Pisorisporiales, a new order of aquatic and terrestrial fungi for Achroceratosphaeria and Pisorisporium gen. nov. in the Sordariomycetes.

Four morphologically similar specimens of an unidentified perithecial ascomycete were collected on decaying wood submerged in fresh water. Phylogenetic analysis of DNA sequences from protein-coding and ribosomal nuclear loci supports the placement of the unidentified fungus together with Achroceratosphaeria in a strongly supported monophyletic clade. The four collections are described as two new species of the new genus Pisorisporium characterised by non-stromatic, black, immersed to superficial perithecial ascomata, persistent paraphyses, unitunicate, persistent asci with an amyloid apical annulus and hyaline, fusiform, cymbiform to cylindrical, transversely multiseptate ascospores with conspicuous guttules. The asexual morph is unknown and no conidia were formed in vitro or on the natural substratum. The clade containing Achroceratosphaeria and Pisorisporium is introduced as the new order Pisorisporiales, family Pisorisporiaceae in the class Sordariomycetes. It represents a new lineage of aquatic fungi. A sister relationship for Pisorisporiales with the Lulworthiales and Koralionastetales is weakly supported by Bayesian inference and maximum likelihood analyses. The systematic position of Pisorisporium among morphologically similar perithecial ascomycetes is discussed.

INTRODUCTION

The genus Achroceratosphaeria was described for perithecial ascomycetes that are morphologically similar to Ceratosphaeria and Pseudohalonectria of the Magnaporthaceae (Réblová et al. 2010).

In the phylogeny inferred from sequences of the small and large subunits of nuclear ribosomal DNA (nuc18S and nuc28S rDNA) Achroceratosphaeria has been placed within Sordariomycetes incertae sedis; it was nested in a weakly supported clade as sister to the Lulworthiales and Koralionastetales containing fungi from predominantly marine habitats (Kohlmeyer 1997, Kohlmeyer et al. 2000, Campbell et al. 2005, 2008). Achroceratosphaeria comprises two freshwater and one terrestrial species characterised by minute, immersed, subhyaline to pale brown ascomata with a fragile, hyaline to pale brown protruding neck, tapering paraphyses, unitunicate stipitate asci with a nonamyloid apical annulus and eight hyaline, septate, ellipsoidal to fusiform ascospores. The asexual morph is unknown.

Four specimens of an unidentified fungus were collected on deciduous wood submerged in fresh water in France and Belgium during the years 2006–2014. They are characterised by non-stromatic, immersed to superficial papillate perithecial ascomata, persistent paraphyses, unitunicate asci with an amyloid apical annulus and hyaline, fusiform, cylindrical to cymbiform, transversely multiseptate ascospores with numerous guttules. No conidia were formed in vitro or on the natural substratum containing ascomata. In ascospore morphology, the unknown fungus resembles members of Ceratosphaeria, Ceratosphaerella and Pseudohalonectria of the Magnaporthaceae (Shearer 1989, Huhndorf et al. 2008).

Preliminary analysis of the three phylogenetic markers nuc18S rDNA, nuc28S rDNA and the second largest subunit of RNA polymerase II (rpb2) revealed that three strains of the unidentified fungus are closely related to Achroceratosphaeria. A sister relationship with the Lulworthiales and Koralionastetales as a basal group to the Hypocreomycetidae was suggested. In the Hypocreomycetidae four lineages contain mostly aquatic fungi. Marine fungi characterised by considerable morphological and ecological diversity are accommodated in the Lulworthiales/Koralionastetales clade. Other marine fungi are placed in the Halosphaeriaceae of the Microascales and in four families introduced for genera of the so-called TBM clade 'Torpedospora/Bertia/Melanospora' (Kohlmeyer 1972, Spatafora et al. 1998, Schoch et al. 2007, Jones et al. 2014), while Savoryellales comprises usually lignicolous species found in freshwater and brackish water habitats (Boonyuen et al. 2011). Other nonstromatic freshwater fungi are placed in the Sordariomycetidae in the Annulatascaceae (Wong et al. 1998, Ho & Hyde 2000, Campbell & Shearer 2004) and in other numerous small or monotypic genera of uncertain position (Hyde et al. 1997, 1999, 2000, Ho et al. 1999, Ranghoo et al. 2000, 2001, Raja et al. 2003, Vijaykrishna et al. 2005, Zelski et al. 2011a, b, Ferrer et al. 2012, Liu et al. 2012). The family Papulosaceae placed in the Sordariomycetidae originally comprised a single species growing on saltmarsh plants (Winka & Eriksson 2000). Based on molecular sequence data, the two freshwater genera Brunneosporella and Fluminicola (Wong et al. 1999, Ranghoo et al. 2001) were shown to be closely related to Papulosa (Réblová 2013).

This study aims to investigate and clarify the ordinal and familial relationships of Achroceratosphaeria and the unidentified freshwater fungus in the Sordariomycetes employing molecular sequence characters from protein-coding and ribosomal nuclear loci.

MATERIALS AND METHODS

Herbarium material and fungal strains

Dry ascomata were rehydrated with water; material was examined with an Olympus SZX12 dissecting microscope, and hand-sectioned centrum material (including asci, ascospores and paraphyses) was mounted in Melzer’s reagent, Lugol, 90 % lactic acid, aqueous cotton-blue (1 mg/mL), and blue or black Waterman ink. Hand sections of the ascomatal wall were studied in 3 % KOH or heated chloral-lactophenol. All measurements were made in Melzer’s reagent. Means ± standard deviation (SD) based on 20–25 measurements, excluding maxima and minima, are given for dimensions of asci and ascospores. Images were captured by differential interference (DIC) or phase contrast (PC) microscopy using an Olympus DP70 Camera operated by Imaging Software Cell on an Olympus BX51 compound microscope.

Multi-ascospore isolates were obtained from fresh material of three collections (PRM 924377-924379) with the aid of a spore isolator (Meopta, Prague, Czech Republic). Ascospores and asci were spread on water agar, ascospores germinated within 48 h. Germinating ascospores were transferred and isolates were grown on water agar, potato-dextrose agar (PDA, Oxoid) and potato-carrot agar (PCA, Gams et al. 1998). Colonies were examined after 7, 21 and 30 d incubation at 25 °C in the dark. The ex-type culture is maintained at CBS (CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands). Type and other herbarium material are deposited in PRM herbarium (National Museum in Prague, Czech Republic). The Online auction colour chart (2004) was used as the colour standard.

DNA extraction, amplification and sequence alignment

Cultures used for DNA isolations were grown as previously described by Réblová et al. (2011) and DNA was extracted following the protocols of Lee & Taylor (1990). Procedures for amplifying and sequencing the nuc18S, nuc28S and rpb2 were performed as described in Réblová et al. (2011). Sequences were edited using Sequencher v. 5.0 software (Gene Codes Corp., Ann Arbor, MI, USA).

GenBank accession numbers for newly sequenced taxa and other homologous sequences of members of the Sordariomycetes and Leotiomycetes retrieved from GenBank are listed in Table 1. Sequences were manually aligned in BioEdit v. 7.0.9.0 (Hall 1999). The nuclear ribosomal loci were aligned according to the secondary structure of Saccharomyces cerevisiae Meyen ex E.C. Hansen in order to improve the decisions on homologous characters and introduction of gaps (Gutell 1993, Gutell et al. 1993, www.rna.ccbb.utexas.edu). These procedures and alignment of the rpb2 sequences were performed as described in Réblová & Réblová (2013).

Table 1

A list of fungi, isolate information and new sequences determined for this study and those retrieved from GeneBank. GenBank accession numbers in bold were generated for this study.

Classification	Taxon	Source	GenBank accession numbers
			nuc28S	nuc18S	rpb2
Sordariomycetes
Annulatascaceae	Annulatascus velatisporus	A70–18	AY316354	–	–
	Annulusmagnus triseptatus	CBS 131483, CBS 128831	GQ996540	JQ429242	JQ429258
	Ascitendus austriascus	CBS 131685	GQ996539	GQ996542	JQ429257
Boliniales	Camaropella pugillus	SMH 3846	EU481406	–	–
	Camarops microspora	CBS 649.92	AY083821	DQ471036	DQ470937
	Cornipulvina ellipsoides	SMH 1378	DQ231441	–	–
Calosphaeriales	Calosphaeria pulchella	CBS 115999	AY761075	AY761071	GU180661
	Jattaea algeriensis	STE-U 6399, CBS 120871	EU367457	EU367462	HQ878603
	Togniniella microspora	CBS 113648	AY761076	AY761073	GU180660
Chaetosphaeriales	Chaetosphaeria ciliata	ICMP 18253	GU180637	GU180614	GU180659
	Chaetosphaeria curvispora	ICMP 18255	GU180636	AY502933	GU180655
Coniochaetales	Coniochaeta discoidea	SANK 12878, CBS 158.80	AY346297	AJ875179	AY780191
	Coniochaeta ostrea	CBS 507.70	DQ470959	DQ471007	DQ470909
Coronophorales	Bertia moriformis	SMH 3344, SMH 4320	AY695261	–	AY780151
	Chaetosphaerella phaeostroma	SMH 4585	AY346274	–	AY780172
Diaporthales	Diaporthe phaseolorum	FAU 458, NRRL 13736	U47830	L36985	AY641036
	Gnomonia gnomon	CBS 199.53	AF408361	DQ471019	DQ470922
	Valsa ambiens	AR 3516	AF362564	DQ862056	DQ862025
Etheirophoraceae	Etheirophora blepharospora	JK 5397A	EF027723	–	EF027731
	Etheirophora unijubata	JK 5443B	EF027725	EF027718	EF027733
	Swampomyces armeniacus	JK5059C	EF027728	EF027721	–
	Swampomyces triseptatus	CY2802	AY858953	AY858942	–
Glomerellales	Glomerella cingulata	MCA 2498, FAU 513	DQ286199	M55640	DQ858455
	Kylindria peruamazonensis	CBS 838.91	GU180638	GU180609	GU180656
	Monilochaetes infuscans	CBS 379.77	GU180645	GU180619	GU180658
	Reticulascus clavatus	CBS 125296	GU180643	GU180622	–
Hypocreales	Pseudonectria rousseliana	AR 2716, CBS 114049	U17416	AF543767	DQ522459
	Trichoderma viride	GJS 89–127, IFFI 13001	AY489726	AF525230	EU252006
	Virgatospora echinofibrosa	CBS 110115	AY489724	AY489692	EF692516
Juncigenaceae	Juncigena adarca	JK 5235A	EF027726	EF027719	EF027734
	Fulvocentrum aegyptiacus	CY 2973	AY858950	AY858943	–
	Fulvocentrum clavatisporium	LP 83	AY858952	AY858945	–
Koralionastetales	Koralionastes ellipticus	JK 5769	EU863585	EU863581	–
	Pontogeneia microdictyi	JK 5748	–	EU863582	–
Lulworthiales	Kohlmeyeriella tubulata	PP 1105, PP 0989	AF491265	AY878997	–
	Lindra thalassiae	JK 5090A	DQ470947	DQ470994	–
	Lulwoana uniseptata	PP 7333, CBS 16760	FJ176904	AY879034	–
	Lulwoidea lignoarenaria	AFTOL 5013, IFO 32135	FJ176903	AY879010	–
	Lulworthia fucicola	PP 1235, C 21–1	AF491270	AF050481	–
	Rostrupiella danica	BBH 16759	DQ394094	–	–
	Spathulospora antarctica	JK 3530	–	AY380315	–
Magnaporthales	Ceratosphaeria lampadophora	CBS 117555	AY761084	AY761088	–
	Gaeumannomyces graminis	AR 3401, M 57	AF362557	JF414874	–
	Magnaporthe grisea	Ina168, 70–15	AB026819	DQ493955	–
	Muraeriata africana	GKM 1084	EU527995	–	–
Melanosporales	Melanospora tiffanii	ATCC 15515	AY015630	AY01561	AY015637
	Melanospora zamiae	ATCC 12340, CBS 421.87	AY046579	AY046578	DQ368634
Microascales	Ceratocystis adiposa	CCFC 212726, CBS 600.74	AY281101	EU984263	–
	Ceratocystis fimbriata	C89, Cef 0801, CBS 374.83	U17401	HQ908495	DQ368641
	Corollospora maritima	AFTOL 5011, JK 4834	FJ176901	U46871	DQ368632
	Custingophora olivacea	CBS 335.68	AF178566	JX070460	GU180665
	Graphium penicillioides	C 1505, CBS 506.86	AF222500	DQ471038	DQ470938
	Knoxdaviesia proteae	CMW 3936, CBS 486.88	AF221011	AY271804	–
	Lignincola laevis	AFTOL 737, A169–1D	U46890	AF050487	DQ836886
	Microascus trigonosporus	BS 218.31, ATCC52470	DQ470958	DQ471006	AF107792
	Petriella setifera	CCFC 226737, CBS 385.87, CBS 110344	AY281100	U43908	DQ368640
Ophiostomatales	Ophiostoma piliferum	DAOM 226737, CBS 129.32, CBS 158.74	AY281094	AJ243295	DQ470905
	Ophiostoma stenoceras	CBS 139.51	DQ836904	DQ836897	DQ836891
Papulosaceae	Brunneosporella aquatica	HKUCC 3708	AF132326	–	–
	Fluminicola coronata	HKUCC 3717	AF132332	–	–
	Papulosa amerospora	JK 5547F	DQ470950	DQ470998	DQ470901
Pisorisporiales	Achroceratosphaeria potamia	CBS 125414	GQ996538	GQ996541	KM588908
	Achroceratosphaeria sp.	HKU(M) 5224	AF132325	–	–
	Pisorisporium cymbiforme	CBS 138884	KM588904	KM588901	KM588907
	Pisorisporium cymbiforme	PRM 924378	KM588902	KM588899	KM588905
	Pisorisporium cymbiforme	PRM 924379	KM588903	KM588900	KM588906
Sordariales	Gelasinospora tetrasperma	CBS 178.33	DQ470980	DQ471032	DQ470932
	Lasiosphaeria ovina	SMH 1538, CBS 958.72	AF064643	AY083799	AY600292
	Sordaria fimicola	SMH 4106, MUCL 937, CBS 723.96	AY780079	X69851	DQ368647
Savoryellales	Ascotaiwania lignicola	NIL 00005	HQ446364	HQ446284	–
	Canalisporium exiguum	SS 00809	GQ390281	GQ390266	–
	Savoryella lignicola	NF 00204, NTOU 791	HQ446378	HQ446299	–
Torpedosporaceae	Torpedospora ambispinosa	CY3386	AY858946	AY858941	–
	Torpedospora radiata	JK5252C	EF027730	EF027722	EF027737
Xylariales	Anthostomella torosa	JK 5678E	DQ836902	DQ836895	DQ836885
	Graphostroma platystoma	CBS 270.87	DQ836906	DQ836900	DQ836893
	Xylaria hypoxylon	AFTOL 51	AY544648	AY544692	DQ470878
Leotiomycetes
Helotiales	Leotia lubrica	AFTOL 1, isolate unknown for nuc18S	AY544644	L37536	DQ470876
	Microglossum rufum	AFTOL 1292	DQ470981	DQ471033	D Q 4 7 0 9 3 3

The single-locus datasets (nuc28S: 1 923 characters and 77 sequences, nuc18S: 1 805 characters and 68 sequences, rpb2 segments 5–7: 1 213 characters and 48 sequences) were examined for topological incongruence among loci. For each individual locus, 500 bootstrap replicates were generated with RAxML-HPC v. 7.0.3 (Stamatakis et al. 2005, Stamatakis 2006) and compared visually for topological conflict between supported clades in phylogenetic trees. A conflict between two loci was assumed to occur when a clade appeared monophyletic with bootstrap support of ≥ 75 % in one tree, but was supported as non-monophyletic in another (Mason-Gamer & Kellogg 1996). Individual, conflict-free alignments were concatenated to combine sequences for subsequent phylogenetic analyses. The multiple sequence alignment is deposited in TreeBASE (Study no. 16406).

Phylogenetic analysis

Phylogenetic relationships of the unidentified fungus were resolved by an analysis of nuc18S, nuc28S and rpb2 sequences of representatives of 19 orders or individual families of the Sordariomycetes. We analysed the first 2/3 of the 5’ half of the nuc28S, the almost entire nuc18S, and segments 5–7 of rpb2. Bases 1–148 of the nuc18S, 1–85 of the nuc28S, and 1–58 of the rpb2 alignments at the 5’-end and 1 457–1 923 of the nuc28S alignment at the 3’-end were excluded from analyses because of incompleteness of the majority of the available sequences. The combined dataset was partitioned into several subsets of nucleotide sites, i.e. nuc28S, nuc18S, and first, second and third codon positions of rpb2. Two members of the Leotiomycetes, Leotia lubrica and Microglossum rufum were used to root the multilocus phylogeny.

The program MrModeltest2 v. 2.3 (Nylander 2008) was used to infer the appropriate substitution model that would best fit the model of DNA evolution for each sequence dataset and each partition of the combined datasets. Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to estimate phylogenetic relationships. ML analysis was performed with RAxML-HPC v. 7.0.3 with a GTRCAT model of evolution. Nodal support was determined by non-parametric bootstrapping (BS) with 1 000 replicates.

BI analysis was performed in a likelihood framework as implemented in MrBayes v. 3.0b4 software package to reconstruct phylogenetic trees (Huelsenbeck & Ronquist 2001). For the combined nuc18S, nuc28S and rpb2 dataset we used for each partition the GTR+I+G substitution model. Two Bayesian searches were performed using the default parameters. Analyses were run for 10 M generations, with trees sampled every 1 000 generations. Tracer v. 1.6.0. (Rambaut et al. 2013) was used to confirm convergence of trees and burn-in. The first 50 000 trees, which represented the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating posterior probabilities (PP) of recovered branches (Larget & Simon 1999).

RESULTS

Phylogenetic results

In the ML analyses (conducted by RAxML) of individual nuc28S, nuc18S and rpb2 loci, the three strains of the unidentified fungus grouped always with two species of Achroceratosphaeria in a strongly supported monophyletic clade distantly related to the known orders and families of the Sordariomycetes. The clade is introduced as the new order Pisorisporiales. Analyses of individual nuclear ribosomal and protein-coding loci place Pisorisporiales at different positions in the Sordariomycetes, however none of the internodes received significant statistical support. The nuc28S locus supports the placement of Pisorisporiales as a basal group in the Sordariomycetes. The phylogenies derived from individual nuc18S and rpb2 loci consistently place Pisorisporiales within the Sordariomycetes. In the nuc18S tree, the Pisorisporiales are located basal to the Hypocreomycetidae, while in the rpb2 locus they are at the base of the Sordariomycetidae. All other families and orders of the Sordariomycetes formed well-supported monophyletic clades in analyses of all three individual loci.

The final alignment consisted of 79 combined nuc18S, nuc28S and rpb2 sequences of members of the Sordariomycetes, each with 4 941 characters after introduction of gaps. The alignment had 2 551 distinct alignment patterns (ML analysis); the ML tree is shown in Fig. 1. The Sordariomycetes are shown as a robust monophyletic clade (100 % ML BS / 1.0 PP) comprising three strongly supported lineages, the Sordariomycetidae, Hypocreomycetidae and Xylariomycetidae. The Pisorisporiales are nested in a weakly supported clade as sister to the Lulworthiales (100/1.0) and Koralionastetales (100/1.0). The whole clade is situated basal (65/0.87) to the Hypocreomycetidae. The other three taxonomic groups of the Hypocreomycetidae that contain predominantly fungi from aquatic habitats form strongly supported monophyletic clades, i.e. the Halosphaeriaceae (100/1.0), Savoryellales (100/1.0), and the complex of marine genera (95/1.0) comprising the Etheirophoraceae (100/1.0), Juncigenaceae (100/1.0) and Torpedosporaceae (100/1.0) of the TBM clade (76/1.0).

Fig. 1

Multilocus phylogenetic analysis of the nuc18S-nuc28S-rpb2 sequences of the Sordariomycetes. Phylogram inferred from the ML analysis with RAxML using a GTRCAT model of evolution. Only high branch support is shown at the nodes, maximum likelihood bootstrap support (ML BS) ≥ 75 % and Bayesian posterior probability (PP) ≥ 95 %). Taxa labelled in blue correspond to groups whose members predominantly occur in freshwater or marine habitats besides the generally aquatic Pisorisporiales, Lulworthiales, and Koralionastetales clades.

TAXONOMY

DNA sequences of nuclear ribosomal and protein-coding loci of specimens obtained from freshwater habitats in this study were shown to represent a new genus Pisorisporium and order in the Sordariomycetes based on phylogenetic analysis. Morphological examination showed that two species were present, described here as P. cymbiforme and P. glaucum. For the latter species DNA sequences could not be obtained but morphologically and ecologically it fits clearly within the newly described genus, while it is morphologically distinct from the first species.

Réblová & J. Fourn., ord. nov. — MycoBank MB810338

Type family. Pisorisporiaceae Réblová & J. Fourn.

Ascomata perithecial, non-stromatic. Ostiole periphysate. Ascomatal wall leathery to fragile, brown, partly carbonaceous. Hamathecium of true paraphyses. Asci unitunicate, persistent, with an amyloid or non-amyloid apical ring. Ascospores hyaline, transversely multiseptate. Asexual morph unknown. Saprobic on wood.

Réblová & J. Fourn., fam. nov. — MycoBank MB810339

Type genus. Pisorisporium Réblová & J. Fourn.

Ascomata non-stromatic, immersed to superficial, papillate or with a long neck, venter subglobose to conical, upright or lying obliquely or horizontally, neck central rarely eccentric. Ostiole periphysate. Ascomatal wall leathery to fragile, partly carbonaceous in the outer layers, pigmented dark brown, opaque to light brown to subhyaline, comprising two layers. Paraphyses abundant, persistent, cylindrical. Asci unitunicate, 8-spored, with a pronounced amyloid or non-amyloid apical annulus, cylindrical-clavate, persistently attached to the ascogenous hyphae at maturity. Ascospores fusiform, cylindrical to cymbiform slightly tapering towards the ends, hyaline, transversely multiseptate, lacking a mucilaginous sheath or appendages, often with numerous guttules. Asexual morph unknown.

Réblová & J. Fourn., gen. nov. — MycoBank MB810340

Type species. Pisorisporium cymbiforme Réblová & J. Fourn.

Etymology. Pisorum (Latin), meaning peas in a pod, referring to the numerous guttules arranged in a chain within ascospores; spora (Latin), referring to the ascospores.

Ascomata non-stromatic, immersed, gradually erumpent to superficial, solitary or in small groups or rows, papillate or with a short beak, glabrous, venter subglobose to broadly conical, laterally or basally flattened, upright or lying obliquely or horizontally. Ostiole periphysate. Ascomatal wall fragile, partly carbonaceous in the outer layer, 2-layered. Paraphyses persistent, septate, hyaline, arising from the bottom and sides in the ascomatal cavity. Asci unitunicate, 8-spored, cylindrical-clavate, short-stipitate, with a pronounced thimble-shaped amyloid apical annulus, persistently attached to the ascogenous hyphae at maturity. Ascospores fusiform, cylindrical to cymbiform, sometimes falcate, hyaline, transversely multiseptate, lacking a mucilaginous sheath or appendages, smooth-walled, with numerous guttules. Asexual morph unknown.

Notes — The paraphyses were present abundantly, they are fragile, easily broken in squash mounts, making it difficult to determine their length. They are cylindrical, arranged in parallel at the bottom of the ascomata and among the asci, tapering, sparsely branched and often intertwined in the upper half. The outer ascomatal wall is carbonaceous, grading outwards into 2–3 layers of subhyaline to pale brown, polyhedral to angular cells that probably account for the finely roughened appearance of the wall in both species.

Réblová & J. Fourn., sp. nov. — MycoBank MB810341; Fig. 2

Fig. 2

Pisorisporium cymbiforme. a, b. Ascomata arranged in small groups or in rows; c, d. vertical sections of the ascomatal wall; e–g. asci; h, i. ascospores; j. paraphyses (a, b, g, i, j from PRM 924377 holotype; c, d, h from PRM 924379; e, f from PRM 924378); e–i: DIC; j: PC. — Scale bars: a, b = 200 μm; c, d = 20 μm; e–j = 10 μm.

Etymology. Cymbiform (Latin), meaning boat-shaped (a long rowboat), referring to the shape of the ascospores.

Ascomata non-stromatic, immersed, gradually erumpent to superficial, solitary or in small groups of 2–4, or in rows, venter (240–)290–380 μm diam, 220–280 μm high, subglobose to broadly conical, dark brown to black, sometimes laterally or basally flattened, glabrous, finely roughened, upright or lying obliquely to horizontally, papillate or with a beak 30–110 μm high, conical or subcylindrical, central to lateral, opening by a rounded pore. Ostiole periphysate. Ascomatal wall fragile, carbonaceous, (12–)14–26 μm thick, becoming thicker in the neck c. 20–30 μm, 2–layered; outer layer consisting of brown, polyhedral cells of textura prismatica with opaque walls and lumina reduced to occluded; outwards grading into 2–3 layers of subhyaline to pale brown, polyhedral to angular cells of textura angularis c. 4–6 μm thick, collapsing in old ascomata and forming a persistent subhyaline amorphous coating; inwards grading into several layers of thin-walled, pale brown to hyaline, flattened cells. Paraphyses abundant, persistent, septate, hyaline, sparsely branched in the upper half and intertwined, c. 3.5–5.0 μm wide, tapering to c. 3.0 μm. Asci (180–)190–207 × 11–13(–14) μm (mean ± SD = 199.7 ± 5.4 × 12.5 ± 1.2 μm), cylindrical-clavate, obtuse to broadly rounded apically, 8-spored; apex with an amyloid apical annulus 2.7–3.2 μm wide, 1.9–2.3 μm high. Ascospores 40–45(–48)× (3.8–) 4.3–4.8(–5.0) μm (mean ± SD = 43.7 ± 1.9 × 4.5 ± 0.3 μm), cymbiform to fusiform to cylindrical, slightly tapering towards the ends, hyaline, smooth, (8–)12– 16–septate, nonconstricted at the septa, each cell with a large guttule, arranged 2-seriately in the ascus.

Culture characteristics — Colonies slow growing, 18–22 mm diam on PDA after 21 d at 25 °C. Aerial mycelium beige (oac816) near the centre of the colony and on the inoculum block, white (oac909) towards the margin, felty, margin entire. Sporulation absent. Aerial mycelium composed of thin-walled, hyaline, unbranched or sparsely branched hyphae, 2.0–3.0 μm diam. Chlamydospores not observed.

Specimens examined. France, Midi-Pyrénées, Ariège, Rimont, valley of La Maille brook, c. 550 m asl, submerged decorticated wood of Alnus glutinosa, 9 May 2014, J. Fournier J.F. 14046 (holotype PRM 924377, culture ex-type CBS 138884); ibid., 2 Apr. 2013, submerged decorticated wood of Fraxinus excelsior, J. Fournier, J.F. 13067, J.F. 13070, PRM 924378, PRM 924379.

Notes — The two collections PRM 924378 and PRM 924379 were acquired from the same branch submerged in water. The ascospores in PRM 924378 were slightly smaller, 34–39 × 4.0–4.7(–5.0) μm (mean ± SD = 36.9 ± 1.5 × 4.4 ± 0.4 μm), and asci slightly longer, 187–210(–230) × 11.5–13.5 (–14) μm (mean ± SD = 201.4 ± 8.8 × 13.1 ± 1.3 μm) than in the type specimen. The ascospores of all three collections germinated easily on water and PDA agar within 48 h, cultures derived from PRM 924378 and PRM 924379 are no longer viable.

Réblová & J. Fourn., sp. nov. — MycoBank MB810342; Fig. 3

Fig. 3

Pisorisporium glaucum. a, b. Ascomata arranged in small groups or in rows; c. vertical section of the ascomatal wall; d. ascogenous hypha with attached bases of asci; e–g. asci; h. thimble-shaped apical annulus staining blue in Lugol; i–k. ascospores (a–k from PRM 924380 holotype); e, f, h, i, k: DIC; d, g, j: PC. — Scale bars: a, b = 200 μm; c = 20 μm; d–g, i–k = 10 μm; h = 5 μm.

Etymology. Glaucus (Latin), meaning blue, referring to the intense blue amyloid reaction of the apical annulus.

Ascomata non-stromatic, immersed, gradually erumpent to superficial, solitary or in small groups of 2–5, or in rows, venter 270–390 μm diam, 250–350 μm high, subglobose to broadly conical, dark brown to black sometimes laterally or basally flattened, glabrous, upright or lying obliquely to horizontally, papillate or with a beak 50–180 μm high, conical or subcylindrical, central to lateral, opening by a rounded pore. Ostiole periphysate. Ascomatal wall fragile, carbonaceous, 20–32 μm thick, becoming thicker in the neck c. 45–58 μm, 2-layered; outer layer consisting of brown, polyhedral cells of textura prismatica with opaque walls and lumina reduced to occluded; outwards grading into 2–3 layers of subhyaline to pale brown, polyhedral to angular cells of textura angularis; inwards grading into several layers of thin-walled, pale brown to hyaline, flattened cells. Paraphyses abundant, persistent, hyaline, septate, sparsely branched in the upper half and intertwined, c. 3.0–5.5μm wide, tapering to 2.0–2.5 μm. Asci 190–245 × 12–15 μm (mean ± SD = 209.7 ± 12.8 × 12.8 ± 1.2 μm), cylindrical-clavate, obtuse to broadly rounded apically, 8-spored; apex with an amyloid thimble-shaped apical annulus 3.0–3.2 μm wide, 2.0–2.3 μm high. Ascospores (52–)55–67(–72) × 4.5–5.5 μm (mean ± SD = 59.5 ± 4.5 × 4.9 ± 0.3 μm), fusiform to subcylindrical, falcate, slightly tapering towards the ends, hyaline, smooth, 10–14-septate, non-constricted at the septa, each cell with a large guttule, arranged 2–3-seriately in the ascus.

Specimen examined. BELGIUM, Hainaut Province, Wellin, Halma, Ry des Glands brook, 26 Sept. 2006, on driftwood of Acer pseudoplatanus, J. Fournier J.F. 06232 (holotype PRM 924380).

Notes — Pisorisporium glaucum is easily distinguishable from P. cymbiforme by longer and slightly wider ascospores and longer asci. The number of septa of the ascospore is in both species comparable and varies from 10 to 16. This species has not been cultivated at the time of its collection and DNA sequences could not be obtained due to insufficient number of ascomata that would be required for successful DNA extraction. Such procedure would cause destruction of the type material.

DISCUSSION

The combined analysis of nuc18S-nuc28S-rpb2 sequences (Fig. 1) led to the discovery that the three strains of P. cymbiforme and Achroceratosphaeria form a strongly supported monophyletic clade (100/1.0), which is distantly related to freshwater and marine ascomycetes of the Annulatascaceae, Halosphaeriaceae, Papulosaceae, Savoryellales, marine genera of the TBM clade, now classified as the Etheirophoraceae, Juncigenaceae, Torpedosporaceae and Falcocladiaceae (Jones et al. 2014), and other morphologically similar fungi. The newly recognised clade containing Achroceratosphaeria and Pisorisporium represents a distinct taxonomic group at the ordinal level within the Sordariomycetes based on the evidence of molecular sequence data. However, its relationship with other orders could not be elucidated with good statistical support. Pisorisporiales is nested in an unsupported clade as sister to the Lulworthiales and Koralionastetales situated basal to the Hypocreomycetidae.

The placement of the Lulworthiales, including Spathulosporales, and Koralionastetales within the Sordariomycetes based on DNA data had been ambiguous (Eriksson & Winka 1997, Spatafora et al. 1998, Kohlmeyer et al. 2000, Jones et al. 2009). Their sister relationship with the Hypocreomycetidae is supported in the 3-, 4- and 6-gene phylogenies by BI and ML methods (Schoch et al. 2007, Spatafora et al. 2007, Zhang et al. 2007), whereas the maximum parsimony, weighted parsimony and ML methods of the 4-gene analysis support their placement as a basal group in the Sordariomycetes (Zhang et al. 2007). The current position in combination with the Pisorisporiales may suggest a new subclass lineage in the Sordariomycetes.

Without molecular data, it is, in fact, challenging to place Pisorisporium in any of the accepted families and genera of the Sordariomycetes. Members of Pisorisporium grow on decaying deciduous wood submersed in fresh water. They are characterised by minute, immersed ascomata arranged in small groups or in rows oriented with the grain of wood, gradually erumpent by water erosion of the substrate and becoming superficial. Ascomata are upright but often grow obliquely or almost horizontally, which may be caused by the water flow. Paraphyses are fragile, arranged parallel among the asci, continuously tapering, becoming sparsely branched and intertwined above the ascal apices (Fig. 4). In ascospore and to some extent ascus morphology, Pisorisporium resembles members of Ceratosphaeria, Ceratosphaerella and Pseudohalonectria of the Magnaporthaceae. They are similar in overall morphology of fusiform, cylindrical to cymbiform, multiseptate, hyaline ascospores, but the three latter genera differ from Pisorisporium in a non-amyloid reaction of the apical annulus, long, sometimes flexuous protruding necks of ascomata and asexual morphs, i.e. harpophora-like and phialophora-like asexual morphs experimentally linked to Ceratosphaeria and the presumed Didymobotryum-like asexual morph of Ceratosphaerella (Shearer 1989, Réblová 2006, Huhndorf et al. 2008). Moreover, species of Pseudohalonectria and Ceratosphaeria phialidica differ from Pisorisporium by cylindrical to cymbiform asci with ascospores arranged in a fascicle or rarely 4-seriately, while in Pisorisporium, Ceratosphaeria and Ceratosphaerella the ascospores are predominantly 2-seriate within the ascus.

Fig. 4

Pisorisporium spp. a–d. Pisorisporium glaucum. a, b. paraphyses in Melzer’s reagent, arrow indicates filaments that are sparsely branched and intertwined above ascal apices; c. paraphyses and three asci in blue waterman ink; d. apical annulus in blue waterman ink. — e, f. Pisorisporium cymbiforme. e. apical ring in black waterman ink; f. apical annulus in Melzer’s reagent (a–d from PRM 924380; e, f from PRM 924378); a, b: PC; c–f: DIC. — Scale bars: a–c = 20 μm; d–f = 10 μm.

The amyloid reaction of the apical annulus is not quite consistent among orders of ascomycetes; in the Sordariomycetes it occurs predominantly in members of the Xylariales, i.e. Amphisphaeriaceae, Diatrypaceae and Xylariaceae. The positive blue to dark reaction of iodine solutions, i.e. Lugol and Melzer’s reagents, due to the presence of starch-like polysaccharides in fungal microscopic structures is generally termed amyloid or euamyloid. The apical annulus of both species of Pisorisporium can be termed amyloid; it turns blue in Melzer’s reagent and in Lugol’s solution irrespective of whether a pre-treatment with KOH was applied. Regarding the amyloidity of the ascal apical structures and chemical reactions with other dyes like Congo red, toluidine blue or blue ink, we noticed a difference between Pisorisporium on one hand and members of the Xylariaceae and other taxa on the other. Only in Pisorisporium the apical annulus is readily stained by these chemicals (Fig. 4). However, such coloration, commonly encountered in many sordariaceous genera with chitinoid (non-amyloid) apical annulus, does not occur in genera with a known amyloid apical annulus. Our observation may imply that the apical annulus of Pisorisporium is composed of other components than commonly encountered in members of the Xylariales. Clarification of the chemical compounds responsible for this discrepancy is beyond the scope of the present paper. However, the fact itself is interesting and worth being reported.

Two genera of the Amphisphaeriaceae, Crassoascus and Iodosphaeria, can be compared with Pisorisporium based on morphology of ascospores, asci and the amyloidity of the apical annulus (Samuels et al. 1987, Barrasa et al. 1993). Members of Crassoascus differ from Pisorisporium by the flat apical annulus and fusiform, multiseptate, versicolorous ascospores with brown middle cells and hyaline end-cells, sometimes with hyaline cap-like appendages (Barr 1993, Barrasa et al. 1993, Catania & Romero 2012). Iodosphaeria can be distinguished from Pisorisporium by non-papillate ascomata associated with a repent, spreading network of brown hyphae, with a flat top from which radiate numerous, flexuous, unbranched hairs, asci with a flat apical annulus and suballantoid, rarely ellipsoid, non-septate ascospores and asexual morphs belonging to Ceratosporium and Selenosporella (Samuels et al. 1987, Barr 1993, Hsieh et al. 1997, Catania & Romero 2012).

Iodosphaeria aquatica is the only species that does not conform to the description of that genus; it resembles Pisorisporium with regard to the aquatic habitat, glabrous ascomata and septate, fusiform ascospores arranged 2–3-seriately within the ascus (Hyde 1995). Iodosphaeria aquatica differs from Pisorisporium by ascomata that are immersed beneath a blackened clypeus, the ascomatal wall, which is composed of thin-walled, brown angular cells and by 1-septate ascospores that have mucilaginous appendages at each pole. Molecular analysis of partial nuc28S rDNA sequences of I. aquatica revealed that the fungus is unrelated to the Sordariomycetes and it is preliminarily placed in the Dothideomycetes among genera with ascolocular development of the ascomata (strain HKUCC 166, nuc28S GenBank accession: AF452044, Jeewon et al. 2003). However, no information is available about whether this is a sequence obtained from the ex-type strain or the DNA was isolated from different material of I. aquatica.

Based on morphological characters and habitat, it is difficult to find similarities among members of the Koralionastetales, Lulworthiales and Pisorisporiales. Lulworthiales was established by Kohlmeyer et al. (2000), when it was discovered that the Halosphaeriales are polyphyletic comprising two distinct evolutionary lineages of marine fungi with terrestrial ancestors (Spatafora et al. 1998). Members of the Lulworthiales are predominantly marine ascomycetes, but some also inhabit niches in estuarine environments. They include saprobes on driftwood and intertidal wood, sea grasses, saltmarsh plants, coral rocks or parasites of uncalcified Rhodophyta or Phaeophyta. They are characterised by filiform, one- to multiseptate ascospores with apical mucus-containing chambers or gelatinous sheath (except species of Lindra), early deliquescing asci and the absence of a hamathecium in mature ascomata, while young ascomata contain pseudoparenchyma (Kohlmeyer 1997, Nakagiri & Tadayoshi 1997, Campbell et al. 2005, Koch et al. 2007).

Asexual morphs of Lulworthiales belong to nine hyphomycetous dematiaceous genera with usually coiled conidia; they were assigned to the order based on molecular DNA data or the link between sexual and asexual morph was proven experimentally (Nakagiri & Tubaki 1983, Nakagiri 1984, Campbell et al. 2005, Jones et al. 2008, 2009, Abdel-Wahab et al. 2010).

The Koralionastetales were separated from the Lulworthiales by Campbell et al. (2008) to include fungi occurring obligatorily in marine habitats. They are characterised by a centrum containing paraphyses and periphyses and ellipsoid, fusiform to filiform ascospores without any apical structures and with typical formation of antheridia on germ tubes. Members of Koralionastes live on coral rocks, while Pontogeneia is a parasite of marine Phaeophyta. Their asexual morphs are unknown.

The type species of Achroceratosphaeria (A. potamia) and Pisorisporium (P. cymbiforme), originate in the same territory in the Ariège department in Midi-Pyrénées less than 1 km apart. The La Maille locality, where P. cymbiforme was repeatedly collected in 2013 and 2014, is a deep valley bordering steep slopes at the foot of the Arize massif, with a thick deciduous forest with high humidity. Trees and shrubs grow densely also along the shadowy La Maille brook. Decaying branches, twigs and larger logs fall regularly in the water flow, which provide a rich substrate in this locality. The La Maille brook may dry up at the end of season, leaving the otherwise submerged wood and driftwood exposed to air for several weeks or even months. Not far from here, in the Le Baup stream, of which the La Maille brook is a tributary, was collected A. potamia, another member of the Pisorisporiales.