Monitoring of the Iberian wolf using fecal DNA

The Iberian wolf (Canis lupus signatus) in the Iberian Peninsula faced a severe population decline in the mid-20th century due to human persecution. Since then, the population has increased because of enhanced legal protections. However, the species is still categorized as “Near Threatened” because of hunting and a lack of coordinated management. Studies have revealed low heterozygosity and strong linkage disequilibrium among Iberian wolves, indicating low levels of genetic diversity. Therefore, monitoring the population is important for management towards its long-term persistence. Traditional wildlife monitoring methods, such as the use of radio-collars, are usually expensive and labor intensive. Furthermore, these methods are invasive as they often require trapping and handling of wolves. Non-invasive methods, such as fecal DNA analysis, have become an important tool for tracking elusive animals and detecting patterns of population structure, population sizes and behavior. However, fecal DNA is often degraded and has a limited quantity of endogenous DNA. Therefore, optimizing protocols for extracting and amplifying DNA from feces is important. In this study, 312 fecal samples believed to represent Iberian wolves in northern Spain and Portugal were analysed. The study aimed to test the efficiency of using fecal samples for wildlife monitoring by determining the species and the sex of the samples. Canid specific sex primers were used to amplify a sequence on the X-chromosome and Y- chromosome for sex determination. For species identification, a 350-bp fragment of the hypervariable region of the mitochondrial control region (D-loop) was PCR amplified and sequenced. Mitochondrial DNA was successfully amplified for 180 samples (57,7 %). There was no difference in the proportion of fecal samples collected throughout the year from which mtDNA could be extracted. Sex chromosome DNA was obtained for 123 out of 312 successfully extracted samples (39,4 %). Feces preserved in 70 % ethanol showed a higher DNA extraction success compared to those preserved in NAP buffer. Sanger sequencing results revealed 138 samples as Iberian wolf, 18 as dogs, and 3 as red foxes. The sex distribution of the samples could be determined from 95 feces, identifying 48 males and 47 females. The even distribution of male and female indicates that feces are a reliable source for non-invasive monitoring of populations. It also suggests that both sexes use scent marking to the same degree. This is in contrast to previous reports suggesting that scent marking is mainly territorial behavior among male wolves. This study shows that fecal sampling for genetic studies can be an efficient approach in wolf monitoring and that sex determination of wolves can provide useful information regarding reproductive and behavioral patterns.