Biodiversity in Oyster Reefs: A DNA Barcoding Approach Sydney Hoffmann, Fiona Mackenzie, Catherine McNamara& Cody Onufrock Long Beach High School Abstract Oyster reefs are a keystone species which are slowly becoming extinct due to the decreasing pH of the ocean and over-harvesting. The hypothesis is that the oyster reef will attract unique biodiversity. Our objectives are to study the biodiversity of the reefs on either side of the pole and to identify the specimens of the reef using taxonomic keys and DNA barcoding. We chose to barcode 13 of 25 samples, and 4 of them were successfully sequenced. One of our sequenced samples had no results for both the forward and reverse. Of our 3 remaining species, 2 of them came back as 81% matches for Monocorophium insidiosum, and the other one came back as a 97% match for Flatromenis proxima. Introduction Oyster reefs are slowly but surely going functionally extinct (Soniat, 2004). As the pH level of the water in bays and oceans decreases, oysters are struggling to reproduce. Due to the lack of reefs, the biodiversity of benthic invertebrates and infaunal reef dwellings have also struggled. Large factors in the widespread decline include destructive dredging, pollution, and the spread of disease through the introduction of non-native oyster species (Madsen, 2011), (Rudolf, 2011). As ''ecosystem engineers'' oyster reefs also help protect our shorelines from erosion, storms and provide a habitat - and food source - for fish, crabs, birds, and others (Madsen, 2011). The oysters help with overall purification and clarification of the waters which allows different plants to grow within the waters, one example of this is eelgrass. Another importance of oyster reefs is that they provide homes and hiding places for organisms that are seeking refuge from predation. This project aims to gain understanding of the biota that utilizes the reefs. The hypothesis is that the oyster reef will attract unique biodiversity. Materials and Methodology 2 in wide PVC pipe We collected samples from the oysters on 10, 100 and 1000 micropipettes the oyster side of the pond and for the Agarose gel muck side we stuck a half meter of the PVC Microcentrifuge pipe in the mud to collect organisms. Then Insect COI primer we used taxonomic keys to identify our Wash buffer organisms. We proceeded to Isolate DNA Distilled water from the specimens, amplify our specimens by Lysis solution PCR, analyze by gel electrophoresis and send our PCR products in for sequencing. Results Out of our original 25 samples, 13 of them we attempted to barcode. After going through the barcoding protocol, only 4 of them were able to be sequenced. When our sequences came back, sample 9I came back with no available sequencing data. Sample 4D ended up having a 97% match to Flatromenis proxima, and both species 17Q and 22V had an 81% match to Monocorophium insidiosum. Sample 4D Sample 17Q Sample 22V Sample 15O Oyster reef when it was just put in the pond. Discussion Based on our findings, we can conclude that the oyster reefs attract biodiversity in the pond. The monocorophium insidiosum is an amphipod that builds tubes of mud on weeds in shallow subtidal waters, like a pond. Although the Flatromenis proxima was a match for one of our organisms using BLAST, it is not likely to be the organism collected because it had quite a few mismatches. There were also limited resources available to describe the Flatromenis proxima ecology. And we plan to re-barcode this sample. Our specimen may also be novel species, because none have a 100% match. Acknowledgements We would like to thank the Cold Spring Harbor DNA Laboratory and Cody Onufrock for assisting us with this project. Gel electrophoresis

References Soniat, T. M., Finelli, C. M., & Ruiz, J. T. (2004). Vertical structure and predator refuge mediate oyster reef development and community dynamics. Journal of Experimental Marine Biology and Ecology, 310(2), 163-182. Rudolf, J. C. (n.d.). Oyster Reefs Are Vanishing From Overharvesting. Retrieved February 1, 2017 Madsen, A. (n.d). Oysters in Danger of Extinction | Nature | The Earth Times. Retrieved February 13, 2017.