DNA barcoding and phylogenetic analysis of dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) in wildlife and wildlife-livestock systems of Namibia

Taxonomical keys have been used in the identification of dung beetle species due to its cost effectiveness and accessibilities. However, morphological identification of dung beetles remains a challenging task as a result of variable morphological differences among the species. The identification of dung beetles by the use of molecular markers helps in avoiding inaccuracy and appears promising to solve the problem of identifying species. The aim of this study was to determine the DNA barcoding and phylogenetic analysis of dung beetles in wildlife and wildlife-livestock ecosystems of Namibia. Sampling of dung beetles was done using baited pitfall traps and some species were handpicked from the abundant bovine dung pads. The collected dung beetles were preserved in DNA/RNA shield (Zymo Research), and kept in a freezer (-20 ℃) before the initial transportation. The genomic DNA was isolated using blood and tissue Quick-DNA Miniprep Plus Kit (Zymo Research). Two sets of universal primers based on the cytochrome c oxidase gene I (COI) barcodes selected for use. The sequence FASTA files were aligned using multiple alignments ClustalW software, and the phylogenetic relationships by Neighbor joining analysis was performed using the MEGA12 software. A total of 62 DNA barcodes of dung beetle species were accomplished from which 15 species from 27 sequenced samples were identified with the percentage of 95–100%, while 35 dung beetle sequences were not retrieved from the database. Indicating that most of the sequences found in wildlife and wildlife-livestock systems did not have reference sequences publicly entered into the database. The Neighbour-Joining phylogenetic tree showed 9 species of the genera Onthophagus , Milichus , and Caccobius of the tribe Onthophagini separated from each other and grouped together with the bootstrap value (> 95%). The clustering examination divided the 32 species into three main clades, while two species appeared separately from the three clades. This constituted the first report of DNA barcodes for Namibian dung beetles. The DNA barcoding and phylogenetic analysis application for molecular identification in dung beetles provides valuable and accurate information, which is useful in understanding their diversity.