Tick Densities and Infection Prevalence on Coastal Islands in Massachusetts, USA: Establishing a Baseline.

Tick-borne diseases and a tick-induced red meat allergy have become increasingly common in the northeastern USA and elsewhere. At the scale of local communities, few studies have documented tick densities or infection levels to characterize current conditions and provide a baseline for further monitoring. Using the town of Nantucket, MA, as a case study, we recorded tick densities by drag sampling along hiking trails in nature preserves on two islands. Nymphal blacklegged ticks (Ixodes scapularis Say) were most abundant at shadier sites and least common in grasslands and scrub oak thickets (Quercus ilicifolia). Lone star ticks (Amblyomma americanum L.) were common on Tuckernuck Island and rare on Nantucket Island, while both tick species were more numerous in 2021 compared to 2020 and 2022. We tested for pathogens in blacklegged nymphs at five sites over two years. In 2020 and 2021, infection levels among the four Nantucket Island sites averaged 10% vs. 19% for Borrelia burgdorferi, 11% vs. 15% for Babesia microti, and 17% (both years) for Anaplasma phagocytophilum, while corresponding levels were significantly greater on Tuckernuck in 2021. Our site-specific, quantitative approach represents a practical example of how potential exposure to tick-borne diseases can be monitored on a local scale.

Target-site EPSPS Pro-106-Ser mutation in Conyza canadensis biotypes with extreme resistance to glyphosate in Ohio and Iowa, USA.

Documenting the diversity of mechanisms for herbicide resistance in agricultural weeds is helpful for understanding evolutionary processes that contribute to weed management problems. More than 40 species have evolved resistance to glyphosate, and at least 13 species have a target-site mutation at position 106 of EPSPS. In horseweed (Conyza canadensis), this p106 mutation has only been reported in Canada. Here, we sampled seeds from one plant (= biotype) at 24 sites in Ohio and 20 in Iowa, screened these biotypes for levels of resistance, and sequenced their DNA to detect the p106 mutation. Resistance categories were based on 80% survival at five glyphosate doses: S (0×), R1 (1×), R2 (8×), R3 (20×), or R4 (40×). The p106 mutation was not found in the19 biotypes scored as S, R1, or R2, while all 25 biotypes scored as R3 or R4 had the same proline-to-serine substitution at p106. These findings represent the first documented case of target-site mediated glyphosate resistance in horseweed in the United States, and the first to show that this mutation was associated with very strong resistance. We hypothesize that the p106 mutation has occurred multiple times in horseweed and may be spreading rapidly, further complicating weed management efforts.

No evidence for early fitness penalty in glyphosate-resistant biotypes of Conyza canadensis: Common garden experiments in the absence of glyphosate.

Strong selection from herbicides has led to the rapid evolution of herbicide-resistant weeds, greatly complicating weed management efforts worldwide. In particular, overreliance on glyphosate, the active ingredient in RoundUp®, has spurred the evolution of resistance to this herbicide in ≥40 species. Previously, we reported that Conyza canadensis (horseweed) has evolved extreme resistance to glyphosate, surviving at 40× the original 1× effective dosage. Here, we tested for underlying fitness effects of glyphosate resistance to better understand whether resistance could persist indefinitely in this self-pollinating, annual weed. We sampled seeds from a single maternal plant ("biotype") at each of 26 horseweed populations in Iowa, representing nine susceptible biotypes (S), eight with low-level resistance (LR), and nine with extreme resistance (ER). In 2016 and 2017, we compared early growth rates and bolting dates of these biotypes in common garden experiments at two sites near Ames, Iowa. Nested ANOVAs showed that, as a group, ER biotypes attained similar or larger rosette size after 6 weeks compared to S or LR biotypes, which were similar to each other in size. Also, ER biotypes bolted 1-2 weeks earlier than S or LR biotypes. These fitness-related traits also varied among biotypes within the same resistance category, and time to bolting was inversely correlated with rosette size across all biotypes. Disease symptoms affected 40% of all plants in 2016 and 78% in 2017, so we did not attempt to measure lifetime fecundity. In both years, the frequency of disease symptoms was greatest in S biotypes and similar in LR versus ER biotypes. Overall, our findings indicate there are no early growth penalty and possibly no lifetime fitness penalty associated with glyphosate resistance, including extremely strong resistance. We conclude that glyphosate resistance is likely to persist in horseweed populations, with or without continued selection pressure from exposure to glyphosate.

High Levels of Glyphosate Resistance in Conyza canadensis from Agricultural and Non-Agricultural Sites in Ohio and Iowa.

Glyphosate is an important herbicide worldwide, but its efficacy has been compromised where weed species have evolved glyphosate resistance. To better understand evolutionary outcomes of continued and strong selection from glyphosate exposure, we characterized variation in resistance in self-pollinating Conyza canadensis (horseweed) in Ohio and Iowa, where glyphosate resistance was first reported in 2002 and 2011, respectively. In 2015, we collected seeds from a total of 74 maternal plants (biotypes) from no-till soybean fields vs. non-agricultural sites in each state, using one representative plant per site. Young plants from each biotype were sprayed with glyphosate rates of 0x, 1x (840 g ae ha-1), 8x, 20x, or 40x. Resistant biotypes with at least 80% survival at each dosage were designated as R1 (1x), R2 (8x), R3 (20x), or R4 (40x). Nearly all Ohio agricultural biotypes were R4, as were 62% of biotypes from the non-agricultural sites. In Iowa, R4 biotypes were clustered in the southeastern soybean fields, where no-till agriculture is more common, and 45% of non-agricultural biotypes were R1-R4. Our results show that resistance levels to glyphosate can be very high (at least 40x) in both states, and that non-agricultural sites likely serve as a refuge for glyphosate-resistant biotypes.

Growth and fecundity of fertile Miscanthus × giganteus ("PowerCane") compared to feral and ornamental Miscanthus sinensis in a common garden experiment: Implications for invasion.

Perennial grasses are promising candidates for bioenergy crops, but species that can escape cultivation and establish self-sustaining naturalized populations (feral) may have the potential to become invasive. Fertile Miscanthus × giganteus, known as "PowerCane," is a new potential biofuel crop. Its parent species are ornamental, non-native Miscanthus species that establish feral populations and are sometimes invasive in the USA. As a first step toward assessing the potential for "PowerCane" to become invasive, we documented its growth and fecundity relative to one of its parent species (Miscanthus sinensis) in competition with native and invasive grasses in common garden experiments located in Columbus, Ohio and Ames, Iowa, within the targeted range of biofuel cultivation. We conducted a 2-year experiment to compare growth and reproduction among three Miscanthus biotypes-"PowerCane," ornamental M. sinensis, and feral M. sinensis-at two locations. Single Miscanthus plants were subjected to competition with a native grass (Panicum virgatum), a weedy grass (Bromus inermis), or no competition. Response variables were aboveground biomass, number of shoots, basal area, and seed set. In Iowa, all Miscanthus plants died after the first winter, which was unusually cold, so no further results are reported from the Iowa site. In Ohio, we found significant differences among biotypes in growth and fecundity, as well as significant effects of competition. Interactions between these treatments were not significant. "PowerCane" performed as well or better than ornamental or feral M. sinensis in vegetative traits, but had much lower seed production, perhaps due to pollen limitation. In general, ornamental M. sinensis performed somewhat better than feral M. sinensis. Our findings suggest that feral populations of "PowerCane" could become established adjacent to biofuel production areas. Fertile Miscanthus × giganteus should be studied further to assess its potential to spread via seed production in large, sexually compatible populations.

Effects of over-expressing a native gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) on glyphosate resistance in Arabidopsis thaliana.

Widespread overuse of the herbicide glyphosate, the active ingredient in RoundUp®, has led to the evolution of glyphosate-resistant weed biotypes, some of which persist by overproducing the herbicide's target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the shikimic acid pathway for biosynthesis of aromatic amino acids, lignin, and defensive compounds, but little is known about how overproducing EPSPS affects downstream metabolites, growth, or lifetime fitness in the absence of glyphosate. We are using Arabidopsis as a model system for investigating phenotypic effects of overproducing EPSPS, thereby avoiding confounding effects of genetic background or other mechanisms of herbicide resistance in agricultural weeds. Here, we report results from the first stage of this project. We designed a binary vector expressing a native EPSPS gene from Arabidopsis under control of the CaMV35S promoter (labelled OX, for over-expression). For both OX and the empty vector (labelled EV), we obtained nine independent T3 lines. Subsets of these lines were used to characterize glyphosate resistance in greenhouse experiments. Seven of the nine OX lines exhibited enhanced glyphosate resistance when compared to EV and wild-type control lines, and one of these was discarded due to severe deformities. The remaining six OX lines exhibited enhanced EPSPS gene expression and glyphosate resistance compared to controls. Glyphosate resistance was correlated with the degree of EPSPS over-expression for both vegetative and flowering plants, indicating that glyphosate resistance can be used as a surrogate for EPSPS expression levels in this system. These findings set the stage for examination of the effects of EPSPS over-expression on fitness-related traits in the absence of glyphosate. We invite other investigators to contact us if they wish to study gene expression, downstream metabolic effects, and other questions with these particular lines.

The potential for crop to wild hybridization in eggplant (Solanum melongena; Solanaceae) in southern India.

UNLABELLED: • PREMISE OF THE STUDY: In India and elsewhere, transgenic Bt eggplant (Solanum melongena) has been developed to reduce insect herbivore damage, but published studies of the potential for pollen-mediated, crop- to- wild gene flow are scant. This information is useful for risk assessments as well as in situ conservation strategies for wild germplasm.• METHODS: In 2010-2014, we surveyed 23 populations of wild/weedy eggplant (Solanum insanum; known as wild brinjal), carried out hand-pollination experiments, and observed pollinators to assess the potential for crop- to- wild gene flow in southern India.• KEY RESULTS: Wild brinjal is a spiny, low-growing perennial commonly found in disturbed sites such as roadsides, wastelands, and sparsely vegetated areas near villages and agricultural fields. Fourteen of the 23 wild populations in our study occurred within 0.5 km of cultivated brinjal and at least nine flowered in synchrony with the crop. Hand crosses between wild and cultivated brinjal resulted in seed set and viable F1 progeny. Wild brinjal flowers that were bagged to exclude pollinators did not set fruit, and fruit set from manual self-pollination was low. The exserted stigmas of wild brinjal are likely to promote outcrossing. The most effective pollinators appeared to be bees (Amegilla, Xylocopa, Nomia, and Heterotrigona spp.), which also were observed foraging for pollen on crop brinjal.• CONCLUSION: Our findings suggest that hybridization is possible between cultivated and wild brinjal in southern India. Thus, as part of the risk assessment process, we assume that transgenes from the crop could spread to wild brinjal populations that occur nearby.

Genetic diversity and population structure of wild/weedy eggplant (Solanum insanum, Solanaceae) in southern India: implications for conservation.

UNLABELLED: • PREMISE OF THE STUDY: Crop wild relatives represent important genetic resources for crop improvement and the preservation of native biodiversity. Eggplant (Solanum melongena), known as brinjal in India, ranks high among crops whose wild gene pools are underrepresented in ex situ collections and warrant urgent conservation. Knowledge of outcrossing rates and patterns of genetic variation among wild populations can aid in designing strategies for both in situ and ex situ preservation.• METHODS: We used 14 microsatellite (simple sequence repeat) markers to examine genetic diversity, population structure, and outcrossing in 10 natural populations of wild/weedy eggplant (S. insanum = S. melongena var. insanum) and three cultivated populations in southern India.• KEY RESULTS: Multilocus FST analyses revealed strong differentiation among populations and significant isolation by distance. Bayesian model-based clustering, principal coordinate analysis, and hierarchical cluster analysis grouped the wild/weedy populations into three major clusters, largely according to their geographic origin. The three crop populations were similar to each other and grouped with two wild/weedy populations that occurred nearby. Outcrossing rates among the wild/weedy populations ranged from 5-33%, indicating a variable mixed-mating system.• CONCLUSION: Geographic isolation has played a significant role in shaping the contemporary patterns of genetic differentiation among these populations, many of which represent excellent candidates for in situ conservation. In two cases, close genetic affinity between cultivars and nearby wild/weedy populations suggests that gene flow has occurred between them. To our knowledge, this is the first study investigating population-level patterns of genetic diversity in wild relatives of eggplant.

Fitness of crop-wild hybrid sunflower under competitive conditions: implications for crop-to-wild introgression.

Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle perspective. Additionally, due to effects of exogenous selection, a given hybrid generation may be especially well-suited to hastening introgression under particular environmental conditions.

A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.

Illegal gene flow from transgenic creeping bentgrass: the saga continues.

Ecologists have paid close attention to environmental effects that fitness-enhancing transgenes might have following crop-to-wild gene flow (e.g. Snow et al. 2003). For some crops, gene flow also can lead to legal problems,especially when government agencies have not approved transgenic events for unrestricted environmental release.Creeping bentgrass (Agrostis stolonifera), a common turf grass used in golf courses, is the focus of both areas of concern. In 2002, prior to expected deregulation (still pending), The Scotts Company planted creeping bentgrass with transgenic resistance to the herbicide glyphosate,also known as RoundUp, on 162 ha in a designated control area in central Oregon (Fig. 1).Despite efforts to restrict gene flow, wind-dispersed pollen carried transgenes to florets of local A. stolonifera and A. gigantea as far as 14 km away, and to sentinel plants placed as far as 21 km away (Watrud et al. 2004).Then, in August 2003, a strong wind event moved transgenic seeds from wind rows of cut bentgrass into nearby areas. The company's efforts to kill all transgenic survivors in the area failed: feral glyphosate-resistant populations of A. stolonifera were found by Reichman et al.(2006), and 62% of 585 bentgrass plants had the telltale CP4 EPSPS transgene in 2006 (Zapiola et al. 2008; Fig. 2).Now, in this issue, the story gets even more interesting as Zapiola & Mallory-Smith (2012) describe a transgenic,intergeneric hybrid produced on a feral, transgenic creeping bentgrass plant that received pollen from Polypogon monspeliensis (rabbitfoot grass). Their finding raises a host of new questions about the prevalence and fitness of intergeneric hybrids, as well as how to evaluate the full extent of gene flow from transgenic crops.

Hybridization alters early life-history traits and increases plant colonization success in a novel region.

Hybridization is hypothesized to promote invasiveness, but empirical tests comparing the performance of hybrid taxa versus parental taxa in novel regions are lacking. We experimentally compared colonization ability of populations of wild radish (Raphanus raphanistrum) with populations of advanced-generation hybrids between wild radish and cultivated radish (Raphanus sativus) in a southeast Texas pasture, well beyond the known invasive range of hybrid radish. We also manipulated the strength of interspecific competition to better generalize across variable environments. In both competitive environments, hybrid populations produced at least three times more seeds than did wild radish populations, a distinction that was driven by greater hybrid seedling emergence, earlier hybrid emergence, and more hybrid seedlings surviving to flower, rather than by greater individual fecundity. Flowering duration in hybrids was less negatively affected by competition than it was in wild radish, while early emergence was associated with subsequent high seed output in both biotypes. Our data show that hybridization can enhance colonization success in a novel region and, by comparison with previous studies, that the life-history traits enhancing hybrid success can differ across regions, even for lineages originating from the same hybridization event. These results imply a much larger arena for hybrid success than previously appreciated.

Selection on seedling emergence timing and size in an annual plant, Helianthus annuus (common sunflower, Asteraceae).

PREMISE: Variation in seedling emergence timing is considered adaptive over the long term in wild populations, but early emergence can result in a fitness advantage. To explore the adaptive significance of seedling emergence timing, it should be studied under realistic conditions and in the context of other traits that influence fitness. METHODS: In a common garden, we monitored maternal families from seed to flowering (including over winter) with intra- and interspecific competition. We assessed the effects of emergence timing and plant size on survival to anthesis in different genetic backgrounds and under varying competition. KEY RESULTS: We found genetic variation for emergence (probability and timing), size, and survival to anthesis. We also found negative selection, both phenotypic and genetic, on emergence time, such that early emergers (day 8) had almost twice as great a predicted probability of surviving as later emergers (day 28). Size had strong positive effects on survival and, furthermore, the beneficial effects of early emergence may be mediated through size. Maternal family and competitive environment can also affect selection on emergence timing. CONCLUSIONS: Our results indicate that early emergence is related to greater survival in wild sunflower, although there may be little direct selection on this trait; rather, its importance may be mediated by its effects on highly adaptive traits associated with size. Also, the effects of early emergence may vary across genetic backgrounds and competitive conditions, facilitating the maintenance of variation for this trait across a diverse landscape.

Transgenes for insect resistance reduce herbivory and enhance fecundity in advanced generations of crop-weed hybrids of rice.

Gene flow from transgenic crops allows novel traits to spread to sexually compatible weeds. Traits such as resistance to insects may enhance the fitness of weeds, but few studies have tested for these effects under natural field conditions. We created F 2 and F 3 crop-weed hybrid lineages of genetically engineered rice (Oryza sativa) using lines with two transgene constructs, cowpea trypsin inhibitor (CpTI) and a Bt transgene linked to CpTI (Bt/CpTI). Experiments conducted in Fuzhou, China, demonstrated that CpTI alone did not significantly affect fecundity, although it reduced herbivory. In contrast, under certain conditions, Bt/CpTI conferred up to 79% less insect damage and 47% greater fecundity relative to nontransgenic controls, and a 44% increase in fecundity relative to the weedy parent. A small fitness cost was detected in F 3 progeny with Bt/CpTI when grown under low insect pressure and direct competition with transgene-negative controls. We conclude that Bt/CpTI transgenes may introgress into co-occurring weedy rice populations and contribute to greater seed production when target insects are abundant. However, the net fitness benefits that are associated with Bt/CpTI could be ephemeral if insect pressure is lacking, for example, because of widespread planting of Bt cultivars that suppress target insect populations.

Species-specific SSR alleles for studies of hybrid cattails (Typha latifolia x T. angustifolia; Typhaceae) in North America.

PREMISE: Studies of hybridizing species are facilitated by the availability of species-specific molecular markers for identifying early- and later-generation hybrids. Cattails are a dominant feature of wetland communities, and a better understanding of the prevalence of hybrids is needed to assess the ecological and evolutionary effects of hybridization. Hybridization between Typha angustifolia and T. latifolia produce long-lived clones, known as Typha ×glauca, which are considered to be invasive. Although morphological variation in cattails makes it difficult to recognize early- and later-generation hybrids, several dominant, species-specific RAPD markers are available. Our goal was to find codominant, species-specific markers with greater polymorphism than RAPDs, to identify later-generation hybrids more efficiently. • METHODS: We screened nine SSR (simple sequence repeat) loci that were described from populations in Ukraine, and we surveyed 31 cattail populations from the upper Midwest and eastern USA. • KEY RESULTS: Seven SSR loci distinguished the parent taxa and were consistent with known species-specific RAPD markers, allowing easier detection of backcrossing. We used linear discriminant analysis to show that F(1) hybrid phenotypes were intermediate between the parent taxa, while those of backcrossed plants overlapped with the hybrids and their parents. Log(leaf length/leaf width), spike gap length, spike length, and stem diameter explained much of the variation among groups. • CONCLUSIONS: We provide the first documentation of backcrossed plants in hybridizing cattail populations in Michigan. The diagnostic SSR loci we identified should be extremely useful for examining the evolutionary and ecology interactions of hybridizing cattails in North America.

When divergent life histories hybridize: insights into adaptive life-history traits in an annual weed.

*Colonizing weed populations face novel selective environments, which may drive rapid shifts in life history. These shifts may be amplified when colonists are hybrids of species with divergent life histories. Selection on such phenotypically diverse hybrids may create highly fecund weeds. We measured the phenotypic variation, strength of natural selection and evolutionary response of hybrid and nonhybrid weeds. *We created F(1) hybrids of wild radish, an early flowering, small-stemmed weed, and its late-flowering, large-stemmed, crop relative (Raphanus spp.). Replicate wild and hybrid populations were established in an agricultural landscape in Michigan, USA. The consequences of three generations of natural selection were measured in a common garden experiment. *Hybrid populations experienced strong selection for larger, earlier flowering plants whereas selection was relatively weak on wild populations. Large plant size evolved two to three times faster in the hybrid populations than in wild populations, yet hybrid populations did not evolve earlier flowering. Strong selection on size and phenotypic correlations between age at reproduction and size may have limited the response of flowering phenology. *Our findings demonstrate hybridization between species with divergent life histories may catalyse the rapid evolution of certain adaptive, weedy traits while tradeoffs limit the evolution of others.