ABSTRACT Dietary plasticity is an important issue for conservation biology as it may be essential for species to cope with environmental changes. However, it still remains scarcely addressed in the literature, potentially because diet studies have long been constrained by methodological limits. The advent of molecular approaches now makes it possible to get a precise picture of diet and its plasticity, even for endangered and elusive species. Here we focused on the greater horseshoe bat (Rhinolophus ferrumequinum) in Western France, where this insectivorous species has been classified as ‘Vulnerable’ on the Regional Red List (2016). We applied an eDNA metabarcoding approach on 1986 fecal samples collected in six maternity colonies at three sampling dates. We described its diet and investigated whether the landscape surrounding colonies and the different phases of the maternity cycle influenced the diversity and the composition of this diet. We showed that R. ferrumequinum feed on a highly more diverse spectrum of prey than expected from previous studies, therefore highlighting how eDNA metabarcoding can help improving diet knowledge of a flying elusive endangered species. Our approach also revealed that R. ferrumequinum diet is composed of two distinct features: the core diet consisting in a few preferred taxa shared by all the colonies (25% of the occurrences) and the secondary diet consisting in numerous rare prey that were highly different between colonies and sampling dates (75% of the occurrences). Energetic needs and constraints associated with the greater horseshoe bat life-cycle, as well as insect phenology and landscape features, strongly influenced the diversity and composition of both the whole and core diets. Further research should now explore the relationships between R. ferrumequinum dietary plasticity and fitness, to better assess the impact of core prey decline on R. ferrumequinum populations viability. FILE DESCRIPTION Information concerning the samples and the positive and negative controls multiplexed in the MiSeq Runs 5 to 9 This XLSX file contains the sample IDs, the sample types, the PCR IDs, the PCR replicate numbers, the locality names, the predator species and the fastq file names for each PCR products multiplexed in the five different Illumina MiSeq runs. File name: Sample_informations.xlsx MiSeq raw sequences of the COI minibarcode from the faecal pellets of bats (Run5) This ZIP file contains the Run5 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal pellet in triplicate using the MiSeq platform. The 1271 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 475 multiplexed samples and the 8 positive and 94 negative controls are provided in the following XLSX file titled: Sample_Information.xlsx. Note: the 186 PCR3 replicates from the localities BEA and SGE are available in the ZIP file MiSeq_Reads_COI_Bat_faecal_pellets_Run9.zip File name: MiSeq_Reads_COI_Bat_faecal_pellets_Run5.zip MiSeq raw sequences of the COI minibarcode from the faecal pellets of bats (Run6) This ZIP file contains the Run6 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal pellet in triplicate using the MiSeq platform. The 1440 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 466 multiplexed samples and the 8 positive and 130 negative controls are provided in the following XLSX file titled: Sample_Information.xlsx. File name: MiSeq_Reads_COI_Bat_faecal_pellets_Run6.zip MiSeq raw sequences of the COI minibarcode from the faecal pellets of bats (Run7) This ZIP file contains the Run7 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal pellet in triplicate using the MiSeq platform. The 1464 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 475 multiplexed samples and the 8 positive and 103 negative controls are provided in the following XLSX file titled: Sample_Information.xlsx. File name: MiSeq_Reads_COI_Bat_faecal_pellets_Run7.zip MiSeq raw sequences of the COI minibarcode from the faecal pellets of bats (Run8) This ZIP file contains the Run8 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal pellet in triplicate using the MiSeq platform. The 1464 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 475 multiplexed samples and the 8 positive and 103 negative controls are provided in the following XLSX file titled: Sample_Information.xlsx. File name: MiSeq_Reads_COI_Bat_faecal_pellets_Run8.zip MiSeq raw sequences of the COI minibarcode from the faecal pellets of bats (Run9) This ZIP file contains the Run9 FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for each faecal pellet in triplicate using the MiSeq platform. The 499 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 281 multiplexed samples (including 29 samples from another project) and the 11 positive and 172 negative controls are provided in the following XLSX file titled: Sample_Information.xlsx. Note: the 372 PCR1 & PCR2 replicates from the localities BEA and SGE are available in the ZIP file MiSeq_Reads_COI_Bat_faecal_pellets_Run5.zip File name: MiSeq_Reads_COI_Bat_faecal_pellets_Run9.zip Raw abundance tables of the COI minibarcode from the faecal pellets of bats before data filtering (Run5 to 9) This ZIP file contains five TXT files showing the number of reads for each of the 17,998 distinct variants (OTUs) and each of the 6138 PCR products of the samples (n=2015) and controls sequenced in the MiSeq Runs 5 to 9 before the data filtering. File name: Raw_COI_Bat_Faecal_Pellets_abundance_Runs5to9_before_filtering.zip Abundance table of the COI minibarcode from the faecal pellets of bats after data filtering (Run5 to 9) This XLSX file contains the number of reads for each of the 7206 distinct variants (OTUs) and each of sample (n=2014) after the data filtering using (1) the thresholds based on the negative and positive controls (Tcc & Tfa) and (2) the validation using the three technical replicates. File name: COI_Bat_Faecal_Pellets_abundance_Runs5to9_after_filtering.xlsx Final abundance table of the COI minibarcode for the prey of Rhinolophus ferrumequinum only (Run5 to 9) This XLSX file contains the number of reads for each Rhinolophus ferrumequinum samples (n=1034) and each of the 679 validated prey taxa (OTUs) after taxonomic affiliations check and redundancy removals. File name: Final_COI_Rhino_Prey_abundance_Run5to9.xlsx Landscape data table used to build the PCA File name: Landscape_variables.xlsx