Phylogeny and Taxonomy of the Ophiostoma piceae Complex and the Dutch Elm Disease Fungi

Doug McNew

Bark beetles (Coleoptera: Scolytidae) infesting Norway spruce trees are known to be associated with fungi, especially species of Ophiostoma sensu lato and Ceratocystis. However, very little is known about these fungi in Poland. In this study, we examined the ophiostomatoid species associated with seven species of bark beetles developing in Norway spruce (Picea abies). Fungi were isolated from the beetles and their galleries at ten sites in southern Poland. We identified a total of 2,769 fungal isolates that belong to 19 ophiostomatoid species. These 19 species included a total of 16 associations between fungi and bark beetles that had not been previously recorded. The isolated fungal species were similar to those previously reported. The species spectrum and relative abundance of fungal associates were similar in seven bark beetle species, despite some differences between species. Except for Pityophthorus pityographus, all bark beetles species were frequently associated with ophiostomatoid fungi. The most commonly encountered fungal associates of bark beetles were the following: Ophiostoma ainoae, O. bicolor, O. piceae sensu lato and Grosmannia piceiperda. The results support the hypothesis that pathogenic ophiostomatoid fungi are important for aggressive bark beetles to kill trees. The most virulent fungal associate of bark beetles, Ceratocystis polonica was most commonly associated with Ips typographus. The most frequent ophiostomatoid species isolated from the beetles and galleries of P. pityographus were O. ainoae and Graphium fimbriisporum.

Mycologia, 93(1), 2001, pp. 111-136. ? 2001 by The Mycological Society of America, Lawrence, KS 66044-8897 Phylogeny and taxonomy of the Ophiostoma piceae complex and the Dutch elm disease fungi well 1994), and convergence on similar morphologies has greatly confused generic and species delimitations of both teleomorphs and anamorphs (Spatafora and Blackwell 1994). One of the largest groups of insect dispersed pyrenomycetes is the genus Ophiostoma,which is now recognized as distinct from Ceratocystisbased on anamorphs and biology (Hoog and Scheffer 1984, Harrington 1981, 1987). Considerable confusion remains with generic limits for anamorphic fungi dispersed by insects, many of which form droplets of conidia at the apex of compact conidiomata called synnemata (Okada et al 1998). Some synnema-forming species have affinities to OphiostomaH. & P. Sydow, especially to the common sapwood-colonizing species 0. piceae (Munch) H. & P. Sydow and the Dutch elm disease fungus O. ulmi (Buisman) Nannf. (Okada et al 1998). Synnematous anamorphs of Ophiostoma species have been placed in the genus Graphium Corda, though it is now thought that Graphium species are anamorphs of the Microascales (Okada et al 1998), and the genus name Pesotum Crane & Schoknecht is available for the anamorphs of Ophiostoma species. Pesotum was based on the anamorph of 0. ulmi and was characterized by the synanematous anamorph as well as a SporothrixHektoen & Perkins ex Nicot & Mariat synanamorph (Crane and Schoknecht 1973). Sporothrix conidiophores are micronematous, with conidiogenous cells that have prominent denticles at the point of conidium detachment (Hoog 1974, 1993). Secondary conidia are frequently formed from the primary Sporothrixconidia, and the name Hyalodendron Diddens has been used for such anamorphs. However, the name Hyalodendron should be reserved for basidiomycetous anamorphs, and there appears to be little need for distinguishing Sporothrix species with secondary conidium production (Hoog 1993). Species of Hyalorhinocladiella Upadhyay & Kendrick form micronematous conidiophores similar to those of Sporothrixbut without prominent denticles (Hoog 1993), and the conidiogenous cells of Hyalorhinocladiella resemble those of Pesotum species. Synnema forming anamorphs with phialidic conidium production were placed in Phialographium Upadhyay & Kendrick, but Hoog and Scheffer (1984) and Wingfield et al (1991) do not distinguish Phialographium from Graphium (Pesotum sensu Okada et Thomas C. Harrington' Doug McNew Joseph Steimel Departmentof Plant Pathology,Iowa StateUniversity, AmesIowa, USA50010 Deborah Hofstra2 Roberta Farrell Departmentof Biology,WaikatoUniversity,Hamilton, New Zealand Abstract: The Ophiostoma piceae complex forms a monophyletic group of insect-dispersed pyrenomycetes with synnemata (Pesotum) and micronematous (Sporothrix) synanamorphs. Other species of Ophiostoma outside of the 0. piceae complex that form synnemata lack the Sporothrixstate. The nine recognized species within the 0. piceae complex are delimited by synnema morphology, growth rate at 32 C, mating reactions and sequences of the internal transcribed spacer (ITS) region of the rDNA operon. Phylogenetic analysis of the ITS region suggests two major clades in the complex, one that causes bluestain in primarily coniferous hosts and the other on primarily hardwood hosts. In the coniferous group are 0. piceae, 0. canum, 0. floccosum and the recently described 0. setosum (anamorph Pesotum cupulatum sp. nov.). In the hardwood group are 0. querci, 0. catonianum, and the Dutch elm disease fungi: 0. ulmi, 0. novo-ulmi and 0. himal-ulmi. Restriction fragment length polymorphisms of the ITS region are shown to be a convenient diagnostic tool for delimiting these species. Key Words: Bluestain, Dutch elm disease, Graphium, Leptographium,Pesotum, Phialographium, Sporothrix INTRODUCTION Adaptations for insect dispersal (ascomata and conidiomata with sticky spore drops) have arisen frequently in the evolution of the ascomycetes (BlackAccepted for publication September 29, 2000. 1 Email: tcharrin@iastate.edu 2 Current address: National Institute of Water and Atmospheric Research, PO Box 11-115, Hillcrest, Hamilton New Zealand. 111 112 MYCOLOGIA al 1998). Pesotum-type conidiophores with a single row of cells for the stipe (mononematous conidioand a of series metulae at the phores) stipe apex are in & Melin placed Leptographium Lagerb. (Harrington 1988). Okada et al (1998) used Pesotum for all synnema forming species with affinities to Ophiostoma, even those species with no Sporothrix synanamorph. This paper focuses on those Ophiostoma species with Pesotum anamorphs sensu Crane and Schoknecht (1973), i.e., those that also have Sporothrix synanamorphs, such as 0. piceae. Ophiostoma piceae was originally described by Munch (1907) from sapstain in pine and spruce but is now known as a complex of closely related Ophiostoma species with pigmented, synnematous anamorphs. Included in the complex are 0. querci (Brasier and Kirk 1993) and the Dutch elm disease fungi, 0. ulmi, 0. novo-ulmi and 0. himal-ulmi (Brasier and Mehrotra 1995). We found four other species closely tied to the 0. piceae complex. We used DNA sequences of the internal transcribed spacer (ITS) region of the nuclear rDNA and mating studies to identify lineages and potential intersterility and physiological comgroups, and morphological were used to delimit parisons species in the complex. MATERIALSAND METHODS Isolates.-The isolates most intensively studied and considered as part of the 0. piceae complex are listed in TABLEI. Isolates are maintained in the collection at Iowa State University. For morphological comparisons, isolates were grown at room temperature (21-24 C) and lighting for 10-14 d on malt extract agar (MEA, 1.5% Difco malt extract and 2.0% agar) or pine twig medium (MEA with a section of debarked Pinus strobus twig added to the molten agar medium, Harrington 1992). Isolates grown for DNA extraction were cultured on MYE broth (2% malt extract, 1% yeast extract). Temperaturegrowth assay.-Isolates were grown at 32 C on MEA because growth at this temperature was shown to distinguish 0. piceae from 0. querci (Brasier and Stephens 1993). Plates were inoculated using a plug of agar and mycelium (#1 cork borer, from the advancing margin of a colony) placed upside down in the middle of the plate. The plates were incubated upside down in the dark. After 7 d, the plates were removed from the incubator and two radial measurements taken for each plate. The assaywas repeated twice at separate times, and the mean extent of radial growth determined from the three separate experiments. Pairings.-Tests for sexual compatibility were conducted on MEA with pine twigs. Two methods of pairing were used. In most cases, a single isolate was grown on the agar medium for 1 wk and then spermatized with a conidial/mycelium slurry (Brasier and Kirk 1993, Harrington and McNew 1997). In other cases, two isolates were co-inoculat- ed with colonized agar plugs placed ca 5 mm apart. Single ascospore strains were obtained from an ascospore mass at the tip of a perithecium by suspending the mass in a light oil and streaking onto MEA plates (Harrington and McNew 1997). PCR, DNA sequencing, and RFLP analysis.-The ITS1-F (5'-CTTGGTCATTTAGAGGAAGTAA-3') primers and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (Gardes and Bruns 1993, White et al 1990) were used to amplify the ITS region. Either extracted DNA (DeScenzo and Harrington 1994) at 10-100 ng per reaction or scraped mycelium with spores (Harrington and Wingfield 1995) were used as template for the polymerase chain reaction (PCR). The reaction mixture (100 gFL final volume) contained 2.5 units Taq DNA polymerase (Promega Inc., Madison, Wisconsin), 1X PCR re- action buffer, 1.5 mM MgC12,200 pLMDNTPs, 5% (V/V) DMSO, and 0.25 or 0.50 FM of each primer. Reaction mixtures were placed into a precooled (4 C) thermocycler (MJ Research, Inc., Watertown, Massachusetts). Cycling conditions were an initial denaturation at 95 C for 95 s, followed by 35 cycles of 52 C for 40 s (annealing), 72 C for 2 min (extension), and 95 C for 35 s (denaturation). There was a final extension step of 10 min at 72 C. Amplicons were either sequenced using the primers ITS1-F and ITS4 or were digested with restriction enzymes following the manufacturer's recommendations. Sequencing was performed on a ABI PRISM 377 Genetic Analyzer (Perkin-Elmer Inc., USA) at the DNA Synthesis and Sequencing Facility (Iowa State University, Ames, Iowa) after purification using QIAquick PCR purification Kits (Qiagen Inc., USA) or Microcon-100 Microconcentrators (Amicon, Inc., USA). The ITS sequences of 58 isolates of taxa in the 0. piceae complex and an outgroup taxon (isolate C327 of Ophiostoma ips from New York, USA) were aligned manually and analyzed using PAUP 4.0 (Swofford 1998). A total of 616 unordered characters, including gaps, were utilized, with gaps considered a "fifth base" and all characters with equal weight. Sixty-four of the characters were parsimony-informative, and 491 characters were constant. Heuristic searches used stepwise (simple) addition and tree-bisection-reconnection. Bootstrap support (Felsenstein 1985) was determined from 1000 replications. Polymorphisms in restriction sites of the ITS region were identified among species in the 0. piceaecomplex using the online tool Webcutter 2.0 (http://www.ccsi.com/firstmarket/ cutter/cut2.html;Max Heiman, copyright1997). The restriction enzyme HaeII (Gibco BRL, Inc., USA) produced the greatest number of polymorphisms among species; however,some species showed similar RFLP patterns with this enzyme. The restriction enzyme DdeI (Gibco BRL, Inc., USA) was used to differentiate some taxa. For either enzyme, the unpurified PCR product (17.5 JiL) was combined with 2.0 FLLof 10X buffer (supplied with the enzyme) and 0.5 pL of restriction enzyme (5 units). Digestion was allowed to proceed for 1-16 h at the appropriate incubation temperature for the enzyme employed. Restriction fragments and a 100-bp ladder (Gibco BRL, Inc., USA) were separated by electrophoresis for 3 h at 75 volts in 2% agarose gels (Bio-Rad, Inc., USA) with a TBE PICEAE HARRINGTONET AL: OPHIOSTOMA [89 mMTris,89 mMboricacid,2 mMEDTA(pH 8.0)] buffer system.Gelswere stainedwith ethidiumbromideand illuminatedwithultravioletlight.Molecularweightsof the DNAfragments were determinedusing digitalimagesand GelReader Uni2.0.5 (NationalCenterfor Supercomputing Applications, versityof Illinois,Urbana-Champaign). RESULTS A wide range of synnema morphologies was seen among the isolates studied. All isolates forming both synnema and Sporothrixstates were tolerant of cycloheximide (Harrington 1981) and had similar ITS sequences. The ITS sequence analysis grouped these isolates with bona fide isolates of 0. piceae and 0. querci (FIG.1). Nine species in the 0. piceae complex are recognized based on the ITS sequences (FIG.1), mating reactions (TABLEI), cultural characteristics, and morphology (FIGS.2-36, TABLEII). Other synnema-forming species, including Ophiostomaspecies excluded from the 0. piceae complex (TABLEIII) and isolates of G. penicillioides, the type species for Graphium, did not form a Sporothrixsynanamorph (FIGS. 37-48). Their ITS sequences, where available, aligned poorly with sequences of 0. piceae and 0. querci. Culture characteristics.-The distinguishing features of the species recognized in the 0. piceae complex are listed in TABLEII. Morphological features are determined on MEA or MEA with pine twig medium, and a relatively weak medium is needed to discern these characteristics. Aroma is useful for distinguishing 0. querci and 0. setosum from the other species, and this feature is also best determined on MEA. Growth at 32 C is a distinguishing feature for species in the complex (TABLEI), but accurate temperature control is needed. Protoperithecia, which are distinctly light-brown in 0. querci, are most reliably produced 1 wk after a conidial mass from a synnema is streaked over MEA or MEA with pine twig medium. The concentric rings of aerial mycelium that are diagnostic for the Dutch elm disease fungi (Brasier and Mehrotra 1995), 0. querci, and 0. setosum are also best seen on MEA. Phylogenetic analyses.-Most of the sequences of the ITS region of other Ophiostomaspecies [e.g., O. pluriannulatum (Hedgcock) H. & P. Sydow, 0. stenoceras (Robak) Melin & Nannf., and 0. europhioides(Wright & Cain) Solheim] could not be unambiguously aligned with the ITS sequences of the 0. piceae complex, so only members of the 0. piceae complex were included in analyses. Of the species outside of the 0. piceae complex, the ITS sequence of 0. ips (Rum- 113 bold) Nannf. (AF198244) was reasonably similar, and O. ips was used as an outgroup taxon. Two major clades in the 0. piceae complex were inferred from parsimony analysis of the ITS sequences (FIG. 1). Ophiostomapiceae s.s., 0. canum, 0. floccosumand 0. setosumformed a moderately supported group of species that are primarily found on conifer (Pinaceae) wood. Ophiostoma querci, 0. catonianum, 0. himal-ulmi, 0. ulmi and 0. novo-ulmi were found in a strongly supported branch of primarily hardwood (angiosperm) inhabiting fungi. The inferred hardwood-inhabiting clade was also seen in neighborjoining analysis (Swofford 1998), as was the branch containing 0. himal-ulmi and 0. querci, but the conifer-inhabiting species did not resolve as a single clade. In most cases, the well supported branches in the phylogenetic analysis (FIG. 1) were of isolates with unique cultural or morphological characteristics (TABLEII). The ITS sequences of 0. piceae and 0. canum were identical, though all of the examined 0. piceae isolates had cylindrical to obovoid conidia, and the two examined isolates of 0. canum had globose conidia at the tip of synnemata (FIG. 13). The synnema stipes of 0. piceae isolates were consistently black, while some synnemata of 0. canum had brown stipes. Ophiostomafloccosum formed red-brown synnemata, frequently with lateral knobs (FIG. 16). Another branch in the ITS tree contained isolates of the recently describe 0. setosum (Uzunovic et al 2000), which is characterized by darkly pigmented synnemata with the vertical cells of the stipe extending into the conidial mass, forming a cup-shaped structure (FIGS.21-23). As expected, the Dutch elm disease fungi 0. ulmi and 0. novo-ulmi were grouped as closely-related sister species (FIG. 1). The 0. himal-ulmi ITS sequence was closer to that of 0. quercithan to that of 0. ulmi. The 0. querciclade included the only available isolate of 0. catonianum and anomalous isolates from New Zealand and Viet Nam. Ophiostomafagi had an ITS sequence identical to 0. querci and is considered a synonym. PCR-RFLP diagnostics.-Based on the ITS sequences used in FIG. 1, we identified restriction enzymes that would differentiate among the species in the 0. piceae complex, except that no attempt was made to differentiate among the three Dutch elm disease species. Most species could be differentiated based on HaeII digestions (TABLE IV, FIG. 49). The HaeII restriction pattern for 0. ulmi, 0. novo-ulmi and 0. himal-ulmi is the same, but it is distinct from that of 0. querci and the other species (TABLE IV, FIG. 50). Ophiostomapiceae and 0. setosum have the same re- TABLE I. Isolate numbers, accession numbers for ITS sequences, substrate, origin, mating type and growth at 32 C of isolates of the O Species Ophiostomapiceae Ophiostoma canum Ophiostomafloccosum Ophiostomasetosum Isolate3 C82 C967 C968 C987 C992 C993 C1087 C1103 C1201 C1246 C1209 C114 C1088 C424 C425 C988 C989 C1010 C1011 C1012 C1013 C1086 C1160 C1198 C933 C985 C986 C1037 C1194 C1200 GenBank AF198226 AF198217 AF198229 AF198228 AF198231 AF198230 Other no./collector or supplier A21 H2181/Webber H2009/Gibbs 3NZ-S519 3NZ-S501-5 4NZ-125 CBS 108.21/Munch (From the type) CBS 600.85 (as 0. perfectum) CBS 102355 CBS 102356/Worrall 19NZ-1384 NFRI 60-165, NCC-60-16/Kaiarik CBS 133.51/KAirik CBS 102360, RS1 RS2 CBS 102361, 103 CBS 102362, 2NZ-35 Gr4+/Webber Grl 3 +/Webber Gr36-/Webber GrlO-/Webber CBS 799.73/Kaarik (Authentic for type) CBS 102363 SSI202/Blanchette 3NZ-S509 CBS 102357, 2NZ-29 8NZ-211 CBS 102358 CBS 102359 Substrate Pseudotsuga Picea Pinus Pinus Pinus Pinus Cat hair Tsuga Pseudotsuga Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pseudotsuga Tsuga Pinus Pinus Pinus Pinus Pseudotsuga Tsuga Country Idaho, U United K United K New Zea New Zea New Zea German German Washing New Yor New Zea Sweden Sweden Californ Californ New Zea New Zea United K United K United K United K Sweden Washing Washing New Zea New Zea New Zea New Zea Washing Washing TABLE I. Continued Species Ophiostoma querci Isolatea GenBank C426 C799 C934 C935 C936 C969 AF198238 C970 AF198239 C984 C1085 AF198237 C1159 C1283 Ophiostoma catonianum Pesotum sp., near 0. querci Pesotum sp., near 0. querci Pesotum sp., near O. querci Ophiostoma ulmi Ophiostoma novo-ulmi Ophiostoma himal-ulmi C1084 C1203 C1214 C1215 C55 C1089 C1182 AF198243 AF198242 AF198241 AF198240 AF198232 C1186 C48 C510 C913 C943 C1184 C1185 C1187 C1183 AF198233 C1306 AF198234 Other no./collector or supplier TAB103/Wingfield TAB191/Blanchette 103/Blanchette 108/Blanchette 109/Blanchette CBS 102352, H1042/Scard and Webber CBS 102353, H1039/Scard and Webber 2NZ-26 CBS 236.32/Loos (as Ophiostoma fagi, from the type) CBS 102354 CBS 277.54, ATCC 24586, JCM 9745/Kiarik (as Graphium aureum) CBS 263.35/Goidanich (from the type) 18NZ-1281 144-E/Blanchette NZum8/Blanchette UM C-20/Blanchette CBS 137.36 (as 0. ips) CBS 102.63, IMI 101223,JCM 9303/Holmes and Heybroek CBS 296.87, WCS 811/Elgersma UM C56, Aggressive W-Z/Blanchette AF198236 AF198235 Jacobs Jacobs CBS 377.79/Meulemans CBS 298.87, WCS 637/Heybroek CBS 297.87, WCS 802/Scheffer CBS 374.67, ATCC 36176, ATCC 36204/Heybroek HP27/Brasier Substrate Country Abies or Pseudotsuga Pinus Pinus Pinus Quercus South Af Washingt New Zea New Zea New Zea United K Quercus United K Pinus Fagus New Zea Germany Pseudotsuga Pinus Washingt Sweden Pyrus Ulmus Italy New Zea Viet Nam New Zea Minnesot Ulmus Netherlan Ulmus Ulmus Netherlan United K Iowa, US Illinois, U Illinois, U Belgium Russia Netherlan India Ulmus India Pinus Aquilaria crassna Nothofagus Ulmus Ulmus Ulmus Ulmus Ulmus Ulmus Ulmus Isolate numbers from the collection of the senior author. Designations of mating type as either A/B or ? were based on compatibilities with tester strains provided by C. Brasier, A. Uzuno nomenclature. Isolates producing perithecia and ascospores without pairings ("mixed") are mixtures (heterokaryons or mixtures of my c Extent of radial growth on MEA after 7 d at 32 C. a b 116 MYCOLOGIA 114- Sweden 62- i1088 - Sweden canum m -1 1087 - Germany* 967- Britain e 1103 - Germany 77 968- Britain 82 - Idaho,USA 1201 - Washington, USA 0 . Pceae - 1246 - New York, USA 987 - New Zealand 992 - New Zealand 56 993 - New Zealand - 1209 - New Zealand -1012- Britain 1086 - Sweden* 1010 - Britain 1011 - Britain 1013 - Britain 100 5 bp r424 75 - California,USA 425 - California, USA - Washington, USA _ E-~-~1160 1198- Washington, USA 988 - New Zealand 989 - New Zealand 1194 - Washington, USA 50 1200 - Washington, USA ~-- 96 96 | - 986 - New Zealand 1037 - New Zealand 933- New Zealand I- O. floccusum 0. setosum - 985 - New Zealand 55- Minnesota, USA 99 1089 1182- Netherlands 0. ulmi . m 1186 - Netherlands - 48 - Britain 1184 - Belgium 93 1185-Russia 96 1187- Netherlands 0. novo-ulmi Q~99 913- Illinois, USA 943- Illinois, USA 510 - Iowa, USA ~ -- 76 95 1183-India 1306 -India I . himal-ulmi -1085 - Germany, 0. fagi* 1283- Sweden 969 - Britain 984- Britain 970- Britain 68 799- Washington, USA 0. querci 1159 - Washington, USA - 426 - South Africa 936 - New Zealand 72 - 934 - New Zealand 935 - New Zealand 84 1215 - New Zealand - 1203 - New Zealand Pesotum spp. -1214 - Vietnam 1084- Italy*- 0. catonianum 327 - New York, USA - 0. ips FIG. 1. One of four most parsimonious trees of the Ophiostomapiceae complex based on 616 characters, including gaps, of the ITS-1, 5.8S, and ITS-2 regions of the rDNA operon. The tree is rooted to Ophiostomaips. Branches with strong support (> 75%) are bolder, with bootstrap values - 50% indicated above the branches. Total tree length - 181 steps, consistency index = 0.8122, retention index = 0.9663. Asterisk indicates a culture from the holotype. 1177 HARRINGTON ET AL: OPHIIOSTOMA PICEAE C14 C striction pattern with HaeII, but these two species can be differentiated based on DdeI restriction fragments (TABLEIV, FIG.49). in xn - ONvix i)C)r-4r.. Mating reactions.-Tester isolates of each mating type of 0. piceae, 0. querci and an unidentified species found here to be 0. fioccosum were provided by J. Webber, and A. Uzunovic provided tester strains of 0. setosum. Isolates of the Dutch elm disease fungi were not tested, but most other isolates of the 0. piceae complex were identified as either of the two mating types based on pairings with these and other selected tester strains (TABLEI). Most species formed perithecia and ascospores within two weeks of pairing, but 0. floccosum isolates, when paired with isolates of opposite mating type, formed perithecia only after 4-6 wk, if at all. Isolates of 0. piceae generally formed perithecia and ascospores when paired with isolates of opposite mating type. The two isolates of 0. canum tested did not produce perithecia when paired with each other but produced a few perithecia with aborted asci and misshapen ascospores with mating type A strains of 0. piceae (FIG. 14). No protoperithecia or perithecia were seen in our isolates of 0. setosum (TABLE I) when paired among themselves or with other isolates of the 0. piceae complex. Howevei; these isolates were interfertile with a mating type A strain of 0. setosum when incubated at 20 C for 4-8 wk (Uzunovic et al 2000). Ophiostoma querci isolates readily produced perithecia and ascospores when isolates of opposite mating type were paired. The isolate of 0. fagi from the holotype also mated with 0. quercitesters. An isolate deposited in CBS as G. aureum (TABLE I) also paired with isolates of 0. querci;however, we do not think this isolate represents G. aureum. An isolate from the holotype of 0. catonianum, which is reportedly homothallic (Goidanich 1935), did not produce perithecia on its own and failed to mate with any other isolates. cc v 64 0) Z0Z 00 un 0 ct 0 0)0 z00 00) 0) cn v 05(rn0 z sz 0.) vr. EJc 0 Ogcnr o C 0 4 C -~~tU~~ Z 0) 0-) u n v 0 04 00) 0. - 4 ;.o V 6. 0 r. +,~0 .0) . bC) 10Gr. 0C iV ;4 b OuO' 11 CI 11 C) 0 0 4 0)u S C). o ES.. z l u 01 lz 0) S * T * 000 0 00 4 4..) 0 ) 0 ) 0 ) 0 ) 0 ) 0 )m y, k ~t- .h k "C .2 "C -. .~ .x "C .1 -t 3 0 0 0 00u 0 0 u 0o C/l * 0000000 0) (: U Td 0000000o 00 0 c, 0 .73 . * .3 00 0 0) 0 0) S4 Cc: cnII I 1I 0, 00 0c Ec 0 0) * .1 ., - E . 1.4 . 0 0q 4 .5 x01< u0) 000 L. 0) I1 4 I In000 zzxz zzz 00 I I I- 05 .5 0a i r0 C-)) 0) 0 00 ct 0) 0) L4 .5 4 j 0.. ~ 0 0) 0 0.. 0) 0) S E (1) TAXONOMY ;..q CZ '0 1f 0) 50 ?.. 1. 4 0 W E 0 50 04Ii 00 k ko 64 6. , -C3 64 cq -Ck 9~f :0 I .5( 4 4 U Irr $.. .5 S 0 -4 3 I I u C0 0, D ' L Species of the Ophiostoma piceae complex.-Based on ITS sequence analysis, mating reactions, tests for growth at 32 C, and morphology, the following species are recognized in the complex. 0 0: 5 Ophiostomapiceae (Mulnch) H. & P. Sydow, Ann. MyFIGS.2-12 col. 17: 43. 1919. Forstw.5: Land. Naturw. Ceratostomella piceaeMiinch, 547. 1907. piceae (Miinch) Bakshi,Trans.Br. Mycol. Geratocystis Soc. 33: 113. 1950. - 4 cr 0t E C'sII u H ? ,~E 0) 11 .~ 0 oE - 118 MYCOLOGIA 6 IA 3 r. - '... r 10 . -re e ,i, , o,I, 1 11 12 ,b If ' '% ^^:-^ FIGS.2-12. Ophiostomapiceae. 2, 3. Protoperithecia. 4. Perithecium. 5. Ostiolar hyphae. 6. Ascospores. 7, 8. Synnemata. 9-12. Conidiophores and conidia of the Sporothrixsynanamorph. Arrows in 9 and 10 indicate apex and side walls of conidiogenous cells, showing prominent denticles. 2, 3 from strain C967; 4-6 from a pairing of 967 and C1087; 7-12 from strain C1087, from the HOLOTYPE. Scale bars: 2, 3 = 25 pim; 4, 8 = 100 ,um; 5, 6, 9-12 = 10 pm; 7 = 50 pum. Anamorph. Pesotum piceae Crane & Schoknecht, Am. J. Bot. 60: 348. 1973. Graphiumpiceae (Crane & Schoknecht) Wingfield & Kendrick, Mycol. Res. 95: 1331. 1991. Commentary. Lectotype (BPI 595978 = FP 14395) and other material (BPI 595979, BPI 595977) from Picea abies sapwood in Germany, collected by Munch, was deposited in BPI, apparently by Hedgcock. Although there is some mold growth on these specimens, the lectotype has, in addition to synnemata and perithecia, the Sporothrix synanamorph (with prominent denticles) typical of the 0. piceae com= plex. The culture from the type (CBS 108.21 C1087) forms synnemata on wood and is of the B mating type (TABLEI). This species is most commonly found on members of the Pinaceae throughout Europe, North America, and Japan (Brasier and Kirk 1993, Halmschlager et al 1994, Kim et al 1999, Morelet 1992, Pipe et al 1995). We have also identified isolates of 0. piceae on the exotic Pinus radiata from New Zealand (TABLEI) and Chile. Both the teleomorph and anamorphs of this spe- HARRINGTONET AL: OPHIOSTOMA PICEAE cies lack distinctive features, which has led to the proposal of numerous synonyms, all of which we have excluded from 0. piceae. Synnemata stipe lengths of 0. piceae and 0. quercioverlap, but those of 0. piceae are larger (Hamschlager et al 1994). The size of the ascospores (2.5-4.5 X 1.5-2 FLm)and range in size of synnematous conidia (5-15 X 1-3.5 jxm) (Upadhyay 1981) of 0. piceae overlap with the sizes of most other species in the complex. However, ITS sequences (FIG. 1, Kim et al 1999), mating reactions (TABLE I, Brasier and Kirk 1993), dark protoperithecia, the inability to grow at 32 C (TABLEII, Brasier and Stephens 1993), and absence of a distinctive aroma or concentric rings on MEA (TABLEII) distinguish isolates of 0. piceae from the more common and widespread 0. querci.The synnemata of 0. piceae are also similar to those of 0. setosum, but the cup-like, conidial-bearing structure at the top of the synnemata of 0. setosum distinguishes this species from the rest of the 0. piceae complex. Red-brown synnemata distinguish 0. floccosum from 0. piceae and other members of this complex. Although 0. canum has the same ITS sequence as 0. piceae, the former is distinguished by its globose conidia and brown synnematous stipe. Ophiostoma canum (Munch) H. & P. Sydow, Ann. Mycol. 17: 43. 1919. FIG. 13 = Ceratostomella cana Mfinch, Naturw.Z. Forst.Landw. 5: 558. 1907. = cana (Munch)Moreau,Rev.Mycol.(Paris) Ceratocystis Col. 17:22. 1952. Suppl. Anamorph.Pesotum canum (Upadhyay & Kendrick) Okada& Seifert,Can.J. Bot. 76: 1503. 1998. PachnodiumcanumUpadhyay& Kendrick,Mycologia, 67: 802. 1975. Commentary. The lectotype of 0. canum (BPI595767 = BPI 2107) and two other specimens (BPI 595765 = BPI 2110 and BPI 595766 = FP 14394) from Munch were deposited in BPI by Hedgcock. The lectotype has perithecia and ascospores, while the other two specimens have synnemata and a Sporothrixanamorph but no perithecium. In addition to the isolates listed in TABLEI, we have examined Norwegian isolates NFRI 1652/2 (C1478) and NFRI 97-33/47 (C1477), both from Tomicus minor Hartig or stained Pinus sylvestrissapwood around galleries of this beetle, and globose conidia are also formed by these isolates. Mathiesen (1950, Mathiesen-Kaarik 1960) noted the association of 0. canum with T minor, and this beetle association distinguishes 0. canum ecologically from 0. piceae. MathiesenKaarik (1960) also noted physiological differences between 0. canum and 0. piceae. To date, 0. canum 119 is known only from pine in Europe, from Scandinavia and Germany. Mathiesen (1950) and Upadhyay (1981) noted ascospores 5-6 X 1.5-2.5 ILmand 4-6.5 X 1.5-3 jLm, respectively, in 0. canum, somewhat larger than those in 0. piceae. The globose conidia of 0. canum are particularly distinctive (FIG. 13) and separate it from all other members of the complex. Although we found that the ITS sequence of 0. canum is the same as that of 0. piceae, Kim et al (1999) reported that PCR primers specific for the ITS region of 0. piceae were unable to amplify this fragment in 0. canum. Hausner et al (1993) found that 259 of 261 aligned bases of the 26S rDNA gene were identical between a culture from the holotype of 0. piceae and a Norwegian isolate of 0. canum. We have not been able to produce perithecia or ascospores with the two isolates (TABLEI) of 0. canum that we have studied closely, perhaps because these two isolates are of the same mating type. However, we were able to produce a few perithecia when either of these two isolates were paired with mating type A testers of 0. piceae. Ascospores produced from these hybrid crosses were generally inviable, but viable ascospore progeny were recovered from one perithecium of the cross between isolate C967 of O. piceae and isolate C114 of 0. canum. Ten viable progeny were recovered, with globose conidia of 0. canum and the elliptical conidia of 0. piceae seen among the progeny. The fact that the 0. canum isolates are only partially interfertile with 0. piceae strains suggests that there is an intersterility barrier between these two species and further supports recognition of 0. canum as distinct (Brasier 1993, Harrington and McNew 1998). Ophiostomafloccosum Mathiesen, Svenka Bot. Tidskr. 45: 219. 1951. FIGS. 15-20 - Ceratocystisfloccosa (Mathiesen)Hunt, Lloydia,19:36. 1956. Anamorph.Pesotum aureum (Hedgcock) McNew et Harrington,comb. nov. - GraphiumaureumHedgcock, Mo. Bot. Gard.Rept. 17: 94. 1906. Commentary. Although well described by Mathiesen (1951), this species has been rarely reported by others, probably because it was thought to be a morphological variant of 0. piceae. Also, perithecia and ascospores are not commonly seen in cultures of O. floccosum, and perithecia often take many weeks to form in mating tests. We have examined specimens and cultures of this species from Europe, North America, Australia, and New Zealand (TABLEI), and also isolates from Korea. All of these isolates were from Pinaceae. TABLE III. Ophiostomaspecies with Pesotum-like anamorphs but without a Sporothrixsynanamorph Anamorph Teleomorph 0. ainoae Solheim 0. araucariae (Butin) de Hoog & Scheffer Pesotum-like Pesotum-like 0. arborea(Olchow. & Reid) Yamaoka & Wingf. 0. brunneo-ciliatumMathiesen-Kaarik Pesotum-like 0. cainii (Olchow. & Reid) Harrington Pesotum-like 0. clavatum Mathiesen 0. clavigerum (Robinson-Jeffrey & Davidson) Harrington 0. columnaris (Olchow. & Reid) Seifert & Okada 0. cucullatum Solheim 0. davidsonii (Olchow. & Reid) Solheim Pesotum-like Teleomorph not known Pesotum-like Graphiocladiella clavigerum Upadhyay =Pesotum clavigerum Okada & Seifert Pesotum-like Pesotum-like Pesotum-like 0. galeiformis (Bakshi) Mathiesen-Kaarik Graphium fragrans Mathiesen-Kiarik =Pesotum fragrans Okada & Seifert Pesotum-like 0. introcitrina (Olchow. & Reid) Hausner, Reid & Klassen Hyalopesotum introcitrinum Upadhyay & Kendrick -Pesotum introcitrinum Okada & Seifert 0. ips (Rumbold) Nannf. Pesotunm-like 0. japonicum Yamaoka & MJ. Wingf. 0. obscura (Davidson) von Arx 0. olivaceum Mathiesen Pesotum-like Pesotum-like Pesotum-like 0. olivaceapinii (Davidson) Seifert & Okada Pesotum-like Re Solheim (1986), Okada Butin (1968), Hausner (1998) Okada et al (1998), Ol Upadhyay (1981) Mathiesen-IKaarik(1953 dhyay (1981) Okada et al (1998), Ol Upadhyay (1981) Hunt (1956), Mathiese Okada et al. (1998), R (1968), Upadhyay (1 Okada et al (1998), Ol Upadhyay (1981) Okada et al (1998), So Okada et al (1998), Ol Upadhyay (1981) Mathiesen-Kairik (1953 Bakshi (1951), Mathies (1998) Hausner et al (1993), ecki and Reid (1974) and Kendrick (1975) Okada et al (1998), Ru (1981) Yamaoka et al (1997) Davidson (1935), Hunt Hunt (1956), Mathiese Olchowecki and Reid Davidson (1971), Okad (1981) HARRINGTON ET AL: OPHIOSTOMAPICEAE c~ c 00 004 - Td OCh o 0 Q. Q o m0-q 00 00 00 o r-. -eQ 1 00 3 Td t ~L T4 0. 0d c T- O t Td . c3o 0355 ^ fflb C. (J- cO * z 60 S QA Q~ 00 .: 0 0d~ oTQ "0U Q) Qc 'T- 0. E :S00w ~."0' C Q~ e a 3 r .5 . L 0d ~e -~ 0 ed ill E Q~ II Q I Q 0 CZ (Z .i I Y.5 "0 E "8 $ 0 0 Q C4-Q, r. .r~ Q, Cf "0I Q 0 00 I llc 0 i r. C.,r E 0 Q, Q HL 0 0 0 0 0 0 0 00Q i I Hi I H 64 "0 Q "0 1u $Q *0 v 0 C/) P4 7:; v3 04 r. I~l1 (Z3 -;.- 0 0: u 0 r. L- 121 No holotype for 0. floccosum was designated by Mathiesen, but herbarium material is apparently available in Statens Skogsforskningsinstitut Experimentalfaltet, Sweden (Hunt 1956). A culture from Kaarik deposited in CBS (799.73) is considered authentic for the type but does not produce perithecia and ascospores, unless mated with - (minus) mating type strains. Tester strains of an intersterility group identified byJ. Webber proved to be 0. floccosum in morphology and ITS sequence. We deposited a dried specimen (BPI 746439) with perithecia and ascospores that resulted from pairing CBS 799.73 (from the holotype) and C1013 (a tester strain from Britain provided by Webber). Mathiesen (1951) noted the relatively short ostiolar hyphae of this species when compared to O. piceae. We see little difference between the two species (FIGS.5, 15), though ascospore masses at the top of perithecia are relatively small for 0. floccosum. Synnemata of 0. floccosum are distinctly red-brown, and lateral knobs on synnemata (FIG. 16) are common in all of the cultures of 0. floccosum that we have examined. Also, secondary synnemata are frequently found emanating from the conidial mass on top of primary synnemata. Conidial masses on synnemata are yellow compared to white in fresh spore masses of other species in the complex. Hedgcock (1906) described Graphium aureum from stained sapwood of Pinus strobusin Wisconsin. His description includes white to yellow conidial masses and stipes that are white at first, changing to yellow and often to dark brown at the base. The yellow conidial masses described by Hedgcock (1906) are typical for 0. floccosum, though our cultures generally produce red-brown stipes instead of yellow to brown stipes. Hedgcock's type material is available (BPI 448701 and BPI 448702), and both specimens contain red-brown synnemata and black protoperithecia, which are also common for 0. floccosum. A Sporothrix-likeanamorph was noted by Hedgcock and was observed in the BPI specimens. Thus, we believe that Hedgcock's Graphium species is the anamorph of O. floccosum, and we have transferred his fungus to Pesotum. A culture deposited by Kiarik (CBS 277.54 = ATCC 24586 = JCM 9745 = C1283) as G. aureum mated with 0. querci tester strains as discussed above and does not represent G. aureum. Ophiostoma setosum Uzunovic, Seifert, Kim & Breuil, Mycol. Res. 104:490. 2000. Anamorph.Pesotum cupulatumMcNew et Harrington FIGS.21-26 sp. nov. Coloniae olivaceae-brunneaede infra, in agaro extracti malti diam. 55 mm attingenspost 11 dies ad 25 C, odore 122 MYCOLOGIA ......~~~~~~~~~~~~~~~~~~~~~~~~~~~~~t ;......, ,. ...i :'.,...~, '-~..i: ~'" ~~~~~~~~~~ .c.i.. ~ $..: , '~ . ,.~.~t~ . .-~ -? ?I , -..~- ?'~ :l..~-.' . .*?1~~? ......":'-?,".. .-. L'? ~'-;~;,. ~ ~~~~~~~~~~~~~~~~~~~~~~~~.:i~ ~...,. ~~~~~~~~ ~::":''- *'.~".~ :' .~:?... . . I I. I FIGS. 13-20. f U. .t Ophiostoma canum and 0. floccosum. 13, 14. 0. canum. 13. Synnema apex with globose conidia. 14. Aborted asci and ascospores from a crossing of 0. canum and 0. piceae. 13 from the LECTOTYPE (BPI 595765); 14 from a cross of strain C114 and strain C967. 15-20. 0. floccosum. 15. Ostiolar hyphae. 16, 17. Synnemata. 18, 19. Conidiophores and conidia of Sporothrixsynanamorph. Arrows in 18 indicate prominent denticles on apex and side walls of conidiogenous cells. 20. Primary conidium from Sporothrixsynanamorph, with numerous denticles at apex where secondary conidia were attached. 15 from a cross of strain C1013 and C1086; 16-20 from strain 1086. Scale bars: 13-15 = 10 Rm; 16 = 100 gim; 17 = 50 ,xm; 18-20 = 10 tum. 123 HARRINGTON ET AL: OPHIOSTOMAPICEAE 22 -i ?. . :.-.".. .K?aK^^^^^^^ .^h; w :*'fl,';:' .:'.^ _^^ w ^ - \ | l, * ... j r s * .y; C;" r FIGS.21-26. Pesotum cupulatum, from the HOLOTYPE, anamorph of Ophiostoma setosum. 21-23. Synnemata. 24-25. Conidiophores and conidia of the Sporothrixsynanamorph. Arrows in 24 and 25 indicate apex of conidiogenous cells, showing prominent denticles. 26. Conidia from synnema. Scale bars: 21-23 = 50 JLm;24-26 = 10 ,im. dulci. Incremento nullo ad 32 C. Synanamorpha primaria stipite 80-820 Ixm longo, cum cellulis fuscae protrudens in zonam conidiogenam, calyx formans. Conidia unicellularia hyalina, cylindrica vel obovoidea, 3.0-5.5 ,Im X 1.0-2.0 jim. Synanamorpha altera, simili Sporotrichiin denticules conspicui ubi sporae portatae et quoque in catena acropeta conidia holoblastica efferens. Colonies on malt extract agar at 25 C attaining a diameter of 55 mm in 11 d, appearing olive-green to gray from above, olive-green to brown from below, with concentric rings developing on the upper surface of the mycelium with age, with a distinctly sweet aroma. Cultures not inhibited by cycloheximide. No growth at 32 C on malt extract agar. Hyphae mostly submerged, aerial hyphae appressed, hyaline, smooth-walled, 1.0-3.0 ,xm wide, to brown, smooth or rough-walled, 2.5-4.0 ixm wide. Conidiophores of two types: synnemata (FIGs. 21-23) single or in groups of two to three, simple or rarely branched, macronematous. Stipe dark brown to black at base, becoming lighter towards apex, 80-820 ,xm (mean 290 jLm) long, base 10-40 jxm (mean 23 ,xm) wide, tapering to 6-32 uim (mean 16 jim) near the apex and flaring to 14-120 ,xm (mean 45 ,xm). Many of the synnema stipe cells extend into the mass of conidiogenous cells and conidia, forming a cup of brown to black, setae-like structures. Conidiogenous cells proliferating sympodially with inconspicuous conidial scars. Synnematous conidia (FIG. 26) hyaline, one- 124 MYCOLOGIA TABLE IV. Restrictionfragmentsof the amplifiedITSproduct from species in the Ophiostoma piceaecomplex Ddel HaeII Species fragments(bp) fragments(bp) 0. piceae/O. canum O. floccosum 0. setosum 0. querci/O. catonianum 0. ulmi/O. novo-ulmi/ 0. himal-ulmi 400, 280, 400, 520, 520, 200, 120 200, (120)a 200, 120 200 170 290, 290, 270, 290, 290, 270, 190 270, 190 (190)a, 100 270, 190 270, 190 Band sizes in parentheses are for two co-migrating bands. a celled, mostly guttulate, cylindrical to obovoid with rounded apices and slightly tapering bases, which may be truncate to rounded, 3.0-5.5 ILm (mean 4.0 jim) long and 1.0-2.0 jLm (mean 1.6 Jm) wide, accumulating in a white, gelatinous mass at the apex of the synnema. Micronematous conidiophores typical of the genus Sporothrix(FIGS.24, 25), arising terminally or laterally from hyphae, 11-156 jm (mean 73 jLm) long, 1.0-4.0 Ijm wide at the base, tapering slightly at the apex, sometimes septate. Conidia produced at the apex of the conidiogenous cell (FIGS.24, 25), which has prominent denticles, 0.5-1.5 tim long, where conidia have abscised along a rachis 1.5-21 jLm (mean 5.5 jim) long. Conidiogenous cells may proliferate at or below the rachis. Sporothrixconidia hyaline, smooth-walled, 6.5-24 jim (mean 12.0 jim) long, 2.0-3.5 Jim (mean 2.5 jim) wide, with a rounded apex and tapering base, often giving rise to secondary conidia at the apex and having denticles at the point of dehiscence of the secondary conidia. Secondary conidia hyaline, smooth-walled, guttulate, cylindrical to obovoid, 3.0-8.0 jLm (mean 5.5 Jim) long, 1.0-2.5 jLm(mean 2.0 jim) wide, with a rounded apex and tapering at the base. No protoperithecium or perithecium seen. HOLOTYPE. USA. WASHINGTON: Aberdeen, from stained wood of Pseudotsuga menziesii, Dec 1997, T Harrington, BPI 746441, from isolate C1194 (CBS 102358). Etymology. Refers to the cup-like structure at the top of the synnemata. Commentary.Cultures representing the two mating types of a new Ophiostoma species (0. setosum) from Canada were kindly provided to us by A. Uzunovic (Uzunovic et al 2000) after our initial mating studies were completed. Pairings of our P cupulatum isolates with the tester strains of 0. setosum showed that all of our isolates were of the same mating type (B, TABLEI). After repeated pairings at 20 C, the isolates of P. cupulatum from the USA and New Zealand (TABLE I) were able to mate with the tester strains provided by Uzunovic. Morphologically, our isolates and the Canadian isolates (Uzunovic et al 2000) are identical. Synnemata of 0. setosum are similar in size and pigmentation to those of 0. piceae and 0. querci,but the apex of the synnemata of 0. setosumis unique in its cup-like structure formed by extension of the external stipe cells into the conidial mass (FIGS.21-23). The concentric rings of aerial mycelium found in most isolates and the distinctively sweet aroma of cultures of 0. setosum also distinguish this species from 0. piceae (TABLEII) . Ophiostoma setosum fails to grow at 32 C, as does 0. piceae. In ITS sequence, 0. setosum is close to 0. piceae, and we have found 0. setosum only on wood of conifers (Pinaceae), the most common substrate of 0. piceae. We originally isolated 0. setosum from stained wood of Pinus radiata in New Zealand. Additional isolations were made from lumber of Psuedotsuga menziesii and Tsuga sp. from Washington, USA and from Pinus radiata wood from California. Uzunovic et al (2000) identified isolates from Picea, Pinus, and Tsuga in British Columbia, Canada and Oregon, USA. Thus, the species appears endemic to the Pacific Coast of North America on various Pinaceae and was likely introduced to New Zealand. Synnemata of this species are commonly found in association with 0. piceae, 0. querci, and 0. floccosum on bluestained wood. Ophiostoma querci (Georgevitch) Nannf., in Melin and Nannf., Sven. Skogsvardsforen Tidskr. 32: 408. 1934. FIGS. 27-36 = CeratostomellaquerciGeorg6vitch, C. R. Acad. Sci. Paris 183: 759. 1926. = Ceratocystisquerci (Georg6vitch) Moreau, Rev. Mycol. (Paris)Suppl. Col. 17: 22. 1952. =Ophiostoma fagi (Loos) Nannf., in Melin & Nannf., Sven. Skogsvardsforen Tidskr.32: 408. 1934. Ceratostomellafagi Loos, Arch. Mikrobiol. 3: 376. 1932. = Ceratocystisfagi (Loos) Moreau, Rev. Mycol. (Paris) Suppl. Col. 17: 22. 1952. ?= OphiostomaroborisGeorgescu & Teodoru, Anal. Inst. Cerc. Exp. For.,Ser. 1, 11: 207. 1948. = Ceratocystis roboris (Georgescu & Teodoru) Potlajchuk, Nov. Sist. Niz. Rast. 22: 154. 1985. ?= Ophiostomavalachicum Georgescu & Teodoru, Anal. Inst. Cerc. Exp. For., Ser. 1, 11: 198, 1948. Ceratocystisvalachicum (Georgescu & Teodoru) Potlajchuk, Novosti Sist. Niz. Rast. 22: 155. 1985. ?= Ophiostomakubanicum Sherbin-Parfenenko, Rak. Sos. Bol. List. Porod (Moscow) p. 49. 1953. Ceratocystiskubanicum (Sherbin-Parfenenko) Potlajchuk, Novosti Sist. Niz. Rast. 22: 153. 1985. Anamorph. ?Graphium roboris Georgescu & Teodoru, Anal. Inst. Cerc. Exp. For., Ser. 1, 11: 213. 1948. 125 HARRINGTON ET AL: OPHIOSTOMAPICEAE % \i ' . .p I 33 ' 4;3 . .. _ m .. *E ili-Ai '.-s- .-I ?;?;?I :cl . L IL FIGS.27-36. Ophiostoma querci. 27, 28. Protoperithecia. 29. Perithecium. 30. Perithecial neck with drops of ascospores (arrowheads). 31. Ostiolar hyphae. 32. Ascospores. 33, 34. Conidiophores and conidia of the Sporothrixsynanamorph. Arrows in 33 and 34 indicate apex of conidiogenous cells, showingprominent denticles. 35. Synnema.36. Conidiafrom synnema. 27, 28, 33-36 from strainC969;29-32 from a pairingof 969 and C970. Scale bars:27, 28 = 25 ,um;29 = 100 ,um;30 = 220 ,um; 31-34, 36 = 10 aRm;35 = 50 ,um. ?= Graphium kubanicum Potlajchuk & Schekunova, NovostiSist. Niz. Rast.22: 153. 1985. Commentary.Unfortunately, no type material or culture was preserved by Georgevitch, and no Latin description was made. However, this name has been commonly applied to the so-called "hardwood" form of the 0. piceaecomplex, and we continue to apply the name in this sense. Morelet (1992) designated neotype material (PFN 1463) for 0. querci,which was derived from mating two French isolates (CBS 366.93 and CBS 367.93). We have deposited a dried specimen (BPI 746440) with perithecia and ascospores resulting from a cross of two British isolates, C969 and C970. Ophiostoma querci is difficult to distinguish morphologically from 0. piceae, though ITS sequencing shows it to be only distantly related (FIG.1). It is common to find small, glistening drops of ascospores along the neck of 0. querci perithecia (FIG. 30), 126 MYCOLOGIA which are due to exudations of ascospores as the perithecial neck elongates. We have not seen this in perithecia of the other species in the 0. piceae complex, but we have seen such ascospore droplets along the neck of 0. pluriannulatum perithecia. Most isolates of 0. querciand 0. piceae form protoperithecia on MEA, but those of 0. querci are a light, goldenbrown color (FIGS. 27, 28), while those of 0. piceae are black (FIGS. 2, 3). Most isolates of 0. quercihave a nut-like aroma when growing on MEA, in contrast to an indistinct aroma in cultures of 0. piceae (TABLE II). Also, 0. querci isolates grow at 32 C, while O. piceae isolates do not (Brasier and Stephens 1993, TABLE I). Many isolates of 0. querci form concentric rings of aerial mycelium on MEA, similar to isolates of the Dutch elm disease fungi (TABLEII). In pairings, 0. querci produces perithecia and ascospores when isolates of opposite mating types are paired. An isolate (CBS 236.32) of 0. fagi from the holotype had the ITS sequence of 0. querciand mated with this species. It is, therefore, considered a synonym of 0. querci,as was suggested by Brasier (1993). Three other Ophiostoma species (0. roboris, 0. kubanicum, and 0. valachicum) from Quercusin eastern Europe are likely synonyms of 0. querci (Przybyl and de Hoog 1989), but there appears to be no authentic material available of these three species (Przybyl and de Hoog 1989). From descriptions by Potlajczuk and Schekunova (1985), 0. roboris,0. kubanicum, and 0. valachicum are indistinguishable from 0. querci.Both 0. roborisand 0. kubanicum have been isolated from Quercus from central Europe to Azerbaijan (Brasier and Kirk 1993). An isolate of 0. roborisfrom Azerbaijan by Guseinov paired with testers of 0. quercito produce perithecia and ascospores (TABLEI), as was reported by Brasier and Kirk (1993). Both Morelet (1992) and Okada et al (1998) list Pesotum pirinum as the anamorph of 0. querci. We consider 0. catonianum and its anamorph, P pirinum, as distinct from 0. querci and 0. piceae, leaving no clear anamorph name for 0. querci. An isolate deposited as Graphium aureum (CBS 277.54, C1283) had the ITS sequence of 0. querci,and it paired with our tester strains of 0. querci.However, examinations of the holotype specimen and description of G. aureum (Hedgcock 1906) show that it is the anamorph of 0. floccosum, as noted above, and CBS 277.54 was misidentified as G. aureum. Should 0. roborisprove to be a validly described synonym of 0. querci, then the anamorph name Graphium roborisis available as a basionym for the anamorph of 0. querci. Ophiostoma querciappears to be a widespread species and is commonly found on conifers and hardwoods throughout Europe and North America (Brasier and Kirk 1993, Halmschlager et al 1994, Kim et al 1999, Morelet 1992, Pipe et al 1995). In addition to isolates from South Africa and New Zealand listed in TABLE I, we have identified isolates of 0. querci from P radiata in Australia, Eucalyptus sp. in Uruguay and Pinus sp. in Korea. In Europe, it apparently occurs more frequently on hardwoods, especially oak, than on conifers (Brasier and Kirk 1993, Halmschlager et al 1994, Morelet 1992, Pipe et al 1995). In North America and the Southern Hemisphere, however, sapwood of Pinaceae is a common substrate. Ophiostoma catonianum (Goid.) Goid., Boll. Stn. Patol. Veg. Roma, N. S., 15: 125. 1935. Ceratostomella catonianum Goid., Atti. Accad. Naz. Linc. Rc. Ser. 6, 21: 199. 1935. = Ceratocystis catonianum(Goid.) Moreau, Rev. Mycol. (Paris) Suppl. Col. 17: 22. 1952. Anamorph. Pesotum pirinum (Goid.) Okada & Seifert, Can.J. Bot. 76: 1504. 1998. = Graphium pirinum Goid., Boll. Stn. Patol. Veg. Roma,N. S., 15: 132. 1935. Synanamorph. Sporothrixpirinum (Goid.) Morelet, Extrait Annales S.S.N.A.T.V.44: 110. 1992. = Hyalodendron pirinum Goid., Boll. Stn. Patol. Veg. Roma, N. S., 15: 136. 1935. Commentary.Although there appears to be no type specimen for 0. catonianum (Hunt 1956), a culture of 0. catonianum deposited by Goidanich is available from CBS (263.35). The ITS sequence of this isolate is unique but near that of 0. querci (FIG. 1). The culture does not form protoperithecia, perithecia, or synnemata, and pairings with testers of the other species in the complex failed to result in perithecia. A Sporothrixanamorph was found in the culture, however. This culture grows very slowly at 32 C, in contrast to 0. querci (TABLEI). Goidanich (1935) described this species from Pyrus (pear) in Italy. His description of 0. catonianum indicates that the perithecial necks are shorter and the ostiolar hyphae longer than those of 0. piceae, 0. querci, and 0. floccosum. He also states that single ascospore and single conidial isolates produce perithecia and ascospores. Thus, 0. catonianum is homothallic, in contrast to the heterothallic mating system of all other species in the complex. Ophiostoma ulmi (Buisman) Nannf., in Melin and Nannfeldt, Svenska Skorgsfor. Tidskr. 32: 408. 1934. - Ceratostomella ulmiBuisman,Tijdskr.Plantenziekt.38: 1. 1932. Ceratocystisulmi (Buisman) Moreau, Rev. Mycol. (Paris) Suppl. Col. 17: 22. 1952. Anamorph. Pesotum ulmi (Schwarz) Crane & Schok- = necht, Am.J. Bot. 60: 348. 1973. HARRINGTONET AL: OPHIOSTOMA PICEAE Graphiumulmi Schwarz,Meded. Phytopathol.Lab. 5: 13. 1922. Commentary. This species, and the following two species, are capable of causing Dutch elm disease, a vascular wilt disease, in inoculated elms (Brasier and Mehrotra 1995). Morphological and cultural characteristics of these three species are contrasted in detail by Brasier and Mehrotra (1995) and need not be reiterated here. The natural geographic range of 0. ulmi is still not clear (Brasier and Mehrotra 1995), but it was apparently introduced into Europe and North America, where dramatic losses to Ulmus species were found. In contrast to 0. querci, synnemata are generally not formed by isolates of 0. ulmi on MEA or MYEA,but addition of twigs or linoleic acid to the medium stimulates synnemata production (Dalpe and Montant 1980). The typically pronounced concentric rings of the mycelium and relatively fast growth at 32 C distinguish this species from 0. novo-ulmi (Brazier and Mehrotra 1995) and 0. querci. Only 0. ulmi isolates grow at a relatively rapid rate at 32 C (TABLEI), and Brasier and Mehrotra (1995) have speculated that it may occur naturally in a tropical region of Asia. Ophiostoma novo-ulmi Brasier, Mycopathologia 115: 155. 1991. Commentary. The natural geographic range of O. novo-ulmi is also in question, though an Asian origin is suggested (Brasier and Mehrotra 1995). Like O. ulmi, 0. novo-ulmi causes a devastating vascular wilt disease in Ulmus species in Europe and North America, and the fungus has been introduced to New Zealand. Isolates of 0. novo-ulmi and 0. ulmi are similar to each other, though differences in mycelium texture, temperature sensitivity, and growth rate (Brasier and Mehrotra 1995) separate these species, which are closely related based on ITS sequences (FIG. 1). Ophiostoma himal-ulmi Brasier & M. D. Mehrota, Mycol. Res. 99: 211. 1995. Commentary.Thus far, 0. himal-ulmi is known only from the Himalayas from branches of Ulmus species attacked by bark beetles (Brasier and Mehrotra 1995). Although thought to be closely related to O. ulmi and 0. novo-ulmi, the ITS sequence data suggest that it is more closely related to 0. querci.Unlike the other Dutch-elm disease fungi, 0. himal-ulmi produces synnmenata on MEA, and morphologically it is difficult to distinguish from 0. querci.However, cultures of 0. himal-ulmi do not produce the nutty aroma of 0. querci and grow slower than 0. querci isolates at 32 C (TABLEI). 127 Synnematous Ophiostoma species without Sporothrix synanamorphs.-A number of Ophiostoma species have been noted to have synnematous anamorphs but no Sporothrixsynanamorph (TABLE III). Ophiostoma novae-zelandiae (Hutchison & Reid) Rulamort was said to rarely form synnemata (Hutchison and Reid 1988a), but we have not seen synnema in a dried culture from the holotype [WIN (M) 869] or in living cultures (UAMH 9556-9) from New Zealand (Hutchison and Reid 1988a). The original description may have been from a culture of more than one species. Perithecia and ascospores as described by Hutchison and Reid (1988a) and as seen in the holotype look like those of 0. pluriannulatum, which is not reported to have synnemata (Hunt 1956, Upadhyay 1981). Reports of synnemata in Ophiostomaspecies other than those in the 0. piceae complex have been of species without known Sporothrix synanamorphs, though many produce Hyalorhinocladiella synanamorphs. These species (TABLEIII) are excluded from the 0. piceae complex. Two such species are discussed below. Pesotum fragrans (Mathiesen-Kaarik) Okada & SeiFIGS. 37-40 fert, Can. J. Bot. 76: 1503. 1998. = Graphiumfragrans Mathiesen-Kiarik,Medd. Statens Inst. 43: 59. 1953. Skogsforskningsinst. An isolate Commentary. deposited as Graphiumfra= = ATCC 24590) by KaaCBS 279.54 (C1224 grans rik likely represents the species. However, this Swedish isolate no longer produces synnema but does produce Leptographium-like or Hyalorhinocladiella-like conidiophores (FIGs. 37-39) that may represent degenerate synnemata. No denticles typical of Sporothrix are seen in the culture. The conidia (FIG. 40) of this species are longer than those of members of the 0. piceae complex. This species does appear to be an anamorph of an Ophiostoma species based on ITS sequence (AF198248), but it does not fall into the 0. piceae complex (data not shown). Similar cultures with almost identical ITS sequences were isolated from pine wood in California, New Zealand and Australia (unpubl). Okada et al (1998) list CBS 219.83 as authentic for the type of G. fragrans, but this isolate was collected by Solheim, not Kaarik, and CBS 279.54 should be authentic for the type. Okada et al (1998) found sympodial proliferation of the conidiogenous cells of synnematous and mononematous conidiophores of CBS 219.83, and they also found that the 18S rDNA sequence of this isolate was similar to that of other Ophiostomaspecies. 128 MYCOLOGIA '' "- Na y.r' I l C WIF'P _ i' . ? ^fr,V *i j FIGS. 37-45. Graphiumfragrans and 0. cucuUatum. 37-40. G. fragrans strain C1224, AUTHENTIC FOR TYPE. 37-39. Conidiophores and conidia. Arrow in 37 indicates abscission scars along the side of apex of conidiogenous cell. 40. Conidia. 41-45. 0. cucullatum. 41-42. Synnemata. 43. Synnema with metulae, phialides, and conidia. 44. Micronematous conidiophores and conidia. 45. Apex of Leptographiumtype conidiophore with metulae, phialides and conidia. 41, 44 from strain 1216 as 0. cucullatum; 42, 43, 45 from strain C1222, AUTHENTIC FOR TYPE of Phialographium erubescens.Scale bars: 37- 40, 43-45 = 10 pm; 41, 42 = 50 pum. HARRINGTONET AL: OPHIOSTOMA PICEAE Ophiostoma cucullatum Solheim, Nord. J. Bot. 6: 202. FIGS.41-44 1986. Anamorph. Phialographium erubescens (Mathiesen-KIarik) Harrington et McNew, comb. nov. Pesotumerubescens Okada,Stud(Mathiesen-Kiarik) ies in Mycology45: 184. 2000. GraphiumerubescensMathiesen-Kiarik,Medd. Statens Skogsforskningsinst Inst. 43: 62. 1953. The ITS Commentary. sequence of 0. cucullatum = NFRI 81-83/2, colfrom isolate C1216 (AF198246, lected by Solheim) differed in only a single base substitution from that of Graphium erubescens (CBS 278.54 = JCM 9747 = C1222, from the holotype) and another culture deposited by KAirik as G. album (CBS 276.54 = JCM 9744 = C1225, ITS sequence = AF198247). This isolate of G. album is morphologically identical to G. erubescensand is distinct from specimens representing G. album as emended by Hedgcock (1906). We believe that Kaarik's isolate of G. album was misidentified and that G. erubescensis the anamorph of 0. cucullatum. The ITS sequences of 0. cucullatum and G. erubescens were not similar to those of other members of the 0. piceae complex. Okada et al (1998) found the 18S rDNA sequence of 0. cucullatum to be similar to that of 0. europhioides, which has a Leptographium anamorph. Likewise, Hausner et al (1993) found the partial 28S rDNA sequence of 0. cucullatum to be similar to those of other Ophiostoma species with Phialographium and Leptographiumanamorphs. All three cultures of 0. cucullatum (C1216, C1222, C1225) failed to form Sporothrixconidiophores on MEA but did form synnemata with loosely- to tightlyfused outer stipe cells (FIGS.41-43), with gradations to mononematous (Leptographium-type) conidiophores (FIG.45). In general, the aerial synnemata are more tightly woven, and the synnemata submerged in the agar medium are a loose collection of Leptographium-like conidiophores. The conidiogenous cells on top of the synnematous and mononematous conidiophores are phialidic based on light microscopy (FIGS.43, 45), as described for the synnemata of 0. cucullatum (Solheim 1986). Okada et al (2000) reported that the conidiogenous cells of P erubescens (isolate CBS 278.54) usually proliferate percurrently but also have an intermediate mode between sympodial proliferation and phialidic ontogeny based on scanning electron microscopy. The anamorph of 0. sagmatosporaealso has conidiogenous cells that are phialidic in light microscopy (Seifert and Okada 1993), and this anamorph was the basis for the synnema-forming genus Phialographium Upadhyay & Kendrick. Okada et al (1998) consider the synnematous anamorphs of 0. sagmatosporaeand 0. cucullatum to be Pesotum species, and 129 they (Okada et al 2000) formally transferred G. erubescens to Pesotum. However, the phialidic conidiogenous cells, the loose aggregation of Leptographium-like conidiophores, and the absence of a Sporothrixanamorph lead us to believe that these species are distinct from Pesotum as originally conceived by Crane and Schoknecht (1973). Further, O. cucullatum and 0. sagmatosporae,like most other Ophiostoma species with Leptographiumor Phialographium anamorphs, have ascospores with thickened outer walls (sheaths) that are cucullate, to rectangular, to triangular, depending on the view (Solheim 1986). In contrast, ascospores in the 0. piceae complex are allantoid to orange section shaped, and are relatively thinwalled, with no visible sheath. Graphium species excludedfrom Ophiostoma.-There are numerous Graphium-likespecies that are related to ascomycetes other than Ophiostoma (Okada et al 1998), and it is beyond the scope of the present paper to discuss these in detail. However, we did examine material of five Graphium species from Hedgcock's specimens in BPI and other CBS cultures, and these examinations suggest that they are not closely related to Ophiostomaspecies. Graphium album (Corda) Sacc., Syll. Fung. 4: 618. 1886. emended Hedgcock, Mo. Bot. Gard. Rept. 17: 96. 1906. = Ceratopodium albumCorda,Ic. Fung. 1: 20. 1837. Commentary.No type material of Corda's specimen is available (Seifert, pers comm), and his illustrations give little indication of the affinities of this species. It was found on Betula in Bohemia and described by Saccardo (1886) as having conidial masses that were white. Hedgcock (1906) emended the species description to include conidial masses white to creamyyellow, or light brown when old and dry, and our examination his specimen (BPI 448665, from Fagus in Arkansas, USA) showed the conidial masses to be yellow-gold. The yellow-gold to light-brown conidial masses of the BPI specimen suggests that G. album as emended by Hedgcock is not an Ophiostomaspecies. However, Hedgcock's fungus may not be G. album, and the disposition of Corda's species remains in question. A culture from Pinus in CBS (276.54, deposited by Kaarik) is listed as G. album, but the culture does not match the BPI specimen or Hedgcock's emended description of G. album. Rather, KYarik'sculture is conspecific with her Graphium erubescens (MathiesenKaarik 1953). As discussed earlier, CBS 276.54 has the ITS sequence of 0. cucullatum (anamorph = Phialographium erubescens). As KIarik's culture is from 130 MYCOLOGIA FIGS.46-48. Graphium rubrum conidiophores and conidia. 46, 47 from strain C1223, AUTHENTIC FOR TYPE of G. rubrum;48 from strain C1221, AUTHENTIC FOR TYPE of G. silanum. Scale bars = 10 ,m. pine, it seems unlikely that it is G. album as described by Corda. Graphium ambrosiigerumHedgcock, Mo. Bot. Gard. Rept. 17: 85. 1906. Commentary.The holotype specimen (BPI 448676) and co-type (BPI 448675) are in poor condition, but both have synnemata with dark stipes and black conidial masses. The species was described from galleries of ambrosia beetles in Pinus arizonica in Arizona, USA. It is possible that Hedgcock was working with a mixture of an Ambrosiella species with a Graphium species. The black conidial masses on the synnema of the G. ambrosiigerumspecimens are not typical for anamorphs of Ophiostomaspecies. Graphium rigidum (Pers.) Sacc., Syll. Fung. 4: 610. 1886. emended Hedgcock, Mo. Bot. Gard. Rep. 17: 92. 1906. Commentary. Seifert (1985) suggested that the name G. rigidum had been frequently used for fungi with black synnemata, and he considered the species a nomen dubium. Siemaszko (1939) considered G. rigidum as a synonym of G. penicillioides, and G. rigidum may be a member of the G. penicillioides complex (Okada et al 1998). A specimen (BPI 448820) from Quercus rubra sapwood from Indiana, USA deposited by Hedgcock in BPI as the emended type of G. rigidum, may be of more than one fungus. The synnemata in the specimen have black stipes from the base up to the yellow-gold conidial masses. Graphium rubrum Rumbold, Phytopathology 24: 300. FIGS.46-47 1934. Commentary. A specimen deposited by Rumbold (BPI 448830) can serve as the holotype of G. rubrum, and it shows black synnematous stipes with pinkishred to yellowish-red conidial masses, and a few syn- nemata have gray conidial masses. A culture from the holotype (CBS 210.34) is no longer producing synnemata but is producing phialide-like structures on short, simple, hyaline conidiophores (FIGS.46-47). Okada et al (2000) also observed phialides in CBS 210.34 using scanning electron microscopy. Rumbold (1934) reported G. rubrum from Quercus, Populus, Liquidambarand Pinus in the USA. Goidanich (1936) described G. silanum from Pinus in Italy, and a culture (CBS 206.37) deposited by him produces no synnemata but does produce phialidelike structures on short, simple, hyaline conidiophores (FIG.48), similar to those of G. rubrum. Also, CBS 206.37 has an ITS sequence identical to that of Rumbold's isolate of G. rubrum. It is possible, however, that CBS 206.37 is not G. silanum, as Goidanich described G. silanum as having a micronematous state with prominent denticles and ramoconidia, and DdeI HaeII M Os Of Op Oq Os Op M FIG.49. Ethidium bromide stained agarose (2%) gel of HaeII and DdeI digestion products of the amplified ITS rDNA region from isolates of Ophiostoma setosum (Os), Ophiostomafloccosum (Of), 0. piceae (Op), and 0. querci (Oq). Co-migrating bands are indicated by arrows. Onehundred base pair ladders (M) are on either side of the gel. HARRINGTON ET AL: OPHIOSTOMAPICEAE HaeII On nO FIG.50. Ethidiumbromide stained agarose (2%) gel of HaeII digestionproductsof the amplifiedITSrDNAregion from isolates of Ophiostomaulmi (Ou), 0. himal-ulmi (Oh), 0. novo-ulmi (On), and 0. querci (Oq). One-hundred base pair ladders (M) are on either side of the gel. CBS 206.37 does not form such a Sporothrix-like anamorph. The ITS sequence (AF198245) of Rumbold's isolate CBS 210.34 did not match well with our sequences of Ophiostomaspecies. A BLAST 2.0 (NCBI) search showed this sequence to be closest to Phialophora gregata (Allington and Chamberlain) Gams, which may be an anamorph of an unknown discomycete (Paulin and Harrington 2000). The 18S rDNA sequence of Rumbold's culture also places it among the discomycetes (Okada et al 2000). Dendrostilbella smaragdina (Alb. & Schw.) Seifert, Stud. Mycol. 45: 185. 2000. Ceratopodiumsmaragdinum A. & S., Consp. Fung., p. 335. 1805. Stilbum smaragdinum Alb. & Schw.:Fr.,Sytem. Mycol. 3: 303. 1832. Tubercularia smaragdinum (Alb. & Schw.) Seifert, Stud. Mycol.27: 127. 1985. Graphium smaragdinum (Alb. & Schw.) Sacc., Syll. Fung. 4: 618. 1886. Commentary. Seifert (1985) examined what was considered authentic material of this species and described the green synnemata and acropleurogenous phialides. Hedgcock (1906) noted green synnematous stipes and gray or green conidial masses, and we also found very dark conidial masses on his specimen (BPI 448617). Based on Seifert's description and Hedgcock's specimen, G. smaragdinum is not a likely member of the 0. piceae complex. This species may have an unnamed Claussenomyces(discomycete) teleomorph (Okada et al 2000). DISCUSSION Although there have been earlier studies distinguishing the so-called conifer form of 0. piceae from the 131 so-called hardwood form, and the Dutch elm disease fungi have been clearly delimited from each other, this is the first comprehensive assessment of the entire complex. The ITS sequences of the nine recognized species were very similar to each other but quite distinct from the ITS sequences of 0. ips, 0. simplex, O. galeiformis, 0. cucullatum, 0. europhioides, O. pluriannulatum, 0. coronatum, O. perfectum, 0. nigrocarpum, 0. stenocerasand other Ophiostomaspecies (data not shown). All members of the 0. piceae complex form a Sporothrixsynanamorph with prominent denticles in addition to the Pesotum synanamorph, and the presence of both synanamorphs sets the complex apart from the rest of Ophiostoma.Thus, this unique combination of anamorphs and the ITS sequence analysis suggest that 0. piceae complex is a monophyletic group. Graphium-like fungi outside the 0. piceae complex.- Synnemata are efficient fruiting bodies for placing conidia in the path of small animals for acquisition and dispersal, and it is not surprising that many evolutionary lines of fungi have converged on such conidiomata. In a recent review of ascomycete-forming fungi with Graphium-like synnemata, Okada et al (1998) found that such synnemata are formed by species of the Microascales (pyrenomycetes) and Chaetothyriales (loculoascomycetes), and they recommend that the genus name Graphium be used only for the species with microascalean affinities, such as G. penicillioides. Further, we found that the ITS sequence of G. rubrum is closer to Phialophora gregata and discomycetes than to pyrenomycetes or loculoascomycetes, and G. rubrum produces a phialidic micronematous state, as does the synnematous Dendrostillbella smaragdina, which also appears to be a dis- comycete anamorph (Okada et al 2000). Species in Ophiostomatolerate cycloheximide at high concentrations (Harrington 1981), and this can aid in separating synnema-forming species with Ophiostomaaffinities from those of other ascomycetes. However, there are other ascomycetes, including some true Graphium species, like G. penicillioides, that are not related to Ophiostomabut do tolerate cycloheximide. Even within Ophiostoma, synnemata may have evolved more than once. Ophiostomais a large genus with a wide variety of anamorphs, and many species have more than one synanamorph. In addition to the Leptographium-type(mononematous, with a stipe and a penicillately branched conidiogenous apparatus) and the Graphium-type (synnematous), some species of Ophiostoma, such as the anamorph of 0. claviger- um, show both types of conidiomata. Graphiocladiella was proposed by Upadhyay (1981) to accommodate anamorphic fungi with both mononematous and syn- 132 MYCOLOGIA nematous conidiomata. Harrington (1988) suggested that such intermediate types could be better accommodated in Graphium (Pesotum) than in Leptographium, but we now suspect that many such species have greater affinity with Leptographium. Variability in conidiogenesis and stipe configuration among the anamorphs of Ophiostomahas led to a multitude of anamorph genera and continued controversy (Harrington 1988, Seifert and Okada 1993, Wingfield et al 1991). In some Ophiostoma species (TABLE III), the synnemata appear to be a loose aggregation of Leptographium conidiophores, without fused stipe cells. In our examination of Ophiostoma species, only members of the 0. piceae complex have proven to produce both synnemata with fused stipe cells and a Sporothrixsynanamorph (micronematous conidiophores with prominent denticles, Hoog 1974, 1993). Micronematous conidiophores are seen in many of the species listed in TABLEIII, but these micronematous conidiophores lack prominent denticles at the conidiogenous cell apex and appear to be nearer to Hyalorhinocladiella (Hoog 1993) or to be degenerative Leptographium-typeconidiophores. The conidiogenous cells of Pesotumand Leptographiumresemble those of Hyalorhinocladiella, and it is possible that the macronematous conidiomata evolved from the Hyalorhinocladiella-likeconidiophores. Some of the synnemata-forming Ophiostoma species with ITS sequences differing greatly from those of 0. piceae include 0. ips, 0. simplex, 0. galeiformis and 0. cucullatum. These species lack a Sporothrix anamorph, though 0. ips forms micronematous, Hyalorhinocladiella-like conidiophores (Hoog 1993). Many of these and other synnemata-forming Ophiostoma species without Sporothrixsynanamorphs form ascospores with sheath-like outer walls, extending to a cucullate brim around the spores (e.g., 0. clavigerum, 0. simplex, 0. galeiformis and 0. cucullatum) or in a box shape (0. ips and 0. ainoae). In contrast, reniform ascospores without thickened outer cell walls are typical for species in the 0. piceae complex. The genus Pesotum was defined by the presence of both synnemata and micronematous conidiophores with denticles, with the synanamorphs of Ophiostoma ulmi as the type species (Crane and Schoknecht 1973). Accepting their definition, we would restrict Pesotum to those anamorphs with affinities to the 0. piceae complex. Other synnematous anamorphs in Ophiostomamay have arisen through convergent evolution, perhaps from Leptographium-type conidiophores. The genus Phialographium is available for synnematous anamorphs of Ophiostomaspecies (e.g., 0. sagmatosporaeand 0. cucullatum) with phialidic conidiogenous cells. Species delimitation and identification in the 0. piceae complex.-Use of a wide array of isolates from around the world and a holistic approach, including mating reactions, phylogenetic analysis, and phenotype (morphological and physiological), allows for a clearer delimitation of the species in the 0. piceae complex. We emphasize phenotype in delimiting species (Harrington and Rizzo 1999), but mating compatibilies and phylogenetic analyses prove valuable in identifying lineages or mating populations where unique phenotypic characters may be found. It is noteworthy that ITS sequences were unable to distinguish 0. piceae from 0. canum, two species that are morphologically and biologically distinct. Other studies (Harrington and Rizzo 1999, Witthuhn et al 2000) have also found that ITS sequences of sympatric sibling species may be identical, and a strictly genealogical species concept cannot be applied if only nuclear rDNA sequences are utilized. Production of the sexual state in culture is relatively easy for species in the 0. piceae complex, and the heterothallic nature of most of the species allows for the identification of mating populations or biological species. Partial interfertility between species, as evidenced by poor ascospore production or aborted asci (Brasier 1993, Harrington and McNew 1998) was seen in some pairings between different species, and this strongly suggests that the species are distinct. In pairings between isolates of the same species, we generally had more success in producing perithecia and ascospores by spermatizing a recipient (female) strain with conidial suspensions of a donor (male) strain than by simply pairing two strains together on a agar medium. Two mating types are seen in O. ulmi, 0. novo-ulmi, 0. himal-ulmi (Brasier and Mehrotra 1995), 0. piceae, 0. querci, (Brasier 1993), 0. setosum (Uzunovic et al 2000), and 0. floccosum, but Goidanich (1935) reported O. catonianum to be homothallic. A number of physiological and morphological characters were found useful in identifying cultures to species (TABLEII). Many of these characters have been used by others (Brasier and Kirk 1989, 1993, Brasier and Stephens 1991, 1993, Hamschlager et al 1994, Pipe et al 1995, Webber and Brasier 1990) to distinguish 0. piceae and 0. querci.Some overlapping morphological characters have been used to distinguish these two species, and these characters, growth at 32 C, and mating reactions with testers are sufficient to identify species in the complex, but a more rapid and objective identification criterion is the RFLP patterns of the amplified ITS region. We did not attempt to distinguish 0. ulmi, 0. novoulmi, and 0. himal-ulmi by the PCR-RFLPtechnique, although ITS sequence divergence should allow de- HARRINGTON ET AL: OPHIOSTOMAPICEAE velopment of a similar protocol for distinguishing these three species. More importantly, the species most commonly found on conifer sapwood (0. piceae, 0. floccosum, 0. querci and 0. setosum) can be identified by ITS-RFLPpatterns. With experience, it is possible to amplify the ITS region using scrapes of mycelium for template DNA. Kim et al (1999) showed that the ITS region of species in the 0. piceae complex can also be amplified directly from conidia at the apex of synnemata, and primers were designed to distinguish 0. piceae and 0. querci.With the RFLP diagnostic technique employed here, more species can be distinguished. Without the need for DNA extraction, the PCR-RFLPtechnique can be completed in a single day, as has been demonstrated for members of Armillaria (Harrington and Wingfield 1995). Phylogenetic analysis of the ITS region supports the distinction between 0. piceae and 0. querci,with 0. piceae grouping with other species (0. canum, 0. floccosum, and 0. setosum) found primarily on conifers. In contrast, 0. quercihas been reported primarily from hardwoods in Europe, as have its relatives, 0. catonianum and the Dutch elm disease fungi. However, 0. querci is apparently common on sapwood of Pinus and other Pinaceae in North America and the Southern Hemisphere. Conifer-inhabiting species of the O. piceae complex.Ophiostomacanum, 0. floccosum, and 0. setosumhave only been isolated from conifers and are more closely related to 0. piceae than to the species found primarily on hardwoods. The ITS sequence of 0. canum is not distinguishable from that of 0. piceae, but lack of interfertility with 0. piceae mating testers and the distinctive conidia of 0. canum set this species apart. Partial interfertility with 0. piceae suggests that both of our isolates of 0. canum are mating type B; thus, we do not appear to have the opposite mating type for production of perithecia and ascospores in culture. The other conifer-inhabiting species appear to be common sapwood staining fungi not associated with any particular insect vector, but 0. canum may be an important symbiont with a specific insect (the bark beetle Tomicusminor,Mathiesen 1950). The collection of thick-walled conidia at the apex of the synnemata may be an adaptation for mycophagy by the bark beetle. Our primary interest in the 0. piceae complex has been in those species that cause bluestain in conifer wood. The most common synnemata-forming species we have encountered in New Zealand and western North America are 0. piceae, 0. floccosum, 0. setosum, and 0. querci.It is relatively common to see two or more of these species on a single chip of wood, and mixed cultures of these species are very fre- 133 quently seen, so these species apparently grow together well, with little inhibition of each other. Such mixed cultures have likely contributed to the confusion over the identification of these species. Unfortunately, the work done on 0. piceae as a bluestain fungus (as reviewed by Seifert 1993) is difficult to interpret now that we realize that the studies could have utilized 0. querci, 0. floccosum or 0. setosum. Hardwood-inhabiting species of the 0. piceae complex.-The taxonomy of 0. querci remains unclear, and the basionym for the species is not ideal. No Latin description, dried specimen or culture was provided by Georgevitch (1926, 1927), but the fact that he described the fungus from oak in Europe (Croatia) makes it likely that his concept of the species matches the modern concept of the "hardwood form" of 0. piceae. Accepting this, Loos' (1932) species, 0. fagi, is a clear synonym. We do not have authentic material for 0. roboris, 0. kubanicum, and 0. valachicum, but these, too, are likely synonyms of O. querci. If 0. roborisis a synonym, then Graphium roboriswould be the earliest available name for the synnematous anamorph of 0. querci.Okada et al (1998) proposed Pesotum pirinum as the anamorph of O. querci,but this is the anamorph name for Ophiostoma catonianum, which we consider distinct from O. querci. We also examined isolates from New Zealand and Vietnam that looked similar to 0. quercibut differed slightly in ITS sequence, and these cultures did not mate with 0. quercitesters. We suspect that more species closely related to 0. querciwill be recognized in the future. The relatedness of 0. querci to the Dutch elm disease fungi 0. ulmi and 0. novo-ulmi was demonstrated by similarity of ITS sequences. These Dutch elm disease fungi are very closely related but distinct species that have been introduced to North America and Europe. They may be native to Asia, though they have not been found there (Brasier 1990, Brasier and Mehrotra 1995). Ophiostoma himal-ulmi does appear to be native to the Himalayas, where it was found associated with bark beetle attacks in elm branches (Brasier and Mehrotra 1995). In inoculation studies, 0. himal-ulmi was able to cause a vascular wilt disease in elm seedlings, and it was thought that this species was closely related to 0. ulmi and 0. novo-ulmi (Brasier and Mehrotra 1995). However, the ITS sequence analysis suggests that 0. himal-ulmi is more closely related to 0. querci. The capacity of 0. querci to cause disease in hardwood species has been debated, and fungi similar to 0. quercihave been frequently associated with declining oak trees in Europe (Brasier 1990, Brasier and Kirk 1989, Brasier and Mehrotra 1995, Harrington MYCOLOGIA 134 1993, Morelet 1992, Oleksyn and Przybyl 1987). Its close relationship to 0. himal-ulmi, 0. ulmi, and 0. novo-ulmi also suggests that it has the potential to cause a vascular disease, and attention should be given to introduction of this species to new environments. We suspect that most species in the 0. piceae complex are native to the Northern Hemisphere. Indeed, the common occurrence of 0. querci on sapwood of Pinaceae in some regions (such as the Southern Hemisphere and North America) may be due to its not being native to these ecosystems. This species and others in the complex will produce synnemata and/ or perithecia on logs, crating, dunnage and other materials commonly discarded from ships, and we believe that the wide distribution of some of these species is due to human activities. ACKNOWLEDGMENTS We acknowledge various herbaria, culture collections and colleagues for providing us with specimens and cultures for study. Such contributions by R. 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