (\( \bar{x} \) = 94 × 103 μm, n = 5), with opening by a pore, central, elongated and laterally compressed, irregular wall, carbonaceous, black, filled with hyaline periphyses. Peridium 14–32 μm wide (\( \bar{x} \) = 23 μm, n= 20), uniform, wider at the apex, heavily pigmented at the apex, composed of 3(–4) layers of textura angularis and textura prismatica, thin-walled cells, cells towards inside lighter than outside, somewhat flattened, inner layer composed of thin, hyaline gelatinous layer, at the apex fusing and indistinguishable from the host tissues. Hamathecium composed of numerous, dense, 2–3 µm wide, filamentous, branched, septate, cellular pseudoparaphyses, embedded in a gelatinous matrix. Asci 78–119 × 11–16 µm (\( \bar{x} \) = 96 × 13 µm, n = 40), 8-spored, bitunicate, fissitunicate, broad cylindrical to cylindrical-clavate, with furcate pedicel, apically rounded, with an ocular chamber. Ascospores 20–32 × 4–7 µm (\( \bar{x} \)= 26 × 6 µm, n = 50), uniseriate, overlapping, hyaline, becoming pale brown at senescence, broad-fusiform, tapering towards the ends, acute ends, 1-euseptate, strongly constricted at the septum, with 1(–2) guttules in each cell, slightly swollen near median septum, with 6–10 µm wide sheath drawn-out to form polar appendages, sheath drawn out, with a pad-like structure, up to 4 μm wide at side. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, olive-brown to dark brown in the middle, dense, circular, convex with papillate surface, fluffy, rough surface, radially furrowed, covered with grey aerial mycelium, oil droplets formed in the middle of culture; reverse dark brown, radiating outwardly.

Material examined: Thailand, Chiang Rai Province, Mae Sai District, dead stems of Clematis fulvicoma, 20 March 2017, C. Phukhamsakda & M. van de Bult, CMTH21 (MFLU 17–1484, holotype); ex-type living culture, MFLUCC 17–2076.

Host: Clematis fulvicoma—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214561; SSU: MT226678; ITS: MT310608; tef1: MT394740; rpb2: MT394688.

Notes: In the phylogenetic analysis, Pseudolophiostoma chiangraiensis (MFLUCC 17–2076) formed a close relationship with P. cornisporum KH 322 (100% ML/1.00 BYPP, Fig. 30). We compared P. chiangraiensis (MFLUCC 17–2076) with P. cornisporum (KH 322) and both have similar characters in being immersed, with only black shiny ostioles visible on the host, and broad-fusiform, 1-euseptate ascospores, with acute ends (Fig. 30). Ascomata of P. chiangraiensis are smaller than P. cornisporum (286 × 239 vs 650–700 × 580–650 μm). In a BLASTn search of GenBank, the ITS sequence had 99% similarity to P. cornisporum KH 322 (NR_158930), while the rpb2 gene was 98% similar to LC312602.

Pseudolophiostoma clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557120; Facesoffungi number: FoF 07292, Fig. 31.

Etymology: The epithet reflects the host, Clematis.

Holotype: MFLU 17–1489.

Saprobic on dead stem of Clematis fulvicoma.Sexual morph: Ascomata 352–370 × 139–243 μm (\( \bar{x} \) = 358 × 220 μm, n = 10), solitary, scattered, immersed, with only black shiny ostioles visible, with globose to compressed, flattened base, coriaceous to carbonaceous at the apex, dark brown to black, with a developed pseudoclypeus, ostiolate. Ostiole 140–159 × 60–80 μm (\( \bar{x} \) = 150 × 70 μm, n = 5), with a crest-like apex and with opening by a pore, central, elongated and laterally compressed, surrounded by a small blackened pseudoclypeus, irregular wall, filled with hyaline periphyses. Peridium 9–28 μm wide (\( \bar{x} \) = 14 μm, n= 20), uniform, wider at the apex, heavily pigmented at the apex, composed of 4(–5) layers of textura prismatica, cells towards the inside lighter, somewhat flattened, inner layer composed of thin, hyaline gelatinous layer, at the apex fusing and indistinguishable from the host tissues. Hamathecium composed of numerous, dense, 2–3 µm wide, filamentous, branched, septate, pseudoparaphyses, situated between and above the asci, embedded in a gelatinous matrix. Asci 67–106 × 10–14 µm (\( \bar{x} \) = 88 × 12 µm, n = 40), 8-spored, bitunicate, fissitunicate, oblong to cylindrical-clavate, with short, furcate pedicel, apically rounded, with an ocular chamber. Ascospores 23–31 × 5–9 µm (\( \bar{x} \) = 26 × 7 µm, n = 50), biseriate or partially overlapping, hyaline, broad-fusiform, tapering towards the ends, round at the end, 1-euseptate, strongly constricted at the septum, with 2(–3) guttules in each cell, slightly swollen near median septum, with 6–10 µm sheath drawn out to form polar appendages, with a lateral pad-like structure, up to 3 μm wide. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, olive gray to dark brown in the middle, dense, circular, umbonate, surface rough, dull, fimbriate, radially furrowed, covered with grey aerial mycelium, oil droplets formed in the middle of culture; reverse: dark brown radiating outwardly.

Material examined: Thailand, Chiang Rai Province, Mae Sai District, dead stems of Clematis fulvicoma, 20 March 2017, C. Phukhamsakda & M. van de Bult, CMTH27 (MFLU 17–1489, holotype); ex-type living culture, MFLUCC 17–2081.

Host: Clematis fulvicoma—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214562; SSU: MT226679; ITS: MN393004; tef1: MT394741; rpb2: MT394689.

Notes: Pseudolophiostoma clematidis (MFLUCC 17–2081) formed a sister clade to P. obtusisporum strains (100% ML/1.00 BYPP, Fig. 26). Pseudolophiostoma obtusisporum is commonly reported on herbaceous plants or palms in Japan (Hashimoto et al. 2018). The morphology of P. clematidis resembles P. obtusisporum except for the larger ascomata with thinner peridium (Fig. 31). A comparison of sequence data revealed that the two species differ in all studied loci (2 bp differences in ITS, 15 bp in tef1 and 13 bp in rbp2). In a BLASTn search of GenBank, the ITS sequence had 99% similarity to the type specimen of Pseudolophiostoma obtusisporum (HHUF 30583), while rpb2 sequence had 98% similarity to LC312605, derived from the same voucher specimen.

This strain was further evaluated for secondary metabolites and biological activities. Pseudolophiostoma clematidis (MFLUCC 17–2081) showed moderate growth of microbes and cytotoxicity in vitro (Macabeo et al. 2020).

Sigarispora Thambug. & K.D. Hyde

Sigarispora was introduced by Thambugala et al. (2015) to accommodate Lophiostoma ravennicum and some lophiostomataceous taxa that formed a separate clade from the type species of Lophiostoma. Sigarispora is characterized by its immersed to semi-immersed ascomata, with a small crest-like ostioles, and brown cigar-shaped, multi-septate ascospores (Thambugala et al. 2015; Wanasinghe et al. 2018). Fourteen species are listed in Index Fungorum (Jayasiri et al. 2015; Index Fungorum 2020). We introduce three new species of Sigarispora from Clematis species, S. clematidicola, S. clematidis and S. montana (Figs. 32, 33, 34).

Fig. 32
figure32

Sigarispora clematidicola (MFLU 20–0419, holotype). a Appearance of ascoma on host surface. b Close up of ascoma. c Vertical section of ascoma. d Ostiolar canal. e Peridium. f Pseudoparaphyses. g–i Asci. j–o Ascospores (o Ascospore in 10% Indian ink; note the boundary of polar appendages). Scale bars: c, d = 100 µm, e, f = 50 µm, gi = 20 µm, jo = 10 µm

Fig. 33
figure33

Sigarispora clematidis (MFLU 20–0417, holotype). a Appearance of ascoma on host surface. b Close up of ascoma. c Section of ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Culture characteristics on MEA. Scale bars: b = 500 µm, c = 200 µm, di = 50 µm, jm = 10 µm

Fig. 34
figure34

Sigarispora montanae (MFLU 20–0418, holotype). a Appearance of ascomata on host surface. b Vertical section of ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f, g Asci. h–k Ascospores. l Germinated ascospore. m, n Culture characteristics on MEA. Scale bars: b = 100 µm, cg = 50 µm, hk = 10 µm

Sigarispora clematidicola Phukhams., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557121; Facesoffungi number: FoF 07293, Fig. 32.

Etymology: The epithet reflects the host Clematis.

Holotype: MFLU 20–0419.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 255–288 × 217–254 μm (\( \bar{x} \) = 276 × 238 μm, n = 10), solitary, scattered immersed, with only black shiny ostioles present, dark brown to black, globose to compressed, coriaceous, rough-walled, sometimes with dark brown hyphae projecting from the peridium, pseudoclypeus, ostiolate. Ostioles 60–135 × 67–156 μm (\( \bar{x} \)= 97 × 102 μm, n = 5), with a crest-like apex, central, elongated and laterally compressed, irregular wall, filled with hyaline periphyses. Peridium 13–48 μm wide (\( \bar{x} \) = 29 μm, n= 20), wider at the apex, thinner at the base, with 6–7 layers of lightly pigmented light brown to brown, thick-walled cells of textura angularis, lighter pigmented cells towards inside, somewhat flattened, inner layer composed of hyaline gelatinous layer, fusing and indistinguishable from the host tissues. Hamathecium numerous, dense, 2–3 µm wide, filamentous, branched, septate, pseudoparaphyses, embedded in a gelatinous matrix. Asci 101–125 × 12–19 µm (\( \bar{x} \) = 115 × 16 µm, n = 40), 8-spored, bitunicate, fissitunicate, broad cylindrical to clavate, with furcate pedicel, rounded at the apex, with an ocular chamber. Ascospores 22–29 × 7–9 µm (\( \bar{x} \) = 26 × 8 µm, n = 50), biseriate or partially overlapping, initially hyaline, becoming yellowish brown at maturity, broad fusiform, tapering towards the end, mostly curved (3–)5-transversely euseptate, constricted at the septa, cells above central septum swollen, guttulate, indentations present, without or with 2–4 µm sheath drawn out to form polar appendages. Asexual morph: Undetermined.

Culture characters: Colonies on MEA, slow-growing, reaching 20 mm diam. after 4 weeks at 25 °C. Culture centrally black dense, circular, flat, umbonate, surface rough; reverse: mycelium strongly radiating into agar, black.

Material examined: Italy, Forlì-Cesena Province, Viale Salinatore—Forlì, dead aerial branch of Clematis vitalba, 23 February 2015, E. Camporesi, IT2389-A (MFLU 20–0419, holotype); ex-type living culture, MFLUCC 16–0446.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214563; SSU: MT226680; ITS: MT310609; tef1: MT394742.

Notes: Based on phylogenetic evidence, the isolates of Sigarispora caudata (KT 530, MAFF 239453), S. clematidicola (MFLUCC 16–0446) and S. clematidis (MFLUCC 16–1368) formed a moderately supported clade (Fig. 26). Sigarispora clematidicola (Fig. 32) and S. clematidis (Fig. 33) share morphological similarity such as brown ascospores with polar appendages, while S. caudata lacks polar appendages (Table 3). Sigarispora clematidicola, differs from S. clematidis in having smaller ascomata, asci and ascospores (Table 3). A pairwise comparison of tef1 sequences of S. clematidis and S. caudata showed 98% similarity with 11 nucleotide differences. The tef1 sequences of S. clematidicola and S. clematidis showed 30 nucleotide differences. A pairwise homoplasy index showed Φw = 1.0 when genealogical correlation model was applied between neighboring strains of clade (c) (Fig. 27c). The result is congruent with the phylogenetic lineages shown in Fig. 26. Thus, S. caudata (KT 530, MAFF 239453), S. clematidicola (MFLUCC 16–0446) and S. clematidis (MFLUCC 16–1368) are significantly different from each other based on molecular as well as morphological data.

Table 3 Morphological comparison of known Sigarispora species

Sigarispora clematidis Phukhams., & K.D. Hyde, sp. nov.

Index Fungorum number: IF557122; Facesoffungi number: FoF 07294, Fig. 33

Etymology: The epithet reflects the host Clematis.

Holotype: MFLU 20–0417

Saprobic on dead stem of Clematis vitalba. Sexual morph: Ascomata 416–493 ×337–386 μm (\( \bar{x} \) = 444 × 369 μm, n = 10), solitary, scattered, immersed, with only black shiny ostioles visible, globose to compressed, coriaceous, dark brown to black, rough-walled, ostiolate. Ostioles 151–187 × 107–170 μm (\( \bar{x} \) = 167 × 129 μm, n = 5), with a crest-like apex and with opening by a pore, central, elongated and laterally compressed, irregular wall, filled with hyaline periphyses, pseudoparenchymatous cells. Peridium 6–46(–76 μm at apex) wide (\( \bar{x} \) = 32 μm, n = 20), wider at the apex, thinner at the base, with lightly pigmented, thick-walled cells of textura angularis, cell towards the inside lighter, somewhat flattened, inner layer composed of thick, hyaline gelatinous layer. Hamathecium composed of numerous, dense, 1.5–2.5 µm wide, filamentous, branched, septate, pseudoparaphyses, situated between and above the asci embedded in a gelatinous matrix. Asci 96–145 × 10–18 µm (\( \bar{x} \) = 118 × 14 µm, n = 40), 8-spored, bitunicate, fissitunicate, clavate, with furcate pedicel, rounded at the apex, with an ocular chamber. Ascospores 22–30 × 6–9 µm (\( \bar{x} \) = 24 × 7 µm, n = 60), biseriate or partially overlapping, broad fusiform, tapering towards the ends, initially hyaline, becoming yellowish brown at maturity, acute ends, mostly curved, 5–6 transversely eusepta, slightly constricted at the septa, cells above central septum swollen, guttulate, indentations present, with 5–10 µm long sheath drawn out to form polar appendages. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, centrally black, dense, circular, flat, umbonate, surface rough, dull, fimbriate, radially furrowed, slightly covered with white aerial mycelium; reverse: mycelium strongly radiating into the agar, black with radiating brown outwardly.

Material examined: UK, Hampshire, Swanick Lake, dead stems of Clematis vitalba, 9 July 2016, E.B.G. Jones, GJ307 (MFLU 20–0417, holotype); ex-type living culture, MFLUCC 16–1368.

Host: Clematis vitalba—(This study).

Distribution: UK—(This study).

GenBank accession numbers: LSU: MT214564; SSU: MT226681; ITS: MT310610; tef1: MT394743.

Notes: See note under Sigarispora clematidicola.

Sigarispora montanae Phukhams., Sue, K.D. Hyde, sp. nov.

Index Fungorum number: IF557124; Facesoffungi number: FoF 07295, Fig. 34.

Etymology: The epithet reflects the host species, Clematis montana.

Holotype: MFLU 20–0418.

Saprobic on dead stems of Clematis montana.Sexual morph: Ascomata 180–230 × 140–200 μm (\( \bar{x} \) = 200 × 160 μm, n = 5), solitary, scattered, semi-immersed, with black shiny ostioles, globose, coriaceous, partial carbonaceous at the apex, dark brown to black, rough-walled, forming a clypeus like character, ostiolate. Ostioles (40–)70–100 × 30–60 μm (\( \bar{x} \)= 80 × 50 μm, n = 5), with a crest-like apex, central, elongated and laterally compressed, irregular wall, filled with hyaline periphyses. Peridium 13–30 μm wide (\( \bar{x} \) = 20 μm, n= 30), wider at the apex, thinner at the base, with 6–7 layers of lightly pigmented light brown to dark brown, thick-walled cells of textura angularis, cells towards the inside lighter, at the outside darker, inner layer composed of thick hyaline gelatinous layer, fusing and indistinguishable from the host tissues. Hamathecium composed of numerous, dense, 1.2–1.5 µm (\( \bar{x} \) = 1.3 μm, n= 30), filamentous, branched, septate, anastomosing, pseudoparaphyses, embedded in a gelatinous matrix. Asci 105–130 × 10–14 µm (\( \bar{x} \) = 120 × 15 µm, n = 20), 8-spored, bitunicate, fissitunicate, broad cylindrical to clavate, with a long, with furcate pedicel, rounded at the apex, with an ocular chamber. Ascospores 20–26 × 5–7 µm (\( \bar{x} \) = 22 × 5 µm, n = 50), biseriate or partially overlapping, broad fusiform, tapering towards the ends, initially hyaline, becoming yellowish brown at maturity, mostly curved, 3(–5) transversely eusepta, constricted at the septa, cells above central septum swollen, indentations present, with 3–5 µm sheath drawn out to form polar appendages. Asexual morph: Undetermined.

Culture characters: Colonies on MEA, reaching 25 mm diam. after 4 weeks at 16 °C. Cultures from above, centrally grey, bearing cream outwardly, dense, circular, flat, umbonate, surface rough; reverse cream with dark brown.

Material examined: China, Yunnan Province, Dali District, on dead stems of Clematis montana, 20 May 2016, C. Phukhamsakda, CMCR1 (MFLU 20–0418, holotype); ex-type living culture, MFLUCC 16–0999.

Host: Clematis montana—(This study).

Distribution: China—(This study).

GenBank accession numbers: LSU: MT214565; SSU: MT226682; ITS: MT310611; tef1: MT394744.

Notes: In the phylogenetic analysis, Sigarispora montanae (Fig. 34) clustered basal to Sigarispora caulium with strong support (100% ML/1.00 BYPP, Fig. 26). Sigarispora montanae has relatively small ascomata and longer pedicels when compared with S. caulium (Thambugala et al. 2015, Table 3). In a BLASTn search of GenBank, the closest matches of tef1 sequence of MFLUCC 16–0999 is 96.5% similar to S. thymi strain MFLU 15–2131 (MG829241). The tef1 sequences of S. montanae had 98% similarity to Sigarispora caulium (MFLUCC 15–0036) with 15 nucleotide differences in the tef1 region (Thambugala et al. 2015; Wanasinghe et al. 2018).

Melanommataceae Winter (= Pseudodidymellaceae)

Melanomma Nitschke ex Fuckel is the generic type (Winter 1885). Members of this family can occur on twigs or bark of various woody plants in terrestrial, marine or freshwater habitats. Melanommataceae is characterized by carbonaceous or coriaceous, gregarious, immersed to erumpent, globose to subglobose, papillate or epapillate, thick-walled, pseudoparenchymatous cells, trabeculate pseudoparaphyses, ascospores uniseriate or biseriate, fusoid to ellipsoidal, or muriform, hyaline or brown, 1 to multi-septate, with or without a mucilaginous sheath. Their asexual morphs can be hyphomycetes or coelomycetes (Sivanesan 1984; Huhndorf 1993; Liew et al. 2000; Tian et al. 2015; Wanasinghe et al. 2018). Wijayawardene et al. (2018) accepted 24 genera, thereafter Wanasinghe et al. (2018) introduced five more genera to Melanommataceae. An analysis of fungal collections on Clematis vitalba revealed a novel genus, Neobyssosphaeria based on a multi-gene phylogeny of LSU, SSU and ITS sequence data for Melanommataceae (Fig. 35).

Fig. 35
figure35

The best scoring RAxML tree with a final likelihood value of − 9510.121854 based on combined LSU, SSU and ITS sequence data for Melanommataceae. The tree is rooted with members of the Cyclothyriellaceae. Seventy-one strains were included in the combined sequence analyses which comprised 2564 characters (921 characters for LSU, 1061 characters for SSU, 582 characters for ITS, including gap regions). The matrix had 602 distinct alignment patterns, with 31.43% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.254800, C = 0.214111, G = 0.278600, T = 0.252489; substitution rates AC = 2.132797, AG = 2.875535, AT = 1.394124, CG = 0.797579, CT = 9.867341, GT = 1.000000; gamma distribution shape parameter α = 0.481484. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study, is indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. The supported values from all analyses are BS ≥ 70%/BYPP ≥ 0.95

Neobyssosphaeria Wanas., E.B.G. Jones & K.D. Hyde, gen. nov.

Index Fungorum number: IF557189; Facesoffungi number: FoF 07281, Fig. 36.

Fig. 36
figure36

Neobyssosphaeria clematidis (MFLU 17–0614, holotype). a, b Appearance of ascomata on Clematis vitalba.c Vertical section through ascoma. d Close up of ostiolar canal. e Cellular pseudoparaphyses. f, g Asci. h–l Ascospores. Scale bars: a = 1 cm, b = 200 µm, c = 100 µm, d, fg = 50 µm, e = 5 µm, hl = 20 µm

Etymology: Name refers to the similarity of its morphology to Byssosphaeria.

Saprobic on decaying wood or herbaceous plants in terrestrial habitats. Sexual morph: Ascomata immersed, ostioles orange, solitary or gregarious, globose to depressed-globose, coriaceous, ostiolate. Ostioles central, papillate, opening by a pore, filled with periphyses with orange pigment around the pore. Peridium thick, multilayered, outer layer composed of reddish brown cells of textura angularis, inner layer composed of thin and hyaline cells of textura angularis. Hamathecium composed of numerous, dense, filiform, branched, anastomosing, transversely septate, trabeculate pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical-clavate, pedicelate with an occular chamber. Ascospores biseriate, broadly fusiform, hyaline, constricted at the septa, guttulate in each cell, with or without a mucilaginous sheath. Asexual morph: Undetermined.

Type species: Neobyssosphaeria clematidis Wanas., Phukhams., E.B.G. Jones & K.D. Hyde

Notes: Neobyssosphaeria is established as a monotypic genus with N. clematidis as the type species. Based on multi-gene analyses, isolate MFLUCC 17–0794 formed a basal lineage to Byssosphaeria, but this placement is not supported by the statistical analyses. Byssosphaeria is characterized by superficial ascomata with bright yellow, orange or red colouration around the ostioles, hairy hypha protruding from the outside of peridium, long pedicellate asci, and hyaline or pale brown ascospores (Barr 1990; Tian et al. 2015). Neobyssosphaeria is similar to Byssosphaeria in its orange apex (Zhang et al. 2012; Hyde et al. 2013). However, Neobyssosphaeria is distinguished by its immersed ascomata with central papilla filled with periphyses, cellular pseudoparaphyses and broad fusiform and hyaline ascospores (Fig. 36). Our taxon failed to produce an asexual morph in culture and therefore it is not possible to compare the asexual characteristics with species of Byssosphaeria. In the BLASTn search of GenBank, the closest match of the LSU region of MFLUCC 17–0794 is Uzbekistanica yakutkhanika (strain MFLUCC 17–0842) with 94.48% similarity (accession number MG829090). Uzbekistanica is however phylogenetically not closely related to our new collection (Fig. 35). Therefore, we believe it is taxonomically prudent to name our collection in a new genus until further studies are carried out with further taxonomic sampling and DNA based sequence analyses.

Neobyssosphaeria clematidis Wanas., Phukhams., E.B.G. Jones & K.D. Hyde, sp. nov.

Index Fungorum number: IF557190; Facesoffungi number: FoF 07282, Fig. 36.

Etymology: Named after the host genus, Clematis.

Holotype: MFLU 17–0614.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 525–550 × 500–520 μm (\( \bar{x} \) = 535 × 510 μm, n = 5), immersed, ostiole part orange, solitary or gregarious, globose to depressd-globose, coriaceous, indistinguishable from host tissue, brown to pale brown, rough-walled, ostiolate. Ostioles 235–270 × 190–230 μm (\( \bar{x} \) = 250 × 200 μm, n = 5), central, papillate, opening by a pore, filled with periphyses, with orange pigment around pore. Peridium 30–50(–70 μm at apex) wide, thick, multilayered, outer layer composed of heavily pigmented, reddish brown cells of textura angularis, inner layer composed of thin and hyaline cells of textura angularis. Hamathecium composed of numerous, 1.6–3 μm (\( \bar{x} \) = 2.3 μm, n = 50), dense, filiform, branched, anastomosing, septate, cellular pseudoparaphyses. Asci 160–210 × 20–30 μm (\( \bar{x} \) = 185 × 25 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical, long pedicellate with a furcated base, clavate, apically rounded, with ocular chamber. Ascospores 55–75 × 8–14 μm (\( \bar{x} \) = 60 × 11 μm, n = 30), biseriate, partially overlapping, broad fusiform, sometimes inequilateral, with acute ends, hyaline, 7-euseptate, constricted at the septa, cell above median septum enlarged, with guttules in each cell, rough-walled, without mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 16 °C. Above cream with orange in the middle, with white edge, medium dense, flattened, umbonate, floccose; reverse: cream, thin, flat, circular.

Material examined: UK, Hampshire, Botley wood, on dead stems of Clematis vitalba, 25 May 2016, E.B.G. Jones, GJ 298 (MFLU 17–0614, holotype); ex-type living culture, MFLUCC 17–0794.

Host: Clematis vitalba—(This study).

Distribution: UK—(This study).

GenBank accession numbers: LSU: MT214566; SSU: MT408594.

Notes: The new fungus morphologically resembles many genera in Pleosporales (e.g. Angustimassarina, Aquastroma, Aquilomyces, Carinispora, Falciformispora, Keissleriella, Lophiopoacea, Lophiostoma, Parabambusicola, Quintaria) in its cylindrical-clavate asci and hyaline, fusiform ascospores with large guttule in each cell (Zhang et al. 2012; Tanaka et al. 2015; Thambugala et al. 2015). However, these taxa are phylogenetically not closely related to Neobyssosphaeria clematidis (see Notes Neobyssosphaeria for further details). Neobyssosphaeria clematidis differs from Byssosphaeria species by its immersed ascomata, lacking hairy hypha protruding from the outside of the peridium (Tian et al. 2015).

Neomassarinaceae Mapook & K.D. Hyde

The type genus of Neomassarinaceae is Neomassarina. The family is phylogenetically related to Sporormiaceae but the characters of the sexual morphs are different. Neomassarinaceae can be characterized by its crest-like ostiolar necks, with a carbonaceous texture, cylindrical to cylindric-clavate asci and broad fusiform ascospores surrounded by a mucilaginous sheath (Thambugala et al. 2015; Tibpromma et al. 2017; Mapook et al. 2020). In this study, a dataset of LSU, SSU, ITS, tef1 and rpb2 were used for phylogenetic analyses (Fig. 2). A collection associated with Clematis formed a clade related to Neomassarinaceae. Thus, we introduce a new genus Pseudohelminthosporium and a first report of the asexual morph in Neomassarinaceae (Fig. 37).

Fig. 37
figure37

Pseudohelminthosporium clematidis (MFLU 171494, holotype). a, b Conidiophores on natural substrate (Clematis sikkimensis). c, d Mononematous conidiophores. e, f Conidiogenous cells and conidia. g–j Conidia (Black basal conidia). k Culture characteristics on MEA. Scale bars: c, d = 500 μm, e = 100 μm, f–j = 50 μm

Pseudohelminthosporium Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557191; Facesoffungi number: FoF 07283, Fig. 37.

Etymology: Referring to its similarity to Helminthosporium.

Saprobic on decaying wood or herbaceous plant material in terrestrial habitats. Sexual morph: Undetermined. Asexual morph: Colonies on host substrates, effuse, black, hairy, scattered, dark brown. Mycelium immersed from the substrate forming dark brown stroma-like aggregations. Conidiophores macronematous, simple, solitary, branched at the apex, stripes straight or flexuous, cylindrical, dark brown to reddish brown, multi-septate, with well-defined small pores at the apex, smooth or verruculose. Conidiogenous cells monotretic or polytretic, integrated, terminal on conidiophores, doliiform to oblong, pale brown. Conidia phragmosporous, acrogenous, broad fusiform or obclavate, dark brown to reddish brown, distoseptate when young, becoming euseptate at maturity, verrucose, dark brown bud scars disjunctions present at the basal position, hyaline, elongate cells at the upper end of conidia, with guttules in each cell.

Type species: Pseudohelminthosporium clematidis Phukhams. & K.D. Hyde

Notes: Pseudohelminthosporium is established as a monotypic genus. Based on the multi-gene analysis (Fig. 2), the isolate MFLUCC 17–2086 formed a basal lineage with Neomassarina species with moderate support (0.93 BYPP). Pseudohelminthosporium is morphologically similar to Helminthosporium (Massarinaceae) in having brown to dark brown phragmosporous conidia (Voglmayr and Jaklitsch 2017). Helminthosporium species have obclavate to rostrate, pale golden brown to brown conidia, with distoseptate and angular lumina. Pseudohelminthosporium is distinguishable by its solitary stipes with monotretic or polytretic conidiogenous cells, phragmosporous, broad fusiform or obclavate conidia, distoseptate when young, becoming euseptate at maturity, with hyaline, elongate cells at the upper end of the conidia and with large guttule in each cell (Fig. 37).

Pseudohelminthosporium clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: FoF 07284; Facesoffungi number: FoF 07284, Fig. 37.

Etymology: The epithet “clematidis” refers to the host substrate.

Holotype: MFLU 17–1494.

Saprobic on decaying wood or herbaceous plant material in terrestrial habitats. Sexual morph: Undetermined. Asexual morph: Colonies on Clematis sikkimensis effuse, black, hairy, scattered, dark brown. Mycelium immersed, on the substrate surface forming stroma-like aggregations of dark brown sheet. Conidiophores 125–435 × 9–20 μm (\( \bar{x} \) = 230 × 12 μm, n = 20), macronematous, simple, solitary, branched at the apex, stipes straight or flexuous, cylindrical, erect, septate, smooth, dark brown to reddish brown, 8–14 septa, brown, well-defined small pores with dark scar at the apex, smooth or verruculose. Conidiogenous cells 17–60 × 9–12 μm (\( \bar{x} \) = 40 × 12 μm, n = 10), monotretic or polytretic, integrated, terminal, becoming intercalary, doliiform to oblong, pale brown. Conidia 63–142 × 16–26 μm (\( \bar{x} \) = 87 × 20 μm, n = 20), phragmosporous, acrogenous, broad fusiform or obclavate, distoseptate at the early state, 3–5-euseptate at maturity, slightly constricted at septa, dark brown to reddish brown, verrucose, gradually tapering to 13 μm at the distal end, with a dark brown to black scar present at the base, subhyaline, elongate cells at the upper end of conidia, with (1–)2 guttules in each cell.

Culture characters: Colonies on MEA reaching 40 mm diam. after 4 weeks at 25 °C. Culture from above, brownish grey, dark green, white in the center, forming cream fluffy mycelium at the edge, dense, umbonate, raised with concave edge, rough, dull, lobate, radially furrowed, with brown pigment slightly diffusing into the agar; reverse dark brown.

Material examined: Thailand, Chiang Rai Province, Mae Sai District, dead stems of Clematis sikkimensis, 24 June 2017, C. Phukhamsakda & M. van de Bult, CMTHDT02 (MFLU 17–1494, holotype); ex-type living culture, MFLUCC 17–2086.

Host: Clematis sikkimensis—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214567; SSU: MT226683; ITS: MT310612; tef1: MT394627; rpb2: MT394690.

Notes: In a BLASTn search of GenBank, the closest match for the LSU sequence of MFLUCC 17–2086 was Preussia terricola strain CBS 317.65 (GQ203725) with 96.77% similarity. The closest match for the ITS region was Forliomyces uniseptata strain MFLUCC 15–0765 (NR_154006). Based on the multi-locus phylogenetic support (Fig. 2), Pseudohelminthosporium formed a separate lineage related to Neomassarina species but lacked backbone support. Therefore, Pseudohelminthosporium is treated as a distinct genus in Neomassarinaceae.

Nigrogranaceae Jaklitsch & H. Voglmayr

Nigrogranaceae was erected to accommodate a well-supported clade of Nigrograna in Pleosporales, with Nigrograna as the generic type (Jaklitsch and Voglmayer 2016). Nigrogranaceae was isolated from the bark of wood and is characterized by its immersed to erumpent ascomata, surrounded by a subiculum with only papillate ostioles seen on the host substrate. The remarkable characteristics of Nigrogranaceae include broad-fusiform to narrowly ellipsoid ascospores with 1–3-euseptate, and pale to chocolate brown ascospores. The asexual morph has pseudoparenchymatous pycnidia and rod-like to ellipsoid, 1-celled, hyaline or subhyaline conidia (Gruyter et al. 2013). Only Nigrograna is accepted in Nigrogranaceae (Wijayawardene et al. 2017, 2018, Fig. 38).

Fig. 38
figure38

The Bayesian 50% majority-rule consensus phylogram based on combined LSU, ITS, SSU and tef1 sequence data for Nigrogranaceae. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with members of Paradictyoarthriniaceae. Thirty-four strains were included in the combined gene sequence analyses which comprised 3202 characters (851 characters for LSU, 474 characters for ITS, 1027 characters for SSU, 850 characters for tef1, including gap regions). The tree from the maximum likelihood analysis had similar topology to the Bayesian analyses. The best scoring RAxML tree had a final likelihood value of − 8012.999545. The matrix had 487 distinct alignment patterns with 20.65% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.244997, C = 0.242630, G = 0.268950, T = 0.243423; substitution rates AC = 1.469986, AG = 2.972149, AT = 1.574187, CG = 0.656303, CT = 11.538665, GT = 1.000000; gamma distribution shape parameter α = 0.62491. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 70% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 70% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Nigrograna Gruyter, Verkley & P.W. Crous

Gruyter et al. (2013) introduced Nigrograna for isolates reported as infectious human pathogen (Serrano et al. 1998; Ahmed et al. 2018). The genus is typified by Nigrograna mackinnonii (Gruyter et al. 2013), and includes 13 species (Index Fungorum 2020) based on phylogenetic analyses of a concatenated dataset of LSU, SSU, ITS and tef1 data (Fig. 38) coupled with morphology. Nigrograna chromolaenae and N. oblique, on Clematis species, are new host records (Figs. 39, 40).

Fig. 39
figure39

Nigrograna chromolaenae (MFLU 17–1487). a, b Appearance of ascomata on Clematis fulvicoma. c Vertical section through ascoma. d Section of peridium. e Pseudoparaphyses. f–h Asci. i–n Ascospores. o Germinated ascospore. p, q Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d–h = 20 µm, i–o = 10 µm

Fig. 40
figure40

Nigrograna obliqua (MFLU 16–0190). a Appearance of ascomata on Clematis vitalba. b Vertical section through ascoma. c Section of peridium. d Pseudoparaphyses. e–h Asci. im Ascospores. n Germinated ascospore. o, p Culture characteristics on MEA. Scale bars: b = 100 µm, ch = 20 µm, in = 10 µm

Nigrograna chromolaenae Mapook & K.D. Hyde, Fungal Divers (2020), new host record

Facesoffungi number: FoF 07297, Fig. 39.

Saprobic on dead stems of Clematis fulvicoma. Sexual morph: Ascomata 135–251 × 87–238 μm (\( \bar{x} \) = 178 × 141 μm, n = 10), on the surface of the host, solitary, gregarious, immersed, the surface of host slightly swollen, subglobose to depressed, coriaceous, dark brown to brown, smooth-walled, papillate. Ostioles 119 × 95 μm, central, dark brown, filled with periphyses. Peridium 9–15 μm wide, multilayered, composed of 5–7 layers of dark brown cells of textura angularis, heavily pigmented at the outer layer, the inner layer composed of hyaline and thin layer. Hamathecium composed of numerous, 0.8–1.5 μm wide (\( \bar{x} \) = 0.8 μm, n = 50), dense, filiform, branched, septate, pseudoparaphyses, anastomosing above asci. Asci 45–60 × 7–9 μm (\( \bar{x} \) = 46 × 7 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindrical-clavate to clavate, with furcate pedicel, apically rounded, ocular chamber visible when immature. Ascospores 11–13 × 3–5 μm (\( \bar{x} \) = 12 × 4 μm, n = 50), biseriate, ellipsoid to broad fusiform, sometimes inequilateral, with rounded ends, cognac brown, (1–)3-septate, constricted at septum, cell above median septum enlarged, with guttule in each cell, smooth-walled, without mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, dark brown, covered with white mycelia in the center, dense, irregular, umbonate, lobate, velvety, with flat parchment-like stromatic sheets; reverse dark brown, lobate.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis fulvicoma, 20 April 2017, C. Phukhamsakda, CMTH25 (MFLU 17–1487); living culture, MFLUCC 17–2079.

Hosts: Chromolaena odorata, Clematis fulvicoma—(Mapook et al. 2020; this study).

Distribution: Thailand—(Mapook et al. 2020; this study).

GenBank accession numbers: LSU: MT214568; SSU: MT226684; ITS: MT310613; tef1: MT394628; rpb2: MT394691.

Notes: Based on phylogenetic analyses (Fig. 38), MFLUCC 17–2079 clustered with Nigrograna chromolaenae (MFLUCC 17–1437). Mapook et al. (2020) introduced N. chromolaenae from a stem of Chromolaena odorata collected in Thailand. Characters of the ex-type strain such as ascomata, asci and ascospores were reported in Mapook et al. (2020) and were similar to our collection (Fig. 39). A comparison of the ITS and tef1 sequence data revealed no significant difference between our new collection and the ex-type strain. Therefore, we introduce Nigrograna chromolaenae on Clematis as a new host record.

Nigrograna obliqua Jaklitsch & H. Voglmayr, Studies in Mycology 85: 59 (2016), new host record

Index Fungorum number: IF817783; Facesoffungi number: FoF 07298, Fig. 40.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 193–450 × 188–294 μm (\( \bar{x} \) = 266 × 258 μm, n = 5), on the surface of the host, solitary, sometimes gregarious, immersed, only black shiny ostioles visible, subglobose to depressed, coriaceous, brown to reddish brown, smooth-walled, papillate. Ostioles 80–165 × 74–96 μm, central, dark brown, filled with periphyses. Peridium 11–27 μm wide, with brown hyphae projecting from the outer layer, composed of 8–10 layers of dark brown cells of textura angularis, heavily pigmented at outer layer, the inner layer hyaline and thin. Hamathecium composed of numerous, 1–1.3 μm (\( \bar{x} \) = 1.2 μm, n = 50), dense, filiform, branched, transverse septate, pseudoparaphyses. Asci 60–101 × 10–16 μm (\( \bar{x} \) = 86 × 12 μm, n = 20), 8-spored, bitunicate, fissitunicate, clavate to broad cylindrical, with furcate pedicel, apically rounded, ocular chamber visible when immature. Ascospores 16–20 × 5–8 μm (\( \bar{x} \) = 18 × 6 μm, n = 50), biseriate, overlapping, ellipsoid to broad fusiform, sometimes inequilateral, with rounded ends, initially hyaline, pale brownish to brown at the maturity, (1–)3-eusepta, constricted at septa, cell above median septum enlarged, the second cell slightly wider than others, with guttule in each cell, smooth-walled, without mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 16 °C. Cultures from above, dark brown, covered with greyish orange mycelia on the surface, dense, irregular, umbonate, lobate, velvety, flat parchment-like stromatic sheets developed, reverse dark brown, fimbriae.

Material examined: Italy, Forlì-Cesena Province, Corniolo—Santa Sofia, dead aerial branch of Clematis vitalba, 17 February 2014, E. Camporesi, IT 1726 (MFLU 16–0190); living culture, MFLUCC 14–0945.

Hosts: Clematis vitalba, Ribes uva-crispa, Salix caprea, Sambucus nigra, S. racemosa—(Jaklitsch et al. 2016; this study).

Distribution: Austria, France, Italy, UK—(Jaklitsch et al. 2016; this study).

GenBank accession numbers: LSU: MT214569; ITS: MT310614.

Notes: Our new collection is morphologically similar and phylogenetically related to the type species of Nigrograna obliqua (strain MF2). MFLUCC 14–0945 is not very different from that of the type species (Fig. 40). Jaklitsch and Voglmayr (2016) introduced four strains of N. obliqua from various shrubs and trees. In the phylogenetic analyses MFLUCC 14–0945 formed a close relationship with the ex-type strain reported from Salix caprea (84% ML/0.99 BYPP). Interestingly, three strains of N. obliqua (BW4, KE and MRP) formed a separate clade from the type strain. However, the morphological data of N. obliqua strains BW4, KE and MRP were not available for comparison. We report N. obliqua on Clematis vitalba as a new host record (Fig. 40).

Occultibambusaceae Dai & K.D. Hyde

Occultibambusaceae is typified by Occultibambusa D.Q. Dai & K.D. Hyde. The species in this family are generally associated with monocotyledon such as Occultibambusa, Seriascoma, and Versicolorisporium. Doilom et al. (2017) reported Neooccultibambusa, another species from Tectona grandis and Brunneofusispora from dead wood in China. Occultibambusaceae is characterized by immersed, solitary to gregarious ascomata with ostioles, broadly cylindrical to clavate asci with broad-fusiform, hyaline to dark brown usually septate ascospores. Pycnidia are reported in the asexual morph of this family (Hatakeyama et al. 2008; Dai et al. 2017).

Brunneofusispora Huang & K.D. Hyde

Brunneofusispora sinensis is the type species. The genus was recorded from dead wood near a river and is characterized by immersed, uniloculate ascomata, cylindrical to clavate and short pedicellate asci, and hyaline to brown, broadly fusiform ascospores (Phookamsak et al. 2019). Brunneofusispora clematidis is reported as a second species of Brunneofusispora on Clematis and is illustrated. An updated phylogenetic analysis of a concatenated dataset of LSU, tef1, ITS and SSU sequence data is presented in Fig. 41.

Fig. 41
figure41

Bayesian 50% majority-rule consensus phylogram based on combined LSU, tef1, ITS and SSU sequence data for Occultibambusaceae. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with members of Ohleriaceae. Twenty-two strains were included in the combined sequence analyses which comprised 3213 characters (851 characters for LSU, 730 characters for tef1, 607 characters for ITS, 1025 characters for SSU, including gap regions). The tree from the maximum likelihood analysis had a similar topology to the Bayesian analyses. The best scoring RAxML tree had a final likelihood value of − 8012.999545. The matrix had 763 distinct alignment patterns with 26.03% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.244085, C = 0.246189, G = 0.275394, T = 0.234331; substitution rates AC = 1.902831, AG = 2.660713, AT = 1.395112, CG = 1.125830, CT = 7.375307, GT = 1.000000; gamma distribution shape parameter α = 0.601072. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study is indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Brunneofusispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557194; Facesoffungi number: FoF 07299, Fig. 42.

Fig. 42
figure42

Brunneofusispora clematidis (MFLU 17–1478, holotype). a, b Appearance of ascomata on Clematis subumbellata. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores. n Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d, e = 50 µm, f, g–i = 20 µm, j–m = 10 µm

Etymology: Name refers to the host Clematis.

Holotype: MFLU 17–1478.

Saprobic on dead stems of Clematis subumbellata. Sexual morph: Ascomata 160–210 × 165–186 μm (\( \bar{x} \)= 185 × 176 μm, n = 5), on the surface of the host, solitary, gregarious, uniloculate, semi-immersed, shiny, globose to depressed, coriaceous, dark brown to black, rough-walled, papillate, ostiolate. Ostioles 96–105 × 86–110 μm, central, dark brown, filled with short periphyses. Peridium 8–17(–28) μm wide, multilayered, composed of 4–5 layers of dark brown cells of textura prismatica, heavily pigmented at outer layer, the inner layer hyaline and thin. Hamathecium composed of medium dense, 1.7–4.5 μm wide (\( \bar{x} \) = 2.7 μm, n = 50), filiform, branched, septate, cellular pseudoparaphyses. Asci 45–117 × 12–24 μm (\( \bar{x} \) = 73 × 17 μm, n = 20), 8-spored, bitunicate, fissitunicate, clavate, with furcate pedicelate, apically rounded, ocular chamber visible when immature. Ascospores 17–35 × 5–10 μm (\( \bar{x} \) = 27 × 9 μm, n = 50), biseriate, broad fusiform to ellipsoid, sometimes inequilateral, ends acute, initially hyaline, pale brown at maturity, 1-euseptate, constricted at septum, with guttule in each cell, smooth-walled with mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, black, with greyish orange aerial mycelia on the surface, dense, irregular, umbonate, lobate, velvety, flat, parchment-like; reverse black, fimbriae.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis subumbellata, 20 April 2017, C. Phukhamsakda, CMTH 14 (MFLU 17–1478, holotype); ex-type living culture, MFLUCC 17–2070.

Host: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214570; SSU: MT226685; ITS: MT310615; tef1: MT394629; rpb2: MT394692.

Notes: In the phylogenetic analysis, Brunneofusispora clematidis clustered with the type species B. sinensis with strong support (100% ML/1.00 BYPP, Fig. 41). Brunneofusispora clematidis can be distinguished from B. sinensis by its thinner peridium layer (8–17(–28) vs 20–45 μm) and longer ascospores (Phookamsak et al. 2019). Comparison of 607 nucleotides across the ITS region reveals 74 bp (12.2%) differences between B. clematidis and B. sinensis. Therefore, B. clematidis is introduced as a new species (Fig. 42).

Paradictyoarthriniaceae Doilom, Liu & K.D. Hyde

Paradictyoarthriniaceae was established to accommodate a hyphomycetes genus Paradictyoarthrinium (P. diffractum, type species) in aquatic and terrestrial habitats (Matsushima 1996; Liu et al. 2015, 2018). Multilocus phylogenetic analyses revealed a rock-inhibiting hyphomycete, Coniosporium olivaceum Link (≡ Sirodesmium olivaceum CBS 395.59) that is related to Paradictyoarthriniaceae (Ruibal et al. 2009). Wanasinghe et al. (2018) introduced the first sexual morph, Xenomassariosphaeria to Paradictyoarthriniaceae. We introduce an additional species of Xenomassariosphaeria on Clematis vitalba in Italy (Figs. 2, 43).

Xenomassariosphaeria Jayasiri, Wanas. & K.D. Hyde

Xenomassariosphaeria was introduced for massariosphaeria-like species that formed a close relationship within Paradictyoarthriniaceae (Tanaka and Harada 2004; Wanasinghe et al. 2018). The genus is characterized by semi-immersed to erumpent, short papillate ascomata, unequal peridium of thick pseudoparenchymatous cells, cylindrical to cylindric-clavate, subsessile to short pedicellate asci, with broad fusiform, hyaline to brown, asymmetric, multiseptate ascospores (Wanasinghe et al. 2018). Based on a multigene analysis of LSU, SSU, ITS, tef1 and rpb2 sequence data (Fig. 2), Xenomassariosphaeria clematidis formed a clade with the type species X. rosae with strong support (100% ML/1.00 BYPP, Fig. 2).

Xenomassariosphaeria clematidis Wanas., Phukhams., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557112; Facesoffungi number: FoF 07350, Fig. 43.

Fig. 43
figure43

Xenomassariosphaeria clematidis (MFLU 16–0119, holotype). a Appearance of ascomata on host surface. b Vertical section of ascoma. c Pseudoparaphyses. d–f Asci. g–l Ascospores. m Germinated ascospore. n, o Cultures characteristics on MEA. Scale bars: b = 100 µm, cf = 20 µm, g–m = 10 µm

Etymology: Epithet reflects the host Clematis.

Holotype: MFLU 16–0119.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 273–283 × 184–208 μm (\( \bar{x} \) = 275 × 190 μm, n = 5), solitary, scattered, immersed, with only black spot visible on the host surface, globose, coriaceous, dark brown to black, rough-walled, ostiolate. Ostioles centrally located, short papillate, with periphysoids. Peridium 12–37 μm wide, composed of 5(–7 at apex) layers of dark brown outer layers of textura angularis, inner layer composed of hyaline gelatinous cells. Hamathecium composed of numerous, 2.3–3.5 μm wide (\( \bar{x} \) = 2.7 μm, n = 50), dense, filiform, septate, branched, cellular pseudoparaphyses. Asci 91–120 × 20–28 μm (\( \bar{x} \) = 107 × 25 μm, n = 20), 8-spored, bitunicate, fissitunicate, clavate to cylindric-clavate, with furcate pedicel, with ocular chamber visible when immature. Ascospores 27–33 × 7–11 μm (\( \bar{x} \) = 30 × 9 μm, n = 50), biseriate, naviculate, narrow towards the lower ends, initially hyaline, becoming yellowish to brown at maturity, 7–8 transversely eusepta, slightly constricted at the septa, third cells from apex usually enlarged, smooth-walled, guttulate and indentations present, without mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 18 °C. Cultures from above, brown radiating yellowish towards the edge, dense, circular, flat, dull, fimbriate, radially furrowed, and slightly covered with white aerial mycelium; reverse black with radiating brown mycelium.

Material examined: Italy, Forlì-Cesena Province, Poggio alla Lastra—Bagno di Romagna, dead aerial stems of Clematis vitalba, 19 January 2013, E. Camporesi, IT1019 (MFLU 16–0119, holotype); ex-type living culture, MFLUCC 14–0923.

Hosts: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214571; ITS: MT310616; tef1: MT394630.

Notes: In the phylogenetic analyses (Fig. 2), strain MFLUCC 14–0923 formed a close relationship with the type species Xenomassariosphaeria rosae (MFLUCC 15–0179) with strong support (100% ML/1.00 BYPP). Xenomassariosphaeria clematidis shares common features with Xenomassariosphaeria in having immersed ascomata with thick papilla, cylindrical or clavate asci, and brown, multiseptate ascospores (Tanaka and Harada 2004; Wanasinghe et al. 2018). Xenomassariosphaeria clematidis is distinguished by its unique obovoid ascospores that are narrow towards the lower end, brown and multi-septate (Fig. 43).

In a BLASTn search of GenBank, the LSU sequence of X. clematidis (strain MFLUCC 14–0923) is 97.5% similar to X. roumeguerei (strain CBS 612.86, MH873692). The ITS region of X. clematidis (strain MFLUCC 14–0923) had 91.5% similarity with X. roumeguerei (strain CBS 612.86, MH862004). Thus, we introduce a novel species, X. clematidis based on morphological and phylogenetic evidence.

Periconiaceae Nann.

Tanaka et al. (2015) verified Periconiaceae belonged in Pleosporales based on modern fungal systematics. Although sequence data is not available for the type species of Periconia (P. lichenoides), the morphological characters of extant Periconia species correspond to P. lichenoides (Mason and Ellis 1953; Tanaka et al. 2015). Periconiaceae is characterized by scattered, immersed to erumpent ascomata, pseudoparaphyses, oblong to cylindrical asci, and broadly fusiform, 1-septate, hyaline ascospores. Asexual characters include synnemata or noosia-like, macronematous, mononematous conidiomata, monoblastic to polyblastic, discrete and branched conidiogenous cells, and globose to ellipsoidal, aseptate, catenate, brown conidia (Tanaka et al. 2015).

Periconia Tode

Over 190 species are listed under Periconia (Index Fungorum 2020). Based on phylogenetic analyses of a concatenated dataset of LSU, ITS, and tef1 sequence data coupled with morphological characters, P. verrucosa is described as a new species from Clematis viticella (Figs. 44, 45).

Fig. 44
figure44

The best scoring RAxML tree with a likelihood value of − 10209.184183 based on combined LSU, ITS and tef1 sequence data for Periconiaceae. The tree is rooted with species of Lentitheciaceae. Forty-seven strains were included in the combined gene sequence analyses which comprised 2974 characters (1320 characters for LSU, 620 characters for ITS, 1034 characters for tef1, including gap regions). The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to Bayesian 50% majority-rule consensus phylogram. The matrix had 865 distinct alignment patterns with 33.43% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.229606, C = 0.269654, G = 0.273255, T = 0.227485; substitution rates AC = 1.424251, AG = 2.110026, AT = 1.548277, CG = 1.179391, CT = 7.719698, GT = 1.000000; gamma distribution shape parameter α = 0.673748. In our analysis, a GTR + I + G model was used for each partition in the Bayesian posterior analysis. The species determined in this study is indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% ML/0.90 BYPP. Thick branches represent significant support values from all analyses (ML ≥ 70%/BYPP ≥ 0.95)

Fig. 45
figure45

Periconia verrucosa (MFLU 17–1516, holotype). a, b Sporodochia on natural substrate. c Close up of sporodochia. d, e Conidiophores. f–i Conidiogenous cells. j–m Conidia. Scale bars: b = 500 μm, ce = 250 μm, f, g = 50 μm, h, i = 10 μm, jm = 5 μm

Periconia verrucosa Phukhams, Ertz, Gerstmans & K.D. Hyde, sp. nov.

Index Fungorum number: IF557143; Facesoffungi number: FoF 07296, Fig. 45.

Etymology: The epithet ‘‘verrucosa’’ refers to the surface of conidia being verrucose.

Holotype: MFLU 17–1516.

Saprobic on dead stems of Clematis viticella. Sexual morph: Undetermined. Asexual morph: Colonies effuse on the natural substrate, scattered, hairy, dark brown. Mycelium partly superficial, semi-immersed, branched, composed of pale brown, septate hyphae. Conidiophores 170–296 × 10–12 μm (\( \bar{x} \) = 225 × 11 μm, n = 20), macronematous, mononematous, solitary, gregarious, scattered, erect, stipes straight or slightly flexuous, with 3–4 short branches at the apex, cylindrical, smooth, dark brown, 2–4-septate, smooth or verruculose. Conidiogenous cells 11–26 × 6–14 μm (\( \bar{x} \) = 16 × 7 μm, n = 20), monoblastic or polyblastic, acropetally proliferating, integrated, terminal, oblong, retrogressive, pale brown. Conidia 7–15 μm (\( \bar{x} \)= 12 μm, n = 50), in branched chains, acrogenous, globose, aseptate, thick-walled, hyaline when immature, dark brown to reddish brown at maturity, verrucose, bud scars or disjunctors present at the site of attachment, easily separating.

Culture characters: Colonies on MEA reaching 30 mm diam. after 2 weeks at 25 °C. Cultures from above, cream or white, mycelia medium dense, circular, umbonate, papillate, fluffy, slightly radiating outwardly; reverse: cream at the centre, radiating outwardly.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, on dead stems of Clematis viticella, 13 June 2017, D. Ertz & C. Gerstmans, BRCV4 (MFLU 17–1516, holotype); ex-type living culture, MFLUCC 17–2158.

Hosts: Artemisia sp., Sasa kurilensis, Clematis viticella—(Tanaka et al. 2015; this study).

Distribution: Belgium, Japan—(Tanaka et al. 2015; this study).

GenBank accession numbers: LSU: MT214572; SSU: MT226686; ITS: MT310617; tef1: MT394631.

Notes: Phylogenetic analysis included LSU, ITS and tef1 sequence data with related sequences retrieved from GenBank (Fig. 44). Periconia verrucosa (MFLUCC 17–2158) formed a strongly supported clade (96% ML/0.99 BYPP) with three unnamed Periconia strains (KT 1820A, KT 1825 and S-900, Tanaka et al. 2015). These strains do not have morphological characters for comparison. A comparison of the ITS nucleotide bases shows that P. verrucosa (MFLUCC 17–2158) has one nucleotide difference with strain S-900 and one nucleotide difference with KT 1820A and KT 1825 in tef1 region. This is regarded as not significant (Jeewon and Hyde 2016). We introduce P. verrucosa as a new species to accommodate this clade of Periconia (Fig. 45).

Phaeoseptaceae Boonmee, Thambug. & K.D. Hyde

Phaeoseptaceae was established for lignicolous fungal lineages on wood. Five genera are included in the family: Decaisnella, Lignosphaeria, Phaeoseptum (generic type), Pleopunctum and putative strains of Thyridaria macrostomoides (Abdel-Wahab and Jones 2003; Zhang et al. 2012; Ariyawansa et al. 2015a; Thambugala et al. 2015; Hyde et al. 2018a; Liu et al. 2019; Phukhamsakda et al. 2019). Phaeoseptaceae members have ascomata immersed in host tissues, short papillate, anastomosed pseudoparaphyses, cylindrical-clavate, long pedicellate asci and broadly fusoid, single or multi-transverse septa, and hyaline to brown ascospores (Hyde et al. 2018a). Phaeoseptaceae had pycnidial or sporodochial characters as asexual morph (Liu et al. 2019). We describe a novel species of Pleopunctum recorded from Clematis in Thailand, based on a multi-gene analysis (Fig. 46) coupled with comparable morphology (Fig. 47).

Fig. 46
figure46

Phylogram generated from maximum parsimony analysis of Phaeoseptaceae based on combined LSU, SSU, ITS, tef1 and rpb2 sequence data. Related sequences are taken from Liu et al. (2019) and retrieved from GenBank. Fifteen strains were included in the analysis of the combined loci and comprised 4077 characters (810 characters for LSU, 1017 characters for SSU, 480 characters for ITS, 895 characters for tef1, 875 characters for rpb2, including gaps). The tree is rooted with Lophiostoma arundinis (CBS 621.86) and L. crenatum (CBS 629.86) in Lophiostomataceae. Maximum parsimony analysis of 717 parsimony informative characters resulted in a most parsimonious tree (CI = 0.797, RI = 0.757, RC = 0.603, HI = 0.203). The best scoring RAxML tree received a final likelihood value of − 12889.704989. The matrix had 769 distinct alignment patterns, with 37.49% undetermined characters and gaps. Estimated base frequencies were: A = 0.241361, C = 0.260527, G = 0.275500, T = 0.222613; substitution rates AC = 1.309734, AG = 3.237095, AT = 1.407451, CG = 1.436805, CT = 9.313126, GT = 1.000000; gamma distribution shape parameter α = 0.742589. In our analysis, GTR + I +  G model was used for each partition in Bayesian posterior analysis. Bootstrap values (BS) from maximum parsimony (MP, left), maximum likelihood (ML, right) higher than 50% BS and Bayesian posterior probabilities (BYPP, below) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95). The ex-type strains are in bold and black. The newly generated sequence is in bold and blue

Fig. 47
figure47

Pleopunctum clematidis (MFLU 17–1499, holotype). a, b Sporodochia on natural substrate. c Vertical section of sporodochia. d Subicular hyphae. ef Cylindrical conidia and lenticular conidia on host substrate. gi Cylindrical conidia. j–n Mature lenticular conidia. o Culture characteristics on MEA. Scale bars = b = 500 μm, c = 100 μm, dn = 20 μm

Pleopunctum Liu, K.D. Hyde & J.K. Liu

Liu et al. (2019) introduced the first asexual morph of Pleopunctum (typified by P. ellipsoideum) in Phaeoseptaceae from decaying wood collected in China. The genus is characterized by sporodochial conidiomata, macronematous, mononematous conidiophores, monoblastic conidiogenous cells and muriform conidia that have a globose basal cell. The multilocus phylogeny (LSU, SSU, ITS, tef1 and rpb2) revealed a new species of Pleopunctum from Clematis based on the morphology and well supported values from multi-gene phylogeny (Fig. 46).

Pleopunctum clematidis Phukhams., Bhat & K.D. Hyde, sp. nov.

Index Fungorum number: IF557139; Facesoffungi number: FoF 07301, Fig. 47.

Etymology: The epithet “clematidis” refers to the host plant, Clematis.

Holotype: MFLU 17–1499.

Saprobic on dead branch of Clematis sikkimensisSexual morph: Undetermined. Asexual morph: Colonies on natural substrate forming sporodochial conidiomata, 168–278 μm wide, superficial, scattered, gregarious, oval, brown, velvety, glistening, orbicular, conidia readily liberated when agitated. Mycelium immersed, septate, smooth-walled, thin-walled, yellowish brown to brown hyphae, 3.5–4.5 μm wide, subicular hyphae short, medium packed. Conidiophores 6.5–15.5 × 2–5 μm, micronematous, mononematous, cylindrical or truncate, erect, smooth or finely verruculose, aseptate, unbranched, often reduced to conidiogenous cells, initially hyaline, brown at maturity. Conidiogenous cells 3–8 × 4–9 μm, holoblastic, monoblastic, integrated, terminal, determinate, cylindrical or slightly truncate, subspherical or ampulliform, hyaline. Conidia dimorphic, solitary, smooth-walled; lenticular conidia 16–33 × 15–23 μm (\( \bar{x} \) = 25 × 20 μm, n = 50), muriform, smooth, broadly ellipsoidal to oval in front view, yellowish brown to brown, slightly constricted at the median septa, inner view composed of one column of 5–7 cells, end cells subhyaline to pale brown, often carrying remnant of conidiogenous cell at base; cylindrical conidia 15–35 × 6–11 μm (\( \bar{x} \) = 20 × 8 μm, n = 30), straight or flexuous, septate, constricted at the septa, consisting of one column, 2–3-septate, doliiform, broad clavate, narrow towards apex, apex rounded, basal cells globose or subglobose, smooth, hyaline.

Culture characters: Colonies on MEA at room temperature (25 °C) reaching 7 cm after 2 weeks. Cultures from above, circular with lobate margin, olive mycelium, white at the margin, white aerial mycelium, smooth at the surface and raised; reverse beige, not sporulating.

Material examined: Thailand, Chiang Rai Province, Doi Tung, on dried stem of Clematis sikkimensis, 2 May 2017, C. Phukhamsakda & M.V. de Bult, CMTHDT08 (MFLU 17–1499, holotype); ex-type living culture, MFLUCC 17–2091.

Host: Clematis sikkimensis—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214573; ITS: MT310618; tef1: MT394632; rpb2: MT394693.

Notes: Pleopunctum clematidis (MFLUCC 17–2091) is similar to extant species of Pleopunctum in having sporodochial conidiomata, holoblastic, monoblastic conidiogenous cells and muriform lenticular conidia (Liu et al. 2019). The strain is distinguishable from other Pleopunctum species by its yellowish brown and smaller lenticular conidia. Additionally, P. clematidis has dimorphic conidia on the natural substrate. The dimorphic conidia type have been documented in Hermatomycetaceae, however, P. clematidis was without subicular hyphae (Tibpromma et al. 2018; Hyde et al. 2019a, Fig. 47). The multi-gene phylogeny of LSU, SSU, ITS, tef1 and rpb2 sequence data revealed that P. clematidis formed a sister lineage to P. ellipsoideum (MFLUCC 19–0390) and P. pseudoellipsoideum (MFLUCC 19–0391) with strong support (100% MP/100% ML/1.00 BYPP, Fig. 46). In a BLASTn search of GenBank, the closest match to MFLUCC 17–2091 is Lignosphaeria fusispora (strain MFLUCC 11–0377, KP888646) with 97.78% similarity in the LSU locus, while the closest match with the ITS sequence is 85.42% similar to KP899140.

Phaeosphaeriaceae Barr

In this family, the taxa are mostly endophytes, pathogens or saprobes in various habitats (Quaedvlieg et al. 2013; Phookamsak et al. 2014). We follow the treatment of Hyde et al. (2020a) and report a novel genus and four new species based on molecular data coupled with morphological evidence.

Chaetosphaeronema Moesz

Chaetosphaeronema hispidulum is the type species (Moesz 1915; Clements and Shear 1931). Chaetosphaeronema is characterized by immersed pycnidia with minute ostioles, enteroblastic, phialidic, determinate, discrete conidiogenous cells and cylindrical, hyaline, 1-septate conidia (Sutton 1980; Hyde et al. 2016). Ophiobolus cirsii (MFLUCC13–0218) is closely related to Chaetosphaeronema as suggested by Zhang et al. (2012). Five species are accepted under Chaetosphaeronema with two strains having sequence data (Phookamsak et al. 2019). Our phylogenetic analysis (Fig. 48) revealed two new species of Chaetosphaeronema from Clematis species, with the sexual morph reported for the genus (Figs. 49, 50).

Fig. 48
figure48figure48

The best scoring RAxML tree with a final likelihood value of − 34209.688899 based on a combined LSU, SSU, ITS and tef1 dataset for Phaeosphaeriaceae. One hundred and fifty-seven strains were included in the combined genes sequence analyses which comprised 3290 characters (822 characters for LSU, 603 characters for ITS, 991 characters for SSU and 874 characters for tef1, including gap regions). The tree is rooted with Staurosphaeria. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to the Bayesian 50% majority-rule consensus phylogram. The matrix had 1355 distinct alignment patterns, with 23.28% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.241166, C = 0.264679, G = 0.235933, T = 0.258221; substitution rates AC = 1.039932, AG = 3.142919, AT = 2.044232, CG = 0.762621, CT = 4.795230, GT = 1.000000; gamma distribution shape parameter α = 0.455752. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. The significant support values from all analyses are BS ≥ 70%/BYPP ≥ 0.95

Fig. 49
figure49

Chaetosphaeronema clematidicola (MFLU 17–1508, holotype). a Appearance of ascomata on Clematis patens. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–h Asci. i–j Ascospores (j Ascospore in 10% India ink). Scale bars: b = 500 µm, c = 200 µm, dh = 100 µm, ij = 50 µm

Fig. 50
figure50

Chaetosphaeronema clematidis (MFLU 17–1504, holotype). a Appearance of conidiomata on Clematis orientalis. b Close up of conidioma on host substrate. c Vertical section through conidioma. d Ostiolar canal. e Section of conidiomatal wall. fi Conidiogenous cells and conidia (f, g conidiogenous cells in cotton blue). j–l Conidia. Scale bars: b = 500 µm, c = 200 µm, d = 50 µm, e = 20 µm, fl = 10 µm

Chaetosphaeronema clematidicola Phukhams., Ertz, Gerstmans & & K.D. Hyde, sp. nov.

Index Fungorum number: IF557196; Facesoffungi number: FoF 07303, Fig. 49.

Etymology: The epithet reflects the host Clematis.

Holotype: MFLU 17–1508.

Saprobic on Clematis patens.Sexual morph: Ascomata 275–480 × 260–405 μm (\( \bar{x} \)= 394 × 330 μm, n = 5), scattered or sometimes clustered, gregarious, semi-immersed, erumpent through host tissue, visible as raised, with only black shiny ostioles visible on the host surface, uniloculate, subglobose to compressed, dark brown to black, ostiolate, papillate. Ostioles 132–248 × 97–120 µm (\( \bar{x} \) = 174 × 110 μm, n = 10), central, campanulate, composed of dark brown to black walled cells, rounded at the apex, with periphyses, ostioles filled with orange pigment at the pore. Peridium 13–35 µm wide (\( \bar{x} \) = 23 μm, n= 20), thicker at apex, composed of several layers of pale brown to dark brown cells of a textura angularis, inner layer lined with subhyaline cells of textura prismatica. Hamathecium of numerous, 2–3.5 µm wide (\( \bar{x} \) = 2.6 μm, n= 50), filamentous, cellular pseudoparaphyses, with distinct septa, embedded in mucilaginous matrix, anastomosing at the apex. Asci 139–208 × 6–7 µm (\( \bar{x} \) = 176 × 6 μm, n = 20), 8-spored, bitunicate, broadly filiform to cylindrical, short, with furcate pedicel, apically rounded, ocular chamber visible when young. Ascospores 134–188 × 1.5–7 µm (\( \bar{x} \) = 162 × 2.5 μm, n = 30), fasciculate, in parallel or spiral, scolecosporous, sometimes breaking at the septa, hyaline to yellowish brown, (18–)20(–23)-septate, not constricted at the septa, smooth-walled, with minute guttules in each cell. Asexual morph: Undetermined

Culture characters: Colonies growing on MEA reaching 40 mm after 4 weeks at 25 °C. Cultures from above, sparse, irregular, filamentous, flattened, smooth surface, with fimbriae edge, cream at the margin, white with pale yellowish at the centre; reverse colony cream at the margin and dark brown at the centre.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis patens C. Morren & Decne., 13 June 2017, D. Ertz & C. Gerstmans, BRCP4 (MFLU 17–1508, holotype); ex-type living culture, MFLUCC 17–2151.

Host: Clematis patens—(This study).

Distribution: Belgium—(This study).

GenBank accession numbers: LSU: MT214574; SSU: MT226687; ITS: MT310619; tef1: MT394633; rpb2: MT394694.

Notes: Chaetosphaeronema clematidicola grouped with Chaetosphaeronema species (Fig. 48) with strong support (100% ML/1.00 BYPP). Chaetosphaeronema clematidicola (MFLUCC 17–2151) is similar to Leptospora (L. rubella) and Pseudoophiobolus based on their ascospore morphology, but MFLUCC 17–2151 differs in having orange colouration at the ostioles (Shoemaker 1976; Phookamsak et al. 2017). A morphological comparison of Chaetosphaeronema clematidicola with Ophiobolus cirsii (MFLUCC 13–0218) showed that it is similar in having fasciculate, cylindrical ascospores (Fig. 49). However, Ophiobolus cirsii has erumpent ascomata without pigment at the ostioles. Petrak (1944) mentioned that the ophiobolus-like characteristic is usually associated with Chaetosphaeronema species. Zhang et al. (2012) showed that Chaetosphaeronema is genetically related to Phaeosphaeriaceae. Phookamsak et al. (2014) subsequently confirmed the taxonomy placement and mentioned that ophiobolus-like species could be the sexual morph of Chaetosphaeronema. The asexual morph of C. clematidicola could not be obtained for morphological comparison.

In the phylogenetic analysis, C. clematidicola (MFLUCC 17–2151) grouped with C. clematidis (MFLUCC 17–2147), another species also on Clematis. A comparison of the ITS region (including of the 5.8S region) showed three nucleotide differences (588/599—98% with no gaps). A comparison of the tef1 region revealed 12 base pair differences (842/874—96% with no gaps). Thus, we keep these isolates as distinct species.

Chaetosphaeronema clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Index Fungorum number: IF557195; Facesoffungi number: FoF 07302, Fig. 50

Etymology: The epithet name “clematidis” refers to the host substrate.

Holotype: MFLU 17–1504

Saprobic on dead branches of Clematis orientalis. Sexual morph: Undetermined. Asexual morph: Conidiomata 175–348 × 115–234 μm (\( \bar{x} \)= 234 × 159 μm, n = 5), pycnidial, solitary, sometimes aggregated, uniloculate, immersed, with only black shiny ostioles visible, globose to compressed, brown to dark brown, coriaceous, thick-walled, ostiolate, with minute papilla. Ostioles 41–139 × 66–85 μm (\( \bar{x} \) = 105 × 73 μm, n = 5), central, oblong, lined with periphyses. Conidiomatal wall 12–32 μm wide (\( \bar{x} \) = 20 μm, n= 20), comprises dark brown to light brown cells of textura angularis, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 6–14 × 1.5–3 μm (\( \bar{x} \) = 10 × 2.5 μm, n = 70), enteroblastic, phialidic, determinate, discrete, cylindrical to subcylindrical, hyaline, canal and collarette minute, smooth-walled, arising from the inner layers of conidioma. Conidia 10–15 × 4–7 μm (\( \bar{x} \) = 12 × 5 μm, n = 100), cylindrical, hyaline, slightly curved, with 1(–2) guttules in each cell, aseptate to 1 septum, smooth-walled.

Culture characters: Colonies on MEA reaching 20 mm diam. after 3 weeks at 25 °C. Cultures from above, cream to pale yellow in the middle, with medium sparse mycelia, circular, umbonate, papillate fairly fluffy, covered with grey aerial mycelium, radially furrowed, dark brown pigment diffusing in the agar; reverse: dark brown at the centre, cream radiating outwardly.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis orientalis L., 13 June 2017, D. Ertz & C. Gerstmans, BRCO1 (MFLU 17–1504, holotype); ex-type living culture, MFLUCC 17–2147.

Host: Clematis orientalis—(This study).

Distribution: Belgium—(This study).

GenBank accession numbers: LSU: MT214575; SSU: MT226688; ITS: MT310620; tef1: MT394634; rpb2: MT394695.

Notes: Chaetosphaeronema clematidis is similar to other Chaetosphaeronema species in having immersed pycnidia, unilocular, with enteroblastic, phialidic, cylindrical, a channel and collarette, minute conidiogenous cells and cylindrical, hyaline conidia (Sutton 1980; Hyde et al. 2016, Fig. 50). It is phylogenetically close to C. achilleae Huang & K.D. Hyde and C. hispidulum (Corda) Moesz, but differs by the lack of setae on top of the ostioles and by larger conidia. In the phylogenetic analysis, the strain formed a close relationship with C. clematidicola (see under C. clematidicola notes for more details).

Dematiopleospora Wanas., Camporesi, E.B.G. Jones & K.D. Hyde

Dematiopleospora is typified by D. mariae Wanas., Camporesi, E.B.G. Jones & K.D. Hyde. The genus is characterized by brown setae filling the ostiolar canal, superficial ascomata and muriform ascospores with pale end cells. There are seven species in the genus (Wanasinghe et al. 2018, Fig. 51).

Dermatiopleospora mariae Wanas., Camporesi, E.B.G. Jones & K.D. Hyde, in Wanasinghe, et al., Cryptog. Mycol. 35(2): 110 (2014), new host record

Index Fungorum number: IF550536; Facesoffungi number: FoF 07304, Fig. 52.

Fig. 51
figure51

The Bayesian 50% majority-rule consensus phylogram based on combined LSU, SSU, ITS and tef1 sequence data for Dermatiopleospora species. Nine strains were included in the combined analyses which comprised 3407 characters (884 characters for LSU, 1033 characters for SSU, 617 characters for ITS and 873 characters for tef1, including gap regions). The tree is rooted with Dlhawksworthia alliariae (MFLUCC 13-0070). Single gene analyses were also performed to compare the topology and clade stability with combined gene analyses. Tree topology generated under the maximum likelihood analysis was similar to Bayesian analyses. The best scoring RAxML tree was obtained with a final likelihood value of − 5814.019143. The matrix had 162 distinct alignment patterns, with 24.34% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.252414, C = 0.229288, G = 0.264353, T = 0.253945; substitution rates AC = 0.248381, AG = 2.072002, AT = 1.746797, CG = 1.084349, CT = 5.244757, GT = 1.000000; gamma distribution shape parameter α = 0.904425. In our analysis, GTR + I + G model was used for each partition in the Bayesian posterior analysis. The species determined in this study is indicated in blue. Bootstrap values (BS) from maximum likelihood (ML, left) of more than 50% BS and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. The significantly supported values from all analyses are BS ≥ 70%/BYPP ≥ 0.95

Fig. 52
figure52

Dermatiopleospora mariae (MFLU 16–0121). a Appearance of ascomata on Clematis vitalba. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–j Asci. k–p Ascospores. Scale bars: a = 500 µm, b = 100 µm, cj = 20 µm, k–p = 10 µm

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 131–210 × 156–300 μm (\( \bar{x} \) = 195 × 240 μm, n = 5), superficial, solitary, scattered, subglobose, flattened at base, dark brown to black, coriaceous, cupulate when dry, ostiolate. Ostioles 35–42 × 50–66 μm, papillate, brown, smooth, comprising short, light brown setae. Peridium 11–24 μm wide (\( \bar{x} \) = 19 μm, n = 20), thick, with 7–9 layers, outer layer heavily pigmented, comprising reddish to dark brown cells of textura angularis, inner layer composed of hyaline thin-walled cells of textura angularis. Hamathecium composed of numerous, 2–4 μm wide, filamentous, branched, septate, cellular pseudoparaphyses. Asci 100–125 × 13–19 µm (\( \bar{x} \) = 111 × 16 µm, n = 40), 8-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, pedicellate, thick-walled at the apex, with minute ocular chamber. Ascospores 20–26 × 7–10 µm (\( \bar{x} \) = 23 × 9 µm, n = 40), partially overlapping, 1–2-seriate, muriform, ellipsoidal to broad fusiform, slightly curved, ends acute, upper part wider than the lower part, 5–6-transversely septate, with 1–3 vertical septa, deeply constricted at the central septum, initially hyaline, becoming yellowish brown at maturity, ends remaining lighter and without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 18 °C. Culture from above, medium dense, circular, margin smooth, white, flat, surface rough; reverse cream radiating outwardly, dark brown in the middle.

Material examined: Italy, Forlì-Cesena Province, Fiumana di Predappio dead and hanging branches of Clematis vitalba, 31 January 2013, E. Camporesi, IT 1037 (MFLU 16–0121).

Hosts: Clematis vitalba, Ononis spinosa—(Wanasinghe et al. 2014; this study).

Distribution: Italy—(Wanasinghe et al. 2014; this study).

GenBank accession numbers: LSU: MT214576; SSU: MT226689; ITS: MT310621; tef1: MT394635.

Notes: Dermatiopleospora mariae MFLU 16–0121 (Fig. 52) grouped with the type strain of D. mariae (MFLUCC 13–0612) with moderate statistical support of 60% ML (Fig. 51). Dermatiopleospora mariae (MFLUCC 13–0612) was originally described from Ononis spinosa in Italy. Morphological characters of our collection are similar to the type strain (Wanasinghe et al. 2014). The ITS sequence of our collection shows five base pair differences, however, the tef1 data is identical to D. mariae (MFLUCC 13–0612).

Leptospora Rabenh.

Leptospora is typified by L. rubella and clustered in Phaeosphaeriaceae (Hyde et al. 2016). Morphological characters of the holotype of Leptospora mentioned that the fungus stains host tissue red to purple and is red at the apical part of ostiolar canal (Rabenhorst 1857; Shoemaker 1976; Crous et al. 2006). Phylogenetic analyses of a combined LSU, SSU, ITS and tef1 dataset revealed one new species and a new host record of Leptospora from Clematis species (Figs. 53, 54).

Fig. 53
figure53

Leptospora clematidis (MFLU 17–1505, holotype). a Appearance of ascomata on Clematis patens. b Vertical section through ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–h Asci. i–l Ascospores. Scale bars: b = 100 µm, c, fh = 20 µm, d, e = 50 µm, il = 10 µm

Fig. 54
figure54

Leptospora thailandica (MFLU 17–1474). a Appearance of ascomata on Clematis subumbellata.b Vertical section through ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–h Asci. i, j Ascospores. k, l Culture characteristics on MEA. Scale bars: b = 100 µm, ch = 50 µm, i, j = 20 µm

Leptospora clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Index Fungorum number: IF557197; Facesoffungi number: FoF 02441, Fig. 53.

Etymology: The specific name “clematidis” refers to the host.

Holotype: MFLU 17–1505.

Saprobic on Clematis patens.Sexual morph: Ascomata 95–245 × 127–247 μm (\( \bar{x} \) = 159 × 202 μm, n = 5), dark brown to black, scattered, sometimes semi-immersed, erumpent through host epidermis, only black shiny dots are visible on the host surface, uniloculate, subglobose, compressed, dark brown to black, coriaceous, ostiolate. Ostioles 28–40 × 38–87 µm, central, pseudoclypeus, with periphyses filling the ostiolar canal, pale brown to brown, with light orange pigment at the pore. Peridium 10–32 µm wide, uniform, composed of 4(–5) layers of cells arranged in textura angularis, brown to dark brown, inner layer lined with sub-hyaline cells of textura angularis. Hamathecium composed of numerous, 2–4 µm wide, filamentous, cellular pseudoparaphyses, with distinct septa, embedded in mucilaginous matrix, anastomosing at the apex. Asci 67–96 × 7–11 µm (\( \bar{x} \) = 78 × 10 μm, n = 30), 8-spored, bitunicate, clavate, with short, furcate pedicel, apically rounded, ocular chamber present when young. Ascospores 17–33 × 3–6 µm (\( \bar{x} \) = 26 × 5 μm, n = 30), narrowly turbinate, rounded at apex, acute at the bottom, hyaline to yellowish brown, 3-septate, cell above median septa slightly enlarged, slightly constricted at the septa, smooth-walled, with minute guttule in each cell, polar appendages visible when immature. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C, from above: sparse, circulate, flattened, surface smooth, with fimbriate edge, cream at the margin, white with pale yellowish at the centre; colony below brown at the margin and centre.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis patens, 13 June 2017, D. Ertz & C. Gerstmans, BRCP1 (MFLU 17–1505, holotype); ex-type living culture, MFLUCC 17–2148, ibid. (MFLU 17–1509, paratypes); ex-paratype living culture, MFLUCC 17–2152.

Host: Clematis patens—(This study).

Distribution: Belgium(This study).

GenBank accession numbers: MFLUCC 17–2148: LSU: MT214577; SSU: MT226690; ITS: MT310622; tef1: MT394636; rpb2: MT394696. MFLUCC 17–2152: LSU: MT214578; SSU: MT226691; ITS: MT310623; tef1: MT394637; rpb2: MT394697.

Notes: Leptospora clematidis shares common characters with Leptospora in having uniloculate ascomata, ostioles with light orange pigment, a peridium of thin-walled cells arranged in textura angularis, and light yellow ascospores (Hyde et al. 2016). Leptospora clematidis has similar morphology to L. galii (KUMCC 15–0521), the strain recorded from Galium sp. in Italy. However, our new species differs from L. galii in having larger ascomata that are subglobose with clavate, furcate asci (Hyde et al. 2016, Fig. 53).

Phylogeny (Fig. 48) reveals that L. clematidis forms a close relationship with L. galii (KUMCC 15–0521) with strong support (100% ML/1.00 BYPP, Fig. 48). A comparison of the ITS region (including 5.8S region) showed 10 nucleotide differences (585/594—98%, with a single gap). A comparison of the tef1 region revealed 15 base pair differences (822/837—98%, with no gaps). Thus, we describe L. clematidis as a distinct species.

Leptospora thailandica Phukhams. & K.D. Hyde, in Hyde et al. Fungal Diversity 80: 100 (2016), new host record

Index Fungorum number: IF552239; Facesoffungi number: FoF 02381, Fig. 54.

Saprobic on dead branches of Clematis subumbellata.Sexual morph: Ascomata 188–229 × 159–179 μm, immersed to erumpent through host tissue, only black shiny dots are visible on the host surface, solitary, scattered, globose to compressed, smooth, brown to dark brown. Ostioles 120–132 × 91–95 μm (\( \bar{x} \) = 125 × 92 μm, n = 5), papillate, oblong, dark brown to light brown, heavily pigmented outer layer, smooth, filled with periphyses, reddish to orange pigment around pore. Peridium 12–18 μm wide, up to 30 μm at the apex, thin-walled, brown to dark brown, pseudoparenchymatous cells, composed of 5–7 layers of textura angularis, inner layers composed of hyaline gelatinous cells. Hamathecium composed of numerous, 2.8–5.5 μm wide (n = 30), broad, branched, filamentous, septate, cellular pseudoparaphyses. Asci 63–107 × 8–13 μm (\( \bar{x} \)= 77 × 10 μm, n = 30), 8-spored, bitunicate, cylindrical to cylindrical-clavate, with short furcate pedicel, apically rounded, ocular chamber visible when immature. Ascospores 40–77 × 2–5 μm (\( \bar{x} \)= 58 × 4 μm, n = 40), fasciculate, scolecosporous, tapering towards the ends, hyaline when immature, pale brown at maturity, with minute guttule in each cell, with (7–)8–17-septate, slightly constricted at the septa, with 3–5 µm sheath drawn out to form bipolar appendages, with a pad-like structure at the apices. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Culture from above brown, sparse, circular, faintly zonate, convex with moderate aerial mycelium, downy, with slightly irregular at margins; reverse brown at the edge, light brown at the centre, dense, margin rough, not pigmented.

Material examined: Thailand, Phayao Province, on dead branches of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH10 (MFLU 17–1474); living culture, MFLUCC 17–2066.

Hosts: Chromolaena odoratain, Clematis patens, Duranta sp.—(Hyde et al. 2016; Mapook et al. 2020; this study).

Distribution: Thailand—(Hyde et al. 2016; Mapook et al. 2020; this study).

GenBank accession numbers: LSU: MT214579; SSU: MT226692; ITS: MT310624; tef1: MT394638.

Notes: Our new collection of Leptospora thailandica (MFLUCC 17–2066) is morphologically similar to the type species (MFLUCC 16–0385) which was reported from Duranta sp. (Hyde et al. 2016). The collection MFLUCC 17–2066 stains the host substrate pinkish red (Fig. 54). Phylogenetic analysis of combined sequence data indicated that L. thailandica (MFLUCC 17–2066) clusters together with the type strain and the strain reported from Chromolaena odorata with strong support (100% ML/1.00 BYPP, Fig. 48). The ITS sequence of our collection shows two base pair differences from the type strain (from 577 characters, including gap regions), while the tef1 region is 100% identical.

Longispora Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557198; Facesoffungi number: FoF 07305, Fig. 55.

Fig. 55
figure55

Longispora clematidis (MFLU 20–0420, holotype). a, b Appearance of ascomata on Clematis vitalba. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Pseudoparaphyses. g–i Asci. j–m Ascospores (m Ascospore in cotton blue). Scale bars: c = 100 µm, d–f, j–m = 20 µm, g–i = 50 µm

Etymology: The generic epithet referring to the long ascospores.

Saprobic on herbaceous plant in terrestrial habitats. Sexual morph: Ascomata solitary, immersed to erumpent, subglobose to compressed, cupulate when dry, brown to dark brown, with brown hyphae projecting from the peridium, ostiolate. Ostioles papillate, oblong, brown to light brown, heavily pigmented at outer layer, smooth, filled with periphyses, with a reddish to orange pigment around the pore. Peridium thick-walled, wider at the apex, comprising brown-walled cells of textura angularis. Hamathecium composed of numerous, branched, filamentous, transversely septate, cellular pseudoparaphyses. Asci 8-spored, bitunicate, cylindrical-clavate, with short pedicel, with a visible ocular chamber. Ascospores fasciculate, scolecosporous, ends rounded, hyaline when immature, pale brown at maturity, multi-septate, deeply constricted at the swollen cell, slightly constricted at the other septa, not separating into part spores. Asexual morph: Undetermined.

Type species: Longispora clematidis Phukhams. & K.D. Hyde

Notes: Longispora is established as a monotypic genus with L. clematidis as the type species. The genus is typical of Phaeosphaeriaceae in having compressed globose, coriaceous, brown to dark brown ascomata, with a reddish to orange pigments around the ostiolar pore, cellular pseudoparaphyses and fasciculate, scolecosporous, pale brown and multi-septate ascospores (Rabenhorst 1857; Crous et al. 2006; Phookamsak et al. 2014). Longispora has morphological characters similar to Leptospora and the sexual morph character of Chaetosphaeronema and Neosetophoma (N. camporesii) in having pigmentation in the ostiolar canal (Hyde et al. 2016, 2020; this study). Moreover, the fasciculate, scolecosporous, ascospores are common in Phaeosphaeriaceae such as in Ophiobolus, Ophiosphaerella or Pseudoophiobolus (Phookamsak et al. 2017). Longispora is distinguishable from other genera having scolecospores in Phaeosphaeriaceae in its cupulate ascomata with colouration around the ostiolar pore, and asci that are cylindrical-clavate, short with a bulbose pedicel, and ascospores that are deeply constricted at the swollen cell.

Based on the multi-gene phylogenetic analysis (Fig. 48), MFLU 20–0420 formed a basal lineage to Leptospora and Populocrescentia with strong support (96% ML/1.00 BYPP, Fig. 48). A BLAST result of the nucleotide sequences showed 98.84% similarity to Phaeosphaeria oryzae (CBS 110110) in the LSU region, while the ITS region showed 89.24% similarity to Populocrescentia forlicesenensis (MFLUCC 14–0651).

Longispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557199; Facesoffungi number: FoF 07306, Fig. 55.

Etymology: The epithet reflects the host Clematis.

Holotype: MFLU 20–0420.

Saprobic on dead stems of Clematis vitalba.Sexual morph: Ascomata 324–340 × 362–368 μm (\( \bar{x} \)= 332 × 365 μm, n = 5), solitary, scattered, immersed to erumpent through host tissue, only black shiny dots are visible on the host surface, subglobose to compressed, cupulate when dry, brown to dark brown, with brown hyphae projecting from the peridium, ostiolate. Ostioles 79–105 × 75–85 μm (\( \bar{x} \) = 94 × 80 μm, n = 5), papillate, oblong, brown to light brown, heavily pigmented at outer layer, smooth, filled with periphyses, with a reddish to orange colouration around the pore. Peridium 15–30(–35) μm wide, up to 50 μm at apex, wider at the apex, comprising 6(–8)-layers of brown-walled cells of textura angularis, outer layer heavily pigmented, with inner region comprising hyaline cells of textura angularis. Hamathecium composed of numerous, 2–4 μm wide (n = 50), branched, filamentous, septate, cellular pseudoparaphyses. Asci 97–157 × 9–15 μm (\( \bar{x} \)= 128 × 12 μm, n = 35), 8-spored, bitunicate, cylindrical-clavate, with short bulbose pedicel, apically rounded, with visible ocular chamber. Ascospores 93–124 × 2–5 μm (\( \bar{x} \)= 110 × 4 μm, n = 40), fasciculate, scolecosporous, ends rounded, hyaline when immature, pale brown at maturity, with minute guttule in each cell, (17–)19–23-septate, swollen near the septa between the 11th or 13th or 14th cell, deeply constricted at the swollen cell, slightly contricted at the other septa, not separating into part spores, indentations present. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 16 °C. Culture slow growing, black, dense, rhizoid, raised with concave edge, rough, irregular at the margins; reverse: black, dense, not pigmented.

Material examined: Italy, Forlì-Cesena Province, Viale Salinatore—Forlì City, on dead aerial branch of Clematis vitalba, 23 February 2015, E. Camporesi, IT2389B (MFLU 20–0420, holotype).

Hosts: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214580; SSU: MT226693; ITS: MT310625; tef1: MT394639.

Notes: Longispora clematidis is characterized by having sessile, cupulate ascomata, reddish orange ostioles, cellular pseudoparaphyses, cylindric-clavate asci with a bulbose pedicel and filiform ascospores, which are multi-septate and swollen between the 11th–14th cells (Fig. 55). We introduce L. clematidis based on morphological and phylogenetic analyses.

Pseudoophiobolus Phookamsak, Wanas., S.K. Huang, Camporesi & K.D. Hyde

Pseudoophiobolus is typified by P. mathieui (Westend.) Phookamsak, Wanas., S.K Huang, Camporesi & K.D. Hyde. The genus was introduced to accommodate an ophiobolus-like taxon that is phylogenetically distant from Ophiobolus sensu stricto (Phookamsak et al. 2017). Pseudoophiobolus is distinguishable from other ophiobolus-like species in having semi-immersed to erumpent, papillate ascomata with pseudoparenchymatous cells, arranged in a textura angularis to textura prismatica, cellular pseudoparaphyses, and fasciculate, scolecosporous multi-septate ascospores with a swollen cell, that do not split into part spores. A new host record of P. rosae on Clematis is presented (Fig. 56).

Fig. 56
figure56

Pseudoophiobolus rosae (MFLU 15–1014). a Appearance of ascomata on Clematis vitalba. b Vertical section through ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–g Asci. h–j Ascospores (j in cotton blue). k Germinated ascospore. l, m Culture characteristics on MEA. Scale bars: a = 500 µm, b = 200 µm, ck = 50 µm

Pseudoophiobolus rosae Phookamsak, Wanas., Phukhams., Camporesi & K.D. Hyde in Phookamsak et al. Fungal Diversity 87: 330 (2017), new host record

Index Fungorum number: IF553928; Facesoffungi number: FoF 03805, Fig. 56.

Saprobic on dried stems of Clematis vitalba. Sexual morph: Ascomata 290–345 × 240–305 μm (\( \bar{x} \)= 317 × 274 μm, n = 5), uniloculate, scattered, solitary, semi-immersed to superficial, ampulliform, globose, cupulate when dried, dark brown to black, with dark brown, septate mycelium at the base, ostiolate. Ostioles 117–135 × 100–117 μm, oblong, apex rounded, short papillate, composed of several layers of dark pseudoparenchymatous cells, with opening by a pore, filled with hyaline periphyses. Peridium 12–30(–36) μm wide, slightly thickened, composed of 6(–9) layers of dark brown cells arranged in textura angularis, pseudoparenchymatous cells, inner layers comprising 2 layers of hyaline cells, arranged in textura angularis. Hamathecium composed of dense, 2–4 μm wide (\( \bar{x} \)= 2.5, n = 50), wide, broad, branched, filamentous, septate, cellular pseudoparaphyses, anastomosing at the apex, embedded in a hyaline gelatinous matrix. Asci 64–153 × 9–14 μm (\( \bar{x} \)= 116 × 12 μm, n = 30), 8-spored, bitunicate, fissitunicate, cylindric-clavate, with short bulbose pedicel, apically rounded, with well-developed ocular chamber. Ascospores (40–)75–110 × 3–5 μm (\( \bar{x} \)= 88 × 4 μm, n = 40), fasciculate, scolecosporous, curved, pale yellowish to yellowish, with rounded ends, tapered towards the lower cells, swollen at the 8th or 10th cell, 17(–22)-septate, not constricted at the septa, smooth-walled. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 40 mm diam. after 4 weeks at 16 °C. Culture medium dense, circular, umbonate, surface smooth, edge erose, thinly hairy, green at the edge, yellowish to cream at the centre; reverse: cream at the margin, brown at the centre, not producing pigmentation in agar.

Material examined: Italy, Arezzo Province, Quota—Poppi City, dead aerial branch of Clematis vitalba, 5 June 2016, E. Camporesi, IT 2983A (MFLU 15–1014); living culture, MFLUCC 16–1364.

Hosts: Clematis vitalba, Rosa canina—(Phookamsak et al. 2017; This study).

Distribution: Italy—(Phookamsak et al. 2017; This study).

GenBank accession numbers: LSU: MT214581; SSU: MT226694; ITS: MT310626; tef1: MT394640.

Notes: Our collection, MFLUCC 16–1364 formed a clade with the type strain of Pseudoophiobolus rosae (MFLUCC 17–1786) with strong support (100% ML/0.99 BYPP, Fig. 48). Our strain is morphologically similar to the type strain of P. rosae which was reported on Rosa canina (Phookamsak et al. 2017, Fig. 56). A nucleotide comparison of the ITS and tef1 regions of MFLUCC 16–1364 and the type strain are 100% identical. Thus, we report a new host record of Pseudoophiobolus rosae on Clematis.

Wojnowiciella Crous, Hern.-Restr. & M.J. Wingf.

Wojnowiciella eucalypti is the type species. The genus is characterized by a papillate, ostiolate conidiomata, ampulliform, phialidic conidiogenous cells, hyaline, aseptate microconidia and brown, multi-septate macroconidia (Wijayawardene et al. 2013; Crous et al. 2015a). The genus has morphological resemblance to Wojnowicia which was introduced by Saccardo (1892). However, Crous et al. (2015a) discussed that the type species of Wojnowicia, W. hirta is compatible with the generic concept of Septoriella. Thus, Wojnowicia was synonymised as a member of Septoriella. Our collection associated with Clematis viticella revealed a novel species W. clematidis from Belgium (Fig. 57).

Fig. 57
figure57

Phylogram obtained from maximum likelihood based on combined LSU, SSU, ITS, tef1 and rpb2 sequence data. The tree is rooted with Galiicola baoshanensis (KUN-HKAS 102234) and Galiicola pseudophaeosphaeria (MFLUCC 14-0524). Related sequences are retrieved from GenBank with 16 strains included in the analysis of the combined loci and comprises 4177 characters (820 characters for LSU, 1012 characters for SSU, 561 characters for ITS, 905 characters for tef1, 879 characters for rpb2, including gaps). The best scoring RAxML tree had a final likelihood value of − 7456.746869. The matrix had 277 distinct alignment patterns with 31.92% undetermined characters and gaps. Estimated base frequencies were: A = 0.244763, C = 0.241642, G = 0.263061, T = 0.250533; substitution rates AC = 1.095229, AG = 1.632728, AT = 1.392298, CG = 0.793970, CT = 6.972828, GT = 1.000000; gamma distribution shape parameter α = 6.674062. Maximum parsimony analysis of 111 parsimony informative characters resulted in a most parsimonious tree (CI = 0.916, RI = 0.858, RC = 0.786, HI = 0.084). In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. Bootstrap values (BS) from maximum parsimony (MP, left), maximum likelihood (ML, right) higher than 50% BS and Bayesian posterior probabilities (BYPP, below) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% ML/0.90 BYPP. Thick branches represent significant support values from all analyses (ML ≥ 70%/BYPP ≥ 0.95). The ex-type strains are in bold and black. The newly generated sequence is in bold and blue

Wojnowiciella clematidis Phukhams., Ertz, Gerstmans & K.D. Hyde, sp. nov.

Index Fungorum number: IF557200; Facesoffungi number: FoF 07307, Fig. 58.

Fig. 58
figure58

Wojnowiciella clematidis (MFLU 17–1517, holotype). a, b Appearance of ascomata on host surface. c Vertical section through ascoma. d Section of peridium. e Pseudoparaphyses. f–h Asci. i–n Ascospores. Scale bars: b, c = 200 µm, d–h = 50 µm, i–n = 20 µm

Etymology: The epithet refers to the host plant, Clematis.

Holotype: MFLU 17–1517.

Saprobic on dried stems of Clematis viticella. Sexual morph: Ascomata 228–240 × 223–250 μm, (\( \bar{x} \) = 236 × 240 μm, n = 5), solitary, gregarious, scattered, immersed to erumpent through host epidermis, black shiny dots are visible on the host surface, uniloculate, subglobose, compressed, dark brown to black, coriaceous, apapillate, ostiolate. Ostioles central, pseudoclypeate, dark brown. Peridium 15–45 µm wide, composed of 7(–9) layers of cells arranged in textura angularis, brown to dark brown, inner layer lined with sub-hyaline cells of textura angularis. Hamathecium composed of numerous, 2–4 µm wide, filamentous, cellular pseudoparaphyses, with distinct septa, embedded in a mucilaginous matrix, anastomosing at the apex. Asci 97–136 × 12–17 µm (\( \bar{x} \) = 116 × 15 μm, n = 30), 8-spored, bitunicate, broadly cylindrical to cylindrical-clavate, with short, furcate pedicel, apically rounded, with well-developed ocular chamber. Ascospores 24–38 × 4–9 µm (\( \bar{x} \) = 29 × 7 μm, n = 30), biseriate, partially overlapping, obovoid to sub-fusiform, rounded at apex, acute at the ends, hyaline to yellowish brown, 4–6-septate, with an oblique or longitudinal septum in the central 2–3 cells, above median septum slightly enlarged, constricted at the cell above median septum, smooth-walled, with minute guttule in each cell, without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Culture from above sparse, circular, flattened, surface smooth, with fimbriae at the edge, cream at the margin, white at the centre, colony from below brown.

Material examined: Belgium, Flemish Brabant, Meise Botanic Garden, Bouchout Domain, dead stems of Clematis viticella, 13 June 2017, D. Ertz & C. Gerstmans, BRCV5 (MFLU 17–1517, holotype); ex-type living culture, MFLUCC 17–2159.

Host: Clematis viticella—(This study).

Distribution: Belgium—(This study).

GenBank accession numbers: LSU: MT214582; SSU: MT226695; ITS: MT310627; tef1: MT394641; rpb2: MT394698.

Notes: Wojnowiciella clematidis (strain MFLUCC 17–2159) is introduced as a novel species based on its distinctive morphology and phylogeny (Figs. 57, 58). The species is similar to other Wojnowiciella species in having immersed to erumpent, dark brown to black, coriaceous, ostiolate, apapillate ascomata, and hyaline to yellowish brown ascospores (Crous et al. 2015a). Wojnowiciella clematidis has similar characters to W. italica (MFLUCC 13–0447), but differs in having cylindrical to cylindrical-clavate asci and obovoid to sub-fusiform ascospore (Hyde et al. 2016). Phylogeny (Fig. 57) revealed that W. clematidis (MFLUCC 17–2159) formed a close relationship with W. dactylidis (CBS 145077) with good support (77% MP/84% ML/1.00 BYPP). Wojnowiciella dactylidis strain CBS 145077 formed a separate clade from the type species (W. dactylidis MFLUCC 13–0735), however the strain CBS 145077, which was isolated from Dypsis sp. only forms microconidia in culture (Crous et al. 2019). A comparison of nucleotides between W. clematidis (MFLUCC 17–2159) and W. dactylidis (CBS 145077) showed that the ITS region (including the 5.8S region) has a single nucleotide difference (550/561—98%, including gaps). The comparison of the tef1 region revealed three base pair differences (869/440, no gaps). Based on current evidence and the lack of asexual morph character from this study, these species are considered as distinct.

Pleosporaceae Nitschke

The family was revisited by Ariyawansa et al. (2015c), with additional taxa and resegregation in Wanasinghe et al. (2017). We introduce a new species and new host records of Stemphylium from Clematis vitalba, based on a morphology and concatenated phylogenetic analysis of LSU, ITS, SSU and gadph sequence data (Fig. 59).

Fig. 59
figure59

The Bayesian 50% majority-rule consensus phylogram based on combined LSU, ITS and gadph data for Stemphylium. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with Curvularia species. Forty-six strains were included in the combined gene sequence analyses which comprised 2052 characters (902 characters for LSU, 555 characters for ITS, 595 characters for gadph, including gap regions). The best scoring RAxML tree had a likelihood value of − 6557.926637. The matrix had 441 distinct alignment patterns with 29.84% undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.245289, C = 0.253669, G = 0.255798, T = 0.245244; substitution rates AC = 1.484723, AG = 3.274866, AT = 0.996508, CG = 1.050822, CT = 5.664441, GT = 1.000000; gamma distribution shape parameter α = 0.781417. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Stemphylium Wallr.

Stemphylium is a dematiaceous hyphomycete genus and the asexual morph has been recorded in Pleospora allies (Ariyawansa et al. 2015c; Woudenberg et al. 2017). Based on morphological studies, multi-gene phylogeny analyses, and ecological evidence, the use of Stemphylium over Pleospora has been recommended (Köhl et al. 2009; McNeill et al. 2012; Rossman et al. 2015; Woudenberg et al. 2017; Wijayawardene et al. 2018). Stemphylium is typified with S. botryosum. Phylogenetic placement of S. botryosum is verified by Woudenberg et al. (2017). Based on the phylogenetic analysis of the combined LSU, ITS and gapdh dataset (Fig. 59), we report a new host record for S. vesicarium and describe a new species S. clematidis from Clematis vitalba (Figs. 60, 61).

Fig. 60
figure60

Stemphylium clematidis (MFLU 16–0176, holotype). a Appearance of ascomata on host surface. b Close up of ascomata. c Vertical section though ascoma. d Peridium. e Pseudoparaphyses. f–h Asci. i–m Ascospores (m Ascospore stained in 10% India ink). Scale bars: a = 500 µm, b = 200 µm, c = 100 µm, dh = 20 µm, im = 10 µm

Fig. 61
figure61

Stemphylium vesicarium (MFLU 16–1109). a Appearance of ascomata on host surface. b Vertical section of ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–h Asci. i–n Ascospores (n Ascospore in 10% Indian ink). o Germinated ascospore. p, q Culture characteristics on MEA. Scale bars: a = 500 µm, b = 200 µm, ch = 50 µm, in = 10 µm, o = 20 µm, p, q = 50 mm

Stemphylium clematidis Wanas., Camporesi & K.D. Hyde, sp. nov.

Index Fungorum number: IF557305; Facesoffungi number: FoF 07327, Fig. 60.

Etymology: Refers to the name of the host.

Holotype: MFLU 16–0176.

Saprobic on dead stem of Clematis vitalba. Sexual morph: Ascomata 190–230 × 290–320 µm (\( \bar{x} \) = 220 × 303 µm, n = 20), solitary, scattered, uniloculate, under epidermal layer of host to superficial, compressed globose to subglobose, coriaceous, black, ostiolate. Ostioles central, papillate, with variable walls, opening by a pore, filled with hyaline periphyses. Peridium 30–43 μm wide (\( \bar{x} \) = 36 μm, n= 20), uniform, composed of 7 layers of textura angularis, black, heavy pigment at outer layer, cells towards the inside lighter, inner layer thin, hyaline gelatinous. Hamathecium composed of numerous, dense, 3–4 µm wide, filamentous, branched, septate, cellular pseudoparaphyses, situated between and above the asci embedded in a gelatinous matrix. Asci 127–172 × 29–38 µm (\( \bar{x} \) = 150 × 35 µm, n = 30), 8-spored, bitunicate, fissitunicate, clavate to broad cylindrical-clavate, with short, with furcate pedicel, apically rounded, with an ocular chamber. Ascospores 24–37 × 12–18 µm (\( \bar{x} \) = 32 × 13 µm, n = 50), biseriate or partially overlapping, broad-fusiform, tapering towards the rounded ends, dictyosporous, 1(–2)-longitudinal euseptate, 7–9-transverse euseptate, constricted at the septa, strongly constricted at the median septum, cells above median septum wider than below, yellowish, with 8–10 µm wide mucilaginous drawn out sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 16 °C. Culture from above cream, orange aerial mycelium, dense, fluffy at the edge, umbonate, rough, lobate; reverse cream radiating white outwardly.

Material examined: Italy, Forli-Cesena Province, Marsignano-Predappio, dead stems of Clematis vitalba, 31 March 2015, E. Camporesi, IT 1537 (MFLU 16–0176, holotype); ex-type living culture, MFLUCC 14–0937.

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214583; SSU: MT226696; ITS: MT310628; gapdh: MT394626.

Notes: Stemphylium clematidis has a pleospora-like sexual morph. The isolate formed a sister clade with S. majusculum, however the species is distinguishable by somewhat larger ascospores (Ramsey 1934; Simmons 1969, Fig. 60). The asexual morph was not obtained from culture. A pairwise comparison of the ITS sequence showed six nucleotide differences out of 555 nucleotides in the ITS regions (including gaps), while gapdh showed five nucleotide differences out of 595 nucleotides. The new strain is introduced as a new species of Stemphylium based on guidelines proposed by Jeewon and Hyde (2016).

Stemphylium rosae (Wanas. et al.) Phukhams. & K.D. Hyde, comb. nov.

Index Fungorum number: IF557603; Facesoffungi number: FoF 0404

Basionym: Pleospora rosae Wanas., Camporesi, E.B.G. Jones & K.D. Hyde, in Wanasinghe et al., Fungal Diversity [153] (2018).

Notes: Based on analyses of combined LSU, ITS and gadph sequence data for Stemphylium (Fig. 60), two isolates of Pleospora rosae and P. rosae-caninae clustered with Stemphylium sensu stricto. These two strains are compatible with the Stemphylium concept. Thus, we synonymize Pleospora rosae under Stemphylium rosae and Pleospora rosae-caninae under Stemphylium rosae-caninae. The nomenclature change is based on one fungus = one name protocol.

Host: Rosa canina—(Wanasinghe et al. 2018).

Distribution: Italy—(Wanasinghe et al. 2018).

Stemphylium rosae-caninae (Wanas. et al.) Phukhams. & K.D. Hyde, comb. nov.

Index Fungorum number: IF557604; Facesoffungi number: FoF 04047

Basionym: Pleospora rosae-caninae Wanas., Camporesi, E.B.G. Jones & K.D. Hyde, in Wanasinghe et al., Fungal Diversity [157] (2018).

Notes: See notes under Stemphylium rosae-caninae.

Host: Rosa canina—(Wanasinghe et al. 2018).

Distribution: Italy—(Wanasinghe et al. 2018).

Stemphylium vesicarium (Wallr.) E.G. Simmons, Mycologia 61(1): 9 (1969), new host record

Index Fungorum number: IF339660; Facesoffungi number: FoF 04472, Figs. 61, 62.

Fig. 62
figure62

Stemphylium vesicarium (MFLUCC 16–0998). a Appearance of asexual morph on the surface of MEA. b, c Close up of conidiophore. d–k Conidia. Scale bars: a = 200 µm, b = 100 µm, c, d = 50 µm, e–k = 20 µm

Saprobic on dead stem of Clematis vitalba. Sexual morph: Ascomata 170–257 × 148–250 μm (\( \bar{x} \) = 220 × 205 µm, n = 10), solitary, scattered, uniloculate, under epidermal layer of host, to superficial, globose to compressed, coriaceous, black, ostiolate. Ostioles central, papillate, with variable walls, opening by a pore, filled with hyaline periphyses. Peridium 25–49 μm wide (\( \bar{x} \) = 40 μm, n= 20), thick, uniform, composed of 7–12 layers of textura angularis, black, heavy pigmented at the outer layer, cells toward the inside cells of lighter, inner layer composed of thin hyaline gelatinous layer. Hamathecium composed of numerous, dense, 1.6–2.25 µm wide (\( \bar{x} \) = 1.8 μm, n= 20), filamentous, branched, septate, cellular pseudoparaphyses, situated between and above the asci embedded in a gelatinous matrix. Asci 103–138 × 16–26 µm (\( \bar{x} \) = 118 × 23 µm, n = 30), 8-spored, bitunicate, fissitunicate, oblong to cylindrical-clavate with short, furcate pedicel, apically rounded, with an ocular chamber. Ascospores 20–30 × 6–12 µm (\( \bar{x} \) = 26 × 10 µm, n = 50), biseriate or partially overlapping, muriform, tapering towards the ends, ends rounded, dictyosporous, 1(–2)-longitudinal euseptate, 4–10-transversely euseptate, constricted at the septa, strongly constricted at the median septum, yellowish, with 5–12 µm wide drawn out mucilaginous sheath. Asexual morph: Colonies effuse on the surface of culture, scattered, hairy, and fluffy. Mycelium white, with 2–5 µm wide, effuse, hyaline, septate. Conidiophores 10–57 × 3–5 μm (\( \bar{x} \) = 35 × 4 μm, n = 20), macronematous, mononematous, simple, branched, stipes straight or flexuous, cylindrical, erect, septate, smooth, dark brown to brown, 3(–6)-septate, with 1–2 primary branches, irregularly branched at the upper parts, brown, smooth. Conidiogenous cells 5–12 × 4–10 μm (\( \bar{x} \) = 9 × 7 μm, n = 20), monotretic, integrated or terminal, on conidiophores, doliiform to oblong, pale brown. Conidia 14–31 × 13–32 μm (\( \bar{x} \) = 21 × 21 μm, n = 30), in branched chains, acrogenous, folded, muriform, dictyosporous, 1(–2)-transversely euseptate, 1–3-horizontal euseptate, constricted at septa, dark brown to brown, bud scars or disjunctors present at the site of attachment, easily separated.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 16 °C. Cultures from above, dark green, fluffy, with dark green aerial mycelium, dense, fluffy at the edge, umbonate, rough, lobate, faintly zonate; reverse: dark green, radiating.

Material examined: Italy, Forli-Cesena Province, Viale Salinatore—Forlì, dead stems of Clematis vitalba, 23 February 2015, E. Camporesi, IT 2983M (MFLU 16–1109); living culture, MFLUCC 16–0998.

Hosts: Abies sp., Allium cepa, A. sativum, Asparagus officinalis, Brassica nigra, Brassica pekinensis, Cirsium sp., Citrus sp., Clematis vitalba, Cremanthodium discoideum, Cremanthodium discoideum, Dahlia pinnata, Dianthus caryophyllus, Lathyrus odoratus, Leucadendron sp., Linum usitatissimum, Lunaria annua, Lunaria rediviva, Malus domestica, Malus sieversii, Mangifera indica, Medicago sativa, Phaseolus vulgaris, Pisum sativum, Populus tomentosa, Pyrus sinkiangensis, Sedum spectabile, Solanum lycopersicum, Tamaric sp., Trigonella foenum-graecum—(Simmons 1969; Simonyan 1981; Câmara et al. 2002; Köhl et al. 2009; Arzanlou et al. 2012; Ariyawansa et al. 2015c; Woudenberg et al. 2017; Farr and Rossman 2020; this study).

Distribution: Australia, Canada, China, Denmark, Germany, India, Italy, Netherlands, New Zealand, Portugal, South Africa, Tunisia, UK (England), USA (California)—(Simmons 1969; Simonyan 1981; Câmara et al. 2002; Köhl et al. 2009; Arzanlou et al. 2012; Ariyawansa et al. 2015c; Woudenberg et al. 2017; Farr and Rossman 2020; this study).

GenBank accession numbers: LSU: MT214584; SSU: MT226697; ITS: MT310629; tef1: MT394642.

Notes: Stemphylium vesicarium (= Pleospora herbarum) is a plant pathogen distributed on a range of wild and cultivated hosts (Köhl et al. 2009; Arzanlou et al. 2012; Woudenberg et al. 2017). Phylogenetic analysis of combined LSU, ITS and gapdh regions show that our isolate clusters with S. vesicarium (Fig. 59). The morphological comparison of our isolate (MFLUCC 16–0998) with S. vesicarium (CPC 29939) which was reported in Woudenberg et al. (2017) showed that our collection has 4–10-transverse eusepta ascospores (Figs. 61, 62). Our collection is similar to the type strian of S. vesicarium (STR) which was described by Simmons (1969). A pairwise comparison of the ITS regions show that our isolate is 100% identical to the type strain of S. vesicarium (CBS 192.86). This is the first report of S. vesicarium on Clematis.

Pseudoberkleasmiaceae Phukhams. & K.D. Hyde

Pseudoberkleasmiaceae was introduced to accommodate a berkleasmium-like species that has phylogenetic stability in Pleosporales (Hyde et al. 2019a). The family comprises Pseudoberkleasmium acacia, Pseudoberkleasmium chiangmaiense and P. pandanicola (the generic type). We describe the first record of P. chiangmaiense on Clematis from Thailand (Figs. 2, 63).

Fig. 63
figure63

Pseudoberkleasmium chiangmaiense (MFLU 17–1496). a Sporodochia on natural substrate. b Vertical section through sporodochia. c Hyphae. d–g Mature lenticular conidia. h Germinated conidia. i, j Culture characteristics on MEA. Scale bars = a = 1 cm, b = 100 μm, c, g–h = 20 μm, df = 10 μm

Pseudoberkleasmium Tibpromma & K.D. Hyde

The monotypic genus Pseudoberkleasmium was introduced with P. pandanicola Tibpromma & K.D. Hyde as the type species. Pseudoberkleasmium is characterized by hyaline, subglobose conidiogenous cells and acrogenous, broadly ellipsoidal to obovoid, muriform, brown or olivaceous green, and guttulate conidia. There is no sexual morph report in this family. A collection on Clematis sikkimensis reveals an additional strain of Pseudoberkleasmium chiangmaiense according to phylogenetic and morphological evidence (Figs. 2, 63).

Pseudoberkleasmium chiangmaiense Y.Z. Lu & K.D. Hyde, in Hyde et al., Fungal Diver (2019), new host record

Index Fungorum number: IF555595; Facesoffungi number: FoF 05310, Fig. 63.

Saprobic on dead stems of Clematis sikkimensis. Sexual morph: Undetermined. Asexual morph: Hyphomycetous, colonies on natural substrate forming sporodochial conidiomata, 109–460 μm wide, superficial, scattered, gregarious, oval, brown, velvety, glistening, orbicular, conidia readily liberated when agitated. Mycelium immersed in the substrate, composed of septate, branched, smooth, hyaline to pale brown, 2.5 μm wide hyphae. Conidiophores 10–25 × 2–5 μm, micronematous, mononematous, cylindrical or truncate, erect, hyaline, smooth-walled. Conidiogenous cells 9–15 × 6–15 μm (\( \bar{x} \) = 12 × 11 μm, n = 20), holoblastic, monoblastic, integrated, terminal, determinate, subglobose, cylindrical or slightly truncate, guttulate, hyaline. Conidia 24–32 × 13–18 μm (\( \bar{x} \) = 27 × 16 μm, n = 50), acrogenous, solitary, broadly ellipsoidal to obovoid, flattened, dictyosporous, muriform, apex rounded, basal cells globose or subglobose, in side view composed of one column of 4–6 cells, guttulate, smooth-walled, brown, usually with conidiogenous cell attached, bud scars or disjunctors present at the site of attachment.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above black, dense, circular, umbonate, papillate, fluffy, slightly radiating, wrinkled folded; reverse black, lifting media up in the centre, wrinkled.

Material examined: Thailand, Chiang Rai Province, Doi Tung, on dried stem of Clematis sikkimensis, 2 May 2017, C. Phukhamsakda & M.V. de Bult, CMTHDT05 (MFLU 17–1496); living culture, MFLUCC 17–2088.

Hosts: Clematis fulvicoma, Undetermined decaying wood—(Hyde et al. 2019a; this study).

Distribution: Thailand—(Hyde et al. 2019a; this study).

GenBank accession numbers: LSU: MT214585; SSU: MT226698; ITS: MT310630; tef1: MT394643; rpb2: MT394699.

Notes: Pseudoberkleasmium chiangmaiense (MFLUCC 17–2088) is similar to the type except for culture characters (Hyde et al. 2019a, Fig. 63), however, our collection was grown on MEA while the type was cultured on PDA. Phylogenetically, P. chiangmaiense (strain MFLUCC 17–2088) formed a clade with the type strain (MFLUCC 17–1809) with strong support (100% ML/1.00 BYPP, Fig. 2). The type strain was isolated from decaying wood collected in Chiang Mai, therefore, we present a new host record of P. chiangmaiense on Clematis sikkimensis (Fig. 63).

Pseudomassarinaceae Phukhams. & K.D. Hyde, fam. nov.

Index Fungorum number: IF557104; Facesoffungi number: FoF 07212, Fig. 64.

Fig. 64
figure64

Pseudomassarina clematidis (MFLU 160493, holotype). a Appearance of ascomata on host surface. b Vertical section of ascoma. c Ostiolar canal. d Section of peridium. e Pseudoparaphyses. f–h Asci. i–m Ascospores. n Ascospore in 10% Indian ink. Scale bars: b = 100 µm, c = 50 µm, dh = 20 µm, in = 10 µm

Saprobic on dried herbaceous plants. Sexual morph: Ascomata immersed, only ostioles visible, uniloculate, obpyriform to subglobose, light brown to brown, coriaceous. Ostioles central, brown to dark brown, carbonaceous, papillate. Peridium thin, multilayered, comprising thin-walled, light brown to brown cells of textura angularis, inner layers comprising hyaline cells. Hamathecium of dense, filiform, branched, pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, thick-walled, oblong, apically rounded, with furcate pedicel. Ascospores biseriate, overlapping, hyaline, broad fusiform, tapering towards the ends, acute at both ends, with transverse septa, with mucilaginous sheath. Asexual morph: Undetermined.

Type genus: Pseudomassarina Phukhams. & K.D. Hyde

Notes: The new family Pseudomassarinaceae is introduced to accommodate a monotypic genus, Pseudomassarina (Fig. 64). Phylogenetic analysis which included related families in Pleosporales, showed that Pseudomassarinaceae formed a distinct lineage related to Amorosiaceae, Halotthiaceae, Lophiostomataceae, Neomassarinaceae, Phaeoseptaceae and Sporormiaceae (Fig. 2). The family is morphologically similar to Lophiostomataceae, Neomassariaceae and Neomassarinaceae in having hyaline, ellipsoid to fusiform, septate ascospores (Thambugala et al. 2015; Ariyawansa et al. 2018; Mapook et al. 2020). Pseudomassarinaceae is distinguished by its immersed, coriaceous ascomata with crest-like, carbonaceous ostioles, oblong and short pedicellate asci and wide mucilaginous sheath surrounding the ascospores.

Pseudomassarina Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557097; Facesoffungi number: FoF 07213, Fig. 64.

Etymology: The genus epithet reflects its morphological similarity to Massarina species.

Saprobic on decaying plants in terrestrial habitats. Sexual morph: Ascomata on surface of the host, visible as black spots, immersed, solitary, uniloculate, obpyriform, brown, rough-walled, coriaceous, with apical ostioles. Ostioles central, brown to dark brown, partially carbonaceous, papillate, filled with periphyses. Peridium multilayered, comprising thin-walled, light brown to brown cells of textura angularis, inner layers comprising hyaline cells. Hamathecium of dense, filiform, branched, transverse septa, pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, thick-walled, oblong, apically rounded, short pedicellate, with an ocular chamber. Ascospores biseriate, overlapping, hyaline, broad fusiform, tapering towards the ends, with transverse septum, guttulate, smooth-walled, with mucilaginous sheath. Asexual morph: Undetermined.

Type species: Pseudomassarina clematidis Phukhams, Camporesi & K.D. Hyde

Notes: Pseudomassarina, is typified by P. clematidis and formed a distinct lineage in Pleosporales (Fig. 2). The morphological comparison showed that our collection has unique characters among related taxa in Pleosporales (Zhang et al. 2012; Hyde et al. 2013; Thambugala et al. 2015; Ariyawansa et al. 2018; Mapook et al. 2020). In a BLASTn search of GenBank, the closest match of the LSU sequence of MFLU 16–0493 is Preussia terricola (strain CBS 317.65) with 97.43% similarity. The closest match with the ITS sequence is Preussia polymorpha (strain CBS 117679) with 84.49% similarity.

Pseudomassarina clematidis Phukhams, Camporesi & K.D. Hyde sp. nov.

Index Fungorum number: IF557098; Facesoffungi number: FoF 07214, Fig. 64.

Etymology: The epithet reflects Clematis.

Holotype: MFLU 16–0493.

Saprobic on dead stems of Clematis vitalba. Sexual morph: Ascomata 150–220 × 80–130 μm (\( \bar{x} \)= 190 × 110 μm, n = 5), on surface of the host, visible as black spots, immersed, only ostioles visible, solitary, scattered, uniloculate, obpyriform to subglobose, light brown to brown, rough-walled, coriaceous, with apical ostioles. Ostioles central, 65–70 × 50–55 μm, brown to dark brown, papillate, opening by a pore, filled with periphyses. Peridium 10–20 μm wide, multilayered, comprising of 4–5 layers of thin-walled light brown to brown cells of textura angularis, inner layers comprising hyaline cells. Hamathecium of dense, 1.3–1.5 μm wide (\( \bar{x} \)= 1.3 μm, n = 50), filiform, branched, septate, pseudoparaphyses. Asci 55–80 × 9–15 μm (\( \bar{x} \) = 60 × 10 μm, n = 30), 8-spored, bitunicate, fissitunicate, thick-walled, oblong, apically rounded, with short, furcate pedicel, ocular chamber clearly visible. Ascospores 16–25 × 3–7 μm (\( \bar{x} \) = 20 × 5 μm, n = 50), biseriate, overlapping, hyaline, broad fusiform, tapering towards the ends, acute at both ends, with 1 transverse septum, with large guttules in each cell, deeply constricted at the septum, cell above septum longer and wider than below cell, smooth-walled, with 4–6 μm wide mucilaginous sheath. Asexual morph: Undetermined.

Material examined: Italy, Forlì-Cesena Province, Fiumicello-Premilcuore, on dead aerial branch of Clematis vitalba, 20 March 2016, E. Camporesi, IT 2335 (MFLU 16–0493, holotype).

Host: Clematis vitalba—(This study).

Distribution: Italy—(This study).

GenBank accession numbers: LSU: MT214586; SSU: MT226699, MT310631; ITS: MT415397; tef1: MT394644; rpb2: MT394700.

Notes: See note under Pseudomassarina.

Pseudolophiotremataceae K.D. Hyde & S. Hongsanan

Pseudolophiotremataceae was introduced by Hongsanan et al. (2018) with Pseudolophiotrema elymicola as the generic type. The multi-locus analysis (Fig. 2) showed that Clematidis italica (MFLUCC 15–0084) clustered with P. elymicola. Thus, we place Clematidis italica (Fig. 65) in Pseudolophiotremataceae.

Fig. 65
figure65

Clematidis italica (MFLU 14–0669, holotype). a Appearance of ascomata on host substrate. b Vertical section of ascoma. c Section of peridium. d Pseudoparaphyses. e–g Asci. h–j Ascospores. Scale bars: a = 200 μm, b = 50 μm, c = 10 μm, d, h–j = 5 μm, eg = 20 μm

Clematidis italica Tibpromma, Camporesi & K.D. Hyde, in Li et al., Fungal Diversity 78: 60 (2016)

Index Fungorum number: IF 551867, Facesoffungi number: FoF 01813, Fig. 65.

Notes: Li et al. (2016) introduced Clematidis italica (strain MFLUCC 15–0084) based on analyses of combined LSU and SSU sequence data. Subsequently, Hashimoto et al. (2017) introduced Pseudolophiotrema elymicola (MAFF 239600) based on analyses of a SSU, ITS, LSU, tef1 and rpb2 dataset (Jaklitsch et al. 2018). In our LSU phylogenetic tree for Pleosporales, Clematidis and Pseudolophiotrema formed a closely related clade with strong support (94% ML/0.98 BYPP, data not shown). Consequently, in a combined dataset of LSU, SSU, ITS, tef1 and rpb2, Clematidis italica (MFLUCC 15–0084) and Pseudolophiotrema elymicola (JCM 13090) formed a well-supported clade (100% ML/1.00 BYPP) basal to Anteagloniaceae (Fig. 2). A similar topology is shown in Hashimoto et al. (2017) wherein P. elymicola formed a distinct clade from Lophiotremataceae. As the epithet “clematidis” is similar to the plant family Clematidaceae/Clemataceae, it is difficult to introduce a generic epithet. Therefore, Pseudolophiotrema elymicola is selected as a generic type for Pseudolophiotremataceae (Hongsanan et al. 2020).

Roussoellaceae Liu, Phookamsak, Dai & K.D. Hyde

Roussoellaceae was introduced by Liu et al. (2014) and family members have steadily increased (Jaklitsch and Voglmayr 2016; Tibpromma et al. 2017, 2018; Wanasinghe et al. 2018; Jiang et al. 2019; Karunarathna et al. 2019; Phookamsak et al. 2019; Mapook et al. 2020). We provide an updated phylogenetic analysis for Roussoellaceae based on a concatenated LSU, ITS, tef1, rpb2, and SSU sequence dataset. New host records and new species occurring on Clematis species in Thailand are described based on morphological characteristics coupled with multigene phylogenetic analyses (Fig. 66).

Fig. 66
figure66

Best scoring RAxML tree with a final likelihood value of − 27212.762040 based on combined LSU, ITS, tef1, rpb2, and SSU sequence data for Roussoellaceae. The tree is rooted with members of Torulaceae. Sixty-nine strains were included in the combined analyses which comprised 4382 characters (819 characters for LSU, 567 characters for ITS, 906 characters for tef1, 1052 characters for rpb2, 1038 characters for SSU, including gaps). The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree from the maximum likelihood analysis had similar topology to the Bayesian 50% majority-rule consensus phylogram. The matrix had 1501 distinct alignment patterns with 38.60% of undetermined characters and gaps. Estimated base frequencies were as follows: A = 0.245133, C = 0.255324, G = 0.268618, T = 0.230925; substitution rates AC = 1.766964, AG = 5.243182, AT = 2.353818, CG = 1.339749, CT = 11.449776, GT = 1.000000; gamma distribution shape parameter α = 0.473912. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The isolates of this study are in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95) at the genus and family levels

Neoroussoella Liu et al.

Neoroussoella was introduced with the single species, N. bambusae Phookamsak, J.K. Liu & K.D. Hyde. Phylogenetic placement of the species in Roussoellaceae was demonstrated by Karunarathna et al. (2019) and Mapook et al. (2020). A multigene phylogenetic analysis (Fig. 66) reveals two novel species N. clematidis and N. fulvicomae, and the first record of N. heveae from a Clematis species.

Neoroussoella clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557113; Facesoffungi number: FoF 07328, Fig. 67.

Fig. 67
figure67

Neoroussoella clematidis (MFLU 17–1467, holotype). a, b Appearance of ascomata on Clematis subumbellata. c Vertical section through ascoma. d Ostiolar canal. e Vertical section through peridium. f Trabeculate pseudoparaphyses. g–i Asci. j-n Ascospores. o, p Culture characters on MEA. Scale bars: b = 200 µm, c = 100 µm, de = 50 µm, fi = 20 µm, jn = 5 µm

Etymology: Name refers to the host genus, Clematis.

Holotype: MFLU 17–1467

Saprobic on dried stems of Clematis subumbellata. Sexual morph: Ascomata 230–250 × 155–160 μm (\( \bar{x} \) = 235 × 158 μm, n = 5), only ostioles present on the surface of host, solitary, gregarious, erumpent, semi-immersed, globose to subglobose, black to rust brown, coriaceous, rough-walled, ostiolate. Ostioles central, 84–90 × 67–70 μm, dark brown to black, papillate, ostiolar canal lined with periphyses. Peridium 12–29 μm wide, outer layer composed of 5–7 layers of brown to chestnut brown cells of textura angularis, with thin, hyaline inner layer. Hamathecium of dense, 0.8–1.5 μm wide (\( \bar{x} \) = 1.3 μm, n = 40), filiform, branched, septate, trabeculate pseudoparaphyses. Asci 62–82 × 4–7 μm (\( \bar{x} \) = 71 × 5 μm, n = 30), (2–)8-spored, bitunicate, fissitunicate, cylindrical, apically rounded, with bulbose pedicel, ocular chamber visible when young. Ascospores 9–11 × 3–5 μm (\( \bar{x} \) = 10 × 4 μm, n = 50), uniseriate, partially overlapping, ellipsoid with rounded ends, olive to yellowish brown, 1-septate, constricted at septum, guttulate in each cell, rough-walled, at maturity, slightly longitudinally ribbed, without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 20 mm diam. after 4 weeks at 25 °C. Culture from above, brown, radiating outwards, dense, irregular in shape, umbonate, dull, edge erose, downy, covered with fairly white mycelium; reverse brown at the middle dark brown at the edge, with domes on the media.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH03 (MFLU 17–1467, holotype); ex-type living culture, MFLUCC 17–2061.

Host: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214587; SSU: MT226700; ITS: MT310632; tef1: MT394645; rpb2: MT394701.

Notes: Neoroussoella clematidis shares common characters with Neoroussoella in having uniloculate ascomata without a clypeus, cylindrical to broad filiform asci with bulbose pedicel and 1-septate ascospores (Liu et al. 2014). Our collection is distinguishable by having immersed and globose ascomata with papillate ostioles (Fig. 67). In the phylogenetic analysis, N. clematidis (MFLUCC 17–2061) formed a strongly supported clade (100% ML/1.00 BYPP, Fig. 66) with N. fulvicoma (MFLUCC 17–2073) which was an asexual morph (Fig. 68). A comparison of the ITS region (including 5.8S region) showed 7.7% base pair differences (with gaps) and 3.4% base pair differences (with gaps) in the tef1 region, which is evidence for new species rank.

Fig. 68
figure68

Neoroussoella fulvicomae (MFLU 17–1481, holotype). a Appearance of conidiomata on Clematis fulvicoma. b Close up of conidioma on host substrate. c Vertical section through conidioma. d Ostiolar canal. e Section of conidioma wall. fh Conidiogenous cells and conidia. im Conidia. n Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d, e = 20 µm, fm = 5 µm

The isolate MFLUCC 17–2061 was evaluated for the potential of secondary metabolites against Bacillus subtillis, Escherichia coli, Mucor plumbeus and Schizosaccharomyces pombe. The strain demonstrated moderate inhibitory activities against Bacillus subtillis and against conidia development in Mucor plumbeus. This isolate is suitable for further evaluation of secondary metabolites.

Neoroussoella fulvicomae Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557114; Facesoffungi number: FoF 07329, Fig. 68.

Etymology: Name refers to Clematis fulvicoma.

Holotype: MFLU 17–1481.

Saprobic on dried stems of Clematis fulvicoma. Sexual morph: Undetermined. Asexual morph: Conidiomata 140–241 × 137–155 μm (\( \bar{x} \)= 207 × 146 μm, n = 5), pycnidial, solitary, sometimes aggregated, uniloculate, immersed, visible as minute, black, shiny ostioles, subglobose, coriaceous, thick-walled, dark brown to brown, with papilla, ostiolate. Ostioles 51–60 × 49–55 μm, central, oblong, papillate. Conidiomatal wall 12–18 μm wide (\( \bar{x} \) = 15 μm, n= 20), thick, outer layer composed of 5–7 layers of brown to light brown cells of textura angularis, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2.5–5 × 1.5–2 μm (\( \bar{x} \) = 4 × 1.7 μm, n = 20), enteroblastic, phialidic, annellidic, determinate, discrete, truncate, smooth-walled, hyaline, arising from the inner layers of conidiomata. Conidia 3–6 × 2–3 μm (\( \bar{x} \) = 4.5 × 2.5 μm, n = 100), oval, slightly curved, hyaline when immature, brown at maturity, with 1(–2) guttules in each cell, aseptate, smooth-walled.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, black, dense, circular, margin undulate, umbonate, fluffy, wrinkled, folded, covered with brown aerial mycelium; reverse black at the centre radiating outwardly.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis fulvicoma, 20 March 2017, C. Phukhamsakda, CMTH17 (MFLU 17–1481, holotype); ex-type living culture, MFLUCC 17–2073.

Host: Clematis fulvicoma—(This study).

Distribution: Thailand(This study).

GenBank accession numbers: LSU: MT214588; SSU: MT226701; ITS: MT310633; tef1: MT394646; rpb2: MT394702.

Notes: In the phylogenetic analysis of combined sequence data Neoroussoella fulvicomae clustered in a separate clade with N. clematidis (100% ML/1.00 BYPP, Fig. 66). Neoroussoella fulvicomae is similar to Neoroussoella in having immersed, uniloculate, globose to subglobose pycnidia, annellidic conidiogenesis cells, and aseptate, 2-guttulate conidia (Liu et al. 2014). Neoroussoella fulvicomae is distinguishable by having papillate ostioles and short conidiogenesis cells and brown conidia with 1–2 guttules in each cell (Fig. 68). Neoroussoella fulvicomae was evaluated for potential of secondary metabolites in the same manner as N. clematidis. Interestingly, isolate MFLUCC 17–2073 does not produce growth inhibitory activity against Bacillus subtillis and Mucor plumbeus.

Neoroussoella heveae Senwanna, Phookamsak & K.D. Hyde, in Phookamsak et al., Fungal Divers [66] (2019), new host record

Index Fungorum number: IF555287; Facesoffungi number: FoF 07330, Fig. 69.

Fig. 69
figure69

Neoroussoella heveae (MFLU 17–1477). a Appearance of conidiomata on Clematis subumbellata specimen. b Close up of conidioma on host substrate. c Vertical section of conidioma. d Ostiolar canal. e Section of conidioma wall. fi Conidiogenous cells and conidia. jl Conidia. m Germinated conidium. n, o Cultures characters on MEA. Scale bars: c = 200 µm, d = 100 µm, e = 20 µm, fl = 5 µm

Saprobic on dried stems of Clematis subumbellata. Sexual morph: Undetermined. Asexual morph: Conidiomata 128–263 × 142–304 μm (\( \bar{x} \)= 177 × 200 μm, n = 5), pycnidial, solitary, gregarious, uniloculate, immersed, visible only as minute ostioles, globose, coriaceous, thick-walled, dark brown to brown, ostiolate. Ostioles 40 × 77 μm, central, papillate, oblong, opening by a pore. Conidiomatal wall 24–31 μm wide (\( \bar{x} \) = 26 μm, n= 20), thick, 7–8 layers, outer layer composed of brown cells of textura angularis, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 2–4 × 1.5–2 μm (\( \bar{x} \) = 3 × 2 μm, n = 50), enteroblastic, phialidic, determinate, discrete, truncate, smooth-walled, hyaline, arising from the inner layers of the conidiomata. Conidia 3.5–4.5 × 2.3–3 μm (\( \bar{x} \) = 4 × 2.5 μm, n = 100), oval, hyaline when immature, olive brown at maturity, slightly curved at the ends, with 1(–2) guttules in each cell, aseptate, smooth-walled.

Culture characters: Colonies on MEA reaching 50 mm diam. after 4 weeks at 25 °C. Cultures from above, dark brown to brown, dense, irregular, margin lobate, umbonate, wrinkled folded, thinly hairy, covered with brown aerial mycelium; reverse black at the centre, radiating outwardly.

Material examined: Thailand, Chiang Rai Province, on dead stems of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH13 (MFLU 17–1477); living culture, MFLUCC 17–2069.

Hosts: Clematis subumbellata, Hevea brasiliensis—(Phookamsak et al. 2019; this study).

Distribution: Thailand—(Phookamsak et al. 2019; this study).

GenBank accession numbers: LSU: MT214589; SSU: MT226702; ITS: MT310634; tef1: MT394647; rpb2: MT394703.

Notes: Neoroussoella heveae was described from a twig of Hevea brasiliensis from northern Thailand (Phookamsak et al. 2019). Isolate MFLUCC 17–2069, recorded on Clematis formed a close relationship (100% ML/1.00 BYPP) with the type strain of N. heveae (MFLUCC 17–0338). Our collection (MFLU 17–1477, Fig. 69) had larger conidia than the type (128–263 × 142–304 vs 90–130 × 115–180 μm). Comparison of ITS sequence data revealed only 1 base pair difference between our isolate and the type. Unfortunately, the tef1 region of the type strain is not available for comparison.

Neoroussoella heveae was evaluated for potential secondary metabolite production in the same manner as N. clematidis. Isolate MFLUCC 17–2069 demonstrated weak inhibitory activities against Bacillus subtillis.

Pararoussoella Wanas., E.B.G. Jones & K.D. Hyde

Pararoussoella (type species P. rosarum) was introduced for species that are distantly related to the type species of Roussoella (R. nitidula Sacc. & Paol.). The genus is characterized by having immersed and globose ascomata with minute black dots of ostioles, cellular pseudoparaphyses, central, papillate, black ostioles, cylindrical to oblong asci, and uniseriate, brown to dark brown ascospores, with irregular, longitudinal striations (Wanasinghe et al. 2018). Multigene phylogenetic analysis (Fig. 66) showed that Roussoella mangrovei is related to Pararoussoella, thus a new combination is proposed.

Pararoussoella mangrovei (Phukhams. & K.D. Hyde) Phukhams. & K.D. Hyde, comb. nov.

Roussoella mangrovei Phukhams. & K.D. Hyde, in Hyde et al., Mycosphere 9(2): 339 (2018)

Index Fungorum number: IF557372; Facesoffungi number: FoF 03923

Notes: Pararoussoella mangrovei formed a related lineage with other Roussoella species. Karunarathna et al. (2019) showed that the strain is related to Pararoussoella (Fig. 66). Therefore, Roussoella mangrovei is transferred to Pararoussoella.

Pseudoneoconiothyrium Wanas., Phukhams., Camporesi & K.D. Hyde

Pseudoneoconiothyrium was described as Neoconiothyrium for fungi associated with Rosaceae plants (Wanasinghe et al. 2018). Neoconiothyrium has been assigned for the fungal strains in Coniothyriaceae (Crous et al. 2017; Hawksworth et al. 2018). Thus, Pseudoneoconiothyrium was proposed for Neoconiothyrium species in Roussoellaceae and is typified by P. rosae (Phukhams., et al.) Phukhams., Camporesi & K.D. Hyde (Fig. 66).

Pseudoneoconiothyrium euonymi (Crous & Akulov) Phukhams. & K.D. Hyde, comb. nov.

Roussoella euonymi Crous & Akulov, in Crous et al., Fungal Systematics and Evolution 1: 204 (2018)

Index Fungorum number: IF557605; Facesoffungi number: FoF 07331

Notes: Pseudoneoconiothyrium euonymi originally clustered with Pararoussoella mukdahanensis (≡ Roussoella mukdahanensis). Based on the phylogenetic analysis (Fig. 66), Pseudoneoconiothyrium euonymi is transferred as a second species of Pseudoneoconiothyrium.

Pseudoroussoella Mapook & K.D. Hyde

Mapook et al. (2020) introduced Pseudoroussoella as a separate lineage for P. euonymi and P. chromolaenae. Phylogenetic analyses (Fig. 66) coupled with morphological characters of collections on Clematis species revealed a first record of P. chromolaenae and P. euonymi on Clematis species (Figs. 70, 71).

Fig. 70
figure70

Pseudoroussoella chromolaenae (MFLU 17–1468). a Appearance of conidiomata on Clematis subumbellata. b Close up of conidioma on host substrate. c Vertical section through conidioma. d Ostiolar canal. e Section of conidioma wall. fi Conidiogenous cells and conidia (f conidiogenous cells in cotton blue). jm Conidia n Geminated conidium. o, p Cultures characters on MEA. Scale bars: b = 500 µm, c = 200 µm, d = 100 µm, e = 20 µm, fm = 5 µm

Fig. 71
figure71

Pseudoroussoella elaeicola (MFLU 17–1465). ac Appearance of ascomata on Clematis subumbellata. d Vertical section through ascoma. e Ostiolar canal. f, g Section of peridium. h Pseudoparaphyses. i–l Asci. mr Ascospores (q verruculose surface). s Germinated ascospore. t, u Culture characters on MEA. Scale bars: a = 500 µm, bd = 200 µm, e = 100 µm, f, g = 20 µm, hl = 50 µm, mr = 10 µm

Pseudoroussoella chromolaenae Mapook & K.D. Hyde, Mapook et al., Fungal Divers (2020), new host record

Index Fungorum number: IF557116; Facesoffungi number: FoF 07332, Fig. 70.

Saprobic on dead stems of Clematis subumbellata. Sexual morph: Undetermined. Asexual morph: Conidiomata 86–100 × 107–112 μm (\( \bar{x} \)= 90 × 110 μm, n = 5), pycnidial, solitary, sometimes aggregated, uniloculate, immersed, with black shiny ostioles visible, globose to subglobose, dark brown to brown, coriaceous, thick-walled, ostiolate. Ostioles 55–70 × 50–67 μm, central, papillate, ovoid. Conidiomatal wall 6–14(–27) μm wide, outer layer composed of 8–10 layers of brown to light brown cells of textura angularis, lined with a hyaline layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 3–7 × 1.5–3 μm (\( \bar{x} \) = 4.5 × 2 μm, n = 30), enteroblastic, phialidic, determinate, discrete, cylindrical to sub-cylindrical, smooth-walled, hyaline. Conidia 4.5–8 × 3–5 μm (\( \bar{x} \) = 6 × 4 μm, n = 50), oval, hyaline when immature, yellowish brown at maturity, slightly curved, with 1(–2) guttules in each cell, aseptate, smooth-walled.

Culture characters: Colonies on MEA reaching 20 mm diam. after 2 weeks at 25 °C. Cultures from above, greyish brown, dense, circular, umbonate, fluffy, covered with white aerial mycelium; reverse dark brown at the central, mycelium cream radiating outwardly.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH04 (MFLU 17–1468); living culture, MFLUCC 17–2062.

Hosts: Chromolaena odorata, Clematis subumbellata—(Mapook et al. 2020; this study).

Distribution: Thailand—(Mapook et al. 2020; this study).

GenBank accession numbers: LSU: MT214590; SSU: MT226703; ITS: MT310635; tef1: MT394648; rpb2: MT394704.

Notes: Phylogenetic analysis (Fig. 66) has shown that the newly collected strain (MFLUCC 17–2062), is related to the ex-type strain of P. chromolaenae (MFLUCC 17–1492) with strong support (100% ML/1.00 BYPP, Fig. 66). The conidiomata of our collection (Fig. 70) are slightly smaller than the ex-type strain (90 × 110 vs 165 × 195 µm). Pairwise comparison of the ITS region (including 5.8S region) showed 100% similarity, while the tef1 region had two base pair differences; these findings are not significantly distinct for the introduction of the strain as a new species (Jeewon and Hyde 2016). Pseudoroussoella chromolaenae strain MFLUCC 17–2062 was evaluated for secondary metabolite production. The collection showed weak inhibitory activities against the growth of Bacillus subtillis.

Pseudoroussoella elaeicola (Konta & K.D. Hyde) Mapook & K.D. Hyde, new host record

Index Fungorum number: IF555291; Facesoffungi number: FoF 07333, Fig. 71.

Saprobic on dead stems of Clematis subumbellata.Sexual morph: Ascomata 375–554 × 375–462 μm (\( \bar{x} \)= 453 × 430 μm, n = 10), on the surface of the host, solitary, gregarious, erumpent, semi-immersed, dark brown hyphae radiating outwards from the peridium wall, globose to depressed-globose, black to dark brown, coriaceous, rough-walled, ostiolar. Ostioles 149–157 × 123–140 μm, central, dark brown to black, papillate, opening by a pore, ostiolate with periphyses. Peridium 17–54(–83 μm at apex) wide, outer layer composed of 8–10 layers of dark brown to light brown cells of textura angularis, the inner layer comprising thin, hyaline layers. Hamathecium of dense, 0.8–1.5 μm wide (\( \bar{x} \) = 1.3 μm, n = 50), filiform, branches, anastomosing above asci, septate, pseudoparaphyses. Asci 60–132 × 7–10 μm (\( \bar{x} \) = 103 × 8 μm, n = 20), (2–)8-spored, bitunicate, fissitunicate, cylindrical to cylindrical-clavate, apically rounded with ocular chamber. Ascospores 10–18 × 5–10 μm ( = 13 × 7 μm, n = 50), uniseriate, sometimes partially overlapping, oval with round ends, brown to yellowish brown, uni-septate, constricted at septum, with guttules in each cell, verruculose, with mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Culture above dark green radiating, edge white, dense, circular, flattened, umbonate, edge entire, fluffy; reverse light brown at the middle, dark brown at the edge.

Material examined: Thailand, Chiang Rai Province, on dead branches of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH01 (MFLU 17–1465); living culture, MFLUCC 17–2059.

Hosts: Chromolaena odorata, Clematis subumbellata, Elaeis guineensis—(Phookamsak et al. 2019; Mapook et al. 2020; this study).

Distribution: Thailand—(Phookamsak et al. 2019; Mapook et al. 2020; this study).

GenBank accession numbers: LSU: MT214591; SSU: MT226704; ITS: MH744730; tef1: MH750239; rpb2: MT394705.

Notes: Pseudoroussoella elaeicola (≡ Roussoella elaeicola) was collected from Elaeis guineensis (Phookamsak et al. 2019). Mapook et al. (2020) recorded this fungus on Chromolaena odorata. In the phylogenetic analysis, R. elaeicola formed a separate clade from the type species of Roussoella (R. nitidula Sacc. & Paol.), thus Mapook et al. (2020) synonymized R. elaeicola under Pseudoroussoella elaeicola. Our new isolate (MFLUCC 17–2059) grouped with another collections reported from Chromolaena odorata and Elaeis guineensis (97% ML/0.99 BYPP, Fig. 70). Our collection is identical to the other collections of this species (Fig. 71). Comparison of the ITS region revealed only one base pair difference between our isolate and the ex-type strain (MFLUCC 15–0276). The pairwise comparison of the available tef1 region with MFLUCC 17–1483 also showed one base pair difference. Therefore, we introduce our collection as a new host record.

Pseudoroussoella elaeicola (MFLUCC 17–2059) was evaluated for the potential of secondary metabolite production. The isolate showed inhibitory activity on biofilm formation by Staphylococcus aureus and showed weak cytotoxicity on L929 murine fibroblasts and human KB3-1 cancer cells (Phukhamsakda et al. 2018).

Sulcatisporaceae Tanaka & K. Hirayama

Sulcatisporaceae was erected for a well-separated clade which included Magnicamarosporium, Neobambusicola and Sulcatispora in Pleosporales (Tanaka et al. 2015). Rupcic et al. (2018) added Pseudobambusicola to Sulcatisporaceae which was collected from a twig of an unidentified plant in Thailand based on the morphology and phylogeny analyses. Sulcatisporaceae is characterized by immersed to erumpent, subglobose to hemisphaerical ascomata, short ostiolar neck, trabecular pseudoparaphyses (Liew et al. 2000), clavate, short pedicellate asci, and broadly fusiform, hyaline, septate ascospores with mucilaginous appendages (Tanaka et al. 2015). Asexual morph is pycnidial conidiomata with various conidia characters (Tanaka et al. 2015; Phukhamsakda et al. 2017a; Rupcic et al. 2018). We provide an updated tree of Sulcatisporaceae based on the combined dataset of LSU, ITS, SSU and tef1 sequence data and propose two new genera from Clematis species collected in Thailand. Additionally, the biological activity of their secondary metabolites is preliminarily reported (Fig. 72).

Fig. 72
figure72

Bayesian 50% majority-rule consensus phylogram based on combined LSU, ITS, SSU and tef1 sequence data for Sulcatisporaceae. The topology and clade stability of the combined gene analyses was compared to the single gene analyses. The tree is rooted with members of the Didymosphaeriaceae. Eighteen strains were included in the combined analyses which comprised 3390 characters (843 characters for LSU, 1025 characters for SSU, 585 characters for ITS, 937 characters for tef1, including gap regions). The best scoring RAxML tree had a final likelihood value of − 11220.180237. The matrix had 709 distinct alignment patterns, with 31.84% of undetermined characters and gaps. Estimated base frequencies were as follows; A = 0.238159, C = 0.264197, G = 0.261408, T = 0.236236; substitution rates AC = 0.958607, AG = 2.023708, AT = 1.033715, CG = 0.741292, CT = 5.717037, GT = 1.000000; gamma distribution shape parameter α = 0.485427. In our analysis, GTR + I + G model was used for each partition in Bayesian posterior analysis. The species determined in this study are indicated in blue. Bootstrap values (BS) greater than 50% BS (ML, left) and Bayesian posterior probabilities (BYPP, right) greater than 0.90 are given at the nodes. Hyphens (-) represent support values less than 50% BS/0.90 BYPP. Thick branches represent significant support values from all analyses (BS ≥ 70%/BYPP ≥ 0.95)

Anthosulcatispora Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557201; Facesoffungi number: FoF 07340, Fig. 73.

Etymology: Anthos-meaning flower, Anthosulcatispora referring to the occurrence on flowering plants.

Fig. 73
figure73

Anthosulcatispora subglobosa (MFLU 17–1473, holotype). a Appearance of conidiomata on Clematis subumbellata. b Vertical section through conidioma. c Section of conidioma wall. dg Conidiogenous cells and conidia. hj Conidia. k Culture characteristics on MEA. Scale bars: b = 100 µm, c = 50 µm, dj = 10 µm

Saprobic on dead stems of herbaceous plants. Sexual morph: Ascomata semi-immersed, blackish, irregular, scattered, uniloculate, glabrous, ostiolate, apapillate. Ostioles dark, circular and sunken. Peridium two-layered, outer layer irregular, comprising dark brown cells of textura angularis and inner layer irregular comprising light brown cells. Hamathecium composed of numerous, filamentous, branched or simple, septate, cellular pseudoparaphyses, embedded in a hyaline gelatinous matrix. Asci 4- or 8-spored, bitunicate, cylindrical to cylindrical-clavate, short pedicellate, apically rounded, with an ocular chamber. Ascospores 1-seriate, brown to dark brown, oblong to ellipsoidal, with rounded ends, 1-septate, slightly constricted at the septum, smooth-walled (Phookamsak et al. 2019). Asexual morph: Conidiomata pycnidial, solitary or gregarious, unilocular, scattered, immersed or erumpent, base flattened, subglobose to compressed, coriaceous, dark brown to reddish brown, with or without ostioles. Pycnidial wall multilayered with cells textura angularis, inner layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells enteroblastic, phialidic, determinate, elongated cylindrical or truncate, smooth-walled, hyaline. Conidia oblong, ends rounded, hyaline, aseptate, guttulate, smooth-walled, with mucilaginous sheath.

Type species: Anthosulcatispora subglobosa Phukhams. & K.D. Hyde

Notes: Anthosulcatispora is introduced for a collection occurring on stems of herbaceous plants (Phookamsak et al. 2019; this study). The genus formed a basal lineage in Sulcatisporaceae with strong support (98% ML/1.00 BYPP, Fig. 72). The sexual morph of Anthosulcatispora was described as Neobambusicola brunnea (Phookamsak et al. 2019). Asexual morphs in Sulcatisporaceae comprised Magnicamarosporium, Neobambusicola, Pseudobambusicola and Sulcatispora (Tanaka et al. 2015; Phukhamsakda et al. 2017a; Rupcic et al. 2018). Anthosulcatispora is distinct within Sulcatisporaceae in having brown ascospores, while the sexual morph in Sulcatispora has hyaline, broadly fusiform ascospores with an entire sheath (Tanaka et al. 2015). The asexual morph of Anthosulcatispora shares similar characters with Neobambusicola and Pseudobambusicola. They all have solitary, unilocular pycnidial, phialidic conidiogenesis and hyaline conidia. Anthosulcatispora is distinct from Neobambusicola and Pseudobambusicola in its subglobose conidiomata, elongated cylindrical or truncate conidiogenous cells and oblong and aseptate conidia, while Neobambusicola and Pseudobambusicola have globose conidiomata with two types of conidia (Crous et al. 2014b; Rupcic et al. 2018).

Anthosulcatispora brunnea (Chen & C. Norphanphoun) Phukhams. & K.D. Hyde, comb. nov.

Index Fungorum number: IF557202; Facesoffungi number: FoF 05708

Basionym: Neobambusicola brunnea Chen & Norphanphoun, in Phookamsak et al., Fungal diversity 95:1–273 (2019)

Notes: Neobambusicola brunnea (MFLU 18–1393) was described based on phylogenetic analysis of a combined LSU and ITS dataset (Phookamsak et al. 2019). The strain formed a clade with the type species of Neobambusicola strelitziae (CBS 138869) with moderate support (87% ML). In our study, the phylogenetic analysis based on the combined LSU, SSU ITS, and tef1 sequence data showed different topology. The strain formed a separate clade from N. strelitziae (CBS 138869) and clustered with our new species Anthosulcatispora subglobosa with strong support (100% ML/1.00 BYPP, Fig. 72). Therefore, Neobambusicola brunnea is transferred to a new genus.

Hosts: Dead stem of herbage—(Phookamsak et al. 2019).

Distribution: China—(Phookamsak et al. 2019).

Anthosulcatispora subglobosa Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557203; Facesoffungi number: FoF 07341, Fig. 73.

Etymology: Refers to the subglobose conidiomata.

Holotype: MFLU 17–1473.

Saprobic on dried stems of Clematis subumbellata. Sexual morph: Undetermined. Asexual morph: Conidiomata 160–210 × 265–345 μm (\( \bar{x} \)= 178 × 291 μm, n = 5), pycnidial, solitary, unilocular, scattered, shiny, immersed or erumpent, flattened base, subglobose to compressed, coriaceous, dark brown to reddish brown, lacking ostioles. Pycnidial wall 18–32 μm wide, thick, multilayered, composed of 10–14 brown layers of textura angularis, inner layer subhyaline, lining bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 5–17(–30) × 2–3.5 μm (\( \bar{x} \)= 13 × 2.5 μm, n = 30), enteroblastic, phialidic, determinate, elongated cylindrical or truncate, smooth-walled, hyaline. Conidia 7–10 × 3–4 μm (\( \bar{x} \)= 8 × 4 μm, n = 50), oblong, rounded ends, hyaline, aseptate, with 1–3 guttules in each cell, smooth-walled, with mucilaginous sheath.

Cultural characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Cultures from above, cream at the centre, radiating, dense, circular, edge lobate, umbonate, papillate, fairly fluffy, covered with white aerial mycelium, black oil drops produced on the surface of cultures; reverse dark brown at the centre, faintly zonate, white at the edge.

Material examination: Thailand, Phayao Province, Phu Sang District, dead stems of Clematis subumbellata, 20 March 2017, C. Phukhamsakda, CMTH09 (MFLU 17–1473, holotype); ex-type living culture, MFLUCC 17–2065.

Host: Clematis subumbellata—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214592; SSU: MT226705; ITS: MT310636; tef1: MT394649; rpb2: MT394706.

Notes: In a BLASTn search of GenBank, the closest match for LSU sequence of Anthosulcatispora subglobosa MFLUCC 17–2065 was Neobambusicola strelitziae (strain CBS 138869), (NG_058125) with 97% similarity, while the closest match for the ITS sequence of MFLUCC 17–2065 was Magnicamarosporium iriomotense (strain HHUF 30125), (NR_153445) with 89% similarity. Pairwise comparison of the ITS region showed that A. subglobosa differs from A. brunnea with 12% differences (76 base pairs difference of 585 base pairs, with gaps). The collection has oblong, hyaline, guttulate, and aseptate, with rounded ends conidia but microconidia were not observed in culture (Crous et al. 2014b; Rupcic et al. 2018). We also noted the formation of oil droplets in culture as a notable character for A. subglobosa (Fig. 73).

Strain MFLUCC 17–2065 was evaluated for potential secondary metabolite production with Bacillus subtillis, Escherichia coli, Mucor plumbeus and Schizosaccharomyces pombe as test organisms. The isolate showed moderate growth inhibitory activities against Bacillus subtillis and Mucor plumbeus, and is thus a suitable candidate for further evaluation.

Parasulcatispora Phukhams. & K.D. Hyde, gen. nov.

Index Fungorum number: IF557204; Facesoffungi number: FoF 01686, Fig. 74.

Fig. 74
figure74

Parasulcatispora clematidis (MFLU 17–1490, holotype). a Appearance of ascomata on host surface. b Close up of ascoma on host substrate. c Vertical section through ascoma. d Ostiolar canal. e Section of peridium. f Trabeculate pseudoparaphyses. g–h Asci. i–l Ascospores. m Culture characteristics on MEA. Scale bars: b = 200 µm, c = 100 µm, d = 20 µm, eh = 50 µm, il = 10 µm

Etymology: Refers to the characteristic features similar to Sulcatispora.

Saprobic on dried stem of herbaceous plants. Sexual morph: Ascomata solitary, gregarious, semi-immersed to erumpent, subglobose to compressed, coriaceous, dark brown to black, ostiolate. Ostioles central, filled with hyaline periphyses. Peridium thin, uniform of flattened cells of textura angularis, thin-walled, cells towards the inside lighter, inner layer composed of thin, hyaline, gelatinous layer. Hamathecium composed of numerous, filamentous, branched, septate, anastomosing, trabeculate pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical-clavate to clavate, asymmetric, with furcate pedicel, apically rounded, with an ocular chamber. Ascospores biseriate or partially overlapping, broad fusiform, hyaline, tapering towards the ends, 1-euseptate, constricted at the septum, smooth-walled, with guttules in each cell, with mucilaginous sheath. Asexual morph: Undetermined.

Type species: Parasulcatispora clematidis Phukhams. & K.D. Hyde

Notes: Parasulcatispora is established as a monotypic genus. In a BLASTn search of GenBank, the closest match for LSU and ITS sequences of MFLUCC 17–2082 is Sulcatispora berchemiae (HHUF 29097, NG_059390) with 98% similarity, while the ITS sequence had 88% similarity to NR_153444. Based on the multi-gene (LSU, SSU, ITS and tef1 regions) analyses, MFLUCC 17–2082 formed a separate lineage within Sulcatisporaceae (Fig. 72). The genus matches Sulcatisporaceae species in having immersed, subglobose to hemisphaerical ascomata, short ostioles, trabeculate pseudoparaphyses (Liew et al. 2000), and fusiform, hyaline, septate ascospores with mucilaginous appendages (Tanaka et al. 2015). Parasulcatispora is similar to Sulcatispora but lacks a pseudoclypeus and has small flattened ascomata with narrower asci and ascospores. The asexual morph failed to develop in culture. We introduce the new genus based on morphology and phylogenetic evidence.

Parasulcatispora clematidis Phukhams. & K.D. Hyde, sp. nov.

Index Fungorum number: IF557205; Facesoffungi number: FoF 07342, Fig. 74.

Etymology: The specific name “clematidis” refers to the host substrate.

Holotype: MFLU 17–1490.

Saprobic on dead stems of Clematis fulvicoma. Sexual morph: Ascomata 160–230 × 230–320 μm (\( \bar{x} \) = 190 × 305 μm, n = 5), solitary, gregarious, semi-immersed to erumpent, subglobose to compressed, coriaceous, dark brown to black, only ostiole visible, ostiolate. Ostioles 84–115 × 116–133 μm (\( \bar{x} \) = 100 × 124 μm, n = 5), shiny, central, smooth-walled, filled with hyaline periphyses. Peridium 22–37 μm wide (\( \bar{x} \) = 19 μm, n= 20), uniform, wider at the apex, composed of 3–4 layers of somewhat flattened cells of textura angularis, thin-walled, cells towards the inside lighter, inner layer composed of thin hyaline gelatinous layer. Hamathecium composed of numerous, dense, 2–3 µm wide, filamentous, branched, septate, anastomosing, trabecular pseudoparaphyses, embedded in a gelatinous matrix. Asci 53–88 × 8–17 µm (\( \bar{x} \) = 72 × 13 µm, n = 30), 8-spored, bitunicate, fissitunicate, cylindrical-clavate to clavate, asymmetric, with furcate pedicel, apically rounded, with an ocular chamber. Ascospores 16–21 × 4–6 µm (\( \bar{x} \) = 17 × 5 µm, n = 40), biseriate or partially overlapping, broad fusiform with acute ends, tapering towards the ends, 1-euseptate, constricted at the septum, upper cell broader than lower cell, smooth-walled, with two guttules in each cell, hyaline, with 4–7 µm wide mucilaginous appendages. Asexual morph: Undetermined.

Culture characters: Colonies on MEA reaching 30 mm diam. after 4 weeks at 25 °C. Culture from above, greyish brown, covered with grey fluffy mycelia, dense, circular, umbonate, dull, undulate, radially furrowed, yellow oil droplets formed in the middle of cultures; reverse grey, radiating outwardly.

Material examined: Thailand, Chiang Rai Province, Mae Sai District, dried stems of Clematis fulvicoma, 20 March 2017, C. Phukhamsakda, CMTH28 (MFLU 17–1490, holotype); ex-type living culture, MFLUCC 17–2082.

Host: Clematis fulvicoma—(This study).

Distribution: Thailand—(This study).

GenBank accession numbers: LSU: MT214593; ITS: MT310637; tef1: MT394650.

Notes: Parasulcatispora clematidis can be distinguished morphologically (Fig. 74) and is supported by the phylogenetic analyses of combined LSU, SSU, ITS and tef1 data (Tanaka et al.