Baseline assessment of mesophotic reefs of West South Atlantic seamounts based on water quality, microbial diversity, benthic cover and fish biomass data

Water quality Water samples were collected by divers within the benthic boundary layer, up to 1 m from the bottom, using two Niskin bottles (10 L each). At least three replicates from Niskin bottles were obtained for each parameter in each of the eight locations. Chlorophyll-a, inorganic nutrients, dissolved organic carbon (DOC), Phosphorus and Nitrogen concentrations and microbial abundance was determined. Chlorophyll a samples were collected using positive pressure filtration of 2 L of water. The filters (Millipore HAWP) were extracted overnight in 90% acetone at 4°C and analyzed by spectrophotometry or fluorimetry. For the inorganic nutrients analyses 1 L of water was frozen and was analyzed at laboratory using the following methods: 1) ammonia by indophenol, 2) nitrite by diazotization, 3) nitrate by reduction in Cd - Cu column followed by diazotization, 4) total nitrogen by digestion with potassium persulfate following nitrate determination, 5) orthophosphate by reaction with ascorbic acid, 6) total phosphorous by acid digestion to phosphate, and 7) silicate by reaction with molybdate. Dissolved organic carbon (DOC), Phosphorus and Nitrogen (shown in Table 1 the sum between the organic and inorganic P and N as Total-P and Total-N, respectively). Microbial abundance was determined from three replicates of seawater per site by flow cytometry with Sybr-green (Life Technologies).

Metagenomics Collected seawater was filtered through Sterivex (0.22 μm) by positive pressure. In total 4 L of seawater were filtered in each Sterivex filter. Microbes collected in Sterivex filters were preserved with SET buffer (20% sucrose, 50 mM EDTA and 0.5 mM Tris-HCl) in liquid nitrogen until DNA extraction procedures at laboratory. Seawater metagenomic DNA extraction was performed following alkaline lysis as previously described 21. Collected corals were also stored in liquid nitrogen until DNA extraction procedures. A protocol using CTAB buffer with 100 mM EDTA and the PowerSoil® purification column was used in order to gather high molecular weight of DNA from small fragment of each coral sample (approx. 1 cm2). High quality DNA extracted from Sterivex filters and from corals was sequenced using GS FLX Titanium kit (Roche) a pyrosequencing technology.

Benthic cover In order to recognize the main topographic features of seamounts’ tops, we used a sidescan sonar (EdgeTech 4100, 100–500kHz) across a linear extent of 110 km with 400 m swaths, covering the Trindade Island shelf and upper slope, as well as in the summits of seamounts Jaseur and Davis. Sonograms were processed with SonarWiz Map4 (V.4.02). Benthic cover was estimated following procedures. Briefly, at each site ten 0.7 m2 photoquadrats were randomly placed. Percent cover was estimated using Coral Point Count with Excel Extension software (CPCe), using 15 randomly distributed points per photograph (225 points per quadrat). Organisms below each point were identified considering the following major benthic categories: turf algae (subdivided into: Jania plus Amphiroa plus other small filamentous algae, and the cyanobacteria Lyngbya sp.), sand, sponge, flashy algae 49, coral (mainly Siderastrea sp., Montastrea cavernosa and the Brazilian endemic Mussismilia hispida. Coral colonies health status were qualified as healthy, presence of tissue necrosis or bleaching) and Crustose coralline algae (CCA) (subdivided into Peyssonelia sp. and others, the latter including mainly Hydrolithon onkodes, Lithophyllum prototypum, Phymatolithon masonianum, Spongites sp.). Reference samples of algae are deposited in the collection of the Rio de Janeiro Botanical Garden Herbarium (RB).

Fish assemblages surveys Fish assemblages were assessed from video records using a remote operated vehicle (ROV) Seabotix® LBV 150S2 equipped with lights and a color video camera and a pair of scaling lasers 5 cm apart (used to estimate fish sizes), and video cameras handled by divers using standard SCUBA (<40 m deep) and mixed-gas techniques (TRIMIX) in open systems (>40 m deep). Fish samples were obtained in the same sites in which water samples were obtained, ranging 25 and 63 m deep. Both ROV and divers records were made in slow movement (not static), close the bottom (about 1 m), and focusing in all available habitats (rhodoliths, interface, reefs, water column) aiming to record the entire reef fish community. Fish counts were performed from frames taken every 10 seconds of video footage. A total of 546 frames from ROV and divers records were used (180 in Trindade Island, 275 in Davis seamount and 91 in Jaseur seamount). Size of fish (total length, TL) was visually estimated using the laser scale and individuals were classified into 10 cm size classes. Fish biomass was estimated using length-weight relationships 50. When no relationship was available for a species, an equation from similarly sized congeners was applied. Fish species were assigned to one of the following trophic guilds based on adult diet literature data 51: herbivores, invertivores, macro carnivores and omnivores. Fish results are presented as relative abundance [number of fishes from a category (size or trophic guild)/total number of fishes from the site] and biomass (Kg/frame).