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Assessing the relative importance of earthworms as granivores under field conditions
Applied Soil Ecology 147 (2020) 103428
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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
Assessing the relative importance of earthworms as granivores under field conditions
T
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Michael J. McTavish , Stephen D. Murphy School of Environment, Resources and Sustainability, University of Waterloo, 200 University Avenue West, Waterloo N2L 3G1, ON, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed predation Field experiment Earthworms Lumbricus terrestris
Plant communities are often seed-limited, and while rodents and birds are considered to be the primary contributors to surface seed removal, there is growing appreciation for the role of soil invertebrates, including earthworms. There have been very few studies assessing the relative importance of earthworms as granivores under field conditions compared to other taxa. We conducted a small-scale experiment in which we used earthworm inoculations and field exclosures to control access to surface-sown grass seed in an open field habitat and monitored impacts on initial grass recruitment. In isolation, earthworms reduced grass recruitment under field conditions (−22% biomass), but their impact was smaller than the reductions that occurred irrespective of earthworm presence when aboveground taxa had access to plots (−80–83%). The results of this small-scale study suggest that although earthworm-seed interactions may be ecologically important overall, in some habitats other taxa may be more influential than earthworms in terms of total surface seed removal.
1. Introduction Many plant communities are suspected to be seed-limited (Clark et al., 2007). In North America, rodents and birds are generally cited as the primary contributors to seed removal (Cassin and Kotanen, 2016; Grant, 1983). More recently, however, soil invertebrates such as earthworms have also been implicated as major seed predators (Cromar et al., 1999; Pufal and Klein, 2013). Interest in the role of earthworms as seed predators and dispersers dates back to the early experiments of Darwin (1881), with a renewed interest in recent years (see review by Forey et al., 2011). Earthworms can ingest seeds actively or coincidentally while burrowing (McCormick et al., 2013) or they may cache seeds too large to be ingested belowground (Eisenhauer and Scheu, 2008; Regnier et al., 2008). Earthworm interactions can have positive effects on some seeds and seedlings (Asshoff et al., 2010; McRill and Sagar, 1973; Milcu et al., 2006; Regnier et al., 2008), but they often reduce total seedling recruitment by digestion of seeds, deep burial of egested or cached seed (McCormick et al., 2013; Regnier et al., 2008), or even herbivory of recently germinated seedlings (Eisenhauer et al., 2010; Griffith et al., 2013). While lab-based Petri dish and microcosm experiments have been useful in learning about earthworm seed preferences and burial (e.g., Clause et al., 2017; Eisenhauer et al., 2008; Grant, 1983; McRill and Sagar, 1973; Quackenbush et al., 2012), earthworm impacts on seeds
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and their interactions with other granivores have seldom been examined experimentally under field conditions; to the best of our knowledge, the only example to date is Cassin and Kotanen (2016). Using field exclosures to manipulate the access of earthworms and other taxa and examine their impacts on surface-sown seeds in a temperate forest, they observed that earthworms removed seeds from the surface under field conditions but that their impacts were smaller than those of rodents. Additional field experiments using different seeds and environments are needed to elucidate the relative importance of earthworm-seed interactions in the field. This information will contribute to our understanding of how earthworms shape plant communities both in their native habitats and as exotic species in the growing number of earthworm-invaded ecosystems (Hendrix et al., 2008). The purpose of this experiment was to assess the relative importance of earthworms as granivores compared to other taxa under field conditions. We conducted a small-scale field experiment in which we used earthworm inoculations and exclosure cages to control access to surface-sown grass seed and monitor the impacts of earthworms and other granivores on seedling recruitment. We used the deep-burrowing, surface-foraging anecic earthworm Lumbricus terrestris L. (sensu Bouché, 1977) because it is a globally widespread and relatively large species suspected to be one of the most ecologically important earthworm granivores and seed dispersers (Asshoff et al., 2010; Grant, 1983).
Corresponding author. E-mail address: [email protected] (M.J. McTavish).
https://doi.org/10.1016/j.apsoil.2019.103428 Received 3 June 2019; Received in revised form 27 October 2019; Accepted 2 November 2019 Available online 11 November 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
Applied Soil Ecology 147 (2020) 103428
M.J. McTavish and S.D. Murphy
Fig. 1. (a) Tilled field beside Columbia Lake, Waterloo where we set up the exclosure field experiment. (b) An exclusion collar on a buried pot to exclude nonearthworm granivores.
2. Materials and methods
seed mix (Scotts Turf Builder® Grass Seed, Sun & Shade Mix) according to the supplier's recommended seeding rate (16 g∙m−2). The mix contained three common lawn grass species: 42% (by abundance) creeping red fescue (Festuca rubra L.), 34% Kentucky bluegrass (Poa pratensis L.), and 24% turf-type perennial ryegrass (Lolium perenne L.). This corresponds to approximate seed additions of 300 F. rubra, 175 P. pratensis, and 250 L. perenne per pot. Seed species differed in width (F. rubra: 1.1 mm, P. pratensis: 0.8 mm, L. perenne: 1.4 mm) and weight (F. rubra: 1.4 mg, P. pratensis: 0.5 mg, L. perenne: 2.6 mg). We used this seed mix because it was already in use for related studies (unpublished data), but our primary interest for this experiment was in total grass recruitment rather than species-specific responses. Half of each of the earthworm-free and earthworm-inoculated pots were randomly covered with cylindrical collars and covers made of 6.4 mm hardware cloth (35 cm diameter, 6.5 cm height) to exclude rodents and birds from accessing the pots (Fig. 1b). The pots received 1 L of tap water after seed addition and 0.5 L every 2–3 days until we observed germination (which occurred after 14 days). Due to the challenges of accurately re-collecting and counting thousands of grass seeds, we used grass biomass as a proxy for seedling recruitment. To limit any confounding effects of the exclusion collars or earthworm presence on grass growth (Scheu, 2003), once we observed germination, we measured by ruler and removed the top 2 cm of soil from each pot and transferred it to plastic plates (30 cm × 30 cm) on an open, indoor plant stand to allow seeds to grow under equal conditions (23 °C, 30% RH, and 12 h light (5000 lx): 12 h dark) with approximately 100 mL of water added every 2–3 days to keep the soils moist. Prior to removing the top 2 cm of soil, we visually checked for earthworm surface casts as qualitative evidence of earthworms being present or absent from earthworm inoculation or exclusion treatments at the end of the experiment. After 16 days we no longer observed any new germination, so we harvested aboveground grass biomass by cutting at the soil surface and dried (72 h at 60 °C) and weighed the collected material. Limiting the growth period prevented identification of specific grass species and resulted in a single, total grass biomass measurement. We assessed the effects of earthworm and other granivore exclusion on grass dry biomass using a Two-Way ANOVA, including the effects of earthworm exclusion, other granivore exclusion, and their interaction. We observed a statistically significant interaction in the model and subsequently analyzed the simple main effects of each treatment using four One-Way ANOVAs to assess the effects of each treatment at each level of the other. We visually inspected residual plots and used Levene's test to assess assumptions of normality and equal variance respectively. All statistical tests were conducted at α = 0.05 in Minitab ® 18.1. Effect sizes are reported as omega-squared (ω2) for single factor ANOVA models and partial omega-squared (ωp2) for multi-factor ANOVA models (Maxwell and Delaney, 2004).
We assessed the interactive effects of earthworms and other granivores on initial seedling recruitment using exclosures to control earthworm access to the plots (earthworms excluded, earthworms allowed) and the access of other granivores such as rodents and birds (others excluded, others allowed) in a 2 × 2 factorial experiment (n = 6 replicate plots per unique treatment combination, total N = 24). We conducted the field trial in early October 2016 in a bare, recently tilled soil surrounded by lawn (Columbia Lake, Waterloo, Ontario, Canada) (Fig. 1a). We chose this site because of its proximity to a nearby river and woodlot as potential sources of wildlife. Additionally, the soil was deemed unlikely to contain high densities of earthworms due to recent disturbance by a manual rototiller and hot, dry conditions resulting from a lack of vegetation (Fig. 1a). Although we did not measure ambient earthworm density, no earthworms or casts were observed in the field before or after the experiment or while digging plots. We were not able to quantitatively monitor wildlife or seed feeding at the site, but potential granivores anecdotally observed at the site include rodents such as eastern grey squirrel (Sciurus carolinensis Gmelin 1788) and eastern chipmunk (Tamias striatus L.), and numerous bird species including American robin (Turdus migratorius L.), northern cardinal (Cardinalis cardinalis L.), and black-capped chickadee (Poecile atricapillus L.). To manipulate and maintain different earthworm densities, we buried 24 plastic nursery pots (28 cm diameter, 28 cm height) 20 cm apart in a single trench. We covered the drainage holes in the bottoms of the pots (2 cm diameter) with window-screen mesh to prevent earthworm movement and backfilled the pots and the surrounding trench excavation with the removed soil. We left approximately 1 cm of the pot rims extending vertically above the soil level to limit earthworm movement in/out of pots while trying to minimize the physical barrier presented to aboveground taxa. Each pot received 0.5 L of tap water to bring soils approximately to field capacity and 5 g of air-dried, crushed sugar maple (Acer saccharum Marshall) and Norway maple (Acer platanoides L.) leaf litter as an initial food source for the earthworms. Pots were then randomly assigned to one of the four earthworm/ other granivore exclusion treatments. To manipulate earthworm density, we purchased mature L. terrestris from a commercial bait vendor (Waterloo, Ontario, Canada) and inoculated half of the pots with groups of five earthworms each to create a high but still realistic population density (81 m−2) able to offset potential losses due to mortality or escape. The combination of the covered drainage holes, the elevated pot rim, and the suspected low ambient earthworm density in the field were intended to keep added earthworms from escaping the pots and prevent any additional earthworms from entering. After a 14-day acclimation period, we removed remaining surface litter and hand-seeded all pots with 1 g of uncoated commercial grass 2
Applied Soil Ecology 147 (2020) 103428
M.J. McTavish and S.D. Murphy
without more detailed wildlife monitoring. Additionally, while the 6.4 mm hardware cloth was likely effective at excluding larger fauna, smaller granivorous invertebrates such as beetles (Cromar et al., 1999) would likely have been unimpeded by these barriers and may have contributed to total seed removal in all plots. It remains unclear why the combined access of earthworms and other taxa did not have a greater impact on recruitment compared to access of other taxa alone. Given that this was a smaller dataset, inherent variation may have hidden an additive effect of different granivores that might be revealed with further experimentation. Alternatively, considering optimal foraging (Mitchell and Brown, 1990) and the effects of resource density on earthworm seed foraging (McTavish and Murphy, 2019), different feeding strategies and decreasing seed densities may limit the effectiveness of how one or both of earthworms and other granivores interact with a shared pool of seeds. Earthworms are also potential prey of various terrestrial and avian animals (Macdonald, 1983), notably including robins known to be at the experimental site (Cameron and Bayne, 2012). Because anecic earthworms such as L. terrestris live belowground but must come to the soil surface to forage (Butt et al., 2003), some may have been fully removed from uncovered pots through predation over the course of the experiment (though we did not quantify earthworm survivorship at the end of the experiment and cannot confirm this), or earthworms detecting tactile or chemical cues (Laverack, 1960) from predators could have reduced time spent foraging at the surface to avoid predation. In our experiment, earthworms in isolation did still have a detectable impact on recruitment and we suspect that they could be more influential as seed predators at certain times of the day or year when other species are less active or in locations with particularly high densities of earthworms or unusually low densities of other taxa (e.g., construction sites, industrially-degraded habitats). We also chose to focus only on the anecic earthworm L. terrestris because it is a largebodied species that forages at the soil surface (i.e., directly where seed is added) and is thought to be one of the most important earthworm granivores (Asshoff et al., 2010; Grant, 1983), but litter-dwelling epigeic earthworms and mineral soil-burrowing endogeic earthworms (sensu Bouché, 1977) can also contribute to seed predation (Asshoff et al., 2010; Eisenhauer et al., 2009a) and a more functionally diverse earthworm community with different feeding behaviours might have greater impacts on overall recruitment. Even without removing large quantities of seeds through predation, earthworm-seed interactions may be ecologically significant in other ways. For example, while larger granivores often prefer larger seeds, earthworms are known to generally prefer or be limited to ingesting smaller seeds (Cassin and Kotanen, 2016; Clause et al., 2011). Indeed, in our experiment, earthworms and other granivores may have selectively affected the performance of specific seeds (which differed in width and mass), though we were unable to compare between the species using our experimental design. Additionally, passage through the earthworm gut and deposition in casts can variably increase or decrease the germination of seeds that survive intact (Clause et al., 2015), though we suspect this was not a factor in this experiment as previous work found no effects of L. terrestris on the germination of these particular grass seeds (unpublished data). Earthworms can also ingest seeds buried beneath the surface that are inaccessible to surface foraging species (Bakker et al., 1996) and deep-burrowing anecic species in particular (such as L. terrestris) can interact with belowground soil seed banks and change the vertical distribution of seeds in the soil profile (Nuzzo et al., 2015; Willems and Huijsmans, 1994; Zaller and Saxler, 2007). Although bulk seed removal may be limited compared to other taxa, these other interactions with seeds paired with longer-term impacts on soil fertility may be more important in influencing overall earthworm impacts on plant communities (Eisenhauer et al., 2009b; Forey et al., 2011; Milcu et al., 2006). The generalizability of our results should be considered with caution as they are the product of a small-scale experiment in a single
Fig. 2. Interaction plot of grass aboveground dry biomass (mg) grown while excluding (dark grey) or allowing (light grey) access of other granivores and L. terrestris (n = 6 per unique treatment combination). Letters denote groupings from Tukey's HSD Test for significant simple main effects of earthworm access (capital letters: other granivores excluded, lower case letters: other granivores allowed). Asterisks (*) denote statistically significant simple main effects of other granivore access. Error bars depict standard deviation (SD).
3. Results Fresh earthworm casts were found at the surface in all earthworminoculated pots and in none of the earthworm-excluded pots when grass biomass was harvested. Aboveground grass biomass was affected by an interaction between the access/exclusion of earthworms and other granivores (Two-Way ANOVA, F1,20 = 8.93, p = 0.007, ωp2 = 0.24). When other granivores were excluded, earthworms reduced grass biomass by 22% (simple main effect, One-Way ANOVA, F1,10 = 14.02, p = 0.004, ω2 = 0.52), but when other granivores were allowed, earthworm access did not affect overall grass biomass (simple main effect, One-Way ANOVA, F1,10 = 0.25, p = 0.629). In contrast, other granivores caused greater reductions of 80–83% irrespective of whether earthworms were excluded (simple main effect, One-Way ANOVA, F1,10 = 266.23, p < 0.001, ω2 = 0.95) or present (simple main effect, One-Way ANOVA, F1,10 = 69.81, p < 0.001, ω2 = 0.85) (Fig. 2).
4. Discussion We found that earthworms did reduce recruitment from seed when other species were excluded, but that this impact was considerably smaller than the reductions in recruitment that occurred when aboveground taxa had access to pots. In addition, when other taxa had access, the total reduction in recruitment was the same irrespective of whether earthworms were present or absent (Fig. 2). Despite a different methodological approach and environment, our results are similar to the findings of Cassin and Kotanen (2016) and may suggest a consistent pattern of earthworm seed impacts under field conditions (at least in this region of North America), though this was a relatively small-scale experiment with a single earthworm species and further evidence is required. In terms of overall impacts on grass recruitment, other taxa were a dominant source of seed removal compared to earthworms. Similar to how Cassin and Kotanen (2016) attributed the removal of their temperate forest seeds primarily to rodents, we suspect that both rodents and – owing to the more open, forest edge habitat – birds were influential at our site. The greater impact of non-earthworm taxa may be a result of higher abundances, seed detection capabilities, or ingestion capacities. We could not determine which species were most active 3
Applied Soil Ecology 147 (2020) 103428
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location and time, though they were highly consistent with the related findings of Cassin and Kotanen (2016). We encourage further experimental investigation of the relative importance of earthworm-seed interactions under field conditions. The approach of measuring recruitment from biomass effectively avoided the counting of thousands of seeds and could easily be expanded in the future by more detailed monitoring of earthworms and other granivores. In addition to increasing the number of locations and experiment duration, we also suggest that future studies consider the individual and interactive effects of different earthworm species and functional groups (Asshoff et al., 2010; Eisenhauer et al., 2009a), use a range of seed species that can be identified as seedlings to allow researchers to evaluate differences in species-specific, community-shaping impacts of different granviores (Nuzzo et al., 2015; Nuzzo et al., 2009), employ camerabased monitoring of larger taxa (Cassin and Kotanen, 2016) combined with pitfall sampling of invertebrates (Cromar et al., 1999; Pufal and Klein, 2013) to account more completely for a broader range of possible granivores, and quantify earthworm survivorship and mass change to determine whether other taxa impede earthworm foraging through avoidance or predation.
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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by a Vanier Canada Graduate Scholarship (Vanier CGS) administered by the Vanier-Banting Secretariat (McTavish) and via grants from the University of Waterloo via the Centre for Ecosystem Resilience and Adaptation (Murphy). References Asshoff, R., Scheu, S., Eisenhauer, N., 2010. Different earthworm ecological groups interactively impact seedling establishment. Eur. J. Soil Biol. 46, 330–334. https://doi. org/10.1016/j.ejsobi.2010.06.005. Bakker, J.P., Poschlod, P., Strykstra, R.J., Bekker, R.M., Thompson, K., 1996. Seed banks and seed dispersal: important topics in restoration ecology. Acta Bot. Neerlandica 45, 461–490. https://doi.org/10.1111/j.1438-8677.1996.tb00806.x. Bouché, M.B., 1977. Strategies lombriciennes. In: Soil Organisms as Components of Ecosystems, Ecological Bulletin, Stockholm. 25. pp. 122–132. Butt, K.R., Nuutinen, V., Sirén, T., 2003. Resources distribution and surface activity of adult Lumbricus terrestris L. in an experimental system. Pedobiologia 47, 548–553. https://doi.org/10.1078/0031-4056-00227. Cameron, E.K., Bayne, E.M., 2012. Invasion by a non-native ecosystem engineer alters distribution of a native predator. Divers. Distrib. 18, 1190–1198. https://doi.org/10. 1111/j.1472-4642.2012.00912.x. Cassin, C.M., Kotanen, P.M., 2016. Invasive earthworms as seed predators of temperate forest plants. Biol. Invasions 18, 1567–1580. https://doi.org/10.1007/s10530-0161101-x. Clark, C.J., Poulsen, J.R., Levey, D.J., Osenberg, C.W., Pfister, A.E.C.A., DeAngelis, E.D.L., 2007. Are plant populations seed limited? A critique and meta-analysis of seed addition experiments. Am. Nat. 170, 128–142. https://doi.org/10.1086/518565. Clause, J., Margerie, P., Langlois, E., Decaëns, T., Forey, E., 2011. Fat but slim: criteria of seed attractiveness for earthworms. Pedobiologia 54, S159–S165. https://doi.org/10. 1016/j.pedobi.2011.08.007. Clause, J., Barot, S., Estelle, F., 2015. Effects of cast properties and passage through the earthworm gut on seed germination and seedling growth. Appl. Soil Ecol. 96, 108–113. https://doi.org/10.1016/j.apsoil.2015.07.007. Clause, J., Forey, E., Eisenhauer, N., Seal, C.E., Soudey, A., Colville, L., Barot, S., 2017. Seed selection by earthworms: chemical seed properties matter more than morphological traits. Plant Soil 413, 97–110. https://doi.org/10.1007/s11104-016-3085-9. Cromar, H.E., Murphy, S.D., Swanton, C.J., 1999. Influence of tillage and crop residue on
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Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
Assessing the relative importance of earthworms as granivores under field conditions
T
⁎
Michael J. McTavish , Stephen D. Murphy School of Environment, Resources and Sustainability, University of Waterloo, 200 University Avenue West, Waterloo N2L 3G1, ON, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Seed predation Field experiment Earthworms Lumbricus terrestris
Plant communities are often seed-limited, and while rodents and birds are considered to be the primary contributors to surface seed removal, there is growing appreciation for the role of soil invertebrates, including earthworms. There have been very few studies assessing the relative importance of earthworms as granivores under field conditions compared to other taxa. We conducted a small-scale experiment in which we used earthworm inoculations and field exclosures to control access to surface-sown grass seed in an open field habitat and monitored impacts on initial grass recruitment. In isolation, earthworms reduced grass recruitment under field conditions (−22% biomass), but their impact was smaller than the reductions that occurred irrespective of earthworm presence when aboveground taxa had access to plots (−80–83%). The results of this small-scale study suggest that although earthworm-seed interactions may be ecologically important overall, in some habitats other taxa may be more influential than earthworms in terms of total surface seed removal.
1. Introduction Many plant communities are suspected to be seed-limited (Clark et al., 2007). In North America, rodents and birds are generally cited as the primary contributors to seed removal (Cassin and Kotanen, 2016; Grant, 1983). More recently, however, soil invertebrates such as earthworms have also been implicated as major seed predators (Cromar et al., 1999; Pufal and Klein, 2013). Interest in the role of earthworms as seed predators and dispersers dates back to the early experiments of Darwin (1881), with a renewed interest in recent years (see review by Forey et al., 2011). Earthworms can ingest seeds actively or coincidentally while burrowing (McCormick et al., 2013) or they may cache seeds too large to be ingested belowground (Eisenhauer and Scheu, 2008; Regnier et al., 2008). Earthworm interactions can have positive effects on some seeds and seedlings (Asshoff et al., 2010; McRill and Sagar, 1973; Milcu et al., 2006; Regnier et al., 2008), but they often reduce total seedling recruitment by digestion of seeds, deep burial of egested or cached seed (McCormick et al., 2013; Regnier et al., 2008), or even herbivory of recently germinated seedlings (Eisenhauer et al., 2010; Griffith et al., 2013). While lab-based Petri dish and microcosm experiments have been useful in learning about earthworm seed preferences and burial (e.g., Clause et al., 2017; Eisenhauer et al., 2008; Grant, 1983; McRill and Sagar, 1973; Quackenbush et al., 2012), earthworm impacts on seeds
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and their interactions with other granivores have seldom been examined experimentally under field conditions; to the best of our knowledge, the only example to date is Cassin and Kotanen (2016). Using field exclosures to manipulate the access of earthworms and other taxa and examine their impacts on surface-sown seeds in a temperate forest, they observed that earthworms removed seeds from the surface under field conditions but that their impacts were smaller than those of rodents. Additional field experiments using different seeds and environments are needed to elucidate the relative importance of earthworm-seed interactions in the field. This information will contribute to our understanding of how earthworms shape plant communities both in their native habitats and as exotic species in the growing number of earthworm-invaded ecosystems (Hendrix et al., 2008). The purpose of this experiment was to assess the relative importance of earthworms as granivores compared to other taxa under field conditions. We conducted a small-scale field experiment in which we used earthworm inoculations and exclosure cages to control access to surface-sown grass seed and monitor the impacts of earthworms and other granivores on seedling recruitment. We used the deep-burrowing, surface-foraging anecic earthworm Lumbricus terrestris L. (sensu Bouché, 1977) because it is a globally widespread and relatively large species suspected to be one of the most ecologically important earthworm granivores and seed dispersers (Asshoff et al., 2010; Grant, 1983).
Corresponding author. E-mail address: [email protected] (M.J. McTavish).
https://doi.org/10.1016/j.apsoil.2019.103428 Received 3 June 2019; Received in revised form 27 October 2019; Accepted 2 November 2019 Available online 11 November 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. (a) Tilled field beside Columbia Lake, Waterloo where we set up the exclosure field experiment. (b) An exclusion collar on a buried pot to exclude nonearthworm granivores.
2. Materials and methods
seed mix (Scotts Turf Builder® Grass Seed, Sun & Shade Mix) according to the supplier's recommended seeding rate (16 g∙m−2). The mix contained three common lawn grass species: 42% (by abundance) creeping red fescue (Festuca rubra L.), 34% Kentucky bluegrass (Poa pratensis L.), and 24% turf-type perennial ryegrass (Lolium perenne L.). This corresponds to approximate seed additions of 300 F. rubra, 175 P. pratensis, and 250 L. perenne per pot. Seed species differed in width (F. rubra: 1.1 mm, P. pratensis: 0.8 mm, L. perenne: 1.4 mm) and weight (F. rubra: 1.4 mg, P. pratensis: 0.5 mg, L. perenne: 2.6 mg). We used this seed mix because it was already in use for related studies (unpublished data), but our primary interest for this experiment was in total grass recruitment rather than species-specific responses. Half of each of the earthworm-free and earthworm-inoculated pots were randomly covered with cylindrical collars and covers made of 6.4 mm hardware cloth (35 cm diameter, 6.5 cm height) to exclude rodents and birds from accessing the pots (Fig. 1b). The pots received 1 L of tap water after seed addition and 0.5 L every 2–3 days until we observed germination (which occurred after 14 days). Due to the challenges of accurately re-collecting and counting thousands of grass seeds, we used grass biomass as a proxy for seedling recruitment. To limit any confounding effects of the exclusion collars or earthworm presence on grass growth (Scheu, 2003), once we observed germination, we measured by ruler and removed the top 2 cm of soil from each pot and transferred it to plastic plates (30 cm × 30 cm) on an open, indoor plant stand to allow seeds to grow under equal conditions (23 °C, 30% RH, and 12 h light (5000 lx): 12 h dark) with approximately 100 mL of water added every 2–3 days to keep the soils moist. Prior to removing the top 2 cm of soil, we visually checked for earthworm surface casts as qualitative evidence of earthworms being present or absent from earthworm inoculation or exclusion treatments at the end of the experiment. After 16 days we no longer observed any new germination, so we harvested aboveground grass biomass by cutting at the soil surface and dried (72 h at 60 °C) and weighed the collected material. Limiting the growth period prevented identification of specific grass species and resulted in a single, total grass biomass measurement. We assessed the effects of earthworm and other granivore exclusion on grass dry biomass using a Two-Way ANOVA, including the effects of earthworm exclusion, other granivore exclusion, and their interaction. We observed a statistically significant interaction in the model and subsequently analyzed the simple main effects of each treatment using four One-Way ANOVAs to assess the effects of each treatment at each level of the other. We visually inspected residual plots and used Levene's test to assess assumptions of normality and equal variance respectively. All statistical tests were conducted at α = 0.05 in Minitab ® 18.1. Effect sizes are reported as omega-squared (ω2) for single factor ANOVA models and partial omega-squared (ωp2) for multi-factor ANOVA models (Maxwell and Delaney, 2004).
We assessed the interactive effects of earthworms and other granivores on initial seedling recruitment using exclosures to control earthworm access to the plots (earthworms excluded, earthworms allowed) and the access of other granivores such as rodents and birds (others excluded, others allowed) in a 2 × 2 factorial experiment (n = 6 replicate plots per unique treatment combination, total N = 24). We conducted the field trial in early October 2016 in a bare, recently tilled soil surrounded by lawn (Columbia Lake, Waterloo, Ontario, Canada) (Fig. 1a). We chose this site because of its proximity to a nearby river and woodlot as potential sources of wildlife. Additionally, the soil was deemed unlikely to contain high densities of earthworms due to recent disturbance by a manual rototiller and hot, dry conditions resulting from a lack of vegetation (Fig. 1a). Although we did not measure ambient earthworm density, no earthworms or casts were observed in the field before or after the experiment or while digging plots. We were not able to quantitatively monitor wildlife or seed feeding at the site, but potential granivores anecdotally observed at the site include rodents such as eastern grey squirrel (Sciurus carolinensis Gmelin 1788) and eastern chipmunk (Tamias striatus L.), and numerous bird species including American robin (Turdus migratorius L.), northern cardinal (Cardinalis cardinalis L.), and black-capped chickadee (Poecile atricapillus L.). To manipulate and maintain different earthworm densities, we buried 24 plastic nursery pots (28 cm diameter, 28 cm height) 20 cm apart in a single trench. We covered the drainage holes in the bottoms of the pots (2 cm diameter) with window-screen mesh to prevent earthworm movement and backfilled the pots and the surrounding trench excavation with the removed soil. We left approximately 1 cm of the pot rims extending vertically above the soil level to limit earthworm movement in/out of pots while trying to minimize the physical barrier presented to aboveground taxa. Each pot received 0.5 L of tap water to bring soils approximately to field capacity and 5 g of air-dried, crushed sugar maple (Acer saccharum Marshall) and Norway maple (Acer platanoides L.) leaf litter as an initial food source for the earthworms. Pots were then randomly assigned to one of the four earthworm/ other granivore exclusion treatments. To manipulate earthworm density, we purchased mature L. terrestris from a commercial bait vendor (Waterloo, Ontario, Canada) and inoculated half of the pots with groups of five earthworms each to create a high but still realistic population density (81 m−2) able to offset potential losses due to mortality or escape. The combination of the covered drainage holes, the elevated pot rim, and the suspected low ambient earthworm density in the field were intended to keep added earthworms from escaping the pots and prevent any additional earthworms from entering. After a 14-day acclimation period, we removed remaining surface litter and hand-seeded all pots with 1 g of uncoated commercial grass 2
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without more detailed wildlife monitoring. Additionally, while the 6.4 mm hardware cloth was likely effective at excluding larger fauna, smaller granivorous invertebrates such as beetles (Cromar et al., 1999) would likely have been unimpeded by these barriers and may have contributed to total seed removal in all plots. It remains unclear why the combined access of earthworms and other taxa did not have a greater impact on recruitment compared to access of other taxa alone. Given that this was a smaller dataset, inherent variation may have hidden an additive effect of different granivores that might be revealed with further experimentation. Alternatively, considering optimal foraging (Mitchell and Brown, 1990) and the effects of resource density on earthworm seed foraging (McTavish and Murphy, 2019), different feeding strategies and decreasing seed densities may limit the effectiveness of how one or both of earthworms and other granivores interact with a shared pool of seeds. Earthworms are also potential prey of various terrestrial and avian animals (Macdonald, 1983), notably including robins known to be at the experimental site (Cameron and Bayne, 2012). Because anecic earthworms such as L. terrestris live belowground but must come to the soil surface to forage (Butt et al., 2003), some may have been fully removed from uncovered pots through predation over the course of the experiment (though we did not quantify earthworm survivorship at the end of the experiment and cannot confirm this), or earthworms detecting tactile or chemical cues (Laverack, 1960) from predators could have reduced time spent foraging at the surface to avoid predation. In our experiment, earthworms in isolation did still have a detectable impact on recruitment and we suspect that they could be more influential as seed predators at certain times of the day or year when other species are less active or in locations with particularly high densities of earthworms or unusually low densities of other taxa (e.g., construction sites, industrially-degraded habitats). We also chose to focus only on the anecic earthworm L. terrestris because it is a largebodied species that forages at the soil surface (i.e., directly where seed is added) and is thought to be one of the most important earthworm granivores (Asshoff et al., 2010; Grant, 1983), but litter-dwelling epigeic earthworms and mineral soil-burrowing endogeic earthworms (sensu Bouché, 1977) can also contribute to seed predation (Asshoff et al., 2010; Eisenhauer et al., 2009a) and a more functionally diverse earthworm community with different feeding behaviours might have greater impacts on overall recruitment. Even without removing large quantities of seeds through predation, earthworm-seed interactions may be ecologically significant in other ways. For example, while larger granivores often prefer larger seeds, earthworms are known to generally prefer or be limited to ingesting smaller seeds (Cassin and Kotanen, 2016; Clause et al., 2011). Indeed, in our experiment, earthworms and other granivores may have selectively affected the performance of specific seeds (which differed in width and mass), though we were unable to compare between the species using our experimental design. Additionally, passage through the earthworm gut and deposition in casts can variably increase or decrease the germination of seeds that survive intact (Clause et al., 2015), though we suspect this was not a factor in this experiment as previous work found no effects of L. terrestris on the germination of these particular grass seeds (unpublished data). Earthworms can also ingest seeds buried beneath the surface that are inaccessible to surface foraging species (Bakker et al., 1996) and deep-burrowing anecic species in particular (such as L. terrestris) can interact with belowground soil seed banks and change the vertical distribution of seeds in the soil profile (Nuzzo et al., 2015; Willems and Huijsmans, 1994; Zaller and Saxler, 2007). Although bulk seed removal may be limited compared to other taxa, these other interactions with seeds paired with longer-term impacts on soil fertility may be more important in influencing overall earthworm impacts on plant communities (Eisenhauer et al., 2009b; Forey et al., 2011; Milcu et al., 2006). The generalizability of our results should be considered with caution as they are the product of a small-scale experiment in a single
Fig. 2. Interaction plot of grass aboveground dry biomass (mg) grown while excluding (dark grey) or allowing (light grey) access of other granivores and L. terrestris (n = 6 per unique treatment combination). Letters denote groupings from Tukey's HSD Test for significant simple main effects of earthworm access (capital letters: other granivores excluded, lower case letters: other granivores allowed). Asterisks (*) denote statistically significant simple main effects of other granivore access. Error bars depict standard deviation (SD).
3. Results Fresh earthworm casts were found at the surface in all earthworminoculated pots and in none of the earthworm-excluded pots when grass biomass was harvested. Aboveground grass biomass was affected by an interaction between the access/exclusion of earthworms and other granivores (Two-Way ANOVA, F1,20 = 8.93, p = 0.007, ωp2 = 0.24). When other granivores were excluded, earthworms reduced grass biomass by 22% (simple main effect, One-Way ANOVA, F1,10 = 14.02, p = 0.004, ω2 = 0.52), but when other granivores were allowed, earthworm access did not affect overall grass biomass (simple main effect, One-Way ANOVA, F1,10 = 0.25, p = 0.629). In contrast, other granivores caused greater reductions of 80–83% irrespective of whether earthworms were excluded (simple main effect, One-Way ANOVA, F1,10 = 266.23, p < 0.001, ω2 = 0.95) or present (simple main effect, One-Way ANOVA, F1,10 = 69.81, p < 0.001, ω2 = 0.85) (Fig. 2).
4. Discussion We found that earthworms did reduce recruitment from seed when other species were excluded, but that this impact was considerably smaller than the reductions in recruitment that occurred when aboveground taxa had access to pots. In addition, when other taxa had access, the total reduction in recruitment was the same irrespective of whether earthworms were present or absent (Fig. 2). Despite a different methodological approach and environment, our results are similar to the findings of Cassin and Kotanen (2016) and may suggest a consistent pattern of earthworm seed impacts under field conditions (at least in this region of North America), though this was a relatively small-scale experiment with a single earthworm species and further evidence is required. In terms of overall impacts on grass recruitment, other taxa were a dominant source of seed removal compared to earthworms. Similar to how Cassin and Kotanen (2016) attributed the removal of their temperate forest seeds primarily to rodents, we suspect that both rodents and – owing to the more open, forest edge habitat – birds were influential at our site. The greater impact of non-earthworm taxa may be a result of higher abundances, seed detection capabilities, or ingestion capacities. We could not determine which species were most active 3
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location and time, though they were highly consistent with the related findings of Cassin and Kotanen (2016). We encourage further experimental investigation of the relative importance of earthworm-seed interactions under field conditions. The approach of measuring recruitment from biomass effectively avoided the counting of thousands of seeds and could easily be expanded in the future by more detailed monitoring of earthworms and other granivores. In addition to increasing the number of locations and experiment duration, we also suggest that future studies consider the individual and interactive effects of different earthworm species and functional groups (Asshoff et al., 2010; Eisenhauer et al., 2009a), use a range of seed species that can be identified as seedlings to allow researchers to evaluate differences in species-specific, community-shaping impacts of different granviores (Nuzzo et al., 2015; Nuzzo et al., 2009), employ camerabased monitoring of larger taxa (Cassin and Kotanen, 2016) combined with pitfall sampling of invertebrates (Cromar et al., 1999; Pufal and Klein, 2013) to account more completely for a broader range of possible granivores, and quantify earthworm survivorship and mass change to determine whether other taxa impede earthworm foraging through avoidance or predation.
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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by a Vanier Canada Graduate Scholarship (Vanier CGS) administered by the Vanier-Banting Secretariat (McTavish) and via grants from the University of Waterloo via the Centre for Ecosystem Resilience and Adaptation (Murphy). References Asshoff, R., Scheu, S., Eisenhauer, N., 2010. Different earthworm ecological groups interactively impact seedling establishment. Eur. J. Soil Biol. 46, 330–334. https://doi. org/10.1016/j.ejsobi.2010.06.005. Bakker, J.P., Poschlod, P., Strykstra, R.J., Bekker, R.M., Thompson, K., 1996. Seed banks and seed dispersal: important topics in restoration ecology. Acta Bot. Neerlandica 45, 461–490. https://doi.org/10.1111/j.1438-8677.1996.tb00806.x. Bouché, M.B., 1977. Strategies lombriciennes. In: Soil Organisms as Components of Ecosystems, Ecological Bulletin, Stockholm. 25. pp. 122–132. Butt, K.R., Nuutinen, V., Sirén, T., 2003. Resources distribution and surface activity of adult Lumbricus terrestris L. in an experimental system. Pedobiologia 47, 548–553. https://doi.org/10.1078/0031-4056-00227. Cameron, E.K., Bayne, E.M., 2012. Invasion by a non-native ecosystem engineer alters distribution of a native predator. Divers. Distrib. 18, 1190–1198. https://doi.org/10. 1111/j.1472-4642.2012.00912.x. Cassin, C.M., Kotanen, P.M., 2016. Invasive earthworms as seed predators of temperate forest plants. Biol. Invasions 18, 1567–1580. https://doi.org/10.1007/s10530-0161101-x. Clark, C.J., Poulsen, J.R., Levey, D.J., Osenberg, C.W., Pfister, A.E.C.A., DeAngelis, E.D.L., 2007. Are plant populations seed limited? A critique and meta-analysis of seed addition experiments. Am. Nat. 170, 128–142. https://doi.org/10.1086/518565. Clause, J., Margerie, P., Langlois, E., Decaëns, T., Forey, E., 2011. Fat but slim: criteria of seed attractiveness for earthworms. Pedobiologia 54, S159–S165. https://doi.org/10. 1016/j.pedobi.2011.08.007. Clause, J., Barot, S., Estelle, F., 2015. Effects of cast properties and passage through the earthworm gut on seed germination and seedling growth. Appl. Soil Ecol. 96, 108–113. https://doi.org/10.1016/j.apsoil.2015.07.007. Clause, J., Forey, E., Eisenhauer, N., Seal, C.E., Soudey, A., Colville, L., Barot, S., 2017. Seed selection by earthworms: chemical seed properties matter more than morphological traits. Plant Soil 413, 97–110. https://doi.org/10.1007/s11104-016-3085-9. Cromar, H.E., Murphy, S.D., Swanton, C.J., 1999. Influence of tillage and crop residue on
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