DOI: 10.5061/dryad.k3j9kd5f5
title: Clay models and eDNA are useful tools for identifying predators of Salamanders

description: Clay models are a popular technique for studying predation in nature due
 to their ease of deployment and minimal disruption of natural processes,
 but a drawback is the ambiguity of identifying predators based on bite
 marks. However, it is possible to amplify and sequence environmental DNA
 (eDNA) from these bite marks and to identify the predators responsible for
 attacking models. In this study, we sought to test the viability of eDNA
 from clay models as a means of identifying predators. We deployed molded
 clay models that resemble Plethodon ventralis Highton (Southern Zigzag
 Salamanders) into the field. We then extracted eDNA from visible bite
 marks, amplified and sequenced the 12S rRNA mitochondrial locus on an
 Illumina MiSeq, and used BLAST to determine the identity of representative
 sequences. We identified likely predators as Procyon lotor L. (American
 Raccoons), Didelphis virginiana Kerr (Virginia Opossums), Turdus
 migratorius L. (American Robins), and Tamias striatus L. (Eastern
 Chipmunks). We believe that this technique is helpful for adding a layer
 of specificity to clay model studies, albeit with a few potential pitfalls
 that we discuss.
description: # Clay Models and eDNA are Useful Tools for Identifying Predators of
 Salamanders
 [https://doi.org/10.5061/dryad.k3j9kd5f5](https://doi.org/10.5061/dryad.k3j9kd5f5) Clay models visually represent organisms and can be placed in natural environments with minimal disturbance to the environment or target species. Deployed clay models are monitored for signs of attack, and dental indentations can then be used to identify potential predators. This study tested whether Environmental DNA, extracted from the surface of attacked clay models, can be used to identify the predator responsible for the attack. ## Description of the data and file structure The DNA sequence data that is provided is based on sequencing of 100 bp of the 12S rRNA mitochondrial marker using fusion versions of vertebrate-specific primers (12S-V5; Riaz et al. 2011). These primers have been verified to provide taxonomic resolution for distinguishing vertebrate fauna in eDNA experiments. This amplicon was sequenced with an Illumina MiSeq Nano PE250 (Illumina, San Diego, CA) with a 20% PhiX spike. All DNA sequence data was processed with the DADA2 (v1.26.0; Callahan et al. 2016) bioinformatic pipeline in R (v4.2.1; R Core Team 2022). The file "eDNA\_OTUs.csv" is a CSV table containing 105 columns that each represent an operational taxonomic unit (OTU). Each OTU is a grouping of DNA sequences that, after filtering for errors, were clustered with 97% similarity using the DECIPHER package in R (Wright, 2016). Thus, 140,514 DNA sequence reads were condensed to 105 significantly unique sequences. The rows of the table represent the counts of an OTU in each of the 48 extraction samples. The file "consensus\_sequences.fasta" is a FASTA file with the consensus sequences for each OTU described above. These sequences are numbered in order of the OTUs. The consensus sequences for each OTU is determined by taking an average of all sequences that are grouped together. This file was used to identify the species using nucleotide BLAST in Genbank (Zhang et al. 2000).