Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment.

Dung beetles are secondary seed dispersers, incidentally moving many of the seeds defecated by mammals vertically (seed burial) and/or horizontally as they process and relocate dung. Although several studies have quantified this ecological function of dung beetles, very few have followed seed fate until seedling establishment, and most of these have focused on the effects of seed burial. We know very little about the effects of horizontal seed movement by dung beetles, though it is generally assumed that it will affect plant recruitment positively through diminishing seed clumping. The objective of our study was to assess the effects of dung beetle activity on the spatial distribution of seeds and seedlings, and on the probability of seedling establishment. In a tropical rainforest in Mexico we carried out two complementary field experiments for each of two tree species (Bursera simaruba and Poulsenia armata), using seeds experimentally imbedded in pig dung and recording their fate and spatial location over time. For both species, dung beetle activity reduced the spatial clumping of seeds and seedlings; however, it did not increase the probability of seedling establishment. We discuss the context- and species-specificity of the combined effects of horizontal and vertical dispersal of seeds by dung beetles, and the need to quantify long-term seedling fates to more accurately determine the effects of seed movement by dung beetles on plant recruitment.

Introduction

Any movement of a seed after deposition by the primary seed dispersal vector constitutes secondary seed dispersal, a common process that affects seed fate and ultimately plant demography [1,2]. In diplochorous systems, where the primary and secondary dispersal vectors are different, a potential advantage of the second phase of dispersal can be the movement of the seeds to microsites that are predictably more adequate for seed survival and/or seedling establishment and survival (i.e., directed dispersal; [3,4]). In tropical forests, a very large proportion of plant species are dispersed primarily by frugivorous animals, mostly birds and mammals [5,6]. In the case of seeds dispersed through mammal defecation, a common diplochorous system occurs, in which dung beetles are often responsible for the secondary dispersal of those seeds [4,7,8].

Dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) disperse seeds accidentally, as they process dung for feeding or ovipositing [9]. The way in which dung beetles process dung depends on the functional group they belong to. They are classified as dwellers, when all their activities are done in the dung pad or immediately underneath it; tunnelers, if they first construct tunnels in the soil under or near the dung pad and then push and pull portions of dung into the tunnels; and rollers, when they first shape dung portions into balls, which they then roll away from the dung pad to a location where they build a tunnel to bury the dung balls [9]. Competition for the ephimerous and patchily-distributed dung is intense, and in tropical rainforests all the fecal material is quickly relocated beneath the soil by tunneler and roller beetles [10]. Dung relocation is responsible for many of the ecological functions attributed to dung beetles [11], including the secondary dispersal of seeds embedded in the dung [7].

The interaction between dung beetles and defecated seeds can have strong effects on seed fate and seedling establishment, but the direction (positive vs. negative) and intensity of these effects are highly species- and context-specific [8,12,13]. For example, burial by beetles is known to greatly diminish the probability of seed predation [7,14-16]. However, while seed burial by beetles may increase seedling establishment for some plant species due to enhanced seed survival [8,12,17,18], others may be affected negatively due to reduced seedling emergence when seeds are buried too deeply [12,13,19]. Even though secondary seed dispersal by dung beetles has been relatively well studied in tropical forests, our ability to generalize on the ultimate net effect of the seed-beetle interaction on plant fitness is still limited, not only due to the context-dependence of the interaction’s result, but also due to the fact that very few studies have followed seed fate until seedling establishment [8,12,13,18,20].

Secondary seed dispersal by dung beetles can include both seed burial (vertical dispersal) and/or horizontal seed dispersal [21], but most studies have focused on the effects of the former. However, it has often been suggested that horizontal movement of seeds by dung beetles, although generally restricted to small distances (< 5 m), may favor plant recruitment by decreasing the degree of spatial clumping in which defecated seeds are deposited by the primary disperser [14,21,22]. Spatial clumping of seeds and seedlings, generally associated with high densities, is known to negatively affect plants through various mechanisms, including decreased germination and increased competition, pathogen attack, predation and herbivory [2,23,24]. To our knowledge, only one study has so far quantified a decrease in the spatial clumping of seedlings of two species, following dung beetle activity, with an associated positive effect on the probability of seedling establishment for one of the species, but not for the other one [20]. However, this study used mixtures of both seed species in each dung pile, and seedling establishment could have been affected by interspecific competition [20]. Thus, considering that: (i) the outcome of the seed-beetle interaction is species- and context-specific, (ii) very few studies have followed seed fate until seedling establishment, and (iii) only one study has quantified the effects of horizontal seed dispersal by dung beetles on seedlings, we believe that additional information is necessary before we can reach a more general conclusion on when secondary dispersal by dung beetles has positive effects on plant regeneration, and when not.

Our main objective was to assess, for two tree species, the effects of secondary seed dispersal by dung beetles, with emphasis on the horizontal movement of seeds, on the spatial distribution of seeds and seedlings, and on the probability of seedling establishment. Our hypothesis was that dung beetle activity would favor seedling establishment because it reduces seed and seedling clustering through horizontal relocation of the seeds present in dung. To test this hypothesis we carried out field experiments to assess the following predictions: (i) dung beetle activity increases the horizontal distance between adjacent seeds deposited in dung, regardless of seed burial; (ii) dung beetle activity increases the distance between established seedlings; (iii) dung beetle activity increases the probability of seedling establishment.

Methods

Study site

The study was carried out in the Los Tuxtlas Biological Station (LTBS), in the Mexican state of Veracruz (18°35'5" N, 95°4'34" W; ca. 150 m a.s.l.). The LTBS, a protected area established in 1967, encompasses 640 ha of tropical rainforest that constitute, since 1998, one of the core areas of the 155,122-ha Los Tuxtlas Biosphere Reserve [25]. All field activities were conducted under full permission of the authorities of the LTBS, Institute of Biology, National Autonomous University of Mexico. No endangered or protected species were collected. Mean annual temperature is 24.1°C and mean annual rainfall is 4201 mm, with a short drier period (< 100 mm per month) from March to April, and a long wetter period (≥ 100 mm per month) from May through February, the latter representing ca. 95% of the total annual rainfall [26].

Despite intense forest loss and fragmentation occurring in the Los Tuxtlas region, the LTBS and the other core areas of the Biosphere Reserve are an important refuge for animals, including dung beetles, with at least 34 species reported for LTBS [27], as well as many mammals [28,29]. Among mammals present at LTBS that are primary dispersers of seeds through defecation, figure most prominently the herbivorous-frugivorous howler monkeys (Alouatta palliata; [14]), but also other important omnivorous-frugivorous species such as coatis (Nasua narica), raccoons (Procyon lotor), tayras (Eira barbara), peccaries (Pecari tajacu) and kinkajous (Potos flavus) [30].

Effects of dung beetle activity on seed and seedling spatial distribution and on seedling establishment

To assess the effects of dung beetle activity on seeds and seedlings we carried out two complementary field experiments focusing on two tree species that are abundant in our study site (G. Ibarra-Manríquez, pers. comm.): Bursera simaruba (L.) Sarg. (Burseraceae) and Poulsenia armata (Miq.) Standl. (Moraceae), hereafter referred to by their genus names. Both experiments were carried out independently for each species.

Bursera is a dioecious canopy tree; it is a light-demanding species that grows mostly in tree-fall gaps, reaching heights of 30 m [31]. Fruiting occurs between October and May; fruits are dehiscent drupes that contain a single seed covered by a juicy aril [31,32]. Bursera seeds are 7–9 mm long, 5–7 mm thick and wide, and germinate in ~ 2 weeks [33]. Poulsenia is a monoecious late-successional tree species that can reach heights of 20–40 m [34,35]. Fruiting occurs between May and November; the fruit is soft and berry-like, containing 9–17 seeds [32]. Poulsenia seeds are 8–10 mm long, 5–7 mm thick and wide, and germinate in ~ 4 weeks [33].

We chose to focus on Bursera and Poulsenia because they can be dispersed through defecation by mammals (e.g., Alouatta palliata; [14]), because their seeds have no dormancy [36], and due to the ease of obtaining the necessary number of seeds for the experiments. Furthermore, the seeds of these two species are of a size large enough to allow marking (see below, Experiment 1), but small enough that beetles at our study site will secondarily disperse many of them while processing dung.

First, we carried out Experiment 2 and then Experiment 1; however, since the former corresponds to the seedling stage and the latter to the seed stage, we describe them in life-cycle order. All experimental seeds of Bursera and Poulsenia were obtained from freshly fallen fruits underneath parent trees in April and June 2017, respectively. For both species, half of the seeds were used within 2 days of collection to set up Experiment 2; the rest of the seeds were air-dried in the shade and stored at room temperature until the setup of Experiment 1 (six months of storage for Bursera seeds and four months for Poulsenia seeds). We did not test the viability of the seeds prior to our experiments, and seed storage might have negatively affected it. However, in Experiment 1 we did not require seeds to be viable as we only recorded seed condition and position after 48 h (see below).

In the experiments we used fresh domestic pig dung (collected from a nearby household on the same day it was used) to make experimental dung piles containing seeds, thus mimicking primary seed dispersal through mammal defecation. We used domestic pig dung because we needed large quantities of dung for our experiments, and because it is efficient in attracting rainforest dung beetles [37].

Experiment 1. Seeds: Secondary dispersal and spatial distribution

We established 30 experimental sites in the forest understory (≥ 50 m apart and ≥ 10 m from fruiting adults of the focal plant species). In each site we had 3 circular plots 50 cm in diameter, with 2–3 m between adjacent plots. The border of each plot was delimited by burying a 30 cm wide metallic mosquito netting strip 10 cm into the soil (S1 Fig). From the inside of each plot we removed the few existing seedlings. However, we kept the leaf litter to avoid affecting the behavior of roller dung beetles, which often choose a spot hidden under litter to build their tunnel (E. Andresen, pers. obs.), and thus litter removal may cause them to roll the dung balls larger distances. Each plot was randomly assigned to one of the following treatment levels: (1) 50 g of dung with 20 seeds embedded in it and access to dung beetles (+Feces+Beetles), (2) 50 g of dung with 20 seeds but no access to dung beetles (+Feces−Beetles), (3) no dung, 20 seeds placed directly on the forest floor (−Feces−Beetles). Since the amount of seeds present in the defecations of rainforest mammals can vary tremendously, depending on the plant and animal species (e.g., [8,38,39]), we used seed numbers that can commonly be found in howler-monkey dung piles (e.g., [40,41]). Furthermore, to better mimic the generally smaller size of individual dung piles of frugivorous rainforest mammals that fall on the forest floor (5–25 g; [22,41,42]) we divided the 50 g of dung into 4 equal portions (S1A Fig), each containing 5 seeds. Portions were placed in the center of plots, with ~ 3 cm between portions; this layout was also used for seeds without dung (S1B Fig). In order to measure seed movement, we marked each seed by threading a 30 cm-long piece of fishing line through it (S1C and S1D Fig). After placing dung and seeds in the plots we covered the two control plots (+Feces−Beetles and −Feces−Beetles) with mosquito netting to exclude dung beetles (S1E Fig), but left the plots with dung beetle access uncovered (+Feces+Beetles). After 48 h, when all dung in the plots with dung beetle activity had disappeared from the soil surface (S1F Fig), we used a 2 cm grid to map the location of each seed in the three plots (S1G Fig); for seeds that were buried we mapped its location projected to the surface. To describe the short-term fate of the seeds we classified each seed into one of three categories: (1) seed intact, when seeds were unharmed; (2) seed predated, when seed remains were found; and (3) seed removed, when the seed could not be found. For seeds intact we also recorded if the seed was visible on the soil surface, under leaf-litter, or buried. In the case of buried and horizontally moved seeds we measured the vertical and horizontal distances to the nearest centimeter. Dung beetle movement was limited by the plot’s fence, i.e., seeds could not be dispersed beyond the fence. While this allowed us to find most seeds, it probably caused some underestimation of horizontal distances (see Discussion). This experiment was carried out during the rainy season (October 2017), first with Poulsenia seeds, and after two weeks, the same sites and plots were used to repeat the experiment with Bursera seeds.

Experiment 2. Seedlings: Spatial distribution and probability of establishment

We used a similar experimental setup as above, but without thread-marking the experimental seeds, to allow for germination. We used the same 30 sites as in Experiment 1, but with different plots for each plant species. For Bursera, the experiment started in April 2017 (one of the driest months) and for Poulsenia in June 2017 (the beginning of the rainy season). Control plots were immediately covered with mosquito netting, while the plot with dung beetle activity was kept open. After 48 hours the latter plots were also covered, to have the same conditions affecting seed and seedling fates in all treatments. The netting remained throughout the experiment to avoid seed rain and to minimize seed removal by granivorous animals and seedling loss due to herbivory. Once seedling establishment occurred (20 and 32 days after the setup of the experiments for Bursera and Poulsenia, respectively; S1H and S1I Fig) we checked each plot once a week for 15 weeks. During each check we registered established seedlings of the focal species and we mapped the location of each seedling using the 2 cm grid. We assumed that all seedlings of the focal plant species that we recorded, originated from our experimental seeds because: (i) all plots were > 10 m away from any fruiting adult, and (ii) in a previous study in the same sites and with the same treatments, only two seedlings of Bursera and two of Poulsenia established, overall, from the soil seed bank during a time period of 8 months [33]. Finally, we acknowledge that using seeds extracted from fruits may yield different results compared to using seeds that have passed through the digestive system of a mammal. However, we expect that whatever difference there might be in terms of seed germination would equally have affected our three treatment levels.

Data analyses

To measure the spatial distribution of seeds (Experiment 1) and seedlings (Experiment 2), following Lawson et al. [20] we used the Clark-Evans nearest neighbor index R [43]: , where is the average observed distance from an individual to its nearest neighbor in the plot, and is the expected mean distance between neighbors if the distribution were random. When R < 1 the spatial distribution of individuals is clumped, when R = 1 it is random, and when R > 1 it is overdispersed [43].

For Experiment 1 we lost one Poulsenia plot with the +Feces+Beetles treatment. The nearest neighbor index data were analyzed by fitting linear mixed models (LMMs), one for each species. Treatment (+Feces+Beetles, +Feces−Beetles, and −Feces−Beetles) was the fixed factor, while site was included as a random factor.

For Experiment 2, following Lawson et al. [20] we analyzed the values of the nearest neighbor index observed during the week of peak seedling abundance, which was determined separately for each species-treatment combination. We chose this approach because for Bursera the temporal pattern of seedling emergence differed strongly among treatment levels, and mortality of seedlings occurred rapidly after emergence (S2 Fig). The weeks of peak abundance were as follows: Bursera, +Feces+Beetles: 4 wk, +Feces−Beetles: 9 wk, −Feces−Beetles: 10 wk; Poulsenia, +Feces+Beetles: 11 wk, +Feces−Beetles and −Feces−Beetles: 6 wk. The nearest neighbor index data for seedlings was analyzed in the same way as for seeds (LMM) in the case of Poulsenia; for Bursera, due to singularity problems during model fitting, seemingly caused by almost zero variance estimation for the random effect, we excluded the random factor following Bolker [44] and fitted a simple linear model.

To analyze seedling establishment, we fitted Cox regression models with mixed effects (i.e., frailty models), following [45]. In these models we included treatment (+Feces+Beetles, +Feces−Beetles, and −Feces−Beetles) as the fixed factor, and site and plot as random factors. For these survival analyses we used as response variable the number of days elapsed until seedling establishment occurred for each seed; when this event did not occur at the end of the experiment, we considered this observation as a right-censored datum (e.g., [46]).

Data analyses were carried out using the R statistical environment (v. 3.5.2; [47]). The nearest neighbor indices were calculated using the function clarkevans.test of package ‘spatstat’ [48]. Models for the nearest neighbor index were fitted using functions lm of package ‘stats’ [47], and lmer of package ‘lme4’ [49]. Models for seedling establishment were fitted with function coxme of package ‘coxme’ [50]. In all models, treatment effect was tested through a Wald Chi-square test using the Anova function in the ‘car’ package [51]. We obtained marginal mean and standard error values, and carried out post hoc tests using the function emmeans of the package ‘emmeans’ [52]. We adjusted P-values in all post hoc tests using the False Discovery Rate method [53], because this method controls for false positives while also minimizing false negatives (e.g., [54]). All processed and raw data used in this study are provided in Supporting Information.

Results

Experiment 1. Seeds: Secondary dispersal and spatial distribution

The percentages of experimental seeds lost due to disappearance and predation were very low (Bursera: 0.5% and 0.2%, respectively; Poulsenia: 2.1% and 0.2%, respectively). For seeds classified as ‘intact’, most were seeds buried by beetles (Bursera: 57.2% ± 26.7%; Poulsenia: 54.1% ± 26.4%; mean ± SD), followed by seeds visible on the soil surface (Bursera: 32.4% ± 25%; Poulsenia: 34.1% ± 25.3%), and by seeds hidden under the leaf litter (Bursera: 10.4% ± 11.5%; Poulsenia: 11.8% ± 12.4%). For buried seeds, mean depth for both species was 5 cm, but the distribution was asymmetrical with most seeds buried shallowly, although some were buried deeply (Bursera, N = 341, min = 1 cm, max = 30 cm, median = 3 cm; Poulsenia, N = 308, min = 1 cm, max = 25 cm, median = 2 cm, S3 Fig).

Of all seeds classified as ‘intact’, 97% of Bursera seeds and 98% of Poulsenia seeds were moved horizontally at least 2 cm by dung beetles. The mean dispersal distance was 6 cm for both species, again with an asymmetrical distribution (Bursera, N = 577, min = 2 cm, max = 24 cm, median = 5 cm; Poulsenia, N = 556, min = 2 cm, max = 25 cm, median = 5 cm, S3 Fig).

As we predicted, for both species the mean nearest distance between two seeds was higher in plots with dung beetle activity (Bursera: 1.23 cm ± 0.61 cm; Poulsenia: 1.15 cm ± 0.51 cm), compared to plots with dung added but beetles excluded (Bursera: 0.003 cm ± 0.02 cm; Poulsenia: 0.0 cm) and to plots with no dung or beetles (Bursera: 0.13 cm ± 0.57; Poulsenia: 0.0 cm). The treatment had a significant effect on the spatial distribution of both seed species (Bursera, χ2 = 137.46, df = 2, P < 0.001, Fig 1A; Poulsenia, χ2 = 310.66, df = 2, P < 0.001, Fig 1B). Although in all cases the nearest neighbor index values were <1, indicating spatial clustering of seeds, dung beetle activity significantly reduced the degree of seed aggregation, compared to both treatment levels with no dung beetles (Bursera, +Feces+Beetles vs. +Feces–Beetles: t = 10.60, df = 87, P < 0.001, +Feces+Beetles vs.–Feces–Beetles: t = 9.64, df = 87, P < 0.001; Poulsenia, +Feces+Beetles vs. +Feces–Beetles: t = 15.30, df = 86, P < 0.001, +Feces+Beetles vs.–Feces–Beetles: t = 15.30, df = 86, P < 0.001). The two control treatment levels were not significantly different from each other (Bursera, +Feces–Beetles vs.–Feces–Beetles: t = -0.95, df = 87, P = 0.34; Poulsenia, +Feces–Beetles vs.–Feces–Beetles: t = 0, df = 86, P = 1).

Fig 1

Box-plots of the nearest neighbor index for seeds and seedlings of two plant species.

The index was measured for Bursera seeds (A) and seedlings (C) and Poulsenia seeds (B) and seedlings (D) in plots (50-cm-diameter) with three treatment levels: 50 g of feces and dung beetle access (+Feces+Beetles), 50 g of feces and dung beetle exclusion (+Feces−Beetles), and no feces or dung beetles (−Feces−Beetles). In the first two treatments 20 seeds were mixed in the dung, and in the last treatment seeds were placed on the soil surface. Independent experiments were carried out for seeds and seedlings. Seedling results are from data observed during the week of peak seedling abundance, which was determined separately for each species-treatment level combination (see text for details). Circles represent outliers; different letters above bars indicate statistical differences.

Experiment 2. Seedlings: Spatial distribution and probability of establishment

As observed for seeds, for both plant species we found that the nearest neighbor distance between seedlings was higher in plots with dung beetle activity (Bursera, 3.67 cm ± 1.43 cm; Poulsenia, 4.39 cm ± 2.14 cm; S4 Fig), than in plots with dung added but beetles excluded (Bursera, 2.79 cm ± 1.84 cm; Poulsenia, 2.56 cm ± 1.84 cm) and plots with no dung or beetles (Bursera, 2.34 ± 1.54; Poulsenia, 1.86 cm ± 1.29 cm). The treatment had a significant effect on the spatial distribution of seedlings of both species (Bursera, F = 5.57, df = 2, P = 0.006, Fig 1C; Poulsenia, χ2 = 31.88, df = 2, P < 0.001, Fig 1D). As with seeds, dung beetle activity significantly reduced spatial aggregation of seedlings when compared to both control plots (Bursera, +Feces+Beetles vs. +Feces–Beetles: t = 2.26, df = 57, P = 0.04, +Feces+Beetles vs.–Feces–Beetles: t = 3.30, df = 57, P = 0.005; Poulsenia, +Feces+Beetles vs. +Feces–Beetles: t = 3.89, df = 53, P < 0.001, +Feces+Beetles vs.–Feces–Beetles: t = 5.50, df = 53, P < 0.001). The two control treatment levels were not significantly different from each other (Bursera, +Feces–Beetles vs.–Feces–Beetles: t = 1.25, df = 57, P = 0.21; Poulsenia, +Feces–Beetles vs.–Feces–Beetles: t = 1.10, df = 53, P = 0.27).

Of all experimental seeds, 29% of Bursera and 18% of Poulsenia seeds registered seedling emergence. During the 15 weeks in which we monitored seedling emergence, Poulsenia followed a steady pattern of increase until an asymptote was reached, while for Bursera we observed peaks of emergence followed by seedling death (S2 Fig). The treatment did not have a significant effect on the probability of seedling establishment in the case of Bursera (χ2 = 4.26, df = 2, P = 0.12; Fig 2A). However, it had a significant effect for Poulsenia (χ2 = 69.90, df = 2, P < 0.001); the probability of seedling establishment was higher in the plots with no feces, and it was equally low in the two treatment levels with feces, regardless of dung beetle activity (Fig 2B). In plots with no feces the probability of establishment increased by a factor of 2.70 compared to plots with dung beetle activity.

Fig 2

Kaplan–Meier curves for the probability of seedlings establishing from experimental seeds of two plant species.

During 15 weeks, seedling establishment was monitored for Bursera (A) and Poulsenia (B) in plots containing: 50 g of feces and dung beetle access (solid black lines), 50 g of feces and dung beetle exclusion (dashed black lines), and without feces or dung beetles (dotted black lines). In the first two treatments 20 seeds were mixed in the dung, and in the last treatment seeds were placed on the soil surface. All curves have censored data. Different letters next to each curve indicate statistical differences.

Discussion

Almost all experimental seeds (> 97%) were moved by dung beetles horizontally and more than half the seeds (> 55%) were moved vertically (i.e., buried). As predicted, dung beetle activity was associated with a decrease in the spatial clumping of both seeds and seedlings for both plant species. However, contrary to our expectation, dung beetle activity did not have a positive effect on the probability of seedling establishment. While the first result shows clearly the importance of dung beetle activity in diminishing the aggregation of seeds deposited in fecal clumps by fruit-eating mammals and of the seedlings establishing from those seeds, the second shows that this effect does not necessarily translate into increased seedling establishment. This stresses the need to be cautious when drawing conclusions regarding the effects of dung beetle activity on plant regeneration, based only on their effects on seeds.

Our result on reduced seed aggregation was expected, as several studies have determined and measured horizontal seed movement caused by dung beetles [12,16,21,22,55]. In terms of seedling spatial aggregation, our results are consistent with the only published study (that we are aware of) designed to quantify this consequence of dung beetle activity. In that study, Lawson et al. [20] also found that dung beetle activity decreased seedling spatial clumping in two plant species, Tabernaemontana donnell-smithii Rose and Guazuma ulmifolia Lam. For seedling establishment, they obtained mixed results: while one of the species had a higher percentage of establishment in plots with dung beetle activity, the other one had lower establishment in those plots [20]. In our study, one species had the highest probability of seedling establishment in plots with dung added but beetles excluded, while the other species had significantly more seedling establishment in plots without dung (Fig 2). Lawson’s study used both seed species mixed together in their experiments and the authors argued that their results on seedling establishment might have been affected by competitive interactions between germinating seeds of the two species. Furthermore, they pointed out that given the short duration of their study (4 weeks after experimental setup), they could not disentangle potential mechanisms affecting seedling establishment mediated by dung beetle activity (e.g., effects of dung beetles on seed clumping vs. on seed germination; [20]). We carried out independent experiments for each seed species, and we monitored seedling establishment for a longer time period (15 weeks after onset of establishment), yet we also recognize that several factors could have influenced our results. Both our study and that of Lawson underscore the fact that the outcomes of seed-beetle interactions are species- and stage-specific, and can be affected by many factors [7,8,12,13,33].

So, why did we not find a positive effect of dung beetle activity on short-term seedling establishment? First, vertical seed dispersal by dung beetles might have hindered seedling emergence, counterbalancing a positive effect of decreased clumping due to horizontal dispersal. We found that over half of all experimental seeds in the plots with dung beetle activity were buried. Several studies in tropical forests have shown that, depending on seed species and burial depth, the effect of vertical seed dispersal by dung beetles can promote seedling establishment (through a dominant effect of avoiding seed predation) or it can hinder it (through a dominant effect of non-emergence of germinating seeds; [8,12,13,18,33]). It has been considered that burial depths ≤ 3 cm should favor seedling establishment of rainforest seeds dispersed by dung beetles [7]. In our study, the median burial depth was 2–3 cm, meaning that half of the seeds buried by dung beetles were located at greater depths (S3 Fig) and may have suffered from non-emergence or other types of seed mortality associated with burial [56]. In a parallel study in the same study region, in which we buried seeds of the focal plant species at 3, 5 and 10 cm, seedling establishment decreased with depth [33]. Furthermore, in that study, only 7% of Bursera and 14% of Poulsenia seeds buried at 3 cm emerged as seedlings, compared to 30% for seeds placed on the surface. This clearly indicates that even shallow seed burial depths have a strong negative effect on the recruitment of these two species. However, it could also be that seasonal differences in dung beetle assemblages caused a higher proportion of seeds to be buried and/or buried more deeply in Experiment 2, which was setup in April-June, than in Experiment 1, which was done in October. When we sampled dung beetles, we found higher abundances, particularly of large beetles (≥ 10 cm body length), in April than in October (S1 Table).

Second, it is possible that the decrease in seed and seedling spatial aggregation due to dung beetle activity, though statistically meaningful, might not have the necessary effect size to be of ecological significance. Though most seeds were moved horizontally by dung beetles, dispersal distances were short (~ 80% between 2 and 7 cm; S3 Fig). However, given our experimental setup, these distances could, to a certain degree, be underestimates, as the movement of beetles was limited by the edge of the plot, i.e., the plot radius of 25 cm was the maximum possible distance recorded. In our study site, roller dung beetles have been reported to move dung balls (often containing seeds) to a mean distance of 1.2 m, and up to 5 m [14]. On the other hand, tunneler dung beetles bury the dung relatively close to the dung source [10], but that does not mean that they do not cause any horizontal movement of seeds. Tunnelers sometimes push dung portions on the soil surface for a short distance (< 20 cm) before burying it; furthermore, underground tunnels are often dug with an angle < 90° such that seeds moved into the tunnels are displaced both vertically and horizontally (L.A. Urrea-Galeano and E. Andresen, pers. obs.). Given that 95% of all individuals captured in our pitfall traps were tunnelers (S1 Appendix and S1 Table), we believe that most seed movement we observed was carried out by this functional group and that most horizontal distances recorded were therefore accurate. Nonetheless, the few seeds that can be dispersed longer distances by roller dung beetles may be the ones establishing seedlings that have higher long-term survival probabilities. For example, a recent study found that, while dung beetle activity had a negative effect on seedling emergence of one plant species, it increased seedling survival [13]. Although the authors did not interpret the latter result as a possible consequence of reduced seedling clumping, this explanation remains plausible. Thus, studies following the fate of seeds moved by beetles in an unconstrained fashion and assessing seedling recruitment and survival, will be necessary to better understand the role of horizontal seed movement in decreasing the density-dependent processes causing seed/seedling mortality. Furthermore, since the life-stage at which plants suffer density-dependent mortality varies among species and contexts (e.g., [57]), future research will need to carefully determine the necessary duration of studies, in order to accurately assess the effects of reduced seed/seedling clumping caused by dung beetles, on plant fitness.

Related to the point above, it is important to mention that we protected experimental plots with mosquito netting, thus excluding most seed predators and seedling herbivores, while assuming that a positive effect of decreased clumping on seedling establishment might still be evident due to less intense seed/seedling competition and pathogen attack. Yet, density-dependent seed predation and seedling herbivory are known to be frequent processes affecting plant fitness [2,23,24]. Seed predation can often be very high, with 100% seed loss not being uncommon [58]; we used the netting precisely to avoid losing experimental seeds, and thus be able to have enough remaining seeds and seedlings for data analyses. Therefore, by excluding seed predators we also excluded the known positive effect of seed burial by dung beetles, i.e., seed predation avoidance [14-16,59]. To fully understand the effect of dung beetle activity on seed/seedling fates, we will need to design studies that allow us to simultaneously assess each of the positive and negative effects of horizontal and vertical seed dispersal by beetles, while disentangling the effects of both types of seed movement.

Finally, we want to stress once more how species-specific requirements for seed survival, germination, seedling establishment and survival, can strongly influence the results of the beetle-plant interactions. In our study, though seeds of both focal species were similar in size, a plant attribute that strongly affects the short-term fate of seeds after dung beetle activity (e.g., [33,60]), functional seedling attributes differed between species and were perhaps responsible for the different patterns of seedling establishment. For example, the timing of peak seedling establishment differed between treatment levels in Bursera but not in Poulsenia (S2 Fig). The temporal dynamics in Bursera seedlings was determined by their shade-intolerance [31], which caused the death of seedlings shortly after emergence. Poulsenia seedlings, on the other hand, are shade-tolerant [61], but the seeds suffered clear negative effects, both from remaining imbedded in dung (when beetles were excluded) and from being buried (when beetles were active; Fig 2). In the case of seeds that remained in dung, it is possible that merely dung presence could explain differences between the two species, as a few studies have shown that dung itself can have either positive or negative effects on germination or seed/seedling performance, depending on seed species [62,63]. In the case of seeds moved by dung beetles, many of which were buried, characteristics associated with the position, exposure and function of cotyledons can play an important role in determining the probability of seedlings emerging from buried seeds and surviving through the establishment period [64]. In this regard, Poulsenia seedlings are cryptocotylar hypogeal with reserve storage (CHR) while Bursera seedlings are phanerocotylar epigeal with foliaceous cotyledons (PEF; [65]). One study found that out of ten rainforest seed species tested, those that were CHR had the highest seedling establishment from buried seeds, while PEF species had the lowest [7]. In a previous study, CHR Poulsenia seeds buried at 3–5 cm did indeed have more seedling establishment, compared to PEF Bursera seeds [33]. It seems that in the present study, however, the lower seedling establishment observed for Poulsenia seeds in plots with dung beetle activity, compared to those of Bursera, is due, at least partly, to a cause different than seedling functional morphology. For example, differences between species could be due to seasonality in dung beetle activity, since the experiment for Bursera was started in April and the one with Poulsenia in June, a drier and a rainier month, respectively. Secondary seed dispersal by dung beetles has been shown to be affected by seasonality [7], not only because dung beetle assemblages vary seasonally [10], but also because in rainier months softer soils might favor deeper seed burial [21], which in turn might hinder seedling emergence.

In conclusion, our study confirms the important role dung beetles may play, through the horizontal secondary dispersal of seeds, in diminishing seed/seedling aggregation after seeds are deposited by mammals in fecal clumps. However, we did not find evidence indicating that this effect may have consequences for early seedling establishment. Longer-term studies will be necessary to ascertain if over time, the decreased seed/seedling clumping translates into increased seedling or sapling survival probabilities. Furthermore, since vertical and horizontal dispersal by dung beetles can simultaneously affect seeds and seedlings through different and sometimes opposing mechanisms, we must design studies that will allow us to assess each of them accurately, but in conjunction. Finally, given the species- and stage-specific outcome of the interactions between plants and dung beetles, more studies, including many seed species and their different stages of regeneration (seed bank, germination, emergence, establishment, survival), are necessary to fully understand the impact of dung beetle activity on plants.

Dung beetles captured in the Los Tuxtlas Biological Station, Veracruz, Mexico.

Beetles captured using 10 pitfall traps each baited with 50 g of fresh domestic pig dung and opened during 48 hours. Sampling was conducted in April, September and October 2016. Information about dung relocation behavior (tunneler ‘T’, roller ‘R’, dweller ‘D’), diet (predominantly feces ‘F’ or carrion ‘C’), and body measurements (dry weight, body length) are from Díaz & Favila [2009. Escarabajos coprófagos y necrófagos (Scarabaeidae, Silphidae y Trogidae) de la reserva de la biosfera Los Tuxtlas, México. Memorias VIII Reunión Latinoamericana de Scarabaeidología (Coleoptera: Scarabaeoidea). Pp. 34. Xalapa, Veracruz]. *Eurysternus has a unique behavior in which dung is not relocated; unlike typical rollers, feeding takes place directly in the dung source, but unlike typical dwellers, dung balls are made for nesting and are lightly covered by soil near the dung source [Halffter, G., & Edmonds, W. D. 1982. The nesting behavior of dung beetles (Scarabaeinae). An ecological and evolutive approach (Man and the Biosphere Program, Publication 10). Instituto de Ecología, Mexico City].

(DOCX)

Click here for additional data file.

Methodological details of the two experiments carried out to assess the effects of dung beetle activity on the spatial distribution of seeds (Experiment 1) and seedlings (Experiment 2), and on the probability of seedling establishment (Experiment 2).

(A) 50 g of fresh domestic pig dung used in plots with dung added; dung was divided into 4 equal portions, each containing 5 seeds of either Bursera simaruba or Poulsenia armata; (B) seeds inside the plots with no dung added; seeds were placed directly on the soil surface (as indicated by the red arrows); (C and D) experimental seeds of Poulsenia thread-marked with a 30 cm-long fishing line in Experiment 1 (for Experiment 2, seeds were not thread-marked); (E) mosquito netting excluded dung beetles from control plots during the first 48 h (both experiments), and also excluded seed rain and seed/seedling predators from all plots after the first 48 h (Experiment 2); (F) plot with dung beetle activity after 48h of having placed the dung piles containing seeds; no dung remains visible on the soil surface; (G) grid (2 cm) used to map the location of each seed (Experiment 1) and seedling (Experiment 2), to calculate the nearest neighbor index; (H and I) seedlings of Bursera and Poulsenia, respectively, establishing inside plots from experimental seeds.

(DOCX)

Click here for additional data file.

Mean number of seedlings of two plant species registered weekly for 15 weeks.

Bursera simaruba (A) and Poulsenia armata (B) seedlings established in 50-cm-diam plots (N = 30 for each species-treatment level) with three treatment levels: 50 g of dung and beetle access (black continuous line), 50 g of dung and beetle exclusion (black dashed line), and with no dung or beetles (black dotted line). In each plot of the first two treatment levels 20 seeds were mixed in the dung, in the last treatment level seeds were placed on the soil surface. Error bars represent ± 1 SE.

(TIF)

Click here for additional data file.

Frequency distribution of dispersal distances for two seed species.

Vertical (A and B) and horizontal (C and D) dispersal distances for experimental seeds that were secondarily dispersed by dung beetles (seeds that remained in their original position were not dispersed and thus were not included in these graphs). Seed species are Bursera simaruba (A and C) and Poulsenia armata (B and D). Dung beetle activity was restricted to circular plots 25 cm in radius; inside each plot 50 g of fresh pig dung containing 20 seeds of one species was placed.

(TIF)

Click here for additional data file.

Mean nearest neighbor distance for seedlings of two plant species over time.

Distance for seedlings of Bursera simaruba (A) and Poulsenia armata (B) over 15 weeks in plots (N = 30 for each species-treatment level) with three treatment levels: 50 g of dung and beetle access (black continuous line), 50 g of dung and beetle exclusion (black dashed line), and with no dung or beetles (black dotted line). Error bars represent ± 1 SE.

(TIF)

Click here for additional data file.

Methods and results of dung beetle sampling in the Los Tuxtlas Biological Station, Veracruz, Mexico.

(DOCX)

Click here for additional data file.

Data on the nearest neighbor index for seeds of Bursera in experiment 1.

See Expe1RatioBursera in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the coordinates for seeds of Bursera in experiment 1.

See Expe1RawdataBursera_Spatialdistribution in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the secondary seed dispersal for seeds of Bursera in experiment 1.

See Expe1RawdataBursera_Secondaryseeddispersal in metadata file.

(XLSX)

Click here for additional data file.

Data on the nearest neighbor index for seeds of Poulsenia in experiment 1.

See Expe1RatioPoulsenia in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the coordinates for seeds of Poulsenia in experiment 1.

See Expe1RawdataPoulsenia_Spatialdistribution in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the secondary seed dispersal for seeds of Poulsenia in experiment 1.

See Expe1RawdataPoulsenia_Secondaryseeddispersal in metadata file.

(XLSX)

Click here for additional data file.

Data on the nearest neighbor index for seedlings of Bursera during the week of peak seedling abundance in experiment 2.

See Expe2RatioBursera in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the coordinates for seedlings of Bursera during the week of peak seedling abundance in experiment 2.

See Expe2RawDataBursera_Spatialdistribution in metadata file.

(XLSX)

Click here for additional data file.

Data on the germination of seeds of Bursera registered weekly for 15 weeks in experiment 2.

See Expe2SurvivalBursera in metadata file.

(XLSX)

Click here for additional data file.

Data on the nearest neighbor index for seedlings of Poulsenia during the week of peak seedling abundance in experiment 2.

See Expe2RatioPoulsenia in metadata file.

(XLSX)

Click here for additional data file.

Raw data on the coordinates for seedlings of Poulsenia during the week of peak seedling abundance in experiment 2.

See Expe2RawdataPoulsenia_Spatialdistribution in metadata file.

(XLSX)

Click here for additional data file.

Data on the germination of seeds of Poulsenia registered weekly for 15 weeks in experiment 2.

See Expe2SurvivalPoulsenia in metadata file.

(XLSX)

Click here for additional data file.

Data on the dung beetles captured in the Los Tuxtlas Biological Station, Veracruz, Mexico.

See S1 Appendix_RawdataDungbeetle_Sampling in metadata file.

(XLSX)

Click here for additional data file.

Descriptive information about all datasets.

(TXT)

Click here for additional data file.

24 Jun 2019

PONE-D-19-15149

Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment

PLOS ONE

Dear Lina Adonay Urrea Galeano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The three reviewers agreed that the manuscript is well presented and is an important contribution to the effect of dung beetle activity on plant recruitment. However, it is necessary that the authors make the comments suggested by the reviewers for further consideration.

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The three reviewers agreed that the manuscript is well presented and is an important contribution to the effect of dung beetle activity on plant recruitment. However, it is necessary that the authors make the comments suggested by the reviewers for further consideration.

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Reviewer #2: Yes

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Reviewer #1: Yes

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Reviewer #1: In their manuscript “Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment” the authors have tested the effects of horizontal seed movement by dung beetle for two tree species. They found that dung beetle activity reduced the spatial clumping of seeds and seedlings, however, it did not increase the probability of seedling establishment. This study to help fill our knowledge gaps regarding the effects of secondary seed dispersal by dung beetles. The sample design seems adequate and robust to answer their main questions. I also like writing and organization of the manuscripts as well as concise results and discussion. I present below a list of minor comments that will hopefully aid you in improving your manuscript. I therefore really welcome this ms.

L.30- In your abstract and introduction, I think it is necessary that you state more clearly your objectives, rather than “the main goal of study was to help fill this information gap “

L. 31- I recommend adding a sentence here, explained better your field experiments

L. 33 - Be specific: What tree species?

L. 80- I think it is necessary that you state more clearly what the unique contribution of your study is. What is your study diferente from Lawson et al. (2012)?

L. 97- Add elevation of Station

L. 101 - Was the experiment carried out during which season?

L. 107 – 108- References for frugivores are need here, or declare if it is personal observation.

L. 116 – Why did not you use a mixed of human and pig dung?

L. 188 – Were the dung beetles identified using any entomological key?

L. 121 - I would like to know more information about two focal tree species. Are these species dominant in the station? Why do you call these focal species?

L. 106 and L 125: Alouatta palliata or Alouatta palliata mexicana? Try to use the same name.

L.125 - Is Alouatta palliata Mexicana herbivorous or omnivore?

L. 131 - Why as model herbivore? Is the pig dung used as omnivores?

L. 131 - Why you used fresh domestic pig dung rather than of Alouatta palliata? Pigs do not form part of the local biota and are onmnivores (?) rather than frugivores.

L. 137 - I would like to see a photo of experiment in supplemental material.

L. 138 – Why keep the leaf litter?

L.136- Were the plots of all treatments covered?

L. 161- I think that you said the same information in the lines 163 and 164.

L. 165 - Is the dung beetle activity went beyond the plot? Because you said that the plot with dung beetle activity was covered with netting after 48 hours.

L. 215 - What is False Discovery Rate method?

L. 218 - The reason for the dung beetle sampling should be in the methodology

L 219 - 225 – In the results session should have the results regarding their objectives. I suggest you transfer this information on to supplementary material or add a question in your objectives.

L 225 - Why did you put information on Scarabaeidae, Trogidae and Silphidae? In your introduction you state that your study is about dung beetles (scarabaeinae).

L 233 – The sum of Pousenia was 99.9%. Check the values, please.

L. 256, 258, 259 - Put the df

L. 298 - The information “but not in accordance with our prediction” should not be in the results.

L. 316 - In your introduction you describe the tunnelers: they construct tunnels in the

soil beneath the dung pad for burying portions of dung. However, approximately 90% of dung beetle community of your study is tunneler beetles. How do you explain the large amount of seeds moved horizontally (97%)?

L. 325 - I would like you to discuss a bit about what time would be enough. Will this depend on the plant species?

Reviewer #2: Review report PONE-D-19-15149

General comments

The paper covers an interesting topic and certainly fills a gap in knowledge as it is indeed assumed that dung beetles have a positive effect on seed germination and seedling survival. I am not too familiar with the most recent publications in tropical dung beetle ecology, but the paper certainly fills a gap in the current knowledge.

The manuscript is well written in a clear and concise matter and the figures are clear. The used methods are correct, although I do have a suggestion for improving the statistical analysis. I am not sure whether the method is applicable on the dataset that was collected (no raw data were provided), but I’m quite certain that a survival analysis would give more insight in the establishment experiment (experiment 2). Now the authors analyzed the peak emergence data for Bursera, because a large portion of the seedlings died at a given moment. In a survival analysis, the cumulative probability of a certain event (establishment in this case) is calculated. Onofri et al. (2010) wrote a very interesting paper about using survival analysis for the analysis of the germination and emergence of seeds. There is also an R-package available on CRAN with functions for survival analyses (package ‘survival’). By conducting a survival analysis, the authors could assess the differences between treatments over the entire time series instead of using 1 single moment in a long time series.

Data files are provided for all experiments and metadata are provided. However, the raw datasets are not provided, instead derived data (indices, cumulated dung beetle numbers without capture dates) are given. I don’t know the journal’s policy about data availability and the motivation for providing data, but these types of datasets are not very useful. Maybe, the results of this study could become useful in a future study doing a meta-analysis which needs raw data (e.g., real distances instead of the Clark Evans index). If the authors cannot be contacted at that time or they cannot retrieve the raw datasets anymore, sharing the datasets with this paper was not worth the effort.

Also, the dung beetle dataset could provide interesting information for policy makers and conservationalists (e.g., for making red lists), so it would be useful to put it in a suitable database. For biodiversity data, the ‘Global biodiversity information facility (GBIF)’ (www.gbif.org) is the standard database. When publishing data at GBIF, you become the author of the dataset and you get a doi code. So, the dataset can be cited and you get acknowledged for your efforts.

Specific comments

L43: potential advantage to plants of the second phase of… -> remove ‘to plants’ as it is clear that plants are the beneficiary of the process

L61: ‘sign’ sounds a bit weird -> maybe ‘nature’ or ‘(final) result’ ?

L117: Which measures were taken? Which equipment was used and what was the precision of the measurements?

L139: How did you decide to use 20 seeds in dung portions of 50g? How is this related to the seed density in ‘natural’ droppings found in this region?

L152: seed ‘alive’: did you test the viability of the seeds? If not, intact seeds would be a better description.

L159 and following: It is not clear from the text, but did you count the number of Poulsenia and Bursera seedlings originating from the soil seed bank?

L229-232: this part rather belongs to the discussion section.

L232: See my earlier remark. Unless you have tested the retrieved seeds for viability, you should refer to them as intact seeds.

L244-246: this rather belongs to the discussion section

L319-323: It seems that the presence of dung did have a greater effect on the emergence of seedlings than the presence of dung beetles. The fact that dung reduces the germinability of seeds was also found in other studies, e.g., Milotić and Hoffmann (2016). This study was done in a completely different environment and using different plant species (temperate grassland species), but I am not aware of similar studies in a tropical environment.

L338: Another factor to consider is the fact that clean, undigested seeds were used in the experiments. I assume that was practically impossible to use digested seeds as you would have to feed wild animals with seeds and collect the dung afterwards (furthermore, the issue would arise about which species should be used as a ‘digester’ species), but it is worthwhile mentioning this in the discussion. In the natural situation, seeds that passed the gut environment of a frugivore might have an altered germination probability and speed. So, maybe the presence of dung would not be that much of a problem then.

L350 and following: did you test the viability of the seeds prior to the experiments? It could also be that a relatively large proportion of the seeds is dead or dormant. Is there anything known about the germination ecology of these species (e.g., in Baskin and Baskin (2001))?

L393: What is the usual fruiting season for these species or do they carry fruits year-round?

Fig S3: For Bursera, the mean nearest neighbor distance in the +feces+beetles treatment increases over time. Do you have any idea why?

References

Baskin C.C., Baskin J.M. (2001) Seeds: ecology, biogeography and evolution of dormancy and germination. Academic Press, San Diego.

Milotić, T., & Hoffmann, M. (2016). Reduced germination success of temperate grassland seeds sown in dung: consequences for post‐dispersal seed fate. Plant Biology, 18(6), 1038-1047.

Onofri A., Gresta F., Tei F. (2010) A new method for the analysis of germination and emergence data of weed species. Weed Research, 50, 187–198.

Reviewer #3: The manuscript is very well written and brings an important contribution to our understanding of the effect of dung beetle activity of plant recruitment. All sections of the manuscript are very clear and easy to read. I have no major concerns and only did some minor comments here below. Congratulations to the authors. That was a real pleasure to read this manuscript.

L63: Please change “de” into “the” before “probability”

Experiments 1 and 2: Could you provide some information about the fruiting period of the two plant species of the experiments? Were both species present long fruiting period since you could set up experiment 2 three and five months later experiment 1 or did you collect and then freeze the seeds?

In addition, could you add some information about rainfall during experiment 1 and 2? Did you expect variation in the dung beetle activity between these two experiments because of the different time of the year?

L201-202: The proportion of seedling establishment is a continuous variable, right? Why is a binomial structure used in this case?

L561: Legend of Figure S1: “…in 50-cm-diam plots…”

L582: “…50g of feces and beetle…”

L302: “…the results were similar…” (not “where”)

L303: Maybe you can add that you obtained the same pattern of higher seedling establishment of Poulsenia with no feces.

L384: I think it would also be relevant to add that future studies should try to set experiments enabling the disentanglement of the effect of seed burial and clustering (e.g: experiments comparing the seedling establishment of seeds at a same burial depth but at different spatial clustering).

L394-397: This sentence is not very clear. In the second part of the sentence “…possibly due to seed burial”, it is expected you give an explanation on why Poulsenia seeds present negative effects when embedded in dung, but the sentence is confused and you repeated that it is because they are embedded in dung.

Laurence Culot

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Reviewer #1: No

Reviewer #2: Yes: Tanja Milotic

Reviewer #3: Yes: Laurence Culot

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17 Sep 2019

Dear Dr. Guillermo C. Amico,

Thank you for taking into consideration our manuscript entitled “Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment”. After carefully considering all the comments made by the three reviewers, we are submitting our revised manuscript. Our responses (IN CAPITAL LETTERS) to all comments are detailed below. We thank the referees for their constructive suggestions. We believe that the manuscript is considerably improved and look forward to receiving your decision.

Sincerely yours,

L.A. Urrea-Galeano and E. Andresen, on behalf of all co-authors

Reviewer #1: In their manuscript “Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment” the authors have tested the effects of horizontal seed movement by dung beetle for two tree species. They found that dung beetle activity reduced the spatial clumping of seeds and seedlings, however, it did not increase the probability of seedling establishment. This study to help fill our knowledge gaps regarding the effects of secondary seed dispersal by dung beetles. The sample design seems adequate and robust to answer their main questions. I also like writing and organization of the manuscripts as well as concise results and discussion. I present below a list of minor comments that will hopefully aid you in improving your manuscript. I therefore really welcome this ms.

THANK YOU!

L.30- In your abstract and introduction, I think it is necessary that you state more clearly your objectives, rather than “the main goal of study was to help fill this information gap “

HE HAVE WRITTEN MORE CLEARLY THE OBJECTIVE OF OUR STUDY IN THE ABSTRACT AND INTRODUCTION SECTIONS. NOW THE ABSTRACT SAYS: “THE OBJECTIVE OF OUR STUDY WAS TO ASSESS THE EFFECTS OF DUNG BEETLE ACTIVITY ON THE SPATIAL DISTRIBUTION OF SEEDS AND SEEDLINGS, AND ON THE PROBABILITY OF SEEDLING ESTABLISHMENT.” (LINES 30-32). THE INTRODUCTION SAYS: “OUR MAIN OBJECTIVE WAS TO ASSESS, FOR TWO TREE SPECIES, THE EFFECTS OF SECONDARY SEED DISPERSAL BY DUNG BEETLES, WITH EMPHASIS ON THE HORIZONTAL MOVEMENT OF SEEDS, ON THE SPATIAL DISTRIBUTION OF SEEDS AND SEEDLINGS, AND ON THE PROBABILITY OF SEEDLING ESTABLISHMENT.” (LINES 96-98).

L. 31- I recommend adding a sentence here, explained better your field experiments

WE HAVE ADDED SOME TEXT ABOUT THE EXPERIMENTS (LINES 32-35).

L. 33 - Be specific: What tree species?

WE HAVE ADDED THE NAME OF BOTH TREE SPECIES (LINE 34).

L. 80- I think it is necessary that you state more clearly what the unique contribution of your study is. What is your study diferent from Lawson et al. (2012)? WE HAVE ADDED SOME TEXT IN THE INTRODUCTION AND IN THE DISCUSSION POINTING OUT SOME DIFFERENCES BETWEEN OUR STUDY AND LAWSON’S. PLEASE SEE LINES 87-95 (INTRODUCTON) AND LINES 369-377 (DISCUSSION).

L. 97- Add elevation of Station DONE (LINE 110).

L. 101 - Was the experiment carried out during which season? FOR MORE CLARITY WE HAVE ADDED SOME TEXT ABOUT THE MONTHS IN WHICH THE RAINY SEASON OCCURS (LINES 114-115). WE ALSO MENTION NOW DURING WICH SEASON BOTH EXPERIMENTS WERE CARRIED OUT. FOR EXPERIMENT 1 SEE LINES 195-196 AND FOR EXPERIMENT 2 SEE LINES 202-203.

L. 107 – 108- References for frugivores are need here, or declare if it is personal observation. THANKS FOR YOUR OBSERVATION. WE HAVE ADDED THE REFERENCE (LINE 123).

L. 116 – Why did not you use a mixed of human and pig dung? FOR SEVERAL REASONS. FIRST, FOR PERSONAL REASONS, WE DO NOT USE HUMAN DUNG. SECOND, PIG DUNG HAS BEEN FOUND TO BE VERY EFFECTIVE IN ATTRACTING DUNG BEETLES (MARSH ET AL. 2013) AND IT WAS EASY TO OBTAIN THE LARGE QUANTITIES WE NEEDED FOR EXPERIMENTS. SINCE EXPERIMENTS WERE CARRIED OUT WITH PIG DUNG, WE DECIDED TO USE THE SAME BAIT FOR SAMPLING THE DUNG BEETLE COMMUNITY. WE HAVE ADDED THIS INFORMATION IN THE TEXT THAT CAN BE FOUND IN THE SUMPLEMENTARY INFORMATION; WE HAVE REMOVED THIS INFORMATION FROM THE MAIN MANUSCRIPT FOLLOWING ONE OF YOUR SUGGESTIONS.

L. 188 – Were the dung beetles identified using any entomological key? ALFONSO DÍAZ, ONE OF THE CO-AUTHORS, WHO IS EXPERT IDENTIFYING DUNG BEETLES DID THE IDENTIFICATION. IN THE SUPLEMENTARY INFORMATION WE HAVE THE FOLLOWING INFORMATION ABOUT IT: “All individuals were counted and identified in the Laboratorio de Ecoetología at the Instituto de Ecología A.C., Xalapa,Veracruz, Mexico. Also, we used a dung beetle collection with specimens collected from LTBS and Los Tuxtlas region.”

L. 121 - I would like to know more information about two focal tree species. Are these species dominant in the station? Why do you call these focal species? WE HAVED ADDED MORE INFORMATION FOR BOTH PLANT SPECIES IN THE METHODS SECTION (LINES 128-129; 132-139). WE CALL THEM FOCAL SPECIES SIMPLY BECAUSE OUR STUDY FOCUSED ON THEM; IT IS VERY COMMON TO REFER TO THE STUDY SPECIES AS THE FOCAL SPECIES.

L. 106 and L 125: Alouatta palliata or Alouatta palliata mexicana? Try to use the same name. THANKS FOR YOUR OBSERVATION. TO AVOID CONFUSION WE NOW USE THE NAME “ALOUATTA PALLIATA”, AS USED BY ESTRADA & COATES-ESTRADA (1991). (LINES 121; 141).

L.125 - Is Alouatta palliata Mexicana herbivorous or omnivore? ALOUATTA PALLIATA IS HERBIVOROUS-FRUGIVOROUS; WE HAVE ADDED SOME INFORMATION ON THIS IN LINE 120. WE HAVE ALSO ADDED THIS SAME INFORMATION FOR THE OTHER SEED-DISPERSING MAMMALS THAT CAN BE FOUND AT OUR STUDY SITE (LINE 121).

L. 131 - Why as model herbivore? Is the pig dung used as omnivores? WE HAVE REMOVED THIS TEXT AND HAVE SIMPLIFIED OUR EXPLANATION OF WHY WE USED PIG DUNG; PLEASE SEE LINES 158-160.

L. 131 - Why you used fresh domestic pig dung rather than of Alouatta palliata? Pigs do not form part of the local biota and are onmnivores (?) rather than frugivores. IDEALLY WE WOULD HAVE USED THE DUNG OF A FOREST ANIMAL. HOWEVER, THE LOGISTICS OF FINDING ENOUGH DUNG FOR THE EXPERIMENTS MADE THIS AN UNREALISTIC PROPOSITION. UNLIKE OTHER SPECIES OF ALOUATTA, A. PALLIATA AT LOS TUXTLAS DEFECATES FROM UP IN THE CANOPY AND MUCH OF THE DUNG NEVER REACHES THE GROUND; THE SMALL AMOUNTS THAT DO, ARE DIFFICULT TO COLLECT. COMPARED TO OTHER STUDIES THAT USE COW OR HORSE DUNG (E.G. LAWSON ET AL. 2012 USED HORSE DUNG), WE THINK THAT PIG DUNG IS PROBABLY MORE SIMILAR TO DUNG THAT CAN BE FOUND IN THE FOREST.

L. 137 - I would like to see a photo of experiment in supplemental material. PHOTOS HAVE BEEN ADDED; PLEASE SEE THE SUPPLEMENTARY INFORMATION.

L. 138 – Why keep the leaf litter? TO AVOID INTERFERING WITH THE DUNG-RELOCATION BEHAVIOR OF ROLLER DUNG BEETLES. WE HAVE ADDED THE FOLLOWING EXPLANATION IN METHODS: “…we kept the leaf litter to avoid affecting the behavior of roller dung beetles, which often choose a spot hidden under litter to build their tunnel (E. Andresen, pers. obs.), and thus litter removal may cause them to roll the dung balls larger distances.” LINES 166-169.

L.136- Were the plots of all treatments covered? IN EXPERIMENT 1 WE ONLY COVERED THE PLOTS WHERE DUNG BEETLES WERE EXCLUDED. PLOTS WITH DUNG BEETLE ACCESS WERE LEFT OPEN. WE HAVE ADDED SOME TEXT TO CLARIFY (LINES 182-184).

L. 161- I think that you said the same information in the lines 163 and 164. THANKS FOR YOUR OBSERVATION. WE HAVE DELETED THE REPEATED TEXT (LINES 200-201).

L. 165 - Is the dung beetle activity went beyond the plot? Because you said that the plot with dung beetle activity was covered with netting after 48 hours. DUNG BEETLES COULD NOT RELOCATE DUNG/SEEDS OUTSIDE THE PLOT. ALL THE PLOTS WERE DELIMITED BY A FENCE. AFTER 48 H ALL DUNG HAD DISAPPEARED DUE TO DUNG BEETLE ACTIVITY, AND PLOTS WERE COVERED WITH NETTING TO AVOID SEED RAIN AND TO PREVENT LOSS OF SEEDS AND SEEDLINGS THROUGH THE ACTION OF SEED PREDATORS AND HERBIVORES. WE HAVE ADDED THE FOLLOWING TEXT AT THE END OF THE DESCRIPTION OF EXPERIMENT 1 (LINES 193-195): “Dung beetle movement was limited by the plot’s fence, i.e., seeds could not be dispersed beyond the fence. While this allowed us to find most seeds, it makes our estimates of horizontal dispersal conservative (see Discussion).” AND AGAIN, WE MENTION THIS POINT IN THE DISCUSSION (LINES 402-405).

L. 215 - What is False Discovery Rate method? IT IS A METHOD FOR ADJUSTING P VALUES FOR MULTIPLE COMPARISONS, FOCUSING ON CONTROLLING THE FALSE DISCOVERY RATE (PROPORTION OF ALL REJECTIONS THAT ARE FALSE POSITIVES), RATHER THAN THE NUMBER OF FALSE POSITIVES PER SE, AS BONFERRONI CORRECTION DOES (JAFARI & ANSARI-POUR 2019). THIS CORRECTION CONSTITUTES AN ADEQUATE COMPROMISE BETWEEN CONTROLLING FOR FALSE POSITIVES AND FOR FALSE NEGATIVES. WE HAVE ADDED A SHORT TEXT ABOUT THIS IN LINES 259-260.

L. 218 - The reason for the dung beetle sampling should be in the methodology. THANKS FOR YOUR OBSERVATION. WE HAVE DELETED THIS INFORMATION FROM RESULTS AND ADDED IT TO SUPPLEMENTARY MATERIAL BASED ON YOUR OBSERVATION BELOW.

L 219 - 225 – In the results session should have the results regarding their objectives. I suggest you transfer this information on to supplementary material or add a question in your objectives. WE SAMPLED DUNG BEETLE AS COMPLEMENTARY INFORMATION, I.E. TO HELP US DISCUSS THE RESULTS OBTAINED FROM OUR MAIN EXPERIMENTS. SO, WE DECIDED TO TRANSFER THIS INFORMATION TO SUPPLEMENTARY MATERIAL.

L 225 - Why did you put information on Scarabaeidae, Trogidae and Silphidae? In your introduction you state that your study is about dung beetles (scarabaeinae). OUR LIST ONLY INCLUDES SCARABAEINAE. WE DO CITE A REFERENCE THAT INCLUDES THESE OTHER FAMILIES, AS WE USED IT TO OBTAIN SOME OF THE ECOLOGICAL DATA PRESENTED IN THE TABLE. IT IS TRUE THAT WE HAD LISTED ONE SPECIES (TEAMSCARABORUM OCAMPO) WHICH USED TO BE INCLUDED IN THE SCARABAEINA BUT IS NOW IN ANOHTER FAMILY AND SUBFAMILY (HYBOSORIDAE, HYBOSORINAE). SO, IN THE REVISED VERSION OF OUR TABLE WE HAVE REMOVED THIS SPECIES. SEE SUPPLEMENTARY MATERIAL.

L 233 – The sum of Pousenia was 99.9%. Check the values, please. THANKS FOR THE OBSERVATION. HE HAVE RE-CHECKED AND MADE THE CORRECTION (LINE 269).

L. 256, 258, 259 - Put the df. DONE (LINES 289-294). WE ALSO ADDED DF IN THE RESULTS OF EXPERIMENT 2 (LINES 318-323).

L. 298 - The information “but not in accordance with our prediction” should not be in the results. WE HAVE DELETED THIS SENTENCE (LINE 331).

L. 316 - In your introduction you describe the tunnelers: they construct tunnels in the soil beneath the dung pad for burying portions of dung. However, approximately 90% of dung beetle community of your study is tunneler beetles. How do you explain the large amount of seeds moved horizontally (97%)? WE HAVE RE-WRITTEN THIS PART IN THE INTRODUCTION, TO DESCRIBE BETTER THE DUNG-RELOCATION BEHAVIOR OF TUNNELERS AND ROLLERS; PLEASE SEE LINES 55-59. ALSO, IN THE DISCUSSION WE HAVE ADDED THE FOLLOWING TEXT: “On the other hand, tunneler dung beetles bury the dung relatively close to the dung source [10], but that does not mean that they do not cause any horizontal movement of seeds. Tunnelers sometimes push dung portions on the soil surface for a short distance (<20 cm) before burying it; furthermore, underground tunnels are often dug with an angle <90°, such that seeds moved into the tunnels are displaced both vertically and horizontally (L.A. Urrea-Galeano and E. Andresen, pers. obs.).” LINES 407-412.

L. 325 - I would like you to discuss a bit about what time would be enough. Will this depend on the plant species? WE AGREE THAT THIS TIME WILL DEPEND ON THE PLANT SPECIES. WE HAVE REWRITTEN THE END OF OUR FIRST PARAGRAPH OF THE DISCUSSION AND DO NOT MENTION HERE THIS ISSUE ANYMORE (PLEASE SEE LINES 355-357); HOWEVER WE MENTION IT NOW IN A DIFFERENT PART, AND HAVE ADDED THIS TEXT: “Furthermore, since the life-stage at which plants suffer negative density-dependent mortality varies among species and contexts, future research will need to carefully determine the necessary duration of studies, in order to accurately assess the effects of reduced seed/seedling clumping caused by dung beetles, on plant fitness.” SEE LINES 424-428.

Reviewer #2: Review report PONE-D-19-15149

General comments

The paper covers an interesting topic and certainly fills a gap in knowledge as it is indeed assumed that dung beetles have a positive effect on seed germination and seedling survival. I am not too familiar with the most recent publications in tropical dung beetle ecology, but the paper certainly fills a gap in the current knowledge. THANK YOU!

The manuscript is well written in a clear and concise matter and the figures are clear. The used methods are correct, although I do have a suggestion for improving the statistical analysis. I am not sure whether the method is applicable on the dataset that was collected (no raw data were provided), but I’m quite certain that a survival analysis would give more insight in the establishment experiment (experiment 2). Now the authors analyzed the peak emergence data for Bursera, because a large portion of the seedlings died at a given moment. In a survival analysis, the cumulative probability of a certain event (establishment in this case) is calculated. Onofri et al. (2010) wrote a very interesting paper about using survival analysis for the analysis of the germination and emergence of seeds. There is also an R-package available on CRAN with functions for survival analyses (package ‘survival’). By conducting a survival analysis, the authors could assess the differences between treatments over the entire time series instead of using 1 single moment in a long time series. THANK YOU FOR THIS SUGGESTION. WE HAVE READ THE PAPER FROM ONOFRI ET AL. (2010) AND SOME OTHER LITERATURE ABOUT THIS ISSUE, AND WE DECIDED TO FOLLOW YOUR SUGGESTION AND TO RE-ANALYSE OUR DATA FOR SEEDLING ESTABLISHMENT (EXPERIMENT 2) USING A SURVIVAL ANALYSIS THAT CONSIDERS RANDOM EFFECTS (FOLLOWING AUSTIN 2017). THUS, WE HAVE ELIMINATED FROM THE MANUSCRIPT ALL THE INFORMATION OF OUR PRIOR ANALYSES DONE WITH GLMMS. NOW, YOU CAN FIND IN THE ANALYSIS AND RESULTS SECTIONS JUST THE INFORMATION ABOUT THE SURVIVAL ANALYSIS (LINES 244-250 AND LINES 328-335). WE HAVE ALSO CHANGED FIGURE 2, WHICH NOW CONTAINS THE SURVIVAL CURVES (LINES 337; 343-344). IT IS IMPORTANT TO NOTE THAT OUR RESULTS FROM THE SEEDLING ESTABLISHMENT WITH THE SURVIVAL ANALYSIS ARE CONSISTENT WITH THOSE OBTAINED WITH THE GLMMS. THEREFORE, OUR GENERAL CONCLUSIONS ABOUT THE EFFECT OF DUNG AND BEETLE ACTIVITY ON THIS RESPONSE VARIABLE ARE THE SAME AS BEFORE.

Data files are provided for all experiments and metadata are provided. However, the raw datasets are not provided, instead derived data (indices, cumulated dung beetle numbers without capture dates) are given. I don’t know the journal’s policy about data availability and the motivation for providing data, but these types of datasets are not very useful. Maybe, the results of this study could become useful in a future study doing a meta-analysis which needs raw data (e.g., real distances instead of the Clark Evans index). If the authors cannot be contacted at that time or they cannot retrieve the raw datasets anymore, sharing the datasets with this paper was not worth the effort. YOU ARE RIGHT. WE HAVE PROVIDED THE RAW DATA NOW IN SUPPORTING INFORMATION.

Also, the dung beetle dataset could provide interesting information for policy makers and conservationalists (e.g., for making red lists), so it would be useful to put it in a suitable database. For biodiversity data, the ‘Global biodiversity information facility (GBIF)’ (www.gbif.org) is the standard database. When publishing data at GBIF, you become the author of the dataset and you get a doi code. So, the dataset can be cited and you get acknowledged for your efforts. YOU ARE RIGHT. WE HAVE PROVIDED THE RAW DATA NOW IN SUPPORTING INFORMATION.

Specific comments

L43: potential advantage to plants of the second phase of… -> remove ‘to plants’ as it is clear that plants are the beneficiary of the process DONE (LINE 45)

L61: ‘sign’ sounds a bit weird -> maybe ‘nature’ or ‘(final) result’ ? INSTEAD OF “SIGN”, WE WROTE “DIRECTION” (LINE 65)

L117: Which measures were taken? Which equipment was used and what was the precision of the measurements? THANKS FOR THE OBSERVATION. ACTUALLY, WE DID NOT MEASURE DUNG BEETLES. IT WAS A MISTAKE TO WRITE THIS. WE GOT MEASUREMENTS FROM DÍAZ AND FAVILA 2006. WE HAVE RE-WRITTEN THIS SENTENCE. SEE TEXT IN SUPPLEMENTARY INFORMATION. THIS PART IS NOW IN SUPPLEMENTARY INFORMATION, FOLLOWING ONE COMMENT FROM REVIEWER 1.

L139: How did you decide to use 20 seeds in dung portions of 50g? How is this related to the seed density in ‘natural’ droppings found in this region? IN METHODS WE HAVE ADDED THE FOLLOWING TEXT TO ADDRESS THIS POINT: “Since the amount of seeds present in the defecations of rainforest mammals can vary tremendously, depending on the plant and animal species (e.g., [8,38,39], we used seed numbers that can commonly be found in howler-monkey dung piles (e.g., [40,41])”. PLEASE SEE LINES 173-176.

L152: seed ‘alive’: did you test the viability of the seeds? If not, intact seeds would be a better description. WE DID NOT TEST THE VIABILITY OF THE SEEDS. WE HAVE CHANGED THE WORD “ALIVE” FOR “INTACT” THROUGHOUT THE MANUSCRIPT (LINES 189, 190).

L159 and following: It is not clear from the text, but did you count the number of Poulsenia and Bursera seedlings originating from the soil seed bank? TO ADDRESS THIS POINT WE HAVE ADDED THE FOLLOWING TEXT IN THE METHODS SECTION: “We assumed that all seedlings of the focal plant species that we recorded, originated from our experimental seeds because: (i) all plots were > 10 m away from any fruiting adult, and (ii) in a previous study in the same sites and with the same treatments, only two seedlings of Bursera and two of Poulsenia established, overall, from the soil seed bank during a time period of 8 months [33].” SEE LINES 211-216.

L229-232: this part rather belongs to the discussion section. THANK YOU FOR THE OBSERVATION. WE HAVE RE-WRITTEN THIS PARAGRAPH. LINES 266-267.

L232: See my earlier remark. Unless you have tested the retrieved seeds for viability, you should refer to them as intact seeds. DONE (LINE 268).

L244-246: this rather belongs to the discussion section. WE MOVED THIS INFORMATION TO THE DISCUSSION SECTION (LINES 402-405).

L319-323: It seems that the presence of dung did have a greater effect on the emergence of seedlings than the presence of dung beetles. The fact that dung reduces the germinability of seeds was also found in other studies, e.g., Milotić and Hoffmann (2016). This study was done in a completely different environment and using different plant species (temperate grassland species), but I am not aware of similar studies in a tropical environment. THANKS FOR THE OBSERVATION. WHAT YOU SAY IS RIGHT AND WE NOW INCLUDE SOME TEXT ON THIS TOWARDS THE END OF OUR DISCUSSION (LINES 454-457).

L338: Another factor to consider is the fact that clean, undigested seeds were used in the experiments. I assume that was practically impossible to use digested seeds as you would have to feed wild animals with seeds and collect the dung afterwards (furthermore, the issue would arise about which species should be used as a ‘digester’ species), but it is worthwhile mentioning this in the discussion. In the natural situation, seeds that passed the gut environment of a frugivore might have an altered germination probability and speed. So, maybe the presence of dung would not be that much of a problem then. THANKS FOR YOUR OBSERVATION. WE SEE THIS MORE AS A METHODOLOGICAL LIMITATION; SO, RATHER THAN INCLUDING IT IN THE DISCUSSION (WHICH HAS ICREASED IN LENGTH IN THE REVISED MANUSCRIPT), WE MENTION THIS ISSUE IN THE METHODS, AT THE END OF THE DESCRIPTION OF EXPERIMENT 2, WHERE WE HAVE ADDED THIS TEXT: “Finally, we acknowledge that using seeds extracted from fruits may yield different results compared to using seeds that have passed through the digestive system of a mammal. However, we expect that whatever difference there might be in terms of seed germination would equally have affected our three treatment levels.” SEE LINES 216-219.

L350 and following: did you test the viability of the seeds prior to the experiments? It could also be that a relatively large proportion of the seeds is dead or dormant. Is there anything known about the germination ecology of these species (e.g., in Baskin and Baskin (2001))? AS MENTIONED ABOVE, WE DID NOT TEST THE VIABILITY OF SEEDS. FROM LITERATURE WE FOUND THAT SEEDS OF THESE TWO SPECIES ARE NOT DORMANT; WE HAVE ADDED THIS INFORMATION IN METHODS (LINES 141-142). IF THE SPECIES HAVE NATURALLY LOW SEED VIABILITY, IT WOULD HAVE AFFECTED ALL OUR TREATMENTS SIMILARLY, AND THUS THE PATTERNS OBSERVED, IN TERMS OF THE EFFECTS OF DUNG BEETLES, WOULD HAVE BEEN THE SAME.

L393: What is the usual fruiting season for these species or do they carry fruits year-round? FRUITING SEASON FOR BURSERA IS FROM OCTOBER TO MAY AND FOR POULSENIA IS FROM MAY TO NOVEMBER. WE HAVE ADDED THIS INFORMATION IN THE METHODS SECTION (LINES 133; 137).

Fig S3: For Bursera, the mean nearest neighbor distance in the +feces+beetles treatment increases over time. Do you have any idea why? BURSERA SEEDLINGS DIED SHORTLY AFTER ESTABLISHING, DUE TO SHADE-INTOLERANCE; WE BELIEVE THAT THE INCREASE IN NEIGHBOR DISTANCE WAS SIMPLY A RESULT OF THERE BEING FEWER SEEDLINGS. PLEASE, NOTE THIS FIGURE HAS CHANGED FROM Fig S3 TO S4 Fig.

Reviewer #3: The manuscript is very well written and brings an important contribution to our understanding of the effect of dung beetle activity of plant recruitment. All sections of the manuscript are very clear and easy to read. I have no major concerns and only did some minor comments here below. Congratulations to the authors. That was a real pleasure to read this manuscript. THANK YOU!

L63: Please change “de” into “the” before “probability” DONE (LINE 67).

Experiments 1 and 2: Could you provide some information about the fruiting period of the two plant species of the experiments? WE HAVE ADDED THIS INFORMATION IN THE METHODS SECTION (LINES 133; 137).

Were both species present long fruiting period since you could set up experiment 2 three and five months later experiment 1 or did you collect and then freeze the seeds? FOR BOTH EXPERIMENTS WE COLLECTED ALL EXPERIMENTAL SEEDS ONCE BY THE TIME EXPERIMENT 2 STARTED (SEEDLING EXPERIMENT). SINCE WE NEEDED FRESH SEEDS FOR THIS EXPERIMENT (TO AVOID ANY INTERFERENCE IN GERMINATION), WE STARTED WITH THIS EXPERIMENT FIRST USING HALF OF THE SEEDS COLLECTED. THE REST OF SEEDS WAS DRIED AND STORED FOR EXPERIMENT 1 (SEEDS EXPERIMENT). ALTHOUGH FOR EXPERIMENT 1 WE COULD HAVE OBTAINED FRESH SEEDS DIRECTLY FROM TREES, WE DECIDED TO USE THE SEEDS ALREADY STORED TO FACILITATE THE MARKING PROCESS OF SEEDS REQUIRED FOR THIS EXPERIMENT. WE HAVE ADDED SOME TEXT IN THE METHODS SECTION TOO (LINES 146-155).

In addition, could you add some information about rainfall during experiment 1 and 2? WE HAVED ADDED INFORMATION ABOUT THE RAINFALL PATTERNS (LINES 113-115), AND WE ALSO MENTION THE SEASON DURING WHICH EACH EXPERIMENT WAS DONE (LINES 195-196 AND 202-203).

Did you expect variation in the dung beetle activity between these two experiments because of the different time of the year? BASED ON OUR DUNG BEETLE SAMPLING (SEE ALSO ESTRADA & COATES-ESTRADA, 1991) WE KNOW THAT THERE COULD BE A VARIATION IN THE MEAN NUMBER OF SPECIES AND INDIVIDUALS CAPTURED PER TRAP, WHICH IS RELATED WITH THE TIME OF THE YEAR. THUS, WE COULD EXPECT A VARIATION OF THE DUNG BEETLE ACTIVITY BETWEEN THESE TWO EXPERIMENTS. HOWEVER, DUE TO THE LOW PERCENTAGE OF ROLLERS CAPTURED BY THE TIME EXPERIMENT 2 AND EXPERIMENT 1 STARTED, WE DO NOT BELIEVE THAT THIS VARIATION COULD AFFECT OUR GENERAL CONCLUSION REGARDING THEIR EFFECT ON THE SPATIAL DISTRIBUTION OF SEED AND SEEDLINGS.

L201-202: The proportion of seedling establishment is a continuous variable, right? Why is a binomial structure used in this case? SEEDLING ESTABLISHMENT CAN BE ANALYZED USING A BINOMIAL DISTRIBUTION IN WHICH THE NUMBER OF TRIALS IS THE INITIAL NUMBER OF SEEDS AND THE SUCCESSES ARE THE NUMBER OF ESTABLISHED SEEDLINGS. THE PARAMETER ESTIMATED FROM SUCH MODEL IS P, THE PROPORTION OF SUCCESSES (PROPORTION OF SEEDLINGS ESTABLISHED IN THIS CASE). HOWEVER, FOLLOWING A SUGGESTION BY REVIEWER 2 WE HAVE RE-ANALYSED THESE DATA WITH A SURVIVAL ANALYSIS TO INCLUDE THE DATA FROM OVER THE ENTIRE EXPERIMENT, NOT JUST THE PEAK OF ESTABLISHMENT.

L561: Legend of Figure S1: “…in 50-cm-diam plots…” DONE (LINE 683). PLEASE NOTE THAT Figure S1 IS NOW S2 FIG.

L582: “…50g of feces and beetle…” THANKS FOR THE OBSERVATION. HOWEVER, WE HAVE DELETED THIS FIGURE FROM THE MANUSCRIPT AND REPLACED IT WITH A FIGURE OF SURVIVAL CURVES, ACCORDING TO THE WAY IN WHICH WE RE-ANALYSED THESE DATA.

L302: “…the results were similar…” (not “where”) WE HAVE RE-WRITTEN THIS PARAGRAPH (LINES 328-335) BASED ON A COMMENT FROM REVIEWER 2.

L303: Maybe you can add that you obtained the same pattern of higher seedling establishment of Poulsenia with no feces. THANK YOU FOR THIS OBSERVATION. HOWEVER, SINCE WE RE-ANALYSED THESE DATA USING A SURVIVAL ANALYSIS, WE HAVE DELETED THIS INFORMATION FROM THE TEXT.

L384: I think it would also be relevant to add that future studies should try to set experiments enabling the disentanglement of the effect of seed burial and clustering (e.g: experiments comparing the seedling establishment of seeds at a same burial depth but at different spatial clustering). DONE (LINES 441-442)

L394-397: This sentence is not very clear. In the second part of the sentence “…possibly due to seed burial”, it is expected you give an explanation on why Poulsenia seeds present negative effects when embedded in dung, but the sentence is confused and you repeated that it is because they are embedded in dung. THANKS FOR YOUR OBSERVATION. WE HAVE RE-WRITTEN THIS SENTENCE FOR CLARITY (LINES 452-454).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Oct 2019

PONE-D-19-15149R1

Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment

PLOS ONE

Dear Dra. Lina Adonay Urrea Galeano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments (if provided):

The reviewers coincide that comments have been addressed and manuscript has improved considerably. However, there are some minors concerns that the authors should address.

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Reviewers' comments:

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Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #2: This revised version of the manuscript has improved considerably. The methods are written down in a way it is possible to reproduce the experiment, and I really appreciate the pictures that are included as an appendix. The whole text is well written, and the images are clear. All suggestions and corrections I proposed were addressed properly, and questions were answered. After carefully reading the manuscript, inspecting the graphs and appendices, no new issues raised, so I would advise to publish this paper.

Reviewer #3: You responded satisfactorily to most of my comments and I think that the manuscript has been well improved.

I still think that you can expect a different secondary seed dispersal activity between seasons, and therefore between the experiment 1 and 2. You may have a low proportion of rollers, but the tunnelers also influence the burial depth, which consequently can affect the probability of seedling emergence. This difference does not discredit your work, but I think that a mention of this possible bias should be mentioned in the discussion.

Please find a few minor comments based on the reading of this new version of the manuscript:

L197-198: What do you mean by “conservative” in this case. The restricted area of your experiments can bias the results towards an underestimation of the horizontal distances. Is it what you mean? If so, I suggest to rephrase. If not, please clarify or delete the sentence and keep this information for the discussion.

L205: I suggest writing “one of the driest month”

L2016: I suggest writing “the beginning of the rainy season”

**********

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Reviewer #1: No

Reviewer #2: Yes: Tanja Milotic

Reviewer #3: Yes: Laurence Culot

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9 Oct 2019

Dear Dr. Guillermo C. Amico,

We have read and addressed all the comments made by Dr. Laurence Culot (Reviewer #3); please see our responses (in CAPITAL LETTERS) to the comments detailed below. Once again, we thank you and the referees for all comments done to improve our manuscript.

Sincerely yours,

L.A. Urrea-Galeano and E. Andresen, on behalf of all co-authors

Reviewer #1: (No Response)

Reviewer #2: This revised version of the manuscript has improved considerably. The methods are written down in a way it is possible to reproduce the experiment, and I really appreciate the pictures that are included as an appendix. The whole text is well written, and the images are clear. All suggestions and corrections I proposed were addressed properly, and questions were answered. After carefully reading the manuscript, inspecting the graphs and appendices, no new issues raised, so I would advise to publish this paper. THANK YOU!

Reviewer #3: You responded satisfactorily to most of my comments and I think that the manuscript has been well improved. THANK YOU!

I still think that you can expect a different secondary seed dispersal activity between seasons, and therefore between the experiment 1 and 2. You may have a low proportion of rollers, but the tunnelers also influence the burial depth, which consequently can affect the probability of seedling emergence. This difference does not discredit your work, but I think that a mention of this possible bias should be mentioned in the discussion. THANKS FOR YOUR OBSERVATION, WE AGREE. WE HAVE MENTIONED THIS ISSUE IN TWO PARTS IN THE DISCUSSION. IN LINES 401-406 WE HAVE WRITTEN THIS: “However, it could also be that seasonal differences in dung beetle assemblages caused a higher proportion of seeds to be buried and/or buried more deeply in Experiment 2, which was setup in April-June, than in Experiment 1, which was done in October. When we sampled dung beetles, we found higher abundances, particularly of large beetles (≥ 10 cm body length), in April than in October (S1 Table).” ALSO, IN LINES 478-483 WE HAVE ADDED THIS TEXT: “For example, differences between species could be due to seasonality in dung beetle activity, since the experiment for Bursera was started in April and the one with Poulsenia in June, a drier and a rainier month, respectively. Secondary seed dispersal by dung beetles has been shown to be affected by seasonality [7], not only because dung beetle assemblages vary seasonally [10], but also because in rainier months softer soils might favor deeper seed burial [21], which in turn might hinder seedling emergence.” FINALLY, IN S1 TABLE WE HAVE ADDED INFORMATION ON THE NUMBER OF INDIVIDUALS CAPTURED IN EACH OF THE SAMPLINGS, SO THAT THE POTENTIAL EFFECT OF SEASONALITY CAN BE BETTER APPRECIATED.

Please find a few minor comments based on the reading of this new version of the manuscript:

L197-198: What do you mean by “conservative” in this case. The restricted area of your experiments can bias the results towards an underestimation of the horizontal distances. Is it what you mean? If so, I suggest to rephrase. If not, please clarify or delete the sentence and keep this information for the discussion. YES, UNDERESTIMATION IS WHAT WE MEAN; WE HAVE REWRITTEN THE SENTENCE FOR CLARITY (LINES 197-198). NOW IT READS: “While this allowed us to find most seeds, it probably caused some underestimation of horizontal distances (see Discussion).”

L205: I suggest writing “one of the driest months” DONE (LINE 205)

L2016: I suggest writing “the beginning of the rainy season” DONE (LINE 206)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Oct 2019

Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment

PONE-D-19-15149R2

Dear Dra. Lina Adonay Urrea Galeano,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Guillermo C. Amico

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

15 Oct 2019

PONE-D-19-15149R2

Horizontal seed dispersal by dung beetles reduced seed and seedling clumping, but did not increase short-term seedling establishment

Dear Dr. Urrea Galeano:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Academic Editor

PLOS ONE