Molecular phylogeny and a new Iranian species of Caudospora (Sydowiellaceae, Diaporthales)

Abstract

For the first time, molecular phylogenetic data on the peculiar diaporthalean genus Caudospora are available. Macro- and microscopic morphology and phylogenetic multilocus analyses of partial nuc SSU-ITS-LSU rDNA, cal, ms204, rpb1, rpb2, tef1 and tub2 sequences revealed two distinct species of Caudospora, which are described and illustrated by light and scanning electron microscopy. Caudospora iranica is described as a new species from corticated dead twigs of Quercus sp. collected in Iran. It differs from the generic type, C. taleola, mainly by coarsely verrucose ascospores. The asexual morph of C. taleola on natural substrate is described and illustrated. Caudospora taleola is neotypified, and it is recorded from Iran for the first time. Phylogenetic analyses of a multigene matrix containing a representative selection of Diaporthales from four loci (ITS, LSU rDNA, rpb2 and tef1) revealed a placement of Caudospora within Sydowiellaceae.

Starbäck (1889) introduced the genus Caudospora for a single peculiar species, C. taleola, which is characterized by perithecia immersed in a brown corticolous pseudostroma delimited by a distinct blackish zone, a whitish ectostromatic disc bearing few converging dark ostioles, cylindrical asci and hyaline, two-celled ascospores with an appendage at each end and 2–3 median appendages. The single known species, C. taleola, is confined to various species of Quercus spp., where it is widely distributed in Europe and North America (Rogers 1984). Ecologically, C. taleola is a saprotroph or weak pathogen of oaks, which can cause canker diseases (Phillips & Burdekin 1992).

Whereas morphological features clearly place C. taleola within Diaporthales, its generic as well as familial affiliation within the order remained controversial. Munk (1957) provided a detailed morphological description, noted that the genus is very distinct and found a high similarity of its centrum to that of Sydowiella fenestrans (Sydowiellaceae). Wehmeyer (1933) included Caudospora in the genus Diaporthe, whereas Müller & Arx (1962) transferred it to Hercospora. This generic classification was accepted by Kobayashi (1970) and Barr (1978). Rogers (1984) re-examined C. taleola from natural substrate and characterized the anamorph in pure culture. He concluded that Caudospora should not be considered synonymous with Hercospora because of its verrucose ascospores and appendages. Moreover, the anamorph of H. tiliae, the type species of Hercospora, is rabenhorstia-like whereas that of C. taleola is phomopsis-like. Rogers (1984) concluded that C. taleola is related to certain species of Diaporthe (D. leiphaemia var. raveneliana) based on the verrucose ascospores and anamorph. However, this connection has never been proven in molecular studies, and it is also doubtful whether D. leiphaemia var. raveneliana really belongs to Diaporthe. Until now, in the lack of sequence data the phylogenetic position of Caudospora within Diaporthales is uncertain.

During a survey of diatrypaceous fungi in Iran, a collection resembling Caudospora was revealed from dead corticated twigs of Quercus sp., which markedly differed from C. taleola in coarsely verrucose ascospores. Several fresh collections of C. taleola from Austria, France and Iran provided the opportunity for pure culture isolation and sequencing to clarify the phylogenetic affinities of Caudospora and the species status of the Iranian collection with verrucose ascospores, the results of which are here presented.

Materials and methods

Isolates and specimens

All isolates used in this study originated from ascospores of fresh specimens. Numbers of strains including NCBI GenBank accession numbers of gene sequences generated in the present study are listed in Tables 1 and 2. Strain acronyms other than those of official culture collections are used here primarily as strain identifiers throughout the work. Details of the specimens used for morphological investigations are listed in the Taxonomy section under the respective descriptions. In addition, the following strain of Hapalocystis berkeleyi was sequenced as outgroup taxon: UK, England, London, Royal Botanic Gardens Kew (RGB), on corticated branches of Platanus ×hispanica, 11 November 2008, H. Voglmayr D84 (WU 39959, living culture CBS 124568). Isolates were deposited in the Iranian Fungal Culture Collection of the Iranian Research Institute of Plant Protection (IRAN…C) and at the Westerdijk Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS culture collection), and dry specimens in the Herbarium of Iranian Research Institute of Plant Protection (IRAN…F) and the Fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU).

Tab. 1.

Strains and NCBI GenBank accessions used in the phylogenetic analyses of the combined SSU-ITS-LSU-cal-ms204-rpb1-rpb2-tef1-tub1 matrix. All sequences were generated during the present study.

Taxon	Strain	Voucher	Country	Host/substrate	GenBank accession numbers
					SSU-ITS-LSU	cal	ms204	rpb1	rpb2	tef1	tub2
Caudospora iranica	D189 = IRAN 2552 C = CBS 143507	IRAN 16716 F, WU 39950	Iran	Quercus sp.	MG495960	MG495951	MG495970	MG495979	MG495988	MG495997	MG496004
C. taleola	D185 = CBS 143508	WU 39951	Austria	Quercus robur	MG495961	MG495952	MG495971	MG495980	MG495989	MG495998	MG496005
C. taleola	D186	WU 39952	Austria	Quercus robur	MG495962	MG495953	MG495972	MG495981	MG495990	MG495999	MG496006
C. taleola	D187	WU 39953	Austria	Quercus robur	MG495963	MG495954	MG495973	MG495982	MG495991	MG496000	MG496007
C. taleola	D188 = IRAN 2551 C	IRAN 16715 F, WU 39954	Iran	Quercus sp.	MG495964	MG495955	MG495974	MG495983	MG495992	MG496001	MG496008
C. taleola	D197	WU 39955	France	Quercus robur	MG495965	MG495956	MG495975	MG495984	MG495993	MG496002	MG496009
C. taleola	D198	WU 39956	France	Quercus robur	MG495966	MG495957	MG495976	MG495985	MG495994	–	MG496010
C. taleola	D216	WU 39957	Austria	Quercus petraea	MG495967	MG495958	MG495977	MG495986	MG495995	–	MG496011
Hapalocystis berkeleyi	D84 = CBS 124568	WU 39959	UK	Platanus ×hispanica	MG495968	MG495959	MG495978	MG495987	MG495996	MG496003	MG496012

Tab. 2.

Strains and NCBI GenBank accessions used in the phylogenetic analyses of the combined ITS-LSU-rpb2-tef1 matrix. Sequences in bold were generated during the present study.

Taxon	Strain	Voucher	Host/substrate	GenBank accession numbers
				ITS	LSU	rpb2	tef1
Alnecium auctum	CBS 124263	WU 30206	Alnus glutinosa	KF570154	KF570154	KF570170	KF570200
Ambarignomonia petiolorum	CBS 121227	BPI 844274	Liquidambar styraciflua	EU254748	EU255070	EU219307	EU221898
Amphiporthe hranicensis	CBS 119289	BPI 843515	Tilia platyphyllos	EU199178	EU199122	EU199137	–
Apiognomonia errabunda	CBS 109747	AR 2813	Fagus sylvatica	DQ313525	NG_027592	DQ862014	DQ313565
Apoharknessia insueta	CBS 111377	CPC 1451	Eucalyptus pellita	JQ706083	AY720814	–	–
Asterosporium asterospermum	CBS 112404	–	Fagus sylvatica	–	AB553745	–	–
Auratiopycnidiella tristaniopsidis	CBS 132180	CBS H-20932	Tristaniopsis laurina	JQ685516	JQ685522	–	–
Cainiella johansonii	Kruys 731	UPS F-567263	Dryas octopetala	JF701922	JF701920	–	–
Caudospora iranica	D189 = IRAN 2552 C	IRAN 16716 F	Quercus sp.	MG495960	MG495960	MG495988	MG495997
Caudospora taleola	D185	WU 39951	Quercus robur	MG495961	MG495961	MG495989	MG495998
Caudospora taleola	D188 = IRAN 2551 C	IRAN 16715 F	Quercus sp.	MG495964	MG495964	MG495992	MG496001
Calosporella innesii	AR 3639	BPI 840945	Acer pseudoplatanus	JF681965	EU683071	–	–
Celoporthe dispersa	CBS 118781	PREM 58897	Syzygium cordatum	AY214316	HQ730854	–	HQ730841
Chapeckia nigrospora	CBS 125532	BPI 863766	Betula sp.	JF681957	EU683068	–	–
Chrysocrypta corymbiae	CBS 132528	CPC 19279	Corymbia sp.	JX069867	JX069851	–	–
Chrysoporthe cubensis	CBS 118654	PREM 58788	Eucalyptus sp.	JN942342	JN940856	–	GQ290137
Coniella fragariae	CBS 172.49	CBS H-10697	Forest soil	AY339317	AY339282	KX833472	KX833663
Coniella obovata	CBS 111025	CBS H-22703	Leaf litter	AY339313	KX833409	KX833497	KX833692
Coniella straminea	CBS 149.22	STE U 3932	Fragaria sp.	AY339348	AY339296	KX833506	KX833704
Coryneum depressum	AR 3897	BPI 843585	Quercus cerris	–	EU683074	–	–
Coryneum modonium	AR 3558	BPI 749131	Castanea sativa	–	EU683073	–	–
Coryneum umbonatum	AR 3541	BPI 872021	Quercus cerris	–	EU683072	–	–
Crinitospora pulchra	CBS 138014	CBS H-21729	Mangifera indica	KJ710466	KJ710443	–	–
Cryphonectria parasitica	ATCC 38755	–	Castanea dentata	AY141856	EU199123	–	EU222014
Cryptodiaporthe aesculi	CBS 109765	BPI 748430	Aesculus hippocastanum	EU199179	AF408342	EU199138	–
Cryptosporella hypodermia	CBS 116866	BPI 748432	Ulmus minor	EU199181	AF408346	EU199140	–
Cytospora chrysosperma	CFCC 89630	BJFC-S978	Salix psammophila	KF765674	KF765690	KF765706	KP321972
Cytospora hippophaes	CFCC 89640	–	Hippophae rhamnoides	KF765682	KF765698	KF765714	KP310865
Cytospora nivea	CFCC 89643	–	Salix psammophila	KF765685	KF765701	KF765717	KP310863
Diaporthe eres	CBS 138594	BPI 892912	Ulmus sp.	KJ210529	–	–	KJ210550
Discula destructiva	CBS 109771	BPI 1107757	Cornus nuttallii	EU199186	AF408359	EU199144	–
Disculoides eucalypti	CBS 132183	CBS H-20935	Eucalyptus sp.	JQ685517	JQ685523	–	–
Ditopella ditopa	CBS 109748	BPI 748439	Alnus glutinosa	DQ323526	EU199126	EU199145	–
Endothia gyrosa	CBS 112915	–	Quercus palustris	AF046905	AY194114	–	–
Erythrogloeum hymenaeae	CBS 132185	CBS H-20937	Hymenaea courbaril	JQ685519	JQ685525	–	–
Gaeumannomyces graminis	CBS 235.32	–	Oryza sativa	JX134669	JX134681	–	JX134695
Gnomonia gnomon	CBS 199.53	–	Corylus avellana	DQ491518	AF408361	EU219295	EU221885
Gnomoniopsis racemula	CBS 121469	BPI 871003	Epilobium angustifolium	EU254841	EU255122	EU219241	EU221889
Greeneria uvicola	FI1 2007	–	Vitis sp.	HQ586009	GQ870619	–	–
Hapalocystis berkeleyi	CBS 124568	WU 39959	Platanus ×hispanica	MG495968	MG495968	MG495996	MG496003
Harknessia eucalypti	CBS 342.97	–	Eucalyptus regnans	AY720745	AF408363	–	–
Harknessia molokaiensis	CBS 114877	–	Eucalyptus robusta	AY720749	AY720842	–	–
Harknessia weresubiae	CBS 113075	CBS H-9903	Eucalyptus sp.	AY720741	AY720835	–	–
Hercospora tiliae	CBS 109746	BPI 748440	Tilia tomentosa	–	AF408365	–	–
Immersiporthe knoxdaviesiana	CBS 132862	PREM 60740	Rapanea melanophloeos	JQ862770	JQ862760	–	–
Juglanconis appendiculata	CBS 142389	WU 35960	Juglans nigra	KY427145	KY427145	KY427195	KY427214
Juglanconis juglandina	ME23	WU 35965	Juglans nigra	KY427150	KY427150	KY427203	KY427222
Juglanconis pterocaryae	MAFF 410079	TFM FPH 3373	Pterocarya rhoifolia	KY427155	KY427155	KY427205	KY427224
Kohlmeyeriopsis medullaris	CBS 117849	–	Juncus roemerianus	KM484852	FJ176854	–	–
Lamproconium desmazieri	AR 3525	BPI 748445	Tilia sp.	–	AF408372	–	–
Luteocirrhus shearii	CBS 130776	PERTH 08439362	Banksia baxteri	KC197021	KC197019	–	–
Macrohilum eucalypti	CBS 118551	–	Eucalyptus sp.	DQ195781	DQ195793	–	–
Melanconiella chrysodiscosporina	CBS 125597	WU 31859	Carpinus betulus	JQ926238	JQ926238	JQ926310	JQ926376
Melanconiella hyperopta	CBS 132231	WU 31836	Carpinus betulus	JQ926285	JQ926285	JQ926351	JQ926418
Melanconiella spodiaea	SPOD	WU 31854	Carpinus betulus	JQ926300	JQ926300	JQ926366	JQ926433
Melanconis alni	CBS 122310	BPI 872035	Alnus viridis	EU199195	EU199130	EU199153	–
Melanconis marginalis	CBS 109744	BPI 748446	Alnus rubra	EU199197	AF408373	EU219301	EU221991
Melanconis stilbostoma	D143	WU 35970	Betula pendula	KY427156	KY427156	KY427206	KY427225
Occultocarpon ailaoshanense	CBS 129147	BPI 879254	Alnus nepalensis	JF779848	JF779852	JF779857	JF779862
Ophiodiaporthe cyatheae	BCRC 34961	HAST 1364	Cyathea lepifera	JX570889	JX570891	JX570893	KC465406
Ophiognomonia melanostyla	CBS 128482	BPI 879257	Tilia cordata	JF779850	JF779854	JF779858	–
Phaeocytostroma ambiguum	CBS 128562	–	Zea mays	FR748042	FR748101	–	FR748074
Phaeodiaporthe appendiculata	CBS 123821	WU 32449	Acer campestre	KF570156	KF570156	–	–
Pleuroceras tenellum	CBS 121082	BPI 871059	Acer rubrum	EU199199	EU255202	EU199155	EU221907
Prosopidicola mexicana	CBS 113529	CBS-H 7948	Prosopis glandulosa	AY720709	–	–	–
Pseudoplagiostoma eucalypti	CBS 124807	CBS H-20303	Eucalyptus urophylla	GU973512	GU973606	–	GU973542
Pseudoplagiostoma oldii	CBS 124808	CBS H-20300	Eucalyptus camaldulensis	GU973534	GU973609	–	GU973564
Pseudoplagiostoma variabile	CBS 113067	CBS H-20304	Eucalyptus globulus	GU973536	GU973611	–	GU973566
Pustulomyces bambusicola	MFLUCC 11-0436	MFLU 13–0369	Bambusa sp.	KF806752	KF806753	–	KF806755
Rossmania ukurunduensis	AR 3484	BPI 747566	Acer ukurunduense	–	EU683075	–	–
Rostraureum tropicale	CBS 115757	PREM 57519	Terminalia ivorensis	AY167436	AY194092	–	–
Sillia ferruginea	CBS 126567	BPI 843619	Corylus avellana	JF681959	EU683076	–	–
Sirococcus tsugae	CBS 119626	BPI 871167	Tsuga mertensiana	EU199203	EU199136	EU199159	EF512534
Stegonsporium acerophilum	CBS 117025	WU 28050	Acer saccharum	EU039982	EU039993	KF570173	EU040027
Stenocarpella macrospora	CBS 117560	MRC 8615	Zea mays	FR748048	EU754219	–	–
Stilbospora macrosperma	CBS 115073	WU 24708	Carpinus betulus	EU039965	MG495969	KF570195	EU039999
Sydowiella fenestrans	CBS 125530	BPI 843503	Chamerion angustifolium	JF681956	EU683078	–	–

Light microscopy

Dead corticated branches were examined with the aid of an Olympus SZH or a Nikon SMZ 1500 stereomicroscope. Microscopic observations were made in tap water except where noted. Sections were cut manually with a sterilized razorblade and microscope mounts were examined with a Nikon 80i equipped with a Canon digital camera or a Zeiss Axio Imager.A1 equipped with a Zeiss Axiocam 506 colour digital camera. Measurements were taken with the help of DinoCapture 2.0 or Zeiss ZEN Blue Edition softwares. Measurements are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses; in addition, the means (x̄) are given.

Scanning electron microscopy (SEM)

For SEM of ascospores, perithecial contents were re-hydrated in a drop of distilled water on a cover slide and thoroughly cleaved with a fine forceps and preparation needles to release the ascospores from the asci. After evaporation of the water at room temperature, the cover slides were mounted on Cambridge stubs, sputter coated with gold, and examined in a Jeol JSM-6390 scanning electron microscope at 10 kV.

Pure culture isolation

Single ascospore isolates were made by spreading ascospores on 1.5 % potato dextrose agar (PDA, Difco), or on 2 % corn meal agar (CMA, Sigma-Aldrich) plus 2 % w/v dextrose (CMD) supplemented with 200 mg/l penicillin G and streptomycin sulphate (Sigma-Aldrich). Germinated ascospores were then transferred to PDA or CMD plates, sealed with laboratory film and incubated at room temperature. Colony morphology, colour (Rayner, 1970) and growth rate were determined on PDA at 24 °C.

DNA extraction, PCR and sequencing

Growth of liquid cultures and extraction of genomic DNA was done according to Voglmayr & Jaklitsch (2011), using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany). The following loci were sequenced for phylogenetic analyses: The complete ITS region and D1 and D2 domains of 28S nuc rDNA region (ITS-LSU) were amplified with primers V9G (Hoog & Gerrits van den Ende 1998) and LR5 (Vilgalys & Hester 1990); a ca 0.6–0.7 kb fragment of the calmodulin (cal) gene with primers CAL-228F (Carbone & Kohn 1999) and CAL2Rd (Groenewald et al. 2013); a ca 1 kb fragment of the guanine nucleotide-binding protein subunit beta (ms204) gene with primers MS-E1F1n1 (5’ AAGGGNACYCTSGAGGGCCAC 3’) and MS-E5R2n (5’ CCASAGCATGGTGGTRCCRTC 3’); a ca 1.1 kb fragment of the RNA polymerase II subunit 1 (rpb1) gene with primers RPB1-6R1asc (Hofstetter et al. 2007) and RPB1-Af (Stiller & Hall 1997); a ca 1.1 kb fragment of the RNA polymerase II subunit 2 (rpb2) gene with primers dRPB2-5f and dRPB2-7r (Voglmayr et al. 2016a); a ca 1.3–1.5 kb fragment of the translation elongation factor 1-α (tef1) gene with primers EF1728F (Carbone & Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005) or EF1-2218R (Rehner & Buckley 2005); and a ca 1.5 kb fragment of the beta tubulin (tub2) gene with primers T1 and T22 (O’Donnell & Cigelnik 1997). PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr & Jaklitsch (2008). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington) and the PCR primers; in addition, the following primers were used: ITS-LSU region: ITS4 (White et al. 1990), LR3 (Vilgalys & Hester 1990); tub2: BtHV2r (Voglmayr et al. 2016b, 2017) and BtHVf (5’ AACTGGGCMAAGGGYCAYTACAC 3’). Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyzer, Applied Biosystems). GenBank accession numbers of the newly generated sequences are given in Tab. 1.

Phylogenetic analyses

All alignments were produced with the server version of MAFFT (http://mafft.cbrc.jp/alignment/server/), the E-INS-i iterative refinement method and a gap opening penalty of 1.4 for the ITS-LSU and tef1, and with default settings for the other loci. All alignments were checked and refined using BioEdit version 7.0.9.0 (Hall 1999).

To reveal the phylogenetic position of Caudospora within Diaporthales, ITS-LSU, rpb2 and tef1 sequences of two accessions of C. taleola and of the new Iranian species sequenced in the present study were aligned to representative sequences of Diaporthales selected and downloaded from GenBank according to the phylogeny of Senanayake et al. (2017a). The finial matrix contained 75 representatives of Diaporthales, with Kohlmeyeriopsis medullaris and Gaeumannomyces graminis (Magnaporthales) selected as outgroups (Voglmayr et al. 2017); the strain details and GenBank accession numbers of sequences are given in Tab 2. The resulting combined four-loci sequence matrix contained 5188 nucleotide positions (2148 from ITS-LSU, 1210 from rpb2, 1830 from tef1).

For phylogenetic relationships within Caudospora, a combined matrix of nine loci was produced, containing 8683 nucleotide positions (1665 from SSU-ITS-LSU, 710 from cal, 1017 from ms204, 1151 from rpb1, 1160 from rpb2, 1433 from tef1, 1547 from tub2). According to the results of phylogenetic analyses of the four-loci matrix, Hapalocystis berkeleyi was selected as outgroup.

Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro & Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. The matrix was partitioned for the individual gene regions, and substitution model parameters were calculated separately for them.

Maximum parsimony (MP) analyses were performed with PAUP v. 4.0a157 (Swofford 2002). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MINBRLEN. For the combined four-loci matrix, a parsimony ratchet approach was applied. A nexus file was prepared using PRAP v. 2.0b3 (Müller 2004), implementing 1000 ratchet replicates with 25 % of randomly chosen positions upweighted to 2, which was then run with PAUP. The resulting best trees were subsequently loaded in PAUP and subjected to heuristic search with TBR branch swapping (MULTREES option in effect, steepest descent option not in effect). Bootstrap analysis with 1000 replicates was performed using 5 rounds of replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping during each bootstrap replicate, with each replicate limited to 1 million rearrangements. MP analysis of the combined nine-loci matrix was done using 1 000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping. Bootstrap analyses with 1 000 replicates were performed in the same way, but using 10 rounds of random sequence addition and subsequent TBR branch swapping during each bootstrap replicate.

Results

Molecular phylogeny

Of the 5188 nucleotide positions included in the four-loci sequence matrix, 1838 were parsimony informative (590 from ITS-LSU, 566 from rpb2, 682 from tef1). The best ML tree (lnL = –48924.555) revealed by the RAxML analysis is shown as phylogram in Fig. 1. Maximum parsimony analyses revealed 12 MP trees 9903 steps long (not shown). In both ML and MP analyses, Caudospora is placed within the Sydowiellaceae with high support, but its closest relatives remain uncertain due to lack of bootstrap support of most nodes within Sydowiellaceae.

Fig. 1.

Phylogram of the best maximum likelihood tree (lnL = -48924.555) revealed by RAxML from an analysis of the combined ITS-LSU-rpb2-tef1 matrix of 75 selected members of Diaporthales, with two members of Magnaporthaceae (Gaeumannomyces graminis, Kohlmeyeriopsis medullaris) selected as outgroup. Familial classification follows Senanayake et al. (2017a). Caudospora (bold) is revealed as a member of Sydowiellaceae with high support. ML and MP bootstrap support above 50 % are given above or below the branches.

Of the 8683 nucleotide positions included in the nine-loci sequence matrix, 72 were parsimony informative (6 from SSU-ITS-LSU, 5 from cal, 15 from ms204, 9 from rpb1, 14 from rpb2, 15 from tef1, 8 from tub2). The MP analysis revealed a single tree 1559 steps long (Fig. 2); it is identical in topology to the best ML tree (lnL = –18010.352) revealed by RAxML. Sister group relationship of Caudospora iranica to C. taleola received maximum and high (93 %) bootstrap support in MP and ML analyses (Fig. 2), respectively, confirming their status as distinct species.

Fig. 2.

Phylogram of the single MP tree 1559 steps long, obtained by PAUP from an analysis of the combined nine-loci matrix (SSU-ITS-LSU, cal, ms204, rpb1, rpb2, tef1, tub2) of Caudospora, with Hapalocystis berkeleyi selected as outgroup according to Fig. 1. MP and ML bootstrap support above 50 % are given at the first and second position, respectively, above or below the branches.

Taxonomy

Caudospora iranica Mehrabi & Voglmayr, sp. nov. – Figs. 3, 5a, b, e.

MycoBank no.: MB 823458

Type. – IRAN. East Azerbaijan Province, Arasbaran, on dead branches of Quercus sp., 11 July 2015, leg. M. Mehrabi (IRAN 16716 F holotype; WU 39950 isotype; ex-holotype culture IRAN 2552 C, ex-isotype culture CBS 143507).

Description. – Pseudostromata 1–3.5 mm diam., immersed in the bark of dead branches (ca. 1.5 cm thick), erumpent, circular to irregular, separate, scattered, sometimes confluent, delimited from surrounding bark by a black line, the latter visible on the bark surface. Ectostromatic discs white, convex, circular to oval, tissue between ostiolar necks distinctly gray to dark grey, powdery. Central column whitish to grey. – Ostioles dark, at the same level as the disc surface, rarely projecting, opening separately. – Perithecia 2–7 per stroma, 300–700 μm diam., subglobose to ovoid, black, monostichous, arranged circinately in brown entostromata, with long ostiolar necks converging towards the ectostromatic disc. – Paraphyses elongate, hyaline, filiform, septate, evanescent. – Asci (170)181–211(230) × 15–18 μm (x̄ = 196 × 16.7 μm; n=20), cylindrical, with short or obsolete stalks and a conspicuous apical ring, containing eight uniseriate ascospores. – Ascospores (18)21–27(32) × (8)10.5–13(15) μm (x̄ = 23.7 × 11.7 μm; n=42), l/w = (1.6)1.8–2.3(2.5), broadly ellipsoid, two-celled, constricted at the septum, hyaline or pale yellowish, coarsely verrucose, in SEM with isolated warts 0.4–1 μm diam., with a tubular gelatinous appendage 10–17 × 1–1.3 μm long at each end and 2–3 (rarely 4) similar median tubular gelatinous appendages arising from near the septum.

Cultural characteristics. – Colonies felty, with irregular margins, first white, later primrose (23,,b) at the surface, often deeply immersed into the agar, reverse primrose, becoming olivaceous black (27,,,,m) in the dark at 24 °C, reaching the margin of a 30 mm Petri-dish after 30 days at 24 °C. No asexual morph seen.

Etymology. – Referring to its origin from Iran.

Habitat. – On dead corticated twigs of Quercus sp.

Distribution. – Only known from the type collection in Iran.

Notes. – Caudospora iranica resembles C. taleola in stroma and ascoma morphology and ascospore appendages. However, there are pronounced differences between these species in the ascospore ornamentation. In C. iranica, the surface of the ascospore wall is covered with prominent isolated warts up to 1 μm diam. that are easily visible by light microscopy, whereas in the latter a finely verruculose ornamentation is scarcely visible in light microscopy without staining; the much smaller (up to 0.4 μm diam.) and densely disposed warts are clearly revealed only in SEM. In addition, asci (196 × 16.7 vs. 162 × 12.1 μm on average) and ascospores (23.7 × 11.7 vs. 23.9 × 9 μm on average) of C. iranica are wider than those of C. taleola.

Caudospora taleola (Fr.) Bih. K. Svenska Vetensk Akad. Handl., Afd. 15(2): 11 (1889). – Figs. 4, 5c, d, f–m.

Basionym. – Sphaeria taleola Fr., Syst. mycol. (Lundae) 2(2): 391 (1823).

Synonyms. – Aglaospora taleola (Fr.) Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 168 (1863).

Chorostate taleola (Fr.) Traverso, Fl. ital. crypt. 1(2): 212 (1906).

Diaporthe taleola (Fr.) Sacc., Atti Soc. Veneto-Trent. Sci. Nat., Padova, Sér. 4 4: 112 (1875).

Hercospora kornhuberi Bäumler, Annln K. K. naturh. Hofmus. Wien 13: 440 (1898).

Hercospora taleola (Fr.) E. Müll., in Müller & von Arx, Beitr. Kryptfl. Schweiz 11(no. 2): 728 (1962).

Melanconis taleola (Fr.) Speg., Atti Soc. Crittogam. Ital., Série 2 3(1): 54 [no. 77] (1881).

Valsa taleola (Fr.) Fr., Summa veg. Scand., Sectio Post. (Stockholm): 391 (1849).

Type. – AUSTRIA. Oberösterreich, Raab, Wetzlbach, on dead branches of Quercus robur, 24 December 2015, leg. H.Voglmayr (WU 39951, neotype here designated, MBT381444; ex-neotype culture D185 = CBS 143508)

Description. – Pseudostromata 1–4 mm diam., immersed in the bark of dead branches (1–3 cm diam.), flat or erumpent, circular to irregular, separate, scattered, sometimes confluent, delimited from surrounding bark by a black line. Ectostromatic discs white, flat, circular to ovoid, tissue between ostiolar necks distinctly gray to dark grey, powdery. Central column whitish to grey. – Ostioles dark, at the same level as the disc surface, rarely projecting, opening separately, sometimes necks of perithecia united below the disc and discharging through a single ostiole. – Perithecia 2–16 per stroma, 300–700 μm diam., subglobose to ovoid, black, monostichous, arranged circinately in brown entostromata, with long ostiolar necks converging towards the ectostromatic disc. – Paraphyses elongate, hyaline, filiform, septate, evanescent. – Asci (140)145–180(207) × 10–13 μm (x̄ = 163 × 12.1 μm; n = 20), cylindrical, with short or obsolete stalks and a conspicuous apical ring, containing eight uniseriate ascospores. – Ascospores (17)20–28(31) × (6.5)8–10(11) μm (x̄ = 23.9 × 9 μm; n = 30), l/w = (2.1)2.3–3.0(3.4), ellipsoid, two-celled, constricted at the septum, hyaline, appearing smooth in light microscopy but distinctly verrucose in SEM with densely disposed warts 0.1–0.4 μm diam., with a hyaline tubular gelatinous appendage 6–17 × 1–1.5 μm long at each end and 2–3 (rarely 4) similar median tubular gelatinous appendages arising from near the septum.

Asexual morph on natural substrate acervular, phomopsis-like. – Conidiomata ca. 1 mm diam., visible as darker spots in surface view, showing the same organisation as the sexual morph, i.e. a central whitish stromatic column with a white ectostromatic disc and a brown pseudostroma delimited from surrounding bark by a black line, preceding the sexual morph; conidiogenous region in section pale olivaceous green. – Conidiophores branched, hyaline, septate, formed on the upper margin of the central column. – Conidiogenous cells phialidic, (6.5)11.5–18.5(26) × (1.4)1.7–2.5(3.5) μm (x̄ = 14.8 × 2.1 μm; n = 70), hyaline, smooth. – Conidia (15)17–25 (32) × 0.7–1.1(1.6) μm (x̄ = 21 × 0.9 μm; n=50), l/w = (14.8)18.2–29.7(37.2), filiform with variable shape from more or less straight, sigmoid, falcate, semicircular to circular, unicellular, hyaline, smooth.

Cultural characteristics. – Colonies felty, irregular, often deeply immersed into the agar, white at the surface and primrose (23,,b) to olivaceous black (27,,,,m) at the reverse in the dark at 24 °C, reaching the margin of 30 mm Petri-dish after 30 days at 24 °C.

Habitat. – On dead corticated twigs of Quercus sp.

Distribution. – Widely distributed in Europe, Asia, North America.

Other specimens examined. – AUSTRIA. Oberösterreich, St. Willibald, Gr. Sallet, on dead branches of Quercus robur, 26 December 2015, leg. H. Voglmayr (WU 39952, culture D186); St. Willibald, Geitzedt, on dead branches of Quercus robur, 31 December 2015, leg. H. Voglmayr (WU 39953, culture D187); Niederösterreich, Mannersdorf im Leithagebirge, Schweingraben, on dead branches of Quercus petraea, 12. March 2016, leg. H. Voglmayr (WU 39957, culture D216). FRANCE. Ardèche (07), Saint-Jean-Roure, Mandon, on dead branches of Quercus robur, 27 December 2015, leg. A. Gardiennet A.G. 15171 (WU 39955, culture D197); Saint-Jean-Roure, Grange de Sagne, on dead branches of Quercus robur, 28 December 2015, leg. A. Gardiennet A.G. 15173 (WU 39956, culture D198). IRAN. East Azerbaijan Province, Arasbaran, on dead branches of Quercus sp., 11 July 2015, leg. M. Mehrabi (IRAN 16715 F, WU 39954, culture IRAN 2551 C).

Notes. – No type specimen of Sphaeria taleola is preserved in the Fries collection at UPS (A. Kruys, pers. comm.), which necessitates neotypification. We here select a well-developed, abundant Austrian collection from the most likely original host, Quercus robur, as neotype, for which a culture and sequence data are available.

To our knowledge, C. taleola has not been previously been recorded from Iran; it is not listed in Ershad (2009). For comparison with C. iranica, see Notes there.

Discussion

Although morphologically undoubtedly a member of Diaporthales (Kirk et al. 2008), the phylogenetic affiliation and familial classification of Caudospora remained unclear up to now in the lack of DNA sequence data. Based on similarities in stromata and perithecia, Müller & Arx (1962) classified Caudospora in Hercospora (Lamproconiaceae), which was challenged by Rogers (1984), who recognized it to be substantially different from Hercospora in verrucose ascospores and a phialidic phomopsis-like asexual morph, vs. the holoblastic conidiation of the rabenhorstia-like asexual morph in Hercospora. However, although Rogers (1984) noted some similarities to the quercicolous Diaporthe leiphaemia var. raveneliana in verrucose ascospore ornamentation, he considered taxonomic rearrangements premature due to insufficient data. In our phylogenies, Caudospora is clearly revealed as a member of Sydowiellaceae (Fig. 1). Remarkably, already Munk (1957) noted a high similarity of centrum morphology of Caudospora to that of Sydowiella fenestrans, to which it is closely related.

After transfer of several lineages originally classified in other families of Diaporthales (e.g. Gnomoniaceae, Melanconidaceae), the Sydowiellaceae have in recent years become a rather heterogeneous assemblage which is difficult to define morphologically (Rossman et al. 2007). It currently comprises about 15 genera occurring as saprotrophs, endophytes and parasites of various herbaceous and woody hosts (Senanayake et al. 2017b). Most genera and species are characterized by reduced, poorly developed stromata containing few to a single perithecium, but there are few exceptions like Sillia and Chapeckia with well-developed multiperitheciate stromata. Also Caudospora has well-developed pseudostromata, which are delimited from the surrounding host tissue by a conspicuous black line, which is so far unique in Sydowiellaceae.

The molecular data reveal that Caudospora iranica and C. taleola are closely related, and they share a similar ecology and the same host genus, Quercus. However, they can be easily separated by ascospore morphology. Caudospora iranica has coarsely verrucose ascospores with isolated warts (Figs. 3h–j, 5a, b, e), whereas the ascospores of C. taleola appear smooth in light microscopy (Fig. 4g, h) but are shown to be finely verrucose in SEM (Fig. 5c, d, f). Ascospore ornamentation of C. taleola revealed by SEM in our investigations matches that illustrated in Rogers (1984: fig. 5) from a North American collection.

Fig. 3.

Caudospora iranica (IRAN 16716 F, holotype). a. stromata on dead branch of Quercus sp.; b. ectostromatic disc; c. transverse sections of a pseudostroma; d. vertical section of a pseudostroma; e, f. asci; g. paraphyses; h–j. ascospores, showing coarsely verrucose ornamentation; k. culture on PDA. Bars: a 3 mm; b 300 μm; c, d 1 mm; e–g 30 μm; h–j 10 μm.

Fig. 5.

a-f. SEM pictures of ascospores of Caudospora iranica (a, b, e) and C. taleola (c, d, f), showing the coarsely and finely verrucose ascospore ornamentation in C. iranica and C. taleola, respectively; the median smooth circular areas in c, d. represent the scars of median gelatinous appendages. g–m. Asexual morph of C. taleola. g, h. conidiomata in transverse section, showing the central stromatic column and olive-green conidial masses; i. conidiomata in vertical section, showing pseudostromata delimited by a black line, the central stromatic column and the olive-green conidial masses produced on the stromatic column; j–l. branched conidiophores with phialides producing filiform conidia (in j. with released elongate and circular conidia); m. conidia. a, b, e. IRAN 16716 F (holotype of C. iranica). c, d, f–m. WU 39951 (neotype of C. taleola). Bars: a–d 5 μm; e, f 1 μm; g 500 μm; h, i 200 μm; j–m 10 μm.

Fig. 4.

Caudospora taleola (IRAN 16715 F). a. stromata on dead branch of Quercus sp.; b. ectostromatic disc; c. transverse sections of a pseudostroma; d. vertical section of a pseudostroma (showing two united necks under ectostromatic disc); e, f. asci; g, h. ascospores; i. culture on PDA. Bars: a 3 mm; b 300 μm; c, d 1 mm; e, f 30 μm; g, h 10 μm.

To our knowledge, the asexual morph of C. taleola is here described and illustrated for the first time in detail from natural substrate. We did not observe an asexual morph in our cultures, but Rogers (1984) obtained a phomopsis-like asexual morph on oatmeal agar, with unicellular, sigmoid to falcate hyaline conidia (5)10–17 × 1–1.5 μm in size produced on phialides. He compared it with the descriptions of Myxosporium taleola and Libertella taleola, which were formally described by Saccardo (1884) as putative asexual morphs of C. taleola, based on data reported by Tulasne & Tulasne (1863) and Fuckel (1871) from associations of sexual and asexual morphs on natural substrate. Rogers (1984) found some differences of his strain in conidial size; especially conidial width was substantially narrower in his isolate than that reported for Myxosporium taleola (16 × 8 μm) and Libertella taleola (20–30 × 4 μm). However, he was unable to resolve whether these differences related to strain differences, differences caused by conidiation in pure culture vs. on natural substrate, or erroneous connections of asexual morphs of species other than C. taleola. The asexual morph from natural substrate investigated by us agrees well with that reported by Rogers (1984) in its phialidic conidiogenous cells produced on short, branched conidiophores, conidial shape and conidial width, but our conidia are somewhat longer (15–32 vs. (5)10–17 μm in Rogers 1984), which may be caused by the different substrate. It therefore seems likely that the erroneous conidial width reported by Tulasne & Tulasne (1863) and Fuckel (1871) is due to misidentification.