Michael Swindle and Michael Whiting, Department of Biology
Introduction
The goal of this project was to expand and clarify the relationships between the various subfamilies of fireflies. Lampyridae (the scientific name for fireflies) is a family within the beetle order, Coleoptera, and include about 2000 species across the world. Lampyridae is currently recognized as having nine separate subfamilies, but the relationships between these groups are unclear. This project was meant to determine clear relationsips between these groups and further determine the location of key species, such as Pterotus obscuripennis, whose phylogenetic position has remained uncertain after many attempts to place it firmly within a specific subfamily. However, this project was modified and pursued in another direction, and the author was instead placed on another project to create a new phylogeny for Mecoptera. Mecoptera, or scorpionflies, are recognizable for having several physical features similar to those of a scorpion, hence their unusual name. This project was then designed to further expand the families within Mecoptera. The Whiting Lab published a phylogeny on Mecoptera and Siphonaptera several years ago and has since acquired many new specimens. Additionally, the technology of gene mapping has significantly expanded, allowing new genes to be added to the phylogeny for comparison and reevaluation of previous work. This new project seeks to use the past information concerning the genes 18S, 28S, EF-1a, and COII and expand it by adding the gene CAD.
Methodology
As the previous work concerning Mecoptera was published in 2002, DNA must be re-extracted to add the gene CAD. DNA will be extracted using a QIAGEN DNeasy Kit. PCRs will be conducted on the CAD gene for all taxa, using the appropriate primers optimized for beetles first, in hopes that there will be some similarities. Such a find would allow the lab to follow a cost-efficient course, as extensive work with CAD has already been performed on beetles by the Whiting Lab. An agarose gel will be run to determine the validity of the product before the product is cleaned using a USB PrepEase PCR Purification plate. Sequencing will take place utilizing BigDye chain terminating chemistry and then purified using Sephadex and submitted to the BYU DNA Sequencing Center. This is currently underway but is still undetermined to consistently work, potentially pushing the lab to design and utilize new primers if the previous ones fail to return accurate or useful information.
Results
As stated previously, the lab is currently in the process of finding effective primers to magnify and then sequence CAD. As the CAD gene is too long for a single set of forwards and backwards primers to effectively map and copy, it is divided into three separate segments after a template run and then uses a nested PCR approach to multiply these three segments. Currently, the lab has been able to utilize beetle-optimized primers to run a PCR with CAD segments 2 and 3, but there have been no successful runs with CAD 1 yet, even after attempting PCR’s with several different primers and protocols. The lab will most probably attempt to create new primers that are optimized specifically for Mecoptera, however, this will not take place until after the new year. The lab is currently waiting to submit a purified reaction to the sequencing center to verify that the recovered CAD 2 and 3 PCR runs were indeed successful and do not turn out to be gibberish.
Discussion
The previous publication from the Whiting lab considered 41 taxa of Mecoptera. However, this new project will attempt to cover over 150 taxa, significantly expanding the previous work and establishing more rigid and consistent separations between the families of Mecoptera. This will also be further developed by the addition of CAD to the previously utilized genes. A greater amount of sequenced DNA information per taxa will go a long ways towards eliciting specific families. Thus, this new project has a similar goal to the previous project on fireflies. Both are attempting to create larger and more in depth phylogenies. However, while Lampyridae is a single family within Coleoptera, this new project is ambitious in its attempt to cover an entire order (restricted only by limited taxa, funding, and time). Even without data on every species within Mecoptera, creating a better framework will make the identification of new species easier and allow for existing species not considered in this specific project to be added to the phylogeny with greater ease and certainty in the future.
Conclusion
So far, the test insects used to determine if the old Coleptera primers would function have turned up consistent results. CAD 2 and 3 should continue to function with regularity, while further work will need to be done to determine optimized primers for the CAD 1 section. Hopefully, this can be determined quickly, such that a complete CAD gene database can be generated for all the taxa. This combined with all the previous data will produce a Mecoptera phylogeny larger than any previously assembled and hopefully more accurate as well.