Preamble: A revolution is currently underway in mycology. Classical taxonomic assumptions based on morphology are giving way to more natural phylogenies based on the DNA sequencing of ribosomal and protein coding genes. Gene sequencing, the actual enumeration of the individual nucleotide bases that comprise the DNA molecule (A, T, C & G), lies at the heart of modern biological research. DNA sequencing can remove much of the subjectivity inherent in classical taxonomy, replacing it instead with a set of objective criteria subject to the rigors of statistical analysis. Since fungi do not leave behind a well preserved fossil record, reconstructing phylogenies has been wholly dependent upon a comparison of morphologies of extant organisms. As compared to plants and animals, fungi have a limited set of morphological features and these often develop in parallel among unrelated groups as adaptations to common environments. Thus, fungal taxonomy has suffered in the past due to different interpretations related to convergent evolution . In short, some organisms that share similar features and were once classified as related, are now being shown to be distantly related and vise versa. DNA sequencing is therefore able to show which morphological features are phylogenetically informative and thus, useful for understanding evolution within the fungi. This has implications for biomedicine, agriculture, pharmaceuticals and biotechnology.

Background: Historically, mycology has been treated as a subdiscipline of botany, but recent molecular evidence, in the form of both ribosomal DNA and protein coding genes, suggests that fungi are actually more closely related to animals than to plants (Baldauf & Palmer 1993). It has recently been estimated that some 1.5 million species of fungi are thought to exist, with about 80,000 species or only 5% having yet having been described, making fungi potentially one of the most speciose groups of organisms on the planet (Hawksworth 2001). Current classification of fungi has been facilitated by a recent  NSF funded, collaborative project termed "Assembling the Fungal Tree of Life", and includes a number of major phyla within the Eumycota, as listed on the Tree of Life Website, and presented in the figure below. As this is a dynamic endeavor, subject to change with new datasets, at the moment, some clades are better resolved than others. The Chytridiomycota and Zygomycota are currently considered polyphyletic (i.e., with multiple origins), whereas the two clades defining the Dikarya, namely the Basidiomycota and the Ascomycota, have been shown to be monophyletic (Berbee & Taylor 2001). According to recent estimates, there are more than 40,000 described species of ascomycetes, belonging to 3,266 genera, grouped in 264 families in 46 orders, thus defining the largest group of fungi (Kirk et al. 2001). Despite a weak fossil record, fungal classification has traditionally been based on a rich number of observable and quantifiable morphological character states as seen under the light microscope among extant organisms. As applied to the Ascomycota, these character states include features associated with: (1) fruitbody or ascoma morphology; (2) ascus type and mode of dehiscence; (3) centrum ontogeny, including hamathecial type; and (4) ascospore type, based on spore septation configurations (amerospore, didymospore, phragmospore & dictyospore: see figure below). Character states associated with (1) thru (3) have traditionally served to define higher taxa (e.g., families, orders and classes), whereas characters associated with ascospore type have often been used to delineate genera and species. The Ascomycota is currently understood to comprise three major subphyla, the Pezizomycotina, the Saccharomycotina, and perhaps the Taphrinomycotina, with several unresolved groups, the Schizosaccharomycetes, Pneumocystidiomycetes, and Neolectomycetes,  as outlined on the Tree of Life Website and detailed on MYCONET.

Research: The specific aim of my research program is to expose biology students to state-of-the-art scientific methodologies, namely gene amplification using the polymerase chain reaction (PCR), DNA sequencing, and bioinformatics, to understand the evolution and phylogeny of the fungi, specifically two families of bitunicate ascomycetes, namely the Hysteriaceae and the Mytilinidiaceae (Hysteriales & Mytilinidiales, Dothideomycetes, Ascomycetes, Eumycota). These fungi are currently being developed as model organisms to address a number of taxonomic and evolutionary questions in ascomycete biology. This is because these fungi possess identical fruitbodies, but contain a diverse array of spore morphologies, used to define genera within each family. Spores range from didymo- to phrago- to dictyo- to scoleco-spores (see figure below), with or without pigmentation, and therefore encompass all the spore types seen among the ascomycetes, only contained within two small families. Specifically, the project aims to use DNA sequencing to: (1) verify the familial placement of genera and assess whether they represent natural groups within the Hysteriaceae & Mytilinidiaceae; (2) to determine which morphological characters within each family are phylogenetically significant and therefore are useful for generic delineation; and (3) to asses whether phylogenies based on molecular character states are concordant with any of the traditional morphology-based classification schemes. This theme relates to how much taxonomic weight should be given to different morphologic features (e.g., spore ontogeny) associated with the sexual process in the fungi. It is expected that findings from this study will be applicable to evolutionary questions in other fungal groups, containing human and plant pathogens.

From left to right: amerospore, didymospore, phragmospore, dictyospore.

Methods: Research is centered on exposing biology majors to a range of scientific methods relating to the molecular systematics of fungi. These methodologies are universal and apply equally well to research in many other scientific fields: (1) Field work: Students engage in field work to collect mature fungal fruitbodies (< 3mm in length) from live bark and decorticated woody substrates. (2) Taxonomy: Students learn to use the primary mycological literature to effectuate proper taxonomic identifications using light microscopy. (3) Microbiology: Students process fruitbodies on inverted Petri plate lids, such that the ejected spores are recovered individually on nutrient agar, thus forming the basis for the establishment of single-spored, genetically pure axenic stock cultures. These cultures are then transferred to stationary flasks to generate material for DNA isolation. (4) Molecular Biology: Students engage in DNA extraction using commercially available kits which involves homogenizing the tissues in Eppendorf tubes, processing the homogenates through various affinity columns and centrifugation. Dilutions of genomic DNA template are then used in PCR to selectively amplify portions of taxonomically conserved genes using conserved primers. These genes include the nuclear small (nuSSU) and large (nuLSU) ribosomal subunits, the internal transcribed spacer regions (ITS1 & 2), and two protein coding genes, the transcription elongation factor (TEF1) and the RNA polymerase II second largest subunit (RPB2). Amplified products are analyzed using gel electrophoresis with known size standards to determine fidelity, yield and purity. Purified PCR products are then commercially sequenced with data accessed on-line. (5) Bioinformatics: DNA sequence data will then be edited and aligned using DNA sequence alignment programs. Additional sequences generated in earlier studies will be retrieved on-line from the GenBank from the databases of the National Center for Biotechnology Information (NCBI). Phylogenetic reconstructions to generate evolutionary trees will be developed using PAUP (Phylogenetic Analysis Using Parsimony) software. These trees will then be compared to the classification of these fungi using classical published morphological evidence relating to fruitbody development and spore septation. What I am seeking for the student is an appreciation of how seemingly different scientific methodologies are integrated to address common questions in modern biology.