Fossil evidence for vampire squid inhabiting oxygen-depleted ocean zones since at least the Oligocene

Oceanic anoxic events record fundamental changes in marine ecosystems and elicit diverse biotic responses, from regional extinctions to radiations and adaptations to low-oxygen habitats in oxygen minimum zones (OMZs). Understanding these dynamics informs predictions under modern deoxygenation. The Recent deep-sea vampire squid (Vampyroteuthis infernalis) inhabits OMZs and shows extraordinary low-oxygen adaptations (low metabolic rate and detritivory). Although Vampyroteuthis has traits bridging Decabrachia and Octobrachia, morphological, molecular and combined studies place it within the octobrachian lineage. A major gap exists because no Cenozoic vampyromorphs had been described, limiting inferences about timing of deep-sea colonization and suggesting a preservation bias (Lazarus effect). Competing hypotheses explain a bathymetric shift of coleoids: (1) competitive/predatory exclusion from shallower habitats pushing taxa deeper, and (2) preferential extinction in shallow environments yielding an apparent deepening through time. The authors propose that active specialization to deep anoxic habitats implies bathymetric variability in origination also matters. Mesozoic gladius-bearing coleoids (Loligosepiina) are octobrachians and represent the vampyromorph branch leading to vampire squids, but their last record is Lower Aptian, leaving at least a 120 My gap to Recent Vampyroteuthis. The enigmatic Oligocene fossil from Hungary, originally described as Necroteuthis hungarica, has had uncertain affinities due to mineralization and a lost holotype. The rediscovered holotype allows chemical and imaging analyses clarifying it as an octobrachian gladius with features linking loligosepiids to Vampyroteuthis. The study aims to (1) re-examine the gladius using SEM, micro-CT, geochemistry, and comparative anatomy to assign its taxonomy, (2) reconstruct Oligocene environments inhabited by this cephalopod, and (3) track the onshore-offshore shift of vampyromorphs from the Early Jurassic to Recent. The Early Oligocene isolation of Paratethys established salinity-stratified, high-productivity, oxygen-depleted basins, potentially triggering deepening and adaptation of vampyromorphs. This work assesses whether deep-sea association with dysoxic/anoxic conditions in Vampyroteuthis is ancient or recent.

Background work identifies OMZ-associated adaptations in modern Vampyroteuthis (low metabolism and detritivory) and places it among Octobrachia based on morphology and multi-gene phylogenies. The post-Cretaceous absence of vampyromorph fossils has been framed as a Lazarus effect due to preservation bias or reduced range/abundance. Two hypotheses have been proposed for coleoid bathymetric shifts: (i) competitive/predatory exclusion from shallow seas prompting deep colonization and (ii) higher extinction rates in shallow waters producing an apparent deepening trend. Mesozoic loligosepiids (Triassic–Aptian) are considered ancestral to vampire squids, with many records associated with hypoxic/anoxic shelf settings (Toarcian OAE, Callovian La Voulte, Aptian OAE1). Cenozoic records were lacking prior to this study, obscuring timing of deep-sea specialization. The study leverages this context to test for an Oligocene deep-water, low-oxygen adaptation.

Specimen and geological context: The holotype gladius of Necroteuthis hungarica (M59/4672, Hungarian Natural History Museum) originates from the Csillaghegy Brickyard near Budapest. Two formations occur in tectonic contact: the laminated, anoxic Tard Clay Formation and the overlying, bioturbated Kiscell Clay Formation. Lithology of the matrix (laminated argillaceous rock) and calcareous nannoplankton assemblage (Reticulofenestra ornata, R. umbilicus, Discoaster nodifer; absence of R. abisecta) place the specimen in the Tard Clay Fm., NP22–lower NP23 (Early Oligocene, Kiscellian). Regional palaeogeography indicates restricted Central Paratethys basins with stratified water columns and organic-rich shale deposition; palaeodepth exceeded 400 m and locally reached ~800 m. Imaging and morphology: Macrophotography documented overall gladius dimensions and features. Micro-computed tomography (phoenix v|tome|x L 240; 200 kV, 250 μA, 2500 projections, voxel size 80 μm, 0.5 mm Cu filter) produced 3D datasets evaluated in VG Studio Max 2.2. µ-CT assessed internal architecture and thickness distribution to distinguish gladius vs. cuttlebone (absence of ventral chambered portion). Scanning electron microscopy (JEOL-6380LV at 20–30 kV, 1.7–10k×) examined lamination and ultrastructure; samples were Au-coated and imaged in low/high vacuum. Geochemistry: Fourier transform infrared spectroscopy (Thermo Nicolet iN10 FTIR, micro-ATR with Ge crystal; 675–4000 cm−1, 2 cm−1 resolution) characterized mineral and organic phases. Reference spectra for gypsum and (hydroxyl)apatite were taken from RRUFF. FTIR targeted miniature dark fossil fragments and surrounding matrix. Stable isotopes: Bulk carbonate δ13C and δ18O were measured with MAT253 IRMS coupled to Kiel IV; 40–100 μg powders, phosphoric acid digestion at 70 °C, calibrated against NBS18 and working standards; precision 0.02‰ (C), 0.04‰ (O). A 4 cm transect around the gladius was sampled at 2.5 mm increments. Organic carbon δ13Corg was measured on MAT253 coupled to Flash2000 EA; residues after HCl digestion (900–1300 μg) combusted at 1000 °C; calibrated to PDB using USGS standards; precision 0.11‰, SD 0.106‰. Micropaleontology and paleoecology: Washed sediment (0.063–2 mm) was examined for microfossils (Olympus SZ61 stereomicroscope, Zeiss Axiolab 5; SEM confirmation). Taxonomy followed published protocols. Benthic foraminiferal dissolved-oxygen index (BFOI) was calculated using proportions of oxic and dysoxic indicator taxa following Kaiho’s classification, with BFOI = [O/(O+D)] × 100 when at least one oxic species occurs. Taphonomic assessment and counts also included organic-walled cysts and algae. Calcareous nannoplankton was studied optically (1000×) and by SEM; slides prepared per standard methods. Comparative anatomy and systematics: Gladius morphology (median field, lateral fields, hyperbolar zones, conus, posterior process) was compared to loligosepiids, other octobrachians (teudopseids, prototeuthids), and Vampyroteuthis using published treatises to assess phylogenetic affinities.

- The rediscovered holotype of Necroteuthis hungarica is a nearly complete, dorsally exposed gladius 146 mm long and 60 mm wide with a triangular median field, lateral fields, hyperbolar zones, a conus, and a posterior process. - Micro-CT shows no ventral chambered portion, excluding a sepiid cuttlebone; gladius thickness increases posteriorly, reaching ~2 mm in the conus. - SEM reveals multi-laminated structure composed of amorphous matter, typical of gladii in decabrachian and octobrachian coleoids. - FTIR detects organic functional groups (−CH3, −CH2−; bands at 2963, 2930, 2878 cm−1) and identifies gypsum and (hydroxyl)apatite, indicating predominant replacement of original β-chitin by apatite during early diagenesis; diagnostic amide bands are obscured by mineral signals. - Micropaleontology: Wash residues contain framboidal pyrite, fish remains, organic-walled acritarchs (Leiosphaeridia, Tasmanites), dinocysts, and small benthic foraminifera (Caucasina, Miliammina, Fursenkoina, Bulimina). Foraminifera comprise 44% of microfossils; organic-walled algae/acritarchs 56% (Tasmanites ~80% of that fraction). Benthic assemblage (9 taxa; Shannon index 0.74; n=68) is dominated by Caucasina oligocaenica (80.4%) and Fursenkoina subacuta (12.4%). BFOI = −48 indicates dysoxic to near-anoxic bottom waters with dissolved oxygen 0.1–0.3 ml/l; shell ultrastructure shows thin, densely porous tests consistent with hypoxia tolerance. - Calcareous nannoplankton is dominated by Reticulofenestra lockeri, Coccolithus pelagicus, and Reticulofenestra ornata; preservation ranges from excellent to degraded; diversity low–medium (6–11 spp. per sample; 17 total), without reworking. - Stable isotopes (bulk carbonate) around the gladius: δ13Ccarb −3.05 to −2.08‰; δ18Ocarb −6.70 to −5.71‰, consistent with low-salinity surface water calcification of coccoliths and strong water column stratification. δ13Corg ranges from −27.07 to −26.46‰; samples directly at the gladius −26.74 to −26.73‰. Overlying Kiscell Clay (with sepiid Archaeosepia) shows less negative δ13Ccarb (avg −0.78‰) and δ18Ocarb (avg −2.78‰), indicating more oxygenated, less stratified conditions. - Environmental reconstruction: The Tard Clay Fm. was deposited in bathyal depths (>400 m; up to ~800 m regionally) within a salinity-stratified basin with high primary productivity (TOC ~3%) and bottom-water dysoxia/anoxia to euxinia. - Systematics and evolutionary context: Gladius characters (short, weakly arcuate hyperbolar zones; posterior process; convex margins) support assignment of Necroteuthis to Vampyroteuthidae and close affinity to Vampyroteuthis over loligosepiids or teuthids. This represents the first Cenozoic vampyromorph record, bridging a ~120 My gap. - Comparative ecology: Necroteuthis inhabited oxygen-depleted bathyal habitats, whereas coeval sepiids occupied shallower, better-oxygenated settings and were constrained from greater depths by gas-filled cuttlebone mechanics.

The morphological suite of Necroteuthis aligns it more closely with Vampyroteuthis than with other octobrachians or decabrachians, indicating it is a vampyromorph within Vampyroteuthidae. Geochemical and micropalaeontological proxies demonstrate deposition under strongly stratified, hyposaline surface waters and dysoxic to anoxic bottom waters, with high primary productivity and limited bioturbation. This establishes that a vampyromorph occupied bathyal, oxygen-depleted habitats in the Early Oligocene Central Paratethys. Placing this in a temporal framework, Jurassic and Early Cretaceous loligosepiids are commonly associated with hypoxic or anoxic shelf settings (Toarcian OAE, La Voulte, Aptian OAE1), suggesting early adaptation to low oxygen but within shallower epicontinental seas. The absence of vampyromorphs from later Cretaceous shallow-water Lagerstätten and constraints from taphonomy imply a bathyal shift may have occurred by the end of the Early Cretaceous, though deep-sea records are sparse. The Oligocene Necroteuthis provides direct evidence that the shift to deep, oxygen-limited habitats had occurred by at least the Oligocene. OMZ development in the Central Paratethys likely facilitated ecological specialization by reducing predation/competition and offering elevated detrital food supply at OMZ boundaries. Comparison with overlying Kiscell Clay fauna and isotopes underscores ecological partitioning: sepiids in better-oxygenated, shallower waters vs. vampyromorphs in deeper, dysoxic to anoxic settings. This partitioning aligns with functional constraints (cuttlebone implosion limits) and supports a survival strategy for vampyromorphs through major oceanic crises by exploiting low-oxygen refugia. The findings address the research questions by (1) assigning Necroteuthis to Vampyroteuthidae via gladius anatomy, (2) reconstructing bathyal, anoxic Oligocene habitats, and (3) documenting an onshore–offshore evolutionary trajectory tied to OMZ expansion.

Necroteuthis, an Oligocene vampyromorph from the Central Paratethys, is a close relative of the extant Vampyroteuthis and occupied bathyal, oxygen-depleted environments within a strongly stratified basin. This demonstrates that vampyromorph adaptation to low-oxygen deep-sea habitats existed at least since the Oligocene, likely rooted in Mesozoic low-oxygen tolerances on epicontinental shelves. The lineage’s exploitation of OMZ-linked refugia may have facilitated survivorship across Mesozoic and Cenozoic oceanic crises, including the K–Pg boundary. Due to the sparse fossil record, the divergence timing between deep-sea Vampyromorphina and shallow-water Loligosepiina remains unresolved. Future work should target additional deep-sea OAE-related deposits and refine phylogenetic frameworks with new fossil gladii and molecular clock calibrations to narrow divergence estimates and test the timing and drivers of bathyal specialization.

- The vampyromorph fossil record remains extremely sparse (Lazarus effect), with this study relying on a single rediscovered holotype, limiting statistical generalization. - Poor preservational potential of coleoids and rarity of well-preserved deep-sea sediments constrain detection of Cretaceous–Cenozoic records, potentially biasing inferences about timing of bathyal shift. - Diagnostic organic signals in FTIR are partly masked by diagenetic minerals; original chitin is largely replaced, restricting direct biomolecular confirmation. - The precise divergence time between deep-sea Vampyromorphina and shallow-water Loligosepiina cannot be estimated with current data. - Environmental reconstructions derive from bulk proxies and local stratigraphic context; regional variability in Paratethys basins may not be fully captured.