Roger Villanueva

Research on cephalopod early ontogeny has significantly advanced in recent decades, including embryo organogenesis and neurogenesis and early behavioural adaptations, particularly in commercially important and coastal species. Within this context, here we compiled current knowledge on the collection, handling and care under experimental conditions and monitoring in the field of cephalopod eggs and egg masses. It covers field observations and egg collection methods, as well as laboratory incubation protocols for eggs maintained with and without maternal care. It is emphasized how abiotic and biotic factors, including temperature, salinity, oxygen and maternal condition, shape embryonic development and hatchling survival and viability. Monitoring methods for cephalopod egg masses in the wild and the effects of natural threats such as storms and predators are reviewed. Anthropogenic impacts such as pollution, fisheries and climate change are also discussed. Technological advances have enabled finer analysis of neural development as the embryo grows, while ethical considerations regarding embryonic sentience capacity and welfare conditions are becoming central to current experimental designs. Based on current knowledge, it is recommended to apply ethical considerations and the 3Rs principle (Replacement, Reduction, Refinement) for cephalopod embryos following the onset of organogenesis. Thus, a list of potential indicators of health and welfare that could be used for assessing and monitoring cephalopod embryos and hatchlings is proposed. Overall, this synthesis aims to serve as a guide for advancing egg collection and laboratory incubation methodologies, as well as adopting ethical handling protocols to improve cephalopod embryo care and quality.

Meropelagic octopuses hatch as planktonic paralarvae, being the subject of progressive morphological and behavioural changes ending with settlement on the seafloor as juveniles. The comparative morphological study of digestive systems can help to understand the adaptation to particular niches during this challenging plankton-benthos transition. Here, the morphometric development of the digestive system of the common octopus (Octopus vulgaris) is described through 3D microscopy techniques over the first two months of life. This morphological development is compared with those of adults and the holobenthic cuttlefish Sepia officinalis, as well as with the holopelagic squids Loligo vulgaris and Illex coindetii. Based on present and published results, we suggest four stages for the early development of O. vulgaris: (1) post embryonic, (2) early paralarval, (3) advanced paralarval and (4) early juvenile. The digestive system development was variable over time, with faster growth during the first days of planktonic life and after settlement. All the paralarvae-bearing species showed very proportionally enlarged posterior salivary glands at hatching compared both to more developed conspecifics and to the cuttlefish hatchling. This could reflect a potential common feeding mechanism through external digestion and probably a compensatory mechanism for an early digestive gland underperformance. The proportionally smallest digestive system has been found in L. vulgaris, mainly because of their remarkably smaller digestive glands. All species showed major changes in digestive organs between hatchlings and developed phases, indicating how different ecological contexts over ontogeny are reflected in their digestive structures. Graphical abstract

The settlement phase is a challenging period for meropelagic octopus, as they adapt to their new life in the sea bottom after a planktonic period. Their ecology and trophic interactions with the surrounding fauna in the wild are practically unknown. To understand their predatory role in the littoral zone, the diet of recently settled Octopus vulgaris from a Mediterranean sandy bottom was studied through molecular methods. Amphipods were present in all the analysed stomach contents, with a total of 20 amphipod taxa recorded as prey. Jassa slatteryi and Microprotopus maculatus were the most commonly found amphipod species. Hydrozoans, decapod crustaceans, cephalopods and bivalves followed amphipods in importance. Niche breadth index assigned to small recently settled octopus the role of a specialised predator, increasing the diversity of prey as they grow. Larger juveniles displayed a higher number of prey taxa suggesting a decreasing predatory specialisation with octopus size and an ontogenetic shift in trophic ecology from diet mainly based on amphipods to a richer diet. The considerable arm elongation and the strong muscular arm crown development after settlement probably allow the juvenile benthic octopus to develop ambush predation and speculative hunting, increasing the range of potential benthic prey species along its adaptation to the benthos. Graphic abstract

Cryptic species complexes represent an important challenge for the adequate characterization of Earth’s biodiversity. Oceanic organisms tend to have greater unrecognized cryptic biodiversity since the marine realm was often considered to lack hard barriers to genetic exchange. Here, we tested the effect of several Atlantic and Mediterranean oceanic barriers on 16 morphospecies of oceanic squids of the orders Oegopsida and Bathyteuthida using three mitochondrial and one nuclear molecular marker and five species delimitation methods. Number of species recognized within each morphospecies differed among different markers and analyses, but we found strong evidence of cryptic biodiversity in at least four of the studied species (Chtenopteryx sicula, Chtenopteryx canariensis, Ancistrocheirus lesueurii, and Galiteuthis armata). There were highly geographically structured units within Helicocranchia navossae that could either represent recently diverged species or population structure. Although the species studied here can be considered relatively passive with respect to oceanic currents, cryptic speciation patterns showed few signs of being related to oceanic currents. We hypothesize that the bathymetry of the egg masses and duration of the paralarval stage might influence the geographic distribution of oceanic squids. Because the results of different markers and different species delimitation methods are inconsistent and because molecular data encompassing broad geographic sampling areas for oceanic squids are scarce and finding morphological diagnostic characters for early life stages is difficult, it is challenging to assess the species boundaries for many of these species. Thus, we consider many to be in the “grey speciation zone.” As many oceanic squids have cosmopolitan distributions, new studies combining genomic and morphological information from specimens collected worldwide are needed to correctly assess the actual oceanic squid biodiversity.

Cephalopod beaks are essential for prey acquisition and fragmentation during feeding. Thus, it is expected that ecological pressures affect cephalopod beak shape. From a practical perspective, these structures are also used to identify gut contents of marine megafauna, such as toothed whales, sharks, seabirds, and large pelagic fishes. Here, we investigated the relative importance of ecological pressures and phylogenetic relatedness in the evolution of beak shape using a wide range of Mediterranean cephalopod species. Phylogenetic analyses based on complete mitogenomes and nuclear ribosomal genes provided a well-supported phylogeny among the 18 included cephalopods. Geometric morphometric and stable isotope methods were implemented to describe interspecific beak shape and trophic niche variability, respectively. Phylogenetic signal was detected in the shape of both parts of the beak (upper and lower). However, lower beak shape was more distinct among closely related species, in line with the empirical notion that lower beak morphology is more useful as an identification tool in cephalopods. Interestingly, no association between beak shape and trophic niche (stable isotope values) was found. These results suggest that the evolution of cephalopod beak shape as quantified here is mainly driven by phylogenetic relationships, while feeding habits play a minor role. Graphical abstract

Oceanic squids of the order Oegopsida are ecologically and economically important members of the pelagic environment. They are the most diverse group of cephalopods, with 24 families that are divergent morphologically. Despite their importance, knowledge of phylogenetic relationships among oegopsids is less than that among neritic cephalopods. Here, we provide the complete mitogenomes and the nuclear 18S and 28S ribosomal genes for 35 selected oceanic squids, which were generated using genome skimming. We performed maximum likelihood and Bayesian inference analyses that included 21 of the 24 oegopsid families. In our analyses, the architeuthid, chiroteuthid and enoploteuthid family groups, which have been proposed previously based on morphological and natural history characteristics, were retrieved as monophyletic. The morphologically divergent Cranchiidae formed a well-supported clade with families Ommastrephidae and Thysanoteuthidae, with a unique mitochondrial gene order. The family Lycoteuthidae was revealed as paraphyletic and contained Pyroteuthidae. Thus, the two lycoteuthid subfamilies are herein elevated to family level, increasing the number of oegopsid squid families to 25. In order to describe the diversity and evolutionary trends of oegopsid squids accurately, the superfamilies Architeuthoidea, Chiroteuthoidea, Cranchioidea and Enoploteuthoidea are resurrected from the literature, and the superfamilies Cycloteuthoidea, Octopoteuthoidea and Pholidoteuthoidea are proposed. The phylogenetic positions of Gonatidae, Histioteuthidae and Onychoteuthidae were not stable in our phylogenetic analyses and are not assigned to a superfamily. This study supports the utility of genome skimming to solve the phylogenetic relationships of oceanic squids.

The entire skin surface of octopus embryos, hatchlings and juveniles bears scattered tufts of tiny chitinous setae within small pockets, from which they can be everted and retracted. Known as Kölliker’s organs (KO), they disappear before the subadult stage. The function of these structures during the early life of the octopus is unknown, despite having been first described nearly two centuries ago. To investigate these organs further, general trends in size of KO distribution and density were analyzed in hatchlings and juveniles of 17 octopod species from all oceans, representing holobenthic, holopelagic and meropelagic modes of life. The size of the KO is fairly constant across species, unrelated to mode of life or hatchling size. The density of KO is similar on ventral and dorsal body surfaces, but hatchlings of smaller size tend to have a higher density of KO on the aboral surface of the arms. Analysis of a series of post-hatchingOctopus vulgarisshows KO size to be constant throughout ontogeny; it is therefore a consistent structure during the octopus early life from planktonic hatchling to benthic juvenile. New KO are generated on the skin of the arm tips during the planktonic period and initial benthic lives of juveniles. Their density, on both the mantle and arms, gradually decreases as the octopus grows. In older benthic juveniles, the KO degrades, losing its setae and the base of its follicle becomes exposed as a nearly circular stump of muscle. It is estimated that fully everted KO increase the body surface area by around two-thirds compared to when the KO are retracted. This modular mechanism of body surface extension and roughness probably influences flow-related forces such as drag and propulsion of the moving surface of the young octopus while it is of small size with a relatively large surface area. In addition, the distribution of these organs on the aboral surface of the arms of the octopus and their birefringent properties suggest a role in camouflage. Further research is needed to test these hypotheses of KO function in live animals.

Historically, marine oceanic open environments have been considered without barriers to dispersal, and the subsequent speciation of lineages from distant areas. As a consequence, many marine pelagic invertebrates are considered as monotypic cosmopolitan taxa, sometimes even including divergent geographic morphotypes. However, this view has been consistently challenged in the last decades by the discovery of many cryptic species complexes among pelagic marine “cosmopolitan” invertebrates. Despite their vast ecological importance, oceanic squids of the orders Oegopsida Orbigny, 1845 and Bathyteuthida Lindgren, 2010 are seldomly molecularly tested for cryptic biodiversity covering wide areas. Here, we barcoded specimens belonging to 12 oceanic squid species sampled during several Atlantic oceanic cruises covering waters from Brazil to Iceland, and the Mediterranean Sea. For assessing the presence of cryptic lineages, we studied the uncorrected p-distances at the intra- and interclade level and performed molecular species delimitation methods, such as the Poisson Tree Processes and the Generalized Mixed Yule Coalescent approach. Within Atlantic waters, we found cryptic biodiversity in five species: Abraliopsis morisii (Verany, 1839), Ancistrocheirus lessueuri (Orbigny 1842), Chtenopteryx sicula (Verany 1851), Galiteuthis armata Joubin, 1898 and Helicocranchia pfefferi Massy 1907. Atlantic individuals of Pterygioteuthis gemmata Chun, 1908 represent a divergent lineage of those from New Zealand. The divergence values among cryptic lineages of individuals of the same nominal species range from 2.2 to 17%, likely representing different stages of divergence since each putative speciation phenomena. In total, 50% of the tested species revealed cryptic lineages, which indicates that oceanic squid biodiversity is underestimated and it is necessary to develop more studies to assess the diversity of these animals at a global scale.