DOI: 10.6084/m9.figshare.29400578.v1
title: <b>Dietary diversity of the Amami rabbit endemic to insular evergreen forests</b>

description: This dataset details the <b>research methodology and results for analyzing the dietary habits of the Amami rabbit</b>based on fecal samples. The study aims to contribute to a better understanding of the ecological role and conservation needs of this endangered species.<b>Sample Collection</b>: 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).<b>DNA Extraction</b>: 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).<b>Library Construction and psbA Gene Sequencing</b>: 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).<b>Data Analysis</b>: 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.<br>