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

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.


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.

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 R , 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).
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 mm 3 was removed from the specimen and added to a reaction tube with 5 µl of sterile distilled water (dH 2 O). 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 R G2 Hot Start Colorless Master Mix (PROMEGA; GoTaq R Hot Start Polymerase in 2 × Colorless GoTaq R 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 dH 2 O 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 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.  (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 100 th generation. The distribution of loglikelihood 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

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 (

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.

Type Genus
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.

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(Dennis, , 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. 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.

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.