DOI: 10.5061/dryad.wh70rxwxb
title: Seeds go up and down: The role of dung beetles in soil seed movement in the Southern Atlantic Forest of Argentina

description: The vertical movement of seeds performed by dung beetles from the soil
 bank and in animal feces influences seed germination and the temporal
 dynamic of forest regeneration. While this process has been explored at
 the community level, the individual role of species is less understood.
 Here, we investigated the role of dung beetle size and seed size in this
 vertical movement under experimental conditions. We performed experiments
 using a gradient of dung beetle sizes and three sizes of artificial seeds
 (plastic beads) in two situations: inside feces (secondary dispersal) and
 buried in the ground (soil bank). Through regression analysis, we related
 dung beetle size, seed size, and the initial position of seeds on the soil
 bank to the final position of seeds and their potential germination. For
 seeds in the soil bank, upward movement and exhumation were mainly of
 medium and large seeds, initially located at shallower depths, with larger
 beetles being primarily responsible for this movement. The downward
 movement was similar for all seed sizes. In dung seeds, the percentage of
 small seeds buried gradually increased with beetle size, while larger
 beetles made the main contribution for medium and large seeds. Besides,
 all seed sizes were buried at an average maximum bury depth of nearly 4 cm
 (the limit of the germination zone). The relative contribution of species
 depended on the interaction between dung beetles and seed sizes. Moreover,
 large dung beetles were essential for burying large seeds in the southern
 Atlantic Forest.
description: <strong><em>Study area and collection of dung
 beetles</em></strong> We conducted this
 study with dung beetle species native to the Southern Atlantic Forest of
 Argentina (25° 44’ S and 54° 30’ W). The region is characterized by a warm
 seasonal climate, with annual temperatures between 17 and 22°C and a mean
 annual precipitation of 2000 mm, with no dry season (Oliveira &
 Fontes, 2000). We worked with eight copro-necrophagous beetle species
 representing the weight and size gradient for the whole assemblage
 (ranging from 0.007 g to 1.7 g.) (Table 1) (Giménez Gómez et al.,
 2022). We capture live dung beetles in late spring of
 2015 (December) using ten pitfall traps in two habitat types: native
 forests in protected areas and nearby mature pine plantations. Traps were
 a plastic cup of 12 cm in diameter and depth. We baited five traps with
 human dung and five with decomposing chicken because they are the most
 efficient resource for capturing dung beetles (Gardner et al., 2008). We
 separated the traps by 50 m between them to minimize the interference
 among them (Larsen & Forsyth, 2005). We used the same sites and
 traps to capture individuals until we finished the experiments. Once
 captured, we took the beetles to the laboratory, keeping them alive until
 we performed experiments. The collected individuals were weighed and
 measured in the laboratory.
 <strong><em>Manipulative
 experiments</em></strong> The objective of
 the experiments was to determine the role of dung beetles in the vertical
 movement of seeds in the soil, considering the initial location and size
 of the seeds and the body size of the beetles. An illustrative figure of
 the experiments can be found in Figure 2 and Figure S1. The experiments
 were composed of two parts: 1)    <em>Soil
 seeds</em>, we explored the role of dung beetles in the vertical
 movement (up and down) of the seeds in the seed bank;
 2)    <em>Dung seeds</em>, we explore the role of
 dung beetles in the burial of seeds contained in feces.
 For the experiments, we used cylinders 40 cm deep and 30 cm in
 diameter filled with 35 cm soil (Figure S1). In all cases, we used
 spherical plastic beads as artificial seeds. Plastic beads as a
 replacement for natural seeds have been used successfully in other studies
 (Griffiths et al., 2015; Koike et al., 2012). The sizes of the plastic
 beads used in this study were 2, 6 and 8 mm in diameter, representing the
 size range of 84% of the seeds of woody plant species dispersed by animals
 in the region, excluding very large seeds (Vespa et al., 2014) (Figure
 1). To study the vertical movement of seeds in the seed
 bank (soil seeds), we buried 240 g of plastic beads between 0 – 8 cm; 60 g
 of orange spherical plastic beads between 0 to 2 cm, 60 g of green beads
 between 2 to 4 cm, 60 g of red beads between 4 to 6 cm, and 60 g of blue
 beads between 6 to 8 cm (Figure 2, left). For each colour/level, the 60 g
 were divided into three groups of 20 g using beads of different sizes:
 1746 small beads of 2 mm, 215 medium beads of 6 mm, and 73 large beads of
 8 mm (Figure 2, left). In total, each cylinder contained 8136 beads in the
 artificial seed bank. We used this number of beads to ensure that
 differences, if there were, in the role of dung beetles in vertical seed
 movement based on beetle size could be observed.To study the burial of
 seeds in the feces (dung seeds), we placed 60 g of white spherical plastic
 beads (also divided into three groups of 20 g as above) in 100 g of howler
 monkey dung on the ground surface (Figure 2, left).
 Then, we placed ten individuals of the same dung beetle species
 in each experiment. All individuals were not fed for 24 hours before the
 experiment. We covered the system with a mesh with 2 mm pores for 72 hours
 to keep the beetles inside. At the end of this time, we
 moved the beetles and the soil surface, centimetre by centimetre,
 recording the depth at which we found each plastic bead, its size and
 colour (Figure 2, right). We performed between two and three replicates
 for each dung beetle species depending on the number of individuals
 available (19 replicates in total) (Table 1). Beetles were not reused
 between replicates. <strong>Data
 analysis</strong> <strong><em>Soil
 seeds experiment</em></strong> To analyse
 seed movement in the soil, we first calculated the percentage of beads
 moved upward and downward for each size and initial depth; then, we
 compared these percentages using a chi-square analysis. We performed a
 two-factor ANOVA to compare the percentage of beads exhumed (emerged to
 the surface) according to initial depth and bead size.
 To analyse the effect of vertical seed movement on the potential
 probability of germination and subsequent seedling emergence, we
 considered a threshold of 4 cm as the critical depth. Seeds buried at
 greater depths have a low probability of germination and seedling
 emergence (Urrea-Galeano et al., 2019). Considering this, we defined two
 zones: 1) viable zone, above 4 cm depth and 2) non-viable zone, below 4 cm
 depth. Then, we performed two regression analyses using the total length
 of the beetles as explanatory variable and as response variables: 1) the
 percentage of beads moved from the viable zone to the non-viable zone, and
 2) the percentage of beads moved from the non-viable zone to the viable
 zone. We fitted all data to linear, exponential, and sigmoidal models.
 Then, we selected the best fit for each case based on the Akaike criterion
 of information. In cases that did not meet the assumptions of normality
 and homoscedasticity, the data were ln transformed.
 <strong><em>Dung Seeds
 experiment</em></strong> To explore the
 role of dung beetles on seed burial, we first performed a regression
 analysis using the percentage of buried seeds according to size as the
 dependent variable and the total length of the beetles as the independent
 variable. We also performed a regression analysis for the depth to which
 the seeds are buried; in this case, we used the average depth to which the
 seeds were buried for each seed size as the dependent variable.
description: # Seeds go up and down: The role of dung beetles in soil seed movement in
 the Southern Atlantic Forest of Argentina
 [https://doi.org/10.5061/dryad.wh70rxwxb](https://doi.org/10.5061/dryad.wh70rxwxb) ## Description of the data and file structure ### Files and variables #### File: Data.xlsx **Description:** On the first sheet, the file contains the species of dung beetle used in each replicate, the initial location of the plastic beads, their size (small: 2 millimeters (mm), medium: 6 mm and large: 8 mm) and the final depth (centimeters). On the second sheet, the file contains the metadata.  ##### Variables * Number of counted plastic beads ## Code/software You need excel or similar software.