A Field‐Deployable eDNA Metabarcoding Workflow Including De Novo Reference Assembly for Characterising Understudied Biodiversity Hotspots

Molecular Ecology Resources, vol. 26, pp. e70122

Abstract

Field-deployable DNA metabarcoding offers a transformative approach to biodiversity research and monitoring, yet its application remains limited due to technical constraints and a lack of reference data in poorly studied ecosystems. Combining isothermal Recombinase Polymerase Amplification (RPA) and Oxford Nanopore sequencing, we introduce a two-step approach that uses non-invasive species barcoding to directly generate reference sequences for use in environmental DNA (eDNA) metabarcoding, and enables real-time, PCR-free and cost-effective molecular assessment of ecological communities in the field. Using an endemic and understudied tropical amphibian assemblage as a model, we demonstrate the functionality of this novel workflow. De novo generation of a reference sequence library from amphibian skin swab samples significantly improved the accuracy and taxonomic resolution of sequence assignments from eDNA samples, particularly on the species level, in turn allowing a characterisation of fine-scale patterns in community composition. Beyond generating new RPA-compatible amphibian metabarcoding primers, our results show that combining field-based eDNA metabarcoding with the offline assembly of a local reference database can directly bridge existing data gaps in molecular biodiversity monitoring, providing a scalable solution to accelerate biodiversity assessments in data-deficient ecosystems. This workflow paves the way for broader deployment of molecular tools in global biodiversity hotspots-particularly in remote and resource-limited tropical regions-to directly contribute critical baseline data, and support conservation efforts in regions where they are most urgently needed.

Authors 7

1. Jesse Erens corresponding Aachen Marine Evolution and Ecology Group Naturalis Biodiversity Center Leiden The Netherlands

   Naturalis Biodiversity Center · Universität Trier

   Affiliation as printed

   Biogeography Department Trier University Trier Germany

   Habitats Conservation Hostert Luxembourg

   Marine Evolution and Ecology Group, Naturalis Biodiversity Center Leiden the Netherlands

2. Christopher Heine

   Universität Trier

   Affiliation as printed

   Biogeography Department Trier University Trier Germany

3. Stefan Lötters

   Universität Trier

   Affiliation as printed

   Biogeography Department Trier University Trier Germany

   Habitats Conservation Hostert Luxembourg

4. Henrik Krehenwinkel

   Universität Trier

   Affiliation as printed

   Biogeography Department Trier University Trier Germany

5. Andrew J. Crawford

   Universidad de Los Andes

   Affiliation as printed

   Department of Biological Sciences Universidad de los Andes Bogotá Colombia

6. Luis Alberto Rueda‐Solano

   University of Magdalena

   Affiliation as printed

   Facultad de Ciencias Básicas, Universidad del Magdalena Santa Marta Colombia

7. Amadeus Plewnia

   Universität Trier

   Affiliation as printed

   Biogeography Department Trier University Trier Germany

   Habitats Conservation Hostert Luxembourg