Closing the reference gap for Australo-Papuan rodents: a 431-mitogenome library for environmental DNA and species discovery

As biodiversity declines at an unprecedented rate, rapid and scalable approaches to monitoring species are increasingly needed, particularly in poorly known and highly threatened groups. Environmental DNA (eDNA) is increasingly enabling high-throughput monitoring to inform conservation. However, species-level monitoring through eDNA relies on comprehensive reference databases and short, species-discriminating ‘mini-barcode’ loci, resources that are lacking for many taxa. The application of eDNA for species-level monitoring is therefore constrained by these gaps. Here, we use Australo-Papuan rodents (subfamily Murinae: tribe Hydromyini) as a model system to address these limitations, given their high species diversity, conservation concern, and distribution across geographically complex regions. Among animals, eDNA monitoring typically depends on mitochondrial markers. Leveraging taxonomically-verified museum specimens, we develop a 431-mitogenome reference library representing ~72% of described species diversity, and use this database to assess the species-level resolution of candidate mini-barcodes. We show that common 12S rRNA mammalian eDNA markers cannot reliably discriminate between Hydromyini species (resolving 23-33% of tested species). In contrast, a Rodentia-specific Cytb marker resolves 66% of tested species, and two novel ATP6 Hydromyini markers, presented here, resolves 93% through a multi-locus approach. This reference library represents a >40-fold increase in available complete mitogenomes for Australo-Papuan rodents, enabling eDNA monitoring in this highly threatened group. More broadly, we demonstrate the value of pairing comprehensive reference resources with empirical marker assessment to reveal where eDNA based approaches may fail to distinguish species. Understanding these limitations is essential to realising the promise of eDNA as a reliable tool for species-level monitoring and conservation.