Additions to Phaeosphaeriaceae (Pleosporales): Elongaticollum gen. nov., Ophiosphaerella taiwanensis sp. nov., Phaeosphaeriopsis beaucarneae sp. nov. and a new host record of Neosetophoma poaceicola from Musaceae

Abstract A novel ascomycetous genus, Elongaticollum, occurring on leaf litter of Hedychium coronarium (Zingiberaceae) in Taiwan, is described and illustrated. Elongaticollum is characterized by dark brown to black, superficial, obpyriform, pycnidial conidiomata with a distinct elongate neck, and oval to oblong, hyaline, aseptate conidia. Phylogenetic analyses (maximum likelihood, maximum parsimony and Bayesian) of combined ITS, LSU, SSU and tef1-α sequence data revealed Elongaticollum as a distinct genus within the family Phaeosphaeriaceae with high statistical support. In addition, Ophiosphaerella taiwanensis and Phaeosphaeriopsis beaucarneae are described as new species from dead leaves of Agave tequilana and Beaucarnea recurvata (Asparagaceae), respectively. Neosetophoma poaceicola is reported as a new host record from dead leaves of Musa acuminata (Musaceae). Newly described taxa are compared with other similar species and comprehensive descriptions and micrographs are provided.

We are investigating the diversity of microfungi on leaf litter in the tropics with the aim of clarifying their taxonomy based on morphology coupled with multi-gene phylogeny. As a part of this study, we have collected and isolated four taxa from Taiwan, which belong to the family Phaeosphaeriaceae. We present herein comprehensive morphological descriptions and an in-depth phylogenetic investigation of the newly introduced species.

Sample collection, morphological studies and isolation
Decaying leaf litter samples of Agave tequilana F.A.C. Weber (Asparagaceae), Beaucarnea recurvata Lem. (Asparagaceae), Hedychium coronarium J.Koenig (Zingiberaceae), and Musa acuminata Colla (Musaceae) were collected from Dahu Forest Area in Chiayi, Taiwan and taken to the laboratory in Zip lock plastic bags. Specimens were examined with a LEICA EZ4 stereomicroscope. Micro-morphological characters were determined using AXIOSKOP 2 PLUS compound microscope and images were captured with a Zeiss AXIOCAM 506 COLOR digital camera. Observations and photomicrographs were made from materials mounted in water. Permanent slides were preserved in lactoglycerol, sealed by applying nail-polish around the margins of cover slip. All measurements were made with ZEN2 (blue edition) and images used for figures were processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA).
Single ascospore and conidial isolation was carried out following the method described in Phookamsak et al. (2014). The single germinated spore was picked up and transferred to potato dextrose agar (PDA) and incubated at 25 °C in natural light. Subsequent sub-culturing was done carefully to obtain pure culture and ensure absence of contaminants. Culture characteristics were observed after three weeks. Colonies were photographed and colonial characters were noted and described. Type specimens of new taxa were deposited at the herbarium of Mae Fah Luang University (MFLU) and National Chiayi University Herbarium (NCYU). Living cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and National Chiayi University Culture Collection (NCYUCC). Faces of Fungi and Index Fungorum numbers were provided as in Jayasiri et al. (2015) and Index Fungorum (2020).

DNA extraction and PCR amplification
Total genomic DNA was extracted from scraped fresh fungal mycelium using the DNA extraction kit E.Z.N.A Fungal DNA Mini Kit (D3390-02, Omega Bio-Tek) following the manufacturer's protocol. The DNA product was kept at 4 °C for DNA amplification and maintained at -20 °C for long term storage. DNA was amplified by polymerase chain reaction (PCR) for four genes, the large subunit (28S, LSU), small subunit (18S, SSU), internal transcribed spacers including the 5.8s rDNA (ITS1-5.8S-ITS2) and translation elongation factor 1 alpha (tef1-α). The partial LSU gene was amplified by using the primer combination LR0R and LR5 (Vilgalys and Hester 1990;Rehner and Samuels 1994); partial SSU was amplified with NS1 and NS4 (White et al. 1990), nuclear ITS was amplified with primers ITS5 and ITS4 (White et al. 1990), and tef1-α gene was amplified using the primers EF1-983F and EF1-2218R (Rehner et al. 2001). Amplification reactions were performed in 25 µl of total reaction that contained 9.5 µl of sterilized water, 12.5 µl of 2×Power Taq PCR MasterMix (Tri-I Biotech, Taipei, Taiwan), 1 µl of each forward and reverse primers and 1 µl of DNA template. The PCR thermal cycle program of ITS, LSU, SSU and tef1-α gene was processed initially at 94 °C for 3 minutes, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 50 seconds, elongation at 72 °C for 1 minute and a final extension at 72 °C for 10 minutes and a holding temperature of 4 °C. The PCR products were analyzed by 1.5% agarose gels containing the Safeview DNA stain (GeneMark, Taipei, Taiwan) to confirm their expected molecular weight. PCR products were purified and sequenced with primers mentioned above by Tri-I Biotech, Taipei, Taiwan. Nucleotide sequences were deposited in GenBank (Table 1).

Phylogenetic analysis
Phylogenetic analyses were performed using a combined LSU, SSU, ITS and tef1-α sequence dataset. Newly generated sequence data were initially subjected to blast search in NCBI to obtain the closest matches in GenBank. Sequences generated from this study were analyzed with related taxa in the family Phaeosphaeriaceae, which were obtained from GenBank and from recently published data (Bakhshi et al. 2019;Hyde et al. 2019;Maharachchikumbura et al. 2019;Yang et al. 2019;Mapook et al. 2020) ( Table 1). The combined dataset consisted of 168 sequences including our newly generated sequences. Multiple alignments were automatically made with MAFFT v. 7 at the web server (http://mafft.cbrc.jp/alignment/server), using default settings (Katoh and Standley 2013). The alignment was refined manually with BioEdit v. 7.0.5.2 (Hall 1999), where necessary.
Evolutionary models for phylogenetic analyses were selected independently for each locus using MrModeltest v. 3.7 (Posada and Crandall 1998) under the Akaike Information Criterion (AIC). Phylogenetic trees were obtained from Randomized Accelerated Maximum Likelihood (RAxML), maximum parsimony analysis (MP) and   (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (MCMC). Six MCMC chains were run simultaneously, starting from random trees for 3,000,000 generations. Trees were sampled every 100 th generation for a total of 30,000 trees. The first 6,000 trees were discarded as the burn-in phase of each analysis. Posterior probabilities (Rannala and Yang 1996) were determined from a majority-rule consensus tree generated with the remaining 24,000 trees. Phylograms were visualized with FigTree v1.4.0 (Rambaut 2012) and annotated in Microsoft Power Point (2010). Sequences of the new strains generated in this study are deposited in GenBank. The final alignment and trees were deposited in TreeBASE, submission ID: 26088.
Culture characteristics. Colonies on PDA reaching 30 mm diameter after 3 weeks at 20-25 °C, colonies medium sparse, circular, raised, surface slightly rough with entire edge, margin entire, colony from above: light brown to grey at the margin, dark brown at middle, dark brown to black at the center; reverse, light brown to yellowish at the margin, brown at middle, dark brown to black at the center; mycelium light brown to grey with tufts; not producing pigments in PDA. Notes. The genus Elongaticollum differs from other asexual morphs in Phaeosphaeriaceae in dark brown to black, superficial, obpyriform, pycnidial conidiomata with distinct elongate necks (80-100 µm) and a globose base and oval to oblong, hyaline, aseptate conidia ( Figure 2). Multi-gene phylogenetic analyses (LSU, SSU, ITS, tef1-α), show Elongaticollum strains constitute a highly supported independent lineage nested between Setophoma sensu lato and Neostagonosporella (97% ML, 80% MP, 1.00 BYPP, Figure 1). However, the asexual morph of Setophoma can be distinguished from Elongaticollum in having setose conidiomata without elongate necks and oblong to ellipsoidal conidia, whereas, Elongaticollum have conidiomata with distinct elongate necks and lacking setae and oval to oblong conidia (De Gruyter et al. 2010;Phookamsak et al. 2014). Despite some Setophoma species not having setae (i.e. S. antiqua, S. endophytica, and S. yunnanensis) , Elongaticollum species can be distinguished by its superficial conidiomata with elongate necks.
The asexual morph of Neostagonosporella differs from Elongaticollum in having multiloculate conidiomata without distinct elongate necks and two types of conidia (macroconidia: subcylindrical to cylindrical, transversely multi-septate, hyaline and microconidia oval, ellipsoidal or long ellipsoidal, aseptate, hyaline), whereas Elongaticollum has uni-loculate conidiomata with distinct elongate necks and oval to oblong conidia (Figure 2  Notes. Ophiosphaerella was introduced by Spegazzini (1909) to accommodate O. graminicola Speg. as the type species. The species of this genus are characterized by papillate ascomata bearing fissitunicate, cylindrical asci frequently narrower near the base, with a short furcate pedicel and filamentous, pale brown, multi-septate ascospores without swollen cells or separating into part spores. Barr (1987) placed Ophiosphaerella in Phaeosphaeriaceae and this was confirmed by Zhang et al. (2009Zhang et al. ( , 2012 and Hyde et al. (2013) based on molecular phylogeny. Most Ophiosphaerella species are often found as pathogens or saprobes worldwide on Poaceae and Cyperaceae (Câmara et al. 2000). Currently, twelve Ophiosphaerella species are listed in Index Fungorum (2020). In this study, we introduce Ophiosphaerella taiwanensis from Agave tequilana F.A.C. Weber (Asparagaceae) as a new species. Etymology. Named after Taiwan, where this fungus was collected.
Culture characteristics. Colonies on PDA reaching 25 mm diameter after 3 weeks at 20-25 °C, colonies medium sparse, circular, raised, surface slightly rough with entire edge, margin well-defined, colony from above: gray to light brown at the margin, gray to cream at the center; reverse, gray to light brown at the margin, dark brown to black at the center; mycelium whitish gray with tufting; not producing pigments in PDA.  shows our strain (Ophiosphaerella taiwanensis, NCYUCC 19-0152), cluster with other Ophiosphaerella species, in particular with close affinity to Ophiosphaerella agrostidis with high bootstrap support (88% ML, 70% MP, 0.99 BYPP, Figure 1). Morphological characters of our collection (NCYUCC 19-0152) differ from Ophiosphaerella agrostidis in having periphyses in the ostiole, 12-13 septate ascospores and host occurrence (Asparagaceae). Ophiosphaerella agrostidis was introduced by Câmara et al. (2000) on Agrostis palustris (Poaceae), and is lacking periphyses, comprises 15-septate ascospores . A comparison of the 619 nucleotides across the tef1-α gene region of Ophiosphaerella taiwanensis and O. agrostidis (MFLUCC 11-0152) reveals 17 base pair differences (2.74%). Ramaley. The genus is typified by P. glaucopunctata and characterized by having immersed, sub-epidermal, globose to subglobose to pyriform ascomata, cylindric asci and septate, punctate or verrucose ascospores (Câmara et al. 2003;Phookamsak et al. 2014;Thambugala et al. 2014;Tibpromma et al. 2017). Currently, 17 Phaeosphaeriopsis species are accepted in Index Fungorum (2020). In this paper, Phaeosphaeriopsis beaucarneae is introduced from Beaucarnea recurvata (Asparagaceae) as a new species and the sexual/asexual morph connection between strains isolated from the natural habitat was established based on molecular sequence data. Etymology. Name reflects the host Beaucarnea recurvata Lem., from which the holotype was collected.

Discussion
The taxonomy of Phaeosphaeriaceae has been subjected to several changes in recent years. Traditionally, morphology-based identification was the main means for identifying Phaeosphaeriaceae species (Barr 1979(Barr , 1992Tomilin 1993). However, species identification has been revolutionized by the application of molecular based approaches incorporating DNA sequence data in Phaeosphaeriaceae Tennakoon et al. 2016;Wanasinghe et al. 2018;Bakhshi et al. 2019;Chethana et al. 2020;Hyde et al. 2020). Phaeosphaeriaceae species are adapted to a wide range of ecological environments and are present in soils, fresh and marine habitats and cause infections in humans (Yuan 1994;Phookamsak et al. 2014Phookamsak et al. , 2017Ahmed et al. 2017;Maharachchikumbura et al. 2019;Valenzuela-Lopez et al. 2019). Members of the Phaeosphaeriaceae have also been recorded from both temperate and tropical countries (i.e. Austria, Belgium, Bulgaria, Canada, China, Germany, Italy, Japan, Norway, Poland, Thailand, Sweden, Switzerland) and from different host families (i. e. Acoraceae, Arecaceae, Cyperaceae, Asparagaceae, Brassicaceae, Fabaceae, Poaceae, Marantaceae) (Shoemaker and Babcock 1989;Phookamsak et al. 2014Phookamsak et al. , 2019Wanasinghe et al. 2018;Maharachchikumbura et al. 2019;Farr and Rossman 2020). Due to their cosmopolitan distribution, in the last few years, many researchers have paid significant attention to the Phaeosphaeriaceae Tennakoon et al. 2016;Wanasinghe et al. 2018;Bakhshi et al. 2019;Hyde et al. 2020).
The fungi that decay leaf litter are highly diverse and may be host-specific (Parungao et al. 2002). Several studies have examined the succession of leaf degrading communities and found unique sets of species on different types of litter (Promputtha et al. 2002Duong et al. 2008). Additional ecological studies are therefore needed to establish whether these fungi are generalists or specialists. This study provides evidence to indicate the fungal diversity in leaf litter, even within a single family, Phaeosphaeriaceae. Additional work is necessary to identify if the newly described species are host specific.