Abstract

Introduction

The family Phaeosphaeriaceae is a large and important family of Pleosporales, initially introduced by Barr (1979) with Phaeosphaeriaoryzae I. Miyake as the type species (Miyake 1909). The taxonomy of members within this family has often been confused with those of the Leptosphaeriaceae (Müller 1950, Holm et al. 1957, Munk 1957, Zhang et al. 2009, Phookamsak et al. 2014) and it is sometimes difficult to distinguish species. Criteria which have previously been used to differentiate species have been based mostly on the morphology of the peridial wall, asexual characteristics and host association (Eriksson 1967, 1981, Lucas and Webster 1967, Leuchtmann 1984, Shoemaker 1984, Barr 1987, Shoemaker and Babcock 1989, Shearer et al. 1990, Khashnobish and Shearer 1996, Câmara et al. 2002) and taxonomic schemes followed are those of Kirk et al. (2008), Zhang et al. (2009), Hyde et al. (2013), Phookamsak et al. (2014a) and Abd-Elsalam et al. (2016). However, this delimitation of taxa in Phaeosphaeriaceae and Leptosphaeriaceae, based solely on the above-mentioned features, is not feasible. Recent studies showed that it is very difficult to discriminate them only by such characters, because numerous new members have been introduced to these two families and these species are not significantly different in these features, but they can be differentiated by phylogenetic analysis (Zhang et al. 2012, Hyde et al. 2013, Ahmed et al. 2014, Ariyawansa et al. 2015a, 2018, Bakhshi et al. 2018). Hence there is a need to use the multigene sequence data analyses to infer relationships.

Barr (1979) originally introduced 15 genera in this family and subsequent researchers have revised this number (Barr 1992, Eriksson and Hawksworth 1993, Kirk et al. 2001, 2008, Lumbsch and Huhndorf 2007, 2010). The taxonomic placement of genera within this family has been changed in recent years based on phylogenetic analyses (Zhang et al. 2012, Hyde et al. 2013, Wijayawardene et al. 2014, Phookamsak et al. 2014a, 2017, Wanasinghe et al. 2018). Taxonomic revision of the genera in Phaeosphaeriaceae resulted in 28 genera based on morphology and phylogenetic evidence (Phookamsak et al. 2014a). Since 2014, many new genera have been introduced based on molecular data (Ariyawansa et al. 2015b, Ertz et al. 2015, Crous et al. 2015a, 2015b, 2017a, Jayasiri et al. 2015, Li et al. 2015, Phukhamsakda et al. 2015, Rossman et al. 2015, Tibpromma et al. 2015, 2017, Abd-Elsalam et al. 2016, Hernández-Restrepo et al. 2016, Hyde et al. 2016, 2017, Tennakoon et al. 2016, Wijayawardene et al. 2016, Ahmed et al. 2017, Huang et al. 2017, Karunarathna et al. 2017, Phookamsak et al. 2017, Bakhshi et al. 2018, Senanayake et al. 2018, Wanasinghe et al. 2018). The placement of some older genera has been reconfirmed with DNA sequence (Phookamsak et al. 2017, Senanayake et al. 2018). However, there are still a few genera lacking molecular data, such as Bricookea, Dothideopsella, Eudarluca, Phaeostagonospora and Tiarospora. At present, this family includes more than 800 species in 61 genera (25 genera are known only from asexual morphs) (Index Fungorum 2018, Wijayawardene et al. 2017, 2018). Many genera were introduced to accommodate a single or a few species in Phaeosphaeriaceae. Only 14 genera in the Phaeosphaeriaceaecontained 10–50 species, while Ophiobolus and Phaeosphaeria comprised more than 150 species. However, most species in Ophiobolus and Phaeosphaeria lack molecular data to confirm their phylogenetic affinities.

We are studying fungi on bamboo which is the main food for panda in Sichuan Province of China (Tang et al. 2007, Wang et al. 2017). The purpose of this paper is to introduce a new genus with one species in Phaeosphaeriaceae recovered from Phyllostachysheteroclada Oliv. Combined multigene (LSU, SSU, ITS and TEF 1-α) analyses confirm its phylogenetic position in Phaeosphaeriaceae. A comprehensive comparison with similar genera and detailed descriptions and illustrations are provided.

Materials and methods

Results

Phylogenetic analyses

In this phylogenetic analysis, we include all representative sequences of genera in Phaeosphaeriaceae and other representative genera and species in Pleosporineae and Massarineae. The final concatenated dataset containing 138 ingroup taxa within the suborder Pleosporineae, included 56 currently existing genera in Phaeosphaeriaceae, with 3559 characters including gaps (917 characters for LSU, 1046 for SSU, 681 for ITS and 915 for TEF 1-α). Single gene datasets of LSU, SSU, ITS and TEF 1-α were initially analysed and checked for topological congruence but these were not significantly different (data not shown). Support values of MP, ML and BI analyses (equal to or higher than 70% for MPBP and MLBP and 0.95 for BYPP) are shown in Fig. ​Fig.11 which is the best scoring tree generated from ML. The phylogenetic trees generated from ML analyses were similar to previous phylogenies including Phaeosphaeriaceae (Phookamsak et al. 2014a, b, 2017, Jayasiri et al. 2015, Li et al. 2015, Liu et al. 2015, Phukhamsakda et al. 2015, Tibpromma et al. 2015, 2016, 2017, Hyde et al. 2016, Mapook et al. 2016, Ahmed et al. 2017, Huang et al. 2017, Karunarathna et al. 2017, Thambugala et al. 2017, Ariyawansa et al. 2018, Bakhshi et al. 2018, Senanayake et al. 2018, Wanasinghe et al. 2018).

Open in a separate window

Figure 1.

Phylogram generated from maximum likelihood analysis (RAxML) based on combined LSU, SSU, ITS and TEF 1-α sequenced data of taxa from the family Phaeosphaeriaceae and other representative species in Pleosporineae and Massarineae. The tree is rooted to Cyclothyriellarubronotata (CBS 121892), C.rubronotata (CBS 141486), Didymosphaeriarubi-ulmifolii (MFLUCC 14-0024) and D.variabile (CBS 120014). Bootstrap support values of maximum parsimony and maximum likelihood (MPBP, left; MLBP, middle) equal to or greater than 70% and Bayesian posterior probabilities (BYPP, right) equal to or greater than 0.95 are provided. The type strains were highlighted in bold and the newly generated sequences are highlighted in red.

The best scoring RAxML tree with the final optimisation had a likelihood value of -32702.569414. The matrix had 1387 distinct alignment patterns and 32.39% in this alignment is the gaps and completely undetermined characters. Estimated base frequencies were as follows: A=0.244424, C=0.233850, G=0.265929, T=0.255797, with substitution rates AC=1.171601, AG=2.805496, AT=2.145028, CG=0.771605, CT=6.035018 and GT=1.000000. The gamma distribution shape parameter α=0.167161 and the Tree-Length=5.334112. The maximum parsimony dataset consisted of 3559 characters, of which 2580 characters were constant, 217 were parsimony-uninformative and 762 were parsimony-informative. All characters were of type ‘unord’ with equal weight. The parsimony analysis resulted in a thousand equally most parsimonious trees with a length of 5829 steps (CI = 0.270, RI = 0.654, RC = 0.177, HI = 0.730). Bayesian posterior probabilities were determined by MCMC and the final average standard deviation of split frequencies was 0.009939.

Neostagonosporellasichuanensis clusters in the family Phaeosphaeriaceae with strong support (100% MLBP/100% MPBP/1.00 BYPP) and nucleotide sequences from all strains are the same and it confirms that our three collections are the same species. The multigene analyses show that N.sichuanensis is phylogenetically close to the genus Setophoma and Edenia and separated from the remaining genera of the family in a distinct clade with moderate bootstrap support.

Taxonomy

Neostagonosporella

Keywords: Fungi, Pleosporales, Phaeosphaeriaceae

C.L. Yang, X.L. Xu & K.D. Hyde gen. nov.

Index Fungorum number: IF555713

Facesoffungi number: FoF 05490

Type species.

Neostagonosporellasichuanensis C.L. Yang, X.L. Xu & K.D. Hyde

Etymology.

Name reflects the morphological similarity to the genus Stagonospora.

Description.

Parasitic on living to nearly dead stems and branches of bamboo. Sexual morph: Ascostromata coriaceous, visible as raised to superficial on host, gregarious, multi-loculate, ellipsoidal, globose to subglobose or irregular in shape, dark brown to black, glabrous. Locules globose to subglobose, with a centrally located ostiole, lacking periphyses. Peridium multi-layered, of brown to dark brown, pseudoparenchymatous cells of textura angularis. Hamathecium comprising trabeculate, anastomosed pseudoparaphyses. Asci 8-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, short pedicellate, apically rounded with an ocular chamber. Ascospores overlapping bi-seriate, hyaline, cylindrical to fusiform, septate, smooth-walled, surrounded by a distinct mucilaginous sheath. Asexual morph: Coelomycetous. Conidiostromata pycindial, coriaceous, superficial, dark brown to black, fusiform to long fusiform or rhomboid, multi-loculate, solitary, glabrous. Pycnidia globose to subglobose, ostiolate. Pycnidial wall comprising multi-layered, of dark brown to black, pseudoparenchymatous cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells ampulliform to subcylindrical, smooth, hyaline, enteroblastic, phialidic, arising from inner layer of pycnidial wall. Macroconidia hyaline, subcylindrical to cylindrical, septate, nearly equidistant between septa, smooth-walled, sometimes surrounded by a mucilaginous sheath when immature. Microconidia hyaline, varied in shape, aseptate, smooth-walled, with small guttulate.

Notes.

Stagonospora resembles Neostagonosporella in asexual status, but Stagonospora differs in having generally uni-loculate conidiomata, a thick-walled pycnidial wall, doliiform, holoblastic conidiogenous cells with several percurrent proliferations at the apex and mostly smooth to verruculose conidia (Quaedvlieg et al. 2013, Hyde et al. 2016). Phylogenetic analyses based on a concatenated LSU, SSU, ITS and TEF 1-α sequence data (Fig. ​(Fig.1)1) show that Neostagonosporella is closely related to Setophoma and Edenia within Phaeosphaeriaceae. There are some significant differences in morphology between these genera and these are summarised in Table ​Table2.2. Six species are currently accepted in Setophoma and two species in Edenia and both of them occur on different grasses but only our new collections are parasitic on bamboo. Comparison of DNA sequence data across four gene regions reveals base pair differences as shown in Table ​Table3.3. Phylogenetic analyses also clearly differentiate these taxa (Fig. ​(Fig.1).1). It is the first time that species with massarineae-like morphology occurring on bamboo, were found in the Phaeosphaeriaceae. Based on molecular phylogeny, the new genus is introduced in Phaeosphaeriaceae to accommodate a massarineae-like taxon.

Table 2.

Morphological comparison of Neostagonosporella, Setophoma and Edenia.

Morphology	Neostagonosporella	Setophoma	Edenia
	(Type: N.sichuanensis)	(Type: S.terrestris)	(Type: E.gomezpompae)
Ascostromata	Multi-loculate, globose to subglobose or irregular	Uni-loculate, globose
Locules	Globose to subglobose, with a central ostiole, lacking periphyses	Globose, with a central ostiole
Pseudoparaphyses	Narrow, septate, trabeculae, longer than asci	Broad, septate, prominently branched, constricted at septa, sometimes anastomosing
Asci	Cylindrical to cylindric-clavate, short-pedicellate	Cylindrical or subcylindrical, fasciculate, pedicellate
Ascospores	Bi-seriate, hyaline, cylindrical to fusiform, smooth-walled, transversely multi-septate	Uni- to multi-seriate, light brown or red brown, fusiform, sometimes verruculose, 2–3-septate
Conidiostromata	Multi-loculate	Uni-loculate
Pycnidia	Globose to subglobose, smooth, ostiolate	Globose to subglobose, setose, with papillate ostiolate
Conidia	Two types. Macroconidia subcylindrical to cylindrical, transversely multi-septate, hyaline. Microconidia oval, ellipsoidal or long ellipsoidal, aseptate, hyaline	One type. Ellipsoidal to subcylindrical to subfusoid, aseptate, hyaline	One type. Ellipsoidal or slightly narrowed at base, aseptate, subhyaline
Others	On PDA, grey white, reverse dark brown. Hyphae developing by different angle branched and without forming rope-like strands	On PDA, iron-grey-olivaceous, reverse same. Hyphae undescribed	On PDA, pinkish-white, reverse reddish-brown, velvety to floccose. Hyphae frequently developing by 90° angle branched and forming rope-like strands
References	This study	de Gruyter et al. 2010, Quaedvlieg et al. 2013, Phookamsak et al. 2014a, b, Crous et al. 2016, Thambugala et al. 2017	González et al. 2007, Sun et al. 2013

Open in a separate window

Table 3.

Comparison of DNA sequence data Parastagonosporella vs Edenia and Setophoma.

Gene region	Parastagonosporella vs Edenia	Parastagonosporella vs Setophoma
LSU	12/819 (1.47%)	13/818 (1.6%)
SSU	NA*	4/981 (0.4%)
TEF	47/869 (5.41%)	43/868 (5%)
ITS	89/515 (17.28%)	66/515 (12.8%)

Open in a separate window

*SSU is not available for Edenia

Neostagonosporella sichuanensis

Keywords: Fungi, Pleosporales, Phaeosphaeriaceae

C.L. Yang, X.L. Xu & K.D. Hyde sp. nov.

Index Fungorum number: IF555714

Facesoffungi number: FoF 05491

Figs 2 , 3

Open in a separate window

Figure 2.

Neostagonosporellasichuanensis (MFLU 18-1212, holotype). a аppearance of ascostromata on host b ascostroma c, d vertical section of ascostroma e, f close up of ascoma g peridium h trabeculate pseudoparaphyses and asci i–k asci l bitunicate asci, note ocular chamber m, n, q, r ascospores with mucilaginous sheath o, s germinated ascospores in lactate cotton blue reagent p, t colonies on PDA (p-from above, t-from below). Scale bars: 1 cm (a); 1 mm (b); 200 μm (c, d); 100 μm (e, f); 20 μm (g–k); 10 μm (l–o, q–s).

Open in a separate window

Figure 3.

Neostagonosporellasichuanensis (MFLU 18-1220, paratype). a appearance of conidiomata on host b, c vertical section of conidioma d pycnidia e peridium f, g conidiogenous cells and developing conidia h–l conidia m germinated conidium. Scale bars: 1 cm (a); 200 μm (b–d); 20 μm (e, f); 10 μm (g–m).

Type.

CHINA, Sichuan Province, Ya’an City, Yucheng District, Kongping Township, Alt. 1133 m, 29°50.14'N 103°03'E, on living to nearly dead branches of Phyllostachysheteroclada Oliv. (Poaceae), 8 April 2016, C.L. Yang and X.L. Xu, YCL201604001 (MFLU 18-1212/SICAU 16-0001, holotype), ex-type living culture, MFLUCC 18-1228/SICAUCC 16-0001; Sichuan Province, Ya’an City, Yucheng District, Yanchang Township, Alt. 951 m, 29°43.57'N 103°04.74'E, on nearly dead stems of Phyllostachysheteroclada Oliv. (Poaceae), 9 April 2017, C.L. Yang and X.L. Xu, YCL201704001 (MFLU 18-1220/SICAU 17-0001, paratype), ex-type living culture, MFLUCC 18-1231/SICAUCC 17-0001; Sichuan Province, Ya’an City, Lushan County, Longmen Township, Alt. 949 m, 30°15.74'N 102°59.27'E, on nearly dead branches of Phyllostachysheteroclada Oliv. (Poaceae), 12 September 2017, C.L. Yang and X.L. Xu, YCL201709002 (MFLU 18-1223, paratype).

Etymology.

in reference to Sichuan Province where the specimens were collected.

Description.

Associated with stem spot disease on living to nearly dead stems and branches of Phyllostachysheteroclada (Poaceae). Sexual morph: Ascostromata (0.5–) 1–2 (–4.5) × 0.8–1.3 mm long (x¯ = 1.9 × 1 mm, n = 50), 230–340 μm high (x¯ = 290 μm, n = 20), ellipsoidal, globose to subglobose or irregular in shape, immersed in host epidermis, becoming raised to superficial, coriaceous, solitary to gregarious, multi-loculate, erumpent through host tissue, with dark brown to black, glabrous, ostiole, usually generating subrhombic to rhombic pale yellow stripes at ascostromatal fringe. Locules 230–300 μm high (x¯ = 264 μm, n = 20), 330–460 μm diam. (x¯ = 393 μm, n = 20), clustered, gregarious, globose to subglobose, with a centrally located ostiole, lacking periphyses. Peridium 18–35 μm wide (x¯ = 27 μm, n = 20), composed of several layers of small, brown to dark brown pseudoparenchymatous cells of textura angularis, with inner hyaline layer, slightly thin at base, thick at sides towards apex, upper part fused with host tissue. Hamathecium composed of 1–2 μm (x¯ = 1.59 μm, n = 50) wide, filiform, septate, trabeculate, anastomosed pseudoparaphyses, embedded in a hyaline gelatinous matrix. Asci 90–125 × 12.5–14 μm (x¯ = 108.1 × 13.3 μm, n = 40), 8-spored, bitunicate, fissitunicate, cylindrical to cylindric-clavate, short pedicellate, 7.8–14 μm long (x¯ = 11 μm, n=20), apically rounded with an ocular chamber. Ascospores 30–35 × 6–7 μm (x¯ = 31.9 × 6.6 μm, n = 50), overlapping bi-seriate, hyaline, cylindrical to fusiform or subcylindric-clavate, with rounded to acute ends, narrower towards end cells, sometimes narrower at lower end cell, straight or slightly curved, 5–8 transversely septa, mostly 7-septate, slightly constricted at septa, nearly equidistant between septa, guttulate, smooth-walled, surrounded by a mucilaginous sheath, 5–9 μm thick (x¯ = 6.9 μm, n = 30). Asexual morph: Coelomycetous. Conidiostromata 9–13 × 1–2 mm long (x¯ = 11.2 × 1.6 mm, n = 10), 320–350 μm high (x¯ = 332 μm, n=10), fusiform to long fusiform or rhomboid, coriaceous, superficial, dark brown to black, multi-loculate, solitary, scattered, glabrous. Pycnidia 180–240 μm high (x¯ = 209 μm, n = 20), 170–240 μm diam. (x¯ = 210 μm, n = 20), globose to subglobose, ostiolate. Pycnidial wall 12–18 (–23) μm wide (x¯ = 15 μm, n = 20), comprising multi-layered, brown to dark brown pseudoparenchymatous cells, of textura angularis, paler towards inner layers, slightly thin at base, thick at sides towards apex, upper part fused with host tissue. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 3–5.5 (–7) × 3–4 μm (x¯ = 4.17 × 3.29 μm, n = 20), ampulliform to subcylindrical, smooth, hyaline, enteroblastic, phialidic, formed from inner layer of pycnidial wall. Macroconidia (32.5–) 33.5–40 (–44) × (5–) 5.5–7 (–7.5) μm (x¯ = 37.5 × 6.2 μm, n = 40), subcylindrical to cylindrical, narrowly rounded at both ends, sometimes curved, 7–13 transversely septa, nearly equidistant between septa, hyaline, smooth-walled, guttulate, sometimes surrounded by a mucilaginous sheath when immature. Microconidia (3–) 3.5–4 (–5) × (1–) 1.5–2 (–3) μm (x¯ = 3.9 × 1.9 μm, n = 50), oval, ellipsoidal or elongate-ellipsoidal, aseptate, rounded at both ends, hyaline, smooth-walled, with small guttulate.

Culture characteristics. Ascospores germinating in sterilised water within 24 hours at 25°C, with germ tubes developed from each cell of ascospores, mostly from middle and end of spores. Colonies on PDA circular, with concentric circles, grey white in outer side, fawn in reverse side, grey in inner side, dark brown on back side. Conidial germination similar to ascospores. Conidiomata formed on PDA at 25°C after 75 days, pycnidial, solitary to gregarious, raised on agar, black dots, pyriform, globose to subglobose, or irregular, uniloculate, covered by white or grey hyphae. Conidia two types, macroconidia and microconidia and both longer than ones on host. Macroconidia (30–)40–48(–60.5) × (4–)5–6 μm (x¯ = 43.8 × 5.2 μm, n = 50), hyaline, 4–7-septate, occasionally 3-septate, hyaline. Microconidia (3.5–)4–6(–12) × (1–)1.5–2(–3) μm (x¯ = 5.3 × 1.9 μm, n = 50), aseptate, hyaline.

Discussion

Neostagonosporella has a unique suite of characters that differentiate it from other genera in Phaeosphaeriaceae, such as multi-loculate ascostromata and trabeculate pseudoparaphyses. Trabeculate pseudoparaphyses have been shown to be uninformative at the higher taxonomic levels (Liew et al. 2000), but appear to be informative at the genus level. Neostagonosporella is the only genus of Phaeosphaeriaceae with this type of pseudoparaphyses. Phaeosphaeriaceous taxa have diverse morphological characteristics and the familial placement of some genera could not be resolved based on a concatenated phylogeny of three to four loci, because some genera contain only 1-2 described species (Crous et al. 2015a, 2015b, 2017a, Jayasiri et al. 2015, Phukhamsakda et al. 2015, Tibpromma et al. 2015, 2017, Abd-Elsalam et al. 2016, Hernández-Restrepo et al. 2016, Hyde et al. 2016, 2017, Wijayawardene et al. 2016, Ahmed et al. 2017, Karunarathna et al. 2017, Phookamsak et al. 2017, Bakhshi et al. 2018, Wanasinghe et al. 2018).

Species of Phaeosphaeriaceae have been found on various hosts and substrates, including plants, lichens, mushrooms, algae, human, soil and air (Saccardo 1883, Berlese and Voglino 1886, Phookamsak et al. 2014a, Ahmed et al. 2016, Karunarathna et al. 2017, Zhang et al. 2017, Joshi et al. 2018). However, most Phaeosphaeriaceous genera occur on plants of more than 65 host families, the majority of them being monocotyledons and herbaceous plants, such as Arecaceae, Asparagaceae, Compositae, Juncaceae, Leguminosae, Poaceae, Ranunculaceae, Restionaceae and Rosaceae etc. (Taylor and Hyde 2003, Quaedvlieg et al. 2013, Crous et al. 2015b, Hyde et al. 2016, Tibpromma et al. 2016a, Karunarathna et al. 2017, Phookamsak et al. 2017, Wanasinghe et al. 2018). Our new genus exists on Poaceae and at least 30 genera are reported within this family. Currently, 11 genera are observed only on Poaceae: Amarenomyces, Bricookea, Camarosporioides, Dactylidina, Embarria, Melnikia, Neosphaerellopsis, Phaeopoacea, Sulcispora, Vagicola and Yunnanensis, all of them being recently established except for Amarenomyces, Bricookea and Sulcispora (Eriksson 1981, Barr 1982, Shoemaker and Babcock 1989, Trakunyingcharoen et al. 2014, Ariyawansa et al. 2015b, Hyde et al. 2016, Wijayawardene et al. 2016, Karunarathna et al. 2017, Thambugala et al. 2017, Wanasinghe et al. 2018). Amongst them, all hosts are short herbaceous plants and there are no bamboo plants recorded so far, with the exception of a few species of Ophiobolus and Phaeosphaeria in the old literature (Penzig and Saccardo 1897, Miyake and Hara 1910). A large number of bamboo forests (more than 130 species) are distributed throughout Sichuan (Yi 1997) and, most likely, many Phaeosphaeriaceae species are waiting for exploration and discovery.

Supplementary Material

Acknowledgements

Notes

References