Phylogenetic placement of Iodosphaeriaceae (Xylariales, Ascomycota), designation of an epitype for the type species of Iodosphaeria, I. phyllophila, and description of I. foliicola sp. nov.

The Iodosphaeriaceae is represented by the single genus, Iodosphaeria, which is composed of nine species with superficial, black, globose ascomata covered with long, flexuous, brown hairs projecting from the ascomata in a stellate fashion, unitunicate asci with an amyloid apical ring or ring lacking and ellipsoidal, ellipsoidal-fusiform or allantoid, hyaline, aseptate ascospores. Members of Iodosphaeria are infrequently found worldwide as saprobes on various hosts and a wide range of substrates. Only three species have been sequenced and included in phylogenetic analyses, but the type species, I. phyllophila, lacks sequence data. In order to stabilize the placement of the genus and family, an epitype for the type species was designated after obtaining ITS sequence data and conducting maximum likelihood and Bayesian phylogenetic analyses. Iodosphaeria foliicola occurring on overwintered Alnus sp. leaves is described as new. Five species in the genus form a well-supported monophyletic group, sister to the Pseudosporidesmiaceae in the Xylariales. Selenosporella-like and/or ceratosporium-like synasexual morphs were experimentally verified or found associated with ascomata of seven of the nine accepted species in the genus. Taxa included and excluded from Iodosphaeria are discussed.

INTRODUCTION

Iodosphaeria was introduced by Samuels to accommodate the type species, I. phyllophila based on Lasiosphaeria phyllophila (Mouton 1900), and a second species, I. ripogoni. Seven additional species have been added to the genus: I. aquatica, I. arundinariae, I. honghensis, I. hongkongensis, I. podocarpi, I. polygoni, and I. tongrenensis (MycoBank, www.mycobank.org, accessed on 7 Apr. 2021). Morphological and molecular analyses of I. aquatica have led to uncertainty in its taxonomic placement (Hsieh , Kang , Taylor & Hyde 1999). Jeewon showed the genus to be paraphyletic when they included I. aquatica and another Iodosphaeria sp. in their molecular analyses. A key to all known species of Iodosphaeria was presented in Marasinghe .

Sexual morphs are comprised of superficial, black, globose to subglobose, non-stromatic ascomata with flat apices and covered with long, brown, flexuous hairs, a two-layered ascomal wall, cylindrical, 8-spored asci sometimes with an amyloid apical ring or ring is lacking, and ellipsoidal, allantoid or fusiform, hyaline, aseptate ascospores with or without a gelatinous sheath. Selenosporella- and ceratosporium-like synasexual morphs have been reported on the surface of ascomata in I. tongrenensis (Li ), on field-collected material of I. phyllophila (Bell & Mahoney 2016) and I. ripogoni (Samuels , Bell & Mahoney 2016) and in cultures of I. phyllophila (Samuels ). Ceratosporium-like conidia were observed on the host surface in I. honghensis (Marasinghe ). Members of Iodosphaeria are infrequently found worldwide as saprobes on a variety of hosts and a wide range of substrates including dead branches, stems, vines, leaves and petioles.

Iodosphaeria was initially placed in the Amphisphaeriaceae (Samuels ) based on its synasexual morphs and amyloid ascal ring, but Barr (1990) later transferred it to the Lasiosphaeriaceae based on ascomal wall anatomy and centrum similarities. It was placed in the Trichosphaeriaceae by Réblová (1999). Hilber & Hilber (2002) created a new family, Iodosphaeriaceae, for the genus. The family, represented by I. honghensis and I. tongrenensis, is placed in the Xylariales based on morphology and phylogeny (Marasinghe ).

Only three species, I. aquatica, I. honghensis and I. tongrenensis, have been sequenced and included in phylogenetic analyses. Because the type species has never been sequenced, ambiguity surrounds the placement of the genus and family. The type material of I. phyllophila, described from decaying leaves of Betula alba and Corylus from Belgium, is sparse and over 120 years old, so attempts to obtain molecular data from this material would be futile. The goals of this study were to designate an epitype of I. phyllophila from the country of origin, obtain sequence data from the epitype and provide a stable phylogenetic placement for Iodosphaeria and Iodosphaeriaceae. During our investigation, two specimens of unknown Iodosphaeria from Canada were discovered and compared with the known species.

MATERIALS AND METHODS

Taxon sampling and morphological examination

Fresh specimens were collected in the field, dried and stored in paper packets. No attempts were made to obtain these specimens in culture. All specimens are deposited in Meise Botanic Garden (BR, Meise, Belgium), the Fungarium of the Illinois Natural History Survey (ILLS, Champaign, Illinois, USA) and the New Zealand Fungarium (PDD, Auckland, New Zealand). Other specimens were obtained from the Plant Pathology Herbarium, Cornell University (CUP, Ithaca, New York, USA), New Brunswick Museum (NBM, Saint John, Canada), and Staatliche Naturwissenschaftliche Sammlungen Bayerns (M, Munich, Germany).

Ascomata were squash-mounted in distilled water and micromorphological structures were examined on an Olympus BX51 compound microscope using differential interference or phase contrast microscopy. Images were processed using Adobe Photoshop 2021 (Adobe Systems Inc., Mountain View, California). A minimum of 30 measurements was taken for all morphological structures using Olympus cellSens Standard v. 1.14 image analysis software after digital capture with an Olympus DP70 or a XC50 5.0 megapixel digital camera using Olympus Imaging Software Cell^D. Sections of the ascomal wall were prepared at 25 μm thickness using a Physitemp BSF-3 freezing stage mounted on a Leica SM2000 sliding microtome.

Molecular data

DNA was extracted directly from ascomata using an E.Z.N.A.® Microelute Genomic DNA kit (Omega Bio-tek, Norcross, Georgia, USA) following the manufacturer’s instructions. The entire internal transcribed spacer (ITS) region and the first 600 bp of the 5’ end of 28S nuclear large subunit (LSU) were PCR amplified using known primers (Vilgalys & Hester 1990, White , Rehner & Samuels 1995). PCR reactions contained 12.5 μL GoTaq® Green Master Mix (Promega, Madison, Wisconsin, USA), 2.5 μL of BSA (bovine serum albumin, New England Biolabs, Ipswich, MA), 2.5 μL of DMSO (dimethyl sulfoxide, Fisher Scientific, Pittsburgh, PA), 1.5 μl of each 10 mM primer, and 3–5 μl genomic DNA. PCR amplification of ITS and LSU was performed on a Bio-Rad C1000 thermal cycler under the following conditions: initial denaturation at 94 ཐC for 2 min, followed by 40 cycles of 94 ཐC for 30 s, 41 ཐC for 45 s, and 72 ཐC for 1 min with a final extension step of 72 ཐC for 10 min. After verification on an ethidium bromide-stained 1 % TBE agarose gel, PCR products were purified with a Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA). Purified PCR products were used in 11 μL sequencing reactions to sequence both strands with BigDye Terminators v. 3.1 (Applied Biosystems, Foster City, CA) in combination with the following ITS primers: ITS1F, ITS2, ITS3, ITS4, and LSU primers: LR0R, LR3. Sequences were generated on an Applied Biosystems 3730XL high throughput capillary sequencer at the Roy J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign (Champaign, Illinois, USA). Consensus sequences for each gene were assembled with Sequencher v. 5.4 (Gene Codes Corp., Ann Arbor, Michigan, USA) and each sequence was subjected to an individual BLASTn analysis to verify its identity. PCR amplifications of rpb2 and tef1-α were unsuccessful.

Phylogenetic analyses

The ITS-LSU for newly sequenced taxa was added to the combined ITS-LSU-rpb2-tef1-α alignment from Réblová . The two protein-coding genes were kept in the analyses to provide backbone support for higher level taxa. Portions of the 5’ and 3’ ends of each gene were excluded from all analyses due to missing data in most taxa and aligned using MUSCLE (Edgar 2004). PartitionFinder2 (Lanfear et al. 2016) was used to determine the best-fit model for each dataset, which was the SYM+I+G model for ITS, and the GTR+I+G model for LSU, rpb2 and tef1-α. A maximum likelihood (ML) analysis with 1 000 bootstrap replicates was performed using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) with a GTRCAT approximation using the CIPRES Science Gateway v. 3.3 portal (Miller ). Bootstrap replicates were performed 1 000 times under the GTR model employing a GAMMA model of rate heterogeneity and the rapid bootstrapping option (Stamatakis ). Clades with bootstrap support (BS) values ≥ 70 % were considered significant and strongly supported (Hillis & Bull 1993). Bayesian Inference (BI) analysis was performed using MrBayes v. 3.2.7 (Huelsenbeck & Ronquist 2001, 2005) under the GTR model on the CIPRES v. 3.3 portal. Constant characters were included and 10 M generations with trees sampled every 1 000th generation were run, resulting in 10 000 total trees. The first 2 500 trees were discarded as burn-in, and Bayesian posterior probabilities (PP) were determined from a consensus tree generated from the remaining 7 500 trees using PAUP v. 4.0b10 (Swofford 2002). Clades with PP ≥ 95 % were considered significant and strongly supported (Larget & Simon 1999, Alfaro ).

RESULTS

Phylogenetic analysis

The ITS-LSU region was sequenced for I. phyllophila, I. ripogoni and the new species (Table 1) and ML and BI analyses were performed on ITS-LSU and ITS-LSU-rpb2-tef1-α datasets of 90 representatives of the Xylariales modified from Réblová . Outgroup taxa were Bactrodesmium abruptum and B. diversum (Savoryellaceae) and Helicoascotaiwania lacustris and Pleurotheciella erumpens (Pleurotheciaceae). Initially, we performed an ITS-LSU analysis (results not shown) to assess the placement of Iodosphaeria and the new species in the Xylariales. The backbone of the ML tree was largely unresolved and familial relationships were not supported. Iodosphaeria, consisting of six strains representing four species, i.e. I. honghensis, I. phyllophila, I. ripogoni and I. tongrenensis, formed a well-supported monophyletic group. However, the new species was placed outside this clade. The next analysis was based on the combined ITS-LSU-rpb2-tef1-α sequences in order to study phylogenetic relationships of Iodosphaeria with other members of the Xylariales. The alignment had 4 002 characters including gaps (ITS = 871 characters, LSU = 766, rpb2 = 1107, tef1-α = 1258) and 2 366 unique character sites (RAxML). This combined four-loci alignment generated a tree with backbone support for familial relationships and correctly placed the new species in the Iodosphaeria clade. Therefore, this ML tree is shown in Fig. 1 and includes 34 well-supported families of the Xylariales. Five Iodosphaeria species form a moderately-supported monophyletic group (78 % BS, 1.0 PP), which is well-supported (100 % BS, 1.0 PP) as a sister clade to the Pseudosporidesmiaceae. Iodosphaeriaceae are not closely related to the Amphisphaeriaceae as previously suggested by Samuels et al. (1987), but rather occupies its own position as a distinct family in the Xylariales. The three representatives of I. phyllophila cluster with moderate support (83 % BS, 1.0 PP) and their ITS sequences, which are 611 bp in length, are 98.2–99.3 % similar with only a single gap. As expected, the specimens from Belgium (ILLS00121493) and France (FC5099-2d) are more closely-related to eachother than they are to the specimen from New Zealand (PDD 56626). The new species occupies a basal position in the Iodosphaeria clade.

Table 1

Specimens used in this study including specimen/strain, type status, GenBank accession numbers and source of sequences; T, E, I, N and P denote ex-type, ex-epitype, ex-neotype and ex-paratype strains.

Taxon	Specimen/Strain	Type Status	GenBank accession numbers				Reference
			ITS	LSU	ref1-α	rpb2
Acrocordiella occulta	CBS 140500	E	KT949893	KT949893	—	—	Jaklitsch et al. (2016)
Amphibambusa bambusicola	MFLUCC 11-0617	T	KP744433	KP744474	—	—	Liu et al. (2015)
Amphisphaeria flava	MFLUCC 18-0361	T	MH971224	MH971234	—	—	Samarakoon et al. (2019)
Amphisphaeria fuckelii	CBS 140409	T	KT949902	KT949902	MH554435	MH554918	Jaklitsch et al. (2016), Liu et al. (2019)
Amphisphaeria thailandica	MFLU 18-0794	T	MH971225	MH971235	—	MK033640	Samarakoon et al. (2019)
Annulohypoxylon michelianum	CBS 119993		KX376320	KY610423	—	KY624234	Kuhnert et al. (2016), Wendt et al. (2018)
Anthostoma decipiens	CBS 133221		KC774565	KC774565	—	—	Jaklitsch et al. (2014)
Anungitiomyces stellenboschiensis	CPC 34726	T	MK876376	MK876415	—	—	Crous et al. (2019a)
Arthrinium hysterinum	ICMP 6889		MK014874	MK014841	MK017951	DQ368649	Pintos et al. (2019), Tang et al. (2007)
Arthrinium pseudoparenchymaticum	SICAUCC 18-0008		MK346319	MK346321	MK359205	MK359207	Wang et al. (2018)
Astrosphaeriella erumpens	S.M.H. 1291		—	AF279410	—	AY641073	Bhattacharya et al. (2000)
Bactrodesmium abruptum	CBS 145967		—	MN699410	MN704315	MN704290	Réblová et al. (2020)
Bactrodesmium diversum	CBS 142448		—	MN699412	MN704317	MN704292	Réblová et al. (2020)
Barrmaelia macrospora	CBS 142768	T	KC774566	KC774566	MF489005	MF488995	Jaklitsch et al. (2014), Voglmayr et al. (2017)
Barrmaelia moravica	CBS 142769	E	MF488987	MF488987	MF489006	MF488996	Voglmayr et al. (2017)
Beltrania pseudorhombica	CBS 138003	T	MH554124	KJ869215	MH554558	MH555032	Liu et al. (2019)
Beltraniopsis neolitseae	CBS 137974	T	KJ869126	KJ869183	—	—	Crous et al. (2014a)
Biscogniauxia nummularia	MUCL 51395	E	KY610382	KY610427	—	KY624236	Wendt et al. (2018)
Brachiampulla verticillata	ICMP 15065	P	MW144418	MW144402	MW147322	MW147336	Réblová et al. (2021)
Brachiampulla verticillata	ICMP 15993		MW144419	MW144403	MW147323	MW147337	Réblová et al. (2021)
Cainia anthoxanthis	MFLUCC 15-0539	T	KR092787	KR092777	—	—	Senanayake et al. (2015)
Camillea obularia	ATCC 28093		AF201714	KY610429	—	—	Wendt et al. (2018)
Castanediella acaciae	CBS 139896	T	KR476728	MH878661	—	—	Crous et al. (2015), Hernández-Restrepo et al. (2017)
Castanediella cagnizarii	MUCL 41095		KC775732	KC775707	KJ476985	—	Becerra-Hernández et al. (2016)
Castanediella ramosa	MUCL 39857		KC775736	KC775711	KJ476989	—	Becerra-Hernández et al. (2016)
Clypeophysalospora latitans	CBS 141463	T	KX820250	KX820261	—	—	Giraldo et al. (2017)
Collodiscula japonica	CBS 124266		JF440974	JF440974	—	—	Jaklitsch & Voglmayr (2012)
Coniocessia cruciformis	CBS 125769	T	MH863750	MH875218	—	—	Vu et al. (2019)
Coniocessia minima	CBS 125765	T	MH863746	MH875214	—	—	Vu et al. (2019)
Cryptosphaeria eunomia var. fraxini	CBS 223.87		KT425231	KT425295	—	KT425361	Trouillas et al. (2015)
Cryptovalsa rabenhorstii	CBS 125574		KC774567	KC774567	—	—	Jaklitsch et al. (2014)
Cylindrium elongatum	CBS 115974		KM231853	KM231733	KM231989	KM232429	Lombard et al. (2015)
Cylindrium grande	CBS 145578		MK876385	MK876426	MK876496	MK876482	Crous et al. (2019a)
Daldinia concentrica	CBS 113277		AY616683	KY610434	—	KY624243	Triebel et al. (2005), Wend et al. (2018)
Delonicicola siamense	MFLUCC 15-0670	T	MF167586	MF158345	—	MF158346	Perera et al. (2017)
Diatrype disciformis	CBS 197.49		—	DQ470964	DQ471085	DQ470915	Spatafora et al. (2007)
Diatrypella vulgaris	CBS 128329		MH864880	MH876328	—	—	Vu et al. (2019)
Entosordaria perfidiosa	CBS 142773	E	MF488993	MF488993	MF489012	MF489003	Voglmayr et al. (2017)
Fasciatispora arengae	MFLUCC 15-0326a		MK120275	MK120300	MK890790	MK890794	Doilom et al. (2018)
Fasciatispora cocoes	MFLUCC 18-1445		MN482680	MN482675	MN481516	MN481517	Hyde et al. (2020)
Furfurella luteostiolata	CBS 143620	T	MK527842	MK527842	MK523302	MK523273	Voglmayr et al. (2019)
Graphostroma platystoma	CBS 270.87		JX658535	DQ836906	DQ836915	KY624296	Zhang et al. (2006), Stadler et al. (2014), Wendt et al. (2018)
Helicoascotaiwania lacustris	CBS 145963	T	—	MN699430	MN704329	MN704304	Réblová et al. (2020)
Hypocopra rostrata	NRRL 66178		KM067909	KM067909	—	—	Jayanetti et al. (2014)
Hyponectria buxi	UME 31430		—	AY083834	—	—	Unpublished
Hypoxylon fragiforme	MUCL 51264	E	KC477229	KM186295	—	KM186296	Stadler et al. (2013)
Idriella lunata	CBS 204.56	T	KP859044	KP858981	—	—	Hernández-Restrepo et al. (2016)
Induratia thailandica	MFLUCC 17-2669	T	MK762707	MK762714	—	MK791283	Samarakoon et al. (2020)
Iodosphaeria foliicola	NBM-F-07096	T	MZ509148	MZ509160	—	—	This study
Iodosphaeria honghensis	MFLU 19-0719	T	MK737501	MK722172	—	MK791287	Marasinghe et al. (2019)
Iodosphaeria phyllophila	PDD 56626		MZ509149	MZ509149	—	—	This study
Iodosphaeria phyllophila	FC 5099-2d		MZ509150	—	—	—	This study
Iodosphaeria phyllophila	ILLS00121493	E	MZ509151	—	—	—	This study
Iodosphaeria ripogoni	PDD 103350		MZ509152	MZ509152	—	—	This study
Iodosphaeria tongrenensis	MFLU 15-0393	T	KR095282	KR095283	—	—	Li et al. (2015)
Kretzschmaria deusta	CBS 163.93		KC477237	KY610458	—	KY624227	Stadler et al. (2013), Wendt et al. (2018)
Leiosphaerella praeclara	CBS 125586		JF440976	JF440976	—	—	Jaklitsch & Voglmayr (2012)
Leptosillia acerina	CBS 143939	E	MK527849	MK527849	MK523310	MK523282	Voglmayr et al. (2019)
Leptosillia macrospora	CBS 143627	E	MK527853	MK527853	MK523314	MK523286	Voglmayr et al. (2019)
Lopadostoma gastrinum	CBS 134632	N	KC774584	KC774584	—	—	Jaklitsch et al. (2014)
Lopadostoma turgidum	CBS 133207	E	KC774618	KC774618	—	KC774563	Jaklitsch et al. (2014)
Melogramma campylosporum	CBS 141086		JF440978	JF440978	—	—	Jaklitsch & Voglmayr (2012)
Microdochium lycopodinum	CBS 125585	T	JF440979	JF440979	—	KP859125	Jaklitsch & Voglmayr (2012), Hernández-Restrepo et al. (2016)
Muscodor yunnanensis	WS38		MG866046	MG866038	—	MG866059	Chen et al. (2019)
Neophysalospora eucalypti	CBS 138864	T	KP004462	KP004490	—	—	Crous et al. (2014b)
Nothodactylaria nephrolepidis	CBS 146078	T	MN562132	MN567639	—	MN556809	Crous et al. (2019b)
Oxydothis metroxylonicola	MFLUCC 15-0281	T	KY206774	KY206763	KY206778	KY206781	Konta et al. (2016)
Oxydothis palmicola	MFLUCC 15-0806	T	KY206776	KY206765	KY206780	KY206782	Konta et al. (2016)
Phlogicylindrium eucalypti	CBS 120080	T	DQ923534	DQ923534	—	MH554893	Summerell et al. (2006), Liu et al. (2019)
Phlogicylindrium uniforme	CBS 131312	T	JQ044426	JQ044445	—	—	Crous et al. (2011)
Pleurotheciella erumpens	CBS 142447	T	—	MN699435	MN704334	MN704311	Réblová et al. (2020)
Pseudapiospora corni	CBS 140736	N	KT949907	KT949907	—	—	Jaklitsch et al. (2016)
Pseudomassaria chondrospora	CBS 125600		JF440981	JF440981	—	—	Jaklitsch & Voglmayr (2012)
Pseudosporidesmium lambertiae	CBS 143169	T	MG386034	MG386087	—	—	Crous et al. (2017)
Pseudotruncatella arezzoensis	MFLUCC 14-0988	T	MG192320	MG192317	—	—	Perera et al. (2018)
Pseudotruncatella bolusanthi	CBS 145532	T	MK876407	MK876448	—	—	Crous et al. (2019a)
Requienella seminuda	CBS 140502		KT949912	KT949912	MK523328	MK523300	Jaklitsch et al. (2016), Voglmayr et al. (2019)
Robillarda sessilis	CBS 114312	E	KR873256	KR873284	—	—	Crous et al. (2015)
Seiridium marginatum	CBS 140403	E	KT949914	KT949914	MK523329	MK523301	Jaklitsch et al. (2016), Voglmayr et al. (2019)
Selenodriella cubensis	CBS 683.96	T	KP859053	KP858990	—	—	Hernández-Restrepo et al. (2016)
Selenodriella fertilis	CBS 772.83		KP859055	KP858992	—	—	Hernández-Restrepo et al. (2016)
Sporidesmium knawiae	CBS 123529	T	FJ349609	FJ349610	—	—	Crous et al. (2008)
Strelitziomyces knysnanus	CBS 146056	T	MN562135	MN567642	—	MN556810	Crous et al. (2019b)
Subsessila turbinata	MFLUCC 15-0831	T	KX762288	KX762289	KX762291	—	Lin et al. (2017)
Vialaea insculpta	DAOM 240257		JX139726	JX139726	—	—	Shoemaker et al. (2013)
Vialaea minutella	BRIP 56959		KC181926	KC181924	—	—	McTaggart et al. (2013)
Xyladictyochaeta lusitanica	CBS 142290	T	KY853479	KY853543	—	—	Hernández-Restrepo et al. (2017)
Xylaria hypoxylon	CBS 122620		KY610407	KY610495	—	KY624231	Wendt et al. (2018)
Zygosporium mycophilum	CBS 894.69		MH859474	MH871255	—	—	Vu et al. (2019)
Zygosporium pseudomasonii	CBS 146059	T	MN562147	MN567654	—	MN556815	Crous et al. (2019b)

Fig. 1.

Maximum likelihood tree generated from a RAxML analysis of combined ITS, LSU, rpb2 and tef1-α sequences of selected members of the Xylariales. Members of Iodosphaeria are given in bold; T, E, N and P indicate ex-type, ex-epitype, ex-neotype and ex-paratype strains. Maximum likelihood bootstrap branch supports ≥ 75 % are shown above or below nodes and thickened branches indicate Bayesian posterior probabilities ≥ 95 %.

Taxonomy

Samuels et al., Mycotaxon 28: 486. 1987.

Sexual morph: Ascomata perithecial, solitary to gregarious, non-stromatic, superficial and easily removed from the substrate, subglobose to globose, black, covered with long, brown, flexuous hairs that project from the ascomata in a stellate fashion, coarse, brown, repent hyphae extending from the base of the ascomata, apex flattened or obtusely rounded, ostiolate, periphysate. Ascomal wall two-layered, outer layer of brown, angular cells, inner layer of hyaline, flattened cells. Paraphyses hyaline, septate, inflated at the base, tapering towards the tip. Asci unitunicate, cylindrical to clavate, with an amyloid ring or ring lacking, 8-spored. Ascospores uniseriate to biseriate, ellipsoidal, ellipsoidal-fusiform or allantoid, aseptate, hyaline, smooth-walled, with or without a mucilaginous sheath. Synasexual morphs: selenosporella-like conidiophores and conidia and/or ceratosporium-like conidia have been observed in culture, on the surface of ascomata and on the repent hyphae. Selenosporella-like. Conidiophores macronematous, mononematous, pigmented, branched, septate. Conidiogenous cells integrated, terminal and discrete, lateral, polyblastic, single or in verticilli, with minute denticles along a short apical rachis; conidiogenesis holoblastic-denticulate. Conidia subcylindrical, slightly curved or straight, hyaline, aseptate. Ceratosporium-like. Conidia arising from aerial hyphae, pigmented, septate, staurosporous with two or more arms.

Taxa included in Iodosphaeria

A.N. Mill. & Réblová, , MycoBank MB 840507. Fig. 2A–G.

Fig. 2.

Iodosphaeria foliicola NBM-F-07096. A. Ascoma on natural substrate. B. Ascoma mounted in water. C, D. Vertical sections of the ascomal wall. E. Paraphyses. F. Asci. G. Ascus apical apices with amyloid rings. H. Ascospores. Scale bars: A, B = 100 μm; C, D = 50 μm; E, F = 20 μm; H = 10 μm; G = 5 μm.

Etymology: Epithet derived from folium (L) leaf, and incola (L) dweller, referring to the substrate on which this species was found.

Typus: Canada, Prince Edward Island, Queens County, Port-la-Joye-Fort, Amherst National Historic Site, 46.1958N, -63.1342W, on overwintered leaves of Alnus sp., 5 Jun. 2011, A. Carter 1555 (holotype NBM-F-07096, isotype ILLS00121496), associated with a selenosporella-like asexual morph; GenBank ITS MZ509148, GenBank LSU MZ509160.

Description: Ascomata globose to subglobose, 220–400 μm diam., superficial, solitary, black, covered with numerous, brown, septate, flexuous, rarely branched hairs, 145–320 × 6–9 μm, thick-walled (walls 1.5–2.5 μm thick), with rounded ends, singly or in fascicles; apex flattened, ostiolate, periphysate. Ascomal wall 50–70 μm wide, two-layered, outer layer composed of pseudoparenchymatous cells forming a textura angularis, composed of 5–8 layers of thin-walled cells, inner cell layers hyaline, outer cell layers becoming smaller and darker brown, outermost layer of thick-walled, melanized, dark brown cells, inner layer composed of 2–3 layers of thin-walled, hyaline, flattened, pseudoparenchymatous cells. Paraphyses sparse, hyaline, septate, of similar length as asci, basal cells inflated, tapering to 3–6 μm wide at apex, disintegrating at maturity. Asci cylindrical, 94–136 × 7.5–10 μm (120 ± 11.0 × 8.5 ± 0.5), 8-spored, apex rounded, short-stipitate, with a wedge-shaped, amyloid apical ring, 1.5–2.3 × 1–1.2 μm. Ascospores oblong to slightly allantoid, ends obtuse, aseptate, eguttulate, hyaline, smooth, 16.5–19.5 × 2.9–4 μm (18 ± 0.8 × 3.5 ± 0.4), without a mucilaginous sheath, uniseriate to biseriate in the ascus.

Habitat and distribution: Found in Canada on mostly the abaxial side of overwintered leaves of Alnus sp.

Additional specimens examined: Canada, Prince Edward Island, Queens County, Port-la-Joye-Fort, Amherst National Historic Site, 46.1958N, -63.1342W, on overwintered leaves of Alnus sp., 21 Jul. 2011, A. Carter 1554 (NBM-F-07095), associated with a selenosporella-like asexual morph.

Notes: Iodosphaeria foliicola is only known from two specimens growing on overwintered Alnus sp. leaves from Prince Edward Island, Canada. It is distinguished from other members of Iodosphaeria by its shorter asci (Fig. 2F) with an amyloid ascal ring (Fig. 2G) and oblong to allantoid, shorter ascospores (Fig. 2H) that lack guttules and a mucilaginous sheath. It closely resembles I. honghensis, but differs in its shorter, eguttulate ascospores (Table 2). Iodosphaeria foliicola, I. phyllophila and I. tarda can occur on dead fallen leaves, but I. foliicola can be separated by its longer asci and shorter, allantoid ascospores. The collector noted a selenosporella-like asexual morph was associated with both collections of I. foliicola, but was not found during our examinations.

Table 2

Morphological characteristics, hosts, geographical distribution and references for species of Iodosphaeria.

Species	Length of asci (μm)	Ascal ring	Ascal ring amyloid	Shape of ascospores	Length of ascospores (μm)	Guttules in ascospores	Asexual morph(s)	Host(s)	Geographical distribution	Reference
Iodosphaeria foliicola	94–136	Present	Yes	Allantoid	16.5–19.5	Absent	Selenosporella-like	Alnus sp.	Canada	This study
I. honghensis	90–130	Present	Yes	Allantoid	18.5–22.5	Present	Ceratosporium-like	Unidentified	China	Marasinghe et al. (2019)
I. hongkongensis	80–102	Absent	n/a	Ellipsoidal	14–22	Absent	Unknown	Archontophoenix alexandrae	Hong Kong	Taylor & Hyde (1999)
I. phyllophila	105–138	Present	Yes	Allantoid	21–26.7	Absent	Selenosporella-like and ceratosporium-like	Alnus sp., Betula alba, Chamaenerion angustifolium, Corylus avellana, Cyathea dealbata, Gahnia sp., Populus sp. Ripogonum scandens, Rubus idaeus, Salix sp.	Belgium, Brazil, French Guiana, Great Britain, New Zealand	Samuels et al. (1987)
I. podocarpi	97–120	Present	Yes	Ellipsoidal	19–20	Absent	Selenosporella-like	Podocarpus parlatorei	Argentina	Catania & Romero (2012)
I. polygoni	150–180	Present	Yes	Ellipsoidal	18–23	Present	Ceratosporium-like	Polygonum chinese	Taiwan	Hsieh et al. (1997)
I. ripogoni	140–185	Absent	n/a	Ellipsoidal	21.5–26	Absent	Selenosporella-like and ceratosporium-like	Ripogonum scandens	New Zealand	Samuels et al. (1987)
I. tarda	70–85	Present	Yes	Ellipsoidal-fusiform	14–20	Absent	Unknown	Corylus sp., Phyllostachys sp.	France, Germany	Candoussau et al. (1996)
I. tongrenensis	150–210	Present	Yes	Ellipsoidal	18.5–22.5	Absent	Ceratosporium-like	Unidentified	China	Li et al. (2015)

Marasinghe et al. [as ‘honghense’], Phytotaxa 420: 276. 2019.

This species is only known from the type specimen (MFLU 19-0719), which was found growing on dead twigs of an unidentified host from Yunnan Province in China (Marasinghe ). It is distinguished from other species in the genus by its cylindrical to allantoid, guttulate ascospores that lack a mucilaginous sheath. A ceratosporium-like asexual morph was found growing on the host surface.

The unpublished tef1-α sequence (GenBank accession MK776957) from MFLU 19-0719, labeled as Iodosphaeria sp., was submitted to GenBank by D.S. Marasinghe & K.D. Hyde. Since the BLASTn search suggests closest relatives in the Hypocreomycetidae it is likely a contaminate; it should be removed from GenBank. However, four other sequences (SSU, ITS, LSU and rpb2) from this same voucher specimen under the original name, I. honghense, published by Marasinghe appear correct, but the taxonomic name should be updated to ‘honghensis’.

J.E. Taylor & K.D. Hyde, Sydowia 51: 128. 1999.This species is only known from four collections, all described as growing on either a dead petiole (holotype) or a dead rachis of a palm, Archontophoenix alexandrae, from Hong Kong (Taylor & Hyde 1999). It is separated from other species of Iodosphaeria by its smaller asci lacking an ascal ring and smaller, ellipsoidal to fusiform ascospores lacking a mucilaginous sheath. The only other species that lacks an apical ring is I. ripogoni, which possesses a mucilaginous sheath surrounding the ascospores. No asexual morph was observed on the host material.

(Mouton) Samuels et al., Mycotaxon 28: 486. 1987.

Basionym: Lasiosphaeria phyllophila Mouton, Bull. Soc. R. Bot. Belg. 39: 48, 1900.

This species is the most commonly collected in the genus, known from at least 20 collections (Dennis 1974, Samuels , MyCoPortal 2021). It was originally described as Lasiosphaeria phyllophila growing on rotten leaves of Betula alba and Corylus from Belgium (Mouton 1900). It has a wide distribution and is also known from Brazil, French Guiana, Great Britain, and New Zealand. It has been found growing on a variety of substrates on numerous hosts including leaves of B. alba, (Mouton 1900), Alnus (Kirchstein 1911) and Corylus avellana (Declercq 2008), dead twigs and fallen debris of Alnus, Populus and Salix (Ellis & Ellis 1985), dead stems of Chamaenerion angustifolium (Dennis 1974) and Rubus idaeus (Declercq 2008), cone scales (Dennis 1974), and on the rachis of Cyathea dealbata, Gahnia sp., and Ripogonum scandens (Samuels ). Iodosphaeria phyllophila is distinguished by its amyloid ascal ring and longer, allantoid, non-guttulate ascospores. Selenosporella- and ceratosporium-like synasexual morphs were reported on field collected material (Bell & Mahoney 2016) and in cultures of I. phyllophila (Samuels ). Iodosphaeria phyllophila and its synasexual morphs have previously been illustrated (Ellis & Ellis 1985, Samuels , Declercq 2008, Senanayake , Bell & Mahoney 2016).

Senanayake revised a collection of I. phyllophila originating from New Zealand (PDD 32622, Samuels ) and incorrectly referred to it as a holotype. The type material is preserved at BR, but since it is over 120 years old, attempts were not made to obtain molecular data directly from this specimen. Instead, a more recent specimen with numerous, fertile ascomata from Belgium was sequenced and chosen as epitype. The ascomata and ascospores of this newly designated epitype specimen have been previously illustrated (Declercq 2008; figs 3C, 4C). Both selenosporella- and ceratosporium-like synasexual morphs were found on and around ascomata of the epitype of I. phyllophila.

Typification: Belgium, Wachtebeke, Reepkes, IFBL C3.35, on stem of Rubus idaeus, 28 Jul. 2007, B. Declercq 07/068 (epitype designated here ILLS00121493; MBT 10002029; GenBank ITS MZ509151, isoepitype GENT), with selenosporella- and ceratosporium-like synasexual morphs, epitype designated for the holotype: Lasiosphaeria phyllophila Mouton, Bull. Soc. Rot. Bot. Belg. 39: 48. 1900. (BR).

Additional specimens examined: France, Las Muros, on scales of Picea cone, 24 Sep. 1997, J. Fournier FC 5099-2a (CUP); Ibid., Sainte-Ogeu, on branch of Vaccinum myrtillus?, 1 Mar. 1992, F. Candoussau FC 5099-2c (CUP), Ibid., Pyrénées Atlantiques, Oloron, Bugangue, 10 Jul. 1994, F. Candoussau FC 5099-2d (CUP). New Zealand, North Island, Auckland, Hūnua Ranges Regional Park, vic. Mangatangi Dam, -37.1158S, 175.2119E, on Cyathea dealbata, 1 Feb. 1989, P.R. Johnson (PDD 56626); Ibid., Swanson, on rachis of Cyathea dealbata, 6 May 1981, G.J. Samuels 81-80 (PDD 45501); Ibid., Waitemata City, Waitakere Range, Marguerite Track, on rachis of Cyathea dealbata, 15 May 1975, G.J. Samuels 75-289 (PDD 36844); Erua Forest, near National Park, on dead wood, 6 Apr. 2005, A. Bell 919 (PDD 83080), with selenosporella-like asexual morph. Spain, Basque region, Gipuzkoa Province, Tolosa, on twig of unidentified shrub, 14 Jan. 1995, F. Candoussau FC 5099-2b (CUP), with ceratosporium-like asexual morph.

Catania & A.I. Romero, Mycosphere 3[‘2’]: 40. 2012.

This species is only known from the type specimen, which was found growing on a branch of Podocarpus parlatorei in Argentina (Catania & Romero 2012). It is unique in having shorter asci with an amyloid apical ring and ellipsoid to navicular or slightly allantoid, shorter ascospores. A selenosporella-like asexual morph was found growing near or among the ascomata.

W.H. Hsieh et al., Mycol. Res. 101: 841. 1997.

This species is only known from the type specimen found on stems of Polygonum chinese in Taiwan (Hsieh ). It is unique in having longer asci and ellipsoidal, guttulate ascospores. A ceratosporium-like asexual morph was found associated with the repent network of brown hyphae occurring at the base of the ascomata.

Samuels et al., Mycotaxon 28: 490. 1987.

This species is known from eight collections, all growing on the woody, vine-like stems of Ripogonum scandens from New Zealand (Samuels , Bell & Mahoney 2016, MyCoPortal 2021). It is distinguished by its lack of an ascal apical ring and its ellipsoidal ascospores that possess a mucilaginous sheath while still in the ascus. The only other species that lacks an apical ring is I. hongkongensis, which also lacks a mucilaginous sheath surrounding the ascospores. Although I. tongrenensis also has ascospores with a sheath, its asci possess an amyloid ascus ring. Selenosporella- and ceratosporium-like synasexual morphs were reported on the coarse, brown, repent hyphae that radiate from the ascomata and cover the surface of the substrate in field collected material of I. ripogoni (Samuels ).

Additional specimen examined: New Zealand, North Island, Mt. Egmont National Park, Stratford, Dawson Falls Road, on dead stems of Ripogonum scandens, 18 Jan. 2013, A. Bell 1205 (PDD 103350), associated with selenosporella-like asexual morph.

(Fuckel) A.N. Mill. & Réblová, , MycoBank MB 840508. Fig. 3A–J.

Fig. 3.

Iodosphaeria tarda Fungi Rhen. Exs., Suppl., 2021. A. Ascomata on decaying leaf. B, C. Ascomata on natural substrate. D. Ascoma mounted in water. E. Vertical section of the ascomal wall. F, G. Setae H. Paraphyses. I, J. Ascus apical apices with amyloid rings. K. Ascospores. Scale bars: A = 1 cm; B, C = 500 μm; D = 100 μm; E, F, H = 20 μm; G = 50 μm; I–K = 5 μm.

Basionym: Sphaeria tarda Fuckel, Fungi Rhen. Exs., Suppl. Fasc. 6: no. 2021, 1867 [Bot. Zeitung 27(6): 97. 1869].

Synonyms: Trichosphaeria tarda (Fuckel) Fuckel, Jahrb. Nassauischen Vereins Naturk. 23–24: 145 (1869–1870) 1870.

Pyrenochaeta tarda (Fuckel) Sacc., Syll. fung. 3: 221. 1884.

Iodosphaeria tarda (Fuckel) M.E. Barr, Mycol. Helv. 8: 14. 1996, Nom. inval. (Art. 41.4., Melbourne).

This species was published in the exsiccatae series Fungi Rhen. Exs. no. 2021 (Fuckel 1870) and is only known from two collections. The isotype has been found on Corylus in Germany, whereas the second specimen (FC 319, Candoussau ) was collected on Phyllostachys from France. The isotype at FH was unavailable for study, but another specimen (M-0312560) of the Fungi Rhen. Exs. No 2021 at M was available. The latter specimen contains a decaying leaf of Corylus sp. with several ascomata arranged mainly along the leaf veins (Fig. 3A). The setae appears at first slightly rigid and erect, covering ascomata (Fig. 3C). Later, setae become decumbent, flexuous and project from the ascomal wall in a stellate fashion (Fig. 3B) leaving the top glabrous. The ascomata are subglobose, without a papilla but do not have the flattened apex that is typical of other species. Instead, the top is obtusely rounded. Iodosphaeria tarda is distinguished by its smaller asci and ascospores that are ellipsoidal-fusiform with a smooth wall. Ascospores in the French collection (FC 319) were reported oblong to nearly allantoid; for description and additional illustrations, see Candoussau . Candoussau examined the specimen Fungi Rhen. Exs. No 2021 (FH), which originated from the Caroline Barbey-Boissier herbarium, labelled Barbey-Boissier 672. Although Candoussau reported that I. tarda had a non-amyloid ascal ring, the specimen at M clearly shows an amyloid ring (Fig. 3I, J). The authors also reported no asexual morph or synasexual morph were found associated with either specimen, which agrees with our observations. Candoussau’s collections were sent to CUP in 2019, but FC 319 was not found there. The illustrations in Candoussau appear to be drawn by Margaret Barr, who proposed the (invalid) transfer to Iodosphaeria. It is possible that she sent FC 319 to MASS, which has been transferred to NY. However, this specimen does not exist at NY (B. Thiers, pers. comm.).

The transfer of the name to Iodosphaeria was invalidly published since Candoussau did not reference the date of the basionym (Art. 41.4, Melbourne; McNeill ). This is now corrected herein and we choose to maintain the same specific epithet.

Additional specimen examined: Germany, Johannisberg, on decaying leaf of Corylus sp., spring, Fuckel, Fungi Rhen. Exs. no. 2021 (isotype, M-0312560).

Q.R. Li et al., Phytotaxa 234: 125. 2015.

This species is only known from the type specimen, which was collected on dead twigs in the Guizhou Province in China (Li ). It is separated from all other species in the genus by its amyloid apical ascal ring and ellipsoidal ascospores that are surrounded by a mucilaginous sheath. Iodosphaeria ripogonii is the only other species known to have ascospores with a sheath, but it lacks an apical ascus ring. A ceratosporium-like asexual morph was observed on the surface of ascomata in I. tongrenensis.

Excluded and doubtful species

K.D. Hyde, Nova Hedwigia 61: 129. 1995.

This species is only known from the type specimen, which was collected on submerged wood in Queensland, Australia. The placement of I. aquatica in the genus was questioned by Hyde (1995) when he described it, others have suggested its taxonomic placement is unclear (Kang , Taylor & Hyde 1999), and Hsieh excluded it based on the glabrous, pyriform, erumpent ascomata often with lateral necks and its aquatic habitat. The LSU sequence of I. aquatica from Jeewon , but listed as dothideomycete sp. in GenBank clearly does not belong in the genus. Its LSU sequence is ~98 % similar to Lentistoma spp. in the Dothideomycetes, but the erumpent, pyriform ascomata and aseptate ascospores of I. aquatica are significantly different from the immersed, subglobose ascomata and 1-septate ascospores with a narrow, bipolar sheath found in Lentistoma (Hashimoto ). Additional material of I. aquatica should be collected and sequenced to properly place this species in the Lophiostomataceae. Likewise, the LSU sequence of Iodosphaeria sp. (AF452045) from voucher specimen HKUCC 3154 (Jeewon ), but listed as dothideomycete sp. in GenBank is 94 % similar to I. aquatica and most likely a member of the Pleosporales.

(Ellis & Everh.) M.E. Barr, Mycotaxon 46: 47. 1993.

Basionym: Trichosphaeria arundinariae Ellis & Everh., N. Amer. Pyrenomyc. 153. 1892.

This species is only known from the type specimen, which was originally described as Trichosphaeria arundinariae growing on an old culm of Arundinaria in Louisiana, USA (Ellis & Everhart 1892). It was transferred to Iodosphaeria by Barr (1993). It can be separated from other species in the genus based on its papillate ascomata, non-amyloid ascal ring, and ellipsoidal to fusiform, verruculose ascospores. Barr (1993) did not observe an asexual morph on the holotype. Iodosphaeria arundinaria deviates from the generic concept based on I. phyllophila in the diagnostic characteristics of ascomata, apical ring and ascospores suggesting certain heterogeneity. Therefore, it is argued that it should be treated as a doubtful species until it is recollected and its relationships are assessed with DNA sequence data.

Discussion

Members of Iodosphaeria possess ascomata that are macroscopically nearly identical in shape, size and overall appearance being subglobose to globose, superficial, and black with long, flexuous brown setae. Species are delimited by a combination of limited microscopic characters including: 1) the length of the asci, particularly whether they are longer or shorter than 150 μm, 2) whether or not the asci possess an apical ring, which is always amyloid if present, 3) the shape (ellipsoidal, ellipsoidal-fusiform or allantoid) and length of the ascospores, and 4) whether or not the ascospores have guttules. Although lengths of asci and ascospores and the presence or absence of an apical ascal ring are definitive, objective characters, the shape of the ascospores and the presence or absence of guttules are more ambiguous, subjective characters. Six of the nine accepted members of Iodosphaeria, exceptions being I. hongkongensis, I. phyllophila and I. ripogoni, are known from only one or two collections. Additional material of these six species needs to be recollected so that intraspecific variation, both molecular and morphological, can be examined to determine the significance of these characters in delimiting species.

Either selenosporella- and/or ceratosporium-like synasexual morphs were found in seven of the nine accepted species of Iodosphaeria. Both appear to be produced directly from the repent hyphae that radiate from ascomata over the surface of the substrate. Although the selenosporella-like synasexual morph produces conidiophores, the ceratosporium-like synasexual morph does not and conidia arise directly from the repent hyphae. Samuels discovered both synasexual morphs in the only species of Iodosphaeria to be obtained in culture, I. phyllophila. These synasexual morphs are dematiaceous hyphomycetes with holoblastic conidiogenesis. Molecular data of Ceratosporium are unavailable and its systematic placement is unknown. Interestingly, species of Ceratosporium form selenosporella-like conidiophores directly on conidia (e.g. Hughes 1964), thus the occurrence of both synasexual morphs in the life cycle of Iodosphaeria is not surprising. A similar phenomenon, when selenosporella-like conidiophores are formed either on conidia or conidiophores of the respective asexual morph, was described for example in Teratosperma (Hughes 1951, Matsushima 1975), Diplococcium (Wang & Sutton 1998, Hernández-Restrepo ) and Endophragmiella (e.g. Hughes 1979). The selenosporella-like morphotype appears polyphyletic in the Ascomycota. The genus Selenosporella (Arnaud 1953, MacGarvie 1968), typified with S. curvispora, was confirmed with molecular data to belong to the Helminthosphaeriaceae (Réblová ). The family is rich in selenosporella-like asexual morphs and synasexual morphs, which have been linked with Endophragmiella, Echinosphaeria, Hilberina and Ruzenia (Awao & Udagawa 1974, Matsushima 1975, Hughes 1979, Sivanesan 1983, Gams 1973, Miller & Huhndorf 2004). The selenosporella-like synasexual morph was described as part of the life cycle of several hyphomycetes such as Acrodictys bambusicola (Diaporthales) (Matsushima 1975), Spadicoides (Xenosphadicoidales) (Kuthubutheen & Nawawi 1991, Castañeda-Ruíz , Réblová ), and also Arachnophora excentrica (Hughes 1979), Polytretophora calcarata (= Spadicoides calcarata, Kuthubutheen & Nawawi 1991) and Quadracaea mediterranea (Lunghini ), whose systematic placements are unknown. In the Xylariales, selenosporella-like fungi have been linked with Oxydothis (Samuels & Rossman 1987) and were also classified in Selenodriella (Castañeda-Ruíz & Kendrick 1990, Hernández-Restrepo ). However, these genera are unrelated to Iodosphaeria (Fig. 1).

Species of Iodosphaeria have been reported from Argentina, Belgium, Brazil, China, French Guiana, Great Britain, Hong Kong, New Zealand, Taiwan and the USA (Samuels , Barr 1993, Hyde 1995, Candoussau , Hsieh , Taylor & Hyde 1999, Catania & Romero 2012, Li , Table 2). Only 27 collections of Iodosphaeria occur in the MyCoPortal (MyCoPortal 2021), 19 of which are either I. phyllophila or I. ripogoni from New Zealand. One collection of Iodosphaeria cf. ripogoni (NY03380508), which Samuels reported as immature and may be a new species, is reported from Hawaii on mistletoe; two previously unidentified specimens from Canada are now described as I. foliicola, and the remaining five specimens are unidentified Iodosphaeria species from Costa Rica and Venezuela. Iodosphaeria occurs on many different hosts including Alnus sp., Archontophoenix alexandrae, Betula alba, Chamaenerion angustifolium, Corylus avellana, Cyathea dealbata, Phyllostachys sp., Podocarpus parlatorei, Polygonum chinense, Populus sp., Ripogonum scandens, Rubus idaeus, and Salix sp.

Several species of Iodosphaeria lack DNA data; only five of the nine species have been sequenced. Molecular data should be obtained from either voucher specimens or freshly-collected material for the remaining four species: I. hongkongensis, I. podocarpi, I. polygoni, and I. tarda, and for the doubtful species, I. arundinariae. This will enable a better understanding of the relationships, distributions, host associations and ecology of members of Iodosphaeria.