- Email: [email protected]
The earthworm Lumbricus terrestris favours the establishment of Lolium perenne over Agrostis capillaris seedlings through seed consumption and burial
applied soil ecology 41 (2009) 360–363
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/apsoil
Short communication
The earthworm Lumbricus terrestris favours the establishment of Lolium perenne over Agrostis capillaris seedlings through seed consumption and burial Manuel Aira *, Trevor G. Piearce Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK
article info
abstract
Article history:
We studied the effects of the anecic earthworm Lumbricus terrestris L. on seed germination of
Received 12 May 2008
two plant species to understand the mechanisms underlying earthworm–seed interactions.
Received in revised form
We set up microcosms with seeds of Lolium perenne L. (large seeds) and Agrostis capillaris L.
18 November 2008
(small seeds), in a loam based compost, with and without earthworms. We buried seeds at
Accepted 26 November 2008
three depths and recorded their germination, we also examined to what depth earthworms buried seeds, and we studied seed viability after transit through the gut of earthworms. Earthworms dramatically reduced the numbers of A. capillaris seeds germinating, but did not
Keywords:
modify the germination of L. perenne seeds. Earthworms did, however, increase the biomass
Earthworms
of L. perenne seedlings. When we buried the seeds, those from A. capillaris rarely germinated
Seed competition
when they were placed below 7 cm, unlike L. perenne seeds. Earthworms buried the seeds of
Seed size
L. perenne to a depth of 15 cm. Passage of seeds through the gut of earthworms had a
Seed burial
detrimental effect on A. capillaris seed viability, while L. perenne seeds remained unaffected.
Seed digestion
While our results show that earthworm activity can strongly influence grass seed germination and plant establishment in microcosm experiments, further work is needed to asses the implications of earthworm–seed interactions for vegetation dynamics in the field. # 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Earthworms are known to function as ecosystem engineers (sensu Jones et al., 1994), by modifying soil physico-chemical and biological properties through the structures they build: burrows, casts and middens. Although it is widely accepted that earthworms have beneficial effects on plant growth through several mechanisms (reviewed in Blouin et al., 2006), there is a lack of knowledge on how earthworms affect the processes regulating seed germination and establishment. A
mean of 860000 seeds ha 1 year 1 has been recorded in casts of earthworms (Edwards and Bohlen, 1996), suggesting that these animals may play a major role in seed dispersal. It has been stated that casts of the earthworms Lumbricus terrestris L. and Aporrectodea longa Ude are safe places for germination for Trifolium dubium (Thompson et al., 1993). Surface casts can be quite stable structures, with limited access for water and air which favours seed dormancy and delays germination (Grant, 1983). Earthworms seem to discriminate between seeds of different plant species, ingesting them preferentially accord-
* Corresponding author. Present address: Departamento de Ecoloxı´a e Bioloxı´a Animal., Facultade de Bioloxı´a, Universidad de Vigo, Vigo E36310, Espan˜a. E-mail address: [email protected] (M. Aira). 0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2008.11.007
applied soil ecology 41 (2009) 360–363
ing to their size more than their chemical characteristics (McRill and Sagar, 1973; Piearce et al., 1994). Milcu et al. (2006) showed that plant recruitment was strongly influenced by the earthworm L. terrestris, the earthworm’s activity favouring large over small seeded plant species. Earthworms may affect seed germination in two ways. First, earthworms may accelerate or delay germination (Grant, 1983). Second, the viability of ingested seeds can increase (McRill and Sagar, 1973) or decrease (Decae¨ns et al., 2003). However, it is not known how earthworms influence seedling performance. The objective of this study was to investigate how the earthworm L. terrestris interacts with seeds, modifying their germination and establishment. First, we set up a microcosm experiment to study how L. terrestris affects the germination of two plant species (A. capillaris L. and L. perenne L.) differing in seed size (small and large seeds respectively). Second, we studied the ability of seeds from both plant species to germinate at different depths; third, we set up another microcosm experiment to study to what depths L. terrestris buries seeds in the soil profile. Finally, we studied how passage through the gut of L. terrestris modifies the viability of seeds from both plant species
2.
Materials and methods
2.1.
Microcosm experiment
We chose two plant species, A. capillaris (small seeds, 1.7 0.4 mm, n = 10) and L. perenne (large seeds, 5.3 1.1 mm, n = 10). Both kinds of seeds are ingested by L. terrestris (Piearce et al., 1994). Seeds were purchased from Emorsgate Wild Seeds (www.wildseed.co.uk). We set up 30 pots (16 cm diameter 16 cm height) that were filled to 90% of their capacity with John Innes Potting Compost No 2 (composed of seven parts loam, three parts peat and two parts sand by volume; plus fertilizer constituents). Although the depth of the pots was small compared with the size of L. terrestris burrows (Edwards and Bohlen, 1996), it was enough to test our hypothesis since seeds from the selected plant species were not able to germinate below 14 cm depth, as we found in this study. We added a mature individual of L. terrestris (3.4 0.1 g fresh weight) to each of 15 pots and sowed 50 seeds of both plant species in each of the pots, and in 15 worm-free pots (controls). To avoid earthworm escape we placed a plastic Petri dish at the bottom of the pot, covering the drainage holes, and covered the top of the pot with a mesh (0.1 mm) which was fixed with elastic bands. Pots were placed outside in a 5 6 pot array and randomly rearranged every 2 days to compensate for any environmental effect. The number of germinated seeds was counted once a week in order to detect earthworm effects on seed germination, the experiment starting on 23 March 2006 and lasting 9 weeks. There was no germination of other plant species in any pot. After 9 weeks, we harvested, washed and dried (60 8C) the established seedlings (shoot + roots) to determine their biomass.
2.2.
Burial experiments
In order to study the ability of seeds of A. capillaris and L. perenne to germinate at different depths in the soil profile we
361
set up test tubes (3 cm diameter 17 cm deep) containing John Innes Potting Compost No 2. We buried the seeds at one of three depths: surface (0 cm), medium (7 cm) and deep (14 cm). There were four tubes per depth and plant species, and four seeds per tube and depth. The tubes were randomly placed in a grid inside a scientific incubator (20 8C), and checked weekly during 9 weeks. Moisture in the substrate was kept constant by addition of tap water. To determine the depth to which earthworms bury seeds in the soil we constructed observation chambers. These chambers were made of two glass walls (48 25 cm) that were separated by a piece of PVC (2 cm wide) which divided the chamber into four individual compartments (8 cm wide). This design allowed us to open each chamber and remove earthworms, avoiding any disturbance to the soil profile inside the compartment, and additionally to record to what depth earthworms buried the seeds (the position of the seeds being revealed when they germinated). We set up three chambers and the 12 compartments were filled to 90% of their capacity with John Innes Potting Compost No 2; then we added one mature L. terrestris (4.2 0.4 g fresh weight) to each compartment. After 4 days for acclimatization, we randomly added 10 seeds of A. capillaris or 10 seeds of L. perenne to the surface of each compartment. The three chambers were randomly placed inside a scientific incubator (20 8C), and after 4 days (no seeds on the surface) we removed the earthworms from the compartments. Germination was recorded weekly over 9 weeks. Soil moisture was kept constant by addition of tap water. We characterized seed burial as surface (0–5 cm), medium (5–15 cm) and deep (15–25 cm).
2.3. gut
Seed viability after passing through the earthworm
To determine how passage through the gut of L. terrestris affected viability of seeds of A. capillaris and L. perenne. We put a mature L. terrestris (3.9 0.3 g fresh weight) with its gut empty (2 days in isolation without food) into each of 20 plastic Petri dishes (10 cm diameter) lined with moistened filter paper. Ten seeds per plant species and dish were mixed with a small amount of John Innes Potting Compost No 2 to provide more natural conditions than would offering seeds alone. Ten of the dishes received 10 seeds of A. capillaris and the remaining dishes 10 seeds of L. perenne. The dishes were randomly placed inside a scientific incubator (20 8C). Once each earthworm had consumed the compost with the seeds, the earthworm was removed and the dish was examined carefully to ensure that all seeds had been ingested; then earthworms were placed again in the dishes and the dish contents examined daily to recover worm casts, which were placed individually in Eppendorf tubes and seed germination recorded at weekly intervals over 9 weeks.
2.4.
Statistical analysis
Effects of time on germination as affected by earthworms (with and without L. terrestris) were examined with repeated measures ANOVA (RM-ANOVA). Total number of seedlings and mean dry plant biomass (shoot + roots) were analyzed by fitting generalized linear models (GLM) to the data. Earthworm
362
applied soil ecology 41 (2009) 360–363
(with and without) and plant species were fixed as factors. The same procedure was used to evaluate the ability of seeds of two plant species to germinate at different depths (surface, medium and deep) in the absence (test tube experiment) and presence of earthworms (chamber experiment) and to determine the effect on seed viability of passage through the gut of L. terrestris. In all cases the number of germinated seeds was analyzed using a Poisson distribution and log link, and mean dry biomass using a normal distribution and identity link.
3.
Results
3.1.
Microcosm experiment
Seeds of L. perenne germinated mainly in the first 4 weeks of incubation, whereas seeds of A. capillaris started germination after 5 weeks of incubation, resulting in a significant interaction between plant species and time (RM-ANOVA, F7,392 = 296.47, P < 0.0001). Earthworms did not influence time to germination. After 9 weeks of incubation, twice as many L. perenne seeds had germinated as A. capillaris seeds (85 1% and 44 2% respectively; Fig. 1a; GLM, x2 = 318.01, d.f. = 1, P < 0.0001). Earthworm activity reduced overall germination of seeds; (69 3% and 60 4% without and with earthworms respectively (GLM, x2 = 24.62, d.f. = 1, P < 0.0001). However, seeds of L. perenne were unaffected, whereas the germination of seeds of A. capillaris was reduced by 13% (Fig. 1a; interaction earthworm plant species, GLM, x2 = 13.98, d.f. = 1, P < 0.001). The biomass of L. perenne seedlings was 30 times higher than that of A. capillaris (Fig. 1b; GLM, x2 = 740.92, d.f. = 1, P < 0.0001). The presence of L. terrestris did not influence the biomass of A. capillaris, but doubled the biomass of L. perenne (GLM, x2 = 76.11, d.f. = 1, P < 0.0001), resulting in a significant interaction between earthworm and seed size (Fig. 1b; GLM, x2 = 77.14, d.f. = 1, P < 0.0001).
3.2.
Burial experiments
Burial reduced seed germination (GLM, x2 = 114.52, d.f. = 2, P < 0.0001), more seeds of L. perenne germinating at 7 and 14 cm
depth than those of A. capillaris (Table 1; interaction plant species depth of burial, GLM, x2 = 11.58, d.f. = 1, P < 0.001). Burial of seeds by L. terrestris differed between plant species (Table 1); in addition, seeds of L. perenne germinated 2.7 times more frequently than seeds of A. capillaris (GLM, x2 = 15.18, d.f. = 1, P < 0.0001). Depth of burial dramatically reduced the germination of seeds (GLM, x2 = 180.27, d.f. = 2, P < 0.0001). However, this reduction depended on seed species, because very few seeds of A. capillaris germinated at 5–15 and 15–25 cm depth, whereas seeds of L. perenne readily germinated at 5– 15 cm depth (Table 1; interaction seed size x depth of burial, GLM, x2 = 16.34, d.f. = 1, P < 0.001).
3.3. gut
Seed viability after passing through the earthworm
Earthworms ingested all the seeds of both plant species. Transit through the gut of L. terrestris strongly affected viability of seeds (GLM, x2 = 419.87, d.f. = 1, P < 0.0001). Seeds of L. perenne germinated 6 times more than seeds of A. capillaris (1.3 0.2 and 7.8 0.3 seeds/earthworm for A. capillaris and L. perenne respectively).
4.
Discussion
Earthworms dramatically reduced germination of seeds of A. capillaris (up to 1.4 times), whereas they did not affect germination of seeds of L. perenne. Milcu et al. (2006) found that L. terrestris reduced the recruitment of small seeds (23%) whereas it promoted that of large seeds (40%), which agree with our results. Earthworms did not change patterns of germination of seeds of either plant species, which does not accord with the delaying/acceleration of seed germination reported by Grant (1983) and Decae¨ns et al. (2003). Time spent growing after germination was longer for L. perenne seeds, and seedlings will, where earthworms were present, have received beneficial effects of earthworm activity which is consistent with reported increases in grassland plant species (reviewed in Wurst et al., 2008). However, nutrient supply in the compost
Fig. 1 – Differences in (a) number of seeds germinated and (b) mean dry biomass (shoot and roots) of seedlings of Agrostis capillaris (open symbols) and Lolium perenne (closed symbols) growing in the presence and absence of the earthworm Lumbricus terrestris. Mean W S.E.
363
applied soil ecology 41 (2009) 360–363
Table 1 – Number of seeds of Agrostis capillaris and Lolium perenne germinating after being buried at different depths (0, 7 and 14 cm) in test tubes, and after being buried at different depths (0–5, 5–15 and 15–25 cm) by the earthworm Lumbricus terrestris in incubation chambers. Mean W S.E. Burial in test tubes Depth of burial 0 cm 7 cm 14 cm
Burial by L. terrestris
A. capillaris
L. perenne
Depth of burial
A. capillaris
40 0.5 0.3 0
40 1.5 0.3 0.5 0.3
0–5 cm 5–15 cm 15–25 cm
3.1 0.4 0.3 0.2 0
used will of course have been substantially greater than in the field. It will have been to the advantage of A. capillaris seeds to be preferentially ingested because ingestion, if the seed is not digested, confers several potential benefits. First, earthworm casts are nutrient rich structures (Edwards and Bohlen, 1996) that provide spaces in the vegetation that are relatively free from competition. In this respect, casts of L. terrestris are favourable sites for germination of seeds of T. dubium (Thompson et al., 1993). Second, transit through the gut of L. terrestris increased the viability of seeds of Poa trivialis, Bellis perennis and T. repens (McRill and Sagar, 1973), but Decae¨ns et al. (2003) found that the earthworm Martiodrilus sp. (an anecic earthworm) reduced the viability of ingested seeds between 70% and 97%. However, casts may not be deposited on the soil surface; in fact, casts in this experiment were always deposited below the surface. Our data show that being ingested was not an advantage for A. capillaris seeds due to detrimental effects of transit through the gut and reduced germination due to burial. The reduced seed germination that we observed accords with the reduced germination of seedlings of Senecio vulgaris and Poa annua due to burial of seeds by the earthworms L. terrestris and A. longa (Thompson et al., 1993). We found that the earthworm L. terrestris heavily influenced the germination and establishment of two plant species. It did not accelerate or delay germination, but it enhanced the performance of L. perenne seeds, and strongly reduced the performance of A. capillaris seeds. This reduction resulted from the joint action of burial and transit through the gut of the earthworm. While our results show that earthworm activity can strongly influence grass seed germination and plant establishment under simplified microcosm experimental conditions, further work is needed to asses the implications of earthworm–seed interactions for vegetation dynamics in the field. Furthermore, successful establishment of plants from seeds under the competitive conditions of closed swards is likely to be much less common than where there are patches of bare soil available, as existed in our experiments. In such closed swards surface wormcasts are likely to be especially
L. perenne 5.8 0.3 3.2 0.3 0.2 0.2
important sites for plant recruitment (McRill and Sagar, 1973; Piearce et al., 1994), particularly for plants with small seeds and relatively low nutrient reserves.
Acknowledgements Manuel Aira was financially supported by a postdoctoral fellowship from Xunta de Galicia, and now by the Parga Pondal Program.
references
Blouin, M., Barot, S., Lavelle, P., 2006. Earthworms (Millsonia anomala, Megascolecidae) do not increase rice growth through enhanced nitrogen mineralization. Soil Biol. Biochem. 38, 2063–2068. Decae¨ns, T., Mariani, L., Betancourt, N., Jime´nez, J.J., 2003. Seed dispersion by surface casting activities of earthworms in Colombian grasslands. Acta Oecol. 24, 175–185. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Grant, J.D., 1983. The activities of earthworms and the fates of seeds. In: Satchell, J.E. (Ed.), Earthworm Ecology from Darwin to Vermiculture. Chapman and Hall, London, pp. 107–122. Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosystem engineers. Oikos 69, 373–386. McRill, M., Sagar, G.R., 1973. Earthworms and seeds. Nature 243, 482–1482. Milcu, A., Schumacher, J., Scheu, S., 2006. Earthworms (Lumbricus terrestris) affect plant seedling recruitment and microhabitat heterogeneity. Funct. Ecol. 20, 261–268. Piearce, T.G., Roggero, N., Tipping, R., 1994. Earthworms and seeds. J. Biol. Educ. 28, 195–202. Thompson, K., Thomas, C., Radley, J.M.A., 1993. The effect of earthworms and snails in a simple plant community. Oecologia 95, 171–178. Wurst, S., Allema, B., Duyts, H., van der Putten, W.H., 2008. Earthworms counterbalance the negative effect of microorganisms on plant diversity and enhance the tolerance of grasses to nematodes. Oikos 117, 711–718.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/apsoil
Short communication
The earthworm Lumbricus terrestris favours the establishment of Lolium perenne over Agrostis capillaris seedlings through seed consumption and burial Manuel Aira *, Trevor G. Piearce Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK
article info
abstract
Article history:
We studied the effects of the anecic earthworm Lumbricus terrestris L. on seed germination of
Received 12 May 2008
two plant species to understand the mechanisms underlying earthworm–seed interactions.
Received in revised form
We set up microcosms with seeds of Lolium perenne L. (large seeds) and Agrostis capillaris L.
18 November 2008
(small seeds), in a loam based compost, with and without earthworms. We buried seeds at
Accepted 26 November 2008
three depths and recorded their germination, we also examined to what depth earthworms buried seeds, and we studied seed viability after transit through the gut of earthworms. Earthworms dramatically reduced the numbers of A. capillaris seeds germinating, but did not
Keywords:
modify the germination of L. perenne seeds. Earthworms did, however, increase the biomass
Earthworms
of L. perenne seedlings. When we buried the seeds, those from A. capillaris rarely germinated
Seed competition
when they were placed below 7 cm, unlike L. perenne seeds. Earthworms buried the seeds of
Seed size
L. perenne to a depth of 15 cm. Passage of seeds through the gut of earthworms had a
Seed burial
detrimental effect on A. capillaris seed viability, while L. perenne seeds remained unaffected.
Seed digestion
While our results show that earthworm activity can strongly influence grass seed germination and plant establishment in microcosm experiments, further work is needed to asses the implications of earthworm–seed interactions for vegetation dynamics in the field. # 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Earthworms are known to function as ecosystem engineers (sensu Jones et al., 1994), by modifying soil physico-chemical and biological properties through the structures they build: burrows, casts and middens. Although it is widely accepted that earthworms have beneficial effects on plant growth through several mechanisms (reviewed in Blouin et al., 2006), there is a lack of knowledge on how earthworms affect the processes regulating seed germination and establishment. A
mean of 860000 seeds ha 1 year 1 has been recorded in casts of earthworms (Edwards and Bohlen, 1996), suggesting that these animals may play a major role in seed dispersal. It has been stated that casts of the earthworms Lumbricus terrestris L. and Aporrectodea longa Ude are safe places for germination for Trifolium dubium (Thompson et al., 1993). Surface casts can be quite stable structures, with limited access for water and air which favours seed dormancy and delays germination (Grant, 1983). Earthworms seem to discriminate between seeds of different plant species, ingesting them preferentially accord-
* Corresponding author. Present address: Departamento de Ecoloxı´a e Bioloxı´a Animal., Facultade de Bioloxı´a, Universidad de Vigo, Vigo E36310, Espan˜a. E-mail address: [email protected] (M. Aira). 0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2008.11.007
applied soil ecology 41 (2009) 360–363
ing to their size more than their chemical characteristics (McRill and Sagar, 1973; Piearce et al., 1994). Milcu et al. (2006) showed that plant recruitment was strongly influenced by the earthworm L. terrestris, the earthworm’s activity favouring large over small seeded plant species. Earthworms may affect seed germination in two ways. First, earthworms may accelerate or delay germination (Grant, 1983). Second, the viability of ingested seeds can increase (McRill and Sagar, 1973) or decrease (Decae¨ns et al., 2003). However, it is not known how earthworms influence seedling performance. The objective of this study was to investigate how the earthworm L. terrestris interacts with seeds, modifying their germination and establishment. First, we set up a microcosm experiment to study how L. terrestris affects the germination of two plant species (A. capillaris L. and L. perenne L.) differing in seed size (small and large seeds respectively). Second, we studied the ability of seeds from both plant species to germinate at different depths; third, we set up another microcosm experiment to study to what depths L. terrestris buries seeds in the soil profile. Finally, we studied how passage through the gut of L. terrestris modifies the viability of seeds from both plant species
2.
Materials and methods
2.1.
Microcosm experiment
We chose two plant species, A. capillaris (small seeds, 1.7 0.4 mm, n = 10) and L. perenne (large seeds, 5.3 1.1 mm, n = 10). Both kinds of seeds are ingested by L. terrestris (Piearce et al., 1994). Seeds were purchased from Emorsgate Wild Seeds (www.wildseed.co.uk). We set up 30 pots (16 cm diameter 16 cm height) that were filled to 90% of their capacity with John Innes Potting Compost No 2 (composed of seven parts loam, three parts peat and two parts sand by volume; plus fertilizer constituents). Although the depth of the pots was small compared with the size of L. terrestris burrows (Edwards and Bohlen, 1996), it was enough to test our hypothesis since seeds from the selected plant species were not able to germinate below 14 cm depth, as we found in this study. We added a mature individual of L. terrestris (3.4 0.1 g fresh weight) to each of 15 pots and sowed 50 seeds of both plant species in each of the pots, and in 15 worm-free pots (controls). To avoid earthworm escape we placed a plastic Petri dish at the bottom of the pot, covering the drainage holes, and covered the top of the pot with a mesh (0.1 mm) which was fixed with elastic bands. Pots were placed outside in a 5 6 pot array and randomly rearranged every 2 days to compensate for any environmental effect. The number of germinated seeds was counted once a week in order to detect earthworm effects on seed germination, the experiment starting on 23 March 2006 and lasting 9 weeks. There was no germination of other plant species in any pot. After 9 weeks, we harvested, washed and dried (60 8C) the established seedlings (shoot + roots) to determine their biomass.
2.2.
Burial experiments
In order to study the ability of seeds of A. capillaris and L. perenne to germinate at different depths in the soil profile we
361
set up test tubes (3 cm diameter 17 cm deep) containing John Innes Potting Compost No 2. We buried the seeds at one of three depths: surface (0 cm), medium (7 cm) and deep (14 cm). There were four tubes per depth and plant species, and four seeds per tube and depth. The tubes were randomly placed in a grid inside a scientific incubator (20 8C), and checked weekly during 9 weeks. Moisture in the substrate was kept constant by addition of tap water. To determine the depth to which earthworms bury seeds in the soil we constructed observation chambers. These chambers were made of two glass walls (48 25 cm) that were separated by a piece of PVC (2 cm wide) which divided the chamber into four individual compartments (8 cm wide). This design allowed us to open each chamber and remove earthworms, avoiding any disturbance to the soil profile inside the compartment, and additionally to record to what depth earthworms buried the seeds (the position of the seeds being revealed when they germinated). We set up three chambers and the 12 compartments were filled to 90% of their capacity with John Innes Potting Compost No 2; then we added one mature L. terrestris (4.2 0.4 g fresh weight) to each compartment. After 4 days for acclimatization, we randomly added 10 seeds of A. capillaris or 10 seeds of L. perenne to the surface of each compartment. The three chambers were randomly placed inside a scientific incubator (20 8C), and after 4 days (no seeds on the surface) we removed the earthworms from the compartments. Germination was recorded weekly over 9 weeks. Soil moisture was kept constant by addition of tap water. We characterized seed burial as surface (0–5 cm), medium (5–15 cm) and deep (15–25 cm).
2.3. gut
Seed viability after passing through the earthworm
To determine how passage through the gut of L. terrestris affected viability of seeds of A. capillaris and L. perenne. We put a mature L. terrestris (3.9 0.3 g fresh weight) with its gut empty (2 days in isolation without food) into each of 20 plastic Petri dishes (10 cm diameter) lined with moistened filter paper. Ten seeds per plant species and dish were mixed with a small amount of John Innes Potting Compost No 2 to provide more natural conditions than would offering seeds alone. Ten of the dishes received 10 seeds of A. capillaris and the remaining dishes 10 seeds of L. perenne. The dishes were randomly placed inside a scientific incubator (20 8C). Once each earthworm had consumed the compost with the seeds, the earthworm was removed and the dish was examined carefully to ensure that all seeds had been ingested; then earthworms were placed again in the dishes and the dish contents examined daily to recover worm casts, which were placed individually in Eppendorf tubes and seed germination recorded at weekly intervals over 9 weeks.
2.4.
Statistical analysis
Effects of time on germination as affected by earthworms (with and without L. terrestris) were examined with repeated measures ANOVA (RM-ANOVA). Total number of seedlings and mean dry plant biomass (shoot + roots) were analyzed by fitting generalized linear models (GLM) to the data. Earthworm
362
applied soil ecology 41 (2009) 360–363
(with and without) and plant species were fixed as factors. The same procedure was used to evaluate the ability of seeds of two plant species to germinate at different depths (surface, medium and deep) in the absence (test tube experiment) and presence of earthworms (chamber experiment) and to determine the effect on seed viability of passage through the gut of L. terrestris. In all cases the number of germinated seeds was analyzed using a Poisson distribution and log link, and mean dry biomass using a normal distribution and identity link.
3.
Results
3.1.
Microcosm experiment
Seeds of L. perenne germinated mainly in the first 4 weeks of incubation, whereas seeds of A. capillaris started germination after 5 weeks of incubation, resulting in a significant interaction between plant species and time (RM-ANOVA, F7,392 = 296.47, P < 0.0001). Earthworms did not influence time to germination. After 9 weeks of incubation, twice as many L. perenne seeds had germinated as A. capillaris seeds (85 1% and 44 2% respectively; Fig. 1a; GLM, x2 = 318.01, d.f. = 1, P < 0.0001). Earthworm activity reduced overall germination of seeds; (69 3% and 60 4% without and with earthworms respectively (GLM, x2 = 24.62, d.f. = 1, P < 0.0001). However, seeds of L. perenne were unaffected, whereas the germination of seeds of A. capillaris was reduced by 13% (Fig. 1a; interaction earthworm plant species, GLM, x2 = 13.98, d.f. = 1, P < 0.001). The biomass of L. perenne seedlings was 30 times higher than that of A. capillaris (Fig. 1b; GLM, x2 = 740.92, d.f. = 1, P < 0.0001). The presence of L. terrestris did not influence the biomass of A. capillaris, but doubled the biomass of L. perenne (GLM, x2 = 76.11, d.f. = 1, P < 0.0001), resulting in a significant interaction between earthworm and seed size (Fig. 1b; GLM, x2 = 77.14, d.f. = 1, P < 0.0001).
3.2.
Burial experiments
Burial reduced seed germination (GLM, x2 = 114.52, d.f. = 2, P < 0.0001), more seeds of L. perenne germinating at 7 and 14 cm
depth than those of A. capillaris (Table 1; interaction plant species depth of burial, GLM, x2 = 11.58, d.f. = 1, P < 0.001). Burial of seeds by L. terrestris differed between plant species (Table 1); in addition, seeds of L. perenne germinated 2.7 times more frequently than seeds of A. capillaris (GLM, x2 = 15.18, d.f. = 1, P < 0.0001). Depth of burial dramatically reduced the germination of seeds (GLM, x2 = 180.27, d.f. = 2, P < 0.0001). However, this reduction depended on seed species, because very few seeds of A. capillaris germinated at 5–15 and 15–25 cm depth, whereas seeds of L. perenne readily germinated at 5– 15 cm depth (Table 1; interaction seed size x depth of burial, GLM, x2 = 16.34, d.f. = 1, P < 0.001).
3.3. gut
Seed viability after passing through the earthworm
Earthworms ingested all the seeds of both plant species. Transit through the gut of L. terrestris strongly affected viability of seeds (GLM, x2 = 419.87, d.f. = 1, P < 0.0001). Seeds of L. perenne germinated 6 times more than seeds of A. capillaris (1.3 0.2 and 7.8 0.3 seeds/earthworm for A. capillaris and L. perenne respectively).
4.
Discussion
Earthworms dramatically reduced germination of seeds of A. capillaris (up to 1.4 times), whereas they did not affect germination of seeds of L. perenne. Milcu et al. (2006) found that L. terrestris reduced the recruitment of small seeds (23%) whereas it promoted that of large seeds (40%), which agree with our results. Earthworms did not change patterns of germination of seeds of either plant species, which does not accord with the delaying/acceleration of seed germination reported by Grant (1983) and Decae¨ns et al. (2003). Time spent growing after germination was longer for L. perenne seeds, and seedlings will, where earthworms were present, have received beneficial effects of earthworm activity which is consistent with reported increases in grassland plant species (reviewed in Wurst et al., 2008). However, nutrient supply in the compost
Fig. 1 – Differences in (a) number of seeds germinated and (b) mean dry biomass (shoot and roots) of seedlings of Agrostis capillaris (open symbols) and Lolium perenne (closed symbols) growing in the presence and absence of the earthworm Lumbricus terrestris. Mean W S.E.
363
applied soil ecology 41 (2009) 360–363
Table 1 – Number of seeds of Agrostis capillaris and Lolium perenne germinating after being buried at different depths (0, 7 and 14 cm) in test tubes, and after being buried at different depths (0–5, 5–15 and 15–25 cm) by the earthworm Lumbricus terrestris in incubation chambers. Mean W S.E. Burial in test tubes Depth of burial 0 cm 7 cm 14 cm
Burial by L. terrestris
A. capillaris
L. perenne
Depth of burial
A. capillaris
40 0.5 0.3 0
40 1.5 0.3 0.5 0.3
0–5 cm 5–15 cm 15–25 cm
3.1 0.4 0.3 0.2 0
used will of course have been substantially greater than in the field. It will have been to the advantage of A. capillaris seeds to be preferentially ingested because ingestion, if the seed is not digested, confers several potential benefits. First, earthworm casts are nutrient rich structures (Edwards and Bohlen, 1996) that provide spaces in the vegetation that are relatively free from competition. In this respect, casts of L. terrestris are favourable sites for germination of seeds of T. dubium (Thompson et al., 1993). Second, transit through the gut of L. terrestris increased the viability of seeds of Poa trivialis, Bellis perennis and T. repens (McRill and Sagar, 1973), but Decae¨ns et al. (2003) found that the earthworm Martiodrilus sp. (an anecic earthworm) reduced the viability of ingested seeds between 70% and 97%. However, casts may not be deposited on the soil surface; in fact, casts in this experiment were always deposited below the surface. Our data show that being ingested was not an advantage for A. capillaris seeds due to detrimental effects of transit through the gut and reduced germination due to burial. The reduced seed germination that we observed accords with the reduced germination of seedlings of Senecio vulgaris and Poa annua due to burial of seeds by the earthworms L. terrestris and A. longa (Thompson et al., 1993). We found that the earthworm L. terrestris heavily influenced the germination and establishment of two plant species. It did not accelerate or delay germination, but it enhanced the performance of L. perenne seeds, and strongly reduced the performance of A. capillaris seeds. This reduction resulted from the joint action of burial and transit through the gut of the earthworm. While our results show that earthworm activity can strongly influence grass seed germination and plant establishment under simplified microcosm experimental conditions, further work is needed to asses the implications of earthworm–seed interactions for vegetation dynamics in the field. Furthermore, successful establishment of plants from seeds under the competitive conditions of closed swards is likely to be much less common than where there are patches of bare soil available, as existed in our experiments. In such closed swards surface wormcasts are likely to be especially
L. perenne 5.8 0.3 3.2 0.3 0.2 0.2
important sites for plant recruitment (McRill and Sagar, 1973; Piearce et al., 1994), particularly for plants with small seeds and relatively low nutrient reserves.
Acknowledgements Manuel Aira was financially supported by a postdoctoral fellowship from Xunta de Galicia, and now by the Parga Pondal Program.
references
Blouin, M., Barot, S., Lavelle, P., 2006. Earthworms (Millsonia anomala, Megascolecidae) do not increase rice growth through enhanced nitrogen mineralization. Soil Biol. Biochem. 38, 2063–2068. Decae¨ns, T., Mariani, L., Betancourt, N., Jime´nez, J.J., 2003. Seed dispersion by surface casting activities of earthworms in Colombian grasslands. Acta Oecol. 24, 175–185. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, 3rd ed. Chapman and Hall, London. Grant, J.D., 1983. The activities of earthworms and the fates of seeds. In: Satchell, J.E. (Ed.), Earthworm Ecology from Darwin to Vermiculture. Chapman and Hall, London, pp. 107–122. Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosystem engineers. Oikos 69, 373–386. McRill, M., Sagar, G.R., 1973. Earthworms and seeds. Nature 243, 482–1482. Milcu, A., Schumacher, J., Scheu, S., 2006. Earthworms (Lumbricus terrestris) affect plant seedling recruitment and microhabitat heterogeneity. Funct. Ecol. 20, 261–268. Piearce, T.G., Roggero, N., Tipping, R., 1994. Earthworms and seeds. J. Biol. Educ. 28, 195–202. Thompson, K., Thomas, C., Radley, J.M.A., 1993. The effect of earthworms and snails in a simple plant community. Oecologia 95, 171–178. Wurst, S., Allema, B., Duyts, H., van der Putten, W.H., 2008. Earthworms counterbalance the negative effect of microorganisms on plant diversity and enhance the tolerance of grasses to nematodes. Oikos 117, 711–718.