DOI: 10.5061/dryad.d7wm37q1v
title: Data from: DNA barcoding identifies cryptic animal tool materials

description: Some animals fashion tools and other constructions out of plant materials
 to aid foraging, reproduction, self-maintenance, and protection. The
 choice of raw materials can affect the structure and mechanical properties
 of the resulting artefacts, with significant fitness consequences.
 Documenting animals’ material preferences is challenging, however, as
 manufacture behaviour is often difficult to observe directly, and
 materials may be processed so heavily that they lack identifying features.
 Here, we use DNA barcoding techniques to identify, from just a few
 recovered tool specimens, the plant species New Caledonian crows (Corvus
 moneduloides) use for crafting elaborate hooked stick tools in one of our
 long-term study populations. The method succeeded where extensive
 fieldwork using conventional approaches had failed, including targeted
 observations, radio-tracking, bird-mounted video-cameras, and behavioural
 experiments with wild and temporarily captive subjects. We believe that
 DNA barcoding will prove useful for investigating many other tool and
 construction behaviours, helping to unlock significant research potential
 across a wide range of study systems.
description: In May 2018, seven crow tools collected at site-3, New
 Caledonia between 2016 and 2017 were subsampled, two subsamples were taken
 from each tool, and these were homogenized in 2 ml tubes with two tungsten
 beads using the FastPrep-24™ 5G Benchtop Homogenizer for up to one minute,
 or with QIAGEN Tissuelyser II for 6 minutes at 25 Hz. DNA extraction then
 proceeded according to the DNeasy Plant Mini Kit (QIAGEN, Manchester UK),
 except the lysis step was extended to 60 minutes. For
 species identification, we amplified ~500 bp regions of the chloroplast
 trnL-UAA intron [trnLc-d] and a ~600 bp nuclear ribosomal region
 containing the internal transcribed spacer regions ITS1 and ITS2
 [ITS5p-8p]. Amplification was in 20 µl reactions using approximately 100
 ng of genomic DNA, 10 x reaction buffer (Bioline BIOTAQ Reagent Buffer),
 30 nmol MgCl<sub>2</sub>, 4 nmol dNTPs, 7.5 pmol primers, and
 Bioline BIOTAQ DNA polymerase (0.5 units). Negative controls and DNA
 extraction blanks were included in each PCR to check for potential
 contamination. Thermocycling was performed on a Bio-Rad Tetrad 2 (Bio-Rad,
 Hamburg, Germany) under the following conditions. For trnL: initial
 denaturation (94 °C for 4 min), followed by 35 cycles of denaturation (94
 °C for 45 s), annealing (55 °C for 45 s) and extension (72 °C for 120 s),
 and a final extension of 10 min at 72 °C; and for ITS: initial
 denaturation (94 °C for 2 min), followed by 30 cycles of denaturation (94
 °C for 60 s), annealing (55 °C for 60 s) and extension (72 °C for 90 s),
 and a final extension of 5 min at 72 °C. Two independent PCRs were carried
 out for each subsample. Successfully amplified PCR products were cleaned
 using ExoSap-IT© (USB Corporation, Cleveland, Ohio, USA). Sequencing was
 resolved on an AB 3730xl Sequencer at Edinburgh Genomics (Scotland, UK).
 Sequences were checked and edited with reference to chromatograms using
 Sequencher v 5.4.1 (Gene Codes Corporation, Ann Arbor, MI, USA).
 Sequences obtained for all tool samples were lodged with GenBank
 under accession numbers MT366813–MT366819 (<em>trnL</em>) and
 MT366951–MT366952, MT366955–MT366959 (ITS). The resulting sequence
 haplotype for each region was used for performing BLASTn searches against
 the National Centre for Biotechnology Information Nucleotide nonredundant
 database (4 July 2018) to obtain the best 100 matches (excluding
 environmental samples).  Based on putative
 identification from the BLASTn searches, we also collected reference leaf
 samples of <em>Mimusops elengi </em>(n = 3) and its closest
 locally occurring relative, <em>Planchonella cinerea </em>(n =
 2). Leaves were collected from separate trees at or near site-3 and
 site-1. In March 2019, DNA extraction, PCR amplification and sequencing
 for the reference samples were performed exactly as described above for
 the tool samples. The resulting DNA sequences for the reference data were
 lodged with GenBank under accession numbers MT366823–MT366824
 (<em>trnL</em>) and MT366953–MT366954 (ITS) for <em>P.
 cinerea</em>, and MT366820–MT366822 (<em>trnL</em>) and
 MT366960–MT366962 (ITS) for <em>M. elengi</em>. Phylogenetic
 trees were generated using these samples plus the top 100 BLASTn matches
 (excluding environmental samples). Since taxa from both Sapotaceae and
 Theaceae were present in the top matches for trnL, <em>Acanthogilia
 </em>gl<em>oriosa </em>(GenBank accession EU348374), a
 polemonioid Ericale was included as the outgroup for
 <em>trnL</em>. Since all the taxa in the top matches for ITS
 were from the Sapotaceae, subfamily Sapotoideae, <em>Sarcosperma
 laurinum</em> (GenBank accession AM408055), which has previously
 been shown to be sister to the rest of the family, was included as an
 outgroup for ITS. Sequences were aligned using the
 MUSCLE algorithm. Maximum-likelihood (ML) trees were constructed using the
 .pml function in the package phangorn, implemented in R. The most
 appropriate model of DNA substitution was selected using the AIC and the
 function modelTest. We used the HKY model with the proportion of invariant
 sites and rate variation optimized using the function optim.pml. Support
 for the branching topology was evaluated with 1,000 bootstrap replicates.
 Separate trees were generated for chloroplast (<em>trnL</em>)
 and nuclear (ITS) regions.
description: See README file for usage. Data in Mesquite format and includes
 alignments and consensus tree. There is one Mesquite file per DNA barcode.
 All Genbank numbers are listed in the sequence headers and branch
 labels.