Dietary diversity of the Amami rabbit endemic to insular evergreen forests

This dataset details the research methodology and results for analyzing the dietary habits of the Amami rabbitbased on fecal samples. The study aims to contribute to a better understanding of the ecological role and conservation needs of this endangered species.Sample Collection: Fecal samples from the Amami rabbit were collected from Amami Oshima Island, Japan, at two distinct periods and locations:August 18, 2022: Two samples (A-rab-1, A-rab-2) were collected from the Yakugachi River basin (28.22° N, 129.34° E).October 28, 2023: Four samples (A-rab-3, A-rab-4, A-rab-5, A-rab-6) were collected from the Asato forest road (28.22° N, 129.34° E).DNA Extraction: DNA was extracted from the collected fecal samples using the following protocol:Freeze-drying: Samples were freeze-dried using a VD-250R Freeze Dryer (TAITEC).Homogenization: Freeze-dried samples were crushed at 1,500 rpm for 2 minutes using a Multi-Beads Shocker (Yasui Kikai).Lysis: Lysis Solution F (Nippon Gene) was added to the homogenized samples and incubated at 65°C for 10 minutes.Centrifugation and Supernatant Separation: Samples were centrifuged at 12,000 x g for 2 minutes, and the supernatant was collected.Purification: Purification Solution (Nippon Gene) and chloroform were added to the separated supernatant and thoroughly mixed.Second Centrifugation: The mixture was centrifuged at 12,000 x g for 15 minutes, and the supernatant was again collected.DNA Purification: DNA was further purified from this solution using a Lab-Aid824s DNA Extraction kit (ZEESAN).PCR Inhibitor Removal: PCR inhibitors were removed using a DNeasy PowerClean Pro Cleanup Kit (Qiagen).Library Construction and psbA Gene Sequencing: The psbA gene, a commonly used marker for plant identification, was targeted for sequencing.DNA Quantification: DNA solution concentration was measured using Synergy LX (Agilent Technologies) and the QuantiFluor dsDNA System (Promega).Library Construction: Libraries were constructed using the 2-step tailed PCR method.Library Quantification and Quality Control: The concentration of the prepared libraries was measured using Synergy H1 (Agilent Technologies) and the QuantiFluor dsDNA System. Library quality was confirmed using a Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies).Sequencing: Sequencing was performed on a MiSeq system (Illumina) under 2x300 bp conditions using a MiSeq Reagent Kit v3 (Illumina).Data Analysis: Bioinformatic analysis of the sequencing data was performed as follows:Primer Matching and Trimming: Read sequences perfectly matching the primer sequence at the start were extracted using fastx_barcode_splitter from FASTX-Toolkit (v0.0.14). The primer sequence and 50 bases at the 3' end were then removed using fastx_trimmer from FASTX-Toolkit.Quality Filtering: Sequences with a quality value less than 20 were removed using sickle (v1.33). Sequences with a length of 40 bases or less, along with their paired sequences, were discarded.Paired-End Read Joining: Paired-end reads were joined using FLASH (v1.2.11) with a minimum overlap of 10 bases.Chimeric and Noise Sequence Removal: Chimeric and noise sequences were removed using the dada2 plugin within Qiime2 (v2023.7), which also generated representative sequences and an ASV (Amplicon Sequence Variant) table.Phylogenetic Estimation: Representative sequences were compared against the NCBI nt database using BLASTN (v2.13.0) to estimate phylogenetic order. Default parameters were used for BLASTN.Data Visualization: Data in .qzv format was converted to viewable data using the tools exportplugin of Qiime2.