Effects of high- vs. low-intensity herbicide management on Phragmites australis propagule pressure in a brackish wetland of California

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Phragmites australis , or common reed, is a relatively recent invader of brackish and freshwater ecosystems across North America. In California’s Suisun Marsh—a 46,950-hectare network of public and private wetlands— P. australis has dramatically expanded over the past two decades. This expansion reduces habitat quality for waterfowl and other species, as dense stands displace native wetland plants. Herbicide-based management, primarily using glyphosate, has been employed in some areas of the marsh for over a decade, while other areas remain untreated or were only recently treated. To investigate the effects of management, we collected inflorescences from 11 high-intensity treatment parcels (≥ 9 years of spraying) and 9 low-intensity parcels (0–3 years). Random Forest classification identified large patches of P. australis across satellite imagery, allowing us to estimate propagule pressure as seeds per square meter. High-intensity parcels produced fewer seeds on average than low-intensity parcels, due largely to differences in the area occupied by P. australis (t = − 5.307, df = 313.93, p 0.05; t = − 1.52, df = 18, p > 0.05). These findings highlight the effectiveness of consistent herbicide use in reducing P. australis seed output and emphasize the need for coordinated, watershed-scale strategies to prevent the establishment of new patches. Herbicide propagule pressure resistance viability Phragmites Figures Figure 1 Figure 2 Figure 3 Introduction Wetlands provide critical habitat to many migratory and endemic species, as well as essential nutrient cycling, flood control, and recreational ecosystem services (Bergstrom et al. 1990 ; Fennessy et al. 2008 ; Gokce 2019 ). Brackish and freshwater wetlands around the United States are becoming increasingly dominated by the perennial reed Phragmites australis (Cav.) Trin ex Steud (hereafter: P. australis ) with varied management outcomes (Meyerson et al. 2009 ; Getsinger et al. 2013 ; Hazelton et al. 2014 ; Rohal et al. 2019 ; Conrad et al. 2023 ). The dense patches produced by P. australis have negative impacts on native plant biodiversity and nutrient dynamics, costing public and private interests more than $ 4.5 million (USD) annually in management costs (Martin & Blossey 2013 ; Hazelton et al. 2014 ; Uddin et al. 2017; Lindsay et al. 2022; Yuckin et al. 2023 ). Although native subspecies exist in North America, the predominant stands in recent decades are identified as the invasive Eurasian haplotype M (Saltonstall 2003 ; Lambert et al. 2016 ; Lindsay et al. 2022). While studies in other regions of North America and the world have clarified our understanding of P. australis expansion, its reproduction and management outcomes on the Pacific coast are obscure. The brackish wetlands of Suisun Marsh in the Sacramento-San Joaquin Delta of California exemplify the expansion of invasive P. australis on the Pacific coast (Grossinger et al. 1998 ; Meyerson et al. 2009 ; Whitcraft et al. 2011 ; Grewell et al. 2014 ; Lambert et al. 2016 ; Conrad et al. 2023 ; Hagani et al. 2023 ). As one of the largest contiguous wetlands on the Pacific coast of North America, Suisun Marsh is managed heavily for waterfowl habitat, providing a large stopover area for migrant birds along the Pacific Flyway. Suisun Marsh also offers year-round critical habitat for endangered and endemic species such as the Delta Smelt ( Hypomesus transpacificus ), Salt-marsh Harvest Mouse ( Reithrodontomys raviventris ), and Ridgway’s Rail ( Rallus obsoletus ) (Shapiro 1974 ; Sustaita et al. 2011 ; Takekawa et al. 2011 ; Smith et al. 2019; Hagani et al. 2023 ). Between 2000 and 2018, the 46,950-ha wetland experienced more than a 200% increase in P. australis cover despite the extensive efforts of land managers and the Suisun Resource Conservation District (SRCD), the agency responsible for managing control efforts of private landowners marsh-wide (Suisun RCD; Hagani et al. 2023 ). These efforts are principally herbicide-based, with mowing, disking, and burning as complementary or alternative methods (Conrad et al. 2023 ; Hagani et al. 2023 ). The patchwork of private, public, and non-profit land has resulted in a lack of uniformity in P. australis control across Suisun Marsh (Conrad et al. 2023 ; Hagani et al. 2023 ). Understanding how management outcomes differ between treatment regimes may inform cohesive future management in brackish wetlands. Based on genetic and remote sensing analysis, non-native P. australis colonization in North America is thought to be primarily established sexually via wind-dispersed seeds followed by clonal patch expansion (McCormick et al. 2010 ; Hagani et al. 2023 ). Rhizomes are clonal underground stems that produce shoots and contribute to the expansion of individual patches, however, they do not increase genetic diversity and are less likely than viable seeds to initiate new patches (Baldwin et al. 2010 ; McCormick et al. 2010 ; Kettenring & Whigham 2018 ). Sexual propagation can increase genetic variation across and within stands of P. australis , a likely contributor to higher rates of seed viability in an invasive population of Chesapeake Bay due to cross-pollination (Kettenring et al. 2010 ; Kettenring et al. 2011 ; Hazelton et al. 2014 ). It is unclear whether the same relationship exists within the populations of P. australis in Suisun Marsh, but if so, it has the potential to create a positive feedback loop in which greater genetic variability increases seed viability, and vice versa. Land managers continue to use herbicides for P. australis and other invasive species because of their efficacy on young shoots and new patches that drive rapid expansion (Mozdzer et al. 2008 ; Hazelton et al. 2014 ; Martin & Blossey 2013 ). More mature stems are damaged but do not always die from herbicide spraying. The damage endured by above-ground stems and leaves impacts the synthesis of essential amino acids via disruption of the shikimate pathway that limits energy and proteins available for seed production (Lacroix & Kurrasch 2023 ). Following decades of herbicide treatment with little abatement, remaining patches of P. australis in Suisun Marsh may have developed some resistance to the compounds being used due to intense selective pressure, as shown in other species (Sammons & Gaines 2014 ; Gaines et al. 2020 ). Based on our understanding of other tenacious populations of P. australis , we surmise that producing more viable seeds in areas of high patch density leads to rapid establishment and expansion of the P. australis haplotype M, which may also apply to Suisun Marsh. Here, we look to answer three main questions: How has past treatment impacted P. australis propagule pressure? How has past treatment impacted P. australis seed viability? Has the high-intensity treatment led to a detectable amount of herbicide resistance developing? By answering these questions, we hope to better inform land managers' decision-making and contribute to more successful management outcomes in the future. Methods Study Area Seeds were collected from public and private land parcels across Suisun Marsh (38.1475 N, 122.0053 W). The marsh consists mostly of managed wetland ponds protected from semi-diurnal tides by levees with some tidal wetlands and sloughs open to natural tidal influence. Salinity is generally highest in managed ponds, followed by canals, and then sloughs, with a mean salinity between 1 and 10 ppt (Schacter et al., 2021 ). Managed ponds are flooded and drained based on landowner needs, with most flooding occurring in September and early October to create habitat for the migratory season of many waterfowl. This is followed by a spring leaching cycle from February to May, after which managed ponds are dried up and invasive plant management is undertaken from July through September (Grewell et al. 2014 ; Conrad et al. 2023 ; Hagani et al. 2023 ). Although 2,000–3,000 ha of managed land is being restored to tidal marsh to benefit these species, the vast majority is managed to support migrating and wintering waterfowl (Hagani et al. 2023 ). Herbicide treatments of P. australis with glyphosate-based products occur only in managed wetlands when ponds are dry to avoid impacts on non-target species. To cover large areas with some accuracy, most spraying is done aerially with helicopters or drones, but other methods of applying herbicide are also used (such as backpack sprayers). Mowing, disking, and burning are also employed on occasion, but we only took account of herbicide spraying in designating treatment areas as high- or low-intensity management because we did not have access to records on the prevalence of the other activities. Seed Collection Land parcels were selected for seed collection based on the years of glyphosate-based herbicide treatment as determined from historical data provided by SRCD, which has records of the acreage of spraying per parcel per year from 2000 to the present. Parcels with 9 or more years of herbicide spraying were classified as high-intensity treatment, while those parcels with 3 years or less of spraying were classified as low-intensity treatment. Five of the “low-intensity” treatment parcels had had no treatment at all during the period since 2000. Seeds were collected in late August 2022 from 11 sites of high-intensity treatment and 9 of low-intensity treatment, with multiple sites in some large parcels (Fig. 1 ). Five inflorescences were collected from 5 patches in each site for a total of 25 inflorescences per site and 250 inflorescences per treatment. All but one of the collection sites were in managed wetlands, while Joice Island (JI) is a tidal wetland that has never seen herbicide treatment. During collection, a 0.5m x 0.5m quadrat was used to measure inflorescence density in 5 randomly selected areas across each patch. The long and short axes of each patch were measured to estimate each patch's area. Propagule Pressure Each inflorescence was weighed and stripped of all spikelets. Spikelets were then weighed separately from the stem. For each inflorescence, an aliquot of 60 spikelets was counted and weighed to estimate the mass of a single spikelet, and this value in turn was used to estimate the total number of spikelets on the inflorescence. Three spikelets from each inflorescence were also dissected to determine the average number of seeds per spikelet. A raster file of P. australis cover in Suisun Marsh provided by the Random Forest classification of 2018 NAIP imagery was used to estimate the percentage of P. australis per m 2 of marsh in each parcel (Hagani et al. 2023 ). The propagule pressure of P. australis per m 2 of marsh was calculated as follows: Random Forest Classification We used the classifications derived by Hagani et al. ( 2023 ) to estimate the area of P. australis per area of managed wetland in each land parcel. The classifications were built upon aerial imagery collected by the National Agricultural Imagery Program (NAIP) from 2003 to 2020. NAIP imagery includes three color bands (red, blue, and green) and a near-infrared band. In California, these images were collected every three years pre-2009 and every 2 years since. Eight classifications were generated within this timeframe: 2003, 2006, 2009, 2012, 2014, 2016, 2018, and 2020. For each classification year, representative P. australis patches were selected from the image and manually classified (Tadros et al. 2020 ) to use as training and validation data. Separate polygons were also manually selected to serve as “non- P. australis ” data. Random forest classifiers (Breiman 2001; Cutler et al. 2007 ) were built in R Studio (Liaw and Wiener 2002 ; R Core Team 2021 ; R Studio Team 2021 ) using a random 70% of the training data, with the remaining 30% withheld for model evaluation (Paz-Kagan et al. 2019 ). Classifications were generated with the color bands, the near-infrared band, and an additional predictor variable for every year following 2003, which described the distance of every pixel from the nearest P. australis pixel classified on the previous image. For each classification year, five random forest models were run based on a different random selection of training and validation data, and accuracy metrics (User’s accuracy, producer’s accuracy, overall accuracy, and Kappa’s statistic; Fielding and Bell 1997 ; Kraemer 2015 ) were averaged. The singular random forest classifier, which produced the best accuracy metrics, was then used to create a map of predicted P. australis each year in Suisun Marsh with the package “raster” in R (version 3.6–11; Hijmans and van Etten 2012 ). The final output of this process was a fine-scale (1-2m) wall-to-wall raster of Suisun Marsh with values of “1” to represent P. australis and “0” to represent non- P. australis for each classification year. Seed Germination Spikelets from each inflorescence were counted out in groups of 60 and then combined by patch so that each patch had 300 spikelets from 5 inflorescences in the germination trial. Spikelets were initially washed for two minutes in a deionized water bath with 10% by volume household bleach to prevent mold growth. Petri dishes of 150 mm were then filled halfway with autoclaved sand. Deionized water was added to moisten the sand, and any excess water was poured off. Filter paper was used to cover the sand and allowed to equilibrate to the moisture. The spikelets were spread onto the prepared Petri dishes in a layer 1 spikelet thick, after which the Petri dishes were sealed with parafilm. The seeds were then cold-stratified for 2 weeks at 4˚C in the dark according to the method of Kettenring et al. ( 2010 ). Once stratified, seeds were placed in an Intellus Control System growth chamber (Percival Scientific Inc., Perry, IA) set to an 11:9 h day/night cycle mimicking temperature fluctuations and daylight hours of Suisun Marsh in late spring and early summer, ramping linearly for 2 hours from 13˚C to 25˚C. The petri dishes were checked for germination and rotated every other day for 2 weeks. Germinated seeds were counted and removed. Germination was defined as the observation of a radicle emerging. After two weeks of germination trials, the ungerminated seeds were discarded. Herbicide Resistance Germinated seeds were grown in a greenhouse for 1.5 months in June and July 2022. Plants were kept at ambient temperature in Santa Clara, CA with ambient lighting. Plug trays were filled with potting soil and then placed into a larger tray kept flooded with water, whose salinity was managed to remain at 5.7 ppt, mimicking conditions found in Suisun Marsh. Plants were trimmed above the first healthy leaf to standardize for biomass and leaf area. The remaining leaf on each plant was then wiped with 2% glyphosate (Round-Up, Bayer Corporation, Whippany, NJ). P. australis plants were visually checked one week after herbicide application and damage was rated on a 0–5 scale, with 0 being no damage and 5 being total death (Ogg et al. 1991; Dear et al. 2003 ). We also took note of whether resprouts began growing from plants post-herbicide application. Results Propagule Pressure The propagule pressure of P. australis was 17,986 seeds per m 2 of marsh, on average, in low-intensity treatment parcels and 8,178 seeds per m 2 of marsh, on average, in high-intensity treatment parcels, a 54.5% reduction in seed production (t = -5.307, df = 313.93, p < 0.0001). The difference results largely from the lower percentage of P. australis cover in land parcels classified as high-intensity treatment. Parcels subject to low-intensity treatment had 7.42% P. australis cover, on average, compared with 3.39% cover in parcels of high-intensity treatment. This was a 54.3% decrease in P. australis cover across the marsh from low- to high-intensity treatment land parcels, which includes 59 additional land parcels in Suisun marsh where seeds were not collected (t = -3.23, df = 74, p < 0.05). On average, inflorescences were similar in weight between the two treatment groups, with 1.1 g inflorescences, on average, in the low-intensity treatment parcels and 0.98 g in the high-intensity treatment parcels (t = -1.036, df = 415.4, p > 0.05). There was no significant difference in the number of seeds contained within each spikelet (~ 6 seeds/spikelet), nor stem density (~ 40 stems/m 2 ) between the high- and low-intensity herbicide treatments (t = 1.051, df = 513.11, p = 0.4782; t = -1.2161, df = 441.93, p = 0.2245). Joice Island (JI) had the highest estimated propagule pressure with 44,212 seeds produced per m 2 of marsh (Fig. 2 ). This was the only tidal wetland site sampled that had never experienced herbicide treatments due to regulations on the use in tidally influenced areas. The sampled area had 9.33% P. australis cover and the mean weight of seed material per inflorescence was the greatest compared with all other sites at 1.89 g. The publicly managed Crescent Unit (CRU) had the lowest estimated propagule pressure with only 344 seeds per m 2 of marsh, attributable to smaller inflorescences (0.485 g) and low stem density (8.80 stems per m 2 ). Seed Germination Germination rates ranged from 0 to 35% which varied between patches of the same site. The mean germination rate was not significantly different between high- and low-intensity herbicide treatment sites with an average of 7.81% and 7.15% germinating, respectively (Fig. 3 ., t = 0.349, df = 84.27, p > 0.05). Seed viability ranged widely across all sites with the with East Lower Joice (ELJ), 11.1%; Mid Lower Joice, (MJL) 11.5%; North Lower Joice (NLJ), 17.1%; and Joice Island (JI), 22.9%) representing the highest percentages while other sites, such as Delta King (DK) and Grizzly Fairview (GFV) produced fewer viable seeds, on average, with 0.043% and 0.23% germinating, respectively. Herbicide Resistance The average damage rating of young plants grown from seeds collected in high- and low-intensity treatment sites was not significantly different, suggesting no difference in herbicide resistance (t = -1.52, df = 18, p > 0.05). There was also no difference in the percentage of resprouts between high- and low-intensity treatment plants that survived the glyphosate treatment (t = 0.38, df = 18, p > 0.05). Discussion Seed production was reduced in land parcels that experienced high-intensity herbicide spraying (≥ 9 years), with this reduction in propagule pressure primarily driven by decreased P. australis cover, though factors such as inflorescence weight and stem density also influenced overall production. The overall expansion of P. australis has been rapid over the past two decades, however, remote sensing analysis reveals that 61 land parcels across Suisun Marsh experienced a decrease in cover between 2003 and 2020, as reported by Hagani et al. ( 2023 ). The regular use of glyphosate-based herbicides to limit the establishment and expansion of P. australis patches is a likely contributor to the decrease in these land parcels, given our results. Successful control of P. australis is less challenging for small, newly established “satellite” patches, whereas larger, established patches are more productive and difficult to control (Mozdzer et al. 2008 ; Kettenring et al. 2011 ; Hazelton et al. 2014 ; Martin & Blossey 2013 ). Therefore, land parcels that have been managed with herbicides for a longer duration may have prevented satellite patches from expanding their area via rhizome to become unmanageable. Corroborating our findings, another study evaluating the management outcomes of an introduced P. australis population in the Great Salt Lake found a marked reduction in seeds produced per m 2 following treatment with glyphosate and mowing at different times of the year (Rohal et al. 2019 ). Herbicide application was carried out in either early July or late August, the same period during which most management and maintenance operations are performed in Suisun Marsh. Our results show that land managed with herbicides can still host considerable populations of P. australis , as Lower Joice Island has higher coverage than the mean of most other high-intensity treatment sites (Table 1). Despite their coverage, the majority of land parcels under both high- and low-intensity treatment produced an estimated hundreds or thousands of viable P. australis seeds per m 2 of marshland (Fig. 2 ). Consistent with previous research on the topic, this suggests that seed propagation is a notable contributor to new patch initiation (McCormick et al. 2010 ; Kettenring & Whigham 2018 ; Hagani et al. 2023 ). This presents continuous pressure as the dispersal of P. australis in managed land parcels is influenced by the number of unmanaged neighboring parcels (Hagani et al. 2023 ). Due to the long distances possible with wind dispersal, management is most effective at a watershed or regional scale, as is advocated for by other authors on the subject of P. australis control methods (Takekawa et al. 2011 ; Hazelton et al. 2014 ; Hagani et al. 2023 ). Seed viability is a consequential element in the establishment of P. australis , and may be difficult to contend with in a noxious population, as our results showed no significant effect of glyphosate on seed germination rate (Fig. 3 ). Rohal et al. ( 2019 ) investigated the introduced Great Salt Lake population to quantify glyphosate impacts on seed viability which did not differ significantly from the control that received no treatment. Though not specifically looking at P. australis , Nurse et al. ( 2015 ) focused on the impacts of same-year glyphosate application on seed viability of Hairy cupgrass ( Eriochloa villosa ), an annual grass that can reduce crop yields in the Midwestern United States. They saw no changes in seed production, however, they reported a significant decrease in seed weight and germination rate. These results are the opposite of what we found in our results on P. australis production, though our focus was not same-year glyphosate application, but rather regular treatment over multiple years. Viable seed production can be controlled in some invasive species such as E. villosa , but our results support those of Rohal et al. ( 2019 ) that indicate lower susceptibility of P. australis to these effects. While seed viability was not significantly impacted by the treatment regime in this case, the variability in viability across all sites regardless of treatment suggests other environmental or genetic factors. Influences affecting the viable seed production in P. australis are not fully understood, however, likely contributors to increased germination rates are genetic diversity and patch size (Kettenring et al. 2010 ; Kettenring et al. 2011 ). Thorough research of the introduced Chesapeake Bay population found slight positive relationships between both of these factors and seed viability. Future genetic analysis of patches with high and low seed viability may help illuminate the potential of positive feedbacks that result in more new patches of P. australis . Nutrient loads have also been evaluated as drivers of seed viability, and although a significant relationship was noted with inflorescence production per plant, the same was not true for viable seed production in the Chesapeake Bay. Resistance to glyphosate in introduced populations of P. australis has yet to be investigated thoroughly, though the management implications of resistance are of utmost importance. Due to its prevalence as a weed control agent since the late 20th century, at least 24 species of weeds were documented to have developed some resistance in 2014, which has since reached 57 species as of 2023 (Sammons & Gaines 2014 ; Landau et al. 2023). The mechanism identified in resistance involves one to three point mutations in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is essential in the shikimate pathway targeted by glyphosate herbicides (Sammons & Gaines 2014 ; García et al. 2019 ). The EPSPS mutation has not been identified in P. australis , but it was detected once during a small-scale investigation of P. australis resistance in a population of Indiana Dunes National Park, with inconclusive results (Nikkel et al. 2021 ). Future genetic analysis of populations in Suisun Marsh is vital to understanding the state of resistance. It is clear from our findings that regular application of glyphosate is at least partially effective in controlling P. australis in Suisun Marsh by reducing cover. Still, many other factors are involved in the continued spread that have received little attention on the West Coast of North America. Even if cover is shown to decrease with continued herbicide application, the varied results in seed viability suggest that land managers are contending with uncontrolled environmental and genetic factors that heavily influence the success of new patches established via seed. Until our understanding of positive feedback and invasion mechanisms is clarified, management of P. australis will be most effective through combined, comprehensive, and contiguous control efforts across land parcels. Declarations Competing Interests The authors have no competing financial or non-financial interests to disclose. Funding This work was primarily funded by the Delta Stewardship Council (Contract #DSC-21004) to VM, with assistance from a Santa Clara University Hayes Fellowship to GR. Author Contributions VM, MW, and GR contributed to the study conception and design, material preparation, data collection, and analysis. Herbicide resistance trials were primarily carried out by MW and VM. Remote sensing analysis was performed by JH. The first draft of the manuscript was written by GR and MW, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgments We would like to thank the Suisun Resource Conservation District for their assistance in contacting landowners for sampling permission, specifically Dr. John Takekawa. We also thank the Delta Stewardship Council for primary funding to VM, and the Santa Clara University Hayes Fellowship that provided supplementary funding to GR. We would also like to thank Dr. Karin Kettenring for her consultation on methods. Data Availability The datasets generated and/or analysed during the current study are available in the phrag repository that is publicly available, https://github.com/gaberodkey/phrag/tree/7528eb506bacc3199863d1f8d5e4b92868b4b311 References Baldwin AH, Kettenring KM, Whigham DF (2010) Seed banks of Phragmites australis-dominated brackish wetlands: relationships to seed viability, inundation, and land cover. Aquat Bot 93(3):163–169. https://doi.org/10.1016/j.aquabot.2010.06.001 Bergstrom JC, Stoll JR, Titre JP, Wright VL (1990) Economic value of wetlands-based recreation. Ecol Econ 2(2):129–147. https://doi.org/10.1016/0921-8009(90)90004-E Burdick DM, Konisky RA (2003) Determinants of expansion for Phragmites australis, common reed, in natural and impacted coastal marshes. 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Available at: http://CRAN.R-project.org/package=raster (Accessed May 31, 2022) Kettenring KM, Whigham DF (2009) Seed viability and seed dormancy of non-native Phragmites australis in suburbanized and forested watersheds of the Chesapeake Bay, USA. Aquat Bot 91(3):199–204. https://doi.org/10.1016/j.aquabot.2009.06.002 Kettenring KM, McCormick MK, Baron HM, Whigham DF (2010) Phragmites australis (common reed) invasion in the Rhode River subestuary of the Chesapeake Bay: disentangling the effects of foliar nutrients, genetic diversity, patch size, and seed viability. Estuaries Coasts 33:118–126. https://doi.org/10.1007/s12237-009-9241-1 Kettenring KM, McCormick MK, Baron HM, Whigham DF (2011) Mechanisms of Phragmites australis invasion: feedbacks among genetic diversity, nutrients, and sexual reproduction. J Appl Ecol 48(5):1305–1313. https://doi.org/10.1111/j.1365-2664.2011.02024.x Kettenring KM, Whigham DF (2018) The role of propagule type, resource availability, and seed source in Phragmites invasion in Chesapeake Bay wetlands. Wetlands 38:1259–1268. https://doi.org/10.1007/s13157-018-1034-5 Kraemer HC Kappa coefficient in Wiley StatsRef: Statistics reference online (Hoboken, NJ:, Wiley (2015) 1–4. https://doi.org/10.1002/9781118445112.stat00365.pub2 Lacroix R, Kurrasch DM (2023) Glyphosate toxicity: in vivo, in vitro, and epidemiological evidence. Toxicol Sci 192(2):131–140. https://doi.org/10.1093/toxsci/kfad018 Lambert AM, Saltonstall K, Long R, Dudley TL (2016) Biogeography of Phragmites australis lineages in the southwestern United States. Biol Invasions 18:2597–2617. https://doi.org/10.1007/s10530-016-1164-8 Liaw A, Wiener M (2002) Random forests. R News 2:18–22. http://CRAN.R-project.org/doc/Rnews/ Lindsay DL, Freeland J, Gong P, Guan X, Harms NE, Kowalski KP, Lance RF, Oh D, Sartain BT, Wendell DL (2023) Genetic analysis of North American Phragmites australis guides management approaches. Aquat Bot 184:103589. https://doi.org/10.1016/j.aquabot.2022.103589 Martin LJ, Blossey B (2013) The runaway weed: costs and failures of Phragmites australis management in the USA. Estuaries Coasts 36:626–632. https://doi.org/10.1007/s12237-013-9593-4 McCormick MK, Kettenring KM, Baron HM, Whigham DF (2010) Extent and reproductive mechanisms of Phragmites australis spread in brackish wetlands in Chesapeake Bay, Maryland (USA). Wetlands 30:67–74. https://doi.org/10.1007/s13157-009-0007-0 Meyerson LA, Saltonstall K, Windham L, Kiviat E, Findlay S (2000) A comparison of Phragmites australis in freshwater and brackish marsh environments in North America. Wetlands Ecol Manage 8:89–103. https://doi.org/10.1023/A:1008432200133 Meyerson LA, Saltonstall K, Chambers RM, Silliman BR, Bertness MD, Strong D (2009) Phragmites australis in eastern North America: a historical and ecological perspective. Salt marshes under global siege, 57–82. https://books.google.com/books?hl=en& lr=&id=Vs-IDwAAQBAJ&oi=fnd&pg=PA57&dq=Suisun+phragmites&ots=eKDDMTqget&sig=E1S_axZYYC5zI7b_255Z0to4SZE#v=onepage&q=Suisun%20phragmites&f=false Moyle PB, Manfree AD, Fiedler PL (2013) The future of Suisun Marsh as mitigation habitat. San Francisco Estuary Watershed Sci 11(3). https://doi.org/10.15447/sfews.2013v11iss3art10 Mozdzer TJ, Hutto CJ, Clarke PA, Field DP (2008) Efficacy of imazapyr and glyphosate in the control of non-native Phragmites australis. Restor Ecol 16(2):221–224. https://doi.org/10.1111/j.1526-100X.2008.00386.x Nikkel J, Pash R, Choi Y, Stanic R (2021) Investigation of glyphosate resistance in Phragmites australis in the Indiana Dunes National Park. Botany 2021 Conference, Virtual Format. https://doi.org/10.13140/RG.2.2.34540.28802 Nurse RE, Darbyshire SJ, Simard MJ (2015) Impact of post-anthesis glyphosate on woolly cupgrass seed production, seed weight and seed viability. Can J Plant Sci 95(6):1193–1197. https://doi.org/10.4141/cjps-2015-166 Ogg Jr AG, Ahmedullah MA, Wright GM (1991) Influence of repeated applications of 2, 4-D on yield and juice quality of concord grapes (Vitis labruscana). Weed Sci 39(2):284–295. https://doi.org/10.1017/S0043174500071629 Paz-Kagan T, Silver M, Panov N, Karnieli A (2019) Multispectral approach for identifying invasive plant species based on flowering phenology characteristics. Remote Sens 11:953. https://doi.org/10.3390/rs11080953 R Core Team (2021) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available at: https://www.Rproject.org. RStudio: Integrated development for R. RStudio, PBC, R Studio Team, Boston MA (2021) Available at: http://www.rstudio.com/ Rohal CB, Cranney C, Hazelton ELG, Kettenring KM (2019) Invasive Phragmites australis management outcomes and native plant recovery are context dependent. Ecol Evol 9(24):13835–13849. https://doi.org/10.1002/ece3.5820 Rohal CB, Adams R, Reynolds C, Hazelton LK, E. L. G., Kettenring KM (2021) Do common assumptions about the wetland seed bank following invasive plant removal hold true? Divergent outcomes following multi-year Phragmites australis management. Appl Veg Sci 24(4):e12626. https://doi.org/10.1111/avsc.12626 Saltonstall K (2003) Genetic variation among North American populations of Phragmites australis: implications for management. Estuaries 26:444–451. https://doi.org/10.1007/BF02823721 Saltonstall K, Stevenson JC (2007) The effect of nutrients on seedling growth of native and introduced Phragmites australis. Aquat Bot 86(4):331–336. https://doi.org/10.1016/j.aquabot.2006.12.003 Sammons RD, Gaines TA (2014) Glyphosate resistance: state of knowledge. Pest Manag Sci 70(9):1367–1377. https://doi.org/10.1002/ps.3743 Schacter CR, Peterson SH, Herzog MP, Hartman CA, Casazza ML, Ackerman JT (2021) Wetland availability and salinity concentrations for breeding waterfowl in Suisun Marsh, California. San Francisco Estuary Watershed Sci 19(3). https://doi.org/10.15447/sfews.2021v19iss3art5 Shapiro AM (1974) SUISUN MARSH, CALIFORNIA. J Res Lepidoptera 13(3):191–206. https://www.researchgate.net/profile/Arthur-Shapiro-4/publication/292022104_Butterflies_of_the_Suisun_Marsh_California/links/5732851c08ae9ace84048106/Butterflies-of-the-Suisun-Marsh-California.pdf Simberloff D (2009) The role of propagule pressure in biological invasions. Annu Rev Ecol Evol Syst 40:81–102. https://doi.org/10.1146/annurev.ecolsys.110308.120304 Smith KR, Kelt DA (2019) Waterfowl management and diet of the salt marsh harvest mouse. J Wildl Manag 83(8):1687–1699. https://doi.org/10.1002/jwmg.21752 Suisun Resource Conservation District . Suisun RCD. (2024) https://suisunrcd.org/ Sustaita D, Quickert PF, Patterson L, Barthman-Thompson L, Estrella S (2011) Salt marsh harvest mouse demography and habitat use in the Suisun Marsh, California. J Wildl Manag 75(6):1498–1507. https://doi.org/10.1002/jwmg.187 Tadros MJ, Al-Assaf A, Othman YA, Makhamreh Z, Taifour H (2020) Evaluating the effect of Prosopis juliflora, an alien invasive species, on land cover change using remote sensing approach. Sustainability 12:5887. https://doi.org/10.3390/su12155887 Takekawa JY, Woo I, Gardiner R, Casazza M, Ackerman JT, Nur N, Leonard L, Spautz H (2011) Avian communities in tidal salt marshes of San Francisco Bay: a review of functional groups by foraging guild and habitat association. San Francisco Estuary Watershed Sci 9(3). https://doi.org/10.15447/sfews.2011v9iss3art4 Uddin MN, Robinson RW (2017) Changes associated with Phragmites australis invasion in plant community and soil properties: A study on three invaded communities in a wetland, Victoria, Australia. Limnologica 66:24–30. https://doi.org/10.1016/j.limno.2017.07.006 Whitcraft C, Grewell BJ, Baye PR (2011) 5. Flora and Ecological Profile of Native and Exotic Estuarine Wetland Vegetation by Hydrogeomorphic Setting at Rush Ranch, Suisun Marsh. profile San Francisco Bay Natl Estuar Res Reserve, 79–144. https://www.researchgate.net/publication/279181056_Flora_and_ecological_profile_of_native_and_exotic_estuarine_wetland_vegetation_by_hydrogeomorphic_setting_at_Rush_Ranch_Suisun_Marsh Yuckin SJ, Howell G, Robichaud CD, Rooney RC (2023) Phragmites australis invasion and herbicide-based control changes primary production and decomposition in a freshwater wetland. Wetlands Ecol Manage 31(1):73–88. https://doi.org/10.1007/s11273-022-09902-3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7474318","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512629246,"identity":"a7318daa-94d0-4a84-aa41-a6ebc8fa9afe","order_by":0,"name":"Michael Weatherford","email":"","orcid":"","institution":"Santa Clara University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Weatherford","suffix":""},{"id":512629247,"identity":"8cae3f54-6eb2-4de2-b21c-d5e73be4d4ab","order_by":1,"name":"Gabriel Rodkey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYFACHhDBbADEB4AMCRlStLAlgLTwkKKFxwDOxQvk3c8efFxRYW3ML5Hz+dWNGgseBvbDRzfg02J4Ji/Z8MyZdDPJGbnbrHOOAR3Gk5Z2A6+Whhwzyca2wzYGN3K3GeewAbVI8Jjh19L/xvwnSIv9jZxnxjn/iNAiL5FjxgjUYmYgkcP8OLeNCC0GEu+SJRvOpBtLnHlmxpzbJ8HDRsgv8v25Bz82VFgb9rcnP/6c861Ojp/98DH8thyAsQQS2CRANBs+5WBbGmAs/gPMHwipHgWjYBSMgpEJANWSRylOR6W2AAAAAElFTkSuQmCC","orcid":"","institution":"Santa Clara University","correspondingAuthor":true,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Rodkey","suffix":""},{"id":512629248,"identity":"6aa4bca2-b114-4275-b140-1dce5094569f","order_by":2,"name":"Jason Hagani","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Hagani","suffix":""},{"id":512629249,"identity":"8778ef78-41b9-4d88-80c0-8032c3763e2f","order_by":3,"name":"Virginia Matzek","email":"","orcid":"","institution":"Santa Clara University","correspondingAuthor":false,"prefix":"","firstName":"Virginia","middleName":"","lastName":"Matzek","suffix":""}],"badges":[],"createdAt":"2025-08-27 19:17:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7474318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7474318/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91562607,"identity":"dcfb696b-bcf4-4259-a200-e54ca1783703","added_by":"auto","created_at":"2025-09-17 18:57:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5760014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAreas of sample collection across Suisun Marsh. \u003c/strong\u003eLabels bordered in white represent the entire land parcel; labels bordered in black represent sites within a single land parcel (LJ, MZ, GI). The red rectangle on the inset map represents the map area.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7474318/v1/e1bddfff62bc7b93d5917b8e.png"},{"id":91560207,"identity":"ac010bad-bad2-4a03-9a0d-7a70c028d244","added_by":"auto","created_at":"2025-09-17 18:41:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProduction of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. australis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seeds from high- and low-intensity herbicide treatment land parcels. \u003c/strong\u003eA) Averages include all replicates within each treatment; n\u003csub\u003elow\u003c/sub\u003e = 225, n\u003csub\u003ehigh\u003c/sub\u003e = 294. B) Averages represent replicates from each land parcel; n = 5\u003cstrong\u003e \u003c/strong\u003efor all land parcels, except n\u003csub\u003eIC \u003c/sub\u003e= 4, n\u003csub\u003eGI, MZ\u003c/sub\u003e = 10, and n\u003csub\u003eLJ\u003c/sub\u003e = 15. Error bars represent ±SEM.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7474318/v1/71c556fc472a06eafb3f18db.png"},{"id":91560216,"identity":"40964acd-8828-4053-9287-0d6b0ae28396","added_by":"auto","created_at":"2025-09-17 18:41:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGermination rates of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. australis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seeds from high- and low-intensity treatment land parcels. \u003c/strong\u003eA) Averages represent replicates from each treatment; n\u003csub\u003elow\u003c/sub\u003e = 54, n\u003csub\u003ehigh\u003c/sub\u003e = 49. B) Averages represent replicates from each site; n = 5\u003cstrong\u003e \u003c/strong\u003efor all sites, except n\u003csub\u003eIC \u003c/sub\u003e= 4. Error bars represent ±SEM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7474318/v1/5da2b1df6dd4d3adc996c4d1.png"},{"id":96913553,"identity":"f9eed041-40cb-4d18-9d3f-8cf1f4c2aad1","added_by":"auto","created_at":"2025-11-27 14:02:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7157510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474318/v1/9b71feee-c235-4e76-9d5c-6cf0a9ec9828.pdf"}],"financialInterests":"","formattedTitle":"Effects of high- vs. low-intensity herbicide management on Phragmites australis propagule pressure in a brackish wetland of California","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWetlands provide critical habitat to many migratory and endemic species, as well as essential nutrient cycling, flood control, and recreational ecosystem services (Bergstrom et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Fennessy et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gokce \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Brackish and freshwater wetlands around the United States are becoming increasingly dominated by the perennial reed \u003cem\u003ePhragmites australis\u003c/em\u003e (Cav.) Trin ex Steud (hereafter: \u003cem\u003eP. australis\u003c/em\u003e) with varied management outcomes (Meyerson et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Getsinger et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rohal et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Conrad et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The dense patches produced by \u003cem\u003eP. australis\u003c/em\u003e have negative impacts on native plant biodiversity and nutrient dynamics, costing public and private interests more than \u003cspan\u003e$\u003c/span\u003e4.5\u0026nbsp;million (USD) annually in management costs (Martin \u0026amp; Blossey \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Uddin et al. 2017; Lindsay et al. 2022; Yuckin et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although native subspecies exist in North America, the predominant stands in recent decades are identified as the invasive Eurasian haplotype M (Saltonstall \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lambert et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lindsay et al. 2022). While studies in other regions of North America and the world have clarified our understanding of \u003cem\u003eP. australis\u003c/em\u003e expansion, its reproduction and management outcomes on the Pacific coast are obscure.\u003c/p\u003e\u003cp\u003eThe brackish wetlands of Suisun Marsh in the Sacramento-San Joaquin Delta of California exemplify the expansion of invasive \u003cem\u003eP. australis\u003c/em\u003e on the Pacific coast (Grossinger et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Meyerson et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Whitcraft et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Grewell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lambert et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Conrad et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As one of the largest contiguous wetlands on the Pacific coast of North America, Suisun Marsh is managed heavily for waterfowl habitat, providing a large stopover area for migrant birds along the Pacific Flyway. Suisun Marsh also offers year-round critical habitat for endangered and endemic species such as the Delta Smelt (\u003cem\u003eHypomesus transpacificus\u003c/em\u003e), Salt-marsh Harvest Mouse (\u003cem\u003eReithrodontomys raviventris\u003c/em\u003e), and Ridgway\u0026rsquo;s Rail (\u003cem\u003eRallus obsoletus\u003c/em\u003e) (Shapiro \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Sustaita et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Takekawa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Smith et al. 2019; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Between 2000 and 2018, the 46,950-ha wetland experienced more than a 200% increase in \u003cem\u003eP. australis\u003c/em\u003e cover despite the extensive efforts of land managers and the Suisun Resource Conservation District (SRCD), the agency responsible for managing control efforts of private landowners marsh-wide (Suisun RCD; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These efforts are principally herbicide-based, with mowing, disking, and burning as complementary or alternative methods (Conrad et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The patchwork of private, public, and non-profit land has resulted in a lack of uniformity in \u003cem\u003eP. australis\u003c/em\u003e control across Suisun Marsh (Conrad et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Understanding how management outcomes differ between treatment regimes may inform cohesive future management in brackish wetlands.\u003c/p\u003e\u003cp\u003eBased on genetic and remote sensing analysis, non-native \u003cem\u003eP. australis\u003c/em\u003e colonization in North America is thought to be primarily established sexually via wind-dispersed seeds followed by clonal patch expansion (McCormick et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Rhizomes are clonal underground stems that produce shoots and contribute to the expansion of individual patches, however, they do not increase genetic diversity and are less likely than viable seeds to initiate new patches (Baldwin et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; McCormick et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kettenring \u0026amp; Whigham \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Sexual propagation can increase genetic variation across and within stands of \u003cem\u003eP. australis\u003c/em\u003e, a likely contributor to higher rates of seed viability in an invasive population of Chesapeake Bay due to cross-pollination (Kettenring et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kettenring et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is unclear whether the same relationship exists within the populations of \u003cem\u003eP. australis\u003c/em\u003e in Suisun Marsh, but if so, it has the potential to create a positive feedback loop in which greater genetic variability increases seed viability, and vice versa.\u003c/p\u003e\u003cp\u003eLand managers continue to use herbicides for \u003cem\u003eP. australis\u003c/em\u003e and other invasive species because of their efficacy on young shoots and new patches that drive rapid expansion (Mozdzer et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Martin \u0026amp; Blossey \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). More mature stems are damaged but do not always die from herbicide spraying. The damage endured by above-ground stems and leaves impacts the synthesis of essential amino acids via disruption of the shikimate pathway that limits energy and proteins available for seed production (Lacroix \u0026amp; Kurrasch \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Following decades of herbicide treatment with little abatement, remaining patches of \u003cem\u003eP. australis\u003c/em\u003e in Suisun Marsh may have developed some resistance to the compounds being used due to intense selective pressure, as shown in other species (Sammons \u0026amp; Gaines \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gaines et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on our understanding of other tenacious populations of \u003cem\u003eP. australis\u003c/em\u003e, we surmise that producing more viable seeds in areas of high patch density leads to rapid establishment and expansion of the \u003cem\u003eP. australis\u003c/em\u003e haplotype M, which may also apply to Suisun Marsh. Here, we look to answer three main questions:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eHow has past treatment impacted \u003cem\u003eP. australis\u003c/em\u003e propagule pressure?\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHow has past treatment impacted \u003cem\u003eP. australis\u003c/em\u003e seed viability?\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHas the high-intensity treatment led to a detectable amount of herbicide resistance developing?\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eBy answering these questions, we hope to better inform land managers' decision-making and contribute to more successful management outcomes in the future.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Area\u003c/h2\u003e\u003cp\u003eSeeds were collected from public and private land parcels across Suisun Marsh (38.1475 N, 122.0053 W). The marsh consists mostly of managed wetland ponds protected from semi-diurnal tides by levees with some tidal wetlands and sloughs open to natural tidal influence. Salinity is generally highest in managed ponds, followed by canals, and then sloughs, with a mean salinity between 1 and 10 ppt (Schacter et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Managed ponds are flooded and drained based on landowner needs, with most flooding occurring in September and early October to create habitat for the migratory season of many waterfowl. This is followed by a spring leaching cycle from February to May, after which managed ponds are dried up and invasive plant management is undertaken from July through September (Grewell et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Conrad et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough 2,000\u0026ndash;3,000 ha of managed land is being restored to tidal marsh to benefit these species, the vast majority is managed to support migrating and wintering waterfowl (Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Herbicide treatments of \u003cem\u003eP. australis\u003c/em\u003e with glyphosate-based products occur only in managed wetlands when ponds are dry to avoid impacts on non-target species. To cover large areas with some accuracy, most spraying is done aerially with helicopters or drones, but other methods of applying herbicide are also used (such as backpack sprayers). Mowing, disking, and burning are also employed on occasion, but we only took account of herbicide spraying in designating treatment areas as high- or low-intensity management because we did not have access to records on the prevalence of the other activities.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSeed Collection\u003c/h3\u003e\n\u003cp\u003eLand parcels were selected for seed collection based on the years of glyphosate-based herbicide treatment as determined from historical data provided by SRCD, which has records of the acreage of spraying per parcel per year from 2000 to the present. Parcels with 9 or more years of herbicide spraying were classified as high-intensity treatment, while those parcels with 3 years or less of spraying were classified as low-intensity treatment. Five of the \u0026ldquo;low-intensity\u0026rdquo; treatment parcels had had no treatment at all during the period since 2000. Seeds were collected in late August 2022 from 11 sites of high-intensity treatment and 9 of low-intensity treatment, with multiple sites in some large parcels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Five inflorescences were collected from 5 patches in each site for a total of 25 inflorescences per site and 250 inflorescences per treatment. All but one of the collection sites were in managed wetlands, while Joice Island (JI) is a tidal wetland that has never seen herbicide treatment.\u003c/p\u003e\u003cp\u003eDuring collection, a 0.5m x 0.5m quadrat was used to measure inflorescence density in 5 randomly selected areas across each patch. The long and short axes of each patch were measured to estimate each patch's area.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePropagule Pressure\u003c/h3\u003e\n\u003cp\u003eEach inflorescence was weighed and stripped of all spikelets. Spikelets were then weighed separately from the stem. For each inflorescence, an aliquot of 60 spikelets was counted and weighed to estimate the mass of a single spikelet, and this value in turn was used to estimate the total number of spikelets on the inflorescence. Three spikelets from each inflorescence were also dissected to determine the average number of seeds per spikelet.\u003c/p\u003e\u003cp\u003eA raster file of \u003cem\u003eP. australis\u003c/em\u003e cover in Suisun Marsh provided by the Random Forest classification of 2018 NAIP imagery was used to estimate the percentage of \u003cem\u003eP. australis\u003c/em\u003e per m\u003csup\u003e2\u003c/sup\u003e of marsh in each parcel (Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The propagule pressure of \u003cem\u003eP. australis\u003c/em\u003e per m\u003csup\u003e2\u003c/sup\u003e of marsh was calculated as follows:\u003c/p\u003e\u003cp\u003e\u003cimg 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UOJ3IvNINP5wzbjc/hD9qgj/FFkpwIrcmquG2cQS0W86rPxyEZJTRPbW6Yh8wvcYPSeGhZOJx2v8Ro4n74jf97R9xL3u5ebQMTyYvYuxzKkHVYmX+Sa78lSNfcJGcmKD6srTp6TbxRf9ncsvax7eSs9XVFC6giPdS5q6wds2LaGGCNkbPXW4RtqwqE1Obrmz9Ct432+zoQ91UV7PSnlqVFYY9poyKcV6loiZGSkjddmGTmn0kz7ycnu/ui/qnbW+/eyAZ2J0dwm1tlUYVPt7HDUC+z6NgYZSKsz0KD+3SWU1pRflnqoA3ktuUjWl+mzyRXjYk76S37Ykwufm8NUWULG9hF4I6NTerxUDpGMB9bh+QBSpu1dJRrEAZ0Sy0SoAYR86OKpXtSbg3bPvc8qtLKtXSZmj3OpeaUxA4qwYEGKUAp5kpTJlWYlX5msCKtWjnsg0vCTlxC/f/OFb36MNlxAoYkJYGODGgahktYD/xqi9n19ntPpIelMfOnpTedyO/YDwqZIczadVGDds2jDK9Ok29BCwza3u2e66WBvLh3aFmN4IQKFjFZLyRx0TuKL3/z0rSA3fB8q1J3MFdLAgmC6n6KKWonmKiPAHbPnShuubgtkV2iKLtZHoFpA6P80Kkk4Ztv95uNFn+33ELfrH+Zj4sEYRu2ggtTMpE6CvBGIeOOvDjKH1UDhFysKBqPTTQ+lFX8CbuvRVi3xPjSXmIaGmTs+JVWv5NP7fZuBF19CPuDpS+uiHsobYVpmJkJONdoBLTTkmMCagRfi8J1r60fPITXrBl+iM+v7D2zpE25ENjCkkdwi94F+GxfQfQRX9MhobHm3+yKO7vvRiY3oDzaLe8Nj3xZX35Fy9hXxlemUayyTz6aJmIvf5opb7yPuAF20+C7rSK3+kR3VFLV3Wmph+/g1RtqdPOi3l+0zrMKdX/gep9zNoSFz7EWyiX+Y73Kc30CzqG2m7CCmvP7GcRq9MY+VKuqS51J/oaM4g5G694zR65U9kFNElrcrrej8ippi41o82laNX/gd5Xu93/BRYHFHOR0R/kFO+PmaKhFjVG7t+ErG5hDxuvsfuxzDUlUyqpAv/q3iLBB//SHAZ39nwI+FpEZcFJOJnP5kxY18860IUG+kZ+Ie4Sex2pZN69sV/Gi+DC9c/cuHIj3ZUcMeQbA28IgFfuur+SWKtSDIUZpFfmhRK8YNZ7LQ0DGHrnRoxI/v3Ujuy7bPWSdzkWPhofMTHosn7OvCkGtxbho7Zt5oIVySDLQBGITks930fTDiXAzMz1e9XbCJMk0CygYpyvERmfsfNz0e279Y51XRhayM6NL6UWpgYETRp07e68BsoFM4Z00oh/M2F9ffL3YmtmMXQOIxvrulRL6OCZZwA5qZ9DdLPuO08vhXZU0gy63w/GaSctpweXitZ0UCzctsoJiphRPdrAtaGW4baYVK3YLtzO2lMJ0b273ZjOxWm/iMJIfPrFsxq/Ug0vbBxjJhj/i64dYar5sISAzJbnjvUNG46Dh9E6iWbKUQMB4a8aRiKHUd6fj5Qo3If6rNs+IdwL3bGGtqwYPGePG6wfl50PWyBNjTSJ7Z6NAr32cdAArk9dZkA34YthcgJP9rI2T3y4/L5DUVLWlkjkdJHjwk/KqOm0VQE/8iISwEsz9sFaP3xWaB6QxeQc2q4ec6UwHq5m2Om1S/6DqtLveofDjzI157O1h+XkH8IjU0NGrmw1V+PHwF38N1WN3yaiH92Xd7mP0c+pWFkZRFSwdSvlefAW1KcAWxylVglfYjKYwoZFsYB7EdTq5oKeiXmUFtiPaZagt7MVxldzoZ6/kDszKe8/N0ek+QQJhWMT2XnrlIy8gfU8rdhYD71PsoNk5YC9A9trN7JAdR6xpmaAanTn0qAWZEGi+n8qDdOv2G1agAzaph4Y5+/r9Hm/JchPf9CWEVNDPNsfVLPl6DVba9vY7IumHpijpU7vXx2PT4fsUutZIHHo6FL1sfAYuTc4QXLwFk0l3qGjxRddZGq+ou45JylN/M1ZB9qoVv/4r3VOaEnucDG9Hkn++oDnaz8gZDj43Osortqzd9XspdsgGqhy4OaDZBSDF5pe8oM8meuBc9XQ0iXB++Pdl0TcSdurdXDAUDSnv2PSUdo2OzZmt7CtNuznTXQmFnSz+allqjqzx3ZmSsdSzY/oku/hujTta0wqRR9RGYYgylZsTSwp2/NRHD+2sME+8og5tsZ0cD5DlxewSMsT98AhAU9wcpSH8a1sWqN76aoeb3QVfrtpD3Lt5RuAyyOtlWlxjmXoxx4jS0DZ/5WCZMdxKb3im549nAnDkxErGscJLewvNavxbhrTK8749GDKJ+dXhAQpk2UwOySG86aacwSuSWlXtzorhKI2kWb6uF1+32Ee3nJDGj22v3i8T0ApsX3QlGbi1xNoZ4BlU7QKCIXLBAUjdoypApzyly+n93+AVYP93d0WXOovL3dEjAmRcCVM7DMSgYsJ+2z6Z4ERLlfNt2TPgQlttFg54RGdzz4C5Nzaj+9hW9w7E0sAJv4SFwrdwYs/SiHDtgTRIA1k4wBKwdgfyhlGWx7cU7uWrESdtsZ9gYLGQ+pBBYsjK+dfrhzKkrIBriFmkLajz8LSyOFpjlWLQsylLM/7zCXyigBjCcE7QuOuPv9JSNVQfpMmWwGlO/k0+WtxCEXEQf7Rj9sGmcifvpESXzS15fkm0IbYgQMCzUG13Em5J3OArW2WtbUvikZ8TYjr0UC1/K4UCk4unDOSzZSrJuSCPprFhcwK38PxkVFSns6VVBS48A2YGefkIIzGY9VVx9Tsk3rPBc+pgawtMOlSlC1GkMJueVLr2l1kgXW5nGx8roFMuL5AUwZb9r+srIVZ9R7r4it2kO+v3D1SNBas1x8PmBaJHATk38c0VP96aImwe4aDk1vUaR6CbZyVDRh5hUOs678V5QxUE7Tl8330yh4i4LhH6uiA7SpUUtqYlreF4jvBuQmdQQUg7mQzpPGmgA1YHUANzORyoepcVaFKIvleRyQIUPFAFVEnXaDfmMniWZ7s6nnMP0JklypuIbNVvw5+o35vZHq+CtwI74EVe78QXDlneU8MBkMVVga3pHivfgfwOBILJrtDJhBP9sAuDUXGBNApcjh/V8WbLAbPfkJ92LDNwlIUREcNsAea0BtEfLZcmb31tVM4SApiQVoUiOQWmyCOotnGr9jJxdesuB6UfHuvrFJGqnAkzvdkKJCpZdIRY7zbcl6qKYKkOQLbBPsWeJ66SNlN+Djzbel1GJVNDTB8rIAT0IDEJPT9WHDx/q1rMT3h/6vkbRj/tro/fMXVIkEzRSwIOA8KiBx3N/zcozX1hioA+FbPVzKJ2yrDa5xe/LQ0Tl80PHOrgvBUuzy7sDYloX5qV4YCakHebvBCNgL3iUHJmwM++TD4wBG1VSUiD6+7e9zbzVHZ5ZWta75pNliJudNoOTUQ/qd4Pv7Jf4FlB2oR4bALa/SyBl7Pfj37d7pivzUvUdgNmG0FDa5Uwlg6BTqlnpzpNifOer4ZSmPeeGkT+wOBdnDJ2UHztGD19J3MAv6VSlp8+2E3Y/uwyZwp9rkwsauPPYuy3u+bJ/tkhFHbgteGEwo3QUUTvoULZXisNfgeqJP3PXiIWAdsAGr5C/7iO1ipolklsGTXUvbDpnr/hZoY3j+TMBroGy0KjrlPQAkTr4/Pk+vYdNNQTlz/lSjo7b9Nh1NF7TVdcKeg3crMte3OWtwm1pAGGpYdvzKBtlX6zvLzSGFkMs+d+lOGEJqXmkV/K/xkwAaIc8vF59Pa3s6x03rioC3Y3dlO7JPevLpxPxjxAToH6jANrygOczoTeCK5P+hhBhj/GzwZsyfVmv71QSxRpFy4PT5LieWAQp56XOyz2kAnW4HV4RfGQmv9Dub2b0vLvJwGQfV83llA5irrUePvsvD7tzAcw22DPwG+mPIXynAs6BVMC5eNFlMp5ivoDHV6ehNRm26iKeDP/C4Pd8osvSoGZ19CG7k/fCLvYCX1oYea3g45mSYuNoCh8hZqCbOa7t7550rib9KQM4qYHTOlxk2X6H6pkHpoOgDeYPSZgwWeMs6FtSqT8kr/DhESJ07qsmzK1d93uZNHJJCt8An8A8se6/DXev4p6x45bPb3UseuEEy1Ah3SRg4jnuA3rbJwN9jedxz9XapayLMxXFLl/5WYWf3LDpIi15IpchNOVl6OeRE1i/ywWekkBb0HEtIhe+GV3n7jEaOgMMlmO8bgidBmf207jTbDcbAY1dTFBF5XDb8kEFVHyaGkY0wIn2Rf3sAam+XwOzSYya2vehM6bEeZDIbPGxzd2H5WrUsakXUfIiSPDUd7RFYndaPWk7UMZEAe7kFsNOECZj3i6GEaqaaALvqLD9KSSXqi7jjr8HRHSDHA3y9mGD1HsJCTHFd+Jfon1S0h7+kOOUd3b27zjDQTItSB8wG1uZT1pqo7llqxyuWjFs3SoUYqwIKIWKAivKf1HCtoBzT+HwRpuiJRT5ItFVAYluB6n+1+5FMg+efVNcN1zQ4b99BTf5MMH1pBTQuzvKCLAvZ+6jie6QHOAz1GZ2BDelJ8H+4AVBaFwM8cpoKXvNVOQvOQcGi3IAD+syxGfRC6m+wTtkloUtQV7x3YJidOQJssh0ITCNDwWTAxjoN4Rycv22KQQca3zd0GHfswd73HqyrOfQiPortuVBMF8PacmfMLDBDb/eXwOICfhIfe6UTteXDdL8K5MdTk95WYKZjY/ViS3hb8HlzwX8SlRd3keSYATW/TvQiCo7nhCT1ehbVGEMzVAQpAe8epqBo/SqgT26pG6Azn/J0cy4ZUHVNgqYCJwaroqQ0BHhrrQ74epIMwVOGRnIoTw9VpARR6QKn+Cmn30Lt4mU4B23BgIMbgcxgHpB4gP/TEy6R31hg556EztEWOOS75W2A97wVULETmCmus+gBRIMHg3N4PHCuA1DkJwf0v/phLf9GVn8mrl1dhdk1reQ9O+LXMmO1MYg+X8pPyf/nmW4dhT5Z8hhxUGCYjmUAf2OYTzh6AnPi1cDJLINFwCVK5H/qoApMaqho+VWn+ebTMLITMWQ28HeBPkQdMimZ1GwypUFLTWT2Q0ssCfXHtPUCtwF2ezMCaVm10J5fAUlShQHu4wGLRWvrStV/JSMwwO4JljvF0c9sPEOXhqAfkcd9u2UhAjK4upJKeC16B41J7yJlHRzFJAsOx5oytvM1ScUa4NwsBmacAbOzZ2TLnHkfYEm3aZV2vonOqJ6NnTS1Mivbp2Cauxomt4+NFvATqtw/rFSIZ/Ad2RNOyZYEXYzU5QU+SC06Pai3SWEK8oku9Irdlk+BfCi1jxSeElOFTLJRTqfsws4E2e0ze0ss6QFOaJP2IaSTeREYWe4MLauBgm7Ho4htePJiG5IDoiOVSIWvPek0jqtdSu1qw5qmDQVyE0/nx6DPmxGmzTYo9rg6462fF866bh8kwHl60z0MPYkyNA6O3eP7mpx48GidxxP/Av6/budX+OyKEdSyhoMzN6+LoPmbkIZYJYLhGaCKZKfEUV8ameoOZG2d4nlG+DFqu0jwggxy/KXoxCD7Tgbrz2+c1z159Nxw9La9arAJqw/y42y80nM7Vz6MGGl++XSX6WYnSifGeh5MHrmoJYonxa4/VA+WAZfJqcX0QgAdtjOYIne42Bh/qVPShIeelQFj2yqsIZRJzgiISLbv4A1FEg43Yri62WFy/fKBIdKqkOr4UtBigvF9MXg95QJuz83BfVqpbjZFCZo9+BaaxNNX7cKAVCruYWAvao80Id9BTxyMsyGARaWSAA+s+mKjkroHc8BTyj0J9deLddR7SQPelRXQfTKQXXF3yzBKrymvCAFMa+vM0bBjcqh4ogavNtpAVa8ExFbO/k3gBvqNxVlpJ0SNdAHvXgJ3PekhD2ZaGBOpr6cyYHLMBGHPkzDuKeVoYEY9n/37biBECTAbOrT5kx8hQoQIESJEiBAhQoQIESJEiBAhQoQIESJEiBAhQoQIESJEiBAhQoQIESLk38r/ARC+FK1SlTzNAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n\u003ch3\u003eRandom Forest Classification\u003c/h3\u003e\n\u003cp\u003eWe used the classifications derived by Hagani et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) to estimate the area of \u003cem\u003eP. australis\u003c/em\u003e per area of managed wetland in each land parcel. The classifications were built upon aerial imagery collected by the National Agricultural Imagery Program (NAIP) from 2003 to 2020. NAIP imagery includes three color bands (red, blue, and green) and a near-infrared band. In California, these images were collected every three years pre-2009 and every 2 years since. Eight classifications were generated within this timeframe: 2003, 2006, 2009, 2012, 2014, 2016, 2018, and 2020. For each classification year, representative \u003cem\u003eP. australis\u003c/em\u003e patches were selected from the image and manually classified (Tadros et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) to use as training and validation data. Separate polygons were also manually selected to serve as \u0026ldquo;non-\u003cem\u003eP. australis\u003c/em\u003e\u0026rdquo; data. Random forest classifiers (Breiman 2001; Cutler et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) were built in R Studio (Liaw and Wiener \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; R Core Team \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; R Studio Team \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using a random 70% of the training data, with the remaining 30% withheld for model evaluation (Paz-Kagan et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Classifications were generated with the color bands, the near-infrared band, and an additional predictor variable for every year following 2003, which described the distance of every pixel from the nearest \u003cem\u003eP. australis\u003c/em\u003e pixel classified on the previous image. For each classification year, five random forest models were run based on a different random selection of training and validation data, and accuracy metrics (User\u0026rsquo;s accuracy, producer\u0026rsquo;s accuracy, overall accuracy, and Kappa\u0026rsquo;s statistic; Fielding and Bell \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kraemer \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) were averaged. The singular random forest classifier, which produced the best accuracy metrics, was then used to create a map of predicted \u003cem\u003eP. australis\u003c/em\u003e each year in Suisun Marsh with the package \u0026ldquo;raster\u0026rdquo; in R (version 3.6\u0026ndash;11; Hijmans and van Etten \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The final output of this process was a fine-scale (1-2m) wall-to-wall raster of Suisun Marsh with values of \u0026ldquo;1\u0026rdquo; to represent \u003cem\u003eP. australis\u003c/em\u003e and \u0026ldquo;0\u0026rdquo; to represent non-\u003cem\u003eP. australis\u003c/em\u003e for each classification year.\u003c/p\u003e\n\u003ch3\u003eSeed Germination\u003c/h3\u003e\n\u003cp\u003eSpikelets from each inflorescence were counted out in groups of 60 and then combined by patch so that each patch had 300 spikelets from 5 inflorescences in the germination trial. Spikelets were initially washed for two minutes in a deionized water bath with 10% by volume household bleach to prevent mold growth. Petri dishes of 150 mm were then filled halfway with autoclaved sand. Deionized water was added to moisten the sand, and any excess water was poured off. Filter paper was used to cover the sand and allowed to equilibrate to the moisture. The spikelets were spread onto the prepared Petri dishes in a layer 1 spikelet thick, after which the Petri dishes were sealed with parafilm. The seeds were then cold-stratified for 2 weeks at 4˚C in the dark according to the method of Kettenring et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Once stratified, seeds were placed in an Intellus Control System growth chamber (Percival Scientific Inc., Perry, IA) set to an 11:9 h day/night cycle mimicking temperature fluctuations and daylight hours of Suisun Marsh in late spring and early summer, ramping linearly for 2 hours from 13˚C to 25˚C. The petri dishes were checked for germination and rotated every other day for 2 weeks. Germinated seeds were counted and removed. Germination was defined as the observation of a radicle emerging. After two weeks of germination trials, the ungerminated seeds were discarded.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHerbicide Resistance\u003c/h2\u003e\u003cp\u003eGerminated seeds were grown in a greenhouse for 1.5 months in June and July 2022. Plants were kept at ambient temperature in Santa Clara, CA with ambient lighting. Plug trays were filled with potting soil and then placed into a larger tray kept flooded with water, whose salinity was managed to remain at 5.7 ppt, mimicking conditions found in Suisun Marsh. Plants were trimmed above the first healthy leaf to standardize for biomass and leaf area. The remaining leaf on each plant was then wiped with 2% glyphosate (Round-Up, Bayer Corporation, Whippany, NJ). \u003cem\u003eP. australis\u003c/em\u003e plants were visually checked one week after herbicide application and damage was rated on a 0\u0026ndash;5 scale, with 0 being no damage and 5 being total death (Ogg et al. 1991; Dear et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). We also took note of whether resprouts began growing from plants post-herbicide application.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003ePropagule Pressure\u003c/h2\u003e\u003cp\u003eThe propagule pressure of \u003cem\u003eP. australis\u003c/em\u003e was 17,986 seeds per m\u003csup\u003e2\u003c/sup\u003e of marsh, on average, in low-intensity treatment parcels and 8,178 seeds per m\u003csup\u003e2\u003c/sup\u003e of marsh, on average, in high-intensity treatment parcels, a 54.5% reduction in seed production (t = -5.307, df\u0026thinsp;=\u0026thinsp;313.93, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The difference results largely from the lower percentage of \u003cem\u003eP. australis\u003c/em\u003e cover in land parcels classified as high-intensity treatment. Parcels subject to low-intensity treatment had 7.42% \u003cem\u003eP. australis\u003c/em\u003e cover, on average, compared with 3.39% cover in parcels of high-intensity treatment. This was a 54.3% decrease in \u003cem\u003eP. australis\u003c/em\u003e cover across the marsh from low- to high-intensity treatment land parcels, which includes 59 additional land parcels in Suisun marsh where seeds were not collected (t = -3.23, df\u0026thinsp;=\u0026thinsp;74, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On average, inflorescences were similar in weight between the two treatment groups, with 1.1 g inflorescences, on average, in the low-intensity treatment parcels and 0.98 g in the high-intensity treatment parcels (t = -1.036, df\u0026thinsp;=\u0026thinsp;415.4, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was no significant difference in the number of seeds contained within each spikelet (~\u0026thinsp;6 seeds/spikelet), nor stem density (~\u0026thinsp;40 stems/m\u003csup\u003e2\u003c/sup\u003e) between the high- and low-intensity herbicide treatments (t\u0026thinsp;=\u0026thinsp;1.051, df\u0026thinsp;=\u0026thinsp;513.11, p\u0026thinsp;=\u0026thinsp;0.4782; t = -1.2161, df\u0026thinsp;=\u0026thinsp;441.93, p\u0026thinsp;=\u0026thinsp;0.2245).\u003c/p\u003e\u003cp\u003eJoice Island (JI) had the highest estimated propagule pressure with 44,212 seeds produced per m\u003csup\u003e2\u003c/sup\u003e of marsh (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This was the only tidal wetland site sampled that had never experienced herbicide treatments due to regulations on the use in tidally influenced areas. The sampled area had 9.33% \u003cem\u003eP. australis\u003c/em\u003e cover and the mean weight of seed material per inflorescence was the greatest compared with all other sites at 1.89 g. The publicly managed Crescent Unit (CRU) had the lowest estimated propagule pressure with only 344 seeds per m\u003csup\u003e2\u003c/sup\u003e of marsh, attributable to smaller inflorescences (0.485 g) and low stem density (8.80 stems per m\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSeed Germination\u003c/h2\u003e\u003cp\u003eGermination rates ranged from 0 to 35% which varied between patches of the same site. The mean germination rate was not significantly different between high- and low-intensity herbicide treatment sites with an average of 7.81% and 7.15% germinating, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e., t\u0026thinsp;=\u0026thinsp;0.349, df\u0026thinsp;=\u0026thinsp;84.27, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Seed viability ranged widely across all sites with the with East Lower Joice (ELJ), 11.1%; Mid Lower Joice, (MJL) 11.5%; North Lower Joice (NLJ), 17.1%; and Joice Island (JI), 22.9%) representing the highest percentages while other sites, such as Delta King (DK) and Grizzly Fairview (GFV) produced fewer viable seeds, on average, with 0.043% and 0.23% germinating, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHerbicide Resistance\u003c/h2\u003e\u003cp\u003eThe average damage rating of young plants grown from seeds collected in high- and low-intensity treatment sites was not significantly different, suggesting no difference in herbicide resistance (t = -1.52, df\u0026thinsp;=\u0026thinsp;18, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There was also no difference in the percentage of resprouts between high- and low-intensity treatment plants that survived the glyphosate treatment (t\u0026thinsp;=\u0026thinsp;0.38, df\u0026thinsp;=\u0026thinsp;18, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeed production was reduced in land parcels that experienced high-intensity herbicide spraying (\u0026ge;\u0026thinsp;9 years), with this reduction in propagule pressure primarily driven by decreased \u003cem\u003eP. australis\u003c/em\u003e cover, though factors such as inflorescence weight and stem density also influenced overall production. The overall expansion of \u003cem\u003eP. australis\u003c/em\u003e has been rapid over the past two decades, however, remote sensing analysis reveals that 61 land parcels across Suisun Marsh experienced a decrease in cover between 2003 and 2020, as reported by Hagani et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The regular use of glyphosate-based herbicides to limit the establishment and expansion of \u003cem\u003eP. australis\u003c/em\u003e patches is a likely contributor to the decrease in these land parcels, given our results. Successful control of \u003cem\u003eP. australis\u003c/em\u003e is less challenging for small, newly established \u0026ldquo;satellite\u0026rdquo; patches, whereas larger, established patches are more productive and difficult to control (Mozdzer et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kettenring et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Martin \u0026amp; Blossey \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, land parcels that have been managed with herbicides for a longer duration may have prevented satellite patches from expanding their area via rhizome to become unmanageable. Corroborating our findings, another study evaluating the management outcomes of an introduced \u003cem\u003eP. australis\u003c/em\u003e population in the Great Salt Lake found a marked reduction in seeds produced per m\u003csup\u003e2\u003c/sup\u003e following treatment with glyphosate and mowing at different times of the year (Rohal et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Herbicide application was carried out in either early July or late August, the same period during which most management and maintenance operations are performed in Suisun Marsh.\u003c/p\u003e\u003cp\u003eOur results show that land managed with herbicides can still host considerable populations of \u003cem\u003eP. australis\u003c/em\u003e, as Lower Joice Island has higher coverage than the mean of most other high-intensity treatment sites (Table\u0026nbsp;1). Despite their coverage, the majority of land parcels under both high- and low-intensity treatment produced an estimated hundreds or thousands of viable \u003cem\u003eP. australis\u003c/em\u003e seeds per m\u003csup\u003e2\u003c/sup\u003e of marshland (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consistent with previous research on the topic, this suggests that seed propagation is a notable contributor to new patch initiation (McCormick et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kettenring \u0026amp; Whigham \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This presents continuous pressure as the dispersal of \u003cem\u003eP. australis\u003c/em\u003e in managed land parcels is influenced by the number of unmanaged neighboring parcels (Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to the long distances possible with wind dispersal, management is most effective at a watershed or regional scale, as is advocated for by other authors on the subject of \u003cem\u003eP. australis\u003c/em\u003e control methods (Takekawa et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hazelton et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hagani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeed viability is a consequential element in the establishment of \u003cem\u003eP. australis\u003c/em\u003e, and may be difficult to contend with in a noxious population, as our results showed no significant effect of glyphosate on seed germination rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Rohal et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) investigated the introduced Great Salt Lake population to quantify glyphosate impacts on seed viability which did not differ significantly from the control that received no treatment. Though not specifically looking at \u003cem\u003eP. australis\u003c/em\u003e, Nurse et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) focused on the impacts of same-year glyphosate application on seed viability of Hairy cupgrass (\u003cem\u003eEriochloa villosa\u003c/em\u003e), an annual grass that can reduce crop yields in the Midwestern United States. They saw no changes in seed production, however, they reported a significant decrease in seed weight and germination rate. These results are the opposite of what we found in our results on \u003cem\u003eP. australis\u003c/em\u003e production, though our focus was not same-year glyphosate application, but rather regular treatment over multiple years. Viable seed production can be controlled in some invasive species such as \u003cem\u003eE. villosa\u003c/em\u003e, but our results support those of Rohal et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) that indicate lower susceptibility of \u003cem\u003eP. australis\u003c/em\u003e to these effects.\u003c/p\u003e\u003cp\u003eWhile seed viability was not significantly impacted by the treatment regime in this case, the variability in viability across all sites regardless of treatment suggests other environmental or genetic factors. Influences affecting the viable seed production in \u003cem\u003eP. australis\u003c/em\u003e are not fully understood, however, likely contributors to increased germination rates are genetic diversity and patch size (Kettenring et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kettenring et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Thorough research of the introduced Chesapeake Bay population found slight positive relationships between both of these factors and seed viability. Future genetic analysis of patches with high and low seed viability may help illuminate the potential of positive feedbacks that result in more new patches of \u003cem\u003eP. australis\u003c/em\u003e. Nutrient loads have also been evaluated as drivers of seed viability, and although a significant relationship was noted with inflorescence production per plant, the same was not true for viable seed production in the Chesapeake Bay.\u003c/p\u003e\u003cp\u003eResistance to glyphosate in introduced populations of \u003cem\u003eP. australis\u003c/em\u003e has yet to be investigated thoroughly, though the management implications of resistance are of utmost importance. Due to its prevalence as a weed control agent since the late 20th century, at least 24 species of weeds were documented to have developed some resistance in 2014, which has since reached 57 species as of 2023 (Sammons \u0026amp; Gaines \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Landau et al. 2023). The mechanism identified in resistance involves one to three point mutations in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is essential in the shikimate pathway targeted by glyphosate herbicides (Sammons \u0026amp; Gaines \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Garc\u0026iacute;a et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The EPSPS mutation has not been identified in \u003cem\u003eP. australis\u003c/em\u003e, but it was detected once during a small-scale investigation of \u003cem\u003eP. australis\u003c/em\u003e resistance in a population of Indiana Dunes National Park, with inconclusive results (Nikkel et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Future genetic analysis of populations in Suisun Marsh is vital to understanding the state of resistance.\u003c/p\u003e\u003cp\u003eIt is clear from our findings that regular application of glyphosate is at least partially effective in controlling \u003cem\u003eP. australis\u003c/em\u003e in Suisun Marsh by reducing cover. Still, many other factors are involved in the continued spread that have received little attention on the West Coast of North America. Even if cover is shown to decrease with continued herbicide application, the varied results in seed viability suggest that land managers are contending with uncontrolled environmental and genetic factors that heavily influence the success of new patches established via seed. Until our understanding of positive feedback and invasion mechanisms is clarified, management of \u003cem\u003eP. australis\u003c/em\u003e will be most effective through combined, comprehensive, and contiguous control efforts across land parcels.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no competing financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was primarily funded by the Delta Stewardship Council (Contract #DSC-21004) to VM, with assistance from a Santa Clara University Hayes Fellowship to GR.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eVM, MW, and GR contributed to the study conception and design, material preparation, data collection, and analysis. Herbicide resistance trials were primarily carried out by MW and VM. Remote sensing analysis was performed by JH. The first draft of the manuscript was written by GR and MW, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe would like to thank the Suisun Resource Conservation District for their assistance in contacting landowners for sampling permission, specifically Dr. John Takekawa. We also thank the Delta Stewardship Council for primary funding to VM, and the Santa Clara University Hayes Fellowship that provided supplementary funding to GR. We would also like to thank Dr. Karin Kettenring for her consultation on methods.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the \u003cem\u003ephrag\u003c/em\u003e repository that is publicly available,\u003c/p\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/gaberodkey/phrag/tree/7528eb506bacc3199863d1f8d5e4b92868b4b311\u003c/span\u003e\u003cspan address=\"https://github.com/gaberodkey/phrag/tree/7528eb506bacc3199863d1f8d5e4b92868b4b311\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaldwin AH, Kettenring KM, Whigham DF (2010) Seed banks of Phragmites australis-dominated brackish wetlands: relationships to seed viability, inundation, and land cover. 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Flora and Ecological Profile of Native and Exotic Estuarine Wetland Vegetation by Hydrogeomorphic Setting at Rush Ranch, Suisun Marsh. profile San Francisco Bay Natl Estuar Res Reserve, 79\u0026ndash;144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchgate.net/publication/279181056_Flora_and_ecological_profile_of_native_and_exotic_estuarine_wetland_vegetation_by_hydrogeomorphic_setting_at_Rush_Ranch_Suisun_Marsh\u003c/span\u003e\u003cspan address=\"https://www.researchgate.net/publication/279181056_Flora_and_ecological_profile_of_native_and_exotic_estuarine_wetland_vegetation_by_hydrogeomorphic_setting_at_Rush_Ranch_Suisun_Marsh\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuckin SJ, Howell G, Robichaud CD, Rooney RC (2023) Phragmites australis invasion and herbicide-based control changes primary production and decomposition in a freshwater wetland. Wetlands Ecol Manage 31(1):73\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11273-022-09902-3\u003c/span\u003e\u003cspan address=\"10.1007/s11273-022-09902-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Herbicide, propagule pressure, resistance, viability, Phragmites","lastPublishedDoi":"10.21203/rs.3.rs-7474318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7474318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBrackish wetlands are vital habitats for migratory and endemic species in coastal areas, supporting nutrient cycling, flood management, and recreation. \u003cem\u003ePhragmites australis\u003c/em\u003e, or common reed, is a relatively recent invader of brackish and freshwater ecosystems across North America. In California\u0026rsquo;s Suisun Marsh\u0026mdash;a 46,950-hectare network of public and private wetlands\u0026mdash;\u003cem\u003eP. australis\u003c/em\u003e has dramatically expanded over the past two decades. This expansion reduces habitat quality for waterfowl and other species, as dense stands displace native wetland plants. Herbicide-based management, primarily using glyphosate, has been employed in some areas of the marsh for over a decade, while other areas remain untreated or were only recently treated. To investigate the effects of management, we collected inflorescences from 11 high-intensity treatment parcels (\u0026ge;\u0026thinsp;9 years of spraying) and 9 low-intensity parcels (0\u0026ndash;3 years). Random Forest classification identified large patches of \u003cem\u003eP. australis\u003c/em\u003e across satellite imagery, allowing us to estimate propagule pressure as seeds per square meter. High-intensity parcels produced fewer seeds on average than low-intensity parcels, due largely to differences in the area occupied by \u003cem\u003eP. australis\u003c/em\u003e (t = \u0026minus;\u0026thinsp;5.307, df\u0026thinsp;=\u0026thinsp;313.93, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However, we found no significant differences in seed germination or herbicide resistance between the two groups (t\u0026thinsp;=\u0026thinsp;0.449, df\u0026thinsp;=\u0026thinsp;101.95, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; t = \u0026minus;\u0026thinsp;1.52, df\u0026thinsp;=\u0026thinsp;18, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings highlight the effectiveness of consistent herbicide use in reducing \u003cem\u003eP. australis\u003c/em\u003e seed output and emphasize the need for coordinated, watershed-scale strategies to prevent the establishment of new patches.\u003c/p\u003e","manuscriptTitle":"Effects of high- vs. low-intensity herbicide management on Phragmites australis propagule pressure in a brackish wetland of California","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 18:40:59","doi":"10.21203/rs.3.rs-7474318/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60820d29-7174-4ab2-a141-e2585ec87df1","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T00:35:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 18:40:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7474318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7474318","identity":"rs-7474318","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}