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DNA Barcoding of the First Recorded American Burying Beetle, Nicrophorus Americanus, in Clark County, Arkansas J Journal of the Arkansas Academy of Science Volume 70 Article 21 2016 DNA Barcoding of the First Recorded American Burying Beetle, Nicrophorus americanus, in Clark County, Arkansas J. Kelly Ouachita Baptist University, [email protected] T. Files Ouachita Baptist University N. Reyna Ouachita Baptist University B. Baley Ouachita Baptist University Follow this and additional works at: http://scholarworks.uark.edu/jaas Part of the Entomology Commons, and the Population Biology Commons Recommended Citation Kelly, J.; Files, T.; Reyna, N.; and Baley, B. (2016) "DNA Barcoding of the First Recorded American Burying Beetle, Nicrophorus americanus, in Clark County, Arkansas," Journal of the Arkansas Academy of Science: Vol. 70 , Article 21. Available at: http://scholarworks.uark.edu/jaas/vol70/iss1/21 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Journal of the Arkansas Academy of Science, Vol. 70 [2016], Art. 21 DNA Barcoding of the First Recorded American Burying Beetle, Nicrophorus americanus, in Clark County, Arkansas J. Kelly*, T. Files, B. Baley and N. Reyna Department of Biology, Ouachita Baptist University, 410 Ouachita Street, Box 3792, Arkadelphia, AR 71998-0001 Correspondence: [email protected] Running Title: DNA Barcoding of the American Burying Beetle, N. americanus, in Clark County, AR Abstract and Wildlife Service (USFWS) to report the accidental capture of an endangered species outside of its’ known The American Burying Beetle (ABB), Nicrophorus range. That specimen would be awarded to Ouachita americanus, is a red-listed endangered species under Baptist University by letter from an Endangered the Endangered Species Act (16 U.S.C. 1531 et seq.). Species specialist. Two subsequent individuals were We serendipitously recorded 3 individuals of this noted in the next evening sample which were species during a forensic study in the summer of 2013. measured, sexed and released. Those captures were These specimens represent the first known records for also reported. We suspended the field collections at Clark County, AR and the southeastern-most record in that point to avoid the capture of further endangered the State since the extirpation of the species from the ABB after consulting with the USFWS. Our region in the late 1800’s. Two males and one female specimens were captured in a mature growth forest just were collected, photographed, sexed and measured. east of the City of Arkadelphia, AR. This location is One male specimen was deceased upon discovery. The consistent with the known preference of habitat as remaining two individuals were released. The U.S. stated in Lomolino and Creighton (1996) and (Sikes Fish and Wildlife Service (USFWS) was notified of the 2005). accidental catch and death of an endangered species as Reports of ABB capture are sporadically received required and the specimen was awarded to Ouachita by the USFWS but most tend to be inaccurate within Baptist University for further study. The deceased the genus based on common field identification errors. specimen was used for DNA barcode sequence Other closely related Nicrophorus species captured in analysis. A 400bp section of the cytochrome c oxidase our 2013 study included N. orbicolis and N. I (COI) gene was amplified using gene specific primers tomentosus. Our accidental capture report was met and then sequenced using Sanger sequencing methods. with initial skepticism until detailed descriptions and a Sequence analysis revealed the collected beetle to be crude phone camera photo were submitted (Figure 1). 98.5% identical to the ABB voucher sequence and was ABB are readily identifiable by their bright orange to 86% similar to other Nicrophorus species. Taken red coloration on the head and pronotum, a feature together the DNA sequence analysis results and lacking in the other Nicrophorus species (Sikes and taxonomic identification both support the identification Peck, 2000). ABB are also typically much larger than of our specimen. their closely related species and are the largest of the Silphid beetles in North America (Anderson and Peck Introduction 1985). The biology and distribution of the ABB is well established into four ecoregions within the United We discovered three specimens of the American States, including the northwest region within the State Burying Beetle (ABB), Nicrophorus americanus of Arkansas (USFWS 2008). during a forensic study in the summer of 2013 in Clark A few previous studies have examined the genetic County, Arkansas. Two males and one female were variation of the ABB in Arkansas (Kozol et al. 1994) discovered on experimental pig and rat carrion over a and (Szalanski et al. 2000) and we knew that genetic three night period. Burying beetles are commonly comparisons were available for the ABB in the iPlant associated with carrion (Scott 1998). A male specimen DNA Subway (Goff et al. 2011). We decided to was found deceased on the second night and we confirm the identity of our specimen using DNA realized it was an endangered ABB species. We barcoding methods to eliminate any consideration of a immediately made contact with the United States Fish false identification in the field. This was risky due to us Journal of the Arkansas Academy of Science, Vol. 70, 2016 118 Published by Arkansas Academy of Science, 2016 118 Journal of the Arkansas Academy of Science, Vol. 70 [2016], Art. 21 DNA Barcoding of the American Burying Beetle, N. americanus, in Clark County, AR (Accession ID: KX687862). Sequence Analysis and Alignment Sequence chromatograms were edited and assembled using the iPlant: DNA subway editing tool (http://dnasubway.iplantcollaborative.org/, Goff et al. 2011). These alignments were examined by eye to detect potential base calling errors, particularly at the beginning and ends of traces. Potential errors were checked in the trace files and corrected (shortened) as necessary. Figure 1 provides the shortened trace file submitted for comparison. Sequence alignments and similarity were generated using MUSCLE [v. 3.8.31], (Robert, 2004) as implemented by DNA Subway. Figure 1. Deceased American Burying Beetle male on rat carrion Additional, alignments, comparisons and dot plot were in a bucket trap in Clark County, AR. generated directly using the Basic Local Alignment Search Tool [BLAST 2.3.1] (Altschul et al. 1997) on having only one individual to work with so we decided the NCBI website (http://blast.ncbi.nlm.nih.gov/ to run a test study with the closely related Nearctic Blast.cgi). Our BLAST results were compared to the burying beetle, Nicrophorus orbicolis. This study Nicrophorus americanus voucher specimen. demonstrated that DNA extraction was best achieved using the material near the joints of the legs of the Results and Discussion beetle. Our DNA barcoding effort was designed based upon the results of that study (Kelly and Jackson, DNA Barcode Analysis unpub). DNA barcoding uses a 650 base pair region of DNA associated with the mitochondrial cytochrome Materials and Methods oxidase subunit 1 gene (COI) to create a genetic DNA barcode (Ratnasingham and Herbert 2013, Sikes and DNA extraction, PCR and Sequencing Venables 2013). Identification is achieved by DNA extraction and PCR were conducted using comparing the sequenced data or genetic barcode to a the DNA Barcode Amplification Kit (Carolina reference library containing known taxa or voucher Biological Inc. Cat# 211385) without modifications. sequences (Ratnasingham and Herbert 2007). This kit was specially designed to be a robust DNA A 600 base pair region of the Clark County ABB extraction procedure and was made under the specimen was isolated and sequenced using recommendations of the International Barcode of Life conventional DNA barcode protocols (Goff et al. project and the iPlant Collaborative Bar Code Project 2011). However this sequence was trimmed to a 352 (Goff et al. 2011). A single beetle leg was used for base pair section (Figure 2) which was used for DNA extraction. PCR was done using the analysis and identification. Trimming was performed Animal/Insect primer mix (Carolina Biological Cat# due to several misreads at the ends of each tail of the 211513). This primer mix is a cocktail of primers that ABB sequence. While there was reliable data with target a 650 base pair segment of the mitochondrial good homology in these areas the occasional presence cytochrome c oxidase subunit 1 (COI) gene. The of misread basses, designated with an n, would insect specific primers LepF1A (5’- potentially make this data un-reliable. We did not feel ATTCAACCAATCATAAAGATATTGG-3’) and confident in including misreads in our submission. LepR1 (5’TAAACTTCTGGATGTCCAAAAAATCA- 3’) are included in this cocktail (Herbert et al. 2004). Both primers contained the M13 (5’- GTAAAACGACGGCCAGT-3’) sequence on their 5’ end. M13 specific primers were used for DNA sequencing. PCR amplicon purification and single direction sequencing were performed by GENEWIZ, Figure 2. FASTA trimmed data for the Clark County 2013 inc. This sequence was submitted to NCBI GenBank American Burying Beetle specimen (GenBank ID: KX687862). Journal of the Arkansas Academy of Science, Vol. 70, 2016 119 http://scholarworks.uark.edu/jaas/vol70/iss1/21 119 Journal of the Arkansas Academy of Science, Vol. 70 [2016], Art. 21 J. Kelly, T. Files, B. Baley and N. Reyna However, it should be noted that misreads are common Conclusions at the beginning and ends of DNA sequenced with Sanger sequencing methods. Due to the scarcity of our The aim of this study was to confirm that we had material, we were unable to submit additional samples discovered and properly identified three specimens of for sequencing.
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