The Genus Oidiodendron: Species Delimitation and Phylogenetic Relationships Based on Nuclear Ribosomal DNA Analysis

The genus Oidiodendron: species delimitation and phylogenetic relationships based on nuclear ribosomal DNA analysis

Sarah Hambleton that this group has diverged significantly from the Department of Biological Sciences, University of two other genera in the family. Relationships in the Alberta, Edmonton, Alberta, Canada T6G 2E9 monophyletic group were correlated with anamorph state produced; three spp., and B. stria- Keith N. Egger Myxotrichum tosporus, all with distinct or reported Oidiodendron Natural Resourcesand Environmental Studies, states, nested with while University of Northern British Columbia, Prince anamorphic species, species George,British Columbia, Canada V2N 4Z9 of Myxotrichum with other anamorph states were ex- cluded. In addition, sequence divergence measures Randolph S. Currahl between the meiotic and mitotic species clustered in Department of Biological Sciences, University of two monophyletic clades were found to be compa- Canada T6G 2E9 Alberta, Edmonton, Alberta, rable to the intraspecific levels for Oidiodendronspp. These results support the initial hypothesis that Oi- diodendron is closely related phylogenetically to the Abstract: Nuclear ribosomal DNA sequences (ITS genus Myxotrichumand suggest that the generic con- region) of fifteen species in the hyphomycete genus cept of Byssoascus needs reexamination. Oidiodendron and ten species from the 4 genera in Key Words: ascomycetes, ericoid mycorrhiza, mo- the Myxotrichaceae, Byssoascus, Gymnostellatospora, lecular systematics, Myxotrichaceae, Onygenales Myxotrichum, and Pseudogymnoascus, were analysed to: (i) reveal the levels of intra- versus interspecific sequence variation within the genus Oidiodendron, INTRODUCTION clarify species delimitation and examine the useful- ness of some morphological characters used for iden- Species of Oidiodendron Robak (1932) (Hyphomy- tification; (ii) assess the possible conspecificity of doc- cetes) are commonly recovered from humus or de- umented ericoid mycorrhizal strains of Oidiodendron; caying wood and bark. The genus is readily identified and (iii) test the hypothesis based on morphological by the distinctive arborescent conidiogenous appa- inference that the genus Oidiodendron belongs with ratus. Erect, dematiaceous conidiophores terminate the genus Myxotrichumin the Myxotrichaceae (Ony- in a complex branching head of fertile hyphae that genales). Comparison of molecular and morpholog- segment basipetally into arthrospores. The mono- ical data for multiple strains of 0. griseum, 0. tenuis- graph of Barron (1962) provides a thorough discus- simum and 0. maius revealed that conidiophore sion of conidiogenesis with descriptions of nine spe- length and the production of a diffusing pigment are cies. The most comprehensive key to the genus in- not reliable key characters for the genus. Several his- cludes 13 species (Domsch et al., 1980) and a further torically important ericoid mycorrhizal strains, doc- six have been described in the Index of Fungi, Inter- umented as 0. griseum, were reidentified as 0. maius. national Mycological Institute, Vol. 1-6, 1940-1996. Parsimony analyses of 23 Oidiodendronstrains showed The genus is putatively placed within the Myxotri- that three highly supported monophyletic groups, chaceae (Onygenales) since the only teleomorphic each one consisting of a pair of species, are resolved species known to have an Oidiodendron state (Myxo- within the genus. A low level of sequence divergence trichum arcticum, M. cancellatum, M. setosum, and between the species in these pairs suggests conspe- Byssoascus striatosporus)belong to this family (Hawks- cificity for each pair. Other interspecific relationships worth et al., 1995, p. 320). were not well-supported by bootstrap values. Parsi- Though some Oidiodendronspecies have additional mony analysis of a second dataset composed of mi- distinctive features, the primary characters used for totic and meiotic taxa showed that Oidiodendron, My- identification are conidium size, shape and ornamen- xotrichum and Byssoascus form a well-supported tation as well as conidiophore length and cultural monophyletic group within the Myxotrichaceae, and morphology. All these vary even on one culture me- dium and the ranges can overlap among species (Bar- Accepted for publication May 21, 1998. ron, 1962). Such variability in morphology leads to 1 Corresponding author, email: [email protected] confusion about species delimitation and uncertainty

854 HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OlDIODENDRON 855 as to which characteristics should be emphasized for simum and 0. maius) using sequence analysis of the identification purposes. internal transcribed spacer regions of the nuclear Three culturally similar species with intergrading rRNA gene (ITS-1 and ITS-2) as a basis for species features are Oidiodendron maius, 0. griseum, and 0. delimitation. The data were then used to assess the tenuissimum, the first two of which have been docu- stability of some key morphological characters for mented as ericoid mycorrhizal fungi and isolated identification purposes, notably conidiophore length from field-collected roots. In our recent study of the and pigment production. Secondly, morphological root endophytes of the Ericaceae in Alberta, Canada, assessment and RFLP analysis were used to examine a large number of Oidiodendron isolates was recov- whether five historically important ericoid mycorrhi- ered (Hambleton and Currah, 1997). Most were zal Oidiodendronstrains are conspecific. The third ob- identified as 0. maius, possessing the very long co- jective was to test the hypothesis that species of Oi- nidiophores and undulating branches of fertile hy- diodendronrepresent taxa within the Myxotrichaceae. phae that are distinctive for the species. Some strains were intermediate between 0. maius and 0. griseum MATERIALSAND METHODS based on conidiophore characteristics, though the conidia more closely matched those of 0. maius. Molecular.-Fungal strains chosen for this study (TA- Restriction fragment length polymorphism (RFLP) BLE I) included 15 species of Oidiodendron (14 de- analysis of PCR-amplified ITS regions was used to scribed species and one new species revealed by the identify strains of 0. maius that were morphologically analysis), seven species of Myxotrichum, and a single atypical (Hambleton and Currah, 1997). The ubiq- strain for each of Byssoascus striatosporus, Gymnostel- uitous presence of 0. maius in the roots of Ericaceae latospora japonica and Pseudogymnoascus roseus. All of boreal habitats, and the difficulties with identifi- four genera in the Myxotrichaceae were represented. cations based on morphology, led to speculation that The only well-recognized species in the family not historical records identifying 0. griseum as an ericoid included were M. ochraceumBerkeley & Broome, G. mycorrhizal fungus may have been confounded by frigida Uchiyama, Kamiya & Udagawa, and three spe- ambiguous features. cies for which there are no cultures available, M. Pigment production on agar media is another aeruginosum Montagne, M. bicolor(Ehrenberg) Fries, character that may not be as informative as once and M. berkeleyiApinis. Ex-type and authentic strains thought. The production of a dark brown diffusing were used where possible as species reference stan- pigment cited in the original description of 0. fus- dards for both the molecular and morphological cum (Robak, 1932, p. 251) (=0. tenuissimum; analyses. Hughes, 1958), which was used as a key character for Fungi were grown on thin plates of E-strain agar an intraspecific cultural grouping in the key of (Egger and Fortin, 1990) for 3 wk in the dark at Domsch et al. (1980), was also reported for 0. gri- room temperature. Eight plugs of mycelium, 0.5 cm seum by Tokumasu (1973) and Hambleton and Cur- diam with a minimum of agar attached, were used rah (1997). for DNA extraction following the chloroform/isopro- For one of the ericoid endophytic isolates of Ham- panol protocol of Gardes and Bruns (1993) with mi- bleton and Currah, and for a strain examined by Tok- nor modifications (Hambleton and Currah, 1997). umasu as well, the identification was more problem- Primers ITS1 and ITS4 (White et al., 1990) were atic because the upper range of the conidiophore used to amplify a portion of the nuclear ribosomal lengths was greater than described in the original de- RNA genes (rDNA) including both ITS spacer scription of 0. griseum (Melin and Nannfeldt, 1934, regions and the 5.8S subunit. PCR reaction volumes p. 440). Barron (1962) had already revised the co- of 100 ,iL contained PCR buffer, 25 mM MgCl2, 50 nidiophore length range for 0. griseum upwards with !1M each of dGTP, dATP, dTTP, and dCTP (Boehrin- the result that it overlapped with the ranges of both ger Mannheim Biochemica), 0.4 pJMof each primer 0. tenuissimum and 0. maius. Unfortunately, in this and 2 units of Taq DNA polymerase. One FLLof di- case, RFLP analysis was unable to differentiate 0. gri- luted DNA was used as the template for each reaction seum from 0. tenuissimum (Hambleton, unpub. except for a DNA-free, negative control. PCR ampli- data). Further, RFLP analysis of the genus as a whole fications were carried out on a DNA Thermal Cycler suggested that for some pairs of species, morpholog- (Gene E. Techne Ltd., Princeton, New York) using ical variation might only be significant at the subspe- the following cycle parameters: 94 C denaturation for cies level. 1 min, 55 C annealing for 1 min, 72 C extension for The first objective of this study was to clarify species 2 min. The total number of cycles was 30 with an concepts within the genus Oidiodendronwith empha- initial denaturation step of 2 min at 94 C and a final sis on the three target species (0. griseum, 0. tenuis- extension at 72 C for 7 min. Amplified PCR products 856 MYCOLOGIA

TABLEI. Name, strain number, origin, the analysis that each strain was included in and GenBank accession number of fungi used for sequence analysis Data GenBank Species Straina Origin"1 matrix' No. OidiodendronRobak 0. cerealis (Thum.) Barron UAMH 1522 peat soil; ON Canada. Oid, Myx AF062788 0. chlamydosporicumMorrall UAMH 6520 T, soil ex boreal forest; SK Canada. Oid, Myx AF062789 0. citrinum Barron UAMH 1525 T, soil ex cedar bog; ON Canada. Oid, Myx AF062790 0. echinulatum Barron IMI 110132 A, soil ex cedar bog; ON Canada. Oid, Myx AF062791 (UAMH 8467) 0. flavum Szilvinyi UAMH 1524 soil ex cedar bog; ON Canada. Oid, Myx AF062792 0. griseum Robak CBS 249.33 A, wood pulp; Sweden. (Melin & Nann- Oid, Myx AF062793 (UAMH 1403) feldt, 1934) UAMH 1693 Douglas fir timber; BC Canada. Oidd AF062794 UAMH 4080 wood chips and bark ex logging truck; Oidd AF062795 AB Canada. (received as 0. tenuissimum) DAOM 51071 Pinus contorta;AB Canada. Oid AF062796 (UAMH 8528) UAMH 8925 ex roots Vaccinium myrtilloides;AB Cana- Oid AF062797 da. (Hambleton & Currah, 1997) 0. maius Barron UAMH 1540 T, soil ex cedar bog; ON Canada. (Bar- Oid, Myx AF062798 ron, 1962) (received as 0. griseum) DAOM 184107 ex roots Vaccinium corymbosum;PQ Cana- Oid" AF062799 (UAMH 8529) da. (Couture et al., 1983) UAMH 8921 ex roots Vaccinium myrtilloides;AB Cana- Oidd AF062800 da. (Hambleton & Currah, 1997) UAMH 8922 ex roots Vaccinium vitis-idaea; AB Cana- Oidd AF062801 da. (Hambleton & Currah, 1997) 0. periconioidesMorrall DAOM 197506 T, soil; SK, Canada. Oid, Myx AF062802 (UAMH 8527) 0. pilicola Kobayasi UAMH 7526 forest soil; Sweden. Oid, Myx AF062787 0. rhodogenumRobak UAMH 1405 A, sludge in pulp strainers; Norway. Oid, Myx AF062803 0. scytaloidesGams & S6derstrom UAMH 6521 T, forest soil; Sweden. Oid, Myx AF062804 0. setiferumUdagawa & Toyazaki UAMH 5715 T, house dust; Japan. Oid, Myx AF062805 0. tenuissimum (Peck) Hughes CBS 238.31 T of 0. fuscum, wood pulp; Norway. (Ro- Oid, Myx AF062807 (UAMH 8511) bak, 1932) (= 0. tenuissimum, Barron, 1962) CBS 920.73 forest soil; Sweden. Oid AF062808 (UAMH 8512) 0. truncatum Barron UAMH 1399 T, soil ex mixed woods; ON Canada. Oid, Myx AF062809 0. sp. nov. CBS 315.95 leaf litter; Canary Islands Spain. Oid, Myx AF062806 (received as 0. tenuissimum) (UAMH 8513) MyxotrichumKunze M. arcticum Udagawa, UAMH 7565 T, forest soil; Alaska USA. Myx AF062810 Uchiyama & Kamiyae M. cancellatum Phillipse UAMH 1911 frozen blueberry pastry; NJ USA. Myx AF062811 M. carminoparumRobak UAMH 1597 T, wood pulp; Norway. Myx AF062812 M. chartarum (Nees) Kunze UAMH 1997 soil; Japan. Myx AF062813 M. deflexum Berkeley UAMH 6365 soil; ON Canada. Myx AF062814 M. setosum (Eidam) UAMH 3835 soil; AB Canada. Myx AF062815 Orr & Plunkettc

were purified using Wizard PCR Preps columns (Pro- formed using standardized methods, then processed mega, Madison, Wisconsin) following the manufac- and analysed on a ABI 373A automatic DNA sequenc- turer's instructions. er (Perkin-Elmer: Applied Biosystems, Foster, Califor- Automated DNA sequencing reactions were per- nia) following the protocols suggested by the manu- HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OIDIODENDRON 857

TABLEI. Continued

Data GenBank Species Straina Originb matrixc No. M. stipitatum(Lindfors) UAMH 1510 N, sand dune; England. Myx AF062816 Orr & Kuehn Byssoascusstriatosporus UAMH3572 T, soil; ON Canada. Myx AF062817 (Barron& Booth) von Arxe Gymnostellatosporajaponica UAMH8899 white spruce log, decay stage 5, fire site, Myx AF062818 Udagawa,Uchiyama & Kamiya AB, Canada. Pseudogymnoascusroseus Raillo UAMH 9163 ex roots Abies lasiocarpa;AB Canada. Oid, Myx AF062819 a Fungi were obtained from culture collections and original isolation work of S.H. Culture collection origin is noted for strainsnot previouslyat UAMH.CBS = Centraalbureauvoor Schimmelcultures,Baarn, The Netherlands.DAOM = Canadian Collection of Fungal Cultures,Ottawa, Canada. IMI = InternationalMycological Institute, Egham, United Kingdom.UAMH = Universityof AlbertaMicrofungus Collection and Herbarium,Edmonton, Alberta, Canada. h Ex-type(T), authentic (A) or neotype (N) strainswere used as standardsfor the species whereverpossible. c Strainswere included in one or both data matrices:Oid = Oidiodendron,Myx = Myxotrichaceae. d Strainsof Oidiodendronexcluded from the branch-and-boundanalysis of the Oidiodendrondata matrix. CTeleomorphic species with an Oidiodendronstate.

facturer. Sequences of complementary strands were from the comparison of 500 uncoded aligned nucle- determined for those strains serving as the reference otides at two taxonomic levels. At the intraspecific standards of the target species as well as any strains level, strains within the monophyletic clades contain- whose initial sequence was ambiguous. Consensus se- ing the ex-type strains of 0. griseum, 0. tenuissimum quences were determined for these and used in the and 0. maius were compared. At the interspecific lev- analyses. Preliminary alignments of DNA sequences el, pairwise comparisons were made between meiotic were performed using the automatic DNA sequencer and/or mitotic species. software package SeqEd ver. 1.0. Final alignments Data matrices were analysed using the maximum were optimized by hand. parsimony program PAUP 3.1.1 (Swofford, 1993). Two data matrices were used for analysis. The first Parsimony trees were constructed from a bootstrap alignment (Oidiodendron analysis) was generated us- analysis of each data matrix; 1000 replicate searches ing the sequences of all 23 strains of Oidiodendron were carried out using the heuristic search algorithm, listed in TABLEI, with Pseudogymnoascusroseus as the tree bisection-reconnection branch swapping, and outgroup taxon. The second alignment (Myxotricha- random stepwise sequence addition. For the Oidio- ceae analysis) was generated using the sequences of dendron data matrix, a second analysis using the the 15 Oidiodendron spp. and the 10 teleomorphic branch and bound search algorithm was performed. species of the Myxotrichaceae listed in TABLEI. For To permit timely execution of the data analysis, the 0. griseum, 0. tenuissimum, and 0. maius only the dataset was slightly reduced by excluding two strains ex-type strains were included. The last five nucleotide of 0. griseum (UAMH 1693, 4080) and three of 0. positions at the 3'0 end of the 18S small subunit maius (UAMH 8529, 8921, 8922). were used as the starting point of the aligned matri- Restriction fragment length polymorphism analysis ces. Sequences were coded to remove ambiguities in was carried out on five historically important Oidiod- the alignment, in the manner described by Bruns et endron strains that have been isolated from ericoid al. (1992), and gaps were counted as a fifth character. mycorrhizas, deposited in culture collections and For both P. roseus and G. japonica the first 58 bases documented in the literature (TABLEII). Methods were coded as missing since the alignment was too followed those previously detailed in Hambleton and ambiguous to perform with confidence. Though cod- Currah (1997) using four restriction enzymes RsaI, ing did not significantly affect the overall topology of AluI, HhaI, and Hinfl. Two separate extractions of the resulting trees, it was used as a check for the va- different subcultures were tested for these five strains lidity of the support indices. Sequences have been to provide confidence in the results. UAMH 8529 was in deposited GenBank (TABLEI) and the alignments also included in the Oidiodendronsequence analysis. used are deposited in TreeBASE (http://herbar- ia.harvard.edu/treebase/). Morphological.-All strains originally received as Oi- Sequence dissimilarity measures were determined diodendron griseum (UAMH 1403, 1693, 4080, 6514, 858 MYCOLOGIA

TABLEII. Strainnumber and origin of previouslydocumented ericoid mycorrhizalfungi that have been identified as Oi- diodendron maius using RFLP analysis Straina Origin Receivedas UAMH6514 ex roots Loiseleuria procumbens;AB, Canada. (Stoykeand Currah,1991) 0. griseum UAMH7022 ex roots Gaultheriashallon; BC, Canada. (Xiao and Berch, 1992; one of five 0. griseum 0. griseumstrains deposited from the study) ATCC66504 (UAMH8442) ex roots Rhododendronsp cv. Pink Pearl;Ireland. (Douglaset al, 1989) 0. maius CBS 334.52 (UAMH8507) ex roots Ericaceae;Sweden. (Burgeff,1961) 0. griseum DAOM184107b (UAMH 8529) ex roots Vacciniumcorymbosum; PQ Canada.(Couture et al., 1983;one of 0. griseum three 0. griseumstrains deposited from the study) a ATCC= AmericanType Culture Collection, Rockville,Maryland, USA. CBS = Centraalbureauvoor Schimmelcultures, Baarn,The Netherlands.DAOM = CanadianCollection of FungalCultures, Ottawa, Canada. UAMH = Universityof Alberta MicrofungusCollection and Herbarium,Edmonton, Alberta, Canada. Culture collection origin is noted for strainsnot previouslyat UAMH. b This strainwas also included in the sequence analysis.

7022, 8507, 8529, 8925), 0. tenuissimum (UAMH strains) and Pseudogymnoascus roseus comprised 540 8511, 8512, 8513, 8528), and O. maius (UAMH 1540, bases of which 218 were variable and 79 were parsi- 8442, 8921, 8922) (TABLESI, II) were examined for mony informative. The analysis was run with P roseus seven morphological characters: colonial morpholo- and Gymnostellatosporajaponica (both lack an Oidiod- gy; the production of diffusing pigment on agar; the endron anamorph state) as the outgroup but each range in length of conidiophores; and size, shape, one placed the root at the same position (data not color and surface texture of the conidia. These data shown). Attempts were made to include a taxon from were compared to the original descriptions of these one of the other three families in the Onygenales species (TABLEIII). (Gymnoascaceae, Arthrodermataceae, Onygenaceae) and colonial Pigment production morphology in the analysis. Although these families are hypothe- were characterized on dextrose (PDA: potato agar sized to represent the closest sister group to the My- Difco-Bacto) and malt agar (MEA: 2% malt extract, xotrichaceae according to traditional classification Difco-Bacto). Cereal (CER) slide culture mounts schemes, the sequences of selected taxa were too di- (Sigler, 1993), 16-28 d old, were used to measure vergent to be aligned successfully. Since the identity conidiophore length and characterize the conidia. of the true sister to the is un- Conidiophore length ranges were based on at least group Myxotrichaceae clear, P roseus, from within the was chosen as 25 random choices per slide. To standardize the re- family, taxon. sults, only the portion of the conidiophore that was outgroup darkly pigmented was included in the measurement. The 50% majority rule consensus tree generated Pigmentation was typically present for the entire by the bootstrap analysis is presented in FIG. 1 (308 length of the conidiophore and ceased at the septum steps). One of three most parsimonious trees (279 below the lowermost conidiogenous branches (dis- steps; consistency index 0.65) generated by the tance varied), which are hyaline. Size, shape, color branch and bound search algorithm is shown in FI(G. and surface texture of conidia (under oil, at X 1000) 2. Branch lengths are proportional to the number of were documented for 10 mature conidia randomly nucleotide changes along that branch. The overall chosen on the slide. topology of the two trees is the same and, while spe- Other Oidiodendronspecies and teleomorphic taxa cies relationships are clarified in FIG. 2, the lack of were grown in CER slide culture preparations in or- resolution in the bootstrap analysis indicates that der to compare the observed anamorphic character- some of these branches are not well supported. istics with herbarium documentation (University of Although the inferred phylogenies did not resolve Alberta Collection and Ed- Microfungus Herbarium, all the interspecific relationships in the genus, the ex- Alberta) and relevant discussions in the lit- monton, type strains of the target species of 0. 0. erature et and griseum, (Orr al., 1963; Sigler Carmichael, maius and 0. tenuissimum were delimited as distinct 1976; Currah, 1985; Udagawa et al., 1993, 1994). in separate monophyletic clades (FIG. 1). Similarly, several pairings of species with high bootstrap sup- RESULTS port and low interspecific sequence divergence were Oidiodendron analysis.-The aligned DNA se- revealed: 0. griseum/ 0. flavum, 0. maius/ 0. citrin- quence data matrix of 15 Oidiodendron species (23 urn, and 0. chlamydosporicum/O0. scytaloides (FI(. 2). HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OIDIODENDRON 859

0. pilicola 0. periconioidesT 0. echinulatumA 0. truncatumT 0. griseum (1693) O. griseum (1403)A "griseum" 0. griseum(4080) clade 0. griseum (8528) 0. griseum (8925) 0. flavum , O. cerealis 0. rhodogenumA 0. citrinumT 0. maius (8921) 0. maius "maius" (8922) clade 0. maius (8529) 0. maius (1540)T O.sp. nov. 0. tenuissimum(8511) T "tenuissimum" 0. setiferumT clade 0. tenuissimum(8512) 0. chlamydosporicumT - L.. 0. scytaloides T Pseudogymnoascus roseus

FIG. 1. Majority rule consensus tree (308 steps) resulting from 1000 bootstrap replications of maximum parsimony analysis of the Oidiodendron data set using the heuristic search algorithm of PAUP 3.1.1. Bootstrap values above 50% are given adjacent to the corresponding node. UAMH numbers are given to identify multiple strains for a species where needed and ex-type (T) and authentic (A) strains are indicated.

Sequence divergence between species outside these ing pigment, UAMH 1693 and UAMH 8925; and pairings ranged from 6 to 10%, except for 0. echin- UAMH 8528, which was received as 0. tenuissimum. ulatum/O. cerealisat 4%. Percent sequence dissimilarity between these strains The "griseum" clade (bootstrap 98; FIG. 1) com- was at most 1%. 0. flavum clusters with the 0. grn- prised UAMH 1403, an authentic strain; UAMH seum isolates and differs from the ex-type strain at 7 4080; two strains that produced a dark amber diffus- nucleotide positions (1.4%; FIG. 2). 860 MYCOLOGIA

0. pilicola 5 0. rhodogenum A

'hinulatumA

- 0. cerealis

-16 --- 0. truncatumT

10. griseum (1403) A 2 . griseum(8528) _ 1 0. griseum (8528) <1.5%

7 citrinum 1% 10l ||h12 O. maius (1540)T 24" 24li--- - - 0. sp. nov.

1 0. tenuissimum (8511)T

6 4 0. setiferum T <2.5%

6 0. tenuissimum(8512)

-11O 0. periconioides T

I 0. chlamydosporicum T -1 -3 0.8% O. scytaloides T

52 Pseudogymnoascus roseus

FIG. 2. One of three most parsimonious trees (279 steps, CI = 0.65) resulting from a maximum parsimony analysis of the Oidiodendrondata set (excluding two strains of 0. griseum and three of 0. maius) using the branch and bound algorithm of PAUP 3.1.1. Numbers above the branches indicate the number of nucleotide changes along each branch. UAMH numbers are given to identify multiple strains for a species where needed and ex-type (T) and authentic (A) strains are indicated. Sequence divergence measures for four monophyletic clades are indicated. HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OIDIODENDRON 861

The "maius" clade (bootstrap 100; FIG. 1) com- gence overall between species of Myxotrichumranged prised UAMH 1540, the ex-type strain, as well as from 6 to 12%. three strains isolated from ericoid mycorrhizas, RestrictionFragment Length PolymorphismAnalysis.- UAMH 8921, UAMH 8922, and UAMH 8529, which The RFLP patterns of all five strains in TABLEII, and was received as 0. Percent sequence dissim- griseum. for both extractions, were identical and matched ilarity between these strains was at most 0.8%. 0. ci- those published by Hambleton and Currah (1997) trinum also clusters with 0. maius and differs from for 0. maius. the ex-type strain at 5 nucleotide positions (1%; FIG. 2), of which one is an insertion/deletion event which Morphology.-A summary of the information from is probably due to replication slippage. the original species descriptions for the characters The "tenuissimum" clade (bootstrap 60; FIG. 1) measured in the morphological examination of comprised UAMH 8511, the ex-type strain; a second strains of Oidiodendron griseum, 0. tenuissimum and strain, UAMH 8512; and the ex-type strain of 0. se- 0. maius is given in TABLEIII. Results of the mor- tiferum. Percent sequence dissimilarity of the latter phological assessment of multiple strains of these spe- two with UAMH 8511 was less than or equal to 2.2% cies is given in TABLEIV; strains are grouped accord- (FIG.2) with 10 and 7 nucleotide differences respec- ing to their reidentification based on the molecular tively. analyses. Slight variations between the media used for Aside from these groupings, 0. chlamydosporicum the original diagnoses and the commercial prepara- and 0. scytaloides formed a closely related species tions used in this study may have affected colony pair (FIGS. 1, 2; bootstrap 100) with only 4 nucleotide characteristics somewhat. The upper end of the differences between them (0.8%), of which three are range in conidiophore length was considered more insertion/deletion events (two were coded out in the informative since a small proportion of the conidio- data matrix). phores in all strains were extremely short (<50 ,im). For the ex-type strains of all three species, the re- Myxotrichaceaeanalysis. -The aligned DNA sequence sults for each character were within the range of vari- data matrix generated using the 25 species listed in ability initially described (compare TABLESIII, IV). TABLE I comprised 547 bases of which 227 were vari- The results for most other strains were also congru- able and 129 were parsimony informative. The 50% ent within each of the three species after grouping majority rule consensus tree generated by the boot- based on the molecular assessment as summarized in strap analysis with Pseudogymnoascus roseus as out- TABLEIV. UAMH 8507, 0. maius, did not produce group taxon is presented in FIG. 3 (446 steps). Oi- conidia on agar or in slide culture. UAMH 8513, Oi- diodendron, Byssoascus, and Myxotrichum formed a diodendronsp., was different from all other strains for monophyletic group within the Myxotrichaceae, conidium color, conidium ornamentation and cul- while Gymnostellatosporajaponica clustered with the tural morphology. outgroup taxon P roseus, with high support (boot- A few strains were atypical for conidiphore length: strap 100). M. deflexum was basal to the rest of the UAMH 8528 and 8925, long for 0. griseum at 100/ species in the "Myxotrichum" ingroup. Sequence di- 150 to 225/250 ,xm; UAMH 8511, short for 0. ten- vergence between P roseus and G. japonica was 8.2% uissimum at less than 100 tm; UAMH 8529 and 8921 but much higher at 19% when either species was short for 0. maius at 100 to 200/250 ,um. Conidia of compared to M. deflexum. 0. griseum and 0. tenuissimum were distinctly pale Several terminal clusters of species received high brown when grouped in masses, but less distinct in- support. The species pairs 0. chlamydosporicum/0. dividually. Conidia of 0. maius were more frequently scytaloides, 0. tenuissimum/ 0. setiferum, 0. griseum/ hyaline but sometimes pale brown in masses. Conid- 0. flavum, and 0. maius/0. citrinum, observed in ium surface texture was smooth for 0. maius while the Oidiodendronanalysis, were repeated here. Three finely roughened for the other two species, though species of Myxotrichum clustered with anamorphic this distinction was not apparent except at high mag- taxa: M. arcticum with 0. griseum/O. flavum (boot- nification. strap 96), M. cancellatum with 0. echinulatum (boot- Conidium size exhibited a high degree of overlap strap 97), and M. setosum with 0. truncatum (boot- but was useful for 0. maius when paired with conid- strap 89). For the first two, the percent sequence di- ium shape. As well as the subglobose and ovoid co- vergence from their associated Oidiodendron species nidia seen in the other two species, in 0. maius the is very low (FIG. 3). Three other Myxotrichumspecies variable shapes included cylindrical conidia, and odd- formed a well supported divergent clade (bootstrap shaped conidia formed by septation and disarticula- 100) and sequence divergence amongst these species tion of the fertile hyphae across branching points. was much higher at 7.0 to 8.5%. Sequence diver- Colony size for all strains on MEA and PDA was 862 MYCOLOGIA

0. pilicola 0. periconioides 0. echinulatum 0.6% M. cancellatum (Oidiodendron) 0. truncatum 4.6% M. setosum (Oidiodendron) 0. cerealis 0. chlamydosporicum 0. scytaloides 0. rhodogenum 0. citrinum 0. maius 0. tenuissimum 0. setiferum 0. sp. nov. 0. griseum 0.60.6% M. arcticum (Oidiodendron)= 0. flavum 2% Byssoascus striatosporus (Oidiodendron) M. carminoparum (Malbranchea) M. chartarum (Malbranchea) I M. stipitatum (noanamorph) M. deflexum (infrequentarthroconidia) Pseudogymnoascus roseus (Geomyces) Gymnostellatospora japonica (Ovadendron4ike) FIG.3. Majority rule consensus tree (446 steps) resulting from 1000 bootstrap replications of maximum parsimony analysis of the Myxotrichaceae data set using the heuristic search algorithm of PAUP 3.1.1. Bootstrap values above 50% are given adjacent to the corresponding node. Anamorph states for the teleomorphic taxa are mapped on the tree and sequence divergence measures for four meiotic/mitotic species pairs are given.

restricted to approximately half of the petri plate, re- on MEA that was diagnostic but only after prolonged gardless of age. The production of an amber diffus- incubation (four wk or more); no diffusing pigment ing pigment into the rest of the agar was observed was produced on PDA. on both media for two strains of 0. griseum (UAMH Anamorph states for species in the Myxotrichaceae 1693 and 8925) but was absent for the others. For have been reported as and were observed as follows: both strains of 0. tenuissimum, pigment production Myxotrichumcarminoparum, M. chartarum, M. deflex- was strong on MEA but slight on PDA. Strains of 0. um, M. stipitatum: absent to infrequent arthroconi- maius produced an orange-brown diffusing pigment dia to chains of arthroconidia assignable to the genus HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OIDIODENDRON 863

TABLEIII. Comparison of morphological characteristics of Oidiodendrongriseum, 0. tenuissimum and 0. maius taken from the original descriptions of the type specimens

0. griseum 0. tenuissimum 0. maius Melin and Nannfeldt 1934 (= O. fuscum) Robak 1932 Barron 1962

Conidiophore length 40-150 jim (90-100) to 300 JLm(110-150) to 500 ,um (250-350) Conidium size 1.6-2 X 2-3.6 ,im 1.2-2.2 X 1.6-3.6 ,im 2-2.5 X 2.5-4 jim Conidium shape globose, subglobose, ovoid to globose, subglobose, ovoid globose, subglobose, short-cy- short-cylindric lindric Conidium color hyaline to dilute greenish-grey hyaline to brownish- green hyaline Conidium surface texture smooth to roughened not indicated smooth to roughened MEA: colonial morphology olive-grey to greenish grey brown to grey brown brown grey PDA: colonial morphology not indicated not indicated pale-grey to dirty-white

TABLEIV. Summary of morphological data for strains of Oidiodendron griseum, 0. tenuissimum and 0. maius used in sequence or RFLP analysis, grouped according to their reidentification based on the molecular analyses Diffus- Conidio- ing pig- phore Conidium mente length Conidium Conidium Conidium surface MEA/ Colonial morphology (}Lm)a size (jim)a shapeb colorc textured PDAf MEA/PDAf 0. griseum UAMH 1403g + + G, sG, shC pbr fine rough -/- MEA: submerged growth olive- UAMH 1693 + + G, sG pbr fine rough A/A black at center, hyaline margin; UAMH 4080 + + G, sG, shC pbr fine rough -/- conidiogenous layer grey-brown UAMH 8528 100-225 + shC pbr fine rough -/- PDA: varied, similar to 0. maius UAMH 8925 150-250 + shC pbr fine rough A/A but with a smoother surface texture 0. maius UAMH 1540g + + shC, C, B hyl-pbr smooth O/- MEA: orange-brown cast to agar UAMH 6514 + + shC, C, B hyl-pbr smooth O/- and colonies after 3+ wk; UAMH 7022 + + shC, C, B hyl-pbr smooth 0/- conidiogenous layer brownish UAMH 8442 + + shC, C, B hyl smooth O/- (except 8529, lighter grey- UAMH 8507 - - - - O/- brown, smoother) UAMH 8529 100-200 + shC, C, B hyl-pbr smooth O/- PDA: submerged mycelium dark; UAMH 8921 100-250 + shC, C, B hyl smooth O/- brownish-grey, granular UAMH 8922 + + shC, C, B hyl-pbr smooth O/- surface texture 0. tenuissimum UAMH 8511g few, < 100 + G, sG pbr fine rough A/sl MEA: varied, dark grey-brown UAMH 8512 + + G, sG pbr fine rough A/- PDA: mostly submerged, grey-purplish-black

0. sp. UAMH 8513 80-200 2.0 X 2-3.2 G dkbr echinulate A/sl MEA: dark brown, granular PDA: dark brown, raised center

a + = within ranges given in original species description. b G = globose, sG = subglobose, shC = short-cylindric, C = cylindric, B = odd shapes due to branching of fertile hyphae. c pbr = pale brown, hyl = hyaline, dkbr = dark brown. d Examined under oil at 1000X magnification. e A = dark amber pigment, 0 = orange-brown pigment after 4+ wk, sl = slight amount of amber pigment,-= pigment absent. f MEA = malt agar, PDA = potato dextrose agar. g Ex-type or authentic strain for the species. 864 MYCOLOGIA

Malbranchea; M. arcticum, M. cancellatum, M. seto- only one study recovered 0. maius (TABLEII). RFLP sum, Byssoascus striatosporus: Oidiodendron; Gymno- analysis confirms the morphological assessment that stellatosporajaponica: lacking in the type specimen, these strains are all conspecific with 0. maius. Conid- Ovadendron-like in this strain; P roseus: Geomyces. ium shape and ornamentation, and cultural morphology are consistent with the ex-type strain of 0. maius. UAMH 8507 has degenerated and no longer DISCUSSION forms conidia but culturally it exhibits similarities to Intraspecific analysis of Oidiodendron.-The com- 0. maius (orange-brown diffusing pigment on MEA). bined molecular and morphological examination of Hambleton and Currah (1997) showed that the multiple strains of three species in this study high- RFLP patterns of 0. maius and 0. griseum are distinct lights some species delimitation problems. Oidioden- from each other for the four restriction enzymes dron griseum, 0. tenuissimum and 0. maius, as cir- used. Within the genus Oidiodendron, these RFLP cumscribed, share similar cultural morphology and profiles of 0. maius are diagnostic for the species conidium color, and exhibit overlapping characteris- though the same cannot be said for 0. griseum (Ham- tics for conidium size, shape and surface texture (TA- bleton, unpubl. data). BLEIII). In spite of these morphological similarities, All of the major records of ericoid mycorrhizal Oi- the inferred phylogeny generated from the analysis diodendronisolates are now confirmed as strains of 0. of ITS sequences indicates that each species is dis- maius (Burgeff, 1961; Couture et al., 1983; Douglas tinct phylogenetically; the ex-type strains serving as et al., 1989; Stoyke and Currah, 1991; Xiao and species standards are resolved in three different Berch, 1992; Hambleton and Currah, 1997) though monophyletic clades (FIG. 1). Bootstrap support for for two of those studies not all of the deposited the cluster of strains within the clades containing strains were examined (Couture et al., 1983; Xiao these reference strains is robust for 0. griseum and and Berch, 1992). The importance of this clarifica- 0. maius (98 and 100 respectively) and in addition, tion is two-fold. The strains that have been used in the measures of sequence dissimilarity for the two experiments aimed at elucidating the role of the fun- species are very low (1% and 0.8% respectively). gus as a mycobiont (Dalpe, 1986; Abuzinadah and Conidiophore length has been considered a useful Read, 1989; Leake and Read, 1990; Haselwandter et key character, especially for 0. maius which is typified al., 1990; Xiao and Berch, 1995) have been those iso- by very tall conidiophores (up to 500 Vm) and long, lated by Couture et al. (1983) (though specific strain undulate fertile hyphae at the apex. 0. griseum is de- identity was not always cited) or by Xiao and Berch scribed as having very short conidiophores, most less (1992), giving the species 0. griseum an undeserved than 100 Jim, with shorter, straight fertile hyphae, reputation as a mycobiont. Results may also have while 0. tenuissimum is intermediate in conidiophore been misinterpreted when bonafide strains of 0. gri- length. In this study, conidiophore measurements seum were not observed to form ericoid mycorrhizas were made under standardized growth conditions, in pot cultures (for instance, UAMH 1403 in Douglas and those of the ex-type strains matched published et al., 1989). measurements. While the morphological and molec- Secondly, the results indicate that 0. maius is the ular assessments of most of the isolates sampled were only Oidiodendron species to have been confirmed congruent, the "maius" clade and the "griseum" from field-collected mycorrhizas. Hambleton and clade, as delimited by sequence analysis, both contain Currah (1997) reported very few strains of 0. griseum strains exhibiting atypical conidiophore ranges. (for example, UAMH 8925) in their study but spec- The "maius" clade contains an isolate (UAMH ulated that this extremely low recovery indicated that 8529) derived from the first study to document 0. these were rhizosphere fungi that occasionally sur- griseum as an ericoid mycorrhizal fungus (Couture et vived the root sterilization process. Preliminary re- al., 1983). The identification of this strain as 0. gri- sults of experiments in progress to test whether this seum is not surprising since conidiophore lengths for strain forms mycorrhizas in axenic resynthesis with UAMH 8529 (one of three strains from that study, all the host species indicate that it does not (Hambleton, reported to look similar) are less than 200 jm, rather unpubl. data). than up to 500 jLmas is typical for 0. maius. Another The "griseum" clade contains strains isolated from ericoid isolate, UAMH 8921, also produces conidio- a wider range of substrates, of which two produce a phores which are relatively short, yet in the molecular striking amber diffusing pigment on PDA and MEA. analysis these two strains cluster with 0. maius with For one of these, UAMH 8925, and for the isolate less than 1% sequence divergence. received as 0. tenuissimum, UAMH 8528, conidio- There are three other studies documenting the iso- phore lengths ranged up to 250/225 jxm. The mo- lation of 0. griseumfrom roots of the Ericaceae, while lecular analysis, then, shows that conidiophore HAMBLETON ET AL.: PHYLOGENETIC RELATIONSHIPS OF OIDIODENDRON 865 length is not a reliable character for these species: 0. FIGS. 1, 2 but a lack of resolution of these interspe- maius may produce very short conidiophores in the cific relationships is evident from the number of range of 0. griseum and 0. tenuissimum and vice-ver- branches which received bootstrap support of less sa. Since conidiophore length range for 0. tenuissi- than 50. The relatively low consistency index of the mum is reported as intermediate, all three species tree in FIG. 2 indicates that data from a more con- overlap for this character. served region, such as the 28S subunit, are needed In the original description of 0. tenuissimum (= to resolve the phylogeny. In contrast, several species 0. fuscum; Hughes, 1958, p. 790) one important fea- pairs at the terminal nodes are supported by high ture noted as absent from 0. griseum was the pro- bootstrap values and also exhibit low sequence dis- duction of a dark, diffusing pigment on agar. Unfor- similarity measures, similar to those observed at the tunately, surface ornamentation of the conidia was intraspecific level. not mentioned, and indeed this is a difficult charac- For 0. griseum/O. flavum, 0. maius/O0. citrinum, ter to resolve for such small conidia using the light and 0. chlamydosporicum/O. scytaloides,sequence di- microscope. Barron (1962), after identifying numer- vergence between the paired taxa is very low, less ous strains isolated from soil as 0. tenuissimum, noted than 1.5%, and bootstrap support is high (98 or 100) that the species was highly variable as to pigment pro- for their clustering. 0. flavum and 0. citrinum are duction but could be distinguished from 0. griseum distinguished from their paired species primarily by by dark and echinulate conidia. Although UAMH a yellow conidium color (Barron, 1962), whereas the 8513, received as 0. tenuissimum, demonstrates these difference between 0. chlamydosporicumand 0. scy- conidium characteristics and is also positive for pig- taloides is based on the size of chlamydospores and ment production, it diverges significantly from the conidia (Gams and S6derstr6m, 1983). The molecu- "tenuissimum" clade in the phylogenetic analyses lar data suggest that these morphological criteria are (FIGS. 1, 2). too variable to be used as key characters or are only In the ex-type strain UAMH 8511, and UAMH significant at the subspecies level. 8512, the conidia were pale brown and only slightly In the "tenuissimum" clade, 0. setiferum is more roughened and in these respects similar to 0. gri- closely related phylogenetically to the ex-type strain seum. Based on the divergent sequences and on other of 0. tenuissimum than is a second strain, and though distinctive morphological features, UAMH 8513 is the sequence divergence is still under 2.5% for that considered to represent an undescribed species. The species, bootstrap support for the clade is low. In production of a dark diffusing pigment on agar me- many respects the description of 0. setiferumis simi- dia is not a species specific attribute since it has been lar to that of 0. tenuissimum but the species is unique observed for several species in addition to 0. tenuis- in its production of dematiaceous setae at the apex simum and 0. griseum, but because production varies of the conidiophore (Udagawa and Toyazaki, 1987). depending on agar type, it still may be useful for sort- More sequence data are needed for both species in ing species groups. order to evaluate the cohesiveness of their inferred Overall, the results indicate that there are few mor- relationship. phological characters that can be used reliably to In this study molecular characters have been used identify Oidiodendrongriseum, 0. tenuissimum and 0. to clarify taxonomic decisions that were made based maius and yet the ITS analysis provides unambiguous on morphology and to impose a phylogenetic per- support for their validity as distinct phylogenetic spe- spective on species concepts in the genus Oidioden- cies. Scanning electron microscopy of the conidia dron. Such an approach involves making decisions may provide new useful characters. In our prelimi- about the level of sequence variation that character- nary examination of this approach, differences in izes each taxonomic level. Seifert et al. (1995) have surface texture have been consistent with the molec- surveyed sequence divergence measures reported for in the ITS of rDNA. ular analysis for strains of 0. griseum and 0. maius. ascomycetous fungi regions The Careful observations of colonial features, in particu- wide variation and often overlapping measures for lar color, also appear predictive but these may be dif- intra- versus interspecific values is thought to be due ficult to standardize for general use. to inconsistency in the rate of evolution of these regions (Seifert et al., 1995). Therefore taxonomic Interspecificrelationships within Oidiodendron.-Mo- rankings based on molecular analysis should be at- lecular analysis reveals that within the genus Oidiod- tempted with caution and linked to an assessment of endron there is a high degree of sequence substitu- other characters common to the taxa within mono- tion in the ITS region of the rDNA. Sequence diver- phyletic groups. gence varies from 6 to 10% among species at the A comparison of the divergence measures of other internal nodes of the phylogenetic trees shown in ascomycetous taxa based on both ITS1 and ITS2 with 866 MYCOLOGIA the 5.8S gene, such as 6.3% for Metarhizium aniso- here suggest that a separate monotypic genus is not pliae (Curran et al., 1994), 10.5% for Beauveria brong- appropriate for this taxon as it clusters with high sup- niartii (Neuveglise et al., 1994) and 0% for three spe- port with the other Oidiodendronspecies in both the cies of Penicillium (LoBuglio et al., 1994), indicates Oidiodendronand the Myxotrichaceae analyses. that the intraspecific values for Oidiodendrongriseum and 0. maius (1%) are relatively conservative. By Anamorph-teleomorphrelationships.-Within the My- contrast, the much higher interspecific sequence di- xotrichaceae, species of Oidiodendron, Myxotrichum vergence measures for Oidiodendron (6 to 10%) and and Byssoascus form a well-supported monophyletic Myxotrichum (6 to 12%) suggest that sequence diver- group that has diverged significantly from the two gence is useful for distinguishing phylogenetic other genera in the family. We judged Pseudogym- groups at the two taxonomic levels in this group of noascus roseus and Gymnostellatosporajaponica to be fungi. equally effective as outgroup taxa because they were Without the resorting of strains based on the mo- unalignable to the rest of the data matrix for the first lecular analysis, the intra- and interspecific measures 58 nt and were less divergent from each other than for the genus Oidiodendronwould have overlapped. from M. deflexum, the most basal species of the in- In our view, this resorting is validated by the corre- group. In addition, neither produces an Oidioden- lation of several morphological characters and my- dron anamorph state. corrhizal lifestyle with the phylogenetic species Relationships of the teleomorphic taxa within the groupings of 0. griseum and 0. maius (TABLE IV). monophyletic clade appear correlated with the ana- Similarly, Kuhls et al. (1997) reported intraspecific morph state produced. Myxotrichumdeflexum, which divergence between 15 strains of Hypocreaschweinitzii produces infrequent alternate arthroconidia, is basal, as 6.1%. When strains were sorted into groups based while the three Myxotrichum species with either no on sequence type, the divergence within each group anamorph or with a Malbranchea-likeanamorph state was at most 0.3%, which supported morphological (M. carminoparum, M. chartarum and M. stipitatum) and isozyme data suggesting that H. schweinitziicom- cluster together in a well-supported clade excluding prises several phylogenetic species. all species of Oidiodendron. Byssoascus striatosporus, Based on the low sequence divergence and an eval- M. arcticum, M. cancellatum, and M. setosum, all with uation of the morphological criteria used to delimit distinct or reported Oidiodendron anamorph states, these 0. and 0. species, chlamydosporicum scytaloides are nested with the Oidiodendronspecies. are assessed as conspecific. Chlamydospore produc- Both M. arcticum and M. cancellatum exhibit less tion is a attribute and the recorded variation unique than 1% sequence divergence with a paired anamor- in chlamydospore and conidium dimensions are sim- phic species (FIG.3). Based on earlier arguments, this ilar to the levels of size variation seen in the genus result could indicate conspecific identity for these as a whole. Similarly, 0. maius and 0. citrinum are taxa. M. arcticumproduces an Oidiodendronstate that assessed to be conspecific. The yellow conidium color is morphologically typical of 0. griseum though some may be a phenotypic response to characteristics of conidiophores form a geniculate head with single co- the original substrate, since for some strains it has nidia produced over the surface (Udagawa et al., been observed to fade in storage but this morpho- 1994). The conidial state of M. cancellatum was ob- logical variant could be given variety status. While served to be very similar to the strain of 0. echinu- similar justification could be used for merging 0. fla- latum used in this study but neither produced the vum with 0. griseum, judgment is reserved pending conidia for the latter analysis of more strains. distinctly roughened reported In both more strains need to be ex- The taxonomic placement of 0. cerealis has been species. cases, amined before the subject of some debate, such that strains are cur- any judgments concerning conspecif- rently found deposited also as Stephanosporium cer- icity are made. ealis (Thfim) Swart. Either placement can be found The position of Byssoascus striatosporuswithin the in the hyphomycete literature [compare Domsch et "Myxotrichum" clade is interesting because it sug- al. (1980, p. 518) with Ellis (1971, p. 35)]. Barron gests that the generic concepts of Myxotrichumand (1962) placed the species in Oidiodendron because Byssoascus need to be reexamined. Byssoascus stria- conidiogenesis follows the same ontogeny as other tosporusforms ascomata atypical for the family (Cur- species in the genus. Later, Swart (1965) emphasized rah, 1985) in which the fruiting body (telaperidium) the unusual lens-shaped conidia with a dark equato- is a cobweb-like envelope of thin-walled hyphae rial band in erecting a new combination, S. cerealis, scarcely differentiated from the vegetative hyphae. to deal with several conspecific strains that had been Species of Myxotrichum form a distinctive reticulo- given names in three genera. The analyses presented peridium made up of a mesh of thick-walled, demat- HAMBLETONET AL.: PHYLOGENETICRELATIONSHIPS OF OIDIODENDRON 867 iaceous peridial hyphae, often ornamented with pig- ACKNOWLEDGMENTS mented appendages. The authors wish to thank Lynne Sigler, Univ. of Alberta was Byssoascus striatosporus (a monotypic genus) Microfungus Collection and Herbarium, for helpful discus- erected by von Arx (1971) but Sigler and Carmichael sions on the taxa studied; Pat Murray, Dept. of Biological (1976) recommended that the taxon be transferred Sciences DNA Synthesis Lab (Univ. of Alberta), for per- to Myxotrichum. In addition to the marked similari- forming the ABI sequencing protocols; and the anonymous ties in ascospore morphology and cellulolytic ability reviewers who provided thoughtful comments on an earlier version of the for this work was of the two genera, this recommendation was based manuscript. Support provided the Grants on observed ascospore production amongst blunt, by Biodiversity Program jointly sponsored by the Alberta Dept. of Environmental Protection, Fish and dematiaceous, thick-walled hyphae on cereal agar, Wildlife Trust Fund and the Univ. of Alberta Dept. of Bio- of ascom- similar to the peridial hyphae Myxotrichum logical Sciences; and by the Natural Sciences and Engi- ata. Due to the lack of a distinct peridium, though, neering Research Council of Canada with a postgraduate the transfer was not adopted in subsequent mono- scholarship to SH and operating grants to KNE and RSC. graphs in which fruiting body characteristics were at the level and Kim- emphasized genus (Benny LITERATURE CITED brough, 1980; Currah, 1985). Historically, M. deflexum was renamed as Eidamella Abuzinadah, R. A., and D. J. Read. 1989. The role of pro- teins in the nutrition of deflexa ( also a monotypic genus) by Benjamin (1956) nitrogen ectomycorrhizal with the statement that it did not fit the then-current plants. V. Nitrogen transfer in Birch (Betula pendula) grown in association with mycorrhizal and non-mycor- concept of the genus Myxotrichum, but more recent rhizal fungi. New Phytol. 112: 61-68. treatments do not concur et (Orr al., 1963; Benny Barron, G. L. 1962. New species and new records of Oi- Ben- and Kimbrough, 1980; Currah, 1985). Though diodendron. Canad. J. Bot. 40: 589-607. jamin's criteria were not clearly defined, they were Benjamin, R. K. 1956. A new genus of the Gymnoascaceae apparently based on whether appendages are differ- with a review of the other genera. Aliso 3: 301-328. entiated from the peridial hyphae. However, append- Benny, G. L., and J. W. Kimbrough. 1980. A synopsis of the age characteristics have since been used for interspe- orders and families of plectomycetes with keys to genera. 1-91. cific distinctions. Data from more strains and from Mycotaxon 12: Bruns, T. D., R. Vilgalys, S. M. Barns, D. Gonzalez, D. S. more conserved of DNA need to be evaluat- regions Hibbett, D. J. Lane, L. Simon, S. Stickel, T. M. Szaro, ed to assess the phylogenetic stability of ascomatal W. G. Weisburg, and M. L. Sogin. 1992. Evolutionary characters for inferring relationships in this group. relationships within the fungi: analyses of nuclear small Sigler and Carmichael (1976) noted similarities be- subunit rRNA sequences. Molec. Phylogenet. Evol. 1: tween the Oidiodendron state of Byssoascus striatospo- 231-241. rus and that of M. setosum in which the conidia are Burgeff, H. 1961. Mikrobiologiedes Hochmoores.Gustav Fi- scher 197 described as barrel-shaped. Though conidiophore Verlag, Stuttgart, Germany. pp. Couture, M., J. A. Fortin, and Y. Dalpe. 1983. Oidiodendron production was poor for the strain of M. setosum ex- griseum Robak: an endophyte of ericoid mycorrhiza in it is in the amined, interestingly paired phylogenetic Vaccinium spp. New Phytol. 95: 375-380. analysis with 0. truncatum (4% sequence diver- Currah, R. S. 1985. Taxonomy of the Onygenales: Arthro- gence), with relatively high support. In 0. truncatum dermataceae, Gymnoascaceae, Myxotrichaceae and conidia are barrel-shaped rather than the more com- Onygenaceae. Mycotaxon 24: 1-216. monly seen ovoid or cylindrical shapes. Curran,J., F. Driver,J. W. 0. Ballard, and R.J. Milner. 1994. of Metarhizium: of ribosomal DNA The molecular analysis confirms the initial hypoth- Phylogeny analysis sequence data. Mycol. Res. 98: 547-552. esis that the hyphomycete genus Oidiodendron is, as Dalp6, Y. 1986. 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