Genomic-guided conservation actions to restore the most endangered conifer in the Mediterranean Basin

Species with extremely small population sizes are critically endangered due to reduced genetic diversity, increased inbreeding, and the added threat of hybridization. Genomic tools significantly advance conservation by revealing genetic insights into endangered species, notably in monitoring frameworks. Sicilian fir is the most endangered conifer in Europe with only 30 adult trees spread across an 84-hectare area. Using 20,824 SNPs from RAD-seq employing the silver fir genome assembly and a custom 120 SNP-array, we evaluated genetic diversity, mating patterns, and effective population size in adult trees, 118 natural seedlings, and 2,064 nursery seedlings from past conservation actions. We assessed introgression from neighboring non-native fir plantations and established an intra-population assisted gene flow program selecting the most genetically dissimilar individuals and investigating the outcome through simulations. Genomic analysis unveiled significant genetic diversity among adult Sicilian firs, comparable to non-endangered Mediterranean firs with larger populations. However, the genetic diversity of the forthcoming generation declined due to high self-fertilization, leading to marked inbreeding (Fis = 0.38) and an alarmingly low effective population size (Ne = 6). Nursery seedling monitoring revealed similar selfing rates but significant introgression ([~]50%) from non-native firs. Although intra-population assisted gene flow could help to mitigate genetic loss, it may not alleviate the species vulnerability to imminent environmental challenges, perpetuating the risk of an extinction vortex. Hence, investigating the impact of Sicilian fir population decline and selfing on inbreeding depression, along with exploring the potential of hybrids for genetic load alleviation and future adaptation, is crucial for effective conservation strategies. This study stands as a compelling model for guiding conservation strategies in similarly imperiled species characterized by extremely small populations.