Families and genera of diaporthalean fungi associated with canker and dieback of tree hosts

Abstract

In this study we accept 25 families in Diaporthales based on phylogenetic analyses using partial ITS, LSU, rpb2 and tef1-α gene sequences. Four different families associated with canker and dieback of tree hosts are morphologically treated and phylogenetically compared. These include three new families (Diaporthostomataceae, Pseudomelanconidaceae, Synnemasporellaceae), and one new genus, Dendrostoma (Erythrogloeaceae). Dendrostoma is newly described from Malus spectabilis, Osmanthus fragrans and Quercus acutissima having fusoid to cylindrical, bicellular ascospores, with three new species namely D. mali, D. osmanthi and D. quercinum. Diaporthostomataceae is characterised by conical and discrete perithecia with bicellular, fusoid ascospores on branches of Machilus leptophylla. Pseudomelanconidaceae is defined by conidiogenous cells with apical collarets and discreet annellations, and the inconspicuous hyaline conidial sheath when mature on Carya cathayensis, compared to morphologically similar families Melanconidaceae and Juglanconidaceae. Synnemasporellaceae is proposed to accommodate fungi with synnematous conidiomata, with descriptions of S. toxicodendri on Toxicodendron sylvestre and S. aculeans on Rhus copallina.

INTRODUCTION

Diaporthales represents an important order in Sordariomycetes containing taxa that are mainly isolated as endophytes, saprobes or plant pathogens on various hosts. The order is characterised by perithecia with elongate beaks, often forming within stromatic tissues, deliquescent paraphyses and asci that generally deliquesce, become detached from the perithecial wall when mature, and have a characteristic refractive apical annulus (Rossman et al. 2007). Members of diaporthalean fungi are responsible for several diseases causing severe damage in plants with economic importance. The most notorious is chestnut blight caused by Cryphonectria parasitica (Cryphonectriaceae) that devastated American chestnut (Castanea dentata) populations in North America (Anagnostakis 1987, Gryzenhout et al. 2006). Other common diseases include ash anthracnose due to Gnomoniella fraxinii and birch canker caused by Cryptosporella platyphylla (Gnomoniaceae) (Redlin & Stack 1988, Fan et al. 2016a), stem-end rot of citrus fruits infected by Diaporthe citri and walnut canker by Diaporthe rostrata (Diaporthaceae) (Huang et al. 2013, 2015, Fan et al. 2015b, Guarnaccia & Crous 2017), willow and walnut canker disease caused by Cytospora chrysosperma (Cytosporaceae) (Fan et al. 2014, 2015a), birch dieback disease resulting from Melanconis stilbostoma (Melanconidaceae) (Fan et al. 2016b), walnut dieback disease by Juglanconis juglandina and J. oblonga (in Juglanconidaceae) (Voglmayr et al. 2017), foliar diseases of Eucalyptus by Harknessia spp. (Harknessiaceae) (Crous et al. 2012), and foliar, fruit and stem diseases by Coniella spp. (Schizoparmaceae) (Alvarez et al. 2016, Marin-Felix et al. 2017).

The classification of Diaporthales has changed drastically over the past decades because of the plasticity and variability in morphology. The order Diaporthales and Valsales were first introduced by Nannfeldt (1932), based on subfamilies Eu-Diaportheen and Valseen in Diaportheaceae proposed by Von Höhnel (1917). Later, Diaporthaceae and ‘Valsaceae’ (now Cytosporaceae, and referred to as such below) were recognised in the Diaporthales by Von Arx & Müller (1954). Kobayashi (1970) proposed Diaporthaceae (including Valsa = Cytospora) in a wide concept including all taxa accepted in Diaporthales by Barr (1978). Wehmeyer & Hanlin (1975) accepted three families within this order, including non-allantoid spored Gnomoniaceae and Diaporthaceae separated on the presence or absence of a stroma, and Cytosporaceae with allantoid ascospores. Barr (1978) arranged four families (Gnomoniaceae, Melanconidaceae, Pseudovalsaceae and Cytosporaceae) in Diaporthales based on beak position of ascomata and thin or firm ascospore walls without special emphasis on allantoid or non-allantoid ascospores. Families within the Diaporthales have been segregated by several mycologists to utilise various criteria: stromatic tissues, arrangement of ascomata in the stroma or substrate, and ascospore shape, e.g., four families (Cytosporaceae, Endoxylaceae, Gnomoniaceae and Melanconidaceae) by Monod (1983), three families (Cytosporaceae, Melanconidaceae and Phyllachoraceae) by Cannon (1988), while Hawksworth et al. (1995) merged the Cytosporaceae, Gnomoniaceae and Melanconidaceae proposed by Barr (1990) to Cytosporaceae and Melanconidaceae. These changes and confusions suggested that phenotypic characters alone were unable to provide sufficient evidence to resolve phylogenetic and evolutionary patterns among these fungi.

Molecular studies on fungi began in the early 1990s, and since then ribosomal DNA sequence data were accepted as the standard gene loci for fungi (Berbee & Taylor 1992, Schoch et al. 2012). Zhang & Blackwell (2001) recognised three lineages in Diaporthales, while Castlebury et al. (2002) postulated six major lineages, namely the Cryphonectria-Endothia complex, Cytosporaceae s.str., Diaporthaceae s.str., Gnomoniaceae s.str., Melanconidaceae s.str. and the Schizoparme complex. When Rossman et al. (2007) reviewed the Diaporthales, nine families were recognised, i.e., Cryphonectriaceae, Cytosporaceae, Diaporthaceae, Gnomoniaceae, Melanconidaceae, Pseudovalsaceae, Schizoparmeaceae, Sydowiellaceae and Togniniaceae. Kirk et al. (2008) added Melogrammataceae and listed 10 families in this order, whereas Jaklitsch & Voglmayr (2012) placed Melogrammataceae within Xylariales rather than Diaporthales. Subsequently, the Pseudoplagiostomataceae, Harknessiaceae, Macrohilaceae and Tirisporellaceae were also added to the Diaporthales (Cheewangkoon et al. 2010, Crous et al. 2012, 2015, Suetrong et al. 2015). Voglmayr & Jaklitsch (2014) resurrected Stilbosporaceae, while the Togniniaceae and Tirisporellaceae were reallocated to the Togniniales and Tirisporellales (Gramaje et al. 2015, Jones et al. 2015). Later, Lamproconiaceae and Juglanconidaceae were proposed as new families in this order (Norphanphoun et al. 2016, Voglmayr et al. 2017). The recent outline of Diaporthales published by Senanayake et al. (2017) used morphological and phylogenetic evidence to introduce seven new families and accepted a total of 21 families in the order. In spite of these changes, the phylogenetic placement of many genera in the Diaporthales remains unknown, and many families still wait to be elucidated.

During the trips to collect forest pathogens that cause canker or dieback diseases in China, several diaporthalean taxa associated with various disease symptoms were collected in Jiangxi and Zhejiang Provinces, China. Because the higher-level phylogeny of many genera within the Diaporthales remains largely unresolved, this project was initiated to address this issue. In this paper, we propose three new families and one new genus as well as several new species.

MATERIALS AND METHODS

Isolation

Fresh specimens of diaporthalean fungi were collected from infected branches of seven hosts during collection trips in China (Table 1). A total of 20 isolates were established by removing a mucoid spore mass from ascomata or conidiomata, spreading the suspension on the surface of 1.8 % potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating conidia/ascospores were removed and plated onto fresh PDA plates. Specimens and isolates were deposited in the Key Laboratory for Silviculture and Conservation of the Ministry of Education in the Beijing Forestry University (BJFU) and the working Collection of X.L. Fan (CF) housed at the BJFU. Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Table 1.

Details of the strains included for molecular study.

Species	Culture	Location	Host	GenBank accession numbers
				ITS	LSU	rpb2	tef1-α
Apiosporopsis carpinea	CBS 771.79	Switzerland	Carpinus betulus	NA	AF277130	NA	NA
Apiosporopsis sp.	Masuya 11Af2-1	Japan	Alnus firma	NA	AB669034	NA	NA
Apoharknessia insueta	CBS 111377	Brazil	Eucalyptus pellita	JQ706083	AY720814	NA	NA
	CBS 114575	Colombia	Eucalyptus sp.	NA	AY720813	NA	NA
Asterosporium asterospermum	MFLU 15-3555	Italy	Fagus sylvatica	NA	MF190062	MF377615	NA
	CBS 112404	Italy	Fagus sylvatica	NA	AB553745	NA	NA
	KT2138	Japan	Fagus crenata	NA	AB553744	NA	NA
Auratiopycnidiella tristaniopsidis	CBS 132180 = CPC 16371	Australia	Tristaniopsis laurina	JQ685516	JQ685522	NA	NA
Cainiella johansonii	Kruys 731	Sweden	Dryas octopetala	NA	JF701920	NA	NA
Chapeckia nigrospora	AR 3809	USA	Betula sp.	JF681957	EU683068	NA	NA
Chiangraiomyces bauhiniae	MFLUCC 17-1669	Thailand	Bauhinia sp.	MF190118	MF190064	MF377604	NA
	MFLUCC 17-1670	Thailand	Bauhinia sp.	MF190119	MF190065	MF377603	NA
Chrysocrypta corymbiae	CBS 132528	Australia	Corymbia sp.	JX069867	JX069851	NA	NA
Coniella diplodiella	CBS 111858 = CPC 3708	France	Vitis vinifera	AY339323	AY339284	KX833423	KX833603
Coniella koreana	CBS 143.97	Korea	NA	KX833584	AF408378	KX833490	KX833684
Coniella musaiensis var. hibisci	AR 3534 = CBS 109757	South Africa	Hibiscus sp.	KX833589	AF408337	NA	KX833689
Coniella straminea	CBS 149.22 = CPC 3932	USA	Fragaria sp.	AY339348	AF362569	KX833506	KX833704
Coniella wangiensis	CBS 132530 = CPC 19397	Australia	Eucalyptus sp.	JX069873	JX069857	KX833509	KX833705
Coryneum depressum	AR 3897	Austria	Quercus cerris	NA	EU683074	NA	NA
Coryneum modonium	AR 3558	Austria	Castanea sativa	NA	EU683073	NA	NA
Coryneum umbonatum	AR 3541	Austria	Quercus cerris	NA	EU683072	NA	NA
	MFLUCC 15-1110	Italy	Quercus sp.	MF190121	MF190067	MF377610	NA
	MFLUCC 13-0658	Italy	Quercus sp.	MF190120	MF190066	MF377609	NA
Cryphonectria macrospora	AR 3444 = CBS 109764	Russia	Quercus mongolica	EU199182	AF408340	EU220029	NA
Cryphonectria nitschkei	AR 3433 = CBS 109776	Russia	Quercus mongolica	DQ120761	AF408341	NA	NA
Cryphonectria parasitica	ATCC 38755	USA	Castanea dentata	AY141856	EU199123	DQ862017	EU222014
Cryptodiaporthe aesculi	CBS 109765 = AFTOL-ID 1238	Austria	Aesculus hippocastanum	DQ323530	AF408342	EU199138	GU354004
	AR3640 = CBS 121905	USA	Aesculus hippocastanum	EU254994	EU255164	EU219269	DQ313558
	LCM 447.01	Germany	Aesculus hippocastanum	GU367076	NA	GU367110	GU354002
Cryptosporella betulae	AR 3524 = CBS 109763	Austria	Betula pendula	EU199180	AF408375	EU199139	EU221884
Cryptosporella hypodermia	AR 3552	Austria	Ulmus minor	EU199181	AF408346	EU199140	NA
Cryptosporella suffusa	AR 3496 = CBS 109750	Austria	Alnus incana	EU199207	AF408376	EU199163	EU221945
Cytospora cenisia	AR 3522 = CBS 109752	Austria	Juniperus communis	NA	AF408385	NA	NA
Cytospora chrysosperma	CFCC 89600	China	Sophora japonica	KR045623	KR045623	KU710951	KU710915
Cytospora elaeagni	CFCC 89633	China	Elaeagnus angustifolia	KF765677	KF765693	KU710956	KU710919
Cytospora leucostoma	CFCC 50468	China	Betula platyphylla	KT732949	KT732968	NA	NA
Cytospora nivea	AR 3512	Austria	Salix purpurea	NA	AF408367	NA	NA
Cytospora sacculus	AR 3416= CBS 109756	Russia	Quercus mongolica	NA	AF408386	NA	NA
	AR 3426 = CBS 109777	Austria	Quercus robur	NA	AF408387	NA	NA
Dendrostoma mali	CFCC 52102*	China	Malus spectabilis	MG682072	MG682012	MG682032	MG682052
Dendrostoma osmanthi	CFCC 52106*	China	Osmanthus fragrans	MG682073	MG682013	MG682033	MG682053
	CFCC 52107*	China	Osmanthus fragrans	MG682074	MG682014	MG682034	MG682054
	CFCC 52108*	China	Osmanthus fragrans	MG682075	MG682015	MG682035	MG682055
	CFCC 52109*	China	Osmanthus fragrans	MG682076	MG682016	MG682036	MG682056
Dendrostoma quercinum	CFCC 52103*	China	Quercus acutissima	MG682077	MG682017	MG682037	MG682057
	CFCC 52104*	China	Quercus acutissima	MG682078	MG682018	MG682038	MG682058
	CFCC 52105*	China	Quercus acutissima	MG682079	MG682019	MG682039	MG682059
Diaporthe decedens	AR 3459 = CBS 109772	Austria	Corylus avellana	KC343059	AF408348	NA	NA
Diaporthe detrusa	AR 3424 = CBS 109770	Austria	Berberis vulgaris	KC343061	AF408349	NA	KC343787
Diaporthe eres	AR 3538 = CBS 109767	Austria	Acer campestre	KC343075	AF408350	NA	KC343801
Diaporthella corylina	CBS 121124	China	Corylus sp.	KC343004	NA	NA	NA
Diaporthella sp.	CN 5	Italy	Corylus avellana	KP205483	NA	NA	NA
	CN13	Italy	Corylus avellana	KP205484	NA	NA	NA
Diaporthosporella cercidicola	CFCC 51994	China	Cercis chinensis	KY852492	KY852515	NA	NA
	CFCC 51995	China	Cercis chinensis	KY852493	KY852516	NA	NA
	CFCC 51996	China	Cercis chinensis	KY852494	KY852517	NA	NA
Diaporthostoma machili	CFCC 52100*	China	Machilus leptophylla	MG682080	MG682020	MG682040	MG682060
	CFCC 52101*	China	Machilus leptophylla	MG682081	MG682021	MG682041	MG682061
Disculoides eucalypti	CPC 17650	Australia	Eucalyptus sp.	JQ685517	JQ685523	NA	NA
Disculoides eucalyptorum	CBS 132184 = CPC 17648	Australia	Eucalyptus viminalis	NR120090	JQ685524	NA	NA
Ditopella ditopa	AR 3423 = CBS 109748	Austria	Alnus glutinosa	EU199187	EU199126	EU199145	NA
Erythrogloeum hymenaeae	CPC 18819	Brazil	Hymenaea courbaril	JQ685519	JQ685525	NA	NA
Gnomonia gnomon	CBS 199.53	Italy	Corylus avellana	AY818956	AF408361	EU219295	EU221885
Harknessia eucalypti	CBS 342.97	Australia	Eucalyptus regnans	AY720745	AF408363	NA	NA
Harknessia molokaiensis	AR 3578 = CBS 109779	USA	Eucalyptus robusta	NA	AF408390	NA	NA
Hercospora tiliae	AR 3526	Austria	Tilia tomentosa	NA	AF408365	NA	NA
Hyaliappendispora galii	MFLUCC 16-1208	Italy	Galium sp.	MF190149	MF190095	NA	NA
Juglanconis appendiculata	D96	Austria	Juglans nigra	KY427139	KY427139	KY427189	KY427208
Juglanconis juglandina	ME23	Austria	Juglans nigra	KY427150	KY427150	KY427200	KY427219
Juglanconis oblonga	ME14	USA	Juglans cinerea	KY427151	KY427151	KY427201	KY427220
Juglanconis pterocaryae	ME20	Japan	Pterocarya rhoifolia	KY427155	KY427155	KY427205	KY427224
Lamproconium desmazieri	MFLUCC 14-1047	Russia	Tilia cordata	KX430132	KX430133	NA	MF377592
	MFLUCC 15-0870	Russia	Tilia tomentosa	KX430134	KX430135	MF377605	MF377591
Lasmenia sp.	CBS 124123	Puerto Rico	Nephelium lappaceum	GU797406	JF838338	NA	NA
	CBS 124124	Puerto Rico	Nephelium lappaceum	JF838336	JF838341	NA	NA
Luteocirrhus shearii	CBS 130776	Australia	Banksia baxteri	NR120254	NG042770	NA	NA
Macrohilum eucalypti	CPC 10945	New Zealand	Eucalyptus sp.	DQ195781	DQ195793	NA	NA
	CPC 19421	Australia	Eucalyptus piperita	KR873244	KR873275	NA	NA
Melanconiella ellisii	BPI 878343	USA	Carpinus caroliniana	JQ926271	JQ926271	JQ926339	JQ926406
Melanconiella hyperopta	AR 3832 = CBS 131492	Austria	Carpinus betulus	JQ926278	JQ926278	NA	NA
Melanconiella spodiaea	MSH	Austria	Carpinus betulus	JQ926298	JQ926298	JQ926364	JQ926431
Melanconis alni	AR 3748	Austria	Alnus viridis	EU199195	EU199130	EU199153	NA
Melanconis betulae	CFCC 50471	China	Betula albosinensis	KT732952	KT732971	KT732984	KT733001
Melanconis itoana	CFCC 50474	China	Betula albosinensis	KT732955	KT732974	KT732987	KT733004
Melanconis marginalis	AR 3442 = CBS 109744	Canada	Alnus rubra	EU199197	AF408373	EU219301	EU221991
Melanconis stilbostoma	CFCC 50475	China	Betula platyphylla	KT732956	KT732975	KT732988	KT733005
Nakataea oryzae	CBS 243.76	NA	NA	KM484861	DQ341498	NA	NA
Ophiodiaporthe cyatheae	YMJ1364	China	Cyathea lepifera	JX570889	JX570891	JX570893	NA
Pachytrype princeps	Rogers S	USA	NA	NA	FJ532382	NA	NA
Pachytrype rimosa	FF1066	Costa Rica	NA	NA	FJ532381	NA	NA
Paradiaporthe artemisiae	MFLUCC 14-0850	Italy	Artemisia sp.	MF190155	MF190100	NA	NA
	MFLUCC 17-1663	Italy	Artemisia sp.	MF190156	MF190101	NA	NA
Phaeoappendispora thailandensis	MFLUCC 13-0161	Thailand	Quercus sp.	MF190157	MF190102	MF377613	NA
Phaeodiaporthe appendiculata	CBS 123821 = D77	Austria	Acer campestre	KF570156	KF570156	NA	NA
	CBS 123809 = D76	Austria	Acer campestre	KF570155	KF570155	NA	NA
Phragmoporthe conformis	AR 3632 = CBS 109783	Canada	Alnus rubra	DQ323527	AF408377	NA	NA
Plagiostoma euphorbiae	CBS 340.78	Netherlands	Euphorbia palustris	EU199198	AF408382	DQ368643	NA
Plagiostoma salicellum	AR 3455 = CBS 109775	Austria	Salix sp.	DQ323529	AF408345	EU199141	EU221916
Prosopidicola mexicana	CBS 113530	USA	Prosopis glandulosa	AY720710	NA	NA	NA
	CBS 113529	USA	Prosopis glandulosa	AY720709	KX228354	NA	NA
Pseudomelanconis caryae	CFCC 52110*	China	Carya cathayensis	MG682082	MG682022	MG682042	MG682062
	CFCC 52111*	China	Carya cathayensis	MG682083	MG682023	MG682043	MG682063
	CFCC 52112*	China	Carya cathayensis	MG682084	MG682024	MG682044	MG682064
	CFCC 52113*	China	Carya cathayensis	MG682085	MG682025	MG682045	MG682065
Pseudoplagiostoma eucalypti	CBS 124807	Venezuela	Eucalyptus urophylla	GU973512	GU973606	NA	NA
	CBS 116382	Thailand	Eucalyptus camaldulensis	GU973514	GU973608	NA	NA
Pseudoplagiostoma oldii	CBS 115722	Australia	Eucalyptus camaldulensis	GU973535	GU973610	NA	NA
Pseudoplagiostoma variabile	CBS 113067	Uruguay	Eucalyptus globulus	GU973536	GU973611	NA	NA
Pyricularia grisea	Ina168	NA	NA	AB026819	AB026819	NA	NA
Rossmania ukurunduensis	AR 3484	Russia	Acer ukurunduense	NA	EU683075	NA	NA
Sillia ferruginea	AR 3440 = CBS 126567	Austria	Corylus avellana	JF681959	EU683076	NA	NA
Stegonsporium protopyriforme	CBS 117041	Austria	Acer pseudoplatanus	NR126119	EU039992	NA	NA
Stegonsporium pyriforme	CBS 124487	UK	Acer heldreichii	KF570160	KF570160	KF570190	NA
Stilbospora macrosperma	CBS 121883	Austria	Carpinus betulus	JX517290	JX517299	KF570196	NA
	CBS 121695	Netherlands	Carpinus betulus	JX517288	JX517297	NA	NA
Sydowiella depressula	CBS 813.79	Switzerland	Rubus sp.	NA	EU683077	NA	NA
Sydowiella fenestrans	AR 3777 = CBS 125530	Russia	Chamerion angustifolium	JF681956	EU683078	NA	NA
Synnemasporella aculeans	CFCC 52094*	China	Rhus chinensis	MG682086	MG682026	MG682046	MG682066
	CFCC 52095*	China	Rhus chinensis	MG682087	MG682027	MG682047	MG682067
	CFCC 52096*	China	Rhus chinensis	MG682088	MG682028	MG682048	MG682068
	AR 3878 = CBS 126566	USA	Rhus glabra	NA	EU255134	NA	NA
Synnemasporella toxicodendri	CFCC 52097*	China	Toxicodendron sylvestre	MG682089	MG682029	MG682049	MG682069
	CFCC 52098*	China	Toxicodendron sylvestre	MG682090	MG682030	MG682050	MG682070
	CFCC 52099*	China	Toxicodendron sylvestre	MG682091	MG682031	MG682051	MG682071

Note: CBS: Westerdijk Fungal Biodiversity Institute (CBS-KNAW Fungal Biodiversity Centre), Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Center, Beijing, China; CPC: Culture collection of Pedro Crous, The Netherlands; MFLU: Mae Fah Luang University herbarium, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Thailand; NA: not applicable. All the new isolates used in this study are marked by an asterisk (*) and the strains from generic type species are in bold.

Morphology

Species identification was based on morphological features of the ascomata or conidiomata produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Cross-sections were prepared by hand using a double-edge blade under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci and 30 conidia/ascospores were measured to calculate the mean size and standard deviation (SD). Microscopic photographs were captured with a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software NIS-Elements D Package v. 3.00. Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Colony diameters were measured, and the colony colours described after 3 wk according to the colour charts of Rayner (1970).

DNA extraction, amplification and sequencing

Genomic DNA was extracted using a modified CTAB method, with fungal mycelium harvested from PDA plates with cellophane (Doyle & Doyle 1990). The ITS region was amplified with the primers ITS1 and ITS4 (White et al. 1990), the LSU region with the primers LR0R and LR5 (Vilgalys & Hester 1990), the rpb2 region with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999), and the tef1-α gene with the primers EF1-728F and EF1-986R (Carbone & Kohn 1999). The PCR mixture for all regions consisted of 1 μL genomic DNA, 3 mM MgCl₂, 20 μM of each dNTP, 0.2 μM of each primer and 0.25 U BIOTAQ DNA polymerase (Bioline). Conditions for PCR of ITS and LSU genes constituted an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 45 s at 51 °C and 1 min at 72 °C, and a final denaturation step of 8 min at 72 °C, while the tef1-α gene was performed using an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 45 s at 56 °C and 1 min at 72 °C, and a final denaturation step of 8 min at 72 °C. For the rpb2 amplification, conditions consisted of five cycles of 45 s at 95 °C, 45 s at 56 °C and 2 min at 72 °C, then five cycles with a 53 °C annealing temperature and 30 cycles with a 50 °C annealing temperature. The DNA sequencing was performed using an ABI PRISM^® 3730XL DNA Analyzer with BigDye^® Terminater Kit v. 3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Molecular data analyses

DNA sequences generated by each primer combination were used to obtain consensus sequences using SeqMan v. 7.1.0 in the DNASTAR Lasergene Core Suite software (DNASTAR Inc., Madison, WI, USA). Reference sequences were selected based on ex-type or ex-epitype sequences available from relevant published literature (Rossman et al. 2007, Cheewangkoon et al. 2010, Crous et al. 2012, 2015, Suetrong et al. 2015, Norphanphoun et al. 2016, Hongsanan et al. 2017, Senanayake et al. 2017, Voglmayr et al. 2017) (Table 1). All sequences were aligned using MAFFT v. 6 (Katoh & Toh 2010) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), MrBayes v. 3.1.2 for Bayesian Inference (BI) analysis (Ronquist & Huelsenbeck 2003), and PhyML v. 7.2.8 for Maximum Likelihood (ML) analysis (Guindon et al. 2010). The first analyses were performed on the combined multi-gene dataset (ITS, LSU, rpb2, tef1-α) to compare isolates of Diaporthales species to ex-type sequence data from recent studies (Table 1).

A partition homogeneity test (PHT) with heuristic search and 1 000 replicates was performed using PAUP v. 4.0b10 to test the discrepancy among the ITS, LSU, rpb2 and tef1-α sequence dataset in reconstructing phylogenetic trees. Maximum parsimony (MP) analysis was run using a heuristic search option of 1 000 search replicates with random-additions of sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5 000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Maximum likelihood (ML) analysis was performed with a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated with a bootstrapping (BS) method of 1 000 replicates (Hillis & Bull 1993).

MrModeltest v. 2.3 was used to estimate the best nucleotide substitution model settings for each gene (Posada & Crandall 1998). Bayesian inference (BI) was performed based on the DNA dataset from the results of the MrModeltest, using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Two MCMC chains were run from random trees for 1 000 M generations and stopped when average standard deviation of split frequencies fell below 0.01. Trees were saved each 1 000 generations. The first 25 % of trees were discarded as the burn-in phase of each analysis, and the posterior probabilities (BPP) were calculated from the remaining trees (Rannala & Yang 1996). Nakataea oryzae (CBS 243.76) and Pyricularia grisea (Ina168) were selected as outgroups in all analyses (Fan et al. 2016b). Phylograms were shown using FigTree v. 1.3.1 (Rambaut & Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (www.treebase.org; accession number: S22175).

RESULTS

Molecular phylogenetic analyses

The alignment based on the sequence dataset (ITS, LSU, rpb2 and tef1-α) included 122 ingroup taxa, comprising 3 261 characters in the aligned matrix. Of these, 1 562 characters were constant, 184 variable characters were parsimony-uninformative and 1 515 characters were parsimony informative. The MP analysis resulted in 119 equally most parsimonious trees (TL = 8 082, CI = 0.385, RI = 0.761, RC = 0.293) and the first tree is shown in Fig. 1. For BI analyses, the general time reversible model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (GTR + I + G) was determined to be the best for the ITS, LSU and tef1-α loci by MrModeltest, while the most appropriate model for the rpb2 locus was the Tamura-Nei model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (TrN + I + G). The MP and ML bootstrap support values above 70 % are shown at the first and second position, respectively. Branches with significant Bayesian posterior probability (≥ 0.95) in Bayesian analyses were thickened in the phylogenetic tree. The phylogram based on four genes indicated 24 known lineages, representing 22 known families and two unknown taxa lacking typification studies, namely Diaporthella and Phaeoappendispora. Four new lineages belonging to the Diaporthales, distinct from all known taxa, are herein described as three new families and a new genus in Erythrogloeaceae (Fig. 1).

Fig. 1.

Phylogram of Diaporthales resulting from MP analysis based on combined ITS, LSU, rpb2 and tef1-α. MP and ML bootstrap support values above 70 % are shown at the first and second position. Thickened branches represent posterior probabilities above 0.95 from BI. Type species are in bold. Strains obtained in the current study are in blue. — Scale bar = 200 changes.

Taxonomy

Diaporthostomataceae X.L. Fan & C.M. Tian, fam. nov. — MycoBank MB823983

Etymology. Name derived from the type genus, Diaporthostoma.

Type genus. Diaporthostoma.

Sexual morph: Pseudostromata immersed in host bark and slightly erumpent from host tissues. Ectostromatic disc ovoid to ellipsoid, yellowish to dark grey. Central column beneath the disc more or less conical. Stromatic zones lacking. Perithecia conical, surrounding the ectostromatic disc. Ostioles single, dark grey to black. Paraphyses deliquescent. Asci 8-spored, with an apical ring. Ascospores hyaline, fusoid, bicellular. Asexual morph: not observed.

Notes — The current phylogenetic analyses placed the new family Diaporthostomataceae in a highly supported clade (MP/ML/BI = 100/100/1) closely related to Diaporthosporellaceae. Diaporthostomataceae is morphologically distinct from Diaporthosporellaceae (Yang et al. 2018) by discrete perithecia and fusoid, straight to curved ascospores with a median septum.

Diaporthostoma X.L. Fan & C.M. Tian, gen. nov. — MycoBank MB823984

Etymology. Name derived from the morphological similarity with the genus Diaporthe.

Type species. Diaporthostoma machili X.L. Fan & C.M. Tian.

Sexual morph: Pseudostromata immersed in host bark, slightly erumpent from the bark surface. Ectostromatic disc yellowish to dark grey, nearly flat, ovoid to ellipsoid. Central column beneath the disc more or less conical. Stromatic zones lacking. Perithecia conical, surrounding the ectostromatic disc, regularly scattered. Ostioles single, dark grey to black. Paraphyses deliquescent. Asci oblong to cylindrical-clavate, 8-spored, 2–3-seriate, with a more or less distinct apical ring. Ascospores hyaline, smooth, fusoid, multiguttulate, straight to curved, bicellular, with an inconspicuous median septum. Asexual morph: not observed.

Diaporthostoma machili X.L. Fan & C.M. Tian, sp. nov. — MycoBank MB823985; Fig. 2

Fig. 2.

Morphology of Diaporthostoma machili from Machilus leptophylla. a, b. Habit of pseudostromata on branches; c. transverse section through pseudostroma; d. longitudinal section through pseudostroma; e, f. asci and ascospores; g. ascospores. — Scale bars: a = 1 mm; b–d = 100 μm; e–g = 10 μm.

Etymology. Name derived from the host genus, Machilus.

Sexual morph: Pseudostromata immersed in host bark, slightly erumpent, 400–700 μm diam. Ectostromatic disc yellowish to dark grey, nearly flat, ovoid to ellipsoid, 120–140 μm diam. Central column beneath the disc more or less conical. Stromatic zones lacking. Perithecia conical, surrounding the ectostromatic disc, regularly scattered, 380–420 μm diam. Ostioles single, dark grey to black, 65–85 μm diam. Paraphyses deliquescent. Asci oblong to cylindrical-clavate, 8-spored, 2–3-seriate, with a more or less distinct apical ring, (26–)30–38(–40) × 6–8 μm (x̄ = 33.5 ± 1.5 × 7 ± 0.5 μm, n = 20). Ascospores hyaline, smooth, fusoid, multiguttulate, straight to slight curved, bicellular, with a median septum, not constricted at the septum, 11–14 × 2–2.5 (x̄ = 12 ± 1.1 × 2.3 ± 0.2 μm, n = 30) μm. Asexual morph: not observed.

On PDA, cultures are initially white, becoming dark vinaceous after 3 wk. The colonies are flat with an irregular edge, producing white, sparse aerial mycelium; texture initially uniform, producing a dark brick-coloured circular ring on the margin after 3 d; sterile.

Host & Distribution — On Machilus leptophylla in China.

Materials examined (all on twigs and branches of Machilus leptophylla). China, Zhejiang Province, Hangzhou City, Linan, Tianmu Mountain, N30°19′18.21″ E119°26′18.21″, 354 m asl, 20 Apr. 2017, Q. Yang & Z. Du (holo-type CF 2017475; living ex-type culture CFCC 52100); Zhejiang Province, Hangzhou City, Linan, Tianmu Mountain, N30°19′17.33″ E119°26′15.60″, 350 m asl, 20 Apr. 2017, Q. Yang & Z. Du (CF 2017479; living culture CFCC 52101).

Notes — Diaporthostoma machili is the type species of Diaporthostoma, and is thus far only known to occur on Machilus leptophylla. Morphologically, it is characterised by the scattered, conical perithecia and fusoid, straight to curved ascospores with a median septum, which differs with other species in Diaporthales.

Erythrogloeaceae Senan. et al., Stud. Mycol. 86: 258. 2017. emend.

Type genus. Erythrogloeum Petr.

Sexual morph: Pseudostromata small to large, erumpent, consisting of an inconspicuous ectostromatic disc generally with orange colour, semi-immersed to superficial, causing a more or less pustulate bark surface. Central column beneath the disc more or less conical. Stromatic zones lacking. Perithecia umber to fuscous black, covered with orange to umber pseudostromatic tissue, surrounding the ectostromatic disc, with small to long ostioles that emerge within the ectostromatic disc. Paraphyses deliquescent. Asci 8-spored, with an apical ring, grouped at the base with other asci, becoming detached from the perithecial wall. Ascospores hyaline, fusoid to cylindrical, bicellular. Asexual morph: foliicolous, associated with leaf spots. Conidiomata epiphyllous, subepidermal, sometimes eustromatic, acervular or subglobose, brown to black or yellow-orange, amphigenous, opening by irregular rupture, wall of 2–6 layers of orange-brown textura angularis, exuding slimy orange masses of conidia. Conidiophores hyaline to amber. Conidiogenous cells lining the inner cavity of conidioma, hyaline to olivaceous, smooth, subcylindrical to ampulliform, tapering to a long, thin neck, at times apical part elongated into a long neck, proliferating several times percurrently near apex, with flaring collarettes, or apex truncate, with minute periclinal thickening. Conidia hyaline to olivaceous, smooth, guttulate or not, thin-walled, ellipsoid, fusoid, ovoid to somewhat obclavate, straight to curved, apex subobtuse, obtusely rounded, base truncate, with prominent marginal frill, or dimorphic, intermixed in same conidiomata. Macroconidia broadly ellipsoid to obovoid, hyaline, smooth, granular to guttulate, thick-walled, apex obtuse, base flattened. Microconidia hyaline, smooth, guttulate, fusoid-ellipsoid, acutely rounded at apex, truncate at base (emended from Senanayake et al. 2017).

Notes — The family Erythrogloeaceae was recently introduced by Senanayake et al. (2017) to accommodate Chrysocrypta, Disculoides and Erythrogloeum species having epiphyllous acervuli, and subcylindrical to ampulliform conidiogenous cells. These authors did not report any sexual morph associated with these genera. During our investigation, phylogenetic inferences using DNA sequences from some materials with a sexual morph placed these samples in a highly supported clade (MP/ML/BI = 100/100/1) in the Erythrogloeaceae. The family Erythrogloeaceae is emended here to include the morphological features of the new sexual morphs observed during our study. These fungi have typical diaporthalean perithecia with clavate asci, and fusoid to cylindrical, bicellular ascospores.

Dendrostoma X.L. Fan & C.M. Tian, gen. nov. — MycoBank MB823986

Etymology. Name derived from pseudostromata emerging from woody host tissue.

Type species. Dendrostoma mali X.L. Fan & C.M. Tian.

Sexual morph: Pseudostromata small to large, distinct, circular, erumpent, consisting of an inconspicuous, usually orange ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface. Ectostromatic disc flat or concave, orange, surrounded by bark flaps. Central column beneath the disc more or less conical. Stromatic zones lacking. Perithecia conspicuous, umber to fuscous black, embedded in orange to umber pseudostromatic tissue, regularly scattered, surrounding the ectostromatic disc, with small to long ostioles that emerge within the ectostromatic disc. Ostioles flat in the disc or sometimes slightly projecting, cylindrical, sometimes obscuring the disc, covered by an orange, umber to fuscous black crust. Paraphyses deliquescent. Asci fusoid, 8-spored, 2–3-seriate, with an apical ring, becoming detached from the perithecial wall. Ascospores hyaline, fusoid to cylindrical, symmetrical to asymmetrical, straight to curved, bicellular, with a median septum, constricted at the septum, smooth, multiguttulate. Asexual morph: observed on PDA. Conidiomata pycnidial, hemispherical, somewhat erumpent, coated with aerial mycelium. Conidiophores hyaline to amber. Conidiogenous cells enteroblastic, polyphialidic, hyaline, verruculose, ampulliform to doliiform. Conidia hyaline, aseptate, ovoid to ellipsoid, or fusoid.

Notes — The current phylogenetic analyses placed the new genus Dendrostoma in a highly supported clade (MP/ML/BI = 100/100/1) closely related to other genera in Erythrogloeaceae (Senanayake et al. 2017). Dendrostoma is described based on the typical diaporthalean perithecia with clavate asci and fusoid to cylindrical, bicellular ascospores. This study describes one genus and four species from China, and the host association appears to provide an important character for reliable identification. However, further collections are needed to confirm the host ranges and geographical distributions.

Dendrostoma mali X.L. Fan & C.M. Tian, sp. nov. — MycoBank MB823987; Fig. 3

Fig. 3.

Morphology of Dendrostoma mali from Malus spectabilis. a–c. Habit of pseudostromata on branches; d. transverse section of perithecia; e. longitudinal section through perithecia; f–i. asci and ascospores; j, k. ascospores; l, m. conidiomata on PDA; n. conidiophores and conidiogenous cells; o. conidia. — Scale bars: a = 1 mm; b–e, l, m = 500 μm; f–k, n, o = 10 μm.

Etymology. Name derived from the host genus, Malus.

Pseudostromata erumpent, consisting of an inconspicuous orange ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface, 1300–2100 μm diam. Ectostromatic disc flat or concave, orange, or brown to black, sometimes concealed by ostioles, surrounded by bark flaps, 350–800 μm diam. Central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, umber to fuscous black, regularly scattered, surrounding the ectostromatic disc, 300–500 μm diam. Ostioles 2–6 per disc, flat in the disc or sometimes slightly projecting, cylindrical, covered by an orange, umber to fuscous black crust, 70–100 μm diam. Paraphyses deliquescent. Asci fusoid, 8-spored, biseriate, with an apical ring, 40–60(–65) × 7–10(–11) μm (x̄ = 47 ± 5.3 × 8.5 ± 1.1 μm, n = 10). Ascospores hyaline, fusoid to cylindrical, smooth, multiguttulate, often containing two guttules per cell, symmetrical to asymmetrical, straight to slightly curved, bicellular, with a median septum distinctly constricted, 12–14 × 3–4 μm (x̄ = 13 ± 1 × 3.4 ± 0.3 μm, n = 30). Conidiomata pycnidial, hemispherical, somewhat erumpent, coated with white aerial mycelium, 1200–2500 μm, conidial masses extruding from the ostioles. Conidiophores hyaline, occasionally amber at the base, aseptate. Conidiogenous cells enteroblastic, polyphialidic, with 1–2 integrated loci, hyaline, verruculose, ampulliform to doliiform. Conidia hyaline, aseptate, ovoid to ellipsoid, apex obtuse, 3–4.5 × 2–2.5 μm (x̄ = 3.6 ± 0.5 × 2.2 ± 0.2 μm, n = 30).

On PDA, cultures are white. Colonies are flat with regular edge; texture initially uniform, producing concentric circles after 3 wk with sparse conidiomata irregularly distributed on the agar surface.

Host & Distribution — On Malus spectabilis in China.

Material examined. China, Zhejiang Province, Hangzhou City, Linan, Tianmu Mountain, N30°19′02.62″ E119°26′34.33″, 320 m asl, on twigs and branches of Malus spectabilis, 21 Apr. 2017, Q. Yang & Z. Du (holotype CF 2017445; living ex-type culture CFCC 52102).

Notes — Dendrostoma mali is the type species of Dendrostoma, and presently is only known on Malus spectabilis. It can be distinguished from other known Dendrostoma spp. by the fusoid to cylindrical ascospores, and the ovoid to ellipsoid conidia with obtuse apices. Dendrostoma mali is assumed to be host specific which needs to be confirmed by additional studies.

Dendrostoma quercinum X.L. Fan & C.M. Tian, sp. nov. — MycoBank MB823989; Fig. 4

Fig. 4.

Morphology of Dendrostoma quercina from Quercus acutissima. a, b. Habit of pseudostromata on branches; c. transverse section of pseudostroma; d. longitudinal section through pseudostroma; e–g. asci and ascospores; h, i. ascospores; j, k. conidiomata on PDA; l. conidiophores and conidiogenous cells; m. conidia. — Scale bars: a = 1 mm; b–d, j, k = 500 μm; e–i, l, m = 10 μm.

Etymology. Name derived from the host genus, Quercus.

Pseudostromata erumpent, consisting of an inconspicuous orange ectostromatic disc, semi-immersed to erumpent, causing a pustulate bark surface, 800–1500 μm diam. Ectostromatic disc flat or concave, orange, or brown to black, sometimes concealed by ostioles, surrounded by bark flaps, 500–1100 μm diam. Central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, umber to fuscous black, regularly scattered, surrounding the ectostromatic disc, 250–500 μm. Ostioles 3–8 per disc, flat in disc or sometimes slightly projecting, cylindrical, covered by an orange, umber to fuscous black crust, 100–150 μm diam. Paraphyses deliquescent. Asci fusoid, 8-spored, 2–3-seriate, with an apical ring, 53–70 × 9.5–10 μm (x̄ = 60.5 ± 4 × 9.8 ± 0.3 μm, n = 10). Ascospores hyaline, fusoid to cylindrical, smooth, multi-guttulate, often containing two guttules per cell, symmetrical to asymmetrical, straight to slightly curved, bicellular, with a median septum distinctly constricted, 16–22(–24) × 3–4 μm (x̄ = 20 ± 1.7 × 3.5 ± 0.4 μm, n = 30). Conidiomata pycnidial, hemispherical, somewhat erumpent, covered with cinnamon aerial mycelium, 900–2200 μm diam; conidial masses extruding from ostioles. Conidiophores hyaline, occasionally amber at the base, aseptate. Conidiogenous cells enteroblastic, polyphialidic, with 1–2 integrated loci, hyaline, verruculose, ampulliform to doliiform. Conidia hyaline, aseptate, fusoid, acute at each end, 10.5–14 × 2.5(–3) μm (x̄ = 12 ± 1 × 2.5 ± 0.2 μm, n = 30).

On PDA, cultures are white, becoming hazel in the centre after 2 wk. The colonies are flat with regular edge; texture initially uniform, becoming dense in the centre after 2 wk, producing circular conidiomata at the margin of the compact centre.

Host & Distribution — On Quercus acutissima in China.

Materials examined (all on twigs and branches of Quercus acutissima). China, Zhejiang Province, Hangzhou City, Hangzhou Botanical Garden, N30°15′13.25″ E120°06′56.33″, 49 m asl, 17 Apr. 2017, Q. Yang & Z. Du (holotype CF 2017461; living ex-type culture CFCC 52103); Zhejiang Province, Hangzhou City, Hangzhou Botanical Garden, N30°15′12.52″, E120°06′57.02″, 50 m asl, 17 Apr. 2017, Q. Yang & Z. Du (CF 2017462; living culture CFCC 52104); Hangzhou City, Hangzhou Botanical Garden, N30°15′13.77″ E120°06′59.93″, 46 m asl, 17 Apr. 2017, Q. Yang & Z. Du (CF 2017470; living culture CFCC 52105).

Notes — Dendrostoma quercinum can be distinguished from D. mali and D. osmanthi by its larger ascospores (16–24 × 3–4 μm), and DNA sequence data.

Dendrostoma osmanthi X.L. Fan & C.M. Tian, sp. nov. — MycoBank MB823990; Fig. 5

Fig. 5.

Morphology of Dendrostoma osmanthi from Osmanthus fragrans. a, b. Habit of pseudostromata on branches; c. transverse section of pseudostroma; d. longitudinal section through pseudostroma; e–g. asci and ascospores; h. ascospores; i. conidiomata on PDA; j. conidiophores and conidiogenous cells; k. conidia. — Scale bars: a = 1 mm; b–d, i = 500 μm; e–h, j, k = 10 μm.

Etymology. Name derived from the host genus, Osmanthus.

Pseudostromata erumpent, consisting of an inconspicuous orange ectostromatic disc, semi-immersed to superficial, causing a pustulate bark surface, 1200–1400 μm diam. Ectostromatic disc flat or concave, orange, brown to black, sometimes concealed by ostioles, surrounded by bark flaps, 500–1100 μm diam. Central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, umber to fuscous black, regularly scattered, surrounding the ectostromatic disc, 300–400 μm diam. Ostioles 4–16 per disc, flat in the disc or sometimes slightly projecting, cylindrical, covered by an orange, umber to fuscous black crust, 80–130 μm diam. Paraphyses deliquescent. Asci fusoid, 8-spored, biseriate, with an apical ring, 55–65 × 7.5–9.5(–10) μm (x̄ = 59 ± 2.1 × 8.7 ± 0.7 μm, n = 10). Ascospores hyaline, fusoid to cylindrical, smooth, often containing two guttules per cell to multiguttulate, symmetrical to asymmetrical, straight to slightly curved, bicellular, with a median septum distinctly constricted, 11.5–14.5 × 3.5–4 μm (x̄ = 13 ± 1.2 × 3.7 ± 0.2 μm, n = 30). Conidiomata pycnidial, hemispherical, somewhat erumpent, coated with cinnamon aerial mycelium, 900–2200 μm diam, with translucent conidial droplets emerging from ostioles. Conidiophores hyaline, aseptate. Conidiogenous cells enteroblastic, polyphialidic, with 1–2 integrated loci, hyaline, ampulliform to doliiform. Conidia hyaline, aseptate, fusoid, acute at each end, 7.5–10.5(–12) × 2–2.5 μm (x̄ = 9.5 ± 1.2 × 2.3 ± 0.2 μm, n = 30).

On PDA, cultures are white, becoming slight isabelline after 2 wk. The colonies are flat with irregular edge; texture initially uniform, producing concentric circles after 2 wk with sparse conidiomata irregularly distributed on the agar surface.

Host & Distribution — On Osmanthus fragrans in China.

Materials examined (all on twigs and branches of Osmanthus fragrans). China, Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°17′00.77″ E119°43′28.70″, 139 m asl, 23 Apr. 2017, Q. Yang & Z. Du (holo-type CF 2017473; living ex-type culture CFCC 52106); Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N33°17′00.35″ E119°43′26.70″, 154 m asl, 23 Apr. 2017, Q. Yang & Z. Du (CF 2017476; living culture CFCC 52108); Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°17′30.67″ E119°43′20.30″, 139 m asl, 23 Apr. 2017, Q. Yang & Z. Du (CF 2017474; living culture CFCC 52107); Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N33°17′32.33″ E119°43′22.15″, 154 m asl, 23 Apr. 2017, Q. Yang & Z. Du (CF 2017477; living culture CFCC 52109).

Notes — The bark surface of Osmanthus fragrans is pustulate, with the fungus appearing to be pathogenic to this host. Dendrostoma osmanthi is similar to D. mali but differs by having fusoid to cylindrical ascospores that are distinctly constricted at the median septum. The phylogenetic inferences indicated this species as an individual well-supported clade (MP/ML/BI = 100/99/1) in the genus Dendrostoma.

Pseudomelanconidaceae C.M. Tian & X.L. Fan, fam. nov. — MycoBank MB823991

Etymology. Name derived from the type genus, Pseudomelanconis.

Type genus. Pseudomelanconis C.M. Tian & X.L. Fan.

Asexual morph: melanconium-like. Conidiomata in bark, acervular, with an inconspicuous ectostromatic disc causing a more or less pustulate bark surface. Central column beneath the disc more or less conical and becoming pale brown or olive at maturity. The marginal part of ectostroma comprises conidiophores and their basal cell layers. Conidiophores aseptate, unbranched, cylindrical, hyaline to pale brown, smooth-walled. Conidiogenous cells annellidic. Conidia ellipsoid to elongate pyriform, brown at maturity with hyaline sheath. Sexual morph: not observed.

Notes — The asexual morph of the new family Pseudomelanconidaceae is similar to members of Melanconiellaceae, Melanconidaceae and Juglanconidaceae (Fan et al. 2016b, Voglmayr et al. 2017), but differs mainly by having conidiogenous cells with discreet annellations and an inconspicuous hyaline conidial sheath when mature. The phylogenetic inferences resolved this family as an individual group with well-supported value (MP/ML/BI = 100/100/1) from other families of Diaporthales.

Pseudomelanconis C.M. Tian & X.L. Fan, gen. nov. — MycoBank MB823992

Etymology. Name derived from pseudo- (false-, in Greek) and the genus name Melanconis.

Type species. Pseudomelanconis caryae C.M. Tian & X.L. Fan.

Asexual morph: melanconium-like. Conidiomata in bark, acervular, immersed in host bark to erumpent. Ectostromatic disc inconspicuous, causing a more or less pustulate bark surface. Central column beneath the disc more or less conical. The marginal part of the central column comprises conidiophores and their basal cell layers. Conidiophores unbranched, aseptate, cylindrical, hyaline to pale brown, smooth-walled, sometimes reduced to conidiogenous cells. Conidiogenous cells annellidic, sometimes with apical collarette. Conidia hyaline when immature, becoming brown at maturity, ellipsoid to oblong, aseptate, multiguttulate, with distinct hyaline sheath, becoming inconspicuous when mature. Conidial wall smooth on the outer surface, with inconspicuous to distinct, sometimes confluent irregular verrucae on the inner surface. Sexual morph: not observed.

Pseudomelanconis caryae C.M. Tian & X.L. Fan, sp. nov. — MycoBank MB823993; Fig. 6

Fig. 6.

Morphology of Pseudomelanconis caryae from Carya cathayensis. a, b. Habit of conidiomata on branches; c. transverse section of conidioma; d. longitudinal section through conidioma; e–g. conidiophores and conidiogenous cells; h. conidia. — Scale bars: a = 1 mm; b–d = 500 μm; e–h = 10 μm.

Etymology. Named after the host genus from which it was isolated, Carya.

Asexual morph: melanconium-like. Conidiomata acervular, 500–800 μm diam, immersed in host bark to erumpent, covered by brown to blackish exuding conidial masses at maturity. Central column beneath the disc more or less conical. Conidiophores unbranched, aseptate, cylindrical, hyaline to pale brown, smooth-walled, 14–30 μm. Conidiogenous cells annellidic, occasionally with distinct annellations and collarettes. Conidia hyaline when immature, becoming greyish sepia to olivaceous, ellipsoid to oblong, multiguttulate, aseptate, (12.5–)13–15(–16) × 4–5 μm (x̄ = 14 ± 1.1 × 4.5 ± 0.3 μm, n = 30), with distinct hyaline sheath, 0.5–1 μm diam, becoming inconspicuous when mature. Conidial wall smooth on the outer surface. Sexual morph: not observed.

On PDA, cultures are initially white, becoming grey olivaceous. The colonies are flat, with irregular margins; texture initially uniform, becoming compact in centre after 3 wk. Conidiomata sparse, producing black conidial droplets, irregularly distributed over the agar surface.

Host & Distribution — On Carya cathayensis in China.

Materials examined (all on twigs and branches of Carya cathayensis). China, Zhejiang Province, Hangzhou City, Linan, Tianmu Mountain, N30°18′48.85″ E119°26′36.99″, 288 m asl, 21 Apr. 2017, Q. Yang & Z. Du (holotype CF 2017466; living ex-type culture CFCC 52110); Zhejiang Province, Hangzhou City, Linan, Tianmu Mountain, N30°18′49.19″ E119°26′37.24″, 281 m asl, 21 Apr. 2017, Q. Yang & Z. Du (CF 2017467; living culture CFCC 52111); Hangzhou City, Linan, Tianmu Mountain, N30°18′48.77″ E119°26′36.56″, 287 m asl, 21 Apr. 2017, Q. Yang & Z. Du (CF 2017468; living culture CFCC 52112); Hangzhou City, Linan, Tianmu Mountain, N30°18′49.14″ E119°26′30.44″, 285 m asl, 21 Apr. 2017, Q. Yang & Z. Du (CF 2017469; living culture CFCC 52113).

Notes — Pseudomelanconis caryae is the type species of Pseudomelanconis, and only occurs on Carya cathayensis in China. Isolates were identified as P. caryae based on their characteristic morphology, host, and DNA phylogeny (MP/ML/BI = 100/100/1). Juglanconis oblonga is similar to P. caryae, but it can be distinguished by larger brown to blackish conidia (18–22.7 × 9.2–12), and distinctly integrated annellations, as well as DNA sequence data (Voglmayr et al. 2017). Melanconis juglandis var. caryae was recorded from Carya cathayensis, which was considered as a distinct species by Wehmeyer (1941). However, it differs from P. caryae primarily by hyaline alpha (10.5–14 × 5–7 μm) and beta conidia (2–2.5 × 0.8–1 μm; Wehmeyer 1941). Wehmeyer (1937) also transferred Melanconiella pallida from Carya spp. to Melanconis, which differs from P. caryae in dark brown, subspherical to ovoid or oblong-cylindrical conidia (18–26.5 × 13.3–16.5 μm). Although Pseudomelanconis has acervular conidiomata covered by a pustulate conidial mass on the bark surface similar to Melanconis and Juglanconis, DNA sequence data confirmed them to represent a distinct phylogenetic lineage. Results of recent molecular phylogenetic investigations revealed a remarkably high diversity of corticolous melanconium-like fungi in Diaporthales (Fan et al. 2016b, Voglmayr et al. 2012, 2017).

Synnemasporellaceae X.L. Fan & J.D.P. Bezerra, fam. nov. — MycoBank MB823994

Etymology. Name derived from the type genus, Synnemasporella.

Type genus. Synnemasporella X.L. Fan & J.D.P. Bezerra.

Pseudostromata appearing upon the bark surface as pustules containing small groups of a few ostioles emergent through the adherent periderm, covered by a whitish pulverulence. Stromatic zones lacking. Perithecia spherical or flattened, with long necks, thickly clustered beneath the ectostromatic disks. Asci clavate. Ascospores biseriate, fusoid-ellipsoid, two-celled, hyaline, usually with a short, hyaline, bristle-like appendage at each end. Conidiomata synnematal or pycnidial. Synnemata determinate, parallel, consisting of slender, cylindrical black stalks and a spherical, capitate, shiny black mass of conidia which was cut off from the ends of the numerous entwined hyphae of the stalk; conidiogenous cells zone concave. Pycnidia with a central circular ostiole, hemispherical, immersed, somewhat erumpent. Conidiophores aggregated, straight to curved. Conidiogenous cells aggregated, hyaline, straight to curved, cylindrical. Conidia cylindrical to clavate, with a discrete hilum, smooth, pale brown.

Notes — The new family Synnemasporellaceae is proposed to accommodate fungi without the typical characters of any of the two-celled, hyaline-spored, stromatic genera. Also, the synnematal and pycnidial conidiomata differ widely from melanconium-like fungi, having pale brown conidia with a distinct hilum (Wehmeyer 1933). The phylogenetic inferences resolved this family as a well-supported clade (MP/ML/BI = 98/98/1) between the families Juglanconidaceae and Apiosporopsidaceae. Members of the new family differ from Juglanconidaceae and Apiosporopsidaceae (Senanayake et al. 2017, Voglmayr et al. 2017) mainly in the type of the host plant association, disease symptoms, ascomatal and/or conidiomatal characters, shape of ascospores, conidiogenous cells and conidia, and distinct synnemata.

Synnemasporella X.L. Fan & J.D.P. Bezerra, gen. nov. — MycoBank MB823995

Etymology. Name derived from the synnematous conidiomata.

Type species. Synnemasporella toxicodendri X.L. Fan & J.D.P. Bezerra.

Sexual morph (based on Wehmeyer 1933): Pseudostromata appearing upon the surface as pustules containing small groups of a few ostioles emergent through the adherent periderm, or as larger dense fascicles of elongate-cylindrical ostioles, erumpent through a whitish pulverulent disk. Ectostromatic disc often obliterated by the erumpent ostioles, many of which are covered by a whitish pulverulence. Perithecia spherical or somewhat flattened, with long slender necks, thickly clustered beneath the ectostromatic disks. Asci clavate. Ascospores biseriate, long fusoid-ellipsoid, two-celled, hyaline, constricted at the septum, usually with a short, hyaline, bristle-like appendage at each end. Asexual morph: Conidiomata synnematal or pycnidial. Synnemata long and determinate, erumpent, growing from the host tissue, pale brown, straight to curved, parallel, conidiogenous cells zone concave and dark, with some host tissue at the base of synnema. Pycnidia with a central circular ostiole, hemispherical, immersed, somewhat erumpent. Conidiophores aggregated, aseptate, straight to curved. Conidiogenous cells aggregated, hyaline, straight to curved, cylindrical, arranged alongside one another, each producing one conidium. Conidia cylindrical to oblong-cylindrical, with a discrete hilum, smooth, multiguttulate, pale brown.

Synnemasporella aculeans (Schwein.) X.L. Fan & J.D.P. Bezerra, comb. nov. — MycoBank MB823996; Fig. 7

Fig. 7.

Morphology of Synnemasporella aculeans from Rhus chinensis. a–c. Habit of synnemata on branches; d, e. longitudinal section through synnema; f, m, n. conidiophores and conidiogenous cells; g, h, o. conidia; i, j. habit of pycnidia on branches; k. transverse section of pycnidium; l. longitudinal section through pycnidium. — Scale bars: a–c = 1 mm; d, j–l = 500 μm; e = 100 μm; f–h, m–o = 10 μm; i = 5 mm.

Basionym. Sphaeria aculeans Schwein., Trans. Amer. Philos. Soc. 4: 204. 1832.

Synonym. Cryptodiaporthe aculeans (Schwein.) Wehm., Monogr. Gen. Diaporthe Nitschke & Segreg., Univ. Michigan Stud., Sci. Ser. 9: 212. 1933.

Sexual morph (based on Wehmeyer 1933): Pseudostromata appearing upon the surface as pustules containing small groups of a few ostioles emergent through the adherent periderm, or as larger dense fascicles of elongate-cylindrical ostioles, erumpent through a whitish pulverulent disk, 0.3–1 mm diam, covered by a whitish pulverulence. Stromatic zones lacking. Perithecia spherical or flattened, with long slender necks, thickly clustered beneath the ectostromatic disks, 260–480 × 250–400 μm. Asci clavate, 47–65 × 5–8 μm. Ascospores biseriate, long fusoid-ellipsoid, two-celled, hyaline, constricted at the septum, 12–18 × 2.5–3 μm, and usually with a short, hyaline, bristle-like appendage at each end, 2–2.5 μm length. Asexual morph: Conidiomata synnematal or pycnidial. Synnemata long and determinated, growing from the host tissue, pale to brown, straight to curved, parallel, with convex and dark conidiogenous cells zone, and some host tissue at the base of synnema, 1100–1500 μm high, 200–400 μm diam. Conidiophores aggregated, aseptate, straight to curved, 20–30 μm. Conidiogenous cells aggregated, hyaline, straight to curved, cylindrical, arranged alongside one another at the end of the synnemata, each producing one conidium. Conidia oblong-cylindrical, with a distinct hilum, smooth, multiguttulate, hyaline when young and becoming pale brown at maturity, 8–10(–11) × 3–3.5 μm (x̄ = 9.3 ± 0.9 × 3.2 ± 0.3 μm, n = 30). Pycnidia with a central circular ostiole, hemispherical, immersed, somewhat erumpent, containing an irregular one-chambered locule with black conidial mass, 700–1000 μm. Conidiophores aggregated, aseptate, straight to curved, 20–35 μm. Conidiogenous cells aggregated, hyaline, straight to curved, cylindrical, arranged alongside one another at the base of the pycnidia, each producing one conidium. Conidia ovoid to oblong-fusoid, one-celled, hyaline, multiguttulate, (6.5–)7–8.5(–9) × 2.5–3(–3.5) μm (x̄ = 7.6 ± 0.6 × 3 ± 0.3 μm, n = 30).

On PDA, cultures are initially white, becoming straw on the margin after 3 wk. The colonies are felty with regular edge; texture initially uniform, producing concentric circle on the margin after 3 d; sterile.

Host & Distribution — On Rhus copallina, R. diversiloba, R. glabra, R. javanica, R. typhina and R. vernix in Japan and USA (Wehmeyer 1933, Kobayashi 1970, Mejía et al. 2011), and on R. chinensis in China.

Materials examined (all on twigs and branches of Rhus chinensis). China, Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°15′32.83″ E119°43′30.73″, 47 m asl, 22 Apr. 2017, Q. Yang & Z. Du (CF 2017464; living culture CFCC 52094); Jiangxi Province, Dexing City, Phoenix Lake, N28°56′15.20″ E117°35′32.12″, 40 m asl, 8 Apr. 2017, B. Cao (CF 2017401; living culture CFCC 52096); Jiangxi Province, Dexing City, Phoenix Lake, N28°56′14.11″ E117°35′32.84″, 41 m asl, 8 Apr. 2017, B. Cao (CF 2017400; living culture CFCC 52095).

Notes — Synnemasporella aculeans is proposed as a new combination in the new genus Synnemasporella based on the description of Cryptodiaporthe aculeans, which was introduced producing perithecial ascomata, and an asexual morph producing sporodochial and/or pycnidial conidiomata (Wehmeyer 1933). Wehmeyer (1933) placed C. aculeans provisionally in Cryptodiaporthe and suggested this genus as a ‘heterogeneous group of species which will probably be segregated into several genera when the relationships of its species are better known’, highlighting that C. aculeans could be proposed as a new genus based on its atypical morphological features (see notes of Synnemasporellaceae). Sogonov et al. (2008) treated Cryptodiaporthe (type C. aesculi) as a synonym of Plagiostoma, which was followed by Mejía et al. (2011) and Voglmayr et al. (2017). Recently, Senanayake et al. (2017) separated the genera Plagiostoma and Cryptodiaporthe in their phylogenetically inferences, and pointed out that Gnomoniaceae comprised 24 genera, including Plagiostoma and Cryptodiaporthe. In the current study, the phylogram indicated that our isolates clustered into the same clade (MP/ML/BI = 80/100/1) with the only available culture of C. aculeans (AR3878). Based on the phylogenetic inferences and morphological features, we transferred C. aculeans to the new genus Synnemasporella, as a new combination S. aculeans. Synnemasporella aculeans is similar to S. toxicodendri, but differs from it in having shorter synnemata (1100–1500 μm vs 1200–1800 μm), a convex conidiogenous cells zone on top of synnemata, and larger, oblong-cylindrical conidia (8–11 × 3–3.5 μm).

This species also represents two types of conidiomata in Diaporthales, namely pycnidia and synnemata. This uncommon phenomenon was also recorded by Wehmeyer (1933), who observed the production of sporodochia on twigs of Rhus. Wehmeyer (1933) also reported the production of conidiomata when cultures were grown on agar, ‘showing all intergradations between true pycnidia and true sporodochia’.

Synnemasporella toxicodendri X.L. Fan & J.D.P. Bezerra, sp. nov. — MycoBank MB823997; Fig. 8

Fig. 8.

Morphology of Synnemasporella toxicodendri from Toxicodendron sylvestre. a, b. Habit of synnemata on branches; c, d. longitudinal section through synnema; e. conidiophores and conidiogenous cells; f, g. conidia. — Scale bars: a, b = 1 mm; c, d = 100 μm; e–g = 10 μm.

Etymology. Name derived from the host genus, Toxicodendron.

Conidiomata synnematal. Synnemata long and determinate, growing from host tissue, pale to brown, straight to curved, parallel, with flat to slightly concave and dark conidiogenous cells zone and some host tissue at the base of synnema, 1200–1800 μm high, 150–300 μm diam. Conidiophores aggregated, aseptate, straight to curved, reduced to conidiogenous cells, 20–30 μm. Conidiogenous cells aggregated, hyaline, straight to curved, cylindrical, arranged adjacent to one another at the end of the synnema, producing one conidium each. Conidia cylindrical to oblong-cylindrical, with a discrete hilum, smooth, multiguttulate, pale brown, 6–8 × 2.5–4 μm (x̄ = 7.3 ± 0.6 × 3.1 ± 0.3 μm, n = 30). Sexual morph: not observed.

On PDA, cultures are initially white, becoming sepia on the bottom after 3 d. The colonies are felty with an irregular edge; texture initially uniform, producing concentric circles after 3 wk; sterile.

Host & Distribution — From Toxicodendron sylvestre in China.

Materials examined (all on twigs and branches of Toxicodendron sylvestre). China, Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°15′32.84″ E119°43′31.21″, 54 m asl, 22 Apr. 2017, Q. Yang & Z. Du (holotype CF 2017481; living ex-type culture CFCC 52097); Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°15′32.21″ E119°43′31.55″, 51 m asl, 22 Apr. 2017, Q. Yang & Z. Du (CF 2017483; living culture CFCC 52099); Zhejiang Province, Hangzhou City, Linan, Xijing Mountain, N30°15′31.12″ E119°43′30.77″, 52 m asl, 22 Apr. 2017, Q. Yang & Z. Du (CF 2017482; living culture CFCC 52098).

Notes — Structures of S. toxicodendri were observed growing on diseased wood of Toxicodendron sylvestre in China, and so far, only occurs on T. sylvestre. Morphologically, it can be distinguished from S. aculeans and other genera in Diaporthales because it is characterised by the higher synnemata (1200–1800 μm) growing from the host tissue, with flat to slightly concave and dark conidiogenous cells zone on the top. The sexual morph of this species is not known and further collections are required to resolve its life cycle.

DISCUSSION

In this study we propose three new families, namely Diaporthostomataceae, Pseudomelanconidaceae, Synnemasporellaceae, and a new genus, Dendrostoma (Erythrogloeaceae), including three new species. The new materials studied here were collected in Zhejiang Province, China. This province was chiefly selected due to Tianmu Mountain, which is considered as a biodiversity hotspot with a high diversity for forest species (Zhou 1995). In the current study, all specimens were collected from symptomatic branches and twigs associated with canker or dieback disease of Anacardiaceae (Rhus chinensis, Toxicodendron sylvestre), Fagaceae (Quercus acutissima), Juglandaceae (Carya cathayensis), Lauraceae (Machilus leptophylla), Oleaceae (Osmanthus fragrans) and Rosaceae (Malus spectabilis), suggesting that many additional undiscovered species of diaporthalean fungi exist in China.

The classification of Diaporthales presented here integrates results from prior analyses (Castlebury et al. 2002, Rossman et al. 2007) and discoveries of new taxa from many other research groups (Cheewangkoon et al. 2010, Crous et al. 2012, 2015, Suetrong et al. 2015, Norphanphoun et al. 2016, Senanayake et al. 2017, Voglmayr et al. 2017). Diaporthales are mostly characterised by diaporthalean perithecia with an elongate beak and unitunicate asci with a characteristic refractive apical annulus when mature (Rossman et al. 2007). However, these characters are difficult to fully study in vivo due to the absence of various morphological morphs. As a result, the family concepts have thus been unstable, with many species being transferred from one genus or family to another (Rossman et al. 2007, Hongsanan et al. 2017, Senanayake et al. 2017). Based on newly generated molecular data, the current systematic framework provides good support for this order and related families (Castlebury et al. 2002, Rossman et al. 2007, Crous et al. 2015, Senanayake et al. 2017, Voglmayr et al. 2017). The current study revised the Diaporthales and accepted 25 families in the order (Table 2). However, some nodes remain weakly supported, and genera such as Diaporthella and Phaeoappendispora require further collection and study (Senanayake et al. 2017).

Table 2.

Placement of families in Diaporthales by different authors.

Nannfeldt (1932)	Von Arx & Müller (1954)	Wehmeyer & Hanlin (1975)	Barr (1978)	Rossman et al. (2007)	Senanayake et al. (2017)	This paper (2018)
Diaportheaceae	Diaportheaceae	Diaportheaceae	Diaportheaceae	Cryphonectriaceae	Apiosporopsidaceae	Apiosporopsidaceae
	Valsaceae	Gnomoniaceae	Gnomoniaceae	Diaportheaceae	Apoharknessiaceae	Apoharknessiaceae
		Valsaceae	Melanconidaceae	Gnomoniaceae	Asterosporiaceae	Asterosporiaceae
			Valsaceae	Melanconidaceae	Auratiopycnidiellaceae	Auratiopycnidiellaceae
				Pseudovalsaceae	Coryneaceae	Coryneaceae
				Schizoparmeaceae	Cryphonectriaceae	Cryphonectriaceae
				Sydowiellaceae	Cytosporaceae	Cytosporaceae
				Togniniaceae	Diaporthaceae	Diaporthaceae
				Valsaceae	Erythrogloeaceae	Diaporthosporellaceae
					Gnomoniaceae	Diaporthostomataceae
					Harknessiaceae	Erythrogloeaceae
					Juglanconidaceae	Gnomoniaceae
					Lamproconiaceae	Harknessiaceae
					Macrohilaceae	Juglanconidaceae
					Melanconidaceae	Lamproconiaceae
					Melanconiellaceae	Macrohilaceae
					Prosopidicolaceae	Melanconidaceae
					Pseudoplagiostomataceae	Melanconiellaceae
					Schizoparmeaceae	Prosopidicolaceae
					Stilbosporaceae	Pseudomelanconidaceae
					Sydowiellaceae	Pseudoplagiostomataceae
						Schizoparmeaceae
						Stilbosporaceae
						Sydowiellaceae
						Synnemasporellaceae

As the morphological features in Diaporthales are highly diverse, phylogenetic studies have been useful to elucidate the diversity in this group, and the inclusion or exclusion of taxa in this order. In this study we proposed a new genus in Erythrogloeaceae, namely Dendrostoma, which is characterised by typical diaporthalean perithecia with clavate asci, and fusoid to cylindrical, bicellular ascospores. The family Erythrogloeaceae was recently proposed by Senanayake et al. (2017) to accommodate Chrysocrypta, Disculoides and Erythrogloeum based on morphological features and phylogenetic analyses. Members of this family are mainly characterised by acervular conidiomata, hyaline to olivaceous conidia, and the presence of macro- and microconidia. Although Senanayake et al. (2017) did not observe any sexual morph in this family, we did collect a sexual morph in the present study, and thus emended the family description accordingly.

The new family Diaporthostomataceae is introduced here based on phylogenetic inferences and morphology of its members, which are mainly characterised by conical and discrete perithecia, and bicellular, fusoid ascospores. The new family is morphologically distinct from its sister family Diaporthosporellaceae, which is also distinguished from other diaporthalean families by irregularly uniseriate, allantoid or subreniform ascospores, phialidic conidiophores, and cylindrical to ellipsoidal, aseptate conidia (Yang et al. 2018). Members of Diaporthosporellaceae are known from twigs and branches of Cercis chinensis (Yang et al. 2018), and representatives of Diaporthostomataceae occur on Machilus leptophylla, both occurring in China.

Pseudomelanconidaceae is described here (on Carya cathayensis in China) based on phylogenetic inferences, and on few morphological features which distinguish it from members of Melanconium-related Melanconiellaceae, Melanconidaceae and Juglanconidaceae. Senanayake et al. (2017) introduced Melanconiellaceae to accommodate the previous unresolved Melanconiella clade in Diaporthales. Melanconiella species were previously observed to be highly host-specific to the host family Betulaceae, and confined to the north temperate zone, namely Europe and North America (Voglmayr et al. 2012). Du et al. (2017) extended the host and geographic range to include Cornus controversa and Juglans regia in China. The Melanconidaceae was recently revised by Senanayake et al. (2017), and included a single genus, Melanconis, which is characterised by perithecial ascomata having 8-spored asci, hyaline, ellipsoid, 1-septate ascospores, and acervular conidiomata with hyaline to brown, ellipsoid or subglobose conidia. Members of this family include saprobic and plant pathogenic species in North America and Europe (Senanayake et al. 2017). Juglanconidaceae was introduced by Voglmayr et al. (2017) and it comprises a single genus, Juglanconis, which occurs on dead, corticated twigs and branches of Juglandaceae species. Morphologically, members of this family have perithecial ascomata, octosporous asci with an apical ring, hyaline, bicellular ascospores with or without gelatinous appendages, and acervular conidiomata with brown conidia with gelatinous sheaths (Senanayake et al. 2017, Voglmayr et al. 2017).

The molecular phylogenetic analyses also revealed another well-supported family, Synnemasporellaceae, which is closely related to Juglanconidaceae (Fig. 1). We identified one species of this clade as Cryptodiaporthe aculeans based on the ambiguous asexual features of this species, which can produce synnemata and pycnidia. Interestingly, C. aculeans was ‘arbitrarily placed in the genus Cryptodiaporthe’ by Wehmeyer (1933), who also suggested that this species could be described as a new genus ‘since it does not show the typical characters of any of the two-celled, hyaline-spored, stromata genera’. Recent studies, however, did not include Cryptodiaporthe aculeans in their phylogenetic analyses (Sogonov et al. 2008, Mejía et al. 2011, Senanayake et al. 2017). In the present study, this fungus was transferred to Synnemasporella as a new combination, based on the original description of Wehmeyer (1933), and the only sequenced culture (AR3878) available in GenBank. Synnemasporellaceae comprises fungi distinguished by the presence of spherical or flattened perithecia with long necks, clavate asci, fusoid-ellipsoid, two-celled, hyaline ascospores, usually with a short, hyaline, bristle-like appendage at each end, and synnematal and/or pycnidial conidiomata producing cylindrical to clavate, smooth, pale brown conidia.

As shown in this study, future studies addressing the family-level organization of the Diaporthales should routinely include data for protein-coding genes, especially rpb2 and tef1-α. It is hoped that the classification proposed here will also provide an updated phylogenetic framework that will facilitate further revision of the Diaporthales.