Mycologia - Taylor & Francis Online

Mycologia, 99(6), 2007, pp. 884–894. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897

Tubeufia asiana, the teleomorph of Aquaphila albicans in the Tubeufiaceae, Pleosporales, based on cultural and molecular data Clement K.M. Tsui1

Helicoma chlamydosporum. This is the first molecular phylogenetic study to place a nonhelicosporous species within the Tubeufiaceae and to show that helical conidia were lost at least once within the family. Key words: Acanthostigma, aquatic, freshwater, phylogeny, systematics, Taphrophila, Tubeufia, Tubeufiaceae

Department of Botany, 3529–6270 University Boulevard, University of British Columbia, Vancouver, BC V6T 1Z4 Canada

Somsak Sivichai BIOTEC-Mycology, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand

INTRODUCTION

Amy Y. Rossman

Aquaphila Goh, K.D. Hyde & W.H. Ho is a small anamorphic genus consisting of the type, A. albicans Goh, K.D. Hyde & W.H. Ho, and a second species, A. ˜ eda Ruı´z, Guarro & Gene´. The edentata R.F. Casten two species were described from decaying wood (Goh and Hyde 1998, Casten ˜ eda Ruı´z et al 2000). Aquaphila albicans produces hyaline, multiseptate, falcate conidia that resemble the macroconidia of Fusarium Link but it differs in having sympodial, denticulate conidiogenous cells. In their conidiogenous cells Aquaphila species resemble species of Helicomyces Link and Dactylaria Sacc. (Goh and Hyde 1998). Before this study a teleomorph for Aquaphila had not been reported and its relationship to other fungi was unknown. During a biodiversity survey of fungi on submerged wood in Thailand, A. albicans and an adjacent meiotic ascomycete were isolated. The meiotic ascomycete produced superficial, dark brown, globose, soft-textured pseudothecia covered with setae. Its asci were bitunicate, cylindroclavate, and the ascospores were fusoid to cylindrical, slightly curved, hyaline to pale brown with multitransverse septa. These characteristics agreed largely with the circumscription of the Tubeufiaceae. Tubeufia Penz. & Sacc. sensu Barr (1980) is characterized by brightly colored to dark brown ascomata with or without setae, by bitunicate asci and by fusiform, hyaline ascospores (Barr 1980). Two other genera in the Tubeufiaceae, Acanthostigma de Not. and Taphrophila Scheuer, resembled the meiotic ascomycete in having ascomata with setae (Scheuer 1991, Re´blova´ and Barr 2000). Previous analysis of ribosomal SSU, ITS and partial LSU data has shown that Tubeufia sensu Barr (1980) and most species of the anamorphic genera Helicoma Corda, Helicomyces, and Helicosporium Nees were close relatives, clustering with 100% bootstrap support in a clade designated ‘Tubeufiaceae s. str.’ (Tsui and Berbee 2006). After comparison with descriptions or

Systematic Botany & Mycology Laboratory, Room 304, B011A, 10300 Baltimore, Maryland 20705

Mary L. Berbee Department of Botany, 3529–6270 University Boulevard, University of British Columbia, Vancouver, BC V6T 1Z4 Canada

Abstract: The teleomorph of Aquaphila albicans was discovered on submerged wood collected in Thailand. Its black, soft-textured, setose ascomata, bitunicate asci and hyaline to pale brown, multiseptate ascospores indicated an affinity to Tubeufiaceae (Dothideomycetes). After morphological or molecular comparisons with related species in Tubeufia, Acanthostigma and Taphrophila, it is described and illustrated as a new species, T. asiana Sivichai & K.M. Tsui, sp. nov. Finding this Tubeufia teleomorph was surprising, given the falcate conidia of its A. albicans anamorph, which superficially resemble the conidia of Fusarium and not the coiled, helicosporous conidia of other species in Tubeufiaceae. We assessed the phylogenetic relationships of A. albicans-T. asiana with ribosomal sequences from SSU and ITS and partial LSU regions by parsimony and Bayesian analysis. An initial set of 40 taxa representing a wide range of ascomycete families and their SSU sequences from GenBank showed A. albicans-T. asiana to be nested within the Tubeufiaceae with 100% bootstrap support. Their placement was inferred with ITS and partial LSU ribosomal sequences. The nearly identical ITS sequences of two isolates of A. albicans and one isolate of Tubeufia asiana united these fungi as a monophyletic group with 100% bootstrap support and further nested them, with 88% bootstrap support, in a clade containing Helicoon gigantisporum and Accepted for publication 10 September 2007. 1 Corresponding author. E-mail: [email protected]

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TSUI ET AL: A TUBEUFIA TELEOMORPH FOR AQUAPHILA ALBICANS authentic specimens and cultures of species and genera in the Tubeufiaceae, we describe the ascomycete collected from a river in Thailand as a new species in Tubeufia. We had suspected that the A. albicans was the anamorph of the new Tubeufia because isolates from each were almost identical in pure culture. We analyzed sequences from the SSU, 5.8S, ITS regions and partial LSU regions to determine the relationships among A. albicans, the new Tubeufia, and other species in Tubeufiaceae. MATERIALS AND METHODS

Taxon sampling.—Tubeufia and A. albicans were isolated from decaying wood in freshwater habitats (TABLE I). Single spore isolation was performed in which the asci and ascospores within the Tubeufia ascomata were transferred onto CMA (cornmeal agar, Difco) and then at least eight germinated ascospores were transferred to a new plate. The resulting cultural morphology was compared. The conidia of A. albicans also were cultured on CMA. The cultures were maintained on CMA and PDA (potato-dextrose agar, Difco) at 20 C under white fluorescent light and deposited in BCC and CBS. The isolate of Tubeufia cylindrothecia AR4206 was isolated from ascospores and deposited at CBS (CBS120503). Additional cultures of Acanthostigma perpusillum were borrowed from the University of Alberta Microfungus Collection and Herbarium (UAMH), Canada, and the Belgian Coordinated Collections of Microorganisms (MUCL). Sporulation of cultures was induced by inoculation onto SNA (synthetic nutrient-poor agar) (KH2PO4, 1 g; KNO3, 1 g; MgSO4.7H2O, 0.5 g; KCl, 0.5 g; glucose, 0.2 g; sucrose, 0.2 g; yeast extract, 0.5 g; agar, 20 g; distilled water, 1 l). Authentic and type specimens of Taphrophila cornu-capreoli were borrowed from Mycological Herbarium (IMI), CABI, Egham. DNA extraction, PCR amplification and sequencing.—Fungal mycelium was scraped from Petri dishes, and DNA was extracted with a DNeasyH Plant Mini Kit (QIAGEN Inc., Mississauga, Ontario) according to the instructions supplied. Small subunit (SSU) rDNA was amplified with SL1 (Landvik et al 1997) and CITS5 (complementary sequence of ITS5) (White et al 1990). ITS and partial LSU regions of rDNA were amplified with primers ITS1F and TW13 (http://plantbio.berkeley.edu/,bruns/primers.html#its). A total of 12.5 mL diluted DNA was amplified in a total volume of 25 mL with puReTaqTM Ready-To-GoTM PCR beads (Amersham Biosciences Corp., Piscataway, New Jersey) with 10 mL of H2O and 1.25 mL of each of the two primers (10 mM). The amplification was done in a GeneAmpH PCR system 9700 (Applied Biosystems, Foster City, California) with this program: 5 min of denaturation at 95 C, 35 cycles at 95 C for 20 s, 55 C for 30 s, 72 C for 40 s, a final extension at 72 C for 7 min and a 4 C soak. The PCR product was precipitated and sequencing reactions were carried out with ABI PRISMH BigDyeH Terminator v3.1 Cycle Sequencing Kit (PE Applied Biosystems, Foster City, California) with primers SL1, CITS5 (Inderbitzin et al

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2001), NS2, NS4 (White et al 1990), NS19, MB1 and MB2 (sequences available through http://plantbio.berkeley. edu/,bruns/tours/primers.html#18s) for SSU region. ITS and partial LSU regions were sequenced with primers ITS1F, ITS4, CTB6 (White et al 1990) and TW13. The sequences were determined at the Nucleic Acid Protein Service unit at the University of British Columbia. Fragments were assembled with ABI PRISMH AutoAssemblerTM v.1.4 (Applied Biosystems, Foster City, California). GenBank accession numbers of sequences are DQ341091–DQ341103 (TABLE I). Phylogenetic analyses.—Sequences were aligned with ClustalX (Thompson et al 1997) (distributed by the authors, ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) and adjusted manually with Se-Al v.1 d1 (Rambaut 1999). The alignment initially was analyzed with PAUP* 4.0b10 (Swofford 2003). Maximum parsimony (MP) analyses were conducted with a heuristic search with 100 random-sequence addition replicates and tree bisection-reconnection branch swapping algorithms, with MAXTREES set at 200. All characters were equally weighted and unordered, and gaps were treated as missing data. Bootstrap support for the branches was based on 1000 MP replicates with a single sequence addition replicate for each bootstrap replicate. Bayesian inference of phylogeny was calculated with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001). Modeltest 3.7 was used to select the general time reversible model of substitution and among site variation described by a gamma distribution (Posada and Crandall 1998). The final dataset was partitioned into ITS and LSU regions. Four simultaneous Markov chains were run from random starting trees for 1 000 000 generations, and trees were sampled every 100 generations (generating 10 001 trees). The first 5000 trees were discarded as burn-in; hence inferences of posterior probability were calculated from 5001 trees.

TAXONOMY

Tubeufia asiana Sivichai & K.M. Tsui sp. nov. FIGS. 1–6 Ascomata 180–300 3 160–280 mm, atrobrunnea ad nigra, setosa, ca. 60 mm longa, 6–8 mm lata, gregaria, superficialia, globosa ad subglobosa. Asci 100–120 3 15–18 mm, bitunicati, cylindro-clavati, octospori, pedicellati. Paraphyses septatea, ramificantes. Ascosporae 34–53 3 4.5–6.5 mm, biseriates, fusiformes, hyalinae vel dilute brunneae, 7– septatae.

HOLOTYPE: THAILAND, Prachin Buri Province, Khao Yai National Park, stream at km 29.2, on wooden test block (Acacia oblonga) submerged 5 mo, 15 Mar 1997, S. Sivichai (BIOTEC SS260). Etymology: in Latin, asiana means Asia, with reference to the continent where the fungus was isolated. Ascomata 180–300 3 160–280 mm, globose to subglobose, dark brown to black with brown, straight, thick-walled, setae up to 60 3 6–8 mm, tapering to an acute apex, gregarious, superficial. Peridium outer layer cells black, angular; inner layer cells pale brown

886 TABLE I.

MYCOLOGIA Taxa used in this investigation GenBank accession numbers Fungal isolates

Asexual fungi Aquaphila albicans Aquaphila albicans Helicoma ambiens Helicoma chlamydosporum Helicoma conicodentatum Helicoma dennisii Helicoma perelegans Helicoma morganii Helicoma muelleri Helicoma palmigenum Helicoma pulchra Helicoma sp. Helicoma vaccinii Helicoma violaceum Helicomyces bellus Helicomyces lilliputeus Helicomyces macrofilamentosus Helicomyces roseus Helicomyces roseus Helicomyces roseus Helicomyces scandens Helicomyces torquatus Helicoon gigantisporum Helicosporium abuense Helicosporium citreo-viride Helicosporium gracile Helicosporium griseum Helicosporium griseum Helicosporium guianensis Helicosporium indicum Helicosporium linderi Helicosporium lumbricoides Helicosporium pallidum Helicosporium pallidum Helicosporium panachaeum Helicosporium phragmitis Helicosporium sp. Helicosporium sp. Helicosporium talbotii Helicosporium vegetum Helicosporium vegetum Helicosporium vegetum Helicosporium vegetum Ascomycetes Acanthostigma perpusillum Tubeufia asiana Tubeufia amazonensis Tubeufia cerea Tubeufia cerea AFTOL Tubeufia cylindrothecia Tubeufia cylindrothecia Tubeufia helicoma Tubeufia helicoma Tubeufia helicoma (as Tubeufia paludosa) Tubeufia paludosa (as Tubeufia helicomyces)

Sources of culture

ITS regions

LSU

BCC3520 BCC3543 UAMH 10533 CBS 160.69 UAMH 10534 NBRC 30667 ATCC 22621 CBS 281.54 CBS 964.69 NBRC 32663 MUCL 39827 BCC 3512 CBS 216.90 CBS 222.58 CBS 113542 NBRC 32664 HKUCC 10235 BCC 8808 CBS 283.51 BCC 3381 CBS 113453 CBS 189.95 BCC 3550 CBS 101688 NBRC 7098 CBS 284.54 UAMH 1694 CBS 961.69 UAMH 1699 CBS 374.93 NBRC 9207 JCM 9265 CBS 962.69 UAMH 10535 CBS 257.59 CBS 271.52 CBS 269.52 UBC F14999 MUCL 33010 CBS 941.72 BCC8125 BCC 3332 NBRC 30345

DQ341098* DQ341096* AY916451 AY916466 AY916450 AY916455 AY916463 AY916468 AY916453 AY916480 AY916457 AY916484 AY916486 AY916469 AY916475 AY916483 — AY916481 AY916464 — — AY916472 AY916467 AY916470 AY916478 AY916485 AY916473 AY916474 AY916479 AY916477 AY916454 AY916476 AY916460 AY916462 AY916471 AY916461 AY916487 AY916456 AY916465 AY916488 AY916491 AY916490 —

DQ341102* DQ341101* AY856916 AY856875 AY856869 AY856897 AY856912 AY856876 AY856877 AY856898 AY856872 AY856905 AY856879 AY856880 AY916088 AY856899 AY849942 AY856910 AY856881 AY787932 AY916089 AY856882 AY856904 AY916085 AY856894 AY916086 AY856902 AY856884 AY856891 AY856885 AY856895 AY856889 AY856886 AY856913 AY916087 AY856887 AY856893 AY856870 AY856874 AY856883 AY856909 AY856907 AY856896

UAMH 7237 BCC3463 ATCC 42524 NBRC 9014

AY916492 DQ341097* AY916458 AY916489 — — DQ341095* — AY916452 — AY916459

AY856892 DQ341100* AY856911 AY856903 DQ471982 AY849965 DQ341103* AY856888 AY856917 AY849967 AY856873

BCC 3559 CBS120503 JCM2739 UBC F13877 HKUCC 9118 MUCL 15702

TSUI ET AL: A TUBEUFIA TELEOMORPH FOR AQUAPHILA ALBICANS TABLE I.

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Continued GenBank accession numbers Fungal isolates

Tubeufia sp. Tubeufia sp. Tubeufia sp.

Sources of culture BCC 3585 BCC 3580 MLTS 119

ITS regions

LSU

AY916482 — —

AY856908 DQ296554 DQ296555

*

new sequences determined in this investigation. ATCC: American Type Culture Collection, Manassas; CBS: Centraalbureau voor Schimmelcultures, Netherlands; BCC: Biotec Culture Collection, Thailand; MUCL: Belgian Coordinated Collections of Micro-organisms; JCM: Japan Collection of Microorganisms; NBRC: NITE Biological Resource Center, Japan; UAMH: University of Alberta Microfungus collection and Herbarium; UBCF: University of British Columbia Herbarium.

FIGS. 1–5. Interference contrast micrographs of Tubeufia asiana. 1. Ascoma dark brown to black covered with setae (arrows). 2–3. Hyaline to pale brown, fusiform ascospores with rounded ends. Squash mount of asci and pseudoparaphyses. 4– 5. Cylindrical-clavate bitunicate asci. Bars: 1 5 50 mm, 2–5 5 10 mm.

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MYCOLOGIA superficial, consisting of septate, branched, hyaline, smooth, thin-walled hyphae. Conidiophores hyaline, arising singly as lateral branches from procumbent hyphae, up to 60 mm long, 4–5 mm wide, simple or branched, thin-walled and smooth, indistinctly septate, flexuous or geniculate. Conidiogenous cells monoblastic or polyblastic, sympodial with cylindrical denticles, hyaline, ca. 2 3 2–2.5 mm, many integrated as tiny pegs, other as simple lateral sympodial cells. Conidia 50–75 3 7.5–10 mm, holoblastic, solitary, borne acrogenously on denticles, hyaline or pale yellowish, predominantly fusoid to sickle-shaped, sometimes sigmoid, rarely straight, obclavate, thinwalled, smooth, 6–14 euseptate, not constricted at septa, densely guttulate, conically rounded at both ends, slightly broad at base, basal cell obconical, pedicellate. Conidial secession schizolytic. Materials examined: THAILAND, Prachin Buri Province, Khao Yai National Park, stream at km 29.2, on wooden test block (Acacia oblonga) submerged 6 mo, 12 Jun 1997, S. Sivichai (BIOTEC SS354); — Nakorn Ratchassima Province, Khao Yai National Park, stream at Tad Ta Phu, on wooden test block (Dipterocarpus alatus) submerged 2 mo, 13 Oct 1997, S. Sivichai (BIOTEC SS393); — stream at Tad Ta Phu, on wooden test block (Xylia dolabriformis) submerged 6 mo, 25 Mar 1997, S. Sivichai (BIOTEC SS478).

FIG. 6. A line drawing showing the morphological characters of Tubeufia asiana. A. Asci. B. Ascospores. C. Setae of ascomata. Bars 5 10 mm.

to subhyaline, elongate. Asci 100–120 3 15–18 mm, bitunicate, cylindroclavate, rounded apical and 8spored. Pseudoparaphyses hyaline, filiform, branched, hyphae-like extending above asci, ca. 2 mm wide. Ascospores 34–53 3 4.5–6.5 mm, hyaline to pale brown, cylindrical fusiform, with seven transverse septa. Cultures. Ascospores germinated on CMA within 36 h and produced germ tubes from both ends. Colonies on CMA slow growing, up to ca. 10 mm diam in 10 d, compact, velutinous, no aerial mycelium, dark green-brown, consisting of hyphae that were septate, branched, smooth-walled, subyaline to pale yellowbrown, thick-walled and smooth. Anamorph formed after 12 d. Conidiophores short, 0–1-septate, cylindrical, hyaline, thin-walled, 10–12 mm 3 ca. 3 mm. Conidia identical with those collected on the natural substratum. Known distribution: Thailand. Anamorph: Aquaphila albicans Goh, K.D. Hyde & W.H. Ho, Mycol Res 102:587–592, 1998. FIGS. 7–13 Colonies on natural substratum effuse, translucent or pale yellowish, chalky white when dry. Mycelium partly immersed in woody substratum and partly

Known distribution: Australia, Brunei, Malaysia, Philippines and Thailand (Goh and Hyde 1998). Comments. Single ascospores from ascomata yielded an anamorph similar to A. albicans on the test blocks and to A. albicans colonies grown in pure culture from conidia (Goh and Hyde 1998). Of the 42 Tubeufia species listed in Index Fungorum, nine have been transferred to other genera and one was synonymized within Tubeufia (http://www.indexfungorum.org/ names/names.asp; Crane et al 1998, Kodsueb et al 2004). Tubeufia asiana is distinguished from most other described species of Tubeufia by having thickwalled, black, unbranched setae, an Aquaphila anamorph, and hyaline to pale brown ascospores. Tubeufia asiana and Tubeufia dactylariae are similar in their hyaline to pale brown ascospores and nonhelicosporous conidia (Chang 2003) but T. dactylariae lacks black setae (TABLE II). Tubeufia asiana resembles T. claspisphaeria, T. pachythrix and T. stromaticola in ascospore size and number of septa (Rossman 1979, Kodsueb et al 2004) but these other species lack both the brown ascospores and the black setae. Tubeufia claspisphaeria does have black setae, but they are hooked shaped (Kodsueb et al 2004). RESULTS

Phylogenetic analyses.— We determined new SSU sequences for Aquaphila (GenBank accession num-

TSUI ET AL: A TUBEUFIA TELEOMORPH FOR AQUAPHILA ALBICANS

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FIGS. 7–13. Interference contrast micrographs of Aquaphila albicans. 7. Colony on test block. 8. Conidiophores on hyaline hypha. 9. Conidiophore with developing conidium. 10–13. Conidia, hyaline, fusoid with 4–6 septa. Bars: 7 5 200 mm, 9 5 20 mm, 8, 10–135 10 mm.

bers DQ341091, DQ341093) and Tubeufia (DQ341092) and BLAST for GenBank returned the best match sequences from species in Helicoma, Helicomyces and Helicosporium. As in Tsui and Berbee (2006) Cenococcum geophilum Fr. was used as outgroup. MP analysis of the dataset containing 43 taxa from a wide range of ascomycete families resulted in 16 parsimonious trees that required 987 steps (CI 5 0.62, RI 5 0.76, RC 5 0.47). Out of 1714 characters 1234 were constant and 308 were parsimony informative. (For the alignment and a representative tree see TreeBase accession number SN3513.) Aquaphila and Tubeufia clustered within Tubeufiaceae s. str. with 100% bootstrap support. However, as in Tsui

and Berbee (2006), the SSU dataset provided little resolution of relationships within Tubeufiaceae. New ITS and partial LSU sequences (TABLE I) therefore were determined and aligned with closely related taxa within Tubeufiaceae s. str. (Tsui et al 2006). We also included additional LSU rDNA sequences of fungi generated from the AFTOL project as well as the latest determined sequences from GenBank (Kodsueb et al 2006). Helicoma isiola R.T. Moore (AY856890) and Cenococcum geophilum (AY112935) were chosen as outgroups (Tsui et al 2006). The final dataset consisted of 58 taxa, 683 characters from ITS and 638 characters from partial LSU (TreeBase SN3514). Trees from a maximum

55–65 3 3 mm, 5–9-septate, in fascicle, narrowly cylindricfusiform, hyaline 40–70 3 2.4–2.8 mm, 10–15septate, filiform, hyaline

45–75 3 10–15 mm, cylindrical

100 mm diam, brown, setae black, branched or unbranched

28–40 3 10–12 mm, ellipsoid to oval

67–130 3 12–15 mm, cylindrical-clavate

unknown

unknown

Helicosporium aureum Corda Linder Mirandina sp.

Helicomyces sp.

unknown

100 3 11 mm, cylindrical

Rossman 1979

unknown

40–63 3 5–7.5 mm, 10–14septate, long fusiform, 4–8-seriate, hyaline 40–95 3 2–3.5 mm, 10–14septate, long fusiform, 5–8seriate, hyaline 21–30 3 2.5–3.6 mm, 5-septate, long fusiform, hyaline

Rossman 1979

unknown


Chang 2003

Dactylaria sp.

22–27 3 3–3.5 mm, 5–9-septate, unknown fusiform, 4-seriate, hyaline

Kodsueb et al 2004

unknown

Re´blova´ and Barr 2000


Scheuer 1991/IMI 334247




Re´blova´ and Barr 2000/UAMH 7237

BIOTEC SS354, SS393, SS478

References/materials examined

Aquaphila albicans

Anamorphs


77–79 3 14–16 mm, clavate

NA

34–53 3 4.5–6.5 mm, 5–7septate, fusiform, biseriate, hyaline to pale brown 34–64 3 5–8 mm, 3–8-septate, elongate-fusiform, curved, hyaline 32–36 3 6–7 mm, 7–8-septate, long fusiform, hyaline to pale brown 40–57 3 4–5 mm, multiseptate, long fusiform, hyaline 52–66 3 5.5–7.5 mm, multiseptate, long clavate 30.5–42 3 5–6 mm, 5–8-septate, long-fusiform, 4-seriate, hyaline

Ascospores

Yellow with short thick-walled setae 150–155 mm diam 3 100–110 mm high, reddish brown to dark brown, setae dark brown, unbranched 120–150 mm diam 3 120–140 mm high, dark brown, setae dark brown 150–230 mm diam 3 150–250 mm high, brown, setae dark brown, unbranched 165–310 mm diam 3 180–300 mm high, brown, setae dark brown, unbranched 40–60 mm diam, yellowish brown, setae black, dichotomously branched 60–100 mm diam, blackish brown, setae unbranched

NA: information not available.

Acanthostigma scopulum Cooke & Ellis Peck Taphrophila cornu-capreoli Scheuer Taphrophila hebridensis Dennis Re´blova´ & M.E. Barr Taphrophila trichella Sacc., E. Bommer & M. Rousseau Re´blova´ & M.E. Barr

Acanthostigma minutum Fuckel Sacc.

Acanthostigma revocatum Sacc.

Tubeufia pachythrix Rehm Rossman Tubeufia stromaticola Henn. Rossman Acanthostigma perpusillum de Not.

Tubeufia dactylariae H.S. Chang

Tubeufia claspisphaeria Kodsueb

Asci

180–300 mm diam 3 160–280 mm 100–120 3 15–18 mm, high, dark brown, setae straight, cylindrical-clavate dark brown, unbranched 180–350 mm diam, dark brown to 93–127 3 12–18 mm, black, setae hooked, dark brown cylindrical-clavate unbranched 80–100 3 9–12 mm, 100–120 mm diam 3 150–200 mm high, brown, no setae clavate to cylindrical Yellow with long tapering setae NA

Ascomata

A synopsis of taxa morphologically closely related to Tubeufia asiana in the Tubeufiaceae

Tubeufia asiana

TABLE II.

890 MYCOLOGIA

TSUI ET AL: A TUBEUFIA TELEOMORPH FOR AQUAPHILA ALBICANS parsimony analysis of the dataset required 2067 steps (CI 5 0.46, RI 5 0.68, RC 5 0.31). Out of 1321 characters 762 were constant and 397 were parsimony informative. The 50% majority rule tree obtained from 5001 Bayesian trees is provided (FIG. 14). The combined data from ITS regions and the 39 region of the 28S gene divided species within the Tubeufiaceae into nine clades, each receiving strong bootstrap support (.75%) and 95% or more posterior probability. Tubeufia asiana and the two conidial isolates of A. albicans clustered with 100% bootstrap support and Bayesian probability and nested within group G consisting of Helicoon gigantisporum Goh & K.D. Hyde and Helicoma chlamydosporum Shearer with good bootstrap support (more than 80%) and high posterior probability (.95%). As in Tsui et al (2006) no single clade corresponded well to the current circumscriptions of Helicoma, Helicomyces or Helicosporium, or to the established teleomorphic genera. Most clades contained species from more than one anamorphic genus. Only clades A, C and H were restricted to species from a single anamorphic genus. Even different isolates of the same species did not cluster together. Three isolates of Helicomyces roseus Link were distributed in different clades. DISCUSSION

Tubeufia asiana as the teleomorph of Aquaphila albicans.—The determination that T. asiana and A. albicans, type of the genus, are manifestations of the same species is based on similar morphology of the anamorph and cultures produced by ascospores of T. asiana. In addition the ITS and partial LSU rDNA of T. asiana and A. albicans have almost identical ITS sequences and clustered together with strong bootstrap support. This is the first report of a teleomorph for a species in Aquaphila. We decided against describing Tubeufia asiana as a species in Acanthostigma de Not. because an isolate of the type species of Acanthostigma, A. perpusillum (University of Alberta Microfungus Collection and Herbarium 7237), clustered phylogenetically within Tubeufia (Tsui et al 2006). This suggested that Acanthostigma probably should be synonymized under Tubeufia. We re-identified a supposed second isolate of A. perpusillum (MUCL41721, Kodsueb et al 2006) as Capronia parasitica (Ellis & Everh.) E Mu¨ll., Petrini, P.J. Fisher, Samuels & Rossman based on both sequence identity and on morphology of its anamorph. Isolate MUCL41721 is in the Chaetothyriales and is unrelated to our new species. We verified these results by re-ordering both the UAMH and the MUCL isolates listed above and resequencing their ITS

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regions. In both cases the new sequences were identical to the original ones in GenBank, confirming our conclusions. According to the circumscription of Re´blova´ and Barr (2000) pseudothecia of Acanthostigma bear setae but those of Tubeufia do not and T. asiana has unbranched setae. Our results call into question the use of ascomatal setae to distinguish genera. Based on morphology alone our new species would fit well in Acanthostigma and so we compared it to described Acanthostigma species. Tubeufia asiana is most similar to A. scopulum and A. minutum with respect to ascospore dimensions, but their ascospores are hyaline and their conidia are helicoid, while ascospores of T. asiana are pale brown and its conidia are falcate. Tubeufia asiana also shares morphological characters with Taphrophila Scheuer including noncollapsing ascomata covered with dichotomously branched setae, bitunicate asci and multiseptate, elongate, fusiform ascospores (Scheuer 1991, Crane et al 1998). Taphrophila currently includes five species but, due to lack of availability of either cultures or copious herbarium specimens, no representative has ever been included in a molecular study and we were unable to include one in this paper. Tubeufia asiana differs from Taphrophila in having unbranched setae on dark brown ascomata, and 5–7-septate ascospores that are fusiform, hyaline to brown. Tubeufia asiana is most similar to Taphrophila hebridensis, but the ascospores differ in color and have fewer septa (TABLE II). A. albicans anamorph differs from others in the Tubeufiaceae.—The present investigation confirms that Tubeufiaceae s. str. includes nonhelicosporous, mitosporic fungi, suggesting the production of helical conidia was lost at least once within the family. The conidia of A. albicans are fusoid, curved or rarely straight, instead of being curved more than 180u. A Monodictys and an Asteromella-like anamorph have been reported for Tubeufia amazonensis Samuels, Rossman & E. Mu¨ll. (Samuels et al 1979), and Tubeufia dactylariae produced a Dactylaria-like anamorph with long, hyaline, fusiform conidia with schizolytic secession (Chang 2003). However the relationship of these anamorphs to the Tubeufiaceae has not been verified with molecular data. A lack of correlation among anamorphic genera and clades has been the general pattern in the Tubeufiaceae and it reflects evolutionary convergence among the characters used for generic definition (FIG. 14; Tsui et al 2006). Even so the grouping of A. albicans with Helicoon gigantisporum and Helicoma chlamydosporum was surprising because their conidia are markedly dissimilar. Helicoon gigantisporum pro-

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FIG. 14. Likelihood tree from Bayesian analysis showing the phylogenetic positions of Tubeufia, Aquaphila and other taxa within the Tubeufiaceae inferred from ITS and partial LSU (-lnl 10965). Bootstrap percentage values (.75) generated from 1000 replicates from maximum parsimony and posterior probabilities (.95%) from Bayesian analysis (marked by *) are shown above branches.

TSUI ET AL: A TUBEUFIA TELEOMORPH FOR AQUAPHILA ALBICANS duces brown, three-dimensional, barrel-shaped conidia, while Helicoma chlamydosporum produces black chlamydospores (Shearer 1987, Goh and Hyde 1996b). Many helicosporous fungi within the Tubeufiaceae are known from freshwater habitats as is Aquaphila (Goh and Hyde 1998). Because helical conidia have been considered to be an adaptation to aquatic habitats (Goh and Hyde 1996a) their absence from Aquaphila is puzzling. Unlike its conidial shape conidiogenesis in A. albicans is similar to that of its relatives. Like Helicomyces A. albicans has hyaline conidiophores that develop from repent hyphae and denticulate, sympodial conidiogenous cells (Goh and Hyde 1998). The Mirandina anamorph of Taphrophila cornucapreoli (Scheuer 1991) and the Dactylaria-like anamorph from Tubeufia dactylariae (Chang 2003) as well as the Aquaphila in this study share the same mode of conidiogenesis (de Hoog 1985). In the Tubeufiaceae molecular phylogenies often have been inconsistent with morphological identification of species as well as genera. As in a previous analysis two isolates of T. paludosa did not group together (Tsui et al 2006) although isolate MUCL15702 clustered near an isolate of its anamorph, Helicosporium phragmitis. Our analysis provides yet another example of inconsistency; Rossman (1977, 1987) suggested T. helicomyces and T. cylindrothecia should be synonyms of T. paludosa. However the two sequenced isolates of T. cylindrothecia are not monophyletic and neither clusters with either isolate of T. paludosa. Because cultures extype are not readily available resolution of these problems of species identification and delimitation will be challenging. CONCLUSION

Our analyses show the presence of a nonhelicosporous anamorph within the Tubeufiaceae and support the connection between Aquaphila albicans and the new species Tubeufia asiana. The close phylogenetic relationship among Aquaphila albicans with its fusoid conidia and a Helicoon and a Helicoma with coiled conidia provides a striking example of the lack of correlation between conidial characters and clades in the Tubeufiaceae. ACKNOWLEDGMENTS

CKM Tsui is grateful to the Croucher Foundation for the award of a postdoctoral fellowship. NSERC provided support through an operating grant to Mary Berbee. TRF/BIOTEC special Program for Biodiversity Research and Training grants BRT 143016 and BRT 145006 is thanked for supporting freshwater fungal research in

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Thailand. We are grateful for type and authentic specimens and cultures for identification and confirmation by IMI, MUCL and UAMH; for technical assistance provided by SeaRa Lim and Patrik Inderbitzin; and for photographic assistance of the Electron Microscopy Unit at UBC.

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