By Kevin E. Noonan —

Cephalopods are fascinating creatures, and their primary living representative — the octopus — has recently been the subject of the Academy Award winning documentary "My Octopus Teacher."  They are clearly intelligent (as set forth in Peter Godfrey-Smith's Other Minds: The Octopus, the Sea, and the Deep Origins of Consciousness), but it is an intelligence so different from human intelligence that it was an inspiration for the alien first-contact film, Arrival.

Outside the coleoid group, the only living relative are animals of the genus Nautilus, the sole surviving externally shelled cephalopod since the Paleozoic (541 to 252 million years ago).  That shell is itself iconic, as both objet d'art and a living illustration of the Fibonacci series in the ratios of the whorls from which the shell is made.

Recently, an international group of researchers (predominantly working in China)* published a paper entitled "The genome of Nautilus pompilius illuminates eye evolution and biomineralization," in the July edition of Nature Ecology & Evolution, elucidating the Nautilus genome.  (The octopus genome has been determined previously; see "Octopus Genome Sequenced".)  In stark contrast with the octopus genome, the Nautilus genome is parsimonious, the authors characterizing it as "compact, minimalist" with few genes and "slow evolutionary rates in both non-coding and coding regions."  Genetic innovations in Nautilus involve gene loss, with expansion and contraction independently of certain gene families resulting in the "pinhole" eye which functions without either cornea or lens.  These distinctions with their coleoid cephalopod cousins provide the possibility that genomic comparisons could provide insights into how evolution shaped both cephaloid types, according to the authors.

These researchers report that the Nautilus genome comprises 731 megabasepairs, which is the smallest of the cephalopods, being 2.5-7 fold smaller than their extant coleoid relatives.  One feature the authors called "strikingly different" in Nautilus DNA is found in transposable elements (TE), known to be "the driving force in shaping genomic architecture and evolution."  TEs comprise 31% of the genome, with retrotransposons, including LINE (long interspersed nuclear element) SINE (short interspersed nuclear element), and LTR (long terminal repeats) elements constituting only a minor portion (6.5%); in contrast, these elements are "a prominent presence" in the coleoid cephalopod genome.  Moreover, further analysis indicated that an ancient DNA transposon "burst" occurred once in the Nautilus lineage and that there has been no further expansion of TEs in the Nautilus genome (coleoid cephalopods have had more evolutionarily recent instances of such bursts).  Nautilus shares this characteristic with other mollusks, which the authors say suggest slow evolutionary rates in non-coding regions of the Nautilus genome.

Genetic analyses identified 17,710 protein-coding sequences in the Nautilus genome, which is only 53-61% of the number of putative gene sequences found in octopus and squid genomes.  On the gene family level this is reflected by "a huge contraction of orthologous gene families" in the Nautilus genome (204 gene families contracted versus only 9 gene families that have expanded).  The authors note that 18 domains of centromere protein B were specifically expanded in Nautilus compared with other cephalopods; this protein is thought to "play[] crucial roles in host genome integrity and replication fidelity through the repression of retrotransposons and centromere formation in yeast or humans" consistent with the frequencies of TE sequences found in the Nautilus genome.

On a more global level, comparisons of the Nautilus genome to related (and not-so-related) species was used to assess phylogenetic relationships, using 423 single-copy orthologues from 16 animal genomes as shown in the figure:

Divergence from the coleoid cephalopods was estimated by these studies to have occurred around the Silurian-Devonian boundary at about 423 million years ago (Ma), consistent with other estimates from geological evidence and "molecular clock" inferences.  On a finer scale, these researchers report that N. pompilius populations expanded about 23 Ma ("at the turn of the Miocene") in a "stepwise manner," which expansion then stopped as a result of climate changes (glaciation) at the Mid-Pleistocene Transition (around 1 Ma), and finally fell precipitously around 0.4 Ma during another intense glaciation period termed the Mid-Brunhes Event.  These authors conclude that this pattern indicates N. pompilius to be sensitive to "extreme environmental fluctuations" (although it is not evident that they are particularly more sensitive than other aquatic species).  Paradoxically octopus species and certain bony fishes thrived during this period according to the paper, suggesting "ecological competition" (i.e.,"nature red in tooth and claw") had much to do with the population record of N. pompilius during these times.

Turning to the pinhole eye, these researchers report the presence of "nearly all" of the genes encoding the core regulatory complex of transcription factors thought to govern lens formation in coleoid cephalopods (PAX6, SIX3/6 and SOX2, among others) in Nautilus, consistent with lens development in the early Cambrian era (541-485 Ma).  In Nautilus, an important regulatory gene for lens production (in vertebrates as well as coleoid cephalopods), Nrl/Maf, is missing and genes encoding lens-specific crystallin genes are "contracted" compared to other mollusks like octopus that have lens-equipped eyes.  Other specific crystallin genes found expanded in coleoids are missing in the Nautilus genome.  Nautilus is known for behaviors (such as coming towards the surface to feed under lower light conditions) indicating visual perception, and were found by these researchers to possess one photoreceptive r-opsin gene and one retinochrome gene, which these authors assert represents "the minimal opsin gene number among known metazoans" (albeit conceding that N. pompilius is almost certainly colorblind).  The authors note that light intensity is a more critical faculty for animals like N. pompilius that migrate vertically between depths at sea, and that such sensitivity depends on the 11-cis retinal chromophore which isomerizes in response to light.  The N. pompilius genome contains a retinochrome encoding such an isomerase, and in addition these researchers identified an ortholog of a gene family (ten genes) named retinal pigment epithelium-specific protein 65 kDa present in vertebrates that is also found in the Nautilus genome.  These genes contain a conserved iron binding site, an "active cavity" site, and a "hydrophobic tunnel" consistent with catalytic activity.  The significance of these finding is explained by the authors as:

From a perspective of evolutionary adaptation, the appearance of the pinhole eye is one adaptive breakthrough essential to the nautilus lifestyle of vertical depth migrations, allowing the organism to acquire spatial vision and rapidly cope with hydrostatic pressure within the eye through opening the pupil to seawater.  Overall, multiple genomic innovations including gene losses, independent contraction and expansion of specific gene families and presence of associated regulatory networks seem to work in unison to drive the evolution of the pinhole eye in nautilus.

The paper provides a schematic diagram of the Nautilus pinhole eye to put these genetic analyses in context:

Turning to the other characteristic morphological feature of Nautilus, its shell, the authors note that it is made up of aragonite (an orthorhombic crystal of calcium carbonate, CaCO₃) and shell matrix proteins (SMP) used as a substrate.  The authors report finding genes encoding a total of 78 SMPs with expression pattern concentrated in the shell mantle (72%).  Genetic comparisons revealed that 21 Nautilus SMPs shared sequence similarity with analogous genes from bivalves and gastropods and found several conserved domains in these protein across mollusk species, including "laminin, chitin-binding and carbonic anhydrase domains."  The authors posit these regions as part of a "core biomineralization toolkit" conserved across external shell-expressing mollusks.  On the other hand, 52 of the 78 SMPs encoded in the Nautilus genome were either new or Nautilus-specific, and these researchers speculate that most of the unique SMPs evolved independently in different mollusk species including Nautilus.  Indeed, there is one important SMP, Nautilin-63, that these researchers found showed low sequence similarity even within the Nautilus genus.  And the top ten SMPs enriched in the Nautilus mantle contain an evolutionarily novel poly(Gly or Gly-Ala) repetitive motif; also detected encoded in these proteins were several repetitive low-complexity domains (RLCDs), in common with other shelled mollusks, that the inventors suggest "could be a unifying principle for molluscan biomineralization, especially for nacre formation."

Finally, the paper discusses the Nautilus immune system, which contains a "highly complex yet comprehensive innate immune components" enabling Toll-like receptor and tumor necrosis factor receptor signaling that regulates "apoptosis, inflammation and immune defences."  Also detected were genes encoding IL17R, H-lectin, and IL1, which the authors assert "supports the assumption that nautilus has preserved a more complete repertoire of immune molecules than other cephalopods."  An assessment of gene numbers for immune system-specific genes showed expansion of C-type lectin genes (81) compared to coleoid species (wherein 12-33 genes are encoded) and some of these genes have been independently duplicated.  Also found to be specifically expanded are interferon-inducible GTPases, genes also known to be implicated in immune function.  These genetic features of the immune system-involved genes in Nautilus (and comparisons with coleoid species) is illustrated in this figure:

The authors conclude with this synopsis of their findings:

Genomic evidence reveals that nautilus has undergone lineage-specific innovations in both body plan and behaviour since the Cambrian and retained these extraordinary features after a long evolutionary history.  In particular, vertical depth migration in Nautilus and other chambered cephalopods is one of several critical and common strategies needed to avoid predators and budget energy; these may have helped the survival of these species ever since.  The emergence of the pinhole eye is a great innovation for switching from directional to spatial vision and rapidly change hydrostatic pressure, making vertical depth migration possible.

Our findings highlight that co-evolutionary loss of core regulatory transcription factors may have driven the evolution of the pinhole eye.  Moreover, our proteomic and transcriptomic data suggest that an ancient "core biomineralization toolkit" and new RLCDs coordinately directed the construction of the chamber shell, which has evolved into the buoyancy apparatus needed to adapt to a critical life mode.  Taken together, the draft genome of N. pompilius together with multiomics provide a valuable insight into not only the adaptive innovations of the ancestor of cephalopods but also the dynamic evolution of coleoids.

* Key Laboratory of Tropical Marine Bio-resources and Ecology and Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou; Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou; Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou; MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei; Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo; Biomarker Technologies Corporation, Beijing; State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, Guangzhou; Department of Neuroscience and Developmental Biology, University of Vienna.

By Kevin E. Noonan —

The domestic dog (Canis lupus familiaris) has been the subject of many genetic studies, particularly since the dawning of the age of Whole Genome Sequencing in the late 20th Century.  These studies have elucidated some interesting facts about "man's best friend," some of which have been discussed here (see "The Genetic Basis of Coat Variation in Dogs"; "Leg Length Variation in Dogs and its Relevance to Human Mutations"; "From Toy Poodle to Rottweiler: Why Is Fido So Small (or Large)?";  "Selection for Facial Features in Domestic Dogs: The Evolution of Cuteness") and comparators to related species (see "Red Fox Genome Sheds Light on Domesticated Dogs (and Maybe Humans)").

But as with other organisms, a great deal of the genetic sequence set forth early in the 21st Century was incomplete due to technical limitations. Chief amongst these was difficulty in obtaining reliable sequence information on sequences repeated in many places in the genome, which interfered with reliable assembly of sequenced portions (termed "contigs") into longer (ideally, chromosomal-length) linear sequence assemblies (termed "scaffolds).  Improvements in sequencing technology now permit better sequence determinations for high GC and highly repetitive regions.

The existence of a prior reference genome (produced from a female boxer named Tasha), combined with these new sequencing tools, was used by an international team of researchers* in a paper published in March of this year in the Proceedings of the National Academy of Sciences (USA) entitled "Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes."  These scientists reported elucidation of the genomic sequence from a female Great Dane named Zoey, wherein they were able to identify and eliminate (in large part) incorporation of false contigs due to repetitive ends and fragment ends that map to multimer genomic locations.  Using alignment with the reference dog genome revealed gaps between contigs as earlier reported in dog genomic DNA.  They report finding 373 additional sequence regions spanning contigs having N50 of 30kb and a total length of 10.5 Mbp.  The resulting aligned scaffolds were assigned to dog chromosomes to constitute a genomic sequence for the dog genomic complement of 78 chromosomes.

From this sequence the researchers were able to identify 22,182 protein-coding gene models; full-length matches with the reference canine genome were found for only 84.9% (18,834) of all protein-coding gene models, and almost full-length alignments were found for 93% (20,670) of the models; in addition they identified 49 protein-coding genes not present in the earlier canine reference genome.  Also annotated were 7,049 long noncoding RNAs that included 84 with no or only partial alignment to the reference canine genome.  From this comparison they further were able to appreciate that the assembly from the Great Dane genome spanned the majority of sequence gaps not having been sequenced in the reference dog genome.  Mapping of these gaps showed that 2,151 gaps (16.8% of gaps) overlapped the transcription start site of a predicted protein-coding gene and were found to be extremely GC rich (67.3%).  Further, a subset of the resolved gaps, having a median 80.95% GC content, were found to be preferentially localized (i.e., at a frequency greater than random chance) at transcription start sites and recombination hotspots.  About 12% (1,457 of 12,304 on the autosomes) of gap segments were found to be located within 1 kbp of a hotspot (the expected percentage of such location was closer to 3%).  These hotspot-adjacent segments were extremely GC rich; 5,553 such identified segments had a GC content greater than that what would be expected from random sequence permutations.  The total extent of these extreme GC segments spanned 4.03 Mbp in the Great Dane sequence assembly, were found to have "a median length of 531 bp, a median GC content of 80.95%, and are located much closer to transcription start sites (median distance of 290 bp) and recombination hotspots (median distance of 68.7 kbp) than expected by chance."

Analysis of the Great Dane genome assembly with the reference canine genome identified 16,834 deletions (median size: 207 bp) and 15,621 insertions (median size: 204 bp) in Great Dane DNA.  Genetic assessment of these sequences revealed predominantly the presence of two forms of "retrotransposon insertion/deletion polymorphisms" which included dimorphic canine short interspersed elements (SINECs) (16,221 copies) and dimorphic long interspersed element-1 sequences (LINE-1_Cfs) (1,121 copies).  The 3' flanking sequence for the LINE-_Cfs elements suggested "multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations."

The researchers further reported a length distribution of the detected variants having "a striking bimodal pattern, with clear peaks at ∼200 bp and ∼6 kbp, consistent with the size of SINEC and LINE-1 sequences," as shown in this Figure:

The location of these sequences was associated with insertions and deletions, the researchers reporting that inspection of the sequences in the 150-250-bp range (dimorphic SINEC elements) were found at 7,298 deletion and 6,071 insertion sites, and that "LINE-1 sequences accounted for 339 deletions and 581 insertions longer than 1 kbp." When combined with data from the reference canine genome there were at least 16,221 dimorphic SINEC and 1,121 dimorphic LINE-1 sequences identified.  "Hallmarks of retrotransposition" were identified at these sites, wherein the SINEC and LINE-1 sequences were flanked by "target site duplications having a 15 bp median size with the elements ending in poly(A) tracts having median lengths of 9 bp to 12 bp."

To test whether these sequences were capable of retrotransposition, a particular sequence was cloned that had intact open reading frames encoding the ORF1p and ORF2p predicted proteins and lacked mutations expected to disrupt protein function.  This element was introduced into human cells in vitro and shown to be capable of retrotransposition, as illustrated in this Figure:

This element also capable of mobilizing both the canine SINEC elements and analogous human Alu elements.  The researchers speculated that this result was consistent with ongoing retrotransposon activity as a driver of canine genetic variation.

The researchers set forth a synopsis of their results as follows.  They had identified 49 predicted protein-coding genes from the Great Dane assembly that were not found in the canine genome, as well as 2,151 protein-coding gene models having a transcription start position located in a gap in the reference genome sequence.  The existence of high GC-content sequences in canine promoter regions distinguishes the preferential location of recombination events in dogs, which lack a functional PRDM9 gene known to mediate recombination in other mammals; see Paigen & Petkov, 2018, "PRDM9 and its role in genetic recombination," Trends Genet. 34, 291–300, and Auton et al., 2013, "Genetic recombination is targeted towards gene promoter regions in dogs," PLoS Genet. 9, e1003984.  The researchers assert that "[t]he presence of extremely GC-rich segments likely reflects a key aspect of canine genome biology" as a consequence of these findings.  Further, a comparison of the number of single nucleotide variants (i.e., differences) found in Zoey and Tasha (3.57 million) was lower than found in similar comparison between humans (4.1-5.0 million).  In contrast, the levels of LINE-1 and SINEC dimorphism between these two dog genomes was "disproportionately large," there being "an ∼17-fold increase in SINE differences (16,221/915) and an eightfold increase in LINE differences (1,121/128) compared to the numbers found among humans" (indeed, the researchers report that "more dimorphic SINEs were found between these two breed dogs than have been found in studies of thousands of humans").  They note that these results are consistent with prior studies, including Wang & Kirkness, 2005, "Short interspersed elements (SINEs) are a major source of canine genomic diversity," Genome Res. 15, 1798–808.

The authors conclude by saying:

[O]ur study suggests that retrotransposition is an ongoing process that continues to affect the canine genome.  We provide proof-of-principle evidence that dog genomes contain LINE-1 and SINEC elements that are capable of retrotransposition in a cultured cell assay.  We also identified two LINE-1 lineages with the same 3′ transduced sequence associated with multiple elements, suggesting the presence of multiple canine LINE-1s that are capable of spawning new insertions.  Additionally, analysis of 3′ transduction patterns suggests the presence of additional active LINE-1s in canines that have yet to be characterized.  Thus, a full understanding of canine evolution and phenotypic differences requires consideration of these important drivers of genome diversity.

* From  the Department of Biological Sciences, Bowling Green State University; the Department of Human Genetics, Department of Computational Medicine and Bioinformatics, and the Department of Internal Medicine, University of Michigan; the Université Côte d'Azur, CNRS, INSERM, Institut de Recherche sur le Cancer et le Vieillissement de Nice, Nice; the Université de Rennes 1, CNRS, Institut de Génétique et Développement de Rennes−UMR 6290, Rennes; and the Department of Biomedical Sciences, Cornell University, Ithaca, NY 14850.