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. 2020 May 26;11:906. doi: 10.3389/fmicb.2020.00906

Patellariopsidaceae Fam. Nov. With Sexual-Asexual Connection and a New Host Record for Cheirospora botryospora (Vibrisseaceae, Ascomycota)

Anuruddha Karunarathna 1,2,3,4,5, Derek Peršoh 6, Anusha H Ekanayaka 4,5, Ruvishika S Jayawardena 5, K W Thilini Chethana 5, Ishani D Goonasekara 3,4,5, Ratchadawan Cheewangkoon 2,7, Erio Camporesi 8,9, Kevin D Hyde 3,4,5, Saisamorn Lumyong 1,7,10,*, Samantha C Karunarathna 1,3,4,*
PMCID: PMC7264944  PMID: 32528427

Abstract

Helotiales is a polyphyletic order of Ascomycetes. The paucity of relevant molecular data and unclear connections of sexual and asexual morphs present challenges in resolving taxa within this order. In the present study, Patellariopsidaceae fam. nov., the asexual morph of Patellariopsis atrovinosa, and a new record of Cheirospora botryospora (Vibrisseaceae) on Fagus sylvatica (Fagaceae) from Italy are discussed based on morphology and molecular phylogeny. Phylogenetic analyses based on a combined sequence dataset of LSU and ITS were used to infer the phylogenetic relationships within the Helotiales. The results of this research provide a solid base to the taxonomy and phylogeny of Helotiales.

Keywords: Ascomycetes, Cheirospora botryospora, Leotiomycetes, Pezizomycotina, sporodochium

Introduction

The Leotiomycetes (Pezizomycotina) is a very diverse class and was erected when the super-class Leotiomyceta was split in to seven classes by Eriksson & Winka (Eriksson and Winka, 1997). Leotiomycetes currently comprises 13 orders, out of which eight are monotypic, while over 200 genera are represented by one species only (Baral, 2016; Wijayawardene et al., 2018; Ekanayaka et al., 2019; Johnston et al., 2019). Among the orders in Leotiomycetes, Helotiales consists of the highest number of genera, incertae sedis within the familial rank (ca. 90–151) (Baral, 2016; Quijada et al., 2018; Wijayawardene et al., 2018). Hawksworth (2001) estimated that Helotiales consists of 70,000 species. Only 2,334 species belonging to 423 genera in 25 families have been recorded in Helotiales. This constitutes half of all known species in Leotiomycetes (Ekanayaka et al., 2019).

Recent phylogenetic studies based on ribosomal DNA analyses have reported the polyphyletic nature of Helotiales (Ekanayaka et al., 2019; Johnston et al., 2019). The lack of knowledge between asexual and sexual morph connections complicates the systematics of this order (Wang et al., 2006b). Many helotialean fungi are known based on a sexual morph, with their asexual morphs being either undiscovered or assumed to have been lost in evolution (Wang et al., 2006b). On the other hand, it is suggested that asexual morphs from various environmental samples are members of Helotiales, with no mention of their sexual morphs (Sutton and Hennebert, 1994; Marvanova et al., 1997).

Helotiales is the largest group of non-lichen forming ascomycetes and occur in a wide range of niches (Ekanayaka et al., 2017; Wijayawardene et al., 2017). The members of Helotiales are recorded as plant pathogens, endophytes, nematode-trapping fungi, mycorrhizae, fungal parasites, terrestrial and aquatic saprobes, root symbionts and wood rot fungi (Wang et al., 2006a).

The objectives of this study are to introduce a new family with their sexual-asexual inter-connection and to provide a new host record for Cheirospora in Vibrisseaceae.

Materials and Methods

Plant Sample Collection, Morphological Studies and Isolation of Pure Culture

Dead aerial branches of Fagus sylvatica L. (Fagaceae) and Corylus avellana L. (Betulaceae) were collected from Passo la Calla, Stia (province of Arezzo [AR]) Italy and Fiumicello di Premilcuore (province of Forlì-Cesena [FC]) Italy, respectively. Specimens were preserved and observed following the method of Karunarathna et al. (2017). Hand-cut sections of the fruiting structures were mounted in water for microscopic studies and photomicrography. Specimens were examined with a Nikon ECLIPSE 80i compound microscope and photographed with a Canon EOS 600D digital camera fitted to the microscope. Measurements of morphological characteristics were made with the Tarosoft (R) Image Frame Work program and images used for figures were processed with Adobe Photoshop CS3 Extended version 10.0 (Adobe Systems, United States).

Single spore isolation was carried out following the method described in Chomnunti et al. (2014). Germinated spores were individually transferred to potato dextrose agar (PDA) plates and grown at 10–16°C. Colony color and other characteristics were observed and measured after 1 week and 3 weeks. The specimens were deposited in the Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand. Living cultures were deposited in Mae Fah Luang Culture Collection (MFLUCC). Facesoffungi (FoF) and Index Fungorum numbers (IF) were acquired as in Jayasiri et al. (2015) and Index Fungorum (2019).

DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA was extracted from fresh fungal mycelium grown on PDA media at 16°C for 4 weeks using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux®, Hangzhou, China) following the instructions of the manufacturer.

The DNA amplification was performed by polymerase chain reaction (PCR). A partial sequence of the LSU rRNA gene region was amplified using the primer pair LR0R and LR5 (Vilgalys and Hester, 1990). The internal transcribed spacer regions (ITS1, 5.8S, ITS2) were amplified using the primer pair ITS5 and ITS4 (White et al., 1990). PCR was carried out following the protocol of Phookamsak et al. (2014). The quality of PCR products was checked by gel electrophoresis on 1% agarose gels stained with ethidium bromide. The amplified PCR fragments were sent to a commercial sequencing provider (Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China). The sequence data acquired were deposited in GenBank (Table 1).

TABLE 1.

Taxa used in the phylogenetic analyses and their corresponding GenBank numbers (Newly generated sequences are indicated in black bold).

Species Strain/ GenBank
Voucher No. Accession No.
ITS LSU
Acidomelania panicicola 61R8 KF874619 KF874622
Amicodisca sp. KUS_F51377 JN086692 JN033389
Aquapoterium pinicola ATCC MYA-4213 NR_111345 NG_056957
Arachnopeziza aurata KUS-F52038 JN033393 JN086696
Arachnopeziza aurelia KUS-F51520 JN033409 JN086712
Ascocoryne cylichnium KUS_F52351 JN086709 JN033406
Ascocoryne sarcoides HKAS 90651 MK591999 MK584973
Bryoclaviculus campylopi PDD:101074 JX393084
Bryoglossum gracile DAOM178087 AY789285
Bulgariella pulla DHP 15-215 KU845540 KU845536
Cadophora fastigiata CBS 869.69 MH859469 MH871247
Cadophora malorum A163 AY249057 AY249080
Cheirospora botryospora CPC 24607 KR611872 KR611894
Cheirospora botryospora MFLUCC 17-1399 MN535816 MN535856
Chlorosplenium chlora BHI_F737a MG553994
Chlorosplenium chlora BHI_F736a MG553993 MG553993
Crucellisporium umtamvunae CBS 125742 MH863659 MH875124
Dicephalospora huangshanica MFLU 18-1828 MK591979 MK584979
Discinella boudieri HB4326 KC412001
Drepanopeziza ribis CBS 200.36 MH855774 MH867284
Drepanopeziza salicis CBS 405.64 MH858467 MH870102
Encoeliopsis rhododendri CBS 905.69 MH859479 MH871259
Geniculospora grandis CBS 261.84 MH873440 MH861735
Godronia ribis CBS 163.66 MH858761 MH870393
Graddonia coracina ILLS60491 JN012009 JQ256423
Haplographium delicatum CBS 196.73 MH872362 MH860659
Heterosphaeria linariae MFLU 15-2764 MK591955 MK585000
Heterosphaeria patella G.M. 2014-08-04-1 MF196187
Hyaloscypha bicolor CBS 144009 MH018932 MH018943
Hyaloscypha vitreola CBS 126276 MH863954 MH875413
Hydrocina chaetocladia CCM F-10890 KC834062 KC834031
Hymenotorrendiella madsenii ICMP 15648 KJ606676 AY755336
Lachnum abnorme KUS-F52080 JN033395 JN086698
Lambertella seditiosa WU 32446 KF499362
Loramyces juncicola CBS 293.52 MH857043 MH868576
Loramyces macrosporus CBS 235.53 MH857170 MH868710
Mitrulinia ushuaiae PDD:105643 KX273438 KX273439
Mollisia cinerea CBS 128349 JF514855 MH876343
Neopyrenopeziza MFLU 16-0599 NR_163783 MK592001
nigripigmentata
Patellariopsis atrovinosa G.M. 2014-06-15-1 KY462814 KY462814
Patellariopsis atrovinosa G.M. 2016-05-04-1 KY970066 KY970066
Patellariopsis atrovinosa MFLUCC 17-1411 MN535817 MN535857
Patellariopsis dennisii CBS 174.66 MH858765 MH870396
Patellariopsis dennisii G.M.2017-09-04.3 MK120898 MK120898
Peltigeromyces sp. HB 6432 KX090803
Phialocephala scopiformis CBS 468.94 NR_119460
Phialocephala urceolata UAMH 10827 NR_111285
Pulvinata tomentosa MFLU 18-1819 MK591965 MK584938
Rhexocercosporidium carotae CBS 418.65 MH858647 MH870289
Rutstroemia longipes TNS F-40097 AB926073 AB926142
Tetracladium marchalianum CBS 266.84 MH861736 MH873441
Trimmatostroma betulinum MFLU 15-2991 MK591956 MK584993
Trimmatostroma salicis MFLU 18-0702 MK584996
Unguicularia unguiculata NK322 HG326612
Varicosporium delicatum CCM F-19494 JQ412864 KC834036
Vibrissea flavovirens MBH39316 AY789427 AY789426
Vibrissea truncorum AFTOL_ID 1322 FJ176874
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Sequencing of the ITS region of strain MFLUCC 17-1411 was failed due to an intron, of about 1.4 kb in length, positioned between the binding site of primer ITS5 and the start of the ITS region. To obtain a double-stranded ITS sequence, a piece of sporodochium < 0.5 mm3 was removed from the specimen and added to a reaction tube with 5 μl of sterile distilled water (dH2O). The soaked specimen was frozen (−20°C) and thawn (+20°C) for five times and 0.5 μl of the solution was used for amplification. Based on initially obtained sequence information, a forward primer (Karu_F01: 5′-CAATGATCAAAGCAGTTGCG-3′) was designed, which has similar properties as the ITS4 primer and binds to the intron sequences close its 3′-end. The PCR reaction included 0.5 μl of the DNA-containing solution, 0.25 μl of each primer (Karu_F01 and ITS4; 10 μM, each), 5.25 μl of sterile dH2O and 6.25 μl of the GoTaq® G2 Hot Start Colorless Master Mix (PROMEGA; GoTaq® Hot Start Polymerase in 2 × Colorless GoTaq® Reaction Buffer (pH 8.5), 400 μM dNTPs, 4 mM MgCl2). The PCR commenced with 3 min denaturation at 95°C, followed by 33 amplification cycles (27 s at 94°C, 60 s at 56°C, and 90 s at 72°C) and a final elongation at 72°C for 7 min. The PCR products were cleaned by successive incubation at 37°C for 30 min and 80°C for 15 min after adding 0.2 μl exonuclease I (20.000 U/ml), 0.2 μl Shrimp-Alkaline-Phosphatase (1.000 U/ml; both New England Biolabs) and 1.6 μl sterile dH2O to 5 μl of PCR product. Purified PCR products were sequenced by the sequencing service of the Ruhr-Universität Bochum using a Genetic Analyzer 3130xl (Applied Biosystems).

Phylogenetic Analyses

Phylogenetic analyses were conducted separately based on LSU and ITS gene sequence data. Reference sequences (Table 1) of representative families in Leotiomycetes were retrieved from GenBank. The related sequences were obtained from a BLAST search and from recently published data (Ekanayaka et al., 2019). Individual datasets for LSU and ITS genes were aligned using the default settings of MAFFT V.7.0361 (Katoh et al., 2018) and improved manually where necessary using Bioedit. Aligned gene regions were concatenated using Bioedit v.7.2 (Hall, 1999) and analyzed.

Initial alignment of LSU region included 7163 base pairs and ITS region included 6619 base pairs. In the phylogenetic analysis, LSU and ITS regions consisted of ambiguously aligned regions. Hence, manual alignment was performed where necessary and some unambiguous regions were removed from the analysis. The removed regions of LSU data set are 0–2658, 2738–2888, 2897–2955, 3037–3220, 3285–3338, 3421–3464, 3723–3730, 4029–4103, 4129–4562, 4581–7163. The excluded regions of the ITS data set are 0–146, 149–173, 181–2164, 2199–2234, 2253–2307, 2714–2923, 2936–2971, 2986–3052, 3065–3113, 3132–3270, 3331–6619. In the final alignment, LSU and ITS regions consist of 898 and 587 bp, respectively.

Phylogenetic constructions of combined gene trees were performed using maximum likelihood (ML), maximum parsimony (MP) and bayesian inference (BI) criteria. Maximum likelihood trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al., 2008; Stamatakis, 2014) in the CIPRES Science Gateway platform (Miller et al., 2010) using the GTR+I+G model of evolution. The robustness of the most parsimonious tree was estimated based on 1000 bootstrap replications.

Maximum parsimony analysis was carried out in PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford, 2002) using the heuristic search option, random stepwise addition, and 1000 replicates, with maxtrees set at 1000. Descriptive tree statistics for parsimony such as Tree Length [TL], Consistency Index [CI], Retention Index [RI], Relative Consistency Index [RC] and Homoplasy Index [HI] were calculated for trees generated under different optimality criteria. The Kishino Hasegawa tests (Kishino and Hasegawa, 1989) were performed to determine whether the trees inferred under different optimality criteria were different.

Evolutionary models for phylogenetic analyses were selected independently for each locus using MrModeltest v. 3.7 (Nylander, 2004) under the Akaike Information Criterion (AIC) implemented in both PAUP v. 4.0b10 and MrBayes v. 3.

Bayesian inference analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist, 2001) to evaluate posterior probabilities (BYPP) (Rannala and Yang, 1996; Zhaxybayeva and Gogarten, 2002) by Markov Chain Monte Carlo sampling (BMCMC). Kimura 2-parameter model coupled with discrete gamma distribution with a proportion of invariant site (TrN+I+G) was applied for LSU gene region and symmetrical model with discrete gamma distribution coupled with a proportion of invariant sites (TIM2ef+I+G) was applied for ITS gene region. Two parallel runs were conducted, using the default settings, but with the following adjustments: Four simultaneous Markov chains were run for 2,000,000 generations and trees were sampled every 100th generation. The distribution of log-likelihood scores indicated the stationary phase for each search and were used to decide if extra runs were required to achieve convergence, using Tracer v. 1.6 (Rambaut et al., 2014). The first 20% of the generated trees represented the burn-in phase and were discarded. The remaining trees were used to calculate posterior probabilities of the majority rule consensus tree.

Phylograms were visualized with FigTree v1.4.0 (Rambaut, 2012) and edited in Microsoft Power Point (2016) and Adobe Illustrator CS5 (Version 15.0.0, Adobe, San Jose, CA, United States). The finalized alignment and the tree were deposited in TreeBASE, submission ID: 248922.

Results

Phylogenetic Analyses

Phylogenetic trees obtained from LSU and ITS single gene analyses as well as the combined gene analyses share similar overall topologies at the generic level and are in agreement with previous studies (Johnston et al., 2014; Crous et al., 2015; Ekanayaka et al., 2019). The concatenated LSU and ITS dataset consisted of 58 taxa.

The RAxML analysis of the LSU dataset yielded a best scoring tree (Figure 1) with a final ML optimization likelihood value of −16138.064322. The matrix had 758 distinct alignment patterns, with 19.46% of undetermined characters or gaps. Parameters for the GAMMA+P-Invar model of the LSU and ITS were as follows: Estimated base frequencies; A = 0.247456, C = 0.223115, G = 0.280101, T = 0.249328; substitution rates AC = 1.847670, AG = 2.484717, AT = 1.566081, CG = 1.283952, CT = 5.746161, GT = 1.000000; proportion of invariable sites I = 0.458978; gamma distribution shape parameter α = 0.633036. The maximum parsimony dataset consisted of 1490 characters, of which 860 were constant, 475 parsimony-informative and 155 parsimony-uninformative. The parsimony analysis of the data matrix resulted in four equally most parsimonious trees with a length of 948 steps (CI = 0.347, RI = 0.556, RC = 0.193, HI = 0.653) in the best tree.

FIGURE 1.

FIGURE 1

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RAxML tree based on a combined dataset of LSU and ITS partial sequence data. Bootstrap support values for maximum likelihood equal to or higher than 70%, maximum parsimony equal to or higher than 70%, and Bayesian posterior probabilities equal to or greater than 0.90 are displayed on the nodes, respectively. Newly generated sequences are indicated in white. The tree is rooted to Lambertella seditiosa and Rutstroemia longipes.

Taxonomy

In this section, Patellariopsidaceae Karun., Camporesi & K.D. Hyde, fam. nov. and the new record of Cheirospora botryospora are described and illustrated. Helotiales includes several families with sporodochial asexual morphs viz. Gelatinodiscaceae, Helotiaceae and Mollisiaceae. A morphological comparison among members of the families in Helotiales is given in Tables 2, 3.

TABLE 2.

Comparison of major asexual morph characteristics of families in order Helotiales.

Family Hyphomycetous conidiomata Conidiophore Conidiogenous cell Conidia
Amicodiscaceae (Ekanayaka et al., 2019) Hyphomycetous/stromatic Hyaline to cinnamon-colored glistening slimy heads, straight or flexuous, dark brown and thick-walled except at the apex Terminal, cylindrical, sympodially proliferate Cylindrical to cylindric-ellipsoidal, hyaline, aseptate, thin-smooth walled.
Discinellaceae (Ekanayaka et al., 2019) Hyphomycetous conidiomata Holoblastic Mostly hyaline, sometimes branched, filiform, globose, or fusoid some form dimorphic conidia
Drepanopezizaceae (Yoshikawa and Yokoyama, 1992; König et al., 2017) Hyphomycetous/acervulus Holoblastic Sometimes two types. Macroconidia- ellipsoid to fusoid, slight curved. Microconidia- ellipsoid to bacilliform
Gelatinodiscaceae (Seaver, 1938; Johnston et al., 2010) Sporodochial Aseptate, hyaline and subglobose
Helotiaceae (Peláez et al., 2011; Jaklitsch et al., 2016) Hyphomycetous, sporodochial or synnematal Macroconidia – holoblastic/Microconidia – phialidic Macroconidia – hyaline, filiform or staurosporous, dark brown, in chains, bulbils or solitary on conidiophores and 3–5-septate. Microconidia rarely pigmented, multicellular and appendaged
Heterosphaeriaceae (Leuchtmann, 1987) Synanamorphic, hyphomycetous acervulus and ceolomycetous
Hyaloscyphaceae (Jaklitsch et al., 2016) hyphomycetous sporodochial Phialidic Aseptate, hyaline or brown, branched and muriform or in chains
Hydrocinaceae (Ekanayaka et al., 2019) Hyphomycetous Long, hyaline, simple or branched, filiform Proliferate, sympodial. Filiform, branched, sometimes septate and fragment into microconidia.
Loramycetaceae (Digby and Goos, 1987; Walsh et al., 2014) anguillospora-like Conidiophores are simple or occasionally branch. Conidiogenous cells are hyaline and straight. Conidia are globose, sub-ellipsoid or sigmoid and hyaline Conidiogenous cells are hyaline and straight. Conidia are globose, sub-ellipsoid or sigmoid and hyaline Conidia are globose, sub-ellipsoid or sigmoid and hyaline
Mollisiaceae (Sutton and Ganapathi, 1978; Butin et al., 1996; Grünig et al., 2002) Sporodochial Hyaline to brown Unicellular, ellipsoid or phragmosporous, hyaline or brown and also in chains
Patellariopsidaceae Sporodochium Cylindrical, straight or slightly curved, branched over the conidiophore, septate, hyaline, expanding toward the apices, smooth Holoblastic, polyblastic, cylindrical, integrated, hyaline, smooth. Sphaerical, acropetal, branched chains. globose to cylindrical mass of small, thick-walled, dark brown, septate, eguttulate, smooth, cheiroid, conidium-complex
Phialocephala urceolata clade Wang, 2009 Hyphomycetous Hyaline to darkly pigmented, septate and mononematous Phialidic and conidiogenous cells are flask to urn-shaped and each with a prominent cylindrical and hyaline collarette Globose, pedicellate and single or adhering in small clusters at the phialide apex
Ploettnerulaceae (Marvanová and Bärlocher, 2001; Goodwin, 2002; Gönczöl and Révay, 2003; Gramaje et al., 2011; Gonçalves et al., 2012; King et al., 2013; Travadon et al., 2015; Duarte et al., 2016; Walsh et al., 2018) hyphomycetous or coelomycetous Hyaline to brown Phialidic Ellipsoid to rod-shaped or filiform with pointed apices and 0–1-septate
Solenopeziaceae (Ekanayaka et al., 2019) Conidiomata hyphomycetous Simple, sparsely branched or absent Cylindrical to subclavate, sometimes apically slightly swollen Hyaline or black, septate, branched, lunate, sometimes formed in a chain and becoming tortuous and appearing as terminal dictyospores, rarely appendaged
Vibrisseaceae (Iturriaga and Israel, 1985; Goh and Hyde, 1998; Goh et al., 1998; Kirschner and Oberwinkler, 2001; Shenoy et al., 2010; Hernández-Restrepo et al., 2012, 2017; Legon, 2012; Crous et al., 2015) hyphomycetous, phialidic and acervulus Straight, cylindrical, hyaline and sometimes branched Holoblastic or polytretic Ellipsoid or irregular in shape and unicellular or up to 7–septate
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TABLE 3.

Comparison of major sexual morph characteristics of families in order Helotiales based on Ekanayaka et al. (2019).

Family Ascomata Excipulum Peridium Paraphyses Asci Ascospores
Amicodiscaceae Apothecial, cupulate, sessile or sub-stipitate, margins covered by hairs Ectal excipulum textura angularis or textura prismatica cells, medullary excipulum loosely arranged hyphae Filiform, cylindrical, septate, simple 8-spored, amyloid, sometimes arising from croziers Ellipsoid to fusoid, aseptate, guttulate, lemon-yellow pigmented
Aquapoterium Unguicularia clade Apothecial, cupulate receptacle, sessile or tipitate, sometimes margins covered with short cylindrical hairs Ectal excipulum textura prismatica cells or a single layer of parallel hyphae with enlarged, globose apices, medullary excipulum reduced or composed of loosely arranged hyphae Filiform, hyaline, obtuse to lavate at apex, septate, smooth-walled, simple or branched 8-spored, amyloid or non-amyloid, cylindric-clavate Ellipsoid to clavate cylindric, hyaline, smooth-walled, 0–1-septate, surrounded by a gelatinous sheath
Arachnopezizaceae Apothecial, covered by hairs Ectal excipulum textura angularis to prismatica cells, medullary excipulum textura prismatica to textura oblita cells Cylindrical, hyaline 8-spored, cylindric clavate, amyloid, arising from croziers Ellipsoid to fusoid, 0–7-septate
Bryoglossaceae Apothecial, clavate to apitate or cupulate to turbinate, long stipitate, gelatinous Ectal excipulum textura porrecta cells, medullary excipulum textura intricata cells Filiform, swollen at the apex 8-spored, amyloid or non-amyloid, arising from croziers Ellipsoid to fusoid, straight, aseptate, guttulate
Bulgariella clade Apothecial or rarely cleistothecial, cupulate, discoid, turbinate or capitate, sessile or stipitate, margins and flanks are covered with hairs Ectal excipulum is composed textura angularis, textura prismatica or textura oblita cells, medullary excipulum is composed of cells of textura intricata or textura oblita cells Filiform, lanceolate or cylindrical 8-spored, cylindric clavate, amyloid or non-amyloid, sometimes arising from croziers Globose, ellipsoid to filiform, septate or aseptate, hyaline or brownish, guttulate
Chlorospleniaceae Apothecial, cupulate or discoid, sessile or substipitate Ectal excipulum textura angularis cells, medullary excipulum textura intricata cells Filiform, septate 8-spored, cylindric clavate, amyloid Ellipsoid to fusoid, hyaline and smooth walled
Colipila clade Apothecial cupulate, covered by long cylindrical hairs Ectal excipulum and medullary excipulum textura prismatica cells Dimorphic, sub cylindrical and not exceed the length of asci, or broadly lanceolate and exceed the length of asci 8-spored, cylindric– clavate, amyloid, arising from croziers Ellipsoid to fusoid
Discinellaceae Apothecial, discoid to cupulate, circular, gelatinous, sometimes covered with hairs Ectal excipulum textura prismatica or textura porrecta cells, medullary excipulum textura intricata to prismatica cells Filiform, branched at the apices 8-spored, cylindrical, amyloid or non amyloid, sometimes arising from croziers Ellipsoid, aseptate, hyaline, without sheath
Drepanopezizaceae Apothecial, cupulate, sessile, mostly immersed A thin layer of textura angularis cells, Apically slightly swollen, straight 4–8- spored, non amyloid Ellipsoid to fusoid, 0–2-septate
Gelatinodiscaceae Apothecial, cupulate or discoid, some are tremelloid, form cerebriform masses which each lobule contains a turbinate apothecium Ectal excipulum textura prismatica to textura angularis to globulosa cells, medullary excipulum textura oblita to textura porrecta or textura intricata cells Filiform, cylindrical, apically swollen, guttulate 8-spored, amyloid, arising from croziers Ellipsoid to fusoid, hyaline, yellowish or brownish, smooth, with a gelatinous sheath, guttulate, 0–5-septate
Godroniaceae Apothecial, urceolate, discoid or cupulate, mostly stromatic, erumpent, sometimes covered with hairs Ectal excipulum textura prismatica to angularis cells, medullary excipulum textura epidermoidea, prismatica to porrecta cells Filiform or lanceolate, simple or branched, sometimes slightly swollen at the apex 8-spored, cylindric clavate, amyloid or non-amyloid Fusoid, hyaline, septate, guttulate
Helotiaceae Apothecial, cupulate, discoid, capitate to clavate, turbinate or globose, sessile or tipitate, margins and flanks smooth or covered with hairs Ectal excipulum textura prismatica, intricata, globulosa-angularis, or toblita cells, medullary excipulum textura intricata or porrecta cells Cylindrical, septate or aseptate, hyaline to yellowish, guttulate 4–8-spored, cylindric-clavate, amyloid or non amyloid, sometimes arising from croziers Ellipsoid, fusoid or filiform, 1–3-septate, rarely ornamented
Heterosphaeriaceae Apothecial, discoid, black, sessile, erumpent, gelatinous Ectal excipulum textura angularis cells, medullary excipulum textura porrecta cells Clavate contains many guttules 8-spored, amyloid, arising from croziers Aseptate, ellipsoid to fusoid, without gel sheath
Hyaloscyphaceae Apothecial, cupulate or discoid, sessile or substipitate, sometimes covered with hairs Ectal excipulum textura globulosa cells, medullary excipulum textura porrecta, intricata to oblita cells Filiform, septate, branched, slightly swollen at the apices 8-spored, cylindric clavate, amyloid, arising from croziers Ellipsoid to fusoid, aseptate or septate, hyaline
Hydrocinaceae Apothecial, cupulate, sessile or substipitate Ectal excipulum textura globulosa cells, medullary excipulum textura porrecta, intricata or oblita cells Filiform, septate, branched, slightly swollen at the apices 8-spored, cylindric clavate, amyloid, arising from croziers Ellipsoid to fusoid, aseptate or septate, hyaline
Lachnaceae Apothecial, cupulate or discoid, sessile or stipitate, margins and flanks are covered with hairs Ectal excipulum textura angularis, prismatica or oblita cells, medullary excipulum textura intricata or textura oblita cells Filiform, lanceolate or rarely cylindrical 8-spored, cylindric clavate, amyloid or non-amyloid, sometimes arising from croziers Globose, ellipsoid to filiform or allantoid, septate or aseptate, hyaline, guttulate
Loramycetaceae Apothecial or perithecial, apothecia cupulate or pulvinate, perithecia sub-globose Ectal excipulum textura prismatica, angularis or globulosa cells, medullary excipulum textura prismatica cells Filiform, septate, unbranched, sometimes apically swollen and pigmented 8-spored, cylindric clavate, amyloid or non-amyloid Fusiform, septate, sometimes with terminal appendages and gel sheath
Mitrulaceae Apothecial, clavate, stipitate Ectal excipulum textura porrecta cells, medullary excipulum textura intricata cells Filiform, cylindrical, with yellow carotenoid droplets 8-spored, cylindric clavate, arising from croziers Fusoid to ellipsoid, straight or curved
Mollisiaceae Apothecial, discoid covered by hairs, Ectal excipulum textura globulosa to angularis cells, medullary excipulum textura prismatica cells Cylindrical or lanceolate, apically swollen, guttulate 8-spored, amyloid, cylindric clavate, mostly arising from croziers Ellipsoid to long filiform, 0–7-septate, guttulate
Patellariopsidaceae Apothecial, discoid, sessile Ectal excipulum textura globulosa to angularis cells, medullary excipulum interwoven refractive hyphae filiform, branched and pigmented at the apices 8-spored, cylindric clavate, amyloid Ellipsoid to fusoid, hyaline, 3–7-septate
Peltigeromyces clade Apothecial, cartilaginous, thin, with a large variety of lobes Records are not available for micro morphological characters
Phialocephala urceolata clade Sexual morphs are not recorded
Ploettnerulaceae Apothecial, cupulate, discoid or urn-shaped, sessile or sub stipitate, sometimes covered with pigmented hairs Ectal excipulum textura globulosa to angularis cells, medullary excipulum textura prismatica cells Filiform, cylindrical or lanceolate, guttulate 8-spored, conical apex, amyloid Ellipsoid to long filiform, 0–3-septate, guttulate
Solenopeziaceae Apothecial cupulate, discoid or pulvinate, sessile or stipitate, sometimes covered with hyaline, whitish, yellow or brown, non-bristle like hairs Ectal excipulum textura angularis, textura prismatica or textura oblita cells, medullary excipulum textura intricata or textura oblita cells Filiform, lanceolate or cylindrical 8-spored, cylindric clavate, amyloid or non-amyloid, sometimes arising from croziers Globose, ellipsoid to fusiform, septate or aseptate, guttulate
Vibrisseaceae Apothecial, cupulate or clavate, sessile to stipitate Ectal excipulum textura angularis to globulosa cells, medullary excipulum reduced or textura oblita cells Filiform, apically slightly swollen, sometimes branched 8-spored, cylindric clavate, long stipitate, sometimes amyloid, arising from croziers
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Patellariopsidaceae Karun., Camporesi and K.D. Hyde, Fam. Nov.

Index Fungorum number: IF556719, Facesoffungi number: FoF06573

Sexual Morph: Ascomata apothecial, discoid, sessile or stipitate. Ectal excipulum composed of cells of textura globulosa to angularis cells. Medullary excipulum composed of interwoven refractive hyphae. Paraphyses filiform branched and pigmented at the apices. Asci 8-spored, cylindric-clavate, amyloid. Ascospores ellipsoid to fusoid, hyaline, 3–7-septate. Asexual morphs: Saprobic on dead branch of Corylus avellana (Betulaceae). Asexual morph: Sporodochium, sub-epidermal or sub-peridermal, solitary. Conidiophores cylindrical, straight or slightly curved, branched over the conidiophore, septate, hyaline, expanding toward the apices, smooth. Conidiogenous cells holoblastic, polyblastic, cylindrical, integrated, hyaline, and smooth. Conidia, sphaerical, proliferating with several, short, lateral, acropetal, branched chains. Primary branches in turn develop secondary branches, which eventually form a globose to cylindrical mass of small, thick-walled, dark brown, septate, eguttulate, smooth, cheiroid, conidium-complex.

Notes

Patellariopsidaceae forms a well-supported (ML 74/BYPP 0.98) clade sister to Chlorospleniaceae, Loramycetaceae, Mollisiaceae and Vibrisseaceae. In Index Fungorum, Patellariopsis is included in Dermateaceae, but Wijayawardene et al. (2017) placed Patellariopsis in Helotiales genera incertae sedis based on morphology. Furthermore, in Ekanayaka et al. (2019), this clade was denoted as separate taxa based on phylogenetic analyses. Hence, we introduce this clade as a new family based on morphology and phylogeny.

Type Genus

Patellariopsis Dennis, Kew Bull. 19(1): 114 (1964)

Patellariopsis Dennis, Kew Bull. 19(1): 114 (1964)

Index Fungorum number: IF556217, Faces of Fungi number: FoF06575

The genus classified under Helotiales genera incertae sedis, Leotiomycetes (Wijayawardene et al., 2018). The type species is Patellariopsis clavispora (Berk. & Broome) Dennis. Five species are recorded in Index Fungorum (2019), P. atrovinosa (A. Bloxam ex Curr.) Dennis, P. carnea G.W. Beaton, P. clavispora (Berk. & Broome) Dennis, P. dennisii (E. Müll. & Hütter) Schläpf.-Bernh., and P. indica A. Pande. We were unable to find any reported described asexual morphs of Patellariopsis in the literature.

Patellariopsis atrovinosa (A. Bloxam ex Curr.) Dennis, Kew Bull. 29(1): 167 (1974)

Index Fungorum number: IF 319233, Facesoffungi number: FoF06574 Figure 2

FIGURE 2.

FIGURE 2

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Asexual morph of Patellariopsis atrovinosa (MFLU 16-2950). (A,B) Appearance of sporodochium on host substrate. (C) Longitudinal section of sporodochium. (D) Conidiophore attached to the host. (E–G) Various stages of conidiogenesis. (H–J) Conidia. (K) Germinated conidium. (L,M) Culture characteristics on PDA (L = from above, M = from below). Scale bars: C = 50 μm; D,E = 20 μm; F–K = 10 μm.

Saprobic on dead branch of Corylus avellana (Betulaceae). Sexual morph: Refer to Dennis (1974). Asexual morph: Sporodochium 33–37 μm high, 278–355 μm diam. (x¯ = 35 × 324 μm, n = 5), sub-epidermal or sub-peridermal, solitary. Conidiophores 41–78 × 1–1.5 μm (x¯ = 58 × 1.3 μm,

n = 20) cylindrical, straight or slightly curved, branched over the conidiophore, septate, hyaline, expanding toward the apices, smooth. Conidiogenous cells 1.5–2 × 1–1.6 μm (x¯ = 1.8 × 1.4 μm, n = 20) holoblastic, polyblastic, cylindrical, integrated, hyaline, smooth. Conidia 1.5–2.7 × 1.5–2.5 μm (x¯ = 2 × 2 μm, n = 40), sphaerical, proliferating with several, short, lateral, acropetal, branched chains. Primary branches in turn develop secondary branches, which eventually form a globose to cylindrical mass of small, thick-walled, dark brown, septate, eguttulate, smooth, cheiroid, conidium-complex.

Colonies growing on PDA becoming 2 cm within 10 days at 16°C, circular, flat, cottony, irregular margin, with less aerial mycelium, olivaceous green to gray from above and dark brown from below.

Material Examined

ITALY, Forlì-Cesena [FC], Fiumicello di Premilcuore, dead aerial branch of Corylus avellana L. (Betulaceae), 5 September 2015, E. Camporesi, IT 3178 (MFLU 16-2950), living cultures, MFLUCC 17-1411.

Cheirospora Moug. & Fr., in Fries, Syst. Orb. Veg. (Lundae) 1: 365 (1825)

Index Fungorum number: IF 7614, Faces of Fungi number: FoF06593

The genus is in Helotiales genera incertae sedis, Leotiomycetes (Wijayawardene et al., 2018). Ekanayaka et al. (2019) placed this genus under Vibrisseaceae. The type species is C. botryospora (Mont.) Berk. & Broome. There are four species in Index Fungorum (2019), C. alni Shabunin., C. betulina (P. Karst.) Kuntze., C. botryospora (Mont.) Berk. & Broome and C. oblonga (Fuckel) Kuntze.

Cheirospora botryospora (Mont.) Berk. & Broome, Ann. Mag. nat. Hist., Ser. 2 5: 455 (1850)

Index Fungorum number: IF 294800, Facesoffungi number: FoF06594 (Figure 3)

FIGURE 3.

FIGURE 3

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Asexual morph of C. botryospora (MFLU 15-2612). (A,B) Appearance of sporodochium on host substrate. (C) Longitudinal section of sporodochium. (D,E) Conidia attached to the conidiophores. (F–J) Conidia. (K) Conidia surrounded by mucilaginous sheath, stained with Indian ink. (L) Germinated conidium. (M,N) Culture characteristics on PDA (M = From above, N = From below). Scale bars: C = 20 μm; D,E = 50 μm; F–J = 10 μm.

Saprobic on dead branches of Fagus sylvatica L. Sexual morph: unidentified. Asexual morph: Sporodochium 1850–1854 μm high, 3728–3732 μm diam. (x¯ = 1852 × 1730 μm, n = 5), sub-epidermal or sub-peridermal, solitary. Conidiophores 171–225 × 3–4 μm (x¯ = 198 × 3.5 μm, n = 20) cylindrical, straight or slightly curved, branched only at the base, septate, hyaline, expanding toward the apices, smooth. Conidiogenous cells 8–7 × 10–11 μm (x¯ = 7.5 × 10.5 μm, n = 20) holoblastic, polyblastic, cylindrical, integrated, hyaline, smooth. Conidia 10–11 × 9–11 μm (x¯ = 10.5 × 10 μm, n = 40), sphaerical, proliferating with several, short, lateral, acropetal, branched chains. Primary branches in turn develop secondary branches which eventually form a globose to cylindrical mass of small, thick-walled, dark brown, septate, eguttulate, smooth, cheiroid, conidium-complex, enclosed in a gelatinous sheath.

Colonies growing on PDA to 2 cm diam. within 10 days at 16°C, circular, flat, cottony, irregular margin, with less aerial mycelium, olivaceous green to gray from above and dark brown from below.

Material Examined

ITALY, Province of Arezzo [AR], Passo la Calla - Stia, dead aerial branch of Fagus sylvatica (Fagaceae), 5 September 2015, E. Camporesi, IT 2609 (MFLU 15-2612), ex-type living culture, MFLUCC 17-1399.

Discussion

The highly divergent morphological, ecological and biological characteristics of Helotiales makes it a focus for taxonomic studies in the Leotiomycetes, as it is one of the most problematic groups for traditional classification and molecular phylogeny (Wang et al., 2006a). It is a poorly studied order, within which about 19–27% of the genera have an uncertain position at the family level (Baral, 2016). Hence, the taxa in Helotiales have already been subjected to several nomenclatural reinterpretations (Wang et al., 2006a). Lantz et al. (2011) revealed that some genera related to members in Helotiales were traditionally placed in Rhytismatales.

Patellariopsidaceae is established herein based on morphological and phylogenetic support. Comparisons of major sexual and asexual morph characteristics of families in Helotiales are provided in Tables 2 and 3. The asexual morph characteristics of this family are unique in having sporodichium with cheiroid conidium complex. The cheiroid conidium complexes are also present in C. botryospora in Vibrisseaceae. Patellariopsidaceae differs from Vibrisseaceae, in having highly branched conidiophores and thicker conidia complexes. Further, the sexual morph of the Patellariopsidaceae shows unique characteristics by having a sessile discoid apothecium, paraphyses with filiform, branched and pigmented apices, cylindric-clavate, amyloid asci and ellipsoid to hyaline septate ascospores. Patellariopsidaceae was further supported by phylogeny. Hence, herein we establish the Patellariopsidaceae under Helotiales.

Most of the Patellariopsis species were recorded from the United Kingdom with few exceptions (Beaton and Weste, 1978; Farr and Rossman, 2020). Patellariopsis atrovinosa on Prunus laurocerasus was also reported from the United Kingdom. In our study, we report the asexual morph of P. atrovinosa on Corylus avellana from Italy. Patellariopsis carnea on dead grass twigs was reported from Australia (Beaton and Weste, 1978). P. clavispora shows a wide host range, which includes Acer sp., Corylus sp., Crataegus sp., Fagus sp., Fraxinus sp., Ligustrum sp., Prunus sp., Quercus sp. and Symphoricarpos sp. from the United Kingdom (Dennis, 1978, 1986) and Mangifera indica from Pakistan (Ahmad, 1978).

Apart from ribosomal RNA sequence data, the use of protein-coding gene phylogenies involving helotialean fungi are slowly emerging (Wang et al., 2006b; Johnston et al., 2014). Most contemporary results suggest that the Helotiales and currently delimited families are not monophyletic and that the highly conserved small subunit (SSU) rRNA gene is not informative enough to resolve these lineages with confidence (Gernandt et al., 2001).

In our phylogenetic analyses, all the Patellariopsis strains available in the GenBank were included. Among them, the phylogenetic placement of P. dennisii (G.M. 2017 09 04.3) is ambiguous. No morphological descriptions are available in the literature for comparison (Ekanayaka et al., 2019; Vu et al., 2019) and the topology obtained in this study is similar to the topology obtained by Ekanayaka et al. (2019). Hence, we suggest the need for having more data to clarify the position of P. dennisii (G.M. 2017-09-04.3). The blast results for the Patellariopsis dennisii (CBS 174.66) strain include several other Ascomycetous fungi. Therefore, Patellariopsis dennisii (CBS 174.66) was excluded in our dataset after the preliminary phylogenetic analyses.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis using multi-gene concatenated sequences is used to determine the recombination level within phylogenetically closely related species. Under this study three P. atrovinosa strains MFLUCC 17-1411, G.M. 2016-05-04.1, G.M. 2014-06-15.1, and P. dennisii G.M. 2017 09 04.3 were subjected to the GCPSR analysis. The analysis failed due to the lack of the informative characters in the highly similar sequences of P. atrovinosa strains MFLUCC 17-1411, G.M. 2016-05-04.1 and G.M. 2014-06-15.1.

Based on phylogenetic analyses, our strain MFLUCC 17-1411 forms a well-supported (ML 100/ MP 100/BYPP 1.00) clade with specimens G.M. 2016-05-04.1 and G.M. 2014-06-15.1 (both P. atrovinosa). The phylogenetic relatedness is supported by the 100% similarity between sequences. The asexual stage of Patellariopsis is not recorded in literature. Hence, no morphological comparison can be done between the strains MFLUCC 17-1411 and P. atrovinosa. However, Meyer & Carrières (Meyer and Carrières, 2007) have described an asexual hyphomycetous Periconia-like association for the sexual morph P. atrovinosa. Nevertheless, no scientific evidence was provided to confirm this association. Hence, we justify MFLUCC 17-1411 belongs to the P. atrovinosa.

Cheirospora botryospora MFLUCC 17-1399 forms a well-supported clade with (ML 100/MP 100/BYPP 1.00) C. botryospora CPC 24607 and this was further supported by morphology. C. botryospora CPC 24607 was isolated from Fagus sylvatica in Germany. Danti et al. (2002) identified an endophytic Cheirospora sp. on Fagus sylvatica from Italy. However, they were unable to identify it to the species level. Therefore, in this study, based on morphology and phylogeny the first report of C. botryospora on F. sylvatica from Italy is provided (Farr and Rossman, 2020).

In this study, the Patellariopsidaceae fam. nov. is introduced with an asexual morph. Furthermore, a new host record for the C. botryospora (Vibrisseaceae) and updated phylogenetic tree for Helotiales are provided.

Data Availability Statement

The datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to anumandrack@yahoo.com.

Author Contributions

AK and KH designed the study. AK performed the morphological study and phylogenetic the data analyses with the help of DP, AE, KC, and RJ. DP did the primer design. SL and SK provided the grant. AK wrote the manuscript. RJ, DP, IG, KC, AE, SK, SL, RC, and KH reviewed and edited the manuscript. All authors reviewed and approved the final manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RJ declared a past co-authorship with the authors AK, DP, AE, RJ, KC, IG, RC, EC, KH, SL, SK to the handling editor.

Acknowledgments

We appreciate the kind support given by the laboratory staff of Center of Excellence in Microbial Diversity and sustainable Utilization, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand; Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand and Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China. The Plant Germplasm and Genomics Center in Germplasm Bank of Wild Species, Kunming Institute of Botany are thanked for the support in doing molecular work. KH would like to thank “the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracena species” (Grant No. DBG6080013) and “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion” (RDG6130001). SK thanks Chiang Mai University for funding his postdoctoral research.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to anumandrack@yahoo.com.

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