Unaddressed hybridization between green (Bufotes viridis) and natterjack toads (Epidalea calamita) can lead to underestimation of genetic heterozygosity and inflated estimates of inbreeding

Abstract Hybridization is a major evolutionary process with significant consequences for the ecology and conservation of species and lineages. However, genetic studies that evaluate the level and influence of hybridization in wild populations of conservation concern remain scarce, despite recognition that unaddressed hybridization can bias results and lead to erroneous conclusions. Here, we explore how hybridization in mixed populations of the natterjack ( Epidalea calamita ) and green toads ( Bufotes viridis ) impacts the estimation of genetic parameters used to evaluate the conservation status of the regionally threatened natterjack toad. In order to determine the population structure of natterjack toads in the Central German Mining District near Leipzig (Germany) we have analysed 594 samples of bufonid eggs and tadpoles from eight different sites. Based on a detailed analysis of microsatellite loci, 392 individuals were determined as E. calamita , 158 as B. viridis and 44 as hybrids. Due to the high proportion of hybrids at some sites, estimates of inbreeding (F IS ) in natterjack toads were biased towards higher values due to the detection of false homozygous individuals when hybrids were not removed from the dataset. Since inbreeding coefficients are a crucial parameter for population genetic analysis and biased estimates can give misleading results, our findings show that unaddressed hybridization can result in inflated estimates of inbreeding. This in turn, can lead to false conclusions that may ultimately misdirect conservation actions. We suggest that, if hybridization is documented for species pairs, it needs to be considered at an early stage when analysing populations.