Separate models were additionally used for each of the Neochetina spp. Stepwise model simplification was performed using likelihood ratio tests. Differences across collection sites were compared, based on 95% CI, using Tukey’s posthoc test in the “multcomp” package . To determine whether the number of individuals released affects genetic diversity, we examined the number of weevils imported to each Florida, Texas, and California in the USA and the ratio of allelic richness retained from the native range in those states. To examine the influence of the number of introduction steps on the genetic diversity in populations of these two biological control agents, we combined the documented importation history and the genetic diversity data . We counted the number of introduction steps based on the number of times the weevils were imported and exported since the initial export from the native range. We used linear models with the number of introduction steps from the native range for each population as a fixed effect with allelic richness or expected heterozygosity as the response variables in separate models. Separate models were used for each of the Neochetina spp.We examined the population genetic structure of N. bruchi and N. eichhorniae to determine whether populations in the different lo‐ cations have diverged from those in the native and introduced ranges since the initial introductions in the 1970s. Additionally, vertical farming racks we explored whether the importation pathways impacted the population genetic structure of these two weevils.
To initially examine whether population divergence has occurred, we conducted pairwise FST and Jost’s D analyses with the R package “popgen report” . Although FST is one of the most utilized metrics in population genetic studies, it can be biased downward for loci with multiple alleles . Thus, we additionally present Jost’s D , which measures the fraction of allelic variation among populations, but can be biased upwards . We used three additional population structure analyses to infer population structure by determining the number of genetic clusters and to assign individuals to their appropriate genetic cluster. We validate the results from these analyses by using information gained from the documented importation history. We used: a discriminant analysis of principal components with the package “adegenet” in R, an iterative reassignment of individuals with the FLOCK software , and a Bayesian approximation with the STRUCTURE software STRUCTURE . The FLOCK software and DAPC do not assume HWE or LE, in contrast to the program, STRUCTURE . As some of the microsatellite loci and populations in this study significantly deviated from HWE and LE , we present the methods for analysis in the STRUCTURE program and the results from STRUCTURE in the Supporting Information Appendix S5. The FLOCK software first randomly divides all of the genotypes into K genetic groups and then reassigns the genotypes at each iteration to the group with the highest probability of belonging, using the multilocus method of maximum likelihood described by Paetkau, Calvert, Stirling, and Strobeck .
FLOCK was run both to provide an estimate of the number of populations and to deter‐ mine which of a potential set of genetic sources is most likely the true source of each introduced population of both species. Default parameter values were used for each k. To identify the most likely sources of an introduced population, we followed a systematic search procedure. In summary, we ran FLOCK with the novel sample and all plausible source samples while k was set at 2. Based on the resulting allocation tables from this run, all of the possible sources that were not mainly allocated to the same cluster as the novel sample were discarded. This same procedure was applied iteratively until only one potential source sample remains. When selecting the initial set of candidate sources for the allocation tables, we discarded the samples that could not be realistically considered potential sources. Those decisions were based mainly a priori on strong historical evidence. The searching procedure is described more formally and in greater detail in Supporting Information Appendix S2. When the searching procedure did not produce an unambiguous output, it was complemented by visualization with a DAPC run with the same samples . We compared the DAPC results and FLOCK runs to the importation history .We confirmed hybridization between N. bruchi and N. eichhorniae by analyzing the species‐specific markers on 12 individuals that had noticeable hybrid‐like markings on their elytra .
One individual from California gave 100% amplification of microsatellite markers designed for N. bruchi and 80% of markers designed for N. eichhorniae, suggesting it may have been a first‐ generation hybrid . A second individual from Uruguay yielded 63% amplification of the markers designed for N. bruchi and 100% of markers designed for N. eichhorniae. Amplification of loci from both species in other individuals from populations in Texas and Uganda suggested possible later generation hybrid backcrosses , with 100% amplification of loci for one species and 30%–40% amplification of markers designed for the other species. As discussed in the methods, none of the species‐specific microsatellite markers developed for N. bruchi cross‐amplified on individuals with species‐specific morphological characteristics of N. eichhorniae and vice versa. Based on the morphological characteristics, we also noticed potential hybrids from the SA: George population, but these individuals did not amplify well for either set of markers likely due to poor DNA extractions. We could not analyze the prevalence of hybrids due to the sampling bias from collectors that selected individuals for each species based on distinct markings that separate the species.Among the most striking results was the confirmation of hybridization between N. bruchi and N. eichhorniae. We found interspecific hybrids in California, Uruguay, and Uganda, ranging from potential first‐generation hybrids to F2 and later generations. Based on the morphological characteristics, we also noticed potential hybrids from South Africa, but these individuals did not amplify well for either set of markers likely due to poor DNA extractions. Inter specific hybrids are likely present in all regions based on the fact that we found a hybrid from a population in Uruguay in the native range, and they may have been introduced from the original collections and releases. However, we could not accurately assess the percent of hybrids per site due to the strong likelihood of sampling bias against hybrids during the collections. Future studies should conduct in‐ depth surveys to examine the prevalence of hybrids in the native and introduced regions and perform hybrid crosses. As these two weevils are used across the globe for the biological control of water hyacinth, it is important to investigate the effect of hybridization on the performance and growth of these weevils. Interspecific hybrid crosses can result in hybrid vigor or hybrid breakdown as well as affect the host‐specificity of a biological control agent . If fitness of hybrids is low, it may be useful to determine the conditions under which hybrids form and try to minimize hybridization in regions where biological control programs are critical for the control of water hyacinth. In addition to the occurrence of hybridization, we found evidence of genetic drift and inbreeding in several populations. From the importation history, there is documented evidence that these weevils went through demographic bottlenecks during the importation and release phases of the biological control programs. Subsequent drift or inbreeding following demographic bottlenecks can lead to in‐ creased homozygosity . We found evidence of genetic drift in the SA: Enseleni and Uruguay populations of N. bruchi and in the SA: Wolseley population of N. eichhorniae. The occurrence of genetic drift in the native range was unexpected as allelic rich‐ ness was the highest in Uruguay and genetic drift typically occurs in populations that have undergone a demographic bottleneck . Alternatively, vertical grow rack these results may have been mediate levels of genetic diversity in Uganda for both species even artifacts of marker scoring , the sampling or though the populations in Uganda had the highest number of steps the markers used in this study . Additional away from the native range. Overall, N. bruchi had similar allelic rich‐ potential evidence of genetic drift or inbreeding was found in the ness and heterozygosity across the eight collection sites. Although California population of N. bruchiwith FIS = 0.15 . We also not significantly higher, the populations of N. bruchi in the native found indications of potential inbreeding in both populations of range and in SA: Enseleni harbored the most alleles.
In N. bruchi and N. eichhorniae in California and in two populations of contrast, there was significant variation in allelic richness and ex‐ N. eichhorniae in the Western Cape of South Africa . Although inbreeding can have detrimental highest allelic richness in the population in Uruguay. Populations of consequences, it has also been known to promote local adaptation each N. bruchi and N. eichhorniae from Uruguay also exhibited the . highest number of private alleles, with eight and 10 private alleles, Integrating the estimates of genetic diversity with the importa‐ respectively. The high allelic richness and many private alleles found tion history for these biological control agents also permitted us to in the population in Uruguay supports the general trends that in‐ examine the consequences of propagule size and introduction protroduced populations typically undergo a loss in genetic diversity cesses on the genetic diversity of introduced populations. We did , but see . introduction steps affected current day genetic diversity in popula‐ We were also able to investigate the potential effects of genetictions of N. bruchi or N. eichhorniae. However, initial propagule sizes admixture on genetic diversity as a result from multiple introduce in this study system may have been higher than a specific threshtions that occurred in this study system. The FLOCK allocation ta‐ old required for an effect to have taken place. Our initial hypothesis bles generally reflected the movement of weevils documented in that populations with more introduction steps away from the native the importation records and additionally clarified genetic range would harbor lower genetic diversity than those populations sources where the importation history was unclear. In places such as South Africa, the importation history was unclear due to intervening importations from multiple locations and multiple introductions across the country. Interestingly, one of the populations of N. bruchi in South Africa, SA: Enseleni, had equivalent allelic richness to the population in the native range. Although the other population had lower allelic richness, the sample size for that population was only six individuals. FLOCK allocation tables helped demonstrate that SA: Enseleni was a composite population with two genetic sub‐clusters. One sub‐cluster was mostly allocated to the Australian genetic cluster, and the other cluster appeared somewhat related to the Australian and Ugandan genetic clusters. Based on the importation history, the latter sub‐cluster was likely derived from Zimbabwe, a population that we did not test. This finding supported the multiple introductions documented in the importation history, and the notion that genetic admixture can increase genetic diversity . Furthermore, genetic admixture may be able to rescue populations that had small initial propagule size or under‐ went demographic bottlenecks Additionally, two out of the three populations of N. eichhorniae in South Africa demonstrated high allelic richness and FLOCK allocation tables found that one of these populations had two genetic sources . Interestingly, FLOCK analyses demonstrated that only one population from USA: Florida contributed to the genetic composition of SA: Wolseley, which also demonstrated high allelic richness. This population also had indica‐ tions of genetic drift and inbreeding, which supports the contrasting forces of genetic admixture and inbreeding, with the latter some‐ times selected for when a population is adapted to the local area . In contrast, the populations of N. eichhorniae in the USA also had high allelic richness, even though FLOCK allocation tables indicate a single introduction from the native range. It appears that only one Uruguayan sub‐cluster contributed to the current day genetic com‐ position of USA: Florida. This was particularly interesting since the importation history indicates two populations from South America were initially imported to USA: Florida. However, the lack of genetic contribution from the two populations in the native range is likely due to the fact that only 10 individuals from Campana Lagoon were imported , due to a low abundance of N. eichhorniae in the Campana Lagoon.