Genetic and genomic analyses are powerful tools in wildlife research. They might be able to yield the same information on, e.g., population size, health, or diet composition as other wildlife research methods, and even provide additional data that would not be possible to obtain by alternative means. If genetic material is obtained non-invasively, this approach might have no or only minimal impact on animal welfare. Noninvasive genetic sampling sensu lato is defined as "obtaining DNA without affecting the physical integrity of the animal through puncturing the skin or other entry into the body".
1. Introduction
The global change and decline of biodiversity require effective species management based on continuous monitoring of trends within wildlife populations
[1]. Monitoring of animal populations can be conducted in numerous ways, for instance, through capture-mark-recapture
[2], camera traps or aerial surveys
[3][4], radio or GPS tagging
[5][6], counting of traces such as faeces and burrows
[7][8], or through genetic assessment
[9].
Genetic monitoring in particular can be a powerful research tool, as it is capable of providing the same information as other methods, for instance, population size estimates
[10][11], species detection
[12][13], individual identification
[14][15], or diet composition
[16][17][18]. Moreover, DNA analyses can deliver multitude of data that might be difficult or impossible to obtain with other methods, e.g., on relatedness among individual animals
[19][20], population structure
[21][22][23], origin of invasive species
[24][25][26][27], hybridization
[28][29][30][31], past and present population sizes
[7][32][33], or gene flow
[26][27][31][34][35][36][37][38][39].
Traditionally, DNA samples have been obtained from blood or tissues
[40]. The advantage of these samples is that they contain high-quality DNA in large quantities, the disadvantage is the invasiveness of these methods, with potentially negative implications for animal welfare
[40]. Fortunately, DNA samples can be also collected in a way that requires no or only minimal physical interaction with the animal. Noninvasive genetic sampling
sensu lato is defined as “obtaining DNA without affecting the physical integrity of the animal through puncturing the skin or other entry into the body”
[41]. This approach was first used approximately 30 years ago, to obtain DNA from hair samples of chimpanzees (
Pan troglodytes)
[42] and faecal samples of brown bears (
Ursus arctos)
[43]. Although faeces and hair remain commonly used sources of non-invasively obtained DNA, it is now possible to obtain genetic sequences also from feathers, saliva, urine, slime, or eggshells
[44]. Furthermore, recent advances in sequencing technologies allow for detecting the presence of a target species or even for describing whole communities by metabarcoding of the so-called environmental DNA (eDNA) samples from water
[45][46] or soil
[47]. In this approach, a sample is amplified using primers for a standard barcode region, such as the mitochondrial COI, and sequenced on a high-throughput sequencing platform
[48].
2. Performance in Comparison with Other Approaches
2.1. Efficacy of Noninvasive Genetic Assessment
A review of 113 wildlife research studies comparing the performance or efficacy of non-invasive genetic assessment and another method (figure 1 and figure 2) found that 94% of the studies reported that noninvasive genetic assessment performs equally well or better than other approaches. Based on the reviewed studies, it can be particularly suitable for species or individual identification, population size estimation, species detection, and as an alternative to invasive DNA sampling (figure 2). The 6% of the studies reporting inferior efficacy of noninvasive genetic sampling demonstrated that its performance strongly depends on the research aims and study design.
figure 1. Sankey diagram with the number of studies grouped according to the type of study (A), source of a non-invasively obtained DNA sample (B), and method compared to the non-invasive genetic assessment (C). The thickness of the lines linking categories is proportional to the number of studies and the colour corresponds to the target category going from left to right.
figure 2. Performance of noninvasive genetic assessment across the 113 studies. (A) The number of studies included in the review published in 1997–2020. (B) The proportion of studies sorted by their type. (C) Sorted by the source of non-invasively obtained DNA sample used. (D) Sorted by the method that noninvasive genetic assessment was compared to.
2.2. Species Bias
Interestingly, carnivores seemed to be particularly popular among the included studies. Similar findings were reported also in previous work assessing research on wildlife
[9][49]. This might be caused by several factors. First, carnivores and large mammals in general are difficult to capture
[50][51]. Secondly, their faces and hair might be relatively easy to find in comparison with those of smaller species
[52][53]. In addition, lastly, some species are more sought-after study subjects than others
[54][55] and attract most of the research and conservation funding
[56]. Accordingly, the suitability of different methods might be understudied in certain animal groups and the same method may have different efficacy depending on the species studied
[57].
2.3. Animal Welfare Considerations
One of the greatest challenges in wildlife research is to successfully monitor the target species or populations while causing minimal disturbance, stress or pain to the studied animal
[40][58]. A huge benefit of noninvasive genetic assessment is the minimal or no impact on animal welfare, because depending on the method, it may not be necessary to even touch the animal
[40]. Several studies included in this work explicitly mentioned no disturbance or harm to animals as an advantage over other methods
[11][15][16][37][59][60][61][62]. However, tissue or blood sampling may not necessarily be an animal welfare issue, when samples are taken from an already deceased animal. An example of this is the use of museum samples or roadkill. Although none of the studies included in this work used this approach, it has been successfully implemented in, for instance, the assessment of genetic structure in the European hedgehog (
Erinaceus europaeus)
[63], kangaroo rat (
Dipodomys panamintinus)
[64], or Alabama red-bellied turtle (
Pseudemys alabamensis)
[65].
Furthermore, it is important to note that not all genetic sampling defined as noninvasive
sensu lato can be considered harmless. Just capture of the animal—without affecting the physical integrity through a needle or another instrument—could be extremely stressful and might lead to capture myopathy
[66][67]. Consequently, whenever possible, one should implement techniques that require no handling or disturbance of wildlife, such as the collection of faeces. In other cases, this would mean using a different approach, e.g., camera traps, which might be even more suitable than genetic assessment for reaching the specific research goals
[68].
2.4. Cost and Time Effort Advantages
Noninvasive genetic assessment could be more cost- and time-effective than both invasive sampling and other survey methods (
figure 3). Nevertheless, the costs and time effort depend on the approach the noninvasive genetic assessment is being compared with, the type of study, and the research design. For instance, field visual or acoustic survey used for population size estimation or species detection could be both less expensive
[69][70] or more expensive
[13][71][72][73][74] than noninvasive genetic sampling and analysis. Similarly, one study reported lower costs of camera traps for population size estimation
[38], while two studies reported the opposite
[75][76]. Another study showed that genotyping from non-invasively obtained DNA samples is more time-consuming and expensive than genotyping from blood or tissue samples
[77], but four studies reported a contradictory calculation
[37][78][79][53]. This variety in findings among the studies stresses the importance of optimal study design.
figure 3. (A) The number of studies reporting lower or higher costs of noninvasive genetic assessment in comparison with another method and studies that did not make this comparison (NA = not assessed). (B) The number of studies reporting lower or higher time effort of noninvasive genetic assessment in comparison with to another method and studies that did not make this comparison (NA = not assessed).
3. Conclusions
The evaluation of the efficacy of noninvasive genetic assessment is an important step toward a wider uptake of this methodology. This work constitutes the first effort to collate the peer-reviewed literature on the performance of noninvasive genetic assessment. The overwhelming majority of studies included in this work supported the notion that noninvasive genetic assessment is a very effective research tool, suitable for a large spectrum of wildlife studies. The recent technological advances in genetic sampling and sequencing methods provide new opportunities for fast, reliable, and cost-efficient wildlife research. Moreover, noninvasive genetic assessment is well-suited to address the increasing demand for effective and efficient research that has minimal impact on animal welfare.