Phytotaxa 480 (3): 251–261 https://www.mapress.com/j/pt/ Copyright © 2021 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.480.3.4 A novel addition to the Pezizellaceae (Rhytismatales, Ascomycota) ANURUDDHA KARUNARATHNA1,2,3,4,5,12, PAWEŁ DZIAŁAK6,13, RUVISHIKA S. JAYAWARDENA5,7,14, SAMANTHA CHANDRANATH KARUNARATHNA1,2,8,9,15, CHANG-HSIN KUO3,16, NAKARIN 2,10,17 1,2,8,9,18 SUWANNARACH , SAOWALUCK TIBPROMMA * & SAISAMORN LUMYONG2,11,19* 1 CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, People’s Republic of China. 2 Research Center of Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand. 3 Department of Plant Medicine, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan, People’s Republic of China. 4 Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand. 5 Centre of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand. 6 Faculty of Geology, Geophysics and Environmental Protection, AGH University of Science and Technology, Al. Mickiewicza 30, 30-059 Kraków, Poland. 7 School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand. 8 World Agroforestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, Yunnan, People’s Republic of China. 9 Centre for Mountain Futures, Kunming Institute of Botany, Kunming 650201, Yunnan, People’s Republic of China. 10 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. 11 Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand. 12 � anumandrack@yahoo.com; https://orcid.org/0000-0003-0956-6636 13 � dzialak@agh.edu.pl; https://orcid.org/0000-0002-8238-1518 14 � ruvi.jaya@yahoo.com; https://orcid.org/0000-0001-7702-4885 15 � samanthakarunarathna@gmail.com; https://orcid.org/0000-0001-7080-0781 16 � chkuo@mail.ncyu.edu.tw; http://orcid.org/0000-0001-9011-6530 17 � suwan.462@gmail.com; https://orcid.org/0000-0002-2653-1913 18 � saowaluckfai@gmail.com; https://orcid.org/0000-0002-4706-6547 19 � scboi009@gmail.com; http://orcid.org/0000-0002-6485-414X *Corresponding author: � scboi009@gmail.com, � saowaluckfai@gmail.com Abstract In previous studies Apiculospora was introduced in Leotiomycetes genera incertae sedis. With our phylogenetic analyses based on large subunit rDNA (LSU) and internal transcribed spacer (ITS1, 5.8S, ITS2), we transfer Apiculospora to Pezizellaceae (Rhytismatales). We introduce Apiculospora penniseti from Pennisetum purpureum. Apiculospora penniseti differs from the closely related A. spartii by the presence of clear apiculi at both apices of the smooth-walled ascospores. Apiculospora penniseti is phylogenetically distinct from A. spartii with moderate support and high BYPP support (69 RAxML/0.97 BYPP). Herein, we discuss the taxonomy and phylogeny of A. penniseti. Keywords: 1 new species, coelomycetes, graminicolous fungi, Leotiomycetes Introduction Graminicolous fungi are highly diverse in tropical and subtropical regions (Ekanayaka et al. 2019, Karunarathna et al. 2019, 2020, Hyde et al. 2020a,b). This is also true of bambusicolous fungi which are not generally regarded as grass fungi (Dai et al. 2017), but often overlap. Taxa belonging to Dothideomycetes and Sordariomycetes are particularly highly abundant in Poaceae (Dai et al. 2017, Thambugala et al. 2017, Hyde et al. 2020b, Karunarathna et al. 2020). Several graminicolous taxa viz. Alishanica miscanthii (Hyde et al. 2020b), Kwanghwana miscanthi (Karunarathna et al. 2020) and Neoroussoella alishanensis (Karunarathna et al. 2019) have been introduced from Taiwan region. Baral and Rama (2015) and Jaklitsch et al. (2016) resurrected Pezizellaceae in Helotiales to accommodate Allophylaria and Calycina. Based on multigene phylogenetic analyses, Pezizellaceae was accommodated in Accepted by Sajeewa Maharachchikumbura: 19 Dec. 2020; published: 22 Jan. 2021 251 Rhytismatales with Calloriaceae, Rhytismataceae and the Hyphodiscus-Chalara clade (Ekanayaka et al. 2019). Pezizellaceae presently contains 23 genera, and among them, only 11 have molecular sequence data (Ekanayaka et al. 2019, Wijayawardene et al. 2020). Wijayawardene et al. (2016) introduced coelomycetous, Apiculospora with A. spartii as the type species under Helotiales, genera incertae sedis. Apiculospora is characterized by one septate conidia with prominent apiculus and enteroblastic, phialidic conidiogenesis (Wijayawardene et al. 2016). Apiculospora was included in Leotiomycetes, genera incertae sedis by Ekanayaka et al. (2019) and later, Hyde et al. (2020a) transferred Apiculospora under Rhytismatales genera incertae sedis based on phylogeny. We are studying the fungi on grasses (Thambugala et al. 2017) and in this study, a new species A. penniseti is introduced from Pennisetum purpureum. Based on morphology and phylogeny, we transfer Apiculospora to Pezizellaceae. A comprehensive description, illustrations, phylogeny and notes of the novel taxon is provided. Materials and methods Sample collection, morphological studies and isolation Decaying Pennisetum purpureum specimens with fungal fruiting bodies were collected from Kwang Hwa, in Chiayi Province of Taiwan region, China. Samples were brought to the laboratory in paper bags and examined. Hand sections of the fruiting structures of the fungi were mounted in water for microscope studies and photomicrography. Specimens were examined as described in Karunarathna et al. (2019). Single spore isolation was carried out using spores suspended on sterile water and by plating the diluted spore suspension as mentioned in Chomnunti et al. (2014). The germination took several weeks, and the isolation failed due to drying off the agar plates and contamination from the other fungal spores in the suspension. Hence, we used glass needle isolation, as described in Goh et al. (1999). The herbarium specimens were deposited in Mae Fah Luang University Herbarium (Herb. MFLU). Living cultures were deposited in the National Chiayi University Culture Collection (NCYUCC). Faces of fungi (FoF) and Index Fungorum numbers (IF) were obtained as in Jayasiri et al. (2015) and Index Fungorum (2020). DNA extraction, PCR amplification and sequencing for fungal isolates Fungi were grown on potato dextrose agar (PDA) at 20 °C for four weeks and the genomic DNA was extracted from fresh fungal mycelium using the DNA extraction kit E.Z.N.A Fungal DNA Mini Kit (D3390-02, Omega Bio-Tek) by following the instructions of the manufacturer. The DNA amplification for the large subunit rDNA (LSU) and internal transcribed spacer (ITS1, 5.8S, ITS2) was performed by polymerase chain reaction (PCR) using the primer pairs LROR/LR5 (Vilgalys & Hester 1990) and ITS5/ITS4 (White et al. 1990) respectively. The PCR amplification was carried out following the conditions given in Karunarathna et al. (2020). Quality of the products from PCR amplification was checked using 1% agarose gels electrophoresis stained with ethidium bromide and the final product of the amplified fragments were sent to the commercial sequencing provider (Tri-I Biotech, Taipei, Taiwan region of China). The acquired nucleotide sequence data in this study were deposited in GenBank (TABLE 1). Phylogenetic analyses Phylogenetic analyses were conducted based on combined genes of LSU and ITS sequence data. The reference nucleotide sequences (TABLE 1) of Pezizellaceae taxa were retrieved from GenBank based on the recently published data (Ekanayaka et al. 2019) and from the NCBI BLAST search relevant to our strain. The single gene sequences were initially aligned using MAFFT V.7.036 (http://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2018) and did the necessary changes manually using Bioedit v.7.2 (Hall 1999). Phylogenetic reconstructions using maximum likelihood (ML) was performed for each gene separately to check for any incongruence in overall topology. The finalized single gene sequences were combined using Bioedit v.7.2 (Hall 1999) and phylogenetic reconstructions of the combined gene trees were performed using maximum likelihood (ML) and Bayesian posterior probability analysis (BYPP) criteria. Maximum likelihood analyses were performed using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008, 252 • Phytotaxa 480 (3) © 2021 Magnolia Press KARUNARATHNA ET AL. Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTR+I+G model of evolution. Evolutionary models for phylogenetic analyses and Bayesian information criterion were obtained using the jModelTest2 on XSEDE (2.1.6) and MrBayes on XSEDE (3.2.7a) respectively in the CIPRES Science Gateway platform (Miller et al. 2010). ITS pairwise comparison with clustering using Ward's method Pairwise similarity ratio of the sequences were obtained by Needleman-Wunsch algorithm provided by Biopython software (Cock et al. 2009). Needleman-Wunsch algorithm (Gotoh 1992) find the best alignment for each pairwise comparison referring to the shortest sequence through the scoring matrix with all the possible alignment patterns while considering the gaps within the sequence. The heat map of the similarity ratio was plotted using the Seaborn plugin (Waskom et al. 2020). FIGURE 1. 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 65% and Bayesian posterior probabilities equal to or greater than 0.90 are displayed on the nodes, respectively. Newly introducing taxon is indicated in white. The tree is rooted to Calloria urticae (MFLU 18-0697) and Dactylaria dimorphospora (CBS 256.70). A NOvEl ADDITION TO ThE PEZIZEllAcEAE Phytotaxa 480 (3) © 2021 Magnolia Press • 253 Results Phylogeny Initial alignments of LSU and ITS included 1374 and 5295 base pairs respectively. The manual alignment was performed where necessary and after trimming the beginning and end of the alignments. In the final alignment, LSU and ITS regions consisted of 897 and 520 base pairs respectively. The phylogenetic trees obtained from single gene analysis of LSU (29 taxa) and ITS (34 taxa) share similar overall topology are in agreement with recent studies (Ekanayaka et al. 2019). The concatenated LSU and ITS data set comprised 36 taxa. The RAxML analysis of the combined dataset yielded a best scoring tree (FIGURE 1) with a final ML optimization likelihood value of -8038.144039. The matrix had 582 distinct alignment patterns, with 20.17% of undetermined characters or gaps. Parameters for the GTR + I + G model of the combined LSU and ITS were as follows: Estimated base frequencies; A = 0.243869, C = 0.235973, G = 0.286686, T = 0.239680; substitution rates AC = 1.526262, AG = 1.843705, AT = 1.535777, CG = 0.733723, CT = 6.112570, GT = 1.000000; proportion of invariable sites I = 0.422920; gamma distribution shape parameter α = 0.604674. Taxonomy Apiculospora penniseti A. Karunarathna & C.H. Kuo, sp. nov. FIGURE 2 Index Fungorum number: IF557871; Facesoffungi number: FoF 09221 Etymology—Refers to the host genus Pennisetum. Saprobic on dried leaves of Pennisetum purpureum. Sexual morph: Undetermined. Asexual morph: Conidiomata 100–160μm high × 70–125μm diam. ( x = 127 × 90μm, n = 5), pycnidial, immersed to erumpent, unilocular, sub-globose, dark brown, spore masses are spread across the host surface immediately above the conidiomata at the maturity. Spore mass tightly attached to the host surface. Conidiomata wall 13–16μm, outer layer, composed of thin-walled, brown cells of textura angularis; inner layer thin-walled, almost reduced to conidiogenesis region. Conidiophores reduced to conidiogenous cells. Paraphyses 1–2µm wide, among conidiogenous cells, light brown, smooth, septate, cylindrical, branched. Conidiogenous cells 5–10 × 2–3μm ( x = 7 × 2μm, n = 20), subcylindrical to ovoid, enteroblastic, phialidic with percurrent proliferation, discrete, indeterminate, hyaline, smooth-walled. Conidia 15–20 × 5–8 μm ( x = 17 × 7 μm, n = 20), ellipsoid to subcylindrical, straight to slightly curved, base truncate, both apices with clear apiculi, 1septate, pale brown to dark brown and notable dark brown region at the septum, guttulate, thick and smooth-walled. Material examined: CHINA, Taiwan region, Chiayi Province, Kwang Hwa, on decaying stems of Pennisetum purpureum Schumach. (Poaceae), 17 March 2018, A. Karunarathna AKTW 19 (MFLU 20-0543, holotype) ex-type culture = NCYUCC 19-0363. Notes: Apiculospora was introduced by Wijayawardene et al. (2016) in Helotiales, genera incertae sedis. Ekanayaka et al. (2019) transferred Apiculospora to Leotiomycetes genera incertae sedis based on multigene phylogeny. Pezizellaceae contains the coelomycetous genus, Porodiplodia (Crous et al. 2018). Porodiplodia contains conidia with fusoid-ellipsoid to sub cylindrical, medium brown, finely verruculose, guttulate, thick-walled, 1-septate and prominently conidia with obtuse apex (at times with central pore) and truncate base. While, Apiculospora consists the spores with ellipsoid to sub cylindrical, pale brown to dark brown, guttulate, thick to smooth walled, 1-septate and with prominent apicules at the both apices and truncate base (Crous et al. 2018). Further our species Apiculospora penniseti contains paraphyces and Porodiplodia also contains paraphyces. Hence, by considering the taxonomy (TABLE 2) and phylogeny (FIGURE 1) of Porodiplodia and Apiculospora, we transfer Apiculospora to Pezizellaceae. The generic description for the Apiculospora and type description along with illustrations were provided in Wijayawardene et al. (2016). Our strain clustered within Apiculospora and formed a separate branch from A. spartii. Apiculospora penniseti significantly differs from A. spartii with the presence of clear apiculi at both apices of the smooth-walled spores and the presence of the paraphyces. Apiculospora penniseti and A. spartii has ellipsoid to subcylindrical, straight to slightly curved conidia with smooth thickened walls. ITS pairwise analysis of the closest strains of A. penniseti showed that 254 • Phytotaxa 480 (3) © 2021 Magnolia Press KARUNARATHNA ET AL. there are considerable differences between the species (FIGURE 3). The ITS pairwise analysis showed that ITS region of the A. penniseti is more similar to A. spartii (MFLU 15-3556) and two strains of Chalara clidemiae (CBS 141319 and CPC 26423). In the phylogenetic tree, A. penniseti and A. spartii form a stable clade with moderate likelihood support and moderate BYPP support (69 RAxML/0.97 BYPP). Discussion Genera in Pezizellaceae contain both sexual and asexual morphs (Ekanayaka et al. 2018). Among the 20 genera, nine have reported asexual morphs (Baral & Rämä 2015, Guatimosim et al. 2016, Wijayawardene et al. 2016, 2017, Ekanayaka et al. 2019). Coelomycetous asexual morphs have been reported from Apiculospora and Porodiplodia (Wijayawardene et al. 2016, Crous et al. 2018). The other asexual morphs are hyphomycetous e.g. Bloxamia, Calycina and Phaeoscypha (Wijayawardene et al. 2017, Ekanayaka et al. 2019) in this family. FIGURE 2. Apiculospora penniseti (MFLU 20-0543, holotype). a,b Appearance of conidiomata on host surface. c,d Conidia on the surface of the host. e,f Section through conidioma. g Pycnidial wall. h Paraphyses. i-l Conidiogenous cells with conidia. m-o Conidia. p,q Conidiomata on the culture. r Conidia from the culture. Scale bars: e = 20 µm g,i,r = 10 µm f = 50 µm h,j–o = 5 µm A NOvEl ADDITION TO ThE PEZIZEllAcEAE Phytotaxa 480 (3) © 2021 Magnolia Press • 255 Several helotialean taxa were among the first hits in the NCBI BLASTn, and we initially started the tree by considering taxa in Helotiales and our strain formed a stable basal clade directly above the out-group. Hence, we considered the other BLASTn hits and a most recent update on Leotiomycetes (Ekanayaka et al. 2019) and tested the phylogenetic placement of our strain within the Pezizellaceae. We have tested the stability of Apiculospora and the new species Apiculospora penniseti within the Pezizellaceae with different out group combinations, without using the out-group and considering only the in-group species and ITS pairwise comparison with clustering using Ward’s method (FIGURE 3). In all the above methods, the clade which includes the Apiculospora and its sister clades showed the highly stable topology. For the phylogenetic analyses, we used all the accepted taxa with molecular data. Based on the BLAST analysis, we included several Chalara strains, which were used in Ekanayaka et al. (2019). In the initial analysis, we tested the placement of our strain in the phylogeny by using all Chalara species in NCBI GenBank. This analysis further confirmed the stability of our strain within Pezizellaceae. FIGURE 3. ITS pairwise comparison with clustering using Ward’s method. Hyde et al. (2020a) showed that two specific Chalara strains MFLU 18-1812 and MFLU 18-1813 are similar to the holotype of Apiculospora spartii (MFLU15-3556) based on morphology and sequence data. In our phylogenetic analyses, those two specific Chalara strains MFLU 18-1812 and MFLU 18-1813 formed a stable clade with Apiculospora spartii (MFLU15-3556). By considering the facts in Hyde et al. (2020a) and our phylogenetic analysis, we identify the strains, MFLU 18-1812 and MFLU 18-1813 as Apiculospora spartii. 256 • Phytotaxa 480 (3) © 2021 Magnolia Press KARUNARATHNA ET AL. TABLE 1. Taxa used in the phylogenetic analyses and their corresponding GenBank numbers (Newly generated sequences are indicated in black bold). GenBank Accession No. Species name Strain/Voucher number LSU ITS Allophylaria subliciformis R.M. 2374 - MH221035 Apiculospora penniseti NCYUCC 19-0363 MT937250 MT937251 Apiculospora spartii MFLU15-3556 MN660233 MN688212 Apiculospora spartii MFLU 18-1812 MK592006 MK584986 Apiculospora spartii MFLU 18-1813 MK592007 MK584987 Bisporella citrina HMAS 275571 - KX781362 Bisporella citrina CBS 139.62 MH869703 - Bisporella citrina 420526MF0079 MH665412 MG719616 Bisporella discedens MFLU 18-0691 MK591996 MK584970 Bisporella discedens MFLU 18-2673 MK591982 MK584952 Bisporella montana HMAS 275566 - NR_153627 Bisporella shangrilana HKAS 90655a MK591998 MK584972 Bisporella shangrilana HKAS 90655b MK591997 MK584971 Bisporella subpallida G.M. 2016-02-14 - KY462818 Bisporella sulfurina G.M. 2015-10-23.6 - MT435017 Bloxamia cyatheicola VIC 42563 NG_058691 NR_153617 Bloxamia cyatheicola VIC42460 KU597759 KU597792 Calloria urticae MFLU 18-0697 MK591969 MK584942 Calycellina populina CBS 247.62 MH869739 MH858147 Calycellina punctata Cantrell GA18 - U57494 Calycellina triseptata CBS 606.77 - MH861105 Calycina herbarum KUS-F51458 JN086693 JN033390 Calycina marina TROM:F26093 KT185670 KT185677 Chalara africana OC0018 FJ176249 - Chalara clidemiae CBS 141319 MH878219 NR_145313 Chalara clidemiae CPC 26423 KX228321 KX228270 Chalara cylindrosperma CBS 658.79 AF222457 MH873005 Dactylaria dimorphospora CBS 256.70 MH871358 MH859594 Orbiliopsis callistea PDD97932 HQ533050 HQ533049 Phialina lachnobrachyoides KUS-F52576 JN086727 JN033424 Porodiplodia livistonae CPC 32154 NG_069575 NR_160355 Porodiplodia vitis CBS144634 NG_070080 NR_163376 Scleropezicula alnicola CBS 200.46 MH867686 MH856161 Triposporium cycadicola CBS 137968 NG_067285 NR_156587 Zymochalara cyatheae CPC 24665 NG_059652 NR_154509 Zymochalara lygodii CPC 24699 NG_059653 NR_154510 A NOvEl ADDITION TO ThE PEZIZEllAcEAE Phytotaxa 480 (3) © 2021 Magnolia Press • 257 258 • Phytotaxa 480 (3) © 2021 Magnolia Press TABLE 2. Synopsis of asexual morph of Porodiplodia livistonae, Apiculospora spartii and Apiculospora penniseti. KARUNARATHNA ET AL. Character Porodiplodia livistonae Apiculospora spartii Apiculospora penniseti Conidiomata 180–250 µm, uni- to multilocular, brown, globose 160–220 μm diam. × 120–140 μm high, pycnidial, immersed to semi erumpent, unilocular, globose, black 101–156 μm diam. × 69–124 μm high, picnidial, immersed to erumpent, unilocular, sub-globose, dark brown Peridium – Outer wall 10–15μm, thin walled, brown textura angularis, inner wall thin, almost reduced to conidiogenesis region 13–16 μm, outer layer, composed of thin-walled, brown cells of textura angularis; inner layer thin-walled, reduced to conidiogenesis region Paraphyces Hyaline, smooth, septate, sub cylindrical with obtuse ends – Light brown, smooth, septate, cylindrical, branched 1–2µm Conidiogenous cell Conidiophores lining inner cavity, sub cylindrical, hyaline, smooth, branched, septate, proliferating percurrently near apex 5–8 × 2–4 μm, sub cylindrical to ovoid, enteroblastic, phialidic with proliferating percurrently, discrete, indeterminate, hyaline, smooth 6–8 × 2–3μm, sub cylindrical to ovoid, enteroblastic, phialidic with percurrent proliferation, discrete, indeterminate, hyaline, smooth Conidia Conidia in short chains (–3), fusoid-ellipsoid to sub cylindrical, medium brown, finely verruculose, guttulate, thick-walled, 1-septate, apex obtuse (at times with central pore), base truncate with central pore, 2 µm diam. 17–25 × 8–11 μm, ellipsoid to sub cylindrical, straight to slightly curved, base truncate, apex with apiculus, 1septate, sometimes constricted at septum, pale brown to dark brown, guttulate, thick wall, verruculose 15–20 × 5–8 μm, ellipsoid to sub cylindrical, straight to slightly curved, base truncate, both apices with clear apiculi, 1-septate, pale brown to dark brown and notable dark brown region at the septum, guttulate, thick and smooth wall. References Crous et al. (2018) Wijayawardene et al. (2016) This study Acknowledgement We appreciate the kind support given by the laboratory staff of Department of Plant Medicine, National Chiayi University (NCYU) for the molecular facilities and sampling facilities, Centre of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand. The authors would like to acknowledge Eleni Gentekaki, Danushka S. Tennakoon, Chih Hao Hsu, Yi-Jyun Chen, Milan C. Samarakoon and Nimali I. de Silva for their help and suggestions. Dr Shaun Pennycookis thanked for checking the nomenclature name. Saowaluck Tibpromma would like to thank the International Postdoctoral Exchange Fellowship Program (number Y9180822S1), CAS President’s International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation and the Yunnan Human Resources, and Social Security Department Foundation for funding her postdoctoral research. Samantha C. 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