Using Environmental DNA to Characterize Amphibian Communities at Sites Infected with Batrachochytrium salamandrivorans in the Netherlands
Batrachochytrium salamandrivorans (Bsal) fungus has the potential to cause high mortality rates in some European salamanders and newts (urodelans) and is in the process of expanding its invasive range in Europe. Therefore, monitoring its distribution and better understanding both the species threatened and the mechanics of infection are essential in mitigating damage Bsal may cause. Environmental DNA has emerged as a promising noninvasive method for detecting both this fungal pathogen and amphibian communities in infected areas. We applied these methods in the province of Gelderland, Netherlands, where the pathogen has previously been detected and is expanding its range, with the goal of characterizing the natural amphibian community present. We sampled 27 bodies of water in the region surrounding the known outbreak sites, determined the presence or absence of Bsal using a targeted quantitative polymerase chain reaction assay, and applied an environmental DNA metabarcoding approach to characterize the amphibian communities using two different primer sets. The 12S vertebrate primer set outperformed the 16S amphibian primer set and detected all expected amphibians in the study area: Bufo bufo, Lissotriton vulgaris, Pelobates fuscus, Pelophylax spp., Rana temporaria and Triturus cristatus. Bsal was detected at 8 of 27 ponds. A distance-based redundancy analysis found a weak but significant relationship between Bsal presence and composition of amphibian communities. This study may provide a basis for future studies on Bsal and its relationship with amphibian communities in Europe, highlighting the need for further research into mechanisms of persistence and transmission between bodies of water.Abstract

Map displaying the amphibian communities detected using the 12S vertebrate and 16S amphibian primer sets at Batrachochytrium salamandrivorans-positive and -negative sites (underlying map not shown because ponds are on private properties). The original two sites where Bsal was detected are included in blue squares. Approximate location of the study site within the Netherlands is shown at the bottom of the figure.

Species accumulation curve showing the interpolated species richness detected by each primer set and by both primer sets combined alongside the extrapolated richness to 50 sites. Shaded regions display the 95% confidence interval. 12S vertebrate primer data has site 22 removed due to failure and therefore has 26 sites.

Distance-based redundancy analysis plot showing the influence of Batrachochytrium salamandrivorans on the differences in amphibian community composition as detected by the 12S vertebrate primer set. CAP1 is the first constrained ordination axis and explains the maximum variation associated with the binary Bsal variable. MDS1 is the first unconstrained ordination axis and explains the general dissimilarities among samples. The influence of the binary Bsal variable on the CAP1 axis is displayed with the loading arrow. Points are jittered for visibility.
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