Metagenomic sequencing of dung beetle intestinal contents directly detects and identifies mammalian fauna

Abstract

O_LICost, time, and expertise constraints limit traditional observation-based comprehensive biodiversity assessment. Therefore, surrogate focal taxa representative of wider biodiversity are commonly used as an imperfect proxy. Contemporary biodiversity assessments are also increasingly benefiting from the combination of high-throughput sequencing and metagenomic methodologies that enable identification of environmental DNA samples. However, there is a need for empirical studies combining the use of surrogate taxa with metagenomic approaches, that promise rapid and efficient biodiversity assessment.\nC_LIO_LIWe here tested for the first time the possibility of using the intestinal contents of wild-collected dung beetles (Scarabaeidae) as a source of mammalian DNA, in a metagenomics proof-of-concept approach to directly detect and identify mammals from an area of savanna-scrub in southern Africa. Dung beetles have been purveyed as an indirect proxy measure of mammalian diversity, owing to their dependence upon vertebrate dung as a food source, and the ease with which they can be comprehensively sampled using simple and repeatable trapping protocols, achievable much faster than vertebrate surveys.\nC_LIO_LIFollowing shotgun sequencing of gut content DNA extractions from ten dung beetle species, we used in silico filters to identify mammals by searching the resulting reads against known mammalian mitochondrial DNA from online sequence repositories, matching 546 paired reads to known mitogenomes held in GenBank, and 634 reads to known mammal barcode sequences held in BOLD. Identified mammalian sequences were consistent with wild and domesticated ungulates known from the sampling site, and included blue wildebeest, plains zebra, and domestic cattle and goat. Four dung beetle samples yielded sufficient sequence data to successfully assemble the near-complete mitogenome of blue wildebeest at up to 21 X mean coverage, despite low initial DNA concentrations, unambiguously corroborating identification.\nC_LIO_LIIt is conceptually and practically possible to rapidly and economically apply metagenomic techniques in dung beetle gut sequencing to detect the presence of mammals upon whose dung the beetles have fed. Since the approach can be readily scaled up, it may prove to be of practical use as a complement to traditional biodiversity assessment methods, and should be tested in usefulness for detecting rare, endangered or cryptic mammal species.\nC_LI