Taxonomic Placement of the Nidulariaceae of Nebraska and Iowa Based on Molecular and Morphological Data Goodmond H. Danielsen, Mark A. Schoenbeck, P. Roxanne Kellar Department of Biology, University of Nebraska at Omaha, Omaha, NE 68182 Introduction Conclusions The fungal family, Nidulariaceae or “bird’s nest fungus,” was extensively studied by two mycologists, Curtis G. Lloyd and Harold J. Brodie, in the early 1900’s and 1970’s, respectively. These authors described many species, creating and continuously updating morphological keys for identification of the family’s five genera. These keys have been updated by mycologists in the Upper Midwest and Pacific Northwest in recent years. Additionally, some molecular work has been completed using mitochondrial and nuclear regions in attempt to find a universal fungal barcode. The purpose of this study is to build on the morphological work already completed by filling gaps in current knowledge on the genera found in Iowa and Nebraska. By testing both specific and not specific primer pairs to amplify the most commonly sequenced gene regions in fungi, phylogenetic trees can be created to compare the species present in Iowa and Nebraska to those found in other parts of the United States. After measuring morphological characteristics of the species found in Iowa and Nebraska and backing up the resulting identifications with molecular data, it is expected that intraspecific and perhaps interspecific variation will be found, resulting in additions to the currently available identification keys. The results of the morphological tree are inconclusive. One branch has high bootstrap support while the others are less than desirable. It is possible that these percentages are the highest possible when using the same species. The outgroup is a species from Crucibulum, a different genus within Nidulariaceae. This was chosen due to the special morphological structure of the bird’s nest fungi. Rooting the tree with a different gasteroid species may yield a better result. The results yielded by the reactions shown in figures 3A and 3B show bands of both expected and unexpected sizes. Those of expected sizes are most likely the regions targeted for amplification. The results yielded by the reactions shown in figure 4 are all bands of the expected size for the region amplified. The control lanes are all empty as expected. Consequently, it is likely that this primer pair and PCR strategy works well for the family Nidulariaceae. Figure 2. Target regions for molecular analysis. The regions chosen for amplification, the ITS and large-subunit (LSU) of the nuclear rRNA gene complex, were chosen due to their frequency in the literature. There are many primer pairs that span most of the nuclear rDNA for most major groups of fungi, including bird’s nest fungi. Placement of highlighted boxes is approximate. Image adapted from the Vilgalys Lab. 3A 3B M *F *R *FR FR Future Direction More data needs to be added to the morphological tree of figure 1 such as traits like hirsuteness of mushrooms, shape of peridioles, presence of striations, etc. Unexpected products seen in gels of figures 3A and 3B need to be worked around. This may require making changes to the PCR strategy. The reactions of figure 4 and the reactions from the remaining specimens must be cleaned and prepared for sequencing at the UNMC DNA Sequencing Core Facility or cloned and sequenced in-house to confirm results. Using the data from the sequences generated from all reactions, create phylogenetic trees to infer evolutionary relationships. Concatenate morphological and molecular data sets. Use all resulting data to add to existing knowledge, in particular existing identification keys. Results Figures 3A and 3B. Amplifications of the ITS (left) and LSU (right) regions of bird’s nest fungi shown with DNA gel electrophoresis. The PCR reactions using specific primers yielded expected results in a few reactions (row 1, lane 7; row 2, lane 4; row 3, lane 4 of both gels). Additionally, most lanes showed additional unpredicted products. In the first lane of each row, there is a ladder (M), followed by sets of three, with some combination of the DNA template (*), the forward primer (F), and the reverse primer (R). The reaction in lane 8 in the bottom row of both gels consists of the forward and reverse primers without the template. 69 52 91 57 54 Special thanks to Claudia M. Rauter, David M. Sutherland, the University of Nebraska at Omaha Fund for Undergraduate Scholarly Experiences (FUSE), the Dr. C.C. and Mabel L. Criss Library 57 M *F *R *FR FR References Brodie, H. J. (2016). The bird’s nest fungi. Canada: University of Toronto Press. Conserved primer sequences for PCR amplification and sequencing from nuclear ribosomal RNA. Retrieved December 1, 2016, from Vilgalys lab, Duke University, http://sites.biology.duke.edu/fungi/mycolab/primers.htm Fay, M., Scates, K., & Ramsey, R. (1981). Trial field key to the species of BIRD’S NEST FUNGI in the Pacific Northwest. Retrieved December 1, 2016, from http://www.svims.ca/council/Bird’s.htm Kuo, M. (2014, February). The bird's nest fungi. Retrieved December 1, 2016, from http://www.mushroomexpert.com/birdsnests.html Lloyd, C. G. (2015). Mycological writings of C. G. Lloyd, volume 2. United States: Andesite Press.   Figure 1. A bootstrap 50% majority-rule consensus phylogenetic tree based on morphological measurements of Cyathus striatus. This tree infers a strong relationship between specimens 5, 8, and 18a. These specimens were all found near each other in the Douglas County area. Other branches with bootstrap support above 50% were also collected from the same approximate geographical area. Figure 4. Amplification of the ITS regions of bird’s nest fungi shown with DNA gel electrophoresis. This gel shows results of PCR with less specific primers. The results show correctly sized bands in the appropriate lanes and no spurious products in any lanes. Labeling scheme is as in figures 3A and 3B. Other Contributor Logos Go Here