Anurans in culturally managed temple and shrine waterscapes in Kyoto: integrated visual, acoustic, and environmental DNA surveys

Temple and shrine gardens are ubiquitous features of historical cities in Japan, yet their ponds and small associated waterbodies remain ecologically understudied and may function as urban refugia for anurans. We conducted a systematic assessment of anuran assemblages across culturally managed waterscapes in the Higashiyama area of Kyoto, Japan, surveying 80 waterbodies across 31 facilities. We combined shore-based visual encounter and acoustic surveys with environmental DNA (eDNA) metabarcoding at 43 waterbodies to improve detection under access and disturbance constraints. We recorded seven anuran species and one newt ( Cynops pyrrhogaster ). Glandirana rugosa was the most frequently detected anuran species, followed by Zhangixalus arboreus and Rana tagoi ; these three species were recorded at more waterbodies than the typical paddy-associated species Dryophytes leopardus and Pelophylax nigromaculatus . Breeding was most often confirmed for Z. arboreus , whereas breeding evidence was less frequent for other species. Field surveys and eDNA were complementary: eDNA yielded additional detections of difficult-to-observe species, particularly R. tagoi , while field surveys were essential for confirming reproduction, for example through observations of foam nests. Univariable binomial generalized linear mixed models of species presence/absence, with facility as a random intercept, suggested species-specific associations with local habitat characteristics, including forest proximity and koi ( Cyprinus carpio ) presence. However, several estimates had wide 95% confidence intervals, and the direction and magnitude of the estimated associations differed among species. These results suggest that culturally managed waterbodies can support a distinctive anuran assemblage within the urban landscape and provide a practical baseline for monitoring and managing these potential biocultural refugia in historical cities.