Developmental gene expression in the eyes of the pygmy squid Xipholeptos notoides.

The eyes of squids, octopuses, and cuttlefish are a textbook example for evolutionary convergence, due to their striking similarity to those of vertebrates. For this reason, studies on cephalopod photoreception and vision are of importance for a broader audience. Previous studies showed that genes such as pax6, or certain opsin-encoding genes, are evolutionarily highly conserved and play similar roles during ontogenesis in remotely related bilaterians. In this study, genes that encode photosensitive proteins and Reflectins are identified and characterized. The expression patterns of rhodopsin, xenopsin, retinochrome, and two reflectin genes have been visualized in developing embryos of the pygmy squid Xipholeptos notoides by in situ hybridization experiments. Rhodopsin is not only expressed in the retina of X. notoides but also in the olfactory organ and the dorsal parolfactory vesicles, the latter a cephalopod apomorphy. Both reflectin genes are expressed in the eyes and in the olfactory organ. These findings corroborate previous studies that found opsin genes in the transcriptomes of the eyes and several extraocular tissues of various cephalopods. Expression of rhodopsin, xenopsin, retinochrome, and the two reflectin genes in the olfactory organ is a finding that has not been described so far. In other organisms, it has been shown that Retinochrome and Rhodopsin proteins are obligatorily associated with each other as both molecules rely on each other for Retinal isomerisation. In addition, we demonstrate that retinochrome is expressed in the retina of X. notoides and in the olfactory organ. This study shows numerous new expression patterns for Opsin-encoding genes in organs that have not been associated with photoreception before, suggesting that either Opsins may not only be involved in photoreception or organs such as the olfactory organ are involved in photoreception.

A Theoretical Comparison of Alternative Male Mating Strategies in Cephalopods and Fishes.

We used computer simulations of growth, mating and death of cephalopods and fishes to explore the effect of different life-history strategies on the relative prevalence of alternative male mating strategies. Specifically, we investigated the consequences of single or multiple matings per lifetime, mating strategy switching, cannibalism, resource stochasticity, and altruism towards relatives. We found that a combination of single (semelparous) matings, cannibalism and an absence of mating strategy changes in one lifetime led to a more strictly partitioned parameter space, with a reduced region where the two mating strategies co-exist in similar numbers. Explicitly including Hamilton's rule in simulations of the social system of a Cichlid led to an increase of dominant males, at the expense of both sneakers and dwarf males ("super-sneakers"). Our predictions provide general bounds on the viable ratios of alternative male mating strategies with different life-histories, and under possibly rapidly changing ecological situations.

Miles down for lunch: deep-sea in situ observations of Arctic finned octopods Cirroteuthis muelleri suggest pelagic-benthic feeding migration.

Deep-sea cephalopods are diverse, abundant, and poorly understood. The Cirrata are gelatinous finned octopods and among the deepest-living cephalopods ever recorded. Their natural feeding behaviour remains undocumented. During deep-sea surveys in the Arctic, we observed Cirroteuthis muelleri. Octopods were encountered with their web spread wide, motionless and drifting in the water column 500-2600 m from the seafloor. Individuals of C. muelleri were also repeatedly observed on the seafloor where they exhibited a repeated, behavioural sequence interpreted as feeding. The sequence (11-21 s) consisted of arm web spreading, enveloping and retreating. Prey capture happened during the enveloping phase and lasted 5-49 s. Numerous traces of feeding activity were also observed on the seafloor. The utilization of the water column for drifting and the deep seafloor for feeding is a novel migration behaviour for cephalopods, but known from gelatinous fishes and holothurians. By benthic feeding, the octopods benefit from the enhanced nutrient availability on the seafloor. Drifting in the water column may be an energetically efficient way of transportation while simultaneously avoiding seafloor-associated predators. In situ observations are indispensable to discover the behaviour of abundant megafauna, and the energetic coupling between the pelagic and benthic deep sea.

Genome skimming elucidates the evolutionary history of Octopoda.

Phylogenies for Octopoda have, until now, been based on morphological characters or a few genes. Here we provide the complete mitogenomes and the nuclear 18S and 28S ribosomal genes of twenty Octopoda specimens, comprising 18 species of Cirrata and Incirrata, representing 13 genera and all five putative families of Cirrata (Cirroctopodidae, Cirroteuthidae, Grimpoteuthidae, Opisthoteuthidae and Stauroteuthidae) and six families of Incirrata (Amphitretidae, Argonautidae, Bathypolypodidae, Eledonidae, Enteroctopodidae, and Megaleledonidae) which were assembled using genome skimming. Phylogenetic trees were built using Maximum Likelihood and Bayesian Inference with several alignment matrices. All mitochondrial genomes had the 'typical' genome composition and gene order previously reported for octopodiforms, except Bathypolypus ergasticus, which appears to lack ND5, two tRNA genes that flank ND5 and two other tRNA genes. Argonautoidea was revealed as sister to Octopodidae by the mitochondrial protein-coding gene dataset, however, it was recovered as sister to all other incirrate octopods with strong support in an analysis using nuclear rRNA genes. Within Cirrata, our study supports two existing classifications suggesting neither is likely in conflict with the true evolutionary history of the suborder. Genome skimming is useful in the analysis of phylogenetic relationships within Octopoda; inclusion of both mitochondrial and nuclear data may be key.

SNP data reveals the complex and diverse evolutionary history of the blue-ringed octopus genus (Octopodidae: Hapalochlaena) in the Asia-Pacific.

The blue-ringed octopus species complex (Hapalochlaena spp.), known to occur from Southern Australia to Japan, currently contains four formally described species (Hapalochlaena maculosa, Hapalochlaena fasciata, Hapalochlaena lunulata and Hapalochlaena nierstraszi). These species are distinguished based on morphological characters (iridescent blue rings and/or lines) along with reproductive strategies. However, the observation of greater morphological diversity than previously captured by the current taxonomic framework indicates that a revision is required. To examine species boundaries within the genus we used mitochondrial (12S rRNA, 16S rRNA, cytochrome c oxidase subunit 1 [COI], cytochrome c oxidase subunit 3 [COIII] and cytochrome b [Cytb]) and genome-wide SNP data (DaRT seq) from specimens collected across its geographic range including variations in depth from 3 m to >100 m. This investigation indicates substantially greater species diversity present within the genus Hapalochlaena than is currently described. We identified 10,346 SNPs across all locations, which when analysed support a minimum of 11 distinct clades. Bayesian phylogenetic analysis of the mitochondrial COI gene on a more limited sample set dates the diversification of the genus to âˆ¼30 mya and corroborates eight of the lineages indicated by the SNP analyses. Furthermore, we demonstrate that the diagnostic lined patterning of H. fasciata found in North Pacific waters and NSW, Australia is polyphyletic and therefore likely the result of convergent evolution. Several "deep water" (>100 m) lineages were also identified in this study with genetic convergence likely to be driven by external selective pressures. Examination of morphological traits, currently being undertaken in a parallel morphological study, is required to describe additional species within the complex.

The complete mitochondrial genome of Octopus vulgaris.

BACKGROUND: The Octopus vulgaris species complex consists of numerous morphologically similar but genetically distinct species. The current publicly available mitogenome of this species has been generated from a specimen collected from Tsukiji Fish Market, Tokyo, Japan. Octopus from the northwestern Pacific Ocean are now considered to be a separate species, Octopus sinensis. For this reason, we hypothesised that the current record of O. vulgaris was sequenced from a specimen of O. sinensis. Here, we sequenced the first complete mitogenome of a specimen of Octopus vulgaris sensu stricto that was collected from the species' confirmed distribution areas in northeastern Atlantic. METHODS AND RESULTS: The complete mitogenome was assembled de novo and annotated using 250 bp paired-end sequences. A single circular contig 15,655 bp in length with a mean read coverage of 1089 reads was reconstructed. The annotation pipeline identified 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNA) and two ribosomal RNAs. A maximum likelihood phylogenetic tree recovered the assembled mitogenome as the sister taxon of a monophyletic group comprising O. sinensis and the previously published mitogenome of "O. vulgaris" from Japan. This confirms that the latter was a Japanese specimen of O. sinensis. CONCLUSION: The mitogenome sequenced here is the first to be published for Octopus vulgaris sensu stricto. It represents an important first step in genetics-informed research on the evolution, conservation, and management of this commercially important species.

Early cephalopod evolution clarified through Bayesian phylogenetic inference.

BACKGROUND: Despite the excellent fossil record of cephalopods, their early evolution is poorly understood. Different, partly incompatible phylogenetic hypotheses have been proposed in the past, which reflected individual author's opinions on the importance of certain characters but were not based on thorough cladistic analyses. At the same time, methods of phylogenetic inference have undergone substantial improvements. For fossil datasets, which typically only include morphological data, Bayesian inference and in particular the introduction of the fossilized birth-death model have opened new possibilities. Nevertheless, many tree topologies recovered from these new methods reflect large uncertainties, which have led to discussions on how to best summarize the information contained in the posterior set of trees. RESULTS: We present a large, newly compiled morphological character matrix of Cambrian and Ordovician cephalopods to conduct a comprehensive phylogenetic analysis and resolve existing controversies. Our results recover three major monophyletic groups, which correspond to the previously recognized Endoceratoidea, Multiceratoidea, and Orthoceratoidea, though comprising slightly different taxa. In addition, many Cambrian and Early Ordovician representatives of the Ellesmerocerida and Plectronocerida were recovered near the root. The Ellesmerocerida is para- and polyphyletic, with some of its members recovered among the Multiceratoidea and early Endoceratoidea. These relationships are robust against modifications of the dataset. While our trees initially seem to reflect large uncertainties, these are mainly a consequence of the way clade support is measured. We show that clade posterior probabilities and tree similarity metrics often underestimate congruence between trees, especially if wildcard taxa are involved. CONCLUSIONS: Our results provide important insights into the earliest evolution of cephalopods and clarify evolutionary pathways. We provide a classification scheme that is based on a robust phylogenetic analysis. Moreover, we provide some general insights on the application of Bayesian phylogenetic inference on morphological datasets. We support earlier findings that quartet similarity metrics should be preferred over the Robinson-Foulds distance when higher-level phylogenetic relationships are of interest and propose that using a posteriori pruned maximum clade credibility trees help in assessing support for phylogenetic relationships among a set of relevant taxa, because they provide clade support values that better reflect the phylogenetic signal.

Mesozoic origin of coleoid cephalopods and their abrupt shifts of diversification patterns.

Coleoids are the most diverse group of cephalopod mollusks. While their origin is date during the Mesozoic, the diversification pattern is unknown. However, two hypotheses have been proposed. The first suggests an increasing diversification rate after the Cretaceous-Paleogene extinction event (K-Pg) as consequence of empty habitats left by the ammonites and belemnites. The second hypothesis proposes a mid-Cenozoic increase in diversification rate related to distributional changes during ice ages and biotic interactions. To test these hypotheses, we estimated a lineage through time (LTT) and the gamma-statistic along with model-based diversification rates. These analyses were conducted on a dated molecular phylogeny for coleoids that we reconstructed using five molecular markers (cytochrome b, 16S rRNA, cytochrome oxidase I, rhodopsin, and PAX-6). Our divergence time estimation suggests that coleoids originated in the Mesozoic Era (Middle Triassic) and that both main clades (Decapodiformes and Octopodiformes) diverged in the Cretaceous/Jurassic Period. The LTT, gamma statistic, and diversification rates inferred with the Bayesian Analysis of Macro-evolutionary Mixtures (BAMM), indicate an acceleration in diversification rate over time since the origin of coleoids. Additionally, BAMM allowed us to detect abrupt increases in diversification rate before and after the K-Pg boundary. Our results partially support both hypotheses as all analyses indicate that the coleoid diversification rate was increasing during the Cenozoic. However, our results also indicate increasing diversification rates before the K-Pg boundary. We propose that the radiation of coleoids has been shaped by an acceleration in diversification rate over time, including exceptional episodes of abrupt increases before and after the K-Pg boundary.

The Modular Architecture of Metallothioneins Facilitates Domain Rearrangements and Contributes to Their Evolvability in Metal-Accumulating Mollusks.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, ß1, ß2, ß3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and ß1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

A phylogenetic approach to understand the evolution of reproduction in coleoid cephalopods.

A central question in the evolution of life-histories is whether organisms reproduce once or repeatedly. For cephalopods, the main differences between semelparous and iteroparous are based on ovulation pattern and spawning type. The different reproductive strategies in coleoid cephalopods could be related to the habitat in which the species dwell (coastal vs. oceanic) and/or to environmental forces, thus, both aspects should be quantitatively evaluated under an evolutionary perspective to reconstruct: (a) the ancestral ovulation type of coleoid cephalopods, and (b) the potential of correlated evolution between ovulation type versus habitat and environment. Ancestral states of ovulation type were estimated using stochastic mapping based on literature data (i.e. synchronous or asynchronous), and this information was combined with a new molecular phylogeny including 165 species. The evolutionary correlation between ovulation type, habitat, and environment was estimated by means of the Markov model comparing the rates of gain and loss. The estimates of ancestral states of ovulation type for coleoid cephalopods resulted in a high probability that Octopodiformes evolved from synchronous ovulation type, and Decapodiformes from asynchronous ovulation type. The three traits evaluated presented phylogenetic signal, although no correlation was found between habitat and ovulation type. Overall, species in stable environments showed a tendency towards synchronous ovulation type, while the asynchronous ovulation pattern was found more frequently in species that live in unstable environments, being this last trait also responsible for triggering the change of ovulation type in some species throughout evolution.

Ocular anatomy and correlation with histopathologic findings in two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) diagnosed with inflammatory phakitis and retinitis.

PURPOSE: Review octopus ocular anatomy and describe the histopathologic findings in three octopuses diagnosed with phakitis and retinitis. ANIMALS: Two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) with a history of ophthalmic disease. METHODS: A literature search was performed for the ocular anatomy section. Both eyes from all three octopuses, and two control eyes, were submitted for histopathologic evaluation. Hematoxylin and eosin stain was used for standard histopathologic evaluation; GMS stain was used to screen for fungi, gram stain for bacteria; and Fite's acid fast stain for acid fast bacteria. RESULTS: Anatomically, the anterior chamber of the octopus has direct contact with ambient water due to an opening in the dorsal aspect of a pseudocornea. The octopus lens is divided into anterior and posterior segments. The anterior half is exposed to the environment through the opening into the anterior chamber. Neither part of the lens has a lens capsule. The retina is everted, unlike the inverted vertebrate retina, and consists of just two layers. Histopathology revealed inflammatory phakitis and retinitis of varying severity in all six eyes of the study animals. No intraocular infectious organisms were recognized but one common octopus eye had clusters of coccidian parasites, identified as Aggregata sp., in extraocular tissues and blood vessels. CONCLUSION: We describe inflammatory phakitis and retinitis in two species of octopuses. The underlying cause for the severe intraocular response may be direct intraocular infection, water quality, an ocular manifestation of a systemic disease, or natural senescence.

Quantitative Proteomic Analysis of the Slime and Ventral Mantle Glands of the Striped Pyjama Squid (Sepioloidea lineolata).

Cephalopods are known to produce an extensive range of secretions including ink, mucus, and venom. Sepiadariidae, a family of small, benthic bobtail squids, are notable for the high volume of viscous slime they emit when stressed. One species, Sepioloidea lineolata (striped pyjama squid), is covered with glands along the perimeter of the ventral mantle, and these structures are hypothesized to be the source of its slime. Using label-free quantitative proteomics, we analyzed five tissue types (dorsal and ventral mantle muscle, dorsal and ventral epithelium, and ventral mantle glands) and the slime from four individuals. In doing so, we were able to determine the relationship between the slime and the tissues as well as highlight proteins that were specifically identified within the slime and ventral mantle glands. A total of 28 proteins were identified to be highly enriched in slime, and these were composed of peptidases and protease inhibitors. Seven of these proteins contained predicted signal peptides, indicating classical secretion, with four proteins having no identifiable domains or similarity to any known proteins. The ventral mantle glands also appear to be the tissue with the closest overall proteomic composition to the slime; therefore, it is likely that the slime originates, at least in part, from these glands.

Comparative Proteomic Analysis of Slime from the Striped Pyjama Squid, Sepioloidea lineolata, and the Southern Bottletail Squid, Sepiadarium austrinum (Cephalopoda: Sepiadariidae).

Sepioloidea lineolata, the striped pyjama squid (family Sepiadariidae), is a small species of benthic bobtail squid distributed along the Southern Indo-Pacific coast of Australia. Like other sepiadariid squids, it is known to secrete large volumes of viscous slime when stressed. In order to identify key proteins involved in the function of sepiadariid slimes, we compared the slime proteome of Sepioloidea lineolata with that of a closely related species, Sepiadarium austrinum. Of the 550 protein groups identified in Sepioloidea lineolata slime, 321 had orthologs in Sepiadarium austrinum, and the abundance of these (iBAQ) was highly correlated between species. Both slimes were dominated by a small number of abundant proteins, and several of these were short secreted proteins with no homologues outside the class Cephalopoda. No mucins were identified within either species' slime, suggesting that it is structurally distinct from mucin polymer-based gels found in many vertebrate and echinoderm secretions. The extent of N-glycosylation in the slime of Sepioloidea lineolata was also studied via glycan cleavage with Peptide: N-glycosidase F (PNGase-F). Although very few (four) proteins showed strong evidence of N-glycosylation, we found that treatment with PNGase-F led to a slight increase in peptide identification rates compared with controls.

Assessing the utility of transcriptome data for inferring phylogenetic relationships among coleoid cephalopods.

Historically, deep-level relationships within the molluscan class Cephalopoda (squids, cuttlefishes, octopods and their relatives) have remained elusive due in part to the considerable morphological diversity of extant taxa, a limited fossil record for species that lack a calcareous shell and difficulties in sampling open ocean taxa. Many conflicts identified by morphologists in the early 1900s remain unresolved today in spite of advances in morphological, molecular and analytical methods. In this study we assess the utility of transcriptome data for resolving cephalopod phylogeny, with special focus on the orders of Decapodiformes (open-eye squids, bobtail squids, cuttlefishes and relatives). To do so, we took new and previously published transcriptome data and used a unique cephalopod core ortholog set to generate a dataset that was subjected to an array of filtering and analytical methods to assess the impacts of: taxon sampling, ortholog number, compositional and rate heterogeneity and incongruence across loci. Analyses indicated that datasets that maximized taxonomic coverage but included fewer orthologs were less stable than datasets that sacrificed taxon sampling to increase the number of orthologs. Clades recovered irrespective of dataset, filtering or analytical method included Octopodiformes (Vampyroteuthis infernalis + octopods), Decapodiformes (squids, cuttlefishes and their relatives), and orders Oegopsida (open-eyed squids) and Myopsida (e.g., loliginid squids). Ordinal-level relationships within Decapodiformes were the most susceptible to dataset perturbation, further emphasizing the challenges associated with uncovering relationships at deep nodes in the cephalopod tree of life.

Updated molecular phylogeny of the squid family Ommastrephidae: Insights into the evolution of spawning strategies.

Two types of spawning strategy have been described for ommastrephid squids: coastal and oceanic. It has been suggested that ancestral ommastrephids inhabited coastal waters and expanded their distribution into the open ocean during global changes in ocean circulation in the Oligocene. This hypothesis could explain the different reproductive strategies in oceanic squids, but has never been tested in a phylogenetic context. In the present study, we assess the coastal-to-open-ocean hypothesis through inferring the evolution of reproductive traits (spawning type) of ommastrephid squids using the phylogenetic comparative method to estimate ancestral states and divergence times. This analysis was performed using a robust molecular phylogeny with three mitochondrial genes (COI, CYTB and 16S) and two nuclear genes (RHO and 18S) for nearly all species of ommastrephid squid. Our results support dividing the Ommastrephidae into the three traditional subfamilies, plus the monotypic subfamily Todaropsinae as proposed previously. Divergence times were found to be older than those suggested. Our analyses strongly suggest that early ommastrephid squids spawned in coastal areas, with some species subsequently switching to spawn in oceanic areas, supporting previous non-tested hypotheses. We found evidence of gradual evolution change of spawning type in ommastrephid squids estimated to have occurred since the Cretaceous.

Molecular clocks indicate turnover and diversification of modern coleoid cephalopods during the Mesozoic Marine Revolution.

Coleoid cephalopod molluscs comprise squid, cuttlefish and octopuses, and represent nearly the entire diversity of modern cephalopods. Sophisticated adaptations such as the use of colour for camouflage and communication, jet propulsion and the ink sac highlight the unique nature of the group. Despite these striking adaptations, there are clear parallels in ecology between coleoids and bony fishes. The coleoid fossil record is limited, however, hindering confident analysis of the tempo and pattern of their evolution. Here we use a molecular dataset (180 genes, approx. 36 000 amino acids) of 26 cephalopod species to explore the phylogeny and timing of cephalopod evolution. We show that crown cephalopods diverged in the Silurian-Devonian, while crown coleoids had origins in the latest Palaeozoic. While the deep-sea vampire squid and dumbo octopuses have ancient origins extending to the Early Mesozoic Era, 242 ± 38 Ma, incirrate octopuses and the decabrachian coleoids (10-armed squid) diversified in the Jurassic Period. These divergence estimates highlight the modern diversity of coleoid cephalopods emerging in the Mesozoic Marine Revolution, a period that also witnessed the radiation of most ray-finned fish groups in addition to several other marine vertebrates. This suggests that that the origin of modern cephalopod biodiversity was contingent on ecological competition with marine vertebrates.

Bathyal feasting: post-spawning squid as a source of carbon for deep-sea benthic communities.

In many oceanic carbon budgets there is a discrepancy between the energetic requirements of deep-sea benthic communities and the supply of organic matter. This suggests that there are unidentified and unmeasured food sources reaching the seafloor. During 11 deep-sea remotely operated vehicle (ROV) surveys in the Gulf of California, the remains (squid carcasses and hatched-out egg sheets) of 64 post-brooding squid were encountered. As many as 36 remains were encountered during a single dive. To our knowledge this is one of the largest numbers of natural food falls of medium-size deep-sea nekton described to date. Various deep-sea scavengers (Ophiuroidea, Holothuroidea, Decapoda, Asteroidea, Enteropneusta) were associated with the remains. Although many of the 80 examined ROV dives did not encounter dead squids or egg sheets (n = 69), and the phenomenon may be geographically and temporally restricted, our results show that dead, sinking squid transport carbon from the water column to the seafloor in the Gulf of California. Based on food fall observations from individual dives, we estimate that annual squid carcass depositions may regionally contribute from 0.05 to 12.07 mg C m-2 d-1 to the seafloor in the areas where we observed the remains. The sinking of squid carcasses may constitute a significant but underestimated carbon vector between the water column and the seafloor worldwide, because squid populations are enormous and are regionally expanding as a result of climate change and pressure on fish stocks. In the future, standardized methods and surveys in geographical regions that have large squid populations will be important for investigating the overall contribution of squid falls to regional carbon budgets.

Whole mitochondrial genome of the Ram's Horn Squid shines light on the phylogenetic position of the monotypic order Spirulida (Haeckel, 1896).

The phylogenetic position of the only known species within the order Spirulida, the Ram's Horn Squid, Spirula spirula, may be the key to resolving relationships within Decapodiformes (squids and cuttlefishes). Spirula spirula possesses several unique features including an internal calcareous chambered shell unlike the familiar cuttlebone of Sepiidae (cuttlefishes). The shell is reduced to a gladius or absent in other decapod clades. To resolve decapodiform phylogenetic relationships we sequenced the mitochondrial genome of S. spirula and Sepiadarium austrinum and analysed these along with other mitochondrial genomes. Sequence analyses found that S. spirula and Sepiidae, the only two extant phragmocone bearing groups, were not sister taxa. Rather, in most analyses S. spirula was placed within a clade containing Bathyteuthoidea and Oegopsida either as the sister taxon to Bathyteuthoidea+Oegopsida or the sister taxon to Bathyteuthoidea only, depending upon the analysis method. Sepiidae was the sister taxon to a clade containing all remaining decapods. Spirulid mitochondrial gene order was identical to that of Octopodiformes, which we recognize as close to that of ancestral molluscs. The phylogenetic position of Idiosepiidae differed among analysis methods of molecular sequence data. However, gene order analysis resolved a highly supported monophyletic relationship containing Idiosepiidae and Sepiolida.

Late Devonian (Frasnian) phyllopod and phyllocarid crustacean shields from Belgium reinterpreted as ammonoid anaptychi.

The taxonomic affinities of fossils from the Frasnian succession of Belgium previously described as phyllopod and phyllocarid crustacean shields are discussed. The rediscovery of the holotype of Ellipsocaris dewalquei, the type species of the genus Ellipsocaris Woodward in Dewalque, 1882, allows to end the discussion on the taxonomic assignation of the genus Ellipsocaris. It is removed from the phyllopod crustaceans as interpreted originally and considered here as an ammonoid anaptychus. Furthermore, it is considered to be a junior synonym of the genus Sidetes Giebel, 1847. Similarly, Van Straelen's (1933) lower to middle Frasnian record Spathiocaris chagrinensis Ruedemann, 1916, is also an ammonoid anaptychus. Although ammonoids can be relatively frequent in some Frasnian horizons of Belgium, anaptychi remain particularly scarce and the attribution to the present material to peculiar ammonoid species is not possible.

Environmental Enrichment Accelerates the Ontogeny of Cryptic Behavior in Pharaoh Cuttlefish (Sepia pharaonis).

We examined effect of environmental enrichment on cuttlefish, the most neutrally advanced invertebrate, to compare species variation of genetic and environmental influences. Cuttlefish were reared from seven to 117 days in one of three environments, namely, "poor" (artificial bottom without objects), "standard" (sandy bottom), and "enriched" (sandy bottom with objects). In Experiment 1, we explored whether enrichment affects the exhibition of crypsis in the cuttlefish. The cuttlefish in the standard and enriched environments spent most of their time at the bottom, exhibiting the mottled or disruptive pattern starting at 27 days of age. On the contrary, those in the poor environment exhibited uniform pattern starting at 87 days of age. Additionally, they repeatedly attempted to dig from 27 to 87 days of age, and moved around by hovering from 77 to 117 days of age. In Experiment 2, we exposed the cuttlefish to six novel substrates every other month after 53 days of age to verify whether enrichment actually affected the maturation of cryptic ability. Cuttlefish from the poor environment tended not to dig into white sandy bottom at 53-55 days of age. Additionally, they did not clearly exhibit appropriate body patterns in response to the six substrates compared to those from the other two environments at 81-83 days of age. However, at 113-115 days of age, most cuttlefish from the three environments exhibited similar cryptic behaviors in response to novel substrates. We conclude that physical enrichment promotes crypsis and accelerates the maturation of this ability in cuttlefish.