However, using 9 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 occuring 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 , while 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 U.S., Brazil, and Italy when compared to 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 lab populations from Japan and Hawaii have half as much pairwise diversity as the wild South Korean population, consistent with as mall lab 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, plant pots with drainage which can occur during a population shrinkage, while 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 be statistically different except for Western U.S. 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 one 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 U.S. 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 geographic 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 U.S. 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 U.S., 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 U.S., we were surprised to discover a lack of structure on a finer scale, either based on latitude or simple geographic distance, given the large number of loci analyzed. In a similar study using 3,484 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 U.S. 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 U.S. 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 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 U.S., 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 U.S., European, Asian, and Brazilian populations varied between 8.86% to 9.02%. However, we were surprised to see that our Italian and Irish samples had much higher values of Fst compared to 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, plastic plants pots we find that our treemix and migration results largely coincide with the proposed invasion pathway inferred from microsatellites , as well as a recent pre-print that reanalyzed invasion pathways with pooled sequencing data . We see that European and U.S./Brazilian populations form two distinct clades, 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 U.S., then the Eastern U.S. and Brazil. Additionally, in the Western U.S., 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 U.S./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 U.S. 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 U.S. 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 outgroup . 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 ofinvasive 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 U.S. 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 non-variable 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.Tuta absoluta is a worldwide economic pest of tomatoes and other solanaceous crops. A member of the gelechiid family, this moth lays eggs on the above-ground portion of the plant, where the hatched larvae will spend their lives creating “mines” throughout the plant tissue before pupating and emerging as adults . At a reproduction rate of up to 10 generations per year, untreated infestations will eventually result in complete death of the plant, leading to up to 100% agricultural loss. Although a large effort has been made to develop and implement integrated pest management programs across different world regions , typical treatments have included heavy use of a variety of insecticides , leading to the rapid appearance of insecticide resistance. As tomatoes represent a massive economic industry, with an estimated 252 million metric tons of tomatoes harvested in 2020 , there is a serious need to understand the invasive biology of this insect and to develop tools for detection and prevention. Tuta absoluta was originally detected in Peru in 1917 but was not recorded as an agricultural pest until the 1960s and 70s when it was discovered in tomato fields in Chile, Argentina, and Venezuela; by the 1990s it was widespread across South America. In 2006, Tuta absoluta appeared in Spain ; since then it has rapidly colonized Europe, Asia, and Africa. It is generally believed that the Peruvian highlands is the ancestral home of T. absoluta, and that the rapid colonization to the rest of Latin America was due to the introduction of Tuta absoluta by human transport of contaminated fruit, although few studies have confirmed this .