Historical effective population size of North American hoary bat (Lasiurus cinereus) and challenges to estimating trends in contemporary effective breeding population size from archived samples

Background Hoary bats (Lasiurus cinereus) are among the bat species most commonly killed by wind turbine strikes in the midwestern United States. The impact of this mortality on species census size is not understood, due in part to the difficulty of estimating population size for this highly migratory and elusive species. Genetic effective population size (Ne) could provide an index of changing census population size if other factors affecting Ne are stable. Methods We used the NeEstimator package to derive effective breeding population size (Nb) estimates for two temporally spaced cohorts: 93 hoary bats collected in 2009–2010 and an additional 93 collected in 2017–2018. We sequenced restriction-site associated polymorphisms and generated a de novo genome assembly to guide the removal of sex-linked and multi-copy loci, as well as identify physically linked markers. Results Analysis of the reference genome with psmc suggested at least a doubling of Ne in the last 100,000 years, likely exceeding Ne = 10,000 in the Holocene. Allele and genotype frequency analyses confirmed that the two cohorts were comparable, although some samples had unusually high or low observed heterozygosities. Additionally, the older cohort had lower mean coverage and greater variability in coverage, and batch effects of sampling locality were observed that were consistent with sample degradation. We therefore excluded samples with low coverage or outlier heterozygosity, as well as loci with sequence coverage far from the mode value, from the final data set. Prior to excluding these outliers, contemporary Nb estimates were significantly higher in the more recent cohort, but this finding was driven by high values for the 2018 sample year and low values for all other years. In the reduced data set, Nb did not differ significantly between cohorts. We found base substitutions to be strongly biased toward cytosine to thymine or the complement, and further partitioning loci by substitution type had a strong effect on Nb estimates. Minor allele frequency and base quality bias thresholds also had strong effects on Nb estimates. Instability of Nb with respect to common data filtering parameters and empirically identified factors prevented robust comparison of the two cohorts. Given that confidence intervals frequently included infinity as the stringency of data filtering increased, contemporary trends in Nb of North American hoary bats may not be tractable with the linkage disequilibrium method, at least using the protocol employed here.