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.

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.

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.