Reduced function in Chamaenerion angustifolium after sub-lethal glyphosate exposure

Reduced function in Chamaenerion angustifolium after sub-lethal glyphosate exposure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reduced function in Chamaenerion angustifolium after sub-lethal glyphosate exposure Lisa Wood, Alexandra Golt, Laurel Berg-Khoo, Brian Hampton This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6011000/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Chamaenerion angustifolium (fireweed) is an ecologically important plant in the northern hemisphere. It provides food across forest openings for many wildlife species including bumblebees, which are important pollinators to North America. Fireweed also acts as a significant food source for honeybees and is used by many North American Indigenous people as food and medicine. In forested areas managed for timber, fireweed is often incidentally exposed to glyphosate-based herbicides (GBH) in post-harvest vegetation management. We studied the response of fireweed to sub-lethal GBH exposure in a controlled experiment and in standard operational field conditions to determine impacts on specific aspects of growth and reproduction of the species. We aimed to determine if GBH-related stress symptoms would significantly impact the fluorescence of fireweed flowers, and/or the nutritional quality of pollen, which would have consequences for pollinators. Results showed that fireweed is negatively impacted by sublethal exposures of GBH including reduced photosynthetic efficiency, reduced height, and reproductive shoot dieback. In operational environments studied, pollen viability was reduced one-year after applications and anther fluorescence was altered. The amino acid concentration of flowers was reduced, and glyphosate residues remained present at low concentrations in floral tissues at two years post-treatment. It was concluded that these changes to fireweed growth and reproduction reduce its function as a primary source of good quality food for pollinators. Biological sciences/Plant sciences/Plant stress responses/Abiotic Biological sciences/Plant sciences/Plant ecology photosynthetic efficiency pollen viability fluorescence amino acids glyphosate residue forest vegetation management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Glyphosate-Based Herbicides in Forest Plants Glyphosate-based herbicides (GBH) are commonly used in Canadian forest management practices, to reduce growth of deciduous tree and shrub species in areas where coniferous trees are harvested and planted, thereby reducing competition for early growth of conifer seedlings [ 1 ]. During application many non-target plants are exposed to GBH due to spray drift or their proximity to targeted plants via the spray cloud/ rain trajectory [ 2 , 3 ]. Residues of glyphosate and aminomethylphosphonic acid (AMPA, a product of glyphosate degradation) have been found in tissues of non-target understory plants in BC forests up to 12-years after initial exposure [ 4 , 5 ], and evidence exists that degradation rates are slower and plant susceptibility greater, in colder climates [ 5 , 6 , 7 ]. Sublethal glyphosate residues in plant tissues have been shown to induce stress symptoms such as chlorosis, stunted growth, reduced leaf area, deformed leaves, and changes to flowers [ 8 , 9 , 10 , 11 , 12 ]. Although GBH has been found to remain in the forest environment and cause changes to plant growth and reproduction [ 11 , 12 ], the current understanding of these changes and their significance remains limited. What is yet to determine is whether the persistence of low concentrations of GBH residues have an impact on the function of plants within their ecosystem, and since each species responds differently to GBH application, it is important that individual species are investigated and their responses documented. Glyphosate (N-(phosphonomethyl) glycine) is a glycine analog that acts as a non-selective herbicide [ 13 ]. It is absorbed across vegetative plant surfaces and is translocated along the same pathways as sugars produced in photosynthesis, allowing it to quickly reach areas of high metabolic activity such as meristematic tissue [ 14 ]. Inside the plant cell glyphosate acts as an inhibitor of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) in the shikimate metabolic pathway by competing with phosphoenolpyruvate for the enzyme’s active site [ 13 , 15 , 16 ]. This inhibition leads to an increased concentration of shikimic acid that may affect protein metabolism, which leads to the inhibition of plant growth and ultimately the death of the plant, depending on the concentration applied [ 17 ]. The shikimate pathway is responsible for producing aromatic amino acids including phenylalanine, tryptophan, and tyrosine, phenolic compounds that have antioxidant activity within the cells of both plants and herbivores, and other important secondary metabolites [ 18 ]; therefore, glyphosate may prevent their production. Wang (2001)[ 17 ] found that the concentration of tryptophan decreased rapidly three days after glyphosate treatment, and the level remained low [ 17 ]. Since tryptophan plays a major role as a precursor in the synthesis of several important plant regulatory chemicals, as well as plant pigments and other biocommunicative chemicals, it follows that a reduction in tryptophan may severely affect the synthesis of these chemicals. However, Wang (2001)[ 17 ] found that the effect of glyphosate on aromatic acid synthesis was inconsistent, as it increased the concentration of some aromatic acids and decreased others, indicating that the production of these metabolites in plants is more complex than is currently understood. Glyphosate and AMPA also appear to prevent production of chlorophyll and promote its degradation, lowering both photosynthetic ability and rate of carbon fixation [ 10 , 19 ]. Because of the known interactions that glyphosate can have with plant chemistry, there is reason to believe that glyphosate may alter ecosystem functions such as producing lower quality food for wildlife. Plants are the base of the food web and life on earth evolved to rely on the nutrition provided by their abundance. A plant’s chemical make-up provides the building blocks for the growth and development of animals [ 20 , 21 , 22 ], therefore, if a plant population is nutritionally compromised in a given space, there are potential health consequences for the rest of the ecosystem relying on those plants. Plant components such as amino acids and proteins, and compounds such as anthocyanins are among the plant chemicals that serve a pivotal role in plant perpetuation and the functional ecology of forest communities. For example, many insects consume pollen for the primary purpose of obtaining protein and fat thereby supporting the growth and development of insect larvae [ 23 , 24 ]. Anthocyanins found in plants, are known for their antioxidative and antimicrobial properties [ 25 ]. Some anthocyanins are also fluorescent compounds that are found in the anthers and pollen grains of select species and act to attract or deter animals, and some were found to protect anthers and pollen from UV-induced damage [ 26 ]. Glyphosate exposure has been found to reduce attraction towards UV light in bumble bees [ 27 ]. Therefore, if antioxidants responsible for fluorescence (often caused by the interaction between the anthocyanins and UV light) are altered by glyphosate treatments, then biocommunication may be compromised, potentially impacting foraging efficiency for wild bees, a keystone functional group in many ecosystems. Study species: Chamaenerion angustifolium Chamaenerion angustifolium [L.) Scop. (Myrtales:Onagraceae)] (fireweed) is a prominent plant species in British Columbia (BC), Canada. Fireweed is widely distributed in the northern temperate and boreal regions of Canada and the northern United States [ 28 ] and is also found in northern parts of Europe [ 20 , 22 ]. It is found in a wide variety of habitats from boreal forests to gravel bars, and from low to subalpine elevation [ 29 , 30 ]. It is an erect perennial herb which can reach a height from 0.5 to 3 m tall, and has alternate, entire lance-shaped leaves reaching from 5 to 20 cm in length [ 29 , 30 ]. Flowering in fireweed occurs continually from June to September. Flowers are magenta or deep pink in colour, have long pedicels and are borne in indeterminate racemes, with the oldest flowers born towards the base and new flowers produced as the shoot grows [ 29 , 28 ]. Each individual flower has four petals, 10 to 15 mm in length, and four lanceolate, spreading sepals that are the same colour as the petals. Each flower has eight stamens, a style that is longer than the stamens, a four-cleft stigma, and a four-loculed ovary [ 29 , 28 ]. The anthers release blue-green or yellow pollen held together with sticky viscin threads [ 28 , 31 ]. The green, fleshy, and slightly concave nectary produces nectar with a high sugar concentration (approximately 77%) and is located on the upper end of the inferior ovary [ 32 ]. In many areas of northern BC, fireweed is one of the first successional species to re-establish after a disturbance such as forest harvesting, natural resource-related vegetation management, or a fire. Fireweed can quickly colonize a disturbed area and recycles early nutrients through the system via its own decaying plant matter, thereby improving soil quality for other plant species [ 33 ]. Dense colonies of fireweed may delay the development of shrubby vegetation, thus assisting in the establishment of coniferous species before competitive shrubs [ 33 ]. Fireweed has been used for many centuries by Indigenous people across North America for food. The majority of First Nations in the interior of BC – including the Nlaka’pamux, Stl’atl’imx, Secwepemc, Carrier, Wet’suwet’en, Sekani, and Nisga’a – collected and ate the raw pith of young stalks, prior to flowering [ 34 ]. Many BC coastal First Nations such as the Sechelt, Squamish, Nuxalk, Haida, and Tsimishian also foraged the pith [ 35 ]. The Saanich First Nations steeped the young leaves to make tea [ 35 ]. Fireweed also had social significance in Haida communities. Patches found close to Haida villages were often owned by certain high-class members, and other individuals had to ask permission to harvest from these patches [ 35 ]. All parts of fireweed plants were (and are) used by Indigenous peoples in western Canada for its anti-inflammatory and antiseptic properties [ 36 , 30 ]. It is important to investigate the impacts of glyphosate-based herbicides (GBH) on fireweed to understand the potential changes to the beneficial ecological and cultural services provided by this species. Fireweed is also an important food source for wildlife. The shoots are consumed by deer, moose, caribou, muskrat, and hares [ 29 , 32 , 37 ]. A multitude of insects, such as bees and flies, use fireweed nectar as it is continuously produced after anthesis and until abscission of the floral parts occurs, which is uncommon in other wildflowers [ 29 , 31 , 32 ]. The most common pollinators for fireweed are bees [ 38 , 32 ]; fireweed is an important plant for the honey industry in Canada [ 29 ]. Study Rationale and Objectives Due to the prevalence of GBH use worldwide [ 13 ] it is important to further investigate how GBH interacts with plants to produce a clearer picture of the functionality of GBH exposed ecosystems. Here we collate the findings of our study of the responses of fireweed ( Chamaenerion angustifolium ) to sublethal GBH treatment in northern British Columbia, Canada. We collected samples of fireweed from operationally managed forest stands one and two years after GBH applications took place and we conducted controlled experimentation using fireweed, to better understand this species’ growth and reproductive potential. Ultimately, we aimed to contribute to the knowledge base on how plant systems function after GBH treatments, studying fireweed as a representative perennial, herbaceous angiosperm. Our specific objectives were: To determine the growth potential of fireweed post GBH application, and to confirm fireweed stress symptoms induced by sublethal glyphosate exposure. To quantify the reproductive capacity and the biocommunicative potential of fireweed flowers, post GBH application. To determine changes in nutrition of fireweed flowers, specifically focusing on amino acids because they are produced downstream of the shikimic acid pathway. Methods We conducted a greenhouse experiment with fireweed at the University of Northern British Columbia (UNBC). We collected fireweed plants, for study and testing, from operationally managed forests during field outings in 2021 and 2022. Growth Chamber Experimentation The specific goal of this experiment was to determine the morphological and physiological response of fireweed to GBH treatment under controlled growth conditions (contributing to Objective 1). Wild fireweed plants were collected from sites on and near the UNBC Prince George campus, as rhizomes with small vegetative shoots immediately following spring emergence. Rhizomes of approximately 15–20 cm lengths were planted in individual 2L pots with a growing medium consisting of 78% peat moss, 15% vermiculite, and 7% perlite with 0.46 mL/L dolomite lime, 0.46mL/L Micromax micronutrient formulation, and 3.65mL/L Osmocote 14-14-14 slow-release fertilizer. All plants were brought into a greenhouse compartment of the I.K. Barber Enhanced Forestry Lab (EFL) at UNBC and allowed to acclimate at 20⁰C for ten days before placement in growth chambers. Plants were divided between three growth chambers (five treatment plants and five controls were placed in each chamber) set to different temperatures (chamber 1 = 20⁰C day/8⁰C night, chamber 2 = 25⁰C day/10⁰C night, chamber 3 = 30⁰C day/15⁰C night) with a 12 hour temperature cycle and 16 hour photoperiod. Plants were frequently monitored and individually watered as needed to ensure no water stress was experienced. Five treatment plants from each growth chamber were taken to an outdoor location, sprayed with a GBH formulation, allowed to dry, and returned to the growth chambers. VisionMax® GBH (540g/L a.e. potassium salt), normally applied at 4 L/ha (540g/L * 4L/ha = 2160 g over 10,000m 2 (1 ha); application rate of 43.2 g/L over 1 ha), was applied at a low concentration to induce a sub-lethal response. We applied 0.2mL of VisionMax® concentrate in 100mL of solution, by hand-held sprayer. Thus, the application rate was 2.5% of the normal rate; 540g/L (0.0002L) /0.1 L = 1.08 g/L application rate over a 1 m 2 or 0.018 g over a 1 m 2 area. Total length of the stem between the root collar and shoot apex and photosynthetic efficiency (PE), assessed by chlorophyll fluorescence of each plant, were measured weekly, the first of these measurements were made prior to treatment. The total overall shoot health was evaluated at the conclusion of the experiment. PE was estimated by calculating the maximum quantum yield of photosystem II (PSII) (Fv/Fm), detected using a portable Hansatech chlorophyll fluorometer. Photosystem II is the most sensitive component within the photosynthetic pathway [ 39 ] and was therefore used as a measure of plant stress response to drought conditions [ 40 ]. To measure PE, the target leaves were first covered with a clip for at least one minute to ensure the portion of the leaf tested was in complete darkness. Then, the Fv/Fm ratio was automatically calculated by the chlorophyll fluorometer by opening the clip to the fluorometer’s sensor, where it detected the minimal fluorescence (Fo), followed by the maximal fluorescence (Fm), which were then related using the following equation: Fv = Fm – Fo [ 40 ]. For healthy plants the Fv/Fm ratio should be between 0.7–0.83 [ 41 ]. Operational Study in 2021–2022: Operational sampling was conducted to meet objectives 2 and 3; to determine if the reproduction, biocommunication, or nutritional components of fireweed were interrupted by GBH applications in the real world. We chose to study pollen viability as a representation of reproductive capacity, floral fluorescence as an indicator or biocommunicative potential, and amino acid content as a marker of nutritional composition. Study Sites The area of study consisted of 15 sites, each site containing paired treated + control areas, in the Prince George Forest Region of BC, Canada (Fig. 1 ). Five of the sites were treated with GBH in 2019, five were treated in 2020, and five were treated in 2021. Each treated site had a paired control site (not treated with GBH) of the same vegetation complex, that was sampled. Samples were collected from these sites during June and July of 2021 and 2022 (Table 1 ). The sites were previously logged using a clear-cut method of harvesting, planted with coniferous trees, lodgepole pine ( Pinus contorta Dougl. ex Loud) and/or hybrid white spruce ( Picea glauca (Moench) Voss x engelmannii Parry ex. Engelm) and treated with a GBH in either 2019, 2020, or 2021 (one-year before sampling) when planted trees were between 5 and 15 years of age. The sites sprayed in 2019 and 2020 were treated with the GBH formula VisionMax® and the sites sprayed in 2021 were treated with the GBH formula GlySil®. VisionMax® (Canadian registration no. 27736 under the Pest Control Products Act) was aerially applied at rates of 3.3–4.0 L/ha (resulting in concentrations of 1.78–2.16 kg a.i. ha-1). GlySil® (Canadian registration no. 29009 under the Pest Control Products Act) was applied aerially at 6.0 L/ha (resulting concentration of application was 2.13 kg a.i. ha-1). Parts of these sites were left untreated (pesticide-free zones) due to the presence of streams to prevent glyphosate contamination and run-off. These untreated areas within the sites or other nearby untreated regions with similar vegetation complexes served as experimental controls. Paired treatment and control areas for each site were identified from forest industry operation maps and were confirmed visually on site through marked treatment lines. Table 1 Sample sites treated with glyphosate-based herbicides in 2019, 2020, and 2021 in northern British Columbia, Canada. Each location contained a paired treated area and non-treated control area. Site Name Latitude (N), Longitude (W) BEC Zone Elevation (m) Total Treatment Area (ha) Date Herbicide Applied OLS61A 54.30553, 122.05421 SBSvk 870 23.6 7-Aug-2021 OLS057 54.30460, 122.01806 SBSvk 770 55.3 7-Aug-2021 RAI063 54.37277, 122.33604 SBSvk 830 27.5 7-Aug-2021 RAI081 54.31566, 122.19036 SBSvk 790 86.8 15-Aug-2021 271-001 53.63264, 121.73552 SBSvk 1005 37.4 1-Aug-2021 2020-01 53.76292, 122.77779 SBSdw3 780 4.4 9-Sept-2020 2020-02 53.76236, 122.77825 SBSdw3 780 4.0 9-Sept-2020 2020-03 53.65097, 122.78725 SBSdw3 730 5.2 9-Sept-2020 2020-04 53.65695, 122.77856 SBSdw3 750 15.5 9-Sept-2020 2020-05 53.66973, 122.77174 SBSdw3 700 6.9 9-Sept-2020 2019-01 53.4454, 122.2919 ESSFwk1 1350 19 16-Aug-2019 2019-02 53.4441, 122.2845 ESSFwk1 1500 1 16-Aug-2019 2019-03 53.3621, 123.1156 SBSdw2 820 10 19-Aug-2019 2019-04 53.3618, 123.1105 SBSdw2 830 9.9 19-Aug-2019 2019-05 54.0434, 123.2552 SBSdw3 810 20 8-Aug-2019 Sites were located in the SBSvk, SBSdw2, SBSdw3, and ESSFwk1 biogeoclimatic ecosystem classification (BEC) zones (Table 1 ). The BEC system was developed and is used within BC to delineate regional differences in topography and dominant vegetation [ 42 ] (Table 1 ). BEC zones and subzones represent divisions that define the climate and dominant vegetation of an area. The first three capital letters represent the zonal information, based on the dominant tree species of the area, and the following two lower case letters describe the subzone. The first letter of the subzone name describes the relative precipitation, and the second letter describes the relative temperature. For example, in the “SBSwk3”, SBS stands for “sub-boreal spruce” and wk3 indicates that the subzone is “wk” or the wet-cool subzone; and the variant is “3” or the third climate variation type identified within this subzone. Each variant is drier, wetter, snowier, warmer, or colder than what is considered typical for the subzone in general [ 43 ]. The SBS is characterized by two dominant tree species – hybrid white spruce and subalpine fir ( Abies lasiocarpa (Hook.) Nutt.), as well as extensive stands of lodgepole pine in drier areas [ 44 ]. The SBS typically has shorter winters and longer growing seasons than boreal areas, allowing for a wide range of subzones. The SBSvk (sub-boreal spruce, very wet cool) zone has the highest annual precipitation and the longest growing season precipitation and is therefore the wettest biogeoclimatic unit in the SBS [ 44 ]. Snowfall data were unavailable, but this subzone has the lowest mean annual temperature of the SBS units, and paper birch ( Betula papyrifera Marshall) spread throughout. The SBSdw3 encompasses a large area to the west of Prince George. The SBSdw3 is warm relative to other subzones with winter precipitation being relatively low and snowpacks with a mean depth of ~ 2m. Growth-limiting factors in this subzone are drought on drier sites and frost on frost-prone and drier sites [ 44 ]. The SBSdw2 borders the SBSdw3 subzone to the south and is drier and warmer than the other subzones in the SBS due to its lower elevation and relatively low precipitation [ 42 ]. The Engelmann spruce – subalpine fir (ESSF) wk1 (Cariboo wet cool) zone occurs above the SBSwk zone between 1200–1500 m elevation. The ESSF is characterized by high precipitation (> 1000 mm), half of which falls as snow [ 45 ]. Within each sample site, we established 10 transects, a minimum of 20 m apart, and each 50 m in length, for collection of fireweed plants. We walked along each transect line and collected a minimum of 10 flowers, from a minimum of five individual plants on each transect (yielding a total collection of ~ 100 flowers per site). We collected newly opened flowers where possible to try to limit environmental factors that could potentially affect flowers over time and as they age, such as light, temperature, wind, and rain. Flowers selected for sampling were cut at the base of the pedicle using small gardening shears. Floral samples were placed in labeled petri dishes and sealed with parafilm to ensure that floral parts, such as anthers and pollen, remained undisturbed and intact for analysis. Samples were placed in a cooler and were transported, stored, and analyzed at UNBC, Prince George, BC. No fireweed flower samples were collected from 271-001 treatment site as none of the fireweed bloomed during the season of collection. Floral Fluorescence Analysis Analysis of fluorescence was conducted on 15 flowers per site immediately after returning from the field to avoid floral degradation (Table 2 , Table 3 ). Photographs were taken using a Leica dissecting microscope with digital camera and Zen Lite software. We used the stereomicroscope adapter system by NIGHTSEA with a royal blue light (wavelength range of 440–460 nm) to induce fluorescence. Stamens were separated from the rest of the flower and placed under the microscope to capture images with all the anthers in focus. We photographed stamen samples under white light (light emitted from microscope) and under the royal blue fluorescence filter. Analysis of resulting colour pixels captured by the photography was conducted using NIS-Elements Imaging Software (Table 2 ). Table 2 Variables measured to assess pixel colour variation in photographed pollen and anthers of fireweed ( C. angustifolium ) using NIS-Elements Imaging Software. Variable Variable description Bright variation The standard deviation of brightness values. Hue typical Describes the most frequent hue in an object of field. Hue variation Describes hue distribution of inner structure of an object of field. Intensity variation Describes the inner structure of an object or field. Mean red, mean green, mean blue Arithmetic mean of pixel intensities of one image component. Mean brightness Arithmetic mean of brightness values of pixels. Mean intensity Arithmetic mean of pixel intensities. Mean saturation Arithmetic mean of saturation values of pixels. Min/max intensity Measures minimum and maximum intensity values of pixels. Table 3 Composite sample replicate numbers of fireweed ( C. angustifolium ) flowers tested with each type of analysis. Flowers collected from forest sites treated with glyphosate-based herbicides and paired control areas in northern British Columbia, Canada. Analytical Test Treated one-year prior Treated two-years prior Controls Glyphosate Residue 15 (3 replicates from 5 sites) 30 (3–5 replicates from 8 sites) 30 (3–5 replicates from 8 sites) Amino Acids None 15 (5 replicates from 3 sites) 15 (5 replicated from 3 sites) Fluorescence 15 flowers (5 plants, 3 flowers per plant) per site 15 flowers (5 plants, 3 flowers per plant) per site 15 flowers (5 plants, 3 flowers per plant) per site Pollen Viability 15 flowers (5 plants, 3 flowers per plant) per site 15 flowers (5 plants, 3 flowers per plant) per site 15 flowers (5 plants, 3 flowers per plant) per site Pollen viability testing Following the imaging of the fresh flowers for fluorescence analysis, pollen was mechanically removed from anthers and Brewbaker and Kwack’s (B and K) medium was prepared for pollen viability testing [ 46 , 47 ]. B and K medium was prepared by dissolving 50 mg boric acid, 150 mg calcium nitrate, 100 mg magnesium sulfate heptahydrate, and 50 mg potassium nitrate in 500 ml of deionized water. This stock solution was then stored at 4°C. Sucrose was dissolved in the solution immediately before viability testing. The amount of sucrose required to produce optimal germination of pollen grains varies between plant species [ 46 , 47 ], and we were unable to find existing literature on the optimal sucrose content for fireweed pollen germination. We initially tested the pollen viability of fireweed using varying amounts of sucrose at 5, 10, 15, 20, 30, 40, 50, 60, and 70%. The optimal concentration of sucrose that induced the highest rate of pollen tube formation in our trial was 15%; therefore, all proceeding viability testing was completed using this concentration of sucrose. Pollen grains became round once placed in B and K medium, and viable grains developed a long pollen tube [ 46 ]. To be classified as viable, the pollen tube produced needed to be longer than the diameter of the pollen grain itself [ 47 ]. Fresh pollen grains were placed on a depression microscope slide. Two drops of B and K medium were added to each slide and pollen grains were mixed into the media using a toothpick. Each slide contained the pollen grains of one flower. Fifteen slides were prepared per site, representing three replicate flowers from each of five individual replicate plants per site (Table 3 ). A cover slip was placed on top of the slide and slides were incubated for 24–36 hours at room temperature, in a Petri dish lined with moist filter paper and sealed with parafilm to maintain humidity. Upon completion of the incubation period, slides were observed using an Eclipse FN1 Nikon microscope at 10× magnification. A microscope camera with NIS-Elements Imaging Software was used to view the pollen grains and capture images. A total of 25 images were captured from each slide in a grid-like manner across the slide moving from the top left corner to the bottom right corner of the slide. The total number of pollen grains per slide were counted, and pollen viability was calculated for each flower and then averaged for control sites and treatment sites. Amino Acids Analysis The remaining flowers collected, varying in number depending on the abundance at each site, were dried in a kiln oven at 80°C for 24 hours in preparation for chemical analyses. Once dried, samples were ground using an IKA A 11 basic analytical mill. Removable parts of the mill were rinsed with water between samples, and the remaining parts were blown out with forced air to minimize the likelihood of cross-contamination between samples. There were fewer fireweed flowers at the sites treated one-year prior to sampling compared to two years prior, therefore samples were only selected to send for amino acid analysis from sites treated two years prior and their corresponding controls. The difference in abundance of fireweed was likely due to the time-since application, fireweed present at sites one-year post treatment were largely in the vegetative phase of growth. Testing for glyphosate-based residues was prioritized over amino acids to ensure that residues were present on the sites identified. Dried and ground fireweed flowers were subsampled to a weight of 0.5 g and sent to Central Testing Laboratories in Winnipeg, Canada, for amino acid testing. Amino acids were analyzed using the AccQ•Tag UPLC Method, which is a precolumn derivatization technique for amino acids. This method derivatizes amino acids, separates the derivatives with reversed-phase UPLC, and quantitates the derivatives based on UV absorbance or fluorescence intensity. The Waters AccQ•Tag Ultra Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC) is an N-hydroxysuccinimide-activated heterocyclic carbamate, a class of amine-derivatizing compounds. The AccQ•Tag Ultra reagent converts both primary and secondary amino acids to stable derivatives. The structure of the derivatizing group is the same for all amino acids, adding both UV absorbance and fluorescent character. Excess reagent hydrolyzes to yield 6-aminoquinoline (AMQ), a non-interfering by-product. Samples were tested for concentrations of: Alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, and valine. We also tested for total crude protein. Glyphosate residue analysis of floral tissues Composite samples were created to meet a minimum 5 g dry matter mass requirements for glyphosate residue analysis of each sample. At least three replicates were made from flowers across each site (Table 3 ). Glyphosate residues in fireweed tissues were analyzed by the Agriculture and Food Laboratory at the University of Guelph using liquid chromatography tandem mass spectrometry (LC-MS/MS). The glyphosate screening process reported each individual component separately, if detected. Prior to analysis, an aqueous extract of a homogenized subsample of plant material was prepared. Sample extracts were acidified and separated using solid-phase extraction. The LC instrument employed a cation guard column for chromatographic separation (Micro-Guard Cation-H cartridge 30 × 4.6 mm), a mobile phase A (0.1% formic acid in nanopure grade H2O) and B (acetonitrile), with a flow rate of 1 ml/min and a total run time of 12 minutes. Retention time for glyphosate was 0.9 minutes. The autosampler temperature was 8°C, injection volume was 50 µl, and column oven temperature was 20 ± 3°C. Validation of results was completed using a five-step detection method to ensure no false positives. Blanks were tested along with samples to check for carry over; no coextracting contaminants were detected, the peak detected in the samples had the same retention time for two ion transitions, the ion ratios were correct in all instances relative to the certified standard used by the laboratory, and there was consistency among sample residues found, indicating reliability. The lab also reported when samples were considered above the minimum detection limit (MDL) of 5 ppb, and above the MDL but below the minimum quantification limit (MQL) of 20 ppb. For our data analysis, these parameters were used to indicate the presence of glyphosate residues. When a sample fell above the MDL of 5 ppb but below the MQL of 20 ppb, we used a conservative value of 6 ppb, so that these positive detections could be acknowledged, but not overestimated. Statistical Analysis The data were analyzed using IBM SPSS 28.1. Normality of distribution was assessed using Shapiro-Wilk significance with a confidence level of 95%. If the data were normal, significant differences between the control and treated were analyzed with ANOVA. If the data were non-parametric, they were assessed for significance between the means using Kruskal Wallis or Mann Whitney U tests. Histograms were used to assess distributions so that any generalized linear models created were fit with the appropriate distribution curve. Controlled Experiment Data Data were grouped by growth chamber and by week to allow comparison between treatment and control groups. Linear regression was used to determine when independent treatment variables (including week#, chamber#, and treatment) had a significant impact on the response variable for normally distributed height data. Generalized linear modelling with a gamma distribution and a log link function was used for photosynthetic efficiency data that were not normally distributed. A percentage of total incidence was calculated for the number of shoot apices that were damaged out of the total plants assessed. Operational Field Data Amino acids were analyzed using principal components (PC) to capture the common variation among the 15 amino acids tested. The first PC was graphed to visually demonstrate differences between control and treated samples. Glyphosate residues, pollen viability, and fluorescence colour properties were analyzed for differences between years post application (1 or 2) and for differences between treatments (control or treated) in fireweed flowers. Since repeated measures were taken across the fluorescence data to appropriately capture the variation that existed, those repeated measures were averaged to true replicate (site level) prior to analyzing for significant differences between controls and treated samples. Results Growth Chamber Experiment Week 1 measurements were made pre-treatment, while weeks 2–5 were post-treatment. Height data was normally distributed, and both one-way ANOVA and regression analysis indicate that plants were significantly impacted by week (t = 5.212, p < 0.001) and treatment (t = -2.265, p = 0.025) but not by chamber condition (t = 0.584, p = 0.561). This result indicates that the range of temperature exposures made little difference on the overall range of growth of fireweed plants, and the treatment and timing of height measurement (number of weeks post treatment) were both important factors in the outcome of plant height (r = 0.442, r 2 = 0.195, p < 0.001). Significant differences in PE were found between treated and control plants across weeks of experimentation post-treatment, especially in newly formed leaves (those formed post GBH application); however, there were no significant differences between growth chambers. Data from growth chambers were used as replicates given that there were no significant differences between them. When only the newest leaves were included in analysis, PE was significantly lower in all treated plants compared to controls in all weeks post-treatment (Mann Whitney U = 267.50, p < 0.001) (Fig. 2 ). In fireweed treated with GBH, many plants responded with dieback of the stem apex (Fig. 3 ). This response was seen in 73% of treated plants by the end of the experiment; we did not observe this dieback in any of the control plants. Shoot apex dieback was first observed ten days after treatment. Operational Forests Results Stamen fluorescence Fireweed stamen and pollen were substantially illuminated by the royal blue light fluorescence filter (wavelength 440–460 nm) (Fig. 4 ). The stamen fluorescence data were normally distributed. There were no significant differences in the measured colour characteristics (Table 2 ) between controls and sites sampled two years post-treatment. However, sites sampled one year post-treatment were significantly different in mean blue pixel intensity (mean blue) (F = 17.957, p = 0.003; Fig. 5 ), the most frequent hue observed (hue typical) (F = 6.681, p = 0.032; Fig. 5 ), and mean pixel saturation value (mean saturation) (F = 18.134, p = 0.003; Fig. 4 ). The range of mean blue decreased, hue typical increased, and mean saturation increased in treated samples (Table 4 ). Table 4 Comparison between fireweed ( C. angustifolium ) flowers from sites treated with glyphosate-based herbicide one year prior to sample collection and control sites, for mean blue pixel intensity, most frequent hue observed (hue typical), and mean pixel saturation value of floral stamen during fluorescence analysis. Fluorescence colour characteristic Glyphosate-treated samples (pixels) Control samples (pixels) Mean blue 4.43 to 5.45 5.75 to 14.06 Mean hue typical 34.02 to 41.86 28.56 to 37.68 Mean saturation 241.31 to 242.13 217.76 to 238.67 Pollen viability The pollen viability data were not normally distributed. Pollen viability differed significantly between control and treated sites (Kruskal Wallis, H = 15.569, p < 0.001), between samples collected one- and two years post-treatment (H = 20.925, p = 0.021), and between samples collected one-year post-treatment and controls (H = 30.602, p < 0.001). No significant difference was observed between samples collected two years post-treatment and controls (H = 9.676, p = 0.303) (Fig. 6 ). Amino Acids Amino acid data were normally distributed. GBH treated fireweed flowers had lower total amino acids than controls (Table 5 ). Upon analysis of 15 individual amino acids in fireweed flowers, and through a principal component analysis of the common variation in these amino acids, we found that fireweed flowers harvested from areas treated with GBH two years prior to sampling generally contained significantly lower amounts of the amino acids (F = 9.267, p = 0.005, n = 28) (Fig. 7 ). The principal component tested accounted for 74.83% of the variation amongst the 15 amino acids. Total crude protein (F = 0.617, p = 0.439), and amino acids glutamic acid (F = 1.523, p = 0.227) and serine (F = 0.377, p = 0.544) were the only compounds insignificantly different between control and treated samples in fireweed flowers. Table 5 Mean concentrations of amino acids tested in C. angustifolium flowers from operational forest cutblocks of northern British Columbia, Canada. Treated flowers were sampled from cutblocks treated with glyphosate-based herbicides two years prior to sampling, and controls were untreated. Amino Acid Control (mg/g) Treated (mg/g) Crude Protein 99.600 105.613 Alanine 4.704 4.439 Arginine 4.489 4.032 Aspartic Acid 7.120 6.701 Glutamic Acid 9.579 9.405 Glycine 4.937 4.447 Histidine 2.043 1.821 Isoleucine 3.951 3.556 Leucine 6.518 6.035 Lysine 5.123 4.709 Phenylalanine 4.489 4.196 Proline 5.045 4.541 Serine 3.615 3.553 Threonine 3.358 3.121 Tyrosine 2.822 2.696 Valine 4.702 4.267 Total amino acids 72.495 67.520 Glyphosate Residue The majority of the composite floral samples tested for glyphosate residues one and two-years post-treatment, from the operational cutblocks, contained glyphosate residues (Fig. 8 ). The mean level of glyphosate residue detected in samples collected one year post-treatment was 19.6 ppb and 51.0 ppb was the highest concentration detected. The mean level present in samples collected two years post-treatment was 18.9 ppb and 62.0 ppb was the highest concentration measured. Glyphosate residues were not detected in control samples; therefore, there was a significant difference detected between the amount of glyphosate residue present between controls, one year post-, and two years post-treatment samples (Kruskal Wallis, H = 20.017, p < 0.001, n = 45). There were no statistical differences between glyphosate residues in plants sampled one- and two-years post-treatment (Mann Whitney U standardized test statistic = 0.021, p = 0.983, n = 30). Discussion Stress symptoms were confirmed in fireweed after exposure to GBH. Shoot dieback combined with overall restricted height growth and reduced photosynthetic efficiency in treated plants during our controlled experiment confirms that stress is induced by GBH treatment in fireweed plants that have been exposed to sub-lethal concentrations. Since stress responses to GBH vary by species [ 12 ], documenting the strategies used by fireweed is important for a better understanding of plant community response to GBH use. Shoot dieback is a specific strategy that only some plant species implement to rid their tissues of contaminants [ 12 ]. Notably, it is the reproductive structures at the shoot apex of the treated fireweed plants that are impacted by the dieback, suggesting that reproductive capacity would be delayed and potentially these plants would yield no fruit or seed production within the first-year post-treatment. Thus, we can conclude that, with respect to objective 1 of our study, growth potential of fireweed is reduced significantly over the first year after application, which in turn reduces its potential to reproduce for at least one year. This is further supported by the operational samples tested which showed significantly reduced pollen viability and fluorescence one-year post-GBH treatment. A reduction in pollen viability, which we used as an indicator for reproductive capacity, was present in fireweed one-year post application indicating that GBH initially had a significant impact on pollen quality. However, the fact that there was no significant difference between controls and samples treated two years prior to collection in terms of pollen viability, indicated that there is likely a recovery in pollen quality over time. This recovery likely varies by species [ 4 ]. Conditions and disturbances that lead to reductions in pollen viability, such as the application of sub-lethal GBH, may greatly reduce the quality of pollen and therefore reduce fruit production and reduce the rewards available to insect pollinators. We can conclude that the reproductive capacity of fireweed is reduced within one-year post-treatment by GBH, based on changes to pollen viability. Further research should include the investigation of female floral components to determine if they are also altered by GBH and whether they follow a similar recovery timeline. Our findings demonstrate that GBH have an impact on the fluorescence of male reproductive structures of forest understory plants. The reduction in the fluorescence emission of blue spectral wavelengths of anthers and pollen within the first-year post GBH treatment potentially impairs the biocommunication between flowers and arthropods, a function that is vital to ecosystem processes like pollination. Bees have trichromatic vision with ultraviolet, blue, and green photoreceptors in their compound eyes [ 26 , 48 ]. In bumblebees ( Bombus spp.) for example, preferential excitation of one or two of the photoreceptor types plays an important role in innate colour preferences [ 48 ] and bumblebees are able to discriminate minute changes in the intensities of colour [ 49 , 50 ]. Therefore, the changes we observed to the mean blue intensity of the anthers and pollen could mean that the blue photoreceptor in a bumblebee’s compound eye would be less likely to detect a flower [ 48 ]. Additionally, the increase in typical hue observed indicates that the dominant wavelength present may no longer be the blue spectral wavelength. Combined with an increase in saturation, it is possible that the presentation of other spectral wavelengths in GBH treated plants are greater than that of the blue wavelength, potentially confusing biocommunication between flowers and pollinators. According to our results, the impact on fluorescence is mostly resolved by the second-year post-treatment indicating morphological recovery of the flower. Further research is required to determine if the changes in fluorescence we observed do result in changes in biocommunication and determine if the changes in fluorescence of fireweed flowers are correlated to changes in concentrations of anthocyanins, or other secondary metabolites, which serve other functions in addition to aiding in biocommunication. Decreased amino acids were noted in our samples two-years post-treatment, indicative of decreased nutritional value. Since we do not have amino acid concentration data from one-year post treatment it is impossible to determine if the amino acid levels are recovering at year two, as was shown with the other characteristics we investigated. Studies have shown decreased levels of nitrogen in plants treated with glyphosate [ 19 ], and nitrogen is essential for the synthesis of amino acids and protein, therefore reduced amino acids are likely related to changes in nitrogen. The change noted in amino acids may have a large impact on insects that derive greater proportions of their diet from pollen and nectar. For example, queen bees eat a great deal of pollen and nectar to build fat reserves for hibernation, and the larvae feed on pollen that is brought to the colony [ 37 ], therefore they may be particularly susceptible to a reduced content. According to work by Barruad et al. (2022) [ 51 ], the performance of Bombus terrestris was explained by pollen amino acid content. Low amino acid content was correlated to low pollen collection:brood mass ratio [ 51 ] and to low body mass [ 52 ]. Based on their assessment, total amino acid concentrations over 200 mg/g are beneficial for bumblebees, and specific amino acids are important in higher quantities for optimal production, such as alanine, leucine, phenylalanine, proline, and tyrosine. The pollen of fireweed flowers may be equal or greater in total amino acids than what is recommended for bee nutrition, as we know it is highly sought after and used by bees in areas of northern BC [ 29 , 32 , 38 , 53 ], but that is not evident in our data. When we compare our results of amino acid content to the results of Barruard et al. (2022) [ 51 ] our samples were much lower in total amino acid content. This could be due to the fact that we tested whole flowers, and not just pollen. Future research should focus on the collection of solely pollen in GBH treated areas to elucidate these findings. Additionally, profiling of fireweed flowers has been conducted in parts of northern Europe for use in nutrition supplements, some showing differences in secondary metabolites based on site conditions [ 20 , 21 , 22 ]. Future investigation should apply this method of chemical profiling to areas treated with GBH to determine how this factor compares to natural environmental variation over larger areas. We confirmed the presence of glyphosate residues in floral tissues one and two-years post-treatment. The amount of glyphosate residue present in floral tissues remained similar between one- and two-years post treatment, likely due to the negative exponential degradation of residues in floral tissues which leads to the majority of the glyphosate residue degrading within the first year after application [ 5 ]. Even though residues may not persist at high levels for greater than one year, our research suggests that the effects of glyphosate residues to plant anatomy and physiology may persist for a longer period, and that these effects are not necessarily linearly correlated to the amount of residue that persists in the tissues. Our data confirms that residues persist in fireweed flowers for at least two years; however, how long these effects last is still unknown. Conclusion Fireweed is often a pioneer species responsible for nutrient cycling in disturbed environments, provides an important source of floral abundance for pollinating insects and birds, and is also an ethnobotanically important medicinal plant. This plant is a prominent component of the herbaceous layer in forests of northern British Columbia, Canada, and shows stress symptoms after sub-lethal exposure to glyphosate-based herbicides used for vegetation management. These symptoms include reduced growth and reproductive capacities. Specifically, height, reproductive shoot apex formation, pollen viability, floral fluorescence, and amino acid content of flowers are all altered for at least one-year post-exposure. These changes to form and make-up have significant implications for the function of the ecosystem in these managed areas, including potential to change biocommunication with insect pollinators, the quality, and/or the quantity of food produced for wildlife and humans. Declarations Acknowledgements This research was funded by the Natural Science and Engineering Research Council of Canada, the Habitat Conservation Trust Foundation and the Weston Family Foundation. The authors would like to thank Deniz Divanli and Kate Rozmarniewich for their assistance in field sampling, data collection and laboratory sample analysis. Author Contributions L.J.W – supervision, funding acquisition, resource provision, field sampling, experimental design, data analysis, manuscript compilation, and revision.A.R.G – field sampling, laboratory analysis, data analysis, writing, revision.L.B-K – carried out experimental protocol, laboratory analysis, data analysis, writing, revision.B.H – field sampling, laboratory analysis, data analysis, writing. Data Availability Statement The datasets generated during the current study are available in the Dryad data repository, DOI: 10.5061/dryad.zgmsbccp8. Ethical Statement No species at risk were targeted for collection in this study, and all plants were obtained from healthy, extensive populations. No genetic manipulation of plants was conducted during this research. No permits, permissions, or licenses were required for plant tissue collection for the purposes of this research. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6011000","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":422122932,"identity":"884638f6-91d6-45b4-afc7-d9711cdf0b05","order_by":0,"name":"Lisa Wood","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACPmYY63gDAzM+lXDABld25gCxWuCsGwnEamFnf/iBsc1Gju/mG7PHBTUM8vwNBB3GYyzB2JZmLHk7x9x4xjEGwxkHCGthkGDcdjhxw+0cM2neBoYEBsJa2B//AGu5eQaiRZ6wFgYziC03eCBaDIhwmJlF4j+gX86klUnPOCZhuJGQFn7+449vfDgDDLHjh7dJF9TYyMsR0gIGCQimBDHqR8EoGAWjYBQQAgBsuDcGMhqT6AAAAABJRU5ErkJggg==","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":true,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Wood","suffix":""},{"id":422122934,"identity":"88822ff6-7202-4b54-8833-ceb91996c4de","order_by":1,"name":"Alexandra Golt","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Golt","suffix":""},{"id":422122937,"identity":"989ee522-0235-45d7-835b-f89421cd31b2","order_by":2,"name":"Laurel Berg-Khoo","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Laurel","middleName":"","lastName":"Berg-Khoo","suffix":""},{"id":422122938,"identity":"1aa20470-71f7-4279-885e-dda7d59d197e","order_by":3,"name":"Brian Hampton","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Hampton","suffix":""}],"badges":[],"createdAt":"2025-02-12 02:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6011000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6011000/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16938-x","type":"published","date":"2025-08-25T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77603220,"identity":"0cbe1633-286a-48f9-a84b-04a5b9b01f1f","added_by":"auto","created_at":"2025-03-03 13:11:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":255738,"visible":true,"origin":"","legend":"\u003cp\u003eMap of field study site locations treated with glyphosate-based herbicides in 2019, 2020, and 2021, within the Prince George Forest District, British Columbia, Canada. Each location contained a paired treated area and non-treated control area.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/1eac9e299ca0fd781014de2b.jpg"},{"id":77603229,"identity":"5b3b7551-cf61-4827-9cf9-aa75c3ca817c","added_by":"auto","created_at":"2025-03-03 13:11:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61255,"visible":true,"origin":"","legend":"\u003cp\u003eFireweed (\u003cem\u003eChamaenerion angustifolium\u003c/em\u003e) plant measurements at week 1 (pre-treatment) and week 2-5 post-treatment for control plants and plants treated with sub-lethal concentration of glyphosate-based herbicide (GBH). Top: Mean chlorophyll fluorescence values (Fv/Fm) measuring photosynthetic efficiency, Bottom: Shoot height measurements between the root collar and shoot apex.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/36b48614a83bf13a973364c4.jpg"},{"id":77603222,"identity":"d342776b-df7b-46a8-b035-0d0387367eb4","added_by":"auto","created_at":"2025-03-03 13:11:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60631,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of reproductive shoot development in control (left) and GBH treated plants (right) 23 days after GBH application date, with notable shoot dieback in the treated plant. Plants were grown in environmentally controlled chambers at the University of Northern British Columbia, Canada.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/cd012a545b2ccece8c62ed7f.jpg"},{"id":77603221,"identity":"6e84b601-1819-4878-92e7-64a753a893f0","added_by":"auto","created_at":"2025-03-03 13:11:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41593,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of fireweed (C. angustifolium) stamen under white light (left) and royal blue (right) light.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/a96cf9dbf9aa76d87834b5c3.jpg"},{"id":77604535,"identity":"239d0e40-046d-437c-a820-f9e4af6b4e9e","added_by":"auto","created_at":"2025-03-03 13:27:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91926,"visible":true,"origin":"","legend":"\u003cp\u003eMean blue pixel intensity, most frequent hue observed (mean hue typical), and mean pixel saturation (+/- 2 SE) of stamen of \u003cem\u003eC. angustifolium \u003c/em\u003eflowers photographed from control samples and glyphosate-based herbicide treated samples one- and two-years post-treatment. Stamen photographed under royal blue (440 – 460 nm) fluorescence (α = 0.05). Samples collected from untreated operational forestry cutblocks (control sites; n = 5) and cutblocks treated in northern BC, Canada, one- (n = 5) and two-years (n = 5) prior to sampling.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/909b208d70086fd4efd643a8.jpg"},{"id":77603354,"identity":"0fdd955e-f8b0-4886-9d7c-6a507455b1e5","added_by":"auto","created_at":"2025-03-03 13:19:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":21749,"visible":true,"origin":"","legend":"\u003cp\u003eMean (+/- 2 SE) pollen viability present in \u003cem\u003eC. angustifolium \u003c/em\u003eflowers collected from operational forestry cutblocks untreated (control sites; n = 5) and treated with glyphosate-based and sampled in northern BC, Canada, one- (n = 5) and two-years (n = 5) post glyphosate treatment (α = 0.05).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/eccd6d6baf973b80f4cc16cf.jpg"},{"id":77603223,"identity":"5331f472-06b3-47db-9c6b-78c32253ad7e","added_by":"auto","created_at":"2025-03-03 13:11:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":31031,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of 15 amino acids found in fireweed (\u003cem\u003eC. angustifolium\u003c/em\u003e) floral tissues, which represented 74.83% of the common variation across these amino acids. Floral samples collected from cutblocks treated with glyphosate-based herbicides two-years prior to sampling, and adjacent control areas in the Omineca region of northern British Columbia, Canada.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/fec9701c9292ae2ba1d1fb1f.jpg"},{"id":77603357,"identity":"a1713659-c6b5-4aab-bb80-6f9f177ff7d2","added_by":"auto","created_at":"2025-03-03 13:19:39","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16353,"visible":true,"origin":"","legend":"\u003cp\u003eMean (+/- 2 SE) glyphosate residue present in \u003cem\u003eC. angustifolium \u003c/em\u003eflowers collected from operational forestry cutblocks untreated (control sites) and treated with glyphosate-based herbicides sampled in northern BC, Canada, one- and two-years post glyphosate treatment (α = 0.05). Three replicate samples were run from each of the three composite samples which contained 60 to 90 individual flowers and buds.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/d0fdcb2312831ea55e5498df.jpg"},{"id":90344983,"identity":"f0dbf8a4-d86f-4634-a4d9-cdbba1950909","added_by":"auto","created_at":"2025-09-01 16:08:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1716144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6011000/v1/88dc19e4-6473-4129-8183-b4303ec1bb85.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reduced function in Chamaenerion angustifolium after sub-lethal glyphosate exposure","fulltext":[{"header":"Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eGlyphosate-Based Herbicides in Forest Plants\u003c/h2\u003e \u003cp\u003eGlyphosate-based herbicides (GBH) are commonly used in Canadian forest management practices, to reduce growth of deciduous tree and shrub species in areas where coniferous trees are harvested and planted, thereby reducing competition for early growth of conifer seedlings [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During application many non-target plants are exposed to GBH due to spray drift or their proximity to targeted plants via the spray cloud/ rain trajectory [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Residues of glyphosate and aminomethylphosphonic acid (AMPA, a product of glyphosate degradation) have been found in tissues of non-target understory plants in BC forests up to 12-years after initial exposure [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and evidence exists that degradation rates are slower and plant susceptibility greater, in colder climates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Sublethal glyphosate residues in plant tissues have been shown to induce stress symptoms such as chlorosis, stunted growth, reduced leaf area, deformed leaves, and changes to flowers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although GBH has been found to remain in the forest environment and cause changes to plant growth and reproduction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the current understanding of these changes and their significance remains limited. What is yet to determine is whether the persistence of low concentrations of GBH residues have an impact on the function of plants within their ecosystem, and since each species responds differently to GBH application, it is important that individual species are investigated and their responses documented.\u003c/p\u003e \u003cp\u003eGlyphosate (N-(phosphonomethyl) glycine) is a glycine analog that acts as a non-selective herbicide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is absorbed across vegetative plant surfaces and is translocated along the same pathways as sugars produced in photosynthesis, allowing it to quickly reach areas of high metabolic activity such as meristematic tissue [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Inside the plant cell glyphosate acts as an inhibitor of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) in the shikimate metabolic pathway by competing with phosphoenolpyruvate for the enzyme\u0026rsquo;s active site [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This inhibition leads to an increased concentration of shikimic acid that may affect protein metabolism, which leads to the inhibition of plant growth and ultimately the death of the plant, depending on the concentration applied [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The shikimate pathway is responsible for producing aromatic amino acids including phenylalanine, tryptophan, and tyrosine, phenolic compounds that have antioxidant activity within the cells of both plants and herbivores, and other important secondary metabolites [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; therefore, glyphosate may prevent their production. Wang (2001)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] found that the concentration of tryptophan decreased rapidly three days after glyphosate treatment, and the level remained low [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Since tryptophan plays a major role as a precursor in the synthesis of several important plant regulatory chemicals, as well as plant pigments and other biocommunicative chemicals, it follows that a reduction in tryptophan may severely affect the synthesis of these chemicals. However, Wang (2001)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] found that the effect of glyphosate on aromatic acid synthesis was inconsistent, as it increased the concentration of some aromatic acids and decreased others, indicating that the production of these metabolites in plants is more complex than is currently understood. Glyphosate and AMPA also appear to prevent production of chlorophyll and promote its degradation, lowering both photosynthetic ability and rate of carbon fixation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBecause of the known interactions that glyphosate can have with plant chemistry, there is reason to believe that glyphosate may alter ecosystem functions such as producing lower quality food for wildlife. Plants are the base of the food web and life on earth evolved to rely on the nutrition provided by their abundance. A plant\u0026rsquo;s chemical make-up provides the building blocks for the growth and development of animals [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], therefore, if a plant population is nutritionally compromised in a given space, there are potential health consequences for the rest of the ecosystem relying on those plants. Plant components such as amino acids and proteins, and compounds such as anthocyanins are among the plant chemicals that serve a pivotal role in plant perpetuation and the functional ecology of forest communities. For example, many insects consume pollen for the primary purpose of obtaining protein and fat thereby supporting the growth and development of insect larvae [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Anthocyanins found in plants, are known for their antioxidative and antimicrobial properties [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome anthocyanins are also fluorescent compounds that are found in the anthers and pollen grains of select species and act to attract or deter animals, and some were found to protect anthers and pollen from UV-induced damage [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Glyphosate exposure has been found to reduce attraction towards UV light in bumble bees [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, if antioxidants responsible for fluorescence (often caused by the interaction between the anthocyanins and UV light) are altered by glyphosate treatments, then biocommunication may be compromised, potentially impacting foraging efficiency for wild bees, a keystone functional group in many ecosystems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy species: Chamaenerion angustifolium\u003c/h2\u003e \u003cp\u003e \u003cem\u003eChamaenerion angustifolium [L.) Scop. (Myrtales:Onagraceae)]\u003c/em\u003e (fireweed) is a prominent plant species in British Columbia (BC), Canada. Fireweed is widely distributed in the northern temperate and boreal regions of Canada and the northern United States [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and is also found in northern parts of Europe [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It is found in a wide variety of habitats from boreal forests to gravel bars, and from low to subalpine elevation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is an erect perennial herb which can reach a height from 0.5 to 3 m tall, and has alternate, entire lance-shaped leaves reaching from 5 to 20 cm in length [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFlowering in fireweed occurs continually from June to September. Flowers are magenta or deep pink in colour, have long pedicels and are borne in indeterminate racemes, with the oldest flowers born towards the base and new flowers produced as the shoot grows [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Each individual flower has four petals, 10 to 15 mm in length, and four lanceolate, spreading sepals that are the same colour as the petals. Each flower has eight stamens, a style that is longer than the stamens, a four-cleft stigma, and a four-loculed ovary [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The anthers release blue-green or yellow pollen held together with sticky viscin threads [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The green, fleshy, and slightly concave nectary produces nectar with a high sugar concentration (approximately 77%) and is located on the upper end of the inferior ovary [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn many areas of northern BC, fireweed is one of the first successional species to re-establish after a disturbance such as forest harvesting, natural resource-related vegetation management, or a fire. Fireweed can quickly colonize a disturbed area and recycles early nutrients through the system via its own decaying plant matter, thereby improving soil quality for other plant species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Dense colonies of fireweed may delay the development of shrubby vegetation, thus assisting in the establishment of coniferous species before competitive shrubs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFireweed has been used for many centuries by Indigenous people across North America for food. The majority of First Nations in the interior of BC \u0026ndash; including the Nlaka\u0026rsquo;pamux, Stl\u0026rsquo;atl\u0026rsquo;imx, Secwepemc, Carrier, Wet\u0026rsquo;suwet\u0026rsquo;en, Sekani, and Nisga\u0026rsquo;a \u0026ndash; collected and ate the raw pith of young stalks, prior to flowering [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Many BC coastal First Nations such as the Sechelt, Squamish, Nuxalk, Haida, and Tsimishian also foraged the pith [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The Saanich First Nations steeped the young leaves to make tea [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Fireweed also had social significance in Haida communities. Patches found close to Haida villages were often owned by certain high-class members, and other individuals had to ask permission to harvest from these patches [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. All parts of fireweed plants were (and are) used by Indigenous peoples in western Canada for its anti-inflammatory and antiseptic properties [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is important to investigate the impacts of glyphosate-based herbicides (GBH) on fireweed to understand the potential changes to the beneficial ecological and cultural services provided by this species.\u003c/p\u003e \u003cp\u003eFireweed is also an important food source for wildlife. The shoots are consumed by deer, moose, caribou, muskrat, and hares [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A multitude of insects, such as bees and flies, use fireweed nectar as it is continuously produced after anthesis and until abscission of the floral parts occurs, which is uncommon in other wildflowers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The most common pollinators for fireweed are bees [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; fireweed is an important plant for the honey industry in Canada [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Rationale and Objectives\u003c/h3\u003e\n\u003cp\u003eDue to the prevalence of GBH use worldwide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] it is important to further investigate how GBH interacts with plants to produce a clearer picture of the functionality of GBH exposed ecosystems. Here we collate the findings of our study of the responses of fireweed (\u003cem\u003eChamaenerion angustifolium\u003c/em\u003e) to sublethal GBH treatment in northern British Columbia, Canada. We collected samples of fireweed from operationally managed forest stands one and two years after GBH applications took place and we conducted controlled experimentation using fireweed, to better understand this species\u0026rsquo; growth and reproductive potential. Ultimately, we aimed to contribute to the knowledge base on how plant systems function after GBH treatments, studying fireweed as a representative perennial, herbaceous angiosperm.\u003c/p\u003e \u003cp\u003eOur specific objectives were:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the growth potential of fireweed post GBH application, and to confirm fireweed stress symptoms induced by sublethal glyphosate exposure.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo quantify the reproductive capacity and the biocommunicative potential of fireweed flowers, post GBH application.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine changes in nutrition of fireweed flowers, specifically focusing on amino acids because they are produced downstream of the shikimic acid pathway.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eWe conducted a greenhouse experiment with fireweed at the University of Northern British Columbia (UNBC). We collected fireweed plants, for study and testing, from operationally managed forests during field outings in 2021 and 2022.\u003c/p\u003e\n\u003ch3\u003eGrowth Chamber Experimentation\u003c/h3\u003e\n\u003cp\u003eThe specific goal of this experiment was to determine the morphological and physiological response of fireweed to GBH treatment under controlled growth conditions (contributing to Objective 1). Wild fireweed plants were collected from sites on and near the UNBC Prince George campus, as rhizomes with small vegetative shoots immediately following spring emergence. Rhizomes of approximately 15\u0026ndash;20 cm lengths were planted in individual 2L pots with a growing medium consisting of 78% peat moss, 15% vermiculite, and 7% perlite with 0.46 mL/L dolomite lime, 0.46mL/L Micromax micronutrient formulation, and 3.65mL/L Osmocote 14-14-14 slow-release fertilizer. All plants were brought into a greenhouse compartment of the I.K. Barber Enhanced Forestry Lab (EFL) at UNBC and allowed to acclimate at 20⁰C for ten days before placement in growth chambers. Plants were divided between three growth chambers (five treatment plants and five controls were placed in each chamber) set to different temperatures (chamber 1\u0026thinsp;=\u0026thinsp;20⁰C day/8⁰C night, chamber 2\u0026thinsp;=\u0026thinsp;25⁰C day/10⁰C night, chamber 3\u0026thinsp;=\u0026thinsp;30⁰C day/15⁰C night) with a 12 hour temperature cycle and 16 hour photoperiod. Plants were frequently monitored and individually watered as needed to ensure no water stress was experienced.\u003c/p\u003e \u003cp\u003eFive treatment plants from each growth chamber were taken to an outdoor location, sprayed with a GBH formulation, allowed to dry, and returned to the growth chambers. VisionMax\u0026reg; GBH (540g/L a.e. potassium salt), normally applied at 4 L/ha (540g/L * 4L/ha\u0026thinsp;=\u0026thinsp;2160 g over 10,000m\u003csup\u003e2\u003c/sup\u003e (1 ha); application rate of 43.2 g/L over 1 ha), was applied at a low concentration to induce a sub-lethal response. We applied 0.2mL of VisionMax\u0026reg; concentrate in 100mL of solution, by hand-held sprayer. Thus, the application rate was 2.5% of the normal rate; 540g/L (0.0002L) /0.1 L\u0026thinsp;=\u0026thinsp;1.08 g/L application rate over a 1 m\u003csup\u003e2\u003c/sup\u003e or 0.018 g over a 1 m\u003csup\u003e2\u003c/sup\u003e area.\u003c/p\u003e \u003cp\u003eTotal length of the stem between the root collar and shoot apex and photosynthetic efficiency (PE), assessed by chlorophyll fluorescence of each plant, were measured weekly, the first of these measurements were made prior to treatment. The total overall shoot health was evaluated at the conclusion of the experiment. PE was estimated by calculating the maximum quantum yield of photosystem II (PSII) (Fv/Fm), detected using a portable Hansatech chlorophyll fluorometer. Photosystem II is the most sensitive component within the photosynthetic pathway [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and was therefore used as a measure of plant stress response to drought conditions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To measure PE, the target leaves were first covered with a clip for at least one minute to ensure the portion of the leaf tested was in complete darkness. Then, the Fv/Fm ratio was automatically calculated by the chlorophyll fluorometer by opening the clip to the fluorometer\u0026rsquo;s sensor, where it detected the minimal fluorescence (Fo), followed by the maximal fluorescence (Fm), which were then related using the following equation: Fv\u0026thinsp;=\u0026thinsp;Fm \u0026ndash; Fo [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For healthy plants the Fv/Fm ratio should be between 0.7\u0026ndash;0.83 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eOperational Study in 2021–2022:\u003c/h3\u003e\n\u003cp\u003eOperational sampling was conducted to meet objectives 2 and 3; to determine if the reproduction, biocommunication, or nutritional components of fireweed were interrupted by GBH applications in the real world. We chose to study pollen viability as a representation of reproductive capacity, floral fluorescence as an indicator or biocommunicative potential, and amino acid content as a marker of nutritional composition.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStudy Sites\u003c/h2\u003e \u003cp\u003eThe area of study consisted of 15 sites, each site containing paired treated\u0026thinsp;+\u0026thinsp;control areas, in the Prince George Forest Region of BC, Canada (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Five of the sites were treated with GBH in 2019, five were treated in 2020, and five were treated in 2021. Each treated site had a paired control site (not treated with GBH) of the same vegetation complex, that was sampled.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSamples were collected from these sites during June and July of 2021 and 2022 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sites were previously logged using a clear-cut method of harvesting, planted with coniferous trees, lodgepole pine (\u003cem\u003ePinus contorta\u003c/em\u003e Dougl. ex Loud) and/or hybrid white spruce (\u003cem\u003ePicea glauca\u003c/em\u003e (Moench) Voss x \u003cem\u003eengelmannii\u003c/em\u003e Parry ex. Engelm) and treated with a GBH in either 2019, 2020, or 2021 (one-year before sampling) when planted trees were between 5 and 15 years of age. The sites sprayed in 2019 and 2020 were treated with the GBH formula VisionMax\u0026reg; and the sites sprayed in 2021 were treated with the GBH formula GlySil\u0026reg;. VisionMax\u0026reg; (Canadian registration no. 27736 under the Pest Control Products Act) was aerially applied at rates of 3.3\u0026ndash;4.0 L/ha (resulting in concentrations of 1.78\u0026ndash;2.16 kg a.i. ha-1). GlySil\u0026reg; (Canadian registration no. 29009 under the Pest Control Products Act) was applied aerially at 6.0 L/ha (resulting concentration of application was 2.13 kg a.i. ha-1). Parts of these sites were left untreated (pesticide-free zones) due to the presence of streams to prevent glyphosate contamination and run-off. These untreated areas within the sites or other nearby untreated regions with similar vegetation complexes served as experimental controls. Paired treatment and control areas for each site were identified from forest industry operation maps and were confirmed visually on site through marked treatment lines.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSample sites treated with glyphosate-based herbicides in 2019, 2020, and 2021 in northern British Columbia, Canada. Each location contained a paired treated area and non-treated control area.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLatitude (N), Longitude (W)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBEC Zone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eElevation (m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal Treatment Area (ha)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDate Herbicide Applied\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLS61A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.30553,\u003c/p\u003e \u003cp\u003e 122.05421\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSvk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7-Aug-2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLS057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.30460, \u003c/p\u003e \u003cp\u003e122.01806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSvk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7-Aug-2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRAI063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.37277, \u003c/p\u003e \u003cp\u003e122.33604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSvk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7-Aug-2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRAI081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.31566, \u003c/p\u003e \u003cp\u003e122.19036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSvk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15-Aug-2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e271-001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.63264, \u003c/p\u003e \u003cp\u003e121.73552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSvk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1-Aug-2021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.76292, 122.77779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9-Sept-2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.76236, 122.77825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9-Sept-2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.65097, 122.78725\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9-Sept-2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.65695, 122.77856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9-Sept-2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.66973, 122.77174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9-Sept-2020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.4454, 122.2919\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eESSFwk1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16-Aug-2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.4441, 122.2845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eESSFwk1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16-Aug-2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.3621, 123.1156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19-Aug-2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53.3618, 123.1105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19-Aug-2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.0434, 123.2552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSBSdw3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8-Aug-2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSites were located in the SBSvk, SBSdw2, SBSdw3, and ESSFwk1 biogeoclimatic ecosystem classification (BEC) zones (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The BEC system was developed and is used within BC to delineate regional differences in topography and dominant vegetation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). BEC zones and subzones represent divisions that define the climate and dominant vegetation of an area. The first three capital letters represent the zonal information, based on the dominant tree species of the area, and the following two lower case letters describe the subzone. The first letter of the subzone name describes the relative precipitation, and the second letter describes the relative temperature. For example, in the \u0026ldquo;SBSwk3\u0026rdquo;, SBS stands for \u0026ldquo;sub-boreal spruce\u0026rdquo; and wk3 indicates that the subzone is \u0026ldquo;wk\u0026rdquo; or the wet-cool subzone; and the variant is \u0026ldquo;3\u0026rdquo; or the third climate variation type identified within this subzone. Each variant is drier, wetter, snowier, warmer, or colder than what is considered typical for the subzone in general [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe SBS is characterized by two dominant tree species \u0026ndash; hybrid white spruce and subalpine fir (\u003cem\u003eAbies lasiocarpa\u003c/em\u003e (Hook.) Nutt.), as well as extensive stands of lodgepole pine in drier areas [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The SBS typically has shorter winters and longer growing seasons than boreal areas, allowing for a wide range of subzones. The SBSvk (sub-boreal spruce, very wet cool) zone has the highest annual precipitation and the longest growing season precipitation and is therefore the wettest biogeoclimatic unit in the SBS [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Snowfall data were unavailable, but this subzone has the lowest mean annual temperature of the SBS units, and paper birch (\u003cem\u003eBetula papyrifera\u003c/em\u003e Marshall) spread throughout. The SBSdw3 encompasses a large area to the west of Prince George. The SBSdw3 is warm relative to other subzones with winter precipitation being relatively low and snowpacks with a mean depth of ~\u0026thinsp;2m. Growth-limiting factors in this subzone are drought on drier sites and frost on frost-prone and drier sites [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The SBSdw2 borders the SBSdw3 subzone to the south and is drier and warmer than the other subzones in the SBS due to its lower elevation and relatively low precipitation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The Engelmann spruce \u0026ndash; subalpine fir (ESSF) wk1 (Cariboo wet cool) zone occurs above the SBSwk zone between 1200\u0026ndash;1500 m elevation. The ESSF is characterized by high precipitation (\u0026gt;\u0026thinsp;1000 mm), half of which falls as snow [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin each sample site, we established 10 transects, a minimum of 20 m apart, and each 50 m in length, for collection of fireweed plants. We walked along each transect line and collected a minimum of 10 flowers, from a minimum of five individual plants on each transect (yielding a total collection of ~\u0026thinsp;100 flowers per site). We collected newly opened flowers where possible to try to limit environmental factors that could potentially affect flowers over time and as they age, such as light, temperature, wind, and rain. Flowers selected for sampling were cut at the base of the pedicle using small gardening shears. Floral samples were placed in labeled petri dishes and sealed with parafilm to ensure that floral parts, such as anthers and pollen, remained undisturbed and intact for analysis. Samples were placed in a cooler and were transported, stored, and analyzed at UNBC, Prince George, BC. No fireweed flower samples were collected from 271-001 treatment site as none of the fireweed bloomed during the season of collection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFloral Fluorescence Analysis\u003c/h3\u003e\n\u003cp\u003eAnalysis of fluorescence was conducted on 15 flowers per site immediately after returning from the field to avoid floral degradation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Photographs were taken using a Leica dissecting microscope with digital camera and Zen Lite software. We used the stereomicroscope adapter system by NIGHTSEA with a royal blue light (wavelength range of 440\u0026ndash;460 nm) to induce fluorescence. Stamens were separated from the rest of the flower and placed under the microscope to capture images with all the anthers in focus. We photographed stamen samples under white light (light emitted from microscope) and under the royal blue fluorescence filter. Analysis of resulting colour pixels captured by the photography was conducted using NIS-Elements Imaging Software (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVariables measured to assess pixel colour variation in photographed pollen and anthers of fireweed (\u003cem\u003eC. angustifolium\u003c/em\u003e) using NIS-Elements Imaging Software.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariable description\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBright variation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThe standard deviation of brightness values.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHue typical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescribes the most frequent hue in an object of field.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHue variation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescribes hue distribution of inner structure of an object of field.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntensity variation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescribes the inner structure of an object or field.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean red, mean green, mean blue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArithmetic mean of pixel intensities of one image component.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean brightness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArithmetic mean of brightness values of pixels.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean intensity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArithmetic mean of pixel intensities.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean saturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArithmetic mean of saturation values of pixels.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMin/max intensity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeasures minimum and maximum intensity values of pixels.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposite sample replicate numbers of fireweed (\u003cem\u003eC. angustifolium\u003c/em\u003e) flowers tested with each type of analysis. Flowers collected from forest sites treated with glyphosate-based herbicides and paired control areas in northern British Columbia, Canada.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalytical Test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreated one-year prior\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreated two-years prior\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlyphosate Residue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (3 replicates from 5 sites)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (3\u0026ndash;5 replicates from 8 sites)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (3\u0026ndash;5 replicates from 8 sites)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino Acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (5 replicates from 3 sites)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (5 replicated from 3 sites)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePollen Viability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 flowers (5 plants, 3 flowers per plant) per site\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePollen viability testing\u003c/h3\u003e\n\u003cp\u003eFollowing the imaging of the fresh flowers for fluorescence analysis, pollen was mechanically removed from anthers and Brewbaker and Kwack\u0026rsquo;s (B and K) medium was prepared for pollen viability testing [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. B and K medium was prepared by dissolving 50 mg boric acid, 150 mg calcium nitrate, 100 mg magnesium sulfate heptahydrate, and 50 mg potassium nitrate in 500 ml of deionized water. This stock solution was then stored at 4\u0026deg;C. Sucrose was dissolved in the solution immediately before viability testing.\u003c/p\u003e \u003cp\u003eThe amount of sucrose required to produce optimal germination of pollen grains varies between plant species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and we were unable to find existing literature on the optimal sucrose content for fireweed pollen germination. We initially tested the pollen viability of fireweed using varying amounts of sucrose at 5, 10, 15, 20, 30, 40, 50, 60, and 70%. The optimal concentration of sucrose that induced the highest rate of pollen tube formation in our trial was 15%; therefore, all proceeding viability testing was completed using this concentration of sucrose.\u003c/p\u003e \u003cp\u003ePollen grains became round once placed in B and K medium, and viable grains developed a long pollen tube [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. To be classified as viable, the pollen tube produced needed to be longer than the diameter of the pollen grain itself [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Fresh pollen grains were placed on a depression microscope slide. Two drops of B and K medium were added to each slide and pollen grains were mixed into the media using a toothpick. Each slide contained the pollen grains of one flower. Fifteen slides were prepared per site, representing three replicate flowers from each of five individual replicate plants per site (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A cover slip was placed on top of the slide and slides were incubated for 24\u0026ndash;36 hours at room temperature, in a Petri dish lined with moist filter paper and sealed with parafilm to maintain humidity. Upon completion of the incubation period, slides were observed using an Eclipse FN1 Nikon microscope at 10\u0026times; magnification. A microscope camera with NIS-Elements Imaging Software was used to view the pollen grains and capture images.\u003c/p\u003e \u003cp\u003eA total of 25 images were captured from each slide in a grid-like manner across the slide moving from the top left corner to the bottom right corner of the slide. The total number of pollen grains per slide were counted, and pollen viability was calculated for each flower and then averaged for control sites and treatment sites.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAmino Acids Analysis\u003c/h2\u003e \u003cp\u003eThe remaining flowers collected, varying in number depending on the abundance at each site, were dried in a kiln oven at 80\u0026deg;C for 24 hours in preparation for chemical analyses. Once dried, samples were ground using an IKA A 11 basic analytical mill. Removable parts of the mill were rinsed with water between samples, and the remaining parts were blown out with forced air to minimize the likelihood of cross-contamination between samples. There were fewer fireweed flowers at the sites treated one-year prior to sampling compared to two years prior, therefore samples were only selected to send for amino acid analysis from sites treated two years prior and their corresponding controls. The difference in abundance of fireweed was likely due to the time-since application, fireweed present at sites one-year post treatment were largely in the vegetative phase of growth. Testing for glyphosate-based residues was prioritized over amino acids to ensure that residues were present on the sites identified.\u003c/p\u003e \u003cp\u003eDried and ground fireweed flowers were subsampled to a weight of 0.5 g and sent to Central Testing Laboratories in Winnipeg, Canada, for amino acid testing. Amino acids were analyzed using the AccQ\u0026bull;Tag UPLC Method, which is a precolumn derivatization technique for amino acids. This method derivatizes amino acids, separates the derivatives with reversed-phase UPLC, and quantitates the derivatives based on UV absorbance or fluorescence intensity. The Waters AccQ\u0026bull;Tag Ultra Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, or AQC) is an N-hydroxysuccinimide-activated heterocyclic carbamate, a class of amine-derivatizing compounds. The AccQ\u0026bull;Tag Ultra reagent converts both primary and secondary amino acids to stable derivatives. The structure of the derivatizing group is the same for all amino acids, adding both UV absorbance and fluorescent character. Excess reagent hydrolyzes to yield 6-aminoquinoline (AMQ), a non-interfering by-product. Samples were tested for concentrations of: Alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, and valine. We also tested for total crude protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGlyphosate residue analysis of floral tissues\u003c/h2\u003e \u003cp\u003eComposite samples were created to meet a minimum 5 g dry matter mass requirements for glyphosate residue analysis of each sample. At least three replicates were made from flowers across each site (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlyphosate residues in fireweed tissues were analyzed by the Agriculture and Food Laboratory at the University of Guelph using liquid chromatography tandem mass spectrometry (LC-MS/MS). The glyphosate screening process reported each individual component separately, if detected. Prior to analysis, an aqueous extract of a homogenized subsample of plant material was prepared. Sample extracts were acidified and separated using solid-phase extraction. The LC instrument employed a cation guard column for chromatographic separation (Micro-Guard Cation-H cartridge 30 \u0026times; 4.6 mm), a mobile phase A (0.1% formic acid in nanopure grade H2O) and B (acetonitrile), with a flow rate of 1 ml/min and a total run time of 12 minutes. Retention time for glyphosate was 0.9 minutes. The autosampler temperature was 8\u0026deg;C, injection volume was 50 \u0026micro;l, and column oven temperature was 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C. Validation of results was completed using a five-step detection method to ensure no false positives. Blanks were tested along with samples to check for carry over; no coextracting contaminants were detected, the peak detected in the samples had the same retention time for two ion transitions, the ion ratios were correct in all instances relative to the certified standard used by the laboratory, and there was consistency among sample residues found, indicating reliability. The lab also reported when samples were considered above the minimum detection limit (MDL) of 5 ppb, and above the MDL but below the minimum quantification limit (MQL) of 20 ppb. For our data analysis, these parameters were used to indicate the presence of glyphosate residues. When a sample fell above the MDL of 5 ppb but below the MQL of 20 ppb, we used a conservative value of 6 ppb, so that these positive detections could be acknowledged, but not overestimated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using IBM SPSS 28.1. Normality of distribution was assessed using Shapiro-Wilk significance with a confidence level of 95%. If the data were normal, significant differences between the control and treated were analyzed with ANOVA. If the data were non-parametric, they were assessed for significance between the means using Kruskal Wallis or Mann Whitney U tests. Histograms were used to assess distributions so that any generalized linear models created were fit with the appropriate distribution curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eControlled Experiment Data\u003c/h2\u003e \u003cp\u003eData were grouped by growth chamber and by week to allow comparison between treatment and control groups. Linear regression was used to determine when independent treatment variables (including week#, chamber#, and treatment) had a significant impact on the response variable for normally distributed height data. Generalized linear modelling with a gamma distribution and a log link function was used for photosynthetic efficiency data that were not normally distributed. A percentage of total incidence was calculated for the number of shoot apices that were damaged out of the total plants assessed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOperational Field Data\u003c/h2\u003e \u003cp\u003eAmino acids were analyzed using principal components (PC) to capture the common variation among the 15 amino acids tested. The first PC was graphed to visually demonstrate differences between control and treated samples.\u003c/p\u003e \u003cp\u003eGlyphosate residues, pollen viability, and fluorescence colour properties were analyzed for differences between years post application (1 or 2) and for differences between treatments (control or treated) in fireweed flowers. Since repeated measures were taken across the fluorescence data to appropriately capture the variation that existed, those repeated measures were averaged to true replicate (site level) prior to analyzing for significant differences between controls and treated samples.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGrowth Chamber Experiment\u003c/h2\u003e \u003cp\u003eWeek 1 measurements were made pre-treatment, while weeks 2\u0026ndash;5 were post-treatment. Height data was normally distributed, and both one-way ANOVA and regression analysis indicate that plants were significantly impacted by week (t\u0026thinsp;=\u0026thinsp;5.212, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and treatment (t = -2.265, p\u0026thinsp;=\u0026thinsp;0.025) but not by chamber condition (t\u0026thinsp;=\u0026thinsp;0.584, p\u0026thinsp;=\u0026thinsp;0.561). This result indicates that the range of temperature exposures made little difference on the overall range of growth of fireweed plants, and the treatment and timing of height measurement (number of weeks post treatment) were both important factors in the outcome of plant height (r\u0026thinsp;=\u0026thinsp;0.442, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.195, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eSignificant differences in PE were found between treated and control plants across weeks of experimentation post-treatment, especially in newly formed leaves (those formed post GBH application); however, there were no significant differences between growth chambers. Data from growth chambers were used as replicates given that there were no significant differences between them. When only the newest leaves were included in analysis, PE was significantly lower in all treated plants compared to controls in all weeks post-treatment (Mann Whitney U\u0026thinsp;=\u0026thinsp;267.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn fireweed treated with GBH, many plants responded with dieback of the stem apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This response was seen in 73% of treated plants by the end of the experiment; we did not observe this dieback in any of the control plants. Shoot apex dieback was first observed ten days after treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOperational Forests Results\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eStamen fluorescence\u003c/h2\u003e \u003cp\u003eFireweed stamen and pollen were substantially illuminated by the royal blue light fluorescence filter (wavelength 440\u0026ndash;460 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The stamen fluorescence data were normally distributed. There were no significant differences in the measured colour characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) between controls and sites sampled two years post-treatment. However, sites sampled one year post-treatment were significantly different in mean blue pixel intensity (mean blue) (F\u0026thinsp;=\u0026thinsp;17.957, p\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), the most frequent hue observed (hue typical) (F\u0026thinsp;=\u0026thinsp;6.681, p\u0026thinsp;=\u0026thinsp;0.032; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and mean pixel saturation value (mean saturation) (F\u0026thinsp;=\u0026thinsp;18.134, p\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The range of mean blue decreased, hue typical increased, and mean saturation increased in treated samples (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between fireweed (\u003cem\u003eC. angustifolium\u003c/em\u003e) flowers from sites treated with glyphosate-based herbicide one year prior to sample collection and control sites, for mean blue pixel intensity, most frequent hue observed (hue typical), and mean pixel saturation value of floral stamen during fluorescence analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorescence colour characteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlyphosate-treated samples (pixels)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl samples (pixels)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean blue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.43 to 5.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.75 to 14.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean hue typical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.02 to 41.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.56 to 37.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean saturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e241.31 to 242.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e217.76 to 238.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePollen viability\u003c/h2\u003e \u003cp\u003eThe pollen viability data were not normally distributed. Pollen viability differed significantly between control and treated sites (Kruskal Wallis, H\u0026thinsp;=\u0026thinsp;15.569, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), between samples collected one- and two years post-treatment (H\u0026thinsp;=\u0026thinsp;20.925, p\u0026thinsp;=\u0026thinsp;0.021), and between samples collected one-year post-treatment and controls (H\u0026thinsp;=\u0026thinsp;30.602, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant difference was observed between samples collected two years post-treatment and controls (H\u0026thinsp;=\u0026thinsp;9.676, p\u0026thinsp;=\u0026thinsp;0.303) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAmino Acids\u003c/h2\u003e \u003cp\u003eAmino acid data were normally distributed. GBH treated fireweed flowers had lower total amino acids than controls (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Upon analysis of 15 individual amino acids in fireweed flowers, and through a principal component analysis of the common variation in these amino acids, we found that fireweed flowers harvested from areas treated with GBH two years prior to sampling generally contained significantly lower amounts of the amino acids (F\u0026thinsp;=\u0026thinsp;9.267, p\u0026thinsp;=\u0026thinsp;0.005, n\u0026thinsp;=\u0026thinsp;28) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The principal component tested accounted for 74.83% of the variation amongst the 15 amino acids. Total crude protein (F\u0026thinsp;=\u0026thinsp;0.617, p\u0026thinsp;=\u0026thinsp;0.439), and amino acids glutamic acid (F\u0026thinsp;=\u0026thinsp;1.523, p\u0026thinsp;=\u0026thinsp;0.227) and serine (F\u0026thinsp;=\u0026thinsp;0.377, p\u0026thinsp;=\u0026thinsp;0.544) were the only compounds insignificantly different between control and treated samples in fireweed flowers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean concentrations of amino acids tested in \u003cem\u003eC. angustifolium\u003c/em\u003e flowers from operational forest cutblocks of northern British Columbia, Canada. Treated flowers were sampled from cutblocks treated with glyphosate-based herbicides two years prior to sampling, and controls were untreated.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreated (mg/g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude Protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99.600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105.613\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.439\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.701\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.405\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.447\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.821\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.951\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.709\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.541\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.553\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.822\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.696\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.267\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal amino acids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e72.495\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e67.520\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eGlyphosate Residue\u003c/h2\u003e \u003cp\u003eThe majority of the composite floral samples tested for glyphosate residues one and two-years post-treatment, from the operational cutblocks, contained glyphosate residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean level of glyphosate residue detected in samples collected one year post-treatment was 19.6 ppb and 51.0 ppb was the highest concentration detected. The mean level present in samples collected two years post-treatment was 18.9 ppb and 62.0 ppb was the highest concentration measured. Glyphosate residues were not detected in control samples; therefore, there was a significant difference detected between the amount of glyphosate residue present between controls, one year post-, and two years post-treatment samples (Kruskal Wallis, H\u0026thinsp;=\u0026thinsp;20.017, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;45). There were no statistical differences between glyphosate residues in plants sampled one- and two-years post-treatment (Mann Whitney U standardized test statistic\u0026thinsp;=\u0026thinsp;0.021, p\u0026thinsp;=\u0026thinsp;0.983, n\u0026thinsp;=\u0026thinsp;30).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStress symptoms were confirmed in fireweed after exposure to GBH. Shoot dieback combined with overall restricted height growth and reduced photosynthetic efficiency in treated plants during our controlled experiment confirms that stress is induced by GBH treatment in fireweed plants that have been exposed to sub-lethal concentrations. Since stress responses to GBH vary by species [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], documenting the strategies used by fireweed is important for a better understanding of plant community response to GBH use. Shoot dieback is a specific strategy that only some plant species implement to rid their tissues of contaminants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, it is the reproductive structures at the shoot apex of the treated fireweed plants that are impacted by the dieback, suggesting that reproductive capacity would be delayed and potentially these plants would yield no fruit or seed production within the first-year post-treatment. Thus, we can conclude that, with respect to objective 1 of our study, growth potential of fireweed is reduced significantly over the first year after application, which in turn reduces its potential to reproduce for at least one year. This is further supported by the operational samples tested which showed significantly reduced pollen viability and fluorescence one-year post-GBH treatment.\u003c/p\u003e \u003cp\u003eA reduction in pollen viability, which we used as an indicator for reproductive capacity, was present in fireweed one-year post application indicating that GBH initially had a significant impact on pollen quality. However, the fact that there was no significant difference between controls and samples treated two years prior to collection in terms of pollen viability, indicated that there is likely a recovery in pollen quality over time. This recovery likely varies by species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Conditions and disturbances that lead to reductions in pollen viability, such as the application of sub-lethal GBH, may greatly reduce the quality of pollen and therefore reduce fruit production and reduce the rewards available to insect pollinators. We can conclude that the reproductive capacity of fireweed is reduced within one-year post-treatment by GBH, based on changes to pollen viability. Further research should include the investigation of female floral components to determine if they are also altered by GBH and whether they follow a similar recovery timeline.\u003c/p\u003e \u003cp\u003eOur findings demonstrate that GBH have an impact on the fluorescence of male reproductive structures of forest understory plants. The reduction in the fluorescence emission of blue spectral wavelengths of anthers and pollen within the first-year post GBH treatment potentially impairs the biocommunication between flowers and arthropods, a function that is vital to ecosystem processes like pollination. Bees have trichromatic vision with ultraviolet, blue, and green photoreceptors in their compound eyes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In bumblebees (\u003cem\u003eBombus\u003c/em\u003e spp.) for example, preferential excitation of one or two of the photoreceptor types plays an important role in innate colour preferences [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and bumblebees are able to discriminate minute changes in the intensities of colour [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therefore, the changes we observed to the mean blue intensity of the anthers and pollen could mean that the blue photoreceptor in a bumblebee\u0026rsquo;s compound eye would be less likely to detect a flower [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additionally, the increase in typical hue observed indicates that the dominant wavelength present may no longer be the blue spectral wavelength. Combined with an increase in saturation, it is possible that the presentation of other spectral wavelengths in GBH treated plants are greater than that of the blue wavelength, potentially confusing biocommunication between flowers and pollinators. According to our results, the impact on fluorescence is mostly resolved by the second-year post-treatment indicating morphological recovery of the flower. Further research is required to determine if the changes in fluorescence we observed do result in changes in biocommunication and determine if the changes in fluorescence of fireweed flowers are correlated to changes in concentrations of anthocyanins, or other secondary metabolites, which serve other functions in addition to aiding in biocommunication.\u003c/p\u003e \u003cp\u003eDecreased amino acids were noted in our samples two-years post-treatment, indicative of decreased nutritional value. Since we do not have amino acid concentration data from one-year post treatment it is impossible to determine if the amino acid levels are recovering at year two, as was shown with the other characteristics we investigated. Studies have shown decreased levels of nitrogen in plants treated with glyphosate [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and nitrogen is essential for the synthesis of amino acids and protein, therefore reduced amino acids are likely related to changes in nitrogen. The change noted in amino acids may have a large impact on insects that derive greater proportions of their diet from pollen and nectar. For example, queen bees eat a great deal of pollen and nectar to build fat reserves for hibernation, and the larvae feed on pollen that is brought to the colony [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], therefore they may be particularly susceptible to a reduced content. According to work by Barruad et al. (2022) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the performance of \u003cem\u003eBombus terrestris\u003c/em\u003e was explained by pollen amino acid content. Low amino acid content was correlated to low pollen collection:brood mass ratio [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and to low body mass [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Based on their assessment, total amino acid concentrations over 200 mg/g are beneficial for bumblebees, and specific amino acids are important in higher quantities for optimal production, such as alanine, leucine, phenylalanine, proline, and tyrosine. The pollen of fireweed flowers may be equal or greater in total amino acids than what is recommended for bee nutrition, as we know it is highly sought after and used by bees in areas of northern BC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], but that is not evident in our data. When we compare our results of amino acid content to the results of Barruard et al. (2022) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] our samples were much lower in total amino acid content. This could be due to the fact that we tested whole flowers, and not just pollen. Future research should focus on the collection of solely pollen in GBH treated areas to elucidate these findings. Additionally, profiling of fireweed flowers has been conducted in parts of northern Europe for use in nutrition supplements, some showing differences in secondary metabolites based on site conditions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Future investigation should apply this method of chemical profiling to areas treated with GBH to determine how this factor compares to natural environmental variation over larger areas.\u003c/p\u003e \u003cp\u003eWe confirmed the presence of glyphosate residues in floral tissues one and two-years post-treatment. The amount of glyphosate residue present in floral tissues remained similar between one- and two-years post treatment, likely due to the negative exponential degradation of residues in floral tissues which leads to the majority of the glyphosate residue degrading within the first year after application [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Even though residues may not persist at high levels for greater than one year, our research suggests that the effects of glyphosate residues to plant anatomy and physiology may persist for a longer period, and that these effects are not necessarily linearly correlated to the amount of residue that persists in the tissues. Our data confirms that residues persist in fireweed flowers for at least two years; however, how long these effects last is still unknown.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFireweed is often a pioneer species responsible for nutrient cycling in disturbed environments, provides an important source of floral abundance for pollinating insects and birds, and is also an ethnobotanically important medicinal plant. This plant is a prominent component of the herbaceous layer in forests of northern British Columbia, Canada, and shows stress symptoms after sub-lethal exposure to glyphosate-based herbicides used for vegetation management. These symptoms include reduced growth and reproductive capacities. Specifically, height, reproductive shoot apex formation, pollen viability, floral fluorescence, and amino acid content of flowers are all altered for at least one-year post-exposure. These changes to form and make-up have significant implications for the function of the ecosystem in these managed areas, including potential to change biocommunication with insect pollinators, the quality, and/or the quantity of food produced for wildlife and humans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science and Engineering Research Council of Canada, the Habitat Conservation Trust Foundation and the Weston Family Foundation. The authors would like to thank Deniz Divanli and Kate Rozmarniewich for their assistance in field sampling, data collection and laboratory sample analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.J.W – supervision, funding acquisition, resource provision, field sampling, experimental design, data analysis, manuscript compilation, and revision.A.R.G – field sampling, laboratory analysis, data analysis, writing, revision.L.B-K – carried out experimental protocol, laboratory analysis, data analysis, writing, revision.B.H – field sampling, laboratory analysis, data analysis, writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the Dryad data repository, DOI: 10.5061/dryad.zgmsbccp8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo species at risk were targeted for collection in this study, and all plants were obtained from healthy, extensive populations. No genetic manipulation of plants was conducted during this research. No permits, permissions, or licenses were required for plant tissue collection for the purposes of this research. A voucher specimen of \u003cem\u003eChamaenerion angustifolium\u003c/em\u003e is deposited in the University of Northern British Columbia (UNBC) herbarium collection (specimen ID: 100-CHAMANG) and is available for public access upon request to the UNBC Faculty of Environment. The specimen was identified by Dr. Lisa Wood, Associate Professor, UNBC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest or competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHunt, J. \u0026amp; Matute, P. Review of glyphosate use in British Columbia forestry. Technical Report#21, FPInnovations Canada. Project number: 301013763. 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E., CHROMOSOME NUMBERS AND TAXONOMY OF SOME CANADIAN \u0026amp; ARCTIC PLANTS. \u003cem\u003eCan. J. Bot.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e(9): 1209\u0026ndash;1218 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/b66-132\u003c/span\u003e\u003cspan address=\"10.1139/b66-132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1966).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"photosynthetic efficiency, pollen viability, fluorescence, amino acids, glyphosate residue, forest vegetation management","lastPublishedDoi":"10.21203/rs.3.rs-6011000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6011000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eChamaenerion angustifolium\u003c/em\u003e (fireweed) is an ecologically important plant in the northern hemisphere. It provides food across forest openings for many wildlife species including bumblebees, which are important pollinators to North America. Fireweed also acts as a significant food source for honeybees and is used by many North American Indigenous people as food and medicine. In forested areas managed for timber, fireweed is often incidentally exposed to glyphosate-based herbicides (GBH) in post-harvest vegetation management. We studied the response of fireweed to sub-lethal GBH exposure in a controlled experiment and in standard operational field conditions to determine impacts on specific aspects of growth and reproduction of the species. We aimed to determine if GBH-related stress symptoms would significantly impact the fluorescence of fireweed flowers, and/or the nutritional quality of pollen, which would have consequences for pollinators. Results showed that fireweed is negatively impacted by sublethal exposures of GBH including reduced photosynthetic efficiency, reduced height, and reproductive shoot dieback. In operational environments studied, pollen viability was reduced one-year after applications and anther fluorescence was altered. The amino acid concentration of flowers was reduced, and glyphosate residues remained present at low concentrations in floral tissues at two years post-treatment. It was concluded that these changes to fireweed growth and reproduction reduce its function as a primary source of good quality food for pollinators.\u003c/p\u003e","manuscriptTitle":"Reduced function in Chamaenerion angustifolium after sub-lethal glyphosate exposure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 13:11:34","doi":"10.21203/rs.3.rs-6011000/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-30T03:05:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T19:33:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-12T10:43:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144446404940374889660542610496450580582","date":"2025-04-08T23:37:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166526722614889908198927026233734276509","date":"2025-04-08T13:38:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-07T05:37:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-07T05:36:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-28T06:23:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-27T07:14:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-12T01:58:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6db3948c-b30c-4d59-ac56-04c58aab1a9e","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44995230,"name":"Biological sciences/Plant sciences/Plant stress responses/Abiotic"},{"id":44995231,"name":"Biological sciences/Plant sciences/Plant ecology"}],"tags":[],"updatedAt":"2025-09-01T16:05:06+00:00","versionOfRecord":{"articleIdentity":"rs-6011000","link":"https://doi.org/10.1038/s41598-025-16938-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-25 15:57:43","publishedOnDateReadable":"August 25th, 2025"},"versionCreatedAt":"2025-03-03 13:11:34","video":"","vorDoi":"10.1038/s41598-025-16938-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16938-x","workflowStages":[]},"version":"v1","identity":"rs-6011000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6011000","identity":"rs-6011000","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}