Journal of Sea Research 71 (2012) 41–49

Contents lists available at SciVerse ScienceDirect

Journal of Sea Research

journal homepage: www.elsevier.com/locate/seares

Burial of Zostera marina seeds in sediment inhabited by three : Laboratory and field studies

M. Delefosse ⁎, E. Kristensen

Institute of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark article info abstract

Article history: The large number of seeds produced by eelgrass, Zostera marina, provides this plant with a potential to disperse Received 4 December 2011 widely and colonise new areas. After dispersal, seeds must be buried into sediment for assuring long-term survival, Received in revised form 25 April 2012 successful germination and safe seedling development. Seeds may be buried passively by sedimentation or actively Accepted 25 April 2012 through sediment reworking by benthic fauna. We evaluated the effect of three polychaetes on the burial rate and Available online 5 May 2012 depth of eelgrass seeds. Burial was first measured in controlled laboratory experiments using different densities of Nereis () diversicolor (400–3200 ind m−2), Arenicola marina (20–80 ind m−2), and the invasive Marenzelleria Keywords: – −2 viridis (400 1600 ind m ). The obtained results were subsequently compared with burial rates of seed mimics in 2 Ecosystem engineer experimental field plots (1 m ) dominated by the respective polychaetes. High recovery of seeds in the laboratory Invasive species (97–100%) suggested that none of these polychaetes species feed on eelgrass seeds. N. diversicolor transported Zostera marina seeds rapidly (b1 day) into its burrow, where they remained buried at a median depth of 0.5 cm. A. marina and Seed burial M. viridis buried seeds by depositing their faeces on top of the sediment. At their highest abundance, A. marina Seed mimics and M. viridis buried seeds to a median depth of 6.7 cm and 0.5 cm, respectively, after a month. The burial efficiency and depth of these species were, in contrast to N. diversicolor, dependent on abundance. Only 2% of seed mimics casted in the field plots were recovered, suggesting that physical dispersion by waves and currents was considerably important for horizontal distribution. However, polychaete affected significantly the vertical distribu- tion of seeds. Overall the effects of these three polychaetes indicate that benthic macroinvertebrates may significant- ly impact eelgrass seed bank at the ecosystem scale. Some species have a positive effect by burying seeds to shallow depths and thereby reducing seed and facilitating seed germination, while other species bury seeds too deep for successful seed germination and seedling development. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Wigand and Churchill, 1988) or dispersed to inappropriate sites (Chambers and Macmahon, 1994; Orth et al., 2006). Once eelgrass As most seagrass species, eelgrass (Zostera marina)usesbothvegeta- seeds are buried in anoxic sediment they will germinate after several tive and sexual reproduction (Orth et al., 2006). The former strategy may months (Olesen, 1999; Orth et al., 2000; Greve et al., 2005)andform be considered most reliable because new shoots can benefitfromparent successfully juvenile plants the following spring if their cotyledon can nutrient supply (Marba et al., 2002). However, vegetative reproduction reach the sediment surface (Churchill, 1983, 1992; Greve et al., 2005). only expands meadows locally and at a slow rate of b30 cm per year Laboratory and field measurements indicate that the cotyledon of Z (Marba and Duarte, 1998; Olesen and Sand–Jensen, 1994). Seeds, on marina seeds can typically reach a maximum length of about 6 cm and the other hand, are produced in large numbers (Orth et al., 2000)and seeds buried below this depth will be lost (Churchill, 1983; Greve et al., can be dispersed from the parent plant over large distance by water 2005; Delefosse et al., in preparation). currents, either along the sea floor (Orth et al., 1994)orbyflotation Seed burial is driven by an array of abiotic and biotic factors. Current (Churchill et al., 1985; Harwell and Orth, 2002). Seed dispersal represents and wave driven resuspension and sedimentation may bury seeds slowly therefore an important mechanism for the establishment of new eelgrass in coastal areas (Berner, 1980). However, these physical processes are meadows at a large spatial scale. Eelgrass seeds deposited on the sedi- irregular and unreliable as they also can resuspend sediment and seeds. ment surface are vulnerable if they are not rapidly buried into the sedi- Benthic macroinvertebrate fauna, on the other hand, may through their ment. They can be consumed by herbivores (Fishman and Orth, 1996; ecosystem-engineering reworking action (Jones et al., 1994; Kristensen et al., 2012) affect seed bank dynamics of seagrasses at a much shorter time-scale (Luckenbach and Orth, 1999; Inglis, 2000; Dumbauld and Wyllie-Echeverria, 2003; Valdemarsen et al., 2011). Experiments have ⁎ Corresponding author at: Campusvej 55, Dk-5230 Odense M, Denmark. Tel.: +45 6550 2333; fax: +45 6550 2786. shown that seeds of other angiosperms are buried efficiently by E-mail address: [email protected] (M. Delefosse). benthic species such as the polychaete Nereis (Hediste) diversicolor

1385-1101/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seares.2012.04.006 42 M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 and tubificid oligochaetes (Grace, 1984; Hughes et al., 2000). No studies have to our knowledge reported the efficiency of benthic macroinvertebrates on eelgrass seed burial. Seed/animal interac- tions are likely to be species-specific and related to seed and animal size, seed nutritional value as well as animal feeding habit and bio- turbation (Forey et al., 2011). In this study, we investigate the effect of three common poly- chaete species on the burial of eelgrass seeds. We have chosen species with different life habits (i.e. size, feeding mode and burrowing activ- ity). The gallery diffusor N. diversicolor lives in ramified burrows rare- ly reaching deeper than 15 cm with multiple connections to the surface of the sediment (Davey, 1994). It either feeds by scavenging the sediment surface or filter feed, but it does not mix the sediment strongly (Duport et al., 2006). The upward conveyor Arenicola marina is a large polychaete (up to 15 g; Valdemarsen et al., 2011) that lives head down in L- or J-shaped burrows. It feeds on sediment at a depth of 20–40 cm and deposits faecal pellets at the surface of the sediment (Cadée, 1976). The feeding/burrowing activities of A. marina popu- lationsmaymixsedimenttoadepthof40cmperyear(Kristensen et al., 2012). The gallery diffusor Marenzelleria viridis lives in burrows that typically reach more than 30 cm into the sediment (Atkins et al., 1987). It is a deposit feeder that collects sediment and with its palps and deposits faecal pellets at the sediment surface (Dauer et al., 1981). Fig. 1. Map showing the study sites in Odense Fjord (Denmark). Our motivation for investigating the effect of these three polychaete species on burial of eelgrass seeds is driven by the fact that A. marina and N. diversicolor are the prevailing benthic macroinvertebrates in 2.2. Laboratory experiment European coastal waters (Cadée, 1976; Scaps, 2002) where eelgrass is threatened. Furthermore, the invasive polychaete M. viridis has become 2.2.1. Sampling and initial preparation asignificant component of the benthic macrofauna in the same regions The density effect of the three polychaetes, N. diversicolor, A. marina with potential repercussions on the population dynamics of A. marina and M. viridis on the burial of eelgrass seeds was tested in the laboratory. and N. diversicolor (Delefosse et al., in press). Finally, field observations Worms were collected in Bregnør Bay by digging and sieving sediment have revealed that these three species occurs close to eelgrass meadows through a 1 mm mesh. Only intact were retrieved and kept in (Valdemarsen et al., 2010; Delefosse et al., in preparation), where most seawater until further use. The sediment that passed through the sieve seeds necessary for eelgrass recovery are deposited (Orth et al., 1994; was saved in buckets for later use in the laboratory. Seawater with salinity Ruckelshaus, 1996). of 20 was collected from a marine field station few kilometres from the The aim of this study is therefore to determine eelgrass seed burial sampling site. efficiency in sediments inhabited by populations of N. diversicolor, Seed bearing shoots of Z. marina were taken from the meadow adja- A. marina and M. viridis. We apply a combined laboratory and field ap- cent to Bregnør Bay in July 2010. The shoots were kept in aerated seawa- proach where the burial rate of seeds and seed mimics by the three ter until seeds matured and detached up to 2 months after collection. species is measured under controlled laboratory conditions and veri- The collected seeds were frozen until further use. Preliminary tests fied at a relevant field site. The significance of the results on seed sur- have shown that freezing has no consequence for seed integrity but vival and seedling success is evaluated at a larger scale using the eliminated storage problems (e.g., fungal infection) and prevented ger- shallow Danish , Odense Fjord, as case study. mination once thawed and introduced in the sediment. Seed mimics were produced from 1.33 mm (SD: 0.07, n=60) in di- ameter nylon cords that were cut into 3.14 mm (SD: 0.15, n=60) long 2. Materials and methods pieces weighing 4.98 mg (SD: 0.23, n=60), matching the size of natural seeds (Wyllie-Echeverria et al., 2003; Delefosse et al., in preparation). 2.1. Study site We found that nylon is the most appropriate material to fabricate seed mimics because it has high specific density (nylon: 1.14 g cm−3) Sampling and field work were conducted from August 2010 to May that makes seed mimics sink as seeds in seawater (Orth et al., 1994). 2011 in Bregnør Bay located on the South East coast of Odense Fjord Nylon cords can be cut easily into the desired length and can as well (55.48° N; 10.61° E) on the island of Fyn, Denmark (Fig. 1). The dominat- be used in a wide range of colours that makes them easier than seeds ing burrowing polychaetes at this location are separated spatially with to sort out from the confounding background colour of the sediment high abundance of N. diversicolor in a near-coastal zone and a high- (Fig. 2). The possibility of large scale production also provided the re- density of A. marina zone in the mid-bay area (Papaspyrou et al., 2007). quired amount of seed mimics for field experiments (Orth et al., 1994). In recent years, a third zone characterised by a growing population The reliability of seed mimics as a tracer for determining the burial of of the invasive polychaete M. viridis has become distinct in the seeds in the sediment was tested by simultaneous introduction of equal outer bay and partly overlapping with the A. marina zone. The proportions of seeds and seed mimics at the surface of the sediment in water depth is b1.2 m, but the N. diversicolor and A. marina zones are all laboratory treatments. The total number introduced matched the av- sporadically exposed to air during low tides and strong easterly winds. erage natural eelgrass seed abundance of about 10,000 seeds m−2 in Otherwise, the environmental conditions in the three zones are similar. Danish eelgrass meadows (Olesen, 1999). The sediment consists of low organic (b1%) sand. The Bay was sparsely vegetated with scattered occurrence of seagrasses Ruppia maritima and 2.2.2. Experimental units Z. marina, but located near (500 m) a 10-hectare coherent Z. marina Two different experimental units were used: core tubes with an in- meadow. ternal diameter (i.d.) of 8 cm and length of 30 cm filled with sediment M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 43

depth of 25 cm using a steel corer (i.d. 15 cm). The sampled sediment was sieved on the site through a 1 mm mesh and the material retained was preserved in 4% formaldehyde buffered in seawater. The animals were in the laboratory sorted from the sediment material and identified to species. The number of individuals of each species was counted and converted into density, i.e. number of individuals ind m−2.

2.3.2. Experimental One experimental station was defined in each of the three fauna zones at Bregnør Bay for determination of seed mimic burial in the field. The experimental stations were separated by about 200 m and each consisted of a permanent 1 m×1 m plot. The plots were marked by inserting four 1 m long iron sticks about 90 cm into the sediment at each corner of the plot quadrate. About 10,000 seed mimics were distributed at the sediment sur- face of each plot in August 2010. The vertical distribution of seed mimics was obtained after 1, 4 and 8 weeks by collecting five 30 cm long cores (diameter of 8 cm) randomly among 100 (10 cm×10 cm) grid squares from each plot. The space left after retrieving the cores Fig. 2. Natural seed (lower left) and nylon seed mimics. Different colours of seed mimics can fi be used in experiments for multiple purposes. Scale bar represents 3 mm. (For interpretation was lled with sediment collected from outside the plot and the location of the references to colour in this figure legend, the reader is referred to the web version of was marked. Subsequent samples were always positioned at least 10 cm this article.) from any previously sampled location. In the laboratory, cores were sliced into 1 cm intervals until 5 cm depth and 5 cm intervals below. to 20 cm depth for N. diversicolor (Nd), M. viridis (Mv) and Control (C), Seed mimics were recovered as mentioned above. and 25 cm (i.d) and 30 cm deep buckets filled with sieved sediment for the large‐bodied A. marina (Am). The cores were covered by 1 mm 2.4. Statistical analyses mesh to prevent escape of worms during the experiment. Cores and buckets were immerged in separate tanks containing aerated seawater The median depth of seeds (DS50%) and seed mimics (DM50%)was at 15 °C (±1 °C) under dark/light cycles (12 h/12 h) during the whole used as a measure of burial by the three polychaete species instead of experiment, except for C that was kept constantly in the dark to prevent the average depth because of non-normality in the vertical distribu- excessive growth of algae. The sediment was left to compact for 2–3days tion (Hughes et al., 2000). before the pre-weighed animals were introduced. The three polychaete To confirm that the burial of seeds was significant (pb0.05) for species were introduced in numbers within the range of their natural faunated treatments in the laboratory, a 1 way ANOVA (factor: abun- density (Delefosse et al., in press). Accordingly, the Nd treatment con- dance) was carried on the median depth (DS50%-lab) between treat- − tained about 400, 800, 1600 and 3200 ind m 2 N. diversicolor, the Am ments for each species and comparison were done by a Dunnett's − treatment about 20, 40, 60 and 80 ind m 2 A. marina and the Mv treat- (1955) test. Linear regression analyses were also carried to verify − ment about 400, 800 and 1600 ind m 2 M. viridis. The animals were left whether seed burial depth was directly related to animal density. to acclimatise for 1 week before the seeds and seed mimics were cast. Additionally, 1 way ANOVAs were used for each animal separately The seed burial development in Nd-3200, Am-40 and Mv-400 treat- to compare the seed and seed mimic recovery, the proportions of ments (n=5) was followed at high temporal resolution as these abun- seeds contained within the top 6 cm of the sediment after 1 month, dances matched those in the field. The other treatments (n=3) were and the percentage of seeds and seed mimics present at the surface only examined for seed burial depth at the end of the experiment. of the sediment at different time. The validity of using seed mimics was tested by comparing their 2.2.3. Seeds and seed mimics burial vertical distribution with that of seeds. The burial depth was consid- Daily to weekly counting of seeds and seed mimics present at the sur- ered similar, if a linear regression between the median depths of face of the sediment were done on Nd-3200, Am-40, and Mv-400 to fol- seeds (DS50%-lab) and seed mimics (DM50%-lab) was close to 1 and low the dynamics of the process involved in the burial of seeds at near significantly different from 0. field faunal abundance. A seed was considered at the surface if more Burial of seed mimics in the field was compared using a 2-way than about 50% of it was visible. After one month, the vertical distribution ANOVA approach (zone×duration) to detect difference of the median of seeds and seed mimics was determined in the sediment of all treat- depth between treatments (DM50%-field). The relation between mean ments. After collecting and counting seeds and seed mimics remaining and variance of the total number of seed mimics of each core from the at the surface, Nd, Mv and C cores were sliced in 1 cm depth intervals. field was used to measure the level of aggregation of seed mimics Each slice was sieved through a 1 mm mesh and all seeds and seed (Jørgensen et al., 2011). mimics retrieved were counted. Animals retrieved were also counted to The statistical tests were done using permutational analysis of vari- determine the survival in each treatment. The large Am experimental ance (Permanova) which accounts for unbalanced design and is robust units were subsampled by retrieving four cores of 8 cm in diameter simul- to non-normal distribution (Anderson et al., 2008). taneously to the bottom of each Am buckets. These 4 cores were treated as described above for the other two species and the results were pooled 3. Results to obtain a single measure per replicate Am treatment. Thus, only 41% of the surface area was actually sampled in each Am treatment unit. 3.1. Laboratory experiments

2.3. Field experiment 3.1.1. Recovery of worms and seeds Individuals of A. marina (1,955 mg ww; SD: 389, n=12) were on 2.3.1. Benthic fauna composition average about 10 times larger than those of N. diversicolor (203 mg; The species composition and abundance of benthic fauna in each of SD: 75, n=40) and M. viridis (180 mg; SD: 56, n=12). All A marina the three zones were determined by collecting 4 replicate samples to a survived the experiment, whereas the recovery for N. diversicolor 44 M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 and M. viridis were 68% and 54%, respectively. Seed and seed mimic in Mv-400 to about 22% in Mv-1600. Seed burial was related significantly recovery from the core slicing was on average 97% in N. diversicolor to density of M. viridis (R2=0.94, p=0.03; Fig. 5)andtheaverage (Nd), M. viridis (Mv), and Control (C) treatments. The recovery monthly seed burial rate was estimated to about 0.00027 cm (SD: corrected for sub-sampling in the A. marina (Am) treatment was ca. 0.00015, n=4) per individual per m-2 at 15 °C. The average median

100% (46% recovery in 41% of the surface area). There was no signifi- depth (DS50%-lab) ranged from 0.22 cm (SD: 0.06, n=5) for Mv-400 to cant difference between seed and seed mimic recovery in any treat- 0.47 cm (SD: 0.06, n=2) for Mv-1600. The proportion of seeds buried ment (p>0.05). within the top 6 cm of the sediment increased with polychaete abun- dance and ranged from 61 to 79% after one month (p b0.001). In general, 3.1.2. Seed burial seeds were not found deeper than 2 cm (Fig. 6). N. diversicolor dragged seeds and seed mimics into their burrow by seizing them with the jaws. Some large individuals managed to carry 2 3.1.3. Comparisons of seeds and seed mimics or 3 seeds at the time and even small (1 mm wide) individuals moved A. marina and M. viridis indifferently buried seeds and mimics and −2 the seeds. Consequently, the 3200 ind m treatment (Nd-3200) ex- the median depth estimated from seeds (DS50%-lab) and mimics amined with high temporal resolution showed that most of the seeds (DM50%-lab) ranged from 0.91 cm for Am-20 to 6.2 cm for Am-80 and (70%) was rapidly removed (b3 h) from the surface of the sediment from 0.25 cm for Mv-400 to 0.47 cm Mv-1600. The correlation between

(Figs. 3 and 4a). The proportion of buried seeds in Nd-3200 only in- DS50%-lab and DM50%-lab had a slope of 0.97 (SD: 0.08) for A. marina creased further to about 80% after one day and remained constant to and 0.98 (SD: 0.21) for M. viridis, which were significantly different the end of the experiment. Although N. diversicolor buried seeds rapidly from 0 (R2=0.99, p=0.002 and R2=0.96, p=0.03 respectively). and effectively (pb0.001; Fig. 5), they only reached a shallow depth. There were slight differences between DS50%-lab and DM50%-lab for The final median depth was on average 0.41 cm (SD: 0.14, n=5; N. diversicolor due to selective removal of seeds from the surface com- Fig. 5) and independent of N. diversicolor density (p=0.16). Some pared to seed mimics. The amount of seed mimics left after 3 h in the seeds were transported down to 8 cm depth, but 70–85% was contained Nd-3200 treatment was about 3 fold higher than that for seeds. The dif- within the top 6 cm of the sediment (Fig. 6). ference diminished through time and the proportion of seeds and A. marina progressively covered seeds by the lateral spreading of un- seed mimics buried were about 84% and 72%, respectively, from day consolidated sediment brought to the surface by defecation (Fig. 4b). This one until the end of the experiment. As for seeds, DM50%-lab was inde- process buried seeds irreversibly and progressively in the Am-40 treat- pendent of N. diversicolor abundance. The slope obtained between ment, resulting in a linear decrease with time of visible seeds at the sed- DS50%-lab and DM50%-lab was significantly different from 0, but only 2 iment surface. Thus, the weekly disappearance rate of surface seeds was reached 0.57 (R =0.70, p=0.02). On average, DM50%-lab was 22% per week (Fig. 3). The seed burial depth by A. marina was clearly 0.33 cm (SD: 0.06, n=5) for N. diversicolor. greater than for the two other polychaetes despite the ca. 10 times larger size of A. marina that was in terms of biomass more than counteracted by 3.2. Field experiments 20–40 times higher abundances of N. diversicolor and M. viridis (Fig. 5).

Burial estimated from the final median depth of seeds (DS50%-lab) was 3.2.1. Infaunal community directly related to the lugworm density (R2=0.99, p=0.0004; Fig. 5) There were 13 different infaunal species identified in the three and monthly individual seed burial estimated from the slope was about zones (Table 1). The dominant species in terms of abundance and bio- 2 0.079 cm (SD: 0.007, n=5) per m at 15 °C. DS50%-lab ranged from mass in the Nd-zone was N. diversicolor (47% of the total abundance) 1.38 cm (SD: 0.11, n=2) in Am-20 to 6.70 cm (SD: 0.38, n=2) in followed by the gastropod Hydrobia (Ecrobia) ventrosa (27%) and the Am-80 after one month. The proportion of seeds contained within the oligochaete Tubificoides benedii (22%). The large-bodied A. marina top-6 cm of the sediment after 1 month decreased significantly from dominated the infaunal biomass in the Am-zone, but its abundance 98% in Am-20 to 40% in Am-80 (pb0.001; Fig. 6). was low compared with chironomids that accounted for about 67% of M. viridis buried seeds by covering them with faecal pellets (Fig. 4c). the total abundance. It should be noticed that M. viridis also was present In the Mv-400 treatment, seed abundance at the sediment surface de- here (6% of the total abundance). M. viridis dominated the Mv-zone creased linearly with time at a rate of 15% per week (Fig. 3). The propor- (43% of total abundance), but it co-occurred with N. diversicolor, Nereis tion of surface visible seeds at the end of the experiment ranged from 40% (Alitta) succinea and Nereis (Alitta) virens. The latter three species to- gether represented about 33% of the total abundance.

3.2.2. Horizontal dispersion of seed mimics Seed mimics that were cast at the plots rapidly reached the sediment surface and the initial proportion within the plot was visually estimated to more than 99%. Subsequently, water movements rapidly moved seed mimics into clusters of several hundred in naturally occurring sediment depressions. The movement of seed mimics (b1cm)wasenforcedby the foraging activity of juvenile fish and shrimp that were present in large numbers (>20 ind m−2 and >100 ind m−2, respectively). The clustering was confirmed by a log(variance) to log(mean) ratio of 2.18 (SE: 0.31, n=9) for seed mimics recovered in the unprotected treatment. A fraction of the seed mimics was still visible at the sediment surface after 3 weeks. The recovery of seed mimics was less than 2% for all 3 zones in- dependently of the time.

3.2.3. Burial of seed mimics Fig. 3. Seed surface clearance in laboratory experiments with Nereis (Hediste) diversicolor There was a strong interaction (pb0.001) between zones and du- at a density of 3200 ind m− 2 (Nd-3200), Arenicola marina at a density of 40 ind m− 2 ration on the median depth of mimics in the field (DM50%-field). (Am-40) and Marenzelleria viridis at a density of 400 ind m−2 (Mv-400) compared to the DM -field was similar for all 3 species ranging from 0.9 to 1.3 cm defaunated control. The average and standard deviation are shown for the proportion of 50% fi seeds left visible at the sediment surface up to one month after introduction (n=5, except after 1 week, (Fig. 7). While, DM50%- eld in the Nd- and Mv-zones for control n = 3). remained constant at about 0.7 cm and 1.5 cm, respectively, with M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 45

Fig. 4. (a) Distribution of visible seeds at the sediment surface 0 (left) and 24 min (right) after casting at a Nereis (Hediste) diversicolor density of ca. 3200 ind m− 2. Seeds were dyed with Rhodamine to improve visibility. (b) Seed (brown) and seed mimics (yellow) near a 5-day old faecal cast of Arenicola marina. (c) Seed (white circled) and seed mimics (dark circled) partly covered by Marenzelleria viridis faecal pellets. Scale bars represent 30 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

time, a significant increase was observed in the Am-zone at a rate of species as indicated by the full recovery of seeds in all laboratory 3 cm per week (R2 =0.97, p=0.02; Fig. 7). treatments. Eelgrass seeds are potentially nutritious (Irving et al., 1988) and are consumed by other macroinvertebrates (Wigand and 4. Discussion Churchill, 1988; Fishman and Orth, 1996), but they are apparently too large (about 3.5×1.5 mm; Delefosse et al., in preparation)forin- 4.1. Seed burial processes gestion by N. diversicolor, A. marina (Jones and Jago, 1993)andM. viridis (Bock and Miller, 1999). Although seeds were not consumed by the Z. marina seeds were buried below the sediment surface by all three species, the burial mechanism involved feeding activities and con- fi three polychaetes species, yet at different depth scales and rates. It rmed the importance of feeding mode for understanding the impli- is important to notice that seeds were not consumed by any of the cations of ecosystem engineering by benthic macroinvertebrates (Quintana et al., 2007; Kristensen et al., 2012). Only N. diversicolor was directly attracted to Z. marina seeds as this species intentionally grabbed and pulled seeds into its burrow and left them just below the sediment surface at a median depth of 0.41 cm. Such behaviour has also been observed for this species with the larger seeds of the common cordgrass, anglica (Hughes et al., 2000). As an omnivore, N. diversicolor actively searches the sediment surface around its burrow for food by extending 30–50% of its body out of the burrow (see video link in Fig. 4; Duport et al., 2006). By doing so, the worm is able to withdraw rapidly with potential food items for safe in- gestion inside its burrow. This behaviour reduces intra- and inter- specific competition with other benthic invertebrates and predation by birds and fish (Rosa et al., 2008). Being unable to swallow the first at- tractive seed, individuals of N. diversicolor repeatedly, and in hope for success, remove all available seeds from the sediment surface, except for those coated with mucus and therefore out of reach of the worms sensory organs. Due to this selective behaviour, seed burial was inde- Fig. 5. Median depths of seeds (DS ) after 1 month in sediment with different densi- 50% pendent of N. diversicolor density, and this species was, even at low ties of the three polychaetes. DS in faunated treatment were all significantly differ- 50% 2 ent from the control (Dunnett's test: pb0.05). Arrows indicate a significant increase abundance (2 worms per 50 cm ), capable of foraging the whole sedi- with polychaete abundances. Average and standard deviation are shown (n=3–5). ment surface efficiently (Duport et al., 2006). 46 M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49

Fig. 6. Vertical distributions of seeds in laboratory systems with Nereis (Hediste) diversicolor (Nd), Arenicola marina (Am) and Marenzelleria viridis (Mv) and in the control after one month. The polychaete abundance (ind m− 2) is indicated on the graphs. Average and standard deviations of seed proportions are reported for 1 cm-depth increment (n=2–5). The proportion of seeds included within the top 6 cm of the sediment is also reported. The full line indicates the sediment–water interface and the dotted lines represented the critical emergence depth of seedling (6 cm).

A. marina covered eelgrass seeds by its massive faecal cast deposi- Table 1 tion at the sediment surface. Faecal production by a single A. marina Species composition in the Nd-zone dominated by Nereis diversicolor, the Am-zone individual is known to reach about 20 g DW per day (Riisgård and dominated by Arenicola marina and the Mv-zone dominated by Marenzelleria viridis. Values are given as average abundance (ind m− 2)±standard deviation (n=4). The Banta, 1998). At the same time surface sediment with seeds is sub- species investigated in this study are marked in bold. ducted into the feeding depression of the A. marina burrow system. Because of its inability to ingest particles larger than 1 mm (Jones Nd-zone Am-zone Mv-zone and Jago, 1993), eelgrass seeds are transported downward unidirec- Arenicola marina 0 50±13 0 tionally and sediment displacement by the feeding activity is there- Chironomidae 28±56 1780 ±1230 99±162 fore a good proxy for seed burial (Valdemarsen et al., 2011). Corophium volutator 127±96 14±28 0 Heteromastus filiformis 0 353±633 113±80 Sediment reworking depth by A. marina has been reported to range − 2 − 1 Hydrobia (Ecrobia) ventrosa 1808±1553 198±359 0 from 0.04 to 0.10 cm m month for a single individual (Riisgård Littorina littorea 99±198 14±28 0 and Banta, 1998; Valdemarsen et al., 2011), which agrees well with Marenzelleria viridis − − 0 169±196 424±98 the estimated seed burial rate of 6.4 cm m 2 month 1 for a population Mya arenaria 28±33 14±28 0 −2 Nereis (Hediste) diversicolor 3164±651 0 28±33 of 80 ind m in the present study. Mixing of sediment by other up- Nereis (Alitta) succinea 0 0 169±226 ward conveyors such as the bamboo worm Clymenella torquata is also Nereis (Allita) virens 0 0 127±96 known to affect seed bank dynamics (Luckenbach and Orth, 1999). It Scoloplos armiger 0 56±113 0 is noteworthy that the feeding mode of A. marina was most efficient fi Tubi coides benedii 1497±1372 184±367 14±28 in seed burial despite its lower area-specific biomass compared to the M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 47

and of different nature. The alteration of surface topography by e.g. faecal mounds and feeding pits of A. marina (Kristensen et al., 2012) may increase the capacity of sediments to trap seeds (Luckenbach and Orth, 1999). However, the rate by which seeds were trapped was slow compared to horizontal dispersal caused by the near- bottom currents induced by wave or tides as indicated by the loss of more than 97% of the seed mimics. By contrast, polychaetes appear to be important in the permanent seed burial since seed mimic burial was significant and more markedly in strongly bioturbated areas (Fig. 7). Such locations may therefore act as hotspots of natural seed burial when the loss from horizontal dispersal is counteracted by gain of similar magnitude from surrounding areas. A general understanding of the balance between seed dispersal and burial is important for successful conservation and restoration of eelgrass beds. However, the horizontal and vertical distribution of seeds is governed by spatial differences in the relative contribution of biological and physical mechanisms at habitat (Steinhardt and Fig. 7. Seed mimics median depth in the field (DM50% field) at 3 different zones dom- inated by Nereis (Hediste) diversicolor (Nd-zone), Arenicola marina (Am-zone) and Selig, 2007) and community levels. Further research is therefore re- Marenzelleria viridis (Mv-zone). Average and standard deviations are shown (n=5). quired to elucidate these differences, which could include large scale field studies using seed mimics as a tool. For example, seed mimics could be applied as a low-cost tool for determining the fate of large seed-casts, and thus help managers decide locations where restoration N. diversicolor and M. viridis, but each of these large individuals may efforts are meaningful (Marion and Orth, 2010). penetrate and forage deeper than the other two species. M. viridis has been reported as a surface deposit feeder ingesting fine 4.4. Ecological implications particles (Dauer et al., 1981; Bock and Miller, 1999), and most of its fae- cal pellets are deposited at the sediment surface. Since it avoids eelgrass The examined polychaetes strongly affect seed bank dynamics seeds, the amount of sediment egested is also for this species a measure through their feeding activities. The process where N. diversicolor captures of the seed burial rate. Bock and Miller (1999) estimated an individual seeds in vicinity of its burrow opening and deposits them less than 1 cm egestion rate for M. viridis of 24–192 mg DW per day which corresponds below the sediment surface provides the seeds with a “safe site” (Orth to a burial rate of 0.02 to 0.16 cm m−2 month−1 for a population of et al., 2006). They are then protected towards potential predators 400 M. viridis. This estimate matches well the monthly burial obtained (Wigand and Churchill, 1988; Fishman and Orth, 1996)andexposedto in our experiment of 0.11 cm m−2 for a similar sized population. redox conditions that are ideal for germination (Moore et al., 1993). Fur- It is worth emphasising that the rate and median depth of eelgrass thermore, when seeds germinate at this depth the young plants may be seed burial is strongly dependent of A. marina and M. viridis abundance assured better anchorage and developing roots have direct access to re- and that such relationship is caused by their inability to select seeds quired nutrients. Seed burial through deposition of faeces by A. marina during particle mixing resulting in burial through passive subduction. and M. viridis will at first have the same beneficial effect, but continuous By contrast, the selective and active search for seeds by N. diversicolor defecation by particularly populations of A. marina will rapidly bury renders the burial by this species independent of abundance despite seeds too deep into the sediment. Germination of seeds and emergence its inability of ingesting the seeds. of seedlings are known to be delayed (Delefosse et al., in preparation) or even prevented (Greve et al., 2005) when seeds are buried below a cer- tain depth (>6 cm). 4.2. Mimics versus seeds Our laboratory results predict that A. marina populations exceeding a density of 10 ind m−2 may bury seeds below the critical 6 cm depth The strong correlation between the burial of seeds and seed over a 10 month period (Table 2), which corresponds to the average mimics in the present study demonstrates the validity of nylon mimics as a surrogate for seeds in experiments involving A. marina and M. viridis. This is obviously a consequence of the non-selective Table 2 process involved in the burial of seeds and seed mimics by these Predicted eelgrass seed burial depth over a period of 10 months at a range of densities two species. Valdemarsen et al. (2011) estimated that a population of Arenicola marina and Marenzelleria viridis. Seed burial rate was calculated based on − 2 the relation between median depth of seeds (DS50%, cm) obtained at 15 °C in the labo- of A. marina (60 ind m ) in the laboratory buried seed mimics at a − ratory as a function of polychaete abundance (Am and Mv, ind m 2; see text). Accord- rate of 0.5 to 3 mm d−1, which is similar to the rate found here for a ingly, the relation used were for A. marina:DS50% =0.079×Am and for M. viridis:DS50% = −1 similar population (0.3−2.1 mm d ). N. diversicolor, on the other 0.00027×Mv. Faecal production was temperature corrected on a monthly basis (Riisgård hand, selectively removed seeds in preference of seed mimics from and Banta, 1998; Valdemarsen et al., 2011). A general temperature correction factor (f) the sediment surface and buried seeds deeper (0.41 cm) compared to was calculated at any given temperature (t) using the relation obtained by Riisgård and Banta (1998) for A. marina (f=(0.24+0.04 t)/(0.24+0.04×15)=0.29+0.047 t). The seed mimics (0.33 cm). This difference is probably caused by the use 10 month burial was estimated by summing up the temperature corrected DS50% for chemical cues to select palatable food items (Evans et al., 1970). Despite each month. this difference in preference, the median depth of seeds and seed Arenicola marina Marenzelleria viridis mimics moved by N. diversicolor were highly correlated. Seed burial − 2 − 2 by this species may therefore be estimated from seed mimics results. Abundance (ind m )DS50% (cm) Abundance (ind m )DS50% (cm) 1 0.57 100 0.20 5 2.8 200 0.40 fi 4.3. Seed distribution in the eld 10 5.7 400 0.79 20 11.4 800 1.6 The field study using seed mimics highlight the role of physical 40 22.8 1600 3.2 and biological factors in seed burial and dispersal under natural con- 60 34.1 2400 4.8 80 45.5 3200 6.3 ditions. Our results indicates that both processes were independent 48 M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 time eelgrass seeds remain dormant from seed release in summer to ger- mination the next spring in Danish sediments (Olesen, 1999; Greve et al., 2005). This prediction agrees well with the sediment reworking depth of 0.5–1cmpermonthfor A. marina (5–10 ind m−2)asmeasuredby Valdemarsen et al. (2011).SeedburialbyM. viridis is less dramatic and only reaches the critical depth after 10 months for high, but not unreal- istic densities of >3000 ind m−2 (Table 2; Essink and Dekker, 2002; Delefosse et al., in press). Thus, both of these species have the potential to affect eelgrass recruitment positively or negatively depending on their abundance. The potential implications of seed burial by the combined activi- ties of N. diversicolor, A. marina and M. viridis on seed-based eelgrass recruitment at an estuary-wide scale can be illustrated using the dis- tribution and abundance of these polychaetes. Data from faunal sur- veys (70 samplings stations) conducted in Odense Fjord in 2008 and 2010 (Delefosse et al., in press) was used to extrapolate our laboratory seed burial rates to the entire estuary assuming that animals used in our laboratory experiments may be considered representative of the popu- lation in this estuary (Delefose et al., unpublished and Kristensen et al., unpublished) It must also be noted that we do not consider the actual density of seeds and ignore seed movements (upward or downward) due to other infaunal species as well as current and wave driven sedi- ment movements. The burial depths were estimated for a 10 months period, and locations with burial down to 6 cm was assigned as posi- Fig. 8. Maps representing Odense Fjord and the combined effect of Nereis (Hediste) tively affected, below 6 cm as negatively affected and not buried as un- diversicolor, Arenicola marina and Marenzelleria viridis on eelgrass seed recruitment over the entire estuary. Maps were drawn to define areas where eelgrass seed recruit- affected. When the burial effects were converted to a GIS based ment was not affected (white), positively affected (grey) or negatively affected (black) “potential seed-based eelgrass recruitment success map”, N. diversicolor by the 3 polychaete species. Seed burial maps were constructed by the inverse distance and M. viridis exhibited similar positive effect on seeds in 38% and 25% of weighting interpolation method (Mitas and Mitasova, 1999) using the GIS software the estuary, whereas A. marina affected the seed-based recruitment MapInfo Professional 8.0. The area above the dash lines was not considered. positively in 15% and negatively in 14% of the estuary (Table 3). By com- bining the three species for the entire estuary, the negatively affected area increased to about 20% due to the additive burial depths (Fig. 8). Strategic Science Foundation (contract # 09‐063190) and the Danish Seed-based eelgrass recruitment was positively affected by the com- Council for Independent Research (contract # 272-08-0577 and bined effects in 30% and not affected in 50% of the 60 km2 estuary. It is # 09-071369). known that large bioturbating mammals and birds locally affect seeds distribution into the sediment (Inglis, 2000; Zipperle et al., 2010), but our results suggest that the smaller but omnipresent polychaetes may References also have a significant impact on the seed bank and thereby colonisation Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: guide to potential of eelgrass at a regional scale. This effect can be positive or neg- software and statistical methods. Primer-e Ltd, Plymouth, UK. ative depending on the abundance and species composition of the ben- Atkins, S.M., Jones, A.M., Garwood, P.R., 1987. The ecology and reproductive cycle of a thic fauna. population of Marenzelleria viridis (Annelida: Polychaeta: Spionidae) in the Tay Es- Supplementary data to this article can be found online at http:// tuary. Proceedings of the Royal Society of Edinburgh. Section B, Biological Sciences 92, 311–322. dx.doi.org/10.1016/j.seares.2012.04.006. Berner, R.A., 1980. Early diagenesis: a theoretical approach. Princeton University, Princeton, NJ, USA. Bock, M.J., Miller, D.C., 1999. Particle selectivity, gut volume, and the response to a step Acknowledgements change in diet for deposit-feeding polychaetes. Limnology and Oceanography 44, 1132–1138. Cadée, G.C., 1976. Sediment reworking by Arenicola marina on tidal flats in Dutch Patricia Gandolfo Castiñeira, Troels Lange and Camilla Schmidt Wadden sea. Netherlands Journal of Sea Research 10, 440–460. Pedersen are thanked for their help in the laboratory and in the Chambers, J.C., Macmahon, J.A., 1994. A day in the life of a seed: movements and fates fi of seeds and their implications for natural and managed systems. Annual Review of eld. Gary Banta is thanked for his comments on the statistical Ecological Systems 25, 263–292. part. We thank Marianne Holmer, Karsten Reise, Jørgen L.S. Hansen Churchill, A.C., 1983. Field studies on seed germination and seedling development in and 2 anonymous reviewers, for valuable comments on an earlier Zostera marina L. Aquatic Botany 16, 21–29. Churchill, A.C., 1992. Growth characteristics of Zostera marina seedlings under anaero- version of the manuscript. This study was supported by the Danish bic conditions. Aquatic Botany 43, 379–392. Churchill, A.C., Nieves, G., Brenovitz, A.H., 1985. Flotation and dispersal of eelgrass seeds by gas bubbles. 8, 352–354. Dauer, D.M., Maybury, C.A., Ewing, R.M., 1981. Feeding behavior and general ecology of Table 3 several spionid polychaetes from the Chesapeake Bay. Journal of Experimental Ma- 2 Faunal effects on seed-based eelgrass recruitment in the 60 km Odense Fjord. The rine Biology and Ecology 54, 21–38. areal proportions (%) affected positively, negatively or left unaffected are shown. Davey, J.T., 1994. The architecture of the burrow of Nereis diversicolor and its quantifi- Note that areas calculated for the combined effect are not equivalent to the sum of cation in relation to sediment water exchange. Journal of Experimental Marine Bi- areas because of cumulative species effects. ology and Ecology 179, 115–129. Delefosse, M., Povidisa, K., Poncet, D., Kristensen, E., Olesen B., in preparation. Ecologi- Species Eelgrass recruitment cal implications of changes in polychaetes population in a shallow Danish estuary. PhD thesis, University of Southern Denmark, Denmark. Not affected Positive Negative Delefosse, M., Banta, G.T., Canal-Vergés, P., Penha-Lopes, G., Quintana, C.O., Nereis (Hediste) diversicolor 62 38 0 Valdemarsen, T., and Kristensen, E., in press. Marenzelleria viridis - a harmful invader Arenicola marina 71 15 14 or just another polychaete. Marine Ecology - Progress Series. fl Marenzelleria viridis 75 25 0 Dumbauld, B.R., Wyllie-Echeverria, S., 2003. The in uence of burrowing thalassinid shrimps on the distribution of intertidal seagrasses in Willapa Bay, Washington, Combined 50 30 20 USA. Aquatic Botany 77, 27–42. M. Delefosse, E. Kristensen / Journal of Sea Research 71 (2012) 41–49 49

Dunnett, C.W., 1955. A multiple comparison procedure for comparing several treat- Moore, K.A., Orth, R.J., Nowak, J.F., 1993. Environmental regulation of seed germination ments with a control. Journal of the American Statistical Association 50, in Zostera marina L (Eelgrass) in Chesapeake Bay: effects of light, oxygen and sed- 1096–1121. iment burial. Aquatic Botany 45, 79–91. Duport, E., Stora, G., Tremblay, P., Gilbert, F., 2006. Effects of population density on the Olesen, B., 1999. Reproduction in Danish eelgrass (Zostera marina L.) stands: size- sediment mixing induced by the gallery-diffusor Hediste (Nereis) diversicolor O.F. dependence and biomass partitioning. Aquatic Botany 65, 209–219. Müller, 1776. Journal of Experimental Marine Biology and Ecology 336, 33–41. Olesen, B., Sand-Jensen, K., 1994. Patch dynamics of eelgrass Zostera marina. Marine Essink, K., Dekker, R., 2002. General patterns in invasion ecology tested in the Dutch Ecology Progress Series 106, 147–156. Wadden Sea: the case of a brackish-marine polychaetous worm. Biological Inva- Orth, R.J., Luckenbach, M., Moore, K.A., 1994. Seed dispersal in a marine macrophyte: sions 4, 359–368. implications for colonization and restoration. Ecology 75, 1927–1939. Evans, S.M., Cram, A., Rogers, F., 1970. Spontaneous activity and responses to stimula- Orth, R.J., Harwell, M.C., Bailey, E.M., Bartholomew, A., Jawad, J.T., Lombana, A.V., tion in the polychaete Nereis diversicolor (O. F. Müller). Marine Behaviour and Moore, K.A., Rhode, J.M., Woods, H.E., 2000. A review of issues in seagrass seed dor- Physiology 3, 35–58. mancy and germination: implications for conservation and restoration. Marine Fishman, J.R., Orth, R.J., 1996. Effects of predation on Zostera marina L seed abundance. Ecology Progress Series 200, 277–288. Journal of Experimental Marine Biology and Ecology 198, 11–26. Orth, R.J., Harwell, M.C., Inglis, G.J., 2006. Ecology of seagrass seeds and seagrass dis- Forey, E., Barot, S., Decaëns, T., Langlois, E., Laossi, K.R., Margerie, P., Scheu, S., persal processes. In: Larkum, A.W.D., Orth, R.J., Duarte, C.M. (Eds.), Seagrasses: Bi- Eisenhauer, N., 2011. Importance of earthworm-seed interactions for the composi- ology, Ecology and Conservation. Springer, The Netherlands, pp. 111–133. tion and structure of plant communities: a review. Acta Oecologica 37, 594–603. Papaspyrou, S., Kristensen, E., Christensen, B., 2007. Arenicola marina (Polychaeta) and Grace, J.B., 1984. Effects of tubificid worms on the germination and establishment of organic matter mineralisation in sandy marine sediments: in situ and microcosm Typha. Ecology 65, 1689–1693. comparison. Estuarine, Coastal and Shelf Science 72, 213–222. Greve, T.M., Krause-Jensen, D., Rasmussen, M.B., Christensen, P.B., 2005. Means of rapid Quintana, C.O., Tang, M., Kristensen, E., 2007. Simultaneous study of particle reworking, eelgrass (Zostera marina L.) recolonisation in former dieback areas. Aquatic Botany irrigation transport and reaction rates in sediment bioturbated by the polychaetes 82, 143–156. Heteromastus and Marenzelleria. Journal of Experimental Marine Biology and Ecol- Harwell, M.C., Orth, R.J., 2002. Long-distance dispersal potential in a marine macro- ogy 352, 392–406. phyte. Ecology 83, 3319–3330. Riisgård, H.U., Banta, G.T., 1998. Irrigation and deposit feeding by the lugworm Arenicola Hughes, R.G., Lloyd, D., Ball, L., Emson, D., 2000. The effects of the polychaete Nereis marina, characteristics and secondary effects on the environment. A review of cur- diversicolor on the distribution and transplanting success of Zostera noltii. Helgo- rent knowledge. Vie et Milieu-Life and Environment 48, 243–257. land Marine Research 54, 129–136. Rosa, S., Granadeiro, J.P., Vinagre, C., Franca, S., Cabral, H.N., Palmeirim, J.M., 2008. Im- Inglis, G.J., 2000. Disturbance-related heterogeneity in the seed banks of a marine an- pact of predation on the polychaete Hediste diversicolor in estuarine intertidal flats. giosperm. Journal of Ecology 88, 88–99. Estuarine, Coastal and Shelf Science 78, 655–664. Irving, D.W., Breda, V.A., Becker, R., Saunders, R.M., 1988. Anatomy and composition of Ruckelshaus, M.H., 1996. Estimation of genetic neighborhood parameters from pollen Zostera marina l—a potential new crop. Ecology of Food and Nutrition 20, 263–274. and seed dispersal in the marine angiosperm Zostera marina L. Evol. 50, 856–864. Jones, S.E., Jago, C.F., 1993. In situ assessment of modification of sediment properties by Scaps, P., 2002. A review of the biology, ecology and potential use of the common ragworm burrowing invertebrates. Marine Biology 115, 133–142. Hediste diversicolor (OF Muller) (Annelida: Polychaeta). Hydrobiol. 470, 203–218. Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosystem engineers. Oikos Steinhardt, T., Selig, U., 2007. Spatial distribution patterns and relationship between 69, 373–386. recent vegetation and diaspore bank of a brackish coastal lagoon on the southern Jørgensen, B., Demétrio, C.G.B., Kristensen, E., Banta, G.T., Petersen, H.C., Delefosse, M., . Estuarine, Coastal and Shelf Science 74, 205–214. 2011. Bias-corrected Pearson estimating functions for Taylor's power law applied Valdemarsen, T., Canal-Verges, P., Kristensen, E., Holmer, M., Kristiansen, M.D., Flindt, to benthic macrofauna data. Statistics & Probability Letters 81, 749–758. M.R., 2010. Vulnerability of Zostera marina seedlings to physical stress. Marine Kristensen, E., Penha-Lopes, G., Delefosse, M., Valdemarsen, T., Quintana, C.O., Banta, Ecology Progress Series 418, 119–130. G.T., 2012. What is bioturbation? The need for a precise definition for fauna in Valdemarsen, T., Wendelboe, K., Egelund, J.T., Kristensen, E., Flindt, M.R., 2011. Burial of aquatic sciences. Marine Ecology Progress Series 446, 285–302. seeds and seedlings by the lugworm Arenicola marina hampers eelgrass (Zostera Luckenbach, M.W., Orth, R.J., 1999. Effects of a deposit-feeding invertebrate on the en- marina) recovery. Journal of Experimental Marine Biology and Ecology 410, 45–52. trapment of Zostera marina L. seeds. Aquatic Botany 62, 235–247. Wigand, C., Churchill, A.C., 1988. Laboratory studies on eelgrass seed and seedling pre- Marba, N., Duarte, C.M., 1998. Rhizome elongation and seagrass clonal growth. Marine dation. Estuaries 11, 180–183. Ecology Progress Series 174, 269–280. Wyllie-Echeverria, S., Cox, P.A., Churchill, A.C., Brotherson, J.D., Wyllie-Echeverria, T., Marba, N., Hemminga, M.A., Mateo, M.A., Duarte, C.M., Mass, Y.E.M., Terrados, J., Gacia, 2003. Seed size variation within Zostera marina L. (Zosteraceae). Botanical Journal E., 2002. Carbon and nitrogen translocation between seagrass ramets. Marine Ecol- of the Linnean Society 142, 281–288. ogy Progress Series 226, 287–300. Zipperle, A.M., Coyer, J.A., Reise, K., Stam, W.T., Olsen, J.L., 2010. Waterfowl grazing in Marion, S.R., Orth, R.J., 2010. Innovative techniques for large scale seagrass restoration autumn enhances spring seedling recruitment of intertidal Zostera noltii. Aquatic using Zostera marina (eelgrass) seeds. Restoration Ecology 18, 514–526. Botany 93, 202–205. Mitas, L., Mitasova, H., 1999. Spatial interpolation. In: Longley, P., Goodchild, M.F., Maguire, D.J., Rhind, D.W. (Eds.), Geographical Information Systems: Principles, Techniques, Management and Applications. Wiley, pp. 481–492.