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

Oceanic anoxic events (OAEs) profoundly impact marine ecosystems, driven by factors like carbon cycle perturbations and reduced ocean ventilation. These events lead to diverse biotic responses, from extinctions to adaptations to low-oxygen environments like oxygen minimum zones (OMZs). The modern vampire squid (*Vampyroteuthis infernalis*) thrives in OMZs, exhibiting unique adaptations such as a low metabolic rate and detritivorous feeding. While its Mesozoic vampyromorph ancestors inhabited shallower waters, the timing of their retreat to bathyal, oxygen-depleted habitats remains poorly understood. A 120-million-year gap in the fossil record hinders understanding this transition. Hypotheses explaining the shift of cephalopods to deeper waters include competitive exclusion from shallower habitats and higher extinction rates in shallower environments. This study aims to address this gap by analyzing a previously enigmatic fossil, *Necroteuthis hungarica*, from the Oligocene of the Hungarian Paleogene Basin (HPB), to determine its systematic affinities, reconstruct its environment, and trace the onshore-offshore shift of vampyromorphs.

Previous research on vampire squid phylogeny has utilized morphological, molecular, and combined approaches, placing *Vampyroteuthis* within the octobrachian lineage. However, the lack of Cenozoic vampire squid fossils has created a significant gap in our understanding of their evolutionary history. This gap, often attributed to the Lazarus effect (preservation bias), inhibits inferences about the timing of their deep-sea colonization. Existing literature on cephalopod bathymetric shifts proposes two main hypotheses: competitive exclusion from shallower waters and higher extinction rates in shallower environments. The study of Mesozoic gladius-bearing coleoids, including loligosepiids, provides insights into the vampyromorph lineage leading to vampire squids. The last known loligosepiids date to the Lower Cretaceous, leaving a 120-million-year gap until the present-day *Vampyroteuthis*. The rediscovery of the holotype of *N. hungarica* offers an opportunity to bridge this gap.

This study employed multiple analytical techniques to characterize *N. hungarica* and its environment. The rediscovered holotype was examined using micro-computed tomography (µ-CT) and scanning electron microscopy (SEM) to determine its morphology and internal structure. Fourier transform infrared spectroscopy (FTIR) analysis determined the chemical composition, specifically identifying the presence of organic material (likely chitin remnants) and diagenetic minerals (apatite and gypsum). Micropaleontological analysis of the surrounding sediment revealed the presence of framboidal pyrite, fish remains, and benthic foraminifera, providing insights into the paleoenvironment. Calcareous nannoplankton analysis further refined the environmental reconstruction. Stable isotope analysis (δ¹³C and δ¹⁸O) of carbonate and organic matter in the sediment provided information about primary productivity, water-column stratification, and oxygen levels. High-resolution sampling around the gladius allowed for detailed analysis of isotopic variations. The data obtained were compared to those from other similar formations and localities to better understand the ecological context.

The µ-CT and SEM analyses confirmed that *N. hungarica* is a gladius, not a cuttlebone, and its morphology shares features with both extinct loligosepiids and the extant *Vampyroteuthis*, particularly short hyperbolar zones and a posterior process. FTIR analysis revealed the presence of organic matter, suggesting preservation of original chitin. The micropaleontological analysis of the surrounding sediment (Tard Clay Formation) indicated dysoxic to anoxic bottom-water conditions and high primary productivity, consistent with a stratified water column. Low BFOI values (-48) strongly support dysoxic conditions with dissolved oxygen between 0.1-0.3 ml/l. The high abundance of calcareous nannoplankton (*Reticulofenestra lockeri*, *Coccolithus pelagicus*, *Reticulofenestra ornata*) and organic-walled algae (*Tasmanites*) in the sediment is typical of low-salinity, high-nutrient environments. Stable isotope data (highly negative δ¹⁸O values in bulk rock samples and δ¹³C values in organic matter) further supported the anoxic bottom-water conditions and marked salinity stratification. Comparison with the overlying Kiscell Clay Formation, containing *Archaeosepia* in a more oxygenated environment, further highlighted the contrasting conditions inhabited by *N. hungarica*. The study also reviewed the occurrence of vampyromorphs in various Mesozoic Lagerstätten, indicating that they often inhabited hypoxic environments. The absence of loligosepiids in other Cretaceous Lagerstätten not associated with OAEs suggests a potential early retreat to deeper waters.

The findings demonstrate that *N. hungarica*, a close relative of the extant *Vampyroteuthis*, inhabited bathyal, oxygen-depleted habitats during the Oligocene. This significantly extends the known range of vampire squid adaptation to low-oxygen conditions back to at least the Oligocene. The analysis suggests that the development of OMZs, particularly in the Central Paratethys during the Early Oligocene, may have acted as a trigger for the range expansion and habitat specialization of vampyromorphs. While Mesozoic loligosepiids also inhabited hypoxic environments, *N. hungarica*'s bathyal habitat represents a clear shift from the shallower, shelf habitats of its ancestors. The combination of reduced competition and predation, along with potentially abundant food supply in OMZs, may have facilitated this transition. The survival of vampyromorphs through major Mesozoic and Cenozoic crises, including OAEs and the K-Pg extinction event, highlights their successful evolutionary strategy in oxygen-depleted niches.

This study presents compelling evidence that vampire squids adapted to oxygen-depleted, bathyal habitats at least since the Oligocene, filling a significant gap in the fossil record. The discovery of *Necroteuthis hungarica* and its environmental context strongly supports the hypothesis that OMZs played a key role in shaping the evolutionary history of vampyromorphs. Future research could focus on further exploring the timing of the deep-sea colonization by analyzing more deep-sea sediments from the Cretaceous and Paleogene, as well as investigating the genetic basis of adaptations to low-oxygen conditions in vampyromorphs.

The study is based on a single, rediscovered holotype specimen of *N. hungarica*. While multiple analytical techniques were used, the limited sample size restricts the generalizability of the findings. The interpretation of the FTIR data is influenced by diagenetic alteration of the gladius, potentially obscuring some details of the original composition. The study's interpretation of the paleoenvironmental conditions relies heavily on micropaleontological and geochemical data from the surrounding sediment. While these data provide strong support for the hypothesized conditions, variations within the sedimentary environment could not be totally excluded.