A. SAMPLE PROCESSING

Sampling was conducted across six main geological features in the Pampa Submarina region off the Pacific coast of Costa Rica, including several hills, knolls, a seamount, and the surrounding abyssal plain. Biological collections were carried out opportunistically during ROV SuBastian dives, guided by real-time observations of biodiversity and habitat characteristics. Specimens were primarily collected using the ROV’s manipulator arms, while ROV nets or the slurp sampler were also used to retrieve delicate or mobile organisms.

Sampling aimed to capture a broad representation of taxonomic diversity across habitats and depth ranges, and will be used to assist with the identification of species in the ROV imagery collected. During the second cruise, efforts focused on collecting specimens that had not been sampled during the first expedition, to complement the existing collection and expand the taxonomic coverage of deep-sea fauna from the region.

Before each ROV recovery, the dive lead designated the team responsible for sample handling and preparation. The wet lab was organized in advance with labeled buckets, appropriate preservative solutions, and sample vials and containers. Upon ROV recovery, samples were transferred carefully into labeled buckets filled with chilled seawater or seawater from the ROV bioboxes in an attempt to maintain their condition. Specimens were handled with clean gloves to prevent contamination or physical damage. All samples were transferred to the cold lab to await further processing.

All specimens were photographed in the wet lab following standardized imaging protocols to ensure consistent documentation as soon as possible after recovery. A clean black or white matte background was used for all photographs, with uniform lighting, a measurement ruler and its label included in each frame. Each specimen was photographed with its label containing its unique sample code clearly visible, capturing dorsal, ventral, and lateral views. Preservation methods were tailored to specific taxa to optimize molecular and morphological integrity (see Preservation Methods below).

Subsampling was performed only after whole-specimen photography was completed. Each subsample received a unique code and was recorded in the shared Google Drive database. Subsamples were also photographed individually, using their respective suffix identifiers (e.g., ...001 in recordNumber. See Labelling section below).

Tools, mats, and equipment were cleaned with ethanol between samples to prevent cross-contamination. Image filenames were cross-referenced with entries in the biological spreadsheet to maintain data traceability between photographs and specimen metadata.

B. PRESERVATION METHODS

Cephalopods: When still alive upon recovery, individuals were euthanized with a 3% ethanol–seawater solution. Muscle tissue was preserved in 95% ethanol for molecular analyses, while additional tissues (e.g., skin, gills, salivary glands, gonads) were preserved in Zymo DNA/RNA Shield or 2% paraformaldehyde for microbiome and histological studies. Adult specimens were fixed in 10% buffered formalin prior to long-term storage in 70% ethanol. Eggs from brooding females were preserved in 95% ethanol.

Other Molluscs: Specimens were photographed and preserved in 95% ethanol.

Corals and Anemones: Branches or tissue samples (approximately 10 cm in length) were preserved in 95% ethanol for DNA analysis, with ethanol refreshed after 24 hours. Additional fragments of corals were air-dried for morphological reference.

Sponges: Sponge tissue was preserved in >90% ethanol, with the preservative refreshed after 24 hours to prevent dilution. In some cases, fragments were air-dried when appropriate for morphological examination.

Crustaceans and Echinoderms: Specimens were photographed following standard macro-organism protocols and preserved in 95% ethanol for morphological and molecular analysis.

Gelatinous Species (e.g., siphonophores, jellyfish): These delicate organisms were preserved in 95% ethanol or fixed in 10% buffered formalin prior to long-term storage in 70% ethanol to maintain structural integrity.

Preservative mixtures were prepared using standardized formulations:

70% Ethanol: 700 mL of 95% ethanol mixed with 300 mL of distilled water.
10% Buffered Formalin: 100 mL of 37–40% formaldehyde diluted in 900 mL of seawater.

C. LABELLING

The dataset follows Darwin Core standards. This dataset is an occurrence core derived directly from the collected samples. The descriptions of all included fields or column names in the dataset are explained in the Parameters Metadata.

eventID

eventID is a unique identifier created by combining CruiseID and DiveID (eventID: e.g., Fkt230602-S0542), where:

CruiseID: Identifier for the Schmidt Ocean Institute expedition (e.g., Fkt230602).
DiveID: Sequential number assigned to ROV SuBastian dives (e.g., S0542).

occurrenceID

Unique identifier for each taxonomic occurrence within the samples:

It follows the structure eventID(CruiseID-DiveID)-SamplingNumber_SequentialRecord (e.g. Fkt230602-S0529-005_1), where:

eventID: CruiseID-DiveID (e.g., Fkt230602-S0529).
SamplingNumber: A sequential number corresponding to the master log of the expedition, where all sampling events were recorded (e.g., 005).
SequentialRecord: A suffix to differentiate different biodiversity occurrences coming from the same eventID and samplingNumber (e.g., _1).

In some cases, one recordNumber (explained below) may correspond to more than one occurrenceID. This occurs when two attached individuals were stored as one sample (e.g., a coral with associated attached fauna). For example, the recordNumber FKt231202-141-B.OCT was assigned to an octocoral specimen with associated attached zoanthids. In this case, both the octocoral and the zoanthids were assigned separate occurrenceID values in the dataset.

Multiple occurrences may also originate from the same sampling event. For example, the occurrenceID Fkt230602-S0529-005_1 and Fkt230602-S0529-005_2 were recovered from the same push core and were subsequently assigned different recordNumber and occurrenceID values. The occurrences can be distinguished by the final sequential suffix in the occurrenceID.

recordNumber

In the field, each biological sample was assigned a unique alphanumeric code (shown in the dataset as recordNumber) following the format:

CruiseID-SamplingNumber-SampleType-SubgroupCode.SubsampleNumber (the latter, if applicable) (e.g., Fkt230602-004-B-OCT.001), where:

CruiseID: Fkt230602 or Fkt231202
SamplingNumber: A sequential number corresponding to the master log of the expedition, where all sampling events were recorded.
SampleType: Coded by the category of the main sampling event (B = Biology; R = Rock; S = Sediment). Additional details on how each sample was collected are provided in the dataset under the samplingProtocol field, which offers further methodological context.
SubgroupCode: Identifies the broad taxonomic group of the specimen (ANE = Anemones, ANT = Black corals, CRU = Crustaceans, ECH = Echinoderms, HYD = Hydrozoa, MOS = Molluscs, OCT = Octocorals, OTH = Others, POR = Sponges, PULP = Octopus, UNK = Unknown, WOR = Worms, ZOA = Zoanthids). However, the detailed and reviewed taxonomic classification is also included in the dataset, following the WoRMS taxonomic hierarchy (kingdom, phylum, class, order, and family) associated with the provided scientificName. The field taxonRank is also provided, indicating the taxonomic level at which the organism was identified.
SubsampleNumber: Applied consecutively for associated or derived samples (if applicable).

Physical labels accompanying each sample included the following information: sample code, tentative taxonomic identification, locality, date (DD/MM/YYYY), geographic coordinates (decimal degrees), depth (m), dive code, and relevant observations (substrate type, color, associated fauna, ROV storage box, and other remarks).