Environmental DNA metabarcoding effectively monitors terrestrial species by using urban green spaces

Ecological corridors and urban parks are created to connect habitats that become isolated due to urban development and integrate ecological, social, and cultural functions. Establishing and implementing the ecological function of urban green structures and conservation plans for urban terrestrial species requires prioritizing developing an understanding of the community structure of urban terrestrial species. However, understanding the appearance and distribution of terrestrial species in urban ecosystems is often neglected or lacking; furthermore, the inaccurate detection of species distribution can create biased estimates of species occupancy. Recently, an applied research method for detecting terrestrial species such as mammals and birds has been proposed based on molecular biology such as environmental DNA (eDNA) metabarcoding. We installed artificial water tanks (mesocosm) in urban green spaces (Urban forests; UFs, Urban ecological corridors; UECs, Urban parks; UPs), applying eDNA metabarcoding as a tool to detect wild terrestrial species, and camera traps (CTs) and field surveys were performed in parallel to verify the results. We detected 22 urban terrestrial species only by eDNA metabarcoding and identified 43 species in total by performing eDNA metabarcoding, CTs, and field surveys in parallel. In addition, we identified 31 species of UFs, 13 species of UECs, and 28 species of UPs of urban terrestrial species using the three species survey methods in parallel. We confirmed that eDNA metabarcoding is very useful for detecting small species that are difficult to detect with the naked eye and internal species vulnerable to disturbance. However, since some species were only detected by CTs or field surveys due to DNA falsenegative errors or the species’ life-history characteristics, it was judged that the species inventory can be effectively collected using several monitoring techniques. • We successfully monitored most of the present terrestrial species using urban green spaces in just seven days, complementing environmental DNA metabarcoding, camera traps, and fieldwork. • In an urban ecosystem, eDNA metabarcoding was performed, in spaces where it was difficult to collect river samples due to large disturbances, with an artificial water tank containing distilled water, and clear spatiotemporal information of the recorded species was provided. • The sensitivity of eDNA metabarcoding was affected depending on the degree of human disturbance. In particular, in the case of terrestrial species within the city, the field survey was still a stronger research technique than eDNA and camera traps. • Urban ecological corridors and urban parks negatively impacted the inter-forest movement and habitation of terrestrial species due to the use of urban residents such as trails, but the use of species was confirmed as much as urban forests; in particular, mammals shared a home-range by dividing day/night time zones with urban residents due to their nocturnal nature. • The introduction of eDNA metabarcoding and camera traps in urban ecosystems can fill gaps in understanding and knowledge about changes in urban biodiversity by inducing the participation of citizens interested in ecology.