Stemflow for detecting mammalian environmental DNA: a case study in a zoo

As mammalian diversity declines, effective biodiversity monitoring tools are urgently needed. To address this issue, we assessed the detectability of mammalian environmental DNA (eDNA) using the eDNA metabarcoding method with stemflow. We collected stemflow samples six times from June 2024 to January 2025 on 11 trees in the Oji Zoo, Kobe, Japan. One tree was inside the cage, which could be directly contacted by captive mammals whereas the others were outside the cages, which could not be directly contacted by captive mammals. Then, we amplified eDNA extracted from the stemflow samples with MiMammal primers targeting the mitochondrial 12S rRNA gene and the PCR products were then sequenced. We detected 25 mammal species eDNA, 16 of which were zoo-kept species and 9 of which were not. Environmental DNA of a captive mammal species which could directly contact the target tree was detected all the six sampling times. According to statistical analysis, there was a significant negative relationship between DNA detection and the distance from each mammal cage to each tree, as well as significant differences in the composition of the detected species between the trees inside and outside the cages. Moreover, the results suggested that significantly fewer species were detected in stemflow collected from trees with rough bark. We hereby show that eDNA analysis using stemflow is useful for detecting mammalian eDNA, indicating that this method has the potential to contribute to understanding the mammalian fauna in the field.