DNA barcoding identifies cryptic animal tool materials Matthew Steele, Linda Neaves, Barbara Klump, James St Clair, Joana Fernandes, Vanessa Hequet, Phil Shaw, Peter Hollingsworth, Christian Rutz To cite this version: Matthew Steele, Linda Neaves, Barbara Klump, James St Clair, Joana Fernandes, et al.. DNA barcoding identifies cryptic animal tool materials. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2021, 118 (29), pp.e2020699118. hal-03285541 HAL Id: hal-03285541 https://hal.inrae.fr/hal-03285541 Submitted on 13 Jul 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License DNA barcoding identifies cryptic animal tool materials BRIEF REPORT Matthew P. Steelea,1, Linda E. Neavesb,c,1, Barbara C. Klumpa,2, James J. H. St Claira, Joana R. S. M. Fernandesa, Vanessa Hequetd, Phil Shawa, Peter M. Hollingsworthb, and Christian Rutza,3 aCentre for Biological Diversity, School of Biology, University of St Andrews, St Andrews KY16 9TH, United Kingdom; bRoyal Botanic Garden Edinburgh, Edinburgh EH3 5LR, United Kingdom; cThe Fenner School of Environment and Society, The Australian National University, Canberra, ACT 2600, Australia; and dInstitut de Recherche pour le Développement, Centre de Nouméa, 98848 Nouméa, New Caledonia, France Edited by Scott V. Edwards, Harvard University, Cambridge, MA, and approved May 2, 2021 (received for review February 11, 2021) Some animals fashion tools or constructions out of plant materials that three study populations target different species despite living to aid foraging, reproduction, self-maintenance, or protection. just a few kilometers apart (12). While we managed to identify raw Their choice of raw materials can affect the structure and proper- materials at two sites (site-1 and site-2), we failed at the third (site- ties of the resulting artifacts, with considerable fitness conse- 3), even after employing a wide range of well-established field quences. Documenting animals’ material preferences is challenging, methods aimed at observing tool manufacture directly (Fig. 1 B however, as manufacture behavior is often difficult to observe di- and C and SI Appendix). rectly, and materials may be processed so heavily that they lack Given the importance of identifying the crows’ preferred plant identifying features. Here, we use DNA barcoding to identify, from species at site-3 for our overall research program, we tried an just a few recovered tool specimens, the plant species New Caledo- innovative artifact-centered approach (Fig. 1D). We extracted nian crows (Corvus moneduloides) use for crafting elaborate hooked DNA from seven hooked stick tools recovered at the site during stick tools in one of our long-term study populations. The method 2016–2017 and amplified two DNA barcoding regions: trnL-UAA succeeded where extensive fieldwork using an array of conventional [∼500 bp (13)] and internal transcribed spacer (ITS) [∼600 bp approaches—including targeted observations, camera traps, radio- (14)]. All samples produced identical haplotypes. Comparison tracking, bird-mounted video cameras, and behavioral experiments against the National Center for Biotechnology Information with wild and temporarily captive subjects—had failed. We believe (NCBI) Nucleotide nonredundant database indicated the tool that DNA barcoding will prove useful for investigating many other samples belonged to the family Sapotaceae, most likely the genus tool and construction behaviors, helping to unlock significant re- Mimusops or Manilkara (trnL: >98% identity; ITS: >95% iden- search potential across a wide range of study systems. tity). The ITS region exhibited greater resolution and indicated ECOLOGY Mimusops elengi as a candidate (96 to 99% identity). With a animal construction behavior | DNA barcoding | nest building | New putative source identified, we collected reference leaf samples of Caledonian crow | tool use M. elengi and Planchonella cinerea, the only closely related spe- cies known to occur locally, and analyzed them using the same here is increasing interest in the plant materials selected by method. The tool samples and M. elengi reference samples Tnonhuman animals to manufacture foraging tools and con- produced identical haplotypes for both DNA barcodes. Fur- structions (1, 2). Animals’ raw-material preferences can affect thermore, the maximum likelihood phylogenetic tree for ITS the structural and functional properties of artifacts, and, in some produced well-supported lineages that clustered tool samples cases, appear to be socially transmitted, contributing to rudimen- and M. elengi reference samples together, within a wider clade of tary material “cultures” (3–5). Two complementary approaches are non-New Caledonian M. elengi sequences (Fig. 1 D, iii). P. cinerea available for identifying plant materials used by wild animals: direct reference samples clustered within the Sapotaceae, but outside the observation of manufacture behavior (“animal-centered”) and ex- Mimusops genus, as expected, confirming that the crow tools from amination of artifacts in isolation from the behavior that created site-3 were made from M. elengi. We subsequently verified that them (“artifact-centered”). The latter, adopted by necessity in ar- wild-caught, temporarily captive New Caledonian crows readily chaeology, is particularly useful when animals cannot be habituated manufacture hooked stick tools from this material (this work was or are otherwise difficult to observe, but can present considerable conducted at site-1, since birds from site-3 proved too difficult to challenges. Artifacts are often heavily processed (lacking features work with in field aviaries). that aid identification, such as leaves or flowers), may be physically The use of DNA barcoding has led to an important break- distanced from the raw materials from which they were produced through for our research program. Reliable raw-material iden- (because the animal transported them), and may comprise a tification is key to uncovering the drivers of the striking regional complex assemblage of materials from different sources (such as in divergence we observed in an important aspect of New Caledo- bird nests). In these cases, material identification has so far relied nian crows’ hooked stick tool-making behavior (15). Specifically, on expert knowledge, which may be difficult and expensive to ac- with a set of three study populations established, and the ability — quire (6). Here, we demonstrate that DNA barcoding the use of to conduct rapid surveys across additional replicate sites, it will standardized DNA regions to identify organic material to species level (7)—provides a robust, cost- and time-efficient solution to these problems. Author contributions: M.P.S., L.E.N., B.C.K., J.J.H.S.C., P.M.H., and C.R. designed research; New Caledonian crows (Corvus moneduloides) are renowned M.P.S., L.E.N., B.C.K., J.J.H.S.C., J.R.S.M.F., V.H., P.S., P.M.H., and C.R. performed research; for their ability to manufacture complex foraging tools (8). When M.P.S., L.E.N., B.C.K., and P.M.H. analyzed data; M.P.S., L.E.N., and C.R. wrote the paper; making a hooked stick tool, they select a forked plant stem, and C.R. led, and secured funding for, the long-term project. remove a suitable branch, trim off any leaves and twiglets, and The authors declare no competing interest. often refine the tool by sculpting the remains of the nodal joint This open access article is distributed under Creative Commons Attribution License 4.0 into a neat terminal hook, stripping bark near the functional end, (CC BY). and bending the tool shaft (9). These processing steps substan- 1M.P.S. and L.E.N. contributed equally to this work. tially alter the appearance of the plant material (Fig. 1A). Im- 2Present address: Max Planck Institute of Animal Behavior, 78315 Radolfzell, Germany. portantly, properties of the raw material affect the morphology 3To whom correspondence may be addressed. Email: [email protected]. of the resulting tools, which in turn affects foraging efficiency This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (9–11). New Caledonian crows are highly selective when choosing doi:10.1073/pnas.2020699118/-/DCSupplemental. plants for hooked stick tool manufacture: we recently discovered Published July 12, 2021. PNAS 2021 Vol. 118 No. 29 e2020699118 https://doi.org/10.1073/pnas.2020699118 | 1of3 Downloaded by guest on July 12, 2021 AB Site-1: Mixed dry forest Tool material: mixed Site-2: Farmland Tool material: Desmanthus virgatus Site-3: Coastal/residential Tool material: Mimusops elengi (this study) 1 km C Initial investigations Research focus on site-2 Aviary experiments Raw material of NC crow tools at site-3 is shown Work focuses on site-2 where wild and Temporarily captive NC crows from site-3 to differ from that at site-2, but field observations, temporarily captive NC crows make are presented with candidate plant species aviary experiments and camera traps fail to hooked stick tools from the non-native M. elengi (along with other locally sourced identify the plant species. perennial shrub D. virgatus. plants) – low task engagement. Bird-mounted video loggersField experiments VHF radio-tracking Miniature cameras are attached to NC A variety of locally sourced plants is presented VHF radio-tags are deployed on crows at site-3 to document foraging to wild NC crows at site-3 alongside naturalistic NC crows at site-3 – challenging behavior – no tool manufacture recorded. foraging tasks – low task engagement. observation conditions.