Seed Size, Not Dispersal Syndrome, Determines Potential for Spread of Ricefield Weeds by Gulls

Citation: Peralta-Sánchez, J.M.; Ansotegui, A.; Hortas, F.; Redón, S.; Martín-Vélez, V.; Green, A.J.; Navarro-Ramos, M.J.; Lovas-Kiss, A.; Sánchez, M.I. Seed Size, Not Dispersal Syndrome, Determines Potential for Spread of Ricefield Weeds by Gulls. Plants 2023,12, 1470. https://doi.org/10.3390/ plants12071470 Academic Editor: Leonid Rasran Received: 1 March 2023 Revised: 22 March 2023 Accepted: 25 March 2023 Published: 27 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Seed Size, Not Dispersal Syndrome, Determines Potential for Spread of Ricefield Weeds by Gulls Juan Manuel Peralta-Sánchez 1,2 , Albán Ansotegui 1,3, Francisco Hortas 4, Stella Redón1,3, Víctor Martín-Vélez 3, Andy J. Green 3, María J. Navarro-Ramos 3, Adam Lovas-Kiss 5and Marta I. Sánchez 1,3,* 1Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Seville, Spain; [email protected] (J.M.P.-S.); [email protected] (A.A.); [email protected] (S.R.) 2Departamento de Microbiología, Universidad de Granada, Avda. Fuentenueva s/n, 18071 Granada, Spain 3Wetland Ecology Department, Estación Biológica de Doñana, EBD-CSIC, Avda. Americo Vespucio 26, 41092 Seville, Spain; [email protected] (V.M.-V.); [email protected] (A.J.G.); [email protected] (M.J.N.-R.) 4Instituto Universitario de Investigación Marina (INMAR), Campus de Excelencia Internacional del Mar (CEI·MAR), Universidad de Cádiz, Avda. República Árabe Saharaui s/n, 11510 Puerto Real, Spain; [email protected] 5Wetland Ecology Research Group, Department of Tisza Research, MTA Centre for Ecological Research-DRI, H-4026 Debrecen, Hungary; [email protected] *Correspondence: [email protected] Abstract: Recent field data suggest that migratory gulls disperse many rice field weeds by gut passage (endozoochory), most of which are dry fruited and widely assumed to have no long-distance dispersal mechanisms, except via human activity. We investigated this mechanism with a feeding experiment, in which seeds of five common rice field weeds (in order of increasing seed size: Juncus bufonius,Cyperus difformis,Polypogon monspeliensis,Amaranthus retroflexus, and the fleshy-fruited Solanum nigrum) were fed to seven individuals of lesser black-backed gulls Larus fuscus held in captivity. We quantified seed survival after collecting faeces at intervals for 33 h after ingestion, then extracting intact seeds and running germination tests, which were also conducted for control seeds. All five species showed high seed survival after gut passage, of >70%. Gut retention times averaged 2–4 h, but maxima exceeded 23 h for all species. Germinability after gut passage was 16–54%, and gut passage accelerated germination in J. bufonius and S. nigrum, but slowed it down in the other species. All species had lower germinability after gut passage compared to control seeds (likely due to stratification prior to the experiment), but the loss of germinability was higher in smaller seeds. There was no evidence that the different dispersal syndromes assigned to the five species (endozoochory, epizoochory or barochory) had any influence on our results. In contrast, mean gut retention time was strongly and positively related to seed size, likely because small seeds pass more quickly from the gizzard into the intestines. Non-classical endozoochory of dry-fruited seeds by waterbirds is a major but overlooked mechanism for potential long-distance dispersal, and more research into this process is likely essential for effective weed management. Keywords: endozoochory; lesser black-backed gull; Larus fuscus; dry-fruited seeds; fleshy-fruited seeds; dispersal syndromes 1. Introduction Seed dispersal by animals is a key process in the ecology and evolution of plants [ 1 ]. It affects recruitment, connectivity and gene flow among populations [ 2 ], with an important role in maintaining plant metacommunities [ 3 , 4 ]. Declines in seed dispersers can have cascading effects and impacts on plant populations, the composition and functional diversity of plant communities, the dynamics of multitrophic systems, and ecosystem functioning [ 5 ]. Plants 2023,12, 1470. https://doi.org/10.3390/plants12071470 https://www.mdpi.com/journal/plants Plants 2023,12, 1470 2 of 17 Animal-mediated seed dispersal is decisive for assuring the persistence and spread of plants under major drivers of biodiversity change such as habitat fragmentation, biological invasions and climate change [6,7]. Many plants can disperse their seeds by gut passage (endozoochory) inside birds or other animals. Major advances have been made in understanding seed dispersal of fleshy-fruited plants by frugivorous birds [ 8 ], but it is often wrongly assumed that only plants with fleshy fruits can be dispersed by endozoochory. This misunderstanding is likely perpetuated by the classical morphological dispersal syndromes applied to plant diaspores, which include a range of abiotic syndromes such as wind, water or gravity, and define an “endozoochory syndrome” based on the presence of a fleshy fruit [9]. However, non-classical endozoochory of plant seeds assigned to other syndromes is an important dispersal mechanism for many plant species [ 9 , 10 ]. For example, only 8% of hundreds of European plant species known to be dispersed by migratory ducks demonstrated endozoochory syndrome, and more research is needed into the internal dispersal of seeds by non-frugivorous birds [ 9 , 11 ]. Understanding the role of non-frugivorous birds in plant dispersal is vital to improve predictions about the consequences of global change, such as how plants respond to climate change or the spread of invasive or weed species. Migratory waterbirds (including gulls, Laridae) are of particular importance because they perform long-distance movements and use anthropogenic habitats affected by global change [ 12 , 13 ]. Given that dispersal syndromes fail to explain what seeds are ingested and egested by waterbirds, as demonstrated by faecal sampling [ 14 ], a key question is how plant traits determine how many seeds survive gut passage, what their retention time is (a predictor of dispersal distance), and what their germination response is after egestion. Experiments feeding seeds to birds in captivity can address such questions, and there have been several studies with ducks and geese (Anatidae) [ 15 – 17 ]. However, to date, no experiments have compared the response to gut passage of seeds from different classical dispersal syndromes, even though waterbirds sometimes feed on +fleshy- and dry fruits at the same time [ 18 ]. Given the overwhelming emphasis in the literature on endozoochory on fleshy fruited plants, it may be hypothesised that the seeds of such plants are better adapted to survive gut passage than those with dry fruits. Among dry-fruited plants, there is evidence from some experiments that smaller seeds have higher seed survival but shorter retention times than larger seeds, although other studies have found contradictory results [15,18,19]. The lesser black-backed gull Larus fuscus, Linnaeus 1758 (LBBG), is a long-distance migratory bird that has increased in numbers across Europe during the last century [ 20 , 21 ] related to the proliferation of landfills and other human-modified habitats. The LBBG is an opportunistic bird with an omnivorous diet connecting anthropogenic and natural habitats at the landscape scale [ 22 ]. Recent studies have shown that wintering LBBG disperse seeds of more than 15 plant species growing in the largest rice field area of Spain by endozoochory, mainly by secondary dispersal of seeds attached to their main prey, the alien red swamp crayfish Procambarus clarkii [ 12 , 23 ]. Most of these plants are terrestrial weeds that lack fleshy-fruits, and during the winter, LBBG can disperse them over distances of up to 100 km or more, even outside migration periods. The dispersal of pest species is one of the main ecological disservices provided by birds with important ecological and economic costs, but remains understudied [15,17]. Here, we assessed the role of LBBG as seed vectors by endozoochory by a controlled experiment in captivity. We used five weed species previously recorded in LBBG faeces and pellets from rice fields, only one of shows endozoochory syndrome [ 12 , 24 ]. Our objectives were to quantify (1) the proportion of seeds of each species that survived gut passage by the LBBG, (2) the seed retention time, and (3) the germinability and germination time after gut passage compared to control seeds. Based on the available literature, the following hypotheses were tested: after gut passage, (1) seeds of the fleshy-fruited species would have the highest seed survival rate, and an increased germinability and faster germination compared to control seeds, consistent with better adaptation for endozoochory than for dry-fruited species; (2) seeds of dry-fruited species would show reduced germinability and Plants 2023,12, 1470 3 of 17 slower germination compared to control seeds; (3) species with smaller seeds would have higher seed survival, but shorter retention time that those with larger seeds. 2. Results 2.1. Survival Intact seeds of the five plant species were recovered from the droppings of gulls ( Table 1 ), but seed survival differed significantly between plant species (GLMM1.1, fixed-factor plant species: χ2 = 57.72, df = 4, p< 0.001). All taxa had an average seed survival of 72–88%, with Polypogon monspeliensis having the highest and Juncus bufonius the lowest (Table 1). Pairwise differences between species in seed survival were significant, except those between Cyperus difformis and J. bufonius or P. monspeliensis, or between Amaranthus retroflexus and J. bufonius (Table 2). Table 1. (a) Percentage of seeds of each plant species that survived gut passage through seven lesser black-backed gulls. (b) Average retention time (in hours) for seven gulls. S.D.: Standard deviation, Min: minimum, Max: maximum. (a) Survival Rate (b) Retention Time Plant Species Average S.D. Min. Max. Median Average S.D. Min. Max. Median Amaranthus retroflexus 76.93 6.95 64.00 85.00 78.50 2.80 3.22 0.25 27.5 2.00 Cyperus difformis 81.86 9.01 69.00 92.00 86.00 2.53 2.66 0.25 23.5 1.00 Juncus bufonius 72.43 9.48 57.50 83.00 72.00 2.47 2.45 0.25 27.5 1.00 Polypogon monspeliensis 87.71 6.38 80.50 96.00 85.00 2.58 2.62 0.25 27.5 1.00 Solanum nigrum 76.71 21.97 31.50 100.00 80.00 3.55 4.00 0.25 27.5 2.00 Table 2. Tukey HSD post-hoc test showing pairwise differences between plant species ( Solanum nigrum , Amaranthus retroflexus, Polypogon monspeliensis, Cyperus difformis and Juncus bufonius ) in the proportion of seeds recovered in gull faeces. Significant p-values are highlighted in bold. Contrast Estimate SE t. Ratio pValue A. retroflexus—C. difformis −0.18 0.06 −2.86 0.036 A. retroflexus—J. bufonius −0.05 0.06 −0.73 0.949 A. retroflexus—P. monspeliensis −0.23 0.06 −3.68 0.002 A. retroflexus—S. nigrum 0.20 0.06 3.18 0.014 C. difformis—J. bufonius 0.13 0.06 2.09 0.228 C. difformis—P. monspeliensis −0.05 0.06 −0.79 0.932 C. difformis—S. nigrum 0.38 0.06 6.00 <0.0001 J. bufonius—P. monspeliensis −0.18 0.06 −2.88 0.034 J. bufonius—S. nigrum 0.25 0.07 3.81 0.002 P. monspeliensis—S. nigrum 0.43 0.06 6.86 <0.0001 For those seeds that survived gut passage, the relative proportion of seeds egested at different retention times varied significantly among species (GLMM1.1, random slope retention time—species: χ2= 61.03, df = 1, p< 0.001, Figure 1). Plants 2023,12, 1470 4 of 17 Plants 2023, 12, x FOR PEER REVIEW 3 of 17 would have the highest seed survival rate, and an increased germinability and faster germination compared to control seeds, consistent with better adaptation for endozoochory than for dry-fruited species; (2) seeds of dry-fruited species would show reduced germinability and slower germination compared to control seeds; (3) species with smaller seeds would have higher seed survival, but shorter retention time that those with larger seeds. 2. Results 2.1. Survival Intact seeds of the five plant species were recovered from the droppings of gulls (Table 1), but seed survival differed significantly between plant species (GLMM1.1, fixedfactor plant species: χ2 = 57.72, df = 4, p < 0.001). All taxa had an average seed survival of 72–88%, with Polypogon monspeliensis having the highest and Juncus bufonius the lowest (Table 1). Pairwise differences between species in seed survival were significant, except those between Cyperus difformis and J. bufonius or P. monspeliensis, or between Amaranthus retroflexus and J. bufonius (Table 2). For those seeds that survived gut passage, the relative proportion of seeds egested at different retention times varied significantly among species (GLMM1.1, random slope retention time—species: χ2 = 61.03, df = 1, p < 0.001, Figure 1). Figure 1. Average proportions of intact seeds recovered from gull faeces, at each sampling time for each plant species. Table 1. (a) Percentage of seeds of each plant species that survived gut passage through seven lesser black-backed gulls. (b) Average retention time (in hours) for seven gulls. S.D.: Standard deviation, Min: minimum, Max: maximum. (a) Survival Rate (b) Retention Time Plant Species Average S.D. Min. Max. Median Average S.D. Min. Max. Median Amaranthus retroflexus 76.93 6.95 64.00 85.00 78.50 2.80 3.22 0.25 27.5 2.00 Cyperus difformis 81.86 9.01 69.00 92.00 86.00 2.53 2.66 0.25 23.5 1.00 Juncus bufonius 72.43 9.48 57.50 83.00 72.00 2.47 2.45 0.25 27.5 1.00 Polypogon monspeliensis 87.71 6.38 80.50 96.00 85.00 2.58 2.62 0.25 27.5 1.00 Solanum nigrum 76.71 21.97 31.50 100.00 80.00 3.55 4.00 0.25 27.5 2.00 Figure 1. Average proportions of intact seeds recovered from gull faeces, at each sampling time for each plant species. 2.2. Retention Time Modal retention time was 1 h for all five plant species (Figure 1), whilst means ranged between 2.5 and 3.6 h, with maxima exceeding 23 h (Table 1). Average retention time was highly correlated with seed size (Figure 1, Table 1, Pearson’s correlation between average retention time against average seed mass: r 2 = 0.95, N = 5, p= 0.004), with S. nigrum showing the highest value and J. bufonius the lowest. C. difformis showed a maximum retention time of 23.5 h, while the other four species reached a maximum retention time of 27.5 h (Table 1, Figure 1). Retention time differed significantly between the five plant species (GLMM2, plant species: χ2 = 102.64, df = 1, p< 0.001). Post hoc tests showed that retention time of S. nigrum differed significantly from that of all other plant species (Table 3). Table 3. Tukey HSD post-hoc test showing pairwise differences between plant species ( Solanum nigrum , Amaranthus retroflexus, Polypogon monspeliensis, Cyperus difformis and Juncus bufonius ) in the retention time of the seeds recovered in faecal samples of lesser black-backed gulls. Significant p-values are highlighted in bold. Contrast Estimate SE t. Ratio pValue A. retroflexus—C. difformis 0.03 0.01 2.47 0.097 A. retroflexus—J. bufonius 0.04 0.01 2.62 0.067 A. retroflexus—P. monspeliensis 0.03 0.01 2.52 0.087 A. retroflexus—S. nigrum −0.08 0.01 −5.92 <0.001 C. difformis—J. bufonius <0.01 0.01 0.24 0.999 C. difformis—P. monspeliensis <0.01 0.01 −0.01 0.999 C. difformis—S. nigrum −0.11 0.01 −8.40 <0.001 J. bufonius—P. monspeliensis <0.01 0.01 −0.25 0.999 J. bufonius—S. nigrum −0.12 0.01 −8.30 <0.001 P. monspeliensis—S. nigrum −0.11 0.01 −8.57 <0.001 2.3. Germinability After gut passage, S. nigrum had the highest germinability (54%), and J. bufonius the lowest (16%, Table 4, Figures 2and 3). In contrast, for control seeds, germinability was >98% for C. difformis and P. monspeliensis, and was lowest for A. retroflexus (46.5%). Plants 2023,12, 1470 5 of 17 Table 4. Differences in germinability (percentage of seeds) after gut passage compared with control seeds. S.D.: Standard deviation. C.I.: 95% Confidence Interval. Experimental Seeds Control Seeds N Average SD CI N Average SD CI Amaranthus retroflexus 1077 28.69 45.26 25.99 31.40 200 46.50 50.00 39.53 53.47 Cyperus difformis 1146 32.55 46.88 29.83 35.26 200 99.00 9.97 97.61 100.39 Juncus bufonius 1014 15.98 36.66 13.72 18.24 200 77.00 42.19 71.12 82.88 Polypogon monspeliensis 1210 24.79 43.20 22.36 27.23 200 98.50 12.18 96.80 100.20 Solanum nigrum 1070 54.21 49.85 51.22 57.20 200 72.50 44.76 66.26 78.74 Plants 2023, 12, x FOR PEER REVIEW 5 of 17 larger seeds (Figures 1 and 2, Pearson’s correlation between germination ratio between treatment and control seed against average seed mass: r2 = 0.82, N = 5, p = 0.035). Figure 2. Differences in proportion of germinated seeds between controls (dark grey) and after gut passage (light grey), in order of decreasing seed size from left to right (Solanum nigrum, Amaranthus retroflexus, Polypogon monspeliensis, Cyperus difformis and Juncus bufonius). Bars represent 95% confidence intervals. All seeds were stratified before the experiment. Figure 2. Differences in proportion of germinated seeds between controls (dark grey) and after gut passage (light grey), in order of decreasing seed size from left to right (Solanum nigrum, Amaranthus retroflexus, Polypogon monspeliensis, Cyperus difformis and Juncus bufonius). Bars represent 95% confidence intervals. All seeds were stratified before the experiment. Plants 2023, 12, x FOR PEER REVIEW 5 of 17 larger seeds (Figures 1 and 2, Pearson’s correlation between germination ratio between treatment and control seed against average seed mass: r2 = 0.82, N = 5, p = 0.035). Figure 2. Differences in proportion of germinated seeds between controls (dark grey) and after gut passage (light grey), in order of decreasing seed size from left to right (Solanum nigrum, Amaranthus retroflexus, Polypogon monspeliensis, Cyperus difformis and Juncus bufonius). Bars represent 95% confidence intervals. All seeds were stratified before the experiment. Figure 3. Cont. Plants 2023,12, 1470 6 of 17 Plants 2023, 12, x FOR PEER REVIEW 6 of 17 Figure 3. Accumulative percentage curve of germinated seeds of five plant species after gut passage (blue) and for control seeds (red). Table 4. Differences in germinability (percentage of seeds) after gut passage compared with control seeds. S.D.: Standard deviation. C.I.: 95% Confidence Interval. Experimental Seeds Control Seeds N Average SD CI N Average SD CI A maranthus retroflexus 1077 28.69 45.26 25.99 31.40 200 46.50 50.00 39.53 53.47 Cyperus difformis 1146 32.55 46.88 29.83 35.26 200 99.00 9.97 97.61 100.39 J uncus bufonius 1014 15.98 36.66 13.72 18.24 200 77.00 42.19 71.12 82.88 Polypogon monspeliensis 1210 24.79 43.20 22.36 27.23 200 98.50 12.18 96.80 100.20 Solanum nigrum 1070 54.21 49.85 51.22 57.20 200 72.50 44.76 66.26 78.74 2.4. Germination Time For both control seeds and after gut passage, seeds of J. bufonius (the species with the smallest seeds) had the longest germination time, and P. monspeliensis had the shortest (Table 5). There was no relation to seed size, since the largest S. nigrum seeds also had relatively long germination times (Table 5). The change in germination time between control and passage seeds differed significantly between plant species (GLMM4.1, random interaction plant species * treatment: χ2 = 584.01, df = 4, p < 0.001, Figure 3). While experimental seeds of A. retroflexus, C. difformis, and P. monspeliensis showed a significant increase in germination time after passage, seeds Figure 3. Accumulative percentage curve of germinated seeds of five plant species after gut passage (blue) and for control seeds (red). The effect of treatment on germinability differed significantly between plant species (GLMM3.1, interaction plant species * treatment: χ2 = 294.71, df = 1, p< 0.001, Figure 2). When the effect of treatment was explored separately for each plant species, germinability was significantly lower after passage than for control seeds for all species (GLMM3.2s, Table 4, Figures 2and 3). However, the difference between controls and gut passage was related to seed size, with the significant loss of germinability after passage decreasing in larger seeds (Figures 1and 2, Pearson’s correlation between germination ratio between treatment and control seed against average seed mass: r2= 0.82, N = 5, p= 0.035). 2.4. Germination Time For both control seeds and after gut passage, seeds of J. bufonius (the species with the smallest seeds) had the longest germination time, and P. monspeliensis had the shortest (Table 5). There was no relation to seed size, since the largest S. nigrum seeds also had relatively long germination times (Table 5). The change in germination time between control and passage seeds differed significantly between plant species (GLMM4.1, random interaction plant species * treatment: χ2 = 584.01, df = 4, p< 0.001, Figure 3). While experimental seeds of A. retroflexus,C. difformis, and P. monspeliensis showed a significant increase in germination time after passage, seeds of J. bufonius and S. nigrum (i.e., the smallest and largest seeds) experienced a significant reduction in germination time after gut passage (Table 6, Figure 3). Plants 2023,12, 1470 7 of 17 Table 5. Germination time (in days) of five plant species after passage through guts of seven LBBG, and for control seeds. A.: Average; M.: Median; S.D.: Standard deviation. C.I.: 95% Confidence Interval. Passage Seeds Control Seeds N A M Max Min SD CI N A M Max Min SD CI Amaranthus retroflexus 309 6.51 5 2 40 5.55 5.15 6.03 92 4.82 4 3 12 2.02 1.77 2.36 Cyperus difformis 373 11.66 8 3 42 8.73 8.15 9.41 198 8.70 9 5 16 1.98 1.80 2.20 Juncus bufonius 162 14.83 7 4 43 13.00 11.73 14.60 154 19.55 16 8 45 10.21 9.18 11.50 Polypogon monspeliensis 300 5.76 4 1 41 6.76 6.26 7.35 197 3.82 3 2 19 2.24 2.04 2.49 Solanum nigrum 579 9.79 8 3 43 5.26 4.98 5.58 145 14.84 15 6 37 6.97 6.25 7.88 Table 6. Differences in germination time between control seeds and seeds after gut passage through seven gulls. Control Passage Plant Species Average SE Average SE Estimate SE Chisq p Amaranthus retroflexus 4.82 0.21 6.51 0.32 0.30 0.05 35.21 <0.001 Cyperus difformis 8.70 0.14 11.66 0.45 0.29 0.03 110.48 <0.001 Juncus bufonius 19.55 0.82 14.83 1.02 −0.28 0.03 102.42 <.001 Polypogon monspeliensis 3.83 0.16 5.76 0.39 0.41 0.04 92.79 <0.001 Solanum nigrum 14.84 0.58 9.79 0.22 −0.42 0.03 253.08 <0.001 3. Discussion This study advances our understanding of endozoochory of seeds by non-frugivorous birds, providing information on parameters influencing effective dispersal, such as seed survival, retention time and germinability. It is the first experimental study of gulls (Laridae) to address their important role as vectors of dry-fruited plants [ 25 ]. Like other waterbirds such as ducks, geese and storks, gulls are important vectors of weeds in rice fields and other agricultural habitats [ 13 , 26 ]. Our research contributes to the understanding of mechanisms of dispersal of weeds and invasive plants, most of which have been assigned to abiotic dispersal syndromes and have been assumed to rely on humans for long-distance dispersal [17,27]. 3.1. Retrieval, Viability and Retention Time of Seeds in Relation to Plant Traits We found that the five weed species (four dry fruited and one fleshy fruited) had high rates of seed survival after gut passage, exceeding 72%. Similar experiments with mallards Anas platyrhynchos and other Anatidae have found some plant species to have much lower rates of seed survival [ 16 , 18 ]. We may even have underestimated seed survival since our experiment ended after 33 h, and some seeds have been recovered after longer periods from Anatidae [ 19 ]. Species with the highest seed survival were dry fruited and of intermediate size (P. monspeliensis and C. difformis). In experiments with Anatidae, smaller seeds generally showed higher survival because they escape from the gizzard into the intestines more quickly, although other characteristics such as hardness, seed shape and phylogeny were also important predictors of seed survival [16], see also [28,29]. Longer retention times may benefit plants by increasing seed dispersal distances, making it more likely that they can reach and colonize previously unoccupied habitat patches. Given the generally high rates of seed survival, our finding that average retention times were strongly related to seed size was to be expected, with larger seeds taking longer to pass into the intestines and then be egested. In Anatidae experiments where species with larger seeds were found to have shorter retention times, this is likely because these species had low seed survival, and the only seeds to be egested were the fraction that passed quickly into the intestines [ 15 , 18 ]. We compared seeds from fleshy-fruited and dry-fruited plants fed simultaneously to birds, and found that seed size, rather than fruit Plants 2023,12, 1470 8 of 17 fleshiness, is the main determinant of retention time. However, when comparing separate experiments, the retention times of seeds are generally much lower for frugivorous than for non-frugivorous birds, as a consequence of the effects of fruit pulp on gut activity [ 30 ]. This suggests that non-frugivorous vectors have a greater potential for long-distance seed dispersal than frugivorous ones. If we had not removed the pulp from S. nigrum seeds prior to our experiments, we may have found different results, with a change in how this species compared with dry-fruited ones. However, our interest was in comparing how the seeds per se compared in their response to gut passage. We found no evidence that, apart from being the largest of the five species, there was anything special about S. nigrum seeds and their gut passage in comparison with the other species assigned to barochory or epizoochory syndromes. This is consistent with Costea et al. [ 31 ], who found that there is a diversity of seed architectures amongst angiosperms that allow different species to survive gut passage, and that there is no evidence for a fundamental difference between seeds from fleshy- or dry-fruited species. When combined with the growing body of field data showing how endozoochory is a frequent and often predictable dispersal mechanism for many species assigned to other dispersal syndromes [ 9 ], our results provide further evidence that seeds of dry-fruited species can have adaptations (or exaptations) for non-classical endozoochory. Species such as A. retroflexus and J. bufonius have been repeatedly shown to disperse by endozoochory in field studies of gulls, Anatidae or other waterbirds [12,32]. Germination experiments confirmed the viability of the egested seeds, although we found germinability to be higher in control seeds in all five species. This result may have changed if the seeds had not been kept at cold temperatures between collection and our experiments, since this stratification may have broken dormancy in control seeds. If the seeds had been kept at ambient temperature, gut passage may have increased germinability compared to control seeds, as observed in some previous studies [ 19 , 33 ]. This is supported by a separate finding that storage time between faeces egestion and seed extraction was correlated with germinability (Figure S1), although this result was confounded with retention time (see Methods). With hindsight, we should have processed faecal samples in a random order with respect to retention time. We did not expect to find the observed positive correlation between seed size and the germinability of egested seeds relative to control seeds, especially as larger seeds had longer average retention times, which would be expected to translate into reduced viability [ 19 , 33 ]. The impact of gut passage on germination time was unrelated to seed size, since the smallest and largest seeds (of J. bufonius and S. nigrum) were the only two for which seeds germinated faster compared to the controls. J. bufonius is easily the most abundant seed in LBBG faeces and pellets from rice fields of the Guadalquivir estuary [ 12 , 24 ], and its rapid germination after gut passage may promote endozoochory given intense intraspecific competition for this weed, which is highly abundant in rice fields, especially between harvest and sowing. It has smaller seeds than the other weeds we studied, but this allows production of more seeds than other species [ 34 , 35 ], with 34,000 seeds/ramet [36]. The relationship between seed size and germinability or germination time is clearly complex and confounded by other seed traits such as dormancy strategies. Kleyheeg et al. 2018 [37] tested 30 wetland plant species and found that germinability increased with seed size for control seeds, but this relationship was less pronounced after artificial digestion. They did not find a significant influence of seed size on time to germination. In contrast, we found that size was more related to germinability after gut passage than before (Figure 3), although this difference was not significant. Some experimental studies have found a negative correlation between seed size and germinability within plant species [29]. Our experimental estimations of seed survival and retention time might differ from real values in natural conditions. Avian diet can have an important influence on these parameters [ 38 ], although our birds were fed on an animal diet, which reflects their preference for crayfish in the field. Captivity can change seed survival and retention time due to the Plants 2023,12, 1470 9 of 17 limited mobility of the birds compared with those active in the field. Kleyheeg, et al. [ 39 ] found that seed survival increased significantly with forced activity in ducks. LBBGs spend long periods of the day inactive, roosting in rice fields or neighbouring habitats [ 40 ], so our experiment may reflect field conditions quite well. Martín-Vélez et al. [ 13 ] developed a spatial seed dispersal model based on GPS data for LBBGs and for field and experimental data for four weed species used in our study (J. bufonius,C. difformis,P. monspeliensis and A. retroflexus). These authors estimated median endozoochory dispersal distances of 690 to 940 m, with the maximum exceeding 150 km. Their model found that seeds would be deposited in suitable habitats for weed establishment such as other rice fields, natural wetlands and other irrigated crops. These geographic distance estimations are longer than those calculated for abiotic dispersal mechanisms such as wind [ 41 ]. For example, the maximum dispersal distance estimated for J. bufonius via wind was 100 m [42]. 3.2. Characteristics of LBBG That Make Them Good Seed Dispersal Vectors The LBBG is a long-distance migrant with a broad flyway, moving from North Europe to northwest Africa [ 43 , 44 ]. It typically combines migration with foraging during frequent and long-migratory stopovers, the so-called fly-and-forage migration strategy [ 44 – 46 ]. Although it does not achieve long daily distances compared with other migratory birds [average instantaneous speed of 38.6 km/h; 44 km/day in autumn and 98 km/day in spring; 44] (average instantaneous speed of 38.6 km/h; 44 km/day in autumn and 98 km/day in spring), the LBBG is still faster and can achieve longer daily distances than other fly- and-forage migrants such as raptors [ 45 ]. On the other hand, LBBG have the ability to find suitable habitats for feeding nearly everywhere along their migratory route due to their opportunistic feeding in inland as well as marine habitats, increasing the possibility that seeds are egested in favourable habitats [ 22 ]. The LBBG may thus be a good vector for long-distance dispersal of weeds, aliens and other plants in a step-by-step manner. Throughout the winter, the LBBG performs daily movements between feeding and roosting sites of up to 80 km [ 22 ] favouring dispersal of seeds from highly anthropized habitats to more natural environments. Due to their common use of landfills, agricultural fields and other human-transformed habitats, the LBBG may be a particularly suitable vector for weeds and invasive species (this study; [22]). The LBBG is an opportunistic feeder that includes many different food items in its diet [ 47 , 48 ]. This omnivorous diet may enable higher seed survival compared to granivorous bird species. Moreover, the structure of the gizzard, responsible for mechanical digestion, plays a key role in seed survival [ 28 ]. In the case of ducks specialized in the digestion of seeds, the gizzard is very thick and muscular, with the presence of grit which improves mechanical digestion of seeds, although grit may also increase germination rate through scarification [ 49 ]. Omnivorous birds such as LBBGs have relatively small gizzards compared to herbivorous or granivorous birds, and are thus more likely to allow seeds to survive gut passage [ 37 ]. This is likely to explain the particularly high recovery rate of intact seeds in our experiment. Overall, gulls can be expected to provide effective seed dispersal with relatively high-quantity and quality components [ 50 ], although further research into the survivorship of dispersed seeds and resulting seedlings is required. 3.3. Ecological and Management Implications of Weed Dispersal by LBBG All five species we studied are noxious agricultural weeds in Europe and other parts of the world, causing important losses to food production globally [ 51 ]. Some of them are also invasive aliens and outcompete native flora. P. monspeliensis,A. retroflexus and C. difformis in particular have evolved under the use of intense herbicide treatments, developing herbicide resistance that results in significant economic costs [ 52 , 53 ]. In India, P. monspeliensis is one of the main weeds of wheat and rice fields [ 54 ], and is spreading rapidly in Spain [ 55 ]. It has invaded saltmarshes in Southern California, outcompeting native Salicornia virginica [ 56 ] and leading to important restoration efforts [ 57 ]. Amaranthus species are among the most serious Plants 2023,12, 1470 16 of 17 41. Bullock, J.M.; Mallada González, L.; Tamme, R.; Götzenberger, L.; White, S.M.; Pärtel, M.; Hooftman, D.A. A synthesis of empirical plant dispersal kernels. J. Ecol. Environ. 2017,105, 6–19. [CrossRef] 42. Tamme, R.; Götzenberger, L.; Zobel, M.; Bullock, J.M.; Hooftman, D.A.; Kaasik, A.; Pärtel, M. Predicting species’ maximum dispersal distances from simple plant traits. Ecol. Evol. 2014,95, 505–513. [CrossRef] 43. Rock, P.; Spence, I. Lesser Black-backed gull. In The Migration Atlas: Movements of the Birds of Britain and Ireland; Wernham, C., Toms, M., Marchant, J., Clark, J., Siriwardena, G., Eds.; T & AD Poyser: London, UK, 2002; pp. 365–368. 44. Klaassen, R.H.; Ens, B.J.; Shamoun-Baranes, J.; Exo, K.-M.; Bairlein, F. Migration strategy of a flight generalist, the Lesser Black-Backed Gull Larus fuscus.Behav. Ecol. Sociobiol. 2012,23, 58–68. [CrossRef] 45. Klaassen, R.H.; Strandberg, R.; Hake, M.; Alerstam, T. Flexibility in daily travel routines causes regional variation in bird migration speed. Behav. Ecol. Sociobiol. 2008,62, 1427–1432. [CrossRef] 46. Baert, J.M.; Stienen, E.W.; Heylen, B.C.; Kavelaars, M.M.; Buijs, R.-J.; Shamoun-Baranes, J.; Lens, L.; Müller, W. High-resolution GPS tracking reveals sex differences in migratory behaviour and stopover habitat use in the Lesser Black-Backed Gull Larus fuscus.Sci. Rep. Ist. Super. Sanita 2018,8, 1–11. [CrossRef] 47. Coulson, J.C.; Coulson, B.A. Lesser Black-backed Gulls Larus fuscus nesting in an inland urban colony: The importance of earthworms (Lumbricidae) in their diet. Bird Study 2008,55, 297–303. [CrossRef] 48. Gyimesi, A.; Boudewijn, T.J.; Buijs, R.-J.; Shamoun-Baranes, J.Z.; de Jong, J.W.; Fijn, R.C.; van Horssen, P.W.; Poot, M.J. Lesser black-backed gulls Larus fuscus thriving on a non-marine diet. Bird Study 2016,63, 241–249. [CrossRef] 49. Figuerola, J.; Green, A.J.; Santamaría, L. Comparative dispersal effectiveness of wigeongrass seeds by waterfowl wintering in South-West Spain: Quantitative and qualitative aspects. J. Ecol. Environ. 2002,90, 989–1001. [CrossRef] 50. Van Leeuwen, C.H.; Villar, N.; Mendoza Sagrera, I.; Green, A.J.; Bakker, E.S.; Soons, M.B.; Galetti, M.; Jansen, P.A.; Nolet, B.A.; Santamaría, L. A seed dispersal effectiveness framework across the mutualism–antagonism continuum. Oikos 2022 ,2022, e09254. [CrossRef] 51. Mandak, B.; Zakravsky, P.; Dostal, P.; Plackova, I. Population genetic structure of the noxious weed Amaranthus retroflexus in Central Europe. Flora 2011,206, 697–703. [CrossRef] 52. Singh, S. Evaluation of some graminicides against Polypogon monspeliensis (L.) Desf. Ind. J. Weed Sci. 2009,41, 96–98. 53. Yadav, D.; Roshan, L.; Punia, S.; Ashok, Y.; Malik, R.; Hasija, R. Efficacy of clodinafop and fenoxaprop in combination with isoproturon against Polypogon monspeliensis and Phalaris minor in wheat. Environ. Ecol. 2011,29, 1256–1259. 54. Singh, A.; Bhullar, M.S.; Yadav, R.; Chowdhury, T. Weed management in zero-till wheat. Ind. J. Weed Sci. 2015,47, 233–239. 55. López-Granados, F.; Peña-Barragán, J.M.; Jurado-Expósito, M.; Francisco-Fernández, M.; Cao, R.; Alonso-Betanzos, A.; Fontenla- Romero, O. Multispectral classification of grass weeds and wheat (Triticum durum) using linear and nonparametric functional discriminant analysis and neural networks. Weed Res. 2008,48, 28–37. [CrossRef] 56. Callaway, J.; Zedler, J.B. Interactions between a salt marsh native perennial (Salicornia virginica) and an exotic annual (Polypogon monspeliensis) under varied salinity and hydroperiod. Wetl. Ecol. Manag. 1997,5, 179–194. [CrossRef] 57. Callaway, J.C.; Zedler, J.B. Restoration of urban salt marshes: Lessons from southern California. Urban Ecosyst. 2004 ,7, 107–124. [CrossRef] 58. Holm, L.; Doll, J.; Holm, E.; Pancho, J.V.; Herberger, J.P. World Weeds: Natural Histories and Distribution; John Wiley & Sons: Hoboken, NJ, USA, 1997. 59. Tranel, P.J.; Trucco, F. 21st-century weed science: A call for Amaranthus genomics. In Weedy and Invasive Plant Genomics; Stewart, C.N., Jr., Ed.; Wiley-Blackwell: Ames, IA, USA, 2009; Volume 53, p. 81. 60. Yarnia, M.; Benam, M.K.; Tabrizi, E.F.M. Allelopathic effects of sorghum extracts on Amaranthus retroflexus seed germination and growth. J. Food Agric. Environ. 2009,7, 770–774. [CrossRef] 61. Marinov-Serafimov, P. Determination of allelopathic effect of some invasive weed species on germination and initial development of grain legume crops. Pestic. Phytomed. 2015,25, 251–259. [CrossRef] 62. McKay, A.; Ophel, K.; Reardon, T.; Gooden, J. Livestock deaths associated with Clavibacter toxicus/Anguina sp. infection in seedheads of Agrostis avenacea and Polypogon monspeliensis.Plant Dis. 1993,77, 635–641. [CrossRef] 63. Kessell, A.; Boulton, J.; Krebs, G.; Quinn, J. Acute renal failure due to Amaranthus species ingestion by lambs. Aust. Vet. J. 2015 ,93, 208–213. [CrossRef] 64. Weston, P.A.; Gurusinghe, S.; Birckhead, E.; Skoneczny, D.; Quinn, J.C.; Weston, L.A. Chemometric analysis of Amaranthus retroflexus in relation to livestock toxicity in Southern Australia. Phytochemistry 2019,161, 1–10. [CrossRef] 65. Wylie, S.J.; Li, H.; Jones, M.G. Yellow tailflower mild mottle virus: A new tobamovirus described from Anthocercis littorea (Solanaceae) in Western Australia. Arch. Virol. 2014,159, 791–795. [CrossRef] 66. Bryson, C.T.; Carter, R. The significance of Cyperaceae as weeds. In Sedges: Uses, Diversity, Systematics of the Cyperaceae; Missouri Botanical Garden Press: St. Louis, MO, USA, 2008; Volume 108, p. 15. 67. Gómez de Barreda, D.; Pardo, G.; Osca, J.M.; Catala-Forner, M.; Consola, S.; Garnica, I.; López-Martínez, N.; Palmerín, J.A.; Osuna, M.D. An overview of rice cultivation in Spain and the management of herbicide-resistant weeds. Agron. J. 2021 ,11, 1095. [CrossRef] 68. Cuba-Díaz, M.; Fuentes, E.; Rondanelli-Reyes, M. Experimental culture of non-indigenous Juncus bufonius from King George Island, South Shetland Island, Antarctica. Adv. Polar Sci. 2015,26, 24–29. [CrossRef] Plants 2023,12, 1470 17 of 17 69. Chauhan, B.S.; Matloob, A.; Mahajan, G.; Aslam, F.; Florentine, S.K.; Jha, P. Emerging challenges and opportunities for education and research in weed science. Front. Plant Sci. 2017,8, 1537. [CrossRef] [PubMed] 70. Hanson, C. Genetic, biological, chemical, and management approaches to crop protection. In Introduction to Crop Protection; Ennis, W.B., Ed.; American Society of Agronomy: Madison, WI, USA, 1979; pp. 89–151. 71. Bartel, R.D.; Sheppard, J.L.; Lovas-Kiss, A.; Green, A.J. Endozoochory by mallard in New Zealand: What seeds are dispersed and how far? PeerJ 2018,6, 19. [CrossRef] 72. Zedler, J.B.; Kercher, S. Causes and consequences of invasive plants in wetlands: Opportunities, opportunists, and outcomes. Crit. Rev. Plant Sci. 2004,23, 431–452. [CrossRef] 73. Chauhan, B.S.; Johnson, D.E. Ecological studies on Cyperus difformis,Cyperus iria and Fimbristylis miliacea: Three troublesome annual sedge weeds of rice. Ann. Appl. Biol. 2009,155, 103–112. [CrossRef] 74. Devlaeminck, R.; Bossuyt, B.; Hermy, M. Seed dispersal from a forest into adjacent cropland. Agric. Ecosyst. Environ. 2005 ,107, 57–64. [CrossRef] 75. Julve, P. Baseflor. Index Botanique, Écologique et Chorologique de la flore de France; Institut Catholique de Lille: Lille, France, 1998. 76. Consejería de Sostenibilidad, Medio Ambiente y Economía Azul. Programa de Emergencias, Control Epidemiológico y Seguimiento de Fauna Silvestre de Andalucía. Seguimiento de Aves Terrestres Amenazadas de Andalucía. Invernada de 2018/2019. Informe Regional; Consejería de Sostenibilidad, Medio Ambiente y Economía Azul, Junta de Andalucía: Seville, Spain, 2020. 77. Snell, R.; Beckman, N.; Fricke, E. Consequences of intraspecific variation in seed dispersal for plant demography. AOB Plants 2019,11, plz016. [CrossRef] 78. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2020. 79. Magnusson, A.; Skaug, H.; Nielsen, A.; Berg, C.; Kristensen, K.; Maechler, M.; van Bentham, K.; Bolker, B.; Brooks, M.; Brooks, M.M. Package ‘glmmtmb’. R J. 2017,9, 378–400. [CrossRef] 80. Lenth, R.V. Emmeans: Estimated Marginal Means, aka Least-Squares Means, R Package Version 1.7.1-1; 2021. Available online: https://cran.r-project.org/web/packages/emmeans/index.html (accessed on 1 October 2022). 81. Wickham, H. Data analysis. In ggplot2; Springer: Berlin/Heidelberg, Germany, 2016; pp. 189–201. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.