Combining Non-Invasive DNA Extraction with Next-Generation Sequencing to Study California Gall Wasps Julian Rasco, Jeremy C
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Combining Non-invasive DNA extraction with Next-Generation Sequencing to study California Gall Wasps Julian Rasco, Jeremy C. Andersen, Rosemary Gillespie, Nicholas J. Mills University of California, Berkeley Background Methods Phylogenetic Reconstruction Based on 28S California oak gall wasps have intrigued researchers for decades 1) Genetic material was isolated from 111 identified and 42 Synergini EMEC1131407 Callirhytis quercuspomiformis EMEC1131427 Heteroecus pacificus due to their striking ability to induce vibrant growth of physically unidentified oak gall wasp specimens (inquilines) EMEC1131442 Neuroterus varians diverse structures from their host’s tissue. The galls’ primary 2) Three genetic loci were targeted for PCR amplification (28S, EMEC1131335A Undet sp EMEC1131416 Disholcaspis plumbella function is to nourish the wasp’s larvae, protecting them while COI, and LWRh) EMEC1131445 Neuroterus varians [1] they are most vulnerable to predation. Most of the work done 3) Amplified fragments were barcoded with unique primer tails EMEC1131424 Dryocosmus minisculus with gall wasps has focused on gall morphology, so very little is 4) Barcoded fragments were sequenced using the Illumina MiSeq EMEC1131423 Dryocosmus minisculus EMEC1131417 Disholcaspis plumbella known about the wasps themselves. For example the underlying Next-Generation Platform sequencing EMEC1131421 Dryocosmus minisculus EMEC1131397 Bassettia ligni mechanism for gall formation is unknown, largely due to a lack of 5) Specimens were re-pinned and returned to the Essig Museum Monophyletic EMEC1131393 Bassettia ligni understanding of the phylogenetic relationships of oak gall wasps. 6) Sequences were aligned to estimate a molecular phylogeny EMEC1131395 Bassettia ligni species This uncertainty in their taxonomy makes the California oak gall 7) Results were compared to published records for verification EMEC1131405B Callirhytis quercuspomiformis 2] EMEC1131404 Callirhytis quercuspomiformis Monophyletic wasps an excellent candidate for research. and to provide species-level IDs when possible EMEC1131405A Callirhytis quercuspomiformis species Cynipini EMEC1131379 Andricus occultatus EMEC1131418 Disholcaspis plumbella (gall formers) EMEC1131420 Disholcaspis plumbella Monophyletic species EMEC1131398B Besbicus conspicuus EMEC1131401 Besbicus conspicuus EMEC1131412 Disholcaspis eldoradensis EMEC1131438 Neuroterus saltatorius EMEC1131398A Besbicus conspicuus EMEC1131436 Neuroterus saltatorius EMEC1131441 Neuroterus varians Monophyletic EMEC1131443 Neuroterus varians species EMEC1131308 Undet sp EMEC1131313Undet sp EMEC1131440 Neuroterus saltatorius EMEC1131428 Heteroecus pacificus EMEC1131437 Neuroterus saltatorius 28S sequence alignment for specimens from the Essig Museum 0.01 Results Future Research 1. Fragments of 28S, COI, and LWRh were amplified from 78, 27, 1. Use the morphological specimens to verify some of the and 124 specimens, respectively misidentifications noted in our analysis 2. Of those loci, only 28S sequenced successfully (32 specimens) 2. Use the 28S results to identify specimens to genus, and then to Examples of California oak galls – photos by Joyce Gross 3. Ten specimens identified as gall formers, are actually inquilines design genus-specific primers for COI and LWRh 4. Only four species were reconstructed as monophyletic 3. Analyze factors that might have influenced the success of Objectives amplification and sequencing (e.g. age, collection method, The Essig Museum of Entomology is home to one of the best DNA concentration, etc.) collections of oak gall wasp species, including specimens collected over 100 years ago. This collection could greatly help illuminate problems in the California oak gall wasp phylogeny and taxonomy. Acknowledgements Here, non-invasive DNA extraction techniques[3][4] are used to This project was made possible by the generous donations of CNR create a digital molecular dataset that will be available for public alumni to the SPUR program. I would also like to thank Peter use. This dataset will be used to provide species-level Oboyski for his mentorship & for allowing use of the museum’s identifications for specimens in the Essig Museum and other specimens. Finally, my greatest thanks to Joyce Gross, Kathy Natural History Museums as well as pave the way for future Example of oak gall wasp specimen prior to DNA extraction (left), Schick, Diane Erwin, and members of the Gillespie lab for their research regarding mechanisms of gall formation. during (center), and after (right) guidance & assistance with the project. REFERENCES [1] Stone, G. N. and Schönrogge, K. (2003) The adaptive significance of insect gall morphology. [3] Andersen, J.C. & Mills, N.J. (2012) DNA extraction from museum specimens of parasitic TRENDS in Ecology and Evolution. 18: 512-522 hymenoptera. PLoS One 7: e45549 [2] Russo, R.A. (1979). Plat Galls of the California Region. The Boxwood Press, Pacific Grove, CA. [4] Gilbert, M.T.P., Moore, W., Melchior, L., & Worobey, M. (2007) DNA extraction from dry museum beetles without conferring external morphological damage. PLoS One 2: e272 .