In 2013, mature P. hysterophorus seeds were collected from plants not controlled with glyphosate at doses normally used (2 L ha⁻¹; 720 g ae ha⁻¹) in areas with perennial crops in two Caribbean Islands. Seeds from Cu-S and Do-S accessions never exposed to glyphosate were collected from adjacent areas and used as a reference control (Table 1). Seeds collected from 25 mature plants were stored under laboratory conditions (25°C) for 2 weeks and then placed in paper bags at 4°C. Approximately 300 seeds of these accessions were sown directly into trays (40 × 60 × 15 cm), containing a mixture of sand and peat (2:1, v/v) and placed in a greenhouse at 28/20°C day/night under a 16 h photoperiod with 850 μmol m⁻² s⁻¹ photon flux density, and 80% relative humidity. At the four leaf stage plants of all accessions were treated with glyphosate at 720 g ae ha⁻¹ using a laboratory spray chamber equipped with a flat fan nozzle (TeeJet 8002 EVS) with a total output volume of 200 L ha⁻¹ water at a pressure of 200 kPa. Four weeks after glyphosate treatment plant survival of the resistant accessions was estimated, and seed produced from surviving plants was collected and stored in paper bags for all subsequent trials. In the case of susceptible accessions (Cu-S and Do-S), no plant survival was observed 4 weeks after glyphosate treatment.

Seeds of putative resistant (Cu-R1, Cu-R2, Cu-R3, Do-R1, Do-R2, Do-R3, and Do-R4) and susceptible (Cu-S and Do-S) of the P. hysterophorus accessions were germinated in trays (12 × 12 × 6 cm) containing the same substrate as described before and placed in a growth chamber of similar environmental conditions controlled as before. One week after germination, individual seedlings were transplanted into pots (6 × 6 × 8 cm) and grown under fluctuating 30/20°C day/night with a 14 h photoperiod and 850 μmol m⁻² s⁻¹ photon flux density, and 80% relative humidity. As glyphosate (EPSPS inhibitor) is used in early post-emergence, at the four leaf stage, resistant and susceptible P. hysterophorus seedlings were treated with increasing glyphosate doses: 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 g ae ha⁻¹ (Roundup Energy 45% w/v, SL, Monsanto Spain). The experiment were conducted with 10 replications (one plant pot⁻¹) of each accession per herbicide dose, and the experiments were repeated twice. Thirty days after herbicide treatment, herbicide effects on plant survival (LD) and above-ground vegetative biomass (GR) were assessed.

Leaf segments (50 mm diameter) were harvested from the youngest fully expanded leaf from a batch of 15 plants per P. hysterophorus accessions at the 4–6 leaf stage (Hanson et al., 2009). Approximately 50 mg of fresh tissue was transferred to 2 mL Eppendorf tubes containing 1 mL of 1 mM NH₄H₂PO₄ (pH 4.4). Glyphosate was added to the tubes at the following concentrations: 0, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, 500, 600, and 1000 μM. The Eppendorf tubes were incubated in a growth chamber during 24 h under the previously described conditions. After 24 h, the tubes were stored at −20°C until analysis. Eppendorf tubes were removed from the freezer and thawed at 60°C for 30 min. Two hundred and fifty micro liters of 1.25 N HCL was added to each tube, and placed at 60°C for 15 min. A 125 μL aliquot from each tube was pipetted into a new 2 mL Eppendorf tube, and 500 μL of periodic acid and sodium metaperiodate (0.25% [wt/v] each) was added. They were incubated at room temperature for 90 min, after which 500 μL of 0.6 N sodium hydroxide and 0.22 M sodium sulfite was added. The contents of all tubes were transferred to glass vials. Samples were measured in a spectrophotometer at 380 nm within 30 min. For each glyphosate concentration and accession, three replications were stablished and repeated twice.

Absorption and translocation study was carried out following the methodology proposed by Cruz-Hipólito et al. (2011) The ¹⁴C-glyphosate was mixed with commercially formulated glyphosate to prepare a solution with a specific activity of 0.834 kBq μL⁻¹ and a glyphosate concentration of 1.8 g ae L⁻¹ (360 g ae ha⁻¹ in 200 L). P. hysterophorus plants at 4-leaf stage were treated with the radiolabeled herbicide by applying one droplet of 1 μL of glyphosate solution (0.834 kBq μL⁻¹) on the adaxial surface of the second leaf in each plant using a micropipette (LabMate). The ¹⁴C-glyphosate unabsorbed in the treated leaf was removed with 3 mL of water: acetone solution (1:1, v/v) 96 h after droplet application. Preliminary assays with two accessions (Cu-R1 and Cu-S) studied had revealed that the glyphosate absorption leveled-off at 96 h after the droplet applications. The rinsate was mixed with 2 mL of scintillation liquid and analyzed by liquid scintillation spectrometry (LSS) (Scintillation Counter, Beckman LS 6500, Fullerton CA). The plants were separated into the treated leaf, rest of the shoot and root after being placed in cellulose cones. The plant tissue was dried at 60°C over 96 h and combusted in a biological sample oxidizer (Packard Tri Carb 307, Perkin-Elmer, Waltham, MA). The ¹⁴CO₂ evolved was trapped and counted in 18 mL of a mixture of Carbo-Sarb E and Permafluor (9:9, v/v) (Perkin-Elmer). Thus, over 95% of the total radioactivity applied was recovered. There were five replications and the experiment was arranged in a completely randomized design, and repeated twice. The proportion of absorbed herbicide was expressed as:

P. hysterophorus plants were treated with a glyphosate rate of 360 g ae ha⁻¹ at 4–6 leaf stage. At 96 h after treatment (HAT), glyphosate and its metabolites, i.e., AMPA (aminomethylphosphonic acid), glyoxylate and sarcosine, were determined by reversed-polarity capillary electrophoresis following the methodology described by Rojano-Delgado et al. (2010). The calibration equations were established using non-treated plants and known concentrations of glyphosate and its metabolites, which were determined from their enclosed areas under the peaks in the electropherogram. The average value for the amount of glyoxylate naturally produced by the plant was subtracted from the average of the produced or reduced amount after treatment of each accession (Rojano-Delgado et al., 2010). The experiment was arranged in a completely randomized design with four replications per accession and repeated three times.

The enzyme extraction was conducted according to the protocol described by Dayan et al. (2015). Five gram of the leaf tissue of all P. hysterophorus accessions (Table 1) were ground to fine powder in a chilled mortar. Immediately after that, the powdered tissue was transferred to tubes containing 100 mL of cold extraction buffer (100 mM MOPS, 5 mM EDTA, 10% glycerol, 50 mMKCl and 0.5 mM benzamidine) containing 70 μL of β-mercaptoethanol and 1% in polyvinylpolypyrrolidone (PVPP). Samples were stirred and subsequently centrifuged for 40 min (18,000 g) at 4°C. The supernatant was decanted into a beaker using a cheesecloth. (NH₄)₂SO₄ was added to the solution to obtain 45% (w/v) concentration, with stirring during 30 min. After that, the mix was centrifuged at 20,000 g for 30 min at 4°C. The previous step was repeated to precipitate the protein in the extracts but in that case with a (NH₄)₂SO₄ concentration of 80% (w/v) stirring for 30 min. Finally, they were centrifuged at 20,000 × g for 30 min at 4°C.

All the pellets were dissolved in 3 mL of extraction buffer and dialyzed in 2 L of dialysis buffer (30 mm, 1000-MWC dialysis tubing at 4°C on a stir plate) over 12 h. The protein concentrations were determined by Bradford assay (Bradford, 1976).

The assay for the determination of EPSPS activity followed the methodology described by Dayan et al. (2015) using the EnzCheck phosphate assay Kit (Invitrogen, Carlsbad, CA) to determine the inorganic phosphate release. The EPSPS activity from the nine accessions was determined in the presence and absence of glyphosate. The glyphosate concentrations used were: 0, 0.1, 1, 10, 100, and 1000 μM to determine the enzyme activity inhibition (I₅₀). The assay buffer was composed of 1 mM MgCl₂, 10% glycerol, and 100 mM MOPS, 2 mM sodiummolybdate and 200 mM NaF. The experiments were conducted with three replications of each accession per glyphosate concentration and repeated three times. EPSPS enzyme activity was expressed as percentage of enzyme activity in presence of glyphosate respect to the control (without glyphosate).

For RNA extraction 100–200 mg of young leaves were taken from plants of each P. hysterophorus accession, and stored at −80°C for the extraction of RNA. Their tissue was ground in liquid nitrogen in a STAR-BEATER 412–0167 mill (VWR International Eurolab S.L., Barcelona, Spain). Total RNA was isolated from leaves as described by Pistón (2013), and the amount and quality were determined in a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Walthman, MA, USA). The synthesis to cDNA was from total RNA being adjusted to the same concentration in all the samples (50 ng μL⁻¹). An iScript^TM cDNA Synthesis Kit (Bio-Rad Laboratories, Inc. CA, USA) at 40 μL reaction volume was used following the manufacturer‘s instructions.

The PCR reactions were carried out with cDNA samples from each of the accession using the primers Bidens-F10 (5′- GGTTGTGGYGGTVTRTTTCC-3′) and Bidens-R11 (5′- GTCCCAASTATCACTRTGTTC-3′) based on EPSPS gene sequences described previously (Alcántara-de la Cruz et al., 2016b). PCR conditions were also as described (Alcántara-de la Cruz et al., 2016b). The PCR on cDNA amplified fragments of 462 bp in length, comprising the region of Thr-102 and Pro-106, which corresponds to the sequence of the EPSPS gene of Arabidopsis Klee et al. (1987), in which point mutations conferring resistance to glyphosate have been associated (Sammons and Gaines, 2014; Yu et al., 2015).

The PCR fragments were cloned in the pGEM®-T Easy Vector System (Promega Biotech Ibérica, SL, Madrid, Spain) and transformed into competent cells of E. coli DH5α (Promega). Transformation was confirmed through PCR using the M13F and M13R primers as described (Alcántara-de la Cruz et al., 2016b). The colonies containing the length of the fragment were sequenced by the STABVIDA sequencing service (Caparica, Portugal). Five biological samples were used per accession providing 15 clones in all for each one. The quality and assembly of cDNA sequences and consensuses were determined employing the programs of SeqMan Pro^TM versión 11(DNASTAR; Wisconsin, USA) and Geneious® versión 8.1.8 (Biomatters Ltd, Auckland, New Zealand). The multiple sequences were aligned by means of the Muscle algorithm incorporated into SeqMan Pro versión 11.

Dose-Response and EPSPS enzyme activity data were subjected to non-linear regression analysis (Seefeldt et al., 1995; Burgos et al., 2013) using a three-parameter log-logistic equation (Equation 1) to determine the glyphosate dose causing 50% reduction in above-ground vegetative biomass (GR₅₀), 50% mortality (LD₅₀), and inhibition of EPSPS activity by 50% (I₅₀).

Where Y is the EPSPS activity, survival or above-ground biomass at herbicide x dose, d is the coefficient corresponding to the upper asymptote, b is the slope of the curve, and g is the herbicide rate at the point of inflection halfway (i.e., LD₅₀, GR₅₀, I₅₀).

Regression analyses were conducted using the drc package (Ritz et al., 2015) for the statistical environment R (R 3.2.4; R Core Team, 2015). Resistance indices were computed as R-to-S GR₅₀ LD₅₀, or I₅₀ ratios. To test for a common GR₅₀, LD₅₀, or I₅₀ for R and S accessions, i.e., Resistance Index equals to 1, a lack-of-fit test was used to compare the model consisting of curves with accessions-specific g values with a reduced model with common g (Ritz et al., 2015).

Analysis of variance (ANOVA) was conducted using Statistix 9.0 (Analytical Software, USA) to test for differences between R and S accessions in shikimate accumulation at 1000 μM glyphosate in the leaf segment; and proportion of the different glyphosate metabolites; proportion of applied ¹⁴C-glyphosate taken up by leaves, and proportions of absorbed ¹⁴C-glyphosate remaining in the treated leaf, translocated to roots and to the rest of the plant at 96 HAT; and basal enzyme activity. Percentage data were previously transformed (arcsine of the square root) to meet model assumptions. Model assumptions of normal distribution of errors and homogeneous variance were graphically inspected. When needed, differences between means were separated using the Tukey HSD test.

Dose-response assays showed the existence of the first case of glyphosate-resistant weeds in the Caribbean (Cuba and Dominican Republic). The two susceptible weeds (Cu-S and Do-S) had similar susceptibility levels (Figures 1, 2; Table 2). The P. hyterophorus accessions from Cuba island had resistance index (RI) values (based on the GR₅₀ and LD₅₀ values) that ranged from 2.7 to 24.6, and 6.1 to 27.5 fold resistance, respectively, while on Dominican Republic island values were between 5.4 to 20, and 6.3 to 22.7 fold resistance, respectively (Table 2).

The fact that plants treated with glyphosate increase shikimic acid accumulation in leaf disks due to the inhibition of EPSPS activity led us to carry out the experiment depicted in Figures 3A,B. Considering the values obtained in vivo (GR₅₀ and LD₅₀) and the shikimic acid accumulation in leaf disks at 1000 μM of glyphosate, the resistance order of the P. hystherophorus accessions was Cu-R1 ≥ Do-R1 > Do-R2 > Cu-R2 > Do-R3 > Do-R4 > Cu-R3 >> Cu-S ≥ Do-S. There were significant differences at 1000 μM glyphosate between R and S accessions of Cuba (p = 0.0013, DF = 3, n = 12) and Dominican Republic (p = 0.0008, DF = 4, n = 15).

There were marked differences in glyphosate absorption between the resistant and susceptible glyphosate P. hysterophorus accessions at 96 h after treatment (HAT) (p = 0.0001, DF = 8, n = 45) (Table 3). All accessions obtain maximum absorption at 96 HAT, and the two susceptible accessions absorbed an average of 80.5%, while the resistance accessions absorbed an average of 59.2% of ¹⁴C-glyphosate which was recovered.

Translocation assays suggest marked differences at 96 HAT between the Cu-S and Do-S accessions compared to the Cu-R1, Cu-R2, Cu-R3, Do-R1, Do-R2, Do-R3, and Do-R4 ones in treated leaf (p = 0.0003, DF = 8, n = 45), rest of the shoots (p = 0.0001, DF = 8, n = 45), and root (p = 0.0004, DF = 8, n = 45) (Table 3). There were no significant differences in translocation between the two susceptible accessions (Cu-S and Do-S) from Caribbean Islands. But there were small significant differences in the resistant accessions (Cu-R1, Cu-R2, Cu-R3, Do-R1, Do-R2, Do-R3, and Do-R4). Nonetheless, the high amount of ¹⁴C-glyphosate in each resistant accession remained in the treated leaf. Due to differences in levels of glyphosate resistance between the P. hysterophous resistant accessions, we suspect that other mechanisms could be involved (Tables 2, 3, Figure 3).

Previous tests demonstrated that the highest glyphosate translocation and metabolism was reached at 96 HAT in the P. hysterophorus accessions (unpublished data). There were significant differences at 96 HAT in glyphosate metabolism levels between accessions (p = 0.0014, DF = 8, n = 36). Glyphosate levels decreased, whereas glyphosate metabolites (AMPA, glyoxylate and sarcosine) increased at 96 HAT in the Cu-R1, Do-R1, Do-R2, Cu-R2, and Do-R3 accessions. Higher glyphosate levels remained in the Cu-R3 and Do-R4 (low resistance), and very high one in the Cu-S and Do-S (susceptible) accessions. In these last accessions, sarcosine was not detected (Table 4). These results can also explain the low level of resistance of the accession (Cu-R3 and Do-R4) with a single resistance mechanism, while the other glyphosate resistant accessions have at least two mechanisms (Tables 3, 4).

The EPSPS enzymes of all the accession plants were inhibited by glyphosate. The I₅₀ (herbicide dose which reduces the enzyme activity to 50%) values were different in all accessions, ranging between approximately 47.65 in Cu-R1, 25.2 in Do-R1, 22.1 in Do-R2, 1.4 in Cu-R2, 1.2 in Do-R3, 1.2 in the Cu-R3, and 1.1-fold resistance in Do-R4 accessions relative to their susceptible accession, respectively (Figure 4, Table 5). These results were in accordance with the in vivo resistance level shown for the different accessions, and suggest that multiple mechanisms in the target-site could be expressed in these accessions.

The basal activity of EPSPS enzyme (without glyphosate) in the resistant accessions was between 0.026 and 0.21 μmol μg⁻¹ protein min⁻¹, while the susceptible accessions (Cu-S and Do-S) were lower with 0.024 and 0.025 μmol μg⁻¹ protein min⁻¹, respectively (Figure 5). There were market differences between accessions in both Cuba (p = 0.0001, DF = 3, n = 12), and Dominican Republic (p = 0.0002, DF = 4, n = 15). The Cu-R1, Do-R1, and Do-R2 exhibited 8.8, 7.2, and 4.8-times higher basal enzyme activities than their susceptible accessions, respectively. For Cu-R2, Do-R3, Do-R4, and Cu-R3 accessions the values were similar to those found for their susceptible accessions, respectively.

A total of 462 bp of the EPSPS gene of P. hysterophorus plants of resistant and susceptible accessions were sequenced. The fragments were aligned and numbered based on a published EPSPS sequence of Arabidopsis thaliana (L.) Heynh. (GenBank: CAA29828.1). The resistant accessions of P. hysterophorus Cu-R1 from Cuba, and Do-R1 and Do-R2 from Dominican Republic, showed an amino acid substitution at position 106 consisting of a Proline to Serine (Figure 6).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.