Methodologies

Mycology: A series of methods are presented, relating to working with hysteriaceous fungi, that may prove equally applicable to working with other groups. Initiation of single-spored genetically pure cultures: Without exception, every molecular phylogenetic study in mycology must begin with a pure or axenic culture of the fungus. Failure to initiate a culture that is faithful to the original material will result in a number of “down-stream” failures, not the least of which are Genbank DNA & protein accessions that do not correspond phylogenetically with the specific epithet of the taxon in question. This has unfortunately also occurred in the literature, where single fungal isolates, used as representative of the genus, family and even order in question, have proved in the end to be a “mis-identified” culture. Of course, if the fungal isolate can fruit in culture or produce a recognizable anamorph in culture, then it is a simple task to verify the culture prior to further analysis. Unfortunately, this is the exception rather than the rule for many fungi. For example, although many anamorphs have been described for the Hysteriaceae Mytilinidiaceae, many isolates do not produce them in culture, even after prolonged incubation lasting many months. Also, in this group coelomycetous anamorphs tend to possess few morphological characters useful for species confirmation. Thus, a certain amount of effort must be put into establishing a single-spored culture suitable for DNA/RNA isolation and eventual deposit in a culture collection. And this requires a certain degree of mycological expertise. Routinely, upon receipt of material from the field or from collaborators overseas, under the stereomicroscope, using a sterile razor blade, I remove a single ascoma. I then affix the fruitbody to the underside of a Petri plate lid, using double edged sticky tape, such that the area of dehiscence (ostiole, apothecia, sulcus etc.) is facing downwards towards the agar surface. I use water agar plates, so that whatever is eventually deposited on the surface of the agar will not grow very fast or far (e.g., weed molds, yeast & bacteria). I never use antibiotics on any mycological media because I want to “see” all that is there, contaminants and all. On the contrary I want to encourage contaminants so that I may be able to avoid or remove them. I then incubate the plate in the dark for up to 96 hrs. which, for this group of fungi, seems to be enough time to rehydrate & begin spore discharge. Most importantly, every 12 hrs., I rotate the Petri plate lid 45°, such that an even series of spore deposits result. There is an interesting difference between unitunicate hymenoascomyetes and bitunicate loculoascomycetes with regard to spore deposits. Unitunicates tend to discharge all of the spores, all at once in a sudden burst, so that the deposits appear densely clumped, hindering the retrieval of individual spores. Bitunicates, on the other hand, tend to shoot their spores one or a few at a time, due to the nature of the fissitunicate dehiscence mechanism of the bitunicate ascus, thus greatly facilitating recovery of individual spores for this group of fungi. Viable ascospores of Hysterium and Hysterographium have been isolated from herbarium material over three years old. Some fungi are recalcitrant to spore release using the methods described above, even after prolonged hydration / incubation, and must be “forced” to liberate their ascospores. What I do is place a single fruitbody in a 1.5mL microfuge tube & add 300uL of sterile water, vortex, aspirate off the water, & repeat several times to clean the surface of the fruitbody of adhering contaminant bacterial, fungal and yeast spores. Sometimes I add 5uL of Tween (5% stock) to the water. Very dilute bleach works OK as well. But then I must wash with water several more times to remove the Tween or bleach. Then with a sterile glass rod I macerate the fruitbody thoroughly to release ascospores and create a dense spore suspension. Then I dilute aliquots 1:10 to as much as 1:100,000,000 in 200uL of sterile water volumes and plate this material out. Use a sterile bent glass rod & Petri plate rotator table to insure an even spread on the agar plate. Use a water agar plate, not a nutrient medium (e.g., PDA), or else everything will germinate & grow out (bacteria and yeast that were not removed initially with water rinses). I usually only plate out the final set of dilution series. Then under the 10X lens I find single isolated spores of the fungus, circle them with a marker, and, under the stereomicroscope, transfer individual spores onto a PDA plate, whereupon they germinate after a day or two. This technique works well for ascomycetes that “refuse” to shoot out their spores. Also, if it’s the “wrong season” (e.g., winter) and the spores are still not mature in the ascus (e.g., as judged by the lack of full septation and pigmentation), they can sometimes be induced to mature by several repeated cycles of freeze-thawing of the material. Hysteriaceous fungi respond well to this, and material collected in the winter months, that do not release their spores, can be induced to do so using this technique (Lohman 1933a). Whatever method one is forced to use, it is very important to actually check the morphology of the individual ascospores prior to their removal onto nutrient media. This is done by pouring very thin water agar plates (< 1mm deep) which allows one to invert the plate and use a compound microscope with the 10X lens to ascertain their morphology. This should not be done under a stereomicroscope, except for those fungi with very large spores (e.g.,Hysterographium fraxini or H. flexuosum). Confirmation of spore morphology, requires the pouring of thin water agar plates, that can be inverted and examined under a compound microscope using the 10X lens. At this magnification, you can clearly make out spore morphology. You need to have a long working distance 10X lens for this to really work. Alternatively, you can use a 4.5X lens and still make out the morphology. Don’t assume a spore deposit represents the fungus at hand until the spores have been morphologically identified under the compound scope. This is very important! Once an isolated spore has been found that corresponds to the morphology of the fungus in question, I circle scribe the bottom of the Petri plate with a thin Sharpie marker. The plate is then transferred back to the stereo microscope and using a sterile needle, under maximum power, a small water agar block is cut out containing the spore and transferred to another Petri plate containing nutrient media (e.g., PDA). I usually plate out half a dozen spores on one plate. After colonies have formed, I check to confirm that they are all identical and then arbitrarily take one to transfer to a PDA slant and designate it the culture for that particular specimen.

 

DNA extraction, amplification and sequencing: Genomic DNA was recovered using the DNeasy® Plant Mini Kit (Qiagen Inc., Valencia, CA, U.S.A.), following the instructions of the manufacturer, but using sterile white quartz sand and a Kontes® battery-powered pestle grinder in 1.5 mL microfuge tubes. The nuSSU was amplified and double-strand sequenced using the primers NS1 and NS4 (White et al. 1990), while amplification of the nuLSU utilised the primers LROR (Rehner & Samuels 1994) and LR7 (Vilgalys & Hester 1990), in addition to the internal sequencing primers LR3R and LR16 (Moncalvo et al. 1993). Final concentrations for 50 µL PCR amplification reactions were as follows: 1.0 µM of each forward and reverse primer, 2.0 mM MgCl2, 200 µM dNTP, 1X Promega GoTaq® Flexi Reaction Buffer, 1.25 U of Promega GoTaq® Polymerase, and 2 µL template DNA diluted tenfold. For the nuSSU and nuLSU, PCR reaction parameters were as follows: a 95 °C pre-melt for 3 min, and 35 cycles of 95 °C for 20 s, 54 °C for 30 s and 72 °C for 60 s, followed by a final extension at 72 °C for 10 min. For TEF1 and RPB2, PCR amplification conditions followed those in Schoch et al. (2006). Primers used for the amplifications and sequencing of these protein coding genes were for TEF1: 983 & 2218R; and for RPB2: fRPB2-5F & fRPB2-7cR. PCR reactions were performed using PCR Master Mix Polymerase from Promega Corporation (Fitchburg, Wisconsin, U.S.A.) and run on an iCycler® from Biorad (Hercules, California, U.S.A.). For the amplification of DNA fragments used to infer the TEF1 amino acid sequence, the following conditions were used: (1) 94 °C for 2 min; (2) five cycles of 94 °C for 40 s, 55 °C for 45 s lowering by 0.8 °C per cycle and 72 °C for 90 s; (3) 30 cycles of 94 °C for 30 s, 52 °C for 45 s and 72 °C for 120 s and (4) a cycle for 10 min at 72 °C. Amplifications of DNA fragments used to infer the RPB2 amino acid sequence utilised the same cycle parameters, except for changes in steps (2) and (3) where the annealing temperatures of 55 °C and 52 °C were changed to 50 °C and 45 °C, respectively. Amplified PCR products were cleaned using the QIAquick® PCR Purification Kit (Qiagen Inc.) and resuspended in water prior to outsourcing for sequencing (Macrogen U.S.A., Inc.).

Phylogenetic analysis: DNA sequences were derived from previous studies (Schoch et al. 2006, Boehm et al. 2009a, Mugambi & Huhndorf 2009), as well as from a number of new accessions generated in the most recent study, Boehm et al. 2009b (see Culture & GenBank page). In this latter study, sequences were aligned using default options for a simultaneous method of estimating alignments and tree phylogenies, SATé (Liu et al. 2009). Protein coding fragments were translated using BioEdit v. 7.0.1 (Hall 2004), and aligned within SATé as amino acid sequence data. These were then aligned with their respective DNA sequences using the RevTrans 1.4 Server (Wernersson & Pedersen 2003).  Newly generated sequences were subsequently added to the core alignment with MAFFT v. 6.713 (Katoh et al. 2009). A congruent super-matrix of five genes (nuLSU, nuSSU TEF1, RPB1 & 2) consisting of 56 % gaps and undetermined characters, across 121 taxa was obtained in Boehm et al. 2009b. The matrix was analysed using maximum likelihood in RAxML v. 7.0.4 (Stamatakis 2006). Data was partitioned by individual gene and, where applicable, by codon, as in Schoch et al. (2009). A most likely tree was obtained after 100 successive searches in RAxML under the GTR model with gamma rate distribution across 11 partitions and starting each search from a randomised tree with a rapid hill climbing option and joint branch length optimisation. Five hundred fast bootstrap pseudoreplicates (Stamatakis et al. 2008) were run under the same conditions and these values are given above each node. The matrix analysed in this study produced 4174 distinct alignment patterns and the most likely tree had a log likelihood of -72114.22899. The average log likelihood over 100 trees was -72117.730727. Three independent Bayesian phylogenetic analyses were performed in MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001) using a uniform [GTR+I+G] model. The Metropolis-coupled Markov chain Monte Carlo (MCMC) sampling approach was used to calculate posterior probabilities (PP). For each Bayesian run four Markov chains were run from a random starting tree for 5 000 000 generations and trees sampled every 100 generations. The first 50 000 generations trees were discarded as burn-in prior to convergence of four of the chains. All three run reached a plateau that converged. One run was chosen to construct a 50 % majority rule consensus tree of all trees remaining after the burn in was discarded. Bayesian Posterior Probabilities with those equal or greater than 50 % are given below each node (for trees see Molecular Systematics page).