Fluid chemistry alters faunal trophodynamics but not composition on the deep-sea Capelinhos hydrothermal edifice (Lucky Strike vent field, Mid-Atlantic Ridge)

The recently discovered deep-sea Capelinhos hydrothermal edifice, ~ 1.5 km of the main Lucky Strike (LS) vent field (northern Mid-Atlantic Ridge), contrasts with the other LS edifices in having poorly-altered end-member hydrothermal fluids with low pH and chlorine, and high metal concentrations. Capelinhos unique chemistry and location offer the opportunity to test the effects of local abiotic filters on faunal community structure while avoiding the often-correlated influence of dispersal limitation and depth. In this paper, we characterize for the first time the distribution patterns of the Capelinhos faunal communities, and analyze the benthic invertebrates (> 250 µm) inhabiting diffusive-flow areas and their trophic structures (δ13C, δ15N and δ34S). We hypothesized that faunal communities would differ from those of the nearest LS vent edifices, showing an impoverished species subset due to the potential toxicity of the chemical environment. Conversely, our results show that: (1) community distribution resembles that of other LS edifices, with assemblages visually dominated by shrimps (close to high-temperature focused-fluid areas) and mussels (at low-temperature diffuse flow areas); (2) most species from diffuse flow areas are well-known LS inhabitants, including the bed-forming and chemosymbiotic mussel Bathymodiolus azoricus and (3) communities are as diverse as those of the most diverse LS edifices. On the contrary, stable isotopes suggest different trophodynamics at Capelinhos. The high δ15N and, especially, δ13C and δ34S values suggest an important role of methane oxidation (i.e., methanotrophy), rather than the sulfide oxidation (i.e., thiotrophy) that predominates at most LS edifices. Our results indicate that Capelinhos shows unique environmental conditions, trophic structure and trophodynamics, yet similar fauna, compared to other LS edifices, which suggest a great environmental and trophic plasticity of the vent faunal communities at the LS.

Understanding the processes structuring faunal assemblages is at the core of community ecology 1 .Regional species pools are determined by speciation, extinction and dispersion interacting with smaller-scale abiotic and biotic processes that ultimately determine species coexistence and community structure at local habitats [2][3][4] .Assessing these community assembly processes is key to predict disturbance impacts on, and resilience of, ecosystems 5 .
In 1977, lush taxon-novel communities fueled by in situ microbial chemoautotrophy were discovered in the Galapagos Rift hydrothermal vents at 2550 m depth 6,7 .Deep-sea vents are often dominated by large foundation chemosymbiotic species not found elsewhere, which promote the establishment of smaller invertebrates 8,9 .Overall, this gives rise to highly productive, biomass-rich communities, orders of magnitude denser than those of the surrounding, energy-limited deep sea (reviewed in [10][11][12] ).More than four decades of exploration have

Habitat characterization based on ROV imagery
Capelinhos edifice has a steep profile with two main structures (Fig. 1, Supplementary material Video S1).The first structure has several "candelabra-like" chimneys, some emitting highly-focused hydrothermal fluids (Fig. 1B,C, Supplementary material Video S1-1:30 min).These chimneys are densely colonized by white microbial mats and sparse shrimp aggregations (probably M. fortunata) close to the focused fluids (Fig. 1C, Supplementary material Video S1-5:00 min).Dense mussel aggregations (including patches of juveniles) and microbial mats occur at the base.The second structure is a single, large, senescent Y-shape chimney.The limited diffuse-flow area found at the top is colonized by a small patch of large mussels (Fig. 1D, Supplementary material Video S1-7:13 min).The base of this structure is colonized by dense mussel beds (covered or not by microbial mats), especially on one of its sides (Fig. 1E, Supplementary material Video S1-8:40 min).The few flanges at the base of each main structure are colonized by large mussels and shrimps (Fig. 1F, Supplementary material Video S1).Crabs, probably Segonzacia mesatlantica (Williams, 1988), and shrimps were present in between and over the mussels (Fig. 1F, Supplementary material Video S1-9:36).Some fishes occur at the vicinity of the chimneys and in peripheral areas (Fig. 1G, Supplementary material Video S1-10:00 min).

Environment, community composition and species diversity
Temperatures over mussel assemblages range from 4.32 to 9.88 °C (Table 1 and Supplementary material Fig. S1).Between-sample variability is relatively high, despite the close proximity of the probes (~ 50 cm), as observed for Fe(II) (0.98-2.40 µM) and even more for ƩS (21.80-44.8µM) (Table 1; Supplementary material Table S2 and Fig. S1).Compared with the habitats colonized by B. azoricus at the Eiffel Tower edifice, the temperatures at Capelinhos are similar to those in cold and intermediate-temperature habitats, while the high Fe(II) and, especially, ƩS concentrations are more similar to those of the warm habitats, highlighting Capelinhos distinct fluid chemistry (Table 1, Supplementary material Table S2 and Fig. S2).

Discussion
Contrary to our expectations, our results suggest a negligible role of the putatively more toxic chemistry of Capelinhos in the distribution and structure of faunal communities compared to those at other LS edifices.The distribution of assemblages along the hydrothermal gradient match the archetypal distribution observed in LS.
For instance, shrimps, likely M. fortunata, where observed at the warmest habitats close to focused fluids, whereas B. azoricus beds were observed at lower-temperature, fluid-diffusive habitats (4.32-9.88°C), as described for the Eiffel Tower edifice 44,[57][58][59][60] .All species found at Capelinhos lower temperature diffusive-fluid habitat, including several taxa identified to genus level and/or as morphotypes (Alfaro-Lucas pers.obs.), inhabit the other LS edifices, sharing the dominance of B. azoricus 28,39,59,61 .Species abundances also suggest a similar assemblage structure at Capelinhos and at the LS Eiffel Tower and Montségur edifices 44,54,55,59 .Specifically, the dominance of B. azoricus, B. seepensis and A. lutzi, as observed at Capelinhos, is indicative of lower-temperature mussel-bed habitats in the Eiffel Tower edifice 59 .The putative harsher chemical environment linked to Capelinhos fluids does not limit species diversity at mussel beds, as hypothesized, being instead comparable to those in the most diverse LS edifices 44 , as supported by the rarefied diversities.Similar results have been reported in mussel beds and tubeworm bushes on the East Pacific Rise, where the assemblages from different chemical environments exhibit similar diversities and community structures 62,63 .At LS, B. azoricus and its associated species colonize lower temperature-habitats within edifices with different chemistries, highlighting their environmental 44 and trophic 64,65 plasticity.However, iron concentrations in the end-member fluid at Capelinhos are the highest of LS (2789.4 ± 84.8 μM) 52,53 , leading to higher iron concentrations in the mixing gradient between seawater and hydrothermal fluid at Capelinhos than to other LS sites 66,67 .Very diluted environments characterized with strong lateral entrainment of seawater are trickier to characterize.Nevertheless, chemical analyses in our study revealed that Fe(II) and ƩS concentrations in cooler diffuse flow areas tend to be higher in Capelinhos than at similar areas with similar temperature and similar mussel assemblages at Eiffel Tower and Montségur edifices (LS) 54,59 .High metal concentrations are hypothesized to limit and even prevent colonization by the species of Bathymodiolus at some nMAR vents 27 .However, our results suggest that neither B. azoricus, nor the associated fauna, are limited at Capelinhos lower temperature-habitats, highlighting their ability to colonize a wide range of chemical environments.The complex 3D biogenic habitat created by B. azoricus likely promotes small scale turbulence and enhances fluid dilution with seawater, buffering against harsh and concentrated chemical environment and fostering a similarly structure of associated fauna 44 , as observed for other vent foundation species elsewhere 9,62,63 ., and a limited abundance of B. azoricus, which are covered with orange metal deposits as observed at Capelinhos (J.Sarrazin pers.comm.).However, species similarity was higher between Capelinhos, LS and Menez Gwen (800 m depth, ~ 100 km from LS) than between Capelinhos and Rainbow, which agrees with recently results showing higher similarity between LS and Menez Gwen 41 .This result could suggest that processes acting at broader scales, such as species dispersal limitation shaping the available colonizing species, may play a stronger role in governing the species composition of mussel assemblages along the ridge 44 than high iron concentrations 27 .In fact, the species diversity of B. azoricus assemblages at Rainbow is similar to those from the richest LS edifices, including Capelinhos, reinforcing the idea that high metal concentration alone may not necessarily lead to a decrease in species diversity.
Despite the similar faunal composition and structure, stable isotopes reveal profound trophodynamic differences between Capelinhos and other LS edifices.nMAR vent communities usually present an upward-diagonallike δ 13 C/δ 15 N structure, with two rather independent compartments: (1) B. azoricus and B. seepensis (with the most negative δ 13 C/δ 15 N isotopic values) being isolated in one end, and (2) bacterivores, detritivores, scavengers and predators (with less negative δ 13 C and positive δ 15 N isotopic values) being spread at the other extreme (e.g., 55,64,65,70 ).Conversely, Capelinhos shows: (1) a rhombus-like δ 13 C/δ 15 N structure, with B. azoricus and B. seepensis at the bottom, the predator G. tesselata at the top, and the grazers P. midatlantica and D. briandi in the left and right corners, respectively; (2) a not so evident isolation of B. azoricus and B. seepensis because dirivultid copepods and the Branchipolynoe found outside their hosts showed closed isotopic values, and (3) an overall less negative δ 13 C and more positive δ 15 N isotopic values, particularly in B. azoricus and B. seepensis.Notable Table 2. Relative mean (± sd) abundance (N) and isotopic composition (δ 13 C, δ 15 N and δ 34 S) of species found at the Capelinhos hydrothermal structure, 1.5 km off the Lucky Strike vent field (nMAR).ET column indicates if the species (S) or morphotype (M) is found at Eiffel Tower edifice in the main Lucky Strike (Alfaro-Lucas et al. 55 ; J.M. Alfaro-Lucas pers.obs.).In brackets the number of samples used to estimate the isotopic composition of species.exceptions are the shrimps A. markensis and M. fortunata that showed similar δ 13 C/δ 15 N isotopic composition between edifices.Less negative δ 13 C values may be indicative of the reductive tricarboxylic acid (rTCA) cycle rather than the Calvin-Benson-Bassham (CBB) cycle 65 .However, rTCA is usually associated with vigorous-fluid flux rather than lower temperature habitats 65 .Furthermore, less negative δ 13 C and more positive δ 15 N ratios have been also observed for B. azoricus at Sintra, a LS edifice where methane is available in higher concentrations 71 .This suggests that methane oxidation (methanotrophy) could be an important energetic pathway at Capelinhos, in addition to CCB (thiotrophy).Although not as evident as in our study, Rainbow assemblages of B. azoricus show a similar rhombus-like δ 13 C/δ 15 N structure, less negative δ 13 C and more positive δ 15 N isotopic values, which have been also attributed to the higher contribution of methanotrophy 65,69 .
The δ 34 S values of Capelinhos fauna, more positive than that of Eiffel Tower, also support the methanotrophy hypothesis.Values over 10‰ are usually associated to consumption of photosynthetic organic matter 72 .However, this source has been consistently discarded in nMAR vent food webs 70 , including those at LS 55,64,65 .Furthermore, Capelinhos is roughly at the same depth and only ~ 1.5 km apart from the other LS edifices.This and the overall low δ 15 N (− 4.70 to 9.11‰) allows discarding a depth/location effect on a putative increase in photosynthetic organic matter suggesting instead chemosynthetic pathways.Methanotrophy instead of thiotrophy may lead to more positive δ 34 S values 73 and Capelinhos fluids are naturally enriched in 34 S, reflecting contrasting subseafloor fluid/rock interactions compared to the other LS edifices 74 .Thus, we suggest the more positive δ 34 S values at Capelinhos are driven by methanotrophy and geological processes influencing fluid composition.Further geochemical and microbiological studies are required to disentangle these intriguing isotopic results, including the measurement of Capelinhos methane concentrations in both end-member and diffuse fluids, and evaluation of the composition of microbial communities compared to those found at other LS edifices.Broadly, our isotopic results: (1) highlight the necessity of characterizing isotopic sources while analyzing multiple isotopes to confidently interpret trophodynamics at chemosynthetic-based habitats, and (2) support the hypothesis that B. azoricus and its associated fauna do not depend on specific trophic pathways or food sources, but rather have a great trophic plasticity allowing them to colonize a wide array of contrasting chemical environments, as previously postulated 44,65,75,76 .

Study area, sampling and sample processing
Capelinhos is located ~ 1.5 km to the east of the main Lucky Strike (LS) area (nMAR) at 1665 m depth (37.28917N, − 32.26388 E) 52 (Fig. 1A) (not to be confused with the Capelinhos volcano on the Faial Island, Azores Archipelago 77 ).The LS hydrothermal vent field (~ 1700 m depth), discovered in 1992, is located on the Lucky Strike Seamount on the Azores Triple Junction 61,78 .It is a basalt-hosted vent field fueled by a magmatic www.nature.com/scientificreports/chamber located at 3-3.5 km depth 78,79 .Hydrothermal activity occurs at > 20 sulfide edifices/structures located around a fossil lava lake of ~ 300 m in diameter situated in between three ancient volcanic cones 78,79 .Edifices emit both high-temperature focused-fluids, ranging from 200 to 340 °C, and low-temperature diffuse venting, and show distinct fluid chemical compositions 52,53 .Nevertheless, evidence suggest a common reaction zone for the entire field, including Capelinhos 53 .
Capelinhos was revisited during the Momarsat 2014 (https:// doi.org/ 10. 17600/ 14000 300) and 2015 (https:// doi.org/ 10. 17600/ 15000 200) cruises on board of the R/V Pourquoi Pas? which visually inspected chimneys, diffuse flow areas and the periphery using the ROV Victor6000.In 2014, three temperature probes were deployed on a large-size mussel bed on a diffuse flow area at the base of one edifice.The probes registered temperatures every 15 min for 9 months, form the 24th July 2014 to the 20th/23rd April 2015.In 2015, prior to faunal sampling associated to mussels, sulfide (ƩS) and iron (Fe(II)) concentrations were measured in situ using the chemical analyzer CHEMINI 80 over the targeted mussel bed.Biological samples were collected using Victor6000's manipulator arm (three to four grabs per sample) and placed in isotherm boxes.After grabs, a suction sampler was used on each sampling area to collect all remaining fauna.Sampled area was estimated analyzing Victor6000's videos with the software ImageJ.Once on board, samples were sieved through 250 µm (directly fixed in 96° ethanol) and 20 µm (fixed with 4% buffered formalin, then in 96° ethanol, not considered in this study) mesh-sizes.Individuals were sorted, identified to the lowest taxonomic level possible using stereo-and binocular microscopes (except for mussel specimens, which were barcoded to identify the species), and counted (only individuals with complete anterior regions).Meiofaunal organisms (e.g., copepods and nematodes) found in the macrofaunal samples were included in the analyses.

Biodiversity and community composition
We estimated species diversity by individual-based rarefaction of Hill numbers (D) 82 of q orders 0, 1 and 2, respectively.D expresses diversity in effective species number, i.e., the number of equally-common species that would represent the observed diversity, thus overcoming the problems of expressing indexes in different scales and respecting the "replication principle" [82][83][84] .When q = 0, 0 D is the species number giving equal weight to abundant and rare species 82,83 .When q = 1, 1 D is the exponential of Shannon index 85 weighting species proportionally to their abundances and providing the effective number of common species in the assemblage 82,83 .When q = 2, 2 D is the Simpson index 86 , which gives more weight to the dominant species and provides the effective number of dominant species in the assemblage 82,83 .We compared Capelinhos rarefied diversities ( 0 D, 1 D and 2 D) to those from Cypress, Y3, Eiffel Tower and Montségur (LS), Atos (Menez Gwen), Thermitière and France (Rainbow) edifices using raw data from Sarrazin et al. 44 .Prior to comparisons, we removed copepod, nematode and ostracod taxa since we identified them at higher taxonomic resolutions than in previous studies.We estimated rarefaction curves and 95% confidence intervals for 1050 individuals and then plotted them using the iNEXT function of the iNEXT package 87 in R V.4.0.2 environment 88 .We compared Capelinhos species composition to those of LS, Menez Gwen, Broken Spur, Rainbow, TAG, Snake Pit, Logatchev and Ashadze-1 nMAR vent fields to disentangle biogeographic affinities based on the presence/absence dataset (including meiofauna) provided by Boschen-Rose and Colaço 41 .For consistency, we removed all taxa not identified at the species level.Moreover, we updated this dataset by including the annelid Ophryotrocha fabriae Paxton & Morineaux, 2009 and the nematode Oncholaimus dyvae Zeppilli et al., 2019, since both species inhabit the LS vent field 55 .We then computed a β-diversity dissimilarity distance matrix using Simpson's pairwise dissimilarity metric (β sim ), which ranges between 0 (no species compositional differences) to 1 (totally dissimilar species composition) 89 .β sim is independent of species richness differences and thus, only accounts for species turnover: it does not identify natural species-poor or unevenly-sampled communities as being highly dissimilar 89,90 .We computed β sim using the function beta.pair in the package betapart in R 91 .We performed a hierarchical cluster analysis (HCA) using the Average-linkage cluster algorithm, the β sim dissimilarity distance matrix and a multiscale bootstrap resampling to calculate the Approximately Unbiased p-values (AU-P) to identify robust clusters, with significant AU-P being set at > 95 92 .We estimated the bootstrap by repeatedly and randomly sampling sites performing the HCA by using the function pvclust in the package pvclust in R 92 .

Figure 1 .
Figure 1.(A).Main hydrothermal fields in the northern Mid-Atlantic Ridge; Capelinhos is located ~ 1.5 km off the main Lucky Strike field.Capelinhos habitats.(B).Active and inactive black smokers with microbial mats at the summit of the structure.(C).Shrimp aggregations (red arrows) probably belonging to Mirocaris fortunata on top of a black smoker.(D).Top of a senescent chimney with a patch of Bathymodiolus azoricus mussels (red arrow) in between its two heads.(E).Dense B. azoricus beds and microbial mats at the base of a senescent chimney.F. Patch of large B. azoricus with sparse shrimps; the CHEMINI analyzer inlet and our checkerboard calibrated target are visible.(G).Cataetyx-like fish (red arrow) on sulphide rubble surroundings.Map made with GeoMapApp 3.6.15(www.geoma papp.org)/CC BY/CC BY (Ryan et al. 51 ).

Figure 3 .
Figure 3. Rarefaction curves and 95% confidence intervals (shaded areas) based on the abundance of the communities at different hydrothermal edifices on the northern Mid-Atlantic Ridge.(A) Species richness.(B) Shannon index.(C) Simpson index.LS, Lucky Strike; MG, Menez Gwen and RB, Rainbow vent fields.LS, MG and RB data from Sarrazin et al.44.

Figure 4 .
Figure 4. Hierarchical cluster analysis of vent communities at hydrothermal fields and Capelinhos edifice (off the main Lucky Strike vent field) on the northern Mid-Atlantic Ridge.Red numbers: Approximately Unbiased p value.Data modified from Boschen-Rose and Colaço 41 .