These two weevils are the most widely used biological control agents of water hyacinth, Eichhornia crassipes , a floating aquatic plant native to South America. Classical biological control of water hyacinth has been implemented across the globe, with some introductions resulting in significant reduction in water hyacinth cover and/or bio‐ mass, including parts of Australia, China, East Africa, the U.S. Gulf Coast, India, Mexico , and the lower elevation regions of South Africa . Releases of N. bruchi and N. eichhorniae from the native range began in the early 1970s, with initial and subsequent releases in 30 and 32 countries, respectively. These weevils have contributed substantially to the control of water hyacinth in at least 13 countries . Through their use as biological control agents, these two weevil species have often undergone multiple and serial introductions . For example, in the United States, weevils of N. eichhorniae released into northern California underwent four sequential importation steps from the original Argentinian population in the native South American region. Native Argentinian weevils were re‐ leased into USA: Florida in the 1970s, and the weevils in USA: Florida were used to found a population in USA: Louisiana, which were then used to found populations in USA: Texas. This USA: Texas population was the source for the northern California population released in the early 1980s . Similarly, in South Africa, there were multiple introductions of each N. bruchi and N. eichhorniae with each new release being sourced from a different location to which they had been introduced for biological control , cannabis drying racks rather than directly from the native range. These multiple introductions from the non‐native range represent serial bottlenecks in population size that could potentially reduce genetic diversity and limit adaptive potential.
Alternatively, these multiple introductions from different source populations could increase genetic diversity through genetic admixture of the different source populations . The latter may occur particularly if each source population had sufficient time to diverge or adapt to the region of introduction, resulting in increased genetic differentiation from its source population. Based on the importation history and documented releases of these two biological control agents, we proposed several hypotheses addressing our five study questions in turn. We hypothesized that hybrids of these two species would occur, as they have frequently been co‐introduced to the same geographic regions and individuals with morphological characteristics of both species have been found . We hypothesized that genetic diversity would be the highest in the native range compared to regions where the weevils were introduced as biological control agents. Similarly, we hypothesized that the populations from the native range would have more rare alleles than the introduced regions. We hypothesized that allelic richness would reflect the number of individuals released . Specifically, as the initial propagule size of N. bruchi was greater than that of N. eichhorniae in USA: Florida, we ex‐ pected that populations of N. bruchi in USA: Florida would be less likely than populations of N. eichhorniae to lose rare alleles and exhibit reduced allelic richness in the introduced range compared to the native range. In cases where multiple introductions were prominent, particularly regarding the introduced populations of N. eichhorniae in South Africa, we hypothesized that the geneticad mixture would increase genetic diversity and buffer against the negative effects of serial bottlenecks. On a global scale, regarding the number of serial introductions, we hypothesized that populations with more introduction steps away from the native range would harbor lower genetic diversity than those with fewer steps. As the initial releases occurred over 40 years ago, we hypothesized that despite originating from the same initial populations that most introduced populations would have diverged genetically from the native range and each other.Importation and release history were obtained from peer‐reviewed literature, government reports , shipment letters , unpublished quarantine records , and published quarantine records .
However, there were many gaps, as details in the importation and release history of biological control agents are often missing or not easily accessible to the public including the number of adults surviving shipments, the number used for mass‐rearing after quarantine inspection, the number ultimately released, the localities of the releases, and whether multiple releases occurred. From the shipment letters and quarantine reports pertaining to the initial exports from Argentina to USA: Florida , it appears that samples from at least two populations of N. bruchi and N. eichhorniae were collected from Argentina and released in USA: Florida. Initial shipments of N. bruchi received in 1974 to the USA: Florida quarantine consisted of 156 and 1,050 surviving adult weevils from collections in Campana Lagoon and Dique Lujan, Buenos Aires, Argentina, respectively. However, it is unclear whether or not individuals from Campana were used for mass rearing, based on notes about possible infections by nematodes. Additional shipments from these collection sites appear to have occurred around this same time, but it cannot be confirmed whether they were used for augmenting the populations that were eventually released. Samples from two populations of N. eichhorniae were collected and shipped in 1971, with the number of surviving adults arriving in the quarantine in USA: Florida documented as 10 from Campana Lagoon and 156 from Santa Fe, Argentina, with these collection sites c. 300 miles apart. An additional third population of N. eichhorniae may have been received, containing a mixture of 219 weevils from Campana and Dique Lujan Buenos Aires and arriving in 1975 . However, these reports indicated potential nematode and fungal infestation in this later shipment, and again it was not clear whether or not offspring from these weevils were included in augmentation of laboratory colonies or released. In 1980, following the quarantine and mass‐rearing periods in USA: Florida, 50 N. bruchi adults were released from USA: Florida in Wallisville Reservoir, Texas, USA . N. eichhorniae were found in this same reservoir as a consequence of westward migration from a biological control site in Louisiana .
In 1981, 500 adults of N. eichhorniae were imported from Louisiana populations and released in Wallisville, Texas . A total of 7,500 N. eichhorniae and 2,823 N. bruchi from the populations in Wallisville Texas were then released across four locations in the Sacramento–San Joaquin River Delta in California . All other importation data pertinent to this study are summa‐ rized in Figure 1 and further detailed in the Supporting Information Appendix S1.Potential microsatellite loci for N. bruchi and N. eichhorniae were identified using a Perl script, PAL_FINDER_v0.02.03 , and Primer3 to analyze 150‐bp paired‐end Illumina sequences from extracted DNA enriched for mi‐ crosatellite loci at the Savannah River Ecology Laboratory . From this, primers were designed for 48 loci, using only those with tri‐ and tetranucleotides and those with at least six repeats. For each species, the final loci for analysis were tested on DNA extractions from 24 adult weevils ranging across several collection sites. A set of 10 and 11 microsatellite loci for N. bruchi and N. eichhorniae, respectively, met the criteria of selection, that is, purerepeat, polymorphism, and amplification by PCR. Following amplifi‐ cation by PCR, eight and 10 loci, respectively , were kept for the statistical analysis due to the high occurrence of null alleles in two loci for N. bruchi and one locus in N. eichhorniae. After the initial screening, weed growing rack primers were combined in three multiplex reactions per individual for each species. For each 96‐well plate, we included a negative control and an internal control of aliquoted DNA from an individual weevil that was used on every plate for the respective species. PCR multiplex reactions were run separately for the two species to avoid cross‐contamination. Pig‐tails were added to the 5′ end of each reverse primer, and one of four different universal tails was added to the 5′ end of each forward primer . The system of universal tailed primers was used to introduce a fluorescent dye during the PCR according to Blacket et al., and Culley et al. . Initial single plex and subsequent multiplex PCRs were in a final volume of 10 μl containing 50–70 ng of DNA, 5 μl of Qiagen Multiplex PCR Master Mix, 0.2 μM of reverse primer, 0.05 μM of for‐ ward primer, and 0.2 μM of the corresponding fluorescent primer using fluorescence‐labeled oligos , and the addition of 2 μl of Qiagen Multiplex Q‐solution for several of the multiplex reactions . PCR was performed at the following conditions: 95°C for 15 min; 35 cycles of 94°C for 30 s, the optimum annealing temperature of each primer for 1.5 min, 72°C for 1 min, and a final extension of 30 min at 60°C. Following successful amplification, 0.5 μl of the amplified prod‐ uct was added to 11 μl of solution containing 10.5 μl Hi‐Di formamide and 0.5 μl Liz size standard. Fragment lengths were measured in comparison with the GeneScan™ LIZ® 600 Size Standard v. 2.0 and genotyped on an Applied Biosystems 3730XL DNA Analyzer at the DNA Sequencing Facility at the University of California Berkeley. Fragment lengths were manually scored and binned using the Microsatellite Plug‐in for Geneious Pro v. 5.6.2 . We re‐ran multiplex reactions and subsequently re‐genotyped samples if clear peaks were not obtained in the first run. Genotype scores were checked with the program MICRO‐ CHECKER v. 2.2.3 to identify possible null alleles and genotyping errors due to stuttering and large allele dropout .We re‐examined the relevant raw genotype data and either corrected the peak calls, or removed individuals that had poor quality peaks based on the recommenda‐ tions of MICRO‐CHECKER. Genotype scores from the two wee‐ vil species were divided into two datasets for each species as the microsatellite markers did not overlap for weevils with diagnostic morphological characteristics for N. bruchi and N. eichhorniae. The dataset for N. bruchi consisted of genotype scores for 171 weevils from eight independent collection sites among five countries. The second dataset for N. eichhorniae consisted of genotype scores for 267 weevils from 11 independent collection sites among seven countries . Final genotype scores for each individual, species, and collection site are in the Supporting Information Appendix S4. We used the program GenAlex and the R packages, “poppr” v. 2.5.0 and “adegenet” to convert genotyping results into formats suitable for analysis in R . We calculated the null allele frequency from the final datasets in the R package “pop‐ genreport” . As some statistical tests assume linkage equilibrium and Hardy–Weinberg equilibrium , we assessed deviations from LE with “poppr” and deviations from HWE across all sites for each locus with the package “pegas” . We constructed genotype accumulation curves with the R packages “poppr” and “vegan” to test whether sufficient sampling had been performed for each species and collection site.To evaluate whether co‐introduction of these two related weevils species resulted in hybridization, we first identified individuals for each species that had ambiguous markings on the elytra that contrasted the typical morphological characteristics for that species . Then, we tested both sets of species‐specific microsatel‐ lite markers on 12 weevils with ambiguous morphological characteristics, as well as on weevils that had the typical species‐specific morphological characteristics for comparison. Hybridization is inferred from at least two of the species‐specific markers from each species amplifying in the same individual .As bottlenecks in population size can reduce genetic heterozygosity through processes of genetic drift and inbreeding, we estimated the average observed and expected heterozygosity, deviations from HWE , and the average “inbreeding coefficient” for each collection site across all loci with the R package “diveRsity” . Here, we use FIS to estimate increases in ho‐ mozygosity due to genetic drift caused by a larger population being separated into sub‐populations, rather than due to consanguineous mating . Thus, we used “g2” to test for inbreeding within populations of each weevil species by using 1,000 permutations in the R package “InbreedR”. In populations with inbreeding, g2 is significantly greater than zero, indicating correlated heterozygosity among pairs of loci . We compared total and aver‐ age allelic richness and the number of private alleles among collection sites . To compare genetic diversity among the introduced and native populations, we tested for the effects of population on genetic diversity by fitting linear mixed models with the lmerfunction in the lme4 package . Implementing an LMM accounts for the variability of the mi‐ crosatellite loci by modeling locus as a random effect, and collection site as a fixed effect with allelic richness or expected heterozygosity as the response variables in separate models.