ROH were identified using VCFtools (63). ROH spanning regions smaller than 100 kb were ignored. Highly similar patterns of ROH were also inferred using PLINK v1.90b6.7 (64) with the following options: --homozyg-window-snp 200 --homozyg-window-het 3 --homozyg-window-missing 50. Overall, we found that PLINK identified a greater number of short ROH (100 kb to 1 Mb) relative to VCFtools and also inferred fewer medium ROH (1 to 10 Mb) in the Ethiopian wolf genome (fig. S7). We used ROH identified by VCFtools in further analyses.

The proportion of the genome within ROH (FROH) was calculated as the total length of ROH of 100 kb or longer within an individual divided by the length of the genome (2,203,764,842 nucleotides). The X chromosome was included in the calculation for females only (total genome length = 2,327,633,984 nucleotides). To test for enrichment of homozygous deleterious variants within ROH, we followed the method of Szpiech et al. (45). The fraction of the genome within ROH and the fraction of damaging and benign homozygous genotypes inside ROH were calculated in each individual. Following Equation 10 of Szpiech et al. (45), we fit linear models to test whether variant impact (benign versus deleterious) and FROH were significant predictors (β2 and β3, respectively) for the proportion of nonreference homozygotes within ROH.

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