Chilling with cephalopods: Temperature-responsive RNA editing in octopus and squid.

The molecular mechanisms that generate the developmental and physiological complexity found within cephalopods are not well understood. In this issue of Cell, Birk et al. and Rangan and Reck-Peterson show that cephalopods differentially edit their RNA in response to temperature changes and that this editing has consequences on protein function.

Temperature-dependent RNA editing in octopus extensively recodes the neural proteome.

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

Molecular Basis of Chemotactile Sensation in Octopus.

Animals display wide-ranging evolutionary adaptations based on their ecological niche. Octopuses explore the seafloor with their flexible arms using a specialized "taste by touch" system to locally sense and respond to prey-derived chemicals and movement. How the peripherally distributed octopus nervous system mediates relatively autonomous arm behavior is unknown. Here, we report that octopus arms use a family of cephalopod-specific chemotactile receptors (CRs) to detect poorly soluble natural products, thereby defining a form of contact-dependent, aquatic chemosensation. CRs form discrete ion channel complexes that mediate the detection of diverse stimuli and transduction of specific ionic signals. Furthermore, distinct chemo- and mechanosensory cells exhibit specific receptor expression and electrical activities to support peripheral information coding and complex chemotactile behaviors. These findings demonstrate that the peripherally distributed octopus nervous system is a key site for signal processing and highlight how molecular and anatomical features synergistically evolve to suit an animal's environmental context.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

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.

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.

Gene network analyses of larvae under different egg-protecting behaviors provide novel insights into immune response mechanisms of Amphioctopus fangsiao.

Amphioctopus fangsiao was a representative economic species in cephalopods, which was vulnerable to marine bacteria. Vibrio anguillarum was a highly infectious pathogen that have recently been found to infect A. fangsiao and inhibit its growth and development. There were significant differences in the immune response mechanisms between egg-protected and egg-unprotected larvae. To explore larval immunity under different egg-protecting behaviors, we infected A. fangsiao larvae with V. anguillarum for 24 h and analyzed the transcriptome data about egg-protected and egg-unprotected larvae infected with 0, 4, 12, and 24 h using weighted gene co-expression networks (WGCNA) and protein-protein interaction (PPI) networks. Network analyses revealed a series of immune response processes after infection, and identified six key modules and multiple immune-related hub genes. Meanwhile, we found that ZNF family, such as ZNF32, ZNF160, ZNF271, ZNF479, and ZNF493 might play significant roles in A. fangsiao immune response processes. We first creatively combined WGCNA and PPI network analysis to deeply explore the immune response mechanisms of A. fangsiao larvae with different egg-protecting behaviors. Our results provided further insights into the immunity of V. anguillarum infected invertebrates, and laid the foundation for exploring the immune differences among cephalopods with different egg protecting behaviors.

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.

The gold-ringed octopus (Amphioctopus fangsiao) genome and cerebral single-nucleus transcriptomes provide insights into the evolution of karyotype and neural novelties.

BACKGROUND: Coleoid cephalopods have distinctive neural and morphological characteristics compared to other invertebrates. Early studies reported massive genomic rearrangements occurred before the split of octopus and squid lineages (Proc Natl Acad Sci U S A 116:3030-5, 2019), which might be related to the neural innovations of their brain, yet the details remain elusive. Here we combine genomic and single-nucleus transcriptome analyses to investigate the octopod chromosome evolution and cerebral characteristics. RESULTS: We present a chromosome-level genome assembly of a gold-ringed octopus, Amphioctopus fangsiao, and a single-nucleus transcriptome of its supra-esophageal brain. Chromosome-level synteny analyses estimate that the chromosomes of the ancestral octopods experienced multiple chromosome fission/fusion and loss/gain events by comparing with the nautilus genome as outgroup, and that a conserved genome organization was detected during the evolutionary process from the last common octopod ancestor to their descendants. Besides, protocadherin, GPCR, and C2H2 ZNF genes are thought to be highly related to the neural innovations in cephalopods (Nature 524:220-4, 2015), and the chromosome analyses pinpointed several collinear modes of these genes on the octopod chromosomes, such as the collinearity between PCDH and C2H2 ZNF, as well as between GPCR and C2H2 ZNF. Phylogenetic analyses show that the expansion of the octopod protocadherin genes is driven by a tandem-duplication mechanism on one single chromosome, including two separate expansions at 65 million years ago (Ma) and 8-14 Ma, respectively. Furthermore, we identify eight cell types (i.e., cholinergic and glutamatergic neurons) in the supra-esophageal brain of A. fangsiao, and the single-cell expression analyses reveal the co-expression of protocadherin and GPCR in specific neural cells, which may contribute to the neural development and signal transductions in the octopod brain. CONCLUSIONS: The octopod genome analyses reveal the dynamic evolutionary history of octopod chromosomes and neural-related gene families. The single-nucleus transcriptomes of the supra-esophageal brain indicate their cellular heterogeneities and functional interactions with other tissues (i.e., gill), which provides a foundation for further octopod cerebral studies.

Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain.

BACKGROUND: Transposable elements (TEs) widely contribute to the evolution of genomes allowing genomic innovations, generating germinal and somatic heterogeneity, and giving birth to long non-coding RNAs (lncRNAs). These features have been associated to the evolution, functioning, and complexity of the nervous system at such a level that somatic retrotransposition of long interspersed element (LINE) L1 has been proposed to be associated to human cognition. Among invertebrates, octopuses are fascinating animals whose nervous system reaches a high level of complexity achieving sophisticated cognitive abilities. The sequencing of the genome of the Octopus bimaculoides revealed a striking expansion of TEs which were proposed to have contributed to the evolution of its complex nervous system. We recently found a similar expansion also in the genome of Octopus vulgaris. However, a specific search for the existence and the transcription of full-length transpositionally competent TEs has not been performed in this genus. RESULTS: Here, we report the identification of LINE elements competent for retrotransposition in Octopus vulgaris and Octopus bimaculoides and show evidence suggesting that they might be transcribed and determine germline and somatic polymorphisms especially in the brain. Transcription and translation measured for one of these elements resulted in specific signals in neurons belonging to areas associated with behavioral plasticity. We also report the transcription of thousands of lncRNAs and the pervasive inclusion of TE fragments in the transcriptomes of both Octopus species, further testifying the crucial activity of TEs in the evolution of the octopus genomes. CONCLUSIONS: The neural transcriptome of the octopus shows the transcription of thousands of putative lncRNAs and of a full-length LINE element belonging to the RTE class. We speculate that a convergent evolutionary process involving retrotransposons activity in the brain has been important for the evolution of sophisticated cognitive abilities in this genus.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

Transcriptome profiling based on larvae at different time points after hatching provides a core set of gene resource for understanding the immune response mechanisms of the egg-protecting behavior against Vibrio anguillarum infection in Amphioctopus fangsiao.

Mollusks have recently received increasing attention because of their unique immune systems. Mollusks such as Amphioctopus fangsiao are economically important cephalopods, and the effects of their egg-protecting behavior on the larval immune response are unclear. Meanwhile, little research has been done on the resistance response of cephalopod larvae infected with pathogenic bacteria such as Vibrio anguillarum. In this study, V. anguillarum was used to infect the primary hatching A. fangsiao larvae under different egg-protecting behaviors for 24 h, and a total of 7156 differentially expressed genes (DEGs) were identified at four time points after hatching based on transcriptome analysis. GO and KEGG enrichment analyses showed that multiple immune-related GO terms and KEGG signaling pathways were enriched. Protein-protein interaction networks (PPI networks) were used to search functional relationships between immune-related DEGs. Finally, 20 hub genes related to multiple gene functions or involved in multiple signaling pathways were identified, and their accuracy was verified using quantitative RT-PCR. PPI networks were first used to study the effects A. fangsiao larvae after infection with V. anguillarum under different egg-protecting behaviors. The results provide significant genetic resources for exploring invertebrate larval immune processes. The data lays a foundation for further study the immune response mechanisms for invertebrates after infection.

Sex-specific role of the optic gland in octopus maya: A transcriptomic analysis.

The optic glands (OG) of cephalopods are a source of molecules associated with the control of reproductive traits and lifecycle events such as sexual maturation, reproductive behavior, feeding, parental care, and senescence. However, little is known about the role of the optic gland in Octopus maya adults during mating and egg laying. RNA sequencing, de novo transcriptome assembly, ubiquity and differential expression analysis were performed. First, we analyzed the expression patterns of transcripts commonly associated with OG regulatory functions to describe their possible role once the maturation of the gonad is complete. The transcriptomic profiles of the optic gland of both sexes were compared with emphasis on the signaling pathways involved in the dimorphism of reproductive traits. Results suggest that in the OG of males, the reproductive condition (mated or non-mated) did not affect the general expression profile. In contrast, more differentially expressed genes were observed in females. In mated females, the mRNA metabolic process and the response to norepinephrine were enriched, suggesting a high cellular activity in preparation for the laying of the embryos. Whereas in egg-laying females, energetic and metabolic processes were the most represented, including the oxidation-reduction process. Finally, the gene expression patterns in senescence females suggest a physiological response to starvation as well as upregulation of genes involved retrotransposon activity. In conclusion, more substantial fluctuations in gene expression were observed in the optic glands of the fertilized females compared to the males. Such differences might be associated with the regulation of the egg-laying and the onset of senescence.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

Immature trematodes of Lecithochirium sp. (Digenea: Hemiuridae) in the California two-spot octopus (Octopus bimaculatus) from Mexico.

Immature trematodes of Lecithochirium sp. are recorded for the first time as parasites of the California two-spot octopus Octopus bimaculatus from Bahía de los Ángeles, Baja California, Mexico. Thirty-nine O. bimaculatus were examined for trematodes and a total of 100 immature specimens of Lecithochirium sp. were recorded from the crop of seven infected octopuses. Based on these records, O. bimaculatus may act as a second intermediate or paratenic host for these parasites. Partial sequences of the 28S (region D1-D3) ribosomal gene corroborate the identifications based on morphological characters. DNA sequences of the 28S gene from GenBank were analyzed to include the immature samples of Lecithochirium sp. within a hemiurid phylogenetic framework. All immature specimens of Lecithochirium sp. were recovered as monophyletic and Pulmovermis cyanovitellosus was identified as the sister species of Lecithochirium sp. However, due to the lack of molecular data for species of the genus Lecithochirium, these phylogenetic inferences must be taken with caution. Therefore, the morphological and molecular data obtained here provide a foundation for future work to develop a systematic comparison among- and within-species of the genus Lecithochirium. Additionally, the present records of Lecithochirium in O. bimaculus add to the knowledge of the parasite fauna of cephalopods.

Transcriptome profiling based on protein-protein interaction networks provides a set of core genes for understanding the immune response mechanisms of the egg-protecting behavior in Octopus ocellatus.

Protection via of the immune system is indispensable to the life of organisms. Within an immune network, problems with a given link will affect the normal life activities of the organism. Octopus ocellatus is cephalopod widely distributed throughout the world's oceans. Because of its unique nervous system and locomotive organs, research on this species has gradually increased in recent years. Many immune response mechanisms associated with behaviors of O. ocellatus are still unclear. Moreover, as a factor affecting the normal growth of O. ocellatus, egg protection has rarely been considered in previous behavioral studies. In this study, we analyzed the transcriptome profile of gene expression in O. ocellatus larvae, and identified 5936 differentially expressed genes (DEGs). GO and KEGG enrichment analyses were used to search for immune-related DEGs. Protein-protein interaction networks were constructed to examine the interactions between immune-related genes. Fifteen hub genes involved in multiple KEGG signaling pathways or with multiple protein-protein interaction relationships were obtained and verified by quantitative RT-PCR. We first studied the effects of egg protection on the immunity of O. ocellatus larvae by means of protein-protein interaction networks, and the results provide valuable genetic resources for understanding the immunity of invertebrate larvae. The data serve as a foundation for further research on the egg-protecting behavior of invertebrates.

New microsatellite loci for estimating genetic diversity and structure in Octopus hubbsorum from Nayarit, México.

BACKGROUND: Octopus hubbsorum Berry, 1953 is the most important species for commercial fishing in the Mexican Pacific. However, there is a lack of information regarding population structure that could have important management implications. We tested 44 microsatellite loci in O. hubbsorum by cross-amplification from O. bimaculatus. METHODS AND RESULTS: Genetic diversity and structure was tested over 30 octopus sampled from Santa Cruz de Miramar (Nayarit, México). A total of 11 loci were successfully amplified. All loci were polymorphic with the number of effective alleles ranging from 2.13 to 23.14, while three loci significantly deviated from Hardy-Weinberg equilibrium. No significant LD was observed between pairs of loci (P ≥ 0.05). The application of the new markers in a O. hubbsorum population from Santa Cruz de Miramar Nayarit, México, did not showed Wahlund or isolate breaking effects due to the mixing of distinct populations. CONCLUSIONS: The loci were useful to estimate levels of pairwise relatedness and to discard the presence of recent demographic bottlenecks in the population. We consider that eight microsatellites are adequate from the 11 amplified loci.

The octopus genome and the evolution of cephalopod neural and morphological novelties.

Coleoid cephalopods (octopus, squid and cuttlefish) are active, resourceful predators with a rich behavioural repertoire. They have the largest nervous systems among the invertebrates and present other striking morphological innovations including camera-like eyes, prehensile arms, a highly derived early embryogenesis and a remarkably sophisticated adaptive colouration system. To investigate the molecular bases of cephalopod brain and body innovations, we sequenced the genome and multiple transcriptomes of the California two-spot octopus, Octopus bimaculoides. We found no evidence for hypothesized whole-genome duplications in the octopus lineage. The core developmental and neuronal gene repertoire of the octopus is broadly similar to that found across invertebrate bilaterians, except for massive expansions in two gene families previously thought to be uniquely enlarged in vertebrates: the protocadherins, which regulate neuronal development, and the C2H2 superfamily of zinc-finger transcription factors. Extensive messenger RNA editing generates transcript and protein diversity in genes involved in neural excitability, as previously described, as well as in genes participating in a broad range of other cellular functions. We identified hundreds of cephalopod-specific genes, many of which showed elevated expression levels in such specialized structures as the skin, the suckers and the nervous system. Finally, we found evidence for large-scale genomic rearrangements that are closely associated with transposable element expansions. Our analysis suggests that substantial expansion of a handful of gene families, along with extensive remodelling of genome linkage and repetitive content, played a critical role in the evolution of cephalopod morphological innovations, including their large and complex nervous systems.

Population genetics and comparative mitogenomic analyses reveal cryptic diversity of Amphioctopus neglectus (Cephalopoda: Octopodidae).

This study presented 96 cox1 and 76 cox3 genes of Amphioctopus neglectus populations. Three distinct lineages were formed from phylogenetic trees and networks constructed using haplotypes. Mitogenomes of A. neglectus-a and A. neglectus-b as the representatives of two lineages separated from population genetics were sequenced to compare with A. neglectus at the genome-level. Amphioctopus neglectus-a showed significant differences with A. neglectus, mainly reflected in gene length, intergenic regions and the secondary structure of tandem repeat motifs. Notably, two sequence deletions in mitogenomes of the two representative species were detected in different positions of major non-coding regions, which were the most distinct differences with A. neglectus. Pairwise genetic distances and the phylogenetic analysis supported the relationship of (A. neglectus-a + (A. neglectus + A. neglectus-b)). This study suggested that A. neglectus-a should be considered as a potential cryptic species of this complex, while A. neglectus-b needed further verification to be defined.

The complete mitochondrial genomes of two octopods of the eastern Pacific Ocean: Octopus mimus and 'Octopus' fitchi (Cephalopoda: Octopodidae) and their phylogenetic position within Octopoda.

The complete mitochondrial genomes of two important octopus species from the eastern Pacific were sequenced, obtaining their complete nucleotide sequences. Octopus mimus is the most important commercially catched species along the eastern Pacific, from Mexico to Chile, whereas 'Octopus' fitchi is a pigmy species with uncertain taxonomic genus. The mitogenomes of Octopus mimus and 'Octopus' fitchi were 15,696 and 15,780 base pairs (bp) in length with an A + T composition of 75.5% and 75.8%, respectively. Each genome contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, as well as a control region. Gene order is maintained as reported for other species of the Octopodidae. The phylogenetic analysis based on the concatenated thirteen protein-coding genes confirms that O. mimus belongs to the genus Octopus, which is supported by the genetic distance (11-16%) whereas the position of 'O'. fitchi within this group it is not supported. The analysis also indicated that the phylogenetic position of 'O'. fitchi is closer to Callistoctopus than to the Cistopus or the Amphioctopus clades. Based on the tree topology and the high genetic distance observed (24-25%), we suggest that 'O'. fitchi might represent a different genus.