Mycologia, 97(4), 2005, pp. 804–811. ᭧ 2005 by The Mycological Society of America, Lawrence, KS 66044-8897

A phylogenetic study of the Haligena (Halosphaeriales, )

Jariya Sakayaroj1 INTRODUCTION Department of Microbiology, Faculty of Science, Prince Haligena Kohlm. was described by Kohlmeyer (1961), of Songkla University, Hat Yai, Songkhla, 90112, Thailand with the type species H. elaterophora Kohlm. The National Center for Genetic Engineering and unique characteristic of the species was the long bi- Biotechnology, 113 Thailand Science Park, polar strap-like appendages and multiseptate asco- Paholyothin Road, Khlong 1, Khlong Luang, Pathum spores that characterize and clearly distinguish the Thani, 12120, Thailand genus from other members of the Ka-Lai Pang (Kohlmeyer 1961). A number of species later were Department of Biology and Chemistry, City University assigned to the genus: H. amicta (Kohlm.) Kohlm. & of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, E. Kohlm., H. spartinae E.B.G. Jones, H. unicaudata Hong Kong SAR School of Biological Sciences, University of Portsmouth, E.B.G. Jones & Le Camp.-Als. and H. viscidula King Henry Building, King Henry I Street, Kohlm. & E. Kohlm. ( Jones 1962, Kohlmeyer and Portsmouth, PO1 2DY, UK Kohlmeyer 1965, Jones and Le Campion-Alsumard Souwalak Phongpaichit 1970). Shearer and Crane (1980) transferred H. spar- tinae, H. unicaudata and H. viscidula to Halosarpheia Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, because of their hamate polar appendages that un- Thailand coil to form long thread-like structures. Recent phy- logenetic studies showed that they are not related to E.B. Gareth Jones Halosarpheia and were transferred to Magnisphaera J. National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Campb. et al and Ascosalsum J. Campb. et al (Ander- Paholyothin Road, Khlong 1, Khlong Luang, Pathum son et al 2001, Campbell et al 2003). Haligena amicta Thani, 12120, Thailand is distinct from Haligena in having appendages that arise from the episporium at various points in the spore wall ( Johnson et al 1987). In Haligena append- Abstract: The genus Haligena (Halosphaeriales, As- ages are polar, arising as outgrowths of the ascospore comycota), with two accepted species, is encountered wall. Thereforea new genus Appendichordella R.G. frequently in marine habitats, especially on wood in Johnson et al was introduced to accommodate H. temperate regions. Phylogenetic analyses of Haligena amicta ( Johnson et al 1987). elaterophora (type species) and H. salina were under- Another species that has been accepted in Hali- taken, with partial large subunit ribosomal DNA se- gena is H. salina C.A. Farrant & E.B.G. Jones, which quences, to determine their relationships with other originally was identified as a Remispora-like species closely related genera in the order. The genus was ( Jones 1985, Farrant and Jones 1986). Haligena sal- shown to be polyphyletic within the Halosphaeriales ina differs from the type species in ascospore size, with the type species forming a basal clade to the septation and especially appendage morphology; ap- order. Haligena salina constituted a sister clade with pendages spoon-shaped at the base, initially coiled weak support of Neptunella longirostris in all analyses. and attached closely to the spore wall and separating Haligena elaterophora and H. salina differ significant- to form a long thread-like filament (Farrant and ly in the nature of their ascospore appendages: wider, Jones 1986). Thus only two species, H. elaterophora more sticky and strap-like in H. elaterophora and and H. salina, are retained in Haligena. Therefore spoon-shaped at the point of attachment; in H. salina in this study relationships of Haligena elaterophora they are longer and narrower, finely drawn out fila- and H. salina and their affinity to other genera in ments. A new genus, Morakotiella, is introduced to the Halosphaeriales are investigated with sequences accommodate H. salina. of the large subunit ribosomal DNA (LSU rDNA). Key words: Halosphaeriales, LSU rDNA, molecu- lar systematics, Morakotiella MATERIALS AND METHODS Accepted for publication 23 March 2005. Fungal isolates and culture characteristics.—Fungal cultures 1 Corresponding author. Email: [email protected] were obtained from City University of Hong Kong Culture

804 SAKAYAROJ ET AL: HALIGENA 805

TABLE I. Fungal isolates sequenced for this study Species Culture No.a Origin GenBank accession No. Haligena elaterophora PP4705 Friday Harbour, USA AY864845 Haligena elaterophora JS147 Portsmouth, England AY864846 Morakotiella salina CY3437 Friday Harbour, USA AY864843 Morakotiella salina BCC12781 Marloes, South Wales AY864844 a CY is from City University of Hong Kong Culture Collection, PP is from Portsmouth University Culture Collection, BCC and JS are from BIOTEC Culture Collection.

Collection (CY) and Portsmouth University Culture Collec- and Saagaromyces ratnagiriensis (AF539470). Inclusion and tion (PP) (TABLE I). Additional cultures were obtained by exclusion of all insertion regions had no effect on the tree single-spore isolations from woody material collected at topology in all analyses. Therefore the insertion regions Marloes, South Wales, and Portsmouth, England. Isolates were included in all analyses. are maintained in the BIOTEC Culture Collection and cod- The phylogenetic analyses were performed with PAUP ed as BCC and JS numbers (TABLE I). All cultures were 4.0b10 (Swofford 2002) with maximum parsimony analysis grown in seawater glucose-yeast extract-peptone broth applying heuristic searches with this setting: 100 replicates (GY P) (Abdel-Wahab et al 2001) on a rotary shaker at 200 of random stepwise addition of sequence and tree-bisection- rpm at 25 C. reconnection (TBR) branch-swapping algorithm. Gaps were treated as missing data; all characters were given equal DNA extraction, amplification and sequencing.—Fungal bio- weight. The consistency indices (CI), retention indices (RI) mass was harvested through cheesecloth, washed several and rescaled consistency indices (RC) were calculated for times with sterile distilled water, frozen in liquid nitrogen each tree generated. Weighted parsimony analysis was per- and ground into fine powder with a mortar and pestle. formed with a step matrix to weight nucleotide transfor- Ground fungal mycelia of 50-100 mg were placed into 400 mations based on the reciprocal of the observed transition ␮L lysis buffer (O’Donnell et al 1997). DNA was extracted to transversion (ti : tv) ratio, which was estimated by maxi- following the instruction of a Nucleospin௡ Plant DNA ex- mum likelihood score setting in PAUP* (Swofford 2002). traction kit (Macherey-Nagel, Germany). Moreover characters were reweighted according to their RC The partial LSU rDNA was amplified with primers LROR- with PAUP* default setting for reweighting character. Boot- LR7 (Bunyard et al 1994) and JS1-JS8 (Landvik 1996). PCR strap analysis (Felsenstein 1985) was performed for un- reactions were carried out in total volume of 50 ␮L con- weighted and weighted parsimony with full heuristic search taining 50 ng DNA template, 1ϫ PCR buffer, 1.5 mM on 1000 replicates (10 replicates of random stepwise addi- MgCl , 2 mM dNTPs, 0.2 ␮M each primer and 0.5 units of 2 tion of sequence and TBR branch-swapping algorithm). Taq Polymerase (DyNAzyme௢ II DNA Polymerase Kit, The proportion of invariable sites, gamma distribution FINNZYMES, Finland). Amplification cycles were per- shape parameter and base frequency were estimated from formed following the procedure of Pang et al (2003). The maximum likelihood score setting in PAUP*. Maximum PCR products were purified with NucleoSpin௡ Extract Kit likelihood analysis was employed with a heuristic search (as (Macherey-Nagel, Germany), following the manufacturer’s is stepwise addition sequence and TBR branch-swapping al- instructions. PCR products were sequenced directly by the gorithm) according to these estimated values. Bio Service Unit (BSU) laboratory with BigDye on an ABI Bayesian phylogenetic inference was calculated with 377 automated sequencer (Perkin Elmer). Forward and re- MrBayes 3.0b4 with general time reversible (GTR) model verse primers: JS1, NL4, JS5, JS8, LROR, NL3 and NL4R, of DNA substitution and a gamma distribution rate varia- were used for the sequencing reactions (Bunyard et al 1994, tion across sites (Huelsenbeck and Ronquist 2001). Four Landvik 1996). Each sequence was checked for ambiguous Markov chains were run from random starting trees for bases and was assembled with Bioedit 5.0.6 (Hall 2001). 2 000 000 generations and sampled every 100 generations. Sequence alignment and phylogenetic analyses.—The consen- The first 100 000 generations were discarded as burn-in of sus sequences for each species were multiple aligned by the chain. A majority rule consensus tree of all remaining Clustal W 1.6 (Thompson et al 1994) along with other se- trees, as well as the posterior probabilities, was calculated. quences obtained from the GenBank database. The dataset The alignments were deposited in TreeBase: study accession was refined visually in Se-Al v1.0a1 (Rambaut 1999) and number ϭ S1228, matrix accession numbers ϭ M2135, Bioedit 5.0.6, 6.0.7 (Hall 2001, 2004). Daldinia concentrica M2136. (Bolton) Ces. and Xylaria hypoxylon (L) Grev. were chosen as outgroup for all analyses. Two insertion regions were ob- served, one at a position 835-1035 of Halosarpheia trullifera RESULTS (AF396875), H. unicellularis (AF396876), H. salina (AY094182), H. salina (AY864843) and H. salina The dataset consists of 1705 total characters, 1081 (AY864844) and the other at a position 1148-1243 of Hal- characters are constant, 322 characters are parsimony osarpheia unicellularis (AF396876), H. fibrosa (AY094183) informative (18.8%) and 302 variable characters are 806 MYCOLOGIA parsimony uninformative (17.7%). The maximum Ultrastructurally spore wall composed of two lay- parsimony analysis resulted in two most parsimonious ers: electron-dense episporium and a wide electron trees (MPTs) 1399 steps long (CI ϭ 0.602, RI ϭ transparent mesosporium. Appendage fibrillar, 0.607, RC ϭ 0.366). The difference between these bounded by a delimiting membrane. two MPTs is in the branching pattern of Magnis- Ascomata immersed or partly immersed or super- phaera spartinae (FIGS. 1, 2). The weighted parsimony ficial, globose, subglobose, ostiolate, black, perithe- (step matrix of 1.38) resulted in two MPTs, which cial wall coriaceous. Neck short, cylindrical and per- gave the same topology as unweighted maximum par- iphysate. Asci thin-walled, unitunicate, pedunculate, simony, 1641.06 steps long, CI ϭ 0.607, RI ϭ 0.612 fusiform to clavate, deliquescing early. Catenophyses and RC ϭ 0.372. The weighted parsimony (charac- present or absent. Ascospores 1-septate, ellipsoidal, ters reweighted) resulted in a single MPT with a tree slightly constricted, hyaline, appendaged. Append- length of 623.77 steps, CI ϭ 0.863, RI ϭ 0.804 and ages polar, initially wrapped around the ascospore RC ϭ 0.693 (FIG. 1). wall, later separating to form long filaments that are The estimated proportion of invariable sites, gam- spoon-shaped at the place of attachment to the spore ma distribution shape parameter and base frequency wall, attenuate, channeled, more than 50 ␮m long. of the gene are respectively 0.127930, 0.505699, A: Ascospore wall under TEM two-layered, outer epis- 0.25680 C: 0.22268 G: 0.29729 T: 0.22323. A single porium electron-dense, inner wall layer mesosporium maximum likelihood tree (Ϫln L ϭ 9341.95372) pro- less electron-dense, at each pole wall bulging outward duced overall topology similar to other analyses, but with electron material within the mesosporium and it differed in the position of Nohea umiumi (tree not beneath the episporium. Appendage origin not de- shown). termined but bounded by a thin electron-dense de- Bayesian inference provided a topology similar to limitating membrane attached to ascospore apices by other analyses. Although a minor difference in the fine threads. Appendage substructure fibrillar to position of Nohea umiumi was noted, this difference amorphous, electron-dense. Under SEM appendage does not affect the overall topology of the tree and comprising fine fibrillar material running the length the conclusions drawn. of the appendages becoming amorphous and some- The position of Haligena within the Halosphaeri- times deliquescing. ales clearly was supported by all analyses (FIGS.1,2) Typus. Morakotiella salina (C.A. Farrant & E.B.G. and shown to be polyphyletic. Two isolates of H. ela- Jones) Sakayaroj terophora and three isolates of H. salina were shown Etymology. ‘‘Morakot’’ refers to Professor Morakot as separate but monophyletic clades with high boot- Tanticharoen, Director BIOTEC Thailand, for her strap values. Haligena elaterophora always was shown continued support of fungal in Thailand on a basal branch to the rest of the Halosphaeriales and ‘‘ella’’ ϭ diminutive with 82% bootstrap values and 100% posterior prob- abilities support in weighted parsimony and Bayesian Morakotiella salina (C.A. Farrant & E.B.G. Jones) inference, respectively (FIGS. 1, 2). All three H. salina Sakayaroj comb. nov. FIGS. 8–12. sequences grouped together with high bootstrap sup- Basionym: Haligena salina C.A. Farrant, E.B.G. Jones. port as a sister clade of Neptunella longirostris with Bot J Linn Soc 93:405. 1986. 75% bootstrap obtained from parsimony analysis and Holotype: IMI 297765. below 95% posterior probabilities from Bayesian analysis. DISCUSSION

TAXONOMY Ascospore appendage morphology and ontogeny are significant characters used to delimit genera of ma- Morakotiella Sakayaroj, gen.nov. rine ascomycetes. Campbell et al (2003), in their Typus generis: M. salina (C.A. Farrant & E.B.G. treatment of Halosarpheia species with bipolar un- Jones) Sakayaroj. furling appendages, stated that ‘‘transmission elec- Ascomata globosa, subglobosa, immersa vel partim im- tron microscope and scanning electron microscope mersa, ostiolata, papillata, coriacea, nigra, collo hyaline. Ca- tenophyses praesentes. Asci clavati vel fusiformis, pedicel- studies on a limited number of species to date do not lati, unitunicati, leptodermi, pristine deliquescentes. Ascos- indicate any heterogeneity in structure or ontogeny’’ porae 1-septatae, ellipsoidalis, hyalina, ad septa constrictae, and that ‘‘all the appendages are reported to develop verrucosus pagina, appendices bipolares. Appendages fi- the same way, by extrusion through pores in the ep- lum, denique, polares, as basim cochleariformes, attenu- isporium wall.’’ These statements do not consider the atae, canaliculatae, ad extensionem apicis, sporae affixae. diversity in structure of the polar appendages. For SAKAYAROJ ET AL: HALIGENA 807

FIG. 1. A single most parsimonious tree resulted from weighted parsimony analysis (characters reweighted), from partial LSU rDNA sequences. Bootstrap values higher than 50% from weighted parsimony (characters reweighted) are given above the branches. Bar indicates 10 character state changes. example, the development of ascospore appendages of the water exerts a profound influence on the mor- from a pore in Magnisphaera spartinae (E.B.G. Jones) phology of the appendages. J. Campb. et al differs significantly from that of the Likewise the appendage structure of Haligena spe- pore fields in Saagaromyces ratnagiriensis (S.D. Patil cies differ. In H. elaterophora and H. salina the ap- & Borse) K.L. Pang & E.B.G. Jones ( Jones and Moss pendages initially are wrapped around the ascospores 1980, Baker et al 2001). In Cucullosporella mangrovei and then uncoil to form long polar appendages (K.D. Hyde & E.B.G. Jones) K.D. Hyde & E.B.G. (FIGS. 3–12). These characteristics are relevant when Jones, the substructure of the appendage comprises comparing the appendages of H. salina and Panorbis two distinct elements: folded fibrogranular electron- viscosus. However H. salina and P. viscosus differ at dense material and fine fibrils (Alias et al 2001). the ultrastructural level and molecular results (FIGS. However we have much to learn about how append- 1, 2). Haligena salina originally was identified as a ages in species with bipolar unfurling appendages are Remispora-like species (Farrant and Jones 1986), how- formed, as appreciated by Campbell et al (2003). ever, it cannot be referred to Remispora because they While distinct fibers are apparent in the appendages are distantly related morphologically and phyloge- within the delimiting membrane in asci in some spe- netically (tree not shown). Appendages of H. salina cies, in others the appendage may be extruded as a also resemble the appendages of Halosphaeria appen- gel-like material, only later to aggregate to form fi- diculata but they differ at the ultrastructural level: in bers (Nakagiri and Ito 1994, E.B.G. Jones, unpubl). H. salina equatorial appendages are lacking and ap- Another consideration is the condition under which pear amorphous, while in H. appendiculata they are appendages are formed as it was shown for the ap- present and are reticulate, with a substructure com- pendages of Aniptodera salsuginosa Nakagiri & Tad. posed of both electron-dense and less electron-dense Ito (Nakagiri and Ito 1994). In this case, the salinity material (Hyde et al 1994). Our recent molecular re- 808 MYCOLOGIA

FIG. 2. Bayesian analysis of partial LSU rDNA sequences. Posterior probabilities higher than 95% are indicated above the branches. Bar indicates 0.1 substitution/site.

sults confirm that they are distantly related (FIGS.1, this character no longer can be considered as a uni- 2). fying feature. Spatafora et al (1998) argued that ev- Marine ascomycetes have adapted to life in the ma- anescent asci are a continuation of the lineage arising rine environment in a number of ways: as indicated from entomopathogenic ascomycetes. However deli- by their great diversity in morphology, such as early quescent asci are common in a number of unrelated deliquescing asci, lack of apical apparatus and vari- taxa that have made the transition from terrestrial to ously appendaged ascospores ( Jones 1995). Polar the marine environment: e.g. Halonectria E.B.G. and equatorial ascospore appendages aid in the en- Jones (); Amylocarpus Curr. (Leotio- trapment and attachment to suitable substrata by mycetidae) and Dryosphaera Jørgen Koch & E.B.G. their sticky nature and in increasing the surface area Jones (). for attachment ( Jones 1994). Delineation of genera within the Halosphaeriales The deliquescent nature of the asci of many ma- has relied heavily on the ascospore and appendage rine ascomycetes was considered as a unifying char- ontogeny, due to their great variation in morphology acter of the Halosphaeriales by many authors (Cain ( Jones 1994). This similarity is often the result of en- 1972, Berbee and Taylor 1992, Blackwell 1994, Spa- vironmental adaptation to life in the marine milieu tafora and Blackwell 1994). With the transfer of Lul- ( Jones 1995, Shearer 1993). This has led to many worthia and Lindra to a new order, the , marine genera being circumscribed incorrectly (e.g. SAKAYAROJ ET AL: HALIGENA 809

FIGS. 3–12. 3. Light micrographs of ascus containing ascospores of Haligena elaterophora ( JS147). 4–7. Ascospores multi- septate with bipolar, long, strap-like appendages. 8. Light micrographs of black, globose ascomata of Morakotiella salina (BCC12781). 9, 11. Ascospores one-septum with tightly coiled appendages around the spores. 10. Ascus cylindrical-clavate 12. Ascospore forms a long thread-like appendage after released into water. Bars: FIGS. 3–7, 9, 11, 12 ϭ 20 ␮m; 8 ϭ 100 ␮m; 10 ϭ 10 ␮m. the inclusion of such species as Arenariomyces trifur- The molecular study by Campbell et al (2003) and catus and Nereiospora comata in Corollospora). Ultra- Pang et al (unpubl) support the exclusion of Mag- structural studies clearly demonstrated distinct differ- nisphaera spartinae and Ascosalsum unicaudatum ences in ascospore appendage ontogeny, observa- from Haligena, as does our recent molecular obser- tions later supported by molecular sequences of the vations. SSU rDNA (Campbell et al 2002). Haligena elaterophora can be differentiated from H. Haligena was shown to be clearly delineated within salina by both morphological and molecular evi- the Halosphaeriales, but it is polyphyletic, with H. dence. Haligena elaterophora possess smooth, multi- salina distantly related to the type species, H. elater- septate ascospores with constriction and the append- ophora. Haligena elaterophora constitutes the basal ages are wider, strap-like, and polymorphic (FIGS.4– clade to the order with high support for all analyses. 7). Ascospores of H. salina are smaller than those of 810 MYCOLOGIA

the former species; they have a warty ascospore wall 8a. Ascospores cylindrical narrow less that 5 ␮m surface (in the original collection) composed of an wide, wall smooth ...... Ascosalsum ␮ electron-transparent mesosporium and an electron- 8b. Ascospores broad wider than 20 m, shorter, opaque episporium, continuous beneath the append- wall verrucose ...... Magnisphaera 9a. Ascospore appendages coiled and arise as out- ages (FIGS. 6, 10 in Farrant and Jones 1986). Its ap- growths of spore wall ...... 10 pendages are long, narrower, drawn out and attenu- 9b. Ascospore appendages hamate, arising from a pore ated at their tips and distinctly spoon-shaped at their field ...... 11 point of attachment. Pseudoparenchymatous cells in 10a. Appendages wide (wider than 20 ␮m) strap- the ascoma centrum of H. elaterophora break up to like ...... Haligena form catenophyses while these cells might be absent 10b. Appendages narrow (width less than 10 ␮m or present in some collections of H. salina. However thread-like ...... Morakotiella we did not observe catenophyses in material from 11a. Ascospore appendages arise through a cup-like which our isolates were derived. structure ...... Cucullosporella The three isolates of H. salina formed a mono- 11b. Appendages arise through a pore filed ...... 12 12a. Asci persistent ...... Halosarpheia phyletic clade and showed a high number of base 12b. Asci deliquesce early ...... 13 substitutions that caused a long branch length in iso- 13a. Ascospores fusoid to ellipsoidal, mostly over 25 ␮m late AY094182. The closest sister taxon of H. salina long, catenophyses present ...... Natantispora is Neptunella longirostris, but they are not congeneric 13b. Ascospores ellipsoidal, mostly under 25 ␮m long, and have weak phylogenetic support (FIGS. 1, 2). catenophyses present or absent ...... Panorbis They significantly differ in the morphology of the as- Keys to the halosphaeriaceous taxa with unfurling comata and ascus structure: in H. salina the ascus polar appendages ( Jones 1995, Campbell et al 2003) deliquesce early, while in N. longirostris the ascus is are unsatisfactory because of the overlapping char- persistent and with an apical pore. acters of many of the genera. In addition ultrastruc- Haligena salina differs from other genera with un- tural studies of spore appendage ontogeny are avail- coiling appendages by the mode of attachment of the able only for a few of these genera (Alias et al 2001, appendage to the ascospore wall; appendages are Baker et al 2001). coiled around the spore, spoon-shaped at the point of attachment, channeled along its length, amor- phous with distinct striations running the length of ACKNOWLEDGMENTS the appendage (visible under SEM) and arising as an We thank these people for their support: Dr Julian I. Mitch- outgrowth of the spore wall. Based on these morpho- ell and Prof Lilian Vrijmoed for supporting cultures and logical features and molecular data, a new genus Mo- laboratory facilities. This study was financially support by rakotiella, is proposed for H. salina. BRT Grant No. R࿞245002, LGS scholarship, BIOTEC, Grad- uate School Prince of Songkla University and the Croucher Foundation (to Dr Pang). KEY TO THE GENERA IN THE HALOSPHAERIALES WITH POLAR UNFURLING APPENDAGES

1a. Parasitic on crabs, appendages coiled around as- LITERATURE CITED cosporic ...... Trichomaris 1b. Saprobes on marine plants, wood ...... 2 Abdel-Wahab MA, Pang KL, El-Sharouny HM, Jones EBG. 2001. Halosarpheia unicellularis sp. nov. (Halosphaeri- 2a. Ascospores with a single polar appendage .. 3 ales, Ascomycota) based on morphological and molec- 2b. Ascospores with bipolar appendages ...... 4 ular evidence. Mycoscience 42:255–260. 3a. Asci persistent, with retraction of the cytoplasm, at Alias SA, Moss ST, Jones EBG. 2001. Cucullosporella man- the apex, apical pore present ...... Tirispora grovei, ultrastructure of ascospores and their append- 3b. Asci deliquescing early, no apical pore .. Ophiodeira ages. Mycoscience 42:405–411. 4a. Asci persistent with retraction of cytoplasm 5 Anderson JL, Chen W, Shearer CA. 2001. Phylogeny of Hal- 4b. Asci deliquescing early without retraction of osarpheia based on 18S rDNA. Mycologia 93:897–906...... cytoplasm 6 Baker TA, Jones EBG, Moss ST. 2001. Ultrastructure of ascus ␮ 5a. Ascospore dimensions wider than 20 m, asci with and ascospore appendages of mangrove Halo- long pedicels ...... Saagaromyces sarpheia ratnagiriensis (Halosphaeriales, Ascomycota). ␮ 5b. Ascospore dimensions narrower than 20 m, ascus Can J Bot 79:1307–1317. pedicel short ...... Aniptodera Berbee ML, Taylor JW. 1992. Convergence in ascospore dis- 6a. Ascospores hyaline ...... 13 charge mechanism among pyrenomycete fungi based 6b. Ascospores brown ...... Phaeonectriella on 18S ribosomal RNA gene sequence. Molec Phylogen 7a. Ascospores 2 or more ...... 8 Evol 1:59–71. 7b. Ascospores 1-septate ...... 9 Blackwell M. 1994. Minute mycological mysteries: the influ- SAKAYAROJ ET AL: HALIGENA 811

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