Propagule resistance of an invasive Poaceae as a trait of its invasiveness

Biological invasions and consequent mass development of aquatic macrophytes constitute a significant threat to aquatic environments. As a consequence, species invasiveness is becoming of great interest. Urochloa arrecta is a mass development invasive Poaceae that has caused many impacts in freshwaters in Brazil. Studies have described its remarkable tolerance to stressful conditions, but propagules resistance to desiccation while in dispersion is unknown. Here, we analyzed through a microcosm experiment U. arrecta’s small propagules regeneration after desiccation and without any sediment—thus simulating a transportation scenario. As expected, the longer the time of stress, the lower the propagule regeneration performance. Even so, the macrophyte propagules can survive up to six days off of water and any sediment. Even when the propagules seemed unviable, there was some degree of regeneration. As a consequence of the results, we state that removal and transportation of U. arrecta should be controlled considering the propagule viability.


Introduction
A crucial factor for invasion's success is propagule pressure (Simberloff et al. 2013).The higher the propagule pressure, the greater the likelihood of successful invasion by the invasive organism (Lockwood et al. 2005).In this context, if the propagule of an invasive species can endure extreme environmental conditions, it implies that colonization by this invasive species could take place long after significant disturbances have occurred.Invasive macrophyte species can undergo massive development, thus posing a significant threat to aquatic ecosystems (Thiemer et al. 2021).Many traits explain the colonization and invasiveness of macrophyte species, one of which is resistance to drought, particularly in species with an amphibious life form (Hussner et al. 2021).
It is common to observe small fragments of invasive macrophytes colonizing new sites, which can even disperse outside of the water.For instance, grasses such as Urochloa arrecta (Hack.ex T.Durand & Schinz) Morrone & Zuloaga (the tropical signalgrass) were brought from Africa to Latin America on slave ships and used as bedding material (see also Ferreira et al. 2016).However, there are no records of intentional cultivation during transportation.Still, the resistance of macrophyte propagules while in dispersion is not widely discussed.In this study, we conducted a microcosm experiment to analyze the resistance of tropical signalgrass propagules and their regeneration following periods of desiccation stress without any substrate, simulating a scenario resembling overland transport of propagules.This invasive amphibious Poaceae species has successfully invaded numerous freshwater environments in Brazil, posing a significant threat to ecosystems from the South Atlantic Forests (Galvanese et al. 2022) to the Amazon Biome (Fares et al. 2020).The stress caused by this invasive species is primarily due to its ability to outcompete native flora and even impact the local ichthyofauna (Carniatto et al. 2013).
Urochloa arrecta's propagules, once detached from the whole plant, can be carried through water bodies, facilitating easy dispersal and colonization of different environments, since each node present on stems can generate new roots, sprouts and leafs (Bando et al 2016).The tropical signalgrass has been shown to tolerate up to 26 days of desiccation when planted and rooted in sediment-filled trays (Michelan et al. 2010).However, the propagule tolerance of this species without sediment and water, simulating anthropogenic dispersion outside of water, is still unknown.Therefore, based on the fact that fragments of this species have already demonstrated notable drought tolerance (Michelan et al. 2010), we hypothesize that the regeneration of propagules without any sediment would also occur.If this is the case, this observation of invasion is crucial in informing caution regarding biomass removal and overland transportation of the tropical signalgrass.

Methods
Tropical signalgrass whole individuals were collected from the Guaraguaçu River (South of Paraná state, Brazil) in October 2021 and transported to the greenhouse at Federal University of Paraná for pilot tests and acclimatization.Our experiment began in April 2022, when 60 propagules were subjected to desiccation (Fig. 1A).The desiccation treatments involved subjecting two propagules (intermediate and apical stem parts) with two nodes each to durations of 0, 2, 4, 6, 10, and 15 days without water and outside of any sediment, simulating severe environmental stress during overland dispersal.Each treatment had five replicates, and after the desiccation periods, the propagules were planted in trays measuring 30 × 50 × 10 cm, filled with sediment (approximately 3 kg of soil with compost) and tap water (Fig. 1B).The trays were watered every two days for 35 days to observe plant growth and regeneration after the stress period.Throughout the experiment, the minimum and maximum average temperatures were 12.3 °C and 21.3 °C, respectively.The number of days for regeneration was determined based on the time required for measurable growth and sprout development in regenerating plants.
We evaluated propagule ability in terms of sprouting, growing, and rooting, which served as indicators of regeneration (Barrat-Segretain and Bornette 2000; Michelan et al. 2010).After the 35 days, we measured the maximum aerial and root length of the macrophytes, the number of sprouts on each propagule, as well as the root and aerial dry biomasses.To assess the impact of treatments on these measured traits, we performed ANOVAs with permutations, considering the lack of assumptions required for this type of analysis.Additionally, since the initial size of the propagule can influence macrophyte growth and other trait developments (Bando et al. 2016), we also took into account the initial mean size of the propagules as a random factor.Graphs and Fig. 1 were created using R software version 4.2.2 (R Core Team 2022) and Corel 11.

Results
After two days of desiccation, the propagules were already severely dry and showing signs of senescence, indicating initial propagule death.However, upon planting and watering, propagules that were subjected to up to 6 days of desiccation were able to survive.Even after nearly one week of desiccation and nutrient deficiency, all propagules from the 0-, 2-, and 6-day treatments produced at least one sprout, demonstrating propagule regeneration.The 4-day desiccation treatment also showed a high resistance rate, with only one unit failing to produce sprouts.
All variables, except for root dry biomass, were significantly affected by the desiccation duration according to ANOVA models.Additionally, only aerial length was influenced by the initial mean size of the propagules.Our graphs clearly illustrate a decrease in the development of all variables as the number of desiccation days increased (Fig. 2).The 6-day desiccation treatment showed lower development in all variables compared to the 0-, 2-, and 4-day treatments.In contrast, the 10-and 15-day desiccation treatments did not exhibit any regeneration, suggesting that these extended periods of water and nutrient stress can be fatal to tropical signalgrass propagules.

Discussion
We have demonstrated that tropical signalgrass propagules can survive up to six days under conditions simulating overland transportation (i.e., unrooted fragments without water).This indicates that the propagules are resistant and capable of surviving in highly stressful situations during dispersion through anthropogenic actions, birds, or other means of transportation without water.Indeed, a six-day period provides sufficient time for long-distance transportation of this plant via human structures such as boats or ships.It is important to note that our results cannot be directly compared to those of Michelan et al. (2010).In their experiment, the fragments were already planted in sediment, simulating drought in rivers or lakes.They found that, under those conditions, the tropical signalgrass can survive up to 26 days without water.In contrast, our study simulated the colonization of unrooted propagules.
When considering the results from both our study and Michelan et al. (2010), it becomes evident that sediment plays a critical role in sustaining the resistance of the invasive grass following drought stress.Here, all performance indicators analyzed (except for root biomass) were negatively affected by the treatments (Fig. 2), which is similar to Michelan et al (2010) work.However, after 6 days all of the variables measured presented values next to zero, indicating no regeneration after 6 days without water and sediment.The presence of sediment may allow roots to grow and facilitate nutrient uptake during periods of stress.Various environmental conditions, such as sediment, can affect macrophyte propagule resistance and survival (Kuntz et al. 2014;Hoffmann et al. 2015;Li et al. 2015).For instance, Hoffmann et al. (2015) found that increasing sediment levels softened the mortality of E. nuttallii fragments after desiccation.
As with many invasive species, we emphasize the need for the precautionary principle and increased control of biological material transportation, as propagules are resistant and can easily invade new locations, even when they appear unviable (as small senescent fragments).Anthropogenic transportation can carry propagules over very long distances, crossing geopolitical borders as well (Anderson et al. 2014;De Ventura et al. 2016).When an invasive species reaches regions far away from its native habitat, favorable conditions may facilitate its establishment and spread (e.g., through the Enemy Release Hypothesis, see Keane and Crawley 2002;Torchin et al. 2002Torchin et al. , 2003;;Mitchell and Power 2003).The invasiveness of a macrophyte is linked to its potential for rapid colonization of new environments, based on its successful establishment and spread (Ricciardi and Cohen 2007;Rejmánek 2011).Many invasive species demonstrate resistance to various factors, enabling them to adapt to new environmental conditions and successfully colonize new locations (Santamaria 2002).This resilience contributes to their invasiveness.The tropical signalgrass is indeed an invasive grass with rapid evolutionary adaptations (see Bora et al. 2020), and it should be closely monitored and carefully managed in aquatic ecosystems due to its invasiveness and detrimental impacts on native communities (Sato et al. 2021;Galvanese et al. 2022).Furthermore, such invasive plants are becoming a significant issue in many Brazilian rivers, from the south (where we conducted our study) to the Amazon (Fares et al. 2020).
Aside from propagule resistance, it is worth mentioning that almost all the traits we analyzed were negatively affected as the duration of desiccation increased.The only variable not influenced by our treatments was root dry biomass.This lack of effect might be due to a root elongation strategy rather than biomass accumulation.Root elongation occurs when plants face unfavorable conditions, such as high salinity (Bora et al. 2020).It can help with sediment aeration, which assists in dealing with salinity (Li et al. 2019).In our study, the low root dry biomass observed even in the control treatment might be responsible for the lack of a statistical relationship between this variable and our desiccation treatments.
In this work, we indicate that the tropical signalgrass propagules removal of water without an appropriate destination is an ineffective management attitude when the period of time off of water and sediment is less than ten days.After ten days, our experiment revealed a complete lack of regeneration response in the species' propagules when they were of water and without sediment.The tropical signalgrass is an amphibious plant, meaning it can survive for some time without water.However, we argue that the species' response differs when the propagules are not planted in sediment (as observed in Michelan et al. 2010).It is vital to base biosecurity protocols and management on empirical evidence, such as the results from our study, to ensure environmental conservation and prevent the spread of invasive species.
Nevertheless, desiccation strategies aimed at containing the spread of invasive macrophytes may not always be appropriate.Even though we did not observe any signs of propagule survival in this highly invasive plant after the 10-day treatment, we recommend that management measures should remain stringent and account for an extended period of desiccation as a safe approach.Our results clearly indicate the species' propagule resistance duration under highly stressful conditions during dispersion.

Fig. 1
Fig. 1 U. arrecta's propagules submitted to desiccation and absence of sediment (A), and the propagules after the treatments, in trays with water and sediment (B)

Fig. 2
Fig. 2 Performance variables (Mean ∓ Standard Deviation) of tropical signalgrass under varying durations of desiccation treatment (0, 2, 4, 6, 10, and 15 days without water and sediment).F and P-values obtained in ANOVA with permutations are shown in each graph.Propagule initial size was not statistically different for any of the variables analyzed