Marine Fungi

Evolutionary Relationships Between Aquatic Anamorphs and Teleomorphs II:

Tricladium and Varicosporium

 

Jinx Campbell¹, Carol Shearer² and Ludmila Marvanová³

 

¹Department of Coastal Sciences, University of Southern Mississippi, Ocean Springs, Mississippi, USA, 39564

²Department of Plant Biology, University of Illinois, Urbana, Illinois, USA, 61801

³Masaryk University, Faculty of Science, Tvrdeho 14, 602 00 Brno, Czech Republic

 

CCM = Czech Collection of Microorganisms

 

Abstract           

Tricladium, with 21 species, is the largest genus of aquatic hyphomycetes. It encompasses species with dematiaceous as well as mucedinaceous colonies. Conidiophores range from simple and identical with conidiogenous cells, to well developed and profusely branched. Conidiogenesis is thalloblastic; conidiogenous cells proliferate percurrently or sympodially. Conidia, although typically with two alternate primary lateral branches only, may have up to four laterals, sometimes with secondaries on the proximal primaries; branch insertion is unconstricted or constricted, sometimes unilaterally or only slightly. Molecular analyses on the 28S rDNA of 33 isolates, including 12 species of Tricladium, 5 species of Varicosporium, 2 species of Fontanospora and one species of Variocladium, place these hyphomycetes within Helotiales, except Tricladium caudatum, which appears within Rhytismatales. Tricladium is polyphyletic and placed in five clades; Varicosporium is polyphyletic and placed in three clades; Fontanospora is polyphyletic within a single clade; Variocladium is not placed with any of the other included species.

 

Results

             Initial analyses were performed on ~250 species with representatives from 17 orders of ascomycetes, and with basidiomycetes as outgroup taxa (results not shown).  All species of Tricladium, Fontanospora, Variocladium and Varicosporium were placed among the Leotiomycetes. Further analyses were run with 74 taxa from Leotiomycetes and Lecanoromycetes and with Pezizales as outgroup taxa.

             Tricladium is polyphyletic and placed in five clades. One clade contains T. attenuatum, T. biappendiculatum, T. patulum, T. minutum, Fontanospora eccentrica, F. fusiramosa, and Varicosporium giganteum.  Five of these species have pale colonies, T. minutum has brown colonies, and V. giganteum has dark colonies.  Other than T. and T. minutum, which have percurrent conidiogenous cell proliferation, all have sympodial proliferation. This clade also contains the teleomorph Hymenoscyphus scutula. The second clade contains T. castaneicola, T. terrestre, T. splendens and T. obesum. These species all have dark colonies; conidiogenous cell proliferation is either sympodial or percurrent. This clade also contains the teleomorphs Helicodendron conglomeratum and Lambertella tubulosa. The third clade contains T. chaetocladium, T. curvisporum and V. delicatum. These species have either pale or dark colonies and conidiogenous cell proliferation is either sympodial or percurrent. This clade also contains the teleomorph of Tricladium chaetocladium (Hydrocina chaetocladia). The fourth clade consists of T. angulatum and V. scoparium, both of which have pale colonies and sympodial conidiogenous cell proliferation. This clade also contains the teleomorphs Chlorencoelia sp, Heyderia abietis, Fabrella tsugae and Sclerotina sclerotiorum. The fifth clade contains T. caudatum and the teleomorph Rhytisma acerinum. Variocladium giganteum is not placed with any of the other included hyphomycetes.

 

References

Bremer K. 1988. Evolution 42:795–803.

Bremer K. 1994. Cladistics 10:295–304.

Bunyard BA, Nicholson MS and Royse DJ. 1994. Mycologia 86:762–772.

Descals E, Marvanová L and Webster J. 1998. Canadian Journal of Botany 76:1647–1659.

Dyko BJ. 1978. Transactions of the British Mycological Society 70:409–416.

Eriksson T. 1998. AutoDecay. ver 4.0. Stockholm: Department of Botany, Stockholm University.

Felsenstein J. 1985. Evolution 39:783–791.

Huelsenbeck JP and Ronquist F. 2001. Bioinformatics 17:754–755.

Iqbal SH. 1974. Biologia (Lahore) 20:17–21.

Kegel W. 1906. Berichte der Deutschen Botanischen Gesellschaft 24:213–216.

Kishino H and Hasegawa M. 1989. Journal of Molecular Evolution 29:170–179.

Marvanová L and Descals E. 1996. Mycotaxon 60:455–469.

Nawawi A. 1985. Botanical Journal of the Linnean Society 91:51–60.

Nawawi A and  Kuthubutheen AJ. 1988. Transactions of the British mycological Society 90:482–487.

Nawawi A, Webster J and Davey RA. 1977. Transactions of the British mycological Society 68:59–63.

Qiagen. 2004. DNeasyTM plant mini handbook. USA: QIAGEN Inc.

Qiagen. 2002a. Taq PCR handbook for Taq PCR master mix kit. USA: QIAGEN Inc.

Qiagen. 2002b. QIAquick spin handbook for QIAquick PCR purification kit. USA: QIAGEN Inc.

Raja HA, Campbell J and Shearer CA. 2003. Mycotaxon 88:1–17.

Shimodaira H and Hasegawa M. 1999. Molecular Biology and Evolution 16:1114–1116.

Swofford DL. 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.

   Sinauer Associates, Massachusetts.

Vilgalys R and Hester M. 1990. Journal of Bacteriology 172:4238–4246.

 

Materials and Methods

             Collection, isolation and characterisation. Collections were made from streams in Europe and Canada. A loopful of fresh foam was streaked onto a microscope slide coated with a thin layer of malt extract agar (0.1 - 2 % MEA plus 100 mg/L Chloramphenicol or penicillin/streptomycin) and kept at 10 -15°C. After 24 - 48 h germinating conidia were transferred to MEA + antibiotic (100 mg/L Chloramphenicol or penicillin/streptomycin) plates and stored at 10 - 18°C. Cultures were checked for bacterial and fungal contaminants, subcultured to MEA plates and incubated at 15C.

             DNA extraction, sequencing and cladistic analysis. Mycelia were harvested directly from MEA plates, incubated with 200 units of lyticase and genomic DNA extracted using Qiagen's DNeasy Plant Mini Kit (Raja et al 2003, Qiagen 2004). The 5' end of the 28S ribosomal gene was amplified with Taq PCR Master Mix Kit (Qiagen 2002a) using fungal primers LROR (Bunyard et al 1994) and LR6 (Vilgalys and Hester 1990). The PCR products were cleaned up with Qiaquick PCR Purification Kit (Qiagen 2002b) and sequenced directly. Cladistic analyses were performed in PAUP* 4.0b10 (Swofford 2002) using maximum parsimony, weighted parsimony and maximum likelihood criteria, with heuristic searches, random starting trees, random stepwise addition on 100 replicates, gaps treated as missing and TBR branch-swapping algorithm. Bayesian inference of phylogeny was performed with MrBayes (Huelsenbeck and Ronquist 2001). The alternative topologies were tested using the Kishino-Hasegawa (1989) and Shimodaira-Hasegawa (1999) maximum likelihood tests. Bootstrap analyses (Felsenstein 1985) were performed on 1000 replicates and decay indices (Bremer 1988, 1994) were calculated in AutoDecay (Eriksson 1998).

 

Acknowledgements

 

Many thanks go to Allison Kennedy for assistance with DNA extraction and amplification, to Dr. Monika Drimalkova for photographic assistance, and to the Czech Collection of Microorganisms for use of their facilities.

 

Appreciation is expressed to Tidelands Trust Fund, Mississippi Department of Marine Resources, and Mississippi-Alabama Sea Grant Consortium (MASGC) for financial support.