Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire and Genomic Organization

Nearly one-fifth of ancestral eumetazoan genes are novelties lacking recognizable homologs outside animals or containing new protein domains or domain combinations absent in other eukaryotes.

“Nearly one-fifth of the genes of the ancestor are eumetazoan novelties in the sense that they have no recognizable homologs outside of animals, or contain new protein domains and/or domain combinations that are not found in other eukaryotes.”

The Nematostella genome has a gene repertoire, exon-intron structure, and large-scale gene linkage more similar to vertebrates than to flies or nematodes.

“The anemone genome is surprisingly complex, with a gene repertoire, exon-intron structure, and large-scale gene linkage more similar to vertebrates than to flies or nematodes.”

Only 33 genes are found in Nematostella and both Drosophila and C. elegans but not in any vertebrate, representing putative deuterostome or vertebrate loss.

“In contrast, only 33 genes are found in Nematostella and both Drosophila and C. elegans but not in any vertebrate, representing putative deuterostome or vertebrate loss, indicating a much lower degree of gene loss in the vertebrates than in the ecdysozoan model systems.”

The human genome has more reciprocal best-scoring BLAST orthologous pairs with Nematostella (6,989) than with Drosophila (5,772), C. elegans (4,846), or Ciona intestinalis (6,313).

“Surprisingly, the human genome has many more such orthologous pairs with Nematostella (6,989) than with nonvertebrate bilaterians, including Drosophila (5,772), C. elegans (4,846), and even the invertebrate chordate Ciona intestinalis (6,313).”

Combining homology-based and ab initio methods with over 146,000 ESTs predicted about 27,000 complete or partial protein-coding transcripts.

“Combining homology-based and ab initio methods with sequences from over 146,000 expressed sequence tags, we predicted ~27,000 complete or partial protein-coding transcripts in the genome (32).”

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After correcting for sampling, each pair of haplotypes differs at 0.65% of nucleotide positions.

“After correcting for sampling, we estimated that each pair of haplotypes differ at 0.65% of nucleotide positions (32).”

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The remainder of eumetazoan introns appear to be shared only by animals.

“Althought many eumetazoan introns are evidently of ancient eukaryotic origin (46) --for example, nearly 26% of human and Nematostella introns are conserved with Arabidopsis, and 24% with Cryptococcus --the remainder appear to be shared only by animals.”

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Of the 7,766 ancestral eumetazoan gene families, 72% (5,626) are represented in complete genomes of all three major modern eumetazoan lineages.

“Of the 7,766 ancestral eumetazoan gene families, only 72% (5,626) are represented in the complete genomes of all three major modern eumetazoan lineages: cnidarians (i.e., Nematostella), protostomes (i.e., Drosophila and/or C. elegans), and deuterostomes (requiring presence of at least two of pufferfish, frog, and human).”

7,766 putatively orthologous gene families were constructed, anchored by reciprocal best-scoring BLAST alignments between anemone and one or more of fly, nematode, human, frog, or fish.

“To approximate the gene repertoire of the eumetazoan ancestor we constructed 7,766 putatively orthologous gene families that are anchored by reciprocal best-scoring BLAST alignments between genes from anemone and one or more of fly, nematode, human, frog, or fish [S].”

Over 500 families of transposable elements were discovered, including DNA transposons and both LTR and non-LTR retrotransposons.

“Over 500 families of them were discovered in the genome, including DNA transposons and both LTR and non-LTR retrotransposons (Table S2.3).”

Metaphase spreads indicate a diploid chromosome number of 2N=30.

“Metaphase spreads indicate a diploid chromosome number of 2N=30 (Fig S2.4).”

Long branch lengths indicating increased sequence divergence were found along fly, nematode, and sea squirt lineages.

“Long branch lengths, indicating increased levels of sequence divergence, were found along the fly, nematode, and sea squirt lineages.”

The typical locus in the draft genome is in a contiguous gap-free stretch of nearly 20 Kb.

“The typical locus in the draft genome is in a contiguous gap-free stretch of nearly 20 Kb.”

Sea anemone sequences appear to be evolving at a rate comparable to or somewhat lower than vertebrates.

“The sea anemone sequences, however, appear to be evolving at a rate comparable to, or even somewhat lower than, vertebrates.”

Phylogeny of Metazoa inferred using 337 single-copy genes places Nematostella and Hydra as a monophyletic group that branched off the metazoan stem before bilaterian radiation.

“As expected, the two cnidarians Nematostella and Hydra form a monophyletic group that branched off the metazoan stem prior to the radiation of bilaterians.”

More than 12,000 predicted transcripts are in robust eumetazoan gene families and supported as orthologs of other animal genes.

“More than 12,000 of these are found in robust eumetazoan gene families, and are therefore supported as orthologs of genes in other animals (see below).”

More than 25% of the genome is made of repetitive elements that are fossilized copies of transposable elements.

“More than 25% of the genome is made of repetitive elements that are fossilized copies of transposable elements.”

Weighted parsimony analysis of 2,645 intron positions inferred intron gain and loss events across metazoan genomes.

“Figure 3b shows intron gain (left) and loss (right) events inferred by weighted parsimony analysis of 2,645 intron positions that lie within highly conserved protein sequence in two or more animals, the flowering plant Arabidopsis, and the relatively intron-rich fungus Cryptococcus neoformans (32).”

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Slightly more than 10% of EST contigs have significant alignments to multiple scaffolds, providing an estimate of assembly redundancy from separate assembly of divergent haplotypes.

“Slightly more than 10% of the EST contigs have significant (95% identify, 75% length) alignments to multiple scaffolds (32), providing an estimate of the redundancy of the assembly, which appears to arise from the occasional separate assembly of divergent haplotypes.”

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The subfamilies established on the eumetazoan stem are still maintained in modern vertebrates.

“nearly 750 million years later, are still maintained in modern vertebrates”

Within animals, intron gains range from 8-22% relative to the content of the eumetazoan ancestor.

“Within animals, intron gains range from 8-22% relative to the content of the eumetazoan ancestor.”

Nidogen represents a Type III novelty, a new pairing of ancient domains.

“others such as nidogen are novel pairings of ancient domains (Figure 5c)”

LIM-homeobox transcription factors resulted from fusion of ancient LIM protein-interaction and homeobox DNA-binding domains on the eumetazoan stem.

“the LIM-homeobox transcription factors are the result of a fusion of the ancient LIM protein-protein interaction and homeobox DNA-binding domains on the eumetazoan stem”

Matrix metalloproteases are Type II novelties.

“Matrix metalloproteases also were invented as Type II novelties”

These 98 human segments span 89% of the base pairs of the human genome.

“These segments span 89% of the base pairs of the human genome.”

Tolloid is a Type III novelty, formed by a new pairing of ancient domains.

“some, such as tolloid are novel pairings of ancient domains (Type III)”

Fyn arose later, on the tetrapod stem, through gene duplication.

“Fyn itself is a more recent invention derived on the tetrapod stem by gene duplication”

Within each conserved segment, local gene order is considerably scrambled.

“Within each segment, however, local gene order is considerably scrambled.”

Neuronal-activity genes are numerous among the novelties.

“genes with neuronal activities are abundant among its novelties”

98 human segments were identified that each contain descendants of 40 or more ancestral eumetazoan genes and do not appear to have undergone recent breaks or fusions.

“To remove confounding signals from recent rearrangements, we used comparisons with the genomes of other chordates to identify 98 human segments large enough that they each contain descendants of 40 or more ancestral eumetazoan genes, that do not appear to have undergone recent breaks or fusions (Figures 4a, S7.1) (32).”

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Netrin and semaphorin are novelties lacking clear homology to ancient eukaryotic proteins.

“guidance cues such as netrin and semaphorin that mediate adhesion are novelties with no clear homology to ancient eukaryotic proteins”

Secreted ligands and antagonists in the Wnt and TGF-beta pathways are novelties.

“We find that in both pathways, the secreted ligands and their antagonists (wnt, SFRP, BMP, chordin etc.) are novelties (Figure 5b)”

Mesoderm-development genes of bilaterians are overrepresented among eumetazoan novelties.

“genes known to be involved in mesoderm development in bilaterians are also enriched among eumetazoan novelties”

Wnt-activated downstream transcription factors are ancient.

“Transcription factors that are activated downstream of Wnt signaling are ancient”

Type I receptors of the TGF-beta pathway arose as pairings of novel animal domains with ancient domains (Type II novelties).

“Type I receptors of the TGFbeta pathway arose as a pairing of novel animal domains with ancient domains (Type II novelties)”

An ancestral linkage group includes the eumetazoan Hox cluster of homeobox transcription factors that regulate anterior-posterior identity in bilaterians.

“An ancestral linkage group of particular interest includes the eumetazoan Hox cluster of homeobox transcription factors that regulate anterior-posterior identity in bilaterians.”

Novel genes show significant enrichment in signal transduction, cell communication/adhesion, and developmental processes.

“we find the novel genes to be significantly enriched for signal transduction, cell communication and adhesion, and developmental processes (32)”

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Of 4,402 ancestral eumetazoan gene families represented in the largest anemone scaffolds and human segments, more than 30% (1,336) participate in a conserved linkage group.

“Of the 4,402 ancestral eumetazoan gene families represented in the largest anemone scaffolds and human segments (i.e., in the genomic regions large enough to permit statistically significant analysis, and therefore eligible for consideration in our analysis), more than 30% (1,336) participate in a conserved linkage group.”

The complete Oxford grid shows all 13 putative ancestral eumetazoan linkage groups.

“The complete Oxford grid showing all 13 PALs is shown in Table S7.2.”

Some lineages, notably fly, nematode, and sea squirt, have experienced significant intron loss, each discarding 50-90% of inferred ancestral eumetazoan introns.

“In contrast to intron gains, which seem to occur more or less uniformly across animal phyla, some lineages appear to have experienced significant intron loss, notably fly, nematode, and sea squirt, which have each discarded 50-90% of inferred ancestral eumetazoan introns.”

TGF-beta signaling transcription factors are novel.

“but the ones involved in TGF-beta signaling are novel”

Collagen and laminin are Type II novelties that appeared on the eumetazoan stem.

“Basement membrane proteins such as collagen and laminin arose as Type II novelties along the stem leading to the Eumetazoa”

These genes had already duplicated and diversified on the eumetazoan stem, establishing subfamilies.

“they had already duplicated and diversified on the eumetazoan stem to establish the subfamilies”

The Wnt receptor frizzled is a Type I eumetazoan novelty.

“The receptor in the Wnt pathway, frizzled, also arose as a Type I eumetazoan novelty”

Dickkopf is classified as a Type II novelty.

“some are Type II novelties (dickkopf)”

Nearly 80% (6,182/7,766) of ancestral eumetazoan genes have clearly identifiable relatives outside animals, including fungi, plants, slime molds, ciliates, or other public datasets.

“Nearly 80% (6,182/7,766) of the ancestral eumetazoan genes have clearly identifiable relatives (i.e., proteins with significant sequence homology and conserved domain architecture) outside of the animals, including fungi, plants, slime molds, ciliates, or other species available from public datasets (32).”

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The eumetazoan ancestor already possessed the genes in question.

“not only were these genes present in the eumetazoan ancestor”

Notch proteins contain two Notch domains found only in metazoans plus ancient eukaryotic ankyrin and EGF domains.

“Notch proteins have two Notch domains found only in metazoans in addition to ancient eukaryotic ankyrin and EGF domains”

A more sensitive approach can assign more than twice as many ancestral genes to a putative ancestral eumetazoan linkage group.

“A more sensitive approach can assign more than twice as many ancestral genes to a PAL (32).”

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The ancestral eumetazoan genome included core genes needed for complex neural and muscular function.

“as well as the core gene set required to execute sophisticated neural and muscular function”

DNA transposons are fourfold more abundant than retrotransposons in terms of their bulk contribution to the sea anemone genome size.

“In terms of their bulk contribution to the genome size, DNA transposons are fourfold more abundant than retrotransposons (Table S2.3).”

The 5'-CACAG termini of anemone EnSpm-1_NV and EnSpm-2_NV transposons differ from the 5'-CACTA termini of plant transposons.

“their 5'-CACAG termini differ from the 5'-CACTA termini of the plant transposons.”

We assigned one or more PFAM domains to 15,102 human genes and 12,202 Nematostella genes.

“We were able to assign one or more PFAM domains to 15,102 human genes and 12,202 Nematostella genes.”

Only nine ortholog sets contained one gene from each of the twelve whole genomes plus a Mnemiopsis sequence.

“Only nine ortholog sets contained one gene from each of the twelve whole genomes plus a Mnemiopsis sequence.”

From the manual review, the expected number of bona fide Nematostella genes is 18,345.

“Adding up these expected numbers gives us an estimate of 18,345 bona fide Nematostella genes.”

Representatives of all reported superfamilies and groups of eukaryotic DNA transposons, excluding the Transib superfamily and the Mariner group of the Mariner superfamily, are present in the anemone genome.

“Representatives of all reported superfamilies and groups of eukaryotic DNA transposons (14-16), excluding the Transib superfamily and the Mariner group of the Mariner superfamily, are present in the anemone genome.”

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Nebulin and tropomodulin are Type II sarcomere novelties.

“Other sarcomere proteins are Type II novelties (e.g.nebulin and tropomodulin)”

The eumetazoan ancestral genome was rich in introns.

“it was intron rich”

The sea anemone genome contains two novel groups of hAT transposons, hAT5 and hAT6, characterized by 5-bp and 6-bp target site duplications, respectively.

“the sea anemone genome, in addition to the canonical hAT transposons contains two novel groups, hAT5 and hAT6, characterized by 5-and 6-bp TSDs, respectively.”

The ORFs coding for transposases, reverse transcriptases, and other transposon-specific proteins in sea anemone transposable elements are not severely damaged by mutations.

“their ORFs coding for transposases, reverse transcriptases, and other transposon-specific proteins are not severely damaged by mutations.”

Different classes of autonomous DNA transposons in the sea anemone genome are represented by just a few families.

“different classes of autonomous DNA transposons are represented by just a few families.”

The families of transposable elements in the sea anemone genome are composed of a small number of copies, ranging from 1 to about 5,000.

“These families are composed of a small number of copies (from 1 to ~5,000)”

This type of reversal in the best C-value curve does not appear in human-Drosophila, human-Caenorhabditis, or Drosophila-Caenorhabditis comparisons.

“This type of reversal does not appear in human-Drosophila, human-Caenorhabditis , or Drosophila-Caenorhabditis comparisons (data not shown).”

16 Pfam domains previously found exclusively in vertebrates, but not in other phyla of bilateria or other eukaryotes, are present in the Nematostella genome.

“16 Pfam domains previously exclusively found only in vertebrates, but not in other phyla of bilateria (or other eukaryotes), are present in Nematostella genome, including:”

Dsh, Arx, and neuralized are Type II novelties, combining old domains with new animal-specific domains.

“some are parings of old domains with novel animalspecific domains (such as Dsh, Arx, neuralized)”

Of the PFAM domains present in human, mouse, dog, chicken, frog, and fugu, Nematostella has more in common than any of Ciona, fruit fly, or nematode.

“Of the PFAM domains present in human, mouse, dog, chicken, frog and fugu, Nematostella has more in common than any of Ciona, fruit fly, or nematode.(Figure S3.4)”

By this method, 337 ortholog sets were identified that had one gene representing each of the twelve whole genome datasets.

“By this method, 337 ortholog sets were identified that had one gene representing each of the twelve whole genome datasets.”

In general, the gene families showing strong expansions along the human and Nematostella lineages are different.

“In general, the gene families showing strong expansions along the two lineages are different.”

A similar algorithm applied to the ENSEMBL annotation of the human genome detected 504 recent expansions with 1,317 genes.

“A similar algorithm applied to the ENSEMBL annotation of the human genome detected 504 recent expansions with 1,317 genes.”

Proteins closest to the anemone Troyka, hAT5, and hAT6 transposon-encoded proteins in GenBank are encoded by transposable elements characterized by the same unusual lengths of target site duplications.

“using reverse transcriptase/integrase and transposase encoded by the anemone Troyka, hAT5, and hAT6 transposons as queries in TBLASTN searches against GenBank DNA sequences, we found that proteins closest to the queries (>30% protein identity) are encoded by TEs characterized by the same unusual lengths of TSDs.”

Many neurogenesis genes, such as homeobox and bHLH transcription factors, trace back to ancient eukaryotic genes carrying their signature domains.

“Numerous genes known to be involved in neurogenesis, such as members of the homeobox and basic helixloop helix transcription factor families (Emx, Otp, Otx; achaete-scute), can be traced to ancient eukaryotic genes with these signature domains”

The human genome has more genes with a mutual best hit in Nematostella than in the proteomes of Ciona, fruit flies, or nematodes.

“The human genome has more genes with a mutual best hit in Nematostella than in the proteomes of Ciona, fruit flies or nematodes.(Figure S3.2)”

The eumetazoan ancestor had more than 1,500 genes apparently novel compared with other eukaryotic kingdoms.

“The eumetazoan ancestor possessed over fifteen hundred genes that are apparently novel relative to other eukaryotic kingdoms”

Over 3% of the anemone genome is made of fossilized copies of self-synthesized Polinton DNA transposons whose transposition depends on the Polinton-encoded DNA polymerase and integrase.

“Over 3% of the anemone genome is made of fossilized copies of self-synthesized Polinton DNA transposons whose transposition depends on the Polinton-encoded DNA polymerase and integrase (17).”

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Different classes of anemone retrotransposons, including Gypsy, DIRS, Penelope, and CR1, are composed of more than 50-100 families each.

“different classes of anemone retrotransposons, including Gypsy, DIRS, Penelope, and CR1, are composed of more than 50-100 families each”

Dystrophin, syntrophin, beta-dystrobrevin, and beta-sarcoglycan, sarcolemmal DPC components, are Type I novelties.

“Various components of the dystrophin-associated protein complex (DPC) in the sarcolemma such as dystrophin, syntrophin, beta-dystrobrevin and beta-sarcoglycan are Type I novelties”

In Nematostella, there is no evidence for recent selection for retention of new genes created by tandem duplication with the PF000001 (rhodopsin family) domain.

“Hence, in Nematostella, there is no evidence for recent selection for retention of new genes greated by tandem duplication with PF000001.”

• AssumptionThe authors state this as an assumption: Any gene has an equal probability of getting duplicated and fixed in the population.

Many animal-specific genes have sequences without readily recognizable counterparts outside animals.

“a significant number of animal-specific genes contain sequences with no readily recognizable counterparts outside of animals”

In the human genome, 112 genes with the PF000001 domain are observed in recent tandem expansions, with an expected value of 29 ± 5.3, consistent with strong recent selective retention of such receptors within vertebrates or mammals.

“In the human genome, on the other hand, 112 such genes are observed, with an expected value of 29 +-5.3, consistent with a strong recent selective retention of such receptors (olfactory and visual) within vertebrates or mammals.”

• AssumptionThe authors state this as an assumption: Any gene has an equal probability of getting duplicated and fixed in the population.

The eumetazoan ancestor possessed linked gene blocks still found together in human and anemone genomes.

“the ancestor had blocks of linked genes that remain together in the modern human and anemone genomes”

The anemone genome harbors two families of Tx1-like non-LTR retrotransposons, Tx1-1_NV and Tx1-2_NV, inserted in 5S rRNA and U2 smRNA, respectively, at target sites identical to those of different Tx1 elements in fish, frog, and lancelet.

“the anemone genome harbors two families of Tx1-like non-LTR retrotransposons, Tx1-1_NV and Tx1-2_NV, inserted in 5S rRNA and U2 smRNA, respectively, at target sites identical to those of different Tx1 elements in fish (18), frog and lancelet.”

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Members of the IS4EU superfamily have been found in other species, including lancelet, following their identification in the anemone genome.

“Following identification of the IS4EU TEs in the anemone genome, members of this superfamily have been also found in other species, including lancelet.”

Troyka, a novel type of LTR retrotransposon distantly related to the Gypsy superfamily, is characterized by 3-bp target site duplications.

“Troyka, a novel type of LTR retrotransposons distantly related to the Gypsy superfamily, is characterized by 3-bp target site duplications (TSDs)”

Nematostella genes are more similar to vertebrate genes than to fly and nematode genes, based on Bayesian branch length estimation and percent sequence identity.

“We compared predicted protein sequences from Nematostella to those from other metazoan and outgroup genomes, and find that Nematostella genes are more similar to vertebrate genes than to fly and nematode genes using bayesian branch length estimation and an analysis of percent sequence identity.”

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The oldest transposable element families in the sea anemone genome are less than 15% divergent from their consensus sequences.

“elements from the oldest families are less than 15% divergent from their consensus sequences”

Some novel genes arose by domain shuffling, creating new combinations of domains also found in other eukaryotes on the animal stem.

“Some are the result of domain shuffling, bringing together on the animal stem new combinations of domains that are shared with other eukaryotes”

Of the 7,766 ancestral metazoan gene clusters, 1,619 are composed of a single gene from each of the six representative metazoan genomes: human, fish, frog, Nematostella, fruit fly, and nematode.

“Of the 7,766 ancestral metaozan gene clusters, 1,619 are composed of a single gene from each of the six representative metazoan genomes listed in Supplement S4: human, fish, frog, Nematostella, fruit fly and nematode.”

En/Spm (CACTA) transposons, previously believed to populate only plant genomes, reside in the anemone genome.

“Even, En/Spm (also called CACTA) and transposons, which were believed to populate plants genomes only (14), reside in the anemone genome.”

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The Nematostella genome contains many proteins with domain architectures (combinations of PFAM domains) that are shared exclusively with vertebrate genes.

“The Nematostella genome contains many protines with domain architectures (combinations of PFAM domains) that are shared exclusively with vertebrate genes.(Figure S3.3)”

Manual review of 60 Nematostella genes indicates that about one third of all genes in the FM1.0 set could be expected to be rejected by manual reviews.

“the results of the sampling indicate that about one third of all genes in the FM1.0 set could be expected to be rejected by manual reviews.”

The clustering of scaffolds and chromosome segments defines 15 large PALs, each with descendants of more than one hundred ancestral eumetazoan genes.

“This clustering of scaffolds and chromosome segments defines 15 large PALs, each with descendants of more than one hundred ancestral eumetazoan genes.”

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Sea anemone shares more bidirectional BlastP hits with human than other bilaterians, except vertebrates.

“Despite early divergence, sea anemone shares more hits with human, than other bilaterians, except vertebrates.”

In 1,523 of these clusters, the human gene had a mutual best hit to a Nematostella gene, forming clusters of four orthologous genes.

“In 1,523 of these clusters, the human gene had a mutual best hit to a Nematostella gene, forming clusters of four orthologous genes.”

98 segments of the human genome appear to be uninterrupted by inter-chromosomal translocations or fusions when compared to the genomes of other chordates.

“we identified 98 segments of the human genome which appear to be uninterrupted by inter-chromosomal translocations or fusions when compared to the genomes of other chordates.”

9,947 highly reliable intron splice sites were identified by the requirements used in the analysis.

“9,947 highly reliable intron splice sites were identified by these requirements.”

2,347 clusters of orthologous genes in all bilaterian orthologous clusters have an unambiguous 1:1:1 member relationship in human, Drosophila melanogaster, and C. elegans.

“We next identified 2,347 clusters of orthologous genes in all bilaterian orthologous clusters with an unambiguous 1:1:1 member relationship in human, Drosophila melanogaster (fly), and C. elegans.”

There is extensive conservation of linkage relationships among genes using the conservative statistical criteria described above.

“Having demonstrated that there is extensive conservation of linkage relationships among genes using the conservative statistical criteria described above”

• AssumptionThe authors state this as an assumption: The null model treats the genes in scaffolds and segments as independently drawn from the 7,766 ancestral genes.

2264 Pfam domains are present in all 6 vertebrates with known genomes: human, mouse, dog, chicken, frog and fugu.

“2264 Pfam domains present in all 6 vertebrates with known genomes:human, mouse, dog, chicken, frog and fugu.”

4,342 introns from seven genomes were identified at 2,645 aligned positions that contain an intron in at least one of the seven orthologs.

“4342 introns from the seven genomes at 2645 aligned positions which contain an intron in at least one of the seven orthologs.”

Panther ontology annotations of the inferred ancestral gene set show significant over- and under-representations in each of the three categories of novelty (novel sequence, novel domain, and novel combination of domains).

“Panther ontology annotations of the inferred ancestral gene set have been tested for enrichment in each of the three categories of novelty (novel sequence, novel domain, and novel combination of domains), as described in section S8, and significant over-and under-representations have been tabulated here”

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4,951 highly reliable introns were identified by these requirements.

“4,951 highly reliable introns were identified by these requirements.”

Calpain appears as a novel domain combination of ancient domains.

“Calpain, a protease that regulates the aggregation of Talin, Paxillin and FAK around the receptor appears as a novel domain combination of ancient domains.”

Focal adhesion kinase (FAK) appears as a Type II novelty in eumetazoans.

“Focal adhesion kinase (FAK) is a cytosolic component that appears as a Type II novelty in eumetazoans”

About 81% of human introns (4,403 of 5,435) are shared with Nematostella.

“Remarkably, about 81% of the human introns (4,403 of 5,435) are shared with nematostella.”

3055 ancestral genes, or 40% of the ancestral genes, are assigned to one of these PALs.

“3055 ancestral genes, or 40% of the ancestral genes are assigned to one of these PALs.”

2264 Pfam domains are present in all 6 vertebrates.

“Figure S3.4: 2264 Pfam domains present in all 6 vertebrates”

28.6% of all introns shared by human and Nematostella (and hence present in their LCA) are also shared by Arabidopsis.

“28.6% of all introns shared by human and Nematostella (and hence present in their LCA) are also shared by Arabidopsis.”

Fly and nematode genomes have been modified by sequence divergence, gene and intron loss, and genomic rearrangement.

“These results imply that the genome of the eumetazoan ancestor was similarly complex, and that fly and nematode genomes have been modified via sequence divergence, gene and intron loss, and genomic rearrangement.”

The genome of the eumetazoan ancestor was similarly complex to the Nematostella genome.

“These results imply that the genome of the eumetazoan ancestor was similarly complex, and that fly and nematode genomes have been modified via sequence divergence, gene and intron loss, and genomic rearrangement.”

Subsequent diversification in cnidarian and bilaterian lineages was associated with new regulatory linkages and higher-level integration of preexisting pathways and networks.

“Subsequent diversification in the cnidarian and bilaterian lineages was therefore associated with new regulatory linkages and higher-level integration of these preexisting pathways and networks.”

Characteristics of the eumetazoan ancestor such as flagellated sperm, gastrulation, multiple germ layers, true epithelia, a lined gut, a neuromuscular system, multiple sensory systems, and fixed body axes can be inferred because they are conserved in modern descendants.

“Yet we can infer many of its characteristics --flagellated sperm, development through a process of gastrulation, multiple germ layers, true epithelia lying upon a basement membrane, a lined gut (enteron), a neuromuscular system, multiple sensory systems, and fixed body axes --since they are conserved features retained by its modern descendants.”

Many genes and gene families previously assumed to be chordate or vertebrate innovations are actually much more ancient.

“These results strongly suggest that many genes and gene families previously assumed to be chordate or vertebrate innovations are actually much more ancient, and conversely that the fruit fly, soil nematode, and sea squirt lineages have experienced higher levels of divergence and gene loss than Nematostella, consistent with phylogenetic the analysis described below.”

Both anemone and vertebrate genomes retain more ancestral eumetazoan features than sequences from flies and nematodes.

“This surprising similarity between Nematostella and vertebrates is a recurring theme of our analysis, indicating that both the anemone and vertebrate genomes retain more ancestral eumetazoan features than sequences from flies and nematodes.”

The dataset nominally contains up to four haplotypes at each locus.

“Our dataset thus nominally contains up to four haplotypes at each locus.”

The reconstructed ancestral gene set is necessarily incomplete because genes highly diverged or lost in descendants cannot be captured.

“Our reconstructed ancestral gene set is necessarily incomplete since we cannot capture genes that were present in the eumetazoan progenitor but became highly diverged or lost in one or more descendents, nevertheless it provides a starting point for further analysis.”

Fungi are not by themselves a sufficient outgroup for characterizing history of eumetazoan introns because putative ancient eukaryotic introns shared by animals and plants have been lost in fungi.

“Note that although fungi and animals are phylogenetically closer to each other than either group is to plants, fungi are not by themselves a sufficient outgroup for characterizing the history of eumetazoan introns, since there are putative ancient eukaryotic introns shared by modern animals and plants that have evidently been lost in fungi (46).”

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Analysis does not support extrapolating accelerated molecular evolution documented in flies and echinoderms to all invertebrates.

“While accelerated rates of molecular evolution have been documented in flies and echinoderms (43) relative to vertebrates, our analysis does not support the extrapolation of these higher rates to all invertebrates.”

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This analysis is protected from gene modeling artifacts because erroneous predictions near splice sites would disrupt alignment and remove such sequences.

“Note that this analysis is protected from the effects of gene modeling artifacts, since erroneous predictions in the vicinity of splice sites would disrupt alignment, thereby removing such sequences from consideration.”

From a genomic perspective, the eumetazoan ancestor more closely resembled modern vertebrates and anemones.

“Thus from a genomic perspective, the eumetazoan ancestor more closely resembled modern vertebrates and anemones.”

The assembly captures about 95% of known protein-coding content.

“Comparison of the assembled sequence with open reading frames derived from expressed sequence tags (ESTs, see below) shows that the assembly captures ~95% of the known protein coding content (32).”

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Expression domains in Nematostella occur with apparent bilateral symmetry, including a left-right plane of symmetry.

“The expression domains occur with apparent bilaterial symmetry, i.e., reflecting distinct directed axes both along and perpendicular to the main body axis, and with a left-right plane of symmetry (27)(28)(29).”

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Fruit fly, soil nematode, and sea squirt lineages have experienced higher levels of divergence and gene loss than Nematostella.

“These results strongly suggest that many genes and gene families previously assumed to be chordate or vertebrate innovations are actually much more ancient, and conversely that the fruit fly, soil nematode, and sea squirt lineages have experienced higher levels of divergence and gene loss than Nematostella, consistent with phylogenetic the analysis described below.”

These conserved linkages are the most ancient known outside prokaryotic operons.

“These are the most ancient conserved linkages known outside of prokaryotic operons.”

Nematostella is not a true broadcast spawner because females lay eggs in a jelly mass fixed to substrate.

“Nematostella, however, is not a true broadcast spawner, since while males release sperm into the water, females lay tens to hundreds of eggs encased in a jelly mass that becomes fixed to a benthic substrate.”

A crude molecular clock interpolation suggests the eumetazoan ancestor lived about 670-820 Mya.

“Using our branch lengths, a very crude molecular clock interpolation based on the eukaryotic time scales of Douzery et al. (42) suggests that the eumetazoan ancestor lived ~670-820 Mya (32).”

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• AssumptionRests on an assumption: The rate of protein evolution was constant on the eumetazoan stem for the molecular clock estimate.

Vertebrates have a much lower degree of gene loss than ecdysozoan model systems.

“In contrast, only 33 genes are found in Nematostella and both Drosophila and C. elegans but not in any vertebrate, representing putative deuterostome or vertebrate loss, indicating a much lower degree of gene loss in the vertebrates than in the ecdysozoan model systems.”

Other studies minimizing long branch attraction by additional taxa generate a phylogeny agreeing with our more limited taxon sampling but larger gene set.

“In other studies (e.g., (42)), the effect of long branch attraction is minimized by including additional taxa that break up long branches, generating a phylogeny that agrees with our more limited taxon sampling but larger gene set.”

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The draft assembly is sufficiently long-range to permit useful synteny analysis because half of predicted genes are in scaffolds containing 48 or more genes.

“Nevertheless, since half of the predicted genes are in scaffolds containing 48 or more genes, the present draft assembly is sufficiently long-range to permit useful analysis of synteny with other species, as shown below.”

Each of these families represents a single gene in the eumetazoan ancestor whose descendants survive in recognizable form in both cnidarians and bilaterians.

“Each family thus represents a single gene in the eumetazoan ancestor whose descendents survive in recognizable form as modern genes in both cnidarians and bilaterians.”

Introns shared between Nematostella and vertebrates and/or other bilaterians are most parsimoniously interpreted as conserved ancient eumetazoan introns.

“Introns that are shared between Nematostella and vertebrates and/or other bilaterians are most parsimoniously interpreted as conserved ancient eumetazoan introns (44).”

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A new method was developed to search for statistically significant conserved linkage groups that does not rely on gene order.

“To overcome this limitation we developed a new method to search for statistically significant conserved linkage groups that does not rely on gene order.”

Animals have a unique complement of these conserved gene families due to family expansion and contraction on the eumetazoan stem.

“Although these eumetazoan gene families are conserved with other eukaryotes, animals have a unique complement due to family expansion/contraction on the eumetazoan stem.”

The observation of conserved linkage groups is most easily explained as remnants of large ancestral chromosomal segments containing hundreds of genes that evolved without obvious constraint on gene order within each block.

“The observation of conserved linkage groups is most easily explained as the remnants of large ancestral chromosomal segments containing hundreds of genes that have evolved without obvious constraint on gene order within each block.”

Seven putative ancestral eumetazoan linkage groups link anemone scaffolds to multiple regions of the human genome in a manner consistent with multiple large-scale duplication events along the vertebrate lineage.

“Seven of the PALs link anemone scaffolds to multiple regions of the human genome in a manner consistent with multiple large-scale duplication events along the vertebrate lineage (reviewed, for example, in (53)).”

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Each class is most easily interpreted as segments of present-day Nematostella and human genomes descending from the same chromosome of the eumetazoan ancestor, defining a putative ancestral eumetazoan linkage group.

“Each class of scaffolds and chromosome segments is most easily interpreted as collecting together segments of the present day Nematostella and human genomes that descend from the same chromosome of the eumetazoan ancestor, and therefore defines a putative ancestral eumetazoan linkage group (PAL).”

These combinations arose through gene fusion or domain shuffling during eumetazoan stem evolution.

“due to gene fusions and/or domain shuffling events on the eumetazoan stem”

FAK is targeted to focal adhesions in eumetazoans because an animal-specific focal adhesion targeting domain was added to the ancient kinase domain.

“focal adhesion kinase (FAK) is targeted to focal adhesions in eumetazoans because of the addition of an animal specific focal adhesion targeting domain to the ancient kinase domain”

These seven putative ancestral eumetazoan linkage groups represent the ancestral preduplication linkage of these regions.

“These seven PALs represent the ancestral (preduplication) linkage of these regions.”

This is a lower bound on the true extent of remnant ancient linkage groups because analysis is limited by scaffold length and conservative statistical criteria.

“This is a lower bound on the true extent of the remnant ancient linkage groups, since our analysis is limited by the length of the Nematostella scaffolds and the use of conservative statistical criteria.”

Relaxing gene order constraints dramatically increases the number of such segments expected by chance, reducing power to detect more ancient conserved genome organization.

“Further relaxing gene order constraints dramatically increases the number of such segments expected by chance, reducing the power of this approach to detect even more ancient conserved genome organization in the face of intrachromosomal rearrangements.”

Hox genes in Nematostella and other cnidarians are expressed in spatial patterns consistent with an ancient role in embryonic development.

“Hox genes in Nematostella and other cnidarians are also expressed in spatial patterns consistent with an ancient role in embryonic development (54)(55)(56).”

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The ancestry of Type II novel genes traces to the eukaryotic radiation through their ancient domains.

“The ancestry of these genes can be traced back to the eukaryotic radiation through their ancient domains”

A general trend in signaling pathway evolution may have been co-option of cytosolic signaling components into pathways regulated by newly invented ligands and receptors.

“A general trend in the evolution of signaling pathways may have been the co-option of cytosolic signaling components into pathways that could be regulated by newly invented ligands and receptors”

• AssumptionThe authors state this as an assumption: Processes conserved between living species were likely functional in their common ancestor.

Because cnidarians and bilaterians share nearly complete signaling pathways, the novel eumetazoan genes were probably already integrated into these systems in the eumetazoan ancestor.

“The presence of essentially complete signal transduction pathways in the common gene set of cnidarians and bilaterians strongly suggests that the integration of novel eumetazoan genes into these systems was largely complete in the eumetazoan ancestor”

• AssumptionThe authors state this as an assumption: Processes conserved between living species were likely functional in their common ancestor.

This conserved linkage is the oldest known synteny outside prokaryotic operons.

“the oldest known conserved synteny outside of prokaryotic operons”

Retrotransposition of retrotransposons in the anemone genome has not been as efficient as propagation of DNA transposons, despite the high diversity of retrotransposons.

“It appears that retrotransposition of retrotransposons, despite their high diversity, has not been as efficient as propagation of DNA transposons in the anemone genome.”

The newly acquired repertoire of expanded gene families in humans almost certainly plays a key role in defining vertebrates and mammals.

“This newly acquired repertoire almost certainly plays a key role in defining vertebrates and mammals.”

Tx1-like elements form a novel clade of non-LTR retrotransposons that differs from L1 clade elements by strong target-site specificity.

“We suggest that Tx1-like elements form a novel clade of non-LTR retrotransposons differing from the L1 clade elements by the strong target-site specificity.”

Nematostella is the first metazoan in which Polintons constitute a substantial portion of the genome.

“It makes Nematostella the first metazoan with Polintons constituting a substantial portion of the genome (17).”

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The estimate of 18,345 bona fide Nematostella genes may be an overestimate because some genes with lower C-values are at the edges of short scaffolds and may be double-counted.

“Even this may be an overestimate, since quite a few of the genes with lower c-values are at the edges of short scaffolds, and their other half may be picked up by another scaffold, causing 2 annotations for a single gene.”

Fruit fly and nematode genomes are poor proxies for the ancestral eumetazoan genome, as they lost introns, genes, and linkages.

“their genomes are relatively poor models for the ancestral eumetazoan genome, having lost introns, genes, and gene linkages”

The variety of different types of DNA transposons found in the anemone genome is the highest among eukaryotic species studied so far.

“The variety of different types of DNA transposons found in the anemone genome is the highest among eukaryotic species studied so far.”

Nematostella has lost 11% of its introns since the last common ancestor with human.

“we estimate the loss in Nematostella since the last common ancestor (LCA) with human as 158 / (158 + 1258) = 11%.”

• AssumptionThe authors state this as an assumption: Intron losses have occurred independently in the human and Nematostella lineages.
• AssumptionThe authors state this as an assumption: The probability of independent intron insertion events at the same location is negligible.

Ancient cytosolic proteins talin, paxillin, Grb2, Sos, and Crk were brought under the control of two novel receptors, integrin a and integrin b.

“Various ancient cytosolic proteins (talin, paxillin, Grb2, Sos and Crk) have been brought under the control of two novel receptors, integrin a and integrin b (the former being a Type I novelty and the latter a Type II novelty).”

The method of counting orthologs and the null model control for independent tandem gene duplicates that could otherwise inflate observed orthologs when no conserved synteny remains.

“This method of counting orthologs, and this null model control naturally for independent tandem gene duplicates which could otherwise artifactually inflate the number of observed orthologs in circumstances where there is no remnant of conserved synteny, because tandem duplicates arising independently should be contained in a single reconstructed ancestral gene cluster.”

• AssumptionThe authors state this as an assumption: The null model treats the genes in scaffolds and segments as independently drawn from the 7,766 ancestral genes.

The nematode has gained a considerable number of introns, as also reported by Logsdon 2004.

“This suggests a considerable gain of introns in the nematodes, as also reported by [Logsdon 2004].”

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These collections of modern sequences are interpreted as descended from the same chromosomes or chromosomal segments of the common ancestor of eumetazoa, referred to as putative ancestral linkage groups (PALs).

“We interpret these collections of modern sequences to be descended from the same chromosomes, or chromosomal segments of the common ancestor of eumetazoa, and refer to them therefore as putative ancestral linkage groups, or PALs.”

• AssumptionThe authors state this as an assumption: The null model treats the genes in scaffolds and segments as independently drawn from the 7,766 ancestral genes.

Discontinuities in the distribution of orthologous gene positions within human chromosome arms, identified by a hidden Markov model, are most easily explained by chromosomal fusions or large-scale rearrangements in the human lineage that are recent relative to the time scale of gene order evolution.

“These discontinuities are most easily explained by chromosomal fusions or large-scale re-arrangements in the human lineage which are recent compared to the time scale of gene order evolution.”

Most core components of the FAK and Fyn/Shc integrin signaling pathways were present in the eumetazoan ancestor.

“A detailed look at integrin signaling (Figure5d) reveals that most of the core components of the FAK and Fyn/Shc pathways were present in the eumetazoan ancestor.”

Filtering out weak hits is important because their presence would significantly increase the false positive rate in detecting segments of conserved linkage.

“Note the importance of the filtering out weak hits in this method, as the presence of such hits would significantly increase the false positive rate in the detection of segments of conserved linkage.”