Introduction

Seed dispersal is vital to allow plant species to maintain their populations and survive the impact of environmental changes, such as climate change, habitat fragmentation and habitat loss (Nathan et al., 2008). In wetlands, plants can disperse their seeds via multiple possible mechanisms, including anemochory, hydrochory and zoochory. Anemochory involves seed dispersal via wind, and can be facilitated by seed traits that decrease the terminal velocity of seeds by means of wings or hairs (Soomers et al., 2013). Hydrochory is transport by water, and is most important for plants along shorelines with seeds with high buoyancy capacity due to traits such as impermeable seed coats and the presence of spongy tissues (Dehgan & Yuen, 1983; Nilsson et al., 2010; Van Leeuwen et al., 2014; Soons et al., 2017). Zoochory is seed dispersal via animal vectors—either by ingestion, transport and egestion (endozoochory) or attachment to the hairs, feathers or skin of an animal (ecto- or epizoochory). Endozoochory is the quantitatively more important zoochory mechanism in wetlands (Green et al., 2023), and depends on interactions among a wide range of seed traits that together increase ingestion rates, and seed survival and retention times during gut passage (Tóth et al., 2023; van Leeuwen et al., 2023).

Plant seeds can also be transported sequentially by multiple dispersal mechanisms before they germinate and establish somewhere as seedlings (i.e., polychory, or diplochory in the case of two vectors, Vander Wall & Longland, 2004). Two dispersal mechanisms can provide more benefits to seeds than only one, allowing plants to avoid local competition or pathogens (Hamilton & May, 1977). Vander Wall & Longland (2004) give examples of diplochory such as pines dispersed at first by wind and later scatter-hoarded by vertebrates, or seeds dispersed by ballistic propulsion and subsequently dispersed by ants. Wetland plants provide other examples. Seeds can be blown from their mother plant before they enter the water to subsequently be dispersed via hydrochory, potentially increasing seed dispersal distances several times over (Säumel & Kowarik, 2010; Soomers et al., 2013). Seeds may also first float on the water before they are ingested by dabbling ducks and other waterbirds that disperse them via endozoochory (Tóth et al., 2023; Urgyán et al., 2023). Sequential dispersal of seeds via multiple mechanisms typically disperses seeds farther and potentially to more novel habitats than seed dispersal by single vectors (Vander Wall & Longland, 2004; van Leeuwen et al., 2017).

Despite its importance, diplochory is relatively understudied because most research focuses on single mechanisms, encouraged by the popular use of morphological dispersal syndromes that typically assign each plant species to a single syndrome (e.g., hydrochory for many wetland plants). This is despite the extensive empirical evidence demonstrating that these syndromes do not provide a reliable basis for predicting dispersal mechanisms in the field, especially for zoochory and long-distance dispersal (Green et al., 2022). We address this knowledge gap for diplochory by studying the combination of endozoochory and hydrochory for wetland plants. Endozoochory in wetland ecosystems can occur after seed ingestion by large herbivorous mammals, reptiles, fish (Horn et al., 2011; Albert et al., 2015; Valido & Olesen, 2019) and waterbirds (Green et al., 2023). Herein, we focus on waterbirds because they are often the quantitatively most important seed dispersing animals in wetlands, intentionally ingest seeds, and move over great distances during diurnal and annual cycles (Kleyheeg et al., 2017, Martín-Vélez et al., 2021).

Important seed-dispersing waterbird species such as mallards (Anas platyrhynchos Linnaeus, 1758), lesser black-backed gulls (Larus fuscus Linnaeus, 1758) and greylag geese (Anser anser Linnaeus, 1758) divide their time between terrestrial and aquatic habitats, and disperse seeds from terrestrial, aquatic or shoreline habitats (Kleyheeg et al., 2017; Martín-Vélez et al., 2021; Navarro-Ramos et al., 2024). This implies that seeds of terrestrial and shoreline plant species may often be ingested while floating on water, and also may often be egested into open water which may not be a suitable habitat for germination and growth. Seeds that are defecated into the water may potentially either sink and be incorporated into the wetland seed bank until conditions are suitable for germination (Leck & Schutz, 2005; Espinar et al., 2023), or they may still be able to float and wash ashore to a more suitable location for germination (Soons et al., 2017). A retained ability to float after passage of a digestive tract may, therefore, increase the chance of terrestrial and shoreline plants to reach suitable habitat for germination. However, to date, there is no information on what happens to the buoyancy capacity of wetland plant seeds after endozoochory. This makes it hard to predict the fate of plant seeds egested after endozoochory by waterbirds: will they be able to float and reach microsites that may be more suitable for germination along the shoreline, or are they more likely to sink and be incorporated into the seed bank?

Here, we experimentally determined the buoyancy capacity of seeds of 41 species of wetland plants before and after they were exposed to simulated digestive processes by waterbirds. We applied a recently developed bioassay to simulate avian gut passage (Van Leeuwen et al., 2023), which standardizes methodology across plant species and avoids the use of experimental animals. We selected these plant species because they lacked a fleshy fruit cover, represent a wide range of plant traits (i.e., seed size, mass, shape), are native to The Netherlands and are common in areas where dabbling ducks forage. The selection represents a wide variety of species with soil moisture requirements ranging from fully aquatic to shorelines to fully terrestrial. We tested four hypotheses: (1) undigested (control) seeds of plant species previously assigned a hydrochory dispersal syndrome float longer than control seeds of plants with other syndromes, because the hydrochory syndrome is based on seed traits expected to predict buoyancy (Van der Pijl, 1982; Vargas et al., 2023); (2) digestive processes reduce seed buoyancy capacity during an early phase of digestion in the gizzard, and even more so after intestinal digestion has also taken place. Because plant species growing along shorelines depend most on seed dispersal via floating to reach suitable microhabitats (Soons et al., 2017), we furthermore hypothesized that (3) plant species growing along wetland shorelines have seeds that float longer than seeds of fully aquatic (i.e., floating or submerged plants) and/or terrestrial plant species; and (4) seeds of these shoreline species also float longer after exposure to digestive processes than seeds of fully aquatic and/or terrestrial plant species. We expected lowest seed buoyancy capacity prior to and after simulated digestion for fully aquatic plants, because for species germinating under water it may be more adaptive to have seeds that sink (Van den Broek et al., 2005; Soons et al., 2017). Understanding the potential of plant seeds to successfully combine dispersal mechanisms to extend their dispersal distances and reach particular microsites is crucial for improving our predictions on plant establishment in an era of global change (Ozinga et al., 2009).

Methods

Plant species selection

We experimentally assessed seed buoyancy capacity for 41 wetland plant species before and after exposure of the seeds to simulated avian digestive processes. Many (59%) of these species are already known to be dispersed by waterbirds (Anatidae, Table 1). The selected plant species ranged from fully terrestrial plants (which we define as those having Ellenberg F value 3–6), to plants growing on and along the shoreline edges (Ellenberg F value 7–9) and plants that grow under fully aquatic conditions (Ellenberg F value 10–12, Table 1). Ellenberg F values and dispersal syndromes were obtained from the ECOFACT (Hill et al., 1999) and Baseflor databases (Julve, 1998). The seeds were supplied by Biodivers B.V. (Oudewater, the Netherlands) in 2018, who collected them in natural areas in the Netherlands (with permission). All seeds were stored dry in the dark at 4°C until the experiments started in the spring of 2019. All used seeds were visually assessed while counting them, which ensured that they were in normal and representative conditions before use.

Table 1 Species tested for buoyancy before (Control) and after simulated digestion (Partial and Full digestion), presenting means (± sd), medians and maxima for days taken to sink
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Experimental simulations of avian digestive processes

To assess the effects of simulated gut passage on seed buoyancy, we compared the buoyancy capacity among control seeds that had not been digested, seeds that had experienced simulated passage through the avian gizzard (hereafter “partial digestion”), and seeds that experienced complete passage through both the avian gizzard and the intestines (hereafter “full digestion”, Fig. 1). To obtain seeds that had experienced partial or full digestion, we simulated the avian digestive processes according to a recently published bioassay (Van Leeuwen et al., 2023). This method simulates the five key conditions that ingesta experience upon entering the digestive tract of waterbirds: elevated temperature, mechanical forces, low pH conditions, lack of oxygen and enzymatic digestive processes.

Fig. 1
figure 1

a) Representation of wetland landscape with plant species distributed according to their moisture requirements (Ellenberg F) from fully terrestrial soils, to plants growing along the shorelines, and including fully aquatic plants. Seeds dispersed towards aquatic habitats are indicated by flying seeds; b) Graphical representation of the buoyancy experiment for control seeds, partial (i.e., gizzard simulation) and fully digested seeds (gizzards followed by intestinal simulation). All species had two experimental replicates, hence for each plant species 240 seeds were used

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In brief [see Van Leeuwen et al. (2023) for details], the simulations were as follows. To simulate the avian body temperature, the experiment was entirely carried out in a climate chamber that was controlled at 42°C (avian body temperature). To simulate mechanical digestion in the avian gizzard, 100 seeds per species were placed at a time in a pipetting balloon of natural rubber together with 50 g silica grit (2–4 mm diameter) and 17 mL gizzard solution. To create anoxic conditions, the top of the balloon was sealed and filled with N2-gas. The balloon was then compressed sidewards 20 times per minute with amplitudes of 20 mm, while slowly rotating. After 2 h, surviving seeds were collected and a fraction of the seeds was used to assess buoyancy under the partially digested treatment (see next section), and another fraction (20 seeds per species) was subjected to simulations of intestinal digestion. For intestinal digestion simulations, we used 50 mL polypropylene tubes that were rotated in a VWR Tube Rotator filled with 50 mL of intestine solution to simulate gut passage. These 20 retrieved seeds were then used to assess how buoyancy was affected by full digestion.

Assessing seed buoyancy

For all 41 plant species, we tested the buoyancy of 120 seeds divided between the three different treatments: control seeds, partially digested seeds (only gizzard) and fully digested seeds (gizzard plus intestine). Buoyancy was assessed by placing 20 seeds per species per treatment in separate plastic beakers of 400 mL that were filled with 300 mL dechlorinated tap water with a conductivity between 400 and 500 µS/cm (which is realistic for the habitat of the used plant species). The beakers were covered with plastic lids to prevent evaporation and stored at 10°C in a dark cooled chamber. The number of seeds still floating was counted daily for 14 weeks. Upon counting, the water was stirred for 5 s with a 15 cm-long spoon to break the potentially unrealistic role of water surface tension that helps seeds to remain buoyant (van der Broek et al., 2005; Infante-Izquierdo et al., 2023). Sunken seeds were only removed if they started to germinate. All species were studied simultaneously in one-time period and in duplicates (i.e., two times 20 seeds were tested per species per treatment). At the end of the experiment (i.e., after 14 weeks), seeds were categorized as “floating” and given numeric value of “0”, or “sinking” with a value of “1”.

Statistical analyses

To test our first hypothesis on the effects of dispersal syndrome on the buoyancy of control seeds, we calculated the total number of the 40 control seeds that still floated at the end of the 14-week experiment for each plant species. This generated one value per plant species. We similarly calculated the median for the number of days that the control seeds floated prior to sinking for each plant species (excluding seeds that remained floating at the end of the experiment), which also generated one value per plant species. Then we compared these values between two categories (species with a hydrochory syndrome, and species with other syndromes) in two Student’s t tests.

To test our second, third and fourth hypotheses, we used multiple generalized linear mixed-effects models that were fitted with the packages “lme4” and “glmmTMB” in R software version 3.6.3 (Bates et al., 2015, Brooks et al., 2017, R Core Team, 2023). In all models, experimental replicates and plant species were included as random-effect intercepts. Normality of model residuals was assessed with quantile–quantile plots and by plotting residuals against fitted values. Model residuals were analysed for possible heteroscedasticity, overdispersion and zero-inflation with package “DHARMa” in R (Hartig, 2022). Tukey’s Honest Significant Difference tests were used to test for differences among factor levels.

In Models 1A, 2A and 3A, seed buoyancy was the binary dependent variable (i.e., 0 = “still floating after 14 weeks” and 1 = “sunk after 14 weeks”, Table 2). In Model 1A, we tested hypothesis 2, by including the explanatory variable “treatment” (factor with three levels: control, partial digestion, and full digestion) to test whether digestion affected buoyancy of plant species. In Model 2A and 3A, we tested hypotheses 3 and 4: whether seed buoyancy differed among plant species growing at different moisture conditions, for undigested control seeds and fully digested seeds respectively. We based the analyses on Ellenberg F values, excluding three species for which Ellenberg F values were not available. Because we did not expect a linear effect of Ellenberg F value, but hypothesized contrasts between fully aquatic, shoreline and terrestrial plant species, we included “Ellenberg F value" as a fixed factor with three levels (F = 10–12 wet to submerged, 7–9 moist to wet, and 3–6 dry to moist) in these models.

In Models 1B, 2B and 3B, the dependent variable was the time in days until a seed sank, modelled as a zero-inflated Poisson distribution for count data (the zero-inflated function from package DHARMa revealed significant zero-inflation: ratioObsSim = 41.095, P < 0.0001). Seeds still floating when the experiment ended after 98 days were excluded from the analysis. The model structures of the independent variables were identical to those in models 1A, 2A and 3A, respectively. To assess effects of the treatments at the species-specific level, we additionally (1) compared the proportion of seeds still floating after 14 weeks between control seeds and fully digested seeds in X2-tests and (2) compared the distributions of the number of days that each seed floated between control and fully digested seeds using Student’s t tests.

Results

Influence of dispersal syndrome on proportion of seeds floating

For control seeds of 41 wetland plant species, on average a proportion of 0.36 were still floating at the end of the 14-week experiment. This proportion was higher for plant species assigned a hydrochory syndrome (n = 15, 0.67 ± 0.36 SD) than for plant species with other syndromes (n = 26 species, 0.18 ± 0.31, Chi-squared test, X2 = 27.515, P < 0.01, Fig. 2a). However, there was considerable overlap between syndromes, and some species assigned other syndromes had extremely high buoyancy, whereas some species with a hydrochory syndrome had low buoyancy (Fig. 2a). Seeds with a hydrochory syndrome (38.2 ± 22.4 mean ± SD) floated on average 8.7 days longer before sinking than seeds of plant species with other syndromes (29.5 ± 24.1 mean ± SD), but due to the large standard deviations this was not significantly different (Student’s t test, t = 1.03, P = 0.31, Fig. 2b).

Fig. 2
figure 2

(a) Mean proportion of seeds that still floated after 14 weeks for all plant species with a hydrochory dispersal syndrome, and all plant species with other syndromes (including barochory, anemochory and epizoochory). A higher proportion of seeds still floated after 14 weeks for plant species with a hydrochory dispersal syndrome (Chi-squared test, X2 = 27.5, simulated P < 0.01). (b) Mean number of days before seeds sank for all plant species with a hydrochory syndrome and all plant species with other syndromes (excluding seeds that still floated after 14 weeks). There was no significant difference (Student’s t test, t = 1.03, P = 0.31). Total numbers of species for each dispersal syndrome category are indicated in brackets. Boxes represent the 25th and 75th percentiles, and the line inside indicates median values. Upper and lower bars represent maximum and minimum values, excluding outliers which are indicated with coloured triangles. Dots represent plant species. Categories that share the same capital letter are not significantly different

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Influence of digestive processes on seed buoyancy

A total of 18,448 seeds (65.3%) were recovered intact after simulated gut passage, and 4800 seeds of 41 plant species were tested for buoyancy (Table 1). In contrast to the 36% of all control seeds that were still floating after 14 weeks, for the partially and fully digested seeds, only 0.13% and 0.06% were still floating, respectively. Post-hoc Tukey’s HSD tests showed that the proportion of seeds still floating differed significantly between control seeds and either of the two gut-passage treatments (P < 0.0001, Table 2), while no significant difference was observed between partially and fully digested seeds for the proportion of seeds still floating after 14 weeks (hypothesis 2, P = 0.81, Z = 0.61, Model 1A, Fig. 3a, Table 2). However, a small but significant difference indicated that partially digested seeds floated slightly longer before sinking than fully digested seeds (Model 1B, Table 2).

Table 2 Results of generalized linear mixed-effects models (GLMMs) using binomial and zero-inflated Poisson error distributions to test effects of treatment (control, partial and full avian digestion) and different plant moisture requirements (i.e., Ellenberg F values) on 1) whether seeds sank or not and 2) time taken to sink
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Fig. 3
figure 3

Effects of digestive treatments on the buoyancy of plant seeds in relation to soil moisture requirements based on Ellenberg F values, expressed as (a) proportion of all seeds that still floated after 14 weeks and (b) number of days taken for plant seeds to sink (excluding seeds that still floated after 14 weeks). Boxes represent the 25th and 75th percentiles, and the line inside indicates median values. Upper and lower bars represent maximum and minimum values, excluding outliers which are indicated with coloured triangles. See Table 2 for statistical analyses

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The effects of the treatments on the proportion of seeds floating at the end of 14-week experiment differed widely among plant species, although buoyancy was never increased by gut passage (Table 3). Seventeen plant species showed no significant effect of treatment on how many seeds remained floating (usually because no control seeds remained floating), whereas 24 species (59%) had significantly fewer seeds still floating after full digestion compared with controls (Table 3). For all plant species, we found a large effect of the full digestion treatment compared to the control with respect to the mean number of days that a seed required to sink (Table 3).

Table 3 Mean proportion of seeds that still floated after 14 weeks and mean number of days that seeds floated before sinking, according to plant species and three experimental treatments (controls, partial and full digestion)
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Control seeds also floated significantly longer (median = 28 days, excluding those which remained floating at the end of the 14-week experiment) compared to partially digested seeds (median = 0 day) and fully digested seeds (median = 0 day; P < 0.0001, Model 1B, Fig. 3b, Table 2). Seeds that were partially digested remained floating for more days than fully digested seeds (P < 0.0001, Model 1B, Table 2). For control seeds, 73% were still floating after 1 month compared to only 13% of partial and fully digested seeds combined.

Influence of habitat type on seed buoyancy

Variation in seed buoyancy among plant species with different Ellenberg F value categories was tested for control seeds (hypothesis 3) and fully digested seeds (hypothesis 4). For control seeds, the proportion of seeds that had sunk after 14 weeks was significantly higher for species categorized as terrestrial (i.e., F = 3–6) than for either shoreline (F = 7–9), or fully aquatic species (F = 10–12, P < 0.0001, Model 2A, 3A, Table 2, Fig. 3a). However, there was no significant difference between shoreline and aquatic species for undigested control seeds (P = 0.92, Model 2A) or seeds after full digestion (P = 0.92, Model 3A, Fig. 3a, Table 2). The lower floatability of terrestrial plant seeds was particularly evident for control seeds (Fig. 3a). For either control and full digestion treatment, seeds of fully aquatic, shoreline and terrestrial plants did not significantly differ in the number of days that they remained floating (Model 2B, 3B, Table 2, Fig. 3b). This is likely associated with strong interspecific variation and large standard deviations within and among species (Table 3).

Discussion

We found a strong, consistent pattern that wetland plant seeds subjected to simulated gut passage have a reduced ability to float and disperse on the water surface. Dispersal of these species via endozoochory by waterbirds is a common process, and often occurs because seeds are ingested while floating and are thus readily accessible to dabbling ducks or other waterbirds (Almeida et al., 2022; Green et al., 2023; Tóth et al., 2023). Hence, our results suggest that in nature seeds will often first disperse by floatation (hydrochory), then move inside flying waterbirds (endozoochory) and then sink after egestion in faeces. After being dispersed by these mechanisms, seeds may continue to be transported by water movements or other biological vectors such as fish (Mulder et al., 2021), or they may then enter seed banks in sediments until conditions become suitable for germination (Espinar et al., 2023).

In our experiments, plant species previously assigned a hydrochory dispersal syndrome—based on assessment of their seed morphologies—typically had higher buoyancy capacity than species given other classical syndromes (confirming hypothesis 1). This lends support to the idea that seed buoyancy can be predicted largely from seeds traits such as air chambers, low density tissues and/or waxy substances (Nilsson et al., 2010; Carthey et al., 2016; Vargas et al., 2023). Nevertheless, several species assigned other syndromes also had high floatability, some even higher than species with a hydrochory syndrome (Table 1; Fig. 2a). For example, Bidens spp. had very high floatability, despite having an epizoochory syndrome. Peucedanum palustre (L.) Moench is assigned an anemochory syndrome, but also had very high floatability. This suggests this shoreline species may be particularly effective at combining wind and water dispersal. Thalictrum spp. are assigned the barochory syndrome, but still had very high floatability. This suggests that seed dispersal syndromes can be helpful in predicting dispersal mechanisms in some situations, but we should be aware that many species also disperse well via other mechanisms, or use combinations of dispersal mechanisms (Green et al., 2022; González-Varo et al., 2024).

After exposure to avian digestive processes, seeds of almost all plant species showed a major loss of buoyancy capacity even after partial digestion, including plant species with hydrochory syndromes and plant species growing along shorelines. The slightly stronger effect of full digestion than partial digestion on the number of days seeds could still float confirms our hypothesis 2. This also suggests that seeds that have been digested for shorter durations or less completely—for instance, if dispersed via very short retention times or after regurgitation (Van Leeuwen et al., 2017; Navarro-Ramos et al., 2022)—may still retain somewhat higher potential to float.

For undigested control seeds, hydrochory potential was clearly related to soil moisture requirements of the plant species, and terrestrial plants floated for shorter periods than shoreline or fully aquatic plants. Terrestrial plants are less likely to be assigned hydrochory syndromes, and less likely to be adapted to disperse via water, although their seeds may often be blown or washed into wetlands. Our results indicate they can readily float for days or weeks (but not months), potentially facilitating endozoochory. This helps to explain the large number of terrestrial plants that disperse by waterbird endozoochory (Tóth et al., 2023; Urgyán et al., 2023). Plants growing along shorelines and fully aquatic plants were also the most likely to have seeds that were still floating after 14 weeks, and their seeds floated for longest before sinking (partly confirming hypotheses 3 and 4). This is in line with the observation that shoreline species germinate best in the moist soils typical of shorelines (Soons et al., 2017). The manner in which seeds of aquatic and shoreline plants can float for several months allows them to be dispersed by migratory ducks that feed on these seeds long after they left the mother plant (Urgyán et al., 2023). For our third and fourth hypotheses we expected shoreline plants to have evolutionary benefits of retaining buoyancy after endozoochory. Indeed, even after full digestion 19 species (46%) retained some floatability, of which four species were still able to float for an average of > 20 days (i.e., Carex paniculata L., Carex pseudocyperus L., Lycopus europaeus L., Lysimachia vulgaris L., Table 1). These four species have in common that they have Ellenberg F values of 8 or 9, are typical shoreline species and are all assigned a hydrochory dispersal syndrome. In lentic aquatic ecosystems, floating seeds can disperse up to 1000 m due to wind interaction at the water surface (Soomers et al., 2010, 2013; Sarneel et al., 2014) whereas in lotic waters, dispersal distances can be even longer. All these species grow closely to the shoreline, suggesting that these species are better adapted to retain buoyancy after digestion. However, it seems that only species with very high buoyancy capacity prior to digestion also retained some of this buoyancy capacity after digestion. This may potentially be a result of strong selection on having a high buoyancy capacity prior to digestive processes rather than on selection to retain this capacity after endozoochory. Alternatively, there may be stronger selection on these species to float to the shoreline after endozoochory, which generates interesting hypotheses to further address in future studies.

Loss of buoyancy after digestion can often be explained by increases in water permeability caused by damage to seed architecture during gut passage (Costea et al., 2019). Seed traits required for remaining buoyant such as air sacks, waxy substances, impermeable seed coat or low-density tissues (Nilsson et al., 2010; Carthey et al., 2016; Vargas et al., 2023) may be damaged or lost during gut passage. However, seeds that sink can still have a chance to become established, and seed buoyancy is not advantageous in all circumstances (Schneider & Sharitz 1988; Danvind & Nilsson 1997; Boedeltje et al., 2003; Boedeltje et al., 2004). For instance, seeds may remain in the seed bank until conditions are more suitable for germination (Espinar et al., 2004, 2005, 2023). Hydrochory of seeds remains possible for non-floating seeds if they roll on the sediment in lotic systems (Soons et al., 2017), and sunken seeds can be dispersed over long distances by fish endozoochory (Mulder et al., 2021). Furthermore, after endozoochory seeds can also be egested on land or along shorelines instead of on water, especially since dabbling ducks and geese like to rest out of the water along shorelines or in fields close to water bodies (Tóth et al., 2023; Navarro-Ramos et al., 2024). Thus, we should not overestimate the importance of seed floatation for plant fitness. Still, there could be an interesting trade-off between the morphological seed traits required for remaining buoyant and those required for successful gut passage (such as being small, hard, with high-density tissues and more compact, Van Leeuwen et al., 2023). A detailed study of variation in seed traits in relation to the trade-off among hydrochory and endozoochory among the 41 plant species, and how they relate to the variation we observed in buoyancy before and after gut passage, would be of interest.

Diplochory has the potential to greatly increase dispersal distances of seeds and other propagules (Vander Wall & Longland, 2004), but this study illustrates how different dispersal mechanisms may occur in a specific order during diplochory events. Hydrochory is likely to be the dominant dispersal mechanism for seeds of aquatic and shoreline plants within a given wetland, whereas endozoochory is vital for dispersal to new habitats, and long-distance movements of tens or hundreds of kilometres (Martín-Vélez et al., 2021; Urgyán et al., 2023; Navarro-Ramos et al., 2024). Hydrochory is more readily followed by endozoochory than vice versa. Plant species that normally rely strongly on hydrochory by floating on the water surface and germinating along the shorelines are more likely to benefit from waterbird endozoochory if they can germinate under submerged conditions, and/or are growing in wetlands with variable water levels (e.g., with drawdowns in dry summer months). Further assessment of the generality of our findings should include analyses of trade-offs among seeds traits in relation to various dispersal vectors, and the effects of gut passage on seed floatation at higher salinities, since waterbirds often disperse seeds into coastal brackish or saline wetlands (Almeida et al., 2022), where egested seeds may be more likely to float.