From roads to biobanks: Roadkill animals as a valuable source of genetic data

Abstract

To protect biodiversity we must understand its structure and composition including the bacteria and microparasites associated with wildlife, which may pose risks to human health. However, acquiring this knowledge often presents challenges, particularly in areas of high biodiversity where there are many undescribed and poorly studied species and funding resources can be limited. A solution to fill this knowledge gap is sampling roadkill (animals that die on roads as a result of collisions with circulating vehicles). These specimens can help characterize local wildlife and their associated parasites with fewer ethical and logistical challenges compared to traditional specimen collection. Here we test this approach by analyzing 817 tissue samples obtained from 590 roadkill vertebrate specimens (Amphibia, Reptilia, Aves and Mammalia) collected in roads within the Tropical Andes of Ecuador. First, we tested if the quantity and quality of recovered DNA varied across roadkill specimens collected at different times since death, exploring if decomposition affected the potential to identify vertebrate species and associated microorganisms. Second, we compared DNA stability across taxa and tissues to identify potential limitations and offer recommendations for future work. Finally, we illustrate how these samples can aid in taxonomic identification and parasite detection. Our study shows that sampling roadkill can help study biodiversity. DNA was recovered and amplified (allowing species identification and parasite detection) from roadkill even 120 hours after death, although risk of degradation increased overtime. DNA was extracted from all vertebrate classes but in smaller quantities and with lower quality from amphibians. We recommend sampling liver if possible as it produced the highest amounts of DNA (muscle produced the lowest). Additional testing of this approach in areas with different environmental and traffic conditions is needed, but our results show that sampling roadkill specimens can help detect and potentially monitor biodiversity and could be a valuable approach to create biobanks and preserve genetic data.

Fig 1. DNA quantification by taxonomic class.

Variation in the amount of DNA extracted from 817 tissue samples obtained from specimens of four vertebrate classes collected during roadkill surveys in the Napo region of Ecuador from 2020 to 2021. Different letters above the plot indicate significant differences between the groups.

Fig 2. Variation in the obtained quantity DNA from roadkill specimens at different estimated times since death across the four studied taxonomic classes.

Symbols represent the observed data with the line and shaded area representing the estimated relationship and 95% CI respectively from a linear regression model (Table 3). Samples were collected during a roadkill survey in the Napo region of Ecuador from 2020 to 2021.

Fig 3. DNA quantification by tissue.

Variation in the amount of DNA extracted from different tissues collected from roadkill vertebrates during surveys in the Napo region of Ecuador from 2020 to 2021. Different letters above the plot indicate significant differences between the groups.

Fig 4. DNA integrity.

Variation in the degradation of DNA extracted from intestine tissues collected at varying times time since death (A) and that yield different quantities of DNA (B). Samples were obtained from roadkill vertebrates during surveys in the Napo region of Ecuador completed in 2020–2021. Different letters above the plot indicate significant differences between the groups.

Fig 5. Amplification of the targeted COI region for vertebrate taxonomic identification.

DNA electrophoresis from the eight tested vertebrate samples containing isolated genomic DNA (A) and the amplified COI (B). Molecular weight (MW); Negative control (CN). The identification number of each sample (ID) is shown in the top line.

Fig 6. Amplification of molecular target from Leishmania spp.

DNA electrophoresis from eight samples of vertebrate liver obtained from roadkilled specimens, containing isolated genomic DNA (A), or the amplified 18S SSU rRNA of Leishmania spp. (B).