Hiding in plain sight: The secret contribution of the solitary ascidian Herdmania grandis to temperate reef nitrous oxide production

Large solitary ascidians, like Herdmania grandis (Heller), can dominate the benthic substrates of subtropical and temperate reefs; however, their influence on nitrogen cycling, particularly nitrous oxide (N2O) production, is unknown. Here, we incubated individual H. grandis and compared fluxes of dissolved inorganic and gaseous nitrogen species to fluxes from reef sediments. Nitrous oxide production rates per individual ascidian (21 ± 8 nmol ind h−1) are the highest reported for any marine invertebrate. An individual ascidian produced more N2O than 1 m2 of inter‐reef sediment (1.7 ± 1.7 nmol m−2 h−1). Ascidian mediated N2O production was found to occur under nutrient depleted conditions. The addition of 15N labeled organic material showed that the microbiota associated with H. grandis is capable of both nitrification and denitrification, but the contribution of these pathways to N2O production could not be ascertained. As the ecology of temperate reefs change, any range expansion of H. grandis will increase coastal N2O production.

Given their dominance on temperate reefs, ascidians are likely to play an important role in local nutrient cycling (López-Legentil et al. 2015); however, few studies investigating their impact on nitrogen (N) cycling exist. Early observations demonstrated that ascidians excrete ammonium (NH 4 + ) which can fuel algal productivity (Goodbody 1957), but very little is known about how ascidians influence other N cycling pathways, for example, the capacity of their microbiota (i.e. the microbial community living in and on the animal) to produce the greenhouse gas N 2 O (Stief et al. 2009;Heisterkamp et al. 2010).
Our current understanding about benthic invertebrate organisms and N cycling is largely based on research involving bivalves (Stief et al. 2009;Welsh et al. 2015;McCarthy et al. 2019), sponges (Hoffmann et al. 2009;Fiore et al. 2013), and corals (Middelburg et al. 2015), with only a few studies targeting ascidians (Heisterkamp et al. 2010;Evans et al. 2017). This is surprising given the abundance of ascidians across temperate reef systems, and the fact that the range of large solitary ascidians appears to be expanding as oceans warm (Gewing et al. 2019).
Here, we conducted a pulse chase stable isotope labeling experiment to quantify ascidian mediated N cycling rates on a temperate reef in the GSR, specifically addressing the role of ascidians in mobilizing organic N and their contribution to N 2 O production. As a comparison, sediment N cycling rates were also measured.

Study site
Specimens of H. grandis were collected from Surgeons Reef, an oligotrophic coastal temperate reef (Northwest Solitary Island) in the Solitary Islands Marine Park, NSW, Australia (30.0178 S, 153.2714 E), in April 2019 (Fig. 1). This reef is dominated by the solitary ascidian H. grandis with coverage ranging from 0 to over 200 individuals per m 2 of reef area. Sandy sediments (10 cm 2 by 5 cm depth) were collected with a perspex corer from the inter-reef sediments at the same site and pooled in a shallow crate.
Ascidians and sediments were transported back to the National Marine Science Centre (Coffs Harbour, NSW) for the experiments. Animals were gently brushed to remove large external epiphytes from the tunic before being placed in the holding tanks. The ascidians and sediments were preincubated for 7 d in 1000 litter flow-through tanks (3 L min −1 of 20 μm filtered seawater) at the same temperature (23 C) as the collection site and under a 12 : 12 light : dark cycle. Seawater used for the preincubation and experiment was of very similar quality to the ambient seawater at the collection site. During the preincubation, the ascidians were fed 1 liter of cultured algae (Proteomonas sulcata) once on the fourth day of the 7-d preincubation period to ensure they remained healthy for the trial.

Experimental design
The ascidians and sediments were incubated in sealed chambers with 15 N enriched particulate (40.1 μmol L −1 15 N-PON-freeze-dried algal product, Cambridge Isotope Laboratories, lot no. CNLM-455-1), or dissolved organic N (108.2 μmol L −1 15 N-DON-amino acid mixture, Cambridge Isotope Laboratories, lot no. NLM-2161-1). Nine 8 liter circular perspex chambers were filled with filtered seawater at in situ temperature. One ascidian individual was placed in each chamber. Three chambers received 15 N-PON, three received 15 N-DON, and three received no amendment (controls). Sediments had the same treatments, but in smaller chambers (0.25 L). An additional incubation in which 15 N-PON and 15 N-DON were incubated in seawater only was carried out to quantify leaching of NO 3 − and NH 4 + from the tracer itself.
Chambers were incubated under fluorescent lights for 6 h and the experiment was repeated in the dark with new animals.

Sample collection and analysis
Dissolved oxygen (DO) was measured every hour in the chambers with a PreSens optode. Water samples for concentration and 15 N determination in NH 4 + and NO 3 − were collected with a plastic syringe (2 × 60 mL) from taps in the chamber lid at 0, 3, and 6 h. For the 15 N 2 samples, water samples were collected and added to duplicate 12 mL exetainers without a headspace and immediately injected with mercuric chloride solution (20 μL $ 8% w/v). Samples for N 2 O concentration were collected at each time point with a gas tight syringe (3 × 6 mL) from each chamber and added into 12 mL exetainers that had been previously flushed with helium (He). During sampling chamber water was replaced with seawater through a second tap in the chamber lid. Following the incubations, the ascidians and sediments were flushed with filtered seawater (0.5 h) and then frozen at −20 C. Specimens were defrosted, weighed, and dissected into the tunic (external and internal surfaces) and internal organs (basket, stomach, and gonads combined), freeze-dried, cut into fine pieces, and weighed (0.6-0.8 mg) into tin capsules for bulk δ 15 N analysis. Sediments were dried in an oven (60 C), ground into a fine powder and weighed (100 mg) into tin capsules for bulk δ 15 N analysis. The %N and δ 15 N of the ascidian compartments were measured via elemental analysis (Thermo Finnigan Flash EA 112, coupled to a Thermo Delta V Plus IRMS via Thermo Conflo III) (precision AE 1% and 0.15‰ respectively).
Concentrations of NH 4 + and NO 3 − (i.e., NO 2 − + NO 3 − ) were analyzed via a Lachat QuickChem 8000 Flow Injection Analyser. Headspace N 2 O concentrations were determined by gas chromatography combined with a micro-electron capture detector (Sturm et al. 2015) (Agilent 7890A gas chromatograph [GC] fitted with a Gerstel multipurpose autosampler). The net nutrient fluxes and N 2 O fluxes were determined as: where the initial concentration in each incubation vessel (μmol L −1 ) is conc i , the final nutrient concentration is conc f (μmol L −1 ), the incubation volume (L) is V, A is the dry weight of the ascidian or the sediment surface area (m 2 ), and t is time (hours). The net flux of nutrient from the 15 N-PON and 15 N-DON amendments in water only (i.e., no ascidians) was subtracted off the rates for the 15 N-PON and 15 N-DON treatments to account for water column processing and/or leaching of nutrients from the isotopic label. The sediment rates are given in μmol m −2 h −1 , and the rates in the ascidian treatments are in units of μmol g −1 h −1 (rates for N 2 O are in nmol). To aid comparison between the ascidian and sediment rates, we converted the ascidian rate to an areal rate. To do this, the rate measured in each chamber was multiplied by the total average dry weight of all the ascidians used in the experiment to give units of nmol animal −1 h −1 . The abundance of ascidians on Surgeons Reef (Northwest Solitary Island) can vary from 1 to 200 per m 2 , so a conservative estimate of 10 m −2 was used. The μmol animal −1 h −1 was multiplied by 10 to give units of μmol m −2 h −1 . Rates were averaged over the light and dark incubations.
For 15 N-N 2 , 2 mL of pure He was added to the exetainer vials containing the sample, 10 μL of this was subsequently analyzed for 29 N 2 and 30 N 2 on a Thermo Trace GC Ultra with a 25 m × 0.32 mm PoraPLOT Q column interfaced to a Thermo Delta V Plus IRMS (precision AE 0.15‰). The instrument precision for 30 N 2 was poor due to interference from NO in the IRMS ion source (even following correction with standard gases of known isotopic composition). As such, the rate of total 15 N-N 2 production was calculated via the production of 29 N 2 only (Lewicka-Szczebak et al. 2013). The labeling of the N pool for denitrification, which is required for the calculation, was directly determined by measuring the ratio of 15 N/ 14 N in NO 3 − in chamber water at the end of the trial. The production of Kelly et al.
Ascidian mediated nitrous oxide production on temperate reefs  , and total N 2 production was the sum of 15 N-N 2 and 14 N-N 2 production. This approach will overestimate denitrification rates if anammox is active.
The δ 15 N of NO 3 − was determined by the denitrifier protocol (Sigman et al. 2001), and the δ 15 N of NH 4 + was determined using the hypobromite/azide protocol (Zhang et al. 2007

Results
The average dry weight of the animals was 23.8 AE 8.1 g and there was no significant difference (p > 0.05) between the weights of animals used in the difference treatments of the controls. Day/night differences in fluxes were insignificant (p > 0.05) for most parameters with the exception of N 2 O. As such, we combined the day/night treatments so that there were six rather than three replicates. The ascidians consumed significantly more DO (p < 0.05, n = 18, df = 1, F = 162) than the sediments, but the amendment type (i.e., control, 15 N-PON and 15 N-DON) (Fig. 2) did not influence DO flux. Ammonium flux was similar between sediments and ascidians. For the ascidians, the flux of NH 4 + was significantly higher when 15 N-PON and 15 N-DON were present relative to the control (p < 0.05, n = 6, df = 2, F = 13.4). Sediment NH 4 + flux in the 15 N-DON amended sediments was also positive and significantly higher (p < 0.05, n = 6, df = 2, F = 19.8) than the control or 15 N-PON amended sediments (Fig. 2C,D). Production of NO 3 − was significantly higher when ascidians were present relative to the sediments (p < 0.05, n = 18, df = 1, F = 26.5). Nitrate fluxes were positive in the ascidian treatments, but unlike NH 4 + did not differ between the amendments or control. The areal production of N 2 O was significantly higher in the ascidian incubations compared to the sediments (p < 0.05, n = 18, df = 1, F = 73.3) (Fig. 2G,H). In the ascidian incubations, the presence of 15 N-PON and 15 N-DON did not significantly increase N 2 O production (p > 0.05) relative to the control (Fig. 2G). In the ascidian control incubation, that is, without organic N addition, the average daily flux of N 2 O was 0.9 AE 0.4 nmol g −1 h −1 or 21 AE 8 nmol ind −1 h −1 . At 10 individuals per m 2 , this equates to 209 AE 77 nmol m −2 h −1 . For the sediments, the average daily flux of N 2 O was 1.7 AE 1.7 nmol m −2 h −1 . The flux of N 2 O was significantly higher in the light than in the dark (p < 0.05, n = 9, df = 1, F = 15.5) (data not shown). Labeled 29 N 2 was detected in both the ascidian and sediment incubations receiving 15 N amendments, indicative of denitrification. We cannot rule out that anammox was active, in which case the calculated rate of denitrification would be overestimated. The rates of total N 2 production were low and statistically similar between ascidian and sediment treatments (Table 1). In the treatments receiving 15 N-DON and 15 N-PON, both 15 N-NH 4 + and 15 N-NO 3 − were detected, indicating that mineralization and nitrification, respectively, were occurring. The rate of nitrification was significantly higher when DON was added (Table 1). Combining all the different N pools, the recovery of added 15 N was 27% and 37% in the ascidian treatments receiving 15 N-DON and 15 N-PON, respectively. In the treatment receiving 15 N-DON, most of the recovered 15 N was in the NH 4 + pool (65% of recovered 15 N), followed by the tunic (23%) (Table 2). Similarly, most of the recovered 15 N was in the NH 4 + pool of the 15 N-PON treatment (89%). In both the 15 N-DON and 15 N-PON treatments, 15 N-NO 3 − was detected indicating the presence of active nitrification. When 15 N-PON was added to sediment incubations the largest proportion of 15 N was found in the sediments, reflecting the recovery of the added particulate material. The recovery of 15 N in NO 3 − was < 2% of the added label in either of the sediment treatments (Table 1) indicating low rates of nitrification. Isotopically labeled 15 N-NH 4 + was only detected in the sediments receiving 15 N-DON.
Overall, 27% and 78% of the added 15 N was recovered in the 15 N-DON and 15 N-PON sediment treatments.

Discussion
In this study, we have identified that the Ascidian H. grandis is capable of producing significant quantities of N 2 O. There are two main sites for N 2 O production in marine invertebrates; these are on the organism's surfaces and within their stomach (Stief et al. 2009;Heisterkamp et al. 2013). Nitrification and denitrification can occur in either location (Svenningsen et al. 2012) although denitrification is more likely to occur in the anoxic stomach. Another possible pathway is nitrifier denitrification, that is, the reduction of nitrite (NO 2 − ) to N 2 O carried out by ammonium oxidizing bacteria under oxygen limitation (Poth and Focht 1985;Wrage et al. 2001). Denitrification was confirmed in the ascidian treatments receiving 15 N by the production of 15 N-N 2 (Table 1), but rates were much lower than reported for other benthic invertebrates (Smyth et al. 2013;Welsh et al. 2015;Erler et al. 2017). Consequently the specific N 2 O yield (i.e., N 2 O/(N 2 O + N 2 ) was large (44% and 21% in the 15 N-PON and 15 N-DON amendments, respectively) compared to other studies (Heisterkamp et al. 2013;Welsh et al. 2015;Erler et al. 2017). The specific N 2 O yield for the sediment was < 1% for either amendment.
A high specific N 2 O yield suggests that denitrification is inefficient, or that nitrification and/or nitrifier denitrification are the dominant N 2 O production pathways. Nitrification (i.e., the production of 15 N-NO 3 − ) was confirmed when 15 N-DON and 15 N-PON were added to the ascidians. In fact, 15 N-NO 3 − accounted for almost 8% of the recovered 15 N in the 15 N-DON treatment and was thus orders of magnitude higher than denitrification, indicating that it is more likely to be a pathway of N 2 O production than denitrification. We also cannot rule out nitrifier denitrification as a contributor to N 2 O, as it is carried out by the same microbes performing NH 4 + oxidation.
We have found that H. grandis is a significant source of N 2 O relative to reef sediments. The average rate of N 2 O production in the ascidian control without organic N addition, on a dry weight basis, was 0.92 AE 0.34 nmol g h −1 . This N 2 O production rate is within the range of other invertebrates measured in the literature (Table 3); however, when we factor in their large size, the N 2 O production per individual H. grandis is the highest recorded for any marine invertebrate to date. The average areal N 2 O flux for the ascidians in the control treatment was 209 AE 77 nmol m −2 h −1 , two orders of magnitude higher than the sediment N 2 O flux (based on 10 animals per m 2 ). Extrapolation of the chamber incubation results to areal fluxes is problematic as we do not know the distribution of animals on different section of the reef or on other reefs. However, an important point is that based on the experimental incubations, a single ascidian of average weight produces more N 2 O than 1 m 2 of inter-reef sediment. Therefore, the presence of ascidians is likely to have a major influence on ecosystem N 2 O dynamics.
Importantly, N 2 O production was active in the ascidian controls (i.e. not receiving added nitrogen). The similarity in N 2 O production between the control animals and those receiving organic N may simply be a function of the short experimental duration. Other studies have clearly demonstrated that increased N availability stimulates N 2 O production, but the response can take days rather than hours (Garate et al. 2019). All ascidians were acclimated in oligotrophic flow-through seawater and were only fed once during the 7-d preincubation period. Therefore, the ascidians in the control were producing N 2 O with N assimilated during the preincubation period. In other words, H. grandis can produce appreciable quantities of N 2 O under relatively nutrient limited conditions.
The ability of the ascidians in the control treatment to produce N 2 O may be partly related to their ability to assimilate and process dissolved organic N, which was clearly demonstrated in the 15 N-DON amendment where 15 N was recovered in NH 4 + , NO 3 − , and on the tunic ( Table 2). The ability of ascidians to produce N 2 O under nutrient limited conditions is also important from an ecological perspective. The rate of N 2 O production by H. grandis (0.21 μmol m −2 h −1 ) is not that much less than the average N 2 O flux from subtropical estuarine sediments (0.59 μmol m −2 h −1 ) (Murray et al. 2015). Note that this comparison does not account for the three-dimensional structure of reefs, so a m 2 of reef viewed from above could actually contain many m 2 of effective surface area of ascidians. The implication is that temperate reefs on Australia's continental shelf are potentially as important to N 2 O production as the estuaries; however, we do not know how much area temperate reefs cover, or the densities of H. grandis on these reefs.
Another consideration in the comparison between estuarine and ascidian mediated N 2 O production is that the average rate derived from Murray et al. (2015) includes estuarine systems both with and without inorganic N inputs, whereas the comparable rate measured in the H. grandis incubations occurred without additional N supply. Assuming that both estuarine sediments and ascidian dominated reefs increase N 2 O production if N is supplied, we would expect the ascidian system to generate more N 2 O than sediment dominated systems (as shown in our comparison between reef sediments and ascidians). If N 2 O produced by benthic organisms is not consumed by denitrification in sediments then it will eventually be released to the atmosphere. The sediment incubations showed production of N 2 O and therefore are not going to consume the N 2 O produced by the ascidians. Our data suggest that current coastal estimates of N 2 O production will be underestimated if temperate reef habitat is not included.
This experiment was not carried out for long enough to see the added organic material significantly increase N 2 O production. However, NH 4 + production and nitrification were observed following organic amendment addition, implying that given enough time, increased organic N availability will increase N 2 O production. Any increases in N availability on temperate reefs (inorganic or organic) are therefore likely to cause further increases in N 2 O production. Further work is needed to quantify the abundance of H. grandis on temperate reefs, determine if its range is expanding as a result of ocean warming, quantify how temperature and food availability will affect N 2 O fluxes, and better understand the processes that are responsible for N 2 O production.