Benchmarks in antimicrobial peptide prediction are biased due to the selection of negative data.

Antimicrobial peptides (AMPs) are a heterogeneous group of short polypeptides that target not only microorganisms but also viruses and cancer cells. Due to their lower selection for resistance compared with traditional antibiotics, AMPs have been attracting the ever-growing attention from researchers, including bioinformaticians. Machine learning represents the most cost-effective method for novel AMP discovery and consequently many computational tools for AMP prediction have been recently developed. In this article, we investigate the impact of negative data sampling on model performance and benchmarking. We generated 660 predictive models using 12 machine learning architectures, a single positive data set and 11 negative data sampling methods; the architectures and methods were defined on the basis of published AMP prediction software. Our results clearly indicate that similar training and benchmark data set, i.e. produced by the same or a similar negative data sampling method, positively affect model performance. Consequently, all the benchmark analyses that have been performed for AMP prediction models are significantly biased and, moreover, we do not know which model is the most accurate. To provide researchers with reliable information about the performance of AMP predictors, we also created a web server AMPBenchmark for fair model benchmarking. AMPBenchmark is available at http://BioGenies.info/AMPBenchmark.

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.

ampir: an R package for fast genome-wide prediction of antimicrobial peptides.

SUMMARY: Antimicrobial peptides (AMPs) are the key components of the innate immune system that protect against pathogens, regulate the microbiome and are promising targets for pharmaceutical research. Computational tools based on machine learning have the potential to aid discovery of genes encoding novel AMPs but existing approaches are not designed for genome-wide scans. To facilitate such genome-wide discovery of AMPs we developed a fast and accurate AMP classification framework, ampir. ampir is designed for high throughput, integrates well with existing bioinformatics pipelines, and has much higher classification accuracy than existing methods when applied to whole genome data. AVAILABILITY AND IMPLEMENTATION: ampir is implemented primarily in R with core feature calculation methods written in C++. Release versions are available via CRAN and work on all major operating systems. The development version is maintained at https://github.com/legana/ampir. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coupled Genomic Evolutionary Histories as Signatures of Organismal Innovations in Cephalopods: Co-evolutionary Signatures Across Levels of Genome Organization May Shed Light on Functional Linkage and Origin of Cephalopod Novelties.

How genomic innovation translates into organismal organization remains largely unanswered. Possessing the largest invertebrate nervous system, in conjunction with many species-specific organs, coleoid cephalopods (octopuses, squids, cuttlefishes) provide exciting model systems to investigate how organismal novelties evolve. However, dissecting these processes requires novel approaches that enable deeper interrogation of genome evolution. Here, the existence of specific sets of genomic co-evolutionary signatures between expanded gene families, genome reorganization, and novel genes is posited. It is reasoned that their co-evolution has contributed to the complex organization of cephalopod nervous systems and the emergence of ecologically unique organs. In the course of reviewing this field, how the first cephalopod genomic studies have begun to shed light on the molecular underpinnings of morphological novelty is illustrated and their impact on directing future research is described. It is argued that the application and evolutionary profiling of evolutionary signatures from these studies will help identify and dissect the organismal principles of cephalopod innovations. By providing specific examples, the implications of this approach both within and beyond cephalopod biology are discussed.

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.

Shotgun Proteomics Analysis of Saliva and Salivary Gland Tissue from the Common Octopus Octopus vulgaris.

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.

Liver proteome response of pre-harvest Atlantic salmon following exposure to elevated temperature.

BACKGROUND: Atlantic salmon production in Tasmania (Southern Australia) occurs near the upper limits of the species thermal tolerance. Summer water temperatures can average over 19 °C over several weeks and have negative effects on performance and health. Liver tissue exerts important metabolic functions in thermal adaptation. With the aim of identifying mechanisms underlying liver plasticity in response to chronic elevated temperature in Atlantic salmon, label-free shotgun proteomics was used to explore quantitative protein changes after 43 days of exposure to elevated temperature. RESULTS: A total of 276 proteins were differentially (adjusted p-value < 0.05) expressed between the control (15 °C) and elevated (21 °C) temperature treatments. As identified by Ingenuity Pathway Analysis (IPA), transcription and translation mechanisms, protein degradation via the proteasome, and cytoskeletal components were down-regulated at elevated temperature. In contrast, an up-regulated response was identified for NRF2-mediated oxidative stress, endoplasmic reticulum stress, and amino acid degradation. The proteome response was paralleled by reduced fish condition factor and hepato-somatic index at elevated temperature. CONCLUSIONS: The present study provides new evidence of the interplay among different cellular machineries in a scenario of heat-induced energy deficit and oxidative stress, and refines present understanding of how Atlantic salmon cope with chronic exposure to temperature near the upper limits of thermal tolerance.

Combined Transcriptomic and Proteomic Analysis of the Posterior Salivary Gland from the Southern Blue-Ringed Octopus and the Southern Sand Octopus.

This study provides comprehensive proteomic profiles from the venom producing posterior salivary glands of octopus (superorder Octopodiformes) species. A combined transcriptomic and proteomic approach was used to identify 1703 proteins from the posterior salivary gland of the southern blue-ringed octopus, Hapalochlaena maculosa and 1300 proteins from the posterior salivary gland of the southern sand octopus, Octopus kaurna. The two proteomes were broadly similar; clustering of proteins into orthogroups revealed 937 that were shared between species. Serine proteases were particularly diverse and abundant in both species. Other abundant proteins included a large number of secreted proteins, many of which had no known conserved domains, or homology to proteins with known function. On the basis of homology to known venom proteins, 23 putative toxins were identified in H. maculosa and 24 in O. kaurna. These toxins span nine protein families: CAP (cysteine rich secretory proteins, antigen 5, parthenogenesis related), chitinase, carboxylesterase, DNase, hyaluronidase, metalloprotease, phospholipase, serine protease and tachykinin. Serine proteases were responsible for 70.9% and 86.3% of putative toxin expression in H. maculosa and O. kaurna, respectively, as determined using intensity based absolute quantification (iBAQ) measurements. Phylogenetic analysis of the putative toxin serine proteases revealed a similar suite of diverse proteins present in both species. Posterior salivary gland composition of H. maculosa and O. kaurna differ in several key aspects. While O. kaurna expressed the proteinaceous neurotoxin, tachykinin, this was absent from H. maculosa, perhaps reflecting the acquisition of a potent nonproteinaceous neurotoxin, tetrodotoxin (TTX) produced by bacteria in the salivary glands of that species. The dispersal factor, hyaluronidase was particularly abundant in H. maculosa. Chitinase was abundant in both species and is believed to facilitate envenomation in chitinous prey such as crustaceans. Cephalopods represent a largely unexplored source of novel proteins distinct from all other venomous taxa and are of interest for further inquiry, as novel proteinaceous toxins derived from venoms may contribute to pharmaceutical design.

Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.

A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.

Cryptic diversity and spatial genetic variation in the coral Acropora tenuis and its endosymbionts across the Great Barrier Reef.

Genomic studies are uncovering extensive cryptic diversity within reef-building corals, suggesting that evolutionarily and ecologically relevant diversity is highly underestimated in the very organisms that structure coral reefs. Furthermore, endosymbiotic algae within coral host species can confer adaptive responses to environmental stress and may represent additional axes of coral genetic variation that are not constrained by taxonomic divergence of the cnidarian host. Here, we examine genetic variation in a common and widespread, reef-building coral, Acropora tenuis, and its associated endosymbiotic algae along the entire expanse of the Great Barrier Reef (GBR). We use SNPs derived from genome-wide sequencing to characterize the cnidarian coral host and organelles from zooxanthellate endosymbionts (genus Cladocopium). We discover three distinct and sympatric genetic clusters of coral hosts, whose distributions appear associated with latitude and inshore-offshore reef position. Demographic modelling suggests that the divergence history of the three distinct host taxa ranges from 0.5 to 1.5 million years ago, preceding the GBR's formation, and has been characterized by low-to-moderate ongoing inter-taxon gene flow, consistent with occasional hybridization and introgression typifying coral evolution. Despite this differentiation in the cnidarian host, A. tenuis taxa share a common symbiont pool, dominated by the genus Cladocopium (Clade C). Cladocopium plastid diversity is not strongly associated with host identity but varies with reef location relative to shore: inshore colonies contain lower symbiont diversity on average but have greater differences between colonies as compared with symbiont communities from offshore colonies. Spatial genetic patterns of symbiont communities could reflect local selective pressures maintaining coral holobiont differentiation across an inshore-offshore environmental gradient. The strong influence of environment (but not host identity) on symbiont community composition supports the notion that symbiont community composition responds to habitat and may assist in the adaptation of corals to future environmental change.

Genomic evidence for West Antarctic Ice Sheet collapse during the Last Interglacial.

The marine-based West Antarctic Ice Sheet (WAIS) is considered vulnerable to irreversible collapse under future climate trajectories, and its tipping point may lie within the mitigated warming scenarios of 1.5° to 2°C of the United Nations Paris Agreement. Knowledge of ice loss during similarly warm past climates could resolve this uncertainty, including the Last Interglacial when global sea levels were 5 to 10 meters higher than today and global average temperatures were 0.5° to 1.5°C warmer than preindustrial levels. Using a panel of genome-wide, single-nucleotide polymorphisms of a circum-Antarctic octopus, we show persistent, historic signals of gene flow only possible with complete WAIS collapse. Our results provide the first empirical evidence that the tipping point of WAIS loss could be reached even under stringent climate mitigation scenarios.

Spatially varying selection between habitats drives physiological shifts and local adaptation in a broadcast spawning coral on a remote atoll in Western Australia.

At the Rowley Shoals in Western Australia, the prominent reef flat becomes exposed on low tide and the stagnant water in the shallow atoll lagoons heats up, creating a natural laboratory for characterizing the mechanisms of coral resilience to climate change. To explore these mechanisms in the reef coral Acropora tenuis, we collected samples from lagoon and reef slope habitats and combined whole-genome sequencing, ITS2 metabarcoding, experimental heat stress, and transcriptomics. Despite high gene flow across the atoll, we identified clear shifts in allele frequencies between habitats at relatively small linked genomic islands. Common garden heat stress assays showed corals from the lagoon to be more resistant to bleaching, and RNA sequencing revealed marked differences in baseline levels of gene expression between habitats. Our results provide new insight into the complex mechanisms of coral resilience to climate change and highlight the potential for spatially varying selection across complex coral reef seascapes to drive pronounced ecological divergence in climate-related traits.

Morphological stasis masks ecologically divergent coral species on tropical reefs.

Coral reefs are the epitome of species diversity, yet the number of described scleractinian coral species, the framework-builders of coral reefs, remains moderate by comparison. DNA sequencing studies are rapidly challenging this notion by exposing a wealth of undescribed diversity, but the evolutionary and ecological significance of this diversity remains largely unclear. Here, we present an annotated genome for one of the most ubiquitous corals in the Indo-Pacific (Pachyseris speciosa) and uncover, through a comprehensive genomic and phenotypic assessment, that it comprises morphologically indistinguishable but ecologically divergent lineages. Demographic modeling based on whole-genome resequencing indicated that morphological crypsis (across micro- and macromorphological traits) was due to ancient morphological stasis rather than recent divergence. Although the lineages occur sympatrically across shallow and mesophotic habitats, extensive genotyping using a rapid molecular assay revealed differentiation of their ecological distributions. Leveraging "common garden" conditions facilitated by the overlapping distributions, we assessed physiological and quantitative skeletal traits and demonstrated concurrent phenotypic differentiation. Lastly, spawning observations of genotyped colonies highlighted the potential role of temporal reproductive isolation in the limited admixture, with consistent genomic signatures in genes related to morphogenesis and reproduction. Overall, our findings demonstrate the presence of ecologically and phenotypically divergent coral species without substantial morphological differentiation and provide new leads into the potential mechanisms facilitating such divergence. More broadly, they indicate that our current taxonomic framework for reef-building corals may be scratching the surface of the ecologically relevant diversity on coral reefs, consequently limiting our ability to protect or restore this diversity effectively.

Adaptive venom evolution and toxicity in octopods is driven by extensive novel gene formation, expansion, and loss.

BACKGROUND: Cephalopods represent a rich system for investigating the genetic basis underlying organismal novelties. This diverse group of specialized predators has evolved many adaptations including proteinaceous venom. Of particular interest is the blue-ringed octopus genus (Hapalochlaena), which are the only octopods known to store large quantities of the potent neurotoxin, tetrodotoxin, within their tissues and venom gland. FINDINGS: To reveal genomic correlates of organismal novelties, we conducted a comparative study of 3 octopod genomes, including the Southern blue-ringed octopus (Hapalochlaena maculosa). We present the genome of this species and reveal highly dynamic evolutionary patterns at both non-coding and coding organizational levels. Gene family expansions previously reported in Octopus bimaculoides (e.g., zinc finger and cadherins, both associated with neural functions), as well as formation of novel gene families, dominate the genomic landscape in all octopods. Examination of tissue-specific genes in the posterior salivary gland revealed that expression was dominated by serine proteases in non-tetrodotoxin-bearing octopods, while this family was a minor component in H. maculosa. Moreover, voltage-gated sodium channels in H. maculosa contain a resistance mutation found in pufferfish and garter snakes, which is exclusive to the genus. Analysis of the posterior salivary gland microbiome revealed a diverse array of bacterial species, including genera that can produce tetrodotoxin, suggestive of a possible production source. CONCLUSIONS: We present the first tetrodotoxin-bearing octopod genome H. maculosa, which displays lineage-specific adaptations to tetrodotoxin acquisition. This genome, along with other recently published cephalopod genomes, represents a valuable resource from which future work could advance our understanding of the evolution of genomic novelty in this family.

The evolution and origin of tetrodotoxin acquisition in the blue-ringed octopus (genus Hapalochlaena).

Tetrodotoxin is a potent non-proteinaceous neurotoxin, which is commonly found in the marine environment. Synthesised by bacteria, tetrodotoxin has been isolated from the tissues of several genera including pufferfish, salamanders and octopus. Believed to provide a defensive function, the independent evolution of tetrodotoxin sequestration is poorly understood in most species. Two mechanisms of tetrodotoxin resistance have been identified to date, tetrodotoxin binding proteins in the circulatory system and mutations to voltage gated sodium channels, the binding target of tetrodotoxin with the former potentially succeeding the latter in evolutionary time. This review focuses on the evolution of tetrodotoxin acquisition, in particular how it may have occurred within the blue-ringed octopus genus (Hapalochlaena) and the subsequent impact on venom evolution.

Epipodial Tentacle Gene Expression and Predetermined Resilience to Summer Mortality in the Commercially Important Greenlip Abalone, Haliotis laevigata.

"Summer mortality" is a phenomenon that occurs during warm water temperature spikes that results in the mass mortality of many ecologically and economically important mollusks such as abalone. This study aimed to determine whether the baseline gene expression of abalone before a laboratory-induced summer mortality event was associated with resilience to summer mortality. Tentacle transcriptomes of 35 greenlip abalone (Haliotis laevigata) were sequenced prior to the animals being exposed to an increase in water temperature-simulating conditions which have previously resulted in summer mortality. Abalone derived from three source locations with different environmental conditions were categorized as susceptible or resistant to summer mortality depending on whether they died or survived after the water temperature was increased. We detected two genes showing significantly higher expression in resilient abalone relative to susceptible abalone prior to the laboratory-induced summer mortality event. One of these genes was annotated through the NCBI non-redundant protein database using BLASTX to an anemone (Exaiptasia pallida) Transposon Ty3-G Gag Pol polyprotein. Distinct gene expression signatures were also found between resilient and susceptible abalone depending on the population origin, which may suggest divergence in local adaptation mechanisms for resilience. Many of these genes have been suggested to be involved in antioxidant and immune-related functions. The identification of these genes and their functional roles have enhanced our understanding of processes that may contribute to summer mortality in abalone. Our study supports the hypothesis that prestress gene expression signatures are indicative of the likelihood of summer mortality.

Triploid Atlantic salmon shows similar performance, fatty acid composition and proteome response to diploids during early freshwater rearing.

There is currently renewed interest in farming triploid Atlantic salmon. Improving farming requires identifying triploid specific phenotypic and physiological traits that are uniquely derived from ploidy per se and developed under optimal growing conditions. This study investigated firstly, the impact of ploidy on growth performance and whole body composition of Atlantic salmon at different early freshwater stages [34dph (days post-hatching) alevin, 109dph fry, and 162dph parr] and secondly, whether phenotypic differences at these stages were reflected in protein samples collected from whole fish, white muscle or liver tissue. Female diploid and triploid Atlantic salmon (n=3) were first fed at 35dph and then maintained by feeding to satiation on commercial feeds. Triploids were significantly lower in weight at the late alevin and fry stages but matched diploid weight at the parr stage. The whole-body lipid content was significantly higher for triploids at the parr stage, while the whole-body lipid class profile was broadly similar and was largely not affected by ploidy. Comparative label-free shotgun proteomic analysis did not detect significant alterations in protein expression between diploids and triploids at any growth stage. The present results indicate that ploidy under optimal growing conditions and during early freshwater stages only result in small phenotypic differences in weight and whole body lipid content that were not reflected at the proteome level. These findings suggest that optimal husbandry conditions for freshwater Atlantic salmon are similar between ploidies, at least for all-female populations.

An Accessible Proteogenomics Informatics Resource for Cancer Researchers.

Proteogenomics has emerged as a valuable approach in cancer research, which integrates genomic and transcriptomic data with mass spectrometry-based proteomics data to directly identify expressed, variant protein sequences that may have functional roles in cancer. This approach is computationally intensive, requiring integration of disparate software tools into sophisticated workflows, challenging its adoption by nonexpert, bench scientists. To address this need, we have developed an extensible, Galaxy-based resource aimed at providing more researchers access to, and training in, proteogenomic informatics. Our resource brings together software from several leading research groups to address two foundational aspects of proteogenomics: (i) generation of customized, annotated protein sequence databases from RNA-Seq data; and (ii) accurate matching of tandem mass spectrometry data to putative variants, followed by filtering to confirm their novelty. Directions for accessing software tools and workflows, along with instructional documentation, can be found at z.umn.edu/canresgithub. Cancer Res; 77(21); e43-46. ©2017 AACR.

The complete mitochondrial genome of the pygmy squid, Idiosepius (Cephalopoda: Decapodiformes): the first representative from the family Idiosepiidae.

We report the first complete mitochondrial genome of the pygmy squid, Idiosepius, (Idiosepiidae). The mtDNA genome is 16,183 bp long with an AT content of 75.4%. All conserved metazoan mitochondrial genes are identified with the addition of a 1018 bp non-coding region. Idiosepius gene order most closely resembles that of the bobtail squid Semirossia (Sepiolidae).