Allogenic Effect of Lanice conchilega

Lanice bio-irrigates its tube through piston-pumping activity for 1.5 min generally

every 4 min, thereby transporting about 3 mmol O2 m-2 d-1 into the sediment (Forster

and Graf, 1995). Burrowing benthic animals (mainly polychaeta and Crustacean) also bioirrigate their burrows. However, the mechanism by which benthic animals propel the water current through their burrows or tubes varies considerably within and among taxonomic groups. The exact irrigation cycles therefore differ between species (Kristensen and Kostka, 2005). For example, the burrowing crustacean

Callianassa subterranea bioirrigates its burrow less frequently for 2.6 min every 40

min (Forster and Graf, 1995). However, to determine the amount of oxygen transporting into the sediment, some other factors such as amplitude of the water flow in each ventilation event, the ventilating surface of burrow or tube and density of animals need to be considered (Forster and Graf, 1995; Christensen et al., 2000; Kristensen and Kostka, 2005). Lanice introduces oxygen-rich water in layers where oxygen is absent (Forster and Graf, 1995). This intermittent ventilation results in temporal changes in oxygen concentrations in the upper layers of the sediment (Forster and Graf, 1995), increased oxygen concentrations closer to the Lanice tubes and a deeper OPD (Forster and Graf, 1995; Braeckman et al., 2010). Our results confirm that OPD is deeper in the presence of Lanice, and further show a density- dependent effect of Lanice bio-irrigation on the OPD: a significant increase in maximum penetration depth of oxygen in the high Lanice treatment (5.17 ± 0.44mm) was present compared with the low (3.69 ± 0.17mm) and control (3.30 ± 0.12mm) treatments (Figure 2; Table S2, Addendum 3). The bio-irrigating effects of Lanice also affected O2 concentrations with a significantly higher oxygen content at the top

sediment layer (mainly 1-4 mm; SIMPER) in high and low Lanice treatments compared to the control sediment (Figure 2; Table S2, Addendum 3). In addition, the periodical piston-pumping activity resulted in oxygen variations over time. We

111 recorded temporal changes in O2 concentration (over a period of 30-35 min) up to a

distance of 1 cm from the tube at 1.5 mm depth. Our results revealed that the oscillations were generally higher in both Lanice treatments compared to the control treatment (Figure 3), although there is no statistically significant difference. This could be caused by the fact that for some measurements, variations in oxygen concentration (the values of CV) were comparable with those in the control treatment. This can be explained by simultaneous piston pumping activity of adjacent animals in their tubes or by a high frequency of occurrence of this pumping activity in one animal resulting in merging individual pulses (Forster and Graf, 1995).

Although not directly measured, this bio-irrigation behaviour can have an effect on the NO3− concentrations in the sediment (Gilbert et al., 1997), which is used as a

substrate for denitrification. This can be done by directly pumping NO3− (Gilbert et al.,

1997) and/or by bringing O2 to deeper layers (Forster and Graf, 1995) thereby

increasing coupled nitrification-denitrification (Gilbert et al., 1997; Fernandes et al., 2012).

The different availability of NO3− between treatments in the low oxygen or anaerobic

deeper layers probably can explain differences observed in diversity indices of nosZ transcripts between treatments at depth. Generally low richness and high diversity (Shannon and inverse Simpson) of nosZ transcripts was observed in the high Lanice treatment compared with the control treatment at depth and these differences were most prominent at the deepest sediment layer (2.5-3 cm) and between the high and low Lanice treatments as well. In a vertical sediment depth profile, nitrate coexists with oxygen in the top layer. In the deeper layers where oxygen is low or absent, the nitrate concentration is an important factor determining similarity or difference in anaerobic denitrifying communities (Liu et al., 2003; Jayakumar et al., 2009). As anoxic conditions continue at depth, nitrate concentrations decrease (Tiquia et al., 2006) while denitrification progresses. This trend coincides with the decrease in diversity of denitrifying organisms and the rate of their activity with increasing sediment depth (Tiquia et al., 2006; Middelburg and Levin, 2009; Jäntti and Hietanen, 2012). During time series incubations of oxygen minimum zone sediments, high diversity of denitrifiers (high Shannon diversity and evenness) has also been found associated with high nitrate concentrations. As denitrification proceeds, nitrate concentrations decrease, coinciding with a lower diversity in denitrifying organisms (Jayakumar et al., 2009). Such trends in nosZ transcript diversity were also observed

112 in control and low Lanice treatments (although not significant): a decrease in nosZ transcript diversity with increasing depth (Figures 4 and 6) probably indicated low NO3− supply for denitrification at depth in control and low Lanice treatments. In

contrast, in the high Lanice treatment, an increasing trend in nosZ transcript diversity (Shannon diversity and inverse Simpson) was observed with increasing depth (Figures 4 and 6). Also in agreement with previously mentioned studies, this can be attributed to the higher availability of NO3− in the high Lanice treatment at depth and

more intensive irrigation activity in this treatment, compared to the low Lanice and control treatments. As mentioned earlier, the most prominent differences were at the deepest sediment layer (2.5-3 cm) between the high Lanice and the other two treatments. Lanice conchilega ventilates its tube in each pumping activity by a volume of water equivalent to 2.4 cm tube length (Forster and Graf, 1995). Therefore, the most suitable condition for denitrification (concomitant high availability of NO3−

and little or no O2 in the surrounding sediment) was met at 2.5-3 cm sediment depth

in the high Lanice treatment, where high nosZ transcript diversity and lowest richness was observed. In the upper layers, high richness values resulted from the presence of a larger number of non-abundant and rare nosZ transcripts mainly belonging to the Alphaproteobacteria (data not shown). This highly diverse pool of transcripts may provide a background reservoir for suboptimal denitrification conditions increasing niche creation (Tilman et al., 1997; Bent and Forney, 2008; Jayakumar et al., 2009). In addition, it should be considered that observed differences between the high

Lanice and the other two treatments at 2.5-3 cm (where the lowest Jaccard similarity

index, less than 50%, was observed; Figure 6) are not due to adaptation of certain transcripts specific to this treatment. 93.5% (231 out of 247) and 94.7 % (234 out of 247) of nosZ transcripts in Hd4 were shared with Cd4 and Ld4, respectively (Figure 6). Therefore, observed differences between treatments are also due to reduction of non-abundant and rare nosZ transcripts in Hd4 compared with Cd4 and Ld4.

In the high Lanice treatment, therefore, the depth of possible denitrification is speculated to shift to the deeper layers compared to the other two treatments following an increase in transcript diversity at 2.5-3 cm. This is consistent with previous studies indicating that in bioturbated sediments denitrification occurs in deeper layers (Bertics et al., 2012) and at higher rates (Gilbert et al., 1997; Webb and Eyre, 2004) compared to non-bioturbated or defaunated sediments.

113 In accordance with a previous study (Bertics et al., 2012) indicating higher variations in denitrification rates in bioturbated sediment, our results on denitrifying transcript distribution showed higher variations in composition of nosZ transcripts in the Lanice treatments compared with the control; and in “depth” with increasing depth (Figure 5). This pattern can also be visualized in diversity indices (Figure 7). In bioturbated sediments, nitrogen cycle processes are most likely driven by the formation of microniches surrounding the burrow systems (Bertics et al., 2012). As we sliced the cores and homogenized the sediment, the microniche formation and biogeochemical differences in the sediment were reflected as higher variations in transcript distribution in Lanice treatments, especially in the high Lanice treatment, and in “depth” with increasing depth.