Marine Fungi

Evolutionary relationships among marine, freshwater

and terrestrial ascomycetes

Jinx Campbell and Carol Shearer

¹Department of Plant Biology, University of Illinois, Urbana, Illinois, USA, 61801

 

Abstract

 

In order to learn more about the phylogeny of freshwater and marine ascomycetes, two different pyrenomycete lineages were studied: species in the genus Halosarpheia, Halosphaeriales; and genera in the Annulatascaceae, Sordariales. Phylogenetic analyses of the 28S rDNA indicate that neither taxonomic group is monophyletic. Morphological features congruent with the molecular based phylogeny, differ between the two taxonomic groups. Ascospore morphology and habitat are more important in Halosarpheia sensu lato, and hamathecium and ascus morphology and behavior are more important in the Annulatascaceae sensu lato.

Introduction

Systematic studies of freshwater and marine ascomycetes began only about fifty years ago. Preliminary evidence suggests that both freshwater and marine habitats have selected for certain lineages of ascomycetes. Currently, we have little knowledge about the phylogenetic relationships among terrestrial, freshwater and marine ascomycetes. In addition, adaptations to the aquatic environment most likely mask some evolutionary relationships among terrestrial, freshwater and marine ascomycetes, and within groups of morphologically similar freshwater and marine ascomycetes.

             In order to learn more about the phylogeny of freshwater and marine ascomycetes, we undertook molecular and morphological studies on two groups of species from different pyrenomycete lineages. The first group of taxa is in the genus Halosarpheia (Halosphaeriaceae, Halosphaeriales). The species are united by the presence of unfurling ascospore appendages. The second group is in the Annulatascaceae (Sordariales). The genera in Annulatascaceae are united by the presence of a large ascus apical ring and long tapering septate paraphyses.

             Molecular sequence data were used to determine: (1) whether each group is monophyletic; (2) which morphological characters are most congruent with molecular phylogeny; and (3) what are the closest ancestors.

Figure 1. Cladogram of MPT from parisomy analysis. Bootstrap support shown above and Bayesian support shown below the branches.

Figure 2. Cladogram of MPT from weighted parsimony analysis. Bootstrap support shown above the branches

Materials and Methods

Collection, isolation and characterization. Submerged woody and herbaceous debris was collected from a variety of freshwater and marine habitats from America, Europe and Asia and incubated on moistened paper toweling in plastic dishes under ambient light (12/12 h light/dark) and temperature conditions (~ 25 C). Substrate was examined for the presence of ascomata immediately after collection and monthly thereafter. Ascomata were removed, examined under the microscope and identified using morphological characteristics. Cultures were obtained from single ascospores or asci, and grown on PYG agar plates at 25C in the dark for 4-6 weeks.

DNA extraction, sequencing. Mycelia were harvested directly from PYG plates, ground in liquid nitrogen with a pestle and mortar and genomic DNA extracted using Qiagen's DNeasy Plant Mini Kit. The 5' end of the 28S ribosomal gene was amplified with Taq PCR Master Mix Kit using fungal primers LROR, LR7 and LR6. The PCR products were cleaned up with Qiaquick PCR Purification Kit and sequenced directly.

Phylogenetic analyses. Our sequences were added to closely related sequences from Genbank, aligned in Clustal X and refined visually in SeAl. Cladistic analyses were performed in PAUP* 4.0b10 using maximum parsimony and weighted parsimony criteria, and distance methods. Heuristic searches were performed using random starting trees, random stepwise addition on 100 replicates, gaps treated as missing and a TBR branch-swapping algorithm. Alternative topologies were tested using the Kishino-Hasegawa maximum likelihood test. Bootstrap analyses were performed on 1000 replicates and Bayesian inference of phylogeny was performed with MrBayes.

Results

The Halosphaeriaceae is monophyletic and consists of species from freshwater, brackish and marine habitats. The closest relative of the Halosphaeriaceae is the Microascales, which consists of terrestrial species. (Figure 1)

Halosarpheia is polyphyletic. The species are separated into 8 distinct clades and distributed throughout the family Halosphaeriaceae (Figure 1).

Annulatascaceae is polyphyletic, with the taxa distributed among four orders. Ceriospora caudae-suis (Amphisphaeriaceae, Xylariales) is placed within the Annulatascaceae sensu stricto, Sordariales (Figure 2).

Discussion

The genus Halosarpheia contains species from freshwater, brackish and marine habitats. The genus is characterized by the presence of unfurling ascospore appendages. Such appendages facilitate the attachment of ascospores to substrates in water. Molecular phylogeny using 28S rDNA data does not support the monophyly of Halosarpheia and indicates that unfurling appendages have evolved by parallel or convergent evolution. The morphological characters that are congruent with molecular phylogeny are ascospore morphology, presence/absence of catenophyses, and habitat.

             The Annulatascaceae is a family of freshwater ascomycetes characterized by long, septate paraphyses and long cylindrical asci with a prominent apical ring. Phylogenetic analyses indicate that the family is polyphyletic. A large ascus apical ring is found in several clades of pyrenomycetes, which indicates this character is a result of parallel or convergent evolution. The morphological characters in congruence with molecular phylogeny that support the delineation of Annulatascaceae sensu stricto are long, cylindrical asci that extend in length in water, a large bipartite apical ring, uniseriate arrangement of ascospores in the ascus, deliquesence of the basal end of the asci, and long tapering, septate paraphyses.

Acknowledgments

Appreciation is expressed to the National Institutes of Health (NIH Grant No. R01 GM-60600) for financial support.

Thanks to Chris Brown for hours of dedicated measuring at the microscope.

 

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