Standardization of a High-Quality Methodological Framework for Long-Term Genetic Monitoring of the French Wolf Population

Abstract

Since the gray wolf was eradicated from large parts of Europe, this species has been recolonizing much of its former distribution, particularly since the past 30 years. Wolves benefit from European legal protection through the Habitats Directive and the Bern Convention, and reporting on the evolution of their populations in each country of Europe is mandatory. To monitor French wolf populations over the long term, a standardized high-quality methodological framework has been developed to analyze data from noninvasively collected samples and assess population diversity. We delineated each step and implemented a laboratory control procedure to analyze 8733 samples harvested within the French distribution range of the species between 2006 and 2022, and provided key quality and diversity indicators. Of these samples, 82.8% were successfully amplified and sequenced for the mitochondrial control region. Subsequently, the wolf samples were genotyped at 22 microsatellite autosomal loci and a sex locus displayed over two independent multiplexes using the multitube approach. The average success rate of polymerase chain reaction per locus was 64.2% across all replicates. The residual genotyping error rates were low compared to those in other studies using non-invasively collected samples, with mean residual allelic dropout rates of 5.8% per locus and mean residual false allele rates of 1.0% per locus. The high-quality dataset identified 1735 individuals in total over the last 15 years, of which 99.9% exhibited a single Italo-Alpine mitotype. Genetic diversity was relatively low, with mean observed heterozygosity of 0.482 and mean expected heterozygosity of 0.519. This supports the natural colonization of the French Alps by a few individuals originating from the remaining Italian populations, which started approximately 30 years ago. By generating high-quality standards and quality control processes, this protocol enhances the cost-efficiency ratio of monitoring French wolf populations and holds high value for managers tasked with the management and conservation of wolf populations in the long term.

Keywords: Canis lupus; France; gray wolf; long‐term monitoring; microsatellite; mitochondrial control region; noninvasive samples.

FIGURE 1

Evolution of the sampling coverage performed to monitor the French gray wolf population from 2006 to 2022 (n = 8733 samples).

FIGURE 2

Flowchart of critical steps and control procedures applied to process gray wolf noninvasive samples. AD, Allelic dropout rate; AD_res, Residual allelic dropout rate; FA_res, Residual false allele rate; HWE, Hardy–Weinberg Equilibrium; MtDNA CR, Mitochondrial Control Region; NIS, Noninvasive samples; PI_sibs, Probability of identity among siblings; QI, Quality Index.

FIGURE 3

Proportion of samples collected for monitoring the French gray wolf population that were successfully analyzed using the mitochondrial control region and the panel of 22 microsatellite loci. CR, Control region. The size of the circles is proportional to the number of samples collected (left circle, n = 8733 samples) and the number of samples with a gray wolf haplotype (right circle, n = 5840). The left circle shows the percentage of samples for which a species could be identified when sequenced at the mitochondrial control region, and the right circle shows the percentage of gray wolf samples that had a Quality Index (QI) > 0.5 and were not contaminated when genotyped with the panel of 22 microsatellite loci.

FIGURE 4

Quality Index values and PCR success per locus for the gray wolf samples collected in France. For each indicator, darker purple bars indicate values estimated using all genotyped samples (n = 5840), while lighter purple bars denote values estimated using only samples with Quality Index (QI) values > 0.5 and without contamination (n = 3797). The mean of the indicator across all loci is represented by the dotted line.

FIGURE 5

Quality indices distribution among gray wolf samples collected in France (n = 5840). Boxplots indicate mean Quality Index (QI) values per sample type and colors correspond to sample type (tissue, blood, hair, scat, or urine).

FIGURE 6

Probability of Identity among siblings (PI_sibs) values calculated using the 1735 different individuals identified. The dotted line delineates the 1.00 × 10⁻² threshold.

FIGURE 7

Genotyping error frequencies per locus in wolf samples. For each indicator, darker purple bars indicate values estimated using all genotyped samples (n = 5840), while lighter purple bars denote values estimated using only samples with Quality Index (QI) values > 0.5 and without contamination (n = 3797) and light blue bars correspond to values estimated on samples from recaptured individuals (n = 2743). The mean of the indicator across all loci is represented by the dotted line.