Population structure and genetic differentiation
The observed differences in flight performance may suggest strong population differentiation, but our genomic analyses revealed that this is not the case. The GATK genotyping pipeline resulted in a total of 32.2 million SNPs. A total of 25.9 million SNPs passed quality control filtering to be used for further analysis. A total of 20.9 million SNPs that were covered in all 43 samples were used for Principal Component Analysis (PCA) and ADMIXTURE (Alexanderet al. , 2009) analysis to determine the genetic structure in the dataset. The PCA generated using the SNPrealte (Zheng et al. , 2012) package showed no evidence for clustering of eastern and western monarchs (Fig. 3), and determined that all samples in the data set belong to one population with K=1 showing the lowest error rate (Fig. 3; Table S2). While eastern and western monarchs could not be separated, three samples from Big Sur, California, seemed to cluster.
Genome-wide genetic differentiation (FST ) and absolute divergence (DXY ) between eastern and western monarchs, calculated in windows of 10kb, were extremely low (Table 1, Fig. 4). Genetic differentiation between monarchs from the three western overwintering sites was likewise low (Tables 2, S3; Fig. S3). The genome-wide differentiation landscapes of eastern and western monarch comparisons were highly correlated with the genome-wide differentiation landscapes of the comparisons between western monarchs from different overwintering sites (Fig. S4). The maximum values ofFS T in all comparisons were also extremely low (Table S3), as was the genetic differentiation within genes (eastern vs. western FST (Genes) = 0.0008 ± 0.0004).FSTZ window outliers were calculated separately for autosomes, Z-chromosome and neo-Z-chromosome, and were very low (Table 3). The extremely low maximum measures of genetic differentiation and the top 1%FSTZ window outliers suggest that there are no regions in the genome with reduced gene flow. This is in contrast with many other species, where islands of differentiation appear to be common (Jiggins, Naisbit, Coe & Mallet, 2001; Martin et al. , 2013; Cruickshank & Hahn, 2014; Nadeau et al. , 2014; Talla, Kalsoom, Shipilina, Marova & Backström, 2017; Irwin et al. , 2018). These results were not driven by window size, as low differentiation was also found for window sizes of 100, 500 and 5,000bp (Fig. S5, Table S4).
In line with these results we also did not identify any fixed nucleotide differences between eastern and western monarchs (Fig. 5). The majority of polymorphisms are shared between eastern and western monarchs, while similar proportions of private polymorphisms are observed in both eastern and western monarchs (Fig. 5). This supports the conclusion that there are no regions in the genome with restricted gene flow or islands of genetic differentiation.