Procedures to genotype problematic non-invasive otter (Lutra lutra) samples

Abstract

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive\ud and rare species such as the Eurasian otter (Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions\ud appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower\ud than in other carnivores.We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0% and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to istinguish the individual unrelated otters (PID=0.001), while 13 loci were needed to distinguish sibling otters (PIDsibs=0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success

Marking behaviour and census of Eurasian otters (Lutra lutra) in riverine habitats: what can scat abundances and non-invasive genetic sampling tell us about otter numbers?

“…However, faecal samples are commonly known to be prone to low DNA concentration and poor DNA quality, potentially leading to genotyping errors and low genotyping success rates (reviewed by Beja-Pereira et al 2009). Other studies applying non-invasive genetic methods on otter scats obtained success rates between 14 and 73%, i. e. on average 46% (e. g. Lanszki et al 2008;Janssens et al 2008;Hájková et al 2009;Bonesi et al 2013;Vergara et al 2014;Lerone et al 2014;Sittenthaler et al 2015;Martin et al 2017). The combination of a comparable high-average genotyping success rate of 57% and an extensive sampling scheme allowed us to draw reliable conclusions regarding otter densities in our study areas.…”

Spatial genetic structure in the Eurasian otter (Lutra lutra) meta-population from its core range in Italy

“…Only the samples that produced three pcr products at these loci and only samples whose replicas resulted in the same typing were retained for subsequent analyses (modified by Koelewijn et al, 2010). The selected samples were then amplified three times for the remaining loci, and only samples which provided 50% or more of positive screening at a locus were considered 'positive' (Lerone et al, 2014). Furthermore, we defined a sample as successfully genotyped if at least six microsatellite loci revealed complete genotype.…”

“…In order to test the robustness of microsatellite assignment, we constructed a consensus genotypes using Gimlet v1.3.3 (Valière, 2002) and estimated the rates of false alleles (fa) and allele drop out (ado) (Taberlet et al, 1996;Frantz et al, 2003;Hájková et al, 2007;Ferrando et al, 2008;Lerone et al, 2014) with a maximum likelihood-based method implemented in Pedant (Johnson & Haydon, 2007a, b). GenALEx v. 6.51b2 (Peakall & Smouse, 2012) was used to identify identical genotypes, the probability of identity (pid) and siblings (pid-sib).…”

“…Since 1990, it was reported that the structure of the Italian otter population was organized in two sub-populations isolated from each other: a small and isolated nucleus living in the Abruzzo and Molise regions (central Italy), and a larger core population along the river networks of the Campania, Basilicata, Apulia and Calabria regions (Southern Italy) (Fusillo et al, 2004;Loy et al, 2004;Marcelli, 2006;Lerone et al, 2014;Balestrieri et al, 2016;Giovacchini et al, 2018). Furthermore, recently, otter presence has been recorded in the North-Eastern of the peninsula, as a consequence of the expansion of Austrian and Slovenian populations (Kranz, 2008, pers.…”

Conservation genetics of otters: Review about the use of non-invasive samples

“…These have shown that fresh mucus and stools are the most appropriate sources for this purpose. Besides, DNA extraction is more efficient using commercial kits, due to the additional purification steps needed with other more affordable methods such as extraction with phenol-chloroform method and the Chelex® 100 method (Goossens et al 2000;Roeder et al 2004;Hajkova et al 2006;Lampa et al 2008;Lerone et al 2014). When a small amount of DNA is obtained, Lampa et al (2008) recommend to perform the amplification in two steps, i.e.…”