Hydrothermal activity lowers trophic diversity in Antarctic sedimented hydrothermal vents

Abstract. Sedimented hydrothermal vents are those in which hydrothermal fluid vents through sediment and are among the least studied deep-sea ecosystems. We present a combination of microbial and biochemical data to assess trophodynamics between and within hydrothermally active and off-vent areas of the Bransfield Strait (1050–1647 m depth). Microbial composition, biomass and fatty acid signatures varied widely between and within vent and non-vent sites and provided evidence of diverse metabolic activity. Several species showed diverse feeding strategies and occupied different trophic positions in vent and non-vent areas and stable isotope values of consumers were generally not consistent with feeding structure morphology. Niche area and the diversity of microbial fatty acids reflected trends in species diversity and was lowest at the most hydrothermally active site. Faunal utilisation of chemosynthetic activity was relatively limited but was detected at both vent and non-vent sites as evidenced by carbon and sulphur isotopic signatures, suggesting that the hydrothermal activity can affect trophodynamics over a much wider area than previously thought.


Section 1. Introduction
As a result of subsurface mixing between hydrothermal fluid and ambient seawater within the sediment, sedimented hydrothermal vents (SHVs) are more similar to non-hydrothermal deepsea habitats than they are to high temperature, hard substratum vents (Bemis et al. 2012, Bernardino et al. 2012).This creates opportunities for non-specialist, soft-sediment fauna to colonise areas of chemosynthetic organic matter production, potentially offering an important metabolic resource in the nutrient-limited deep-sea (Levin et al. 2009, Dowell et al. 2016).To take advantage of this resource, fauna must overcome the environmental stress associated with high-temperature, acidic and toxic conditions at SHVs (Levin et al. 2013, Gollner et al. 2015).The combination of elevated toxicity and in-situ organic matter (OM) production results in a different complement of ecological niches between vents and background conditions that elicits compositional changes along a productivity-toxicity gradient (Bernardino et al. 2012, Gollner et al. 2015, Bell et al. 2016).Hydrothermal sediments offer different relative abundances of chemosynthetic and photosynthetic organic matter, depending upon supply of surface-derived primary productivity, which may vary with depth and latitude, and levels of hydrothermal activity (Tarasov et al. 2005).In shallow environments (<200 m depth), where production of chemosynthetic and photosynthetic organic matter sources can co-occur, consumption may still favour photosynthetic OM over chemosynthetic OM as this does not require adaptions to environmental toxicity (Kharlamenko et al. 1995, Tarasov et al. 2005, Sellanes et al. 2011).Limited information of trophodynamics at deep-sea SHVs indicate that diet composition estimates vary widely between taxa, ranging between 0 -87 % contribution from chemosynthetic OM (Sweetman et al. 2013).Thus, understanding of the significance of chemosynthetic activity in these settings is very limited.
Sedimented hydrothermal vents host diverse microbial communities (Teske et al. 2002, Weber & Jørgensen 2002, Dhillon et al. 2003, Kallmeyer & Boetius 2004, Teske et al. 2014, Dowell et al. 2016).Microbial communities are a vital intermediate between hydrothermal fluid and metazoan consumers, and thus their composition and isotopic signatures are of direct relevance to metazoan food webs.The reduced chemical compounds and heat flux associated with hydrothermal activity provides thermodynamic benefits and constraints to microbial community assembly (Kallmeyer & Boetius 2004, Teske et al. 2014) but also accelerates the degradation of organic matter, giving rise to a wide variety of compounds, including hydrocarbons and organic acids (Martens 1990, Whiticar & Suess 1990, Dowell et al. 2016).
Microbial aggregations are commonly visible on the sediment surface at SHVs (Levin et al. 2009, Aquilina et al. 2013, Sweetman et al. 2013, Dowell et al. 2016) but active communities are distributed throughout the underlying sediment layers, occupying a wide range of geochemical and thermal niches (reviewed by Teske et al. 2014).Sedimented vents may present several sources of organic matter to consumers (Bernardino et al. 2012, Sweetman et al. 2013) and the diverse microbial assemblages can support a variety of reaction pathways, including methane oxidation, sulphide oxidation, sulphate reduction and nitrogen fixation (Teske et al. 2002, Dekas et al. 2009, Frank et al. 2013, Jaeschke et al. 2014, Wu et al. 2014, Inskeep et al. 2015, McKay et al. 2015).Phospholipid fatty acid (PLFA) analysis can be used to describe recent microbial activity and δ 13 C signatures (Kharlamenko et al. 1995, Boschker & Middelburg 2002, Colaço et al. 2007, Jaeschke et al. 2014).Although it can be difficult to ascribe a PLFA to a specific microbial group or process, high relative abundances of certain PLFAs can be strongly indicative of chemoautotrophy (Colaço et al. 2007).
Siboglinum spp. in particular can use a range of resources, including methane or dissolved organic matter (Southward et al. 1979, Schmaljohann et al. 1990, Thornhill et al. 2008, Rodrigues et al. 2013), making SIA an ideal way in which to examine resource utilisation.We also apply the concept of an isotopic niche (Layman et al. 2007) whereby species or community trophic activity is inferred from the distribution of stable isotopic data in isotope space.

Hypotheses
We used a combination of microbial sequencing data and compound specific and bulk isotopic data from sediment, microbial, macro-and megafaunal samples to investigate resource utilisation, niche partitioning and trophic structure at vent and background sites in the Bransfield Strait to test the following hypotheses: 1) Stable isotope signatures will reflect apriori functional designations defined by faunal morphology; 2) Fauna will have distinct niches between vents and background areas; 3) Siboglinid species subsist upon chemosyntheticallyderived OM and 4) Chemosynthetic organic matter will be a significant food source at SHVs.Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -318, 2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.

Sites and Sampling
Samples were collected; during RRS James Cook cruise JC55 in the austral summer of 2011 (Tyler et al. 2011), from three raised edifices along the basin axis (Hook Ridge, the Three Sisters and The Axe, see Fig. 1 Bell et al. (2016) for map) and one off-axis site, in the Bransfield Strait (1024 -1311m depth).We visited two sites of variable hydrothermal activity; Hook Ridge 1 and 2 (Aquilina et al. 2013) and three sites (Three Sisters, the Axe and an Off-Axis site) where hydrothermal activity was not detected (Aquilina et al. 2013).Samples were collected with a series of megacore deployments, using a Bowers & Connelly dampened megacorer (1024 -1311m depth) and a single Agassiz trawl at Hook Ridge (1647m depth).Except salps, all microbial and faunal samples presented here were from megacore deployments.For a detailed description of the megacore sampling programme and macrofaunal communities, see Bell et al. (2016).Sampling consisted of 1 -6 megacore deployments per site, with 2 -5 tubes pooled per deployment (Bell et al. 2016).Cores were sliced into 0 -5cm and 5 -10cm partitions and macrofauna were retained on a 300 μm sieve.Residues were preserved in either 80 % ethanol or 10 % buffered formalin initially and then stored in 80% ethanol after sorting (Bell et al. 2016).Fauna were sorted to species/ morphospecies level (for annelid and bivalve taxa); family level (for peracarids) and higher levels for less abundant phyla (e.g.echiurans).Salps were collected using an Agassiz trawl and samples were immediately picked and frozen at -80 ˚C and subsequently freeze-dried.Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -318, 2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.

Phospholipid Fatty Acids
Samples of 3 -3.5 g of freeze-dried sediment from Hook Ridge 1 & 2, the off-vent site and the Three Sisters were analysed at the James Hutton Institute (Aberdeen, UK) following the procedure detailed in Main et al. (2015), which we summarise below.Samples were from the top 1 cm of sediment for all sites except Hook Ridge 2 where sediment was pooled from two core slices (0 -2 cm), due to sample mass limitations.Lipids were extracted following a method adapted from Bligh (1959), using a single phase mixture of chloroform: methanol: citrate buffer (1:2:0.8v-v:v).Lipids were fractionated using 6 ml ISOLUTE SI SPE columns, preconditioned with 5 ml chloroform.Freeze-dried material was taken up in 400 μL of chloroform; vortex mixed twice and allowed to pass through the column.Columns were washed in chloroform and acetone (eluates discarded) and finally 10 ml of methanol.Methanol eluates were collected in vials, allowed to evaporate under a N2 atmosphere and frozen at -20 ˚C.
PLFAs were derivitised with methanol and potassium hydroxide to produce fatty acid methyl esters (FAMEs).Samples were taken up in 1 mL of 1:1 (v:v) mixture of methanol and toluene. 1 Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -318, 2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.mL of 0.2 M KOH (in methanol) was added with a known quantity of the C19 internal standard (nonadecanoic acid), vortex mixed and incubated at 37 ˚C for 15 min.After cooling to room temperature, 2 mL of isohexane:chloroform (4:1 v:v), 0.3 mL of 1 M acetic acid and 2 mL of deionized water was added to each vial.The solution was mixed and centrifuged and the organic phase transferred to a new vial and the remaining aqueous phase was mixed and centrifuged again to further extract the organic phase, which was combined with the previous.

Biogeosciences
The organic phases were evaporated under a N2 atmosphere and frozen at -20 ˚C.
Samples were taken up in isohexane to perform gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS).The quantity and δ 13 C values of individual FAMEs were determined using a GC Trace Ultra with combustion column attached via a GC Combustion III to a Delta V Advantage isotope ratio mass spectrometer (Thermo Finnigan, Bremen).The δ 13 CVPDB values (‰) of each FAME were calculated with respect to a reference gas of CO2, traceable to IAEA reference material NBS 19 TS-Limestone.Measurement of the Indiana University reference material hexadecanoicacid methyl ester (certified δ 13 CVPDB 30.74 ± 0.01‰) gave a value of 30.91 ± 0.31‰ (mean ± sd, n=51).Combined areas of all mass peaks (m/z 44, 45 and 46), following background correction, were collected for each FAME.These areas, relative to the internal C19:0 standard, were used to quantify the 34 most abundant FAMEs and related to the PLFAs from which they are derived (Thornton et al. 2011).
All bulk isotopic analyses were completed at the East Kilbride Node of the Natural Environment Research Council Life Sciences Mass Spectrometry Facility (EK).Specimens with carbonate structures (e.g.bivalves) were physically decarbonated and all specimens were rinsed and cleaned of attached sediment before drying.Specimens were dried for at least 24 hours at 50˚C and weighed (mg, correct to 3 d.p.) into tin capsules and stored in a desiccator whilst awaiting SIA.Samples were analysed at EK by continuous flow isotope ratio mass spectrometer using a Vario-Pyro Cube elemental analyser (Elementar), coupled with a Delta Plus XP isotope ratio mass spectrometer (Thermo Electron).Each of the runs of CN and CNS isotope analyses used laboratory standards (Gelatine and two amino acid-gelatine mixtures) as well as the international standard USGS40 (glutamic acid).CNS measurements used the internal standards (MSAG2: (Methanesulfonamide/ Gelatine and Methionine) and the international silver sulphide standards IAEA-S1, S2 and S3.All sample runs included samples of freeze-dried, powdered Antimora rostrata (ANR), an external reference material used in other studies of chemosynthetic ecosystems (Reid et al. 2013, Bell et al. Accepted), used to monitor variation between runs and instruments (supplementary file 1).Instrument precision (S.D.) for each isotope measured from the reference material was 0.42, 0.33 and 0.54 for carbon, nitrogen and sulphur respectively.The reference samples were generally consistent except in one of the CNS runs, which showed unusual δ 15 N measurements (S1), so faunal δ 15 N measurements from this run were excluded as a precaution.Stable isotope ratios are all reported in delta (δ) per mil (‰) notation, relative to international standards: V-PDB (δ 13 C); Air (δ 15 N) and V-CDT (δ 34 S).(35 from non-vent sites, 19 from vent sites and 11 from both), 3 megafaunal taxa and sources of organic matter.Samples submitted for carbon and nitrogen (CN) analyses were pooled if necessary to achieve an optimal mass of 0.7 mg (± 0.5 mg).Where possible, individual specimens were kept separate in order to preserve variance structure within populations but in some cases, low sample mass meant individuals had to be pooled (from individuals found in replicate deployments).Optimal mass for Carbon-Nitrogen-Sulphur (CNS) measurements was 2.5 mg (± 0.5 mg) and, as with CN analyses, specimens were submitted as individual samples or pooled where necessary.Samples of freeze-dried sediment from each site were also submitted for CNS analyses (untreated for NS and acidified with 6M HCl for C) Acidification was carried out by repeated washing with acid and de-ionised water.
Specimens were not acidified.A pilot study at EK, and subsequent results, confirmed that the range in δ 13 C measurements between acidified (0.1M and 1.0M HCl) was within the untreated population range, in both polychaetes and peracarids and that acidification did not notably or consistently reduce δ 13 C standard deviation (Table 1).In the absence of a large or consistent treatment effect, the low sample mass, (particularly for CNS samples) was dedicated to increasing replication and preserving integrity of δ 15 N & δ 34 S measurements instead of separating carbon and nitrogen/ sulphur samples (Connolly & Schlacher 2013).
Formalin and ethanol preservation effects can both influence the isotopic signature of a sample.Taxa that had several samples of each preservation method from a single site (to minimise intra-specific differences) were examined to determine the extent of isotopic shifts associated with preservation effects.Carbon and nitrogen isotopic differences between ethanol and formalin preserved samples ranged between 0.07 -1.38 ‰ and 0.40 -1.96 ‰ respectively.Differences across all samples were not significant (Paired t-test, δ 13 C: t = 2.10, df Biogeosciences Discuss., doi:10.5194/bg-2016-318,2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.= 3, p = 0.126 and δ 15 N: t=1.14, df = 3, p = 0.337).Given the unpredictable response of isotopic signatures to preservation effects in this case (which also cannot be extricated from withinsite, intraspecific variation) it was not possible to correct isotopic data.This contributed an unavoidable, but generally quite small, source of error in these measurements.

Statistical Analyses
All analyses were completed in the R statistical environment (R Core Team 2013).Carbon and nitrogen stable isotopic measurements were divided into those from vent or non-vent sites and averaged by taxa and used to construct a Euclidean distance matrix (Valls et al. 2014).This matrix was used to conduct a similarity profile routine (SIMPROF, 10 000 permutations, p = 0.05, Ward linkage) using the clustsig package (v1.0)(Clarke et al. 2008, Whitaker & Christmann 2013) to test for significant structure within the matrix.The resulting cluster assignations were compared to a-priori feeding groups (Bell et al. 2016) using a Spearman Correlation Test (with 9 999 Monte Carlo resamplings) using the coin package (v1.0-24)(Hothorn et al. 2015).Isotopic signatures of species sampled from both vent and non-vent sites were also compared with a one-way ANOVA with Tukey's HSD pairwise comparisons (following a Shapiro-Wilk normality test).
Mean faunal measurements of δ 13 C & δ 15 N were used to calculate Layman metrics for each site (Layman et al. 2007), sample-size corrected standard elliptical area (SEAc) and Bayesian posterior draws (SEA.B, mean of 10 5 draws ± 95% credibility interval) in the SIAR package (v4.2) (Parnell et al. 2010, Jackson et al. 2011).Differences in SEA.B between sites were compared in mixSIAR.The value of p given is the proportion of ellipses from group A that were smaller in area than those from group B (e.g. if p = 0.02, then 2% of posterior draws from

Differences in microbial composition along a hydrothermal gradient
A total of 28,767, 35,490 and 47,870 sequences were obtained from the off-axis site, Hook Ridge 1 and Hook Ridge 2 respectively.Bacteria comprised almost the entirety of each sample, with Archaea being detected only in the Hook Ridge 2 sample (0.008 % of sequences).Hook Ridge 1 was qualitatively more similar to the off-axis site than Hook Ridge 2. Both HR1 and BOV were dominated by Proteobacteria (48 and 61 % of reads respectively; Fig. 1), whereas Flavobacteriia dominated Hook Ridge 2 (43 %, 7 -12 % elsewhere) with Proteobacteria accounting for a smaller percentage of sequences (36 %; Fig. 1).By sequence abundance, Flavobacteriia were the most clearly disparate group between Hook Ridge 2 and the other sites.Flavobacteriia were comprised of 73 genera at Hook Ridge 2, 60 genera at BOV and 63 genera at HR1, of which 54 genera were shared between all sites.Hook Ridge 2 had 15 unique flavobacteriial genera but these collectively accounted for just 0.85 % of reads, indicating that compositional differences were mainly driven by relative abundance, rather than taxonomic richness.
The most abundant genus from each site was Arenicella at BOV and HR1 (7.13 and 5.17 % of reads respectively) and Aestuariicola at HR2 (6.89 % of reads).The four most abundant genera at both BOV and HR1 were Arenicella (γ-proteobacteria), Methylohalomonas (γproteobacteria), Pasteuria (Bacilli) & Blastopirellula (Planctomycetacia), though not in the same order, and accounted for 17.22 and 15.97 % of reads respectively.The four most abundant genera at HR2, accounting for 20.17 were the most relatively abundant across all sites (2.24 -7.14 and 1.67 -5.02 % of reads respectively).

Microbial stable isotopic signatures
A total of 37 sedimentary PLFAs were identified, in individual abundances ranging between 0 -26.4 % of total PLFA (Table 2; Supplementary Fig 1).The most abundant PLFAs at each site were 16:0 (15.73 -26.40 %), 16:1ω7c (11.50 -20.00 %) and 18:1ω7 (4.80 -16.85 %) (Table 2).PLFA profiles from each of the non-vent sites sampled (Off-axis and the Three Sisters, 33 and 34 PLFAs respectively) were quite similar (Table 2) and shared all but one compound (16:1ω11c, present only at the Three Sisters).Fewer PLFAs were enumerated from Hook Ridge 1 and 2 (31 and 23 respectively), including 3 PLFAs not observed at the non-vent sites (br17:0, 10-Me-17:0 & 10-Me-18:0), which accounted for 0.5 -1.2 % of the total at these sites.Hook Ridge 2 had the lowest number of PLFAs and the lowest total PLFA biomass of any site, though this was due in part to the fact that this sample had to be pooled from the top 2 cm of sediment (top 1cm at other sites).
Isotopic signatures of sediment organic matter were similar between vents and non-vents for δ 13 C and δ 15 N but δ 34 S was significantly greater at non-vent sites (p < 0.05, Table 3; Fig. 4).
Variability was higher in vent sediments for all isotopic signatures.Faunal isotopic signatures for δ 13 C and δ 34 S ranged much more widely than sediment signatures and indicate that sediment organics were a mixture of two or more sources of organic matter.A few macrofaunal species had relatively heavy δ 13 C signatures that exceeded -20 ‰ that suggested either a heavy source of carbon or contamination from marine carbonate (~0 ‰).Samples of pelagic salps from Hook Ridge had mean values for δ 13 C of -27.43 ‰ (± 0.88) and δ 34 S of 21.48 ‰ (± 0.74).Several taxa found at both vent and non-vent sites (Hook Ridge or the non-vent sites, Off-axis,

Comparing macrofaunal morphology and stable isotopic signatures
The Three Sisters and The Axe) were assigned to different clusters between sites.A total of eleven taxa were sampled from both vent and non-vent regions, of which four were assigned to different clusters at vent and non-vent sites.Neotanaids (Peracarida: Tanaidacea) had the greatest Euclidean distance between vent/ non-vent samples (11.36), demonstrating clear differences in dietary composition (Fig. 3) but all other species were separated by much smaller distances between regions, ranging 0.24 to 2.69.Raw δ 13 C and δ 15 N values were also compared between vent and non-vent samples for each species (one-way ANOVA with Tukey HSD pairwise comparisons).Analysis of the raw data indicated that δ 13 C signatures were different for neotanaids only and δ 15 N were different for neotanaids and an oligochaete species (Limnodriloides sp.) (ANOVA, p < 0.01, Fig. 3).

Community-level trophic metrics
All site niches overlapped (mean = 50 %, range = 30 -82 %) and the positions of ellipse centroids were broadly similar for all sites (Table 4; Fig  similar but significantly smaller than non-vent ellipses (SEA.B, n = 10 5 , p = < 0.05).There were 358 no significant differences in ellipse area between any non-vent sites.Ranges in carbon sources 359 (dCr) were higher for non-vent sites (Table 4) indicating a greater trophic diversity in 360 background conditions.Nitrogen range (dNr, Table 4) was similar between vents and non-361 vents suggesting a similar number of trophic levels within each assemblage.All site ellipses 362 had broadly similar eccentricity, ranging 0.85 -0.97 (Table 4) but theta differed between vent 363 and non-vent sites (-1.43 to 1.55 at Hook Ridge, 0.67 to 0.86 at non-vent sites).Range in 364 nitrogen sources was more influential at vent sites with Sclerolinum contortum, which had low 365 δ 15 N signatures, had similar to δ 13 C to non-endosymbiont bearing taxa.The strongly depleted 366 PLFA profiles between the off-axis site and the Three Sisters indicated similar bacterial biomass at each of these non-vent sites but that bacterial biomass varied much more widely at Hook Ridge (Table 2).The Hook Ridge 2 sample is not directly comparable to the others as it was sampled from sediment 0 -2 cmbsf (owing to sample mass availability), though the relatively low organic carbon content, hydrogen sulphide flux and taxonomic diversity at this site may support suggestion of a lower overall bacterial biomass (Aquilina et al. 2013, Bell et al. 2016).The very high bacterial biomass at Hook Ridge 1 suggests a potentially very active bacterial community but δ 13 C was qualitatively similar to non-vent sites, implying that chemosynthetic activity was comparatively limited, or that the isotopic signatures of the carbon source (e.g.DIC) and the fractionation associated with FA synthesis resulted in similar δ 13 C signatures.Hook Ridge 1 PLFA composition was intermediate between non-vent sites and Hook Ridge 1 (Supplementary Fig. 2) but sequence composition was quite similar between Hook Ridge 1 and the off-axis site (Fig. 1).A small number of the more abundant PLFAs had notable for differences in relative abundance between vent/ non-vent sites (Table 2).For example, 16:1ω7, which has been linked to sulphur cycling pathways (Colaço et al. 2007) comprised 13.95 -15.19 % of abundance at non-vent sites and 20.00 -23.50 % at vent sites.
However 18:1ω7, also a suggested PLFA linked to thio-oxidation occurred in lower abundance at vent sites (4.80 -11.12 %) than non-vent sites (15.91 -16.85 %).This further suggests that chemosynthetic activity was relatively limited since, although there were differences in microbial signatures of chemosynthetic activity, these were not necessarily consistent between different PLFAs.Several PLFAs had isotopic signatures that varied widely between sites, demonstrating differences in fractionation and/ or source isotopic signatures.The heaviest PLFA δ 13 C signatures were associated with Hook Ridge sites and were quite variable (e.g.16:1ω11t at HR2, δ 13 C = -8.65,~-24 to -25 ‰ elsewhere).This provides strong evidence of isotopic differences in the sources or metabolic pathways used to synthesise these FAs.Heavier carbon isotopic signatures (> -15 ‰) are generally associated with rTCA cycle carbon fixation (Hugler & Sievert 2011, Reid et al. 2013), suggesting that this pathway was active at this site, albeit at probably quite low rates.Conversely, many of the lightest δ 13 C signatures (e.g.19:1ω8, -56.57‰, off-axis site) were associated with the non-vent sites.Siboglinum isotopic data demonstrates that methanotrophy was probably occurring at these sites, and these depleted PLFA isotopic signatures provides further evidence of methanotrophy, in free-living sedimentary bacteria.Chemotrophic bacterial sequences (e.g.Blastopirellula (Schlesner 2015) or Rhodopirellula (Bondoso et al. 2014)) were found at all sites in relatively high abundance, suggesting widespread and active chemosynthesis, though the lack of a particularly dominant bacterial group associated with chemosynthetic activity suggested that the supply of chemosynthetic OM was likely relatively limited.Some PLFAs also had marked differences in δ 13 C signatures, even where there was strong compositional similarity between sites (i.e. the non hook ridge sites).This suggested that either there were differences in the isotopic values of inorganic or organic matter sources or that different bacterial metabolic pathways were active.Between the non-vent sites, these PLFAs included PUFAs such as 18:2ω6, 9 (Δδ 13 C 24.36  (Colaço et al. 2007, Klouche et al. 2009, Boschker et al. 2014) and their presence is consistent with the hydrothermal signature of the sediment microbial community.However, it should be stressed that all PLFAs with larger δ 13 C ranges were comparatively rare and never individually exceeded 5% of total abundance.This provides further evidence of limited chemosynthetic activity at all sites and is consistent with the presence of bacteria associated with methane and sulphur cycling.Microbial signatures, whilst supporting the suggestion of chemosynthetic activity, are not indicative of chemosynthetic OM being the dominant source of organic matter to food webs at any site (hypothesis four).
Siboglinum sp.δ 13 C values (mean -41.43, range -45.73 to -38.10 ‰, n = 8) corresponded very closely to published values of thermogenic methane (-43 to -38 ‰) from the Bransfield Strait (Whiticar & Suess 1990).Biogenic methane typically has much lower δ 13 C values (Whiticar 1999), indicating a hydrothermal source of methane in the Bransfield Strait.Sulphur isotopic signatures were also strongly depleted in Siboglinum sp.(δ 34 S -22.85 ‰, one sample from 15 pooled individuals from the off-axis site), the most depleted measurement of δ 34 S reported for this genus (Schmaljohann & Flügel 1987, Rodrigues et al. 2013).The depleted δ 13 C, δ 15 N and δ 34 S signatures of Siboglinum sp.suggest that its symbionts most likely included methanotrophs, sulphate reducers and diazotrophs (Boetius et al. 2000, Canfield 2001, Dekas et al. 2009).Methanotrophy in Siboglinum spp.has been previously documented at seeps in the NE Pacific (Bernardino & Smith 2010) and Norwegian margin (δ 13 C = -78.3 to -62.2 ‰) (Schmaljohann et al. 1990) and in Atlantic mud volcanoes (δ 13 C range -49.8 to -33.0 ‰) (Rodrigues et al. 2013).Sulphur isotopic signatures in Siboglinum spp.from Atlantic mud volcanoes ranged between -16.8 to 6.5 ‰ (Rodrigues et al. 2013) with the lowest value still being 6 ‰ greater than that of Bransfield strait specimens.Rodrigues et al. (2013) also reported a greater range in δ 15 N than observed in the Bransfield siboglinids (δ 15 N -1.3 to 12.2 ‰ and -10.16 to -7.63 ‰ respectively).This suggests that, in comparison to Siboglinum spp. in Atlantic Mud volcanoes, which seemed to be using a mixture of organic matter sources (Rodrigues et al. 2013), the Bransfield specimens relied much more heavily upon a single OM source, suggesting considerable trophic plasticity in this genus worldwide.Off-vent methanotrophy, using thermogenic methane, (Whiticar & Suess 1990) potentially illustrates an indirect dependence upon hydrothermalism.Sediment methane production is thought to be accelerated by the heat flux associated with mixing of hydrothermal fluid in sediment (Whiticar & Suess 1990) and thus, sediment and Siboglinum isotopic data suggest that the footprint of hydrothermal influence may be much larger than previously recognised, giving rise to transitional environments (Levin et al. 2016, Bell et al. Accepted).Clear contribution of methane-derived carbon to consumer diets was limited predominately to neotanaids, consistent with the relatively small population sizes (64 -159 ind.m 2 ) of Siboglinum sp.observed in the Bransfield Strait (Bell et al. 2016).

Organic Matter Sources
Pelagic salps, collected from an Agassiz trawl at Hook Ridge (1647m), were presumed to most closely represent a diet of entirely surface-derived material and were more depleted in δ 13 C and more enriched in 34 S than sediments (Table 3).Salp samples had a mean δ 13 C of -27.43 ‰, which was also lighter than the majority of macrofauna, both at Hook Ridge and the non-vent sites (Fig. 2) and similar to other suspension feeding fauna in the Bransfield Strait (Elias-Piera et al. 2013).This suggests that fauna with more depleted δ 34 S/ more enriched δ 13 C values are likely to have derived at least a small amount of their diet from chemosynthetic sources, both at vents and background regions.Carbon and sulphur isotopic measurements indicated mixed sources for most consumers between chemosynthetic OM and surface-derived photosynthetic OM.Non-vent sediments were more enriched in 34 S than vent sediments, an offset that probably resulted from greater availability of lighter sulphur sources such as sulphide oxidation at Hook Ridge.Samples of bacterial mat could not be collected during JC55 (Tyler et al. 2011) and without these endmember measurements, it was not possible to quantitatively model resource partitioning in the Bransfield Strait using isotope mixing models (Phillips et al. 2014).Bacterial mats from high-temperature vents in the Southern Ocean had δ 34 S values of 0.8 ‰ (Reid et al. 2013) and at sedimented areas of the Loki's Castle hydrothermal vents in the Arctic Ocean has δ 34 S values of -4.9 ‰ (Bulk sediment; Jaeschke et al. 2014).Therefore it is probable that depleted faunal δ 34 S values represent a contribution of chemosynthetic OM (from either siboglinid tissue or free-living bacteria).Inorganic sulphur can also be a source to consumers when sulphide deposits are utilised by free living bacteria (δ 34 S ranged -7.3 to 5.4 ‰ (Erickson et al. 2009)) and sulphide crusts have been found at Hook Ridge (δ 34 S -28.1 to 5.1 ‰ (Petersen et al. 2004)).There were several species (e.g.Tubificid oligochaetes) that had moderately depleted δ 34 S signatures.Limnodriloides sp. had distinct δ 34 S signatures between sites (7.56 ‰ at vents, -1.21 ‰ at non-vents, Fig. 4) further supporting the hypothesis of different trophic positions between vent/ non-vent regions (hypothesis two).This provides evidence of coupled AOM/sulphate reduction but overall, the contribution of δ 34 S-depleted bacterial production did not seem widespread (further rejecting hypothesis four).
Without samples of all OM sources we cannot quantitatively assert that faunal utilisation of chemosynthetic OM was low in the Bransfield Strait.Although isotopic data were consistent with several OM sources, it seemed unlikely that chemosynthetic OM was a dominant source of OM to the vast majority of taxa.The apparently limited consumption of chemosynthetic OM suggested that either it was not widely available (e.g.patchy or low density of endosymbiontbearing fauna (Bell et al. 2016)), or that the ecological stress associated with feeding in areas of Biogeosciences Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -318, 2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License. in situ production was a significant deterrent to many species (Bernardino et al. 2012, Levin et al. 2013).

A-priori vs. a-posteriori trophic groups
Morphology did not prove to be an accurate predictor of trophic associations, suggesting that faunal behaviour is potentially more important in determining dietary composition than morphology (e.g.having/ lacking jaws).Peracarid species that possessed structures adapted to a motile, carnivorous lifestyle were assigned to a carnivore/ scavenger guild (Bell et al. 2016) but were distributed throughout the food web both at vents and background regions, indicating more diverse feeding strategies than expected.Taxa presumed to be deposit feeders (largely annelids) also had a surprisingly large range of δ 15 N values.This may reflect the consumption of detritus from both 'fresh' and more recycled sources as observed in other nonvent sedimented deep-sea habitats (Iken et al. 2001, Reid et al. 2012) or reflect variability in trophic discrimination related to diet quality (Adams & Sterner 2000).The result is high δ 15 N values in taxa without predatory morphology (e.g.oligochaetes.Tubificid oligochaetes had higher δ 15 N values at the vent sites, suggesting that they fed upon more recycled organic matter, possibly owing to greater microbial activity at vent sites.Bacterial biomass was very variable at the vent sites (86 -535 mg C m -2 , compared with 136 -197 at non-vent sites; Table 2) and so it is possible that at Hook Ridge 1 bacterial assemblages could have had a greater influence upon δ 15 N of organic matter.
Neotanaids from the off-axis site had the most depleted δ 13 C and δ 15 N values of any nonsiboglinid taxon (Fig. 3), suggesting a significant contribution of methane-derived carbon.The clustering of the neotanaids together with endosymbiont-bearing taxa is far more likely to be Biogeosciences Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -318, 2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.
an artifact of the cluster linkage method, introduced by consumption of low δ 13 C methanotrophic sources (e.g.Siboglinum tissue), rather than suggesting symbionts in these fauna (Larsen 2006, Levin et al. 2009).
Several taxa (e.g.neotanaids from the off-axis site and ophiuroids at Hook Ridge) had low δ 15 N values relative to sediment OM, suggesting preferential consumption of chemosynthetic OM (Rau 1981, Dekas et al. 2014).In these taxa, it is likely that the widespread, but patchy bacterial mats or Sclerolinum populations at Hook Ridge (Aquilina et al. 2013) was an important source of organic matter to fauna with low δ 15 N values (e.g.ophiuroids).Fauna from the non-vent sites with low δ 15 N were likely subsisting in part upon siboglinid tissue (Siboglinum sp.).There were no video transects over the off-axis site but footage of the Three Sisters (similar in macrofaunal composition (Bell et al. 2016)) did not reveal bacterial mats (Aquilina et al. 2013), hence it is unlikely that these were a significant resource at non-vent sites.
It is clear that some fauna can exhibit a degree of trophic plasticity, depending upon habitat (supporting hypothesis two).This is consistent with other SHVs where several taxa (e.g.Prionospio sp.-Polychaeta: Spionidae) had different isotopic signatures, depending upon their environment (Levin et al. 2009), demonstrating differential patterns in resource utilisation.
Alternatively, there could have been different δ 15 N baselines between sites, though if these differences were significant, we argue that it likely that more species would have had significant differences in tissue δ 15 N. Standard ellipse area was lower at Hook Ridge than at non-vent sites (Table 4), analogous to trends in macrofaunal diversity and abundance (Bell et al. 2016).This demonstrates that at community level, SEA.B is associated with other macrofaunal assemblage characteristics.This concurrent decline in niche area and alpha diversity is consistent with the concept that species have finely partitioned niches and greater total niche area permits higher biodiversity (McClain & Schlacher 2015).Productivity-diversity relationships, whereby higher productivity sustains higher diversity, have also been suggested in the deep-sea (McClain & Schlacher 2015, Woolley et al. 2016) but in the absence of measurements of in situ organic matter fixation rates at Hook Ridge, it is unclear whether such relationships exist in the Bransfield Strait.Sediment organic carbon content was similar between Hook ridge 1 and non-vent sites (1.35 -1.40 %) but was slightly lower at Hook Ridge 2 (0.97 %) (Bell et al. 2016), which is not consistent with variation in niche area.The decline in alpha diversity and niche area is consistent with the influence of disturbance gradients created by hydrothermalism that result in an impoverished community (McClain & Schlacher 2015, Bell et al. 2016).We suggest that, in the Bransfield Strait, the environmental toxicity at SHVs causes a concomitant decline in both trophic and species diversity (Bell et al. 2016), in spite of the potential for increased localised production.
Community-based trophic metrics (Layman et al. 2007) indicated that, although measures of dispersion within sites were relatively similar between vents and background areas (Table 4), trophic diversity, particularly in terms of range of carbon sources (dCr) and total hull area (TA) was higher at background sites.It was expected that trophic diversity would be greater at Hook Ridge but the greater dCr at non-vent sites (owing to the methanotrophic source) meant that the size isotopic niches at these sites was greater.Range in Nitrogen values (dNr) was also greater at non-vents, driven by the more heavily depleted δ 15 N values of Siboglinum sp. the niche space (where E = 0 corresponds to an equal influence of both carbon and nitrogen) 618 whereas theta (the angle of the long axis) determines which, if any, isotope is most influential 619 in determining ellipse characteristics (Reid et al. 2016).For the non-vent sites, the dominant 620 isotope was carbon, owing to the relatively light δ 13 C of methanotrophic source utilised by 621 Siboglinum.Some sites, particularly the Axe, had several fauna with heavy δ 13 C values (Fig. 5), 622 which could be explained by either contamination from marine carbonate (~0 ‰), as 623 specimens were not acidified, or a diet that included a heavier source of carbon, such as sea ice 624 algae (Henley et al. 2012)
Biogeosciences Discuss., doi:10.5194/bg-2016-318,2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.Averaged species isotopic data were each assigned to one of four clusters (SIMPROF, p = 0.05; Supplementary Figure 3).No significant correlation between a-priori (based on morphology) and a-posteriori clusters (based on isotopic data) was detected (Spearman Correlation Test: Z = -1.34;N = 43; p = 0.18) and consequently, we reject hypothesis one (trophic position determined by morphology).Clusters were mainly discriminated based on δ 15 N values and peracarids were the only taxa to be represented in all of the clusters, indicating high trophic diversity.
Differences in eccentricity are more influenced by the spread of all isotopes used to construct Biogeosciences Discuss., doi:10.5194/bg-2016-318,2016 Manuscript under review for journal Biogeosciences Published: 31 August 2016 c Author(s) 2016.CC-BY 3.0 License.

Fig. 2 -Fig. 3 -
Fig. 1 -Microbial composition (classes) at the off-vent/ off-axis site (BOV) and the two Hook 921Ridge sites (HR1 and HR2).Archaea excluded from figure as they only accounted for 0.008 % 922 of reads at HR2 and were not found elsewhere.923