Recovering whole mitogenome sequences from Eurasian otter ( <i>Lutra lutra</i> ) spraint samples: a metagenomic approach

Abstract Non-invasive genetic monitoring of endangered species and populations is a crucial component of conservation biology, especially for elusive species such as the Eurasian otter (Lutra lutra). Leveraging techniques from the fields of metagenomics and ancient DNA, here we present a laboratory and bioinformatic pipeline for recovering whole mitochondrial genomes (mitogenomes) through shotgun metagenomic sequencing of Eurasian otter spraint (faecal) samples. Using this approach, we successfully assembled whole mitogenomes from 20 out of 27 analysed spraint samples, collected along the river Usk in south Wales (UK). The three recovered haplotypes in these samples were identical to sequences previously documented in high-quality DNA from muscle tissue samples of otters from Wales, validating our approach. We recovered higher mitochondrial depth in spraint samples collected in spring than winter and in samples collected within less than 24 hours. However, our limited sample size prevented us from determining factors which influenced mitochondrial depth within a multivariate framework. In addition to assembling otter mitogenomes and characterizing fungal and bacterial diversity, we also obtained mitogenomes of known prey species of otters, highlighting the value of metagenomic sequencing from non-invasively collected faecal samples.