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DNA-based methods form the cornerstone of contemporary evolutionary biology and they are highly valued tools in conservation biology. The development of non-invasive sampling methods can be crucial for both gathering sample sizes needed for robust ecological inference and to avoid a negative impact on small and/or endangered populations. Such sampling is particularly challenging in working with aquatic organisms, if the goal is to minimize disturbance and to avoid even temporary removal of individuals from their home range. We developed an in situ underwater method of DNA sampling and preservation that can be applied during diving in less than a minute of animal handling. We applied the method on a Herzegovinian population of olm ( Proteus anguinus , Caudata), an endangered aquatic cave-dwelling vertebrate, which makes it an excellent model to test the method under the harshest conditions. We sampled 22 adults during cave-diving and extracted sufficient quantity and quality of DNA from all individuals. We amplified 10 species-specific microsatellite loci, with PCR success varying between 6 and 10 loci (median: 7 loci). Fragment length analyses on 9 loci revealed a single allele at all loci across all individuals. This is in stark contrast to four Croatian populations studied with the same 10 loci previously that showed high within-population genetic variation. Our population and the four Croatian populations were genetically highly divergent. We propose that our method can be widely used to sample endangered aquatic populations, or in projects where the disturbance of individuals must be kept minimal for conservation and scientific purposes.

A new non-invasive in situ underwater DNA sampling method for estimating genetic diversity