The rise of dietary diversity in coral reef fishes.

Diet has been identified as a major driver of reef fish lineage diversification, producing one of the most speciose vertebrate assemblages today. Yet, there is minimal understanding of how, when and why diet itself has evolved. To address this, we used a comprehensive gut content dataset, alongside a recently developed phylogenetic comparative method to assess multivariate prey use across a diverse animal assemblage, coral reef fishes. Specifically, we investigated the diversification, transitions and phylogenetic conservatism of fish diets through evolutionary time. We found two major pulses of diet diversification: one at the end-Cretaceous and one during the Eocene, suggesting that the Cretaceous-Palaeogene mass extinction probably provided the initial ecological landscape for fish diets to diversify. The birth of modern families during the Eocene then provided the foundation for a second wave of dietary expansion. Together, our findings showcase the role of extinction rebound events in shaping the dietary diversity of fishes on present-day coral reefs.

Chromosomal inversions harbour excess mutational load in the coral, Acropora kenti, on the Great Barrier Reef.

The future survival of coral reefs in the Anthropocene depends on the capacity of corals to adapt as oceans warm and extreme weather events become more frequent. Targeted interventions designed to assist evolutionary processes in corals require a comprehensive understanding of the distribution and structure of standing variation, however, efforts to map genomic variation in corals have so far focussed almost exclusively on SNPs, overlooking structural variants that have been shown to drive adaptive processes in other taxa. Here, we show that the reef-building coral, Acropora kenti, harbours at least five large, highly polymorphic structural variants, all of which exhibit signatures of strongly suppressed recombination in heterokaryotypes, a feature commonly associated with chromosomal inversions. Based on their high minor allele frequency, uniform distribution across habitats and elevated genetic load, we propose that these inversions in A. kenti are likely to be under balancing selection. An excess of SNPs with high impact on protein-coding genes within these loci elevates their importance both as potential targets for adaptive selection and as contributors to genetic decline if coral populations become fragmented or inbred in future.

Evolutionary responses of a reef-building coral to climate change at the end of the last glacial maximum.

Climate change threatens the survival of coral reefs on a global scale, primarily through mass bleaching and mortality as a result of marine heatwaves. While these short-term effects are clear, predicting the fate of coral reefs over the coming century is a major challenge. One way to understand the longer-term effects of rapid climate change is to examine the response of coral populations to past climate shifts. Coastal and shallow-water marine ecosystems such as coral reefs have been reshaped many times by sea-level changes during the Pleistocene, yet, few studies have directly linked this with its consequences on population demographics, dispersal, and adaptation. Here we use powerful analytical techniques, afforded by haplotype phased whole-genomes, to establish such links for the reef-building coral, Acropora digitifera. We show that three genetically distinct populations are present in northwestern Australia, and that their rapid divergence since the last glacial maximum (LGM) can be explained by a combination of founder-effects and restricted gene flow. Signatures of selective sweeps, too strong to be explained by demographic history, are present in all three populations and overlap with genes that show different patterns of functional enrichment between inshore and offshore habitats. In contrast to rapid divergence in the host, we find that photosymbiont communities are largely undifferentiated between corals from all three locations, spanning almost 1000 km, indicating that selection on host genes and not acquisition of novel symbionts, has been the primary driver of adaptation for this species in northwestern Australia.

Genomic insights of evolutionary divergence and life history innovations in Antarctic brittle stars.

Understanding the drivers of evolutionary innovation provides a crucial perspective of how evolutionary processes unfold across taxa and ecological systems. It has been hypothesised that the Southern Ocean provided ecological opportunities for novelty in the past. However, the drivers of innovation are challenging to pinpoint as the evolutionary genetics of Southern Ocean fauna are influenced by Quaternary glacial-interglacial cycles, oceanic currents and species ecology. Here we examined the genome-wide single nucleotide polymorphisms of the Southern Ocean brittle stars Ophionotus victoriae (five arms, broadcaster) and O. hexactis (six arms, brooder). We found that O. victoriae and O. hexactis are closely-related species with interspecific gene flow. During the late Pleistocene, O. victoriae likely persisted in a connected deep water refugium and in situ refugia on the Antarctic continental shelf and around Antarctic islands; O. hexactis persisted exclusively within in situ island refugia. Within O. victoriae, contemporary gene flow linking to the Antarctic Circumpolar Current, regional gyres and other local oceanographic regimes was observed. Gene flow connecting West and East Antarctic islands near the Polar Front was also detected in O. hexactis. A strong association was detected between outlier loci and salinity in O. hexactis. Both O. victoriae and O. hexactis are associated with genome-wide increase in alleles at intermediate-frequencies; the alleles associated with this peak appear to be species specific, and these intermediate-frequency variants are far more excessive in O. hexactis. We hypothesise that the peak in alleles at intermediate frequencies could be related to adaptation in the recent past, linked to evolutionary innovations of increase in arm number and a switch to brooding from broadcasting, in O. hexactis.

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.

Is the Southern Ocean ecosystem primed for change or at the cliff edge?

The Southern Ocean is experiencing unprecedented environmental risks and consequences from current climate change. It is unclear how the benthic fauna, which has largely evolved in isolation, will respond to future changes. Knowing how the benthic fauna persisted through repeated extreme glacial-interglacial cycles in the past provides a unique opportunity to inform future predictions. Right now, understanding and preserving current genetic diversity and connectivity between populations will give species the best chance to adapt.

Emerging biological archives can reveal ecological and climatic change in Antarctica.

Anthropogenic climate change is causing observable changes in Antarctica and the Southern Ocean including increased air and ocean temperatures, glacial melt leading to sea-level rise and a reduction in salinity, and changes to freshwater water availability on land. These changes impact local Antarctic ecosystems and the Earth's climate system. The Antarctic has experienced significant past environmental change, including cycles of glaciation over the Quaternary Period (the past ~2.6 million years). Understanding Antarctica's paleoecosystems, and the corresponding paleoenvironments and climates that have shaped them, provides insight into present day ecosystem change, and importantly, helps constrain model projections of future change. Biological archives such as extant moss beds and peat profiles, biological proxies in lake and marine sediments, vertebrate animal colonies, and extant terrestrial and benthic marine invertebrates, complement other Antarctic paleoclimate archives by recording the nature and rate of past ecological change, the paleoenvironmental drivers of that change, and constrain current ecosystem and climate models. These archives provide invaluable information about terrestrial ice-free areas, a key location for Antarctic biodiversity, and the continental margin which is important for understanding ice sheet dynamics. Recent significant advances in analytical techniques (e.g., genomics, biogeochemical analyses) have led to new applications and greater power in elucidating the environmental records contained within biological archives. Paleoecological and paleoclimate discoveries derived from biological archives, and integration with existing data from other paleoclimate data sources, will significantly expand our understanding of past, present, and future ecological change, alongside climate change, in a unique, globally significant region.

Development of an STR panel for a non-native population of an endangered species.

BACKGROUND: The establishment of non-native populations of threatened and legally protected species can have many implications for the areas where these species have been introduced. Non-native populations of threatened species have the potential to be exploited and therefore the subject of legal protection, while conversely, if they have become invasive in their introduced range, there is the likelihood that population control will be carried out to reduce abundance and negative impacts associated with introduced species. From both a legal and invasive species monitoring standpoint, it is important to know how many individuals are present. METHODS AND RESULTS: Short tandem repeats (STRs) were developed for the hog deer, an endangered species that was introduced following European settlement to Victoria, Australia using Illumina MiSeq sequencing technology. These markers were combined with previous STRs characterised for hog deer to create a 29-plex identification system. A total of 224 samples were genotyped across the population in Victoria, and further analyses of null allele frequencies, deviation from Hardy-Weinberg equilibrium, and the removal of monomorphic or low amplifying markers resulted in a final marker panel of 15 loci. Despite low values for number of alleles at each locus (2-4), probability of identity showed sufficient discrimination power, with an average probability of identity at 2.94 × 10-6, and a probability of sibling identity of 8.9 × 10-4 across all sites. CONCLUSIONS: It is feasible to create an informative DNA profiling system that can distinguish between individuals for applications in both wildlife forensic and population control research.

Global drivers of recent diversification in a marine species complex.

Investigating historical gene flow in species complexes can indicate how environmental and reproductive barriers shape genome divergence during speciation. The processes influencing species diversification under environmental change remain one of the central focal points of evolutionary biology, particularly for marine organisms with high dispersal potential. We investigated genome-wide divergence, introgression patterns and inferred demographic history between species pairs of all six extant rock lobster species (Jasus spp.), which have a long larval duration of up to two years and have populated continental shelf and seamount habitats around the globe at approximately 40o S. Genetic differentiation patterns reflected geographic isolation and the environment (i.e. habitat structure). Eastern Pacific species (J. caveorum and J. frontalis) were geographically more distant and genetically more differentiated from the remaining four species. Species associated with continental shelf habitats shared a common ancestry, but are geographically distant from one another. Similarly, species associated with island/seamount habitats in the Atlantic and Indian Oceans shared a common ancestry, but are also geographically distant. Benthic temperature was the environmental variable that explained most of the genetic differentiation (FST ), while controlling for the effects of geographic distance. Eastern Pacific species retained a signal of strict isolation following ancient migration, whereas species pairs from Australia and Africa, and seamounts in the Indian and Atlantic oceans, included events of introgression after secondary contact. Our results reveal important effects of habitat and demographic processes on the recent divergence of species within the genus Jasus, providing one of the first empirical studies of genome-wide drivers of diversification that incorporates all extant species in a marine genus with long pelagic larval duration.

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.

The shell matrix and microstructure of the Ram's Horn squid: Molecular and structural characterization.

Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, the organic matrix. In spite of several studies, these components are mainly known for bivalves and gastropods. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the Ram's Horn squid Spirula spirula. Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared and solid-state nuclear magnetic resonance spectroscopies. NMR identified ß-chitin and additional polysaccharides for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 39 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, or different histones. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods demonstrates that there is no obvious phylogenetic signal in the cephalopod skeletal matrix.

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.

Mitochondrial and nuclear genetic analyses of the tropical black-lip rock oyster (Saccostrea echinata) reveals population subdivision and informs sustainable aquaculture development.

BACKGROUND: The black-lip rock oyster (Saccostrea echinata) has considerable potential for aquaculture throughout the tropics. Previous attempts to farm S. echinata failed due to an insufficient supply of wild spat; however, the prospect of hatchery-based aquaculture has stimulated renewed interest, and small-scale farming is underway across northern Australia and in New Caledonia. The absence of knowledge surrounding the population genetic structure of this species has raised concerns about the genetic impacts of this emerging aquaculture industry. This study is the first to examine population genetics of S. echinata and employs both mitochondrial cytochrome c oxidase subunit I gene (COI) and single nucleotide polymorphism (SNP) markers. RESULTS: The mitochondrial COI data set included 273 sequences of 594 base pair length, which comprised 74 haplotypes. The SNP data set included 27,887 filtered SNPs for 272 oysters and of these 31 SNPs were identified as candidate adaptive loci. Data from the mitochondrial COI analyses, supports a broad tropical Indo-Pacific distribution of S. echinata, and showed high haplotype and nucleotide diversities (0.887-1.000 and 0.005-0.008, respectively). Mitochondrial COI analyses also revealed a 'star-like' haplotype network, and significant and negative neutrality tests (Tajima's D = - 2.030, Fu's Fs = - 25.638, P < 0.001) support a recent population expansion after a bottleneck. The SNP analyses showed significant levels of population subdivision and four genetic clusters were identified: (1) the Noumea (New Caledonia) sample location; (2) the Bowen (north Queensland, Australia) sample location, and remaining sample locations in the Northern Territory, Australia (n = 8) were differentiated into two genetic clusters. These occurred at either side of the Wessel Islands and were termed (3) 'west' and (4) 'east' clusters, and two migrant individuals were detected between them. The SNP data showed a significant positive correlation between genetic and geographic distance (Mantel test, P < 0.001, R2 = 0.798) and supported isolation by distance. Three candidate adaptive SNPs were identified as occurring within known genes and gene ontology was well described for the sex peptide receptor gene. CONCLUSIONS: Data supports the existence of genetically distinct populations of S. echinata, suggesting that management of wild and farmed stocks should be based upon multiple management units. This research has made information on population genetic structure and connectivity available for a new aquaculture species.

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.

Temporal genetic patterns of diversity and structure evidence chaotic genetic patchiness in a spiny lobster.

Population structure of many marine organisms is spatially patchy and varies within and between years, a phenomenon defined as chaotic genetic patchiness. This results from the combination of planktonic larval dispersal and environmental stochasticity. Additionally, in species with bi-partite life, postsettlement selection can magnify these genetic differences. The high fecundity (up to 500,000 eggs annually) and protracted larval duration (12-24 months) and dispersal of the southern rock lobster, Jasus edwardsii, make it a good test species for chaotic genetic patchiness and selection during early benthic life. Here, we used double digest restriction site-associated DNA sequencing (ddRADseq) to investigate chaotic genetic patchiness and postsettlement selection in this species. We assessed differences in genetic structure and diversity of recently settled pueruli across four settlement years and between two sites in southeast Australia separated by approximately 1,000 km. Postsettlement selection was investigated by identifying loci under putative positive selection between recently settled pueruli and postpueruli and quantifying differences in the magnitude and strength of the selection at each year and site. Genetic differences within and among sites through time in neutral SNP markers indicated chaotic genetic patchiness. Recently settled puerulus at the southernmost site exhibited lower genetic diversity during years of low puerulus catches, further supporting this hypothesis. Finally, analyses of outlier SNPs detected fluctuations in the magnitude and strength of the markers putatively under positive selection over space and time. One locus under putative positive selection was consistent at both locations during the same years, suggesting the existence of weak postsettlement selection.

A mitochondrial phylogeny of the family Onychoteuthidae (Cephalopoda: Oegopsida).

The oegopsid squid family Onychoteuthidae was recently revised based on morphology, but sufficient material for a complementary molecular analysis has not been available until now. In the present study, over 250 sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA for 222 individuals were analysed to create a combined phylogeny for the family. Results support monophyly for the family and all seven onychoteuthid genera (including Moroteuthopsis, established herein as the senior genus name for species formerly attributed to Kondakovia); 29 genetically distinct species were recovered, with the BIN (Barcode Index Number) analysis for COI showing good congruence overall with morphological species groupings. No sequences were available for five additional known species, making the total family diversity likely to exceed 34 species. Seven of the BINs formed in this study appear to represent undescribed taxa, suggesting that even in this relatively well-studied family, much additional work remains before a comprehensive understanding of the diversity and evolutionary relationships for the Onychoteuthidae can be achieved.

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.

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.