New eDNA-based insights for the detection of endemic and endangered Apennine yellow bellied toad (Bombina variegata pachypus) and its pathogenic fungus Batrachochytrium dendrobatidis

Abstract Amphibian populations are experiencing a global significant decline due to habitat loss, climate change, disease outbreaks and last but not least, interaction with alien species and/or their parasites. The Apennine yellow-bellied toad ( Bombina variegata pachypus (Bonaparte, 1838)) is endemic to the Italian Peninsula. This subspecies, like many other amphibians, is declining and is listed as Endangered (EN) in the italian red list ( http://www.iucnredlist.org ). This study aimed to use an environmental DNA (eDNA)-based approach to detect the occurrence of both B. v. pachypus and its potential threatening pathogen fungus Batrachochytrium dendrobatidis Lee Berger, 1998 (Chytridiomycetes, hereafter Bd ), at 20 freshwater sampling sites in Central Italy (i.e., five isolated ponds and six rivers). Here, a new TaqMan-based (qPCR) assay has been developed for eDNA-based detection of B. v. pachypus . Additionally, eDNA-based screening for Bd occurrence was conducted. Two sampling replicates per site were carried out in 2021-2022. For the sampling sites along rivers, we collected six fine-scale and 16 large-scale environmental variables that could affect the detection of eDNA from B. v. pachypus and Bd and therefore the occurrence of these species. eDNA of B. v. pachypus was detected in 12 out of 20 sampling sites, whereas Bd was found at one sample site, co-occurring with B. v. pachypus . Environmental DNA of B. v. pachypus was detected at all but one of the rivers surveyed (i.e., Liri River). Nine out of 12 eDNA positive sites represented new occurrences for this amphibian, most of which (n = 8) were located outside Natura 2000 areas. Positive sites included eight out of 14 running water bodies and four out of six standing water bodies; the latter including a pond in a lowland protected area where B. pachypus was considered extirpated since 1997. The outcomes of this study confirm eDNA as a rapid and cost-effective method for large-scale surveys of both the Apennine yellow-bellied toad and its potentially threatening pathogenic fungus Bd .