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Issues in the Benchmarking of Image Analysis Evolutionary Phylogenetics of Cladobranch Sea Slugs Katherine Mock*, Jermaine Mahguib, Ángel Valdés Picture 1: Cuthona sp. Abstract Methods Results Continued DNA was extracted from tissue samples using DNeasy and DNA was extracted from small pieces of tissue samples using DNeasy and Chelex extraction Chelex. PCR was performed on the extractions to amplify protocols. DNeasy Kits use a silica-membrane filtration system to separate DNA. Chelex uses two mitochondrial genes, CO1 and 16S, and one nuclear resin beads that protect the DNA after being released from cells via heating. The solution is gene, H3. The PCR product was electrophoresed in a then centrifuged and the resulting supernatant is a DNA/RNA/water mixture. “used” TBE agarose gel stained with ethidium bromide. The amplified DNA was then purified and sent for These extraction protocols are used in preparation for PCR. Partial sequences of the sequencing. mitochondrial genes CO1 and 16S were amplified along with the nuclear gene H3 using their appropriate primers. Amplifications were carried out using a solution of DI water, additional GenBank sequences were used and the combined MgCl₂, buffer, dNTPs, two primers, taq, and DNA. molecular data (CO1, 16S, H3) was analyzed by Picture 2: Flabellina exoptata MrModelTest to create a phylogenetic tree. Cladobranchs are considered a drug-prolific group. Understanding the evolutionary relationship between cladobranch species will facilitate future drug research. Introduction Cladobranchia are a diverse group of nudibranch sea slugs. They are defined by their many cerata, lateral outgrowths on the surface of their bodies that aid in respiration and metabolic gas The PCR product was electrophoresed in a “used” TBE agarose gel stained with ethidium exchange. The cerata can also be used as a bromide. (See Figure 1) The amplified products were purified and sent for sequencing. defense mechanism. In some cnidarian eating Figure 2: Phylogenetic hypothesis based on combined molecular data (H3,COI,16S) represented by cladobranch species, the nematocysts are stored MrModelTest. The numbers indicate the probabilities at the tips of the cerata to be used at a time of from Bayesian inference. A strong probability is Picture 8: Flabellina pedata defense. represented above 0.9. The monophyly of Tritonia is strongly supported. Figure. 1: Shown is amplified DNA for CO1 gene in Picture 3: Baeolidia moebii Some cladobranchs are able to produce their an agarose gel. The gel is highlighted with own toxins de novo without any dietary influence. The chemical defenses they posses are ethidium bromide and is shown under a UV light. useful in pharmacological research and they are considered a drug-prolific group. Understanding the phylogenetics, evolutionary relationships, between species of cladobranchs is important to aid in drug research. Sequences for CO1, 16S, and H3 were downloaded from GenBank to Geneious. Geneious aligned and concatenated the sequences. MrModelTest was used to determine the Over the course of the summer, molecular congruence between them. The data was analyzed with MrBayes, a program that uses the techniques were utilized to obtain DNA Bayesian inference to calculate the likelihood of familiarity between two species. The tree sequences of target genes (CO1, 16S and H3) for was constructed using these likelihoods (See Figure 2). use in a phylogenetic analysis of representative Picture 10: Austraeolis stearnsi species from varies cladobranch groups. An Picture 9: Dondice banyulensis analysis was run in order to produce a phylogeny Results showing evolutionary relationships among Conclusion cladobranch genera. Sea slug specimens Picture 4: Phyllodesmium longicirrum Approximately 50 sequences from CO1, 16S, and H3 were gathered from GenBank and used preserved in ethanol from the invertebrate to construct the phylogenetic tree. The three genes were combined into a single analysis to DNA was extracted from tissue samples using two extraction protocol: Chelex and DNeasy. animal collections of the California Academy of Sciences and Cal Poly Pomona were used to better represent the relationships between species. Figure 2 shows the phylogenetic PCR was performed on the extracted DNA. The PCR product was electrophoresed in a obtain genomic DNA for amplification of specific genes of interest. DNA was obtained hypothesis based on the data set represented by MrModelTest. The analysis determined the “used” TBE agarose gel stained with ethidium bromide. The DNA was amplified and created through two DNA extraction protocols that were learned: Chelex and DNeasy. Gene maximum likelihood of 200,000 combinations of datasets. bands on a gel. amplification was attempted through use of the polymerase chain reaction, PCR, although technical difficulties encountered throughout the summer lead to the acquisition of only a A dorid nudibranch, diaulula sandiegensis, was used as the control group during the Technical difficulties throughout the summer lead to only a few bands. In order to run the few novel sequences. In order to run our phylogenetic analysis, DNA sequences from many analysis. Outgroups of moderately related specimens are used to establish a root on a phylogenetic analysis, DNA sequences were downloaded from GenBank. The sequences representative cladobranch species were downloaded from GenBank, an online repository phylogenetic tree. The outgroup is shown alone and with no relation to any other were organized according to their genes , aligned, and then concatenated using the program of gene sequences from a wide variety of organisms. These sequences were then organized represented species. A dorid was used because they are the closest related family to the Geneious. MrModelTest determined the best model of evolution and then MrBayes ran by each of our three target genes and aligned using a cladobranchs; diaulula sandiegensis is shown at the top, away from the other species, and Bayesian inference on the concatenated alignments. The phylogenetic tree was created program called Geneious. Geneious was also used to is the root of the tree. with some groups being well supported and others not. concatenate the three gene alignments in preparation for analysis. A program called MrModelTest was used The resulting tree showed many of our representative cladobranch species grouping into to determine the best model of evolution for each of their previously established genera, with some groups being well supported and others not. Acknowledgements the three gene alignments, and a second program called MrBayes was used to run a Bayesian inference This research was supported by an undergraduate research scholarship from the STEM on our concatenated alignment based on their best Grant presented by Dr. Marianna Smith of Citrus College. models of evolution. Picture 5: Babakina anadoni Biological Department at Cal Poly Pomona Picture 6: Tritonia diomedea Picture 7: Tritonia festiva .
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