Octopus genome and evolution of cephalopod neural, morphological novelties [pdf]

The California two-spot octopus genome reveals no evidence of whole-genome duplications, but shows massive expansions in protocadherins and zinc-finger transcription factor gene families previously thought unique to vertebrates. Researchers identified hundreds of cephalopod-specific genes with elevated expression in specialized structures like skin, suckers, and nervous system, alongside extensive mRNA editing in neural genes. The findings suggest that gene family expansions and genomic rearrangements linked to transposable elements drove the evolution of the octopus's large nervous system and morphological innovations.

Abstract Coleoid cephalopods octopus, squid and cuttlefish are active, resourceful predators with a rich behavioural repertoire 1. 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 2 /articles/nature14668 ref-CR2 . To investigate the molecular bases of cephalopod brain and body innovations, we sequenced the genome and multiple transcriptomes of the California two-spot octopus, 1 /articles/nature14668 ref-CR1 , 3 /articles/nature14668 ref-CR3 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 4 /articles/nature14668 ref-CR4 , 5 /articles/nature14668 ref-CR5 , 6 /articles/nature14668 ref-CR6 , 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. 7 /articles/nature14668 ref-CR7 Similar content being viewed by others Main Soft-bodied cephalopods such as the octopus Fig. 1a /articles/nature14668 Fig1 show remarkable morphological departures from the basic molluscan body plan, including dexterous arms lined with hundreds of suckers that function as specialized tactile and chemosensory organs, and an elaborate chromatophore system under direct neural control that enables rapid changes in appearance 1,8. The octopus nervous system is vastly modified in size and organization relative to other molluscs, comprising a circumesophageal brain, paired optic lobes and axial nerve cords in each arm . Together these structures contain nearly half a billion neurons, more than six times the number in a mouse brain 2 /articles/nature14668 ref-CR2 , 3 /articles/nature14668 ref-CR3 . Extant coleoid cephalopods show extraordinarily sophisticated behaviours including complex problem solving, task-dependent conditional discrimination, observational learning and spectacular displays of camouflage 2 /articles/nature14668 ref-CR2 , 9 /articles/nature14668 ref-CR9 1 /articles/nature14668 ref-CR1 , 10 /articles/nature14668 ref-CR10 Supplementary Videos 1 /articles/nature14668 MOESM316 and 2 /articles/nature14668 MOESM317 . To explore the genetic features of these highly specialized animals, we sequenced the Octopus bimaculoides genome by a whole-genome shotgun approach Supplementary Note 1 /articles/nature14668 MOESM313 and annotated it using extensive transcriptome sequence from 12 tissues Methods and Supplementary Note 2 /articles/nature14668 MOESM313 . The genome assembly captures more than 97% of expressed protein-coding genes and 83% of the estimated 2.7 gigabase Gb genome size Methods and Supplementary Notes 1 /articles/nature14668 MOESM313 , 2 /articles/nature14668 MOESM313 , 3 /articles/nature14668 MOESM313 . The unassembled fraction is dominated by high-copy repetitive sequences Supplementary Note 1 /articles/nature14668 MOESM313 . Nearly 45% of the assembled genome is composed of repetitive elements, with two bursts of transposon activity occurring ∼25-million and ∼56-million years ago Mya Supplementary Note 4 /articles/nature14668 MOESM313 . We predicted 33,638 protein-coding genes Methods and Supplementary Note 4 /articles/nature14668 MOESM313 and found alternate splicing at 2,819 loci, but no locus showed an unusually high number of splice variants Supplementary Note 4 /articles/nature14668 MOESM313 . A-to-G discrepancies between the assembled genome and transcriptome sequences provided evidence for extensive mRNA editing by adenosine deaminases acting on RNA ADARs . Many candidate edits are enriched in neural tissues 7 and are found in a range of gene families, including ‘housekeeping’ genes such as the tubulins, which suggests that RNA edits are more widespread than previously appreciated Extended Data Fig. 1 /articles/nature14668 Fig4 and Supplementary Note 5 /articles/nature14668 MOESM313 . Based primarily on chromosome number, several researchers proposed that whole-genome duplications were important in the evolution of the cephalopod body plan 4,5,6, paralleling the role ascribed to the independent whole-genome duplication events that occurred early in vertebrate evolution . Although this is an attractive framework for both gene family expansion and increased regulatory complexity across multiple genes, we found no evidence for it. The gene family expansions present in octopus are predominantly organized in clusters along the genome, rather than distributed in doubly conserved synteny as expected for a paleopolyploid 11 /articles/nature14668 ref-CR11 12 /articles/nature14668 ref-CR12 , 13 /articles/nature14668 ref-CR13 Supplementary Note 6.2 /articles/nature14668 MOESM313 . Although genes that regulate development are often retained in multiple copies after paleopolyploidy in other lineages, they are not generally expanded in octopus relative to limpet, oyster and other invertebrate bilaterians 11 /articles/nature14668 ref-CR11 , 14 /articles/nature14668 ref-CR14 Table 1 /articles/nature14668 Tab1 and Supplementary Notes 7.4 /articles/nature14668 MOESM313 and 8 /articles/nature14668 MOESM313 . Hox genes are commonly retained in multiple copies following whole-genome duplication 15. In O. bimaculoides , however, we found only a single Hox complement, consistent with the single set of Hox transcripts identified in the bobtail squid Euprymna scolopes with PCR . Remarkably, octopus Hox genes are not organized into clusters as in most other bilaterian genomes 16 /articles/nature14668 ref-CR16 , but are completely atomized 15 /articles/nature14668 ref-CR15 Extended Data Fig. 2 /articles/nature14668 Fig5 and Supplementary Note 9 /articles/nature14668 MOESM313 . Although we cannot rule out whole-genome duplication followed by considerable gene loss, the extent of loss needed to support this claim would far exceed that which has been observed in other paleopolyploid lineages, and it is more plausible that chromosome number in coleoids increased by chromosome fragmentation. Mechanisms other than whole-genome duplications can drive genomic novelty, including expansion of existing gene families, evolution of novel genes, modification of gene regulatory networks, and reorganization of the genome through transposon activity. Within the O. bimaculoides genome, we found evidence for all of these mechanisms, including expansions in several gene families, a suite of octopus- and cephalopod-specific genes, and extensive genome shuffling. In gene family content, domain architecture and exon–intron structure, the octopus genome broadly resembles that of the limpet Lottia gigantea 17, the polychaete annelid Capitella teleta and the cephalochordate 17 /articles/nature14668 ref-CR17 Branchiostoma floridae 14 /articles/nature14668 ref-CR14 Supplementary Note 7 /articles/nature14668 MOESM313 and Extended Data Fig. 3 /articles/nature14668 Fig6 . Relative to these invertebrate bilaterians, we found a fairly standard set of developmentally important transcription factors and signalling pathway genes, suggesting that the evolution of the cephalopod body plan did not require extreme expansions of these ‘toolkit’ genes Table 1 /articles/nature14668 Tab1 and Supplementary Note 8.2 /articles/nature14668 MOESM313 . However, statistical analysis of protein domain distributions across animal genomes did identify several notable gene family expansions in octopus, including protocadherins, C2H2 zinc-finger proteins C2H2 ZNFs , interleukin-17-like genes IL17-like , G-protein-coupled receptors GPCRs , chitinases and sialins Figs 1b /articles/nature14668 Fig1 , 2 /articles/nature14668 Fig2 and 3 /articles/nature14668 Fig3 ; Extended Data Figs 4 /articles/nature14668 Fig7 , 5 /articles/nature14668 Fig8 , 6 /articles/nature14668 Fig9 and Supplementary Notes 8 /articles/nature14668 MOESM313 and 10 /articles/nature14668 MOESM313 . The octopus genome encodes 168 multi-exonic protocadherin genes, nearly three-quarters of which are found in tandem clusters on the genome Fig. 2b /articles/nature14668 Fig2 , a striking expansion relative to the 17–25 genes found in Lottia, Crassostrea gigas oyster and Capitella genomes. Protocadherins are homophilic cell adhesion molecules whose function has been primarily studied in mammals, where they are required for neuronal development and survival, as well as synaptic specificity 18. Single protocadherin genes are found in the invertebrate deuterostomes Saccoglossus kowalevskii acorn worm and Strongylocentrotus purpuratus sea urchin , indicating that their absence in Drosophila melanogaster and Caenorhabditis elegans is due to gene loss. Vertebrates also show a remarkable expansion of the protocadherin repertoire, which is generated by complex splicing from a clustered locus rather than tandem gene duplication reviewed in ref. 19 /articles/nature14668 ref-CR19 . Thus both octopuses and vertebrates have independently evolved a diverse array of protocadherin genes. A search of available transcriptome data from the longfin inshore squid Doryteuthis formerly, Loligo pealeii 20 also demonstrated an expanded number of protocadherin genes Supplementary Note 8.3 /articles/nature14668 MOESM313 . Surprisingly, our phylogenetic analyses suggest that the squid and octopus protocadherin arrays arose independently. Unlinked octopus protocadherins appear to have expanded ∼135 Mya, after octopuses diverged from squid. In contrast, clustered octopus protocadherins are much more similar in sequence, either due to more recent duplications or gene conversion as found in clustered protocadherins in zebrafish and mammals . 21 /articles/nature14668 ref-CR21 The expression of protocadherins in octopus neural tissues Fig. 2 /articles/nature14668 Fig2 is consistent with a central role for these genes in establishing and maintaining cephalopod nervous system organization as they do in vertebrates. Protocadherin diversity provides a mechanism for regulating the short-range interactions needed for the assembly of local neural circuits 18, which is where the greatest complexity in the cephalopod nervous system appears . The importance of local neuropil interactions, rather than long-range connections, is probably due to the limits placed on axon density and connectivity by the absence of myelin, as thick axons are then required for rapid high-fidelity signal conduction over long distances. The sequence divergence between octopus and squid protocadherin expansions may reflect the notable differences between octopuses and decapodiforms in brain organization, which have been most clearly demonstrated for the vertical lobe, a key structure in cephalopod learning and memory circuits 2 /articles/nature14668 ref-CR2 . Finally, the independent expansions and nervous system enrichment of protocadherins in coleoid cephalopods and vertebrates offers a striking example of convergent evolution between these clades at the molecular level. 2 /articles/nature14668 ref-CR2 , 22 /articles/nature14668 ref-CR22 As with the protocadherins, we found multiple clusters of C2H2 ZNF transcription factor genes Fig. 3a /articles/nature14668 Fig3 and Supplementary Note 8.4 /articles/nature14668 MOESM313 . The octopus genome contains nearly 1,800 multi-exonic C2H2-containing genes Table 1 /articles/nature14668 Tab1 , more than the 200–400 C2H2 ZNFs found in other lophotrochozoans and the 500–700 found in eutherian mammals, in which they form the second-largest gene family 23. C2H2 ZNF transcription factors contain multiple C2H2 domains that, in combination, result in highly specific nucleic acid binding. The octopus C2H2 ZNFs typically contain 10–20 C2H2 domains but some have as many as 60 Supplementary Note 8.4 /articles/nature14668 MOESM313 . The majority of the transcripts are expressed in embryonic and nervous tissues Fig. 3b /articles/nature14668 Fig3 . This pattern of expression is consistent with roles for C2H2 ZNFs in cell fate determination, early development and transposon silencing, as demonstrated in genetic model systems . 23 /articles/nature14668 ref-CR23 The expansion of the O. bimaculoides C2H2 ZNFs coincides with a burst of transposable element activity at ∼25 Mya Fig. 3c /articles/nature14668 Fig3 . The flanking regions of these genes show a significant enrichment in a 70–90 base pair bp tandem repeat 31% for C2H2 genes versus 4% for all genes; Fisher’s exact test P value <1 × 10−16 , which parallels the linkage of C2H2 gene expansions to β-satellite repeats in humans 24. We also found an expanded C2H2 ZNF repertoire in amphioxus Table 1 /articles/nature14668 Tab1 , showing a similar enrichment in satellite-like repeats. These parallels suggest a common mode of expansion of a highly dynamic transcription factor family implicated in lineage-specific innovations. To investigate further the evolution of gene families implicated in nervous system development and function, we surveyed genes associated with axon guidance Table 1 /articles/nature14668 Tab1 and neurotransmission Table 2 /articles/nature14668 Tab2 , identifying their homologues in octopus and comparing numbers across a diverse set of animal genomes Supplementary Notes 8 /articles/nature14668 MOESM313 , 9 /articles/nature14668 MOESM313 , 10 /articles/nature14668 MOESM313 . Several patterns emerged from this survey. The gene complements present in the model organisms D. melanogaster and C. elegans often showed striking departures from those seen in lophotrochozoans and vertebrates Table 2 /articles/nature14668 Tab2 and Supplementary Note 10 /articles/nature14668 MOESM313 . For example, D. melanogaster encodes one member of the discs large DLG family, a key component of the postsynaptic scaffold. In contrast, mammals have four DLG s, which along with other observations led to suggestions that vertebrates possess uniquely complex synaptic machinery 25. However, we found three DLG s in both octopus and limpet, suggesting that vertebrate and fly gene number differences are not necessarily diagnostic of exceptional vertebrate synaptic complexity Supplementary Note 10.6 /articles/nature14668 MOESM313 . Overall, neurotransmission gene family sizes in the octopus were very similar to those seen in other lophotrochozoans Table 2 /articles/nature14668 Tab2 and Supplementary Note 10 /articles/nature14668 MOESM313 , except for a few strikingly expanded gene families such as the sialic acid vesicular transporters sialins Supplementary Note 10.2 /articles/nature14668 MOESM313 . We did find variations in the sizes of neurotransmission gene families between human and lophotrochozoans Table 2 /articles/nature14668 Tab2 and Supplementary Note 10 /articles/nature14668 MOESM313 , but no evidence for systematic expansion of these gene families in vertebrates relative to octopus or other lophotrochozoans. Although some gene families were larger in mammals or absent in lophotrochozoans for example, ligand-gated 5-HT receptors , others were absent in mammals and present in invertebrates for example, anionic glutamate and acetylcholine receptors . The complement of neurotransmission genes in octopus may be broadly typical for a lophotrochozoan, but our findings suggest it is also not obviously smaller than is found in mammals. Among the octopus complement of ligand-gated ion channels, we identified a set of atypical nicotinic acetylcholine receptor-like genes, most of which are tandemly arrayed in clusters Extended Data Fig. 7 /articles/nature14668 Fig10 . These subunits lack several residues identified as necessary for the binding of acetylcholine 26, so it is unlikely that they function as acetylcholine receptors. The high level of expression of these divergent subunits within the suckers raises the interesting possibility that they act as sensory receptors, as do some divergent glutamate receptors in other protostomes . In addition, we identified 74 27 /articles/nature14668 ref-CR27 Aplysia -like and 11 vertebrate-like candidate chemoreceptors among the octopus GPCR superfamily of ∼330 genes Extended Data Fig. 6 /articles/nature14668 Fig9 . We found, amid extensive transcription of octopus transposons, that a class of octopus-specific short interspersed nuclear element sequences SINEs is highly expressed in neural tissues Supplementary Note 4 /articles/nature14668 MOESM313 and Extended Data Fig. 8 /articles/nature14668 Fig11 . Although the role of active transposons is unclear, elevated transposon expression in neural tissues has been suggested to serve an important function in learning and memory in mammals and flies 28. Transposable element insertions are often associated with genomic rearrangements 29 and we found that the transposon-rich octopus genome displays substantial loss of ancestral bilaterian linkages that are conserved in other species Supplementary Note 6 /articles/nature14668 MOESM313 and Extended Data Fig. 9 /articles/nature14668 Fig12 . Interestingly, genes that are linked in other bilaterians but not in octopus are enriched in neighbouring SINE content. SINE insertions around these genes date to the time of tandem C2H2 expansion Extended Data Fig. 9d /articles/nature14668 Fig12 , pointing to a crucial period of genome evolution in octopus. Other transposons such as Mariner show no such enrichment, suggesting distinct roles for different classes of transposons in shaping genome structure Extended Data Fig. 9c /articles/nature14668 Fig12 . Transposable element activity has been implicated in the modification of gene regulation across several eukaryotic lineages 29. We found that in the nervous system, the degree to which a gene’s expression is tissue-specific is positively correlated with the transposon load around that gene r 2values ranging from 0.49 in the optic lobe to 0.81 in the subesophageal brain; Extended Data Fig. 8 /articles/nature14668 Fig11 and Supplementary Note 4 /articles/nature14668 MOESM313 . This correlation may reflect modulation of gene expression by transposon-derived enhancers or a greater tolerance for transposon insertion near genes with less complex patterns of tissue-specific gene regulation. Using a relaxed molecular clock, we estimate that the octopus and squid lineages diverged ∼270 Mya, emphasizing the deep evolutionary history of coleoid cephalopods 8,30 Supplementary Note 7.1 /articles/nature14668 MOESM313 and Extended Data Fig. 10a /articles/nature14668 Fig13 . Our analyses found hundreds of coleoid- and octopus-specific genes, many of which were expressed in tissues containing novel structures, including the chromatophore-laden skin, the suckers and the nervous system Extended Data Fig. 10 /articles/nature14668 Fig13 and Supplementary Note 11 /articles/nature14668 MOESM313 . Taken together, these novel genes, the expansion of C2H2 ZNFs, genome rearrangements, and extensive transposable element activity yield a new landscape for both trans- and cis- regulatory elements in the octopus genome, resulting in changes in an otherwise ‘typical’ lophotrochozoan gene complement that contributed to the evolution of cephalopod neural complexity and morphological innovations. Methods Data access Genome and transcriptome sequence reads are deposited in the SRA as BioProjects PRJNA270931 and PRJNA285380. The genome assembly and annotation are linked to the same BioProject ID. A browser of this genome assembly is available at http://octopus.metazome.net/ http://octopus.metazome.net/ . Genome sequencing and assembly Genomic DNA from a single male Octopus bimaculoides 31 was isolated and sequenced using Illumina technology to 60-fold redundant coverage in libraries spanning a range of pairs from ∼350 bp to 10 kilobases kb . These data were assembled with meraculous achieving a contig N50-length of 5.4 kb and a scaffold N50-length of 470 kb. The longest scaffold contains 99 genes and half of all predicted genes are on scaffolds with 8 or more genes 32 /articles/nature14668 ref-CR32 Supplementary Note 1 /articles/nature14668 MOESM313 . Genome size and heterozygosity The O. bimaculoides haploid genome size was estimated to be ∼2.7 gigabases Gb based on fluorescence 2.66–2.68 Gb and k -mer 2.86 Gb measurements Supplementary Notes 1 /articles/nature14668 MOESM313 and 2 /articles/nature14668 MOESM313 , making it several times larger than other sequenced molluscan and lophotrochozoan genomes 17. We observed nucleotide-level heterozygosity within the sequenced genome to be 0.08%, which may reflect a small effective population size relative to broadcast-spawning marine invertebrates. Transcriptome sequencing Twelve transcriptomes were sequenced from RNA isolated from ova, testes, viscera, posterior salivary gland PSG , suckers, skin, developmental stage 15 St15 33, retina, optic lobe OL , supraesophageal brain Supra , subesophageal brain Sub , and axial nerve cord ANC Supplementary Note 2 /articles/nature14668 MOESM313 . RNA was isolated using TRIzol Invitrogen and 100-bp paired-end reads insert size 300 bp were generated on an Illumina HiSeq2000 sequencing machine. De novo transcriptome assembly Adapters and low-quality reads were removed before assembling transcriptomes using the Trinity de novo assembly package version r2013-02-25 refs 34 /articles/nature14668 ref-CR34 , 35 /articles/nature14668 ref-CR35 . Assembly statistics are summarized in Supplementary Table 2.2. /articles/nature14668 MOESM313 Following assembly, peptide-coding regions were translated using TransDecoder in the Trinity package. We compared the de novo assembled RNA-seq output to the genome to evaluate the completeness of the genome assembly. To minimize the number of spuriously assembled transcripts, only transcripts with ORFs predicted by TransDecoder were mapped onto the genome with BLASTN. Only 1,130 out of 48,259 transcripts with ORFs 2.34% did not have a match in the genome with a minimum identity of 95%. Annotation of transposable elements Transposable elements were identified with RepeatScout and RepeatModeler 36, and the masking was done with RepeatMasker , as outlined in 37 /articles/nature14668 ref-CR37 Supplementary Note 4.2 /articles/nature14668 MOESM313 . The most abundant transposable element is a previously identified octopus-specific SINE that accounts for 4% of the assembled genome. 38 /articles/nature14668 ref-CR38 Annotation of protein-coding genes Protein-coding genes were annotated by combining transcriptome evidence with homology-based and de novo gene prediction methods Supplementary Note 4 /articles/nature14668 MOESM313 . For homology prediction we used predicted peptide sets of three previously sequenced molluscs L. gigantea , C. gigas , and A. californica along with selected other metazoans. Alternative splice isoforms were identified with PASA 39. Annotation statistics are provided in Supplementary Table 4.1.1. /articles/nature14668 MOESM313 Genes known in vertebrates to have many isoforms, such as ankyrin, TRAK1 and LRCH1 , also show alternative splicing in octopus but at a more limited level. Octopus genes with ten or more alternative splice forms are provided in Supplementary Table 4.1.2 /articles/nature14668 MOESM313 . Calibration of sequence divergence with respect to time The divergence between squid and octopus was estimated using r8s 40 by fixing cephalopod divergence from bivalves and gastropods to 540 Mya . Our estimate of 270 Mya for the squid–octopus divergence corresponds to mean neutral substitution rate of dS ∼2 based on the protein-directed CDS alignments between the species 8 /articles/nature14668 ref-CR8 Supplementary Fig. 6.1.2 /articles/nature14668 MOESM313 and a dS estimation using the yn00 program . Throughout the manuscript we convert from sequence divergence to time by assuming that dS ∼1 corresponds to 135 million years. For example, unlinked octopus protocadherins appear to have expanded ∼135 Mya based on mean pairwise dS ∼1, after octopuses diverged from squid. In contrast, clustered octopus protocadherins are much more similar in sequence mean pairwise dS ∼0.4, or ∼55 Mya . 41 /articles/nature14668 ref-CR41 Quantifying gene expression Transcriptome reads were mapped to the genome assembly with TopHat 2.0.11 ref. 42 /articles/nature14668 ref-CR42 . A range of 76–90% of reads from the different samples mapped to the genome. Mapped reads were sorted and indexed with SAMtools 43. The read counts in each tissue were produced with BEDTools multicov program using the gene model coordinates. The counts were normalized by the total transcriptome size of each tissue and by the length of the gene. Heat maps showing expression patterns were generated in R using the heatmap.2 function. 44 /articles/nature14668 ref-CR44 Gene complement Gene families of particular interest, including developmental regulatory genes, neural-related genes, and gene families that appear to be expanded in O. bimaculoides , were manually curated and analysed. We searched the octopus genome and transcriptome assemblies using BLASTP and TBLASTN with annotated sequences from human, mouse and D. melanogaster . Bulk analyses were also performed using Pfam 45 and PANTHER . We used BLASTP and TBLASTX to search for specific gene families in deposited genome and transcriptome databases for 46 /articles/nature14668 ref-CR46 L. gigantea, A. californica, C. gigas, C. teleta, T. castaneum, D. melanogaster, C. elegans, N. vectensis, A. queenslandica, S. kowalevskii, B. floridae, C. intestinalis, D. rerio, M. musculus and H. sapiens . Candidate genes were verified with BLAST and Pfam 47 /articles/nature14668 ref-CR47 analysis. Genes identified in the octopus genome were confirmed and extended using the transcriptomes. Multiple gene models that matched the same transcript were combined. The identified sequences from octopus and other bilaterians were aligned with either MUSCLE 45 /articles/nature14668 ref-CR45 , CLUSTALO 48 /articles/nature14668 ref-CR48 , MacVector 12.6 MacVector, North Carolina , or Jalview 49 /articles/nature14668 ref-CR49 . Phylogenetic trees were constructed with FastTree 50 /articles/nature14668 ref-CR50 using the Jones–Taylor–Thornton model of amino acid evolution, and visualized with FigTree v1.3.1. 51 /articles/nature14668 ref-CR51 Synteny Microsynteny was computed based on metazoan node gene families Supplementary Note 7 /articles/nature14668 MOESM313 . We used Nmax 10 maximum of 10 intervening genes and Nmin 3 minimum of three genes in a syntenic block according to the pipeline described in ref. 17 /articles/nature14668 ref-CR17 Supplementary Note 6 /articles/nature14668 MOESM313 . To simplify gene family assignments we limited our analyses to 4,033 gene families shared among human, amphioxus, Capitella , Helobdella , Octopus , Lottia , Crassostrea , Drosophila and Nematostella . We required ancestral bilaterian syntenic blocks to have a minimum of one species present in both ingroups, or in one ingroup and one outgroup. To examine the effect of fragmented genome assemblies, we simulated shorter assemblies by artificially fragmenting genomes to contain on average 5 genes per scaffold Supplementary Note 6 /articles/nature14668 MOESM313 . In comparison with other bilaterian genomes, we find that the octopus genome is substantially rearranged. In looking at microsyntenic linkages of genes with a maximum of 10 intervening genes, we found that octopus conserves only 34 out of 198 ancestral bilaterian microsyntenic blocks; the limpet Lottia and amphioxus retain more than twice as many such linkages 96 and 140, respectively . This difference remains significant after accounting for genes missed through orthology assignment as well as simulations of shorter scaffold sizes Supplementary Note 6 /articles/nature14668 MOESM313 ; Extended Data Fig. 9b /articles/nature14668 Fig12 . Scans for intra-genomic synteny, and doubly conserved synteny with Lottia , were performed as described in Supplementary Note 6 /articles/nature14668 MOESM313 . Transposable elements and synteny dynamics The 5 kb upstream and downstream regions of genes were surveyed for transposable element TE content. For genes with non-zero TE load, their assignment to either conserved or lost bilaterian synteny in octopus was done using the microsynteny calculation described above. The number of genes for each category and TE class were as follows: 484 genes for retained synteny and 1,193 genes in lost synteny for all TE classes; 440 and 1,107, respectively, for SINEs; and 116 and 290, respectively, for Mariner. Wilcoxon U -tests for the difference of TE load in linked versus non-linked genes were conducted in R. To assess transposon activity we assigned transcriptome reads aligned to 5,496,558 annotated transposon loci using BEDTools 44. Of these, 2,685,265 loci showed expression in at least one of the tissues. RNA editing RNA-seq reads were mapped to the genome with TopHat 52, and SAMtools was used to identify SNPs between the genomic and the RNA sequences. To identify polymorphic positions in the genome, SNPs and indels were predicted using GATK HaplotypeCaller version 3.1-1 in discovery mode with a minimum Phred scaled probability score of 30, based on an alignment of the 350 bp and 500 bp genomic fragment libraries using BWA-MEM version 0.7.6a. Using BEDTools 43 /articles/nature14668 ref-CR43 , we removed SNPs predicted in both the transcriptome and the genome and discarded SNPs that had a Phred score below 40 or were outside of predicted genes. SNPs were binned according to the type of nucleotide change and the direction of transcription. Candidate edited genes were taken as those having SNPs with A-to-G substitutions in the predicted mRNA transcripts. 44 /articles/nature14668 ref-CR44 Cephalopod-specific genes Cephalopod novelties were obtained by BLASTP and TBLASTN searches against the whole NR database 53 and a custom database of several mollusc transcriptomes Supplementary Note 11.1 /articles/nature14668 MOESM313 . To ensure that we had as close to full-length sequence as possible, we extended proteins predicted from octopus genomic sequence with our de novo assembled transcriptomes, using the longest match to query NR, transcriptome and EST sequences from other animals. Gene sequences with transcriptome support but without a match to non-cephalopod animals at an e-value cutoff of 1 × 10 −3were considered for further analysis. Octopus sequences with a match of 1 × 10 −5or better to a sequence from another cephalopod were used to construct gene families, which were characterized by their BLAST alignments, HMM, PFAM-A/B, and UNIREF90 hits. The cephalopod-specific gene families are listed in the Source Data file for Extended Data Fig. 10 /articles/nature14668 Fig13 . Octopus-specific novelties were defined as sequences with transcriptome support but without any matches to sequences from any other animals <1 × 10 −3 , including nautiloid and decapodiform cephalopods. 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Gui for bioinformatic assistance; S. Shigeno for help with tissue dissection; C. Huffard and R. Caldwell for providing the O. bimaculoides specimen used for genomic DNA isolation; and E. Begovic for genomic DNA preparation. This work was supported by the Molecular Genetics Unit of the Okinawa Institute of Science and Technology Graduate University S.B. and D.S.R. and by funding from the NSF IOS-1354898 and NIH R03 HD064887 to C.W.R. and from the NSF DGE-0903637 to Z.Y.W. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 instrumentation grants S10RR029668 and S10RR027303, and the University of Chicago Functional Genomics Facility, supported by NIH grant UL1 TR000430. Author information Authors and Affiliations Contributions The Chicago and the OIST/Berkeley groups initiated their transcriptome and genome projects independently. In the subsequent collaboration, both groups worked closely on every aspect of the project. Chicago group: C.B.A., Z.Y.W., J.R.P. and C.W.R.; OIST/Berkeley group: O.S., T.M., E.E.-G., S.B. and D.S.R. Corresponding authors Ethics declarations Competing interests The authors declare no competing financial interests. Additional information Genome and transcriptome sequence reads have been deposited in the SRA as BioProjects PRJNA270931 http://www.ncbi.nlm.nih.gov/sra?term=PRJNA270931 and PRJNA285380 http://www.ncbi.nlm.nih.gov/sra?term=PRJNA285380 . A browser of this genome assembly is available at http://octopus.metazome.net/ http://octopus.metazome.net/ . Extended data figures and tables Extended Data Figure 1 RNA editing in octopus. /articles/nature14668/figures/4 a , Approximate maximum likelihood tree of adenosine deaminases acting on RNA ADARs in bilaterians. ADAR1 , ADAR2 , ADAR - like / ADAD , and ADAT tRNA-specific adenosine deaminase were identified in Hsa, Mmu, Cin, Dme, Cte, Lgi, D. opalescens Dop 54 , and Obi with Shimodaira–Hasegawa-like support indicated at the nodes. b , O. bimaculoides ADAR1, ADAR2 and ADAR-like proteins contain one or two double-stranded RNA binding domains dsRBD as well as an adenosine deaminase domain. ADAR1 also has a z-alpha domain. c , Expression profiles of the three ADAR genes found in 12 O. bimaculoides tissues by RNA-seq profiling. d , DNA–RNA differences in O. bimaculoides show prominent A-to-G changes. Histogram illustrates the number of DNA–RNA differences detected between coding sequences in the genome and 12 O. bimaculoides transcriptomes after filtering out polymorphisms identified in genomic sequencing. Differences were binned by the type of change see key in the direction of transcription. A-to-G changes are the most prevalent, particularly in neural tissues and during development, paralleling the expression of octopus ADAR s in c . Other types of changes were also detected at lower levels, possibly resulting from uncharacterized polymorphisms. Extended Data Figure 2 Local arrangement of Hox gene complement in /articles/nature14668/figures/5 O. bimaculoides and selected bilaterians. O. bimaculoides and selected bilaterians. At the top, the four compact Hox clusters of H. sapiens and the single B. floridae cluster are depicted. The D. melanogaster Hox complex is split into two clusters. We included genes in the D. melanogaster locus that are homologues of Hox genes but have lost their homeotic function, such as fushi tarazu ftz , bicoid , zen and zen2 the latter three are represented as overlapping boxes . Hox genes in C. teleta are found on three scaffolds 17. L. gigantea has a single cluster with the full known lophotrochozoan gene complement. In O. bimaculoides many of the scaffolds are several hundred kb long, and no two Hox genes are on the same scaffold. The positions of O. bimaculoides genes approximate their locations on scaffolds. Dashed lines indicate that the scaffold continues beyond what is shown. Scaffold length is depicted to scale with size noted on the left. Genes are positioned to illustrate orthology, which is also highlighted by colour. Extended Data Figure 3 Gene complement and gene architecture evolution in metazoans. /articles/nature14668/figures/6 a , Principal component analysis of gene family counts. O. bimaculoides highlighted in green. Deuterostomes are indicated in blue, ecdysozoans in red, lophotrochozoans in green, and sponges and cnidarians in orange. Xtr, Xenopus tropicalis ; Gga, Gallus gallus ; Tca, Tribolium castaneum ; Dpu, Daphnia pulex ; Isc, Ixodes scapularis ; Ava, Adineta vaga ; Spu, S. purpuratus ; Hma, Hydra magnipapillata ; Adi, Acropora digitifera . For methods, see Supplementary Note 7.4 /articles/nature14668 MOESM313 . b – d , MrBayes 55 tree constrained topology on binary characters of presence or absence of Pfam domain architectures b , introns c , or indels d ; scale bar represents estimated changes per site. For methods, see Supplementary Note 7.3 /articles/nature14668 MOESM313 . Extended Data Figure 4 Protocadherin genes within a genomic cluster are similar in sequence and sites of expression. /articles/nature14668/figures/7 a , Expression profile of the 31 protocadherin genes located on Scaffold 30672 in 12 octopus transcriptomes. Over three-quarters of the protocadherins are highly expressed throughout central brain, OL and ANC, while the others show more mixed distributions. b , Phylogenetic tree highlighting Scaffold 30672 protocadherins in grey bars. c , Expression profile of the 17 protocadherin genes located on Scaffold 9600. Almost all of these protocadherins are most highly expressed in nervous tissues, with the exception of Ocbimv220039316m, which is most highly expressed in the St15 sample. d , Phylogenetic tree highlighting Scaffold 9600 protocadherins in grey bars. As seen in b , protocadherins of the same scaffold tend to cluster together on the tree. Order of the genes in the heat maps a , c follows the ordering on the corresponding scaffold. Extended Data Figure 5 Expansion of interleukin 17 IL17 -like genes. /articles/nature14668/figures/8 a , Phylogenetic tree of interleukin genes in Obi, Cte, Cgi, and Lgi. Mammalian IL1A , IL1B , and IL7 used as outgroups. Human and mouse IL17 s branch from other members of the IL family. Octopus IL s as well as all identified invertebrate IL s group with the mammalian IL17 branch and are named ‘IL17-like’. The 31 octopus genes are distributed across 5 scaffolds: scaffold A Obi A , 23 members; scaffold B Obi B , 4 members; scaffold C Obi C , 2 members; scaffolds D Obi D and E Obi E , 1 member each. b , Expression profile of 31 octopus IL17-like genes. Heat map rows are arranged by order on each scaffold. Blank rows indicate genes not expressed in our transcriptomes. The 27 genes found in our transcriptomes have strong expression in the suckers and skin. The scaffold C genes are enriched in the PSG and the Scaffold D gene is enriched in the viscera. c , Conserved cysteine residues in human IL17 and invertebrate IL17-like proteins. The human IL17 proteins share a conserved cysteine motif comprising 4 cysteine residues, which may form interchain disulfide bonds and facilitate dimerization 56. Octopus IL17-like proteins also contain this four-cysteine motif, highlighted in yellow. One octopus sequence encodes only 3 of these highly conserved cysteine residues. These four cysteines are also present to varying degrees in Lottia , Capitella and Crassostrea sequences. Two additional conserved cysteine residues were found in the octopus sequences and are highlighted in red. The first cysteine residue is found in all invertebrate sequences examined, and none of the mammalian IL17 sequences. Extended Data Figure 6 G-protein-coupled receptors. /articles/nature14668/figures/9 GPCRs, also known as 7-transmembrane 7TM or serpentine receptors, form a large superfamily that activates intracellular second messenger systems upon ligand binding. This figure considers a subset of the 329 GPCRs we identified in O. bimaculoides . The full complement of GPCRs is presented in Supplementary Note 8.5 /articles/nature14668 MOESM313 . a , b , As reported for other lophotrochozoan genomes, the octopus genome contains chemosensory-like GPCR s; 74 GPCR s are similar to the Aplysia chemosensory GPCR s 57 and 11 GPCR s are similar to vertebrate olfactory receptors. c , We identified 4 opsins in the octopus genome from top to bottom : rhodopsin, rhabdomeric opsin, peropsin, and retinochrome. d , The octopus class F GPCRs comprises 6 genes: 5 Frizzled genes and 1 Smoothened gene . e , Thirty octopus genes show similarity to vertebrate adhesion GPCRs. Extended Data Figure 7 /articles/nature14668/figures/10 O. bimaculoides acetylcholine receptor AChR subunits. O. bimaculoides acetylcholine receptor AChR subunits. a , Phylogenetic tree of AChR subunit genes identified in Hsa, Mmu, Dme, Cte, Lgi and Obi. Black asterisk indicates a Dme sequence that groups with alpha 1-4-like subunits despite lacking two defining cysteine residues. b , Expression profiles of octopus AChR subunits. Genes ordered as in the tree a , starting from the grey arrow and continuing anticlockwise. Putative non-ACh-binding subunits are highly expressed in the suckers. One sequence was not detected in our transcriptome data sets. In a and b , red asterisks indicate subunits with the substitution known to confer anionic permissivity 58. c , Divergent octopus subunits lack nearly all residues necessary for ACh binding. Alignment of sequence flanking the cysteine loop yellow of the L. stagnalis ACh-binding protein Lst AchBP , the human and octopus alpha-7 receptor subunits Hsa AchR7, Obi 10697+ , and the 23 divergent AChR subunits. Essential ACh-binding residues on the primary pink and complementary blue side of the ligand-binding domain are indicated , with conservative substitutions in a lighter shade. Outside of the binding residues, residues shared between the alpha-7 subunits are shaded in light grey, with bold letters for conservative substitutions. 26 /articles/nature14668 ref-CR26 Extended Data Figure 8 Active transposable elements and gene expression specificity. /articles/nature14668/figures/11 a , Transposable element expression across 12 tissues. b , Correlation between the total transposable element TE load in bp in the 5 kb regions flanking the gene and the fraction of genes with tissue-specific expression defined as having at least 75% of expression in a single tissue; see Source Data file for this figure . P value indicates the F -statistic for the significance of linear regression H0: r 2 = 0 , with tissues with a P value ≤0.05 indicated in pink. Extended Data Figure 9 Synteny dynamics in octopus and the effect of transposable element TE expansions. /articles/nature14668/figures/12 a , Circos plot showing shared synteny across 6 genomes. Individual scaffolds are plotted according to bp length; scaffolds with no synteny are merged together lighter arcs . Despite the large size of the octopus genome, only a small proportion of the scaffolds show synteny. b , Synteny reduction in octopus quantified based on synteny inference using gene families with at least one representative in human, amphioxus, Capitella , Helobdella , Octopus , Lottia , Crassostrea , Drosophila , and Nematostella. Drosophila , Helobdella and Octopus show the highest synteny loss rates. Branch lengths, estimated with MrBayes 55, reflect extent of local genome rearrangement Supplementary Note 6 /articles/nature14668 MOESM313 . c , Enrichment of overall and specific TE classes base pairs masked around genes from ancient bilaterian synteny blocks, including those absent in octopus see key . Asterisks indicate Mann–Whitney U -test with P value <0.02. d , Transposable element insertion history Jukes–Cantor distance adjusted, see text into the vicinity of genes from ‘lost’ synteny blocks. Note that only one SINE peak is present; a more recent peak visible in ‘All genomic SINEs’ cannot be recovered from those insertions. Extended Data Figure 10 Cephalopod phylogeny and novelties. /articles/nature14668/figures/13 a , Whole-genome-derived phylogeny of molluscs and select other phyla showing the relative position of octopus at the base of the coleoid cephalopods. For methods see Supplementary Note 7.1 /articles/nature14668 MOESM313 . Members of the cephalopod class are indicated in blue, scale indicates number of substitutions per site. b , Phylogenetic tree of reflectin genes. Reflectins are cephalopod-specific genes that allow for rapid and reversible changes in iridescence. Six reflectin genes were identified in the octopus genome. c , d , Novel gene expression across multiple tissues. Bars depict all cephalopod novelties; dark grey indicates sequences with no similarity to non-cephalopod genes using HMM searches see Source Data for this figure . c , Counts of tissue-specific novelties in a given tissue. d , Proportion of expression of novel genes versus total expression in individual tissues. CNS central nervous system combines Supra, Sub, OL and ANC expression data. Extended Data Figure 11 Phylogenetic tree of cadherin genes. /articles/nature14668/figures/14 This is a larger image of Fig. 2a /articles/nature14668 Fig2 . Phylogenetic tree of cadherin genes in Hsa red , Dme orange , Nematostella vectensis mustard yellow , Amphimedon queenslandica yellow , Cte green , Lgi teal , Obi blue , and Saccoglossus kowalevskii purple . I, Type I classical cadherins; II, calsyntenins; III, octopus protocadherin expansion 168 genes ; IV, human protocadherin expansion 58 genes ; V, dachsous; VI, fat-like; VII, fat; VIII, CELSR; IX, Type II classical cadherins. Asterisk denotes a novel cadherin with over 80 extracellular cadherin domains found in Obi and Cte. Supplementary information Supplementary Information download PDF https://static-content.springer.com/esm/art%3A10.1038%2Fnature14668/MediaObjects/41586 2015 BFnature14668 MOESM313 ESM.pdf This file contains Supplementary Text and Data, Supplementary Tables, Supplementary Figures and additional references See Contents for details . PDF 11559 kb Supplementary Figure - Metazoan C2H2 superfamily tree download PDF https://static-content.springer.com/esm/art%3A10.1038%2Fnature14668/MediaObjects/41586 2015 BFnature14668 MOESM314 ESM.pdf Approximate maximum likelihood tree of metazoan C2H2-ZNFs. The tree was built using a near-complete subset of transcriptome-confirmed C2H2-ZNF sequences 1,452/1,790 from octopus dark blue , 539 from human red , 388 from mouse pink , 159 from zebrafish purple , 7 from A. queenslandica yellow , 88 from A. californica aqua , 24 from C. intestinalis dark purple , 65 from S. kowalevskii violet , 44 from T. castaneum orange , 39 from N. vectensis mustard , 87 from L. gigantea teal , and 72 from C. teleta green . PDF 7114 kb Supplementary Figure - Metazoan GPCR superfamily tree download PDF https://static-content.springer.com/esm/art%3A10.1038%2Fnature14668/MediaObjects/41586 2015 BFnature14668 MOESM315 ESM.pdf Approximate maximum likelihood tree of bilaterian GPCRs. The tree was built using the 329 GPCR sequences from octopus dark blue , 185 from human red , 160 from mouse pink , 226 from zebrafish magenta , 204 from S. kowalevskii purple , 125 from L.gigantea aqua , 258 from C. teleta green , and 87 from A. californica grey-blue . For clarity, only a subset of vertebrate olfactory receptors were used. The octopus Class A rhodopsins are centered around 7 o'clock. PDF 229 kb Octopus bimaculoides: Adaptive coloration download MP4 https://static-content.springer.com/esm/art%3A10.1038%2Fnature14668/MediaObjects/41586 2015 BFnature14668 MOESM316 ESM.mp4 The ability to camouflage is present throughout the animal kingdom, but cephalopods uniquely achieve adaptive coloration. Three different structures in the skin contribute to the color of an octopus: chromatophores, iridophores, and leucophores. The reflectin family of proteins reversibly regulates iridescence. Muscles in the skin allow octopuses to alter texture and disrupt the visual boundaries of the body. Most importantly, this complex behavior happens very quickly, allowing octopuses to change their appearance almost instantaneously. The O. bimaculoides genome and transcriptome is reported by Albertin and Simakov et al. Credit: Z. Yan Wang, C. Ragsdale, J. Reynolds, and Schadenfreude the octopus. MP4 36989 kb Octopus bimaculoides: Arms and Suckers download MP4 https://static-content.springer.com/esm/art%3A10.1038%2Fnature14668/MediaObjects/41586 2015 BFnature14668 MOESM317 ESM.mp4 Octopuses are perhaps most easily recognized by their eight appendages. The arms are strong muscular hydrostats, each covered with hundreds of suckers that allow octopuses to exert powerful suction forces. The flexibility and strength of octopus arms is a unique morphological innovation in the animal kingdom. The O. bimaculoides genome and transcriptome is reported by Albertin and Simakov et al. Credit: Z. Yan Wang, C. Ragsdale, J. Reynolds, and Schadenfreude the octopus. MP4 36819 kb Rights and permissions This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ http://creativecommons.org/licenses/by-nc-sa/3.0/ . About this article Cite this article Albertin, C., Simakov, O., Mitros, T. et al. The octopus genome and the evolution of cephalopod neural and morphological novelties. Nature 524 , 220–224 2015 . https://doi.org/10.1038/nature14668 Received: Accepted: Published: Issue date: DOI: https://doi.org/10.1038/nature14668 This article is cited by - Chromosome-level genome assembly of the deep-sea snail Phymorhynchus buccinoides provides insights into the adaptation to the cold seep habitat https://doi.org/10.1186/s12864-023-09760-0 BMC Genomics 2023 - Comparative proteomics of the shell matrix proteins of Nautilus pompilius and the conchiferans provide insights into mollusk shell evolution at the molecular level https://doi.org/10.1007/s00227-023-04244-x Marine Biology 2023 - Identification of LINE retrotransposons and long non-coding RNAs expressed in the octopus brain https://doi.org/10.1186/s12915-022-01303-5 BMC Biology 2022 - Gene transcriptional profiles in gonads of Bacillus taxa Phasmida with different cytological mechanisms of automictic parthenogenesis https://doi.org/10.1186/s40851-022-00197-z Zoological Letters 2022 - Modeled microgravity alters apoptotic gene expression and caspase activity in the squid-vibrio symbiosis https://doi.org/10.1186/s12866-022-02614-x BMC Microbiology 2022 Comments - I will have to read this article more carefully. From my experiences in aquatic animal biology and medicine, the octopus seems to be a potentially excellent animal model for studies of the interaction of consciousness and DNA transcription and translation. Line sheet microscopy conducted at Howard Hughes Medical Institute in Virginia has demonstrated visualizations of neuro-electric impulses in zebrafish larvae. Also the reliability of electroencephalography in the zebrafish has been increasing. The classic studies of Bhasin et al., at Harvard have shown the effects of meditation on DNA transcription/translation. Investigating the interaction of consciousness with the genome needs to be conducted with an invertebrate animal model. Additionally, the octopus can potentially be an excellent animal model for studies of biophotonics.