Phylogenetic Placement and Taxonomic Review of the Genus Cryptosporella and Its Synonyms Ophiovalsa and Winterella (Gnomoniaceae, Diaporthales)

Home , Cryptosporella, Dispora, Gnomonia, Gnomoniaceae, Gnomonia leptostyla

mycological research 112 (2008) 23–35

journal homepage: www.elsevier.com/locate/mycres

Phylogenetic placement and taxonomic review of the genus Cryptosporella and its synonyms Ophiovalsa and Winterella (Gnomoniaceae, Diaporthales)

Luis C. MEJI´Aa,b,*, Lisa A. CASTLEBURYb, Amy Y. ROSSMANb, Mikhail V. SOGONOVa,b, James F. WHITEa aDepartment of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA bSystematic Botany & Mycology Laboratory, USDA Agricultural Research Service, Beltsville, Maryland 20705-2350, USA article info abstract

Article history: The type species of Cryptosporella, C. hypodermia, and Ophiovalsa, O. suffusa, as well as Received 29 December 2006 closely related species were studied using morphological, cultural, and DNA sequence Accepted 18 March 2007 characteristics. DNA sequence data from three different loci (ITS, LSU, and RPB2) suggest Corresponding Editor: Rajesh Jeewon that C. hypodermia and O. suffusa are congeneric within the Gnomoniaceae (Diaporthales). This result is supported by similarities in perithecial, ascal and ascospore morphology, Keywords: and lifestyles characterized as initially endophytic, becoming saprobic as plant tissues Disculina die. Furthermore, both type species produce Disculina anamorphs. A review of the literature Endophyte indicates that the generic name Cryptosporella has priority over Ophiovalsa and its synonym Pyrenomycetes Winterella sensu Reid & Booth (1987). A redescription of the genus Cryptosporella is included, RNA polymerase as well as a description of C. hypodermia, C. suffusa, the type species of Ophiovalsa, a brief Systematics account of the other seven species accepted in Cryptosporella, and a key to species of Cryp- tosporella. Eight new combinations are established: C. alnicola (Fr.) L.C. Mejıá & Castleb., comb. nov.; C. betulae (Tul. & C. Tul.) L.C. Mejıá & Castleb., comb. nov.; C. confusa (Reid & Booth) L.C. Mejıá & Castleb., comb. nov.; C. corylina (Tul. & C. Tul.) L.C. Mejıá & Castleb., comb. nov.; C. femoralis (Peck) L.C. Mejıá & Castleb., comb. nov.; C. suffusa (Fr.) L.C. Mejıá & Castleb., comb. nov.; C. tiliae (Tul. & C. Tul.) L.C. Mejıá & Castleb., comb. nov.; and C. wehmeyeriana (Reid & Booth) L.C. Mejıá & Castleb., comb. nov. ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction branches, their fruiting bodies are inconspicuous, appearing as raised bumps as they develop underneath bark, eventually Species in the genus Cryptosporella,asOphiovalsa and Winter- evident as short, black beaks erumpent through the bark sur- ella (Gnomoniaceae, Diaporthales), are known throughout the face. These species are also encountered as endophytes on temperate regions, especially North America Europe and their hardwood hosts, producing their Disculina anamorphic Japan, as saprobes, endophytes, and occasionally, as patho- states in culture (Barengo et al. 2000; Ganley et al. 2004). gens on hardwood trees especially Alnus, Betula, Corylus, Tilia, The genus Cryptosporella was described by Saccardo (1877) and Ulmus (Barr 1978; Chlebicki 2002; Glawe & Jensen 1986; to distinguish fungi that were classiﬁed as Cryptospora Tulasne Green 2004; Kobayashi 1970; Reid & Booth 1987, 1989; Spauld- & Tulasne (1863), but differed in ascospore shape. At that time ing 1961). Usually sporulating on small, overwintered species of Cryptospora were distinguished by having one to

* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.03.021 24 L. C. Mejı´a et al.

several-celled, long cylindrical ascospores. Based on the type used to store collection information and images and to mea- species Cryptosporella hypodermia, Cryptosporella was defined sure specimen structures as described by Sogonov (2005). by having species with hyaline, one-celled, oval to fusoid as- Cultures derived from recent collections and collections cospores. The distinction of these two genera based on shape made during the course of this study were obtained by means and size of ascospores was accepted by many mycologists of single spore isolation on corn meal agar (CMA, Sigma, (Arx & Mu¨ ller 1954; Barr 1978, 1991; Berlese 1900; Dennis St. Louis, MO) supplemented with antibiotics (1 % solution 1978; Ho¨ hnel 1917, 1918; Munk 1957; Traverso 1906; Wehmeyer 0.2 % streptomycin sulphate, and 0.2 % neomycin). Type spec- 1926). Other scientists (Ellis & Everhart 1892; Winter 1887) rec- imens of C. hypodermia and O. suffusa were sectioned for de- ognized Cryptosporella as a subgenus of Cryptospora and consid- tailed observation of perithecial structures. Small pieces of ered that shared characteristics such as arrangement and the substrata containing perithecia were excised and boiled position of perithecia, perithecial neck, habit, type of stroma, in distilled water for hydration during 90 min and left over- and asci were enough to retain these taxa in a single genus. night. For histological studies, tissue was prepared as in Petrak (1966) erected the genus Ophiovalsa based on Crypto- Torres et al. (2005). In brief, tissue was dehydrated in ethanol, spora suffusa when he realized that Cryptospora Tulasne & embedded in LR White acrylic resin, sectioned in slices of Tulasne 1863 was a later homonym of Cryptospora Karelin & 4 mm using glass knives, and stained in aniline blue (0.1 % Kirilow 1842 in the Brassicaceae. Reid & Booth (1987, 1989) aqueous) followed by toluidine blue (0.1 % aqueous) for 20 s treated C. hypodermia as congeneric with O. suffusa and placed in each stain. these two species and others in the genus Winterella. Discrep- Cultural studies ancies regarding the generic concepts and uncertainty as to which morphological traits to use for differentiating genera Cultures were plated in duplicate on three different media: and species, coupled with poor scientific communication dur- malt extract agar (MEA, Bacto, Becton, Dickinson & Co., ing the 1800s and first half of the 1900s, may have contributed Sparks, MD), potato dextrose agar (PDA, Difco, Becton, Dickin- to several nomenclatural and taxonomic problems related to son & Co., Sparks, MD), and CMA. Agar plugs 5 mm in diam these taxa that still persist today (see Discussion below). from the edge of actively growing colonies were used as inoc- A review of the order Diaporthales by Castlebury et al. (2002) ula for cultural studies. Cultures were grown at 23 C under based on LSU rDNA sequences revealed that O. suffusa and 12 h UV/white light and 12 h of dark. Radial growth measure- Cryptosporella hypodermia are closely related within the Gnomo- ments and phenotypic character observations were made at niaceae, but the details of this relationship were not resolved. 7, 14, and 21 d after plating. Two perpendicular colony diame- Additional DNA sequence data and morphological observa- ter measurements were made for each culture replicate. The tions suggest that several species described in these genera colony diameter presented represents the average of all mea- are congeneric. In order to determine the relationship of surements for a particular species. Colours assigned to colo- Ophiovalsa based on O. suffusa, including Winterella sensu nies are based on the colour chart by Rayner (1970). Reid & Booth (1987) with Cryptosporella based on C. hypodermia, both molecular and morphological evidence were obtained. A DNA extraction and PCR amplification redescription of the genus Cryptosporella and its type species, C. hypodermia, as well as the type species of Ophiovalsa, and One square centimetre of mycelium was scraped from the sur- an account of the genus as a whole is presented. face of actively growing cultures (about one-week old) and used for DNA extractions. Mycelium was lysed using Fast Prep FP120 (Thermo Electron Corporation, Milford, MA) or liquid nitrogen. Material and methods DNA was extracted using DNAeasy Plant mini kit (Qiagen, Valencia, CAUSA) or Puregene DNA Isolation kit (Gentra Systems, Morphological observations Minneapolis, MN) following the manufacturers’ instructions. The ITS regions 1 and 2, including 5.8 S rDNA ITS DNA, were Specimens representing species of Winterella sensu Reid & amplified with primers ITS5 and ITS4 (White et al. 1990). A re- Booth (1987) and the type species Ophiovalsa suffusa and Cryp- gion in the RNA polymerase second LSU (RPB2) was amplified tosporella hypodermia were examined. Morphological observa- with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999). LSU tions included macroscopic appearance and microscopic rDNA was amplified using primers LR0R and LR7 (Rehner & characters, such as size, shape, colour and arrangement of Samuels 1994; Vilgalys & Hester 1990). Amplifications were asci, ascospores, perithecial wall, and perithecial ostiolar tis- carried out in 50-ml reactions on an iCycler thermal cycler sues. Specimens were observed with a Zeiss SV 11 Apo (Carl (Bio-Rad Laboratories, Hercules, CA) under the following reac- Zeiss, NY) dissecting microscope and Zeiss Axiophot micro- tion conditions: 5–15 ng genomic DNA, 200 mM each dNTP, 2.5 scope (Carl Zeiss) with conventional brightfield or Nomarski units Amplitaq (Perkin Elmer), 2 mM of each primer and the sup- differential interference contrast microscopy. Perithecia and plied 10 buffer with 15 mM MgCl2. The thermal cycler pro- pycnidia were placed in a drop of 3 % aqueous potassium hy- gram for ITS was as follows: 2 min at 94 C followed by 35 droxide (KOH) or water on a clean microscope slide. After re- cycles of 30 s at 94 C, 30 s at 55 C, 1 min at 72 C), with a final hydration perithecia were observed and photographed. extension period of 10 min at 72 C. For RPB2 and LSU amplifi- Perithecia and pycnidia were crushed under a glass coverslip cations, Amplitaq Gold (Perkin Elmer) with an initial denatur- to release asci and conidia. Photographs were taken using ation step of 10 min at 94 C and an annealing temperature a Nikon digital camera DXM1200F (Nikon, Melville, NY). Micro- of 58 C were used. The resulting PCR products were treated soft Access 2000 (Microsoft Corporation, Bellevue, WA) was with ExoSAP-IT (USB, Cleveland, OH) following the Phylogenetic placement and taxonomic review 25

manufacturer’s instructions and sequenced with the BigDye (1102 bp) sequences for 24 isolates of gnomoniaceous taxa, in- version 3.1 (Applied Biosystems, Foster City, CA) on an ABI cluding the two type species, Cryptosporella hypodermia and 3100 automated DNA sequencer. PCR primers were used as Ophiovalsa suffusa, and one additional species O. betulae, with sequencing primers for all three genes. Additionally for 2968 total characters. Ninety-seven ambiguously aligned posi- LSU, primers LR3R and LR5 (Vilgalys & Hester 1990; Rehner & tions (1319–1415) were excluded from the ITS gene region, all Samuels 1994) were used as internal sequencing primers. positions were used for the LSU gene, and 40 ambiguously aligned positions (2779–2818) were excluded from the RPB2 Sequence analysis gene encompassing a region with an insertion in the Crypto- sporella/Ophiovalsa taxa. Of the remaining 2831 characters, Sequences were edited using Sequencher version 4.2 for Win- 589 were parsimony informative, 2123 were constant, and dows (Gene Codes Corporation, Ann Arbor, MI). Two datasets 119 were variable, but not parsimony-informative. MP phylo- were prepared. Alignment 1 consisted of ITS, LSU and RPB2 se- genetic analysis of combined alignment 1 resulted in one par- quences from representatives of the major lineages within the simonious tree (length ¼ 2461, CI ¼ 0.427, RI ¼ 0.552, RC ¼ Gnomoniaceae with three species representing the Cryptospor- 0.236, HI ¼ 0.573). Fig 1 shows the MP tree generated for the ella/Ophiovalsa group including both type species, and using combined alignment with MP BS support above and NJ BS sup- species of Melanconis and Cryphonectria as outgroup taxa port below branches. Only BSs 70 % or greater are shown. (Castlebury et al. 2002). This dataset was constructed to con- Results show that the respective type species of Cryptospor- firm the close relationship of C. hypodermia and O. suffusa ella and Ophiovalsa and O. betulae form a monophyletic group with a multiple gene analysis. Alignment 2 consisted of ITS (100 % MP and NJ BSs) within the Gnomoniaceae with Amphi- and RPB2 sequences from all available isolates of species of porthe hranicensis, Discula destructiva, Gnomonia gnomon, G. petio- the Cryptosporella/Ophiovalsa group with outgroup species lorum, Linospora capreae, and Pleuroceras tenellum as a sister identified from the first dataset as closely related and easily group, although BS values do not support this as a strong rela- alignable, including Amphiporthe hranicensis, Discula destruc- tionship (<70 %, Fig 1). Based on these results A. hranicensis, tiva, Gnomonia gnomon, and G. petiolorum. Gene regions were D. destructiva, G. gnomon, and G. petiolorum were chosen as out- aligned separately and concatenated into a single alignment. group taxa in the analysis of all available isolates of Crypospor- Sequences were initially aligned using ClustalX version 1.8 ella/Ophiovalsa. Other monophyletic groups with 100 % BS (Thompson et al. 1997) and manually adjusted in BioEdit support include a group containing the type species of Crypto- Sequence Alignment Editor version 7.0.5.3 (Hall 1999). diaporthe and Plagiostoma with Apiognomonia errabunda. The Each gene was analysed separately through the use of data type species of Gnomonia, G. gnomon, is not supported as partitions and a combined analysis was performed with PAUP strongly grouping with any other currently accepted species 4.0b10 (Swofford 2002). Trees were inferred by the NJ method of Gnomonia. A group of exclusively anamorphic taxa, includ- (Kimura 2-parameter distance calculation) and by MP using ing Sirococcus conigenus and Discula campestris, is supported as the heuristic search option with the random addition se- a monophyletic group (100 %). quence (1K replications) and the branch swapping (tree bisec- The combined alignment 2 comprising all available isolates tion–reconnection) option. For MP analyses, a limit of 1K trees of Cryptosporella and excluding PCR primer binding site re- per random addition sequence was enforced with a MAXTREE gions, consisted of ITS (553 bp), and RPB2 (1103 bp) sequences limit of 10K. For both types of analyses, ambiguously aligned for 25 isolates of Cryptosporella and previously mentioned out- positions were excluded. All characters were unordered and group taxa with 1656 total characters. All positions were used given equal weight during the analysis. Gaps were treated as for the two genes. Of the 1656 characters, 204 were parsimony missing data in the parsimony analysis and the NJ analysis informative, 1282 were constant, and 170 were variable but with missing or ambiguous sites ignored for the affected pair- not parsimony-informative. MP phylogenetic analysis of com- wise comparison. Relative support for branches was esti- bined alignment 2 resulted in 104 equally parsimonious trees mated with 1K BS replications (Felsenstein 1985) and 100 (length ¼ 607, CI ¼ 0.758, RI ¼ 0.792, RC ¼ 0.600, HI ¼ 0.242). random sequence additions per BS replicate for the MP BS Fig 2 shows one randomly chosen MP tree generated for the analysis of alignment 1 (family tree, Fig 1) and 1K BS replica- combined alignment, with MP BS supports above the tions with MULTREES off and ten random sequence additions branches. Only BSs 70 % or greater are shown. per BS replicate for alignment 2 (genus tree, Fig 2). Results (Fig 2) show that O. suffusa (hereafter referred to as C. suffusa) is supported within a monophyletic group (100 %) Voucher material containing C. hypodermia and five other species, including C. alnicola, C. betulae, C. confusa, C. femoralis, and C. wehmeyeri- Voucher material has been deposited at BPI and CBS; the spec- ana when analysed with their closest known relatives as out- imen and culture numbers are listed in Table 1. group taxa. C. betulae is supported (100 %) as most closely related to C. hypodermia. C. alnicola and C. wehmeyeriana are strongly supported as sister species (100 %) with C. confusa Results moderately supported (80 %) with them in a monophyletic group. Although C. suffusa appears basal to the other taxa in Molecular results the tree that is shown (Fig 2), BS values do not support any taxa as basal to the others. The combined alignment 1, excluding PCR primer binding site All species of Cryptosporella included in this analysis con- regions, consisted of ITS (645 bp), LSU (1221 bp), and RPB2 tain an insert of 14 bases in the RPB2 gene that is unique to 26 L. C. Mejıá et al.

Apiognomonia errabunda CBS 109747 95 Cryptodiaporthe aesculi CBS 109765 96 100 Cryptodiaporthe salicella CBS 109755 100 99 100 Gnomonia dispora CBS 205.37 100 100 72 Plagiostoma euphorbiae CBS 340.78 84 Gnomonia cerastis BPI 843494 Ditopella ditopa CBS 109748

Gnomonia fasciculata AR4000 96 90 Gnomonia padicola CBS 845.79 100 Sirococcus clavigignenti-juglandacearum CBS 121081 100 100 Gnomonia leptostyla BPI 747976 100 Amphiporthe hranicensis CBS 119289

Discula destructiva CBS 109771 Gnomonia gnomon CBS 199.53

Gnomonia petiolorum CBS 116866

100 Linospora caprae CBS 372.69 100 Pleuroceras tenella BPI 871059

Cryptosporella betulae CBS109763

100 Cryptosporella hypodermia BPI 748432 100 Cryptosporella suffusa BPI 871231 Discula campestris CBS 328.75

100 Sirococcus conigenus CBS 101225 99 100 Sirococcus piceicola CBS 119621 100 Sirococcus tsugae CBS 119626

100 Cryphonectria macrospora CBS 109764 100 100 Cryphonectria parasitica ATCC 38755 99 100 Melanconis carthusiana BPI 843622 99 100 Melanconis alni BPI 872035 100 Melanconis marginalis BPI 748446 50 changes

Fig 1 – Single most parsimonious tree based on analysis of 2831 bp representing a combination of three different loci (LSU, ITS, and RPB2) of 24 gnomoniaceous taxa using species of Melanconis and Cryphonectria as outgroup taxa (CI [ 0.427, RI [ 0.552, RC [ 0.236, HI [ 0.573, length [ 2461 steps). BS values greater than 70 % are shown above (MP) and below (NJ) each branch. Taxa in bold represent type species of their respective genera. Note that the type species of the genus Ophiovalsa, O. suffusa, here referred to as Cryptosporella suffusa, groups with the type species of the genus Cryptosporella, C. hypodermia.

the Diaporthales (position 2781–2798 of combined alignment 1 data. LSU data (not shown) indicate that W. albofusca and with a consensus sequence of CGSGCAARGASAAG). This is W. aurantiaca belong in the Diaporthales, although outside the based on sampling of more than 30 species representing all ac- Gnomoniaceae. The only available isolate of W. cinctula (CBS cepted genera of Gnomoniaceae, plus type species of genera 137.26) was sequenced and determined to be a representative representing five other families of Diaporthales (unpublished of the Diatrypales. It is not known if this isolate is an authentic data). isolate of W. cinctula or a misidentified culture. The abovemen- Winterella albofusca, W. aurantiaca, W. aurantiaca subsp. val- tioned species are all characterized by multiseptate asco- soides (syn. Ophiovalsa valsoides) and W. cinctula are excluded spores, a character state that is absent in species of from Cryptosporella based on morphological and molecular Cryptosporella as defined in this study. Phylogenetic placement and taxonomic review 27

Cryptosporella betulae BPI 843595

Cryptosporella betulae CBS 121079 96 Cryptosporella betulae CBS 109763

Cryptosporella betulae CBS 121078 99 Cryptosporella betulae BPI 843597 100 Cryptosporella betulae BPI 872328 98 Cryptosporella betulae BPI 843497

99 Cryptosporella hypodermia BPI 748432 99 Cryptosporella hypodermia CBS 171.69 Cryptosporella hypodermia CBS 109753

100 Cryptosporella alnicola BPI 872327 80 Cryptosporella wehmeyeriana BPI 843485

Cryptosporella confusa BPI 843580 Cryptosporella femoralis BPI 872326

93 Cryptosporella femoralis BPI 872325 Cryptosporella femoralis BPI 872325 Cryptosporella femoralis BPI 872324 100 Cryptosporella suffusa AR 4461

Cryptosporella suffusa AR 4462 92 Cryptosporella suffusa AR 4463 Cryptosporella suffusa AR 4464

Cryptosporella suffusa CBS 109750

98 Cryptosporella suffusa BPI 871231 Cryptosporella suffusa CBS 155.47

Cryptosporella suffusa CBS 172.69

Amphiporthe hranicensis BPI 843515

Discula destructiva CBS 109771 Gnomonia petiolorum CBS 116866

Gnomonia gonomon CBS 199.53 10 changes

Fig 2 – One of 104 equally parsimonious trees based on analysis of combined alignment of ITS and RPB2 genes containing (CI [ 0.758, RI [ 0.792, RC [ 0.600, HI [ 0.242, length 607 steps) for 25 isolates of Cryptosporella. BS values above 70 % are shown above each branch. The isolate representing the epitype specimen and culture of the genus Cryptosporella is shown in bold. Thickened branches indicate that the particular branch appears in the strict consensus tree of the 104 trees.

developed stroma. Asci cylindric to clavate with thickened Taxonomy apex, with or without an apical apparatus distinguished by presence of one or two refractive bodies. Acospores hyaline, Cryptosporella Sacc., Michelia 1: 30 (1877). cylindric with rounded tips, tapering or slightly swollen to- Synonyms: Cryptospora Tul. & C. Tul., Sel. Fung. Carp. 2: 144 ward their ends, fusiform, elliptic or femur-like, single- (1863) [non Karelin & Kirilow 1842.] celled, except in Cryptosporella femoralis that are two-celled Winterella (Sacc.) O. Kuntze, Rev. Gen. Pl. 1: 34 (1891). when mature. Ophiovalsa Petr., Sydowia 19: 272 (1966). Habit: As endophytes or sporulating on dead, overwintered Circinate arrangement of black perithecia, initially evi- twigs or branches of trees in the Betulaceae, Tiliaceae, and dent as a slight elevation in bark, later protruding through Ulmaceae. bark periderm either as a mass of perithecial necks or fused Anamorph: Disculina (type species: Disculina vulgaris). into a single ostiolar cavity; with or without a loosely Type species: 28 Table 1 – Isolates in the phylogenetic analyses of Cryptosporella and related members of the Diaporthales, specimen and culture numbers, accession numbers for LSU, ITS, and RPB2 sequences, host, country, and collector. New sequences from this study are in bold Species Specimen Culture LSU in Genbank ITS in Genbank RPB2 in Genbank Host Country Collector

Amphiporthe hracinensis BPI 843515 CBS 119289 EU199122 EU199178 EU199137 Tilia platyphyllos Austria W. Jaklitsch 1755 Apiognomonia errabunda d CBS 109747 AF408334 DQ313525 EU212961 Fagus sylvatica Switzerland M. Monod Cryphonectria macrospora BPI 748428 CBS 109764 AF408340 EU199182 EU220029 Quercus mongolica Russia L. Vasilyeva Cryphonectria parasitica d ATCC 38755 EU199123 AY141856 DQ862017 Castanea dentata USA S. Anagnostakis Cryptodiaporthe aesculi BPI 748430 CBS 109765 AF408342 EU199179 EU199138 Aesculus hippocastanum Austria W. Jaklitsch 1795 Cryptodiaporthe salicella BPI 747938 CBS 109755 AF408345 EU199183 EU199141 Salix sp. Austria W. Jaklitsch 1463 Cryptosporella alnicola BPI 872327 CBS 121074 d EU199204 EU199160 Corylus cornuta USA, MN L. Vasilyeva Cryptosporella betulae BPI 748448 CBS 109763 AF408375 EU199180 EU199139 Betula pendula Austria W. Jaklitsch 1610 Cryptosporella betulae BPI 843595 CBS 121075 d EU199214 EU199170 Betula pendula Austria W. Jaklitsch 2271 Cryptosporella betulae BPI 843597 CBS 121351 d EU199215 EU199171 Alnus alnobetula Austria W. Jaklitsch 2300 Cryptosporella betulae d CBS 121078 d EU199213 EU199169 Betula pendula Scotland S. Green Cryptosporella betulae d CBS 121079 d EU199216 EU199172 Betula pendula Scotland S. Green Cryptosporella betulae BPI 872328 CBS 121073 d EU199217 EU199173 Betula sp. USA, NY L. Vasilyeva Cryptosporella betulae BPI 843497 CBS 121080 d EU199218 EU199174 Betula sp. USA L. Vasilyeva Cryptosporella confusa BPI 843580 CBS 121063 d EU199219 EU199175 Betula papyrifera USA, TN W. Jaklitsch 2208 Cryptosporella femoralis BPI 872326 CBS 121076 d EU199220 EU199176 Alnus rugosa USA, NY L. Vasilyeva Cryptosporella femoralis BPI 872325 cf3 d EU199221 d Alnus rugosa USA, MI G. Adams Cryptosporella femoralis BPI 872325 cf4 d EU199222 d Alnus rugosa USA, MI G. Adams Cryptosporella femoralis BPI 872324 LCM22D d EU199223 d Alnus rugosa USA, ME L. C. Mejia Cryptosporella hypodermia BPI 748432 AR 3552 AF408346 EU199181 EU199140 Ulmus minor Austria W. Jaklitsch 1694 Cryptosporella hypodermia BPI 748433 CBS 109753 d EU199224 EU199177 Ulmus sp. Austria W. Jaklitsch 1497 Cryptosporella hypodermia d CBS 171.69 d EU199225 DQ862018 Ulmus sp. Netherlands H. van der Aa Cryptosporella suffusa BPI 748449 CBS 109750 d EU199207 EU199163 Alnus incana Austria W. Jaklitsch 1556 Cryptosporella suffusa BPI 871231 CBS 121077 EU199124 EU199184 EU199142 Alnus incana Austria W. Jaklitsch 1892 Cryptosporella suffusa d CBS 155.47 d EU199206 EU199162 Alnus glutinosa Netherlands S. Truter Cryptosporella suffusa d AR 4461 d EU199208 EU199164 Alnus sinuata USA, WA S. Lattomus, L. Mejia Cryptosporella suffusa d AR 4462 d EU199209 EU199165 Alnus sinuata USA, WA S. Lattomus, L. Mejia Cryptosporella suffusa d AR 4463 d EU199210 EU199166 Alnus sinuata USA, WA S. Lattomus, L. Mejia Cryptosporella suffusa d AR 4464 d EU199211 EU199167 Alnus sinuata USA, WA S. Lattomus, L. Mejia Cryptosporella suffusa d CBS 172.69 d EU199212 EU199168 Alnus glutinosa Netherlands H. van der Aa 1068 Cryptosporella wehmeyeriana BPI 843485 CBS 121085 d EU199205 EU199161 Tilia sp. USA, NC L. Vasilyeva Discula campestris d CBS 328.75 EU199125 EU199185 EU199143 Acer pseudoplatanus Germany A. John Discula destructiva BPI 1107757 CBS 109771 AF408359 EU199186 EU199144 Cornus nuttallii USA M. Daughtrey Ditopella ditopa BPI 748439 CBS 109748 EU199126 EU199187 EU199145 Alnus glutinosa Austria W. Jaklitsch Gnomonia cerastis BPI 843494 CBS 121084 EU199127 EU199188 EU199146 Acer sp. USA, NY L. Vasilyeva Gnomonia dispora d CBS 205.37 EU199128 EU199189 EU199147 Carya illinoensis M. Wilcox Gnomonia fasciculata BPI 872323 AR 4000 EU199129 EU199190 EU199148 Quercus mongolica Russia L. Vasilyeva Gnomonia gnomon d CBS 199.53 AF408361 AY818956 EU219295 Corylus avellana Italy M. Ribaldi Gnomonia leptostyla BPI 747976 CBS 110136 AF408362 EU199191 EU199149 Juglans nigra USA, IL D. Neely Gnomonia padicola d CBS 845.79 AF277134 EU199192 EU199150 Prunus padus Switzerland M. Monod 508 Mejı C. L. Gnomonia petiolorum BPI 843530 CBS 116866 AY818963 EU199193 EU199151 Liquidambar styraciﬂua USA, TN A. Rossman Linospora capreae d CBS 372.69 AF277143 EU199194 EU199152 Salix caprea Netherlands H. van der Aa

Melanconis alni BPI 872035 AR 3748 EU199130 EU199195 EU199153 Alnus viridis Austria W. Jaklitsch 1796 ´a

Melanconis carthusiana BPI 843622 CBS 121083 EU199131 EU199196 EU199154 Juglans regia Austria W. Jaklitsch 1450 al. et Melanconis marginalis BPI 748446 CBS 109744 AF408373 EU199197 EU219301 Alnus rubra Canada M. Barr 1021A Phylogenetic placement and taxonomic review 29

Cryptosporella hypodermia (Fr.) Sacc., Michelia 1: 30 (1877). Fig 3A–J Synonyms: Sphaeria hypodermia Fr., in Kunze & Schmidt, Mykol. Hefte 2: 49 (1823). Valsa hypodermia (Fr.) Fr., Summ. Veg. Scand. 2: 412 (1849). Cryptospora hypodermia (Fr.) Fuckel, Jahrb. Nassau Ver. Naturkd. 23–24: 192 [‘‘1869’’] (1870). Winterella hypodermia (Fr.) J. Reid & C. Booth, Can. J. Bot. 67: 880 (1989). Sphaeria limminghii West., Bull. Sci. Bruxelles, n.s. 7: 89 (1859). Valsa limminghii (West.) Kickx, Fl. Crypt. Fland.: 323 (1867). Netherlands H. van der Aa USA, NC M. Sogonov 0159 USA, MN M. Ostry Austria H. Anglberger Switzerland O. Holdenrieder Alaska G. Stanosz Cryptosporella limminghii (West.) Sacc., Syll. Fung. 1: 466 (1882). Cryptosporella veneta Sacc., Michelia 1: 31 (1877). Cryptosporella compta var. macrospora Beeli, Bull. Soc. R. Bot. Belg. 56: 58 (1924). Anamorph: Disculina sp. Perithecia initially evident as elevations in bark up to 0.7 mm high. Later perithecia visible as a dry oval to fusoid pustule 0.7 mm long 0.5 mm diam. Erumpent perithecia arranged in Euphorbia palustris Acer rubrum Juglans cinerea Picea abies Picea abies Tsuga mertensiana groups of 6–12, oriented at 45 angle toward surface. Perithecial necks converge centrally and, in many cases, emerge together as a column that protrudes through bark periderm. Perithecial neck column not extending much beyond rupture in bark. Ma- ture perithecia black,shiny,ﬂask-shaped and closely appressed,

EU219292 but separable from one another, diam height ¼ (345–)362– 619(–666) (300–)333–551(–651) mm(mean¼ 504 469, S.D.127, 121, n1 ¼ 12, n2 ¼ 12), perithecial necks (372–)492–650(–890) mm (mean ¼ 591, S.D.216,n ¼ 4) in length, basal diameter(144–)172– 208(–241) mm(mean¼ 192, S.D.27.8,n ¼ 12), and distal diameter (118–)163–213(–241) mm(mean¼ 182, S.D.45.1,n ¼ 6). Ectostroma scanty, embedding neck column where perithecial beaks con- EU199198 verge. Flared ostiolar openings, cup-like in appearance. No entostroma observed. Perithecial wall tissue textura angularis. Venter wall bilaminate, outer region averaging 7.7–15 mm wide (mean ¼ 10.6, S.D. ¼ 1.7 mm, n ¼ 13), of one to two layers of brown, thick-walled cells, cells 15–25 mmdiam(mean¼ 19 mm, S.D. ¼ 3.84, n ¼ 9). Inner region 15–46 mmwide(mean¼ 25 mm, S.D. EU199132 EU199199 EU199155 EU199133 EU199200 EU199156 EU199134 EU199201 EU199157 EU199135 EU199202 EU199158 EU199136 EU199203 EU199159 11.36 mm, n ¼ 13), of four to six layers of hyaline cells. Neck cavity densely filled with periphyses, surrounded by seven to ten layers of thick-walled cells elongated to give appearance of textura angularis or textura epidermoidea when viewed in cross section. Asci cylindric-clavate, floating free at maturity, length breadth ¼ (115–)132–141(–200) (19–)21.3–26.5(–31.3) mm CBS 340.78 AF408382 CBS 121081 (mean ¼ 138 24.1, S.D. 17.7, 3.87, n1 ¼ 24, n2 ¼ 24). Ascus apex thickened when young, with elongate indistinct pore. Eight ascospores per ascus arranged biseriately or obliquely parallel. As- cospores one-celled, hyaline,fusiform,biguttulate, with thick cell d BPI 871059 CBS 121082 d BPI 871248 CBS 101225 BPI 871166 CBS 119621 BPI 871167 CBS 119626 walls, (21.5–)38.0–50.0(–69.5) (5.5–)8.7–11.5(–16.5) mm(mean¼ 43.5 10.0, S.D. 8.5, 2.13, n1 ¼ 139, n2 ¼ 139), l:b (3–)4–5(–8) (mean ¼ 4, S.D.0.92,n ¼ 139) with cell walls appearing smooth at lower magnifications but at 1000 showing undulations or depressions. No colour reaction with Meltzer reagent and KOH. Cultural observations: (based on isolates AR 3552 and CBS 171.69) On PDA after 7 d average colony diam (a.c.d.) 1.7 cm (S.D. ¼ 0.25, n ¼ 8), after 14 d a.c.d. 3.7 cm (SD ¼ 0.30 cm, n ¼ 8), and after 21 d a.c.d. 5.2 cm (S.D. ¼ 0.73 cm, n ¼ 8). Colony appearance after 21 d on PDA smooth to regular margins, thin powdery with slimy pink drops containing conidia. Grey oliva- Plagiostoma euphorbiae Pleuroceras tenellum Sirococcus clavigignenti juglandacearum Sirococcus conigenus Sirococcus piceicola Sirococcus tsugae ceous (#107) rose colony with a dark halo 2.7 cm from centre 30 L. C. Mejıá et al.

Fig 3 – (A–J) Cryptosporella hypodermia. (A) Evidence of ascomata on twigs of Ulmus campestris. (B) Perithecia. (C) Bilaminate venter wall, outer region one to two layers of cells, inner region 4–6 layer of cells. (D) Perithecia, appressed with converging Phylogenetic placement and taxonomic review 31

followed by a honey (#64) halo and hyaline to whitish Valsa rhabdospora de Not., Sfer. Ital. Cent. I: 39 (1863). marginal mycelium. Reverse with a radial growth pattern Cryptospora rhabdospora (de Not.) Sacc., Syll. Fung. 2: 362 (1883). and a black depression in centre of colony, surrounded by Anamorph: Disculina vulgaris (Fr.) B. Sutton, Mycol. Pap. 141:75 a grey olivaceous halo. Conidia hyaline, cylindrical to ellipsoi- (1977). dal, aseptate, (29.0–)34.5–53.5(–64.0) (9.0–)10.0–12.5(–14.5) mm Ascomata evident as scattered elevations in bark with a pla- (mean ¼ 45.5 11.5, SD length 10.5, width 1.5, n ¼ 20). Coni- teau-like form with an average base of 2 mm diam and 0.7 mm diogenous cells holoblastic, annellidic, narrowly cylindrical, high, with or without a darker circular area of 0.7 mm diam. producing a conidium at apex. Perithecia black, in groups of up to 11, oriented parallel or in an- On CMA after 7 d a.c.d. 0.7 cm (S.D. ¼ 0.26, n ¼ 8), after 14 d gles of 45 toward bark surface with necks converging in centre a.c.d. 1.5 cm (S.D. ¼ 0.37 cm, n ¼ 8) and after 21 d a.c.d. 2.2 cm and fused to form a single cavity with a semi-biconic, flat tip- (S.D. ¼ 0.58 cm, n ¼ 8). Colony appearance after 21 d on CMA ped, protruding cone, 0.4 0.1 mm; diam height ¼ (458–)459– with radial mycelium, hyaline to whitish, growing appressed 466(–471) (258–)264–342(–416) mm (mean ¼ 463 314, S.D.6.9, to surface and smooth but irregular margins; reverse similar 89.0, n1 ¼ 3, n2 ¼ 3), Beak 806 mm high 108 mm diam at base, to above. 146 mm diam at apex. Perithecial wall tissue textura angularis. On 2 % MEA after 7 d a.c.d. 0.7 cm (S.D. ¼ 0, n ¼ 8), after 14 d Asci oval to obovoid narrowing to the base and the apex and 1.1 cm (S.D. ¼ 0.19 cm, n ¼ 8) and after 21 d a.c.d. 1.4 cm in some cases given the appearance of been constrained in (S.D. ¼ 0.35 cm, n ¼ 8). Colony appearance after 21 d on 2 % the middle, length width (52–)74.0–84.5(–100) (17.5–)22.0– MEA with smooth, regular to irregular margins, cartilaginous, 26.0(–30.0) mm (mean ¼ 80.0 23.5, S.D. 8.5, 3.2, n1 ¼ 31, greenish black (#124); iron grey (#122) with black inclusions in n2 ¼ 31), eight ascospores per ascus flexuous cylindrical colony reverse. twisted, interwoven, or parallel arranged, rounded at their Lectotype of Sphaeria hypodermia designated by Reid & tips (48.5–)57.5–66.0(–69.5) (3.8–)4.0–4.56(–5.5) mm (mean ¼ Booth 1989: Sweden: Uppsala, as Sphaeria hypodermia, Sclero- 61.5 4.38, S.D. 5.7, 0.4, n1 ¼ 22, n2 ¼ 22) l:b (10.5–)14.0–16.0 myc. Suec. Exs. 32 Herb. Fries. (–16.5) (mean ¼ 14.4, S.D. 1.7, n ¼ 22). No colouration in 3 % Epitype of Sphaeria hypodermia in: Austria: Vienna, 19th dis- KOH, no staining with Meltzer reagent. trict, Lotheissengasse, grid square 7763/2, on Ulmus minor,11 Cultural observations (based on isolates CBS 109750 and CBS Nov 2000, W. Jaklitsch 1694 (BPI 748432 - lectotypus hic designa- 121077): tus, derived culture AR 3552). On PDA after 7 d a.c.d. 1.5 cm (S.D. ¼ 0.3, n ¼ 8), after 14 d Specimens examined: Austria: Niederdonau: Donau-Auen near a.c.d. 3.5 cm (S.D. ¼ 0.8, n ¼ 8), after 21 d a.c.d. 4.3 cm Klosterneuburg, as Cryptosporella hypodermia on Ulmus sp., Apr (S.D. ¼ 0.9, n ¼ 8). Colony appearance after 21 d on PDA smooth F. Petrak 1939, , (Dr F. Petrak Mycotheca Generalis, BPI 601284). and irregular margins, with many drops of water over mycelia. Vienna, 21st district. Marchfeldkanalweg, grid square 7764/2. as Smoke grey (#105) to honey and hazel in the margin. Reverse C. hypodermia on U. minor and U. laevis, 8 Jul 2000, W. Jaklitsch 1497 (BPI 748433, derived culture CBS 109753 ¼ AR3566.) d mycelia with concentric halos (isabelline # 65) with honey Belgium: St Georges Coutrai,asSphaeria limminghii on Ulmus (#64) background and dark brown in the centre. On CMA after campestris, Herb. DeNotaris Rome, Shear Study Collection Types 7 d a.c.d. 0.9 cm (S.D. ¼ 0.2, n ¼ 8), after 14 d a.c.d. 1.2 cm d and Rarities Series I (BPI 800140). Hungary: Pressburg,asC. hypo- (S.D. ¼ 0.3, n ¼ 8), after 21 d a.c.d. 1.3 cm (S.D. ¼ 0.4, n ¼ 8). Colony d dermia,onUlmus campestris, J. A. Baumler (BPI 601289). Germany: appearance on CMA after 21 d with hyaline to salmon (#41) Nassau, Briebrich, on Ulmus sp., Fuckel 1894 (BPI 601287). d The mycelium, growing appressed to the surface, with a slightly Netherlands: Leiden,onUlmus sp., 21 Jun 1923, C. L. Shear, determined by Petrak (BPI 601276). visible salmon halo, and smooth but irregular margin. Reverse similar to above with some dark inclusions appearing in the Cryptosporella suffusa (Fr.) L.C. Mejıá & Castleb., comb. nov. medium. On 2 % MEA after 7 d a.c.d. 0.9 cm (S.D. ¼ 0.2, n ¼ 8), af- Fig 3K–P ter 14 d 1.4 cm (S.D. ¼ 0.2, n ¼ 8), after 21 d 1.9 cm (S.D. ¼ 0.2, MycoBank no.: MB 510391 n ¼ 8). Colony appearance after 21 d in 2 % MEA cartilaginous Basionym: Sphaeria suffusa Fr., Syst. Mycol. 2: 399 (1823). texture, colour hazel with regular honey margins. Reverse Synonyms: Valsa suffusa (Fr.) Fr., Summ. Veg. Scand.: 412 (1846). breaking the agar and grey olivaceous in colour. Cryptospora suffusa (Fr.) Tul. & C. Tul., Sel. Fung. Carpol. 2: 145 Specimens examined: Austria: Tirol: Overtilliach an der Gail, grid square 924/4, on Alnus incana, 29 Aug 2000, W. Jaklitsch 1556 (1863). as Ophiovalsa suffusa (BPI 748449, derived culture CBS Winterella suffusa (Fr.) O. Kuntze, Rev. Gen. Pl. 1: 34 (1891). 109750 ¼ AR3496). Vienna: Marchfeldkanalweg 7764/2, 21st district, Ophiovalsa suffusa (Fr.) Petr., Sydowia, 19: 272 [‘‘1965’’] (1966). on Alnus incana, 19 May 2002, W. Jaklitsch 1892 (BPI 871231, derived Sphaeria cryptosporii Curr., Microsc. J. 3: 271 (1855). culture CBS 121077 ¼ AR 3825). Sphaeria rabenhorstii Berk & Broome, Ann. & Mag. Nat. Hist., ser. The anamorph of Crytosporella suffusa, Disculina vulgaris, 2. 9: 324 (1852). is the type species of the anamorph genus Disculina Ho¨ hn. Valsa commutata Fuckel, Fungi Rhen. 620 (1863). (Sutton 1980).

necks but not fused and flared ostiolar openings. (E) Ascus. (F) Ascospores of the lectotype. (G) Perithecial neck densely filled with paraphyses. (H) Asci of the epitype. (I) Ascospores of the epitype. (J) Conidia. (K–P) Cryptosporella suffusa. (K) Evidence of ascomata in twigs of Alnus incana. (L) Perithecia, note fusion of perithecial necks to form a single ostiolar cavity. (M) Asci. (N–O) Perithecia, note bilaminate venter wall as in C. hypodermia, and perithecial necks densely filled with periphyses. (P) Ascospores. Bars [ (A) 5 mm; (B, D) 100 mm; (C, L) 50 mm; (E, G–H, M–O) 20 mm, (F, I, J, P) 10 mm; (K) 1 mm. 32 L. C. Mejıá et al.

Fig 4 – (A–E) Cryptosporella alnicola. (A) Evidence of ascomata on twigs of Corylus cornuta. (B–E) Asci and ascospores. (F–I) Cryptosporella betulae. (F–G) Asci. (H–I) Ascospores. (J–L) C. confusa. (J) Evidence of ascomata on twigs of Betula papyrifera. (K) Asci. (L) Ascospores. (M–O) C. corylina. (M) Evidence of ascomata on twigs of Corylus avellana (N–O) Asci and ascospores. (P–R) Cryptosporella femoralis. (P–Q) Asci. (R) Ascospores. (S–U) C. wehmeyeriana. (S) Evidence of ascomata on twigs of Tilia sp. (T–U) Asci and ascospores. Bars [ (A, J, M, S) 1 mm; (B–G, K–L, N–R) 20 mm; (H–I) 10 mm.

In addition to Cryptosporella suffusa, the following new Cryptospora suffusa var. nuda Peck, New York State Mus. Rep. 46: combinations are proposed. For descriptions, see Reid & Booth 138 [‘‘1892’’] (1893). (1987). Specimens examined: USA: Minnesota: Itasca State Park, Wilder- ness Drive, on Corylus cornuta Marsh, 15 Aug 2002, L. Vasilyeva Cryptosporella alnicola (Ho¨hn.) L.C. Mejıá & Castleb., comb. as Winterella alnicola (BPI 872327)dCanada: Ontario: Timmins. nov. Fig 4A–E Highway 101, on Alnus sp., 24 Jun 1962, H. D. Griffin as Cryptospora MycoBank no.: MB 510392 alnicola (BPI 627094). British Columbia: Chancellor Mtn, Yoho Na- Basionym: Cryptospora alnicola Ho¨hn., Sitzungsber. Acad. Wiss. tional Park, on Alnus sp., 11 Aug 1962, R. F. Cain as Cryptospora alnicola (BPI 627095). Wien, 123 (Abt.1): 107 (1914). Synonyms: Winterella alnicola (Ho¨ hn.) J. Reid & C. Booth, Can. J. Cryptosporella betulae (Tul. & C. Tul.) L.C. Mejıá & Castleb., Bot. 63: 1323 (1987). comb. nov. Fig 4F–I Phylogenetic placement and taxonomic review 33

MycoBank no.: MB 510393 Maine, North of New Portland, on Alnus rugosa, L. C. Mejıá LCM Basionym: Cryptospora betulae Tul. & C. Tul., Sel. Fung. Carpol. 2: 22 (BPI 872324, derived PCR product LCM22D). 149 (1863). Cryptosporella tiliae (Tul. & C. Tul.) L.C. Mejıá & Castleb., Synonyms: Winterella betulae (Tul. & C. Tul.) O. Kuntze, Rev. Gen. comb. nov. Pl. 1: 34 (1891). MycoBank no.: MB 510399 Valsa tomentella Peck, New York State Mus. Rep. 35: 144 1881 Basionym: Cryptospora tiliae Tul. & C. Tul., Sel. Fung. Carpol. 2: (1884). 150 (1863). Cryptospora tomentella (Peck) Berl. & Vogl., Add. Syll. 1–4: 192 Basionym: Ophiovalsa tiliae (Tul. & C. Tul.) Petr., Sydowia 19: 274 (1886). [‘‘1965’’] (1966). Cryptospora betulae var. tomentella (Peck) Berl., Ic. Fung. 2: 157 Winterella tiliae (Tul. & C. Tul.) O. Kuntze, Rev. Gen. Pl. 1:34 (1889). (1891). Ophiovalsa tomentella (Peck) Petr., Sydowia 19: 275, 1965 (1966). Anamorph: Disculina betulina (Sacc.) Ho¨ hn., Sitzungsber. Akad. Cryptosporella wehmeyeriana (J. Reid & C. Booth) L.C. Mejıá & Wiss. Wien 125 (Abt. 1): 108 (1916). Castleb., comb. nov. Fig 4S–U Specimens examined: USA: New York, Adirondack, on Betula sp. 20 MycoBank no.: MB 510400 June 2002, L. Vasilyeva as Ophiovalsa betulae (BPI 843497, derived Basionym: Winterella wehmeyeriana J. Reid & C. Booth, Can. J. Bot. culture CBS 121080 ¼ AR3889); New York, on Betula sp., 20 Jun 65: 1333 (1987). 2002, L. Vasilyeva as Ophiovalsa betulae (BPI 872328,derived culture Specimens examined: USA: North Carolina: Great Smoky Mts Na- CBS 121073 ¼ AR3863). Austria: Niederoesterreich, Losenheim, tional Park, Cataloocheee vicinity, Caldwell Fork trail, on Tilia Laerchkogel. Mapping grid square 8261/1, on Betula lenta, 5 Jul sp., 23 Apr 2002, L. Vasilyeva as Winterella wehmeyeriana (BPI 2003 W. Jaklitsch 2271 as Winterella betulae (BPI 843595) 843485). District of Columbia: Washington. Dept of Agriculture Grounds, on Tilia sp., 21 Feb. 1903, C. L. Shear as Cryptospora weh- Cryptosporella confusa (J. Reid & C. Booth) L.C. Mejıá & meyeriana (BPI 629315); ibid. (BPI 629319). New York. Lyndonville, Castleb., comb. nov. Fig 4J–L on Tilia sp., Jun 1921, C. L. Shear as Cryptospora wehmeyeriana (BPI MycoBank no.: MB 510393 629320); Alcove, on Tilia americana, Jun 1893, C. L. Shear as Crypto- Basionym: Winterella confusa J. Reid & C. Booth, Can. J. Bot. 65: spora wehmeyeriana (BPI 629318). d Canada: Ontario: London, on 1328 (1987). Tilia sp., June 1893, J. Dearness as Cryptospora wehmeyeriana (BPI Specimens examined: USA: Tennessee: Knoxville, waterfront down- 629317). town, between Calhoun’s and University of Tennessee, on Betula papyrifera, 23 May 2003, W Jaklitsch 2208 as Winterella confusa (BPI 843580, derived cultures CBS 121003 ¼ AR3966 ¼ AR3990); Discussion

Cryptosporella corylina (Tul. & C. Tul.) L.C. Mejıá & Castleb., Here we show evidence from molecular data that Ophiovalsa comb. nov. Fig 4M–O suffusa, the type species of Ophiovalsa, and Cryptosporella hypo- MycoBank no.: MB 510395 dermia, the type species of Cryptosporella, belong to the same Basionym: Valsa corylina Tul. & C. Tul., Sel. Fung. Carpol. 2: 174. genus within the family Gnomoniaceae (Diaporthales) based on (1863). analysis of three different loci (LSU, ITS, and RPB2; Fig 1). Synonyms: Cryptospora corylina (Tul. & C. Tul.) Fuckel, Jahrb. These data corroborate observations that C. hypodermia should Nassau. Ver. Naturkd. 23–24: 192 [‘‘1869’’] (1870). be treated as congeneric with O. suffusa (Reid & Booth 1989); Winterella corylina (Tul. & C. Tul.) O. Kuntze, Rev. Gen. Pl. 1:34 however, these authors placed those two species in Winterella (1891). (Reid & Booth 1987, 1989). Our review of the literature indi- Anamorph: Disculina corylina Ho¨ hn., Ann. Mycol. 16: 108 (1918). cates that the correct name for taxa placed in Winterella sensu Specimens examined: Austria: Kaernten: Kramer Strauch, St Reid & Booth (1987) is Cryptosporella. Margareten im Rosental. Mapping grid square 9452/4, on Corylus As noted first by Kuntze (1891), the generic name Crypto- avellana, 13 Sep 2001, W. Jaklitsch 1811 as Winterella corylina (BPI 843623). spora Tulasne & Tulasne (1863) was antedated by Cryptospora Karelin & Kirilow 1842, a plant genus in the Brassicaceae. Cryptosporella femoralis (Peck) L.C. Mejıá & Castleb., comb. Thus, Kuntze (1891) recognized the name Winterella at the ge- nov. Fig 4P and Q neric level as a replacement for the later homonym Crypto- MycoBank no.: MB 510396 spora. The name Winterella was first used by Saccardo (1883) Basionym: Valsa femoralis Peck, New York State Mus. Rep. 28. for a subgenus of Cryptospora with C. anthostomoides as the 74–75 1874 (1879). type species. It is now known that C. anthostomoides is a locu- Synonyms: Cryptospora femoralis (Peck) Sacc., Syll. Fung. 2: 362 loascomycete and possibly an older taxonomic synonym of (1883). Montagnula (Holm 1992). Thus, the generic name Winterella Winterella femoralis (Peck) O. Kuntze, Rev. Gen. Pl. 1: 34 (1891). cannot be used for diaporthalean species previously placed Ophiovalsa femoralis (Peck) Petr., Sydowia 19: 273 [‘‘1965’’] in Cryptospora. This conclusion is subject to interpretation (1966). about the meaning of the symbol used by Kuntze (1891) in Cryptospora humeralis Dearn. & House, Circ. N. Y. State Mus. 24: his description of Winterella. Holm (1992) and later Rossman 41 (1940). (2002) based on discussions with D. Nicolson (Smithsonian In- Specimens examined: USA: New York: Cranberry Lake, Adirondack stitution, Washington, DC) argued that the symbol x used by mts, on Alnus rugosa, 13 Jun 2002, L. Vasilyeva as Ophiovalsa femoralis (BPI 872326, derived culture CBS 121076 ¼ AR 3868). Michigan: Kuntze (1891) indicates a subgenus and thus Kuntze (1891) Ludington State Park, on Alnus rugosa, 25 Aug 2006, coll. G. Adams, was raising Saccardo’s Cryptospora subg. Winterella (1883) det. L. C. Mejia (BPI 872325, derived PCR products CF3 and CF4); to generic status based on Cryptospora suffusa. However, 34 L. C. Mejıá et al.

Key to species of Cryptosporella

1 Ascospores with one median septum at maturity, ends swollen, thus appearing like a leg bone or femur, (24.0–)45.5–56.0 (–74.0) (3.0–)3.5–4.5(–5.5) mm, l:w (5–)12–14(–18); on Alnus spp. in North America ...... femoralis Ascospores non-septate ...... 2

2(1) Ascospores ellipsoid to fusoid, acute ends, (22.0–)38.0–48.0(–69.5) (5.5–)8.0–11.0(–16.5) mm, l:w (3–)4–5(–8); on Ulmus spp ...... hypodermia Ascospores mostly cylindrical ...... 3

3(2) Ascospores cylindrical with rounded ends ...... 4 Ascospores cylindrical slightly swollen at their ends ...... 5

4(3) Ascospores slightly curved, tapering toward rounded ends, (29.0–)38.0–50.0(–74.0) (4.5–)5.0–6.0(–9.0) mm, l:w (5–)7–10(–13); on Betula spp ...... betulae Ascospores ﬂexuous, interwoven in asci, (49.0–)57.5–66.0(–69.5) 4.0–4.5(–5.5) mm, l:w (10–)14–16(–17); on Alnus spp.suffusa

5(3) Ascospores cylindrical to femuroid; 27–35 5–6.5 mm; on Tilia sp. in Europe*...... tiliae Ascospores cylindrical with slightly swollen ends, greater than 35 mm long ...... 6

6(5) On Alnus spp. and Tilia spp. in North America ...... 7 On Betula spp. and Corylus spp. in North America and Europe ...... 8

7(6) Ascospores (53.5–)62.0–78.5(–99.0) (3.0–)3.5–4.5 mm, l:w (12–)15–21(–28); on Alnus spp. in North America ...... alnicola Ascospores (49.5–)74.0–90.5(–109) (4.0–)5.0–6.0(–7.0) mm, l:w (9–)14–16(–23); on Tilia spp. in North America ... wehmeyeriana

8(6) Ascospores (87.5–)88.5–89.5(–91.0) 3.0–3.5 mm, l:w (25–)26–28(–27); on Betula spp...... confusa Ascospores (21.5–)26.5–75.0(–82.5) (3.5–)4.0–4.5(–10) mm, l:w (5–)7–17(–23); on Corylus spp...... corylina

* Species not observed during the course of this study; description and measurements from Reid & Booth (1987).

Reid & Booth (1987) suggested that the generic name Winterella fusiform (Fig 3E and F, H and I), relatively short and broad com- originated with Kuntze’s (1891) publication with W. suffusa as pared with the other species of Cryptosporella, which tend to be the type species. In our view there is little evidence to support elongate to cylindrical with broadly rounded ends (Figs 3J, M; the latter interpretation. However, this disagreement be- and 4B–I, K and L, N and O, T, U). The ascospores of C. femoralis comes irrelevant because Cryptosporella is an older name for are distinctive in being swollen at each end thus appearing Winterella sensu Reid & Booth (1987) as well as Ophiovalsa. femuroid (Fig 4P). Ascospores of C. tiliae occasionally become When establishing the genus Ophiovalsa for Cryptospora, Petrak femuroid, but are much shorter than those of C. femoralis and (1966) did not mention the work of Kuntze (1891) and Petrak’s are non-septate. Although Reid & Booth (1989) list hardwood generic name Ophiovalsa has been used in the recent literature hosts other than Ulmus spp. for C. hypodermia (as Winterella hypo- (Barr 1978; Kobayashi 1970; Glawe & Jensen 1986). Cryptospor- dermia), most of their specimens examined were on Ulmus and ella provides the oldest name for Ophiovalsa, as well as this fungus was not found to occur on any other host genera. Winterella, sensu Reid & Booth (1987). No single unique morphological character distinguishes species placed in Cryptosporella from other genera in the family Gnomoniaceae. However, these species share a suite of char- Acknowledgements acters, such as perithecia developing below the bark surface, aggregated with converging necks, similar perithecial wall This research was conducted as part of a National Science morphology and structure, and ellipsoid to elongated, non-, Foundation grant in the Partnerships in Enhancing Expertise rarely one-septate, ascospores. The known anamorphs all in Taxonomy program (NSF 03-28364). We are greatly indebted belong to the genus Disculina (Sutton 1980). Most species of to Walter Jaklitsch and Larissa Vasilyeva for their numerous Cryptosporella are endophytic in twigs of the hardwood trees, collections of these and many other diaporthalean fungi. In Alnus, Betula, Corylus, Tilia, and Ulmus, with a saprobic stage addition we thank Gerry Adams for a specimen of Cryptospor- following host tissue death. ella femoralis and Susie Lattomus for the healthy branches of Nine species are accepted in the genus Cryptosporella, seven Alnus sinuata from which C. suffusa was isolated as an of which are included in the RPB2, ITS, and LSU DNA sequence endophyte. analyses (Fig 2). Species in the genus Cryptosporella show a wide range of ascospore shapes and sizes, distinctive colony mor- references phologies, and conidial formation and pigmentation. The species of Cryptosporella can be separated by a combination of host and ascospore size and shape. Most species have non-sep- von Arx JA, Mu¨ ller E, 1954. Die Gattungen der amerosporen tate ascospores, whereas those of C. femoralis become one-sep- Pyrenomyceten. Beitra¨ge Kryptogamenﬂora der Schweiz 11 (1): tate at maturity. The ascospores of C. hypodermia are elliptical to 1–434. Phylogenetic placement and taxonomic review 35

Barengo N, Sieber T, Holdenrieder O, 2000. Diversity of endo- Rayner RW, 1970. A Mycological Colour Chart. Commonwealth phytic mycobiota in leaves and twigs of pubescent birch Mycological Institute, Kew. (Betula pubescens). Sydowia 52: 305–320. Rehner SA, Samuels GJ, 1994. Taxonomy and phylogeny of Glio- Barr ME, 1978. The Diaporthales in North America with cladium analysed from nuclear large subunit ribosomal DNA emphasis on Gnomonia and its segregates. Mycological sequences. Mycological Research 98: 25–634. Memoirs 7: 1–232. Reid J, Booth C, 1987. Winterella. The correct name for Cryptospora Barr ME, 1991. Revisions and additions to the Diaporthales. and Ophiovalsa. Canadian Journal of Botany 65: 1320–1342. Mycotaxon 41: 287–305. Reid J, Booth C, 1989. On Cryptosporella and Wuestneia. Canadian Berlese AN, 1900. Icones Fungorum Omnium Hucusque Cognitorum. Journal of Botany 67: 879–908. vol. 2. Padova. Rossman AY, 2002. Note 3308. Ophiovalsa Petr. In: Eriksson OE, Castlebury LA, Rossman AY, Jaklitsch WJ, Vasilyeva LN, 2002. Baral HO, Currah RS, Hansen K, Kurtzman CP, Laessøe T, A preliminary overview of the Diaporthales based on large Rambold G (eds), Notes on Ascomycete Systematics. Nos 3303- subunit nuclear ribosomal DNA sequences. Mycologia 94: 3579. Myconet 8: 1–54. 1017–1031. Saccardo PA, 1877. Fungi Veneti. Michelia 1: 1–72. Chlebicki A, 2002. Biogeographic relationships between fungi Saccardo PA, 1883. Sylloge Fungorum. vol. 14. Pavia. and selected glacial relict plants. Monographiae Botanicae 90: Sogonov MV, 2005. Software for morphological and molecular 1–230. taxonomic studies of fungi. Abstracts of the Mycological Dennis RWG, 1978. British Ascomycetes, 2nd edn. J. Cramer, Lehre. Society of America and Mycological Society of Japan Joint Ellis JB, Everhard EM, 1892. The North American Pyrenomycetes. Ellis Meeting. 30 July–5 August. Hilo, HI, pp. 193–194. and Everhart, Newfield, NJ. Spaulding P, 1961. Foreign Diseases of Forest Trees of the World. Felsenstein J, 1985. Confidence limits on phylogenies: an [USDA Agricultural Handbook No. 197.] USDA, Washington DC. approach using the bootstrap. Evolution 6: 227–242. Sutton BC, 1980. The Coelomycetes. Fungi Imperfecti with Pycnidia, Ganley RJ, Brunsfeld SJ, Newcombe G, 2004. A community of un- Acervuli and Stromata. Commonwealth Mycological Institute, known, endophytic fungi in western white pine. Proceedings of Kew. the National Academy of Sciences, USA 101: 10107–10112. Swofford DL, 2002. PAUP 4.0b10: phylogenetic analysis using Glawe DA, Jensen JD, 1986. Ophiovalsa in the Pacific Northwest. parsimony. Sinauer Associates, Sunderland, MA. Mycotaxon 25: 645–655. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, Green S, 2004. Fungi associated with shoots of silver birch (Betula 1997. The ClustalX windows interface: flexible strategies for pendula) in Scotland. Mycological Research 108: 1327–1336. multiple sequence alignment aided by quality analysis tools. Hall TA, 1999. BioEdit: a user-friendly biological sequence align- Nucleic Acids Research 24: 4876–4882. ment editor and analysis program for Windows 95/98/NT. Torres MS, White jr JF, Bischoff JF, 2005. Cordyceps spegazzinii sp. Nucleic Acids Symposium Series 41: 95–98. nov, a new species of the C. militaris group. Mycotaxon 94: von Ho¨ hnel F, 1917. System der Diaportheen. Berichte der Deut- 253–263. schen Botanischen Gesellschaft 35: 631–638. Traverso JB, 1906. Fungi pyrenomycetae. Flora Italica Cryptogama 1: von Ho¨ hnel F, 1918. Mykologische fragmente. Annales Mycologici 1–352. 16: 35–174. Tulasne LR, Tulasne C, 1863. Selecta Fungorum Carpologia. vol. 2. Holm L, 1992. On the typification of two pyrenomycete generic Oxford University Press, Oxford [English translation: Grove, names. Systema Ascomycetum 11: 29–30. W.B., 1931]. Karelin G, Kirilow J, 1842. Enumeratio plantarum in desertis Vilgalys R, Hester M, 1990. Rapid genetic identification and Songoriae oerientalis et in jugo summarum Alpium Alatau mapping of enzymatically amplified ribosomal DNA from anno 1841 colectarum. Bulletin de la Societe´ des Naturalistes de several Cryptococcus species. Journal of Bacteriology 172: 4238– Moscou 15: 129–180. 4246. Kobayashi T, 1970. Taxonomic studies of Japanese Diaporthaceae Wehmeyer LE, 1926. A biologic and phylogenetic study of with special reference to their life histories. Bulletin of the the stromatic Sphaeriales. American Journal of Botany 13: Government Forest Experiment Station (Japan) 226: 1–242. 557–645. Kuntze O, 1891. Revisio Generum Plantarum. 1. A. Felix, Leipzig. White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct Liu YL, Whelen S, Hall BD, 1999. Phylogenetic relationships sequencing of fungal ribosomal DNA for phylogenetics. In: among ascomycetes: evidence from an RNA polymerase II Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: subunit. Molecular Biology and Evolution 16: 1799–1808. a guide to methods and applications. Academic Press, San Diego, Munk A, 1957. Danish Pyrenomycetes. A preliminary flora. Dansk pp. 315–322. Botanisk Arkiv 17: 1–491. Winter G, 1887. Pilze. Ascomyceten. Rabenhorts’ Kryptogammen Petrak F, 1966. Uber die Gattung Cryptospora Tul. Sydowia 19: 268–278. Flora von Deutschland, Oesterreich und der Schweiz 1: 1–928.