Neostagonosporellasichuanensis gen. et sp. nov. (Phaeosphaeriaceae, Pleosporales) on Phyllostachysheteroclada (Poaceae) from Sichuan Province, China.

Neostagonosporellasichuanensis sp. nov. was found on Phyllostachysheteroclada collected from Sichuan Province in China and is introduced in a new genus Neostagonosporella gen. nov. in this paper. Evidence for the placement of the new taxon in the family Phaeosphaeriaceae is supported by morphology and phylogenetic analysis of a combined LSU, SSU, ITS and TEF 1-α DNA sequence dataset. Maximum-likelihood, maximum-parsimony and Bayesian inference phylogenetic analyses support Neostagonosporella as a distinct genus within this family. The new genus is compared with related genera of Phaeosphaeriaceae and full descriptions and illustrations are provided. Neostagonosporella is characterised by its unique suite of characters, such as multiloculate ascostromata and cylindrical to fusiform, transversely multiseptate, straight or curved ascospores, which are widest at the central cells. Conidiostromata are multiloculate, fusiform to long fusiform or rhomboid, with two types conidia; macroconidia vermiform or subcylindrical to cylindrical, transversely multiseptate, sometimes curved, almost equidistant between septa and microconidia oval, ellipsoidal or long ellipsoidal, aseptate, rounded at both ends. An updated phylogeny of the Phaeosphaeriaceae based on multigene analysis is provided.

Introduction

The family is a large and important family of , initially introduced by Barr (1979) with I. Miyake as the type species (Miyake 1909). The taxonomy of members within this family has often been confused with those of the (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 and , 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 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 , , , and . 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 . Only 14 genera in the contained 10–50 species, while and comprised more than 150 species. However, most species in and 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 recovered from Oliv. Combined multigene (LSU, SSU, ITS and TEF 1-α) analyses confirm its phylogenetic position in . A comprehensive comparison with similar genera and detailed descriptions and illustrations are provided.

Materials and methods

Sampling and morphological study

The specimens were collected from Ya’an City of Sichuan Province in China, on living to near dead stems and branches of . The samples were kept in Ziplock plastic bags and brought to the laboratory. Fresh materials were examined by using stereo and compound microscopes. Vertical free-hand sections were made by using a razor blade and placed on a droplet of sterilised water on a glass slide (Gupta and Tuohy 2013). Lactate cotton blue reagent was used to observe the number of septa. Micro-morphological characters were examined by using a Nikon ECLIPSE Ni compound microscope fitted to a Cannon 600D digital camera. Fruiting tissues were observed by stereomicroscopy using NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd, China) and photographed by VS-800C (Shenzhen Weishen Times Technology Co. Ltd, China). Measurements were taken using Tarosoft® Image Frame Work v.0.9.7.

Isolation

Single ascospore and conidium isolation was carried out following the method described by Dai et al. (2017). Germinated ascospores and conidia were separately transferred to Potato Dextrose Agar media plates (PDA) and incubated at 25°C and the colonies were observed after 10 days and as outlined by Vijaykrishna et al. (2004) and Liu et al. (2010). Specimens are deposited in Mae Fah Luang University Herbarium (MFLU), Chiang Rai, Thailand and Sichuan Agricultural University Herbarium (SICAU), Chengdu, China. Living cultures are deposited at the Culture Collection at Mae Fah Luang University (MFLUCC) and the Culture Collection at Sichuan Agricultural University (SICAUCC). Facesoffungi and Index Fungorum numbers were registered as in Jayasiri et al. (2015) and Index Fungorum (2018), respectively. New species are established following the recommendations of Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing

Fungal isolates were grown on PDA for seven days at 25°C and genomic DNA was extracted from fresh mycelia, following the protocols of Plant Genomic DNA Kit (Tiangen, China). If cultures were unavailable, fungal DNA was directly extracted from fruiting tissues according to Yang et al. (2017), Wanasinghe et al. (2018) and Zeng et al. (2018). The primers, LR0R and LR5 (Vilgalys and Hester 1990), NS1 and NS4, ITS5 and ITS4 (White et al. 1990) and EF1-983F and EF1-2218R (Rehner 2001) were used for the amplification of the 28S large subunit rDNA (LSU), 18S small subunit rDNA (SSU), internal transcribed spacers (5.8S, ITS) and translation elongation factor 1-α gene region (TEF 1-α), respectively. The amplification reactions were performed as stated by Phukhamsakda et al. (2015). Amplified PCR fragments were purified and sequenced at TsingKe Biological Technology Co., Ltd. (Chengdu, China). Newly generated sequences of LSU, SSU, ITS and TEF 1-α regions are deposited in GenBank.

Molecular phylogenetic analysis

Sequence data, mainly from recent publications (Phookamsak et al. 2017, Wanasinghe et al. 2018), were downloaded for analyses (Table 1). Four taxa (CBS 121892), (CBS 141486), (MFLUCC 14-0024) and (CBS 120014) were chosen as outgroup taxa based on Tanaka et al. (2015) and Jaklitsch and Voglmayr (2016). DNA alignments were performed by using MAFFT v.7.407 online service (Katoh and Standley 2013) and ambiguous regions were excluded with BioEdit version 7.0.5.3 (Hall 1999). Multigene sequences were concatenated by Mesquite version 3.11 (build 766) (Maddison and Maddison 1997–2016). Multigene phylogenetic analyses of the combined LSU, SSU, ITS and TEF 1-α sequence data were obtained from maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) analyses. The alignments were converted to NEXUS file (.nxs) by using ClustalX version 1.81 (Thompson et al. 1997) for MP and BI analyses. The symbols “ABCDEFGHIKLMNOPQRSTUVWXYZ” was deleted in PAUP v. 4.0b10 (Swofford 2002) for preparing data matrix of evaluated evolutionary model by MrModeltest v. 2.2 (Nylander 2004). The best nucleotide substitution model was determined by MrModeltest v. 2.2 (Nylander 2004) and the best-fit model for BI is GTR+I+G under the Akaike Information Criterion (AIC).

Table 1.

Molecular data used in this study and GenBank accession numbers.

Species	Strain/Voucher No.	GenBank Accession No.				Refferences
		LSU	SSU	ITS	TEF 1-α
Acericola italica	MFLUCC 13-0609	MF167429	MF167430	MF167428	-	Hyde et al. 2017
Allophaeosphaeria muriformia	MFLUCC 13-0277	KX910089	KX950400	KX926415	-	Liu et al. 2015
Allophaeosphaeria muriformia	MFLUCC 13-0349	KP765681	KP765682	KP765680	-	Liu et al. 2015
Amarenographium ammophilae	MFLUCC 16-0296	KU848197	KU848198	KU848196	MG520894	Wijayawardene et al. 2016, Phookamsak et al. 2017
Amarenomyces dactylidis	MFLUCC 14-0207	KY775575	-	KY775577	-	Hyde et al. 2017
Ampelomyces quisqualis	CBS 131.31	JX681066	-	AF035781	-	Kiss and Nakasone 1998, Verkley et al. 2014
Ampelomyces quisqualis	CBS 133.32	JX681067	-	-	-	Verkley et al. 2014
Banksiophoma australiensis	CBS 142163	KY979794	-	KY979739	-	Crous et al. 2017
Bhatiellae rosae	MFLUCC 17-0664	MG828989	MG829101	MG828873	-	Wanasinghe et al. 2018
Boeremia exigua	CBS 431.74	EU754183	EU754084	FJ427001	GU349080	Aveskamp et al. 2009, de Gruyter et al. 2009, Schoch et al. 2009
Camarosporioides phragmitis	MFLUCC 13-0365	KX572345	KX572350	KX572340	KX572354	Hyde et al. 2016
Chaetosphaeronema achilleae	MFLUCC 16-0476	KX765266	-	KX765265	-	Hyde et al. 2016
Chaetosphaeronema hispidulum	CBS 216.75	KF251652	EU754045	KF251148	-	de Gruyter et al. 2009, Quaedvlieg et al. 2013
Cyclothyriella rubronotata	CBS 121892	KX650541	-	KX650541	KX650516	Jaklitsch and Voglmayr 2016
Cyclothyriella rubronotata	CBS 141486	KX650544	KX650507	KX650544	KX650519	Jaklitsch and Voglmayr 2016
Dactylidina shoemakeri	MFLUCC 14-0963	MG829003	MG829114	MG828887	MG829200	Wanasinghe et al. 2018
Dematiopleospora cirsii	MFLUCC 13-0615	KX274250	-	KX274243	KX284708	Hyde et al. 2016
Dematiopleospora fusiformis	MFLU 15-2133	KY239030	KY239028	KY239029	-	Huang et al. 2018
Dematiopleospora mariae	MFLUCC 13-0612	KJ749653	KJ749652	KJ749654	KJ749655	Wanasinghe et al. 2014
Didymocyrtis caloplacae	CBS 129338	JQ238643	-	JQ238641	-	Lawrey et al. 2012
Didymocyrtis ficuzzae	CBS 128019	JQ238616	-	KP170647	-	Lawrey et al. 2012, Trakunyingcharoen et al. 2014
Didymocyrtis xanthomendozae	CBS 129666	JQ238634	-	KP170651	-	Lawrey et al. 2012, Trakunyingcharoen et al. 2014
Didymosphaeria rubi-ulmifolii	MFLUCC 14-0024	KJ436585	KJ436587	-	-	Ariyawansa et al. 2014
Didymosphaeria variabile	CBS 120014	JX496139	-	JX496026	-	Verkley et al. 2014
Dlhawksworthia alliariae	MFLUCC 13-0070	KX494877	KX494878	KX494876	-	Hyde et al. 2016
Dlhawksworthia clematidicola	MFLUCC 14-0910	MG829011	MG829120	MG828901	MG829202	Wanasinghe et al. 2018
Dlhawksworthia lonicera	MFLUCC 14-0955	MG829012	MG829121	MG828902	MG829203	Wanasinghe et al. 2018
Dothidotthia aspera	CPC 12933	EU673276	EU673228	-	-	Phillips et al. 2008
Dothidotthia symphoricarpi	CPC 12929	EU673273	EU673224	-	-	Phillips et al. 2008
Edenia gomezpompae	AM04	KM246015	-	KM246160	-	González et al. 2007
Edenia gomezpompae	CBS 124106	FJ839654	-	FJ839619	-	Crous et al. 2009
Edenia sp.	UTHSC: DI16-264	LN907407	-	LT796858	LT797098	Valenzuela-Lopez et al. 2017
Edenia sp.	UTHSC: DI16-260	LN907403	-	LT796855	LT797095	Valenzuela-Lopez et al. 2017
Embarria clematidis	MFLUCC 14-0652	KT306953	KT306956	KT306949	-	Ariyawansa et al. 2015a
Embarria clematidis	MFLUCC 14-0976	MG828987	MG829099	MG828871	MG829194	Wanasinghe et al. 2018
Equiseticola fusispora	MFLUCC 14-0522	KU987669	KU987670	KU987668	MG520895	Abd-Elsalam et al. 2016, Phookamsak et al. 2017
Foliophoma fallens	CBS 161.78	GU238074	GU238215	KY929147	-	Aveskamp et al. 2010, Crous and Groenewald 2017
Foliophoma fallens	CBS 284.70	GU238078	GU238218	KY929148	-	Aveskamp et al. 2010, Crous and Groenewald 2017
Galiicola pseudophaeosphaeria	MFLU 14-0524	KT326693	-	KT326692	MG520896	Phookamsak et al. 2017
Italica achilleae	MFLUCC 14-0959	MG829013	MG829122	MG828903	MG829204	Wanasinghe et al. 2018
Juncaceicola italica	MFLUCC 13-0750	KX500107	KX500108	KX500110	MG520897	Phookamsak et al. 2017
Juncaceicola luzulae	MFLUCC 13-0780	KX449530	KX449531	KX449529	MG520898	Tennakoon et al. 2016, Phookamsak et al. 2017
Leptospora galii	KUMCC 15-0521	KX599548	KX599549	KX599547	MG520899	Phookamsak et al. 2017
Leptospora rubella	CPC 11006	DQ195792	DQ195803	DQ195780	-	Crous et al. 2006
Leptospora thailandica	MFLUCC 16-0385	KX655549	KX655554	KX655559	KX655564	Hyde et al. 2016
Loratospora aestuarii	JK 5535B	GU301838	GU296168	-	-	Schoch et al. 2009
Melnikia anthoxanthii	MFLUCC 14-1010	KU848204	KU848205	-	-	Wijayawardene et al. 2016
"Muriphaeosphaeria" ambrosiae	MFLU 15-1971	KX765264	-	KX765267	-	Hyde et al. 2016
Muriphaeosphaeria galatellae	MFLUCC 14-0614	KT438329	KT438331	KT438333	-	Phukhamsakda et al. 2015
Muriphaeosphaeria galatellae	MFLUCC 15-0769	KT438330	KT438332	-	-	Phukhamsakda et al. 2015
Neocamarosporium lamiacearum	MFLUCC 17-560	MF434279	MF434367	MF434191	MF434454	Wanasinghe et al. 2017
Neosetophoma clematidis	MFLUCC 13-0734	KP684153	KP684154	KP744450	-	Liu et al. 2015
Neosetophoma rosae	MFLUCC 17-0844	MG829035	MG829141	MG828926	MG829219	Wanasinghe et al. 2018
Neosetophoma rosae	MFLU 15-1073	MG829034	MG829140	MG828925	MG829218	Wanasinghe et al. 2018
Neosphaerellopsis thailandica	CPC 21659	KP170721	-	KP170652	-	Trakunyingcharoen et al. 2014
Neostagonospora arrhenatheri	MFLUCC 15-0464	KX910091	KX950402	KX926417	MG520901	Phookamsak et al. 2017, Thambugala et al. 2017
Neostagonospora caricis	CBS 135092	KF251667	-	KF251163	-	Quaedvlieg et al. 2013
Neostagonospora phragmitis	MFLUCC 16-0493	KX910090	KX950401	KX926416	MG520902	Phookamsak et al. 2017, Thambugala et al. 2017
Neostagonosporella sichuanensis	MFLUCC 18-1228	MH368073	MH368079	MH368088	MK313851	This study
Neostagonosporella sichuanensis	MFLUCC 18-1231	MH368074	MH368080	MH368089	-	This study
Neostagonosporella sichuanensis	MFLU 18-1223	MH394690	MH394687	MK296469	MK313854	This study
Neosulcatispora agaves	CPC 26407	KT950867	-	KT950853	-	Crous et al. 2015b
Nodulosphaeria guttulatum	MFLUCC 15-0069	KY496726	KY501115	KY496746	KY514394	Tibpromma et al. 2017
Nodulosphaeria multiseptata	MFLUCC 15-0078	KY496728	KY501116	KY496748	KY514396	Tibpromma et al. 2017
Nodulosphaeria scabiosae	MFLUCC 14-1111	KU708846	KU708842	KU708850	KU708854	Mapook et al. 2016
Ophiobolopsis italica	MFLUCC 17-1791	MG520959	MG520977	MG520939	MG520903	Phookamsak et al. 2017
Ophiobolus artemisiae	MFLUCC 14-1156	KT315509	MG520979	KT315508	MG520905	Phookamsak et al. 2017
Ophiobolus artemisiae	MFLU 15-1966	MG520960	MG520978	MG520940	MG520904	Phookamsak et al. 2017
Ophiobolus disseminans	MFLUCC 17-1787	MG520961	MG520980	MG520941	MG520906	Phookamsak et al. 2017
Ophiobolus italicus	MFLUCC 14-0526	KY496727	-	KY496747	KY514395	Tibpromma et al. 2017
Ophiobolus rossicus	MFLU 17-1639	MG520964	MG520983	MG520944	MG520909	Phookamsak et al. 2017
Ophiobolus rudis	CBS 650.86	GU301812	AF164356	KY090650	GU349012	Liew et al. 2000, Schoch et al. 2009, Ahmed et al. 2016
Ophiobolus senecionis	MFLUCC 13-0575	KT728366	-	KT728365	-	Tibpromma et al. 2015
Ophiosimulans tanaceti	MFLUCC 14-0525	KU738891	KU738892	KU738890	MG520910	Tibpromma et al. 2016b, Phookamsak et al. 2017
Ophiosphaerella agrostidis	MFLUCC 11-0152	KM434281	KM434290	KM434271	KM434299	Phookamsak et al. 2014a
Ophiosphaerella agrostidis	MFLUCC 12-0007	KM434282	KM434291	KM434272	KM434300	Phookamsak et al. 2014a
Ophiosphaerella aquatica	MFLUCC 14-0033	KX767089	KX767090	KX767088	MG520911	Ariyawansa et al. 2015a, Phookamsak et al. 2017
Paraleptosphaeria rubi	MFLUCC 14-0211	KT454718	KT454733	KT454726	-	Ariyawansa et al. 2015b
Paraophiobolus arundinis	MFLUCC 17-1789	MG520965	MG520984	MG520945	MG520912	Phookamsak et al. 2017
Paraophiobolus plantaginis	MFLUCC 17-0245	KY815010	KY815012	KY797641	MG520913	Hyde et al. 2017, Phookamsak et al. 2017
Paraphoma chrysanthemicola	CBS 522.66	GQ387582	GQ387521	KF251166	-	de Gruyter et al. 2010, Quaedvlieg et al. 2013
Paraphoma radicina	CBS 111.79	KF251676	EU754092	KF251172	-	de Gruyter et al. 2009, Quaedvlieg et al. 2013
Parastagonospora dactylidis	MFLUCC 13-0375	KU058722	-	KU058712	-	Li et al. 2015
Parastagonospora italica	MFLUCC 13-0377	KU058724	MG520985	KU058714	MG520915	Li et al. 2015, Phookamsak et al. 2017
Parastagonospora minima	MFLUCC 13-0376	KU058723	MG520986	KU058713	MG520916	Li et al. 2015, Phookamsak et al. 2017
Parastagonospora uniseptata	MFLUCC 13-0387	KU058725	MG520987	KU058715	MG520917	Li et al. 2015, Phookamsak et al. 2017
Parastagonosporella fallopiae	CBS 135981	MH460545	-	MH460543	-	Bakhshi et al. 2018
Parastagonosporella fallopiae	CCTU 1151.1	MH460546	-	MH460544	-	Bakhshi et al. 2018
Phaeopoacea festucae	MFLUCC 17-0056	KY824767	KY824769	KY824766	-	Thambugala et al. 2017
Phaeopoacea phragmiticola	CBS 459.84	KF251691	KY090700	KF251188	-	Quaedvlieg et al. 2013, Ahmed et al. 2016
Phaeosphaeria acaciae	MFLUCC 17-0320	KY768868	KY768870	KY768869	-	Hyde et al. 2017
Phaeosphaeria chiangraina	MFLUCC 13-0231	KM434280	KM434289	KM434270	KM434298	Phookamsak et al. 2014a
Phaeosphaeria musae	MFLUCC 11-0151	KM434278	KM434288	KM434268	KM434297	Phookamsak et al. 2014a
Phaeosphaeria oryzae	CBS 110110	KF251689	GQ387530	KF251186	-	de Gruyter et al. 2010, Quaedvlieg et al. 2013
Phaeosphaeria thysanolaenicola	MFLUCC 10-0563	KM434276	KM434286	KM434266	KM434295	Phookamsak et al. 2014a
Phaeosphaeriopsis dracaenicola	MFLUCC 11-0157	KM434283	KM434292	KM434273	KM434301	Phookamsak et al. 2014a
Phaeosphaeriopsis glaucopunctata	MFLUCC 13-0265	KJ522477	KJ522481	KJ522473	MG520918	Thambugala et al. 2014, Phookamsak et al. 2017
Phaeosphaeriopsis triseptata	MFLUCC 13-0271	KJ522479	KJ522484	KJ522475	MG520919	Thambugala et al. 2014, Phookamsak et al. 2017
Phoma herbarum	AFTOL-ID 1575	DQ678066	DQ678014	-	DQ677909	Schoch et al. 2006
Stemphylium vesicarium	CBS 191.86	GU238160	GU238232	EF452449	DQ471090	Spatafora et al. 2006, Andrie et al. 2008, Aveskamp et al. 2010
Stemphylium botryosum	CBS 714.68	KC584345	KC584603	EF452450	DQ677888	Schoch et al. 2006, Andrie et al. 2008, Woudenberg et al. 2013
Poaceicola arundinis	MFLUCC 14-1060	KX655548	KX655553	KX655558	-	Hyde et al. 2016
Poaceicola arundinis	MFLU 16-0158	MG829057	MG829162	MG828947	MG829229	Wanasinghe et al. 2018
Poaceicola forlicesenica	MFLUCC 15-0470	KX910095	KX950406	KX926422	MG520922	Phookamsak et al. 2017, Thambugala et al. 2017
Poaceicola garethjonesii	MFLUCC 15-0469	KX954390	KY205717	KX926425	MG520923	Phookamsak et al. 2017, Thambugala et al. 2017
Populocrescentia ammophilae	MFLUCC 17-0665	MG829059	MG829164	MG828949	MG829231	Wanasinghe et al. 2018
Populocrescentia forlicesenensis	MFLUCC 14-0651	KT306952	KT306955	KT306948	MG520925	Ariyawansa et al. 2015a, Phookamsak et al. 2017
Populocrescentia rosae	TASM 6125	MG829060	MG829165	-	MG829232	Wanasinghe et al. 2018
Pseudoophiobolus achilleae	MFLU 17-0925	MG520966	-	MG520946	-	Phookamsak et al. 2017
Pseudoophiobolus galii	MFLUCC 17-2257	MG520967	MG520989	MG520947	MG520926	Phookamsak et al. 2017
Pseudoophiobolus urticicola	KUMCC 17-0168	MG520975	MG520996	MG520955	MG520933	Phookamsak et al. 2017
Pseudophaeosphaeria rubi	MFLUCC 14-0259	KX765299	KX765300	KX765298	-	Hyde et al. 2016
Pyrenochaeta nobilis	CBS 407.76	DQ678096	-	EU930011	DQ677936	Ferrer et al. 2006, Schoch et al. 2006
Pyrenophora bromi	DAOM 127414	JN940074	JN940954	JN943666	-	Schoch et al. 2012
Pyrenophora dactylidis	DAOM 92161	JN940087	-	JN943667	-	Schoch et al. 2012
Sclerostagonospora lathyri	MFLUCC 14-0958	MG829066	MG829170	MG828955	MG829235	Wanasinghe et al. 2018
Sclerostagonospora sp.	CBS 118152	JX517292	-	JX517283	-	Crous et al. 2012.
Scolicosporium minkeviciusii	MFLUCC 12-0089	KF366382	KF366383	-	-	Wijayawardene et al. 2013
Septoriella phragmitis	CPC 24118	KR873279	-	KR873251	-	Crous et al. 2015c
Setomelanomma holmii	CBS 110217	GQ387633	GQ387572	KT389542	GU349028	Schoch et al. 2009, de Gruyter et al. 2010, Chen et al. 2015
Setophoma chromolaena	CBS 135105	KF251747	-	KF251244	-	Quaedvlieg et al. 2013
Setophoma sacchari	CBS 333.39	GQ387586	GQ387525	KF251245	-	de Gruyter et al. 2010
Setophoma sacchari	MFLUCC 12-0241	KJ476147	KJ476149	KJ476145	KJ461318	Phookamsak et al. 2014b
Setophoma sacchari	MFLUCC 11-0154	KJ476146	KJ476148	KJ476144	KJ461319	Phookamsak et al. 2014b
Setophoma vernoniae	CPC 23123	KJ869198	-	KJ869141	-	Crous et al. 2014
Staurosphaeria rhamnicola	MFLUCC 17-0813	MF434288	MF434376	MF434200	MF434462	Wanasinghe et al. 2017
Staurosphaeria rhamnicola	MFLUCC 17-0814	MF434289	MF434377	MF434201	MF434463	Wanasinghe et al. 2017
Sulcispora pleurospora	CBS 460.84	-	-	AF439498	-	Câmara et al. 2002
Sulcispora supratumida	MFLUCC 14-0995	KP271444	KP271445	KP271443	-	Senanayake et al. 2018
Tintelnotia destructans	CBS 127737	KY090664	KY090698	KY090652	-	Ahmed et al. 2016
Tintelnotia opuntiae	CBS 376.91	GU238123	GU238226	KY090651	-	Aveskamp et al. 2010, Ahmed et al. 2016
Vagicola chlamydospora	MFLUCC 15-0177	KU163654	KU163655	KU163658	-	Jayasiri et al. 2015
Vrystaatia aloeicola	CBS 135107	KF251781	-	KF251278	-	Quaedvlieg et al. 2013
Wojnowicia italica	MFLUCC 13-0447	KX430001	KX430002	KX342923	KX430003	Hyde et al. 2016
Wojnowicia lonicerae	MFLUCC 13-0737	KP684151	KP684152	KP744471	-	Liu et al. 2015
Wojnowiciella dactylidis	MFLUCC 13-0735	KP684149	KP684150	KP744470	-	Liu et al. 2015
Wojnowiciella eucalypti	CPC 25024	KR476774	-	KR476741	-	Crous et al. 2015a
Wojnowiciella spartii	MFLUCC 13-0402	KU058729	MG520998	KU058719	MG520937	Li et al. 2015, Phookamsak et al. 2017
Xenoseptoria neosaccardoi	CBS 120.43	KF251783	-	KF251280	-	Quaedvlieg et al. 2013
Xenoseptoria neosaccardoi	CBS 128665	KF251784	-	KF251281	-	Quaedvlieg et al. 2013
Yunnanensis phragmitis	MFLUCC 17-0315	MF684863	MF684867	MF684862	MF683624	Karunarathna et al. 2017
Yunnanensis phragmitis	MFLUCC 17-1361	MF684865	MF684864	MF684869	-	Karunarathna et al. 2017

Maximum likelihood analysis was generated by using the CIPRES Science Gateway web server (Miller et al. 2010) and chosen RAxML-HPC BlackBox (8.2.10) (Stamatakis 2014). Maximum parsimony analysis was performed by PAUP v. 4.0b10 (Swofford 2002) with the heuristic search option with 1,000 random sequence additions and tree-bisection reconnection (TBR) as branch-swapping algorithm. All characters were unordered and of equal weight and gaps were regarded as missing data. Maxtrees were set up to 1,000, a zero of maximum branches length was collapsed and all multiple parsimonious trees were saved. Tree length [TL], consistency index [CI], retention index [RI], relative consistency index [RC] and homoplasy index [HI] were determined under different optimality criteria. The robustness was assessed using bootstrap analysis with 1,000 replications (Hillis and Bull 1993). The Kishino-Hasegawa tests were made in order to determine whether trees were significantly different (Kishino and Hasegawa 1989).

Bayesian inference analysis was conducted with MrBayes v. 3.2.2 (Ronquist et al. 2012) and a Bayesian posterior probability (BYPP) was determined by Markov Chain Monte Carlo sampling (MCMC). The Bayesian parameters were set up to “Lset applyto= (all) nst=6 rates=invgamma; prset applyto= (all) statefreqpr=dirichlet (1,1,1,1)”. Six simultaneous Markov chains were set up to 10,000,000 generations and trees were sampled every 100th generation. The programme was automatically terminated when the average standard deviation of split frequencies reached below 0.01 (Maharachchikumbura et al. 2015). The distribution of log-likelihood scores were examined to determine the stationary phase for each search and to decide if extra runs were required to achieve convergence, using Tracer v.1.6 program (Rambaut et al. 2013). The first 10% of generated trees representing the burn-in phase were discarded and the remaining trees were used to calculate posterior probabilities of the majority rule consensus tree.

The tree was made in FigTree v. 1.4.3 (Rambaut 2016) and edited in Adobe Illustrator CS6 (Adobe Systems Inc., United States). The finalised alignment and tree were submitted in TreeBASE, submission ID: 23697 (http://www.treebase.org).

Notes. Ex-type strains are given in bold and the new species in this study is in red. “-” means that the sequence is missing or unavailable.

: Assembling the Fungal Tree of Life; : Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; : Culture Collection of Tabriz University, Tabriz, Iran; : Culture Collection of P.W. Crous; : Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; : J. Kohlmeyer; : Kunming Institute of Botany Culture Collection, Chinese Academy of Sciences, Kunming, China; : Herbarium of Mae Fah Luang University, Chiang Rai, Thailand; : Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; : Tashkent Mycological Herbarium, Institute of Botany and Zoology, Uzbek Academy of Science, Uzbekistan; : Fungus Testing Laboratory of the University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Results

Phylogenetic analyses

In this phylogenetic analysis, we include all representative sequences of genera in and other representative genera and species in and . The final concatenated dataset containing 138 ingroup taxa within the suborder , included 56 currently existing genera in , 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. 1 which is the best scoring tree generated from ML. The phylogenetic trees generated from ML analyses were similar to previous phylogenies including (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).

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 and other representative species in and . The tree is rooted to (CBS 121892), (CBS 141486), (MFLUCC 14-0024) and (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.

clusters in the family 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 is phylogenetically close to the genus and and separated from the remaining genera of the family in a distinct clade with moderate bootstrap support.

Taxonomy

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

Index Fungorum number: IF555713

Facesoffungi number: FoF 05490

Type species.

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. : 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. : 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 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. 1) show that is closely related to and within . There are some significant differences in morphology between these genera and these are summarised in Table 2. Six species are currently accepted in and two species in 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 3. Phylogenetic analyses also clearly differentiate these taxa (Fig. 1). It is the first time that species with -like morphology occurring on bamboo, were found in the . Based on molecular phylogeny, the new genus is introduced in to accommodate a -like taxon.

Table 2.

Morphological comparison of , and .

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

Table 3.

Comparison of DNA sequence data vs and .

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%)

*SSU is not available for

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

Index Fungorum number: IF555714

Facesoffungi number: FoF 05491

Figs 2 , 3

Figure 2.

(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).

Figure 3.

(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 Oliv. (), 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, , on nearly dead stems of Oliv. (), 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, , on nearly dead branches of Oliv. (), 12 September 2017, C.L. Yang and X.L. Xu, YCL201709002 (MFLU 18-1223, paratype).

in reference to Sichuan Province where the specimens were collected.

Associated with stem spot disease on living to nearly dead stems and branches of (). : 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). : 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

has a unique suite of characters that differentiate it from other genera in , 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. is the only genus of 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 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 , , , , , , , and 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 and at least 30 genera are reported within this family. Currently, 11 genera are observed only on : , , , , , , , , , and , all of them being recently established except for , and (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 and 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 species are waiting for exploration and discovery.