Resolving the Lophiostoma bipolare complex: Generic delimitations within Lophiostomataceae.

Lophiostoma bipolare was taxonomically revised based on the morphological observations and phylogenetic analyses of molecular data from nuclear rDNA SSU-ITS-LSU, TUB, tef1, and rpb2 genes. Twenty-nine strains were morphologically similar to Lo. bipolare. A total of 174 sequences were generated from the Lo. bipolare complex. Phylogenetic analyses based on TUB sequence revealed 11 distinct species within the Lo. bipolare complex. Morphological features of the ascospores and the anatomical structure of the ascomata from both field collections as well as axenic culture, which have been reported previously as variable features at intraspecific levels, were compared to evaluate the taxonomic reliability of these features. To clarify the generic position of the 11 species, phylogenetic analyses were done on SSU-ITS-LSU-tef1-rpb2 gene sequences. The Lo. bipolare complex shared phylogenetic relationships with Pseudolophiostoma and Vaginatispora, and formed an additional five distinct clades from other members of Lophiostomataceae. According to its phylogenetic position, Lo. bipolare sensu stricto was distantly related to Lophiostoma s. str., and formed an independent clade within Lophiostomataceae. Lophiostoma bipolare s. str. could be distinguished from the other lophiostomataceous genera by the clypeus around the ostiolar neck and by the thin and uniformly thick peridium. A novel genus described as Lentistoma was established to accommodate this species, and the epitypification of Lentistoma bipolare (basionym: Massarina bipolaris) was proposed. Other lineages of the Lo. bipolare complex could not be separated on the basis of the ascospore size and sheath variations, but were distinguished based on ascomatal features, such as the existence of the clypeus, brown hyphae surrounding the peridium, and the contexture of the peridium, which were stable indicators of generic boundaries in Lophiostomataceae. Four additional new genera with five new species were recognised based on these morphological differences: Crassiclypeus (C. aquaticus), Flabellascoma (F. cycadicola and F. minimum), Leptoparies (Lep. palmarum), and Pseudopaucispora (Pseudop. brunneospora). Three new species were added to Pseudolophiostoma (Pseudol. cornisporum, Pseudol. obtusisporum, and Pseudol. tropicum) and two new species were added to Vaginatispora (V. amygdali and V. scabrispora). The re-evaluation of the validity of several previously recognised genera resulted in the introduction of two new genera with new combinations for Lophiostoma pseudoarmatisporum as Parapaucispora pseudoarmatispora and Vaginatispora fuckelii as Neovaginatispora fuckelii.

Introduction

Lophiostomataceae was established by Saccardo (1883) based on the subfamily Lophiostomeae. Members of the family are readily recognised by their carbonaceous ascomata with the slit-like ostiolar neck. They are saprophytes that grow on herbaceous and woody plants from terrestrial, freshwater, and marine environments (Chesters and Bell, 1970, Holm and Holm, 1988, Barr, 1987, Barr, 1992). The slit-like ostiolar neck and peridium of the ascomata are regarded as variable structures within a single specimen. Chesters & Bell (1970) adapted ascospore features including colour and longitudinal or transverse septation for generic circumscription. However, Holm & Holm (1988) considered ascospore septation as an unimportant characteristic at the generic level, but useful at the species level, and therefore used broad generic concepts for Lophiostomataceae. These broad generic concepts of Lophiostoma have been used by several authors (Barr, 1987, Barr, 1992, Yuan and Zhao, 1994, Checa, 1997, Kirk et al., 2008, Mugambi and Huhndorf, 2009). A recent generic re-evaluation of Lophiostomataceae (Thambugala ) segregated Lophiostoma s. lat. into 16 genera according to the multi-locus phylogenies using small subunit nrDNA (18S; SSU), large subunit nrDNA (28S; LSU), and translation elongation factor 1-α (tef1).

Lophiostoma bipolare is recognised by its striking features of the slit-like ostiolar neck surrounded by a well-developed clypeus, an ascus with a broad ocular chamber, and ascospores bearing an appendage-like sheath (hereafter referred to as the bipolar sheath) (Hyde 1995a). Lophiostoma bipolare has been reported in freshwater (Shearer & Raja 2010) and marine habitats (Hyde ). The species was originally described as a member of Massarina (Hyde 1995a). Although Lo. bipolare slightly differs in morphology from the generic type Lo. macrostomum – which is characterised by a well-developed carbonaceous ascoma, a slit-like ostiolar neck lacking the clypeus, and an ascus with a small ocular chamber (Zhang ) – this species was transferred to the genus Lophiostoma based on the results of the phylogenetic analyses using internal transcribed spacer (ITS) sequences (Liew ). Lophiostoma bipolare was not included in the recent comprehensive study on Lophiostomataceae by Thambugala . Thus, its generic placement remains unresolved.

During our studies of ascomycetous fungi in Japan (Hirayama and Tanaka, 2011, Tanaka et al., 2015, Hashimoto et al., 2017a, Hashimoto et al., 2017b), we obtained strains that were morphologically similar to Lo. bipolare. The main objectives of the present study were to clarify the generic placement of the Lo. bipolare complex and to establish a taxonomic framework of genera in Lophiostomataceae based on the morphological observations and molecular phylogenetic analyses of the sequences of SSU, ITS, LSU, tef1, and rpb2 (the second largest subunit of the DNA-directed RNA polymerase II).

Materials and methods

DNA isolation and amplification

DNA extraction was carried out with an ISOPLANT II kit (Nippon Gene, Japan) based on the manufacturer's protocol. Sequences of SSU, ITS, LSU, TUB, tef1, and rpb2 were amplified by PCR with the following primer pairs: SSU = NS1/NS4, ITS = ITS1/ITS4 (White ), LSU = LR0R/LR7 (Rehner and Samuels, 1994, Vilgalys and Hester, 1990), TUB = T1/Bt2b (Glass and Donaldson, 1995, O'Donnell and Cigelnik, 1997), tef1 = EF1-983F/EF1-2218R (Rehner & Buckley 2005), and rpb2 = fRPB2-5F/fRPB2-7cR (Liu ), respectively. Amplifications were performed in 25 μL volumes consisting of 2 μL DNA, 2.5 μL of 10× TEMPase Buffer I, 10 mM dNTP mix, 1 μL of each primer (20 pM), 25 mM MgCl2, 14.5 μL MilliQ water, and 0.5 μL TEMPase Hot Start DNA polymerase (Ampliqon, Denmark). PCR was carried out on a PC 320 thermocycler (ASTEC, Japan) as follows: 95 °C for 15 min, 35 cycles of 1 min at 94 °C, 1 min at the designated annealing temperature (42.2 °C for SSU, 61.5 °C for ITS, 46 °C for LSU, 50 °C for TUB, 60 °C for tef1, and 58 °C for rpb2), and 1 min at 72 °C, with a final denaturation step of 7 min at 72 °C. The PCR products were sequenced directly at SolGent (South Korea).

Phylogenetic analyses

Newly generated sequences were deposited in GenBank (Table 1). The primary analyses of TUB sequences were applied to 29 strains of Lo. bipolare complex to assess species diversity (Table 1). Secondary analyses were conducted on SSU-ITS-LSU-tef1-rpb2 sequences from 73 taxa of Lophiostomataceae to clarify the generic placement (Table 1, Table 2). Vaginatispora armatispora was excluded from the analyses because the sequences were limited to ITS data only. All sequences were aligned using the MUSCLE algorithm as implemented in MEGA v. 5 (Tamura ). Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian methods. The optimum substitution models for each dataset were estimated using the Kakusan4 program (Tanabe 2011) based on the Akaike information Criterion (AIC; Akaike 1974) for the ML analysis, and the Bayesian Information Criterion (BIC; Schwarz 1978) for the Bayesian analysis. The ML analysis was performed using the TreeFinder Mar 2011 program (Jobb 2011) based on the models selected with the AICc4 parameter (a proportional model among genes and codons). TN93+G was used for TUB in the first dataset. The second dataset used HKY85+G for SSU, TN93+G for LSU, J2ef+G for ITS, F81+G for tef1 first codon position, J1ef+G for tef1 second codon position, J2+G for tef1 third codon position, J2+G for rpb2 first codon position, F81+G for rpb2 second codon position, and J2+G for rpb2 third codon position. Bootstrap proportions (BPs) were obtained via 1 000 bootstrap replicates. Bayesian analysis was performed with MrBayes v. 3.2.2 (Ronquist ) using substitution models containing the BIC4 parameter (i.e., proportional model among loci and among codons). HKY85+G was used for TUB in the first dataset. The second dataset used K80+G for SSU, K80+G for LSU, SYM+G for ITS, F81+G for tef1 first codon position, GTR+G for tef1 second codon position, GTR+G for tef1 third codon position, GTR+G for rpb2 first codon position, F81+G for rpb2 second codon position, and HKY85+G for rpb2 third codon position. Two simultaneous and independent Metropolis-coupled Markov chain Monte Carlo (MCMC) runs were performed for 1 M and 2 M generations with the trees sampled every 1 000 generations for the first and second analyses, respectively. Convergence of the MCMC procedure was assessed from the average standard deviation of split frequencies (< 0.01) and effective sample size scores (all > 100) using MrBayes and Tracer v. 1.6 (Rambaut ), respectively. The first 25 % of the trees were discarded as burn-in, and the remainder were used to calculate the 50 % majority rule trees and to determine the posterior probabilities (PPs) for individual branches. Teichospora rubriostiolata and T. trabicola (Teichosporaceae; Jaklitsch ) were used as outgroups in the secondary analyses. The alignments were submitted to TreeBASE under study number S21190.

Table 1

Specimens, isolates and new sequence accessions used in this study.

Species		Original no.	Specimen no.1	Strain no.	Habitat2	GenBank accession no.
Old name	New name					SSU	ITS	LSU	tef1	rpb2	TUB
Lo. bipolare -1	Crassiclypeus aquaticus	KH 56	HHUF 30566	CBS 143639	F	LC312468	LC312497	LC312526	LC312555	LC312584	LC312613
		KH 91	HHUF 30567	CBS 143640	F	LC312469	LC312498	LC312527	LC312556	LC312585	LC312614
		KH 104	HHUF 30568	CBS 143641	F	LC312470	LC312499	LC312528	LC312557	LC312586	LC312615
		KH 185	HHUF 30569	CBS 143642	F	LC312471	LC312500	LC312529	LC312558	LC312587	LC312616
		KT 970	HHUF 27985H	CBS 143643=JCM 13087=MAFF 239597	F	LC312472	LC312501	LC312530	LC312559	LC312588	LC312617
Lo. bipolare -2	Flabellascoma cycadicola	KT 2034	HHUF 30570H	BCRC FU30901=CBS 143644	T	LC312473	LC312502	LC312531	LC312560	LC312589	LC312618
Lo. bipolare -3	F.minimum	KT 2013	HHUF 30571	BCRC FU30900=CBS 143645	T	LC312474	LC312503	LC312532	LC312561	LC312590	LC312619
		KT 2040	HHUF 30572H	BCRC FU30902=CBS 143646	T	LC312475	LC312504	LC312533	LC312562	LC312591	LC312620
Lo. bipolare -4	Lentistoma bipolare	HKUCC 10069	HHUF 30576	CBS 115370	U	LC312476	LC312505	LC312534	LC312563	LC312592	LC312621
		HKUCC 10110	HHUF 30577E	CBS 115375	U	LC312477	LC312506	LC312535	LC312564	LC312593	LC312622
		HKUCC 8277	HHUF 30575	JCM 14139=CBS 110448	F	LC312478	LC312507	LC312536	LC312565	LC312594	LC312623
		KH 214	HHUF 30578	CBS 143647	F	LC312479	LC312508	LC312537	LC312566	LC312595	LC312624
		KH 216	HHUF 30579	CBS 143648	T	LC312480	LC312509	LC312538	LC312567	LC312596	LC312625
		KH 222	HHUF 30580	CBS 143649	F	LC312481	LC312510	LC312539	LC312568	LC312597	LC312626
		KH 311	HHUF 30581	CBS 143650	F	LC312482	LC312511	LC312540	LC312569	LC312598	LC312627
		KT 2415	HHUF 30573	CBS 143651	T	LC312483	LC312512	LC312541	LC312570	LC312599	LC312628
		KT 3056	HHUF 30574	CBS 143652	T	LC312484	LC312513	LC312542	LC312571	LC312600	LC312629
Lo. bipolare -5	Leptoparies palmarum	KT 1653	HHUF 28983H	CBS 143653=JCM 13089=MAFF 239599	T	LC312485	LC312514	LC312543	LC312572	LC312601	LC312630
Lo. bipolare -6	Pseudolophiostoma cornisporum	KH 322	HHUF 30582H	CBS 143654=JCM 32348	T	LC312486	LC312515	LC312544	LC312573	LC312602	LC312631
Lo. bipolare -7	P. obtusisporum	KH 228	HHUF 30584	CBS 143655	T	LC312487	LC312516	LC312545	LC312574	LC312603	LC312632
		KH 336	HHUF 30585	CBS 143656	T	LC312488	LC312517	LC312546	LC312575	LC312604	LC312633
		KT 2838	HHUF 30583H	CBS 143657=JCM 32349	T	LC312489	LC312518	LC312547	LC312576	LC312605	LC312634
		KT 3098	HHUF 30171	CBS 143941=MAFF 243969	T	LC312490	LC312519	LC312548	LC312577	LC312606	LC312635
		KT 3119	HHUF 30189	CBS 143658=MAFF 243983	T	LC312491	LC312520	LC312549	LC312578	LC312607	LC312636
Lo. bipolare -8	P. tropicum	KH 352	HHUF 30586	CBS 143659	T	LC312492	LC312521	LC312550	LC312579	LC312608	LC312637
		KT 3134	HHUF 30202H	CBS 143660=MAFF 243989	T	LC312493	LC312522	LC312551	LC312580	LC312609	LC312638
Lo. bipolare -9	Pseudopaucispora brunneospora	KH 227	HHUF 30587H	CBS 143661=JCM 32350	T	LC312494	LC312523	LC312552	LC312581	LC312610	LC312639
Lo. bipolare -10	Vaginatispora amygdali	KT 2248	HHUF 30588H	CBS 143662=JCM 32351	T	LC312495	LC312524	LC312553	LC312582	LC312611	LC312640
Lo. bipolare -11	V. scabrispora	KT 2443	HHUF 30589H	CBS 143663=JCM 32352	M	LC312496	LC312525	LC312554	LC312583	LC312612	LC312641

“E”: epitype, “H”: holotype.

“F”: freshwater, “M”: marine, “T”: terrestrial, “U”: unknown.

Table 2

Isolates and GenBank accession numbers of species used in the phylogenetic study.

Species	Strain no.	GenBank Accession no.1
		SSU	ITS	LSU	tef1	rpb2
Alpestrisphaeria terricola	SC-12H	JX985749	JN662930	JX985750	–	–
Biappendiculispora japonica	KT 573H	AB618686	LC001728	AB619005	LC001744	–
	KT 686-1P	AB618687	LC001729	AB619006	LC001745	–
Capulatispora sagittiformis	KT 1934H	AB618693	AB369268	AB369267	LC001756	–
Coelodictyosporium muriforme	MFLUCC 13-0351H	KP899127	KP899136	KP888641	KR075163	–
C. pseudodictyosporium	MFLUCC 13-0451H	–	KR025858	KR025862	–	–
Dimorphiopsis brachystegiae	CPC 22679H	–	KF777160	KF777213	–	–
Guttulispora crataegi	MFLUCC 13-0442H	KP899125	KP899134	KP888639	KR075161	–
	MFLUCC 14-0993P	KP899126	KP899135	KP888640	KR075162	–
Lophiohelichrysum helichrysi	MFLUCC 15-0701H	KT333437	KT333435	KT333436	KT427535	–
Lophiopoacea paramacrostoma	MFLUCC 11-0463H	KP899122	–	KP888636	–	–
L. winteri	KT 740	AB618699	JN942969	AB619017	LC001763	JN993487
	KT 764	AB618700	JN942968	AB619018	LC001764	JN993488
Lophiostoma alpigenum	GKM 1091b	–	–	GU385193	GU327758	–
L. caulium	CBS 623.86	GU296163	–	GU301833	–	GU371791
L. crenatum	CBS 629.86	DQ678017	–	DQ678069	DQ677912	DQ677965
L. heterosporum	CBS 644.86	AY016354	GQ203795	AY016369	DQ497609	DQ497615
L. macrostomoides	CBS 123097	FJ795482	–	FJ795439	GU456277	FJ795458
L. macrostomum	KT 635	AB521731	AB433275	AB433273	LC001752	JN993484
L. quadrinucleatum	GKM 1233	–	–	GU385184	GU327760	–
L. semiliberum	KT 828	AB618696	JN942970	AB619014	LC001759	JN993489
Neotrematosphaeria biappendiculata	KT 1124H	GU205256	–	GU205227	–	–
	KT 975P	GU205254	–	GU205228	–	–
Neovaginatispora fuckelii	CBS 101952	FJ795496	–	DQ399531	–	FJ795472
	KH 161	AB618689	LC001731	AB619008	LC001749	–
	KT 634	AB618690	LC001732	AB619009	LC001750	–
Parapaucispora pseudoarmatispora	KT 2237H	LC100018	LC100021	LC100026	LC100030	–
Paucispora quadrispora	KH 448P	LC001720	LC001733	LC001722	LC001754	–
P. quadrispora	KT 843H	AB618692	LC001734	AB619011	LC001755	–
P. versicolor	KH 110H	LC001721	AB918731	AB918732	LC001760	–
Platystomum actinidiae	KT 521H	JN941375	JN942963	JN941380	LC001747	JN993490
P. compressum	MFLUCC 13-0343	KP899129	–	KP888643	KR075165	–
P. crataegi	MFLUCC 14-0925H	KT026113	KT026117	KT026109	KT026121	–
P. salicicola	MFLUCC 15-0632H	KT026114	KT026118	KT026110	–	–
Pseudolophiostoma vitigenum	HH 26930 H	AB618697	LC001735	AB619015	LC001761	–
	HH 26931I	AB618698	LC001736	AB619016	LC001762	–
Pseudoplatystomum scabridisporum	BCC 22835	GQ925831	–	GQ925844	GU479857	GU479830
	BCC 22836	GQ925832	–	GQ925845	GU479856	GU479829
Sigarispora arundinis	KT 651	AB618680	JN942965	AB618999	LC001738	JN993486
S. caudata	KT 530	AB618681	LC001723	AB619000	LC001739	–
S. ononidis	MFLUCC 15-2667H	KU243126	KU243128	KU243125	KU243127	–
S. ravennica	MFLUCC 14-0005H	KP698415	KP698413	KP698414	–	–
Teichospora rubriostiolata	TR 7H	–	KU601590	KU601590	KU601609	KU601599
T. trabicola	C 134E	–	KU601591	KU601591	KU601601	KU601600
Vaginatispora appendiculata	MFLUCC 16-0314H	KU743219	KU743217	KU743218	KU743220	–
V. aquatica	MFLUCC 11-0083	KJ591575	KJ591577	KJ591576	–	–

“E”: ex-epitype, “H”: ex-holotype, “I”: ex-isotype, “P”: ex-paratype.

Morphology and isolation

All fungal structures were observed in preparations mounted in distilled water. Morphological characters were observed by differential interference and phase contrast microscopy (Olympus BX53) using images captured with an Olympus digital camera (DP21). A total of 29 single-spore isolates were used for morphological observations and phylogenetic analyses (Table 1). The colony characteristics of the cultures grown on potato dextrose agar (PDA; Difco) were observed after 3 wk of growth at 20 °C in the dark. Colours were noted as described by Rayner (1970). To induce sexual or asexual fructification in culture, 5 mm squares of mycelial agar were placed on water agar containing sterilised natural substrates including rice straw and banana leaves. The plates were incubated at 20 °C for 2 wk in the dark. When the substrate was colonised, the plates were incubated at 20 °C under black light blue illumination for 2 mo to observe for sporulation. Cultures were deposited in the Bioresource Collection and Research Center of Food Industry Research and Development Institute, Hsinchu, Taiwan (BCRC); the Japan Collection of Microorganisms (JCM); the Genebank Project NARO, Japan (MAFF); and the Westerdijk Fungal Biodiversity Institute (CBS). Specimens were deposited in the Herbarium of Hirosaki University, Fungi (HHUF).

Results

Phylogeny

Alignment of the first analyses was based on TUB, and consisted of 29 strains with 628 nucleotide positions. Of these positions, 256 were variable and 357 were conserved. Both ML and Bayesian analyses showed 11 distinct operational taxonomic units for the Lo. bipolare complex (Fig. 1).

Fig. 1

Maximum-likelihood (ML) tree of the Lophiostoma bipolare complex based on TUB sequences. An ML bootstrap proportion (BP) greater than 90 % and Bayesian posterior probabilities (PP) above 0.95 are presented at the nodes as ML BS/Bayesian PP. The scale bars represent nucleotide substitutions per site and ascospore size (5 μm).

SSU-LSU phylogenies displayed low resolution at the generic and species levels. SSU-LSU phylogeny also failed to distinguish between Guttulispora, Sigarispora, and Platystomum (Fig. S1A). ITS phylogeny was able to distinguish at the generic and species levels with good resolution, except for Platystomum (Fig. S1B). tef1 phylogeny showed highly supported clades at the species level, while the monophyletic status of genera Lophiostoma, Platystomum, and Vaginatispora were weakly supported (< 70 % ML BS/ < 0.95 Bayesian PP) and Pseudolophiostoma was not reconstructed (Fig. S1C). rpb2 phylogeny was able to distinguish all 12 genera in both analyses, although the dataset included several missing taxa (Fig. S1D).

For the second analyses, ML and Bayesian phylogenetic analyses were conducted using an aligned sequence dataset comprising 935 nucleotide positions from SSU, 1 243 from LSU, 900 from ITS, 885 from tef1, and 1 017 from rpb2. The alignment contained a total of 75 taxa, which consisted of 69 taxa (92 %) in SSU, 75 (100 %) in LSU, 63 (84 %) in ITS, 64 (85.3 %) in tef1, and 44 (58.7 %) in rpb2 (Table 1, Table 2). This combined dataset provided higher confidence values for the generic and species levels than those of the individual gene trees, and a total of 23 genera were reconstructed (Fig. 2, S1). Of the 4 980 characters included in the alignment, 1 387 were variable and 3 524 were conserved. The ML tree with the highest log likelihood (–26083.925) is shown in Fig. 2. The Bayesian likelihood score was –26185.401. The topology recovered by the Bayesian analysis was almost identical to that of the ML tree, except for the positions of Alpestrisphaeria, Coelodictyosporium, and Lophiohelichrysum.

Fig. 2

Maximum-likelihood (ML) tree of Lophiostomataceae based on the SSU-ITS-LSU-tef1-rpb2 sequences. An ML bootstrap proportion (BP) greater than 60 % and Bayesian posterior probabilities (PP) above 0.95 are presented at the nodes as ML BS/Bayesian PP. The circle (●) indicates nodes with 100 % ML BS/ 1.00 Bayesian PP. A hyphen (“-”) indicates values lower than 60 % BP or 0.95 PP. Ex-holotype, isotype, paratype, and epitype strains are indicated with superscripts H, I, P, and E, respectively. The newly obtained sequences are shown in bold and red. The scale bar represents nucleotide substitutions per site.

The phylogenetic analyses showed that 11 of the Lo. bipolare complex appeared polyphyletic (Fig. 1), and were scattered within Lophiostomataceae (Figs 2, S1). The phylogenetic positions of Lo. bipolare (Lo. bipolare-4), including an ex-epitype strain (CBS 115375), was distantly related to Lophiostoma s. str. and was located in a clade separate from other members of Lophiostomataceae (Fig. 2). The results of the phylogenetic analyses suggested that the species should be excluded from Lophiostoma s. str. Lophiostoma bipolare was transferred to a novel, individual genus Lentistoma, and a new combination Lentistoma bipolare was proposed. Other members of the Lo. bipolare complex were scattered to Pseudolophiostoma, Vaginatispora, and four separate clades from known lophiostomataceous genera. Lophiostoma bipolare-1 was resolved as a strongly supported clade (100 % ML BS/ 1.00 Bayesian PP, Fig. 2) and a new generic name Crassiclypeus was proposed for a single novel species, C. aquaticus. Lophiostoma bipolare-2, 3 formed a robust clade (100 % ML BS/ 1.00 Bayesian PP, Fig. 2). A new genus Flabellascoma was introduced for these two species (F. cycadicola and F. minimum). The monotypic genus Leptoparies was introduced for Lep. palmarum (formerly treated as Lo. bipolare-5), which separated from the other lophiostomataceous genera in the phylogenetic tree (Fig. 2). Pseudolophiostoma comprised four species – its type species Pseudol. vitigenum (Thambugala ), as well as Lo. bipolare-6, 7, and 8 (Pseudol. cornisporum, Pseudol. obtusisporum, and Pseudol. tropicum) – forming a strongly supported clade (98 % ML BS/ 1.00 Bayesian PP, Fig. 2). Lophiostoma bipolare-9 represented a basal clade among Lophiostomataceae, for which a new genus and species Pseudopaucispora brunneospora was introduced. A clade containing V. appendiculata, V. aquatica, and Lo. bipolare-10, 11 received strong support (98 % ML BS/ 1.00 Bayesian PP, Fig. 2). Vaginatispora amygdali and V. scabrispora were proposed for Lo. bipolare-10, 11, respectively.

Taxonomy

Our phylogenetic analyses resolved 11 species that were classified in the Lo. bipolare complex (Fig. 1, Fig. 2). These 11 species could not be distinguished solely based on the ascospore morphology due to their close resemblance (Fig. 1). Detailed morphological observations of the ascospores as well as other morphological features (Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13), culture characteristics (Fig. 14) and multi-locus phylogeny differentiated the complex. Seven genera (including five new genera), 11 species (including 10 new species), and one new combination are proposed below. An epitype is designated for Lo. bipolare s. str. (basionym: Massarina bipolaris). Additionally, two new genera and two new combinations are introduced for Lophiostoma pseudoarmatisporum and Vaginatispora fuckelii (See Discussion and Appendix B).

Fig. 3

Crassiclypeus aquaticus.A–C. Appearance of ascomata on substrate. D–F. Ascomata in longitudinal section. G. Peridium of ascoma. H. Ostiolar neck of ascoma. I. Ascus apex. J. Ascus stipe. K, L. Asci. M. Pseudoparaphyses. N–S. Ascospores (arrowheads indicate an internal chamber in S). T–V. Conidiomata in culture. W. Conidioma in longitudinal section. X. Peridium of conidioma. Y–AA. Conidiogenous cells. AB, AC. Conidia. A, G–L, O–S from HHUF 30569. B–F from HHUF 27985 (holotype); M, N from HHUF 30567; T–AC from culture CBS 143640. Scale bars: A, T = 1 mm; B = 300 μm; C, U, V = 100 μm; D–F = 50 μm; K–M, W = 10 μm; G–J, N–S, X–AC = 5 μm.

Fig. 4

Flabellascoma cycadicola. A–C. Appearance of ascomata on substrate. D. Ascoma in longitudinal section. E. Ostiolar neck of ascoma. F. Pseudoparaphyses. G. Peridium of ascoma. H. Ascus apex. I, J. Asci. K–P. Ascospores (arrowheads indicate an internal chamber in P). Q–S. Conidiomata in culture. T, U. Conidiomata in longitudinal section. V. Peridium of conidioma. W, X. Conidiogenous cells. Y, Z. Conidia. A–P from HHUF 30570 (holotype). Q–Z from culture BCRC FU30901 = CBS 143644 (ex-holotype). Scale bars: B = 500 μm; C, S = 100 μm; D, T, U = 20 μm; G, I, J = 10 μm; E, F, H, K–P, V–Z = 5 μm; Q, R = 1 mm.

Fig. 5

Flabellascoma minimum.A–C. Appearance of ascomata on substrate. D, E. Ascomata in longitudinal section. F. Ostiolar neck of ascoma. G, H. Peridium of ascoma. I. Pseudoparaphyses. J, K. Asci. L. Ascus apex. M. Ascus stipe. N–T. Ascospores (arrowheads indicate an internal chamber in T). U–W. Conidiomata in culture. X. Conidioma in longitudinal section. Y. Peridium of conidioma. Z–AB. Conidiogenous cells. AC, AD. Conidia. A, B, D, F, H–K, N, O from HHUF 30572 (holotype); C, E, G, L, M, P–T from HHUF 30571; U–AD from culture BCRC FU30902 = CBS 143646 (ex-holotype). Scale bars: B, U, V = 1 mm; C, W = 100 μm; D, E, X = 20 μm; F–K = 10 μm; L–T, Y–AD = 5 μm.

Fig. 6

Lentistoma bipolare.A–F. Appearance of ascomata on substrate. G. Ascomata in culture. H–J. Ascomata in longitudinal section. K. Peridium. L. Ostiolar neck of ascoma. M. Pseudoparaphyses. N, O. Asci. P–U. Ascospores (arrowheads indicate an internal chamber in U). V. Senescent ascospore. A, E, I, T, U from HHUF 30578; B from HHUF 30574; C, D, O from HHUF 30573; F, M, N, P from BRIP 21489 (holotype); G from culture CBS 115370; H, K, L, S, V from HHUF 30579; J, R from culture CBS 115375 (ex-epitype); Q from culture CBS 143652. Scale bars: A–C, G = 1 mm; D–F = 100 μm; H–J = 50 μm; K–O = 10 μm; P–V = 5 μm.

Fig. 7

Leptoparies palmarum.A–C. Appearance of ascomata on substrate. D–F. Ascomata in culture. G, H. Ascomata in longitudinal section. I. Ostiolar neck of ascoma. J. Peridium of ascoma. K. Ascus apex. L, M. Asci. N. Pseudoparaphyses. O–U. Ascospores (arrowheads indicate an internal chamber in U). A–C, G, I–L, N–Q from HHUF 28983 (holotype); D–F, H, M, R–U from culture CBS 143653 = JCM 13089 = MAFF 239599 (ex-holotype). Scale bars: B, F = 300 μm; C = 100 μm; D, E = 1 mm; G, H = 50 μm; I–N = 10 μm; O–U = 5 μm.

Fig. 8

Pseudolophiostoma cornisporum.A–C. Appearance of ascomata on substrate. D–F. Ascomata in culture. G, H. Ascomata in longitudinal section. I. Ostiolar neck of ascoma. J, K. Peridium of ascoma. L, M. Asci. N. Pseudoparaphyses. O–T. Ascospores. A–C, G, I, J, P, T from HHUF 30582 (holotype). D–F, H, K–O, Q–S from culture CBS 143654 = JCM 32348 (ex-holotype). Scale bars: A, D = 1000 μm; B, E = 200 μm; C, F = 100 μm; G, H = 50 μm; I–N = 10 μm; O–T = 5 μm.

Fig. 9

Pseudolophiostoma obtusisporum.A–D. Appearance of ascomata on substrate. E, F. Ascomata in culture. G–I. Ascomata in longitudinal section. J. Peridium of ascoma. K. Ostiolar neck of ascoma. L, M. Asci. N. Ascus apex. O. Pseudoparaphyses. P–T. Ascospores. A, M, R from HHUF 30189; B, G, J–L from HHUF 30583 (holotype); C, D, H, N from HHUF 30171; E, F, I, O, S from culture CBS 143658 = MAFF 243983; P, T from culture CBS 143941 = MAFF 243969; Q from HHUF 30584. Scale bars: A, E, F = 1 mm; B, C = 200 μm; D = 100 μm; G–I = 50 μm; J–M, O = 10 μm; N, P–T = 5 μm.

Fig. 10

Pseudolophiostoma tropicum.A–E. Appearance of ascomata on substrate. F. Ascomata in culture. G–I. Ascomata in longitudinal section. J. Peridium of ascoma. K. Ostiolar neck of ascoma. L, M. Asci. N. Ascus apex. O. Pseudoparaphyses. P–U. Ascospores. A, B, H, K, Q from HHUF 30586; C–E, G, J, M, N, R–U from HHUF 30202 (holotype); F, I, L, O, P from culture CBS 143659. Scale bars: A, F = 1 mm; B, C = 200 μm; D, E = 100 μm; G–I = 50 μm; J–M, O = 10 μm; N, P–U = 5 μm.

Fig. 11

Pseudopaucispora brunneospora.A–C. Appearance of ascomata on substrate. D. Ascoma in longitudinal section. E. Peridium of ascoma. F. Ostiolar neck of ascoma. G. Pseudoparaphyses. H. Ascus apex. I. Ascus. J–O. Ascospores (arrowheads indicate an internal chamber in O). P–R. Conidiomata in culture. S, T. Conidiomata in longitudinal section (confluent conidioma in T). U. Peridium of conidioma. V, W. Conidiogenous cells. X. Conidia. Y. Germinating conidium. A–F, H–O from HHUF 30587 (holotype); G, P–Y from culture CBS 143661 = JCM 32350 (ex-holotype). Scale bars: A, P, Q = 1 mm; B = 200 μm; C, R = 100 μm; D, S, T = 50 μm; E, F, H, I, U, Y = 10 μm; G, J–O, V–X = 5 μm.

Fig. 12

Vaginatispora amygdali.A–C. Appearance of ascomata on substrate. D, E. Ascomata in culture. F. Ascoma in longitudinal section. G. Ostiolar neck of ascoma. H. Pseudoparaphyses. I. Peridium of ascoma. J. Ascus apex. K, L. Asci. M–R. Ascospores (arrowheads indicate an internal chamber in R). A–C, F–J, L–N, P–R from HHUF 30588 (holotype); D, E, K, O from culture CBS 143662 = JCM 32351 (ex-holotype). Scale bars: B, D = 200 μm; C, E = 100 μm; F = 50 μm; K, L = 10 μm; G–J, M–R = 5 μm.

Fig. 13

Vaginatispora scabrispora.A–C. Appearance of ascomata on substrate. D. Ascomata in culture. E, F. Ascomata in longitudinal section. G. Ostiolar neck of ascoma. H, I. Peridium of ascomata. J. Ascus. K. Ascus apex. L. Ascus stipe. M. Pseudoparaphyses. N–S. Ascospores (arrowheads indicate an internal chamber in S). A–C, E, G, H, K–M, O, P, S from HHUF 30589 (holotype); D, F, I, J, N, Q, R from culture CBS 143663 = JCM 32352 (ex-holotype). Scale bars: A = 1 mm; B–D = 100 μm; E, F = 50 μm; G–J, M = 10 μm; K, L, N–S = 5 μm.

Fig. 14

Colony characters of Lophiostoma bipolare complex used in this study on PDA within 3 wk at 20 °C in the dark (left: upper, right: reverse). A.Crassiclypeus aquaticus (CBS 143643 = JCM 13087 = MAFF 239597, ex-holotype). B.Flabellascoma cycadicola (BCRC FU30901 = CBS 143644, ex-holotype). C.F. minimum (BCRC FU30902 = CBS 143646, ex-holotype). D, E.Lentistoma bipolare (D. CBS 115375, ex-epitype, E. CBS 143647). F.Leptoparies palmarum (CBS 143653 = JCM 13089 = MAFF 239599, ex-holotype). G.Pseudolophiostoma cornisporum (CBS 143654 = JCM 32348, ex-holotype). H.Pseudol. obtusisporum (CBS 143657 = JCM 32349, ex-holotype). I.Pseudol. tropicum (CBS 143659, ex-paratype). J.Pseudopaucispora brunneospora (CBS 143661 = JCM 32350, ex-holotype). K.Vaginatispora amygdali (CBS 143662 = JCM 32351, ex-holotype). L.V. scabrispora (CBS 143663 = JCM 32352, ex-holotype). Scale bar: A–L = 1 cm.

A. Hashim., K. Hiray. & Kaz. Tanaka, gen. nov. MycoBank MB823131.

Etymology: Refers to its well-developed clypeus.

Sexual morph: Ascomata scattered to gregarious, immersed, subglobose. Ostiolar neck elongated, laterally compressed, surrounded by a well-developed clypeus. Peridium composed of elongated, brown cells, surrounded by brown hyphae. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a narrow bipolar sheath. Asexual morph: Conidiomata pycnidial, globose to subglobose, superficial to immersed. Peridium composed of subglobose to rectangular, brown cells. Conidiophores absent. Conidiogenous cells phialidic, ampliform, hyaline, smooth. Conidia subglobose with rounded ends, hyaline, smooth, aseptate.

Type species: Crassiclypeus aquaticus A. Hashim., K. Hiray. & Kaz. Tanaka.

Notes: Crassiclypeus is established to accommodate C. aquaticus, which is characterised by a crest-like, elongated, and laterally compressed ostiolar neck, well-developed peridium surrounded by brown hyphae (Fig. 3D–F), and an ascus with a long stipe (up to 50 μm, Fig. 3K, L).

The genus is superficially similar to Flabellascoma, but differs from the latter by having an ascomatal wall with 1 zone (Fig. 3D–F) and phialidic conidiogenous cells in the conidiomata (Fig. 3Y–AA) (vs. an ascomatal peridium composed of 2 zones and holoblastic conidiogenous cells in the conidiomata; Fig. 4D, W, X). It is also similar to Neotrematosphaeria, but the latter genus has a poorly developed peridium at the base and lacks the clypeated ostiolar neck of the ascomata (Thambugala ). Crassiclypeus has similar morphological features of Lentistoma, such as the clypeated ostiolar neck, but can be distinguished from Lentistoma by the well-developed peridium of the ascomata (up to 70 μm in thickness). Lentistoma is characterised by ascomata with less-developed peridium (up to 45 μm in thickness).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823132. Fig. 3, Fig. 14.

Etymology: Refers to its aquatic habitat.

Sexual morph: Ascomata subglobose, 3–5 grouped, immersed, dark brown to black, 400–780 μm high, 600–1 000 μm diam. Ostiolar neck crest-like, elongated and laterally compressed, 80–100 μm high, 160–300 μm wide, composed of 2.5–7 μm diam, globose, thick-walled, brown to black cells, with hyaline periphyses, surrounded by a well-developed clypeus (up to 390 μm wide). Peridium uniform, (30–)45–70 μm thick at side, composed of 8–9(–15) layers of elongated, thin-walled, 5–18 × 2.5–5 μm, brown cells, surrounded by brown hyphae. Pseudoparaphyses numerous, 1.5–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, clavate, 85–125 × 11–17.5 μm ( = 102.5 × 12.9 μm, n = 96), with a stipe (14–50 μm long,  = 26.3 μm, n = 24), apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 20–32.5 × 5–8 μm ( = 25.3 × 6.7 μm, n = 190), l/w 2.9–4.8 ( = 3.8, n = 190), hyaline, with a septum nearly median ((0.42–)0.45–0.55,  = 0.49, n = 167), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 2–5 μm long at both ends, with an internal chamber at both ends of ascospores. Asexual morph: Conidiomata pycnidial, globose to subglobose, up to 165 μm high, 135–180 μm diam, scattered to 3–6 grouped, superficial to immersed. Peridium 11.5–18.5 μm thick, composed of 2–4 layers of 8–13.5 × 3–4 μm, subglobose to rectangular, brown cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic, 6–12 × 2.5–4 μm, ampliform, hyaline, smooth. Conidia subglobose with rounded ends, 2–3(–4) × 1.2–1.8 μm ( = 2.4 × 1.5 μm, n = 60), l/w 1.2–2.2 ( = 1.6, n = 60), hyaline, smooth, aseptate, guttulate when young.

Culture characteristics: Colonies on PDA attaining 19 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, greenish grey (110; Rayner 1970); reverse dull green (70) (Fig. 14A); red pigment produced in water agar media (Fig. 3T); asexual morph formed.

Materials examined (all on submerged dead twigs of woody plant): Japan, Aomori, Hirosaki, Aoki, near Mohei-pond, 7 Dec. 2002, K. Tanaka & N. Asama, KT 970 (HHUF 27985 holotype designated here; ex-holotype culture CBS 143643 = JCM 13087 = MAFF 239597); ibid., 21 Jul. 2007, K. Hirayama & K. Tanaka, KH 56 (HHUF 30566; ex-paratype culture CBS 143639); ibid., 23 Sep. 2007, K. Hirayama & K. Tanaka, KH 91 (HHUF 30567; ex-paratype culture CBS 143640); ibid., 29 Oct. 2007, K. Hirayama & K. Tanaka, KH 104 (HHUF 30568; ex-paratype culture CBS 143641); ibid., 30 Aug. 2008, K. Hirayama & K. Tanaka, KH 185 (HHUF 30569; ex-paratype culture CBS 143642).

Notes: Crassiclypeus aquaticus was collected from submerged dead twigs of woody plants during summer, fall, and winter. Crassiclypeus aquaticus strains produced a red pigment in water agar medium.

A. Hashim., K. Hiray. & Kaz. Tanaka, gen. nov. MycoBank MB823133.

Etymology: Refers to its ostiolar neck, which resembles a Japanese fan.

Sexual morph: Ascomata scattered, immersed, subglobose to ellipsoidal. Ostiolar neck elongated, laterally compressed. Peridium composed of elongated, brown cells. Pseudoparaphyses septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a narrow bipolar sheath. Asexual morph: Conidiomata pycnidial, globose to subglobose. Peridium composed of subglobose to rectangular, brown cells. Conidiophores absent. Conidiogenous cells holoblastic, cylindrical or ampliform, hyaline, smooth. Conidia subglobose with rounded ends, hyaline, smooth, aseptate.

Type species: Flabellascoma minimum A. Hashim., K. Hiray. & Kaz. Tanaka.

Notes: The genus Flabellascoma is proposed to include F. cycadicola and F. minimum. These two species have well-developed, crest-like ostiolar necks (Fig. 4, Fig. 5) and a uniformly thickened ascomatal wall composed of 2 zones (Fig. 4, Fig. 5), and asci with a short stipe. Flabellascoma is morphologically similar to Pseudolophiostoma in having ascomata with a well-developed, crest-like ostiolar neck, and a peridium of uniform thickness. However, the ascomatal peridium in Pseudolophiostoma is composed of 1 zone (Thambugala , this study Fig. 8, Fig. 9, Fig. 10) rather than the 2 zones in Flabellascoma.

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823134. Fig. 4, Fig. 14.

Etymology: Refers to the generic name of the host plant.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 490–530 μm high, 600–620 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 190–210 μm high, 320–380 μm wide, composed of 3–6 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 45–50 μm thick at side, composed of 2 zones; outer zone 28–38 μm thick, composed of 5–8 layers of rectangular, thin-walled, 10–17 × 3–4 μm, brown cells; inner zone 12–25 μm thick, composed of globose, 1.5–2.5 μm diam, hyaline cells. Pseudoparaphyses numerous, 1–3 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 67.5–88 × 9–12 μm ( = 77.2 × 10.2 μm, n = 20), with a short stipe, apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 17–23 × 4.5–7 μm ( = 20.4 × 5.4 μm, n = 70), l/w 3.0–4.5 ( = 3.8, n = 70), hyaline, with a septum nearly median (0.47–0.55,  = 0.50, n = 70), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 7–10 μm long at both ends, with a lateral pad-like structure within the sheath, with an internal chamber at both ends of ascospores. Asexual morph: Conidiomata pycnidial, globose to subglobose, up to 90 μm high, 50–85 μm diam, 4–10 grouped, superficial. Peridium 7–13 μm thick, composed of 2–4 layers of 6.5–10.5 × 3.5–4 μm, subglobose to rectangular, brown cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells holoblastic, 6–8 × 1.5–2.5 μm, ampliform, hyaline, smooth. Conidia subglobose with rounded ends, 1.5–2.5 × 1.1–2 μm ( = 2.0 × 1.4 μm, n = 60), l/w 1.0–1.8 ( = 1.4, n = 60), hyaline, smooth, aseptate, guttulate when young.

Culture characteristics: Colonies on PDA attaining 20 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, greenish grey (110); reverse grey olivaceous (107) (Fig. 14B); asexual morph formed.

Material examined: Taiwan, Taipei, Wulai, on petiole of Cycas revoluta, 28 Nov. 2005, K. Tanaka, H.S. Chang & G. Okada, KT 2034 (HHUF 30570 holotype designated here; ex-holotype culture BCRC FU30901 = CBS 143644).

Notes: Flabellascoma cycadicola superficially resembles F. minimum, but can be distinguished from the latter by its larger ascospores (17–23 × 4.5–7 μm vs. 12–17.5 × 3.5–5 μm). The two species differ at 15 positions with one gap in their ITS regions.

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823135. Fig. 5, Fig. 14.

Etymology: Refers to its small-sized ascospore.

Sexual morph: Ascomata ellipsoidal to lageniform, scattered, immersed, dark brown to black, 250–320 μm high, 350–500 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 160–310 μm high, 210–280 μm wide, composed of 3–5 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 43–50(–62) μm thick at side, composed of 2 zones; outer zone 18–27(–42) μm thick, composed of 3–5 layers of rectangular, thin-walled, 5–18 × 5–8 μm, brown cells; inner layer 20–25 μm thick, composed of globose, 1–2 μm diam, hyaline cells. Pseudoparaphyses numerous, 1.5–3 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 45–77.5 × 7.5–12 μm ( = 61.9 × 9.3 μm, n = 123), with a stipe, apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 12–17.5 × 3.5–5 μm ( = 14.8 × 4.3 μm, n = 17), l/w 2.6–4.3 ( = 3.4, n = 170), hyaline, with a septum nearly median (0.46–0.54,  = 0.50, n = 170), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 5.5–8 μm long at both ends, with a lateral pad-like structure within the sheath, up to 1.5 μm wide at side, with an internal chamber at both ends of ascospores. Asexual morph: Conidiomata pycnidial, globose to subglobose, up to 125 μm high, 85–110 μm diam, 3–5 grouped, superficial. Peridium 12–17 μm thick, composed of 2–4 layers of 6.5–10 × 3.5–5 μm, subglobose to rectangular, brown cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells holoblastic, 7.5–9 × 2.5–4 μm, cylindrical, hyaline, smooth. Conidia subglobose with rounded ends, 1.9–2.4(–3.2) × 1.3–1.8 μm ( = 2.1 × 1.5 μm, n = 50), l/w 1.1–1.6(–2.1) ( = 1.3, n = 50), hyaline, smooth, aseptate, guttulate when young.

Culture characteristics: Colonies on PDA attaining 17 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, smoke grey (105); reverse sienna (8) (Fig. 14C); asexual morph formed.

Materials examined: Taiwan, Nantou Hsien, Hui Sun Forest Area, Kuan-Dau river, on petioles of Arenga engleri, 26 Nov. 2005, K. Tanaka, C.Y. Chen & G. Okada, KT 2013 (HHUF 30571; ex-paratype culture BCRC FU30900 = CBS 143645); Taipei, Wulai, on pods of Bauhinia purpurea, 28 Nov. 2005, K. Tanaka, H.S. Chang & G. Okada, KT 2040 (HHUF 30572 holotype designated here; ex-holotype culture BCRC FU30902 = CBS 143646).

Notes: ITS sequences of ex-holotype and ex-paratype cultures of F. minima isolated from Arecaceae (Arecales) and Fabaceae (Fabales), respectively, were identical. Although the ascomatal shape was slightly different between the holotype (lageniform, Fig. 5D) and paratype (ellipsoidal, Fig. 5E) of F. minima, the peridial structure of their ascomata and the ascospore size were almost identical. The differences in the ascomatal shape appeared to vary depending on the condition of the substrates. We, therefore, regard these specimens as conspecific.

A. Hashim., K. Hiray. & Kaz. Tanaka, gen. nov. MycoBank MB823136.

Etymology: Refers to its lenticular ascomata.

Sexual morph: Ascomata scattered, immersed, subglobose. Ostiolar neck elongated, laterally compressed, surrounded by a well-developed clypeus. Peridium composed of globose, brown cells. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a narrow bipolar sheath. Asexual morph: Undetermined.

Type species: Lentistoma bipolare (K.D. Hyde) A. Hashim., K. Hiray. & Kaz. Tanaka

Notes: Lophiostoma bipolare was originally described as a species of Massarina (Hyde 1995a). Liew transferred the species to Lophiostoma based on phylogenetic analyses of ITS sequences. This classification was corroborated by subsequent studies (Tanaka and Hosoya, 2008, Hirayama and Tanaka, 2011). Thambugala conducted comprehensive taxonomic revisions in Lophiostomataceae, but Lo. bipolare was not included. Thus, its generic placement remained unresolved. Our phylogenetic study revealed the distant relationship of this species to Lophiostoma s. str. (Fig. 2). Lentistoma is well-characterised and is differentiated from Lophiostoma by its clypeus around the ostiolar neck and by its thinner and uniformly thickened peridium (up to 45 μm in thickness, Fig. 6H–J).

(K.D. Hyde) A. Hashim., K. Hiray. & Kaz. Tanaka, comb. nov. MycoBank MB823137. Fig. 6, Fig. 14.

Basionym: Massarina bipolaris K.D. Hyde, Nova Hedwigia 61: 131. 1995.

Synonym: Lophiostoma bipolare (K.D. Hyde) E.C.Y. Liew et al., Mycologia 94: 812. 2002.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 160–200 μm high, 470–540 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 100–125 μm high, 210–225 μm wide, composed of globose, brown to black cells, with hyaline periphyses, surrounded by a well-developed clypeus (up to 500 μm wide). Peridium uniform, 25–45 μm thick at side, composed of 5–7 layers of rectangular, thin-walled, 12.5–15 × 5 μm, brown cells. Pseudoparaphyses numerous, 1–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (82–)105–140 × 8–15 μm ( = 119.9 × 10.9 μm, n = 30), with a stipe (7.5–18.5 μm long,  = 11.7 μm, n = 11), apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 20–33 × 5.5–9(–11) μm ( = 27.0 × 7.2 μm, n = 216), l/w 2.5–4.8 ( = 3.8, n = 216), hyaline, with a septum nearly median (0.46–0.55,  = 0.50, n = 216), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 5–10 μm long at both ends, with a cap-like structure at tips of the sheath, with an internal chamber at both ends of ascospores. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 16 mm diam within 21 d at 20 °C in the dark, velvety, plane, dull green (110); reverse grey olivaceous (107) (Fig. 14D, E); asexual morph formed.

Materials examined. Australia, Queensland, Kauri Creek, on woody plant, 23 May 2003 (HHUF 30576, dried culture specimen made from culture CBS 115370). China, Hong Kong, Tai Po Country Park, on submerged wood, Aug. 1993, K.D. Hyde (BRIP 21489, holotype); Sai Kung, Highland Reservoir, on submerged wood, 3 May 2003 (HHUF 30575, dried culture specimen made from culture JCM 14139 = CBS 110448); Mt. Nicholson, on woody plant, 9 Sep. 2003 (HHUF 30577, dried culture specimen made from culture CBS 115375, epitype designated here; MBT379010). Japan, Okinawa, Isl. Iriomote, near Kampire waterfall, on dead herbaceous plant, 27 Sep. 2007, K. Tanaka & H. Yonezawa, KT 2415 (HHUF 30573; culture CBS 143651); ibid., on dead twigs of woody plant, 5 Aug. 2012, K. Tanaka, KT 3056 (HHUF 30574; culture CBS 143652); Oomijya river, on submerged dead twigs of woody plant, 22 Nov. 2008, K. Hirayama & K. Tanaka, KH 214 (HHUF 30578; culture CBS 143647); ibid., on submerged dead twigs of woody plant, 12 Jul. 2011, K. Hirayama & K. Tanaka, KH 311 (HHUF 30581; culture CBS 143650); near Maryudu water falls, on herbaceous plant, 21 Nov. 2008, K. Hirayama & K. Tanaka, KH 216 (HHUF 30579; culture CBS 143648); ibid., on submerged dead twigs of woody plant, 21 Nov. 2008, K. Hirayama & K. Tanaka, KH 222 (HHUF 30580; culture CBS 143649).

Notes: Our phylogenetic and morphological studies revealed 11 species scattered among Lophiostomataceae (Fig. 1, Fig. 2). They were originally misidentified as Lo. bipolare based on the morphological resemblance of their ascospores, but a precise morphological observation of the Lo. bipolare complex including its holotype (BRIP 21489) distinguished the Lo. bipolare s. str. from other species of the Lo. bipolare complex on the basis of a clypeus around the ostiolar neck (Fig. 6H–J); an internal chamber at both ends of the ascospores (Fig. 6U); and a bipolar sheath with a cap-like structure at the tips (Fig. 6T). Here, we designated an epitype specimen (HHUF 30577) that was collected from the same country as the holotype specimen. Although the species was previously reported to have been collected from either freshwater or marine habitats (Hyde et al., 2002, Shearer and Raja, 2010), this is the first report of the species from a terrestrial habitat.

A. Hashim., K. Hiray. & Kaz. Tanaka, gen. nov. MycoBank MB823138.

Etymology: Refers to the thin peridium of the ascomata.

Sexual morph: Ascomata scattered, immersed, subglobose. Ostiolar neck elongated, laterally compressed. Peridium relatively thin, composed rectangular, brown cells. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a narrow bipolar sheath. Asexual morph: Undetermined.

Type species: Leptoparies palmarum A. Hashim., K. Hiray. & Kaz. Tanaka.

Notes: Leptoparies is a new monotypic genus characterised by a relatively thinner and non-carbonised peridium, which represents an atypical character for Lophiostomataceae. Leptoparies can be easily distinguished from other genera by the thin peridium composed of rectangular cells and the absence of the surrounding brown hyphae (Fig. 7J). The genus is similar to Capulatispora due to the thin peridium and the ascospores with the drawn-out sheaths; however, Capulatispora differs from Leptoparies due to its short ascus stipe (Tanaka and Hosoya, 2008, Thambugala et al., 2015).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823139. Fig. 7, Fig. 14.

Etymology: Refers to the host plant.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 210–320 μm high, 490–650 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 90–140 μm high, 200–300 μm wide, composed of 6–8 × 3–4 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 25–32 μm thick at side, composed of 3–5 layers of rectangular, thin-walled, 8–10 × 3–7 μm, brown cells, surrounded by brown hyphae. Pseudoparaphyses numerous, 1.5–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (67–)77–118 × 10–14 μm ( = 93.9 × 11.9 μm, n = 20), with a stipe (8.5–18.5 μm long,  = 13.5 μm, n = 20), apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 20–25 × 5–7 μm ( = 23.1 × 6.1 μm, n = 100), l/w 2.8–4.4 ( = 3.5, n = 100), hyaline, with a septum mostly supramedian (0.47–0.55,  = 0.49, n = 100), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 6–8 μm long at both ends, with a lateral pad, up to 1.5 μm wide at side. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 21 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, smoke grey (105); reverse grey olivaceous (107) (Fig. 14F); sexual morph formed.

Material examined: Japan, Kanagawa, Yokohama, Nakaku, near Sankei-garden, on petioles of Trachycarpus fortunei, 9 Mar. 2004, K. Tanaka & Y. Harada, KT 1653 (HHUF 28983 holotype designated here; ex-holotype culture CBS 143653 = JCM 13089 = MAFF 239599).

Notes: Leptoparies palmarum and Flabellascoma minimum can be found on the petioles of palms. The former species is characterised by the larger ascospores (20–25 × 5–7 μm) distinguishing it from the latter species, which has smaller ascospores (12–17.5 × 3.5–5 μm).

Thambug. et al., Fungal Diversity 74: 235. 2015.

Sexual morph: Ascomata scattered, immersed, globose to subglobose. Ostiolar neck elongated, laterally compressed. Peridium composed of rectangular, dark brown to black cells. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a narrow bipolar sheath. Asexual morph: Undetermined.

Type species: Pseudolophiostoma vitigenum (Kaz. Tanaka & Y. Harada) Thambug. et al.

Notes: Thambugala established the monotypic genus Pseudolophiostoma to accommodate its generic type Pseudol. vitigenum, which was found on Vitis coignetiae (Vitaceae), and was collected from a boreal region in Japan. Although this species was considered a member of Lophiotrema (Tanaka & Harada 2003), phylogenetic analyses based on SSU and LSU sequences revealed that this species belonged to Lophiostoma (Hirayama & Tanaka 2011). Later, a monotypic genus Pseudolophiostoma was introduced to segregate L. vitigenum from Lophiostoma s. str. based on phylogenetic analyses (Thambugala ). In our study, three new species (Pseudol. cornisporum, Pseudol. obtusisporum, and Pseudol. tropicum), collected from a subtropical region in Japan, were introduced. These species are also characterised by an ascomatal wall having uniform thickness (Fig. 8, Fig. 9, Fig. 10) and asci with a relatively long stipe (up to 18.5 μm) (Fig. 8, Fig. 9, Fig. 10L, M). Although Pseudolophiostoma possess morphologically similar ascospores to those of Lentistoma, the latter genus is easily differentiated from Pseudolophiostoma by having a clypeated ostiolar neck.

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823140. Fig. 8, Fig. 14G.

Etymology: Refers to the ascospores with acute ends.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 650–700 μm high, 580–650 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 100–190 μm high, 120–140 μm wide, composed of 2–3 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 12–17 μm thick at side, composed of 4–5 layers of rectangular, thin-walled, 8–12 × 3–4 μm, brown cells, surrounded by dark brown hyphae. Pseudoparaphyses numerous, 1.5–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 80–92 × 11–15(–18) μm ( = 86.4 × 13.6 μm, n = 5), with a long stipe (8–15 μm long,  = 11.6 μm, n = 7), apically rounded with an ocular chamber, 8-spored. Ascospores fusiform with acute ends, 21–32 × 4.5–6 μm ( = 26.6 × 5.3 μm, n = 60), l/w (3.9–)4.1–6.3 ( = 5.0, n = 60), hyaline, with a septum nearly median (0.47–0.56,  = 0.51, n = 60), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 2–11 μm long at both ends, with a lateral pad-like structure within the sheath, up 2.5 μm wide at side. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 14–18 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, olivaceous buff (89); reverse olivaceous (48) (Fig. 14G); sexual morph formed.

Material examined: Japan, Okinawa, Isl. Iriomote, near Sonai trail, on dead stem of herbaceous plant, 13 Jul. 2011, K. Hirayama & K. Tanaka, KH 322 (HHUF 30582 holotype designated here; ex-holotype culture CBS 143654 = JCM 32348).

Notes: This species resembles Pseudol. vitigenum by having ascospores with acute ends. Ascospores of the new species are smaller (21–32 × 4.5–6 μm), while those of Pseudol. vitigenum are larger ((30.5–)34–44(–51) × (8–)9–11.5(–13) μm; Thambugala ).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823141. Fig. 9, Fig. 14.

Etymology: Refers to the ascospores with obtuse ends.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 350–400 μm high, 250–350 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 110–200 μm high, 150–250 μm wide, composed of 2–4 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 10–17 μm thick at side and base, composed of 3–4 layers of rectangular, thin-walled, 8–13 × 3–4 μm, brown cells, surrounded by dark brown hyphae. Pseudoparaphyses numerous, 1.5–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (81–)103–140 × 8–15 μm ( = 119.9 × 10.9 μm, n = 30), with a long stipe (7.5–18.5 μm long,  = 11.7 μm, n = 11), apically rounded with an ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, (20–)23.5–31.5 × 4–7 μm ( = 27.3 × 5.5 μm, n = 90), l/w 3.5–6.9 ( = 5.0, n = 90), hyaline, with a septum nearly median (0.47–0.55,  = 0.51, n = 90), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 5–11 μm long at both ends, with a lateral pad-like structure within the sheath, up to 3 μm wide at side. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 22–29 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, lavender grey (125); reverse smoke grey (105) (Fig. 14H); sexual morph formed.

Materials examined: Japan, Okinawa, Isl. Iriomote, near Midara river, on dead stem of herbaceous plant, 22 Nov. 2008, K. Hirayama & K. Tanaka, KH 228 (HHUF 30584; ex-paratype culture CBS 143655); Isl. Ishigaki, Mt. Banna, near small stream, on dead stem of herbaceous plant, 14 Jul. 2011, K. Hirayama & K. Tanaka, KH 336 (HHUF 30585; ex-paratype culture CBS 143656); ibid., on dead stem of herbaceous plant, 14 Jul. 2011, K. Tanaka & K. Hirayama, KT 2838 (HHUF 30583 holotype designated here; ex-holotype culture CBS 143657 = JCM 32349); Tokyo, Ogasawara Islands, Isl. Hahajima, Shizukasawa, on dead stem of Livistona boninensis, 14 Sep. 2012, K. Tanaka, A. Hashimoto, T. Ono & T. Sato, KT 3098 (HHUF 30171; ex-paratype culture CBS 143941 = MAFF 243969); Isl. Chichijima, near Mt. Yoake, on dead stem of Stachytarpheta jamaicensis, 9 Sep. 2012, K. Tanaka, A. Hashimoto, T. Ono & T. Sato, KT 3119 (HHUF 30189; ex-paratype culture CBS 143658 = MAFF 243983).

Notes: Pseudolophiostoma obtusisporum is commonly distributed among subtropical islands in Japan and is found on various herbaceous plants or palm trees. The wall of the ascomata is composed of equal thickness among specimens on herbaceous plants (Fig. 9G) and palm trees (Fig. 9H). Other morphological features, such as the ascospore size, were identical and ITS sequences among these isolates also completely matched. Thus, we regard these isolates as conspecific. Pseudolophiostoma obtusisporum can be distinguished from other Pseudolophiostoma species by its obtuse-ended ascospores (Fig. 9P–T).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823142. Fig. 10, Fig. 14.

Etymology: Refers to the species occurring in the tropical regions.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 530–880 μm high, 550–840 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 100–160 μm high, 100–200 μm wide, composed of 2–4(–7) μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 15–22 μm thick at side and base, composed of 3–4 layers of rectangular, thin-walled, 9–13(–18) × 3–5 μm, brown cells, surrounded by dark brown hyphae. Pseudoparaphyses numerous, 1–1.5 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (75–)85–120 × 10–15 μm ( = 104.4 × 12.3 μm, n = 14), with a long stipe (8–18.5 μm long,  = 11.7 μm, n = 10), apically rounded with an ocular chamber, 8-spored. Ascospores fusiform with acute ends, 23.5–29.5 × 4–6 μm ( = 26.3 × 5.4 μm, n = 70), l/w 4.0–6.3 ( = 4.9, n = 70), hyaline, with a septum nearly median (0.46–0.56,  = 0.51, n = 70), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 6–9 μm long at both ends, with a lateral pad-like structure within the sheath, up 2 μm wide at side. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 24–27 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, greyish blue (97); reverse grey olivaceous (107) (Fig. 14I); sexual morph formed.

Materials examined: Japan, Okinawa, Isl. Iriomote, Takana, on dead stem of herbaceous plant, 13 Jul. 2011, K. Hirayama & K. Tanaka, KH 352 (HHUF 30586 ex-paratype culture CBS 143659); Tokyo, Ogasawara Islands, Isl. Chichijima, Buta coast, on dead stem of Bidens pilosa var. radiata, 15 Sep. 2012, K. Tanaka, A. Hashimoto & T. Sato, KT 3134 (HHUF 30202 holotype designated here; ex-holotype culture CBS 143660 = MAFF 243989).

Notes: In culture, Pseudol. tropicum produced ascomata that were slightly different from those on natural substrates, with a slightly thicker peridium and a well-developed ostiolar neck (Fig. 10F, H, I). Although these differences were observed, the anatomical structure of the ascomatal wall formed in culture were identical to those on natural specimens.

Both Pseudol. tropicum and Pseudol. obtusisporum have ascospores overlapping in size, but can be distinguished by the ascospore shape. Obtuse-ended ascospores were identified as Pseudol. obtusisporum (Fig. 9P–T) and acute-ended ascospores as Pseudol. tropicum (Fig. 10P–U). ITS sequences between these species differed in nine nucleotide positions with three gaps.

A. Hashim., K. Hiray. & Kaz. Tanaka, gen. nov. MycoBank MB823143.

Etymology: Refers to its morphological resemblance to Paucispora.

Sexual morph: Ascomata scattered, immersed, subglobose. Ostiolar neck elongated, laterally compressed. Peridium composed of rectangular, brown cells. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical to clavate, 8-spored. Ascospores fusiform, brown, 1-septate, smooth, with a narrow bipolar sheath. Asexual morph: Conidiomata pseudopycnidial, globose to cylindrical, sometimes deformed, confluent, multiloculate, scattered, semi-immersed, black. Ostiolar neck papillate. Peridium composed of rectangular, brown cells. Conidiophores absent. Conidiogenous cells holoblastic, ampliform to cylindrical, hyaline, smooth. Conidia cylindrical with rounded ends, hyaline, smooth, aseptate, guttulate when young.

Type species: Pseudopaucispora brunneospora A. Hashim., K. Hiray. & Kaz. Tanaka.

Notes: Pseudopaucispora is introduced to accommodate Pseudop. brunneospora, which is characterised by small brown ascospores and pseudopycnidial conidiomata. Pseudopaucispora is superficially similar to Paucispora (Thambugala ). However, Pseudopaucispora has an ascomatal peridium composed of 1 zone and an ascus with a short stipe, while Paucispora is characterised by a peridium composed of 2 zones and an ascus with a relatively long stipe (up to 34 μm in length; Thambugala ).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823144. Fig. 11, Fig. 14.

Etymology: Refers to its brown ascospores.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 210–300 μm high, 215–355 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 145–175 μm high, 95–190 μm wide, composed of 2–4 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium uniform, 15–18 μm thick at side and base, composed of rectangular, thin-walled, 6–16 × 3–4 μm, brown cells. Pseudoparaphyses numerous, 1–1.5 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 67–89 × 7–10.5 μm ( = 75.0 × 8.7 μm, n = 20), with a short stipe (6.5–15 μm long,  = 11.9 μm, n = 20), apically rounded with an ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 14–17 × 3.5–4.5 μm ( = 15.3 × 4.0 μm, n = 50), l/w 3.0–4.7 ( = 3.8, n = 50), brown, with a septum usually supramedian (0.44–0.56,  = 0.48, n = 50), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 6–10 μm long at both ends. Asexual morph: Conidiomata pseudopycnidial, globose to cylindrical, up to 230 μm high, 150–190 μm diam, sometimes deformed, confluent, multiloculate, scattered, semi-immersed, black. Ostiolar neck mainly single, occasionally three, papillate. Peridium 10–18 μm wide, composed of 7.5–16.5 × 3–4 μm, rectangular, brown cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells holoblastic, 10–15 × 2.5–4.5 μm, ampliform to cylindrical, hyaline, smooth. Conidia cylindrical with rounded ends, 2–3(–3.5) × 1–1.3 μm ( = 2.8 × 1.1 μm, n = 50), l/w 1.8–3.1 ( = 2.6, n = 50), hyaline, smooth, aseptate, guttulate when young.

Culture characteristics: Colonies on PDA attaining 9–15 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, pale luteous (11); reverse sienna (8) (Fig. 14J); asexual and sexual morph formed.

Material examined: Japan, Okinawa, Isl. Yonaguni, near Kubura pond, on dead stem of Asteraceae sp., 23 Nov. 2008, K. Hirayama & K. Tanaka, KH 227 (HHUF 30587 holotype designated here; ex-holotype culture CBS 143661 = JCM 32350).

Note: Pseudopaucispora can be easily distinguished from the other Lo. bipolare complex by the brown ascospores, which possesses a sheath without a lateral pad-like structure (Fig. 11J–O).

K.D. Hyde, Nova Hedwigia 61: 234. 1995.

Sexual morph: Ascomata scattered, immersed, subglobose. Ostiolar neck elongated, laterally compressed, with hyaline periphyses. Peridium composed of rectangular, brown cells, surrounded by dark brown hyphae. Pseudoparaphyses numerous, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, 8-spored. Ascospores fusiform, hyaline, 1-septate, with a bipolar or entire sheath. Asexual morph: Undetermined.

Type species: Vaginatispora aquatica K.D. Hyde.

Notes: The genus Vaginatispora was established to accommodate V. aquatica, which was found on submerged twigs of woody plants and was originally characterised by Massarina-like ascomata with much longer ostiolar necks and ascospores bearing an entire sheath (Hyde 1995b). Liew suggested that this genus was related to Lophiostoma, according to the phylogenetic analyses using ITS sequences. However, the authors could not determine the fundamental differences between these two genera. Thus, no taxonomic conclusion regarding whether Vaginatispora was synonymous with Lophiostoma was drawn. Subsequently, the genus was considered to be synonymous with Lophiostoma due to its phylogenetic affinities to the latter genus (Zhang ). Thambugala recently retained Vaginatispora, emphasising the structures of the peridium and asci, as well as based on results of their multi-locus phylogenetic analyses. They accepted V. fuckelii (formerly Lo. fuckelii) as a member of the genus. Although this species was morphologically atypical in the genus because of the 2 zoned peridium, this proposal was accepted by subsequent studies (Wanasinghe et al., 2016, Tibpromma et al., 2017). Our phylogenetic analyses showed a paraphyletic nature of Vaginatispora sensu Thambugala (Fig. 2). We re-circumscribed the genus to include five species with well-developed ascomatal peridium at the sides, while poorly-developed at the base, with numerous brown hyphae around the ascomata, and asci with a broad ocular chamber. Vaginatispora fuckelii is excluded from Vaginatispora and transferred to its own new genus, Neovaginatispora (see Appendix B).

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823145. Fig. 12, Fig. 14.

Etymology: Refers to the generic name of the host plant.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 330–360 μm high, 480–500 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 150–225 μm high, 275–445 μm wide, composed of 3–7 μm diam, globose to elongated, brown to black cells, with hyaline periphyses. Peridium 37.5–62.5 μm thick at side, composed of 9–15 layers of rectangular, thin-walled, 11–13 × 4–5 μm, brown cells, surrounded by dark brown hyphae; 10–17.5 μm thick at base, composed of globose, 6–10 μm diam, pale brown cells. Pseudoparaphyses numerous, 1.5–2.5 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (81–)100–140 × (11.5–)16.5–22.5 μm ( = 115.0 × 18.5 μm, n = 53), with a short stipe (8.5–16 μm long,  = 12.0 μm, n = 10), apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 25–34(–37) × 7–10.5 μm ( = 30.6 × 8.8 μm, n = 120), l/w 2.6–4.6 ( = 3.5, n = 120), hyaline, with a septum nearly median (0.45–0.58,  = 0.51, n = 120), slightly constricted at the septum, smooth, with a narrow sheath. Sheath drawn out 6–8 μm long at both ends, with a lateral pad-like structure within the sheath, with an internal chamber at both ends of ascospores. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 18 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, greenish grey (110); reverse dull green (70) (Fig. 14K); sexual morph formed.

Material examined: Japan, Wakayama, Kinokawa, Kishigawa, Kita, on endocarp of Amygdalus persica, 9 May 2007, S. Hatakeyama, KT 2248 (HHUF 30588 holotype designated here; ex-holotype culture CBS 143662 = JCM 32351).

Notes: Vaginatispora amygdali is morphologically similar to V. armatispora, but ascospores of the latter species are slightly larger (28–39.2 × 7–9.8 μm; Hyde ). ITS sequences of V. amygdali and V. armatispora (AF383955), which were derived from an authentic specimen of the species, differed in 17 positions with five gaps.

This species is difficult to distinguish from other Lo. bipolare complexes based on ascospore features, but detailed features of the ascomata and asci are well-matched to the characteristics present in Vaginatispora.

A. Hashim., K. Hiray. & Kaz. Tanaka, sp. nov. MycoBank MB823146. Fig. 13, Fig. 14.

Etymology: Refers to its verruciform ascospores.

Sexual morph: Ascomata subglobose, scattered, immersed, dark brown to black, 220–340 μm high, 340–360 μm diam. Ostiolar neck crest-like, elongated, laterally compressed, 88–120 μm high, 175–225 μm wide, composed of 3–5 μm diam, globose, brown to black cells, with hyaline periphyses. Peridium 18–28 μm thick at side, composed of 3–5 layers of rectangular, thin-walled, 10–11 × 3–5 μm, brown cells, surrounded by dark brown hyphae (1–1.5 μm wide); 10–17.5 μm thick at base, composed of globose, brown cells. Pseudoparaphyses numerous, 1.5–2 μm wide, septate, branched and anastomosed. Asci bitunicate, fissitunicate, cylindrical-clavate, (77.5–)95–115 × 15–20 μm ( = 102.3 × 16.5 μm, n = 10), with a short stipe (7.5–17.5 μm long,  = 13.0 μm, n = 10), apically rounded with a broad ocular chamber, 8-spored. Ascospores fusiform with obtuse ends, 20–23 × 5–6 μm ( = 21.9 × 5.9 μm, n = 50), l/w 2.9–3.5 ( = 3.3, n = 50), hyaline, with a septum supramedian (0.42–0.49,  = 0.47, n = 50), slightly constricted at the septum, verrucous, with a narrow sheath. Sheath drawn out 5–7 μm long at both ends, with a lateral pad-like structure within the sheath, with an internal chamber at both ends of ascospores. Asexual morph: Undetermined.

Culture characteristics: Colonies on PDA attaining 19 mm diam within 21 d at 20 °C in the dark, velvety, centrally raised, smoke grey (105); reverse smoke grey (105) (Fig. 14L); sexual morph formed.

Material examined: Japan, Okinawa, Isl. Iriomote, near Shiira river (intertidal region), on submerged dead twigs of Rhizophora mucronata, 25 Sep. 2007, K. Tanaka & H. Yonezawa, KT 2443 (HHUF 30589 holotype designated here; ex-holotype culture CBS 143663 = JCM 32352).

Notes: Vaginatispora scabrispora is easily distinguished from other species of Vaginatispora due to its verrucous ascospores (Fig. 13R) and mangrove habitat. This species is phylogenetically related to V. amygdali, but differs from the latter by the smaller sized ascospores (vs. 25–34(–37) × 7–10.5 μm in the latter species).

Discussion

Generic delimitation in Lophiostomataceae

Lophiostoma bipolare has a worldwide distribution in freshwater and marine habitats, and is characterised by ascomata with a slit-like ostiolar neck surrounded by a clypeus and ascospores with a bipolar sheath (e.g. Hyde, 1995a, Hyde et al., 2002, Liew et al., 2002). This species was originally treated as a member of Massarina (Hyde 1995a). Later, Liew transferred this species to Lophiostoma based on the results of molecular phylogenetic analyses using ITS region. The genus Lophiostoma was taxonomically revised on the basis of the phylogenetic analyses of multi-locus genes (Thambugala ). Lophiostoma bipolare was not included in the analyses and thus the taxonomic position of this species has remained unclear. Our phylogenetic analyses, which included 29 strains provisionally identified as Lo. bipolare, indicate that the species is not monophyletic (Fig. 1) and is scattered into seven genera and 11 species within Lophiostomataceae (Fig. 2). The present data also indicate that Lo. bipolare s. str. is phylogenetically distinct from Lophiostoma s. str. and should be separately placed in the novel genus Lentistoma (Fig. 2). Lentistoma is clearly different from other lophiostomataceous genera owing to its well-developed clypeus around the ostiolar neck (Fig. 6H–J). Other Lo. bipolare complexes are scattered among six distinct genera that are morphologically defined and whose monophyly is strongly supported (Fig. 2). As mentioned in previous studies (Chesters and Bell, 1970, Holm and Holm, 1988, Hyde, 1995b), the length of the ostiolar neck and the peridium thickness varies both on natural substrate and in culture (Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 13). The differences in the ascomatal shape are used to differentiate between several lophiostomataceous genera (Thambugala ). However, our results suggest that it may also vary depending on the condition of the substrates (herbaceous or woody plants) within the same species. For example, the ascomata of F. minimum and Len. bipolare found on woody plants were flattened at the base, while those on herbaceous plants were ellipsoidal (Fig. 5, Fig. 6). Although the length of the ostiolar neck, peridium thickness, and ascomatal forms were unstable characteristics depending on different conditions, their peridial features, such as the existence of the clypeus (Crassiclypeus, Lentistoma; Fig. 3, Fig. 6), the brown hyphae surrounding the peridium (Crassiclypeus, Vaginatispora; Fig. 3, Fig. 12, Fig. 13), the contexture of the peridium with 1 zone (Crassiclypeus, Lentistoma, Leptoparies, Pseudolophiostoma, Pseudopaucispora, Vaginatispora; Fig. 3, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13) or with 2 zones (Flabellascoma; Fig. 4, Fig. 5), were always stable even on different hosts or culture conditions (Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 13). These anatomical differences could be useful for generic circumscriptions. Thus, we treated these seven genera as distinct, their monophyly being strongly supported (Fig. 2). Additionally, clear morphological differences were observed in the asexual morphs of Crassiclypeus, Flabellascoma, and Pseudopaucispora; pycnidial conidiomata and phialidic conidiogenous cells (Crassiclypeus; Fig. 3W, Y–AA); pycnidial conidiomata and holoblastic conidiogenous cells (Flabellascoma; Fig. 4, Fig. 5); and pseudopycnidial conidiomata and holoblastic conidiogenous cells (Pseudopaucispora; Fig. 11S, T, V, W).

Resolution of the Lo. bipolare complex led us to reconsider the generic placement of unresolved species and the generic delimitations of the broadly defined genera from previous studies. Thambugala retained Vaginatispora as a natural genus and subsequent studies accepted four species in this genus according to the results of phylogenetic analyses (Wanasinghe et al., 2016, Tibpromma et al., 2017). On the other hand, multi-locus phylogenetic analyses revealed the paraphyletic nature of this genus in the present study (Fig. 2). The morphological observations suggested that the genus was restricted to V. amygdali, V. appendiculata, V. armatispora, V. aquatica, and V. scabrispora, although V. armatispora was not included in our phylogenetic analyses due to the limited availability of the sequence data. Vaginatispora fuckelii is atypical for this genus, because this species possesses a thinner peridium (up to 25 μm in thickness) that is uniformly thick and composed of 2 zones (Thambugala ). Therefore, we propose a new genus, Neovaginatispora, to accommodate this species (see Appendix B). Lophiostoma pseudoarmatisporum was introduced as a species of Lophiostoma s. lat. Lophiostoma pseudoarmatisporum is characterised by fusiform, hyaline ascospores with thin mucilaginous appendages (Li ). The authors did not resolve the generic placement of the species (Hyde ). The species is phylogenetically related to Crassiclypeus, Flabellascoma, Leptoparies, and Paucispora in our phylogenetic trees (Figs 1, S1A–C), but can be distinguished from these genera by the peridium that is composed of 1 zone and an ostiolar neck without the clypeus. Thus, a new monotypic genus, Parapaucispora, is proposed for Par. pseudoarmatispora (see Appendix B). The validity of the genera Alpestrisphaeria, Coelodictyosporium, Guttulispora, Lophiohelichrysum, Platystomum, and Sigarispora remain questionable. Most of these genera were originally divided from Lophiostoma based on insufficient features, such as the form of the ascomata and the ascospore colour and septation, and comprised single species and strain (Thambugala ). Further discovery of more specimens along with additional morphological and molecular data will help to fully elucidate the taxonomic validity of these problematic genera in Lophiostomataceae.

Form and function of the ascospore sheath

Ascospores of the Lo. bipolare complex possess a gelatinous sheath that may help these organisms to attach to plant substrates in aquatic or marine habitats (Shearer, 1993, Hyde and Goh, 2003, Jones, 2006). Several terrestrial ascomycetes with appendaged ascospores have been reported from moist environments near a waterfall (Wanasinghe ), a humid subtropical mountain (Tanaka & Hosoya 2008), and bamboo (Hashimoto ). It is interesting to note that most of the Lo. bipolare complexes were also collected from terrestrial habitats (Table 1). Jones (2006) suggested that these appendaged ascospores adapt to small watery environments in terrestrial habitats.

Ascospore characteristics are particularly useful in species identification of freshwater or marine fungi. Several morphological variations of the ascospore sheath were observed in Lophiostomataceae (Read et al., 1994, Read et al., 1997, Tsui et al., 1999, Au et al., 1999, Hyde et al., 2002). Capulatispora sagittiforme and Lentistoma bipolare have ascospores with bipolar sheaths, providing cap-like structures at the tips (Tanaka and Hosoya, 2008, Thambugala et al., 2015, this study Fig. 6T). Tanaka & Hosoya (2008) indicated that the bipolar appendages with the cap-like structures of the ascospores may contribute to the settlement of the discharged ascospore on the substrate. The lateral, gelatinous, pad-like structure was observed in most Lo. bipolare complexes (Fig. 4, Fig. 5, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 12, Fig. 13), which is suspected to contribute to their attachment to plant substrates in aquatic or marine habitats, as evident in other freshwater fungi (Jones, 2006, Shearer et al., 2009). The presence of the internal chamber or inner spine structure at both ends of the ascospore sheath was observed in Capulatispora, Crassiclypeus, Flabellascoma, Lentistoma, Leptoparies, Pseudopaucispora, and Vaginatispora (Read et al., 1997, Hyde et al., 2002, Tanaka and Hosoya, 2008, this study, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 11, Fig. 12, Fig. 13). Ultrastructural examination of Len. bipolare ascospores suggested that the chamber is comprised of concentrated fibrillar material (Read et al., 1997, Hyde et al., 2002). Although these morphological variations are considered a result of the adaptation to their habitats, their taxonomic importance remains unclear (Hyde ). Our phylogeny showed that these structures may have evolved several times within Lophiostomataceae (Fig. 2). From these results, it appears that the ascospore sheath has less importance to the understanding of the phylogenetic relationships as already reported (Shearer ). Identification with an emphasis on these ascospore features alone may lead to misidentification of morphologically similar genera. A typical example of morphological convergence was reported in the study of “Massarina” ingoldiana s. lat. (Hirayama ), in which the unrelated species complex was comprised of two distinct lineages at the familial level. Precise re-identification of the Lo. bipolare complex based on detailed morphological characteristics (not only ascospores, but also ascomatal features) and molecular evidences will be needed to reveal their generic placements, accurate geographical distributions, ecological traits, and species diversity.

Intraspecific variability of morphological features has traditionally been reported in lophiostomataceous species, even within a single specimen. This complicates their generic and species delimitations (Chesters and Bell, 1970, Holm and Holm, 1988). Although recent studies on Lophiostomataceae using multiple genes revealed new lineages (Thambugala et al., 2015, Li et al., 2016, Wanasinghe et al., 2016, Tibpromma et al., 2017), 16 genera were separated from Lophiostoma based on insufficient morphological features or unclear generic circumscriptions that relied mainly on the molecular phylogenetic topologies. The present data provide insight into the complicated delimitations within the Lophiostomataceae genera and revealed the taxonomic importance of anatomical characteristics of the ascomata, which were previously unclear. To build a stable and comprehensive taxonomic framework, detailed morphological observations based on multiple specimens and comparisons of morphological variability both on natural substrate and in culture, as well as molecular data with high resolution, such as rpb2, will be needed. The data will be helpful to resolve the taxonomic placement of genera within this family and to validate the various problematic genera that currently exist in Lophiostomataceae.