Neonectria and Cylindrocarpon: the Nectria Mammoidea Group and Species Lacking Microconidia

Mycologia, 96(3), 2004, pp. 572–597. ᭧ 2004 by The Mycological Society of America, Lawrence, KS 66044-8897

Neonectria and Cylindrocarpon: the Nectria mammoidea group and species lacking microconidia

David Brayford anamorphs. A key to nectriaceous species of Neonec- International Mycological Institute, Bakeham Lane, tria that have Cylindrocarpon anamorphs that lack mi- Egham, Surrey, England TW20 9TY croconidia and chlamydospores and/or that have a Barry M. Honda N. mammoidea type perithecial wall anatomy is pre- Department of Molecular Biology and Biochemistry, sented. New combinations are proposed for other Simon Fraser University, Burnaby, British Columbia, species formerly included in Nectria that have non- V5A 1S6 Canada microconidial Cylindrocarpon anamorphs: Neonectria cinnamomea, Neo. jungneri, Neo. platycephala, Neo. Feky R. Mantiri phaeodisca and Neo. verrucospora. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Key words: forest pathology, Hypocreales, mito- V5A 1S6 Canada chondrial rDNA, Nectria, Nectriaceae, phylogeny, systematics Gary J. Samuels1 United States Department of Agriculture, Agriculture Research Service, Systematic Botany and Mycology INTRODUCTION Laboratory, Room 304, B-011A, 10300 Baltimore Ave., Beltsville, Maryland 20705-2350 The genus Nectria Fr. (Hypocreales: Nectriaceae) is a classic genus of pyrenomycetes in the sense that its species have been known since it was described early Abstract: Neonectria (Hypocreales: Nectriaceae) spe- in the 19th century, and over the years many species cies having Cylindrocarpon anamorphs that lack mi- from temperate and tropical regions have been in- croconidia and chlamydospores include: Neo. disco- cluded in it. The stereotypical nectria has a brightly phora var. discophora, Neo. discophora var. rubi, stat colored perithecium—usually red—that is seated di- nov. et comb. nov., Neo. lucida, comb. nov., Neo. vir- rectly on a woody substratum (e.g., Booth 1959). idispora, sp. nov. and Neo. westlandica, comb. nov. Closer consideration of the diversity that has been Perithecia of these species are red and perithecial included in Nectria, along with DNA sequence anal- anatomy is of the N. mammoidea type, with a pali- ysis has shown the genus to be polyphyletic. Nectria sade of hypha-like cells in the outer perithecial wall. now is restricted to its type, N. cinnabarina (Tode : These species occur on recently dead or dying trees. Fr.) Fr., and a relatively small number of similar spe- Perithecia of Neo. betulae, sp. nov and Neo. dumontii, cies (Rossman 1989, Rehner and Samuels 1994, Ross- sp. nov. are anatomically and biologically similar to man et al 1999). Many species of Nectria sensu lato those of Neo. discophora. The only known culture of have been placed in other genera and families (Ross- Neo. betulae remained sterile, while Neo. dumontii has man et al 1999, 2001, Schroers 2002). Rossman et al not been cultured; their anamorphs are presumed to (1999) and Mantiri et al (2001) resurrected Neonec- be Cylindrocarpon. Analyses of mit ssu rDNA sequenc- tria Wollenw. for former Nectria species that have Cy- es indicate that Neonectria/Cylindrocarpon is mono- lindrocarpon Wollenw. anamorphs. The only known phyletic. Within the genus, species having N. mam- teleomorphs of Cylindrocarpon are Neonectria. Mantiri moidea type perithecia are paraphyletic. Most species et al (2001) used mitochondrial small-subunit ribo- cluster with Neo. discophora, but Neo. westlandica and somal (mit ssu rDNA) sequences with a selected Neo. trachosa are basal to a clade that includes species group of species to show that Cylindrocarpon, and that do not have a N. mammoidea-type perithecium. therefore Neonectria, is monophyletic. Nectria fuckeliana clusters independently of Neonec- In the present paper we continue describing or re- tria and Nectria. Although reported to have a Cylin- describing species of Nectria s. lat. that have Cylindro- drocarpon anamorph, fresh ascospore isolates of N. fuckeliana did not produce Cylindrocarpon macroco- carpon anamorphs (Brayford and Samuels 1993, Sa- nidia but produced acremonium- or verticillium-like muels and Brayford 1990, 1993, 1994). We discuss species of Neonectria that have been or could have Accepted for publication October 10, 2003. been placed in the Nectria mammoidea group of Nec- 1 Corresponding author. E-mail: [email protected] tria (Booth 1966) because of their perithecial wall

572 BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 573 anatomy. Throughout this paper, these abbreviations surface layers of pseudoparenchymatous cells over are used: N. ϭ Nectria, Neo. ϭ Neonectria. the N. mammoidea palisade. Perithecia of species of Neonectria that have an N. Ascospores in the N. mammoidea Group are typi- mammoidea wall structure often arise from a char- cally ellipsoidal and hyaline, becoming pale brown at acteristic stroma, here termed the N. mammoidea- maturity; they are usually 1-septate, but those of N. type stroma. In this stromal type, compacted hyphae phaeodisca Rossman are phragmosporous (Rossman grow within cells of the cortex. Densely packed, dis- 1983, Samuels and Brayford 1993). The ascospores crete hyphae, ca 3.5 ␮m wide, arise from these stro- usually are smooth but often are spinulose. Typically mal cells and extend perpendicular to the substra- the developing ascospores are surrounded by a thin Ͻ ␮ tum, surrounding the perithecial base (see FIG. 51). ( 1 m) sheath, contraction of which may result in Such a stroma is seen rarely in species that do not the ornamentation. Ascospores with a distinctly wart- have a N. mammoidea wall, such as Nectria vermispora ed, tuberculate or striate ornamentated sheath such Samuels & Brayford (Samuels and Brayford 1993) as found in the Neo. veuillotiana (Sacc. & Roum.) and N. hypoxantha Penz. & Sacc. (Samuels 1976), nei- Brayford & Samuels Group (Brayford and Samuels ther of which has been linked to any anamorph. 1993) have not been observed in taxa of the N. mam- The Cylindrocarpon anamorphs of these fungi lack moidea Group. Discharged ascospores are typically microconidia and chlamydospores and for these rea- pale yellow-brown and are usually spinulose. The re- mains of apical paraphyses are usually not conspicu- sons were included in Cylindrocarpon ‘‘Group 2’’ by ous among mature asci. Booth (1966). All species included in this group were The Cylindrocarpon anamorphs of Group 2 (the N. anamorphs of members of the ‘‘Nectria mammoidea mammoidea Group) form round-ended, slightly ar- Group’’. Following Booth (1959, 1966) this group is cuate macroconidia but lack microconidia and chla- centered on N. mammoidea Phill. & Plowr., a later mydospores (Booth 1959, 1966). However, we have taxonomic synonym of N. discophora (Mont.) Mont. found some exceptions to this generalization. Neo- (ϭ Neo. discophora [Mont.] Mantiri & Samuels). In nectria betulae has a typically N. mammoidea perithe- addition to N. discophora, Booth (1966) included N. cial anatomy, and colony characteristics closely re- mammoidea var. rubi, N. fuckeliana, N. lucida and N. semble Neo. discophora, but cultures lack conidia, westlandica. The new species Neo. betulae, Neo. du- even when freshly isolated. Nectria fuckeliana is re- montii and Neo. viridispora can be added to these. ported to produce a Cylindrocarpon anamorph with Perithecia of Neo. betulae and Neo. dumontii are ana- abundant microconidia (Booth 1959, 1966, Roll-Han- tomically and biologically similar to those of Neo. dis- sen 1962). This species could be assigned to the Neo. cophora, but cultures of the former remained sterile coccinea Group due to its microconidial production and we have not had the opportunity to culture the and colony characteristics and by the fact that peri- latter. thecia of all ages occur in a single cluster (Booth Perithecia of all of these species are red. They of- 1959, 1966), but it is included here because of its N. ten are yellow or orange when young, darkening to mammoidea-like perithecial anatomy. red and brown, or sometimes near black with age and Taxa in this group occur on a wide range of living are red in KOH and yellow in lactic acid. or recently dead, woody plant substrata, including Members of the N. mammoidea Group most dis- palms, gymnosperms and dicotyledonous plants; less tinctively possess characteristic perithecial walls. In frequently they occur on herbaceous tissue. Neonec- longitudinal section the wall is seen to comprise a tria discophora var. rubi is known only from Rubus layer of hyphae that have thickened walls and typi- species where it causes a canker. Perithecia are typically are arranged radially, giving the appearance of cally superficial, solitary or clustered on a character- a palisade (Booth 1959; FIGS. 17, 24, 36, 44, 60). This istic, weakly developed basal stroma as was described wall structure appears in squash mounts as an inter- above. Perithecia typically do not collapse when dry twined layer of thickened hyphal elements, rather but sometimes become cupulate. than as a pseudoparenchymatous structure. As a re- To test the monophyly of Cylindrocarpon and Neo- sult of the uniform palisade layer, the perithecia of nectria we sequenced the mit ssu rDNA gene for rep- N. discophora typically appear smooth and shining resentative species. This expands work reported by Mantiri et al (2001) for Cylindrocarpon and Neonec- (see FIGS. 5, 6) but, in some collections or species, tria. an additional outer layer of globose cells covering the perithecia gives a roughened, frosted or pitted appearance. The perithecial walls of N. westlandica MATERIALS AND METHODS (FIG. 14), Neo. viridispora (FIG. 13) and Neo. dumontii Morphological studies.—Perithecia from herbarium speci- (FIG. 11) are warted distinctly because of additional mens were rehydrated briefly in 3% KOH. Anatomy of peri- 574 MYCOLOGIA No. AF315207 AF315201 AF315199 AF315204 AF315202 AF315205 AF315206 AF315208 GenBank sp. AF315197 trunk AY380924 trunk AY380919 Host sp. AF315209 Substratum/ dead tree sii sii dead tree Quercus Pseudotsuga menzie- Pseudotsuga menzie- Alnus rubra Prunus persica Malus pumila Malus pumila Acer Larix, Betula, year of isolation da, 1991 da, 1991 da, date unknown date unknown 1996 1997 Jamaica, 1971 Wood AF315198 Geographic provenance and son Type of ascospore C. T. Roger- ascospore G. J. Samuels Virginia, U. S. A., 1991 mycelial P. Axelroodmycelial Chilliwack, B. C., Cana- P. Axelrood Pemberton, B. C., Cana- mycelial J. Traquair Learnington, Ont., Cana- mycelial G.ascospore Braun G. J. Samuels Nova Puerto Scotia, Rico, Canada, 1996ascospore Bark of recently G. J. Samuelsascospores G. Virginia, J. U. Samuels S. A., 1991 Scotland, 1992 ascospores G.ascospores J. Samuels G. J. Samuels Scotland, 1992 Thailand, 1997 Bark AY380918 mycelial J. E. Rahe Surrey, B. C., Canada, mycelial F. R. Mantiri Burnaby, B. C., Canada, ascospore G. J. Samuels French Guiana, 1986 Bark of recently isolation Person isolating (Ger- (Bres.) (Pat.) (Penz. sp. mycelial J. Juzwik Ontario, Canada, 1985 Conifer seedlings AY380917 Sacc.) Mantiri & Sa- muels (Sacc. & Roum.) Mantiri & Samuels (Booth & Samuels) Mantiri & Samuels (Mont.) Mantiri & Sa- muels (Tode : Fr.) Fr. Rossman & Samuels lach & Nilsson) Mantiri & Samuels Mantiri & Samuels Neonectria galligena Neonectria magnusiana Neonectria veuillotiana Neonectria coronata Neonectria neomacrospora Neonectria radicicola Neonectria galligena Neonectria discophora Neonectria rugulosa Nectria cinnabarina Nectria fuckeliana Calonectria Neonectria betulae Neonectria jungneri Wol- Identification Cylin- sp. majus reported by var. (Berk. & Br.) Wol- Anamorph Teleomorph tenue lenw. (Sacc.) Wollenw. Brayford Wollenw. (Zinssm.) Scholten mum mum Wollenw.. um Brayford & Samuels (Berk.) Seifert drocarpon cylindroides var. (Wollenw.) Wollenw. lenw. Booth 1966) Cylindrocarpon candidulum Cylindrocarpon coronatum Cylindrocarpon cylindroides Cylindrocarpon cylindroides Neonectria neomacrospora Cylindrocarpon destructans Cylindrocarpon destructans Neonectria radicicola Cylindrocarpon heterone- Cylindrocarpon heterone- Cylindrocarpon ianothele Cylindrocarpon magnusian- Cylindrocarpon rugulosum Tubercularia lateritia Cylindrocladium Cylindrocarpon victoriae a I. Identification and origin of isolates used in the molecular analysis Isolate (1) 19 116 222 (2) (1) 111 160 ABLE C2cun2ab2 CBS 730.87 C. T. R. 71- G. J. S. 92-42 Acremonium-like ( T G. J. S. 91- G. J. S. 86- A. Y. R. 2652 P4c2n22ad FMd2.1 (1) JAT1551 (2) JR0609B-2 GBA1 (2) G. J. S. 96-35 G. J. S. 91- G. J. S. 97- G. J. S. 92-47 None formed in culture BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 575

thecial sections was observed in water or lactic acid by trans- mitted light or by epiﬂuorescence in 0.5% w/v Calcoﬂuor

No. (Sigma Chemical Co., St. Louis, Missouri) in 0.4 M sodium AY380923

GenBank phosphate buffer at pH 8.0, using a Zeiss 05 ﬁlter set. Mea- surements of ascospores were made from water, while those of conidia were made from fresh mounts in lactofuchsin, which made conidial septa more easily seen. Cotton blue stain (Le Gal 1947: 0.5 g cotton blue in 30 g lactic acid, boiled, ﬁltered over 24 h) was used to reveal ascospore or- Host namentation. The three kinds of compound microscopy are

Substratum/ abbreviated: Nomarsky differential contrast (DIC), phase

Ochroma lagopus, wood contrast (PC), fluorescence (FL). Single ascospores were isolated on cornmeal dextrose agar (CMD, Difco cornmeal agar plus 2 g/L dextrose) with the aid of a micromanipulator. Characteristics of cultures were recorded from potato-sucrose agar (PSA, Booth 1971), potato-dextrose agar (PDA, Booth 1971) and on SNAY (SNA, Nirenberg 1976, with filter paper ϩ 0.1% yeast extract). Plates were incubated at 20 C in a 12 h light/dark cycle of daylight fluorescent strip lamps and black light

year of isolation tubes emitting nUV (366 nm). Colony growth was measured

Jamaica, 1971 Rotting wood AY380920 on 90 mm diam Petri dishes containing PSA, 5 mm deep,

Geographic provenance and inoculated with a 4 mm diam mycelial plug after 10 d. Iso- lates were tested for chlamydospore production by incubat- ing 10 mm square blocks of culture taken from SNAY into Roger- 10 mL of sterile water for 2–3 wk at 20 C. The most frequently cited collectors and their cultures

son are abbreviated: Kent P. Dumont (K.P.D.), Richard P. Korf (R.P.K.), Amy Y. Rossman (A.Y.R.), and Gary J. Samuels (G.J.S.). Many Dumont collections were grown in pure culture from ascospores and originally were held in the culture collection of C.T. Rogerson (C.T.R., The New York Botani- Type of ascospores C. T. ascospores G.ascospores J. Samuels G.ascospores J. Samuels Scotland, C. 1992 S. Hodges Newascospores Zealand, 1985 Ecuador, G. 1991 J. Samuels Venezuela, 1971 Tree root Conifer bark Bark AY380921 AY380922 AY380925 isolation Person isolating cal Garden); those cultures now are preserved at BPI. Color terminology follows Rayner (1970). Herbarium designations are taken from Holmgren et al (1990).

Molecular studies.—The sources of the isolates used in this study are given in TABLE I. Among the 22 isolates sampled, nine isolates were sequenced by the authors and the sequences for the other isolates were derived from our pre- vious studies (Mantiri et al 2001). Fungal isolates were main- tained on 1.5% malt-extract agar (Difco) or potato-dextrose agar (Difco). Neonectria phaeodisca Neonectria trachosa Neonectria westlandica Neonectria viridispora Neonectria lucida The mycelia from stored cultures were inoculated into

250 mL V8 broth (V8 juice 200 mL, agar 15 g, CaCO3 3g, distilled water 1 L), harvested after incubation in the dark

Identification at room temperature for 1–2 wk and freeze-dried. Two to five g of freeze-dried mycelium was ground to a fine powder,

sp. sp. sp. suspended in 10 mL of extraction buffer (20 mM EDTA; 50 mM Tris-HCl, pH 8; 2% SDS) and incubated at 65 C for 1 h after the addition of proteinase K to a ﬁnal concentration

Anamorph Teleomorph of 200 ␮g/mL. Cell debris was removed by centrifugation at 15 000 rpm for 20 min at 4 C, after which the supernatant was transferred to a fresh tube containing 2–2.5 volumes of Cylindrocarpon phaeodiscum Samuels & Brayford Cylindrocarpon Cylindrocarpon Cylindrocarpon Cylindrocarpon lucidum Booth cold 95% EtOH. Following an extraction with chloroform— isoamyl alcohol (24:1)—the DNA was precipitated with 3 M a sodium acetate and 2.5 volumes of 95% (v/v) EtOH (Sam- I. Continued brook et al 1989). NaAc was added to a final concentration Isolate Numbers in parentheses are given to identify multiple isolates for a species where needed. of 0.3 M before ethanol precipitation, as an exception in a ABLE G. J. S. 92-45 G. J. S. 85-45 A. Y. R. 2690 T C. T. R. 71-60 C. T. R. 72-71 DNA extraction of Neo. betulae and Neo. viridispora. The 576 MYCOLOGIA precipitated DNA was pelleted by centrifugation, washed variable regions that alternated with three relatively with cold 70% EtOH and resuspended in 50–200 ␮LTE conserved regions (data not shown). buffer. The total length of the sequence alignment was A portion of the mit ssu rDNA coding region was defined 752 positions. Of these, 208 bp were variable and 113 by primers NMS1 and NMS2 (Li et al 1994) and amplified bp (15.03%) were parsimony informative. One of the by the polymerase chain reaction (PCR). Reactions were 164 most-parsimonious trees (MPTs) 357 steps in accomplished in a volume of 50 ␮L containing approxi- length (L) generated by branch and bound search mately 20–60 ng fungal genomic DNA, 200 ␮M each of dNTP (Perkin-Elmer, Norwalk, Connecticut), 1.5 mM algorithm, with consistency (CI), retention (RI) and ␮ ϫ rescaled consistency (RC) indices of 0.78, 0.75 and MgCl2,10 M each primer, 1 reaction buffer (20 mM Tris- HCl, pH 8.4, 50 mM KCl) and 2.5 U Taq DNA Polymerase 0.58, respectively, is shown in FIG. 1. The topologies (Gibco BRL, Burlington, Ontario) and overlain with 50 ␮L of the distance trees with transition:transversion bi- light paraffin oil. Thermal cycling parameters used were an ases weighted at 1:3 and 1:10 were identical to the initial denaturation at 95 C for 2 min, followed by 34 cycles tree with no transition:transversion bias (data not consisting of 95 C for 1 min, 56 C for 1 min and 72 C for shown). 1 min and a final extension at 72 C for 4 min. Members of the genus Neonectria included in this PCR products were purified with Nucleotra௡PCR (Mach- study formed a monophyletic clade, with 74% boot- erey-Nagel, Duren, Germany). All sequences were verified strap support, with respect to outgroups (FIG. 2). The by sequencing both complementary strands using both ingroup consisted of three clades, designated I–III. primers NMS1 and NMS2. Most of PCR products were se- Clade designations correlate with the Cylindrocarpon quenced employing the ABI PRISM௢ Dye Terminator Cycle grouping based on cultural and morphological char- Sequencing Ready Reaction Kit with AmpliTaq௡ DNA Poly- acteristics proposed by Booth (1966). Clade I (boot- merase, following the manufacturer’s protocol (Applied Biosystem, Foster City, California). Some of the purified strap 95%) includes four isolates (two species: Neo. PCR products were sequenced using a Thermo Sequenase neomacrospora and Neo. galligena) having relatively radiolabeled terminator cycle sequencing kit (Amersham low sequence variation (0.2–3.8%, TABLE II). Clade Pharmacia Biotech, Cleveland, Ohio) following the manu- II (bootstrap 95%) includes 11 species: Neo. betulae, facturer’s protocol. Contiguous sequences were assembled Neo. coronata, Neo. discophora, Neo. jungneri, Neo. lu- by the ESEE software package (Cabot 1990). DNA sequenc- cida, Neo. phaeodisca, Neo. rugulosa, Neo. trachosa, es were aligned using the computer program MULTALIN Neo. veuillotiana, Neo. viridispora and Neo. westlandi- version 5.4.1 (Corpet 1988) available on the internet (see ca. Percent sequence dissimilarity among these spe- http://www.toulouse.inra.fr/multalin.html) followed by cies ranged from 0.9 to 7.6% (TABLE II). In this manual adjustment. clade, the first five species mentioned above formed The aligned sequences were analyzed using the maxi- a subclade with very strong bootstrap support mum-parsimony method (MP) found in PAUP* beta ver- (100%). Sequence variation among species in this sion 0b10 for Windows (Swofford 2000). A branch and subclade ranged from 0.9 to 4.3% (TABLE II). Clade bound search was conducted using these settings: Addition III (bootstrap 89%) includes two isolates of Neo. rad- sequence was set to furthest, and zero length branches were set to collapse to yield polytomies. Bootstrap support for icicola. internal branches was evaluated from 100 full heuristic We studied collections and ascospore-derived cul- searches using tree bisection-reconstruction branch swap- tures of Neo. discophora var. discophora from Europe, ping with these settings: MulTrees saved, and steepest de- temperate North America, tropical South America, scent option not in effect. Groups with a frequency greater Indonesia, Jamaica and New Zealand. Within these than 50% were retained in the bootstrap consensus tree. collections we have noted variation in perithecial Neighbor-joining analyses also were performed to explore morphology, especially perithecial size, formation of the effects of different substitution models with transition: a flattened ostiolar disk and in the presence or ab- transversion biases set to 1:3 and 1:10. Gaps were treated as sence of an outer layer of cells giving a smooth or missing in all analyses. roughened appearance. However, we have been un- Fusarium inflexum (GenBank U34519), Nectria cinnabar- able to detect discontinuities in these characteristics ina and Calonectria sp. were chosen as outgroups because or correlations between them and anamorph char- of their apparent close relationship to Neonectria (Rehner acteristics. The cultures and anamorph morphologies and Samuels 1995). are uniform, indicating that there is only one species with a very wide distribution. RESULTS One of the species originally included in the ingroup, Nectria fuckeliana was positioned in outgroups All the isolates yielded a single amplification product in all analyses. This species formed a clade (with 72% range of 539–682 bp in length. Length differences bootstrap support [FIG. 2]) together with Nectria cin- were due mainly to insertions and deletions in two nabarina and Calonectria sp. The collections of N. BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 577

FIG. 1. Phylogenetic relationships of species of Neonectria inferred from mit ssu rDNA sequences. This is one of 164 MPTs (L ϭ 367, CI ϭ 0.78, RI ϭ 0.75, RC ϭ 0.58) resulting from a MP analysis employing the branch and bound algorithm in PAUP*. The numbers of nucleotide changes are indicated above the branches. Numbers in parentheses next to species names (where given) identify multiple isolates for a taxon (see TABLE I). Measures of sequence divergence for the three clades are indicated.

fuckeliana that we cultured and studied are indistin- DISCUSSION guishable from the type specimen of the species in Earlier analysis of sequences of the mit ssu rDNA of all respects. Although N. fuckeliana, a species that a limited but representative set of species of Neonec- occurs on Abies, Larix, Picea and Pinus, is almost in- tria and Cylindrocarpon strongly suggested (i) a one- distinguishable from Neo. discophora in its perithecial for-one relationship between Cylindrocarpon and Neo- anatomy and morphology, it is atypical in the N. nectria and (ii) the monophyly of Neonectria/Cylin- mammoidea Group. Unlike Neo. discophora and all drocarpon (Mantiri et al 2001). other Neonectria species in which apical paraphyses This earlier work also suggested that perithecial disintegrate early and ascospores are pale tan, apical anatomy in Neonectria is not as indicative of relation- paraphyses of N. fuckeliana remain as copious narrow ships among species as is the anamorph, a conclusion ﬁlaments among mature asci (see FIG. 36) and the strongly supported in the present study. Once again, ascospores are hyaline when discharged. We did not Cylindrocarpon Group 1 (macroconidia, microconid- observe macroconidia in any of the fresh ascospore ia, no chlamydospores), Group 2 (macroconidia, no isolates from the specimens of N. fuckeliana. Rather, microconidia, no chlamydospores) and Group 3 (mi- these isolates produced slowly growing, slimy colonies croconidia, macroconidia, abundant chlamydo- with abundant unicellular conidia from acremonium- spores) (respectively I, II and III in FIGS.1,3)of or verticillium-like conidiophores (FIG. 33). Perithe- Booth (1966) are shown to be monophyletic. Species cia did not form in our cultures. of groups 1 and 3, respectively, are homogeneous in 578 MYCOLOGIA

FIG. 2. Phylogenetic relationships of species of Neonectria inferred from mit ssu rDNA sequences. This is a 50% majority rule consensus of 164 MPTs generated from a heuristic analysis of 752 bp for 21 taxa. Bootstrap values greater than 50% calculated from 100 replications are given about the branches. their biology and perithecial morphology and anat- ported to be a wound parasite of conifers and to be omy. Although Group 2 is monophyletic (FIG. 2), associated with dieback (see Booth 1966). Roll-Han- there is diversity in perithecial morphology and anat- sen and Roll-Hansen (1979, 1980) found that N. fuck- omy such that representatives of the clade have been eliana invaded wounds of Picea abies more frequently included in different groups of Nectria, viz. the Nec- than any of the several other fungi that they surveyed tria mammoidea and Nectria veuillotiana Groups but that it had little or no effect on the wood. The (Brayford and Samuels 1993). results of analysis of the mit ss rDNA sequences and That Nectria fuckeliana did not cluster in Neonectria the absence of macroconidia in our cultures lead us was as much a surprise as its lack of Cylindrocarpon to question the link between N. fuckeliana and C. macroconidia. Cylindrocarpon cylindroides Wollenw. cylindroides var. tenue. Although N. fuckeliana is nei- var. tenue Wollenw. is reported to be the anamorph ther Nectria nor Neonectria, we prefer to maintain it of N. fuckeliana (Booth 1966, Roll-Hansen 1962), but in Nectria pending further work with additional iso- according to Booth and Roll-Hansen many isolates lates. The possibility that more than one species falls do not produce macroconidia or the ability to pro- under what has been identiﬁed as N. fuckeliana/Cy- duce macroconidia may be lost after some time in lindrocarpon cylindroides var. tenue should be consid- culture. Even when our isolates were fresh we did not ered. Resolution of this question will require addi- observe macroconidia. Roll-Hansen (1962) also re- tional collections from conifer forests. ported that the species produces perithecia in cul- The stromatic nature of perithecial formation on ture, but none formed in either of our two cultures. host tissues, with perithecia of different ages present A northern temperate species, N. fuckeliana, is re- on the same stroma, the hyaline ascospores, the col- BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 579 ony pigmentation and microconidial anamorph char- Although the N. mammoidea perithecial anatomy acteristics of N. fuckeliana are more characteristic of is distinctive and most species that possess the anat- Neo. coccinea, a species that produces abundant mi- omy are closely related species of Neonectria, the phy- croconidia, than of Neo. discophora. However, peri- logenetic importance of the wall anatomy is evidently thecial anatomy in N. fuckeliana shows strong simi- limited. In addition to N. fuckeliana, this anatomy larity to that of Neo. discophora and its relatives. Nec- also is found in Calostilbe striispora (Ellis & Everh.) tria fuckeliana therefore may form a link between the Seaver (anamorph ϭ Calostilbella calostilbe Ho¨hn.), a Nectria coccinea and Nectria mammoidea Groups species that is not obviously related to Neo. discophora (sensu Booth 1959, 1966) and indicate that there is (see Rossman et al 1999). no clear-cut demarcation between the two groups. In- Teleomorphs of Neo. discophora var. discophora and deed, the other Nectria groups with Cylindrocarpon var. rubi, and Neo. lucida are indistinguishable (cf. anamorphs, viz. the Neo. veuillotiana (Brayford and FIGS. 6 and 11). Neonectria discophora and N. lucida Samuels 1993) and Neo. rugulosa (Samuels and Bray- ford 1994) groups form a morphologically intergrad- can be distinguished only when their anamorphs are ing assemblage of taxa. The only clear discontinuity known. Cultures of Neo. discophora are purple, while is with the N. radicicola Group (Samuels and Bray- those of Neo. lucida are white to tan. DNA sequence ford 1990), which has clearly different perithecial analysis presented here indicates that Neo. discophora anatomy, more similar to that of Calonectria de Not. and Neo. lucida are distinct but closely related, thus than to the other Neonectria/Cylindrocarpon supporting our decision to separate the two taxo- groups. nomically.

TAXONOMY

KEY TO NEONECTRIA SPECIES HAVING NONMICROCONIDIAL CYLINDROCARPON ANAMORPHS

1. Ascospores averaging Յ20 ␮m long ...... 2 1. Ascospores averaging Ͼ20 ␮m long ...... 12 2. Perithecial apex with a fringe of saccate cells ...... Neo. coronata (Penz. & Sacc.) Mantiri & Samuels (Brayford and Samuels 1993) 2. Perithecial apex lacking saccate cells ...... 3 3. Ascospores averaging Ն7 ␮m wide ...... 4 3. Ascospores averaging Ͻ7 ␮m wide ...... 7 4. Perithecia rough to conspicuously warted; on hardwood trees ...... 5 4. Perithecia smooth, on conifers or hardwoods ...... 6 5. Perithecia rough but not conspicuously warted; ascospores (12–)15–20(–25) ϫ (5–)7–8(–9) ␮m, hyaline ...... Neo veuillotiana (Sacc. & Roum.) Mantiri & Samuels (Brayford and Samuels 1993) 5. Perithecia with conspicuous white warts; ascospores 10–16 ϫ 5.5–7.5 ␮m ...... 8. Neo. viridispora 6. On conifers; ascospores smooth, hyaline to indistinctly yellow-brown; colony white to buff, typically acremonium- or verticillium-like, microconidia only forming ...... 5. Nectria fuckeliana 6. On conifers or hardwoods; ascospores spinulose, becoming light but distinctly brown; colony purple, Cylindro- carpon lacking microconidia ...... 2. Neo. discophora 7. Cells at surface of perithecial wall lacking a deﬁnite outline, ca. 5 ␮m across ...... 8 7. Cells at surface of perithecial wall circular to angular, 20–35 ␮m diam ...... 11 8. Causing canker on Rubus; ascospores (11.5–)12.5–17.0(–23.0) ϫ (4.5–)5.5–6.5(–8.0) ␮m, colony purple ...... 3. Neo. discophora var. rubi 8. Usually corticolous or not causing canker on Rubus; ascospores 9–27 ϫ 4–11 ␮m, colony white, ochreous, tan, purple ...... 9 9. Ascospores 10–11 ϫ (3–)4–5 ␮m, spinulose; perithecia pyriform with a ﬂattened apex ...... Neonectria platycephala (Brayford & Samuels) Samuels & Brayford, comb. nov. (ϵ Nectria platycephala Brayford & Samuels, Mycologia 85:625. 1993). 9. Ascospores averaging Ͼ11 ␮m long, spinulose or smooth; perithecia globose and apex rounded or mammiform .. 10 10. Cosmopolitan; ascospores (10–)12–15(–17) ϫ (5.0–)5.5–6.5(–7.0) ␮m, colony purple ...... 2. Neo. discophora var. discophora 10. Mainly tropical; ascospores (9.5–)11.5–15.7(–21.5) ϫ (4–)5–7(–11) ␮m; colonies white to tan .....6. Neo. lucida 11. Ascospores 15–20 ϫ 6–7.5 ␮m, conspicuously warted; perithecial wall pseudoparenchymatous throughout ...... Neonectria verrucospora (Brayford & Samuels) Samuels & Brayford, comb. nov. (ϵ Nectria verrucospora Brayford & Samuels, Mycologia 85:631. 1993) 580 MYCOLOGIA

TABLE II. Pairwise genetic distances between the different Neonectria mitochondrial small subunit rDNA sequences analyzed. Below diagonal: absolute distances (base substitutions between two sequences). Above diagonal: mean distances (adjusted for missing data)

Isolatea 12345678910 1 Fusarium inﬂexum — 0.109 0.077 0.081 0.098 0.098 0.081 0.080 0.091 0.087 2 Nectria cinnabarina 67 — 0.097 0.101 0.107 0.104 0.093 0.091 0.077 0.085 3 Neonectria radicicola (1) 47 60 — 0.008 0.074 0.072 0.063 0.061 0.079 0.083 4 Neo. radicicola (2) 49 63 5 — 0.077 0.076 0.063 0.061 0.082 0.089 5 Neo. galligena (2) 61 67 46 48 — 0.003 0.038 0.036 0.093 0.080 6 Neo. galligena (1) 61 65 45 47 2 — 0.035 0.033 0.093 0.080 7 Neo. neomacrospora (1) 50 58 39 39 25 23 — 0.002 0.089 0.087 8 Neo. neomacrospora (2) 49 57 38 38 24 22 1 — 0.088 0.085 9 Calonectria sp. 53 45 46 48 54 54 52 51 — 0.087 10 Nectria fuckeliana 50 49 48 51 46 46 50 49 50 — 11 Neo. veuillotiana 44 41 23 24 24 24 23 22 40 36 12 Neo. rugulosa 34 34 18 19 20 19 17 16 37 36 13 Neo. coronata 36 38 20 21 22 22 21 20 42 35 14 Neo. trachosa 34 36 18 19 22 21 20 19 39 36 15 Neo. westlandica 38 37 21 22 25 24 23 22 44 37 16 Neo. jungneri 39 38 25 27 28 28 28 27 42 41 17 Neo. discophora 48 52 42 43 41 41 39 39 55 53 18 Neo. phaeodisca 37 40 28 29 29 29 28 27 41 42 19 Neo. betulae 36 39 27 28 28 28 27 26 42 41 20 Neo. viridae 36 43 32 33 33 33 32 31 45 42 21 Neo. lucida 47 48 37 38 39 39 36 35 50 49 a Numbers in parentheses next to species name are isolate numbers given to identify multiple isolates for a species where needed (see TABLE I).

11. Ascospores (15–)17.5–20.5(–22) ϫ 6–9 ␮m, conspicuously warted; perithecial wall ‘‘mammoidea type’’ below a surface region of circular cells ...... 7. Neo. trachosa 12. Ascospores phragmosporous, (38–)50–67 ϫ 10–14 ␮m ...... Neonectria phaeodisca (Rossman) Samuels & Brayford, comb. nov. (ϵ Nectria phaeodisca Rossman, Mycol. Pap. 150:38. 1983; see Samuels and Brayford 1993). 12. Ascospores didymosporous, smaller ...... 13 13. Perithecial apex with a fringe of saccate cells; ascospores (10–)16–24(–37) ϫ (3.5–)5.5–8.5(–11.0) ␮m ...... Neo. coronata (Brayford and Samuels 1993). 13. Perithecial apex lacking saccate cells ...... 14 14. Perithecia smooth, shining; perithecia elongated with a broad and obtuse apex; ascospores hyaline, typically conspicuously striate, less often smooth, (15–)22–29(–36) ϫ (5–)8.5–10.2 ␮m ...... Nenectria jungneri (Henn.) Samuels & Brayford, comb. nov. (ϵ Nectria jungneri Henn., Bot. Jahrb. Syst. 22:75. 1897 (1895); see Samuels and Brayford 1994). 14. Perithecia rough to warted, or nearly smooth but then dull, not shining, globose ...... 15 15. Perithecia pale brown to brown (brick) with ostiolar area dark brown, nearly black; ascospores (23–)26–32(–36) ϫ (5–)8–10(–11) ␮m; often associated with a chrome-yellow conidial mass in nature ...... Neonectria cinnamomea (Brayford & Samuels) Samuels & Brayford, comb. nov. (ϵ Nectria cinnamomea Brayford & Samuels, Mycologia 85:167. 1993). 15. Perithecial red to dark red, not associated with yellow conidia ...... 16 16. Perithecia smooth; ascospores (19–)21–24(–26) ϫ 7–8 ␮m; on Betula, Scotland ...... 1. Neo. betulae 16. Perithecia conspicuously warted ...... 17 17. Ascospores (24–)25–34(–37) ϫ (7.0–)8.5–11.5(–13.0) ␮m, New Zealand ...... 9. Neo. westlandica 17. Ascospores (27–)31–35(–36) ϫ (4–)6.5–8 ␮m, Colombia ...... 4. Neo. dumontii

DESCRIPTIONS OF THE SPECIES lulae superﬁciales perithecii globosae, 15–20 ␮m diam. Asci anguste clavati, 100–135 ϫ 10–15 ␮m, apice anulo refrac- 1. Neonectria betulae Brayford et Samuels, sp. nov. tivo instructi. Ascosporae ellipsoideae vel fusiformes, (19–) FIGS.3,4. 21–24(–26) ϫ 7–8 ␮m, bicellulares, minutae spinulosae, hy- Perithecia globosa, 350–475 ␮m diam, glabra, rubra. Cel- alinae vel pallide luteo-brunneae. Culta in agaro PDA lente BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 581

TABLE II. Extended

11 12 13 14 15 16 17 18 19 20 21 0.077 0.061 0.065 0.062 0.069 0.071 0.088 0.068 0.067 0.066 0.086 0.075 0.062 0.070 0.066 0.068 0.070 0.096 0.074 0.073 0.079 0.084 0.042 0.033 0.037 0.033 0.038 0.046 0.077 0.052 0.050 0.059 0.068 0.044 0.035 0.039 0.035 0.040 0.050 0.079 0.054 0.052 0.061 0.070 0.044 0.037 0.041 0.040 0.046 0.051 0.076 0.054 0.052 0.061 0.071 0.044 0.035 0.041 0.039 0.044 0.051 0.076 0.054 0.052 0.061 0.071 0.042 0.031 0.039 0.037 0.042 0.052 0.072 0.052 0.050 0.059 0.066 0.040 0.029 0.037 0.035 0.040 0.050 0.072 0.050 0.048 0.057 0.064 0.074 0.068 0.078 0.072 0.081 0.078 0.102 0.076 0.079 0.084 0.093 0.066 0.066 0.065 0.066 0.068 0.076 0.098 0.078 0.077 0.078 0.091 — 0.023 0.029 0.036 0.043 0.040 0.063 0.046 0.048 0.050 0.065 13 — 0.024 0.024 0.032 0.036 0.064 0.044 0.043 0.048 0.057 16 13 — 0.029 0.033 0.044 0.066 0.050 0.045 0.052 0.057 20 13 16 — 0.027 0.042 0.068 0.046 0.047 0.050 0.061 24 18 18 15 — 0.044 0.066 0.050 0.048 0.052 0.064 22 20 24 23 24 — 0.076 0.056 0.054 0.057 0.070 34 35 36 37 36 41 — 0.042 0.035 0.037 0.051 25 24 27 25 27 30 23 — 0.009 0.018 0.035 26 23 24 25 26 29 19 5 — 0.015 0.022 27 26 28 27 28 31 20 10 8 — 0.040 36 31 31 33 35 38 28 19 12 22 — crescit, mycelio aerio albo, reversum purpureum. Conidia ner, unpigmented toward the perithecial locule. Peri- absunt. thecial apex a disk formed of vertically oriented hy- HOLOTY PUS: SCOTLAND. Argyllshire: Cowall phal elements with rounded tips, gradually merging Peninsula, Argyll Forest Park, N end of Loch Goil, with the periphyses and continuous with the middle vicinity of Locqoilhead, along trail to Rob Roy’s Cave, region of the perithecial wall. Asci narrowly clavate, elevation 20–100 m, on bark of Betula sp., 12 Apr 100–135 ϫ 10–15 ␮m, apex with a conspicuous re- 1992, leg. D. Brayford & G.J.S. (8012) (G.J.S. culture fractive ring, 8-spored. Ascospores ellipsoid to fusi- 92-47: BPI 802663, IMI 352566). form, (19–)21–24(–26) ϫ 7–8 ␮m, 1-septate, not con- Etymology: Betulae refers to the host genus, Betula. stricted at the septum, finely spinulose, with a 1 ␮m Anamorph: None formed in culture. wide sheath, colorless or very pale yellow-brown, un- Mycelium not visible on host. Perithecia solitary to iseriate with overlapping ends, filling each ascus. gregarious in groups of a few, superficial, globose, 350–475 ␮m diam, nonpapillate, apex with a dark, Characteristics in culture.—Colonies on PSA slow areolate area 90–135 ␮m diam; dark red with ostiolar growing, 20 mm diam after 10 d, aerial hyphae white, area near black, uniformly dark red in 3% KOH, yel- cottony, colony surface violet; reverse with purple dif- low in 100% lactic acid, not collapsing when dry, fusing pigment. Cultures remaining sterile. Colonies smooth, appearing slightly pitted. Cells at surface of grown on autoclaved strips of Betula bark also lacking perithecial wall circular, 15–20 ␮m diam with walls 1 conidia. ␮m thick. Perithecial wall 40–50 ␮m wide, compris- Habitat: Bark of Betula sp. ing three regions: outer region a single layer of cir- Distribution: Scotland. cular, 15 ␮m diam cells with walls 1 ␮m thick, unpig- Commentary: Although a Cylindrocarpon ana- mented; middle region ca 15 ␮m wide, of inter- morph did not form in ascospore isolates of these twined, branched, ca 3.5 ␮m wide hyphal elements specimens, there is little doubt that this species be- arranged Ϯ perpendicular to the perithecial surface, longs to the Nectria mammoidea Group due to its walls ca 1.5 ␮m thick, pigmented; inner region 7–10 perithecial wall anatomy and violaceous pigment in ␮m wide, cells adjacent to the middle wall region PSA cultures. The N. mammoidea-type of anatomy of intertwined, with small, Ϯ elliptic lumina, walls ca 1.5 the perithecial wall is difficult to infer from crush ␮m thick, pigmented, cells of this region becoming mounts because of the superficial layer of circular progressively more fusiform, compressed; walls thin- cells but is suggested by the general perithecial form. 582 MYCOLOGIA

FIGS. 3–14. Perithecia of Neonectria and Nectria species. 3, 4. Neo. betulae G.J.S. 8012. 5, 6. Neo. discophora. 5. G.J.S. 98- 132, 6. G.J.S. 5065. 7. Neo. dumontii K.P.D.-CO 7987. 8, 9. Nectria fuckeliana, respectively BPI 551647 and G.J.S. 90-37. 10. Neo. lucida G.J.S. 2965. 11. Neo. trachosa G.J.S. 8009. 12, 13. Neo. viridispora A.Y.R. 2690. 14. Neo. westlandica G.J.S. 83- 204. Scale bars ϭ 500 ␮m except 6, which ϭ 1 mm. All bright ﬁeld microscopy. BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 583

This species is distinctive in the group because of its surface of perithecium, lumina Ͻ1 ␮m wide and tips large ascospores and because of its apparent lack of rounded, walls 2–2.5 ␮m thick; inner region of peri- anamorph. thecial wall 10–20 ␮m wide, cells lacking a definite outline but with long axis parallel to surface of perithecial wall, wall of cells 2.0–2.5 ␮m thick, cells in- 2. Neonectria discophora (Mont.) Mantiri & Samuels, creasingly more compacted, thin-walled toward the Can. J. Bot. 79:339. 2001 var. discophora perithecial locule; perithecial apex of vertically elon- FIGS.5,6,32 gated cells, continuous with the lateral perithecial ϵ Sphaeria discophora Mont., Ann. Sci. Nat. Bot. II 3: wall, forming a disk around the ostiolar opening, 353. 1835. cells increasingly narrower and merging with the per- ϭ Nectria tasmanica Berk. in Hooker, Botany of the Ant- iphyses within. Stroma arising from cortex of host, arctic Voyage of H.M. Discovery Ships Erebus and Ter- Ϯ ror, part 3. Flora Tasmaniae 2:279. 1860. cells angular to circular in outline, vertically ori- ␮ ϭ Nectria mammoidea W. Phillips & Plowr., Grevillea 3: ented, walls 2–2.5 m thick, continuous with cells of 126. 1875. outer region of perithecial wall. Asci cylindrical, ϵ Creonectria mammoidea (W. Phillips. & Plowr.) Seav- (68–)72–95(–120) ϫ 7–10(–15) ␮m, apex with a re- er, Mycologia: 188. 1909. fractive ring, 8-spored. Ascospores ellipsoidal, (10– ϭ Nectria nelumbicola Henn., Verh. Bot. Vereins Prov. )12–17(–20) ϫ (4–)5–8(–9) ␮m, equally 2-celled, not Brandenburg 40:151. 1898. Fide Ho¨hnel & Weese, constricted at the septum, spinulose, light brown, un- Ann. Mycol. 8:467. 1910. iseriate with overlapping ends, filling each ascus. ϭ Nectria umbilicata Henn., Hedwigia 41:3. 1902. ϭ Nectria mammoidea var. rugulosa Weese, Akad. Wiss. Characteristics in culture.—Colonies 20–30 mm diam Wien Math.-Naturw. Kl., Abt. 1, 125:552. 1916. after 14 d at 20 C on PSA. Colony surface slimy; aerial ϭ Nectria mammoidea var. minor Reinking, Zentralbl. mycelium sparse or floccose, initially white, darken- Bakteriol., Abt. 2, 94:135. 1936. ing to blood/bay or dark violet. Colony reverse ϭ Creonectria discostiolata Chardoń, Bol. Soc. Venez. Ci. strongly pigmented blood color or purple-slate with Nat. 5:341 [241 sic]. 1939. ϭ Nectria pinea Dingley, Trans. Roy. Soc. New Zealand pale vinaceous margin. Slimy sporodochia usually 79:198. 1951. abundant on colony surface and in aerial mycelium, Anamorph: Cylindrocarpon ianthothele Wollenw. var. often forming in concentric rings. On SNA or SNAY majus Wollenw., Z. Parasitenk. (Berlin) 1:161. 1928. growth sparse, lacking pigment, white/buff sporo- FIG.32 dochia forming on agar surface. Conidiogenous cells ϭ Cylindrocarpon ianthothele var. minus Reinking, Zen- formed apically on densely, irregularly branching tralbl. Bakteriol., Abt. 2, 94:135. 1936. clusters of cells borne laterally on broad, thick-walled, ϭ Cylindrocarpon ianthothele var. rugulosum C. Booth, pale brown vegetative hyphae. Conidiogenous cells Mycol. Pap. 104:25. 1966. cylindrical, 12–18 ϫ 3–4 ␮m, with apical thickening ϭ Cylindrocarpon pineum C. Booth, Mycol. Pap. 104:26. and collarette. Macroconidia (2–)3–5(–7) septate, 1966. 43–73 ϫ 4–6 ␮m, cyindrical, but often broader in the Mycelium not visible on host. Perithecia solitary to upper half, uniformly curved or more strongly gregarious or cespitose in groups of a few, superficial curved at the tip, with apical cell rounded and basal or with base partially immersed in substratum on a cell rounded to truncate. Microconidia sparse or ab- minute stroma, stroma at first producing conidia, sent. Chlamydospores not formed. Most strains de- having long, unbranched hyphal hairs, often in fis- veloping fertile perithecia in single-spore cultures, ␮ sures in bark, globose, (260–)300–550(–670) m suggesting homothallism; this ability may decline in diam, nonpapillate or with a broad, mammiform old isolates. apex 150–200 ␮m diam, not collapsed when dry; red Habitat: Bark of twigs and branches of recently to dark red and appearing almost black, smooth, of- dead trees, less frequently on palm trunks. ten shining; ostiolar area often darker red than the Distribution: Cosmopolitan. perithecial body, uniformly dark red in 3% KOH, yel- Illustrations: Wollenweber (1930a, FIG. 536; 1930b, low in 100% lactic acid. Cells at surface of perithecial FIG. 742, as N. mammoidea). Booth (1966, FIG. 16, as wall lacking definite outline, appearing to be inter- N. mammoidea). Kar and Gupta (1977, FIG.2,asN. twined hyphae with lumina irregular in shape, 2–2.5 mammoidea). Dennis (1978, Pl. 33 C, as N. mammo- ␮m in greatest dimension, walls 2–4 ␮m thick. Peri- idea). thecial wall 30–50 ␮m wide, of two intergrading re- HOLOTY PE: CHILE. Juan Fernandez, sur cortice gions; outer region 20–30 ␮m wide, continuous over arborum, date unknown, Bertero 1700 (PC!; ?ISO- the perithecium to form a uniform palisade of hy- TY PE, PC!). phal cells, cells distinctly hyphal, perpendicular to Additional representative specimens examined (out of ca 584 MYCOLOGIA

FIGS. 15–31. Neonectria species. 15–22. Neo. betulae G.J.S. 8012. 15. Perithecia in nature. 16. Median longitudinal section of a mature perithecium showing palisidal nature of the lateral wall. 17, 18. Asci; apex shown in 18. 19–22. Discharged ascospores. 19, 21 in optical section; 20, 22 in surface view showing ﬁne warts. 23–26. Neo. discophora G.J.S. 8018. 23, 24. Median longitudinal section through a perithecium showing the palisidal nature of the lateral wall. 25, 26. Asci with ascospores. Apical discharge mechanism visible in 26. 27–31. Neo. dumontii K.P.D.-CO 7987. 27. Perithecia in nature. 28. Median longitudinal section of a mature perithecium showing the hyphal nature of the wall and large cells at the exterior. 29. Ascus with ascospores. 30, 31. Discharged ascospores. Microscopy: DIC except 16 and 27, stereo microscope and 26, PC. Scale bars: 16, 17, 31 ϭ 50 ␮m; 18–22, 26, 30 ϭ 10 ␮m; 23–26, 28, 29 ϭ 100 ␮m, 27 ϭ 500 ␮m. BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 585

70): AUSTRALIA. TASMANIA: locality unknown, [on bark], Rossman (PDD 44247). BAY OF PLENTY: Whakarewarewa, Archer (K, HOLOTY PE of N. tasmanica; PAD, ISOTY PE of ex Pinus radiata, Sep 1949, G.B. Rawlings (HOLOTY PE of N. tasmanica). VICTORIA: Otway Ranges, vicinity of Lorne, N. pinea, PDD 7510; ISOTY PE IMI 050395). COROMAN- Angahook-Lorne State Forset, along track from Blanket DEL: Thames County, along Kauaeranga R., NE of Thames, Leaf Picnic ground to Cora Lynn Cascades, along Cora on bark of Beilschmiedia tawa, 8 Mar 1973, J.M. Dingley et Lynn Creek, elevation 200–350 m, primarily Eucalyptus for- al (PDD 30629). GISBORNE: Urewera National Park, Lake est, on twigs of recently dead tree, 28 Aug 1999, G.J.S. 8650 Waikaremoana, track to Lake Waikare-iti, on Nothofagus sp., (BPI, culture G.J.S. 00-71); Tarra Valley, Tarra-Bulga Nation- G.J.S. 85-62 & L.M. Kohn (PDD 50072). NORTHLAND: al Park, between Yarram and Traralgon, Tarra Rainforest Hokianga County, Waipoua Forest, vicinity of Te Matua Walk, on bark of recently fallen Eucalyptus sp., 22 Aug 1999, Ngahere kauri tree, on Freycinetia baueriana subsp. banksii, G.J.S. 8561 (BPI 746774). BRAZIL. SANTA CATHARINA: 31 May 1982, G.J.S. 82-93 et al (PDD 43190, IMI 329104). Blumenau, (on decorticated wood), date not known, A. WAIKATO: Waitomo, on bark, 26 Apr 1983, G.J.S. 83-206 Moeller (S, herb Sydow; HOLOTY PE of Nectria umbilicata). et al (PDD 46410, IMI 326258). WESTLAND: Mount Aspir- ‘‘CENTRAL AMERICA.’’ isolated from soil and inoculated ing National Park, Haast Pass, ca 30 km E of Haast Junction, onto apple ‘‘faule’’, 1934/1935, S5360 (B ex BBA, probable Roaring Billy Forest Walk, on bark of ?Podocarpus sp., 28 HOLOTY PE of N. mammoidea var. minor). VENEZUELA. Apr 1985, G.J.S. 85-35 & L.M. Kohn (PDD 50047); Franz ARAGUA: forests at Rancho Grande, on deadwood, eleva- Josef, track to Lake Wombat, on bark of Fuchsia excorticata, tion 1200 m, 30 Apr 1938, C.E. Chardoń 2617 (CUP, HO- 10 Apr 1983, G.J.S. 83-188 & R.H. Petersen (BPI 1109329, LOTY PE of Creonectria discostiolata). ENGLAND. CAM- PDD 46365). SCOTLAND. ARGYLLSHIRE: Cowal Penin- BRIDGESHIRE: Brandon, on Pinus sylvestris stump, 16 Oct. sula, Cowal Peninsula, Argyll Forest Park elevation 50–100 1958, J. Rishbeth (IMI 074992). DEVON: Dartmoor Forest, m, ca 10 km N of Dunoon, on branchlets of Larix sp., 11– on Picea sitchensis, 25 Jul 1975, C.C. Gulliver (IMI 195455). 13 Apr 1992, G.J.S. & D. Brayford 8007 (G.J.S. culture 92- SUSSEX: Dunford House, Midhurst, on Pinus sp., 25 May 36: BPI 802651); Benmore Estate, on Pseudotsuga menziesii 1959, C. Booth (IMI 076785). YORKSHIRE: Clapham, Larix bark and wood of stump, 30 Mar 1935, J. Ehrlich & C.J.C. sp., 3 May 1963, C. Booth (IMI 100408); Buttercombe Chesters, 30.iii.0935, (IMI 052091, Herb. J. Ehrlich 1667); Woods, Stamford Bridge, on Pinus sp. cone, 17 Sep 1961, on Pinus ?sylvestris bark of log, 30 Mar 1935, J. Ehrlich & W.G. Bramley (IMI 089845 Thornton Dale, on Pinus laricis, C.J.C. Chesters (IMI 022815, Herb. J. Ehrlich 1669); Ben- 21 May 1955, W.G. Bramley [IMI 062653]); Cloughton more Forestry Estate, near Dunoon, on Picea excelsa log, 5 Woods, Yorkshire, ?Pinus sp., 17 Apr. 1955, J. Webster & C. Apr 1935, J. Ehrlich & C.J.C. Chesters (IMI 217845); Kin- Booth (IMI 059891). FRANCE. PYRENEÉS ATLANTIQUES: cardine, Maryculter, Pinus sylvestris, 13 Sep 1979, D.W. forest vicinity of Laruns, on ?Corylus avellana, 26 Aug 1978, Minter (IMI 256525). PERTHSHIRE: Braco, wood on Crieff G.J.S. 78-67 & G. Roux (PDD 38803); Isle de la Sauveterre Road, on Picea excelsa log, 31 Mar 1935, J. Ehrlich & C.J.C. de Bearn, elevation 100 m, on Populus nigra, 25 Oct 1998, Chesters (IMI 217844). THAILAND. Khao Yai National Park, G.J.S. & F. Candoussau (BPI 748310, culture G.J.S. 98-132). vicinity of Park Headquarters, trail, on bark of recently dead GUYANA. Cuyuni-Mazaruni Region, VII; Mazaruni Subre- tree, 2 Aug 1997, G.J.S. 8266 & P. Chaverri (G.J.S. culture gion, VII-2, along Koatse R, ca. 2 km E of Pong R., ca. 5 h 97-66, BPI 745816; BioTek, Bangkok). UNITED STATES. walk S of Chinoweing Village, 05Њ28ЈN, 60Њ04ЈW, elevation NORTH CAROLINA: Great Smoky Mountains National 600–650 m, on terminal branchlets of recently dead tree, Park, on bark of ?Quercus sp., 14 Oct 1990, G.J.S. 90-46 & 28 Feb 1987, G.J.S. et al 4952C (G.J.S. culture 87-49: BPI A.Y. Rossman (BPI 1107126); Jackson County, Nantahala 747057, BRG, NY); Base of Mount Wokomung, ca. 5.5 km National Forest, Bull Pen Road to Chattoga Run, Ellicott walk NE of Kopinang Village, in legume-dominated forest, Rock Trail from Fowler Creek, on recently dead Acer rub- 05Њ05ЈN, 59Њ49ЈW, elevation 720 m, on bark of recently fall- rum, 28 Sep 1989, G.J.S. 89-132 et al (BPI 1107267, IMI, en tree, 27 Jun 1989, G.J.S. et al 6269a (G.J.S. culture 89- NY). PENNSYLVANIA: Mercer County, Grove City, in for- 57: NY). INDONESIA. NORTH SULAWESI: Eastern Du- mer city dump at foot of Gilmore Street, on Acer sp., 14 moga-Bone National Park, at confluence of Toraut and Oct 1972, G.J.S. (C.T.R. culture 72-372, NY). VENEZUELA. Tumpah rivers, vicinity of Project Wallace Base Camp, ARAGUA: Forests at Rancho Grande, elevation 1200 m, on 00Њ34ЈN, 123Њ57ЈE, 211 m, on twig, Oct–Nov 1985, G.J.S. deadwood, 30 Apr 1938, C.E. Chardoń 2617 (CUP, HO- 2222A (G.J.S. culture 85-179, NY, IMI 329113); Gn. Muajat, LOTY PE of Creonectria discostiolata); Parque Nacional Hen- Danau Alia, 00Њ45ЈN, 124Њ25ЈE, 1400 m, on bark, 26 Oct. ri Pittier, Parque Nacional Henri Pittier, Rancho Grande´ 1985, G.J.S. 2399 (NY), 2421A (BO, NY). JAMAICA: Border Biological Station, trail to Guacamayo, ca 10Њ21ЈN, 67Њ41W, between Portland and St. Andrew parishes: Traveler’s Rest, elevation 1250–1400 m, 4 Dec 1990, on bark, G.J.S. et al Silver Hill Gap, 25 mi marker from Kingston, on wood, 8 7933 (G.J.S. culture 90-212: BPI 842123, NY, VEN). DIS- Jan 1971, R.P.K. et al CUP-MJ 726 (CUP, NY); PORTLAND TRITO FEDERALE: ca 13 km NE of Colonia Tovar on road PARISH: along trail to Silver Hill Gap, near Woodcutter’s between Colonia Tovar and El Tigre, on bark, 29 Jul 1972, Gap, vicinity of Newcastle, on wood, 9 Jan 1971, R.P.K. et K.P.D.-VE 6531 et al (C.T.R. culture 72-168: NY); vicinity of al CUP-MJ 760 (NY). NEW ZEALAND. AUCKLAND: Wai- El Portachuelo, NE of Colonia Tovar, on bark of tree, 1972, temata City, Waitakere Ranges, Anawhata Road, Chateau K.P.D.-VE 6618 et al (Rogerson culture 71-187: NY). ME- Mosquito Track, on bark of ?Hoheria populnea, 30 Oct 1973, RIDA: Parque Nacional Sierra Nevada, above Tabay, Qda. J.M. Dingley et al (PDD 32631); Piha Road, Cowan Track, Coromoto, La Mucuy 08Њ36ЈN, 71Њ02ЈW, elevation ca 2000 on Ripogonum scandens, 4 Jun 1983, G.J.S. 83-125 & A.Y. m, on bark, 20, 23 Nov 1990, G.J.S. et al 7244A (G.J.S. 586 MYCOLOGIA culture 90-155: BPI, VEN). MONAGAS: SE of Caripe, Ha- trated by Wollenweber 1926, from natural substra- cienda Las Acacias, La Carmelita, on stem of Cecropia sp., tum). 17 Jul 1972, K.P.D.-VE 5253 et al (C.T.R. culture 72-147: Illustrations: Wollenweber (1926: 59, 526, 527). NY). SUCRE: NW of Irapa, trail between Los Pocitos and Booth (1966: FIG. 17). the peak of Cerro Humo, on palm, 12 Jul 1972, K.P.D.-VE Habitat: Associated with cankers on roots and 4769 et al (C.T.R. culture 72-90: NY). TRUJILLO: Parque Nacional Guaramacal, ca 10 km SW of Batatal, La Defensa, crowns of Rubus idaeus and R. fruticosus. along Rio Saguas, Campamiento Granja Bocono, in forest Distribution: England, New Zealand, Northern Ire- along trail to water source, 09Њ18ЈN, 70Њ10ЈW, elevation land, Scotland, Switzerland, Venezuela. 2000 m, on palm fruit, 20, 23 Nov 1990, G.J.S. et al 7447 Specimens Examined: ENGLAND. locality and date un- (BPI 744858, VEN). YARACUY: N of Nirgua, in mountains, known, on Rubus sp, Herb. J. Ehrlich 1518 (IMI 22841). on palm, 7 Jul 1971, K.P.D.-VE 1536 et al (NY, VEN). KENT: on Rubus idaeus cv. Glen Clora, 13.v.1983, G. Thorpe Commentary: As discussed previously (Samuels et (IMI 277659). BATHEASTON: on Rubus fruticosus, iv.1870, al 1990), the better known name N. mammoidea is a Herb. Broome (IMI 52177). SURREY: Merstham, host un- later taxonomic synonym of Neo. discophora. known, viii.1928, R.V. Harris (IMI). NORTHERN IRE- LAND. locality unknown, on Rubus idaeus, 10.viii.1984, Booth (1966) described the Cylindrocarpon ana- comm. R.S. McIlwaine (IMI 288100). SCOTLAND. Loch morph of Neo. discophora (as N. mammoidea) and also Lomond, Inchcailloch, on Rubus fruticosus, R. Watling (IMI recognized several varieties of the species. Of these, 180609). SCOTLAND. Locality unknown, 6.x.0973, collec- we accept only var. rubi (see below). tor unknown, (IMI 52183, Herb. J. Ehrlich 1642, perithecia on oat agar, ex Rubus idaeus); locality unknown, on Rubus 3. Neonectria discophora var. rubi (Osterw.) Bray- idaeus, McIntosh, det. N.A. Alcock (IMI 52181, Herb. J. Ehr- lich 1640); INVERNESSHIRE: Beauly, Beaufort Castle, Ru- ford et Samuels, stat. nov. et comb. nov. bus idaeus, 9 Oct 1925, A. Alcock (IMI 324717), PERTHSHI- ϵ Nectria rubi Osterw., Ber. Deutsch. Bot. Ges. 29:620. RE: Alyth, on Rubus idaeus cv. Glen Moy, 17 May 1988, B. 1911. Williamson, (IMI 325503); Tayside, Kimimuir, Angus, Path ϵ Hypomyces rubi (Osterw.) Wollenw., Phytopathology 3: Head Farm, on Rubus idaeus cv. Glen Moy, 10 Jun 1988, B. 224. 1913. Williamson (IMI 325504); Angus, Newton Park Farm, Rubus Anamorph: Cylindrocarpon ianthothele var. ianthothele idaeus cv. Glen Moy, 10 Jun 1988, B. Williamson (IMI Wollenw., Ann. Mycol. 15:56. 1917. 325505, IMI 325506, IMI 325507). NEW ZEALAND. NEL- Perithecia morphologically and anatomically indistin- SON: Tapawera, Rubus idaeus, 9 Jan 1949, P.R. Fry (IMI guishable from those of Neo. discophora var. discopho- 50403); Tapawera, on Rubus idaeus, 1956, J.M. Dingley (IMI ra, formed on cankers at the roots and crowns of 70017). diseased Rubus canes; ascospores ellipsoid, 1-septate, Cultures studied (all ex Rubus idaeus): UNITED KING- (11.5–)12–16(–23) ϫ (4.5–)5.5–6.5(–8.0), becoming DOM. ENGLAND: location unknown, R.M. Nattrass (IMI pale brown, finely spinulose at maturity. 113917 ϭ CBS 177.27); Plymouth, Wembury, 20 Jun 1978, Characteristics in culture: Colonies on PSA 15–35 A.U. Chapman (IMI 229976, 288100, 324717, 325503, mm diam after 14 d at 20 C. Aerial mycelium floc- 325504, 325505, 325506, 325507, 325509). SCOTLAND: lo- cose, white or honey-colored, becoming dark purple. cation unknown, 1929, H.W. Wollenweber (IMI 113919, CBS In some strains, aerial mycelium sparse or absent, col- 241.29). SWITZERLAND. 1911, A. Osterwalder (IMI onies slimy. Colony reverse-pigmented dark vina- 113918). ceous to vinaceous, in some cases unpigmented or Commentary: This fungus has been associated with with unpigmented sectors. Abundant slimy sporodo- a distinctive basal canker of cultivated Rubus sp. on chia formed on the agar surface and in the aerial several occasions in Europe since 1911 (Brayford mycelium; sporodochia buff, pale violet or dark pur- 1991). The host specificity and pathogenic status of ple, often forming in concentric rings. In some the fungus remain unclear, but the recent disease strains fertile ascocarps developing, especially at the outbreaks in Scotland have been correlated with colony margins of mature cultures. On SNA or SNAY wind damage to the hosts followed by waterlogging. hyphal growth diffuse and spreading through the This suggests that the fungus is either a secondary agar, unpigmented, with scattered buff sporodochia pathogen or only able to cause disease on severely forming on the agar surface. Conidiogenous cells cy- stressed host plants. There is overlapping in the re- lindrical, 10–25 ϫ 2.0–3.5 ␮m, with apical thickening spective anamorphs of Neo. discophora var. discophora and collarette, formed apically on irregularly branch- and var. rubi, but the tendency in strains from Rubus ing clusters of cells, often borne on broad, thick- is to have smaller ascospores, smaller macroconidia walled hyphae. Macroconidia curved, fusoid, 3–5 sep- and slower colony growth than those of the type va- tate, 36–70 ϫ 4–7 ␮m, with rounded apical and basal riety. Due to the association of var. rubi with a char- cells. Sparse microconidia occasionally found but acteristic plant disease, which is unusual for species normally absent. Chlamydospores absent (but illus- in this Neonectria group, and in the absence of DNA BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 587

FIG. 32. Neonectria discophora, Cylindrocarpon ianthothele var. majus G.J.S. 90-212, anamorph from PSA. Fig. 33. Nectria fuckeliana G.J.S. 92-46, acremonium-like anamorph from PSA. Scale bar ϭ 10 ␮m.

sequences for var. rubi, we maintain it as a distinct wide consisting of highly intertwined elements with taxon. walls ca 4 ␮m thick and meandering lumina; inner Weese (1912) was the first to recognize a relation- region 20–30 ␮m wide, cells with elliptic to fusoid, ship between this taxon and Nectria mammoidea (Neo. 5–20 ␮m long, 2–3 ␮m wide lumina and walls 1.5–2 discophora). ␮m thick, pigmented in cells at the exterior, unpigmented in cells toward the locule. Perithecial apex 4. Neonectria dumontii Brayford et Samuels, sp. formed of vertically elongated hyphal elements with ␮ nov. FIGS. 7, 27–31 walls ca 2.5 m thick; elements continuous with the Perithecia solitaria vel gregaria, globosa, 750–1250 ␮m middle region of the perithecial wall and protruding diam, areolata vel discoidea, rubra, stromati immerso insi- through the outer region, becoming narrower and dentia. Asci anguste clavati, (157–)161–238(–250) ϫ merging with the periphyses within. Asci narrowly cla- (11.7–)13.7–20.0 ␮m, anulo apicali refractivo praeditii. As- vate, (157–)161–238(–250) ϫ (11.7–)13.7–20.0 ␮m, cosporae fusiformes, (27.0–)30.7–35.2(–36.0) ϫ (5.5–)6.5– apex with a refractive ring; 8-spored, ascospores bi- ␮ 7.7(–8.0) m, 1-septatae, laeves, hyalinae vel pallide buba- seriate above, uniseriate below, filling each ascus. As- linae. Status anamorphicus ignotis. cospores fusiform, (27.0–)30.7–35.2(–36.0) ϫ (5.5–) HOLOTY PUS: In Colombia, K.P.D.-CO 7987 6.5–7.7(–8.0) ␮m, with one median septum, not con- (NY). stricted, smooth, colorless or very light tan, some- Etymology: Refers to Kent P. Dumont, formerly of times appearing to have a narrow sheath. the New York Botanical Garden, in recognition of the Habitat: Wood, tree fern rachis. many collections of the Hypocreales that he made Distribution: Colombia (Boyaca´, Cundinamarca). during his career. HOLOTY PE: COLOMBIA. DEPARTAMENTO Anamorph: not known. ´ Mycelium on host not visible. Perithecia solitary or BOYACA: Along Tunja-Ramiriquı´-Paéz Road, vicinity gregarious to caespitose in clusters of 4–5, superficial, of kilometer post 20 from the crossing of the Tunja- seated on an immersed stroma with stromal cells Nueva Coloń Road, on wood, 14 Sep 1976, K.P.D.- growing within cortical cells of host, globose, 750– CO 7987 Buritica´ & Umanã (NY, isotype IMI 1250 ␮m diam, perithecial apex with a flattened disk, 352114). often appearing areolate, not collapsing when dry, Additional collection: COLOMBIA. DEPARTA- red, with a darker red apex, uniformly dark red in MENTO CUNDINAMARCA: Between kilometer 3% KOH, yellow in 100% lactic acid; smooth or mi- posts 29–30 from Zipaquira´, on the Zipaquira´-Pacho nutely roughened to scaly. Cells at surface of perithe- Road, elevation ca 10 000 ft, on tree fern, 9 Jun 1976, cial wall circular, 20–30 ␮m diam, walls 2–3 ␮m thick. K.P.D.-CO 4491 et al (NY, IMI 352115). Perithecial wall 65–90 ␮m wide, comprising three in- Commentary: This species is morphologically sim- tergrading regions: outer region, to 30 ␮m wide, in- ilar to Neo. westlandica but differs in having larger cluding scales, consisting of one or two layers of cir- ascospores and a poorly developed N. mammoidea cular, cells with lumina 15–25 ␮m diam, walls ca 2.5 palisade in the lateral wall. Neonectria westlandica is ␮m thick and pigmented; middle region ca 35 ␮m found in New Zealand, whereas Neo. dumontii is 588 MYCOLOGIA

FIGS. 34–44. Nectria fuckeliana. 34, 35. Median longitudinal section through a mature perithecium seated on a stroma. The palisadal nature of the perithecial wall visible in 35. 36. Asci with filamentous apical paraphyses. 37–39. Asci with ascospores. Apical discharge mechanism visible in 37. 40. Discharged ascospores. 34, 35 from G.J.S. 8014; 36–40 from G.J.S. 90-32. 41–44. Neo. trachosa G.J.S. 8009. 41. Perithecia. 42, 43. Median longitudinal section through a perithecium seated on a stroma. Palisadal nature of the perithecial wall with large cells formed at the exterior visible in 43. 44. Part of an ascus with two ascospores. Apical discharge mechanism visible. Microscopy: DIC except 36, PC, and 42, stereo microscope. Scale bars: 34 ϭ 300 ␮m; 35, 43 ϭ 100 ␮m; 36 ϭ 20 ␮m, 37, 38, 40, 44 ϭ 10 ␮m; 39 ϭ 20 ␮m, 41 ϭ 1 mm, 42 ϭ 250 ␮m. known only from Colombia. We have not had an op- dark red with ostiolar area sometimes darker and os- portunity to grow this species in pure culture but pre- tiolar area of young perithecia appearing slightly um- dict that its anamorph will be a species of Cylindro- bilicate and viscid, uniformly red in 3% KOH and carpon. yellow in 100% lactic acid, not collapsing when dry; smooth and oftening shining. Cells at surface of perithecial wall lacking a definite outline, rather appear- 5. Nectria fuckeliana C. Booth, Mycol. Pap. 73:56. ing as intertwined hyphae with thickened walls and 1959. FIGS. 8, 9, 33–40 ϭ circular lumina when seen in optical section. Perithe- Nectria cucurbitula (Tode : Fr.) Fr. f. abietis Roumegu- ␮ e`re, Fungi Gallici exsiccati 1292. 1881, ut ‘‘Nectria cu- cial wall (G.J.S. 8014)75 m wide at the widest part, curbitula Fries f. abietis Sac. [sic].’’ near the perithecial apex, comprising three regions; ϵ Nectria abietis Sacc. [ut Sac.] in Roumegue`re, Rev. outer region 50–60 ␮m wide, of 6 ␮m wide hyphal Mycol. (Toulouse) 3:23. 1 Apr 1881, nom. nud. elements with walls 1.5 ␮m thick and lumina 1 ␮m Anamorph: reported to be Cylindrocarpon cylindro- wide arranged Ϯ perpendicular to the perithecial ides Wollenw. var. tenue Wollenw., Zeitschr. Parasitenk. surface in a N. mammoidea palisade; middle region 1:153. 1928. FIG.33 ca 15 ␮m wide, of intertwined hyphal elements; ele- Mycelium not visible. Perithecia in cespitose clusters ments in section appearing Ϯ elliptic with lumina ca of few to many, often with developing and mature 7.5 ␮m long and 3 ␮m wide, walls 1.5–2 ␮m thick perithecia in the same cluster, superficial on an and pigmented, intergrading to the outer and inner erumpent and often extensive orange stroma, often regions; inner region of elongated, fusiform cells 10– with perithecia scattered on the stroma, globose, 15 ␮m long ϫ 3–6 ␮m wide and walls 1.5–2 ␮m thick (235–)276–346(–404) ␮m diam, nonpapillate; red to and pigmented, becoming progressively thinner and BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 589

unpigmented toward the perithecial locule. Perithe- 551626). CANADA. NEW FOUNDLAND: Bay of Islands, cial apex formed of a central disk ca 100 ␮m diam Coal River, on Abies, 14 Feb 1886, the Rev. A.C. Waghorne of narrow elements; elements arising from the inner (BPI 551622). EUROPE 5 Sep 1925, on dead bark of Pinus region of the perithecial wall, merging with peri- sp., H.W. Wollenweber 7541 (ex herb J.H. Faull ‘‘cause of physes; perithecial apex surrounded by hyphal ele- decay of various conifers’’, BPI 551645). AUSTRIA. Niede- ro¨sterreich: Sonntagberg vicinity of Rosenau, on Abies, Jun ments of the outer region of the perithecial wall. [year unknown], P.P. Strasser (Kryptogamae esx. 965, BPI Cells of the stroma distinctly pseudoparenchymatous, 551620). SILESIA: Carlsbru¨nn, on bark of Abies, Aug 1911, ϫ ␮ ␮ 15–20 ca 10 m, walls ca 3 m thick, pigmented, Niessl (BPI 551621). FRANCE. ‘‘Bas porte du Rhone a merging with the middle region of the perithecial Lyon’’, on bark of ‘‘Abies pectinata’’, Jul 1880, J. Therry (C. wall at the perithecial base. Asci cylindrical to nar- Roumegue`re, Fungi Gallici exsiccati 1292 N. cucurbitula f. rowly clavate, (80–)86–100(–120) ϫ 7–11 ␮m, apex abietis; BPI, CUP, FH, PC) GERMANY: ‘‘Harz im Walde with a refractive ring but frequently appearing sim- zwischen Braunlage und Schierke’’, on bark of Abies excelsa, ple; 4–8-spored, ascospores uniseriate with overlap- 27 Aug 1904, P. Sydow (Sydow, Mycotheca germanica 323: ping ends to biseriate and clustered in the upper half BPI); ‘‘Botanischen Garten’’, on cut branch of Picea excelsa, of the ascus. Ascospores ellipsoidal, (11.0–)12.5– Apr 1883, P. Sydow (Sydow, Mycotheca Marchica 472, as Nec- 15.5(–19.7) ϫ (4.0–)4.7–6.0(–7.0) ␮m, 1-septate, sep- tria cucurbitula: BPI); ‘‘Marchia: Hortus Berolinensis’’, on bark of Picea excelsa, May, P. Magnus (Rabenhorst, Fungi tum median, spinulose to smooth, hyaline to indis- europaei et extraeuropaei 4258, as Nectria cucurbitula: BPI); tinctly yellow brown. Pseudoparaphyses persisting Bergisches Land: Waldbro¨l., on Picea branches, 11 Sep ␮ among mature asci as ca 3 m wide, branched and 1931, A. Schumacher (Flora des Rheinlandes 575, BPI anastomosing ﬁlaments with no free ends. 551618); Berlin, on Picea excelsa, 20 Mar 1883, P. Magnus Characteristics in culture: Colonies on PSA 15–30 (BPI 551634, as N. cucurbitula). Saxony: ‘‘Neckendorfer mm diam after 10 d, irregular or spidery in form, Thal pr. Islebiam’’, on bark of Picea excelsa, Apr 1875, J. with white/buff aerial mycelium, sometimes sparse, Kunze ( Johs. Kunze, Fungi selecti exsiccati 105, as ‘‘Nectria the colony being slimy due to abundant sporulation cucurbitula f. Piceae excelsae [Poir.] Link’’: BPI 551636). in small droplets and pustules. Colonies weakly pig- Frankfurt am Main, on deadwood and bark, date not mented sienna (pale brown) or rust near the inocu- known, Auerswald (BPI 551617); Franconia: vicinity of Sug- lum block but mostly white to buff; reverse with dif- enheim, on bark of pine, date not known, Rehm (Raben- horst, Fungi europaei 1235 as Nectria cucurbitula: BPI); fuse luteous pigment fading to white at the colony Harz: im Walde zwischen Braunlage und Schierke, on bark margin. No odor apparent. Red stromatic structures, of Abies excelsa, 27 Aug 1904, P. Sydow (Sydow, Mycotheca possibly protoperithecia, present in some strains. On germanica 323, BPI 551627); Westfalia: Kr. Siegen, Wald am SNAY colonies spidery and spreading, with sparse ae- Tunnel bei Burgholdinghausen, on Picea excelsa, 30 Apr rial mycelium but abundant sporulation in slimy 1952, A. Ludwig (BPI 551635). NEW ZEALAND. DUNE- droplets. Conidiogenous cells scattered, sessile or on DIN: Tokoiti Forest, on Pinus radiata, 22 Jul 2002, M. Dick sparsely branched cells, often branching at right an- (BPI; culture NZFS 891 ϭ G.J.S. 02-60); second collection, gles, cylindrical or tapering slightly, mostly 30–70 ϫ NZFRI-M 4613, 21 Nov 2001, R. Thun (BPI 842427, culture ϭ 1.5–3.5 ␮m, with a single apical locus, periclinal G.J.S. 02-61 NZFS 839). SOUTHLAND: Dipton Forest, thickening and indistinct collarette. Total length of on Pinus radiata, 20 Nov 2001, M. Dick (NZFRI 4814, BPI, culture G.J.S. 02-67. SCOTLAND. Glenbriers Forest, on Pi- conidiogenous cell and supporting hyphae might be Ͼ ␮ cea excelsa, 27 Mar 1935, J. Ehrlich & N. Wilson 1675 (PDD 100 m. Microconidia abundant, in slimy droplets 11626 ex IMI 022814). Cowal Peninsula, Argyll Forest Park, throughout the colony, hyaline, 0(–1) septate, ellip- N end of Loch Goil, vicinity of Locgoilhead along trail to soidal to cylindrical, sometimes clavate, with indis- Rob Roy’s Cave, elevation 20–100 m, 12 Apr 1992, on trunk tinct basal abscission scar, mostly 3–7 ϫ 2–4 ␮m. Mac- of Abies sp., D. Brayford & G.J.S. 8014 (G.J.S. culture 92- roconidia and chlamydospores not observed. 46: BPI 802662, IMI 352568); second collection, on trunk Illustrations: Richter and Gerlach (1958), Roll- of recently fallen Larix sp., G.J.S. 8016 (BPI 802657, IMI Hansen (1962), Booth (1966, 1979). 352570; culture G.J.S. culture 92-42). Pebbleshire, on Picea Habitat: On bark of conifers including Abies, Larix, sitchensis, Apr 1926, A.E.S. McIntyre (BPI 551642). SWE- Pinus and Picea. DEN. Scania, Boekeberg, on Picea excelsa stump, 24 Apr 1934, Ehrlich & Kemner (BPI 1113363 ex K 52084); Upp- Distribution: Circumboreal. sala, on conifer stump, 22 Jun 1912, C.L. Shear (BPI Type: ‘‘Oestereich (Nassau)’’: ‘‘ad Pinorum ramos 550780). VA¨ STERBOTTEN: Umea par., between Yyttero¨da aridos’’, Fuckel (Herbier Barbey-Boissier 855 ex Fungi and Heleneborg, mixed forest near the road to Vindeln, on rhenani 983 as Nectria cucurbitula, ISOTY PES: NY! Picea abies bark, 27 Oct 1975, O. Eriksson 751027-1 (BPI BPI 551647!). 745076 ex UME 27078). SWITZERLAND. KT. GRAUBUN- Additional specimens examined: BOHEMIA. on Abies pi- DEN: vicinity of Zuoz, along Ova d’Arpiglia, elevation 1700 cea, 1912, J.E. Kaba´t (Fungi bohemici, BPI 551629); on Abi- m, on branchlets of Picea sp., 6 Sep 1990, G.J.S. 90-31 (BPI es excelsa, Nov 1908, F. Buba´k (Fungi bohemici, BPI 1107109, IMI 342667); Landwasser Valley, between Mon- 590 MYCOLOGIA

FIGS. 45–47. Neonectria lucida, morphological diversity in its Cylindrocarpon lucidum anamorph. 64. C.T.R. 71-279. 65. G.J.S. 96-10. 66. G.J.S. 90-180. All from PSA. Scale bar ϭ 10 ␮m.

stein and Wiessen, elevation 1000–1347 m, on Picea sp., elia. ‘‘Nectria abietis Sac.’’ is given as a species in the G.J.S. 90-32 (BPI 1107110, IMI 342668). UNITED STATES. nomenclator in the Roumegue`re reference cited MAINE: Eustis, on Picea rubra, 19 Jul 1935, J.R. Hansbrough above. This name is not accompanied by a descrip- (BPI 551640); Crawford, on Picea rubra, 17 Oct 1936, J.R. tion or reference to a description and, thus, is taken Hansbrough (BPI 551639); second collection (BPI 551638); to be a newly but invalidly published nomen nudum Hulls Cove, Mount Desert, on Abies balsamea, 24 Apr 1935, Gillespie (BPI 551625); Westbrook, on Abies balsamea, Jul based on the Roumegue`re exsiccata. 1897, P.L. Ricker (Flora of Maine. Cryptogams, BPI 551623). Symptomless infection of Picea stems by Nectria NEW YORK: Hamilton County, 7th Lake Region, on Abies fuckeliana was discussed by Roll-Hansen and Roll- balsamica, Aug 1919, Stork (BPI 551624). Hansen (1980). A canker disease of Abies concolor in Commentary: The anamorph of this species is re- California and Oregon caused by N. fuckeliana was ported to be Cylindrocarpon cylindroides var. tenue described by Schultz and Parmeter (1990). (Roll-Hansen 1962, Booth 1966). Although microconidia are always abundant from acremonium- or verticillium-like conidiophores, macroconidia are 6. Neonectria lucida (Ho¨hnel) Samuels et Brayford, said to be sparse or absent in many strains, especially comb. nov. FIGS. 10, 45–47 ϵ after some time in vitro. Macroconidia are cylindrical, Nectria lucida Ho¨hnel, Akad. Wiss. Wien Math. Na- straight or slightly curved with bluntly rounded ends turw. Kl., 1 Abt., 118:289. 1909. and no obvious basal abscission scar, 1–7 septate, Anamorph: Cylindrocarpon lucidum Booth, Mycol. mostly 3–5 septate, 30–80 ϫ 4.5–6.5 ␮m. Chlamydo- Pap. 104:21. 1966. FIGS. 45–47. ␮ spores not observed. Fertile perithecia were obtained Mycelium not visible. Perithecia 430–480 m diam, by Roll-Hansen (1962) by pairing isolates, indicating gregarious in groups of 2–5, globose, nonpapillate heterothallism. None of our ascospore cultures ever but with apex sometimes slightly flattened and mam- produced macroconidia. The absence of macroco- miform, not collapsing when dry, red, with darker nidia in combination with the results of DNA se- red ostiolar area, uniformly dark red in 3% KOH, quencing presented here leads us to suggest that the yellow in 100% lactic acid; wall smooth and shining, connection between N. fuckeliana and the Cylindro- superficial on an immersed stroma; hyphal hairs aris- carpon is not correct. ing from the surface and growing around and nearly There is no description on the label of the Rou- as long as the perithecial height, hairs 6–7 ␮m wide, megue`re exsiccata, so that this specimen could not unbranched, septate, wall 1.0–1.5 ␮m thick, pale yel- be taken to be the place of description of a new form, low. Cells at surface of perithecial wall lacking a def- N. cucurbitula f. abietis. However, there is clear ref- inite outline, hyphal, cells ca. 4 ␮m wide, walls 1.5– erence to ‘‘Sacc. Mich. 1. p. 409.’’ where there is a 2.0 ␮m thick, lumina narrow. Perithecial wall 20–30 valid description of the form. Thus we take the form ␮m wide, of two regions; outer region of short hyphal to have been published validly by Saccardo in Mich- elements in a palisade with long axis perpendicular BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 591 to perithecial surface; outer region continuous over liana, 10 Feb. 1986, G.J.S. 3621 (NY, IMI 325249); along the perithecium to form a uniform palisade of hy- trail from Sau¨l to Mount Galbao, ca 15 km SW of Sau¨l, phal cells; cells of inner region lacking a definite out- Camp 2, elevation 600 m, on twigs of recently fallen tree, line but with long axis parallel to surface of perithe- 14, 18, 29 Jan 1986, G.J.S. 2965 & J. Boise (NY). GUYANA. Cuyuni-Mazaruni Region, VII; Mazaruni Subregion, VII-2, cial wall, cells increasingly more compacted and thin- along Koatse R, ca 2 km E of Pong R., ca 5 h walk S of walled toward the perithecial locule; perithecial apex Chinoweing Village, 05Њ28ЈN, 60Њ04ЈW, elevation 600–650 of vertically elongated cells and continuous with the m, on bark of live mimosoid legume, 28 Feb 1987, G.J.S. lateral perithecial wall, forming a disk around the os- 4976 et al (BPI 842128, BRG, IMI 325856, NY). Mount Wo- tiolar opening, cells increasingly narrower and merg- komung, on ridge leading NW toward summit, ½–1 h walk ing with the periphyses within. Asci cylindrical, 63– from base camp, in well drained forest at lower elevation to 72 ϫ 6.5–10.0 ␮m, apex with a refractive ring, 8- wet forest with palms and tree ferns at higher elevation, spored. Ascospores ellipsoidal, (10–)12–15(–17) ϫ elevation 1150–1300 m, on twig of recently fallen tree, 1 Jul (5.0–)5.5–6.5(–7.0) ␮m, equally 2-celled, not con- 1989, G.J.S. 6380 (G.J.S. culture 89-71, BPI 842129, NY). stricted at the septum, becoming finely spinulose, JAMAICA. HANOVER PARISH: Dolphin Head Mountain, vicinity of Askenish, elevation ca 1000 ft, 22 Jan 1971, on pale brown, uniseriate with overlapping ends, filling bark, R.P.K. et al CUP-MJ 934 (C.T.R. culture 71-107: NY). each ascus. ST. ANDREW PARISH: vicinity of Kingston, along Cane Riv- Characteristics in culture: Colonies 20–30 mm er and slope of Good Hope Mountain, elevation 1000 ft, on diam after 10 d on PSA. Colony surface slimy to felty; bark, 12 Jan 1971, R.P.K. et al CUP-MJ 821 (C.T.R. culture aerial mycelium typically sparse to felty, white to buff 71-49: CUP, Jamaica, NY). NEW ZEALAND. SOUTHLAND: or a shade of brown (umber, bay, chestnut, dark Catlins State Park, Lake Wilkie, on bark of tree, 18 Apr brick, sepia). Colony reverse white to dark brown 1988, G.J.S. 85-27 et al (PDD 50050). PUERTO RICO. Ca- shades. Slimy sporodochia usually abundant on the ribbean National Forest, Luquillo Mountains, trail to El colony surface and in the aerial mycelium, often Toro from Route 186, on bark of recently Cecropia sp., 24 forming in concentric rings. Conidiogenous cells Feb 1996, G.J.S. 8098, H.-J. Schroers & D.J. Lodge (G.J.S. culture 96-35, BPI 475543). UNITED STATES. NEW YORK: formed apically on densely, irregularly branching Pack Forest, on tree stump, 25 Sep 1971, D. Malloch (C.T.R. clusters of cells borne laterally on otherwise undif- culture 71-379, NY). VENEZUELA. AMAZONAS: Cerro de ferentiated vegetative hyphae. Conidiogenous cells la Neblina, along Rıó Mawarinuma, just outside Canõn ϫ ␮ cylindrical, 12–20 4.5–5.5 m, with apical thick- Grande, vicinity of Neblina base camp, 00Њ50ЈN, 66Њ10ЈW, ening and collarette. Macroconidia cyindrical, but of- elevation ca 140 m, on bark of recently dead tree, 27 Apr ten broader in the upper half, uniformly curved or 1984, G.J.S. 1645 (NY, VEN). BO´ LIVAR: ca 118 km S of El more strongly curved at the tip, with apical cell Dorado on El Dorado-Sta. Elena Road, trail up N facing rounded and basal cell rounded to truncate. Macro- slope of Uei-tepui from former military camp Ciento Vein- conidia of most isolates 5-septate, 60–70 ϫ 5–6 ␮m; ticinco, on vine, 5 Aug 1972, K.P.D.-VE 6941 et al (C.T.R. in a second group macroconidia 70–90 ϫ 6–7 ␮m culture 72-180: NY). MIRANDA: Parque Nacional El Avila, S facing slope of La Silla, vicinity of refugio ‘‘No te apures’’, (G.J.S. 71-105, G.J.S. 71-49, G.J.S. 72-71, G.J.S. 85-27, on bark, 30 Jun 1972, K.P.D.-VE 3878 et al (C.T.R. culture G.J.S. 86-178); in a third group macroconidia 70–80 ϫ ␮ 72-71: NY). MERIDA: Parque Nacional Sierra Nevada, above 9.5–10.0 m (G.J.S. 72-180, G.J.S. 86-336, G.J.S. 87- Tabay, Quebrada Coromoto, Estacion La Mucuy, 08Њ36ЈN, 50, G.J.S. 89-71, G.J.S. 90-166). Microconidia absent. 71Њ02ЈW, elevation 2300 m, on bark, Nov 1990, G.J.S. 6763 Chlamydospores not formed. Perithecia forming in et al (G.J.S. culture 90-166: BPI 842125, VEN). MONAGAS: single ascospore cultures of at least some collections, vicinity of Caripe, ‘‘Cueva del Guacharo’’, around canker suggesting homothallism. on living tree, K.P.D.-VE 5319 et al (NY). TRUJILLO: Habitat: Bark of twigs and branches of recently Parque Nacional Guaramacal, on road between Bocono and dead trees, less frequently on lianas. Guaramacal, 8.4 km from Batatal-Bocono Road, Њ Ј Њ Ј Distribution: Indonesia ( Java), Japan, New Zea- 09 15 N,70 13 W, elevation 2350 m, on bark, 22 Nov 1990, G.J.S. 7409 et al (BPI 842127, VEN, G.J. S. culture 90-180). land, tropical America and Caribbean region ( Ja- Commentary: The tan or brown colonies distin- maica). guish Neo. lucida from Neo. discophora. However, HOLOTY PE: INDONESIA. JAVA. Tjibodas, Tjibur- there is considerable variation in macroconidial size rum, an lebenden Zw., 1907–1908, Ho¨hnel (FH-Ho¨h- in Neo. lucida. Macroconidia in some collections are nel 2899!). Additional representative specimens examined (out of the same size as those in purple isolates (Neo. disco- 30): FRENCH GUIANA. ORSTOM research area ‘‘ECER- phora var. discophora); these are interpreted as C. lu- EX’’, kilometer 16 on road between Sinnamary and St. Elie, cidum sensu stricto (Booth 1966). However, macro- on decaying stem of Philodendron sp., 20–29 Feb, 1 Mar. conidia in other collections are considerably larger 1986, G.J.S. 3987 (CAY, IMI, NY); Sau¨l, Circuit Grand Fosse´, and might indicate the existence of distinct taxa with- 03Њ60ЈN, 53Њ20ЈW, elevation 300–350 m, on bark of living in what we currently regard as Neo. lucida. 592 MYCOLOGIA

7. Neonectria trachosa Samuels et Brayford, sp. buff sporodochia, arising from monophialides, scat- nov. FIGS. 11, 41–44, 64 tered and on irregularly branching clusters of cells, Perithecia solitaria vel gregaria, superficialia, globosa, 10–20 ϫ 2–4 ␮m, cylindrical, sometimes swollen and 460–540(–600) ␮m diam, non papillata, rubra vel sangui- constricted near the apex, with periclinal thickening nea, acido lactico lutescentia. Cellulae perithecii superfi- and indistinct collarette. Microconidia not observed. ciales globosae, 20–25 mm diam. Asci cylindrici, 100–140 ϫ ␮ Macroconidia (1–)3–5(–7) septate (mostly 3, one 8- 9–11 m, anulo apicali instructi, octospori. Ascosporae el- septate conidium observed), 3-septate: (32–)38–52 lipsoideae, (15–)17.5–20.5(–22) ϫ (6–)6.7–8.7(–9) ␮m, 1- (–60) ϫ (5.5–)6–7(–7.5) ␮m, 4-septate: (44–)47–55 septatae, non constrictae, conspicue verruculosae, hyalinae; ϫ ␮ ϫ in asco uniseriatae. (–60) 6–7.5 m, 5-septate: (50–)53–63(–65) 6– ␮ ϫ ␮ Anamorphosis Cylindrocarpon sp. 7.5 m, 6-septate: (48–)55–72(–80) 6–7.5 m, 7- ϫ ␮ Coloniae in agaro PSA post 10 dies temperatura 25 C 10– septate: 63–78 7 m, cylindrical to slightly fusoid, 20 mm diam attingentes. Mycelium aerium floccosum, al- curved, less so near the base, with bluntly rounded bum vel bubalinum, reversum etiam album vel bubalinum. apical and basal cells, lacking a distinct abscission Microconidia non formantur. Macroconidia cylindrica vel scar. Chlamydospores not seen. No odor. Perithecia modice fusiformia, latissima in tertio distali, incurvata, not observed in culture. (1–)3–5(–7) septata, plerumque 3-septata et (32–)38–52 Habitat: Conifer bark. ϫ ␮ (–60) (5.5–)6–7(–7.5) m. Apex et basis rotundata. Chla- Distribution: Scotland, known only from the type. mydosporae absentes. HOLOTY PE: SCOTLAND. ARGYLLSHIRE: Cow- HOLOTY PUS: BPI 802661. all Peninsula, Argyll Forest Park, ca 5 km S of Stra- Mycelium not visible. Perithecia solitary to gregarious chur along River Cur, vicinity of Glenbranter Village, in groups of 10 or fewer, superficial, globose, 460– Lauder Broadleaves Walk, elevation ca 50 m, on co- 540(–600) ␮m diam, nonpapillate, apex with a dark, nifer bark, 12 Apr 1992, D. Brayford & G.J.S. 8009 slightly sunken, 85–125 ␮m diam disk; red to dark (BPI 802661, IMI 352560; culture G.J.S. 92-45). red with ostiolar area near black, uniformly dark red Commentary: Neonectria trachosa is characterized in 3% KOH, yellow in 100% lactic acid, not collaps- by its large ascospores, the well-developed, uneven ing when dry; smooth to slightly roughened. Cells at region of circular cells at the exterior of the perithe- surface of perithecial wall circular, 20–25 ␮m diam cial wall that gives the wall a slightly roughened as- with walls 1.5–2 ␮m thick. Perithecial wall ca 100 ␮m pect, and white colonies on agar. The perithecial wall wide, comprising three regions; outer region ca 45 of Neo. westlandica, a species known only from New ␮m wide, cells elliptic to circular, 15–25 ␮m diam, Zealand, also has a well-developed outer region of walls ca 1.5 ␮m thick; middle region ca. 25 ␮m wide, circular cells, but perithecia of this species are coarse- of intertwined and branched hyphal elements ar- ly warted and ascospores are distinctly larger. ranged Ϯ perpendicular to the perithecial surface and with walls 2–3 ␮m thick; inner region 25 ␮m wide, cells adjacent to the middle region intertwined 8. Neonectria viridispora Samuels et Brayford, sp. Ϯ ␮ with lumina elliptic and up to 15 m long, walls nov. FIGS. 12, 13, 48–57, 65 ca 3 ␮m thick and pigmented but becoming pro- Perithecia usque ad quinquagintena caespites formantia, gressively thinner and unpigmented toward the peri- globosa vel late pyriformia, 300–375 ␮m high, 300–375 ␮m thecial locule. Perithecial apex a small disk of parallel diam, rubra, albis verruculosis praedita, basi immersa, ad hyphal elements continuous with the inner region of apicem discoidea. Asci 80–100 ϫ 7–10.5 um, anulo apicali the perithecial wall and protruding through outer re- praediti. Ascosporae ellipsoideae vel fusiformes, 10–16 ϫ ␮ gions. Cells of the stroma pseudoparenchymatous, 5.5–7.5 m, 1-septatae, spinulosae vel laeves, viridescentes. 10–15 ␮m diam, and walls pigmented, 1.5 ␮m thick. Anamorphosis: Cylindrocarpon sp. Coloniae in PSA cultae Asci cylindrical, 100–140 ϫ 9–11 ␮m, apex with con- post decem dies temperatura 20 C 20–25 mm diam attingentes, albae, reverso sanguineae vel umbrinus, vel bubali- spicuous refractive ring; 8-spored, ascospores uniser- nae ad margini. Microconidia nulla. Macroconidia (3–)4– iate, filling each ascus. Ascospores ellipsoid, (15.0–) ϫ ␮ ϫ ␮ 5-septata, (52–)58–67–71) 6.0–6.5 m, cylindrica vel in 17.5–20.5(–22.0) (6.0–)6.7–8.7(–9.0) m, 1-sep- tertio superiore paullo latiora, basi quasi recta sed apicem tate, not constricted at the septum, conspicuously versus incurva, cellula apicali rotundata, nonnumquam tur- warted, with a thin sheath, colorless. gida, cellula basali rotundata vel applanata, pedicello dis- Characteristics in culture: Colonies on PSA 10–20 tincto carenti. Chlamydosporae haud observatae. mm diam after 10 d, with floccose, white aerial my- Holotypus: BPI 842122. celium, remaining white to buff at maturity; colony Mycelium white, arachnoid, formed around perithe- reverse white to buff, lacking purple or dark brown cial bases. Perithecia forming around small cankers, pigmentation. Growth on SNAY sparse and weak. Co- cespitose in groups of up to 50, base immersed in a nidia forming in slimy droplets and small, white to barely erumpent stroma, globose to broadly pyri- BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 593

FIGS. 48–63. Neonectria species. 48–57. Neo. viridispora A.Y.R. 2690. 48, 49. Median longitudinal section through a mature perithecium. 50. Surface of the perithecial wall showing the hyphal nature. 51. Hyphal hairs, typical of the N. mammoidea group, arising from the stroma. 52, 53, 54. Asci. Asci in 54 immature but showing the apical discharge mechanism. 55–57. Ascospores. 55 is the opical section of 56, which shows warts at the surface of the ascospore. 58–63. Neo. westlandica PDD 32634. Microscopy: DIC except 48, 58, 52 FL; 54, 60 PC. Scale bars: 48, 50 ϭ 100 ␮m; 49, 51, 53, 58–61 ϭ 50 ␮m; 52, 54– 57, 62, 63 ϭ 10 ␮m. form, 300–375 ␮m high, 300–375 ␮m wide, apex dis- of the perithecial wall. Cells at surface of perithecial coidal, ca 125 ␮m diam; red but appearing white wall textura epidermoidea, walls 2–3 ␮m thick, ﬁne from large white warts, not changing color in 3% pores joining lumina of adjacent cells. Perithecial KOH, yellow in 100% lactic acid, not collapsing when wall 30–40 ␮m wide, comprising three regions: outer dry, or sometimes collapsing by lateral pinching; region uneven, of disrupted, globose, dead cells that strongly warted, warts formed of cells at the surface form the white layer on the perithecial surface; mid- 594 MYCOLOGIA

FIGS. 64–66. Neonectria species, Cylindrocarpon anamorphs. 64. Neonectria trachosa G.J.S. 8009, from SNAYE. 65. Neo. viridispora A.Y.R. 2690, from PSA. 66. Neo. westlandica G.J.S. 85-45, from PSA. Scale bar ϭ 10 ␮m. dle region 15–20 ␮m wide, of intertwined hyphae pigmented walls and conspicuous septa. Perithecia with walls 1.5–2 ␮m thick; inner region 7–10 ␮m forming commonly around the inoculum block and wide, of flattened, compressed cells with thin, non buried in the agar, scattered singly or in small clus- pigmented walls. Perithecial apex a disk formed of ters, red, globose/ovoid, with warted walls, a discoi- thick-walled hyphal elements arranged in a palisade dal apex, and exuding blue-green ascospores. Micro- with elements ca 5 ␮m wide and walls 1.5–2 ␮m thick conidia absent. Macroconidia forming abundantly on and pigmented; elements becoming progressively the agar surface in buff-colored slimy masses. Coni- narrower toward the ostiolar canal and there merg- diogenous cells cylindrical, 10–20 ϫ 3.0–4.5 ␮m, with ing with the periphyses. The stroma of compact, sep- periclinal thickening and an indistinct collarette, tate, unbranched, parallel hyphae with rounded ends borne apically on irregularly branching cells arising arranged perpendicular to the surface of the host from broad vegetative hyphae or in small scattered and up to 250 ␮m long ϫ 5–7 ␮m wide. Asci narrowly clusters in the aerial mycelium. Macroconidia (3–)4– clavate, 80–100 ϫ 7.0–10.5 ␮m, apex with a refractive 5 septate, (52–)58–67(–71) ϫ 6.0–6.5 ␮m, cylindrical ring, ascospores uniseriate to partially biseriate. As- or slightly broader in their upper third, almost cospores ellipsoid to fusiform, 10–16 ϫ 5.5–7.5 ␮m, straight at the base but becoming more curved to- 1-septate, septum median, spinulose to smooth, hya- ward the apex. Apical cell rounded, sometimes be- line becoming green. coming swollen. Basal cell rounded or flattened, lack- Characteristics in culture: Colonies on PSA 20–25 ing a distinct pedicel. Cells of old macroconidia com- mm diam after 10 d, with white, floccose aerial my- monly becoming inflated, globose, but not develop- celium. Reverse blood-colored to umber, fading ing thick walls. Chlamydospores not observed. through bay to a buff margin. Ochreous pigment Habitat: On bark of Ochroma lagopus (Bombaca- spreading through the medium. Honey to buff slimy ceae). sporodochia forming abundantly on agar surface, Distribution: Ecuador, known only from the type most commonly around the inoculum block. Macro- collection. conidia also forming in slimy, colorless droplets and HOLOTY PE: ECUADOR. vicinity of Santo Domin- clusters in the aerial mycelium. Colonies on CMA 35– go, on Ochroma lagopus, 19 Aug 1991, C.S. Hodges 40 mm diam after 10 d, with sparse, floccose aerial s.n. (BPI 842122, isotype IMI 350698, ex type culture mycelium and sepia to dark brick pigment in reverse. A.Y.R. 2690, CBS 102162). Macroconidia produced in honey to buff sporodo- Commentary: Neonectria viridispora is character- chia forming around the inoculum block and in col- ized by its red perithecia that are covered in nature orless, slimy droplets scattered in the aerial myceli- with coarse, white warts, and by its green ascospores. um. Colonies on SNAY unpigmented, sparse and Perithecia formed in culture are warted, but the spreading, lacking aerial mycelium. Some broad (8– warts remain red. This species is referred to the Nec- 20 ␮m) vegetative hyphae present, with pale brown tria mammoidea Group (Booth 1959) because of its BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 595 characteristic stroma, Cylindrocarpon anamorph, pig- slimy clusters on the colony surface and in the aerial mented spinulose ascospores and perithecial wall mycelium. In some strains a strong actinomycetous that is formed of intertwined, thick-walled hyphae. odor formed. On SNA or SNAY colonies 35 mm diam Green ascospores are unusual in the Nectriaceae, after 21 d, lacking pigment, mycelium dense and known previously only from Viridispora alata (Sa- compact rather than diffuse and spreading. Aerial muels) Samuels & Rossman, V. penicilliferi (Samuels) mycelium sparse, sometimes aggregated in tufts. Samuels & Rossman, and V. diparietispora ( J.H. Miller Small slimy, buff sporodochia forming scattered on et al) Samuels & Rossman, all of which have Penicil- the agar surface. Conidia also forming in slimy drop- lifer van Emden anamorphs (Polishook et al 1991, lets in the aerial mycelium. Conidiogenous cells Samuels 1989, Rossman et al 1999). None of these borne in small groups scattered on the mycelium or fungi has been included in a molecular phylogenetic sometimes on long (110 ␮m), unbranched or spar- analysis, but based on perithecial anatomy and the ingly branched conidiophores, each branch termi- anamorph. They do not appear to be closely related. nating in a single phialide. Phialides cylindrical, 14– 27 ϫ 3–5 ␮m, with periclinal thickening and a small collarette at the apex. Microconidia not observed. 9. Neonectria westlandica (Dingley) Samuels et Bray- Macroconidia (0–6) mostly 3–4 septate, (41–)47– ford, comb. nov. FIGS. 14, 58–63, 66 66(–76) ϫ (5.5–)5.7–7.5(8.0) ␮m, fusiform, evenly ϵ Nectria westlandica Dingley, Trans. Roy. Soc. New curved or slightly more curved near the apex, base Zealand 79:201. 1951. rounded or truncate. Chlamydospores not observed. Anamorph: Cylindrocarpon sp. Habitat: Bark of dicotyledonous trees, less fre- Mycelium not visible. Perithecia solitary to gregarious quently on bark of gymnospermaceous trees. in groups of 3–4, superficial or with base slightly im- Distribution: New Zealand. mersed in a basal stroma, globose, 450–550 ␮m diam, Descriptions and illustrations: Dingley (1951, plate nonpapillate; brownish red with nearly black ostiolar 24, FIG. 7). area, uniformly red in 3% KOH, yellow in 100% lac- HOLOTY PE: NEW ZEALAND. SOUTH ISLAND. tic acid, not collapsing when dry; strongly warted, Westland: Weheka, on Olearia avicenniaefolia, Dec warts to 90 ␮m high. Cells at surface of perithecial 1946, J.M. Dingley (PDD 5129!, ISOTY PE IMI wall and warts conspicuously circular, 15–40 ␮m ␮ ␮ 15560!). diam, walls ca 2 m thick. Perithecial wall 45–55 m Additional specimens examined: NEW ZEALAND. wide, comprising three regions; outer region contin- NORTH ISLAND. Auckland: Waitemata City, Waitakere uous with warts, consisting of a single layer of circular Ranges, Anawhata Road, Chateau Mosquito Track, on bark cells 15–18 ␮m diam with walls 3.5 ␮m thick; middle of Hoheria populnea, 30 Oct 1973, J.M. Dingley & G.J.S. 73- region 18–25 ␮m wide, cells with hyphal characters 242 & B. Segedin (PDD 32634, IMI 352112); Waitakere and arranged perpendicular to the surface of the Ranges, Huia, Parau Track, on bark of Metrosideros sp., 23 perithecium; inner region 10–18 ␮m wide, cells flat- Oct 1980, G.J.S. 80-156A & P.R. Johnston (PDD 41421, IMI tened, walls 1.5–2 ␮m wide, pigmented, becoming 255610). Taranaki: Mount Egmont National Park, Puniho progressively more compressed and thin-walled to- Track, on Pseudopanax crassifolia, 25 Apr 1983, G.J.S. 83- ward the locule. Perithecial apex with a disk ca 150 204, P.R. Johnston & R.H. Petersen (PDD 46408, IMI 352113). SOUTH ISLAND. Westland: N of Hari Hari, Lake ␮m diam, formed of vertically oriented hyphal ele- Ianthe State Forest, on bark of Dacryocarpus cupressinum, ments ca 3.5 ␮m wide at tip, gradually merging with 21 May 1983, G.J.S., T. Matsushima & A.Y. Rossman (G.J.S. the periphyses and continuous with the middle re- culture 83-156; PDD 46336, IMI 326254); Mount Aspiring gion of the perithecial wall. Nectria mammoidea stro- National Park, Haast Pass, ca. 70 km E of Haast Junction, ma sometimes present. Asci clavate, 110–130 ϫ 17– Cameron Flat, on root of indet. tree, 29 Apr 1985, G.J.S. & 23 ␮m, apex with an obscure refractive ring; 8- L.M. Kohn (G.J.S. culture 85-45; PDD 50055). spored, ascospores biseriate above, uniseriate below, Commentary: Dingley (1957) reported microco- filling each ascus. Ascospores ellipsoid to fusiform, nidia in cultures of Neo. westlandica but none were (23.5–)25.0–33.5(–37.0) ϫ (7.2–)8.5–11.5(–13.0) observed in any of our single-ascospore isolates. The ␮m, 1-septate, not constricted at the septum, finely geographical ranges of Neo. westlandica and Neo. dis- spinulose, colorless to pale yellow-brown, with a 1 ␮m cophora overlap in New Zealand. Although the two wide sheath. species are closely related, they are easily distin- Characteristics in culture: Colonies on PSA slow guished by the coarsely warted perithecia and larger growing, 10–25 mm diam after 10 d, either flat and ascospores of Neo. westlandica. Their respective Cylin- slimy or with floccose, white aerial mycelium. Colony drocarpon anamorphs also are morphologically simi- reverse white, cream or vinaceous buff. Strong purple lar, but the colonies of Neo. discophora are faster or brown pigment not observed. Conidia forming in growing and have strong purple slate diffusing pig- 596 MYCOLOGIA ment on PSA, whereas those of Neo. westlandica have Dennis RWG. 1978. British Ascomycetes. J. Cramer, Vaduz. little or no pigment. 585 p ϩ FIGS. 1–31, Plates I–XLIV. Dingley JM. 1951. The Hypocreales of New Zealand II. The genus Nectria. Trans Roy Soc NZ 79:177–202. ACKNOWLEDGMENTS . 1957. Life-history studies of New Zealand species of Nectria Fr. Trans Roy Soc NZ 84:467–477. This work, and all the work of the last author, could not Holmgren PK, Holmgren NH, Barnett LC. 1990. Index have been completed without the encouragement and the Herbariorum Part I: The herbaria of the world. Reg vast literature resource that has been made available to him Veg 120:1–693. over more than 40 years by Dr Clark T. Rogerson. We ap- Kar AK, Gupta SK. 1977. Some wood-inhabiting pyreno- preciate the loan of herbarium material from these herbar- mycetes of West Bengal. Indian Phytopathol 30:330– ia: B, CUP, FH, K, PAD, PC, PDD. Ms Ellen Bloch went 336. above the call of duty to locate specimens in NY. We are Le Gal M. 1947. Rećherche`s sur les ornamentations sporales indebeted to Ms Margaret Dick, New Zealand Forest Ser- des Discomyce`tes opercule´s. Ann Sci Nat Bot se´r 11, 8: vice, Rotorua, who provided us with recent collections of 73–297. N. fuckeliana, and to Dr Charles Hodges, North Carolina Li K-N, Rouse DI, German TL. 1994. PCR primers that allow State University, who provided us with Neo. viridspora. Dr intergeneric differentiation of ascomycetes and their Walter Gams corrected the Latin diagnoses. Drs Amy Ross- application in Verticillium. Appl Environ Microbiol 60: man and Walter Gams provided useful comments on early 4324–4331. drafts of the manuscript. Dr Wendy Untereiner provided Mantiri F, Samuels GJ, Rahe JE, Honda B. 2001. Phyloge- invaluable assistance with editorial details, especially as re- netic relationships in Neonectria species having Cylin- gards DNA sequence analysis. Ms Lutorri Ashley and An- drocarpon anamorphs inferred from mitochondrial ri- drea Zemanova provided technical assistance to G.J.S. Mr. bosomal DNA sequences. Can J Bot 79:334–340. James Plaskowitz prepared some of the plates of illustra- Nirenberg H. 1976. Untersuchungen u¨ber die morpholo- tions. Collecting over the years was supported by The Na- gische und biologische Differenzierung in der Fusari- tional Geographic Society (North Sulawesi) and the U.S. um-Sektion Liseola. Mitt Biol Bundesanst Land- Forstw National Science Foundation. The U.S. National Science Berlin-Dahlem 169:1–117. Foundation supported Prof. R. P. Korf and Dr K. P. Du- O’Donnell K, Cigelnik E. 1997. Two divergent intragenomic mont, to whom the last author is indebted for allowing him rDNA ITS2 types within a monophyletic lineage of the to participate. The many specimens collected by Dr Du- fungus Fusarium are nonorthologous. Mol Phylogenet mont were collected with NSF support. The last author was Evol 7:103–116. supported directly by NSF Grants BSR 8500236, BSR Polishook J, Bills G, Rossman AY. 1991. A new species of 8721877 and PEET Grant 9712308 ‘‘Monographic Studies Neocosmospora with a Penicillifer anamorph. Mycologia of Hypocrealean Fungi: Hypocrea and Hypomyces’’. B.M.H. 83:797–804. and F.M. were supported by grants from the Canadia Na- Rayner RW. 1970. A mycological colour chart. Kew: Com- tional Science and Engineering Research Council. monwealth Mycological Institute. Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analyzed by large subunit rDNA sequences. LITERATURE CITED Mycol Res 98:625–634. , . 1995. Molecular systematics of the Hypo- Booth C. 1959. Studies of Pyrenomycetes: IV. Nectria (Part creales: a teleomorph gene phylogeny and the status I). Mycol Pap 73:1–115. of their anamorphs. Can J Bot 73 (Suppl 1):S816–S823. . 1966. The genus Cylindrocarpon. Mycol Pap 104:1– Richter H, Gerlach W. 1958. U¨ ber die Typus-Art der Gat- 56. tung Cylindrocarpon Wr. und ihre Nectria-hauptfrucht- . 1971. The Genus Fusarium. Kew, United Kingdom: form. Tidschr Plziekt 64:392–395. Commonwealth Mycological Institute. Roll-Hansen F. 1962. Nectria cucurbitula sensu Wollenweber, . 1979. Nectria fuckeliana. CMI Descr Path Fungi Bact its Cephalosporium state, and some other Cephalospori- 624. um sp. from stems of conifers. Meddelelser fra Det Brayford D. 1991. Nectria canker. In: Ellis MA, Converse RH, Skogsforsøksvesen, No. 61, 71:293–312. Williams RN, Williamson B, eds. Compendium of rasp- , Roll-Hansen H. 1979. Microflora of sound-looking berry and blackberry diseases and insects. St. Paul, wood in Picea abies stems. Eur J Forest Pathol 9:308– Minnesota: Amer. Phytopath. Soc. Press. 20 p. 316. , Samuels GJ. 1993. Some didymosporous species of , . 1980. Microorganisms which invade Picea Nectria with non-microconidial Cylindrocarpon ana- abies in seasonal stem wounds. II. Ascomycetes, fungi morphs. Mycologia 85:612–637. imperfecti and bacteria. General discussion, Hymeno- Cabot E. 1990. ESEE (The eyeball sequence editor). New mycetes included. Eur J Forest Pathol 7:396–410. York: Department of Biology, University of Rochestes- Rossman AY. 1983. The phragmosporous species of Nectria ter. and related genera. Mycol Pap 150:1–164. Corpet F. 1988. Multiple sequence alignment with hierar- . 1989. A synopsis of the Nectria cinnabarina group. chiacal clustering. Nucleic Acids Res 16:10 881–10 890. Mem New York Bot Gard 49:253–265. BRAYFORD ET AL: NEONECTRIA AND CYLINDROCARPON 597

, McKemy JM, Pardo-Schultheiss RA, Schroers H-J. red perithecia and striate ascospores. Sydowia 46:75– 2001. Molecular studies of the Bionectriaceae using 161. large subunit rDNA sequences. Mycologia 93:100–110. , Doi Y, Rogerson CT. 1990. Hypocreales. Mem New , Samuels GJ, Rogerson CT, Lowen R. 1999. Genera York Bot Gard 59:1–108. of Bionectriaceae, Hypocreaceae and Nectriaceae (Hy- Schroers H-J. 2002. A monograph of Bionectria (Ascomy- pocreales, Ascomycetes). Stud Mycol 42:1–248. cota, Hypocreales, Bionectriaceae) and its Clonostachys Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular clon- anamorphs. Stud Mycol 46:1–214. ing: a laboratory manual. 2nd ed. Cold Spring Harbor Schultz ME, Parmeter JR. 1990. A canker disease of Abies concolor caused by Nectria fuckeliana. Plant Dis 74:178–180. New York: Cold Spring Harbor Laboratory Press. Swofford DL. 2000. PAUP*: Phylogenetic Analysis Using Samuels GJ. 1976. A revision of the fungi formerly classiﬁed Parsimony (*and other methods). Version 4.0 Beta. in Nectria subg. Hyphonectria. Mem NY Bot Gard 26(3): Sunderland, Massachusetts: Sinauer Associates. 1–126. Weese J. 1912. Studien u¨ber Nectriaceen. (I. Mittilung.). . 1989. Nectria and Penicillifer. Mycologia 81:347–355. Zeitschr Ga¨rungsphysiol 1:126–155. , Brayford D. 1990. Variation in Nectria radicicola and Wollenweber HW. 1926. Fusaria autographice delineata. Ed. its anamorph, Cylindrocarpon destructans. Mycol Res Sec. Berlin. Published by the author. 94:433–442. . 1930a (1924). Fusaria autographice delineata. Ta- , . 1993. Phragmosporous Nectria species with bulis 510–569. Published by the author, Berlin. Cylindrocarpon anamorphs. Sydowia 45:55–80. . 1930b. Fusaria autographice delineata. Tabulis 610– , . 1994. Species of Nectria (sensu lato) with 1110. Published by the author, Berlin.