Assessing the breeding phenology of a threatened frog species using eDNA and automatic acoustic monitoring

Abstract

Background Climate change has driven shifts in breeding phenology of many amphibians, causing phenological mismatches (e.g., predator-prey interactions), and potentially population declines. Collecting data with high spatiotemporal sensitivity on hibernation emergence and breeding times can inform conservation best practices. However, monitoring the phenology of amphibians can be challenging because of their cryptic nature over much of their life cycle. Moreover, most salamanders and caecilians do not produce conspicuous breeding calls like frogs and toads do, presenting additional monitoring challenges. Methods In this study, we designed and evaluated the performance of an environmental DNA (eDNA) droplet digital PCR (ddPCR) assay as a non-invasive tool to assess the breeding phenology of a Western Chorus Frog population (Pseudacris maculata mitotype) in Eastern Ontario and compared eDNA detection patterns to hourly automatic acoustic monitoring. For two eDNA samples with strong PCR inhibition, we tested three methods to diminish the effect of inhibitors: diluting eDNA samples, adding bovine serum albumin to PCR reactions, and purifying eDNA using a commercial clean-up kit. Results We recorded the first male calling when the focal marsh was still largely frozen. Chorus frog eDNA was detected on April 6th, 6 days after acoustic monitoring revealed this first calling male, but only 2 days after males attained higher chorus activity. eDNA signals were detected at more sampling locales within the marsh and eDNA concentrations increased as more males participated in the chorus, suggesting that eDNA may be a reasonable proxy for calling assemblage size. Internal positive control revealed strong inhibition in some samples, limiting detection probability and quantification accuracy in ddPCR. We found diluting samples was the most effective in reducing inhibition and improving eDNA quantification. Conclusions Altogether, our results showed that eDNA ddPCR signals lagged behind male chorusing by a few days; thus, acoustic monitoring is preferable if the desire is to document the onset of male chorusing. However, eDNA may be an effective, non-invasive monitoring tool for amphibians that do not call and may provide a useful complement to automated acoustic recording. We found inhibition patterns were heterogeneous across time and space and we demonstrate that an internal positive control should always be included to assess inhibition for eDNA ddPCR signal interpretations.

Vernal Pool Amphibian Inventories in the Temperate Forests of Eastern North America: Can Environmental DNA Replace the Traditional Methods?

“…This important result has at least two explanations. First, DNA can stay up to three weeks in freshwater bodies [56], with variations based on environmental conditions [57]: species seldomly using vernal pools can be detected over a large time window [33]. The second explanation is related to this window of detection, as active search surveys were conducted during the day over short periods, and thus, limiting the chances of detecting species (underlined by the Chao2 index of the traditional methods showing that a higher proportion of species were detected once).…”

“…Once the eDNA metabarcoding protocol was developed, the laboratory work and data analysis took only days, with a cost per sample of ~CAD$ 33 (for consumables and a Miseq run of 192 samples). Different technologies, such as quantitative or digital PCR, could help in species' specific management plans [33], but metabarcoding can be sufficient on its own or complement the traditional surveys, according to the aims of stakeholders. Regarding the system used here, eDNA could provide the general habitat use data as it reliably detects amphibian species sensitive to habitat modifications and degradations, but difficult to observe with the traditional methods (e.g., N. viridescens, Am.…”

“…[30][31][32]. Notably, recent studies managed to track the temporal variation in amphibians in vernal pools utilizing eDNA, but highlighted that the detections were dependent on the species ecologies and environmental conditions [16,[33][34][35]. If successful, this flexible approach would allow the monitoring of several taxonomic communities (amphibians, arthropods, annelids, etc.)…”

A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanis siamensis) in wet and dry conditions

“…Local factors like rainfall can affect the transport and detection of eDNA in rivers, potentially leading to variations in detection distance and time 15 . Furthermore, climate data, including rainfall, are important in eDNA surveys and data interpretation as rainfall can dilute eDNA concentration and reduce detection probabilities, highlighting the need for repeated sampling and collection of abiotic variables like rainfall 24 . Aucone et al 25 also observed a decrease in eDNA detection in relation to significant heavy rainfall.…”

Patterns of acoustic phenology in an anuran assemblage of the Yungas Andean forests of Argentina

“…Although there is a behavioural continuum between these two extremes (Wells, 1977;. Several works focused on better describing the breeding acoustic strategies that can be found in anuran species (Chen et al, 2023;Donnelly and Guyer, 1994;Forti et al, 2022;Huang et al, 2001;Prado et al, 2005;Prasad et al, 2022;Bertoluci and Rodrigues, 2002) but there is still missing information mainly for Neotropical anurans. Continuous monitoring of anuran advertisement calls along different spatial-temporal scales, can provide valuable information about the different breeding strategies displayed by each species within an assemblage (Moreira et al, 2007).…”