As the US/ Brazilian admixture weight is much less than the European admixture weight, this was likely due to a migration event from the Americas into Irish populations. The other out-of-US admixture event, from the Western United States to South Korea , was seen when m ¼ 6, 8, and 10. F3 statistics all have significantly negative values, and the F4 statistics and are significantly positive, supporting a Western US/South Korea admixture. However, using nine migration edges Treemix reported the reverse direction; as F3 and F4 statistics cannot easily infer directionality, more heavily sampling of the Asian populations or alternate methods may be needed to determine whether flow is occurring in both directions.To determine if invasive populations have experienced loss in genetic diversity, we used the software ANGSD to estimate average pairwise nucleotide diversity in 20 kb increments across the 20 largest contigs of genome for each population. Invasive populations can sometimes exhibit reduced levels of diversity early on in their history due to a founder effect , whereas ancestral populations tend to have the greatest amount of diversity as they have had many generations to accumulate mutations. A Welch one-way test found a significant difference in mean pairwise nucleotide diversity between clusters. We then used pairwise Games-Howell tests and found each cluster to be significantly different , except for the Eastern United States, Brazil, and Italy when compared with each other. As Asia is the ancestral home of D. suzukii, it is no surprise that South Korean wild populations exhibit the highest diversity levels . Similarly, the laboratory populations from Japan and Hawaii have half as much pairwise diversity as the wild South Korean population,drainage pot consistent with a small laboratory population size. The invasive populations display an intermediate level between these extremes. To assess whether invasive populations may have experienced a bottleneck or population shrinkage, we also estimated Tajima’s D in the same genomic intervals.

Extremely positive values can indicate a loss of rare alleles, which can occur during a population shrinkage, whereas extremely negative values can indicate a recent bottleneck followed by rapid expansion . A Welch one-way test again indicated significant differences in mean Tajima’s D between clusters , and pairwise Games-Howell tests found all clusters to betatistically different except for Western US against Brazilian flies. Strains from Hawaii and Japan both had high genome-wide levels of Tajima’s D, indicative of a loss of rare alleles that can occur during a population shrinkage . The remaining populations had neutral values of D, except for Ireland’s relatively high value. Based on this, we conclude there are no strong signals for a recent bottleneck, although the high genome-wide D value for Ireland suggests a recent population shrinkage. As our Irish samples were collected in 2016 only 1 year after its discovery in Ireland, we could be observing the founder’s effect in action .Based on population allele frequencies, we have shown that D. suzukii exhibit population structure based on region and invasion history. In the New World populations, we find that Eastern and Western US samples appear to be distinct populations. While this could be the result of continuous population variation from East through Central to the West coast, it is more likely the case that the two populations experience little gene flow due to strong geographical barriers such as the Sierra Nevada or Rocky Mountain ranges, and the fact that key target fruit crops such as cherries,raspberries, blueberries, and strawberries are primarily grown in states that we sampled . Any genetic exchange between these regions would likely be the result of human activity, such as could be the case with samples collected from Alma Research Farm, Bacon County, Georgia clustering with the Western US populations. As other nearby collections failed to share this signal, the Alma research population could represent a recent and isolated migration event.

Otherwise, we see little evidence of migration events or admixture between the Eastern and Western United States, which is somewhat surprising as the country’s supply of fresh blueberries, cherries, and caneberries are concentrated in a few states and shipped across the country . However, recent changes to cultural management such as more frequent harvesting and post-harvest chilling may be responsible for disrupting the D. suzukii lifecycle and limiting cross-country transport . While we were able to detect population structure between eastern and western locations in the United States, we were surprised to discover a lack of structure on a finer scale, either based on latitude or simple geographical distance, given the large number of loci analyzed. In a similar study using 3484 SNPs in 246 Hawaiian D. suzukii samples, researchers were able to identify three distinct populations roughly seperated by islands . The fact that D. suzukii has been present in Hawaii since 1980, in addition to the isolation by island, are likely the strongest factors in providing enough genetic drift to create such differentiation. As the continental US D. suzukii have only been present since 2008, it may be too early for finer structure to have developed. Alternatively, continual dispersion and transportation of D. suzukii around the United States may be hindering the development of more local structure. Several studies have reported a low probability of D. suzukii surviving when exposed to freezing temperatures, based on cold survival assays of wild-caught specimens , suggesting that flies collected in cold-winter regions such as Washington, Michigan, Maine, and New York could be annual migrants to the area from nearby warmer locations. The lack of north-south population structure supports the hypothesis that flies are regularly re-migrating into colder climates after the harsh winters have ended. Alternatively, flies could be tolerating winters by surviving inside human structures , or by having evolved resistance to freezing temperatures . Studies using D. suzukii collected from different locations have reported different levels of rapid cold-hardening response, suggesting that there could be regional selection present .

If populations in northern regions undergo strong seasonal fluctuations in allele frequencies, such as has been demonstrated in wild D. melanogaster populations collected in Pennsylvania , by only sampling sites in the summer we may be missing signals of population differentiation between the north and south. Likely, some combination of these factors is responsible for the success of D. suzukii in these regions, and further studies will be needed to identify the causes. North-south clines in specific traits such as diapause and circadian rhythms have been previously identified in drosophilids and could be at play here as well . Further analyses using methods such as those recently used to detect SNPs correlated with invasive success could be applied to this dataset to find signals of selection. Fst values between populations from the United States, Brazil, and South Korea were low and agree with previously published Fst estimates based on Pool-Seq data; Olazcuaga et al. 2020 observed that Fst between US, European, Asian, and Brazilian populations varied between 8.86 and 9.02%. However, we were surprised to see that our Italian and Irish samples had much higher values of Fst compared with the other populations, and even to each other. This discrepancy could be due to the small sample sizes we had from Europe; in this scenario, pooling larger number of samples can improve power to estimate Fst, and we instead rely on comparing the relative Fst values between populations for our analysis. High Fst values between our Japanese and Hawaiian populations were expected, however, as these have likely experienced strong drift during their time in captivity. In general, we find that our treemix and migration results largely coincide with the proposed invasion pathway inferred from microsatellites , as well as a recent preprint that re-analyzed invasion pathways with pooled sequencing data . We see that European and US/Brazilian populations form two distinct clades,large pot with drainage emphasizing these regions were invaded by two independent migrations from Asia. Hawaii is the first population to diverge in the Americas, followed by the Western United States, then the Eastern United States and Brazil. Additionally, in the Western United States, we detected a strong signal of admixture from Hawaii, which could be due to multiple or ongoing migration events. We also detected signals of admixture from the Eastern United States/Brazil to Ireland, which matches the predicted initial invasion pathway and suggests multiple migration events. Unique to our analysis, we recover support for admixture of Western US samples in Asia, suggesting that migrations could be ongoing in both directions. Invasive species transport is strongly associated with international trade of live plants and plant products , and indeed agricultural export data supports the possibility of this migration as Japan receives almost 15% of all US blueberry exports, and Oregon recently became the first state to begin shipping blueberries to South Korea in 2012 . It should be noted that while Treemix infers direction of migration, the model can occasionally infer the incorrect direction, particularly when populations are closely related without an available out group .

In conjunction with evidence of this widespread ongoing migration, we observed nucleotide diversity levels of all invasive populations to be only moderately below that of the wild South Korean population, a trend also observed in Fraimout et al. . Typically, recent invasion events are characterized by reduced diversity relative to the ancestral populations due to founder or bottleneck effects . However, successive invasion events can provide relief from any initial bottlenecks by providing increased genetic diversity. This has been observed to occur in multiple animal studies and could lead to increased ability to adapt and evolve to new climates. Correspondingly, in our analysis, we did not find populations with broadly low values of Tajima’s D, suggesting little bottleneck effect. As measures to reduce impacts of invasive species are often hindered by repeated migrations , it will be important to enforce that fruits being exported and imported internationally are free of live D. suzukii as required by the US Department of Agriculture, even though this species is already internationally established.We anticipate that the genomic data provided here will prove useful in many fields of biology beyond the scope of this study. Knowledge of genetic variation and alternate alleles present within a species can be informative for the design of probes and micro RNAs , such as for the purpose of creating gene drives to control invasive species. Gene drive mechanisms to eliminate D. suzukii have been experimentally tested on multiple lines to ensure the miRNAs are broadly effective , but a large dataset of wild population sequencing allow researchers to more confidently select target sites that are nonvariable and thus susceptible to Cas9 targeting . Drury et al., demonstrated that minor natural polymorphisms in target sites reduce gene drive effectiveness in flour beetles, and tools have been developed to help researchers design gRNAs accounting for population variation . Similarly, with the recent development of a CRISPR-Cas9 editing and RNAi knockdown protocols for D. suzukii , prior knowledge of allelic variation will allow researchers to design targeting oligonucleotides more precisely to avoid loci with variability. Most recently, our dataset has been used to study sensory receptor evolution in D. suzukii, giving insights into its evolution toward becoming a major agricultural pest . Other future uses of this trove of genomic data could involve insecticide resistance studies or the development of diagnostic assays for rapid detection in the field.Since domestication efforts began in the early 1900s , higbush blueberry has rapidly become a high-value fruit crop worldwide. Higbush blueberry, compared to hundreds of closely related blueberry species in the Ericaceae, is widely cultivated due to its adaptation to temperate climates, excellent fruit quality, yield, and composition of phytonutrients. As a result for the demand for fresh blueberries as a ”super fruit”, high bush blueberry production has increased 600% during the past three decades and steadily grown to a multi-billion dollar industry. In addition to its short domestication history, high bush blueberry is unique in being one of only three major commercially valuable fruit crops, accompanied by cranberry and the garden strawberry, with wild progenitor species native to North America. Blueberries have a single epidermal layer that expresses a rich profile of anthocyanins during ripening that, in combination with epicuticular wax, generates its characteristic ”powdery blue” color.