Introduction
Transgressive segregation refers to the phenomenon whereby the progeny of a diverse cross exhibit phenotypes that fall outside the range of the parents for a particular trait of interest (Mackay et al. Reference Mackay, Cockram, Howell and Powell2021). At the genetic level, transgressive segregation has often been ascribed to the dispersal of favorable alleles from the parents of a cross to its progeny (de Los Reyes Reference de Los Reyes2019; Mackay et al. Reference Mackay, Cockram, Howell and Powell2021; Rieseberg et al. Reference Rieseberg, Archer and Wayne1999). While transgressive segregation is often mentioned alongside the more widely discussed phenomenon of heterosis, the two are differentiated by the fact that heterosis is most evident in the F1 generation and, by definition, must also show directional dominance (Mackay et al. Reference Mackay, Cockram, Howell and Powell2021). In contrast, transgressive segregation is predominantly expressed in the F2 generation, with segregants that transcend the parental mean in either direction. Plant breeders have long taken advantage of transgressive segregation to select improved cultivars, with many studies reporting transgressive segregants for agronomically important traits of interest, including seed oil content (Alt et al. Reference Alt, Fehr, Welke and Shannon2005), pathogen or disease resistance (Winter et al. Reference Winter, Shelp, Anderson, Welacky and Rajcan2007), and grain yield (Vega and Frey Reference Vega and Frey1980).
In their review of transgressive segregation, Rieseberg et al. (Reference Rieseberg, Archer and Wayne1999) outlined several instances in which we would expect to see transgressive segregation frequently expressed. Based on the assumption that transgressive segregation is underpinned by complementary allelic action, the authors predicted that we should expect it to be most frequently observed in crosses between individuals from self-fertilized species and positively correlated with the genetic divergence of the parental biotypes. The authors also predicted that traits with a history of directional selection are less likely to exhibit transgressive segregation when compared with those that have undergone genetic drift or stabilizing selection. Since the publication of that review, a number of studies have explored these predictions as a framework for understanding how transgressive segregation might influence the processes of adaptation and speciation in a range of natural ecosystems (Bell and Travis Reference Bell and Travis2005; Lamichhaney et al. Reference Lamichhaney, Han, Webster, Andersson, Grant and Grant2018).
With the notable exception of plant breeders and geneticists, transgressive segregation has received comparably little attention in the agricultural literature. The field of weed science in particular would benefit from a deeper understanding of transgressive segregation and its implications for weed management, particularly with respect to crop–weed hybridization, invasive species, and the spread of herbicide resistance among and within populations (Campbell et al. Reference Campbell, Snow and Ridley2006; Clements and Jones Reference Clements and Jones2021; Jasieniuk et al. Reference Jasieniuk, Brûlé-Babel and Morrison1996). Of the few weed science studies that explicitly discussed transgressive segregation (Giacomini et al. Reference Giacomini, Westra and Ward2019; Liu et al. Reference Liu, Kenworthy, MacDonald, Unruh, Trenholm and Leon2019; Zelaya et al. Reference Zelaya, Owen and VanGessel2007), a study of its impact on fitness in slender wild oat (Avena barbata Pott ex Link) is perhaps the most detailed (Johansen-Morris and Latta Reference Johansen-Morris and Latta2006). Through their study of this highly self-fertilizing species, Johansen-Morris and Latta (Reference Johansen-Morris and Latta2006) demonstrated that single hybridization events between genetically divergent biotypes can result in a range of potential outcomes for the progeny, including hybrid vigor, hybrid breakdown, and transgressive segregation. Importantly, the results of this study demonstrated that, while later generations (i.e., F6) were on average less fit than the parents, the novel gene combinations produced resulted in segregants that could outperform the parental biotypes.
Like A. barbata, horseweed (Canada fleabane) [Erigeron canadensis L.; syn.: Conyza canadensis (L.) Cronquist] is a highly self-pollinating, winter annual weed species. It is one of the most widely distributed and problematic weed species throughout much of North America (Weaver Reference Weaver2001) and has evolved resistance to inhibitors of acetolactate synthase and enolpyruvylshikimate-3-phosphate synthase in multiple states and provinces in the United States and Canada (Heap Reference Heap2023; Smisek Reference Smisek1995; Weaver Reference Weaver2001). In a few regions in North America and abroad, E. canadensis has also evolved resistance to the active ingredient paraquat, a photosystem I electron diverter (Heap Reference Heap2023). These cases of resistance have often been associated with horticultural systems, such as a orchards and vineyards, where paraquat has been used for the nonselective control of weed species within and between rows of perennial crops (Moretti et al. Reference Moretti, Sosnoskie, Shrestha, Wright, Hembree, Jasieniuk and Hanson2016; Smisek et al. Reference Smisek, Doucet, Jones and Weaver1998; Yamasue et al. Reference Yamasue, Kamiyama, Hanioka and Kusanagi1992). At present, most of the evidence from studies of paraquat-resistant biotypes suggests that resistance is conferred by a single major gene mechanism that sequesters paraquat away from chloroplasts and into the vacuole (Hawkes Reference Hawkes2014). In contrast to the widespread reports of glyphosate resistance in this species (Beres et al. Reference Beres, Giese, Mackey, Owen, Page and Snow2020; Heap Reference Heap2023; Page et al. Reference Page, Grainger, Laforest, Nurse, Rajcan, Bae and Tardif2018), paraquat resistance is very much localized to a few regions within North America, specifically the U.S. states of California, Mississippi, Delaware, and Oregon and the Canadian province of Ontario.
In this study, we use paraquat-resistant biotypes of E. canadensis as a model system for exploring the role of transgressive segregation in the inheritance of herbicide resistance and the biomass characteristics that are often used in the evaluation of resistance. By examining resistant biotypes from California and Ontario, we address the question of whether similar resistance mechanisms have evolved in these geographically disparate biotypes. In addition, by assessing the progeny of reciprocal crosses between these two resistant biotypes and a susceptible biotype (also from Ontario), we explore the relative impact of genetic divergence and directional selection on the expression of transgressive segregation. Finally, we evaluate how the expression of transgressive segregation changes when the segregating progeny of these crosses are challenged with the original selective agent, paraquat.
Materials and Methods
Plant Material
Three biotypes of E. canadensis, two paraquat resistant (R1 and R2) and one susceptible (S), were selected as parents for reciprocal crossing and dose–response experiments described herein (Table 1). The S biotype was previously used to produce a chromosome-scale draft genome of E. canadensis (Laforest et al. Reference Laforest, Martin, Bisaillon, Soufiane, Meloche and Page2020), while the progenitor populations of the resistant biotypes have been characterized in previous studies (Moretti et al. Reference Moretti, Sosnoskie, Shrestha, Wright, Hembree, Jasieniuk and Hanson2016; Smisek et al. Reference Smisek, Doucet, Jones and Weaver1998).
Table 1. Origins of Erigeron canadensis biotypes.
Dose Response
A dose–response assay was conducted to confirm the response of the parental biotypes to paraquat (Gramoxone, 200 g ai L−1, Syngenta Canada, Guelph, ON, Canada). The experiment was established as a completely randomized design with 17 doses of paraquat (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1,600, 3,200, 6,400, 12,800 and 25,600 g ai ha−1) and four replicates and was repeated in time. The experimental unit consisted of four subsample rosettes of E. canadensis that were approximately 5 cm in diameter. Experimental units were sprayed in an enclosed automatic spray chamber calibrated to deliver 210 L ha−1 at 276 kPa through a stainless-steel even-spray nozzle (8002E SS), 40 cm above the plant canopy. At 14 d after treatment (DAT), the survival of each individual plant within a pot was recorded before harvesting of the aboveground biomass. Plants were considered to be alive if the apical meristem was green in color or if there was evidence of new growth. The four subsamples per pot were harvested and dried together in a forced-air dryer at 65 C for 7 d before being weighed.
Crossing
The three biotypes of E. canadensis (described in Table 1) were used as the parental material in the creation of reciprocal crosses. Seedlings of each biotype were propagated in a greenhouse at the Harrow Research and Development Center with a day/night thermoperiod of 25/20 C, respectively, and a 16-h photoperiod. Once rosettes reached approximately 5 cm in diameter, seedlings of biotypes R1 and R2 were sprayed with 200 g ai ha−1 paraquat in an enclosed spray chamber as previously described. Ten to 14 d later, seedlings of all three biotypes were placed into cold storage for 4 to 6 wk at 4 C to meet any vernalization requirements for the transition to reproductive growth (i.e., bolting). Once removed from cold storage, surviving seedlings were transplanted into larger pots and were left to grow to the reproductive stage in greenhouses under the same conditions as described earlier.
Leaf tissue was collected from each of the prospective parental plants before crossing began. A simple sequence repeat (SSR) marker (HW29) (Okada et al. Reference Okada, Hanson, Hembree, Peng, Shrestha, Stewart, Wright and Jasieniuk2015) was used to genotype each individual, with the parental biotypes known to produce PCR products of varying length (i.e., R1 = 148 to 160 bp, R2 = 190 to 195 bp, and S = 170 to 180 bp). DNA was extracted from approximately 20 mg of that leaf tissue using a Macherey-Nagel NucleoSpin Plant II kits (Macherey-Nagel, Bethlehem, PA, USA) following the manufacturer’s protocol. The PCR reaction cocktail contained the following: 10 μl of 2X Taq FroggaMix master mix, 7.6 μl nuclease-free H2O, 0.7 μl of 4 μM HW29 primer, 0.7 μl of 4 μM EU47 primer, and 1.0 μl (10 ng μl−1) DNA for a total reaction volume of 20 μl. Amplification was performed with the following cycling profile: denaturation: 94 C for 5 min, followed by 35 cycles of 30 s at 94 C; annealing: 53 C for 45 s and 72 C for 45 s, followed by an extension of 10 min at 72 C. PCR products were visualized on a 2.5% agarose gel containing 5% nucleic acid staining solution (RedSafe, FroggaBio, Toronto, ON, Canada) along with a 100-bp DNA ladder (FroggaBio). Only individuals shown to be homozygous with HW29 (i.e., those with only a single band) and with the proper product size for a given parental biotype were used in subsequent crosses.
The crossing methods used in the current study closely follow those outlined by Zelaya et al. (Reference Zelaya, Owen and VanGessel2004). In brief, each cross was initiated by selecting pairs of individuals from the desired set of parental biotypes whose floral initiation was in close synchrony. As noted by Zelaya et al. (Reference Zelaya, Owen and VanGessel2004) and Weaver (Reference Weaver2001), E. canadensis capitula (inflorescences) contain a pistillate ray and perfect disk florets (flowers) (Figure 1). Controlled crossing between individuals is thus achieved by the removal of the disk florets (i.e., emasculation; Figure 1D) using forceps and the transfer of the desired pollen to the ray florets. To assess the efficiency of our emasculations, a capitulum was selected at the top of each plant to serve as a negative control (i.e., was emasculated and covered to prevent outcrossing). Capitula used for crossing were selected at random and emasculated when they reached the appropriate stage of development (Figure 1A). The remaining ray florets were cross-pollinated once a day, every day for 7 to 10 d until the capitula closed, indicating the onset of seed maturation. Reciprocal pollen transfer was achieved by emasculating donor capitula from an individual of the desired parental biotype and brushing these mature perfect disk florets (Figure 1B and 1C) on the ray florets of the emasculated recipient capitulum.
Figure 1. Stages of capitulum development in Erigeron canadensis. (A) The capitulum containing both disk and ray florets at the appropriate stage for emasculation. (B and C) Intact capitulum with mature disk florets for use as pollen donors. (D) The capitulum post-emasculation (i.e., disk florets removed and ray florets remaining).
The number of F1 achenes (hereafter referred to as seeds) produced by each cross ranged from 1 to 20. When mature, F1 seeds were harvested, they were immediately set to germinate in an incubator. Seeds were placed into a petri dish lined with moist blue blotter paper (steel-blue germination blotters, Anchor Paper, St Paul, MN, USA) and incubated under the following conditions: a 25/10 C day/night thermoperiod, 60% relative humidity, and a 14-h photoperiod. Once germinated, seedlings were transplanted and grown in a greenhouse under the same conditions as previously described. All seedlings were genotyped with the HW29 SSR marker (as described earlier), and only those demonstrated to be heterozygous (i.e., two bands), with bands corresponding to the appropriate parental biotypes, were retained. All heterozygous F1 individuals were once again cold acclimated to accelerate bolting. The progeny from separate crosses were segregated by greenhouse compartment, and all individuals were covered with DelNet pollination bags (DelStar Technologies, Austin, TX, USA) before flowering to ensure self-pollination and facilitate the collection of the F2 seed. Seeds from each plant were kept as separate F2 families.
Inheritance of Paraquat Resistance
The F2 progeny arising from the reciprocal crosses among the three parental biotypes were screened at discriminating doses (i.e., the lowest dose that provides 100% mortality of the most susceptible parent in a specific cross based on dose–response survival curves; see section above). These doses were 400 g ai ha−1 for the S × R2 cross and 12, 800 g ai ha−1 and for the R1 × R2 cross. Seedlings of the F2 generation and their parental biotypes were propagated in greenhouse plug flats under the conditions previously described. Once the rosettes reached 5 cm in diameter, experimental units were created by transplanting rosettes as plugs in a new flat that contained an individual plant of each parent for a given cross and an F2 individual produced from each of the reciprocal crosses between these parental biotypes (i.e., 6 or 8 F2 rosettes + 2 parental rosettes per tray). For example, for the R2 × S cross, there were six F2 families created, and 33 replicates were screened at the discriminating dose, resulting in a total of 198 F2 individuals. At 21 DAT, the survival of the rosettes was recorded. The phenotype of surviving F2 individuals closely resembled that of one or the other parental resistant biotypes; no intermediate phenotypes were observed. The aboveground biomass of all individuals surviving at discriminating doses was harvested, and samples were dried in a forced air dryer at 65 C for 7 d before biomass was recorded.
Seed Viability
While crosses between the R1 and S parental biotypes successfully produced an F1 generation, the self-pollination of these F1 consistently failed to produce germinable F2 progeny. When seeds were examined under a microscope at 100× magnification, a noticeable difference in seed integrity was observed between known viable seed and the seeds from these crosses (HH, personal observation). A tetrazolium chloride assay was subsequently used to examine the viability of parental and F2 seed (Peters and Lanham Reference Peters and Lanham2000). Fifty seeds of each F2 family and parental biotype were counted and placed into individual petri plates; there were four replicates of each. A 10-ml volume of a 10 g/L solution of tetrazolium chloride was added, and plates were placed in a growth cabinet at 30 C in complete darkness. After 24 h, the petri plates were removed from the growth cabinet, and seed viability was rated under a dissecting scope.
Biomass Accumulation under Unsprayed Conditions
Seedlings of the three parental biotypes and the two successful F2 generations were propagated under greenhouse conditions as described earlier. Fifty individuals of each F2 family and 100 individuals of each parental biotype were propagated to a size like that utilized in the dose–response and inheritance studies described earlier (i.e., to approximately 5 cm in diameter). At 4 wk after emergence, the aboveground biomass was harvested and dried in a forced-air dryer at 65 C for 7 d before biomass was recorded.
Statistical Analyses
The parental dose–response was conducted as completely randomized design with four replications and two repetitions in time. Survival and aboveground biomass data were used for dose–response analyses using PROC NLIN in SAS v. 9.4 (SAS Institute, Cary, NC, USA). Data were fit to a log-logistic model (Equation 1) (Seefeldt et al. Reference Seefeldt, Jensen and Fuerst1995), where D is the upper response limit bounded at ≤100; C is the lower response limit; LD50 and GR50 are the herbicide doses that result in 50% reduction in survival and aboveground biomass, respectively; and b is the slope at the inflection point.
$$f\left( x \right) = C + {{D - C} \over {1 + {\rm{exp\{ }}b[{\rm{log}}\;(x)\; - {\rm{log}}\;({\rm{L}}{{\rm{D}}_{50}})]\} }}$$
At each discriminating dose, segregation ratios in the F2 generation were analyzed by χ2 test (Hayes and Immer Reference Hayes and Immer1942). The χ2 test for homogeneity was performed to determine whether segregation data could be combined across families. Biomass accumulation of parental and F2 families sprayed with a discriminating dose or from unsprayed conditions were analyzed with ANOVAs. For the unsprayed dataset, a one-way ANOVA was conducted in PROC MIXED with biotype or family as a fixed effect and replicate as a random effect. For the R1 × R2 cross, the biomass accumulation at the discriminating dose was analyzed in a similar manner. In the S × R2 cross, however, the S parent was completely controlled at the discriminating dose. Thus, only the biomass from the R2 parent and the F2 generation were included in the analysis. Finally, to assess the impact of inheritance of the resistance trait on the expression of transgressive segregation, the ANOVA for biomass accumulation at each discriminating dose was repeated using datasets that included only survivors.
Results and Discussion
Response of Parental Biotypes to Paraquat
The phenotypic response of the two resistant biotypes (R1 and R2) to paraquat was notably different. After treatment with paraquat, the older leaf tissue of R2 individuals became necrotic within days, while the young leaves and the apical meristem remained green and continued to produce new tissue (Figure 2). Individuals of the R1 biotype, however, displayed no visual herbicide symptomology. The level of resistance also varied among biotypes, as evidenced by their respective LD50 values (Figure 3). The dose of paraquat required to provide 50% control ranged from 10,749 g ai ha−1 for R1 to 3,511 and 73 g ai ha−1 for R2 and S, respectively. Based on these results, the R1 biotype exhibited a resistance factor of 148-fold while the R2 biotype exhibited a resistance factor of 48-fold, relative to our S control. The response of aboveground biomass was similar to that measured for survival across the studied biotypes (Figure 4). The GR50 of the three biotypes ranged from 832 g ai ha−1 for R1 to 56 and 13 g ai ha−1 for R2 and S, respectively.
Figure 2. Parental resistant biotype (R2) of Erigeron canadensis treated with 1,600 g ai ha −1 paraquat, pictured 24 h after treatment (A) and 14 d after treatment (B).
Figure 3. Survival of three biotypes of Erigeron canadensis (S: triangles, dashed and dotted line; R2: squares, dashed line; R1: circles, solid line) as influenced by paraquat dose. Data points represent the mean survivorship of four plants per experimental unit at 14 d after treatment. Horizontal error bars represent the 95% confidence interval at LD50. Vertical error bars represent the standard error of the mean. A four-parameter log-logistic equation (f(x) = C + (D − C)/1 + exp[b(log x) – log (LD50)]) was fit to R1 (C = 0, D = 99, LD50 = 10,749, b = 3.3), R2 (C = 0, D = 197, LD50 = 3,511, b = 2.06), and S (C = 0, D = 98, LD50 = 73, b = 3.97).
Figure 4. Dose response of three biotypes of Erigeron canadensis (S: triangles, dashed and dotted line; R2: squares, dashed line; R1: circles, solid line) as influenced by paraquat dose. Data points represent the mean biomass of four plants per experimental unit at 14 d after treatment. Horizontal error bars represent the 95% confidence interval at GR50. Vertical error bars represent the standard error of the mean. Dose–response curves were generated via nonlinear regression analysis. A four-parameter log-logistic equation (f(x) = C + (D − C)/1 + exp[b(log x) – log (GR50))]) was fit to R1 (C = 38, D = 104, GR50 = 832, b = 1.3), R2 (C = 35, D = 101, GR50 = 55.8, b = 1.13), and S (C = 7, D = 97, GR50 = 12.9, b = 1.1).
Inheritance of Paraquat Resistance
In this study, we examined the inheritance of paraquat resistance in segregating F2 generations created from the reciprocal crosses of two known paraquat-resistant biotypes and a paraquat-susceptible biotype (Table 1). The self-fertilization of F1 plants from these three reciprocal crosses all produced F2 progeny; however, those of the S × R1 cross were uniformly nonviable. This cross was repeated twice in time, producing a total of 21 F2 families, all of which were nonviable. Results of a tetrazolium chloride assay indicated that 3 of the F2 families from the cross of S × R1 had ≤1% viable seed, whereas the other 18 had no viable seed (Figure 5). In contrast, all parental biotypes germinated consistently, and results of the tetrazolium assay indicated that their seed lots contained 64% to 68% viable seeds (data not shown).
Figure 5. Achenes of Erigeron canadensis at 24 h after treatment with a 1% tetrazolium chloride solution. Top row, left to right are as follows: R1, S, and R2. Second row shows representative achenes from three F2 families arising from the cross of S × R1. Achenes of E. canadensis are on average 1- to 2-mm long (Weaver Reference Weaver2001).
Segregating F2 generations were successfully created from the reciprocal crosses between S and R2 and R1 and R2, and these were examined at appropriate discriminating doses based on the dose responses of their respective parental biotypes (Table 1; Figure 3). At a paraquat dose of 400 g ai ha−1, the survival of F2 individuals arising from the cross of S × R2 approached 67% (133/198) (Table 2). The χ2 test of the pooled F2 indicated that these results deviated from the 3:1 ratio expected under the assumption of monogenic inheritance of the paraquat resistance trait. Similarly, at a paraquat dose of 12,800 g ai ha−1, survival in the F2 progeny of the R1 × R2 cross approached 61% (118/192), and the χ2 test of the pooled F2 also indicated that these results deviated from the expected 3:1 ratio (Table 3). These results differ from previous studies of paraquat resistance in E. canadensis, in which segregation ratios of 3:1 (R:S) were reported (Smisek Reference Smisek1995; Yamasue et al. Reference Yamasue, Kamiyama, Hanioka and Kusanagi1992). When tested against several digenic ratios, results from S × R2 cross fit an 11:5 ratio, while the results from the R1 × R2 did not fit any of the tested ratios (Table 4). A digenic ratio of 11:5 was similarly observed by Okada and Jasieniuk (Reference Okada and Jasieniuk2014) in their study of glyphosate resistance in a Californian biotype of hairy fleabane [Erigeron bonariensis L.; syn: Conyza bonariensis (L.) Cronquist], a species closely related to E. canadensis. In this two-locus model, resistance alleles work additively across loci, and at least two doses of the resistance allele are required to produce the resistant phenotype.
Table 2. Segregation of resistance in F2 families from crosses between Erigeron canadensis biotypes S and R2 at 21 d after treatment with paraquat at 400 g ai ha−1.
a Within each cross, the first parental biotype listed was the pollen donor, and the second was the pollen recipient. Families sharing a letter represent crosses between the same two parental individuals.
Table 3. Segregation of resistance in F2 families from crosses between Erigeron canadensis biotypes R1 and R2 at 21 d after treatment with paraquat at 12,800 g ai ha−1.
a Within each cross, the first parental biotype listed was the pollen donor, and the second was the pollen recipient. Families sharing a letter represent crosses between the same two parental individuals.
Table 4. Segregation of paraquat resistance in F2 populations and expected ratios under four two-locus models and the P-values from χ2 tests for goodness of fit.
Herbicide Resistance and Transgressive Segregation
When the F2 generations created in this study were characterized with respect to their biomass accumulation, it was clear that the mean and range of individual sizes in the F2 generation exceeded those observed in either parental biotype (Figure 6). In the S × R2 cross, for example, not only did the mean biomass accumulation in the F2 exceed that of the parental biotypes by 51% and 63%, respectively, but the range of observed values was nearly double that for either parent. Results for the R1 × R2 cross were nearly identical, with the mean aboveground biomass of the F2 exceeding that of either parent by 50% and 52%, respectively, which indicated a doubling of the range for the trait compared with the parents.
Figure 6. Aboveground biomass of Erigeron canadensis parental biotypes (S, R1, and R2) and the F2 progeny of their successful crosses in the absence of paraquat. Mean values bearing the same letters are not significantly different at P < 0.05 according to Tukey’s honestly significant different test.
When the segregating F2 generations and their parental biotypes were sprayed with discriminating doses of paraquat, there were notable differences in the means and ranges of biomass accumulation after application (Figures 7 and 8). In the S × R2 cross, the selected dose was perfectly discriminatory, and there was no difference in the mean aboveground biomass of the F2 and the resistant parent of the cross (Figure 7A). There was, however, a notable difference in the range of aboveground biomass values in the F2 generation, which exceeded the observed range in the R2 parent by 58%. This result was comparable to the transgressive segregation in biomass accumulation observed under unsprayed conditions (Figure 6). When only the surviving individuals are considered (i.e., those with the resistance trait), the mean aboveground biomass of the F2 generation was 52% greater than that of the R2 parent at 21 DAT (i.e., 97 vs. 64 g plant−1), and the range of values exhibited by the F2 was 61% greater than that observed in R2, further emphasizing the expression of transgressive segregation in the resistant members of the F2 generation (Figure 7B).
Figure 7. Aboveground biomass of Erigeron canadensis parental biotypes S and R2 and their F2 progeny at 21 d after application of paraquat at 400 g ai ha−1. (A) All individuals; (B) only survivors. Mean values within a panel bearing the same letters are not significantly different at P < 0.05 according to Tukey’s honestly significant different test.
Figure 8. Aboveground biomass of Erigeron canadensis parental biotypes R1 and R2 and their F2 progeny at 21 d after application of paraquat at 400 g ai ha−1. (A) All individuals; (B) only survivors. Mean values within a panel bearing the same letters are not significantly different at P < 0.05 according to Tukey’s honestly significant different test.
For the R1 × R2 cross, the selected dose was not completely discriminatory, and this reflected a difficulty in selecting an appropriate dose, given that both parental biotypes exhibited some degree of resistance to paraquat (Figure 3). The biomass accumulation at 21 DAA varied among the F2 and the parental biotypes, such that individuals of the more-resistant parent (i.e., R1) were on average 2.5 and 12 times larger than the F2 and the less-resistant parent (i.e., R2), respectively. In contrast to the results from the S × R2 cross (discussed earlier), the mean and range of biomass accumulation in the F2 generation were intermediate to the two parental resistant biotypes, and there was no evidence for transgressive segregation (Figure 8A). These conclusions did not change when only the surviving individuals were considered (Figure 8B).
Results of the current study highlight three potential outcomes arising from crosses among resistant and susceptible biotypes of E. canadensis: (1) hybrid sterility, (2) transgressive segregation, and (2) inheritance intermediate to parents exhibiting contrasting phenotypes. At present, it is unclear why the hybrid of a susceptible biotype from Ontario and a resistant biotype from California repeatedly failed to produce viable seed. It is possible that gametophytic incompatibility between the genomes of the S and R1 parental biotypes during F1 self-pollination resulted in pollen inviability or in the incomplete development of the zygote (McClure and Franklin-Tong Reference McClure and Franklin-Tong2006; Newbigin et al. Reference Newbigin, Anderson and Clarke1993; Ouyang et al. Reference Ouyang, Liu and Zhang2010). Interestingly, hybrid sterility was not observed in the hybrid of the two resistant biotypes, also originating from California and Ontario. The latter suggests that the observation of hybrid sterility was specific to the combination of parental linages used in the cross and did not reflect broader geographically based incompatibility (Baack et al. Reference Baack, Melo, Rieseberg and Ortiz-Barrientos2015). Further research is required to explore the frequency of this phenomenon in E. canadensis in order to understand its role in shaping intraspecific population dynamics.
Erigeron canadensis is considered to be a highly self-fertilizing species with outcrossing rates ranging between 2% and 13% (Smisek Reference Smisek1995). We would therefore anticipate transgressive segregation to be expressed more frequently in E. canadensis than, for example, in a highly outcrossing species such as common ragweed (Ambrosia artemisiifolia L.) (Friedman and Barrett Reference Friedman and Barrett2008; Rieseberg et al. Reference Rieseberg, Archer and Wayne1999). Our results clearly support this prediction, with all successful crosses exhibiting transgressive segregation in biomass accumulation. In their review of transgressive segregation, adaptation, and speciation, Rieseberg et al. (Reference Rieseberg, Archer and Wayne1999) also predicted that transgressive segregation would be: (1) positively correlated with genetic divergence of the parental lineages and (2) less likely to be observed in lineages with a shared history of directional selection. Results of our study mostly agree with these two predictions; however, our results show conclusions may not be as straightforward depending on the parents’ histories.
Based on geographic distance alone, we anticipate a greater potential for transgressive segregation in the progeny of R1 and R2 than from S and R2 (Table 1). The geographic distance between R1 and R2 approaches 3,500 km and spans a continental divide, whereas S and R2 are only separated by approximately 300 km (Table 1). For most species, this later distance would still represent a significant barrier to gene flow between the regions, yet propagules of E. canadensis have been observed to disperse up to 500 km in a single dispersal event when traveling in the planetary boundary layer (Shields et al. Reference Shields, Dauer, VanGessel and Neumann2006). While this propensity for long-distance dispersal raises the potential for gene flow between S and R2, it is also counterbalanced by the low frequency for outcrossing for plants in close proximity (Smisek et al. Reference Smisek, Doucet, Jones and Weaver1998) and the observation that the vast majority (i.e., >99%) of propagules disperse within 100 m of the parent plant (Dauer et al. Reference Dauer, Mortensen and Vangessel2007). The observation of transgressive segregation in segregating generations of both crosses (in the absence of paraquat) suggests that there was genetic diversity among the parental biotypes studied and this resulted in novel allelic combinations in the progeny.
The geographic distance between biotypes R1 and R2 belies the fact that they share a history of selection with the herbicide paraquat. Both biotypes originated in orchard production systems, in California and Ontario, respectively, where paraquat was applied for weed control multiple times per growing season. It is clear from our dose–response data that, while both biotypes are resistant to paraquat, the Californian biotype (R1) is approximately three times more resistant than the Ontarian biotype (R2). Our results also suggested that the traits conferring resistance in these biotypes were both polygenic, although it remains unclear whether they result from the same molecular mechanism(s). Given this shared history of selection with paraquat, it is plausible to hypothesize that a similar resistance mechanism may have been selected in these biotypes, resulting in a reduction of diversity in the genomic loci where genes involved in resistance are found as well as other genes that are in linkage disequilibrium. This fixation of alleles is predicted to decrease the expression of transgressive segregation in the progeny of lineages that share a history of directional selection (Rieseberg et al. Reference Rieseberg, Archer and Wayne1999). The current study provided a test of such hypothesis, both in the absence and presence of the original selective agent (i.e., paraquat).
In the absence of paraquat, our results clearly refute the prediction that transgressive segregation would be reduced in the progeny of biotypes with a shared history of directional selection. Transgressive segregation not only was evident, but was of similar magnitude in the segregating progeny of the S × R2 and R1 × R2 crosses. However, when paraquat was applied to these same segregating generations, transgressive segregation was absent from the progeny of R1 × R2. While these results (with and without the original selective agent) are seemingly contradictory, we contend that they are in fact supportive of the original prediction of Rieseberg et al. (Reference Rieseberg, Archer and Wayne1999) and are underpinned by the diversity of complementary alleles among biotypes, the number of loci involved, the gene expression under selective agent and without it, the intensity of selection pressure, and fixation of alleles following selection. When the progeny of the R1 × R2 cross were challenged with paraquat, it was at a discriminating dose that was selected to eliminate the R2 biotype. As a result, survivors would have to possess the complete resistance from R1 and/or alleles from R2 that were common to the R1 resistance mechanism. It is also important to note that the resistance in R2 provided less protection than that in R1 and thus may not have reached fixation at all the loci contributing to paraquat resistance in this species. Therefore, we hypothesize that the expression of transgressive segregation in the R1 × R2 progeny in the absence of paraquat stems from the fact that there remain allelic differences between the parental biotypes at the loci conferring paraquat resistance. It is only when paraquat is applied that we can truly see the effects of selection on the fixation of alleles and the subsequent impact on transgressive segregation.
In summary, transgressive segregation in life-history traits contributing to survival and reproduction can play an important role in determining fitness. Segregants that exceed the parental values in such critical traits may represent beneficial new gene combinations that are fitter than their respective resistant parental genotypes. The relative fitness of a particular genotype, however, depends on the environment to which it is exposed (Leon et al. Reference Leon, Dunne and Gould2021), such that transgressive segregants in one environment may not necessarily exceed the parental genotypes in another (Johansen-Morris and Latta Reference Johansen-Morris and Latta2006). Hybridization among diverse biotypes of E. canadensis clearly produces transgressive segregants; a result that supports the prediction that transgressive segregation is frequently expressed in self-fertilizing lineages and is positively correlated with the genetic diversity of the parental genotypes (Rieseberg et al. Reference Rieseberg, Archer and Wayne1999). When these hybrid progenies were exposed to a new environment, one in which past agents of directional selection were not present, segregants were observed in the F2 generation regardless of parental identity or history. In this environment, which would be akin to a year where herbicide rotation was practiced, we would predict that many of the recombinant genotypes would be fitter than either of the parental biotypes. Conversely, if these hybrid progenies were returned to the parental environment with exposure to paraquat, the identity of fittest genotype (i.e., parent or segregant) would depend on the history of directional selection in the parental lineages and the dose to which the hybrid progeny was exposed. When both parental lineages share the same history of directional selection, the likelihood of transgressive segregation is reduced; however, its expression in this case depends on the strength of selection exerted on the progeny.
Acknowledgments
The authors gratefully acknowledge Brad Hanson and Peter Sikkema for their contribution of E. canadensis seed.
Funding statement
The funding for this project was provided by Agriculture and Agri-Food Canada (project J-001751) and (project J-002275).
Competing interests
The authors declare no conflicts of interest.
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