Metabarcoding of Mediterranean monk seal (monachus monachus) scats from the isolated population of Madeira reveals a diverse piscivorous diet

Background: The Mediterranean monk seal is Europe’s most endangered marine mammal, with the Madeira subpopulation remaining critically small and genetically isolated. Reliable diet data is vital for conservation, but traditional methods are invasive and biased, highlighting the need for a non-invasive, high- resolution molecular approach previously unavailable for this population. Aims: Analyze the overall dietary composition of the Madeira monk seal population using DNA metabarcoding. Quantify prey diversity and Frequency of Occurrence (FO%) of all prey species, including novel and cryptic taxa. Investigate dietary overlap with commercial fishing and aquaculture (Sparus aurata) to inform conservation policies. Results – Biodiversity: Detected 60 prey species (39 native, 20 non-native/migratory). First documentation of cartilaginous fishes (Bathytoshia centroura, Bathytoshia lata, Torpedo marmorata) in this population's diet. Diet includes benthic (e.g., Muraena sp, Conger sp), demersal (e.g., Pagellus sp, Dicentrarchus sp), and pelagic (e.g., Scomber sp, Sardina sp) taxa. The Sparidae family dominated the diet, with a FO of 79%. Fisheries overlap: A total of 12 key prey species are also targeted by local artisanal and recreational fisheries, and Sparus aurata is a major aquaculture species in the region. Discussion & Conclusion: Detected previously unrecorded species in Madeira, including Fistularia petimba and Diplodus puntazzo, showcasing fecal DNA’s power to expand the local species list. The Madeira monk seal shows an opportunistic, generalist feeding strategy across benthic, demersal, and pelagic niches. MiFish primers also amplified non-fish DNA, with host DNA present in all samples, and Teira dugesii (the Madeiran wall lizard) in 11/14 specimens. Dual primers improved resolution, adding 20 new diet species. DNA metabarcoding offers superior resolution over traditional diet analysis methods. S. aurata occurrence (78.5%) indicates limited interaction with local aquaculture.