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

<p dir="ltr">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 (<i>Bombina variegata pachypus</i> (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 (<a href="http://www.iucnredlist.org" target="_blank">http://www.iucnredlist.org</a>). This study aimed to use an environmental DNA (eDNA)-based approach to detect the occurrence of both <i>B. v. pachypus</i> and its potential threatening pathogen fungus<i> Batrachochytrium dendrobatidis </i>Lee Berger, 1998<i> </i>(Chytridiomycetes, hereafter <i>Bd</i>),<i> </i>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 <i>B. v. pachypus</i>. Additionally, eDNA-based screening for <i>Bd</i> 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 <i>B. v. pachypus</i> and <i>Bd </i>and therefore the occurrence of these species. eDNA of <i>B. v. pachypus</i> was detected in 12 out of 20 sampling sites, whereas <i>Bd</i> was found at one sample site, co-occurring with <i>B. v. pachypus</i>. Environmental DNA of <i>B. v. pachypus</i> 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 <i>B. pachypus</i> 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 <i>Bd</i>.</p>